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Radiation grafting of NIPAAm and acryloxysuccinimide onto PP films and sequent crosslinking with polylysine
European Polymer Journal 46 (2010) 1074–1083
Contents lists available at ScienceDirect
European Polymer Journal journal homepage: www.elsevier.com/locate/europolj
Radiation grafting of NIPAAm and acryloxysuccinimide onto PP films and sequent crosslinking with polylysine Lorena García-Uriostegui, Guillermina Burillo *, Emilio Bucio Departamento de Química de Radiaciones y Radioquímica, Instituto de Ciencias Nucleares, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, 04510 México D.F., Mexico
a r t i c l e
i n f o
Article history: Received 13 November 2009 Received in revised form 13 January 2010 Accepted 19 January 2010 Available online 25 January 2010 Keywords: Radiation grafting NIPAAm NAS Binary grafting
a b s t r a c t N-isopropylacrylamide (NIPAAm) and N-acryloxysuccinimide (NAS) were grafted from their binary mixtures in tetrahydrofurane (THF) and toluene solutions onto polypropylene (PP) films by the pre-irradiation oxidative method in air. Effects of pre-irradiation dose, dose rate, and monomer concentrations (NAS/NIPAAm) were studied. The grafted copolymers exhibited the lower critical solution temperature (LCST) at around 31 °C. Based on its thermo-reversible behavior, this system has been used for immunoassay, drug delivery, separation processes and immobilization of enzymes. N-acryloxysuccinimide (NAS) has been used as an active ester to bind proteins through amide bond formation with lysine, and because of this property, the grafted copolymer has been crosslinked with polylysine. Techniques used to characterize the films included differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), infrared (FTIR-ATR) and elemental analysis. Results on thermo-sensitivity are presented. This new system could find applications in vesicle immobilizations. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Materials and procedures for protein immobilization are important in the developing technology which uses enzyme catalysis for the synthesis of complex organic substrates [1–3]. The immobilization of membranes on solid supports makes several applications possible such as the use of solid supported membranes as containers for controlled drug release systems. Different methods have been used to immobilize lipid membranes on a solid support. Lipids can be immobilized by covalent binding, by entrapment in a solid matrix or by ionic binding [4–7]. The ionic interactions are advantageous because the electrostatic interaction is less likely to perturb the integrity of the membrane, and the solid support can be washed and reused. These positively charged matrixes are suitable to immobilize cells such as bio-membranes containing nega* Corresponding author. Tel.: +52 55 56224674; fax: +52 55 5622 4707. E-mail address: [email protected] (G. Burillo). 0014-3057/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2010.01.018
tively charged lipids. Hydrogels based on acrylamides and acryloxysuccinimide crosslinked with polylysine were studied by Percot [4] and Pollak [8] as immobilization systems; however, an obvious limitation of the normal hydrogels in some applications is their poor mechanical properties in a highly swollen state. NIPAAm/NAS grafted onto polypropylene, PP-g-(NAS/NIPAAm), when crosslinked with polylysine gives films with good mechanical properties and, because of the reversibility of entrapping and release, have the potential to be used several times. N-isopropylacrylamide (NIPAAm) has been selected for the development of the immobilization system because it is hydrophilic and non-denaturating for many proteins [8], and it has a lower critical solution temperature (LCST) from 30 to 35 °C. Related NIPAAm hydrogel systems and graft copolymers retain the thermosensitivity. This property can be used for controlled release of vesicles immobilized on the gel. Poly(N-acryloxysuccinimide) (PNAS) provides a functional group which is readily displaced by the amino groups of lysine; these amino groups are the
L. García-Uriostegui et al. / European Polymer Journal 46 (2010) 1074–1083
anchoring element of vesicles, and also acts as a crosslinking agent of PNAS. Acryloxysuccinimide NAS has been used as an active ester to bind proteins through bond formation with lysine residues [4,8–11], and it has interesting applications in water purification and biology [12]. Ortega et al. synthesized a new interpenetrating polymer network (IPN) of PNIPAAm/PNAS crosslinked by polylysine [13]. They found a LCST between 29 and 31 °C, with better mechanical properties than the copolymer gels. Grafting of these two monomers, onto PP films by gamma radiation provides an easier method of synthesis along with the good mechanical properties of IPNs. Current investigations and developments are in two main directions: the use of radiation chemical methods to produce biocompatible polymeric materials, and the application of these methods to immobilize bioactive materials in polymeric matrices. There are two main methods of irradiation grafting. The first and simplest radiation chemical method utilizes direct radiation grafting of a vinyl monomer onto a preformed polymer. How-
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ever, because the action of ionizing radiation on matter is unselective, any substance added to the monomer is also radiolyzed and contributes to homopolymerization without grafting effect, especially in case of very reactive monomers. The second grafting method utilizes radiation peroxidized polymers. In this method the polymer is irradiated in the presence of air, and diperoxides and hydroperoxides are formed. Upon heating, these peroxides form radicals which interact with vinyl monomers and initiate the grafting reaction. No homopolymerization occurs in this reaction other than chain transfer to the monomer or by thermal initiation. The thermal dissociation of the hydroperoxide gives rise to an equivalent number of graft copolymer and homopolymer molecules [14,15]. In this work mutual grafting of NIPAAn/NAS onto PP films was realized by gamma irradiation (pre-irradiation oxidative method) followed by crosslinking with polylysine. The swelling behavior and thermosensitivity of the films were characterized.
Scheme 1. Mechanism of grafting.
