J. BIOMED MATER. RES.

VOL. 11, PP. 251-265 (1977)

A New Antithrombogenic Heparinized Polymer HAJIME MIYAMA, NORIHO HARUMIYA, YUICHI MORI, and HIROSHI TANZAWA, Basic Research Laboratories, Toray Industries Inc., 1 1 11 Tebiro, Kamah-ura, Japan

Summary A new antithrombogenic polymer was synthesized by photoinduced graft copolymerization of both a hydrophilic polymer and a cationic polymer to hydrophobic poly(viny1 chloride) copolymer, and by quarternizing and heparinizing the obtained graft copolymer. For polymers of various compositions obtained by the method described above, chemical composition, water absorption, membrane potential, and quantity of adsorbed heparin were determined and antithrombogenicity evaluated zn vim. Thus, it has been found that the polymer of excellent antithrombogenicity has a negative membrane potential, a moderate elution rate of heparin, adsorbs heparin in a quantity of approximately 15 wt-%, and has a degree of water adsorption of about 30 wt-%. Also, measurement of membrane potential was very useful for the estimation of the quantity of the adsorbed heparin and its change with time.

INTRODUCTION In order to develop the medical application of plastics for the internal organs, it is necessary to develop a new polymer having both excellent antithrombogenicity and mechanical properties. From the practical viewpoint, it is best to prepare an antithrombogenic polymer by the chemical modifications of commercially available polymers having the desired mechanical properties. Poly(vinyl chloride) can be easily modified by photografting any polymer to dithiocarbametized poly(viny1 chloride) . I Our preliminary test showed that a polymer obtained by photoinduced graft polymerization of methoxy poly(ethy1ene glycol)monomethacrylate (SM) CH-

E II

-(OCHzCHz)3-OCHa

0 2.51

@ 1977 by John Wiley & Sons, Inc.

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to poly(viny1 chloride) was very hydrophilic and was antithrombogenic to a degree which was comparable to that of silicone rubber. On the other hand, it is well known2 that heparinized materials prepared by ionically binding heparin molecules to cationic sites show good antithrombogenicity. From these facts, the present authors speculated that a graft copolymer consisting of a hydrophobic part, a hydrophilic part, and a cationic part should show excellent antithrombogenicity after being heparinized. As the hydrophobic part, a commercial graft copolymer composed of vinyl chloride, ethylene, and vinyl acetate was chosen because of its good mechanical properties. Also, SM and hT,h'-dimethylaminoethyl methacrylate (DAEM) CHI

I

cH~4-cI1

(CH2)z-N (CH 3)z

0

were chosen as the hydrophilic and cationic parts (which was cationized by quarternization), respectively. As expected, the new polymer prepared by the photoinduced graft copolymerization of SM and DAEM t o the above described hydrophobic polymer showed excellent antithrombogenicity after being quarternized and heparinized. Details of the present study will be described below.

EXPERIMENTAL Preparation of Heparinized Polymer Since feeding conditions for the synthesis of matrix polymer varies as shown in Table I, concentrations and quantities of reagents cannot be specified generally. Therefore, in the following sections, these will be shown by using a typical example. Synthesis of Matrix Polymer

Graftmer Ra (200 g) (graft copolymer composed of 56 molar-% vinyl chloride, 12 molar-yo of vinyl acetate, and 32 molar-% ethylene) made by Japan Geon Co., Ltd. was reacted with 3.2 g of sodium N ,N-diethyldithiocarbamate in 3 liters of N,N-dimethylformamide a t 50 60°C for about 2 hr to introduce dithiocarbamate groups,

-

A NEW ANTITHROMBOGENIC HEPARINIZED POLYMER

253

which were effective for the photoinduced graft copolymerization.1 The reaction mixture was then poured into a mixture of water and methyl alcohol t o precipitate the polymer. ‘After being washed with water and methyl alcohol several times, the polymer was dried under vacuum. The graft copolymerization of 30 g of SM ( M , = 1200) and 26 g of DAEM to 45 g of the Graftmer R 3 containing dithiocarbamate groups was carried out in 1 liter of cyclohexanone by ultraviolet irradiation. The degree of graft copolymerization was controlled by changing irradiation time. The reaction mixture was poured into methyl alcohol. The precipitate was soaked in methyl alcohol for 2 3 days to remove unreacted monomers and then dried under vacuum a t room temperature. Here, a reaction vessel of 1 liter capacity with a 100 W high-pressure mercury lamp placed in the vessel was used,3 where the lamp was covered with a water-cooling jacket made from hard glass. Intensity distribution of the lamp light is shown in Figure 1.

