PROTEIN AND LIPID ADSORPTION BY ACRYLIC

HYDROGELS AND THEIR RELATION TO WATER

WETTABILITY*

FRANK J. HOLLY*
Eye Research Institute of Retina Foundation, 20 Staniford Street,
Boston, Massachusetts 02114, Department of Ophthalmology
(Physiology), Harvard Medical School, Boston, Massachusetts
02115, and Holles Laboratories, Inc., 30 Forest Notch, Cohasset,
Massachusetts 02025

SYNOPSIS
Radiochemical techniques were applied to detennine protein and lipid uptake by acrylic
hydrogels. The water wettability of these gels was previously obtained by using contact angle
goniometry and was found to depend mainly on the composition of the polymer matrix but not
on the equilibrium water content. Three hydrogels, poly(hydroxyethy1 acrylate), poly(gly-
ceryl methacrylate), and poly(acrylamide), had approximately the same equilibrium water con-
tent, therefore the same approximate penetrability with respect to protein. The uptake of both
the protein (bovine serum albumin) and the lipid (cholesteryl oleate) appeared to be directly re-
lated to the water-gel interfacial tension, whereas the ease of removal was inversely related to
water wettability. A cationic preservative, benzalkonium chloride, increased the protein uptake
somewhat for two of the hydrogels. Preexposure to proteinaceous solutions had a pronounced
effect, increasing the lipid uptake by the hydrogels, with the exception of PGMA. The results
obtained appear to support the minimum interfacial free-energy hypothesis of biocompatibility.

INTRODUCTION
The interaction of hydrogels with proteins and lipids is of interest be-
cause it is related to biocompatibility. Hydrogels are used more and more
frequently as coatings for biomaterials to improve their biocompatibility.
The use of contact lenses made of hydrogels is also becoming more and
more popular. Incompatibility of contact lenses with the lacrymal system is
usually manifested by deposit formation over the lenses that interferes with
vision and can be a source of occular irritation. While some of these depos-
its have been shown to contain calcium, the majority of contact lens depos-
its consist of lipids and denatured proteins. Occasionally even hydrogel
lenses acquire such deposits while being worn in the eye. With a more hy-
drophobic substance, the problem can be quite severe.
Even the adsorption of minute amounts of protein at the contact lens-
tear interface can cause problems if the protein denatures to the extent of
*Presented at the Symposium on Medical Polymers: Chemical Problems, August 15-18,
1977, Institute of Macromolecular Sciences, Prague, Czechoslovakia.
tPresent address: Texas Tech University School of Medicine, Department of Ophthalmolo-
gy and Visual Sciences, Thompson Hall, Lubbock, TX. 79430.

Journal of Polymer Science: Polymer Symposium 66, 409 - 417 (1979)

@ 1979 John Wiley & Sons, Inc. 036G8905/79/0066-0409S01.00
410 HOLLY

...........
WATER V A P O R ..............
..................

( OR OCTANE
W AT E R--
c H3 9n ‘F,
..............................
......
CH3 cH~~KOK-----OH-~H-O-~---

.............................
I I. I I C,H3 I I I C.H3

. . . . . D.R. . . ............
..............................
.H y O&EL
.....
FIG. 1. Schematic diagram of hydrogel interfaces (from ref. 3).

becoming antigenic. An allergic reaction resembling vernal conjunctivitis

can occasionally be observed in patients that appears to be caused by con-
tact lens wear [ 11.
It is generally recognized that the adsorption and possible denaturation of
biopolymers and lipids at a solid-air interface depend on the magnitude of
the interfacial tension as well as on other factors. The minimum interfacial
tension hypothesis of biocompatibility [2] presumes that the lower the water-
biomaterial interfacial tension, the less adsorption and protein denaturation
occur and thus the better the biocompatibility of the material will be.
When a biomaterial is alternately exposed to air and aqueous body fluids
as contact lenses are, it becomes more difficult to achieve a minimal interfa-
cial tension at all times. This is so because solids with a low surface-free en-
ergy in air usually have high interfacial tension against water. On the other
hand, materials that have a low interfacial tension when immersed in water
are usually substances with high surface-free energy.
Hydrogels seem to be an exception. They appear to be capable of chang-
ing surface composition by orientational and conformational changes in the
surface polymer chains. Thus hydrogels can have low surface-free energy
when exposed to air, water vapor, or nonpolar liquids by orienting the
most hydrophobic groups (e.g., methyl groups) outward, and they manage
to have a quite low interfacial tension when immersed in water by orienting
the hydrophilic groups on the side chains (e.g., hydroxyl groups) toward
the water phase (Fig. 1). Such a behavior manifests itself in a large contact
angle hysteresis [3].
Due to this phenomenon, the advancing contact angle of water is rather
large on many hydrogels. This contact angle on poly-(Zhydroxyethyl meth-
acrylate) hydrogel (PHEMA) can be as high as that on poly(methy1 meth-
acrylate) (PMMA). Hence the advancing contact angle of water does not
appear to be a good indicator of surface hydrophilicity because it cannot
distinguish between two such materials of widely differing equilibrium
water content (40% vs. 1.5%).
 PROTEIN AND LIPID ADSORPTION 41 1

