Synthesis and characterization of temperature responsive graft copolymers of dextran with poly(N-isopropylacrylamide)

Reactive & Functional Polymers 53 (2002) 19–27 www.elsevier.com / locate / react

Synthesis and characterization of temperature responsive graft copolymers of dextran with poly(N-isopropylacrylamide) Li-Qun Wang a , *, Kehua Tu a , Yuping Li a , Jie Zhang a , Liming Jiang a , Zhihua Zhang b a

Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, PR China b Tobacco Administration Bureau of Zhejiang Province, Hangzhou 310000, PR China Received 25 December 2001; received in revised form 30 April 2002; accepted 5 July 2002

Abstract Poly (N-isopropylacrylamide) (PNIPAAm) was grafted to dextran using ceric ion as redox initiator. The graft copolymers formed temperature responsive materials and can be used to construct polymeric micelles as drug carriers for colon-site specifically delivery. The chemical structure of the graft copolymers was characterized by FTIR, 1 H- and 13 C-NMR spectroscopy. The influence of reaction conditions on the grafting parameters was investigated. It was found that the percentage of homopolymer formation (H%), the grafting efficiency (GE%) and the grafting (G%) of the copolymers increased with increasing the amount of the ceric catalyst used. Extension of the duration of graft reaction increased GE% and G% of the copolymers, suggesting that G% of the copolymers could be readily manipulated by changing copolymerization duration. Higher grafting temperature was in favor of increasing GE% and G%, however, when the reaction temperature was above the LCST of the copolymers, GE% and G% decreased. The optical transmittance of the copolymers in the aqueous solution was examined by UV–Vis instrument. The result showed that the phase transition of the graft copolymer in aqueous solution moved slightly to higher temperature when G% of the graft copolymers decreased. The results of atomic force microscopy and dynamic light scattering measurement indicated that the graft copolymers form micelles in a spherical morphology, and for the copolymer with the G% of 33.8% formed micelles in the mean diameter of less than 30 nm in aqueous solution.  2002 Elsevier Science B.V. All rights reserved. Keywords: Dextran; Poly (N-isopropylacrylamide); Graft copolymers; Temperature response; Micelles

1. Introduction Polymeric micelles, first proposed as drug delivery system in 1984 [1], are still the object of growing scientific attention in drug controlled-delivery. Pharmaceutical research on poly*Corresponding author. Tel. / fax: 186-571-8795-2596. E-mail address: [email protected] (L.-Q. Wang).

meric micelles has been mainly focused on block copolymers generally having hydrophilic– hydrophobic diblock structure [2]. Poly(ethylene glycol) (PEG) is frequently used as the hydrophilic block [3]. Although hydrophobic block changed in a wide chemical composition range, the most preferred hydrophobic polymers are the biodegradable ones, like poly (lactic acid) [4,5], poly(b-benzyl-asparatate) [6]. The am-

1381-5148 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S1381-5148( 02 )00126-8

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phiphilic block copolymers assemble to micelles in aqueous media with the so-called ‘core-shell’ structure. Drugs, especially poorly water-soluble drugs can be incorporated in the core of the micelles by physical entrapment through dialysis or emulsification techniques in order to get a higher entrapment efficiency. However, in both of the techniques organic solvents must be applied. An alternative approach recently is to combine the thermosensitive oligomers to the block copolymers. Poly(N-isopropylacrylamide) (PNIPAAm) was used as thermosensitive block and combined with hydrophilic polymers [3,7]. PNIPAAm exhibits a reversible phase transition in aqueous solution at about 328C, which is known to be the lower critical solution temperature (LCST). The micelles may be constructed when the aqueous solution temperature is raised higher than the LCST of the block copolymer containing PNIPAAm, where PNIPAAm forms the hydrophobic core. Okano and his coworkers evaluated a rapid de-swelling PNIPAAm combtype grafted hydrogel system [8]. PNIPAAm has also been grafted onto various kinds of polymeric chains [9,10], and very recently dextran grafted with poly (N-isopropylacrylamide-coN,N-dimethylacrylamide) copolymers were synthesized and the enzymatic degradation was examined [11]. Recently, we set out the study of using dextran graft copolymers as drug carriers. The choice of dextrans as drug carrier is based on several facts. First, dextrans have been used clinically and safely for more than 5 decades for plasma volume expansion and as antithrombolytic agents. Second, there are many kinds of dextrans commercially available in varied molecular weights and with relatively narrow molecular weight distribution. The degree of branching of the dextrans can be as low as 0.5%, making them ideal molecular model for scientific studies. Third, in addition to high water solubility, dextran polymers are stable under mild acidic and basic conditions [12] and contain large number of hydroxyl groups for conjugation. And finally, dextrans are biodegraded by dextranases present in various organs

