Bioinspired Imprinted PHEMA-Hydrogels for ocular delivery of carbonic anhydrase inhibitor drugs

Article abstract of DOI:10.1021/bm101562v

Hydrogels with high affinity for carbonic anhydrase (CA) inhibitor drugs have been designed trying to mimic the active site of the physiological metallo-enzyme receptor. Using hydroxyethyl methacrylate (HEMA) as the backbone component, zinc methacrylate, 1- or 4-vinylimidazole (1VI or 4VI), and N-hydroxyethyl acrylamide (HEAA) were combined at different ratios to reproduce in the hydrogels the cone-shaped cavity of the CA, which contains a Zn ²⁺ ion coordinated to three histidine residues. 4VI resembles histidine functionality better than 1VI, and, consequently, pHEMA-ZnMA ₂ hydrogels bearing 4VImoietieswere those with the greatest ability to host acetazolamide or ethoxzolamide (2 to 3 times greater network/water partition coefficient) and to sustain the release of these antiglaucoma drugs (50%lower release rate estimated by fitting to the square root kinetics). The use of acetazolamide as template during polymerization did not enhance the affinity of the network for the drugs. In addition to the remarkable improvement in the performance as controlled release systems, the biomimetic hydrogels were highly cytocompatible and possessed adequate oxygen permeability to be used as medicated soft contact lenses or inserts. The results obtained highlight the benefits of mimicking the structure of the physiological receptors for the design of advanced drug delivery systems.

Full text of DOI:10.1021/bm101562v

ARTICLE
pubs.acs.org/Biomac
Bioinspired Imprinted PHEMA-Hydrogels for Ocular Delivery of
Carbonic Anhydrase Inhibitor Drugs
†
,‡,§
†,‡
‡,||
§
§
Andreza Ribeiro,
Francisco Veiga, Delfim Santos, Juan J. Torres-Labandeira, Angel Concheiro,
,§
and Carmen Alvarez-Lorenzo*
†
‡
Department of Pharmaceutical Technology and Centre for Pharmaceutical Studies, Faculty of Pharmacy, University of Coimbra, 3000-
48-Coimbra, Portugal
5
§
Departamento de Farmacia y Tecnología Farmac ꢀe utica, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782-Santiago
de Compostela, Spain
Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, 4050-047-Porto, Portugal
ABSTRACT: Hydrogels with high affinity for carbonic anhydrase
(CA) inhibitor drugs have been designed trying to mimic the active
site of the physiological metallo-enzyme receptor. Using hydro-
xyethyl methacrylate (HEMA) as the backbone component, zinc
methacrylate, 1- or 4-vinylimidazole (1VI or 4VI), and N-hydro-
xyethyl acrylamide (HEAA) were combined at different ratios to
reproduce in the hydrogels the cone-shaped cavity of the CA, which
2þ
contains a Zn ion coordinated to three histidine residues. 4VI
resembles histidine functionality better than 1VI, and, consequently,
pHEMA-ZnMA hydrogels bearing 4VI moieties were those with the
2
greatest ability to host acetazolamide or ethoxzolamide (2 to 3 times greater network/water partition coefficient) and to sustain the
release of these antiglaucomadrugs(50% lower release rateestimated by fittingto the square root kinetics). The use of acetazolamide as
template duringpolymerization did not enhance the affinity of the network for the drugs. Inaddition to the remarkable improvement in
the performance as controlled release systems, the biomimetic hydrogels were highly cytocompatible and possessed adequate oxygen
permeability to be used as medicated soft contact lenses or inserts. The results obtained highlight the benefits of mimicking the
structure of the physiological receptors for the design of advanced drug delivery systems.
’
INTRODUCTION
either, because <5% of the instilled dose is absorbed intraocularly.
The rest pass through the conjuntiva or are removed from the eye
surface by the defense mechanism that partially leads to naso-
Glaucoma is a progressive disease that causes optic nerve head
damage. Currently, its prevalence is a high as 1% in people aged
5
lacrimal drainage; both routes may result in systemic absorption.
