Journal of Radioanalytical and Nuclear Chemistry, Articles, VoL 159, No. 1 (1992) 87--96

R A D I A T I O N S T E R I L I Z A T I O N OF CHITOSAN S E A L A N T FOR V A S C U L A R PROSTHESES J. ROSIAK,* P. ULANSKI,* M. KUCHARSKA,** J. DUTIGEWICZ,** L. JUDKIEWICZ***

* lt,stitute of Applied Raa~ation Chemistry, Technical University of I:6d~ Wrdblewsklego 15, 93-590 Ldd~ (Poland) ** Institute of Man Made Fibers, Technical University of t:,6d~ Zwirki 36, 90-924 ~6d~ (Poland) *** Laboratory of Hemostasls, Institute of Pathology, Medical Academy of &dd~ Narutowicza 96, 91-141 ~6d~ (Poland)

(Received August 9, 1991)

Chitosan was used as a sealant of knitted polyester vascular grafts. Three sterilization methods for ehitosan-coated prostheses were tested: sterilization with ethylene oxide, formaldehyde and irradiation with gamma rays. Radiation sterilization was found to be the most promising of tested methods. The radiation-induced changes in chitosan irradiated in solid slate were investigated. Main chain scission was found as the predominant effect of irradiation. Changes in IR and UV spectra were analyzed. Existence of some post-effects was detected. It seems that the observed increase in biocompatibility of chitosan surface caused by irradiation with sterilizing dose (25 kGy) is due to some structural factor connected'with a decrease in molecular weight.

Introduction

Chitosan is a polysaccharide of natural origin. It is obtained by deacetylation of chitin, a very abundant natural polymer. Chitosan macromolecule consists of glucosamine and N-acetylglucosamine units linked by 1 - 4 glycosidic bond. Chitosan has many practical applications, especially in the field of waste processing, metal ion separation, enzyme immobilization, etc., however, its applications as a biomaterial are the most promising. These applications are based on.its unique biomedical properties, e.g. hemocompatibility, hypocholesterolemic activity, ability to accelerate wound healing and to suppress some leukemia processes.8, 9 In the previous study3 the use of krill chitosan as a coating of knitted polyester vascular grafts was reported. Scaling of the prostheses surface by chitosan eliminates the need of preclotting the grafts in patient's blood before implantation. Preliminary animal tesfs proved the usability of chitosan-coated prostheses. In this paper three sterilization methods of chitosan sealant for vascular grafts are compared in respect of blood compatibility and biocompatibility of sterilized product. Elsevier Sequoia S. A. Lausanne Akad~miai Kiadd, Budapest

J. ROSIAK et al.: RADIATION STERILIZATION OF CHITOSAN SEALANT

As the radiation-induced processes in chitosan are not well known, the radiation sterilization of chitosan is considered in more detail Some preliminary data on this subject were published by MUZZARELLI and TUBERTINI,7 KUME and TAKEHISA6 and ERSHOV et al. 5 In the latter paper, dealing with irradiation in the solid state, the radiation yield of macroradicals was measured (G R ffi 1.4) and the yield of scission was estimated by a viscometric method (Gs = 4.2). However, the unexpected relation between G R and G s (G R < G s) was not explained. The aim of the chemical part of this work was to measure more precisely the radiation yields of scission and crosslinking of chitosan irradiated in various conditions in solid state. Radiation-induced processes were also traced by analysis of changes in IR and U V spectra. The existence of post-effects was investigated. The intensity of radiation degradation of chitosan in solid state and in aqueous solution were compared.

Materials and methods

Chitosan samples were delivered by the Sea Fisheries Institute, Gdynia, Poland. The molecular weights were: M n -- 9.4.105 Daltons, lV~ ffi 1.4.106 Daltons and the degree of deacetylation 90%. DaUon PET vascular grafts were donated by Tricomed R&D Centre of the Knitting Industry, tdd~, Poland. The method of coating of grafts and glass with chitosan was previously described)

Sterilization Chitosan coated grafts, micro slides and glass test-tubes were sterilized with "t-rays in 6~ irradiation chamber with a sterilizing dose of 25 kGy. Sterilization with ethylene oxide was performed in a Medicor GST-21 gas sterilizer for 1 hour. The samples to be sterilized with formaldehyde were placed for 24 hours in a closed vessel containing formaldehyde vapor. Biological tests Anticoagulant properties were analyzed by measurements of whole blood clotting time (WBCT). The presence of anticoagulant or protein denaturating substances leached from the tested chitosan films into the plasma was examined by kaolin clotting time test (KCT). Biocompatibility of chitosan surfaces was analyzed by microscope observation of platelct spreading. Detailed description of the above methods was given in previous w o r k )