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2. Experimental 2.1. Materials All reagents were obtained from Aldrich Chemical USA. NIPAAm, was purified by recrystallization in toluene/hexane (50/50); N-hydroxysuccinimide (NHS), morpholinoethanol sulfonic acid (MES) and polylysine (Mw 400– 2000) were used as received. Acryloyl chloride was distilled in vacuum to remove any trace of inhibitor; chloroform was refluxed for 12 h with MgSO4 and distilled from P2O5, and triethylamine (Net3) was distilled and dried with KOH; tetrahydrofuran (THF) was refluxed for 12 h with Na and distilled from P2O5. 2.2. Synthesis of N-acryloxysuccinimide The N-acryloxysuccinimide (NAS) was synthesized by the Pollak method [8]; N-hydroxysuccinimide (15 g, 0.13 mol) was dissolved in 100 ml of chloroform at 0 °C. Distilled acryloyl chloride (13.04 g, 0.14 mol) was added during a 20 min period to the stirred reaction mixture. After being stirred for an additional 20 min at 0 °C, the solution was washed with cold water saturated NaCl solution, dried with MgSO4, and filtered. Hydroquinone (3 mg) was added to the chloroform solution which was concentrated to a volume of 30 ml using a rotary evaporator and then filtered. Ethyl acetate (60 ml) and n-hexane (200 ml) were added slowly with stirring to the chloroform solution
which was left to stand at 0 °C for several hours. The precipitated, colorless crystals were separated by filtration and washed with a cold mixture of n-hexane and ethyl acetate (4:1), then another portion of n-hexane and ethyl acetate (9:1), and finally with two portions of n-hexane. The crystals were dried in vacuum at room temperature to constant weight; 15.3 g (71%) was obtained, mp 69.5–71 °C. Elemental analysis C, 49.64%; H, 3.90%; O, 37.52%; N, 8.92%.
2.3. Grafting of NAS/NIPAAm onto PP The process for grafting monomers is shown in Scheme 1. The pre-washed and dried PP films (thickness 60 lm and 1 cm 3 cm) were irradiated in air (pre-irradiation peroxidation method) at room temperature with a 60 Co Gamma-Beam source 651-PT source at a dose rate from 2 to 12 kGy h1; and radiation dose from 80 to 120 kGy. The pre-irradiated films were placed in glass ampoules which contained a 50% solution of NAS/NIPAAm in THF/toluene (1/3). The system was degassed by repeated freeze– thaw cycles, and then the ampoules were vacuum sealed. They were then heated in a water bath at several different temperatures and reaction times. Grafted films were extracted by stirring in THF for 24 h in order to remove remaining monomers and the homopolymer not grafted. The grafted films were dried under vacuum. The grafting yield (Yg) was calculated by the equation:
Y g ð%Þ ¼ 100 ðW g W 0 Þ=W 0
Table 1 Molar ratio of NAS/NIPAAm, in the grafted films. NAS/NIPAAm initial (w/w)
Graft (%w)
Dose (kGy)
Dose rate (kGy h1)
NAS/NIPAAm in the film
15/85 15/85 15/85 30/70 30/70
17 24 29 46 112
80 100 100 100 100
– 3 3 3 3
0.77/1 0.33/1 0.22/1 6.0/1 1.5/1
ð1Þ
where Wg and Wo are the mass of the grafted and initial films, respectively. The composition of the copolymers in the chain grafted to PP was measured by elemental analysis (Columbia Analytics USA) (Table 1), and can be deduced from the theoretical plot of composition of polymers as a function of monomer concentrations, from the reactivity of both monomers, and the equation:
Scheme 2. Crosslinking mechanism.
L. García-Uriostegui et al. / European Polymer Journal 46 (2010) 1074–1083
F1 ¼
r1 f12
r 1 f12 þ f1 f2 þ 2f 1 f2 þ r2 f22
ð2Þ
where NAS reactivity r1 = 1.93 and NIPAAM reactivity r2 = 0.27. 2.4. Crosslinking of the copolymers with polylysine Poly (L-lysine) with molecular weight of 400–2000 was used as the crosslinker. The binary grafted film (0.0312 g) was swollen in 5 ml of dimethylformamide (DMF). Polylysine (50 mg) in 10 ml of 2-N-morpholonoethanesulfonic acid (MES) buffer (50 nM, pH 7.4) was added to the polymer and the mixture was incubated at 10 °C during 48 h with constant stirring, then at room temperature overnight [4]. The resulting films were repeatedly washed with water, and vacuum dried. The succinimide group content in the copolymer before and after crosslinking was monitored by FTIR spectroscopy, and indirectly by UV spectroscopy at 260 nm by measuring the hydroxy succinimide formed as a residual compound after the crosslinking reaction Scheme 2. 2.5. Swelling behavior The equilibrium swelling time of the grafted copolymer films was measured gravimetrically. The samples were swollen in distilled water at room temperature for several periods of time. The excess water on the surface of grafted films was wiped off by filter paper and the samples were weighed; the immersion time and drying procedure were repeated until the mass of swollen samples was constant. The swelling percent was defined as follows:
Sð%Þ ¼ 100½ðW s W d Þ=W d
ð3Þ
where Ws and Wd are the mass of the swollen and dry films, respectively.