in the human body, including the lower part of the gastrointestinal tract [13,14]. Thus, they can be used potentially as drug carriers for colonspecific delivery. In this paper we reported the synthesis and characterization of dextran graft copolymers with PNIPAAm. In addition, the phase transition and micelle formation of the graft copolymers in aqueous solution were examined. 2. Experimental section

2.1. Materials Dextran (low fraction with the molecular weight of Mn 53.38310 4 g mol 21 and Mw 5 5.09310 4 g mol 21 measured by GPC) was purchased from Acros, and used without further purification. N-isopropylacrylamide was the product of Tokyo Chemical Industry Co. Ltd., and was purified by recrystallization in toluene / petroleum ether and dried under vacuum at room temperature. Ceric ammonium nitrite was purchased from Aldrich. It was dried at 110 8C before use. All other chemicals were analytical grade and used without further purification.

2.2. Synthesis of the graft copolymers 5 g of dextran was dissolved in 50 ml of deionized water in a three-necked flask, which was kept in a water bath at a pre-settled temperature. The flask was purged with nitrogen for 30 min and the nitrogen atmosphere was maintained throughout the polymerization period. Certain volumes of 0.1 mol l 21 of ceric ammonium nitrate solution in 1 N nitrate acid was added, stirring for 10 min, and then 5 g of NIPAAm was added to the flask, continued the reaction for different time of intervals. The reaction was stopped by addition of 1 N of sodium hydroxyl aqueous solution. The graft copolymer was precipitated in methanol, filtered, washed and extracted with methanol for 24 h in order to get rid of PNIPAAM homo-

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polymers. The product was dried under vacuum at 60 8C for at least 3 days.

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(0.1 g l 21 ) was cast onto a freshly prepared polystyrene coated glass plate under controlled temperature and dried rapidly at 608C.

2.3. Measurements Infrared spectra were recorded using a BRUKER VECTOR 22 Fourier transform infrared instrument. The polymers were dissolved in DMSO and cast onto NaCl plate and thoroughly dried under vacuum at 508C 1 13 H- and C-NMR spectra were obtained using an AVANCE DMX 500 MHz spectrometer with 2 H 2 O as solvent for dextran and the graft copolymers. The molecular weight of dextran was determined by gel permeation chromatography (GPC) on a Waters 510 instrument. Milli-Q water was used as eluent and three columns (G6000PW, GMPW and G3000PW) were applied The phase transition temperature of the polymers in aqueous solution (0.5 g l 21 ) was determined by examination the changes of light transmittance (500 nm) at various temperatures with a Bio810 UV–Vis spectrometer. The sample and the reference cell were thermostated with a temperature controlled circular system. The values of the LCST of the polymer solution were determined at the temperature of 50% decrease in optical transmittance. The size distribution of micelles was determined by dynamic light scattering method. A light scattering spectrometer (BI-90Plus, Brookhaven Instruments Co. USA) equipped with a He–Ne laser was used. 1 ml of aqueous solution of graft copolymer in the concentration of 0.1 g l 21 was moved into a glass cell, which was then put into the measuring compartment of the instrument. The compartment was previously thermostated at 37 8C with a temperature controlled system. The fluctuations of scattering intensity at the scattering angle of 908 were recorded and transformed automatically. The morphology of micelles was taken with an atomic force microscope (AFM) (SPA 300HV/ SPI 3800N, Seiko Co.) in the tapping mode. The aqueous solution of the copolymer

2.4. Definitions of grafting parameters The grafting parameters were defined as follows [15]: H (Homopolymer, %) Weight of homopolymer 5 ]]]]]]]]] Weight of monomer charged GE (Grafting efficiency, %) Weight of polymer grafted 5 ]]]]]]]]] Weight of monomer charged Weight of polymer grafted G (Grafting, %) 5 ]]]]]]]] Weight of graft copolymer For the determination of the above parameters, the weight loss of the polymers during extraction, due to the solubility of the lower fraction of dextran in methanol, was taken into account. The average percentage of weight loss of dextran extracted by methanol for 24 h was found to be 8.2%.

