Radiation processing technology for preparation of fine shaped biomedical materials

Radtat Phys Int J Radtat

Chem Appl

Vol 39, NO Instrum

,

6, pp

571-581.

1992

0146-5724/92

$5 00 + 0 00

Pergamon

Part C

Press

Ltd

Pnnted tn Great Bntatn

RADIATION PROCESSING TECHNOLOGY FOR PREPARATION OF FINE SHAPED BIOMEDICAL MATERIALS M. Kuhmcum,t

M. YOSHIDA,’ M.

ASANO’

and H. YAIWNAKA~

‘Takasakt Radtation Chemistry Research Estabhshment, Japan Atomtc Energy Research Instttute, Takasakt, Gtmma 370-12, Japan and 2School of Medtcme, Gunma Umverstty, Maebasht, Gunma 371, Japan Ah&ret-Radtatton processmg technology for the preparation of fine shaped btomedtcal mater&s was studted from the aspect of a development of the technology and tts apphcatton Electron beam trradtatton technology was apphed to the preparation of fine shaped btomedtcal mater& such as thtn polymer tilms m dtagnosts, m which enzyme and antibody were used as a bioacttve substance Electron beam cast-polymertaatton and electron beam repeat surface-polymenzatton, that are surface trradtatton techmques of homogeneous hydrophthc monomer solutton contatnmg enzymes made tt possible to form

the lmmobllrzed antibody films In thrs technique, the films wrth various thrcknesses (50-SOO~m) were obtained by regulating the electron beam energy The thm polymer films lmmobthnng ant+a-fetoprotem were evaluated from the aspect of rmmunoagents for diagnosis of liver cancer

INTRODUCTlON

There IS a wide variety of biomedtcal material which are foreign to hvmg body and most of which are used m contact wtth hvmg body or serum. The apphcanons of btomedtcal matenals include devices (reagents) or implants for dlagnosts or therapy. Diagnostic biomedical materials such as immunoreagents, which need high sensttivtty and easy handling form, have been prepared by using polymenzatton technique, m which the shaping process was important subject to get fine shaped matenals such as polymer plates, tubes, beads, etc. (Wtsdow, 1976, Woo and Cannon, 1976, Scharpe et al., 1979) In immunoreagent, antibodies were coated or bound on then surfaces The reagents resulting from phystcal coating method have a weak point such as leakage of antibodies, and those resulting from chemical covalent method requrre longer preparatton times and excess amount of antibodies though the leakage does not occur Vanous immunoreagents have been studied for enzyme and radio tmmunoassay. Radiation polymenzatton technique is a very useful tool to produce polymer matenals in various shapes wtthout impunties such as catalyst. The field of electron beam curable coating was launched when polymer chemrsts established that reacttve monomers could be rapidly polymenzed by substituting radlanon techmques for conventional polymerization mitrators. The ionizing and chemical changes caused by absorption of an accelerated electron as well as penetrating properties depend upon the kinetic tIJresent address

Department of Biosctence, The NishtTokyo University, Uenohara, Rttatsuru, Yamanasht 409-01, Japan

energy of the individual electron Modem accelerators are capable of producing electrons with values as high as a few hundred billion electron volt (DeMarteau et al., 1984). For electron beam processing of a typical organic coating or film, electrons in the 150 to 300 keV range have been found to be the most cost effecttve energy (Lauppi, 1990). Thrs work IS concerned with the preparation technique of thin polymer films immobthzing antibody and enzyme. Anti- a-fetoprotem (anti-AFP) and ghrcoamylase were used for tmmohlization, of which a-fetoprotein (AFP) is a tumor-assoctated antrgen. The tmmobitizatton of the enzyme was carried out for the investigation of preparatton condittons.

EXPERIMENTAL

Materials Ethyleneglycol dtmethacrylate (2G), tetraethyleneglycol dimethacrylate (4G), nonaethyleneglycol dtmethacrylate (9G), tetradecaethyleneglycol drmethacrylate (14G), and hydroxyethyl acrylate (HEA) were used as monomer and obtained from Shm Nakamura Chemical Co. Ltd. Glucoamylase (10,000 units/g) was obtained from Negase Sangyo Co. Ltd. AFP, anti-AFP and sheep anti-AFP peroxrdase were obtained from Serotec Ltd. Experrmental procedures A certain amount of monomer solution buffered at pH 4.5 containing glucoamylase (0.5%) put mto a vessel (6 or 60 mm in diameter, 1 cm in height). This vessel was uradiated to get the immobihzed enzyme films with irradratton dose of 1 Mrad by low energy electron beam accelerator (300 keV, 5 mA; 577

