Poly(N-isopropylacrylamide) thermoresponsive cross-linked conjugates containing polymeric soybean oil and/or polypropylene glycol

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Poly(N-isopropylacrylamide) thermoresponsive cross-linked conjugates

containing polymeric soybean oil and/or polypropylene glycol

Abdulkadir Allı, Baki Hazer

*

Zonguldak Karaelmas University, Department of Chemistry, 67100 Zonguldak, Turkey

a r t i c l e

i n f o

Article history:

Received 22 February 2008

Received in revised form 31 March 2008 Accepted 2 April 2008

Available online 8 April 2008

Keywords:

Thermoresponsive polymer Polymeric soybean oil peroxide Macromonomeric initiator Poly(N-isopropylacrylamide) Polypropylene glycol Cross-linked graft copolymer

a b s t r a c t

Synthesis, characterization and solution properties of a new series of the PNIPAM-soybean oil and/or polypropylene glycol, PPG, conjugates (conjugates also referred to as co-net-works) have been described. For this purpose free radical polymerization of NIPAM monomer was initiated by macroinitiators based on PSB and/or PPG in order to obtain PSB-g-PNIPAM, PPG-g-PNIPAM and PSB-g-PPG-g-PNIPAM cross-linked graft copolymers. The autooxidation of soybean oil under air at room temperature rendered waxy soluble polymeric soybean oil peroxide associated with cross-linked parts. The soluble polymeric oil macro-peroxide isolated from the cross-linked part was used to initiate the free radical polymerization of NIPAM to give PSB-g-PNIPAM cross-linked copolymer. To obtain PPG-macromonomeric initiator, PPG-MIM, PPG-bis amino propyl ether with Mn 400 (or 2000) Dalton was reacted with 4,40_{-azo bis cyanopentanoyl chloride and methacryloyl chloride,}

respectively. PPG-MIM also initiated the free radical polymerization of NIPAM at 80 °C to yield PPG-g-PNIPAM cross-linked thermoresponsive product. In order to obtain PSB-g-PPG-g-PNIPAM cross-linked triblock copolymer, NIPAM was polymerized by using the mix-ture of two macroinitiators, PSB and PPG-MIM. PSB contents in the graft copolymers were calculated via elemental analysis of nitrogen in graft copolymers. Thermal analysis, SEM, FTIR and1_{H NMR techniques were used in the characterization of the products. The effect} of polymeric soybean oil, PSB, and/or PPG on the thermal response rate of poly(N-isopro-pylacrylamide, PNIPAM, cross-linked-graft copolymers swollen in water has been investi-gated by means of swelling–deswelling and drug release behaviors against to temperature change. Lower critical solution temperatures (LCST) of the cross-linked PNIPAM conjugates (conjugates also referred to as co-networks) were determined from the curves of swelling degrees versus solution temperatures. The response temperature of the hydrophobically modiﬁed PNIPAM conjugates was reduced to 27 °C, 23 °C and 27 °C for PSB-g-PNIPAM, PPG-g-PNIPAM and PSB-g-PPG-g-PNIPAM, respectively. We have found that the graft copolymers were not pH-responsive. In addition, higher pH ranges cause the hydrolysis of the PSB ester linkages, quickly and makes the cross-linked graft copolymers soluble. The fastest shrinking of the gels was observed by loosing water between 65% and 98% at 50 °C.

Methyl orange (MO), was used as a model drug, loaded into cross-linked graft copolymers to examine and compare the effects of controlled release at lower and higher temperatures of lower critical solution temperature (LCST).

*Corresponding author. Tel.: +90 (372) 2572070; fax: +90 (372) 2574181. E-mail addresses:bkhazer@karaelmas.edu.tr,bhazer2@yahoo.com(B. Hazer).

Contents lists available atScienceDirect

European Polymer Journal

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1. Introduction

Thermoresponsive aqueous polymer systems are known to exhibit large, reversible conformational changes in response to small thermal stimuli[1–7]. Poly (N-isopropylacrylamide), PNIPAM, having both hydro-philic amide groups and hydrophobic isopropyl groups in its sidechains, is one of the most studied thermally sensitive (thermoresponsive) polymers, possessing a low-er critical solution templow-erature, LCST, at 32 °C[8–21]. It is soluble in water at lower temperatures, but precipi-tates out at temperatures higher than 32 °C. The interest in stimuli-responsive polymers, particularly gel polymers, has exponentially increased due to their promising po-tential in a variety of applications for the biomedical and industrial ﬁelds, particularly in the ﬁeld of controlled drug release[1,22–31].

The modiﬁcation of the LCST of temperature-sensitive gels is of primary interest[32–34].

A decrease in the LCST can be achieved by increasing the hydrophobicity of the network while an increase in the LCST can be achieved by increasing the hydrophilicity of the network. Conjugation of PNIPAM is also a useful way to overcome some limitations. For example, in most applications, higher or lower response temperatures of the thermoresponsive polymers are preferred [18]. In addition, conjugation is also useful to combine the advantageous properties of the individual components. For example, hybrid block and graft copolymers of PNI-PAM containing phosphocholine [35a,36], poly(D,L-lac-tide) [37], alginate [38], propyl acrylic acid [39], cystaminebisacrylamide [40], imidazole [41], arginine-glycine-aspartic acid [42], and segmented polystyrene

[2]have successfully synthesized and well characterized. The solution properties of block branched copolymer of PNIPAM [43] and either polystyrene or poly(tert-butyl methacrylate) have been investigated in water with par-ticular emphasis on the temperature response of these copolymers [44]. PNIPAM also used leading hydrogel-brushes[45].