TABLE I

Composition of the Acrylic Hydrogels Studied

t qn H
I
c
I
€I
I
R1

-YC\
0

GEL ABBREVIATION
R1 R2

PHEMA -CH3 -OCH3

PHEA -H -O-CH2-CH2-OR

PGMA -CH -O-CH2-CH-CH2-OH

3 1
OH
PAA -H -NHZ

We have found that the advancing contact angle of water measured when
the material is immersed in n-octane, e,(w/o), is a much better indicator
of hydrophilicity than the water contact angle measured in air [4]. If this
two-phase contact angle, when measured through the aqueous phase, is less
than 90°, then the surface is said to be hydrophilic. When this angle is
greater than 90°, the surface is hydrophobic.
The water-in-octane advancing contact angle is quite high on PHEMA,
88", and usually less for other types of hydrogels. This two-phase contact
angle is about 120" for PMMA and is higher for more hydrophobic solids
such as polyethylene. Thus by the two-phase contact angle criterion of sur-
face hydrophilicity, the hydrogels have a hydrophilic surface, whereas the
surface of solid polymers is hydrophobic.
Table I exhibits the various types of hydrogels, namely, PHEMA, poly(2-
hydroxyethyl acrylate) (PHEA), poly(glycerylmethacry1ate) (PGMA), and
poly(acry1amide) (PAA), that have been investigated. For details of hydro-
gel preparation, consult refs. 3 and 5.
412 HOLLY

advancing 0
0 %

20 30 40 50 60 7b 8-0 90 100
equilibrium water Content i n Percent
FIG. 2. Water wettability of PHEA hydrogels having different values of equilibrium
hydration.

We have previously investigated the effect of equilibrium water content

and the composition of the polymer matrix on surface hydrophilicity [3]. Fig-
ure 2 demonstrates the effect of varying water content on water wettability

0 advancing
60. 1 PHEMA 0 receding

v)
a,

0
50.
a,
D
PHEA
C
'- 40.
-cn
C

-
m
30.
-
m
C

2 20.
L
a,
c

2 10. PAAW

40 50 so 70 a0 90 100
equilibrium water content in percent

FIG. 3. Water wettability of acrylic hydrogels having different matrix composition.

 PROTEIN AND LIPID ADSORPfION 413

TABLE I1

Water Wettability and Equilibrium Water Content of Hydrogels

ADVANCING CONTACT ANGLE RELATIVE CONTACT

HYDROGEL WATER CONTENT *
IN AIQ IN OCTANE ANGLE HYSTERESIS

PHEMA 42% 6 Oo 88' 0.72

PHEA 84% 440 7 go 0.59

PGMA 87% 4 lo 63' 0.76

PAA 8 5% 1 oo 12O 0.60

- .-
*Defined as the ratio of the difference between the advancing and receding angle and the
advancing contact angle.

for a given type of hydrogel, PHEA. Contrary to expectation, the water

wettability does not increase with increasing water content. Similar results
have been obtained with other acrylic hydrogels [3]. Figure 3 contains
water contact angle data obtained on the various types of hydrogels used in
this study. With the exception of PHEMA, the hydrogels had about the
same equilibrium water content (85.3 Az 1.5%), but different hydrophobic
and hydrophilic groups on the polymer backbone. A pronounced depen-
dence of water wettability on the polymer matrix composition can be ob-
served. Table I1 contains data with regard to water wettability, contact
angle hysteresis, and equilibrium water content of these hydrogels.

