Adsorption of Proteins from Artificial Tear Solutions to Poly(methyl

27 Adsorption of Proteins from Artificial Tear Solutions to Poly(methyl methacrylate—2-hydroxyethyl

Downloaded by UNIV OF ROCHESTER on August 31, 2017 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch027

methacrylate) Copolymers FREDERICK H . ROYCE, JR., BUDDY D. RATNER, and THOMAS A. HORBETT University of Washington, Department of Chemical Engineering and Center for Bioengineering, Seattle, WA 98195

Lens hazing and protein deposition are common problems for wearers of soft contact lenses. Previous experiments with hydrophobic-hydrophilic

copolymers

exposed to

plasma

showed protein adsorption to be minimal at intermediate copolymer compositions. Adsorption of proteins from artificial tear solutions to a series of polymers and copolymers ranging in composition from 100% poly(methyl methacrylate) to 100% poly(2-hydroxyethyl

methacrylate)

(PMMA)

(PHEMA)

was

measured. The total protein adsorption due to the three major proteins

in tear fluid (lysozyme,

albumin,

and immuno-

globulins) was at a minimum value at copolymer compositions containing 50% or less PHEMA. proteins from

The elution of the adsorbed

these polymers and copolymers with various

solutions also was investigated to assess the binding mechanism.

S

oft contact lenses, and to a lesser extent hard contact lenses, can accumulate foreign materials on their surfaces and possibly within their polymer

matrices. The deposits are composed primarily of substances present in the

tear fluid (I ). The deposits cloud the lens, cause the wearer discomfort, and may be responsible for a variety of inflammatory conditions including giant papillary conjunctivitis. Manifestations of this syndrome consist of increased mucus, mild itching, decreased lens tolerance, and the development of giant

0065-2393/82/0199-0453$06.00/0 ©1982 American Chemical Society

Cooper et al.; Biomaterials: Interfacial Phenomena and Applications Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

454

BIOMATERIALS: INTERFACIAL PHENOMENA AND APPLICATIONS


papillae i n the u p p e r tarsal conjunctiva (2). O t h e r complications possibly s t e m m i n g f r o m deposits o n soft lenses i n c l u d e corneal vascularization, i n f i l trates, and infections (3). Proteins are the major c o m p o n e n t of lens deposits. O t h e r investigators have observed that on the B a u s c h and L o m b Soflens [cross-linked poly(hyd r o x y e t h y l methacrylate)], p r o t e i n is present at significant levels, and that only a l i m i t e d n u m b e r of the various types of p r o t e i n present i n the tear f l u i d are d e p o s i t e d along w i t h l i p i d s and certain carbohydrates. T h e p r i n c i p l e proteins present i n the tear solution a n d f o u n d i n these deposits are l y sozyme, a l b u m i n , and 7 - G - g l o b u l i n ( i , 7). C a r b o h y d r a t e s may be present i n the f o r m of glycoproteins such as 7 - G - g l o b u l i n s . L i t t l e c a l c i u m is present at the surfaces of contact lenses (4) except w h e n tear c a l c i u m concentration is elevated or there is a tear insufficiency (5). E t h y l e n e d i a m i n e t e t r a a c e t o n i t r i l e ( E D T A ) therefore, does not a i d i n lens deposit removal ( i ) . T h e s e c o n s i d erations suggest that, i n most cases, a material that w o u l d m i n i m i z e p r o t e i n adsorption may i n h i b i t lens c l o u d i n g a n d i m m u n o l o g i c responses. Recent e x p e r i m e n t s indicate that p o l y m e r s that contain a balance of h y d r o p h o b i c (nonpolar) a n d h y d r o p h i l i c (polar) c h e m i c a l groups show m i n i m a l p r o t e i n adsorption a n d c e l l adhesion (6). W i t h the intent of rationally d e s i g n i n g a contact lens material that w o u l d m i n i m i z e p r o t e i n adsorption, the adsorption of l y s o z y m e , a l b u m i n , a n d i m m u n o g l o b u l i n G (IgG) to a series of h y d r o p h o b i c a n d h y d r o p h i l i c p o l y m e r s and c o p o l y m e r s was m e a s u r e d . T h e p o l y m e r s ranged f r o m 100% p o l y ( m e t h y l methacrylate) ( P M M A ) to 100% poly(2-hydroxyethyl methacrylate) ( P H E M A ) . A d s o r p t i o n v a r i e d significantly for each p r o t e i n , as d i d the e l u t a b i l i t y of the proteins f r o m the surfaces.

