~

In vitro toxicity test of 2-cyanoacrylate polymers by cell culture method

Yin-Chao Tseng, Yasuhiko Tabata, Suong-Hyu Hyon, and Yoshito Ikada* Research Center for Medical Polymers and Biomaterials, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606, Japan

The inhibition of Swiss 3T3 cell growth by the microspheres prepared from various 2-cyanoacrylate polymers was investigated to assess their cell toxicity. Poly(ethoxy- ethyl 2-cyanoacrylate) and poly(methy1 2-cyanoacrylate) microspheres inhibited cell growth in a smaller amount than poly- (isobutyl 2-cyanoacrylate) and poly(ethy1 2-cyanoacrylate) microspheres. The extent of cell growth inhibition by the micro- spheres decreased with the increasing mo- lecular weight, regardless of the kind of polymers used. Every kind of the micro-

spheres was degraded releasing formalde- hyde in the culture medium. The cell growth inhibition by the medium contain- ing the microspheres was observed within 24 h for poly(ethoxyethy1 2-cyanoacrylate) and poly(methy1 2-cyanoacrylate). The extent of inhibition was in a linear propor- tion with the amount of formaldehyde re- leased. It is concluded that the cell toxicity of 2-cyanoacrylate polymers is attributed to formaldehyde released upon polymer degradation.

INTRODUCTION

2-Cyanoacrylates (CA) have extensively been studied for surgical applica- tions, such as tissue adhesives' and hemostatic agents.2 In addition, the CA polymers (PCA) are biodegradable and have been used as matrix materials for drug delivery s y ~ t e m s . ~ - ~ Their biodegradation generally takes place more markedly with the decreasing length of their side chain.6t7 The biggest problem of CA is the toxicity. It has been demonstrated that the toxicity is dependent on the rate of polymer degradation, suggesting that the degra- dation products of the polymers are responsible for the t ~ x i c i t y . ~ , ~ , ~ How- ever, the cause of the toxicity of PCA is still controversial. For instance, Lenaerds et al. insist that alcohols generated as a result of hydrolysis of the esters are responsible for the t0xicity.l'

The purpose of this work is to find a clear correlation between the cyto- toxicity and the PCA degradation. In vitro toxicity tests by cell culture have been reported to predict the in vivo toxicity of various biomaterials with con- siderable We use 3T3 cells to this end and determine the cell

*To whom correspondence should be addressed at Research Center for Medical Poly- mers and Biomaterials, Kyoto University, 53, Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, Japan, 606.

Journal of Biomedical Materials Research, Vol. 24, 1355-1367 (1990) 0 1990 John Wiley & Sons, Inc. CCC 0021-9304/90/101355-13$04.00

1356 TSENG ET AL.

growth inhibition by the PCA and compare the results with the release of formaldehyde from the PCA.

MATERIALS AND METHODS

1. Reagents and medium

Methyl 2-cyanoacrylate (MCA), ethyl 2-cyanoacrylate (ECA), isobutyl 2-cyanoacrylate (IBCA), and ethoxyethyl2-cyanoacrylate (EECA) monomer were synthesized from respective 2-cyanoacetate and paraformaldehyde ac- cording to the method of Leonard et a1.6 under its minor modification. A polystyrene (PS) microsphere was prepared by soap-free emulsion poly- merization of ~tyrene.’~ Formaldehyde aqueous solution (35 wt%) was pur- chased from Nakarai Chemicals, Ltd., Kyoto, Japan. A formaldehyde-Test Wako kit for formaldehyde determination was obtained from Wako Pure Chemical Industries, Ltd., Japan.

A culture medium was prepared by supplementing the Dulbecco’s Modi- fied Eagle’s medium (DME, Gibco Laboratories, Life Technologies, Inc.) with 5% and 10% fetal calf serum (FCS, M.A. Bioproducts, Walkersville, MD) and buffered with NaHCO, at pH 7.4.

