Surface Biolization by Grafting Polymerizable Bioactive Chemicals

Chapter 6

Surface Biolization by Grafting Polymerizable Bioactive Chemicals Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on July 11, 2018 at 10:27:32 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Y. Ito, K. Suzuki, and Y. Imanishi Department of Polymer Chemistry, Kyoto University, Kyoto 606-01, Japan

A general method to surface design of materials for biocompatibility was provided. This is based on the surface-grafting of polymerizable biological chemicals on the materials. For the purpose the plasma-pretreated polymerization method was employed and the polymerizable biological chemicals was used or were synthesized. To design blood-compatible materials poly(vinyl sulfonate) and polymerizable thrombin-inhibitor were used. In addition to design cell-adhesive materials the cell-adhesion-peptide, Arg-Gly-Asp-Ser was surface-grafted after connecting with a vinyl group.

Biocompatibility and medico-functionality (elasticity, permeability, etc.) are qualities required in materials for artificial organs (2,2). The former usually depends on the surface properties, and the latter on the bulk properties. Surface modifications to enhance biocompatibility is a useful method in designs which avoid interferring with bulk properties as shown in Figure 1 (1,2). In addition to immobilization of biological macromolecules or cells on the surface (3-5), the surface modification has been mainly considered to be the physico-chemical modifications such as the wettability control. So far very few examples of biomimetic approach using low molecular weight chemicals containing biological activity were carried out (6). Such an approach has some advantages, Le., the designed materials can be more biospecifically active than that designed by the physico-chemical approach and more stable than that containing biological chemicals which are, for example, thermosensitive. We recently developed a new general method to biolize surfaces for biocompatibility, such as blood-compatibility and tissue-compatibility. The basic concept of surface biolization is surface-grafting molecules which is made polymerizable in advance. In the case of polymerizable biological molecules, direct surface-polymerization can be used. Otherwise, a vinyl group can be conjugated to the site which is not related with biological activity of the biological molecules to polymerize. Vinyl sulfonate was used in this study as a polymerizable bioactive molecule because of its heparinoid activity. Other examples such as a thrombin inhibitor and a cell adhesive active peptide, Arg-Gly-Asp-Ser (RGDS), were also employed after conjugating with polymerizable vinyl groups. The chemicals and surface modification method are illustrated in Figure 2. 0097-6156/94/0540-0066$06.00/0 © 1994 American Chemical Society

Shalaby et al.; Polymers of Biological and Biomedical Significance ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

6. ITO ET AL.

67

Surface Biolization by Grafting Surface Medico-functionality |—Elasticity Permeability f—Transparancy

Surface

Biocompatibility

• Physicochemical

•

S u r f a c e energy Wettability

Figure 1. a

vinylsulfonate

Enzymes Other biopolymers

1 This

• Cells

• Biological chemicals

Biomimetic

study

aassification of approaches for biocompatible materials. CHÎ=ÇH

S0 H 3

b

acryloyled CH =CH thrombin inhibitor C=0 (MD-805) 2

CH

c

acryloyled RGDS

3

ClfesCII C=0

ΛΗ c=o NH-CH-CONH- C H CONH-CH-CONH-ÇH-COOH 2

(fcH)4 If C=NH

Ç"2

ψ2

COOH

OH

ΛΗ

2

Glow-discharge

a, b, c , ,

Θ

O-O O-OH

" W////M —

Figure 2.

,

W//7///7/

Surface biolization by plasma-pretreated polymerization method.


68

POLYMERS OF BIOLOGICAL AND BIOMEDICAL SIGNIFICANCE

Surface Design for Blood-Compatibility Polyvinyl sulfonate). When an artificial material comes in contact with blood, thrombi are formed on the surface. The mechanism is illustrated in Figure 3. After the initial stage of blood protein adsorption, the platelet system and the intrinsic coagluation system are activated to form thrombus. Heparin has been the most widely used for inhibition of thrombus formation by inactivation of thrombin which is generated in the coagulation system and catalyzes fibrin formation. In addition to the immobilization of heparin onto materials (3), there have been a number of investigations of the syntheses of heparin-like (heparinoid) polymers (sulfonated polydienes, sulfated chitosan, sulfonated polystyrene, sulfonated dextran, and sulfonated poiyurethanes) (7). However, in these earlier investigations, the synthetic methods were complicated and troublesome, and mechanical properties of the materials have not been taken into account. These properties are very important for practical use. Therefore, surface modified grafts of poly(vinyl sulfonate) by plasmapretreated polymerization was performed on certain mechanically strong materials (8,9). Antithrombogenicity of water-soluble poly(vinyl sulfonate) (PVS) was nearly 7.5% that of heparin, though the activity depended on the molecular weight of PVS. Considering that the blood coagulation time in the presence of PVS was significantly prolonged by the addition of antithrombin III, the antithrombogenic effect was based on the interaction with antithrombin III as illustrated in Figure 4 (8,9). The higher molecualr weight of PVS should more siginificantly interact with antithrombin III to induce a confromational change of the protein because of the continuous sulfonate groups on PVS. PVS was grafted onto the surface of polyurethane, polystyrene, and poly(ethylene terephthalate) films by the plasma-pretreated method. Activated partial thromboplastin time (APTT) of PVS-grafted polyurethane films was greatly prolonged, and a fibrin network was not found at all on the film grafted with PVS in densities higher than 1.6 pg/cm , a level at which the surface is completely covered with PVS. In vitro thrombus formation on the film was suppressed with increasing amounts of grafted PVS, and no thrombus was formed during a 20-min contact with blood on the film with PVS grafted in densities higher than 1.6 Mg/cm . The antithrombogenic mechanism is considered as shown in Figure 5a. 2