Vegetable oils are an economical and environmentally friendly alternative to petroleum for biodegradable poly-mer syntheses in several ways. Soybean oil is an abun-dant and inexpensive vegetable oil having mainly polyunsaturated fatty acids such as linoleic and linolenic acid. Multiple sites of chemical reactivity are intrinsic in soybean based triglycerides, which makes this renewable resource particularly attractive as a potential alternative to petroleum-based monomers[46–48]. Soybean oil, can be polymerized by autooxidation under air oxygen and sunlight via peroxidation, epoxidation and perepoxidation to give polymeric soybean oil peroxide (PSB). Autooxida-tion of polyunsaturated oils affords polymeric oil

perox-ides[46–48]via hydrogen abstraction from a methylene

group between two double bonds. These polymeric oil peroxides, especially polymeric soybean oil (PSB), were used to initiate the free radical polymerization of styrene, methyl methacrylate and butyl methacrylate in our laboratories in order to obtain block/graft copolymers of some vinyl polymers containing polymeric oil segment

[49–51].

Monomeric initiators and macromonomeric initiators (MIM) combine the features of both an initiator and a monomer[52–63]. MIM is a macroinitiator having methac-ryloyl ends.

For example, a MIM-PPG can be prepared from 4,40

-azo-bis-4-cyanopentanoyl chloride, methacryloyl chloride and poly(propylene) glycol bis (2-aminopropyl ether), which can be useful to prepare highly branched PPG-graft copolymers.

We report the synthesis and evaluation of the novel thermoresponsive PNIPAM-conjugates by the polymeriza-tion of NIPAM initiated by PSB and/or MIM-PPG, leading to PSB-g-PNIPAM, PPG-g-PNIPAM and PSB-g-PPG-g-PNI-PAM thermoresponsive polymer conjugates for new appli-cation areas in industry and bioengineering.

2. Experimental section 2.1. Materials

Soybean oil was locally purchased and used as re-ceived. Soybean oil contains (wt%) palmitic acid 7–14, stearic acid 1.4–5.5 oleic acid 19–30, linoleic acid 44– 62, linolenic acid 4.0–11. N-isopropylacrylamide (NIPAM; Aldrich) was puriﬁed by recrystallization from n-hexane and dried under vacuum at room temperature. 4,40

-Azo-bis-4-cyanopentanoic acid (ACPA) was purchased from Fluka AG. Poly(propylene glycol) bis 2-aminopropyl ether (PPG-NH2) (amine groups at both ends of each chain)

with MW 400 and MW 2000 were purchased from Al-drich. All the other chemicals were analytical grade and used without further puriﬁcation. 4,40

-Azobis-4-cyano-pentanoyl chloride (ACPC) was prepared by the reaction of ACPA with phosphorus pentachloride in benzene at 50 °C for 1.5 h according to the procedure cited in the lit-erature[64]. After the solvent was evaporated, the crude product was crystallized (m.p. 95 °C) in ether/hexane (v/ v: 1/1) in a refrigerator.

2.2. Autooxidation of soybean oil

The soybean oil was prepared for the autooxidation according to the procedure reported in the literature[49– 51]. Soybean oil (50 g) spread out in a Petri dish (£ = 16 cm) was exposed to sunlight in the air at room temperature. After ﬁve months, a gel polymer ﬁlm associ-ated with a waxy and viscous liquid was formed. Chloro-form extraction of the crude polymeric oil for 24 h at room temperature allowed separation of the soluble part of the polymeric soybean oil from the gel.

2.3. The Peroxygen analysis

Peroxygen analysis of soluble part fractions was carried out by reﬂuxing a mixture of 2-propanol(50 mL)/acetic acid (10 mL)/saturated aqueous solution of KI (1 mL) and 0.1 g of the polymeric sample for 10 min and titrating the released iodine against thiosulfate solution[65]. Peroxygen content of the soluble part of the polymeric soybean oil was 1.3 wt%.

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2.4. Synthesis of MIM-PPGs

In a typical procedure for the synthesis of MIM-PPG2000, a solution of 2.0 g (6.3 mmol) of ACPC in 50 mL CHCl3 in a round bottom ﬂask with a magnetic stirring

bar was gradually added to the mixture of 25.2 g (12.6 mmol) of PPG-NH22000 and 10 mL of aqueous NaOH

(20 wt%) and stirred for 24 h at room temperature. The mo-lar ratio of ACPC to PPG2000 was 1:2. After the reaction, the mixture was washed with water three times to secure the removal of salts and ACPA from the product. The organ-ic phase was dried with Na2SO4overnight at 0 °C. Solvent

was evaporated. A yellow viscous liquid (macroinitiator) was dried under vacuum and stored at 0 °C until use. The yield was 91%. In the second step, the yellow viscous liquid macroinitiator obtained, in 10 mL of aqueous NaOH (20 wt%) was mixed with methacryloyl chloride in CHCl3

with a molar ratio to the macroinitiator 1:3. The reaction mixture was stirred for 24 h and then washed with water. The product (MIM) was dried with anhydrous Na2SO4.