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2.6. LCST determination The thermosensitive response was studied from 10 to 50 °C by swelling with distilled water (pH 7) at several different temperatures, and the LCST was calculated from the inflection point of the plot of swelling percentage as a function of temperature. Thermosensitivity was defined as the ratio of the swelling percentage of samples at 10 and 40 °C. 2.7. Characterization of grafted films Fourier transform infrared spectra (in total attenuated reflection, FTIR-ATR mode) of the films were recorded using a Perkin–Elmer PARAGON 500; with a SeZn glass in contact with the sample surface. Thermogravimetric analysis in a nitrogen atmosphere was performed with a TGA Q50 (TA Instruments, new Castle, DE) to determine decomposition temperatures. Differential scanning calorimetry (DSC) studies were performed on a TA Instruments Model 2010. Atomic force microscopy (AFM) results were obtained using a JEOL JFPM 4210. A UV/VIS Spectrophotometer (Cary 100 from Varian) was used to determine the residual HNAS groups after the crosslinking reaction; water contact angles on the samples surfaces were determined with a KRUSS, DSA 100 Germany. 3. Results and discussion Fig. 1 shows the grafting yield of NAS/NIPAAm as a function of reaction time, at different pre-irradiation doses, from 80 to 120 kGy. The grafting yield increases with radiation dose, because of increasing formation of peroxy and hydroperoxy groups. These groups will form macro-radicals upon heating which initiate the graft copolymerization (Scheme 1). At 120 kGy the films are easily broken, because of degradation of mechanical properties by the presence of the monomers and radiation degradation of the polypropylene backbone. At this pre-irradiation dose the result
Fig. 1. Grafting yield of NAS/NIPAAa as a function of reaction time, at different pre-irradiation dose: 120 kGy, dd110 kGy, j j 100 kGy, NN 80 kGy; temperature 57 °C, dose rate 3 kGy h1.
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of grafting percentage are erratic and not reproducibles. The optimum reaction time of heating was 6 h, at which the grafting percentage leveled off; all peroxides groups were destroyed, and the radicals formed were reacted. Fig. 2 shows the grafting yield as a function of time at dose rates of 3, 6 and 12 kGy h1. The monomer concentration ratio of 15/85 NAS/NIPAAm and total irradiation dose of 110 kGy remained constant at all three dose rates. The grafting yield decreases with an increase of dose rate due to recombination of radicals before oxygen diffusion takes place. Grafting percentage as a function of reaction time, at different NAS/NIPAAm monomer concentration ratios and at a total radiation dose of 100 kGy and dose rate 3 kGy h1 are shown in Fig. 3. The grafting yield increases with concentration ratio from 15/85 to 30/70 NAS/NIPAAm ratio because of the higher reactivity of NAS macro-radicals; the
reactivity toward copolymerization for NAS is 1.93 and 0.28 for NIPAAm [10]. Monomer concentration ratios greater than 30/70 (NAS/NIPAAm), result in NAS homopolymer formation and lower grafting yield, which could arise because of steric hindrance. Both the grafting yield as a function of NAS concentration at a dose rate of 3 kGy h1 (Fig. 3) and at 6 kGy (Fig. 4) show similar behavior with a maximum grafting percentage observed at about 22% NAS. Thus the optimum concentration to obtain a maximum grafting percentage at different dose rate is around 22%. The theoretical molar ratio of the monomer units in a copolymer of NAS/NIPAAm formed at low doses have been plotted as a function of the initial concentration of the monomers and are shown in Fig. 5. The molar ratio of NAS/NIPAAm in the grafted films is shown in Table 1. The theoretical molar ratio of the monomers in the copoly-
Fig. 2. Grafting yield as a function of time, at different dose rate: 3 kGy h1, jj 6 kGy h1, NN 12 kGy h1. monomer concentration 15/85 NAS/NIPAAm, irradiation dose 110 kGy.
Fig. 3. Grafting percentage as a function of reaction time, at different monomer concentration: 50/50, NN 30/70, jj 15/85 NAS/NIPAAm; radiation dose 100 kGy, dose rate 3 kGy h1.
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Fig. 4. Grafting percentage as a function of reaction time, at different monomer concentration: dd 40/60, NN 30/70, jj 22/78, 15/85 NAS/NIPAAm; radiation dose 100 kGy, dose rate 6 kGy h1.
Fig. 5. Theoretical molar ratio of the monomer units, in the copolymer, as a function of the initial concentration of the monomers.
mer are approximately the same in the case of the graft copolymer at dose up to 80 kGy and a concentration of NAS/NIPAAm of 15/85 (w/w) which corresponds to a molar ratio of 0.37. The amount of NAS in the graft copolymer actually decreases with an increase of grafting percentage due to the higher reactivity of NAS; higher homopolymerization of the NAS monomer takes place with an increase in radiation dose. Fig. 6 shows the equilibrium swelling time of PP-g-(NAS/ NIPAAm) films, at different grafting percentages and preirradiation doses, at a concentration ratio of monomers of 15/85. In this plot we observed samples with different pre-irradiation doses in order to compare swelling behavior at different grafting percentages. The swelling percentage is higher at lower grafting percentage, with a maximum of 14% swelling observed in the films with 29% (100 kGy) grafting .This effect could be due to the ratio of NAS/NIPAAm; this ratio decreases with increasing graft percentage (Table 1). It is known that PNAS is less hydrophilic than PNIPAAm, therefore increasing content of NIPAAm in the copolymer increase the hydrophilicity of the system. Fig. 7 shows the typical temperature-dependence on the contact angle measured for the thermosensitive NIPAAm with a LCST around 32 °C in the binary grafted film.
