Studies on novel radiopaque methyl methacrylate: glycidyl ...

J Mater Sci: Mater Med (2009) 20:S243–S250 DOI 10.1007/s10856-008-3557-4

Studies on novel radiopaque methyl methacrylate: glycidyl methacrylate based polymer for biomedical applications S. Dawlee Æ A. Jayakrishnan Æ M. Jayabalan

Received: 15 June 2008 / Accepted: 21 July 2008 / Published online: 31 July 2008 Ó Springer Science+Business Media, LLC 2008

Abstract A new class of radiopaque copolymer using methyl methacrylate (MMA) and glycidyl methacrylate (GMA) monomers was synthesized and characterized. The copolymer was made radiopaque by the epoxide ring opening of GMA using the catalyst o-phenylenediamine and the subsequent covalent attachment of elemental iodine. The copolymer was characterized by Fourier transform infrared (FTIR) spectra, energy dispersive X-ray analysis using environmental scanning electron microscope (EDAX), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). X-ray visibility of the copolymer was checked by X-radiography. Blood compatibility and cytotoxicity of the newly synthesized copolymer were also evaluated. The iodinated copolymer was thermally stable, blood compatible, non-cytotoxic, and highly radiopaque. The presence of bulky iodine group created a new copolymer with modified properties for potential use in biomedical applications.

1 Introduction Radiopaque polymers find extensive use in medicine and dentistry as prostheses made from such polymers could be S. Dawlee (&) M. Jayabalan Polymer Division, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum 695012, Kerala, India e-mail: [email protected] A. Jayakrishnan Laboratory for Biomaterials Science and Technology, Department of Biotechnology, Bhupat and Jyothi Mehta School of Biosciences, Indian Institute of Technology, Chennai 600 036, Tamil Nadu, India

assessed in a non-invasive manner so as to evaluate their performance. X-radiography is a low cost imaging technique for such an evaluation. Conventional polymers cannot be detected by commonly used imaging techniques like Xray and ultrasound because they contain elements such as C, H, O, and N which exhibit both low electron density and low specific gravity. Therefore, research on methods for increasing the average electron density and the specific gravity of polymers by incorporating heavy elements would lead to the development of novel radiopaque polymers. Radiopaque polymer blends were prepared by incorporating the radio-opacifying agents such as heavy metal powders, inorganic salts of a heavy element, or organic compounds containing a heavy atom substituent as physical mixtures with the polymer [1–3]. A major drawback of this method was the creation of non-homogenous mixtures, resulting in the deterioration of the end product. To overcome this problem, single phase radiopaque polymer-salt complexes were produced by the incorporation of a radiopaque heavy metal salt such as bismuth bromide or uranyl nitrate into an appropriate polymer ligand via chelation [4, 5]. Metal salts of vinyl monomer such as barium and zinc acrylates have been copolymerized with methyl methacrylate to impart radiopacity to the dental implants, but the ionic nature of these resins leads to significant absorption of water and subsequent hydrolysis resulting in the loss of the opacifying atom [6]. Grafting of iodine containing molecules onto preformed high molecular weight polymers have been investigated so as to prepare radiopaque hydrogel spherical particles by acylation of poly(2-hydroxyethyl methacrylate) beads using triiodobenzoic acid, iothalmic acid, and iopanoic acid [7, 8]. In a similar manner, cellulose was made radiopaque by coupling triiodobenzoic acid with the hydroxyl groups of cellulose by Mottu et al. [9] for possible applications as embolic agents. Tecoflex 80A, an aliphatic, commercially

