Radiation induced grafting of methacrylic acid onto silk for the immobilization of antimicrobial...

Die Angewandte Makromolekulare Chemie 112 (1989) 87- 102 (Nr. 2851)

Centre for Biomedical Engineering, Indian Institute of Technology Delhi, New Delhi - 110016, India

Radiation Induced Grafting of Methacrylic Acid onto Silk for the Immobilization of Antimicrobial Drug

for Sustained Delivery

Harpal Singh and P. K. Tyagi

(Received 16 September 1988; revised 16 January 1989)

SUMMARY: Medical grade black braided silk suture and pure mulberry silk twisted yarn were

grafted with methacrylic acid (MAA) using %o gamma radiation. The effect of various synthesis conditions on the grafting was studied. The addition of methanol in water has a marked effect on the degree of grafting, maximum yield of grafting was obtained in an equivolume mixture of water-methanol. The percent grafting increased with the increase in the monomer concentration and in total radiation. For a constant total dose, grafting was found to be higher at lower dose rates. The order of the dependence of the rate of grafting on the dose rate was found to be 0.53 and 0.56 for medical grade black braided silk suture samples and mulberry silk twisted yarn samples, respectively. The immobilization of an antimicrobial drug, 8-hydroxy quinoline (8-HQ) hydrochloride on the grafted pure silk for sustained delivery, its release in vitro and its antimicrobial activity were also studied. The percentage of 8-HQ immobilized onto various grafted silk samples increased with the increase in the degree of grafting. The release of the drug in distilled water was sustained for 32 days. 8-HQ was found to be active against both Gram-positive and Gram-negative bacteria.

ZUSAMMENFASSUNG: Ein medizinischer, schwarzer geflochtener Seidenfaden und ein reiner Maulbeersei-

denzwirn wurden mit Methacrylsaure (MAA) unter Verwendung von Wo-Gamma- strahlung gepfropft. Der Einflufi verschiedener Synthesebedingungen auf die Pfrop- fung wurde untersucht. Die Zugabe von Methanol in Wasser beeinflufit den Pfropf- grad, die maximale Pfropfausbeute wurde in einer Mischung aus gleichen Volumina Wasser und Methanol erhalten. Der Pfropfgrad nahm mit Zunahme der Monomer- konzentration und der Gesamtstrahlungsdosis zu. Bei einer konstanten Gesamtdosis war die Pfropfausbeute bei niedrigeren Dosisleistungen htiher. Die Ordnung der Ab- htingigkeit der Pfropfgeschwindigkeit von der Dosisleistung wurde zu 0,53 fur den medizinischen Seidenfaden und zu 0,56 fur Maulbeerseidenzwirn bestimmt . Die Immobilisierung eines antimikrobiellen Armeimittels, 8-Hydroxychinolin-

@ 1989 HUthig & Wepf Verlag, Base1 ooO3-3146/89/$03.00 87

H. Singh and P. K. Tyagi

hydrochlorid (8-HQ - HC1) auf gepfropfte reine Seide zur dauerhaften Abgabe, seine in-vitro Freisetzung und seine antimikrobielle Aktivitiit wurden auch untersucht . Der Prozentsatz von 8-HQ, das auf verschiedenen gepfropften Seidenproben immobili- siert war, nahm mit der ErhOhung des Pfropfgrades zu. Die Freisetzung des Arznei- mittels in destilliertem Wasser wurde 32 Tage lang aufrecht erhalten 8-HQ war sowohl gegen Gram-positive als auch Gram-negative Bakterien wirksam.

Introduction

Silk has been used as biostable suture in the medical field'.' for a long time. Silk suture has some desirable properties, such as good mechanical strength, knot strength, knot security, biocompatibility, and flexibility3s4. However, due to some serious drawbacks, silk has a restricted use as biostable suture. The main drawback of silk suture is that it cannot be used in the monofilament form because silk monofilaments alone have a very poor mechanical strength. Hence, silk suture is manufactured in the braided multifilament configuration'. Moreover, braided multifilament silk suture causes infection, because there are several interspaces among the filaments. These interspaces provide rooms for bacteria which cause infections6.

Recently, chemical modification of silk through grafting has received con- siderable interest, because grafting of silk by vinyl monomers can improve a number of properties of silk such as viscoelasticity, water repellency, adhesion to a variety of substances, dye uptake, settability, and soil resistance without affecting most of the original properties. Several grafting techniques like chemical, mechanical, photochemical, and radiation have been used for this purpose7-". However, gamma radiation induced graft copolymerization has shown to be the most promising method for biomedical applications, as this method avoids the impurity of chemical initiators which may be toxic to the biological systems. Silk was grafted with methacrylic acid monomer with the aim to immobilize the antimicrobial drugs to avoid bacte- rial infections. The present paper describes the grafting of methacrylic acid onto silk and the immobilization of 8-HQ. Drug release in vitro and its antimicrobial activity are also presented in this paper.