Materials and Methods Polymer Synthesis. Methyl methacrylate (MMA) was obtained from Polysciences, Inc. and was used after vacuum distillation; 2-hydroxyethyl methacrylate (HEMA) was received in highly purified form from Hydromed Sciences, Inc. Ethylene glycol dimethacrylate (EDMA) was obtained from Polysciences, Inc. and was used as received. Cross-linked polymer slabs 5 cm x 8 cm X 0.2 cm were formed by polymerization in a cell faced with Mylar film and separated by a gasket fashioned from Silastic tubing. Thirteen polymers and copolymers were synthesized ranging from 100% PMMA through 100% PHEMA. Aliquots of 1 mL of E D M A (cross-linking agent) and 0.15 g of azobisisobutyronitrile (AIBN, catalyst) were used per 20 mL of M M A/HEM A monomer, with composition expressed as volume percent of monomer less E D M A and AIBN. The cell assembly with the monomer mixture, cross-linking agent, and catalyst was cured in a constant temperature oven at 45°C. Disks of 1 cm diameter were cut on a specially adapted milling machine at high speed ( ~ 2500 rpm), with the polymer slabs affixed to a clean Plexiglas table with double-faced tape. Prior to each experiment, all polymer specimens, contained in mesh cases to prevent scoring, received the following ultrasonic cleaning regime: 30 min in reagent grade petroleum ether, 15 min in reagent grade methanol, another 30 min in reagent grade petroleum ether, and finally, 15 min in reagent grade methanol. The polymers were


27.

ROYCE ET AL.

Adsorption of Proteins from Artificial Tear Solutions

455

then blotted dry between filter paper and placed under vacuum over desiccant for 1 week. Prior to each experiment, all polymers were equilibrated for at least 1 week in a buffer solution (see below), and then transferred to fresh buffer. This ensured complete polymer hydration and elution of any remaining methanol. Equilibrium Water Content. Samples were equilibrated in deionized water for 1 week, blotted and weighed, placed under vacuum for 2 days to remove the water, and reweighed. Water content was computed using the relationship:


(g water)/(g water + g polymer) X 100 = % water content Measurement of Protein Adsorption. The artificial tear solution consisted of lysozyme (1.20 mg/mL), albumin (3.88 mg/mL), immunoglobulin G (IgG) (1.61 mg/ mL), and buffer. Egg white lysozyme was purchased from Sigma Chemical Company (code L-6876). Bovine albumin, crystallized, (code 81-001-2) and immunoglobulin G ("electrophoretically pure"; code 64-140-1) were purchased from Miles Laboratories, Inc. A less purified immunoglobulin fraction was used in some experiments when it was not the protein to be radiolabeled. It was also purchased from Miles Laboratories (Pentex Bovine Gamma Globulins, Labile enzyme free, Fraction I, code 82-042-3). The buffer used throughout these experiments (CPBSz) was 0.01M citric acid, 0.01M phosphate, 0.12M NaCl, 0.02% sodium azide, pH 7.4. Except for the azide, all salts were reagent grade. The protein composition of this artificial tear solution is similar to human tear fluid (7). Measurement of the adsorption of individual proteins to the polymer samples was determined with I-labeled proteins prepared using the ICI method as de scribed previously (8). The protein to be examined was added in small amounts at high specific activity to the tear solution to give a final specific activity of 100-2000 cpm^g. The amount of protein absorbed in μg/cm was calculated from the radio activity of the films by using the specific activity of the tear solution and the planar surface area of the film. In these calculations, film surface area was determined to an accuracy of ± 2% by averaging the diameters and thicknesses (measured with a cathetometer) of three samples and then calculating average surface area. In the adsorption experiments, two samples from each of the 13 polymers were placed in separate test tubes containing CPBSz buffer. An equal volume of protein stock solution (at twice the concentration stated in the results), with the appropriate radiolabeled protein, was then added. Solutions were maintained at 37°C. After 2 h, the adsorption process was terminated by using a dilution-displacement rinse with ambient temperature CPBSz. In this rinsing technique, buffer is run through the test tube containing the polymer adsorbed with protein at about 800 mL/min for approx imately 0.5 min, using a two-hole rubber stopper fitted with glass tubes for entrance and exit of buffer. Following the initial dilution-displacement rinse, the polymers were placed in fresh CPBSz and their radioactivity counted. On the next day, the polymers were again put in fresh CPBSz and recounted to obtain the "overnight soak rinse" adsorption figures. Radioactive counting was performed with a Searle 1025 7-scintillation counter. Elution Experiments. An experiment was performed to investigate qualitatively the nature of chemical binding at the polymer interface through the use of reagents thought to destabilize particular molecular interactions (9). The same polymers used in the albumin adsorption experiment retained enough protein (over 50%) to permit their reuse in the elution experiments. A 2% solution of SDS was used to break hydrophobic bonds. A 2M urea solution was used since it is thought to disrupt hydrogen bonds as well as interfere with hydrophobic bonds. Finally, 2M NaCl was employed to solvate ionic interactions. One sample from each pair of polymers was 125