2. Synthesis of CA polymers

Except for the MCA polymer, the PCA with different molecular weights were prepared by anionic polymerization of the corresponding monomer with triphenyl phosphine of different concentrations as initiat~r.’~ The poly- merization products were purified by precipitation with HCI-acidified hexane for removal of the monomers and other low-molecular-weight compounds. The weight-average molecular weight of the polymers was determined by gel permeation chromatography (GPC).

3. Preparation of PCA microspheres

The poly(methy1 2-cyanoacryla te) (PMCA j microsphere was prepared by polymerization of MCA in the 1.0% aqueous solution containing 2% poly(viny1 alcohol) (PVA) in lo3 M HCl for 4 h under mechanical stirring, ac- cording to the method described by Douglas et a1.16 The weight-average mo- lecular weight of PMCA was estimated from the intrinsic viscosity.’7

The other PCA microspheres were prepared by the solvent evaporation method at 30°C and atmospheric pressure.” Briefly, 1 mL of methylene chloride containing 20 mg of a CA polymer was poured rapidly into 10 mL of 2% aqueous solution of PVA and the mixture was emulsified by sonica- tion at an input power of 70 W for 3 min. The resulting emulsion was agi- tated continuously at room temperature until methylene chloride was thoroughly evaporated. All the microspheres were washed three times with

1357 IN VITRO TOXICITY TEST OF 2-CYANOACRYLATE

cold distilled water by centrifugation at 10,000 rpm and 0°C for 5 min. Then the microspheres were lyophilized and stored at 4°C until use. The result- ing microspheres were all spherical with the same size distribution and an average diameter of 1.5 pm, irrespective of the kind of PCA.

4. Degradation test of PCA microspheres

PCA microspheres were suspended in 1.4 mL of DME containing 5% FCS and penicillin (100 units/mL) in concentrations ranging from 0.25 to 1.6 mg/mL, and kept at 37°C under shaking for predetermined periods of time. The suspension was then used for the cell growth inhibition test which was conducted also for the supernatant collected from the medium containing the microsphere by centrifugation. The amount of formaldehyde in the supernatant was determined with the formaldehyde-Test Wako kit. The determination was reproducible and the limit of detection was 0.1 pg/mL.

5. Cell growth inhibition

Swiss 3T3 cells (American Type Culture Collection) were selected as a typical cell line of fibroblast cells and adapted in vitro before use. Assays were performed always with the cells in their exponential growth phase in culture. A 5 X lo3 quantity of 3T3 cells in 200 pL of DME containing 10% FCS was seeded into a 6.4-mm dish of 96-well multidish culture plates (A/S. Nunc, Kamstrup, Roskilde, Denmark) and incubated for 24 h at 37°C in a 5% C0,-95% air atmosphere. After washing the cell monolayers with the culture medium, 100 pL of DME with 5% FCS and penicillin (100 units/mL) was placed in the dishes. After an addition of 100 pL of the DME-FCS con- taining a PCA microsphere from 25 to 1,600 pg/mL, cells were further incu- bated for 24 h and fixed for 1 h at room temperature by an addition of 50 p L of glutaraldehyde aqueous solution to the culture. Then, the cells were stained with 0.05% methylene blue aqueous solution for 5 min. Moreover, the supernatant was collected from the media containing the microspheres after incubation for 1, 3, 5, 7, 10, and 14 days, and added to the cell/media combination, followed by 24 h incubation. After washing with water, 200 p L of 0.33 N HCl was added to each dish having the dried cell mono- layer and the dye associated with the cells was completely extracted. The absorbance of the dye solutions was measured at 620 nm by Titertek Multi- skan Mcc/340 (Flow Laboratories Inc., USA). The inhibitory effect on the 3T3 cell growth was calculated according to the following formula; Percentage growth inhibition =

i with sample medium 1-i with test medium (Absorbance of cell cultured with sample medium)

Absorbance of cell cultured 1 x 100

Absorbance of cell cultured

1358

RESULTS

1. Release of formaldehyde from PCA microspheres

TSENG ET AL.