Good reversibility of swelling–deswelling behavior at 20 and 40 °C can be observed in Fig. 8; this reversibility improves after three cycles of the swelling–deswelling process because relaxation of the side chains which allows them to become more oriented in the system occurs after several swelling–deswelling cycles. Fig. 9 shows the FTIR spectra of PP films, NAS, NIPAAm and the binary grafted PP-g-NAS/NIPAAm (57% graft). The spectrum of the binary grafted polymer shows the presence of the corresponding groups of the two grafted monomers and also the presence of a band at 1100 cm1corresponding to the ether group resulting from the bond between PP and the NIPAAm formed on the preirradiation oxidative method. Fig. 10 shows the FTIR spectra of PP-g-NAS/NIPAAm 57% graft, before and after crosslinking by polylysine. It is possible to confirm the presence of the polylysine in the system because the bands corresponding to the succinimide group almost disappear (the crosslinking is not a quantitative reaction), and new bands assigned to NH3+ at 3335 and 1510 cm1 did not appear, because they are overlapped with the bands of N–H in 3400 and 1541 cm1. To confirm the crosslinking reaction, the residual HNAS groups could be quantified by UV spectroscopy.
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Fig. 6. Equilibrium swelling time of PP-g-(NAS/NIPAAm) films at different grafting percentages and pre-irradiation doses: NN 29% (radiation dose 100 kGy), j j 89%, and 141% (radiation dose 120 kGy), monomer concentration 15/85 NAS/NIPAAm.
Fig. 7. Contact angle of PP-g-NAS/NIPAAm, as a function of temperature; graft of 107%, radiation dose 100 kGy, dose rate 3 kGy h1, monomer concentration NAS/NIPAAm 15/85.
Fig. 8. Reversibility of swelling–deswelling behavior at 20 and 40 °C, 22% grafted film, radiation dose 100 kGy, monomer concentration 15/85 (NAS/ NIPAAm), dose rate 3 kGy h1.
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Fig. 9. FTIR spectra of different systems: (a) PP film, (b) NAS, (c) NIPAAm, (d) PP-g-NAS/NIPAAm 57% (radiation dose 100 kGy, monomer concentration 30/ 70).
Fig. 10. FTIR spectra of: a) PP-g-NAS/NIPAAm 57% graft (radiation dose 100 kGy, NAS/NIPAAm 30/70), b) PP-g-(NAS/NIPAAm) crosslinked by polylysine.
Fig. 11. DSC thermogram of PP-g-NAS/NIPAAm; (a) 131% graft; (b) 29% graft.
The DSC thermogram of the binary grafted film (131% graft, 120 kGy) is shown in Fig. 11. It shows a melting point
at 165 °C corresponding to PP, a glass transitions at 115 °C due to PNIPAAm and a poorly defined glass transitions at
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Fig. 12. TGA thermogram of (a) PP; (b) NIPAAm; (c) NAS; (d) PP-g-NIPAAm 131% graft.
Fig. 13. AFM of PP-g-NAS/NIPAAm: crosslinked by polylysine (left), grafted (right side).
60 °C due to PNAS. These glass transitions of the monomer are not present in films with low grafting percentage of 29% (100 kGy). TGA thermograms of different systems were observed in Fig. 12, PP has a 10% weight loss at 419 °C and decomposition temperature at 450 °C. This 10% loss weight temperature decreases with an increase in grafting percentage until, at 255 °C and 131% graft, two decomposition temperatures are observed due to PNAS and PNIPAAm, respectively. AFM studies show that the grafted film crosslinked with polylysine exhibits a changed surface morphology and increased roughness (rms), from 186 nm in the binary grafted film without crosslinking to 595 nm after crosslinking (Fig. 13). This change in surface morphology helps to confirm the crosslinking of the NAS groups by the polylisine. SEM determinations did not show any well defined morphology.
broken. Low dose rates are favorable for increasing grafting yield. The optimum conditions of synthesis were radiation dose of 100 kGy, dose rate of 3 kGy h1, and reaction time between 6 and 8 h. The LCST of the new system has the same value as that of PNIPAAm, and the reversibility is good. Crosslinking with polylysine was confirmed by FTIR and morphology changes on the surface by AFM. This system could be used in liposome vesicle immobilization with better advantages due to better mechanical properties (i.e. it could be handling without breakage), faster velocity of response than other forms of network systems and reusability of the films because of good reversibility of the swelling–deswelling process. Acknowledgements The authors thank S. Castillo, F. Garcia, B. Leal and M.Cruz, from ICN UNAM, and to DGAPA UNAM, Grant IN 200208, for economical support.