After evaporation of solvent, it was dried and stored in a refrigerator[63].

2.5. Synthesis of the graft copolymers

A given amount of a macroinitiator (PSB, MIM-PPG400, MIM-PPG2000 or a mixture of PSB and one of MIM-PPGs), N-isopropylacrylamide were charged separately into a Pyr-ex tube with a magnetic stirring bar. Argon was introduced through a needle into the tube for about 3 min to expel the air. The tightly capped tube was put in an oil bath at 80 °C. After a given polymerization time, the tube content was poured into a large amount of methanol to precipitate crude polymer. To remove unreacted monomer from the crude polymer, it was soaked in distilled water for one week by changing the solution with pure water daily at room temperature. The hydrophobic soluble part was also separated from the cross-linked copolymer by soaking the hydrogels in chloroform for one week by changing the solution with pure chloroform daily at room temperature. The puriﬁed cross-linked copolymer was dried under vac-uum at 40 °C.

2.6. Instrumentation

The1H NMR spectra of polymers were recorded on a Bruker AVANCE 400 spectrometer (400 MHz), using CDCl3

as solvent. FTIR spectra were obtained using a Jasco 300 E IR spectrometer. Thermal analysis of the product was carried out by using a Setaram Differential Scanning Cal-orimetry (DSC) DSC-141 series thermal analysis system under nitrogen. Generally, a dried sample was heated at a rate of 10 °C/min from 50 °C to 150 °C under N2

atmo-sphere. The CHNS-932 Model LECO Elemental Analyzer was used for the elemental analysis of C, H and N in the products. Molar fractions (mol%) of comonomer units in PSB-PNIPAM copolymers were calculated using ele-mental analysis data (content of N). Surface topography of the products was carried out by using a JEOL FEG-SEM JSM 6335 F Scanning Electron Microscope (FEG-SEM). The specimens were frozen under liquid nitrogen, then

fractured, mounted, and coated with gold (300 Å) on an Edwards S 150 B sputter coater. The SEM was operated at 15 kV, and the electron images were recorded directly from the cathode ray tube on a Polaroid film. The magni-fication employed was varied up to 15,000; however, 1000, 6000, 10,000 and 15,000 magnifications were useful. Absorbance values of the methyl orange (MO) aqueous solutions with different concentrations were measured by using UNICAM UV/VIS spectrometer, UV 2 at 464 nm.

2.7. Measurement of swelling ratio

The degree of swelling ratio of hydrogels was measured gravimetrically in distilled water in the temperature range from 4 to 40 °C. Before the measurement of swelling ratio, the hydrogel was incubated in distilled water for at least 24 h at every particular temperature, and weighed after blotting the excess surface water. Degree of swelling was deﬁned as follows[63]:

Swelling ð%Þ ¼ 100 ðWs WdÞ=Wd; ð1Þ

where Wsis the weight of swollen hydrogel at a particular

temperature and Wd is the dry weight of hydrogel after

drying under vacuum overnight. 2.8. Measurement of deswelling kinetics

The deswelling kinetics of the polymer samples was studied. The polymer samples were equilibrated in tilled water at 4 °C and they were transferred into new dis-tilled water at 50 °C. At every particular time interval, the samples were removed from the hot water and weighted after wiping off the excess water on the surface with a wet ﬁlter paper. The shrinking of the hydrogels was fol-lowed by the determination of the decrease in the water content of the polymer samples. The weights of the gels were recorded at particular times. The deswelling ratio is deﬁned as[66].

DS ¼ ðWt WdÞ=ðW0ð4CÞ WdÞ ð2Þ

where DS is the deswelling ratio; W0ð4CÞ is the weight of

the gel at equilibrium at 4 °C; W_{t}, is the weight of the gel at a particular time and Wd, is the dry weight of the

polymer sample.

2.9. Chemical release from the cross-linked graft copolymers The chemical release experiments were carried out at 19 and 37 °C, respectively, to investigate the effect of tem-perature sensitive property of these release systems (RS)s on MO release proﬁles. For example, the dry sample was dipped in a saturated aqueous solution of MO and allowed to swell for one day at 19 °C. The sample was then placed in 100 ml of distilled water at a given temperature for in vitro release experiment. The absorbance value of the aqueous solution of MO released from the gel was mea-sured at 464 nm in a UV-VIS spectrometer. And the con-centration of MO in that aliquot was determined by using a standard calibration curve of the absorbance versus the MO concentration at 464 nm. The standard calibration

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curve of the absorbance as a function of the methyl orange concentration at 464 nm has a linear relationship with a correlation coefﬁcient (r) of 0.9999. This linear relation-ship can be quantiﬁed by following equation: A = (23.24c + 76.7) 103_{, where A is the absorbance and c is}

the concentration (mg/mL) of MO. The results were pre-sented in terms of cumulative release as a function of time: Cumulative release ð%Þ ¼ ðMt=M1Þ ð3Þ

where Mtis the amount of MO released from the RS at time

t and M1is the amount of MO pre-loaded in cross-linked

graft copolymers of RS.