The release profiles of formaldehyde from poly(isobu.jl 2-cyanoacrylate) (PIBCA) and poly(ethyl2-cyanoacrylate) (PECA) microspheres in DME with 5% FCS is illustrated in Figures 1 and 2, respectively. It is apparent that formaldehyde is released faster for both the microspheres, as the polymer molecular weight becomes lower. The rate of release from the PIBCA micro- spheres was much lower than that from the PECA microspheres.

The release profiles of formaldehyde are shown in Figures 3 and 4, for PIBCA and PECA microspheres, respectively, when the amount of the mi- crospheres added to the culture medium is changed. For both the micro- spheres, the amount of formaldehyde released increases with the contact time and the amount of the microspheres added. It should be noted that the amount of formaldehyde released from the PECA microspheres is longer than that from the PIBCA microspheres, although the molecular weight of the two PCA is almost the same.

Table I gives the amount of formaldehyde released 24 h after degradation of 100 pg of PCA microspheres. It is seen that the amount of formaldehyde released from poly(ethoxyethy1 2-cyanoacrylate) (PEECA) and PMCA mi-

0 2 4 6 8 10 12 14 16

Contact time (day) Figure 1. Release profiles of HCHO from poly(isobuty1 2-cyanoacrylate) microspheres with different molecular weights (500 pg/mL) . (0) Mw = 3.2 X lo4 and (A) Mw = 6.4 X lo4.

fN VITRO TOXICITY TEST OF 2-CYANOACRYLATE

14

12

h 2 < 10 m v

W B

3 6 E 1 4

2

Q) v) D

0 0 I

0 2 4 6 8 10 12 14 16

Contact lime (day) Figure 2. Release profiles of HCHO from poly(ethy1 2-cyanoacrylate) mi- crospheres with different molecular weights (500 pg/mL). (0) Mw = 1.4 x lo4, (A) Mw = 2.1 x lo4, and (0) Mw = 4.2 x lo4.

3.0 I I I I I 1 I I I

0 2 4 6 8 10 12 14 16

Contact time (day)

Figure 3. Release profiles of HCHO from poly(isobuty1 2-cyanoacrylate) microspheres of different concentrations (Mw = 3.2 x lo4). (0) 8,000 wg/mL, (A) 2,000 pg/mL, and (m) 500 pg/mL.

1359

1360 TSENG ET AL.

5

i

0 2 4 6 8 10 12 14 16

Coniaci lime ( d a y ) Figure 4. Release profiles of HCHO from poly(ethy1 2-cyanoacrylate) mi- crospheres of different concentrations (Mw = 2.1 x lo4), (0) 2,000 pg/mL, (A) 500 Fg/mL, and (0) 125 pg/mL.

TABLE I Amounts of HCHO Released from Poly(2-Cyanoacrylate) Microspheres in DME

Culture Medium Containing 5% FCS after Contact for 24 h ~ ~~

Poly(2-Cy anoacryla te)

~ ~~

HCHO/Poly(2-Cyanoacrylate) - Mw (CLg/lOO F.s)

PMCA PECA PIBCA PEECA

3.8 x lo4

3.2 x 104 2.8 x 104

2.1 x 104 3.621 0.088 0.005 5.320

IN VITRO TOXICITY TEST OF 2-CYANOACRYLATE 1361

crospheres is by far larger than that from two other microspheres. The degradation rate of the microspheres is in the order of PEECA > PMCA 9 PECA > PIBCA.

2. Cell growth inhibition by PCA microspheres

To examine whether the in vitro growth of 3T3 cells is sensitive to formal- dehyde and is suppressed by the presence of microsphere itself, the cells were incubated for 24 h with formaldehyde and the PS microsphere which was widely used in cell culture assays as a nondegradable microsphere. The inhibitory effect of formaldehyde on cell growth was clearly observed in the concentration range higher than 0.03 &well. The addition of PS micro- sphere to this culture system did not have any effect on the cell growth, so far as 100 &well of the microsphere was added. These results indicate that the present test method is applicable to estimating the cell toxicity of formaldehyde.