3. Results and discussion

3.1. Characterization of the graft copolymers by IR and NMR spectroscopy It is generally recognized that ceric redox catalyst can form radicals in cyclic alcohols and glucopyranosyl unit of polysaccharides [16–18]. The free radicals may then initiate the graft polymerization of monomers presented in the solution. The method has been widely used in manufacturing starch graft copolymers, and in the present work, employed in preparing dextran grafted PNIPAAm copolymers. The suggested reaction path is shown in Fig. 1.

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Fig. 1. The graft copolymerization route of dextran with poly(N-isopropylacrylamide).

IR spectra of dextran, PNIPAAm and the graft copolymers are shown in Fig. 2. In the spectrum of the graft copolymer (Fig. 2C), in addition to the characteristic stretching vibrations of hydroxyl group (at 3400 cm 21 and 1023 cm 21 ) corresponding to dextran, one can also

observe the acyl group at 1640 cm 21 and 1530 cm 21 , which are the characteristic absorptions of PNIPAAm. The result testifies the occurring of graft copolymerization. The chemical structure of the graft copolymer was also examined by 1 H-NMR spectroscopy. As shown in the spectrum of the copolymer (Fig. 3C), the characteristic resonance peaks corresponding to the protons in methylene groups and other five methine groups of dextran at 3.40–3.92 ppm and 4.91 ppm are apparently presented. In addition, the signals of protons in PNIPAAm graft also appear in the spectrum at 1.1 ppm (the two methyl groups), 3.8 ppm (isopropyl methine groups), and at about 2.0 and 1.5 ppm (the chain CH 2 and CH groups) respectively. The 13 C-NMR spectra of the dextran, PNIPAAm and their graft copolymer are shown in Fig. 4. In addition to the resonance peaks of the six carbons in dextran at 65.62–97.76 ppm [19], the resonance peaks corresponding to PNIPAAm at 22.64 ppm (methyl carbons in isopropyl groups), 175.29 ppm (carbonyl groups), and at about 41.8–43.1 ppm (the overlapped resonance of the chain methylene and methine carbons and isopropyl methine carbons) are all exhibited in the 13 C-NMR spectrum of the graft copolymer (Fig. 4C).

3.2. Effect of copolymerization conditions on the grafting parameters of the copolymers

Fig. 2. FTIR spectra of dextran (A); PNIPAAm (B) and dextrang-PNIPAAm copolymer (C).

The relation between the amount of initiator used for the graft polymerization and the grafting parameters of the copolymers is listed in

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Fig. 3. 1 H-NMR spectra of dextran (A); PNIPAAm (B) and dextran-g-PNIPAAm copolymer (C).

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Fig. 4.

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C-NMR spectra of dextran (A); PNIPAAm (B) and dextran-g-PNIPAAm copolymer (C).

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Table 1 Effect of the concentration of ceric ion redox initiator on the grafting parameters Concentration of initiator (mol / l)

H%

GE%

G%

0.01 0.02 0.04 0.08 0.16

18.4 61.1 61.3 62.5 64.8

1.40 15.8 17.1 18.5 19.7

3.32 27.9 29.6 31.7 33.3

Table 1. The result indicates that the percentage of homopolymer formation (H%) increases holistically with the increase of the concentration of ceric ion. The grafting efficiency (GE%) and grafting (G%) of the copolymers show the same tendency as H%, i.e., GE% and G% increase with increasing continually the concentration of the initiator to 0.16 mol l 21 . In a contrast experiment, it was found that the formation of PNIPAAm homopolymers in the presence of ceric ion (0.02 mol l 21 ) and absence of dextran was less than 10% after polymerized for 12 h at room temperature. Therefore, the homopolymer formation in the copolymerization system can be mainly attributed to the chain transfer to N-isopropylacrylamide monomers, though a little part of it is definitely attributed to the ceric ion initiation. The dependence of the grafting parameters on the duration of copolymerization is shown in Fig. 5. It is seen that the extent of H% did not change further after 4 h of copolymerization. In contrast, GE% and G% of the copolymers kept in increase even after 8 h. As discussed above, chain transfer process contributes mainly to the formation of PNIPAAm homopolymers. Therefore, the lower concentration of NIPAAm monomers presented in the reaction system after 4 h polymerization might decrease the possibility of chain transfer from copolymers to NIPAAm monomers, which results in an inconspicuous change of PNIPAAm homopolymer after a certain period of copolymerization. The influence of graft reaction temperature on the grafting parameters was also examined. It