2

Polymerizable Thrombin Inhibitor. In order to directly deactivate thrombin without antithrombin ΠΙ as shown in Figure 5b, a thrombin inhibitor was immobilized on polymer surfaces (10). Figure 6 shows a schematic drawing of the interaction of thrombin catalytic site with a thrombin inhibitor, which is named MD-805. Because the carboxylic group of MD-805 was not reported to be related to the inhibition activity, a vinyl group was connected to this group. No siginificant difference in the inhibition constant Ki was found between MD-805 and its vinyl derivative. The polymerizable thrombin inhibitor was then grafted onto a polyurethane film. The film not only deactivated thrombin, but it also reduced platelet adhesion and activation, thus becoming antithrombogenic. Surface Design of Cell-Adhesive Materials Synthesis of RGDS-Immobilized Membrane. Cell adhesion is a ubiquitous process that influences many aspects of cell behavior. For example, proliferation, migration, secretion, and differentiation of cells are triggered by adhesion to matrix. Since Pierschbacher and Ruoslathi found that the RGDS sequence in cell adheison proteins was an active site, a number of investigations into its applications have been carried



*

Intrinsic coagulation activation ^

*

Thrombin generation

Platelet activation

*

Fibrin formation

Thrombus

Figure 3.

Sequence of events during blood-material interactions.

////ιιιιιιιιιιιιιπ^ιιιιι/ιιιπτπτπττιτπ/

Protein adsorption

Platelet adhesion

Time

70


a

Figure 4. Schematic representation of interactions among thrombin, antithrombiin III (ΑΉΙΙ) and heparin (a) or polyvinyl sulfonate) (PVS) (b,c). The interaction is considered to depend on the steric position and continuity of sulfonate groups. The activity of PVS is less than that of heparin because of the difference of steric position of the sulfonate groups. On the other hand, the high molecular weight PVS (c) induces more conformational change of ATIII than the low molecular weight PVS (b) because of the continuity of the sulfonate groups.


6. ITO ET AL.

71

Surface Biolization by Grafting

a

b

Figure 5. Antithrombogenic mechanisms of (a) poly (vinyl sulfonate)-grafted and (b) polymerizable thrombin inhibitor-grafted materials.

modification

Figure 6. Schematic drawing of the interaction of thrombin with a thrombininhibitor, MD-805. The carboxylate group, which is coupled to a vinyl group, is indicated as the modification site.


72


out (22-23). The use of bioactive peptides instead of proteins has two principal advantages. One is that short-chain peptides are more resistant to the denaturation caused by ethanol, heat, and pH variations. Another is that peptides can be integrated in a large amount on the surface to compensate low unit activity when compared to the high molecular weight cell-adhesion proteins. Synthesis of CH2=CH-CONH-(CIfe)5-COOSu (1). 5-Aminhexanoic acid (5 g) and Ca(OH)2 (5 g) were solubilized in water (75 ml). After cooling the solution in an ice bath, 3.5 ml of acryloyl chloride was added dropwise with vigorous stirring over 12 min. After the excess of Ca(OH)2 was removed byfiltration,the solution was acidified by concentrated H Q . The precipitate was washed with water and dried in vacuo. The product (1 g) was suspended in methylene chloride (20 ml), and Nhydroxysuccinimide (670 mg, 1 eq.) and l-ethyi-3-(3-dimethylaminopropyi)carbodiimide (1.34 g, 1.2 eq.) were added to the suspension. After the suspension was stirred for 1 h at 0°C, 15 mi of ethyl acetate was added and the mixture was stirred additionally for 15 min. The mixture was washed with NaHC03 aqueous solution and NaCl aqueous solution and dried on sodium sulfate. The product 1 was recrystallized from methylene chloride-diethyl ether. The yield was 768 mg (54 %). m.p., 122124°C(Lit. 122-123 Q. 0