Experimental

Materials

Medical grade black braided silk suture (size 4) obtained from Johnson & Johnson Company Ltd., Bombay, India, and original degummed pure mulberry silk twisted

88

Radiation Induced Grafting of Methactylic Acid onto Silk

yarn (denier 3478, sericin content 2 - 3%) collected from Central Silk Board of India, Bangalore, were used for all the grafting experiments. Samples of both grades were washed thoroughly with distilled water, dried and stored in a desiccator over anhydrous calcium chloride. Methacrylic acid (MAA) monomer (E. Merck, West Germany) was distilled at 7O0C/2O mmHg and stored at 4'"'. Pure methanol (AR grade, British Drug House, India), was used without any purification. Peptone, beef extract and nutrient agar, all from British Drug House, England were used as obtained. Double distilled water was used in all experiments.

Radiation Source

The irradiation of samples was carried out in a %o gamma chamber supplied by Bhabha Atomic Research Centre Bombay, India.

Grafting Procedure

Graft copolymerization was carried out in standard joint corning glass tubes of 11.5 x 2.8 cm size. All the samples were kept in coming glass tubes for equilibrium swelling in appropriate solvents for 20 h before grafting. Methacrylic acid was added just before the irradiation of the samples. The monomer concentration was kept at 20 vo1.-qo in most of the experiments unless otherwise specified. The system was deoxigenated by slow bubbling of nitrogen gas through the solution for 5 min in an ice bath. The tubes were placed into the radiation chamber for a desired period. After irradiation the tubes were removed and the polymerization was stopped by the addition of a large volume of methanol to the reaction mixture. Adhering polymer was removed by Soxhlet extraction in methanol for 18 h. All samples were then dried to constant weight under vacuum at 50°C. The dry weight of the grafted samples was measured and percentage of grafting was calculated using the following equationf3 :

* loo w2 - w, w, 070 grafting =

W2: Weight of grafted sample, W, : weight of original sample.

Immobilization of 8-Hydroxy Quinoline onto Methacrylic Acid Grafted Silk

8-Hydroxy quinoline (8-HQ), an antimicrobial drug, was immobilized onto methacrylic acid grafted pure mulberry silk twisted yarn samples. Before immobilization the 8-HQ drug was converted to 8-hydroxy quinoline hydrochloride (8-HQ - HCl) by

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the method reported earlierI4. For immobilization studies weighed MAA grafted silk samples were immersed in 0.5% aqueous sodium hydroxide solution for 4 h to convert the carboxylic acid groups to sodium carboxylate. All samples were washed thoroughly and kept in distilled water for 1 h and then immersed in 15% aqueous 8-HQ-HCl solution for 24 h for ion exchange. After 24 h the drug immobilized samples were removed, washed with distilled water and dried under vacuum at 40°C for 24 h. The percent weight add on of 8-HQ was calculated by the following equation:

wd - wl . Qo add on of 8-HQ = w, wd: Weight of drug immobilized sample, W, : weight of unimmobilized sample.

Release of 8-Hydroxy Quinoline in Vitro

Release of 8-HQ in vitro was studied by monitoring its elution into distilled water. 8-HQ ionomer immobilized dry silk samples (90 mg) were immersed in 5.0 ml of distilled water separately and the supernatent was replaced every day with fresh distilled water. The amount of 8-HQ ionomer released into the supernatent was determined by measuring the absorbance of the solution at 310 nm. Absorbance measure- ments were carried out on a Hitachi 160 UV-VIS spectrophotometer.

Preparation of Agar Plates

2% Peptone, 2% beef extract, and 0.5% sodium chloride were mixed in distilled water. The solution was filtered and pH was adjusted to 7.6 by 0.1 N NaOH or 0.1 N HC1. 2% Nutrient agar was added to the filtrate, the mixture was autoclaved for 20 min at 121 "C, 1.05 kg/cm2 pressure and poured 20 ml each into 9.0 cm diameter sterile and covered petridishes, which were stored at 4°C after cooling at room temperature for 30 min.