88

J. ROSIAK et al.: RADIATION STERILIZATION OF CHITOSAN SEALANT

Analytical methods Samples of chitosan were irradiated by 6~ with various doses in closed ampoules (in vacuum or in oxygen) or in open test-tubes. Changes in viscosity, molecular weight and its distribution were measured by HPLC/GPC with differential viscometer/refractometer detection (Knauer, Germany). Aqueous solution containing 0.1M CH3COOH and 0.2M NaCI was used as eluent. The columns used were OHPak: B-800P, B-806/S and B-803/S (polyester packing). Injection volume was 2 . 1 0 -5 dm 3, concentration of polymer 2.5 g/dm 3. Caibmtion was made on the basis of Mark-Houwink constants given by ROBERTS and DOMSZY 1~ (K = 1.81 9 10-3 cm-3/g, ct = 0.93 at 25 ~ IR spectra were registered on SPECORD-M-80 (Carl-Zeiss-Jena). Chitosan films were cast from 3 g/dm 3 solutions in 0.02M formic acid, regenerated by rinsing in 70:30 v/v ethanol/ammonia water for 24 hours and washed thoroughly with distilled water. UV measurements were done on SPECORD-M-40. Solutions containing 2 g/dm 3 of chitosan in 0.04M CHaCOOH were used. Contents of amino group was estimated by the method of DOMSZY and ROBERTS. 2 Details o f measurements concerning radiation-induced effects on aqueous solution of chitosan were given elsewhere. 12 Results and discussion

Biological studies Results concerning the effect of sterilization by various methods on the chitosan films are presented in Tables 1 - 3. The whole blood clotting times (WBCT) on the chitosan surfaces are given in Table 1. Irradiation with the sterilizing dose of 25 kGy does not influence the clotting Table 1 The effects of various surfaces on WBCT (in minutes) Chitosan sterilized by Silicone

Chitosan irradiation

11.1•

20.4•

20.6•

ethylene oxide 25.1•

formaldehyde 17.6•

character of chitosan layer, in the case of sterilization with ethylene oxide, the WBCT is slightly increased. Treatment of samples with formaldehyde leads to reduction of WBCT. 89

J. ROSIAK et al.: RADIATION STERILIZATION OF CHITOSAN SEALANT Table 2 The effect of chitosau and silicone on KCT (s) of platelet-rich plasma (PRP) and platelet-poor plasma (PPP) PRP Shaking time (rain)

0 2O 30 60 120

PPP

silicone

chitosan

silicone

chitosan

irradiated chitosan

EO sterilized ehitosan

HCHOsterilized chitosan

53 52

53 52

57

57

58

56

57

57 59 68

57 56 63

57 59 65

64 60 64

71 77 101

Table 3 The effect of different surfaces on platelet spreading Distribution of platelets (%) Type of surface Type I Silicone Chitosan Irradiated chitosau EO-sterilized chitosan HCHO-sterilized chitosan

2.1 ~ 1.2 1.6 ~- 0.6 2.5 • 0.8 1.0 -,- 1.0 0

Type II 48.7 30.6 69.5 16.8

-,- 8.4 -,- 10.6 • 4.3 • 8.9 0

Type III 34.0 48.0 20.7 54.7 0.7

--- 6.4 • 11.6 • 5.2 • 11.5 --- 1.0

Type IV 15.2 • 19.8 • 7.3 • 27.6 • 99.3 •

7.4 6.5 2.9 6.0 1.0

The KCT values for unsterilized and sterilized samples as well as for reference siliconized glass were measured in order to find whether any anticoagulant compounds are leached out into the plasma, The results presented in Table 2 indicate that no leachable anticoagulant substance is present in untreated chitosan and chitosan sterilized by irradiation or ethylene oxide. Higher values of KCT were obtained in the case of formaldehyde-sterilized samples. Probably some formaldehyde, absorbed in chitosan layer during sterilization, is released and causes denaturation of plasma proteins. In order to compare the biocompatibility of unsterilized and sterilized chitosan film, the platelet spreading on its surface was examined. The percent fractions of each type of platelets (I-IV) found on the tested surfaces are given in Table 3. The best results were obtained for irradiated chitosan. Biocompatibility of its surface is better than that of unirradiated chitosan and it even exceeds the biocompatibility of reference siliconized glass. On the contrary, chemical sterilization of chitosan leads to significant 90

J. ROSIAK et al.: R A D I A T I O N STERILIZATION OF CHITOSAN SEALANT

decrease in biocompatibility. This effect is especially strong for formaldehyde-treated samples. On the basis of the above results, irradiation seems to be the best of tested sterilization methods for chitosan coatings with respect to their biocompatibility and hemocompatibility.