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available, medical grade polyurethane was made radiopaque by coupling a 5-iodine containing molecule, N-(2,6-diiodocarboxy phenyl)-3,4,5-triiodo benzamide onto the polymer backbone and it is expected to find application in vascular stents and catheters, where radiopacity would be an added advantage [10]. Homopolymerization or copolymerization of aromatic iodine containing vinyl monomers with other vinyl monomers such as 2-hydroxyethyl methacrylate (HEMA) or methyl methacrylate (MMA) have also been studied so as to impart radiopacity to polymers [11–19]. Synthesis of iodinated vinyl monomer such as triiodophenyl methacrylate has been reported [13]. This iodinated monomer was highly resistant to homopolymerization and copolymerization, and only polymers having low molecular weight could be formed [13]. This was attributed to the bulky nature of iodinated aromatic nucleus sterically hindering the propagation step during polymerization. Introduction of a spacer arm between the bulky halogenated aromatic nucleus and the vinyl group reduced the steric hindrance as reported by Margel et al. [20]. This resulted in the facile homopolymerization and copolymerization of monomers such as 2-methacryloxyethyl(2,3,5-triiodobenzoate), 2-methacryloxypropyl(2,3,5-triiodobenzoate), and 3-(methacryloylamidoacetamido)-2,4,6 triiodobenzoic acid with HEMA or MMA by dispersion, suspension, and bulk polymerization [20]. Polymers based on GMA have been studied extensively due to the ability of epoxide groups to enter into a large number of chemical reactions. Free radical-mediated random copolymerizations of GMA with conventional monomers have been investigated for their potential applications such as binding enzymes and other biologically active species [21]. In the present investigation, a new radiopaque copolymer based on GMA and MMA was synthesized and characterized for possible biomedical applications. The copolymer was made radiopaque by the epoxide ring opening of GMA, followed by the covalent attachment of the iodine moiety.

2 Materials and methods

J Mater Sci: Mater Med (2009) 20:S243–S250

2.2 Methods 2.2.1 Copolymerization of MMA with GMA A mixture of 0.035 mol of MMA and 0.0105 mol of GMA (70:30 weight ratio) dissolved in 25 ml toluene was taken in a 100 ml RB flask. To this 0.1% benzoyl peroxide (BPO) was added and nitrogen was purged for 30 min. The reaction was then kept at 70°C overnight with constant stirring. After the reaction, the copolymer was precipitated out in 100 ml of hexane. It was washed thoroughly with hexane and dried at room temperature for 24 h. 2.2.2 Iodination of the copolymer In a 100 ml RB flask, 2.53 g copolymer was dissolved in 50 ml of dichloromethane. To this, 0.0578 g o-phenylenediamine was added as the catalyst and 1.35 g iodine dissolved in 150 ml of dichloromethane was then added through a pressure equalized addition funnel in drops. The reaction was then kept at room temperature overnight. After the reaction, iodinated copolymer was precipitated out from methanol. It was washed again with methanol and dried at room temperature for 24 h. The iodinated copolymer was then dissolved in dichloromethane and cast to form films of desired dimensions. 2.3 Characterization of the iodinated copolymer 2.3.1 Determination of epoxy content The concentration of MMA and GMA in the copolymer was determined quantitatively by finding the epoxy content of the copolymer [22]. One gram of the sample was weighed accurately in a 50 ml Erlenmeyer flask. The copolymer was then dissolved in chlorobenzene. Around five drops of 0.1% crystal violet indicator solution was added and titrated against 0.1 N hydrogen bromide in acetic acid. The end point was the appearance of blue green colour.

2.1 Materials

2.3.2 Fourier transform infrared (FTIR) spectra

Iodine and catalyst o-phenylenediamine were of analytical grade and were procured from S.D. Fine chemicals Ltd., Mumbai, India. Benzoyl peroxide was obtained from BDH, Poole, England and was recrystallized from methanol before use. MMA and GMA were purchased from Sigma Chemical Co., St. Louis, MO, USA. MMA and GMA were washed free of the inhibitor using sodium hydroxide solution followed by water, dried over anhydrous sodium sulfate, and distilled under reduced pressure prior to use.

The FTIR spectra of the copolymers were recorded using a FTIR spectrophotometer (Nicolet, Impact 410, USA) as KBr pellets.