An timicrobial Activity Assessment

Antimicrobial activity of the immobilized 8-HQ drug was assessed by inhibition zone technique. The test microorganisms for the study were S. aureus, P. aeruginosa and E. coli. A 50 mg dry weight piece of original, grafted and drug immobilized silk samples were taken, placed separately in the centre of agar plates, inocculated with any one of the strains mentioned above. The plates were incubated at 37°C for 24 h. After 24 h the diameter of the circular area around the test sample was determined.

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Radiation Induced Grafting of Methacrylic Acid onto Silk

The antimicrobial activity of different samples were expressed as the area (in cm2) of the zone of inhibition produced.

Results and Discussion

To obtain a high percent grafting particularly in the simultaneous irradiation, the monomer must be present as close as possible to the active centre created in the polymer back bone. This may be attainable when the monomer can diffuse into the polymer matrix. In the present system, the role of water-methanol solvent mixture is to provide a homogeneous medium for methacrylic acid (MAA) monomer.

Effect of Solvent Composition

Fig. 1 shows the effect of solvent composition on the percent grafting of MAA onto mulberry silk twisted yarn (type A) and medical grade silk suture (type B) samples. It can be seen from the plots that the percent grafting in

50

.- 30 c L

I) & 20

10

0

c

0 10 20 30 LO 50 60 70 80 90 100 Volume of water in solvent I%)

Fig. 1. Amount of grafting vs. volume of water in solvent. Total dose = 0.302 Mrad; dose rate = 56 rad/s. (0) Pure mulberry silk twisted yarn sample; ( 0 ) medical grade silk suture sample.

pure water is 37% and 10.5% for type A and type B samples, respectively. Addition of methanol in water had a marked effect on the degree of grafting of MAA onto silk. The graft content increases with the increase of methanol in water-methanol mixture, and maximum grafting was obtained in 1 : 1

91

H. Sin& and P. K. Tyagi

water-methanol solvent mixture. Methanol has two major effects on the grafting kinetics as the homopolymerization is reduced and the viscosity built up in the surrounding solution is retarded. Percent grafting decreases after increasing the level of methanol in water-methanol mixture beyond fifty percent, and negligible amount of grafting was observed in 100% methanol. A similar behaviour was also reported by Singh et al.14. The higher concentration of methanol appears to be an inhibitor not only for grafting but also for external homopolymerization. From Fig. 1 it is also clear that at a total dose of 0.302 Mrad, in 1 : 1 water-methanol solvent mixture, the percent grafting was found to be higher for type A samples than for type B samples. The type B samples (black braided multifilament silk suture) are black dyed and coated, but type A (pure mulberry silk twisted yarn) samples are neither dyed nor coated. Therefore, the black dye and the coated layer over the type A samples may act as a barrier for the diffusion of monomer into the matrix and result in lower grafting yields. Moreover, type B samples are in multifilament braided configuration and type A samples are in twisted form, and it is a well-known fact that the braided multifilament form is more compact than the multifilament twisted form. This fact is also supported by the higher swelling of type A samples (37%) compared to type B sample (25%) in 1 : 1 water-methanol solvent mixture for 20 h. This clearly indicates that the rate of penetration of the monomer into the matrix of type B samples is slower than into type A samples.

Effect of Dose Rate and Time

Grafting was studied at room temperature at different dose rates viz of 28, 56, and 108 rad/s. The results are shown in Fig. 2. At a constant total dose of 0.302 Mrad grafting was found to be higher at lower dose rates for all the silk samples. A similar behaviour has also been reported for other systemst5. It has been reported earlier that during irradiation a number of free radical (monomeric as well as polymeric) species are formed and their number increases with the increase in dose ratet6. The monomeric radical species may react either with the polymer back bone (grafting) or with the monomer (homopolymerization) ”. During the graft copolymerization process monomer molecules will continuously diffuse into the polymer matrix at a constant rate, irrespective of the irradiation dose rate. Under such conditions the greater availability of free radicals at higher dose rate will increase the rate of homopolymerization. As a consequence the relative rate of grafting will decrease appreciably.

92

Radiation Induced Grqfting of Methactylic Acid onto Silk

6o r

t 01 I 4 I I

0 20 LO 60 80 100 120 Dose rate (rad/sec)

Fig. 2. Amount of grafting vs. dose rate. Total dose = 0.302 Mrad. (0) Pure mulberry silk twisted yarn sample; ( 0 ) medical grade silk suture sample.

The extent of graft copolymerization as a function of time at various dose rates for both type A and type B silk samples is presented in Fig. 3. All the plots pass through the origin indicating that the grafting proceeds without any induction period. All the curves in Fig. 3 show that the percent grafting increased with the irradiation time and leveled off at a certain graft content. The difference, however, caused by different dose rates, is also observed in these curves. According to these results a lower dose rate is more effective to obtain higher graft levels.