Radiation-induced effects in chitosan In order to follow the processes of chitosan transformations caused by irradiation in the solid state, changes of viscosity, molecular weight and its distribution were measured. Plots of intrinsic viscosity, number average and weight average molecular weights as functions of absorbed dose are shown in Fig. la, b, c. The distinct decrease in these parameters indicates that main chain scission is the dominating process. In order to calculate the radiation yields of scission and crosslinking, the dose dependence of number average and weight average molecular weights was transformed to the coordinates resulting from statistical theory of radiation crosslinldng and scission (Eqs (1), (2), Fig. le, f) (1)

M g 1 - Mn~ 1 -- ( G s - GX)" D / 1 0 0 9 N A

M ~ 1 - M~ol = (Gs/2 - 2 . G x ) . D/100 .N A

(2)

where Mne Mwo, M~, M~ denote the number and weight average molecular weight before and after irradiation with dose D; Gs and Gx are the radiation yields of scission ,- 10

t• _

a)

~

1.0

~

08 ~

~ 0.6 O.a

2

~

0 2.O

0.2 l I I.I I ~. o, 10 20 30 40 50 ~ 10

='~o 1.8

6

1,6

2

0.4

I [ I ] i 0 ] ] ] [ I~ 10 20 30 40 50 ~+o ~r- 10 20 30 40 --

e)

f)

,=-

+

+ ~' l L ] l l ,~. 10 20 30 40 50 Dose, kGy

0

0

10 20 30 40 50 Dose, kGy

0

10 20 30 40 50 Dose, kGy

Fig. 1. Viscosity and molecular weight of chitosan irradiated in solid state ( 0 in vacuum, + in air, x in oxygen) as a function of dose: (a) intrinsic viscosity, (b) M., (c) Mw, (d) Mw/M. ratio, (e) (f) reciprocals of M. and Mw

91

J. ROSIAK e't al.: RADIATION STERILIZATION OF CHITOSAN SEALANT

and crosslinking, Radiation yields of sdssion, calculated from the linear correlation, are: G s -- 0.9 for samples irradiated in vacuum, G s = ! 3 in oxygen. Correspond!ng yields of crosslinking are equal to zero. The width of molecular weight distribution, defined as M,/Mn, was equal to 1,5 for unirradiated chitosan; it was increasing rapidly A lOO o" o E t-

80 60

2

1-

40

20

T 84 4000

I

t

i

I

3000

2000

1800

1600

WQve number,

IP

c m -1

A

2

6O 4O

0

2O

o

I

l_

i

I

i

I

I

1600

1400

1700

1000

800

600

400

;

WGve number, cm -~

Fig. 2. IR spectrum of chitosan. Fragments of the spectrum undergoing slight absorbance changes caused by irradiation are marked by arrows

towards 2 during irradiation (Fig. ld). This is in accordance with the statistical theory of degradation. Equations (1) and (2) are strictly true for Mw/Mn -- 2, therefore, some deviation of data points from straight lines is observed for the lowest doses (Fig. le, t3. In order to avoid possible errors resulting from this deviation, only the results for higher doses were taken into account in calculations of radiation yields. The IR spectrum of chitosan is shown in Fig. 2. The spectra of chitosan irradiated in air with the sterilization dose of 25 kGy do not show any significant difference in comparison with the spectra of unirradiated chitosan. That indicates that the chemical structure is not much altered by absorption of such a dose. In order to trace the main 92