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“Curetron”, Nlssm-High Voltage Co Ltd) After lrradlatlon, the lmmobdlzed enzyme films were employed to the enzyme reactlon m maltose solution to get the enzyme activity of the lmmobihzed enzyme films, m which the enzyme actlvlty was defined as the ratio (%) of the glucose formed m the lmmoblhzed enzyme films against to that m the native enzymes A homogeneous monomer solution containing monomer and antlbody (5 mg/ml) m the vessel (16 mm m diameter) was irradiated by low energy electron beam accelerator. After irradlatlon, the Immobilized antibody films were employed to enzyme immunoassay In the case of electron beam repeat surface polymenzation, after irradiation the polymer film formed was taken out from the surface of the monomer solution m the vessel This lrradlatlon process was repeated to get the polymer film lmmoblhzmg antibody. The activity of the lmmobllized

antibody film was measured by enzyme immunoassay method as follows The film was placed in a vessel contammg 1 ml of AFP solution, and incubated at room temperature for 1 h After mcubatlon, the solution was removed, the film was washed three times with the buffer solution, and 1 ml of anti-AFP peroxldase and 1 ml of the buffer solution was added to the vessel, which was incubated at 37°C for 1 h. The film was agam washed three times wth the buffer solution. The enzyme reaction was done with 3 ml of a solution of hydrogen peroxide (0.03%) and o-phenylenedlamme (0.3%) at room temperature for 30mm and terminated by adding 3 ml of 1 M hydrochlonc acid The absorbance of the solution was measured at 492 nm to measure the peroxidase activity

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Fig. 4 Effect of monomer composition on enzyme activity Monomer concentration, 60%; irradiation temperature, 25°C. -_ _, monomer ..-. ~.~~~.~solution ~~~~~~ volume. 0 8 ml

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Rg. 5 Effect of monomer concentration on enzyme actwty m ~mmob~hzed enzyme films wth (0, W) and wthout filter paper (0, Cl) Monomer, 14G (0, n ), HEA-14G (1.1) (0, 0); monomer solution volume, 0 8 ml; rrradlahon temperature, 25°C

RESULTS AND DISCUSSION

Electron beam cast-polymerizatron Electron beam cast-polymermatron technique was useful for the preparation of thin film, m which the thickness of the film obtained was controlled by the volume of monomer solution m the vessel though the thickness of the resulting polymer film, of course, depends on the penetration of electron beam. The enzyme activity of the films was affected by monomer soluuon volume, gtvmg a maximum peak at about 0.8 ml as shown in Fig. 1. The effect of monomer concentratton on the enxyme activity at vanous irradtation temperatures IS shown in Fig. 2. The enzyme activity was affected by monomer concentration, giving a maximum activity at &out 30% monomer concentration. The low enxyrne tu$ivities at low and high monomer concentrations -due to a leakage of the enzymes from

(ng/ml)

Rg. 7 Standard curve for enzyme mmwnoassay of AFP using ~mmobhed antl-AFP fks with 6lter paper. Monomer, HEA-14G (1.1); filter paper, 250 pm, monomer solution volume, 24 the polymer matnx

and a restnction of the enzyme reaction resultmg in a ngtd entrapment of the enzyme mto the polymer matrix. The decrease of trradiation temperatures below 0°C led to the increase of the enzyme activity. ‘flus is able to correspond to the formation of a porous structure in the polymer matrix obtained by radiation polymerization at low temperatures, leading to an increase of surface area of the film. The effect of property (hydrophilic&y) of monomer on the enzyme activity is shown in Fig. 3. The enxyme activity increased with increasing the number (n) of oxyethylene unit in the monomer (polyethleneglycol dtmethacrylate, nG), indicating that the hydrophilictty of the polymer matrix increases with increase of n, by which the diffusion of the substrate in the polymer matnx was affected. The polymer flhns prepared by using the monomers with n = 1-4 had a hydrophrlic property. This was unsuitable for enxytne reaction because the contact between enzymes immobilized mto the polymer matrix and substrate may be restricted. On the other hand, the polymer matrices prepared by using the hydrophilic monomer

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monomer concentration is of optimum condition to tmmobthze the enzymes on the surface of the fibnls of filter paper. As monomer concentration increases, the enzymes Seem to be firmly entrapped m the polymer matrix and on the surface of filter paper, m which the mob&y of the enzyme molecule 1s

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n = 9 or 14 led to the Increase of the enzyme activtty owing to an increase of a mob&y of the enzyme molecule The enzyme acttvtty of the immobthzed enzyme films was also affected by monomer composttton as shown m Ftg 4 HEA 1s a higher hydrophthc acrylate monomer than 14G that is a btfuncttonal hydrophthc crosshnkmg monomer. As can be seen m Ftg 4, a maximum peak of the enzyme activity at certain composttton was observed, meanmg that the enzymes are tmmobrhzed m the polymer matnx by copolymertzation of HEA and 14G, taking optimum molecular conformation state wth