Coupling of RGDS to Product 1. RGDS (150 mg), product 1 (5.0 eq.) and 48.5 ml (1.0 eq.) of triethylamine were dissolved in 10 ml of ^,JV-dimethylformamide and the mixture was stood overnight at room temperature. The coupling product (product c in Figure 1) was purified by LH20 and ODS (MeOH/H20=3/7) columns and freeze-dried. Analysis calculated for C24H39N8O10: C, 47.99 %; H, 6.71 %; N, 18.99 %. Found: C, 48.23 %; H, 6.53 %; N, 18.52 %. Graft-Polymerization of Product c and Cell Adhesion Experiment. A polystyrene (PST) film was glow-discharged (pressure; 0.2 Torr, electric current; 8 mA) over 1 min and immersed in aqueous solutions containing monomers (acrylamide, acrylic acid,iY^3-trimethylammoniumpropyl)acrylamide chloride, and product c of various concentrations for 8 h at 7(FC. The grafted membrane was washed until no changes were observed in the washing solutions by means of pH and ultraviolet measurement. The cell adhesion experiemnts were performed by using sub-cultured mouse fibroblast cells STO as reported previously (13). Cell-Adhesion Activity of the RGDS-Grafted Membrane. Figure 7 summarizes the number of adhered mouse fibroblast cells STO onto various surface-grafted membranes. The glow-discharge treatment increased cell adhesivity. However, the acrylamide- or acrylic acid-grafting reduced the adhesivity markedly. On the other hand, the cationic polymer-grafted membrane had high cell adhesivity. These results indicate that hydrophilic polymer grafting reduced cell adhesion, however, that cationic polymers enhanced the adhesion. The membrane co-polymerized with product c promoted cell adhesion more than that polymerized with cationic monomer, and was comparable to a fibronectin-coated membrane in cell-adhesiveness. In addition, the co-polymerized film enhanced cell spreading more than cationic polymer-grafted film as shown in Figure 8. This study shows that the cell adhesion enhancement of the RGDS-immobilized materials was comparable to the fibronectincoated materials as reported previously (22,13)



with

RGDS

:1 )

with

(4

3

6

+

2

3

3

derivative

ci-

C0NHC H N (CH )

2

CH=CH

2

CH=CH-C00H

2

CH=CH-C0NH

5

25

10

5

10

5

25

1 0

5

H

2.0

1

adhesion

1

—

1

4

—

3-0

4.0

2

Ι-» 1

(x10 cells/cm )

WÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊm—ι

»

1.Q

Cell

Figure 7. Adhesion of mouse fibroblast cells STO onto various surface-grafted polystyrene films.

G l o w - d i s c h a r g e d PST coated with f i b r o n e c t i n (500 Mg/ml)

PST g r a f t e d

PST

(PST)

Glow-discharged

Polystyrene

(wt%)

Monomer concn.

1

74


Figure 8. Photograph of mouse fibroblast cells STO adhered onto (a) polypV(3-trimethylammoniumpropyl)acrylamide chloridej-grafted poly-styrene film and (b) poly[iV-(3-trimethylammonium propyl chloride-co- RGDS)aciylamide]-grafted polystyrene film.


6. ITO ET AL.

Surface Biolization by Grafting

75

References 1. Ito, Y. InSynthesis of Biocomposite Materials: Chemical and Biological Modifications ofNatural Polymers; Imanishi, Y., Ed.; CRC Press: Boca Raton, FL, 1992; pp15-84. 2. Ito, Y.; Imanishi, Y.J.Biomat.Appl. 1992, 6, 293-318. 3. Ito, Y.J.Biomat.Appl.1987, 2, 235-265. 4. Ito, Y.; Imanishi, Y. CRC Crit. Rev. Biocomp. 1989, 5, 45-104. 5. Liu, S. Q.; Ito, Y.; Imanishi, Y.J. Biomed. Mater. Res. in press. 6. Ito, Y.; Liu, L.-S.; Imanishi, Y.J. Biomat. Sci., Polym. Edn. 1991, 2, 123-138. 7. Ito, Y.; Iguchi, Y.; Imanishi, Y. Biomaterials 1992, 13, 131-135. 8. Ito, Y.; Iguchi, Y.; Kashiwagi, T.; Imanishi, Y.J. Biomed. Mater. Res. 1991, 25, 1347-1361. 9. Ito, Y.; Liu, L. -S.; Imanishi, Y.J. Biomed. Mater. Res. 1991, 25, 99-115. 10. Ito, Y.; Liu, L. -S., Matsuo, R.; Imanishi, Y.J. Biomed. Mater. Res. 1992, 26, 1065-1080. 11. Ito, Y. InSynthesis of Biocomposite Materials: Chemical and Biological Modifications ofNatural Polymers; Imanishi, Y., Ed.; CRC Press: Boca Raton, FL, 1992; pp245-284. 12. Imanishi, Y.; Ito, Y.; Liu, L. -S.; Kajihara, M.J. Macromol. Sci. -Chem. 1988, A25, 555-570. 13. Ito, Y.; Kajihara, M.; Imanishi, Y.J. Biomed. Mater. Res. 1991, 25, 13251337. Received May 13, 1993


Surface Biolization by Grafting Polymerizable Bioactive Chemicals

Recommend Documents