4
0-49 years and up to 8% above 80 years old and is one of the
1
,2
An ideal ocular drug delivery system should be able to increase
ocular bioavailability, toprolongthe duration ofdrug action, and to
avoid large fluctuations in ocular drug concentration and ocular
most common causes of blindness. The elevation of the
intraocular pressure (IOP) is the main risk factor for glaucoma
because of compression of the optic nerve fibers against the
lamina cribrosa or ischemia associated to the disturbance of the
blood supply to the nerve. In open-angle glaucoma, there is
impaired flow of aqueous humor through the trabecular mesh-
6
and systemic side effects. In this context, soft contact lenses
(
SCLs) are gaining an increasing attention as combination pro-
ducts able to correct refractive deficiencies and to perform as
7-11
3
sustained delivery systems.
work-Schlemm’s canal venous system. The first choice of
The feasibility of using drug-loaded SCLs depends on whether
the drug and the hydrogel material can be matched so that the lens
uptakes a sufficient quantity of drug and releases it in a controlled
fashion. Most commercially available SCLs show a deficient perfor-
glaucoma treatment is the medical therapy with the goal of
lowering the IOP to a level at which the damage of the optic nerve
ceases to progress. Adrenergic drugs, mainly β-antagonists (such
as timolol) alone or combined with β-agonists (epinephrine) or
R-agonists (brimonidine), cholinergic drugs (pilocarpine),
carbonic anhydrase inhibitors (CAIs; e.g., acetazolamide,
ethoxzolamide), cannabinoids, and prostaglandins have been
12,13
mance because they release ophthalmic drugs too rapidly.
To
overcome this drawback, the following approaches are being
explored: (i) chemically reversible immobilization of drugs through
7,14
4
labile bonds; (ii) incorporation of drug-loaded colloidal systems
1
shown to be useful to decrease the IOP. Systemic delivery, mainly
5-17
into the lens;
(iii) copolymerization with functional monomers
through the oral route, is, however, accompanied by relevant side
effects, which are in most cases associated with the doses required
to achieve therapeutic concentration at the ocular site. Ophthalmic
formulations, such as eye drops, are not exempt of collateral effects
Received: November 9, 2010
Published: February 11, 2011
r 2011 American Chemical Society
701
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|Biomacromolecules 2011, 12, 701–709

Biomacromolecules
ARTICLE
37
Figure 1. Schematic draw of the active site of human carbonic anhydrase II free (A) and after binding acetazolamide (B) as described by Lindskog, and
of the mimicking binding pockets expected to be created in the biomimetic hydrogels (C).
1
8-20
able to interact directly with the drug;
imprinting.
and (iv) molecular
The aim of this work was to design SCLs with high affinity for
CAIs, such as acetazolamide and ethoxzolamide (ETOX), apply-
ing biomimetic principles and the molecular imprinting technol-
ogy. The idea is to create binding pockets in the network
structure that resemble the active site of CA to mime the
noncovalent interactions responsible for the docking of the CAIs
in the physiological receptor. Carbonic anhydrases are metallo-
enzymes that catalyze the conversion of carbon dioxide to
21-25
This last technique aims to organize the compo-
nents of the hydrogel network in such a way that high affinity
binding sites for the drug are created. To do that, the drug is added
before polymerization, and the monomers should arrange as a
function of their ability to interact with the drug molecules. After
polymerization, the drug molecules that have acted as templates are
removed, and the polymer network may exhibit “tailored-active
sites” or “imprinted pockets” with the size and the most suitable
36
bicarbonate ion and protons. Although there are different
26,27
chemical groups to interact again with the drug.
A distinguishing
isoforms, the active site of most of them consists of a cone-
2
þ
key feature of SCLs is their relatively low cross-linking density
compared with common imprinted networks, which can compro-
mise the physical stability of the imprinted cavities due to swelling
after synthesis. Only high affinity cavities can memorize the
structural features of the drug and undergo an “induced fit” in the
presence of the drug recovering the same conformation as upon
shaped cavity that contains a Zn ion coordinated to three
histidine residues in a tetrahedral geometry with a solvent
37
molecule as the fourth ligand (Figure 1A). There are two main
classes of CAIs: (i) the metal-complexing anions and (ii) the
2þ
sulfonamides and their bioisosteres, which bind to the Zn ion
of the enzyme either by substituting the nonprotein zinc ligand to
generate a tetrahedral adduct or by addition to the metal
22,28
polymerization.