3. Results and discussion 3.1. The macroinitiators

The macroinitiators, PSB and MIM-PPGs, were success-fully used in the free radical polymerization of some vinyl monomers in our laboratories, recently. Incorporation of hydrophobic and biodegradable polymeric oil blocks into polymer conjugates could be possible by the polymeriza-tion of vinyl monomers initiated by PSB without any addi-tional catalyst and organic solvent. MIM-PPG is useful to incorporate PPG blocks into a copolymer leading to highly dendrimeric branched and cross-linked graft copolymer. In this work, these macroinitiators were used in the polymer-ization of NIPAM to prepare thermoresponsive polymer conjugates having PSB and/or PPG blocks. The characteris-tics of PSB-macroinitiator: Mn 9280 Dalton, MWD = 2.25

and peroxygen content of the soluble part of the PSB: 1.3 wt%. Only soluble part of the autoxidized soybean oil was used as macroperoxidic initiator in the polymeriza-tion of NIPAM. Mw/Mn of the macromonomeric initiators: 1400/1044 for MIM-PPG400, 4380/3900 for MIM-PPG2000 and their yields were 89 and 78 wt%, respectively. 3.2. Synthesis of the PNIPAM-conjugates

The free radical bulk polymerizations of NIPAM using the macroinitiators yielded novel PSB-g-PNIPAM, PPG-g-PNIPAM, and PSB-g-PPG-g-PNIPAM conjugates containing cross-linked and soluble parts. Cross-linked and soluble graft copolymer fractions were isolated by means of chlo-roform extraction. The gel graft copolymers were consid-ered throughout this work for characterization and for their swelling properties. The soluble parts of the graft copolymers were only used in the structural analysis by

1_{H NMR, which was discussed later. The polymerization}

of NIPAM was initiated by PSB, MIM-PPG or a mixture of PSB and MIM-PPG. Formation of the gel polymers can be seen in Scheme 1a,b and c.Table 1contains feeding ratios, polymerization yield, cross-linked and PSB contents of the polymer conjugates. Crude polymer yield changes from 43 to 84 wt% while cross-linked part isolated changes from 20 to 69 wt% of the crude polymer yield. Amount of macroini-tiator segment in the graft copolymer was proportional with the feeding ratio.

Scheme 1. Polymerization of NIPAM initiated by the macroinitiators by PSB (a), by MIM-PPG (b), by the mixture of PSB and MIM-PPG (c).

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The polymerization conditions of the PSB-g-PNIPAM conjugates in view of the variation of polymer yield, gel ratio and PSB content in the gels were investigated. For example, when the copolymers were compared with the same initial feed ratios, but with different polymerization times (4 h for A-1, A-2 and 5 h for B-3, B-4), the gel poly-mer yield for longer polypoly-merization time was found to be quite higher than the shorter one. However, as it will be discussed later, from the swelling measurements, it is ob-served that longer polymerization time caused formation of a gel with a higher cross-link density, as expected. In addition, the variations of the polymer yield and

cross-linked part by the increase in polymerization time were shown inFig. 1a. The gel formation had an ‘‘induction per-iod” at around polymerization time of 1 h which means that gelation was starting 1 h later. Agreeing with the induction period in the literature, the similar behavior was also reported in the polymerization of the macro-monomeric initiators [61]. Hyperbranched and cross-linked polymers were obtained when the polymerization of NIPAM is initiated by the PSB peroxide initiator. How-ever as PSB concentration decreases in the initial feed, polymer yield together with the gel polymer reaches a pla-teau as expected, N-content of the gel polymer increases

Table 1

Reaction conditions and polymerization results of the synthesis of PNIPAM conjugates

Run No PSB (g) MIM-400 (g) MIM-2000 (g) NIPAM (g) Graft copolymer

Total yield Gel copolymer (g) PSB in gel copolymer (mol%)a

A-1 0.50 – – 0.50 0.74 0.27 61 A-2 0.51 – – 1.00 1.00 0.30 47 A-3 0.50 – – 2.00 1.73 0.50 38 A-4 0.51 – – 5.00 4.57 2.74 22 B-1 0.51 – – 0.15 0.53 0.10 82 B-2 0.50 – – 0.25 0.53 0.19 75 B-3 0.51 – – 0.50 0.77 0.36 59 B-4 0.51 – – 1.00 1.27 0.43 49 F-1 – 0.50 – 0.50 0.43 0.40 – F-2 – 0.50 – 1.00 0.75 0.70 – F-3 – 0.50 – 2.00 1.23 1.14 – F-4 – – 0.51 0.50 0.81 0.66 – F-5 – – 0.50 1.01 1.11 1.03 – F-6 – – 0.50 2.01 1.88 1.64 – E-1 0.51 0.50 – 2.00 2.43 1.94 – E-2 0.51 1.01 – 2.00 2.56 2.15 – E-3 1.01 0.50 – 2.00 2.87 1.78 – E-4 0.51 – 0.50 2.01 2.43 1.94 – E-5 0.50 – 1.01 2.01 2.91 2.33 – E-6 1.00 – 0.51 2.00 2.77 1.91 –

Polymerization times: 4 h for A-series, 5 h for B-, F- and E-series.

a

Calculated from the nitrogen elemental analysis.