4. Conclusions References A new binary grafting system of NAS/NIPAAm has been prepared by the pre-irradiation oxidative method, with an equilibrium swelling time of about 50 min. The grafting efficiency increased with radiation dose, but at radiation dose higher than 100 kGy, the film was rigid and easily
[1] Bedows CG, Gil H, Guthrie JT. The use of graft copolymers as enzyme supports, the preparation and use of polyethylene-co acrylic acid supports. Polym Bull 1980;3:645–53. [2] Chen J-P, Chiu S-H. A poly(N-isopropyl acrylamide-co-Nacryloxysuccinimide-co-2-hydroxyethyl methacrylate) composite
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[9] Ferruti P, Bettelli A, Feré A. High polymers of acrylic and methacrylic esters of N-hydroxysuccinimide as polyacrylamide and polymethacryliamide precursors. Polymer 1972;13:462–4. [10] Yang HJ, Monji N, Hoffman AJ. Preparation of a thermally phaseseparating copolymer, poly(N-isopropylacrylamide-co-Nacryloxysuccinimide), with a controlled number of active esters per polymer chain. Polym Chem 1990;28:219–26. [11] Elbert DL, Hubbell JA. Reduction of fibrous adhesion formation, by a copolymer possessing an affinity for anionic surfaces. J Biomed Mater Res 1998;42:55–65. [12] Relogio P, Charreyre MT, Farinha JPS, Martinho JM, Oichot C. Welldefined polymer precursors synthesized by RAFT polymerization of N,N-dimethylacrylamide/N-acryloxysuccinimide: random and block copolymers. Polymer 2004;45:8639–49. [13] Ortega A, Bucio E, Burillo G. New interpenetrating polymer networks of N-isopropylacrylamide/N-acryloxysuccinimide: synthesis and characterization. Polym Bull 2008;60:515–24. [14] Chapiro A. Radiation chemistry of polymeric systems. England: John Wiley and Sons; 1962. [15] Ivanov VS. Radiation chemistry of polymers. In: Jonge CRHI, series ed. New concepts in polymer science. The Netherlands: Ultrecht; 1992.
Contents lists available at ScienceDirect
European Polymer Journal journal homepage: www.elsevier.com/locate/europolj
Radiation grafting of NIPAAm and acryloxysuccinimide onto PP films and sequent crosslinking with polylysine Lorena García-Uriostegui, Guillermina Burillo *, Emilio Bucio Departamento de Química de Radiaciones y Radioquímica, Instituto de Ciencias Nucleares, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, 04510 México D.F., Mexico
a r t i c l e
i n f o
Article history: Received 13 November 2009 Received in revised form 13 January 2010 Accepted 19 January 2010 Available online 25 January 2010 Keywords: Radiation grafting NIPAAm NAS Binary grafting
a b s t r a c t N-isopropylacrylamide (NIPAAm) and N-acryloxysuccinimide (NAS) were grafted from their binary mixtures in tetrahydrofurane (THF) and toluene solutions onto polypropylene (PP) films by the pre-irradiation oxidative method in air. Effects of pre-irradiation dose, dose rate, and monomer concentrations (NAS/NIPAAm) were studied. The grafted copolymers exhibited the lower critical solution temperature (LCST) at around 31 °C. Based on its thermo-reversible behavior, this system has been used for immunoassay, drug delivery, separation processes and immobilization of enzymes. N-acryloxysuccinimide (NAS) has been used as an active ester to bind proteins through amide bond formation with lysine, and because of this property, the grafted copolymer has been crosslinked with polylysine. Techniques used to characterize the films included differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), infrared (FTIR-ATR) and elemental analysis. Results on thermo-sensitivity are presented. This new system could find applications in vesicle immobilizations. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Materials and procedures for protein immobilization are important in the developing technology which uses enzyme catalysis for the synthesis of complex organic substrates [1–3]. The immobilization of membranes on solid supports makes several applications possible such as the use of solid supported membranes as containers for controlled drug release systems. Different methods have been used to immobilize lipid membranes on a solid support. Lipids can be immobilized by covalent binding, by entrapment in a solid matrix or by ionic binding [4–7]. The ionic interactions are advantageous because the electrostatic interaction is less likely to perturb the integrity of the membrane, and the solid support can be washed and reused. These positively charged matrixes are suitable to immobilize cells such as bio-membranes containing nega* Corresponding author. Tel.: +52 55 56224674; fax: +52 55 5622 4707. E-mail address: [email protected] (G. Burillo). 0014-3057/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2010.01.018
tively charged lipids. Hydrogels based on acrylamides and acryloxysuccinimide crosslinked with polylysine were studied by Percot [4] and Pollak [8] as immobilization systems; however, an obvious limitation of the normal hydrogels in some applications is their poor mechanical properties in a highly swollen state. NIPAAm/NAS grafted onto polypropylene, PP-g-(NAS/NIPAAm), when crosslinked with polylysine gives films with good mechanical properties and, because of the reversibility of entrapping and release, have the potential to be used several times. N-isopropylacrylamide (NIPAAm) has been selected for the development of the immobilization system because it is hydrophilic and non-denaturating for many proteins [8], and it has a lower critical solution temperature (LCST) from 30 to 35 °C. Related NIPAAm hydrogel systems and graft copolymers retain the thermosensitivity. This property can be used for controlled release of vesicles immobilized on the gel. Poly(N-acryloxysuccinimide) (PNAS) provides a functional group which is readily displaced by the amino groups of lysine; these amino groups are the
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anchoring element of vesicles, and also acts as a crosslinking agent of PNAS. Acryloxysuccinimide NAS has been used as an active ester to bind proteins through bond formation with lysine residues [4,8–11], and it has interesting applications in water purification and biology [12]. Ortega et al. synthesized a new interpenetrating polymer network (IPN) of PNIPAAm/PNAS crosslinked by polylysine [13]. They found a LCST between 29 and 31 °C, with better mechanical properties than the copolymer gels. Grafting of these two monomers, onto PP films by gamma radiation provides an easier method of synthesis along with the good mechanical properties of IPNs. Current investigations and developments are in two main directions: the use of radiation chemical methods to produce biocompatible polymeric materials, and the application of these methods to immobilize bioactive materials in polymeric matrices. There are two main methods of irradiation grafting. The first and simplest radiation chemical method utilizes direct radiation grafting of a vinyl monomer onto a preformed polymer. How-
1075
ever, because the action of ionizing radiation on matter is unselective, any substance added to the monomer is also radiolyzed and contributes to homopolymerization without grafting effect, especially in case of very reactive monomers. The second grafting method utilizes radiation peroxidized polymers. In this method the polymer is irradiated in the presence of air, and diperoxides and hydroperoxides are formed. Upon heating, these peroxides form radicals which interact with vinyl monomers and initiate the grafting reaction. No homopolymerization occurs in this reaction other than chain transfer to the monomer or by thermal initiation. The thermal dissociation of the hydroperoxide gives rise to an equivalent number of graft copolymer and homopolymer molecules [14,15]. In this work mutual grafting of NIPAAn/NAS onto PP films was realized by gamma irradiation (pre-irradiation oxidative method) followed by crosslinking with polylysine. The swelling behavior and thermosensitivity of the films were characterized.