EXPERIMENTAL
Protein Absorption and Removal
The protein absorption by acrylic hydrogels was investigated by using '*'I-
labeled bovine serum albumin (BSA) with a molecular weight of 68,000 dal-
tons. Disk-shaped gel samples with 1-cmdiameter were exposed to 0.1%BSA
dissolved in 0.9% saline for 15 hr. The effect of a cationic surfactant,
benzalkonium chloride (BAC), on protein absorption was also investigated,
since this preservative is often used in contact lens solutions as a
bacteriocidal agent. In this experiment, the hydrogel disks were exposed to
0.01%BAC solution for 1 hr before the immersion into the aqueous BSA so-
lution. At the end of the exposure, the gel samples were gently rinsed and
blotted with filter paper, then solubilized in a corrosive lye solution
(Protosol) before being put in scintillation vials containing 5-ml Biofluor
scintillation fluid. The vials were counted in a Searle Mark I11 automatic
scintillation counter.
414 HOLLY

TABLE 111

Protein Absorption by Hydrogels and Its Tenacity

--
-.

AMOUNT OF B S A ABSORBED (pg/cmL) P E R CENT BSA REMOVED

IIYDROGEL
h’0 CLEANING A F T E R CLEANING BY C L E A N I N G

-_----_ by clean hydrogels in 0.1% B S A solution for 1 5 hrs -------

PHEMA 15.!1 1.64 52

PHEA 41.7 29.9 28

PGMA 18.6 8.88 52

PAA 16.1 7.28 55

----- -- by hydrogels exposed to 0 . 0 1 % BAC f o r 1 hour --------------

PHEMA 15.5 6.45 58

PHEA 51.9 31.0 40

PGMA 27.7 7.08 14

PAA 15.8 6.08 62

The ease of removal of absorbed protein was also tested. Separate

hydrogel disks with absorbed BSA were immersed in a cleaning solution
containing a mixture of anionic and nonionic surfactants and placed in an
ultrasonic cleaner for 15 min. After cleaning, the samples were rinsed and
counted in a solubilized form.

Lipid Adsorption and Removal

The lipid adsorption by acrylic hydrogels was investigated by using I4C-
labeled cholesteryl oleate (CHO), a nonpolar lipid typical of the major frac-
tion of meibomian lipids forming the superficial lipid layer of the tear
film. CHO was dissolved in n-hexane at 1% concentration. The hydrogel
samples were exposed to this nonaqueous solution for 15 hr after blotting.
At the end of the exposure time, the samples were rinsed in acetone and
gently blotted with filter paper. The effect of cleaning was also investigated
using separate samples by using the same detergent solution and ultrasonic
cleaner that were used to remove protein. After rinsing with acetone, the
hydrogel samples were cleaned for 15 mint then prepared for counting.
The effect of preabsorbed protein on lipid adsorption was investigated by
exposing gel samples to 1% BSA solution for 1 hr prior to lipid adsorption.
Again the ease of removal of the adsorbed lipid by cleaning was also mea-
sured the same way described above.
 PROTEIN AND LIPID ADSORPTION 41 5

TABLE IV

Lipid Adsorption by Hydrogels and Its Tenacity

AMOUNT OF CHO ADSORBED (pg/cm*) PER CENT CHO REMOVED

HVDROGEL
NO CLEANING AFTER CLEANING BY CLEANING

---------- by clean hydrogels in 1% CHO solution for 1 5 hrs ---------

PHEMA 6.91 2.87 58

PHEA 6.57 1.00 85

PGNA 7.36 1.75 76

PAA 1.06 0.113 89

------------ by hydrogels exposed to 1% BSA for 1 hour ----------------

PHEMA 14.5 0.324 98

PHEA 9.13 0.336 96

PGMA 1.36 0.148 89

PAA 5.76 0.178 97

RESULTS

Protein Adsorption and Removal

It is important to realize that while the water content and therefore the
approximate pore size and permeability of hydrogels PAA, PGMA, and
PHEA are similar, the water content of PHEMA gel is about half as much
as that of the other gels and thus its permeability is considerably lower for
macromolecules.
Table I11 contains the results concerning protein uptake by hydrogels
and the ease of removal of the absorbed protein. If we ignore the results
obtained with PHEMA for the reason stated above, then the extent of
protein adsorption is the highest for PHEA and lowest for PAA with
PGMA in between the two. The removal of the absorbed protein accord-
ing to the amount of protein removed appears to be the greatest for
PAA and the least for PHEA.
The results concerned with the effect of preabsorbed BAC on protein
absorption can be seen in the same table. The protein uptake increas-
ed for PGMA (49%) and for PHEA (24%), whereas the protein uptake by
PHEMA and PAA remained about the same. The amount of protein re-
tained after cleaning does not seem to be affected for any' of the
hydrogels.
416 HOLLY