In such a way, loosely cross-linked imprinted
hydrogels try to mimic the recognition capacity of certain bioma-
cromolecules (e.g., receptors, enzymes, antibodies). Natural evolu-
tion has determined the unique details of protein’s native state, such
as its shape and charge distribution, that enable it to recognize and
interact with specific molecules. On the basis of biomimetic
principles, SCLs endowed with such high affinity imprinted pockets
are expected to be able to load the drug and, subsequently, to sustain
the release. Ultrathin SCLs synthesized applying the molecular
imprinting technology have already demonstrated greater uptake of
timolol and better in vivo control of drug release to the lacrimal fluid
than conventional SCLs and eyedrops, using similar or even lower
doses. The selection of the functional monomers responsible for
the interaction with the drug can be carried out applying analytical
techniques and computational modeling
monomers libraries or according to a configurational biomimesis
based on the chemical functionality of the natural receptors.
38
coordination sphere, generating trigonal-bipyramidal species.
The -NH function of the ionized sulfonamide group replaces
the water molecule bound to zinc and the hydrogen bonds to
the -OH group of threonine 99. One oxygen atom of the sulfon-
amide interacts with the -NH group of treonine 199, whereas
another oxygen points toward the zinc ion (Figure 1B). Other
chemical groups of the CAIs establish van der Waals interactions
29
37
or hydrogen bonds with neighbor amino acids. Therefore,
monomers bearing chemical groups similar to those of the amino
acids involved in the active binding site were chosen to prepare
biomimetic hydrogels: the zinc ions were introduced as metha-
30
crylate salt (ZnMA ); the hydroxyl and amino groups can be
2
25
31,32
supplied by 2-hydroxyethyl methacrylate (HEMA) and N-hy-
droxyethyl acrylamide (HEAA); and 4-vinylimidazole (4VI)
resembles histidine (Figure 1C). 4VI is not commercially avail-
able, and thus the first step was to synthesize it. For comparative
purpose, 1-vinylimidazole (1VI) was also included in the study as
an alternative for 4VI in the mimicking of histidine (Figure 2).
for the screening of
27,33
Byrne et al. have recently shown that the selection of functional
monomers possessing chemical groups similar to those present in
the histamine H1-receptor or in the CD44 protein endows the
hydrogels with high affinity for the antihistamic drug ketotifen
Then, a set of hydrogels with fix content in ZnMA and various
2
23,34
35
fumarate
or for hyaluronic acid, respectively. Imprinted
comonomer combinations was prepared and characterized re-
garding their ability to load and to sustain the release of
acetazolamide and ETOX. Cytocompatibility, degree of swelling,
and other relevant features from the point of view of the use of
the hydrogels as components of SCLs were also evaluated.
hydrogels exhibited higher loading and delayed release compared
with the nonimprinted ones, and it was demonstrated that each
functional monomer relating to the biological binding played a role
23,34,35
in the delayed release.
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Biomacromolecules
ARTICLE
’
EXPERIMENTAL SECTION
Aldrich (St. Louis MO). Purified water was obtained by reverse osmosis
(
Milli-Q, Millipore Ib ꢀe rica SA, Madrid, Spain). Other reagents were
Materials. Acetazolamide (ACT), ETOX, HEMA, ethyleneglycol
analytical grade.
dimethacrylate (EGDMA), zinc methacrylate (ZnMA ), 1VI, urocanic
2
Synthesis of 4(5)-Vinylimidazole (4VI). 4VI was obtained via
acid, and HEAA were from Sigma-Aldrich Chemicals (Madrid, Spain)
39
thermal decarboxylation from urocanic acid according to the literature.
(
(
Figure 2). Azobisisobutyronitrile (AIBN) was from Acros Organic
In brief, anhydrous urocanic acid (2 g, 0.0145 mol) was placed in a short-
necked distilling apparatus. Then, the temperature was slowly increased
to 205 ꢀC under pressure of 18 Torr. The distilled material was trapped
using a glass sink (coldfinger) cooling system. 4VI was obtained as a
crystallized white product in the cold receiver (yield = 12%). Then, it was
stored in the fridge at 4 ꢀC. The obtaining of 4VI was confirmed by
0
0
Geel, Belgium). Zincon monosodium salt (2-carboxy-2 -hydroxy-5 -
sulfoformazylbenzene) and zinc nitrate hexahydrate were from Sigma-
-
1
FTIR-ATR spectroscopy (400-4000 cm , Varian-670-FTIR spectro-
meter equipped with a GladiATR, Madison, WI) and H NMR analysis
1
(
Varian Mercury 300 MHz þ robot spectrophotometer, Madison, WI).
The peaks of 4VI in CDCl were assigned as follows: δ 5.10 (d, J = 12.15
Hz, 1H), 5.65 (d, J = 17.64 Hz, 1 H), 6.60 (m, 1H), 7.02 (s, 1H), 7.61 (s,
3
4
0
1
H), 10.32 (br s, 1H).