Scheme 1. Mechanism of grafting.
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2. Experimental 2.1. Materials All reagents were obtained from Aldrich Chemical USA. NIPAAm, was purified by recrystallization in toluene/hexane (50/50); N-hydroxysuccinimide (NHS), morpholinoethanol sulfonic acid (MES) and polylysine (Mw 400– 2000) were used as received. Acryloyl chloride was distilled in vacuum to remove any trace of inhibitor; chloroform was refluxed for 12 h with MgSO4 and distilled from P2O5, and triethylamine (Net3) was distilled and dried with KOH; tetrahydrofuran (THF) was refluxed for 12 h with Na and distilled from P2O5. 2.2. Synthesis of N-acryloxysuccinimide The N-acryloxysuccinimide (NAS) was synthesized by the Pollak method [8]; N-hydroxysuccinimide (15 g, 0.13 mol) was dissolved in 100 ml of chloroform at 0 °C. Distilled acryloyl chloride (13.04 g, 0.14 mol) was added during a 20 min period to the stirred reaction mixture. After being stirred for an additional 20 min at 0 °C, the solution was washed with cold water saturated NaCl solution, dried with MgSO4, and filtered. Hydroquinone (3 mg) was added to the chloroform solution which was concentrated to a volume of 30 ml using a rotary evaporator and then filtered. Ethyl acetate (60 ml) and n-hexane (200 ml) were added slowly with stirring to the chloroform solution
which was left to stand at 0 °C for several hours. The precipitated, colorless crystals were separated by filtration and washed with a cold mixture of n-hexane and ethyl acetate (4:1), then another portion of n-hexane and ethyl acetate (9:1), and finally with two portions of n-hexane. The crystals were dried in vacuum at room temperature to constant weight; 15.3 g (71%) was obtained, mp 69.5–71 °C. Elemental analysis C, 49.64%; H, 3.90%; O, 37.52%; N, 8.92%.
2.3. Grafting of NAS/NIPAAm onto PP The process for grafting monomers is shown in Scheme 1. The pre-washed and dried PP films (thickness 60 lm and 1 cm 3 cm) were irradiated in air (pre-irradiation peroxidation method) at room temperature with a 60 Co Gamma-Beam source 651-PT source at a dose rate from 2 to 12 kGy h1; and radiation dose from 80 to 120 kGy. The pre-irradiated films were placed in glass ampoules which contained a 50% solution of NAS/NIPAAm in THF/toluene (1/3). The system was degassed by repeated freeze– thaw cycles, and then the ampoules were vacuum sealed. They were then heated in a water bath at several different temperatures and reaction times. Grafted films were extracted by stirring in THF for 24 h in order to remove remaining monomers and the homopolymer not grafted. The grafted films were dried under vacuum. The grafting yield (Yg) was calculated by the equation:
Y g ð%Þ ¼ 100 ðW g W 0 Þ=W 0
Table 1 Molar ratio of NAS/NIPAAm, in the grafted films. NAS/NIPAAm initial (w/w)
Graft (%w)
Dose (kGy)
Dose rate (kGy h1)
NAS/NIPAAm in the film
15/85 15/85 15/85 30/70 30/70
17 24 29 46 112
80 100 100 100 100
– 3 3 3 3
0.77/1 0.33/1 0.22/1 6.0/1 1.5/1
ð1Þ
where Wg and Wo are the mass of the grafted and initial films, respectively. The composition of the copolymers in the chain grafted to PP was measured by elemental analysis (Columbia Analytics USA) (Table 1), and can be deduced from the theoretical plot of composition of polymers as a function of monomer concentrations, from the reactivity of both monomers, and the equation:
Scheme 2. Crosslinking mechanism.
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F1 ¼
r1 f12
r 1 f12 þ f1 f2 þ 2f 1 f2 þ r2 f22
ð2Þ
where NAS reactivity r1 = 1.93 and NIPAAM reactivity r2 = 0.27. 2.4. Crosslinking of the copolymers with polylysine Poly (L-lysine) with molecular weight of 400–2000 was used as the crosslinker. The binary grafted film (0.0312 g) was swollen in 5 ml of dimethylformamide (DMF). Polylysine (50 mg) in 10 ml of 2-N-morpholonoethanesulfonic acid (MES) buffer (50 nM, pH 7.4) was added to the polymer and the mixture was incubated at 10 °C during 48 h with constant stirring, then at room temperature overnight [4]. The resulting films were repeatedly washed with water, and vacuum dried. The succinimide group content in the copolymer before and after crosslinking was monitored by FTIR spectroscopy, and indirectly by UV spectroscopy at 260 nm by measuring the hydroxy succinimide formed as a residual compound after the crosslinking reaction Scheme 2. 2.5. Swelling behavior The equilibrium swelling time of the grafted copolymer films was measured gravimetrically. The samples were swollen in distilled water at room temperature for several periods of time. The excess water on the surface of grafted films was wiped off by filter paper and the samples were weighed; the immersion time and drying procedure were repeated until the mass of swollen samples was constant. The swelling percent was defined as follows:
Sð%Þ ¼ 100½ðW s W d Þ=W d
ð3Þ
where Ws and Wd are the mass of the swollen and dry films, respectively.