Fig. 5. The effect of the duration of graft polymerization on the grafting parameters of H%, GE% and G%.

was found that, in the aqueous media of copolymerization where ceric, ammonium and nitrate ions were presented, the LCST of PNIPAAm and the graft copolymers moved to about 298C. Therefore, the graft reaction was evaluated under 20, 27 and 37 8C respectively. The former two temperatures are below the LCST of PNIPAAm and the graft copolymers, and the letter one is above the LCST of the polymers. As seen in Fig. 6, H%, GE% and G% increase with the increase of the reaction temperature when it is lower than the LCST of PNIPAAm. However, when the graft copolymerization was conducted at the temperature above the LCST of PNIPAAm the three grafting parameters (H%, GE% and G%) drop to lower

Fig. 6. The effect of graft polymerization temperature on the grafting parameters of H%, GE% and G%.

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values. It was also found that the percentage of monomer conversion into polymers became lowered when the reaction temperature was above the LCST of PNIPAAm. It is proposed that the precipitation of the resultant graft copolymers and PNIPAAm due to the phase transition of PNIPAAm might be the main reason for the decrease of the grafting parameters, since the shrink of the polymer chain will inhibit the reaction of N-isopropylacrylamide monomers into polymers.

aqueous solution at 37 8C was found to be much more time dependent and the final balance mean diameter of the micelles is less than 30 nm for the copolymer with the grafting (G%) of 33.8% (Fig. 8). The AFM result shows clearly that the morphology of the constructed micelles at the temperature above the LCST of the copolymer is in spherical shape and the average size of the dried micelles is about 80 nm in diameter (Fig. 9).

3.3. Phase transition behavior and micellization of the graft copolymers Fig. 7 shows optical transmittance of aqueous solution of the graft copolymers at various temperatures. It is seen that the transition range of the graft copolymers becomes much more narrow with the increase of G% of the copolymers, and the transition points of the graft copolymers move slightly to higher temperature when G% of the copolymer becomes lower. The results are in consistent with the find that incorporation of hydrophilic or hydrophobic groups into PNIPAAm chains changed the LCST of the PNIPAAm copolymers [20], and the LCST of PNIPAAm copolymers moved to higher temperature when more hydrophilic segments were incorporated into the molecules [21,22]. The micellization of the graft copolymers in

Fig. 7. Light transmittance changes of the graft copolymers with different G% in aqueous solution as the function of temperature.

Fig. 8. Dependence of the micellization of the graft copolymer on time in aqueous solution (the grafting of the copolymer is 33.7%, the concentration of the copolymer in aqueous solution is 0.5 g l 21 ): 5 min (A); 20 min (B) and 35 min (C).

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with G% of 33.8% forms micelles in the mean diameter of less than 30 nm in aqueous solution. Acknowledgements This project was financially supported by the National Natural Science Foundation of China and the Natural Science Foundation of Zhejiang Province. References

Fig. 9. AFM image of the micelles formed on freshly prepared polystyrene coated glass plate.

4. Conclusions 1. It is confirmed that poly (N-isopropylacrylamide) (PNIPAAm) is grafted to dextran when ceric ion is used as redox initiator, and the grafting (G%) of the copolymers can be manipulated by changing the concentration of ceric ion and / or the duration of the graft polymerization. 2. The result indicates that the percentage of homopolymer formation (H%), the grafting efficiency (GE%) and the grafting (G%) of the copolymers increase when increasing the concentration of the initiator to 0.16 mol l 21 . 3. Higher grafting temperature is in favor of increasing GE% and G%, however, when the reaction temperature is above the LCST of the copolymers, these grafting parameters become lowered. 4. The phase transition of the graft copolymer in aqueous solution moves slightly to higher temperature when the G% of the graft copolymers decreases. 5. The graft copolymers form micelles in a spherical morphology and for the copolymer

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