2


456


used for the SDS elution, while the other sample was used for the urea elution. After these experiments were complete, the polymers were subjected to the NaCl solution. The experiments consisted of an initial radioactive counting, followed by a 1 day soak in the reagent of interest, and transfer to fresh CPBSz for the final radioactive counting. The percent of eluted protein was then calculated.

Results


T h e water content o f P H E M A was the highest of a l l the p o l y m e r s studied (30%). T h e P H E M A / P M M A c o p o l y m e r s had r e d u c e d water contents d e p e n d i n g o n p o l y m e r c o m p o s i t i o n , as illustrated i n F i g u r e 1. F i g u r e 2 s u m m a r i z e s the data for the three p r o t e i n adsorption e x p e r i ments p e r f o r m e d . E a c h c u r v e represents the amount of p r o t e i n b o u n d to the surface after the o v e r n i g h t soak rinse. T h e top curve indicates the total amount o f p r o t e i n a d s o r b e d , a n d is s i m p l y a s u m m a t i o n of the data for the three proteins s t u d i e d . T h e c u r v e was c o m p i l e d u s i n g the data f r o m three separate a d s o r p t i o n e x p e r i m e n t s , each o f w h i c h e x a m i n e d one labeled p r o tein at a t i m e i n the artificial tear s o l u t i o n .

0%

100%

10

20

30

40

50

60

Polymer Composition

70

80

90

100% HEMA 0% MMA

Figure 1. Effect of polymer composition on equilibrium water content for crosslinked PMMA/PHEMA copolymers.


27.

ROYCE ET AL.


457


0.751

0

10

20

100

30 40 n .

50

60

70

80

90

^

Polymer Composition

100 % HEMA 0% M MA

Figure 2. Adsorption of lysozyme ( , • ), albumin ( - • - · , A), IgG ( - • - ·, Ο λ and total protein ( -, O) to PMMA/PHEMA copolymers from artificial tear solutions. F i g u r e 3 presents a useful means of v i e w i n g the adsorption of the three proteins over the range of c o p o l y m e r s , n a m e l y , h o w surface e n r i c h m e n t (SE) varies w i t h c o p o l y m e r c o m p o s i t i o n . Surface e n r i c h m e n t is d e f i n e d as: I μg specific p r o t e i n adsorbed\ SE =

μg total p r o t e i n adsorbed / / μg specific p r o t e i n i n b u l k phase\ ν μ g total p r o t e i n i n b u l k phase /


458


ho

a> «Λ a. ο

aο> ο

3

•

Ο

C α> 2


Ο Ο Ο

II Lu -1.0

0 100

10

20

30

40

50

60

70

Polymer Composition

80

90

100 % ΗΕΜΑ 0 % ΜΜΑ

Figure 3. Surface enrichment for lysozyme ( , • ), albumin and IgG ( artificial tear solutions.