The log-log plot of rate of grafting & (%/h) vs. dose rate I of irradiation is presented in Fig. 4. The rates of grafting at various dose rates for a total dose of 0.302 Mrad were calculated from Fig. 3. The straight lines thus obtained have the slope of 0.56 and 0.53 for type A and B silk samples, respectively. The results can be summarized in the following form:

& a 1°.56 type A 4 a 1°.53 type B

The dependence of the rate of grafting & on the dose rate has been determined in several other systems and a very wide range of dependence, namely between 0.31 and 0.90, has been rep~rted '* . '~ .~~. The values of 0.56 and 0.53 for type A and type B silk samples suggest that the termination of the graft copolymerization proceeds via a bimolecular processm.

93


Time (min)

Fig. 3. Amount of grafting vs. time of irradiation. (- - -) Medical grade silk suture sample; (- ) pure mulberry silk twisted yarn sample; ( 0 ) dose rate = 28 rad/s; (0) dose rate = 56 rad/s; (A) dose rate = 108 rad/s.

Effect of Total Dose

The effect of total dose on grafting has been presented in Fig. 5 . A total dose up to 0.60 Mrad was used at a dose rate of 56 rad/s. The results show that there is a linear increase in the percent grafting with the increasing dose of irradiation up to 0.302 Mrad, beyond this there is a marked fall in the rate of grafting and the tendency to level off. During the graft copolymerization reaction the availability of monomer is higher in the initial stages. Hence, the monomer can diffuse very easily to the grafting sites and grafts smoothly onto the silk. Further, the rate of homopolymer formation is not much affected. Both competing reactions proceed smoothly until a total dose of 0.302 Mrad has been reached. Beyond it a slow decrease in the percent

94

Radiation Induced Grqfting of Methactylic Acid onto Silk

1.6

1.0 t 1.3 1.1 1.5 1.6 1.7 1.8 1.9 2.0 2.1

Log dose rate (rad/s)

Fig. 4. Log rate of grafting vs. log dose rate. (0) Pure mulberry silk twisted yarn sample; ( 0 ) medical grade silk suture sample.

grafting indicates that there is an increase in the rate of homopolymerization as compared to the rate of grafting. Further, the increasing content of grafted poly(methacry1ic acid) may also act as a barrier against the diffusion of monomer into the polymer matrix resulting in the increase of the rate of homopolymerization and decrease in the graft content.

Effect of Monomer Concentration

The relation of the percent grafting to the monomer concentration is shown in Fig. 6. Grafting was carried out at room temperature at a dose rate of 56 rad/s for a total dose of 0.302 Mrad. The results show that there is an increase in the percent grafting with increasing monomer concentration. The results show a very general trend which has also been found in other grafting systems''. During grafting, the monomer continuously diffuses into the polymer matrix and the ability of silk macro radicals to capture the MAA

95

H. Sin& and P. K. Tyagi

80

loo[

Total dose (Mrad)

Fig. 5. Degree of grafting vs. total dose. Dose rate = 56 radls. (0) Pure mulberry silk twisted yarn sample; ( 0 ) medical grade silk suture sample.

would depend on the availability of MAA molecules in their vicinity. At higher monomer concentration, the availability of MAA molecules in their vicinity increased, therefore, graft content increases with the increase in monomer concentration.

During the grafting studies it was found that the degree of grafting is varied upto 6% for the same grafting experiments run on different days, but the shape of the grafting curves is always reproduced. From day to day, however, the curves might appear shifted up or down with respect to degree of grafting axis. For any continuous curve presented in this paper, all experimental points are obtained for samples grafted simultaneously.

Immobilization of 8-HQ onto Silk, its Release and Antimicrobial Activity

The results of the immobilization of 8-HQ drug on various grafted silk samples have been given in Tab. 1. It is evident from the table that the percent immobilization of 8-HQ ionomer onto grafted silk increases with the increase in the graft content of MAA onto silk. This can be expected because

96

Radiation Induced Grqfting of Methacrylic Acid onto Silk

loo r

Monomer concentration ( % I

Fig. 6. Degree of grafting vs. monomer concentration. Total dose = 0.302 Mrad, dose rate = 56 rad/s. (0) Pure mulberry silk twisted yarn sample; ( 0 ) medical grade silk suture sample.

Tab. 1. Amount of immobilized drug on MAA grafted pure silk.