J. ROSIAKet al.: RADIATIONSTERILIZATIONOF CHITOSANSEALANT directions of chemical changes, IR spectra of the samples irradiated with 560 kGy were analyzed. Slight decrease of the broad band between 3600 and 3000 cm -1 is probably due to decay of - OH and - NH 2 groups. Increase of absorbanee in the range 1740 1700 cm -1 indicates that some carbonyl and carboxyl groups are formed. This effect was reported by ERSHOV 5 on the basis of chemical analysis. Slight decrease of three

t

t

~ 020- i i uc

247nmlv290nmt I

,~

o~ 04

~,=247nm j

~o o3

0.10

~ 0.2

0.05

0.1 Q)

0 200

I 250

1 I T D300 400 Wave length, nm

b)

0

0

10

20

I 30

I I 40 50 Dose, kGy

Fig. 3. (a) Absorptionspectraof chitosan irradiated in presence of oxygen; (b) absorbanee at 247 function of dose (O samplesirradiated in vacuum, x in oxygen)

mm as

a

bands at 1152, 1090 and 1035 cm -1 is probably due to decay of C 1 - O - C4 groups. That is in agreement with the basic scheme of polysaccharide degradation; n in this scheme it is assumed, that main chain scission is caused by splitting of 1 - 4 glycosidic bond. The UV spectra of chitosan irradiated with various doses are shown in Fig. 3a. The rise in absorbance at 247 and 290 nm is observed. These changes are probably due to formation of carbonyl and carboxyl groups. The absorbanee increases almost linearly with absorbed dose (Fig. 3b). The most intense changes are observed for samples irradiated in oxygen. The contents of amino group in chitosan were measured before and after irradiation. Absorption of the sterilization dose (25 kGy) causes a slight decrease of amino group contents (by about 0.4% - that is close to experimental error). Irradiation with much higher doses (560 kGy) allows to estimate the radiation yield of amino group decay for chitosan irradiated in air as G( - NH2) -- 8. According to the suggestions of ERSHOV 5 and the general outlines of radiation-induced changes in di- and polysaccharides, u the following degradation 93

J. ROSIAKet al.: RADIATIONSTERILIZATIONOF CHITOSANSEALANT scheme for chitosan irradiated in solid state can be proposed: R- H

7

, R ( C t - C6) + H

R-H+H"

=R'(C l - C 6) + H 2

R ' ( C i , C4) - - " ~ F f + F 2 R - NH 2 + H"

,R" (C2) + NH3

(3) (4)

(5) (6)

where R - H and R - NH 2 denote chitosan macromolecule, R (Cn) is a chitosan macroradical localized on C n carbon atom and F i ' , F 2 are fragments of main~chain after scission. Absorption of ionizing radiation causes generation of radical sites at various carbon atoms of chitosan base units [Reactions (3), (4), (6)]. Only the transformations of radicals localized at C 1 or C 4 carbon atoms lead directly to the splitting of 1 - 4 glycosidic bond, which is equivalent to main chain scission [Reaction (5)]. Reactions of other radicals may result in bond splitting inside the chitosan base units, however, tlaey do not cause main chain scission. Therefore, the radiation yield of scission G s should be lower than the total yield of macroradicals G R created in chitosan. Comparison of our results (Gs = 0.9 in vacuum, G s = 1.1 in air) with GR -~ 1.4 in vacuum or air measured by ERSHOV 5 is a confirmation of the proposed scheme. Existence of post-effects in irradiated chitosan was detected. In all irradiated samples, irrespective of the conditions of storage (in vacuum or air), further reduction of average molecular weights was detected. These data are presented in Table 4. Post-irradiation changes were also observed in UV spectrum.The absorbance at 247 nm rises by about 11% during first 10 days after irradiation (for sample irradiated and stored in air). The occurrence of post-effects seems to be correlated with the existence of microcrystalline structure of chitosan. 4 Radicals trapped in the crystallites can slowly migrate towards their surface, where they undergo rearrangements causing mainly the scission of C 1 - O - C 4 bonds. Such effects are known for other polysaccharides, especially for cellulose. 1 These post-effects should be taken into account when radiation sterilization of chitosan-coated biomedical devices is applied. Radiation yields of scission were measured also for chitosan hydrochloride irradiated in argon-saturated aqueous solution (concentration 1 g/dm3). Comparison of yields of scission allows to estimate that scission is at least 10 times more intense for irradiation in aqueous solution than for irradiation in the solid state. These results indicate that biomedical devices containing chitosan should be irradiated in the solid state (not in water-swollen form) if extensive degradation is not desired. 94

J. ROSIAK et al.: RADIATION STERILIZATION OF CHITOSAN SEALANT

Table 4 Changes in intrinsic viscosity and average molecular weights of irradiated chitosan as a function of storage time Irradiation