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with the increase in the initial feed ratio of NIPAM. Ele-mental analysis of nitrogen in PNIPAM blocks gave NIPAM amount in graft copolymer using the equation shown below:

NIPAM; mol% ¼ ðN=N0Þ 100 ð4Þ

where N is wt% of nitrogen in graft copolymer determined by elemental analysis and N0is wt% of nitrogen content of

pure PNIPAM. PSB content of the PSB-g-PNIPAM graft copolymer can also simply be determined by the equation given below:

PSB; mol% ¼ 100 NIPAM; mol% ð5Þ The variation of the gel polymer yield and its PSB content by the increasing initial feed ratio of NIPAM were given inFig. 1b. Similarily, as the NIPAM in the initial feed in-creases, the NIPAM content in gel polymer increases but the wt% of cross-linked polymer reaches a plateau at around 35 wt%.

For the synthesis of the PPG-g-PNIPAM graft copoly-mers, the polymerization of NIPAM was initiated with either MIM-PPG400 or MIM-PPG2000 at 80 °C, for 5 h to obtain PPG400-g-PNIPAM and PPG2000-g-PNIPAM, respec-tively. Polymerization of NIPAM with macromonomeric initiators gave a mixture of cross-linked PPG-g-PNIPAM graft copolymers, containing PPG units in moderate yields (42–81%w/w). We can conclude that MIM-PPGs are more effective in the free radical polymerization of NIPAM by means of polymer yield and gel yield than PSB macroiniti-ator. Soluble parts of the graft copolymers were removed by chloroform extraction from the mixture to isolate gel polymers. These results are tabulated inTable 1.

For the synthesis of the PSB-g-PPG-g-PNIPAM three block conjugates, a binary mixture of macroinitiators; either PSB/MIM-PPG400 or PSB/MIM-PPG2000 initiated the polymerization of NIPAM to obtain PSB-g-PPG400-g-PNIPAM and PSB-g-PPG2000-g-PSB-g-PPG400-g-PNIPAM, respectively.

Ele-mental analysis of nitrogen was also useful to determine N-content of the graft copolymer containing PPG. In this case, considering the feed ratio, nitrogen content of the PPG having amine terminal groups was discarded from the elemental analysis results of PNIPAM. These graft copolymers contained 49–78 wt% of NIPAM depending on the initial feed ratio (Table 1).

3.3. Characterization of the graft copolymers

The structural analysis of the gel polymer was only car-ried out using FTIR as we discussed below. Only soluble part of the graft copolymers in CDCl3solutions could be

used in proton NMR analysis.1_{H NMR spectra of the}

solu-ble copolymer samples PSB-g-PNIPAM contained charac-teristic peaks: (d, ppm):-CH2of SB at 2.8, 2.4, 1.9, 1.4 and

0.9; the peaks at 4.1–4.4 ppm originate from the protons of the unsaturated oil blocks in methylene groups of the triglyceride. The vinylic protons are detected at 5.3 ppm. In addition, the signals of protons in PNIPAM graft also ap-pear in the spectrum at 1.1–1.4 ppm (–CH3), 2.0–2.2 ppm

(–CH2–), 3.8–4.1 ppm (–CH–). 1

H NMR spectra of the soluble copolymer samples PPG-g-PNIPAM contained characteristic peaks of the –CH3

groups (at d 1.2) and –CH2groups (at d 3.4–3.6) of PPG.

Vi-nyl –CH2groups (at d 5.6). –CH2groups (at d 2.25–2.4) of

ACPA. The signals that appeared at 4.10 ppm due to –NH groups in the macroinimer. In addition, the signals of pro-tons in PNIPAM graft also appear in the spectrum at 1.1-1.4 ppm (–CH3), 2.0–2.2 ppm (–CH2–), 3.8–4.1 ppm

(–CH–).

1_{H NMR spectra of the soluble copolymer samples}

PSB-g-PPG-g-PNIPAM contained characteristic peaks of the –CH3groups (at d 1.2) and –CH2groups (at d 3.4–3.6) of

PPG. Vinyl –CH2groups (at d 5.6). In addition, the signals

of protons in PNIPAm graft also appear in the spectrum at 1.1–1.4 ppm (–CH3), 2.0–2.2 ppm (–CH2–), 3.8–4.1 ppm

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(–CH–). In addition, the peaks at 4.1–4.4 ppm originate from the protons in methylene groups of the triglyceride. The vinylic protons are detected at 5.3 ppm.

The FTIR spectra of the cross-linked graft copolymers were taken as a KBr samples.

The signals carbonyls of amide groups at 1660 cm1_and

–NH- group at 3320 cm1 _{attributed to the PNIPAM}

sequence are also accompanied by the presence of absorp-tion band 2950 cm1_{, characteristic to the PSB segment.}

The signals carbonyls of ester groups at 1740 cm1 _and

ether group at 1160 cm1_{attributed to the PSB.}

FTIR spectrum of the sample of PPG-g-PNIPAM graft copolymer. The signals –CO- group at 1650 cm1 _and

–NH- group at 2950 cm1 _{attributed to the PNIPAM}

sequence are also accompanied by the presence of C–O–C stretching vibration band 1110 cm1_{and, –NH stretching}

vibration band 3500 cm1_{, characteristic of the PPG}

segment.