-

Quarternization and Casting N,N-Dimethylethylaminoethyl groups of the graft copolymer obtained as described above were quarternized in N,N-dimethylformamide a t 50 60°C for 1 -2 hr by using 100 ml of ethyl bromide. An outer surface of a glass tube of 10 mm outer diameter was coated with the solution obtained above and dried to remove solvent. The tube was then soaked in water or a methyl alcoholacetone mixture to swell the polymer. Thus, polymer tubes were prepared and preserved in water.

-

Ef eparinization

-

The tubes prepared as described above were heparinized by soaking them in aqueous solution of containing 2 3% sodium heparin a t 85°C for 1 3 days, washed with distilled water, and preserved in Ringer’s solution.

-

Evaluation of Physical Properties Composition and Water Absorption The tubes dried without heparinization were used for the elementary analysis for C, H, N, C1, and Br. From the analytical values,

2.54

MIYAMA ET AL.

J

400 Wave Length (mp)

L 600

Fig. 1. Intensity distribution of 100 W high-pressure mercury lamp.

composition of the polymer was calculated on the assumption that the degree of quarternization was 100%. The degree of water absorption of the polymer was obtained by calculating the weight of the wet polymer and that of the dry polymer.

$1easurement of Membrane Potential When it was possible to use a flat sample, the size of which was larger than 10 mm x 10 mm, a conventional apparatus4 was used for the measurement of membrane potential. When the flat piece described above could not be taken out of the tube, a specially designed apparatus, shown in Figure 2, was used.

A NEW ANTITHROMBOGENIC HEPARINIZED POLYMER C

A

C

2.55

A

Fig. 2. Apparatus for measurement of membrane potential. (A) KCl electrode as a reference; (B) saturated aqueous KCl; (C) salt bridge; (D) acceptor; (E) silicone tube; (F) glass tube, (G) concentrated aqueous KCl; (H) sample; (I) diluted aqueous KCl.

Estimation of N+ Concentration Values of N+ concentration were estimated by the following four methods. 1) The estimation was made from analytical values of nitrogen of the quarternized polymer on the assumption that the degree of quarternization was 100% and that no decomposition occurred in the casting process. 2) By treating the quarternixed polymer with 0.2 N NaOH aqueous solution at room temperature and washing thoroughly with distilled water, the Br- component of the polymer was substituted by OH- as follows. RSD+Br-

+ NaOH

-j

RSD+OH-

+ NaBr

Here, RSD+Br- expresses the quarternized polymer. To the substituted polymer, 0.1 N NaCl aqueous solution was added. Then the NaOH produced was titrated with HCl.

+ NaCl RSD+ Ci- + NaOH + HCl -+ NaCl + HzO

RSD+OHNaOH

From the titration value obtained as described above, N+ concentration was determined. 3) An aqueous solution of sodium heparin to be used for the heparinization of the quarternized polymer was titrated with 0.06 N AgN03 aqueous solution by using K2Cr04 as an indicator, before and after the heparinization. Thus, the difference in bromine content of the

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polymer before and after the heparinization was obtained. The difference corresponds to the amount of Br- released from the polymer by the heparinization, and therefore to that of N+ sites if it is assumed that all of N+ are completely occupied by -SO3- ions of heparin molecules. 4) The N+ concentration was estimated from the values of membrane potential. For the membrane potential of a polyelectrolyte, the following theoretical expression is given.*

% - l ) ( %____. +l)

+ C In ('+c)

2+1)(%-1)