The cell growth inhibition upon addition of CA polymer microspheres is shown in Figure 5 for the almost similar molecular weight of the polymers. Any significant inhibitory effect on the cell growth is not observed for the PIBCA microsphere, whereas the PECA microsphere inhibits the cell growth in a dose higher than 50 pg/well. The inhibitory effect of the PEECA and the PMCA microsphere increases with an increase in the dose, ap- proaching 100% growth inhibition by a dose of 12.5 pg/well. The order of the inhibitory effect on cell growth is identical to that of the release rate of form- aldehyde from the microsphere.

100 t 0 Z 80 n

f

r C --

60

20 aJ a

0 0.1 1 10 100

Amount of microspheres added (pg/welO

Figure 5. The inhibitory effect of poly(2-cyanoacrylate) microspheres on cell growth (0 h contact and 24 h incubation). (n) poly(ethoxyethy1 2- cyanoacrylate) (Mw = 2.8 x lo4), (0) poly(methy1 2-cyanoacrylate) (Mw = 3.8 x lo4), (0) poly(ethy1 2-cyanoacrylate) (Mw = 2.1 x lo4), and (A) poly- (isobutyl 2-cyanoacrylate) (Mw = 3.2 x lo4).

1362 TSENG ET AL.

3. Effect of the molecular weight of PCA on cell growth inhibition

Cells were incubated for 24 h in the culture medium to which the PECA and the PIBCA microspheres of different molecular weights had been sus- pended. Table I1 shows the result obtained when 100 p g of the micro- spheres in each well was in contact with the medium for 7 and 14 days. The PIBCA microspheres without medium contact in advance does not exhibit any growth inhibitory effect, regardless of the molecular weight of the poly- mer used. However, when the microspheres were in contact with the me- dium for 1 or 2 weeks, a noticeable inhibitory effect was observed. The microsphere of PIBCA with the lower molecular weight inhibited the cell growth stronger than that with the higher molecular weight. The growth inhibitory effect of the PECA microspheres was distinctly higher than that of the PIBCA microspheres. Moreover, cell growth inhibition was observed even without the previous contact for the microsphere prepared from PECA with the molecular weight of 1.4 X lo4 and 2.1 X lo4, in contrast with the mo- lecular weight of 4.2 x lo4. One week after suspension in medium, the cell growth is markedly inhibited by the PECA microsphere, irrespective of the molecular weight. The inhibitory effect increases for any microsphere, as the contact is prolonged. The PECA microspheres kill all cells after 2 weeks contact, irrespective of the molecular weight.

4. Effect of the microsphere amount on cell growth inhibition

The PECA and the PIBCA microsphere of different amounts were al- lowed to be in contact with DME-5% FCS at 37°C for various periods of time up to 14 days and then 3T3 cells were incubated for 24 h in the same me- dium to assess the growth inhibitory effect of the microspheres. The result is illustrated in Figure 6. The inhibitory effect of the microspheres on cell growth increases with the contact time, regardless of the kind of polymers used. However, the inhibitory effect of the PECA microsphere is much more prominent than that of the PIBCA microsphere. An increase of the in- hibitory effect is observed more or less by an increase in the added amount

TABLE I1 The Cell Growth Inhibitory Effect of Poly(Isobuty1 2-Cyanoacrylate) and Poly(Ethy1 2-Cyanoacrylate) Microspheres of Different Molecular Weights after 24 h Incubation

Percentage Growth Inhibition ~

- Mw of PIBCA" Mw of PECA" Contact Time

(day) 3.2 x 104 6.4 x 104 1.4 x 104 2.1 x 104 4.2 x 104

0 0 0 66.9 * 3.2 33.7 c 2.0 0

14 34.7 * 2.7 26.3 t 0.9 100 100 100 7 30.6 f 0.9 6.2 f 2.4 87.9 + 3.9 78.8 t 2.6 72.2 ? 3.4

"100 pg per well.