Fig. 2shows a typical FTIR spectrum of

PSB-g-PPG-g-PNIPAM conjugate (Run no: E3) with the characteristic bands of the related segments.

The amount of the polyether units in the graft copoly-mers can be evaluated by comparing the characteristic bands of PPG segments at 1100 cm1_{. PPG content in gel}

polymer can be expected by comparing the eteric band in FTIR spectrum.Fig. 3shows the decreasing relative inten-sity of the eteric bands of PPG400 content, depending on the feeding ratio of MIM-PPG, in the F-series graft copoly-mers. Similarly, the increasing relative intensity of the eteric bands in the FTIR spectrum has been observed by the increasing molecular weight of the PEG unit in the copolymers (PPG400 with PPG2000).

The morphology of the graft copolymer samples was examined with scanning electron microscopy (SEM).

Fig. 4shows SEM photographs of the cross-sections of PNI-PAM-homopolymer (a) and cross-linked PNIPAM-conju-gates. After grafting, the cross-sectional structures of PNIPAM-conjugates were quite different from those of PNI-PAM-homopolymer. A homogeneous continuos matrix for PNIPAM-homopolymer (a) was observed. In contrast, a miscible continuous phase of PNIPAM conjugates (b and c) was observed. Interestingly, PNIPAM/PPG/PSB conjugate showed porosity like in PNIPAM grafted polyvinylidene ﬂuoride[18].

Thermal analysis of graft copolymers were performed by DSC and TGA. Table 2 shows the thermal analysis

Fig. 2. FTIR spectrum of PSB-g-PPG-g-PNIPAM three block conjugate (Run no: E-3).

Fig. 3. Comparison of the decreasing relative intensity of the etheric bands of PPG400 content in the F-series graft copolymers by decreasing PPG400 in the initial feed ratio.

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results of the graft copolymers andFig. 5shows the DSC traces of the PNIPAM conjugates. Two glass transition tem-peratures, Tg, were observed for each sample much lower

than that of the PNIPAM homopolymer (Tg= 135 °C)[67].

Plasticizer effect of PSB in graft copolymer was observed indicating lower glass transition than that of pure PNIPAM. Hydrogen bonding of the polar groups into the graft copolymers can cause the higher Tgthan that of the

misci-ble hydrophobic parts. However, the highest Tg’s were

ob-served for the samples for E2 (Tg1= 61 °C and Tg2= 103 °C)

and F3 (Tg1= 75 °C and Tg2= 117 °C). Moreover, plasticiser

effect of PSB has been clearly observed by the insertion of PSB blocks to the PPG-PNIPAM diblock copolymer, when

we compare Tg’s of PPG400-g-PNIPAM (0.5/2.0), F3, (75 °C

and 117 °C) with PSB-g-PPG400-g-PNIPAM (0.5/0.5/2.0), E1 (61 °C, 81 °C). In addition, because of the very low molecular weight and the lower content of the PSB blocks and/or PPG blocks into the graft copolymers, Tg’s of PSB

were not present in all graft copolymer samples.

When we look at the DSC traces of the PSB/PG400/PNI-PAM conjugates, we see two Tg’s which may belong to

hydrophobic and hydrophilic segments, separately. In this manner, the dramatic increase in Tgof the sample E-2

hav-ing two times higher amount of PPG than E-1. But increase in the PSB amount in copolymer, did not cause any change in Tgwhen we compare Tg’s of E-1 and E-3. Probably, the

increase of the PPG segments caused an increase in H-bonds between –NH groups of PNIPAM and oxygen atoms of the PPG leading to an increase in Tg.

Decomposition temperatures, Td, of the cross-linked

graft copolymers were similar to that of PNIPAM at around 431 °C[69]. Plasticizing effect of the PSB and PPG blocks reduced the decomposition temperatures of the PNIPAM conjugates 10–20 °C (seeTable 2).

3.4. Swelling behaviors

Swelling degrees of the gels were determined from 4 °C to 40 °C. Temperature dependence of equilibrated swelling ratio of PNIPAM conjugates have been given inFig. 6. The highest swelling degrees were observed at around 4000% when the hydrophobic segment in the gel was the lowest. Increase in the hydrophobic segment content in the gel causes the dramatic decrease in the swelling degree up to 1000%. There is a relationship between swelling ratio

Fig. 4. SEM pictures of the PNIPAM conjugates: (a) PNIPAM-homopolymer, (b) PSB-g-PNIPAM (A-3: 0.5/2.0), (c) PSB-g-PPG-g-PNIPAM (E-4: 0.5/0.5/2.0), (d) PSB-g-PPG-g-PNIPAM (E-5: 0.5/1.0/2.0).