Synthesis of pHEMA Hydrogels. Various sets of monomer
mixtures were prepared with the composition shown in Table 1. ACT
-
4
was added to some mixtures at a concentration of 7.8 ꢀ 10 M to attain
2
þ
a molar ratio of 1:1:3 for drug/Zn /VI. The monomer solutions were
injected into molds constituted by two glass plates pretreated with
dimethyldichlorosilane and separated by a silicone frame of 0.9 mm
4
1
thickness. The molds were placed in an oven at 50 ꢀC for 12 h and then
heated to 70 ꢀC for 24 h more. The hydrogels were removed from the
molds and immersed in boiling water (600 mL) for 15 min to remove
nonreacted components and to facilitate the cutting of the hydrogels as
1
0
0 mm discs. The discs were washed in ultra pure water for 24 h, in NaCl
.9% solution for 24 h and then immersed in water for 15 days (400 mL).
We monitored the removal of unreacted monomers by measuring the
absorbance in the 190-800 nm range of the washing solutions. Finally,
the discs were dried and stored.
Determination of Zinc Content. We prepared a stock solution
of Zincon (1.6 mM) by dissolving 7.4 mg in 250 μL of 1 M NaOH prior
to dilution to 10 mL with water. The solution was kept in the fridge and
2
þ
used in a few days. Zn stock solution (27 mM) was prepared by
dissolving zinc nitrate hexahydrate in 50 mL of 0.1 M nitric acid. The
calibration curve was constructed by adding 25 μL of diluted stock
solution (0.31 to 1.25 mM) to 950 μL of USP borate buffer pH 9.0 and
4
2
2
5 μL of the Zincon stock solution. The blank was prepared
analogously except for the substitution of the metal sample solution
with 0.1 M nitric acid. Absorption spectra were recorded from 400 to 750
nm after 5 min of sample incubation at room temperature. The
absorbances were measured at 620 nm. The intensity of the blue color
4
2
was proportional to the zinc concentration. To quantify the amount of
zinc present in the hydrogels, we transferred dried discs to test tubes
containing 2 mL of 1.0 M nitric acid kept at 70 ꢀC for ∼48 h. Aliquots
(100 μL) of the final solution were diluted with 330 μL of 1.0 M nitric
Figure 2. Amino acids that form part of the active site of carbonic
anhydrase, monomers used to synthesize the hydrogels, and CAI drugs
tested.
Table 1. Composition of the Monomer Mixtures Used to Synthesize the Hydrogels
formulation
HEMA (mL)
ZnMA
2
(g)
HEAA (g)
1VI (mL)
4VI (g)
EGDMA (mL)
AIBN (g)
ACT (g)
0
8
8
8
8
8
8
8
8
8
8
8
0.12
0.12
0.12
0.12
0.12
0.12
0.12
0.12
0.12
0.12
0.12
0.0135
0.0135
0.0135
0.0135
0.0135
0.0135
0.0135
0.0135
0.0135
0.0135
0.0135
A
B
C
D
E
F
G
H
I
0.185
0.185
0.185
0.185
0.185
0.185
0.185
0.185
0.185
0.185
0.173
0.173
0.173
0.173
0.173
0.22
0.22
0.22
0.22
0.14
0.14
0.14
0.14
0.22
0.22
J
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ARTICLE
acid and then mixed with 70 μL of NaOH 5 M to adjust the pH to 4 to 5.
Then, 25 μL of the resulting solution was added to 950 μL of USP borate
buffer pH 9.0 and 25 μL of the Zincon stock solution and incubated at
room temperature for 5 min. The absorbance was recorded at 620 nm,
and the amount of zinc in the sample was estimated from the calibration
curve. All experiments were carried out in triplicate.
at room temperature. The amount of drug released was measured
spectrophotometrically in samples periodically taken and again placed
in the same vessel so that the liquid volume was kept constant. The
network/water partition coefficient, KN/W, which is an index of the
affinity of the drug for the network, was estimated from the total amount
4
3
loaded per gram of gel
Physical and Structural Characterization of pHEMA Hy-
drogels. FTIR-ATR (attenuated total reflection) spectra were re-
"
#
ðVs þ K
VpÞ
N=W
loading
¼
C0
ð3Þ
corded over the range 400-4000 cm- , in a Varian-670-FTIR
spectrometer equipped with a GladiATR (Madison, WI) with diamond
crystal. DSC scans of dried hydrogels (5-10 mg) were recorded by
heating from 25 to 150 ꢀC, cooling to -10 ꢀC, and then heating again
until 300 ꢀC, always at the rate of 10 ꢀC/min, in a DSC Q100 (TA
Instruments, New Castle, DE) with a refrigerated cooling accessory.