IN VITRO TOXICITY TEST OF 2-CYANOACRYLATE 1363

C 0 .- c .- 0

f 3 2

0,

L c .-

m

c Q

Q a

-

100

80

60

40

20

0 2 4 6 8 10 12 14 16 Contact time (dsy)

Figure 6 . The cell growth inhibitory effect of poly(2-cyanoacrylate) mi- crospheres of different concentrations (24 h incubation). poly(ethy1 2- cyanoacrylate) (Mw = 2.1 X lo4): (0) 400 &well, (A) 100 pg/well, and (0) 25 pglwel l . Poly(isobuty1 2-cyanoacrylate) (Mw = 3.2 x l o 4 ) . (@) 1,600 pg/well, (A) 400 pg/well, and (W) 100 &well.

of the microspheres. Especially, the cell growth is entirely inhibited by 400 pg/well of PECA microsphere even for the zero contact time, whereas the PIBCA microsphere does not exhibit any growth inhibitory activity until to 7 days.

The cell growth inhibitory activity of the supernatants obtained from the medium after degradation of the PCA microspheres was investigated and the result is given in Figure 7. The PCA microspheres were allowed to be in

100 c 0 5 80 r c !2 ._

60

40

f 3

c 9,

Q a : 20

0 2 4 6 8 10 12 14 16 12 14 16 Contact firne (day)

Figure 7. The cell growth inhibitory effect of the supernatant of the culture medium (24 h incubation). poly(ethy1 2-cyanoacrylate) microspheres (Mw = 2.1 X 104): (0) 400 pg/well, (A) 100 pg/well, and (0) 25 p$/well. Poly(isobuty1 2-cyanoacrylate) microspheres (Mw = 3.2 x 10 ): (@) 1,600 &well, (A) 400 &well, and (W) 100 &well.

1364 TSENG ET AL.

contact with DME-5% FCS at 37°C for various periods of time up to 14 days and then the supernatant was separated from the microspheres by centrifu- gation. It is apparent that the inhibitory effect of the supernatant on cell growth increases with the contact time and the amount of the microsphere added to the medium. This trend is similar to that of the cell growth inhi- bition by the medium containing the microspheres as shown in Figure 6. Higher growth inhibition by the medium-PECA microsphere mixture than that by the supernatant gives as evidence of the release of formaldehyde from the microsphere. The high inhibitory effect probably results from the continuous exposure of cells to formaldehyde released from the micro- sphere in this assay system. However, such a trend was not observed for the PIBCA microsphere, because of the low released amount of formalde- hyde due to its slow degradation. The above results indicate that the growth inhibition should be caused by formaldehyde the degradation product pres- ent in the supernatant.

DISCUSSION

In a preliminary experiment we tried to do a toxicity test using PCA films, but it was difficult to assess the toxicity with high reproducibility because of poor cell growth on the film surface. Therefore, we explored a new method for the toxicity estimation by using the PCA microspheres. This method en- abled us to readily vary the amount of the PCA to be added to the cell cul- ture system. Moreover, no inhibitory effect on cell growth was observed for the PS microsphere until to 100 pg/well which was sufficiently enough to cover the cell monolayer, indicating that the addition of microsphere itself to the cell culture had no effect on cell growth in this assay.

Release of formaldehyde from the PCA microspheres was investigated, because formaldehyde is a most probable toxic substance in the degradation products of the Indeed, formaldehyde was released from the PCA microspheres upon contact with the aqueous medium (Figs. 3 and 4) and inhibited the growth of 3T3 cells (Fig. 6 and Table 11). A similar trend of cell growth inhibition was observed by the use of supernatant of the culture medium which had been in contact with the microspheres (Fig. 7). In Fig- ure 8, all the results of the cell growth inhibition are plotted against the con- centration of formaldehyde released to the culture medium, together with the inhibitory effect of free formaldehyde. The amount of formaldehyde re- leased from the PEECA and the PMCA microsphere readily reached the level of 100% growth inhibition of 3T3 cells in this assay, since their micro- spheres were rapidly degraded in the culture medium. It is clearly seen from Figure 8 that the cell growth inhibition has a linear correlation with the formaldehyde concentration to indicate that formaldehyde released from the PCA microspheres must be absolutely responsible for inhibiting the cell growth. Lenaerts et al. proposed that only the ester group of PCA was subjected to hydrolysis at high pH's.'' This degradation mechanism is