Storage

Storage (days)time

Intrinsic viscosity (cm3/g)

Mn x 10-3

Mw x 10-3

In vacuum

In vacuum

0 46

269 237

184 162

365 325

In vacuum

In air

0 15

269 246

184 166

365 330

In air

In air

0 10 25 54

229 220 202 175

153 146 134 117

304 291 266 235

In oxygen

In air

0 32

162 134

211 174

214 177

Correlation betwen irradiation effects and biomedical properties The results presented show that the predominant effect of irradiation of chitosan in solid state with sterilizing dose of 25 kGy is the reduction of molecular weight caused by main chain scission. Changes in the amount of functional groups are not strongly marked at this" dose; therefore, it is not possible to consider their correlation with changes in biomedical properties on the basis of experiments reported. The anticoagulant properties of chitosan measured by WBCT and KCT tests are not significantly influenced by changes in molecular weight (in the tested range, that is for M, from 9-105 down to 2.5.105 Daltons). The distinct improvement of biocompatibility (platelet-spreading test) observed for irradiated chitosan surface seems to be caused by the decrease in molecular weight. The effect of microcrystalline structure on these properties is planned to.be studied as well. Conclusions Irradiation was found to be a better method of sterilization of chitosan sealant for vascular prostheses than treatment with ethylene oxide or formaldehyde. It does not singificantly influence the blood compatibility of chitosan and it does not cause leaching any anticoagulant or protein denaturating component into the plasma. The 95

J. ROSIAK et al,: P~M)IATION STERILIZATION OF CHITOSAN SEALANT

biocompatibility of chitosan surface, measured by platelet-spreading test, is efficiently increased by irradiation. The major effect of irradiation of chitosan in soUd state is the main chain splitting. The yields of scission are Gs -- 0.9 in vacuum, G s ffi 1.1 in air and Gs ffi 1.3 in oxygen. This process is not accompanied by crosslinking. Decay of amino groups and formation of carbonyl and carboxy groups was detected, however, at a sterilization dose of 25 kGy these changes are not intense. The increase in biocompatibility in course of irradiation is probably connected with structural changes due to decrease in the size of macromolecules.

The authors (J. R., P. U.) would like to acknowledge the financial support of the International Atomic Energy Agency, Vienna (Research Contract No. 5509/R1/RB and Technical Project POL/1/010). The authors appreciate the financial support of the State Committee for Scientific Research.

References 1. K. BURCZAK, H. DUNSKI, J. ROSIAK, W. PFeKALA,Nukleonika, 30, No 3-4, (1985) 107. 2. J. DOMSZY, G. A. F. ROBERTS, Makromolek, Chemie, 186 (1985) 1671. 3. J. DUTKIEWICZ, L JUDKIEWICZ, A. PAPIEWSKI, M. KUCHARSKA,R. CISZEWSKI, Chitin and Chitosan, Proc. Intern. Conf. on Chitin and Chitosan; G. SKJAK--BRAEK, T. ANTHONSEN, P. SANDFORD(F~Is), Elsevier, London and New York, 1989, 719. 4. J. DUTKIEWICZ, private Communication. 5. B. G. ERSHOV, O. V. ISAKOVA~ S. V. ROCK)SHIN, A. L GAMZAZADE, E. U. LEONOVA; Dokl. Akad. Nauk SSSR, 259, No 5 (1987) 1152. 6. T. KUME, M. TAKEHISA, Proc. Intern. Conf. on Chitin and Chitosan, Sapporo, Japan, 1982, p. 66. 7. R. A. A. MUZZARELLI, O. TUBERTINI, L Radioanal. Chem., I2 (1972) 431. 8. R. A. A. MUZZARELIA, Chitin. Pergamon Press, Oxford, 1977. 9. R. A. A. MUZZARELLI, Chitin, In: The Polysacehafides Vol. 3,G. O. ASPINALL (Ed.) Academic Press, New York, 1984. 10. G. A. F. ROBERTS, J. DOMSZY, Int, Biol. Macromol., 4 (1982) 374. 11. C. VON SONNTAG, The Chemical Basis of Radiation Biology, Taylor & Francis, Lond0n-New YorkPhiladelphia, 1987. 12. P. ULA~4SKI, J. ROSIAK, Radiat. Phys. Chem., 39 (1992) 53.

96