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2.3.3 Energy dispersive X-ray analysis (EDAX) EDAX analysis of the copolymers was conducted using EDAX attached to an Environmental Scanning Electron Microscope (FEI, Quanta 200, The Netherlands).


2.3.4 Thermogravimetric analysis (TGA) The TGA of the polymers were carried out using SDT2960, TA Instruments Inc., USA, thermogravimetric analyzer. Measurements were performed in nitrogen atmosphere at a heating rate of 10°C/min. 2.3.5 Differential scanning calorimetry (DSC) DSC measurements were conducted with DSC-2960, TA Instruments Inc. The calorimeter was calibrated with indium metal as a standard. Measurements were performed in nitrogen atmosphere at a heating rate of 10°C/min. 2.3.6 X-ray opacity X-radiographs were obtained using a standard clinical General Electric X-ray instrument equipped with 2.5 mm aluminium filtration set at 40 KvP with 10 mA current for 0.2 s. The relative X-ray opacity was determined visually by comparison with the opacity exhibited by a standard aluminium wedge exposed on the same radiograph.

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was used as working solution. Cells were cultured as before in multi-well tissue culture plates and when monolayer was attained, culture medium was removed, rinsed with phosphate buffered saline (PBS), and 100 ll each of extracts of copolymer and negative control (high density polyethylene) and 100 ll of diluted phenol (positive control) were added to different pre-labeled wells containing cells. Cells with medium alone served as control. Culture medium (100 ll) was used as reagent blank. Plates were incubated for 24 h at 37°C in 5% carbon dioxide atmosphere. After 24 h, the extracts/medium was removed and 200 ll of MTT working solution was introduced using a multi channel pipette into each well. Plates were wrapped with aluminum foil and incubated at 95% humidified atmosphere at 37°C for 8 h. After removing the reagent solution and rinsing with PBS, 200 ll of isopropanol was added to each well and incubated for 20 min at 37°C in a shaker incubator (Labline Instruments, Melrose Park, USA). The absorbance of the resulting solution in each well was recorded immediately at 570 nm using automated micro plate reader (Bio-Tek Instruments, Vermont, USA).

2.4 Cytotoxicity studies

3 Blood compatibility evaluation

In vitro cytotoxicity testing was done using the direct contact method with the copolymer based on ISO 10993-5 standards [23]. Test sample (iodinated copolymer of GMA and MMA), negative controls (high density polyethylene), and positive controls (copper wire) in triplicate were placed on subconfluent monolayer of L929 mouse fibroblast cells. L929 cells were subcultured from stock culture (National Centre for Cell Sciences, Pune, India) by trypsinization and seeded onto multi-well tissue culture plates (Nunc, Denmark). Cells were fed with Dulbecco’s minimum essential medium supplemented with bovine serum and incubated at 37°C in 5% carbon dioxide atmosphere. When the cells attained a monolayer, the material was kept in contact with the cells in triplicate. After incubation of cells with test samples at 37°C for 24 h, cell culture was examined microscopically (Leica, WILD MPS32, Germany) for cellular response around test sample. Cellular responses were scored as 0, 1, 2, and 3 according to non-cytotoxic, mildly cytotoxic, moderately cytotoxic, and severely cytotoxic. Cytotoxicity of the copolymer was quantitatively assessed further by MTT assay [24] which measures the metabolic reduction of 3-(4,5-dimethylthiazol-2yl)-2,5,diphenyl tetrazolium bromide to a coloured formazan by viable cells. Toxicity was evaluated on the extract of the material (0.1 cm2/ml) in medium containing serum. MTT dissolved at a concentration of 0.05 g/ml in sterile PBS, filtered through a 0.22 lm filter to remove any formazan crystals