Table 2

Thermal properties of the crosslinked graft copolymers

Polymer DSC (°C) TGA (°C) Tg1 Tg2 Tg3 Td1 PSB[32] 44 18 2 420 PNIPAM[53] 135 431 A-1 70 – A-2 50 75 A-3 50 70 A-4 50 75 F1 61 81 420 F2 75 87 425 F3 75 117 430 E1 61 81 430 E2 61 103 430 E3 60 81 425 E4 53 68 420 E6 62 78 420 E5 60 80 410

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and the cross-link density of the hydrogels. When we com-pare the swelling ratios of A-series with B-series, we see that the longer polymerization time gives higher cross-link density. From the plots of swelling degree versus tempera-ture, we found that the LCSTs of the hydrogels were 31 °C, 29 °C and 27 °C for A-4, A-3 and A-2; 27 °C and 29 °C for B-3 and B-4, respectively, corresponding to the increasing contents of the hydrophobic units (PSB). The samples hav-ing higher content of PSB (A-1, B-1 and B-2) do not have thermoresponsive properties.

Incorporating more hydrophilic or hydrophobic mono-mers in the gel compositions can control the phase transi-tion behavior[11,68–72].

PPG homopolymer also exhibits LCST behavior in aque-ous solution[35a]. Unlike the relatively sharp and molecu-lar weight-independent LCST of 32 °C exhibited by linear PNIPAM, the LCST of PPG is a rather broad transition that lies in the 10–20 °C range depending on its degree of repeating unit [17,35b,35c]. In this work, a new type of

PPG-macromonomeric initiator initiated the NIPAM poly-merization leading to highly branched and cross-linked co-network, which is different from that of the conven-tional cross-linkers. Agreeing with this conclusion in the cited literature above, the LCSTs of the hydrogels in this work were determined as 24 °C, 26 °C and 28 °C for F-1, F-2, F-3 and 23 °C, 25 °C, 26 °C for F-4, F-5, F-6, respec-tively. LCST of the hydrogel shifted to higher degrees as PPG content decreases.

The effect of PSB on swelling behavior of the PSB-g-PPG-g-PNIPAM gel was studied by changing the PSB concentra-tion in the gelaconcentra-tion medium. The NIPAM/PPG mole ratio was ﬁxed 2.0/0.5 respectively. The increase in the amount

Fig. 5. DSC traces of PNIPAM conjugates. (For the sample abbreviations, seeTable 1.)

Fig. 6. Temperature dependence of equilibrated swelling ratio of PNIPAM conjugates; (a) A- and B-series, (b) E- and F-series.

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of the PSB unit in PSB-g-PPG-g-PNIPAM graft copolymer caused a signiﬁcant decrease in the thermosensitivity of the copolymer and decreased the hydrophilicity of the whole gel network due to the hydrophobic nature of PSB, which leads to the decreased swelling ratio of hydrogel at temperature below the LCST. Besides, increasing PSB caused decreasing swelling capacities of these hydrogels because of high cross-linking density of the polymer net-work. If free volume in the polymer is too low, water may be unable to penetrate into the polymer matrix to ini-tiate the swelling process. High cross-link density of the network or stiffer polymer backbone intensiﬁes a resis-tance to chain extension, so highly cross-linked polymers with a stiffer backbone will swell less. The transition tem-perature shifted to the left by increasing the PSB content of the gel. High content of PSB in graft copolymer (run nos. E-3, E-6) dramatically reduces thermoresponsivity with LCST at around 27 °C.

The swelling behavior in response to pH changes of B-series (PSB-g-PNIPAM graft copolymer hydrogels) was also studied. For this purpose, swelling experiments were car-ried out in acidic and basic buffer solutions in pH ranges 0–12. Equilibrium swelling behavior of PSB-g-PNIPAM gels as a function of pH at room temperature was plotted in

Fig. 7. We have found that the graft copolymers were not

pH-responsive in acidic solutions. Furthermore, higher pH ranges cause the hydrolysis of the PSB ester linkages, quickly and make the cross-linked graft copolymers solu-ble. As a rule, to use thermoresponsive polymers in a bio-logical system, their phase transition proﬁle should be unaffected by the pH 0–7 [13]. Therefore, this type of new graft copolymers may be promising material for bio-medical use. In addition to this, higher pH ranges cause the hydrolysis of the PSB ester linkages and make the cross-linked graft copolymers soluble. The degradability of the PNIPAM conjugates in basic aqueous solution can also be very important for industrial and biomedical use. 3.5. Deswelling behaviors

To study the shrinking kinetics of the copolymers, the step input on the medium temperature was applied in

the reverse direction. Hydrogels were swollen at 4 °C and shrinking experiments were carried out at 50 °C according to the procedure cited in the reference[73].

Fig. 8shows the curves of the time dependence of the

deswelling ratios of PNIPAM conjugate hydrogels. The fastest shrinking was observed with the NIPAM-rich gel, which reached the equilibrium state ﬁrst. A-4, A-3 and A-2 gels exhibit a dramatically faster response rate to temperature changes. Gel A-4 loses 95% in 3.5 min and over 98% in 5 min. Gel A-3 loses 78% or so of water and gel A-2 loses 84% in 5 min. A-1 loses only 50 wt% of water in 5 min. InFig. 8, it is obvious the B-4 and B-3 exhibit fast shrinking rate and lose water dramatically. These two gels reach the stable water retention within 5 min with less than 65% water in gel, while B-2 and B-1 lose water slowly although they also reach equilibrium quickly with rela-tively high water content.