C=

(?+c)

u+ - uu+ u-

+

Here, Em,R, F , T , X , u,., and C , express membrane potential, gas constant, Faraday constant, temperature (OK), concentration of fixed ion, ionic mobility of cation or anion in the membrane, and concentration of salt i, respectively. When the salt concentration is high, the mobility of the cation becomes equal to that of the anion, that is, C becomes zero, which makes negligible thc second term in eq. (1) for Em. Therefore, according to eq. ( I ) , the fixtld ion concentration X can be calculated from the salt concentration C, and the membrane potential EWL.For the quarternized polymer in the present study, Nf concentration corresponds to X and is therefore easily calculated from membrane potential. Measurement of the Quantity of Adsorbed Heparin

The quantity of adsorbed heparin was obtained by the following three methods. 1) It is known5 that heparin concrntration in solution can be measured by forming a complex between heparin and Azur A. According to the method, the following expression was obtained by mixing 5 ml of 20 25 ppm aqueous Azur A and 3 ml of 0 25 aqueous heparin, and by measuring the absorbance of the mixture

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A NEW ANTITHROMBOCENIC HEPAKINIZEII POLYMER

257

at 535 pm at various concentrations of heparin with a cell of 1 cm thickness. Here, [HI, OD5,,(H), and OD,,,(O) are the concentration of heparin (ppm) and the absorbances of the solution a t 535 pm when the heparin concentration is [H] ppm and that when it is zero, respectively. According to eq. ( 2 ) , the concentration of heparin in the heparin solution to be used for the heparinization of the polymer was determined before and after the heparinization process. Thus, the quantity of heparin adsorbed by the polymer was obtained. 2 ) Under the coexistence of salt, the quantity of adsorbed heparin was obtained not by the above described method, but calculated from the sum of the weight increase of the polymer and the produced quantity of NaBr after the heparinization process. Details of the method will be discussed later. 3) As described above, the quantity of adsorbed heparin could be calculated from values of membrane potential before and after the heparinization.

Measurement of Elution Rate of Heparin in ACD Plasma The elution rates of heparin from the surface of the heparinized polymer tubes were determined by measuring the thrombin time of canine ACD plasma contacted with the surface. The rate was expressed as E’ (g.cm-2-min-1). In some tubes, the measurement was repeated after the in vivo test to estimate the change of the dution rate. Details of the method have been reported elsewhere.6 In vivo Test

A catheter made from the heparinized polymer was inserted into the inferior vena cava of the dog through the right femoral vein. The dog was anesthetized intravenously by pentobarbital sodium. The right femoral vein was ligated at the site of insertion of the catheter. Prior to the implantation into the inferior vena cava, the catheter was filled with sterilized Ringer’s solution, and its distal end was closed with a Teflon plug so that the blood would not enter the lumen of the catheter. After 2 weeks, the dogs were killed by acute exanguination from the aorta and were examined

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by autopsy for the thrombus formation around the catheters. Details of the method were reported previously.6

RESULTS Preparation and Evaluation of Matrix Polymer and Heparinized Polymer Graft copolymerization was carried out by changing feeding composition and polymerization time. The polymers obtained were quarternixed and cast. The polymerization condition and properties of the matrix polymer obtained by the above described method are summarized in Tables I and 11. Here, the amount of N+ in the matrix polymer and the percentage of the adsorbed heparin in the heparinized polymer were measured by the various methods described above. Within experimental error, these values coincided well in spite of the difference of the method. Since the measurement of membrane potential is easier than any other method, it was adopted mainly t o follow the change of the adsorbed heparin during the heparinization as well as the elution of the heparin. TABLE I Polymerization Conditions Feeding Composition ( g ) Exp. No.