Blood compatibility evaluations of the copolymer were carried out using blood from human volunteers. Blood was collected into sodium citrate as the anticoagulant in the ratio 9:1. The consumption of red blood cells (RBC) and white blood cells (WBC) on contact with the sample was analyzed. The copolymer was placed in wells of tissue culture grade polystyrene petridishes and wetted using PBS. Each material was exposed with 1 ml of blood for 30 min under agitation at 75 ± 5 rpm using an environmental bath shaker (Labline Instruments Inc., Illinois, USA) thermostated at 37°C. Samples were withdrawn for analysis immediately after mixing and after 30 min of exposure. A well without any material was used as the reference. The consumption of RBC and WBC was evaluated using a haematology analyzer (Cobas Minos, Roche Diagnostics, France) calibrated using WHO traceable standards. For evaluating the effect of material on blood coagulation, partial thromboplastin time (PTT), and fibrinogen concentration were determined. Fibrinogen content was detected using Coagulation Analyzer (Diagnostica Stago, France) and was based on the principle that, when excess concentration of thrombin was added to diluted plasma, clotting time varied directly with fibrinogen concentration. The automated coagulation analyzer detects the clotting time and a calibration curve was generated using WHO traceable control plasma. From the calibration curve, the concentration of fibrinogen was determined. PTT was

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analyzed on samples exposed to fresh citrated human blood immediately and after 30 min of exposure. Polymeric films were equilibrated for 1 h in PBS, exposed to blood under agitation at 100 rpm using the environmental bath shaker at 37°C. After 1 and 30 min exposure, blood was centrifuged to obtain platelet poor plasma. Plasma was then mixed with cephalin reagent and incubated for 3 min after adding CaCl2 solution to initiate clotting. Clotting time was noted using an automated coagulation analyzer (Diagnostica Stago, France). Blood compatibility evaluations were done according to ISO standards [25].

Fig. 1 Schematic representation of copolymer synthesis

4 Results and discussion Epoxides are well-known carbon electrophiles capable of reacting with various nucleophiles and they undergo regioselective ring opening reactions to form vicinal halohydrins [26]. The regioselective ring opening of epoxides using elemental iodine in the presence of a catalytic amount of o-phenylenediamine affords vicinal iodo alcohols. The major nucleophile in the course of the reaction is the pentaiodide ion (I5 ) and this bulky nucleophile plays a fundamental role in the high regioselectivity

CH3 O C

n CH2

C

O

CH

CH2

m CH2

+

O

CH3

O

C

C

CH

GMA

O

CH3

MMA

CH3

CH3

CH3

CH3

0.1 % BPO, 70 ° C CH2

Toluene

C

CH2

C

O

C

C

O

O CH O

Fig. 2 Schematic representation of synthesis of iodinated copolymer

CH 2

CH O

CH2 CH3

CH 2

C

C

O

O

C

O CH O

CH O

CH2

CH2

CH2 O

I

CH CH2

C

m-1

OCH3

CH3

C

CH 2

C

C

O

C

m-1

OCH3

CH 2

CH2

Copolymer

CH3

CH3

C C

C

CH2

C

O

C

O

C

O

CH2 HO I

CH3

CH2

O HO

O

OCH3

n-1

O

C

CH3

I2 / catalyst RT

C

Copolymer

O

CH 2

CH 2

OCH3

CH2

CH2

C

CH 2

CH3

C

O

O

n-1

123

CH2

n-1

CH CH2

CH3 CH2

OCH3

CH2 Iodinated Copolymer

O

C m-1

C OCH3


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Table 1 Epoxy content of the copolymers Sample

Theoretical value (9103 mol)

Experimental value (9103 mol)