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2.6. LCST determination The thermosensitive response was studied from 10 to 50 °C by swelling with distilled water (pH 7) at several different temperatures, and the LCST was calculated from the inflection point of the plot of swelling percentage as a function of temperature. Thermosensitivity was defined as the ratio of the swelling percentage of samples at 10 and 40 °C. 2.7. Characterization of grafted films Fourier transform infrared spectra (in total attenuated reflection, FTIR-ATR mode) of the films were recorded using a Perkin–Elmer PARAGON 500; with a SeZn glass in contact with the sample surface. Thermogravimetric analysis in a nitrogen atmosphere was performed with a TGA Q50 (TA Instruments, new Castle, DE) to determine decomposition temperatures. Differential scanning calorimetry (DSC) studies were performed on a TA Instruments Model 2010. Atomic force microscopy (AFM) results were obtained using a JEOL JFPM 4210. A UV/VIS Spectrophotometer (Cary 100 from Varian) was used to determine the residual HNAS groups after the crosslinking reaction; water contact angles on the samples surfaces were determined with a KRUSS, DSA 100 Germany. 3. Results and discussion Fig. 1 shows the grafting yield of NAS/NIPAAm as a function of reaction time, at different pre-irradiation doses, from 80 to 120 kGy. The grafting yield increases with radiation dose, because of increasing formation of peroxy and hydroperoxy groups. These groups will form macro-radicals upon heating which initiate the graft copolymerization (Scheme 1). At 120 kGy the films are easily broken, because of degradation of mechanical properties by the presence of the monomers and radiation degradation of the polypropylene backbone. At this pre-irradiation dose the result
Fig. 1. Grafting yield of NAS/NIPAAa as a function of reaction time, at different pre-irradiation dose: 120 kGy, dd110 kGy, j j 100 kGy, NN 80 kGy; temperature 57 °C, dose rate 3 kGy h1.
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of grafting percentage are erratic and not reproducibles. The optimum reaction time of heating was 6 h, at which the grafting percentage leveled off; all peroxides groups were destroyed, and the radicals formed were reacted. Fig. 2 shows the grafting yield as a function of time at dose rates of 3, 6 and 12 kGy h1. The monomer concentration ratio of 15/85 NAS/NIPAAm and total irradiation dose of 110 kGy remained constant at all three dose rates. The grafting yield decreases with an increase of dose rate due to recombination of radicals before oxygen diffusion takes place. Grafting percentage as a function of reaction time, at different NAS/NIPAAm monomer concentration ratios and at a total radiation dose of 100 kGy and dose rate 3 kGy h1 are shown in Fig. 3. The grafting yield increases with concentration ratio from 15/85 to 30/70 NAS/NIPAAm ratio because of the higher reactivity of NAS macro-radicals; the
reactivity toward copolymerization for NAS is 1.93 and 0.28 for NIPAAm [10]. Monomer concentration ratios greater than 30/70 (NAS/NIPAAm), result in NAS homopolymer formation and lower grafting yield, which could arise because of steric hindrance. Both the grafting yield as a function of NAS concentration at a dose rate of 3 kGy h1 (Fig. 3) and at 6 kGy (Fig. 4) show similar behavior with a maximum grafting percentage observed at about 22% NAS. Thus the optimum concentration to obtain a maximum grafting percentage at different dose rate is around 22%. The theoretical molar ratio of the monomer units in a copolymer of NAS/NIPAAm formed at low doses have been plotted as a function of the initial concentration of the monomers and are shown in Fig. 5. The molar ratio of NAS/NIPAAm in the grafted films is shown in Table 1. The theoretical molar ratio of the monomers in the copoly-
Fig. 2. Grafting yield as a function of time, at different dose rate: 3 kGy h1, jj 6 kGy h1, NN 12 kGy h1. monomer concentration 15/85 NAS/NIPAAm, irradiation dose 110 kGy.
Fig. 3. Grafting percentage as a function of reaction time, at different monomer concentration: 50/50, NN 30/70, jj 15/85 NAS/NIPAAm; radiation dose 100 kGy, dose rate 3 kGy h1.
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Fig. 4. Grafting percentage as a function of reaction time, at different monomer concentration: dd 40/60, NN 30/70, jj 22/78, 15/85 NAS/NIPAAm; radiation dose 100 kGy, dose rate 6 kGy h1.
Fig. 5. Theoretical molar ratio of the monomer units, in the copolymer, as a function of the initial concentration of the monomers.
mer are approximately the same in the case of the graft copolymer at dose up to 80 kGy and a concentration of NAS/NIPAAm of 15/85 (w/w) which corresponds to a molar ratio of 0.37. The amount of NAS in the graft copolymer actually decreases with an increase of grafting percentage due to the higher reactivity of NAS; higher homopolymerization of the NAS monomer takes place with an increase in radiation dose. Fig. 6 shows the equilibrium swelling time of PP-g-(NAS/ NIPAAm) films, at different grafting percentages and preirradiation doses, at a concentration ratio of monomers of 15/85. In this plot we observed samples with different pre-irradiation doses in order to compare swelling behavior at different grafting percentages. The swelling percentage is higher at lower grafting percentage, with a maximum of 14% swelling observed in the films with 29% (100 kGy) grafting .This effect could be due to the ratio of NAS/NIPAAm; this ratio decreases with increasing graft percentage (Table 1). It is known that PNAS is less hydrophilic than PNIPAAm, therefore increasing content of NIPAAm in the copolymer increase the hydrophilicity of the system. Fig. 7 shows the typical temperature-dependence on the contact angle measured for the thermosensitive NIPAAm with a LCST around 32 °C in the binary grafted film.