(-•-·,

A ),

A surface e n r i c h m e n t greater than 1.0 means that the specific p r o t e i n prefers the surface to t h e b u l k phase a n d w i l l b e m o r e readily adsorbed. A l t h o u g h a l b u m i n shows l o w surface e n r i c h m e n t , it is present i n solutions at signifi cantly h i g h e r c o n c e n t r a t i o n than l y s o z y m e a n d the g l o b u l i n s . To c o n s i d e r q u a l i t a t i v e l y c h e m i c a l b o n d i n g o f p r o t e i n to the p o l y m e r surfaces a n d the a b i l i t y of various solutions to elute this p r o t e i n layer, three c h e m i c a l l y distinct reagents w e r e chosen. T h e three solutions used w e r e S D S , a surfactant k n o w n to d i s r u p t h y d r o p h o b i c interactions; urea, a polar c o m p o u n d that d i s r u p t s h y d r o g e n bonds a n d h y d r o p h o b i c interactions; and N a C l , an i o n i c c o m p o u n d that s h o u l d d i s r u p t i o n i c bonds. T h e results of the S D S and urea e l u t i o n o f a l b u m i n f r o m the p o l y m e r surface, expressed as p e r c e n t a l b u m i n e l u t e d , are shown i n F i g u r e 4. W i t h i n the l i m i t s of e r r o r o f measurement, none of the r e m a i n i n g p r o t e i n was elutable f r o m the p o l y m e r surface w i t h N a C l . T h e r e m o v a l of over 5 0 % of the a l b u m i n f r o m P M M A b y S D S , c o m p a r e d to less than 1 5 % r e m o v a l f r o m P H E M A b y this reagent, indicates a substantial difference i n the m e c h a n i s m b y w h i c h a l b u m i n ad sorbs to these two p o l y m e r s (that is, h y d r o p h o b i c b o n d i n g may b e m o r e important i n a l b u m i n a d s o r p t i o n to P M M A than to P H E M A ) .

Discussion T h e p o l y m e r s a n d c o p o l y m e r s synthesized for these experiments had e q u i l i b r i u m water contents that v a r i e d w i t h c o m p o s i t i o n . T h e data i n F i g u r e 1 are i n close a g r e e m e n t w i t h p u b l i s h e d values (10). Since the 100% P H E M A s p e c i m e n for this e x p e r i m e n t is of the same material as the Bausch a n d L o m b


27.

ROYCE ET AL.


459

Soflens, t h e literature o n this m a t e r i a l m i g h t b e used as a basis for c o m parison o f e x p e r i m e n t a l results. F i g u r e 2 shows that s i m i l a r adsorption p h e n o m e n a are e x h i b i t e d b y l y s o z y m e a n d a l b u m i n . R e l a t e d w o r k seems to suggest that p o l y m e r s that contain a certain balance o f h y d r o p h o b i c a n d h y d r o p h i l i c c h e m i c a l groups show m i n i m i z e d b i o l o g i c a l i n t e r a c t i o n (for example, l o w p r o t e i n adsorption, low t h r o m b u s d e p o s i t i o n , a n d l o w platelet consumption) (6). T h e adsorption of r a d i o l a b e l e d I g G , h o w e v e r , was m a x i m a l at i n t e r m e d i a t e c o p o l y m e r s . T h i s result has a n u m b e r o f i m p l i c a t i o n s w i t h respect to both the fundamental Downloaded by UNIV OF ROCHESTER on August 31, 2017 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch027

adsorption m e c h a n i s m a n d t h e b i o c o m p a t i b i l i t y o f these materials. O n e m i g h t speculate o n a n u m b e r o f c h e m i c a l a n d p h y s i c a l factors that govern p r o t e i n a d s o r p t i o n behavior. Previous experiments p o i n t to the i m portance of h y d r o p h i l i c i t y as an i n f l u e n t i a l factor (6), but this is certainly not the sole factor. I n this p o l y m e r system, h y d r o p h i l i c i t y (as g o v e r n e d b y t h e surface concentration o f H E M A ) increases l i n e a r l y w i t h t h e b u l k

HEMA

c o m p o s i t i o n (11). T h e surface o f a p r o t e i n w h i c h is also soluble i n aqueous m e d i a is p r o b a b l y of a polar nature. If p r o t e i n adsorption c o u l d b e character i z e d b y polar interactions, t h e n an adsorption t r e n d that w o u l d parallel t h e

50

40H

§

3

30-

c Ε 20H

Adsorption of Proteins from Artificial Tear Solutions to Poly(methyl

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