PGMMA

2.2

2.1 ± 0.1

PIGMMA

0

1.3 ± 0.2

observed. It is due to the attack on the less sterically hindered epoxide carbon [26]. The copolymer was prepared by taking GMA and MMA in the weight ratio 70:30. Copolymer was then iodinated using the catalyst o-phenylenediamine. The mechanism of the reaction is well documented in the literature [26–30]. Schematic representation of the synthesis is as shown in Figs. 1 and 2. The physico-chemical characterizations of the iodinated and non-iodinated copolymers were carried out. The actual composition of GMA–MMA copolymer was determined by finding the epoxy content of the polymer

since GMA is a monomer exhibiting polymerizable methacrylic unsaturation and an epoxy group [31]. The monomer is copolymerized by means of free radical initiators known to attack double bonds. The copolymer was dissolved in a suitable solvent and the resulting solution was titrated directly with a standard solution of hydrogen bromide in glacial acetic acid. The hydrogen bromide reacts stoichiometrically with epoxy groups to form bromohydrins, therefore, the quantity of acid consumed is the measure of epoxy content. The epoxy content of GMA– MMA copolymer in the non-iodinated sample (PGMMA) obtained by the acetic acid–hydrobromic acid method was in agreement with the theoretical value and is as given in Table 1. In the case of iodinated sample (PIGMMA), we expected complete conversion of epoxy groups to iodohydrins and hence, the theoretical value was assumed to be zero. But, after the experiment, the epoxy equivalent in PIGMMA was found to be 0.0013 mol suggesting the

Fig. 3 IR Spectra of iodinated and non-iodinated copolymer

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Fig. 5 EDAX Image of PIGMMA

incomplete conversion of epoxy groups to iodohydrin. Therefore, the total iodine content in the copolymer was theoretically calculated as 13.5%. The FTIR spectrum (Fig. 3) of the copolymer showed the following vibrations. The peak at 1,721 cm-1 in PGMMA is attributed to the ester carbonyl stretching of GMA and the band at 905 cm-1 is due to the asymmetrical stretching of the epoxy group. After iodination in PIGMMA, the intensity of absorbance for epoxy group got substantially diminished, whereas a broad band for hydroxyl functional group appeared at 3,487 cm-1 indicating the substitution of epoxy ring by iodine so as to form iodohydrin. The peak at 907 cm-1 suggests the presence of residual epoxy group and the partial modification could be probably due to a reaction rate diminishing at higher graft ratios, as is usually observed when voluminous agents are used in polymer modification [31].

100

80

(a)

(b)

60

Weight (%)

Fig. 4 EDAX Image of PGMMA

EDAX (Figs. 4 and 5) was carried out for both iodinated and non-iodinated copolymers to detect the presence of iodine moiety. In the case of PGMMA, only two peaks were obtained, one of carbon and the other of oxygen and in PIGMMA, additional peaks were detected in the area 3.5–5 keV which corresponded to the iodine atom. Thermal characteristics of the copolymers were evaluated by DSC and TGA analysis. The DSC traces of PIGMMA and PGMMA are given in Fig. 6. The glass transition temperature (Tg) of the PGMMA increased drastically after halogenation reaction. For PIGMMA, the Tg was found to be 105°C and for PGMMA, it was 58°C. After iodination, due the presence of bulky iodine atoms, the chain mobility got decreased and this accounts for the increase in Tg of PIGMMA. Figure 7 gives TGA traces for PGMMA and PIGMMA. Thermal degradation characteristics of the copolymer got

40

20

0

-20 0

100

200

300

400

500

Temperature (°C)

Fig. 7 TGA thermograms of (a) iodinated and (b) non-iodinated copolymer

0.1

0.0

(a)

Heat Flow (W/g)

-0.1

-0.2

(b)

58 °C

-0.3

105 °C

-0.4

-0.5 0

50

100

150

200

Temperature (°C)

Fig. 6 DSC thermograms of (a) non-iodinated and (b) iodinated copolymer

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Fig. 8 Fibroblast cells around PIGMMA

Absorbance (570 nm)


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1.6

Table 3 Percentage hemolysis in plasma samples after exposure to iodinated copolymer

1.4

Sample

Hemolysis (%)

1.2

PIGMMA

0.05

Reference (tissue culture petridish)