Good reversibility of swelling–deswelling behavior at 20 and 40 °C can be observed in Fig. 8; this reversibility improves after three cycles of the swelling–deswelling process because relaxation of the side chains which allows them to become more oriented in the system occurs after several swelling–deswelling cycles. Fig. 9 shows the FTIR spectra of PP films, NAS, NIPAAm and the binary grafted PP-g-NAS/NIPAAm (57% graft). The spectrum of the binary grafted polymer shows the presence of the corresponding groups of the two grafted monomers and also the presence of a band at 1100 cm1corresponding to the ether group resulting from the bond between PP and the NIPAAm formed on the preirradiation oxidative method. Fig. 10 shows the FTIR spectra of PP-g-NAS/NIPAAm 57% graft, before and after crosslinking by polylysine. It is possible to confirm the presence of the polylysine in the system because the bands corresponding to the succinimide group almost disappear (the crosslinking is not a quantitative reaction), and new bands assigned to NH3+ at 3335 and 1510 cm1 did not appear, because they are overlapped with the bands of N–H in 3400 and 1541 cm1. To confirm the crosslinking reaction, the residual HNAS groups could be quantified by UV spectroscopy.
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Fig. 6. Equilibrium swelling time of PP-g-(NAS/NIPAAm) films at different grafting percentages and pre-irradiation doses: NN 29% (radiation dose 100 kGy), j j 89%, and 141% (radiation dose 120 kGy), monomer concentration 15/85 NAS/NIPAAm.
Fig. 7. Contact angle of PP-g-NAS/NIPAAm, as a function of temperature; graft of 107%, radiation dose 100 kGy, dose rate 3 kGy h1, monomer concentration NAS/NIPAAm 15/85.
Fig. 8. Reversibility of swelling–deswelling behavior at 20 and 40 °C, 22% grafted film, radiation dose 100 kGy, monomer concentration 15/85 (NAS/ NIPAAm), dose rate 3 kGy h1.
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Fig. 9. FTIR spectra of different systems: (a) PP film, (b) NAS, (c) NIPAAm, (d) PP-g-NAS/NIPAAm 57% (radiation dose 100 kGy, monomer concentration 30/ 70).
Fig. 10. FTIR spectra of: a) PP-g-NAS/NIPAAm 57% graft (radiation dose 100 kGy, NAS/NIPAAm 30/70), b) PP-g-(NAS/NIPAAm) crosslinked by polylysine.
Fig. 11. DSC thermogram of PP-g-NAS/NIPAAm; (a) 131% graft; (b) 29% graft.
The DSC thermogram of the binary grafted film (131% graft, 120 kGy) is shown in Fig. 11. It shows a melting point
at 165 °C corresponding to PP, a glass transitions at 115 °C due to PNIPAAm and a poorly defined glass transitions at
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Fig. 12. TGA thermogram of (a) PP; (b) NIPAAm; (c) NAS; (d) PP-g-NIPAAm 131% graft.
Fig. 13. AFM of PP-g-NAS/NIPAAm: crosslinked by polylysine (left), grafted (right side).
60 °C due to PNAS. These glass transitions of the monomer are not present in films with low grafting percentage of 29% (100 kGy). TGA thermograms of different systems were observed in Fig. 12, PP has a 10% weight loss at 419 °C and decomposition temperature at 450 °C. This 10% loss weight temperature decreases with an increase in grafting percentage until, at 255 °C and 131% graft, two decomposition temperatures are observed due to PNAS and PNIPAAm, respectively. AFM studies show that the grafted film crosslinked with polylysine exhibits a changed surface morphology and increased roughness (rms), from 186 nm in the binary grafted film without crosslinking to 595 nm after crosslinking (Fig. 13). This change in surface morphology helps to confirm the crosslinking of the NAS groups by the polylisine. SEM determinations did not show any well defined morphology.
broken. Low dose rates are favorable for increasing grafting yield. The optimum conditions of synthesis were radiation dose of 100 kGy, dose rate of 3 kGy h1, and reaction time between 6 and 8 h. The LCST of the new system has the same value as that of PNIPAAm, and the reversibility is good. Crosslinking with polylysine was confirmed by FTIR and morphology changes on the surface by AFM. This system could be used in liposome vesicle immobilization with better advantages due to better mechanical properties (i.e. it could be handling without breakage), faster velocity of response than other forms of network systems and reusability of the films because of good reversibility of the swelling–deswelling process. Acknowledgements The authors thank S. Castillo, F. Garcia, B. Leal and M.Cruz, from ICN UNAM, and to DGAPA UNAM, Grant IN 200208, for economical support.
4. Conclusions References A new binary grafting system of NAS/NIPAAm has been prepared by the pre-irradiation oxidative method, with an equilibrium swelling time of about 50 min. The grafting efficiency increased with radiation dose, but at radiation dose higher than 100 kGy, the film was rigid and easily
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