Trunk Polymer

DAEM

SM

Polymerization Time (hr)

103 102 113 107 106 110 23 122 121 124 123 125

50 50 50 50 50 50 15 45 45 45 45 45 50 50 50

0 50 50 50 50 40 15 26 26 26 26 26 40 50 50

0 25 25 50 50 60 15 30 30 30 30 30 60 100 25

2.5 2.5 1.5 1.5 3.0 2.5 1.5 4.5 6.0 5.0 4.0 4.5 3.5 2.0 2.0

111

109 105

79.1 72.8 78.6 74.9 70.1 70.7 69.9 77.5 76.2 80.9 79.5 79.0 65.2 64.4 67.1

103 102 113 107 106 110 123 122 121 124 123 125 111 109 105

SNI 0 0 1.7 3.4 9.7 8.3 11.0 7.0 8.1 6.3 7.9 7.5 7.9 15.0 6.3

DAEM

20.1 27.2 19.7 21.7 20.2 21 .o 19.1 15.5 15.7 12.8 12.8 13.5 26.7 20.6 26.6

27-34 28-47 27-34 47-50 35-37 33-59 49 62 97 79 79 79 79-80 72-76 122-1 90

14.0 11.5 15.2 10.5 8.0 10.0 10.5 8.4 7.5 8.2 8.6 11.9 7.5 5.2

Water Absorp- Membrane tion (%) Potential (mV) 0.75 1.02 0.74 0.81 0.76 0.79 0.72 0.58 0.59 0.48 0.48 0.51 1.01 0.77 1.00

(1)

0.44 0.16 0.60

-

0.18 0.42 0.41 0.40 0.51 0.41 -

(2)

~

0.60 0.88

-

0.67 0.07 0.55 0.54 0.76 0.60 0.35 -

(3)

0.47 0.37 0.42 0.43 0.68 0.37 0.41

0.62 0.50 0.71 0.47 0.30 0.40 0.48

(4)

N + Concentration (meq/g)*,b

-

Expressed in milliequivalents of N+/g dry polymer. Values in columns headed (l),(2), (3), and (4) were obtained according to methods ( l ) , (2); ( 3 ) , arid (4) described in the text.

Trunk Polymer

Exp. No.

Composition (wt-%)

TABLE I1 Properties of Matrix Polymers

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Water absorption of the matrix polymer was dependent on the degree of grafting and it decreased to approximately one-half of the original value by the heparinization. Also, when the degree of grafting was the same, the amount of SM grafted contributed much more to the water absorption than did that of DAEM.

Heparinization Condition When the matrix polymer was heparinizcd, decrease of heparin in the heparin solution, the increase of NaBr produced in the solution, the weight increase of the polymer, and the membrane potential of the polymer were measured simultaneously with time. An example of the results is shown in Figure 3, which shows that the decrease of heparin in the heparinizing solution is equal to the sum of the increase of NaBr and the increase of the polymer weight. Also, the membrane potential decreases Kith the progress of heparinization. The amount of NaBr produced was found to be almost

100-

C 50.

IY 0 “00

20

LO

60

80

Heparinizing Time ( h r )

Fig. 3. Decrease of heparin content in heparinizing solution, decrease of membrane potential, increase of NaBr, and increase of polymer weight with heparinizing time (sample No. 110). (A) membrane potential; (B) amount of heparin increase; (C) amount of NaBr produced; (D) increase of polymer weight.

A NEW ANTITHROMBOGENIC HEPARINIZED POLYMER

261

equal to that calculated from the N+ concentration. These facts suggest that the matrix polymer is heparinized according to the following equation:

RSD+Br-

+ Hep-Na+ $ Hep-RSD+ + NaBr

Here, Hep- expresses ionic heparin containing -SO3- ions which are gradually exchanged with Br- of the matrix polymer. This is the reason why the second method was used to find the quantity of the adsorbed heparin. I n Vivo Test and Elution of Heparin The results of in vivo test are shown in Table 111, from which i t is obvious that polymers of excellent antithrombogenicity have a negative membrane potential and a water absorption capacity of about 30Oj,. The membrane potential of the heparinized polymer showing excellent antithrombogenicity (No. 121) were measured before and after the in vivo test for 2 weeks. These values were - 5 . 8 - 7 . 6 and - 5 . 0 - 6 . 3 mV, respectively. The difference was about 1 mV, which corresponds to about 0.4 X g cm-2 . min-l of the elution rate of heparin. Also, the membrane potential of the polymers was followed with time in aqueous KCI solution to find