The deswelling kinetics of the hydrogels after a temper-ature jump from the equilibrated swollen state at 4 °C to the hot water at temperature 50 °C are shown in Fig. 8. As seen here, the fastest shrinking was observed with the NIPAM-rich gel, which reached the equilibrium state ﬁrst. Gel F-1 and F-4 exhibit a dramatically faster response rate to temperature changes. Gel F-4 loses 79% in 3.5 min and over 81% in 5 min. Gel F-2 loses 69% of water and gel F-3

Fig. 7. Equilibrium swelling behavior of PSB-g-PNIPAM gels as a function of pH at room temperature.

Fig. 8. Time dependence of the deswelling ratios of PNIPAM conjugate hydrogels in distilled water at 50 °C. (For the sample abbreviations, see Table 1.)

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loses 65% in 5 min. It is obvious the F-4 gel reach the stable water retention within 5 min with less than 92% water in gel. Gel F-5 loses 87% of water and gel F-6 loses 76% in 5 min.

The fastest shrinking was observed with the hydrogel at gel F-3 and F-6. Gel E-1 loses 55% in 5 min and over 65% in 50 min. Gel E-3 loses 65% in 5 min and over 75% in 50 min. The E-4 and E-6 gels reach the stable water retention with-in 5 mwith-in with less than 45% water with-in gels.

Deswelling ratios of the gels were changing from 0.05 to 0.4 according to the increasing ratio of the hydrophobic moiety. In PSB/PNIPAM series, the gel has highest cross-link density indicated the highest deswelling ratio of 0.7 for B-1 and 0.5 for A-1. In addition, A-1 gel indicated the deswelling ratio as the same as PSB/PPG2000/PNIPAM ser-ies (E-4: 0.5/0.5/2.0 and E-6: 1.0/0.5/2.00). We have also compared the deswelling effect of the PPG400 and PPG2000 in the gels. Deswelling ratio of the PPG2000 was higher than that of PPG400. This shows that deswell-ing properties of the PSB and PPG2000 segments are similar.

3.6. Chemical release behavior

One of the most attractive features of PNIPAM based hydrogels as drug carriers is their intelligent property or auto-adjustable function to external temperature changes. It is important and practical to examine the MO release data from those hydrogels at a temperature below LCST at19 °C and above LCST at 37 °C (the body temperature).

Fig. 9 exhibits the cumulative amounts of MO released

from the PNIPAM hydrogels at 19 °C and 37 °C, respec-tively. At temperatures below the LCST, the release is faster as the polymer chains are expanded. All hydrogels show a burst release of MO. During this burst release within the first 25 min, the cumulative MO release was 66% for F-3, 58% for E-3, 49% for E-1, 53% for E-4, 47% for E-6 and 43% F-6. All the hydrogels exhibited very similar release pro-files. The cumulative MO release during the 315 min study period was 98% for F-3, 96% for 3, 92% for 1, 94% for E-4, 92% for E-6 and 92% F-6. The release profiles at 37 °C are the same as those at 19 °C, but the amounts of the cumu-lative release at the end of the study period (315 min) were lower than those at 19 °C for all PNIPAM hydrogels. At tem-peratures above its LCST, the polymers contract thereby decreasing the mesh size so the rate of drug release is much slower. For release at 19 and 37 °C, the gels released most of the dye within 2 h of starting the experiment. The primary release data exhibited a fast release of MO with the auto-adjustable function to external temperature changes.

Higher than 90 wt% of methyl orange has been released from the gel polymer conjugates at 19 °C while 85 and 70 wt% of methyl orange has been released related to the increasing PSB content in the gel polymer. PSB hydropho-bic segments in the shrinking gel polymer probably bound some MO residue, partially. For example, cumulative release values of MO were 70 wt% for E-3: PSB (1.0)/ PPG400 (0.5)/PNIPAM (2.0); 85 wt% for E-1: PSB (0.5)/ PPG400 (0.5)/PNIPAM (2.0) and 95 wt% for F-3: PPG (0.5)/ PNIPAM (2.0).

4. Conclusions

Soybean oil is a hydrophobic, cheap and renewable sub-stance. Therefore, soybean oil was incorporated into PNI-PAM-gels. Using without any additional catalyst and solvent makes this procedure important for ‘‘green chemis-try”. The gels were not pH responsive in acidic range. As a rule, to use thermoresponsive polymers in a biological sys-tem, their phase transition proﬁle should be unaffected by the pH 0–7[13]. Therefore, this type of new graft copoly-mers may be promising material for biomedical use. In addition to this, higher pH ranges cause the hydrolysis of the PSB ester linkages, quickly and make the cross-linked graft copolymers soluble. The degradability of the PNIPAM conjugates in basic aqueous solution can also be very important for industrial and biomedical use. PPG was also

Fig. 9. Chemical release of methyl orange from the PNIPAM conjugate hydrogels in water at temperature below and upper LCST. (For the sample abbreviations, seeTable 1.)

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inserted into the gel-PNIPAM conjugates using the macro-monomeric initiator based on PPG, via the cross-linking mechanism starting with hyperbranching[52,60]. PNIPAM conjugates containing PPG segments had LCST of 3–6 °C lower than that of pure PNIPAM.

Acknowledgements

This work was ﬁnancially supported by Zonguldak Kar-aelmas University Research Fund and TUBITAK Grant No. 104M128.

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