Chapter 16
Adsorption to Biomaterials from Protein Mixtures Thomas A. Horbett
Downloaded by NORTH CAROLINA STATE UNIV on May 3, 2015 | http://pubs.acs.org Publication Date: July 13, 1987 | doi: 10.1021/bk-1987-0343.ch016
Department of Chemical Engineering, BF-10, and Center for Bioengineering, University of Washington, Seattle, WA 98195
Protein adsorption from relatively complex mixtures is involved in many different applications of biomaterials. A summary of a l l studies involving protein adsorption that were done in the author's laboratory is given, largely in the form of an annotated table. Certain aspects of the methods, results, and conclusions of studies that pertain especially to adsorption from mixtures are also discussed. P r o t e i n adsorption t o implanted b i o m a t e r i a l s i s b e l i e v e d t o p l a y an i m p o r t a n t r o l e i n d e t e r m i n i n g t h e i r b i o c o m p a t i b i l i t y w i t h v a r i o u s b i o l o g i c a l systems and t i s s u e s . In t h e c a r d i o v a s c u l a r s y s t e m t h i s i s p a r t i c u l a r l y c l e a r because t h e i n t r i n s i c c l o t t i n g system c o n s i s t s o f a s e r i e s o f p r o t e i n s which a c t a t i n t e r f a c e s t o i n d u c e f i b r i n c l o t s . In a d d i t i o n , p l a t e l e t thrombus f o r m a t i o n i s s t r o n g l y i n f l u e n c e d by t h e a d s o r p t i o n o f p r o t e i n s t o i n t e r f a c e s . E x t r a v a s c u l a r implants i n s o f t t i s s u e induce t h e c h a r a c t e r i s t i c f o r e i g n body g i a n t c e l l and f i b r o u s c a p s u l e f o r m a t i o n by c e l l u l a r mechansisms. These c e l l u l a r r e s p o n s e s a r e a l s o s t r o n g l y a f f e c t e d by p r o t e i n s a t t h e i n t e r f a c e , a l t h o u g h t h i s i s l e s s w e l l documented. I m p l a n t s i n h a r d t i s s u e s s u c h as bone a r e a l s o v e r y l i k e l y t o be i n f l u e n c e d by a d s o r p t i o n o f p r o t e i n s t o t h e i n t e r f a c e . In t h i s type of implant, adsorbed p r o t e i n s a c t through d i r e c t e f f e c t s on t h e a d h e s i o n and growth o f o s t e o b l a s t s , and a l s o t h r o u g h i n d i r e c t mechanisms s u c h as t h e s u r p r i s i n g a b i l i t y of proteins t o a f f e c t the corrosion of m e t a l l i c implants. In a l l t h e s e a p p l i c a t i o n s o f b i o m a t e r i a l s , adsorption o c c u r s from r e l a t i v e l y complex s o l u t i o n s c o n t a i n i n g many d i f f e r e n t p r o t e i n s . F o r t h i s r e a s o n , much o f t h e e f f o r t i n my l a b o r a t o r i e s has f o c u s e d on t h e b e h a v i o r o f p r o t e i n a d s o r p t i o n a s i t o c c u r s from p r o t e i n m i x t u r e s . I n t h i s p a p e r , a summary o f t h e s e s t u d i e s i s p r o v i d e d w i t h t h e 0097-6156/87/0343-0239$06.50/0 © 1987 American Chemical Society
In Proteins at Interfaces; Brash, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
240
PROTEINS AT INTERFACES
p u r p o s e o f i l l u s t r a t i n g some o f t h e mechanisms by w h i c h complex a d s o r p t i o n p r o c e s s e s a p p e a r t o o c c u r . The r e f e r e n c e l i s t i n c l u d e s a l l p u b l i c a t i o n s f r o m my l a b o r a t o r y t h a t have i n v o l v e d o r been c l o s e l y r e l a t e d t o p r o t e i n a d s o r p t i o n s t u d i e s ( 1 - 2 9 ) . These p u b l i c a t i o n s and t h e i r p r i n c i p a l r e s u l t s a r e summarized and l i s t e d i n Table 1 i n t h e i r order of appearance i n the l i t e r a t u r e , which a p p r o x i m a t e s t h e sequence i n w h i c h t h e y were a c t u a l l y done. P o r t i o n s o f t h i s work have a l r e a d y been r e v i e w e d i n more d e t a i l e l s e w h e r e (26 27) . In what f o l l o w s , c e r t a i n a s p e c t s o f t h e methods, r e s u l t s , and c o n c l u s i o n s of these s t u d i e s are d i s c u s s e d .
Downloaded by NORTH CAROLINA STATE UNIV on May 3, 2015 | http://pubs.acs.org Publication Date: July 13, 1987 | doi: 10.1021/bk-1987-0343.ch016
P
Methods The most f r e q u e n t l y u s e d method i n s t u d i e s o f p r o t e i n a d s o r p t i o n i n t h i s l a b i s t h e 125j p r o t e i n t e c h n i q u e i n w h i c h t h e p r e l a b e l l e d p r o t e i n o f i n t e r e s t i s added t o a m i x t u r e o f o t h e r p r o t e i n s . An in situ radioiodination method was a l s o d e v e l o p e d and e x t e n s i v e l y u s e d i n s e v e r a l attempts at c h a r a c t e r i z i n g the e n t i r e " s p e c t r a " of p r o t e i n s a d s o r b e d t o p o l y m e r s from m i x t u r e s . T e c h n i c a l a s p e c t s o f b o t h methods have been d e s c r i b e d i n d e t a i l (2Â)
.