0.04

1.0 0.8 0.6 0.4 0.2 0.0

PE

PIGMMA

Phenol

Fig. 9 MTT reduction of mouse fibroblast cells with polymer extracts in comparison with negative (high density polyethylenePE) and positive control (dilute phenol) for 24 h

Fig. 10 X-ray images of aluminium wedge of 2 mm thickness (a), iodinated copolymer (b), non-iodinated copolymer (c- Not visible in the X-ray)

altered after iodination. After first stage decomposition, weight of the copolymer remained in the case of PIGMMA and PGMMA were 97.41% and 80.7%, respectively. The thermal stability of the PIGMMA was found to be better than the PGMMA with very little decomposition below 300°C. Consequently with incorporation of bulky iodine atoms in polymer side chains, a novel modified polymer exhibiting new properties was obtained. Preliminary cytotoxicity evaluation was done to assess the potential of the iodinated copolymer for biomedical applications. The samples were found to be non-cytotoxic in nature. Representative microphotograph of fibroblasts

cells around copolymer (scored as zero) is shown in Fig. 8. In order to test the cytotoxicity of the polymer quantitatively, an MTT assay was performed. Quantitative assessment of the cytotoxicity to cells after contact with the material extract showed 91.5% metabolic activity by PIGMMA when compared to cells without the material for 24 h of contact (Figs. 9 and 10). In vitro screening of the copolymer for blood compatibility showed that there was no significant difference in count for RBC and WBC immediately and after 30 min of contact. Evaluations were confined to iodinated copolymer. Determination of fibrinogen content showed that there was no change in fibrinogen concentration when evaluated after 30 min exposure of blood to the samples tested. Partial thromboplastin time indicated that the intrinsic coagulation pathway has not been adversely affected. The values obtained for the copolymer were comparable to the results of reference (Table 2). The hemolysis assay showed that the copolymer was non-hemolytic in nature. The hemolytic potential of the material is defined as the measure of the extent of hemolysis that may be caused by the material when it comes in contact with blood. Table 3 shows the percentage hemolysis of blood in contact with iodinated copolymer at 37°C for 30 min. Radiographic examination of iodinated copolymer showed sharp X-ray images when compared to noniodinated copolymer, thus indicating radiopacity of the iodinated sample. A minimum radiopacity, equivalent to 2 mm of aluminium (as specified by International Standards Organization, ISO 4049-1978) is necessary for detecting sections encountered in polymeric resins. It can be seen from the radiograph that image of PIGMMA film having 2 mm thickness is comparable with that of the aluminium wedge having same dimensions.

Table 2 RBC count, WBC count, fibrinogen content, and PTT of human blood immediately and after 30 min of exposure to iodinated copolymer RBC count (106/mm3)

WBC count (103/mm3)

Fibrinogen concentration (g/dl)

PTT(s)

Before

After

Before

After

Before

After

Before

After

PIGMMA

4.50

4.52

6.5

6.2

3.53

3.53

101.90

113.8

Reference (tissue culture petri dish)

4.57

4.59

6.5

6.3

3.73

3.73

131.4

138.6

Material

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5 Conclusion A new radiopaque copolymer based on methyl methacrylate and glycidyl methacrylate monomers was synthesized and characterized. The copolymer was made radiopaque by the epoxide ring opening of glycidyl methacrylate using the catalyst o-phenylenediamine and the subsequent covalent attachment of elemental iodine. Incorporation of iodine atom to macromolecular chain led to important modifications of polymer properties such as enhanced thermal stability and high radiopacity. Preliminary in vitro screening of the iodinated copolymer for blood compatibility and cytotoxicity has shown that the copolymer was blood compatible and non-cytotoxic. Acknowledgment Thanks are due to Director and Head, SCTIMST for providing the facilities to carry out this work. S. D. acknowledges the financial support from Council for Scientific and Industrial Research, New Delhi in the form of a Senior Research Fellowship.

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