IN VITRO TOXICITY TEST OF 2-CYANOACRYLATE 1365

100

5 80 n

f

c 0

r c .- 60

40 z

al 20

- c al

I I I I 1 I

A A

m a

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

Conceniralion of HCHO (pg/welO

Figure 8. Relationship between the cell growth inhibition and the amount of HCHO released from poly(2-cyanoacrylate) microspheres; (0 ) free HCHO, (A) poly(ethy1 2-cyanoacrylate), and (m) poly(isobuty1 2-cyanoacry- late) microspheres.

not in accord with our findings. We believe that PCA is degraded as a result of depolymerization of PCA, as proposed by numerous research groups7:

$N CN I qN $N

c=o c = o ?= 0 c=o 6 c, 0 0 k k A k

__u CHz-C-CHz-FH t H20 - CH-CH + HCHO + CH2 I I

I

This scheme can give an explanation why PCA with a lower molecular weight exhibits more rapid release of formaldehyde (Figs. 1 and 2). Vezin et al. also have demonstrated that the degradation rate of CA polymers de- pends not only on the kind of the side chain of CA, but also on the polymer molecular eight.^

The IBCA polymer has been known to show a minimal tissue reaction among the PCA used at the present in accordance with our above findings. Very little formaldehyde release from the PIBCA microsphere, which results from its very low degradability, may account for the very low toxicity of the IBCA polymer. It may be concluded that the cellular toxicity of the CA polymers is linearly related to the release rate of formaldehyde. This is in good accordance with the results reported by Leonard et aL6 and Lehman et al.,m which suggest that the toxicity of CA polymers to cell may be caused by the degradation products of the polymers. Wade et al. also have demonstrated that the degradation products of the CA polymers may contribute to histotoxicity in vivo.’l On the other hand, Eiforman et al.

1366 TSENG ET AL.

observed a decreased bacteriostatic activity of CA after polymerization of CA,22-24 and speculated that the residual monomer of CA was responsible for the toxicity. We confirmed the absence of residual monomer in the CA polymers employed here by GPC measurement (data not shown). In ad- dition, we tried to assess the toxicity of CA monomers themselves by this assay system, but could not obtain reliable results because of its very rapid conversion to the polymers in the aqueous medium.

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IN VITRO TOXICITY TEST OF 2-CYANOACRYLATE 1367

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R. C. Com, 0. Corn, and T. Matsumoto, "Osteosynthesis employing isobutyl cyanoacrylate monomer," Int. Surg., 6, 483-487 (1972). R. A. W. Lehman, R. L. West, and F. Leonard, "Toxicity of alkyl 2- cyanoacrylate, 11. Bacterial growth," Arch. Surg., 93, 447-450 (1970). C. W. R. Wade and F. Leonard, "Degradation of poly(methy1 2- cyanoacrylate," J. Biorned. Mater. Res., 6, 215-220 (1972). R. A. Eiferman and J. W. Snyder, "Antibacterial effect of cyanoacrylate glue," Arch. Ophthalrnol., 101, 958-961 (1983). M. Andersen, M.-L. Binderup, P. Eel, H. Larsen, J. Maxild, and S. H. Hansen, "Mutagenic action of methyl 2-cyanoacrylate vapor," Mutation Res., 102, 373-381 (1982). E. C. Rietvold, M. A. Gamaat, and F. Seutter-Berlage, "Bacterial muta- genicity of some methyl 2-cyanoacrylates and methyl 2-cyano-3-phenyl- acrylates," Mutation Res., 188, 97-104 (1987).

545-551 (1979).

Received December 14, 1988 Accepted April 24, 1990

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