Samples Degree of grafting

(9'00) (9'0 add on)

Amount of drug immobilized,

Pure silk Silk-g-MAA Silk-g-MAA Silk-g-MAA

0 42 63 88

2.5 10.3 15.2 25.2

of the higher concentration of carboxylic groups in grafted silk samples for ion exchange. The results of drug release from various samples in distilled water have been presented in Fig. 7. The release of 8-HQ from all grafted samples was sustained up to about 32 days. The amount of drug release per

97


0 L 8 12 16 20 2 1 28 32 Time (days)

Fig. 7. Amount of drug release vs. time. ( 0 ) Ungrafted silk sample with 8-HQ (8-HQ = 2.5%); ( 0 ) grafted silk sample immobilized with 8-HQ (degree of grafting: 42%; 8-HQ: 10.3%); (A) grafted silk sample immobilized with 8-HQ (degree of grafting: 63%; 8-HQ: 15.2%); (0) grafted silk sample immobilized with 8-HQ (degree of grafting: 88%; 8-HQ: = 25.2%).

day increases with the increase in percent grafting, in the initial period the rate of drug release is higher. This may be due to the release of appreciable amount of drug absorbed on the surface of the samples. However, the release of 8-HQ absorbed from drug solution on original silk was sustained only up to about 8-10 days. The results of antimicrobial activity of attached 8-HQ against different strains have been given in Tab. 2. The antimicrobial activity was measured by zone of inhibition produced (in cm2). Original silk and MAA grafted silk samples show no zone of inhibition. But pure MAA grafted silk twisted yarn, onto which 8-HQ was immobilized, showed zones of inhibition against all microorganisms studied and possessed antibacterial properties.

98


Tab. 2. Antimicrobial activity of 8-HQ immobilized silk-g-MAA samples (degree of grafting: 63%).

Microorganism Sample Antimicrobial activity Zone of inhibition (cm2)

Before After 32 days release release

E. coli Pure silk Silk-g-MAA Silk-g-MAA + 8-HQ (25.2%)

Silk-g-MAA Silk-g-MAA + 8-HQ (25.2%)

Silk-g-MAA Silk-g-MAA + 8-HQ (25.2%)

S. aureus Pure silk

P. aeruginosa Pure silk

0 0

19.6 0 0

10.2 0 0 7.1

0 0 2.5 0 0 2.0 0 0 0

Mechanism of Grafting and Drug Immobilization

Silk fibre is a natural protein called fibroin. Fibroin is composed of a number of amino acids, of which the most important are glycine and a-alanine with relatively small amounts of the more bulky side chain amino acids. When the grafting of methacrylic acid onto silk is carried out by gamma irradiation, several free radical sites are produced along the protein chain by the abstraction of hydrogen from secondary carbon atoms, i.e. from a-alanine. The grafting of MAA is initiated from these sites. Besides grafting, homopolymerization of MAA also occurs.

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Grafting

0 0 CH3 0 0 II II I II I1 - -NH-C-CHz-NH-C-C-NH-C-CHz-NH-C- - + CHI = C-COOH

I H

Silk having secondary carbon atoms Methacrylic acid

gamma irradiation

CH3 I 2- I

CH C.

I 0 0 CH3 0 0 I1 II I II II - -NH-C-CHz-NH-C-C-NH-C-CH,-NH-C- - + - -

COOH

Initiation of graft polymerization Initiation and propagation of homopolymerization

Termination I 0 0 CH3 0 0 II II I I1 II

Poly(methacrylic acid) - -NH-C-CHz-NH-C-C-NH-C-CH,-NH-C- - I

H$-C-COOH

CHZ I H3C-C-COOH

Propagation of graft polymerization

Termination

Silk grafted with poly(methacrylic acid)


Immobilization

0 0 CH3 0 0 II I1 I II II

I- NH-C-CH2-NH-C-C-NH-C-CH2-NH-C- - I

I

y2

H3C-C-COOH

Silk grafted with poly(methacry1ic acid)

NaOH treatment I 0 0 CH3 0 0 I/ II I II II


CH2 I I

H3C-C-COO - Na +

Sodium salt of graft copolymer

8-HQ ’ HCI treatment I 0 0 CH3 0 0 II 11 I I1 11


7H2

I H3C-C-COO -

I

Drug immobilized graft copolymer silk

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l9 R. Gouloubandi, A. Chapiro, Eur. Polym. J. 16 (1980) 957 M. T. Razzak, K. Otsuhata. Y. Tabata, J. Appl. Polym. Sci. 33 (1987) 2345 '' M. A. El-Azmirly, A. H. Zahran, M. F. Barakat, Eur. Polym. J. 12 (1976) 195

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