Lipid Adsorption and Removal

The results of the lipid adsorption experiments can be seen in Table IV.
The highest uptake was observed for PGMA, while PAA adsorbed the least
lipid followed by PHEA. The ease of removal as measured by the fraction
of lipid removed by cleaning was the highest for PAA followed by PHEA,
and the lowest for PHEMA. Hence for both protein and lipid, the extent of
adsorption and ease of removal are inversely related if the results obtained
with PHEMA are excepted.
The same table contains the lipid adsorption data for gels that have been
preexposed to 1% BSA solution. The largest increase in lipid uptake was
observed for PAA (443%) and for PHEMA (110%). The lipid uptake by
PHEA only increased by about 40%, while the lipid uptake decreased for
PGMA by more than 80%. In this particular experiment, only PHEA and
PAA acted predictably, i.e., PHEA adsorbed more lipid than PAA and it
was also harder to remove from PHEA than from PAA.

DISCUSSION
Three of the hydrogels studied, PAA, PGMA, and PHEA, had about the
same water content. The surface of the PAA gel was the most hydrophilic
and that of PHEA was the least hydrophilic as determined by the advanc-
ing contact angle of water measured in air. The same order of hydrophilici-
ty is obtained when the water-in-octane contact angle is used as a criterion.
This implies that the water-gel interfacial tension is the lowest for PAA, it
is higher for PGMA, and the highest for PHEA. PHEMA surface is even
more hydrophobic and this gel contained only about half as much water as
the others at equilibrium hydration.
It appears that the albumin uptake was determined by both the degree of
surface hydrophilicity and the penetrability of the gels. The surface effect is
especially pronounced for the three gels with approximately the same water
content. The tendency for decreasing absorption with decreasing water-gel
interfacial tension is clearly seen from the data. On the other hand, the ease
of removal increases with decreasing water-gel interfacial tension, as shown
by the amount of protein that remains absorbed after cleaning, perhaps in-
dicating less denaturation at the interfaces.
The lipid uptake was similar for all gels except for PAA, where it was
much less and also easier to remove. In the presence of protein this advan-
tage seems to diminish for PAA because the lipid uptake becomes much
higher. However, this increased lipid uptake does not adhere as tenaciously
as lipid adsorbed in the absence of protein.
The results of this study appear to support the minimum interfacial ten-
sion hypothesis of biocompatibility and have direct relevance to blood com-
patibility. Direct application of these results to lipoproteinaceous deposit
formation on contact lenses in situ is not possible due to the periodic
 PROTEIN AND LIPID ADSORPTION 417

alternation between lens-tear and lens-air interfaces as a result of blinking.

For such a complicated biosystem, the uptake of lipid and protein by
hydrogels should be studied in dynamic systems that simulate the structure
of the tear film and the blinking conditions. Such experiments are in
progress and will be reported in the near future.
This study was supported by PHS Grant No. EY-00208 from the National Eye Institute,
National Institutes of Health. The hydrogels were obtained from Dr. Miguel F. Refojo.

REFERENCES
[I] M. F. Refojo and F. J. Holly, Cont. Inrraoc. Lens Med. J., 3 , 23 (1977).
[2] J. D. Andrade, Med. Instrum., 7, 110 (1973).
[3] F. J. Holly and M. F. Refojo, in Hydrogels for Medical and Related Applications, J. D.
Andrade, Ed., ACS Symposium Series 31, Washington, D.C., 1976, p. 252.
[4] F. J. Holly and M. F. Refojo, in Colloid and Interface Science, M. Kerker, Ed., Vol. III.,
Academic. New York, 1976, p. 321.
[5] F. J. Holly and M. F. Refojo, J. Biomed. Mazer. Res, 9 , 315 (1975).