Nitrogen was used as purge gas at a flow rate of 50 mL/min. The
calorimeter was calibrated for cell constant and temperature using
indium standard (melting point 156.61 ꢀC, enthalpy of fusion 28.71
J/g) and for heat capacity using sapphire standards. All experiments were
performed in duplicate. Degree of swelling in water was calculated, in
duplicate, as follows
1
ðtotalÞ
3
W
p
where V is the volume of water sorbed by the hydrogel, W is the dried
s
p
hydrogel weight, C
is the concentration of the drug in loading solution,
p
is the volume of dried polymer.
Release profiles up to 60% released were fitted to the square-root
0
and V
4
4
kinetics as follows
Mt
¼ KH 3 t0:5
ð4Þ
M¥
where M represents the amount of drug released at time t and M is the
t
¥
total amount loaded. Each release profile was fitted to the Higuchi
equation; then, the mean and the standard deviation were calculated
from the values obtained for six replicates.
ðWt - W0Þ
Qð%Þ ¼
100
ð1Þ
3
W0
ETOX loading and Release. Dried hydrogels discs (six re-
plicates) were placed in 5 mL of ETOX suspension (0.23 g/L) and
kept for 48 h at room temperature. The ETOX-loaded discs were rinsed
with water; their surfaces were carefully wiped, and the discs were
immediately immersed in 5 mL of NaCl 0.9% at room temperature. The
amount of drug released was measured spectrophotometrically at 303
nm in samples periodically taken up and placed again in the same vessel.
After 360 h, the discs were removed from the medium, rinsed with
ultrapure water, and placed in vials with 5 mL of ethanol/water (70:30)
mixture for 48 h. The drug extracted from the hydrogels was quantified
from the absorbance measured at 303 nm (Agilent 8453, Germany). The
network/water partition coefficient, KN/W, of ETOX was estimated
from the total amount released to the aqueous medium plus that
extracted in ethanol/water medium. ETOX solubility in water (21.38
where W and W represent the weights of a hydrogel disc at the dry state
0
t
and after being immersed in water for a time t. Light transmittance of
fully swollen hydrogels was recorded in triplicate at 600 nm (UV-vis
spectrophotometer, Agilent 8453, Boeblingen, Germany) by mounting
a continuous piece of hydrogel on the inside face of a quartz cell. Oxygen
permeability (Dk) and transmissibility of hydrogels previously swollen
in a 0.9% NaCl solution were measured in triplicate at room temperature
using a Createch permeometer (model 210T, Rehder Development
Company, Castro Valley) fitted with a flat polarographic cell and in a
chamber at 100% of relative humidity.
Cytocompatibility Studies. Dried discs were immersed in phos-
phate buffer (pH 7.4) and autoclaved (121 ꢀC, 20 min). Cytocompat-
ibility tests were carried out in triplicate using the Balb/3T3 Clone A31
cell line (ATCC, LGC Standards S.L.U., Barcelona, Spain) according to
the direct contact test of the ISO 10993-5:1999 standard. The discs were
added in 24-well plates containing 200 000 cells per well in 2 mL of
Dulbecco’s modified Eagle’s medium F12HAM supplemented with 10%
fetal bovine serum and 13 μg/mL gentamicin. The plates were incubated
0
mg/L) was used as C in eq 3 because in the suspension, this
concentration should remain constant. The release rate constants were
estimated using eq 4.
’
RESULTS AND DISCUSSION
at 37 ꢀC, 5% of CO
2
, and 90% of humidity. After 24 h, aliquots (100 μL)
Hydrogels Synthesis and Structural Characterization.
The hydrogels were prepared using HEMA as main component
because of the recognized biocompatibility of pHEMA and its
common use as integrant of SCLs. Several monomers were
copolymerized with HEMA to mimic the active site of CA
of medium were taken in 96-well microplates and mixed with reaction
mixture solution (100 μL, Cytotoxicity Detection Kit, LDH, Roche).