-

-

1

--

-8

c

-6

w

E

-

.-0

c

1:

-4

C

e f

- 2

f 0 * 2 41

0

20

40

60

80

100

120

140

Elution Time (hr)

Fig. 4. Change of membrane potential with elution time in aqueous KCl a t 37°C. (A) Exp. No. 121 (0.2 N ) ; (B) Exp. No. 121 (0.3 N ) ; (C) Exp. No. 121 (0.4 N ) ; (D) Exp. No. 109 (0.2 N).

a

12.2 13 14.5 20.9 29.9 28.9 31 28.1 31.5 31.7 31.4 31.3 37 40.1 44.1

103 102 113 107 106 I10 23 122 121 124 123 125 111 109 105

-5.5 -5.3 -3.1 -2.2 -2.6 -7.0 -5.8 -8.2 -8.2 -7.4 -3.5 -0.8 -7.0

-

N

-

.V

N

-

-8-

6.2

-5.3 -7.3

-10.3

-7.5

-3.3

-

-8

-9

Membrane Potential (mV)

100-120 150-160 80-90 160-170 170-1 80 250-340 300-330 190-220 240-270 190-210 110-180 90- 120 80-90

-

70-80

Thickness (m/l)

8 14 17

20 15 22 23 22 15

10 19

-

-

-

14 14 12 11 17 14 7 -

(2)

12 10 11 11 16 8 11

-

15 17 11 7 7 11

(3)

Adsorbed Heparin (wt-%)*Vb

Poor Poor Poor Poor Excellent Excellent Excellent Excellent Excellent Excellent Excellent Excellent Good Good Good

in vivo Test

Expressed in terms of wt-% of heparin contained/g dry hepaririized polymer. Values in columns headed ( I ) , (2), and (3) were obtained according to methods ( l ) , (2), and (3) described in the text.

NO.

Water Absorption (%I

Exp.

TABLE I11 Properties and Results of in vivo Test of Heparinized Polymers

t.

L-

c3

M

U

z * 9 z9

N

Q, N

A NEW ANTITEIROMBOGENIC HEPARINIZED POLYMER

263

TABLE IV Elution Rate of Heparin in 0.2 M KCla Elution Rate (10-8 g.cm-2-min-1) Exp. No.

Average for 0-24 hr

113 (before in vivo test) 107 (before in vivo test) 121 (before in vivo test) 121 (after in vivo test) 109 (before zn vivo test)

Average for 24-100 hr 0.4 0.4-0.6 1.2 0.9 0.1

0.8 0.6 6.0 0.9 1.7

* Measured from values of membrane potential.

the elution behavior of heparin. Some examples of the change of membrane potential are shown in Figure 4,and elution rates are estimated from the membrane potential measured in Table IV. Also, heparin elution rates of the sample showing excellent antithrombogenicity (No. 121) in the ACD plasma measured before and after in vivo test for 3 weeks are shown in Figurc 5 .

DISCUSSION As described above, the graft copolymer consisting of hydrophobic Graftmer RS, hydrophilic SM, and cationic DAEM showed excellent antithrombogenicity after being heparinized when it has negative L

. 6 6 a -

(Before in vivo test

-

.

0

0

0

x 4 -

.,

W

( A f t e r 3-week in v i v o t e s t )

zt /

.

O:,

200

*

100

1

300

400

5C

Time (rnin.)

Fig. 5. Heparin elution rate E of the sample of excellent antithrombogenicity (No. 121) in ACD plasma before and after in vivo test.

MIYAMA ET AL.