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The effect of monomer concentration on the enzyme acttvity m the presence of filter paper as a porous substance was studied and the result 1s shown m Fig. 5 The enzyme activity of the immobilized enzyme films obtained by the addttton of filter paper at monomer concentration above about 10% was higher than that without addition of filter paper. At lower monomer concentrations below lo%, the difference between the enzyme activttms with and without filter paper was clearly observed. The result means that, m the case of the addition of filter paper, a little amount of monomer is absorbed in filter paper itself, so that a part of the enzymes are not immohhzed. In the addition system of filter paper, the enzyme activity increased, reached a maximum and after that decreased due to increase of monomer concentration. A maximum peak of the enzyme activity at monomer concentration range of 20-30% was observed, suggesting that the monomer content in the monomer solution at 20-30%

of filter paper on activity was studied by using anti-AFP instead of the enzyme The immobthzed anti-AFP films wtth filter paper, were prepared by using small vessel (6 mm m diameter) to develop tmmunoreagents of a dtsc type using electron beam polymenzation technique The relation between absorbance and thickness of filter paper in the tmmobthzed anti-AFP films with filter paper is shown m Fig 6. Absorbance correspondmg to the activity was increased with increase of the thickness of filter paper, suggesting that surface area of the film increase. by addition of thick filter paper. From the expertment m the addmon of filter paper, it was found that the addition of filter paper is able to exhibit the increase of the activtty and the increase of hardness of the films leading to the improvement of handling of the film throughout assay The standard curve for enzyme immunoassay of AFP, which is important for an evaluation of immunoreagents, was obtained by using the tmmobthzed anti-AFP films as shown m Fig 7 The slope IS relatively large, e g the absorbance at 20 ng/ml AFP is 0.4 Because the boundary value of AFP m sera from normal (healthy) and abnormal (pnmary liver cancer) 1s 10-20 ng/ml, the tmmobihzed anti-AFP films can be used for the enzyme immunoassay of AFP Electron beam repeat surface polymerrzatzon

A homogeneous monomer solutton containing hydrophthc monomer and anti-AFP m the vessel (6 mm m diameter) was irradiated under mtrogen atmosphere by the low energy electron beam a*ator After one time trradtation, the film fpmed was taken out from the surface of the mo,nurner solutton in the vessel. This process was r~ated to get the films until the monomer solution exists u-r the vessel It was found that a thm polymer film with a considerable activity was obtained by one time u-radtatton, and similar films with almost the same thickness and absorbance were obtained by repeat surface uradiatton as shown m Fig 8 This preparation technique of thm films is based on the limited penetratmg power of relatively low energy electron beam. A polymerized film formed to a certain thickness 1s the same as the penetration depth of the electron beam and the antibodies are trailed and trapped m the film from the monomer solution phase during irradiation In this technique, it was found that the absorbance m the inner side facing and contactmg wtth the monomer solutton was larger then that m the outer side facmg to the nitrogen atmosphere. This suggests that the antibodies located on the surface of the monomer solution are effectively

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Fine shaped biomedrcal materials immobilized to be tratled and arranged on the inner side, taking a suitable position to antibody-antigen reaction. It is certain that the remaining antibodies in the monomer solution phase does not receive a radiation damage by repeat irradiations of the monomer solution, and always the film with the same absorbance m each uradtatlon is obtained by each irradiation as long as the monomer solution exrsts in the vessel. In the immobilized enzyme films obtained by this techmque, the relation between the enzyme activity of the immobilized enzyme films and the repeated enzyme reacttons gave a constant curve with reasonable enzyme activities m the tmmobilixe enzyme films obtained from 30% monomer concentration, though the data are not shown here. This proved the firm tmmobthzatron by this technique because decrease of the enzyme activity was not observed m the repeated enzyme reactions. The rclatton between the thickness of the tmmobthzed anti-AFP film and electron beam energy is shown m Fig. 9 From this result, it can bc said that the tmmobilixed anti-AFP films wtth various thtcknesses can be speedily obtained by regulating the electron beam energy An increase m conveyer speed does not gave negative results on the absorbance. One

possible reason might be that during immobilization the polymer film formed acts as a screening substance to protect the activity of the antibody under irradiation. Tbe amount of polymer and the thickness of the film increased at irradiation temperature above 0°C and high electron beam energy. This work concerned with radiation processing technology using low energy electron beam accelerator exhibited that the immunoreagents in thin polymer film form can be speedily prepared by repeat u-radiation regulating of electron beam energy. The tmmobihxed anti-AFP films obtained by this technique is applicable for the enzyme immunoassay of AFP; tumor-associated antigen. The minimum detectable concentration was 5 ng/ml, and the assay precision was 5%

RRFERENCRS

DeMarteau W , Gtets P and Lcutx J M (1984) Radiat. Curmg (February), 12

Lauppt Urs V (1990) Radwt. Phys. Chem. 35, 30. Scharw S L.. Cooreman W M and Blomme W J (1979) . I CIA Chem’ 25, 733

Wxulow G. B (1976) Clzn Chem. 22, 1243 Woo J and Cannon D C (1976) Am. J. Path& 66, 854