The use o f p r e l a b e l l e d p r o t e i n s i n a d s o r p t i o n s t u d i e s a p p e a r s t o be a v i r t u a l n e c e s s i t y when c o m p l i c a t e d protein m i x t u r e s s u c h as b l o o d p l a s m a a r e employed. None o f t h e p h y s i c a l methods f o r m e a s u r i n g p r o t e i n a d s o r p t i o n have t h e r e q u i r e d degree of s p e c i f i c i t y , although i t i s p o s s i b l e t h a t F o u r i e r t r a n s f o r m i n f r a r e d s p e c t r o s c o p y may someday p r o v e t o be a b l e t o d i s c r i m i n a t e p r o t e i n s i n some m i x t u r e s . Among b i o c h e m i c a l methods, t h e measurement o f a n t i b o d y u p t a k e by t h e a d s o r b e d p r o t e i n might a t f i r s t a p p e a r t o be b e t t e r t h a n t h e p r e l a b e l l e d p r o t e i n method i n t h a t l a b e l l i n g a r t i f a c t s cannot i n f l u e n c e t h e a d s o r p t i o n p r o c e s s . However, i t i s l i k e l y t h a t n o n - s p e c i f i c a d s o r p t i o n o f a n t i b o d y by t h e s u r f a c e , and changes i n t h e r e a c t i v i t y of the a n t i g e n i c "epitope" i n the adsorbed p r o t e i n due t o o r i e n t a t i o n a l o r c o n f o r m a t i o n a l e f f e c t s , w i l l b o t h i n f l u e n c e a n t i b o d y u p t a k e . These p r o b l e m s l i m i t t h e u s e f u l n e s s o f t h e a n t i b o d y method i n o b t a i n i n g q u a n t i t a t i v e measurements. F u r t h e r m o r e , t h e a n t i b o d y methods a r e i n t r i n s i c a l l y more d i f f i c u l t t h a n p r e l a b e l l i n g methods due t o t h e a d d i t i o n a l r e q u i r e m e n t s t o p r o d u c e and p u r i f y a s p e c i f i c a n t i b o d y and e s t a b l i s h a c a l i b r a t i o n c u r v e w i t h known amounts o f a d s o r b e d a n t i g e n . The p r i n c i p a l d i s a d v a n t a g e o f t h e p r e l a b e l l e d p r o t e i n method i s t h e f a c t t h a t l a b e l l e d p r o t e i n s may not a d s o r b t h e same as t h e u n l a b e l l e d p r o t e i n , t h u s l e a d i n g t o p o t e n t i a l l y l a r g e e r r o r s . In o u r s t u d i e s , we have r e l i e d e x c l u s i v e l y on 125j p r o t e i n s , a l m o s t a l w a y s made w i t h a c h e m i c a l l y v e r y m i l d ICI method. P r e f e r e n t i a l a d s o r p t i o n o f 125j p r o t e i n s made i n t h i s way does not seem t o o c c u r , a t l e a s t as j u d g e d by t h e a b s e n c e o f any e f f e c t o f changes i n t h e r a t i o o f l a b e l l e d t o u n l a b e l l e d p r o t e i n on t h e
In Proteins at Interfaces; Brash, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
In Proteins at Interfaces; Brash, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
low
the
hydrogeIS
of
adsorption
At
2
(Ref. #
Title: __
were
cell
plating
retained
coated (79%
or
efficiency
surfaces serum
containing
γ-globulin
was
proteins.
and,
low.
At
the
MA Ac
Results
studied,
of
concentrations
amounts
proteins
physiologic
among
small
HEMA
&
of
salt,
adsorption
in
adsorption
increased
caused
conclusions gels
in
of
the
order
increases all
the
great
ι
the
in
I
proteins
of
1 Buffered satine;
1 Blood ptasma 1 methacryCic acid, (ÎbiMAc) radiation
and
1 grafted onto sdicone ruBBer
Ή: vinylpyrroCidone
I
strength,
to
Tlution
or
after
but
Treadsorption
12Sf
surfaces
\
|
pH Continued on next page
onto
the
MAAc-HEMA
γ-globulin
ι
9£nttydrin Assay US
isoelectric
1 water; physiologic | 1 hydroTcyethylrnethacrylate (ΉΈ34.Ά),
I
ionic
1
medium
coated
I
1
1
with
Respect
onto Radiation-Grafted Hydrogels and N- Vinyl -pyrrol i don e 1 Adsorption fromi Techniques Surfaces
contact
and adherent.
\
1
(Proteins studied
fiBrinogen, f-gloBulin, and alBumin
1
after
albumin
conclusions
cell
on
Adsorption Methacrylate
more)
low
strongly
was
&
ruBBer; sUicone ruBBer
\
1 -pyrroCidone) fiydrogels on sdicone 1
1 1
1
\
1 metfuurylate) and poly (9^-mny1-2
Results
were
with
1 Adsorption from 1 Techniques
: Implications
Surfaces
Materials
1 %fldiation grafted poly(2 -hydroxyethyl \'Buffer
1
Polymeric
Bovine Plasma Protein Based on Hydroxyethyl
proteins attachment.
gelatin
muscle
Adsorbed cell
embryo
and
Chick
1
(Proteins studied
Adhesion to compatibility
AlBumin, y~g(oBulin and fiBrinogen
Cell Bio
fibrinogen
ι