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membrane potential, as shown in Table 111. This indicates that the sum of negative charges due to -SO3- of the heparin containrd in the heparinized polymer is larger than that of the positive charge due to N+ of the quarternized polymer for the polymer of excellent antithrombogenicity. Table I11 also shows that the water content of about 30% gives the best result for the in vivo test. From Figure 4 and Table IV, i t is obvious that the sample of poor antithrombogenicity has a small elution rate and that the sample of excellent antithrombogenicity (No. 121) has a moderate elution rate even after a long elution time. Also, the sample of high water absorption and of good antithrombogenicity (No. 109) shows a high elution rate a t the initial stage but a smaller rate a t the later stage. The heparin release is considered to be controlled by both the dissociation of the ionic complex between heparin and the cationic site of the polymer, and the diffusion of free heparin which depends on the water content of the polymer. Therefore, in the case of high water content, the consumption of heparin cannot be balanced by the supply of heparin by the dissociation. On the other hand, the low degree of water content makes the diffusion rate of heparin so slow that the dissociation is not a rate-determining step. At the optimum degree of water absorption (about 30%), the consumption of heparin is balanced by the supply of free heparin by the dissociation of the ionic complex. I t is conceivable that the membrane potential could have been changed by the adsorbed protein if the samples were implanted in vivo. This could cause the inaccuracy of the elution rates calculated from the membrane potential. As shown in Table IV, elution rates measured from the membrane potential for the sample No. 121 before and after the in vivo test are 6 . 0 x g m--2 min-' and 0.9 X lo-* g cm+ min-*, respectively, taking the average for the time period 0-24 hr. On the other hand, corresponding values measured in the ACD plasma are approximately 4 x lo-* g cm-2 min-* and 1 x g cm-2 min-I, respectively. Thus, agreement between the two measurements is very good. Therefore, it may be possible to use the membrane potential method to estimate the elution rate of heparin even after the in vivo test. As described in the Results section, the difference in the membrane potential of the heparinized polymer before and after the in vivo test gave the elution rate of 0 . 4 x g cm-2 min-' for the

-

-

-

-

-

-

-

-

-

A NEW ANTITHROMBOGENIC HEPAHINIZEI) POLYMER

265

polymer of excellent antithrombogenicity. Therefore, if it is assumed that heparin is completely eluted in vivo a t the elution rate min-I, a typical heparinized catheter of of 0 . 4 x lo-* g 200 pm thickness containing about 15 wt-% of heparin can elute the heparin for about 8 months in vivo. Therefore, i t is expected that the present heparinized catheter is effective in vivo for several months. Recent in vivo tests have shown that the present polymer remained antithrombogenic for more than 3 months.'

-

-

The authors wish to express their thanks to Urs. M. Hori, N . Ohshima, and Y . Idexuki for their guidance arid help i n the in viuo tests.

References 1. S. Okawara, Kogyo Kaguku Zashi, 69(4), 761 (1966). 2. R. I. Leininger, C. W. Cooper, M. A l . Epst,ein, 11. I>. Falb, and C. K . Grale, Science, 152, 1625 (1966); A. Rembaum, C. P. S. Yen, R. F. Landel, and M. Schen, J . Macronzol. Sci-Chem., A4 (3), 715 (1870). 3. H. Miyama and T. Sato, J . Polym. Sci. A , 1, 2469 (1972). 4. S. J. Baxton, J. Colloid Sci., 2,495 (1947). 5 . T . F. Yen, D. Ilavar, and A. Itembaum, Biochim. Riophys. Acta, 184, 646 (1969). 6. H. Tamawa, Y. Mori, N . Harumiya, H. Miyama, M. Hori, N. Oshirna, arid Y. Idezuki, Trans. Amer. SOC.Artif. Znt. Organs, 19, 188 (1973). 7. Y. Idezuki, H. Wata.nabe, &I. Hagiwara, K. Kanasugi, Y. Mori, S. Nagaoka, M. Hagio, K. Yamamoto, and H. Tanzawa, Trans. Amer. SOC.Artif. Znt. Organs, 21,436 (1975).

Received October 22, 197.5 Revised April 16, 1976

A new antithrombogenic heparinized polymer.

J. BIOMED MATER. RES. VOL. 11, PP. 251-265 (1977) A New Antithrombogenic Heparinized Polymer HAJIME MIYAMA, NORIHO HARUMIYA, YUICHI MORI, and HIROSH...
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