The plates were incubated at 20 ꢀC for 30 min (protected from light). A
stop solution (50 μL) was added to the wells and the absorbance was
measured at 490 nm (BIORAD model 680 Microplate reader). Blank
45
(
Table 1); the biomimetic level is foreseeable to increase from
(culture medium), negative (cells in culture medium), and positive (cells
A to J formulations. A control pHEMA hydrogel without
functional comonomers (formulation 0) was used as a reference
to quantify the role of the comonomers in the binding of the
CAIs. Although 4VI resembles much better than 1VI the func-
tional groups of histidine, both monomers were used to prepare
the hydrogels with the purpose of elucidating the incidence of the
imidazole structure on the affinity for the CAIs. 4VI was
successfully synthesized from urocanic acid, and its structure
in medium with lysis factor) controls were prepared too, and the was
absorbance measured. The cytocompatibility was quantified as follows
Absexp - Absnegativecontrol
cytocompatibility ð%Þ ¼
ð2Þ
Abspositivecontrol - Absnegativecontrol
ACT Loading and Release. Dried hydrogel discs (six replicates)
were placed in 5 mL of ACT 0.2 g/L aqueous solution and kept at room
temperature protected from the light for 48 h. The amount of ACT
loaded was calculated as the difference between the initial amount of
drug in the solution and the amount remaining after loading determined
by UV spectrophotometry at 264 nm (Agilent 8453, Germany). Drug-
loaded discs were rinsed with water, their surface was carefully wiped,
and the discs were immediately immersed in 5 mL of NaCl 0.9% solution
1
was confirmed by H NMR (Figure 3). Urocanic acid showed
chemical shifts at 2.49 and 3.32 ppm (-OH) and 6.32 ppm
(
CHd), whereas 4VI showed two singlet peaks at 7.03 and 7.61
ppm for N-CH and NdCH protons (imidazole), respectively.
The methylene (CH d) and methane (CHd) protons of 4VI
appeared at 5.16, 5.67, and 6.63 ppm according to the
2
46
literature.
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ARTICLE
The hydrogels were intensively washed after synthesis to
remove the template molecules. Both imprinted and nonim-
printed hydrogels underwent the same cleaning procedure.
Because in the natural receptor, zinc directly participates in the
binding of the drug (Figure 1B), we first corroborated the
presence of zinc ions in the networks. It is interesting to note
that even after boiling and immersion in saline medium, zinc ions
were still detected in A to J hydrogel formulations (Table 2).
Nevertheless, remarkable differences among the hydrogels could
be observed; the imidazole monomers were absolutely required
to keep significant amounts of zinc in the network. The small
amount of zinc ions remaining in the hydrogels without 4VI
containing 1VI or 4VI can bind metal ions, including zinc, with
48
a remarkable affinity even in saline medium. Additionally,
we observed that hydrogels bearing 4VI retained twice the
amount of zinc ions (71-90%) than those synthesized with
1VI (38-43%). This finding can be related to the fact that 4VI
mimics better the coordination of zinc to the amine groups of
histidine residues that occurs in the natural CA enzyme. Because
4
7
the affinity of zinc ions for methacrylic acid groups is quite high
and those zinc ions can still coordinate with two imidazole
48
groups, it seems that a quite plausible structure of the binding
pocket is that depicted in Figure 1C. Nevertheless, other con-
figurations of the receptor, such as that with only one imidazole
group, cannot be discarded. The proportion of functional mono-
mers in the hydrogel is too low for being able to gain an insight
into this point using common analytical techniques.
could be due to the fact that ZnMA is not reacting during
2
polymerization or that zinc ions are replaced by counterions in
the absence of 4VI. Because all hydrogels have a similar mono-
meric composition (HEMA being the majority monomer) and
The hydrogels were slightly opalescent because of the pre-
1
00% zinc complexation can only be achieved if methacrylate
sence of ZnMA , particularly those prepared with 4VI (Table 2).
2
mers are effectively copolymerized in the network, it is perfectly
plausible that ZnMA is similarly incorporated to all hydrogels.
Nevertheless, it should be noticed that the thickness of the
hydrogels is larger (three- to four-fold) than that of commercial
SCLs. Therefore, transmittance of thinner hydrogels might make
possible to use some formulations as components of SCLs. The
lower transmittance of the hydrogels prepared with 4VI can be
also related to their lower degree of swelling in aqueous medium
2
Therefore, the decrease in zinc ions can be attributed to the long
extraction process in the presence of saline medium. Removal of
zinc ions by sodium ions is not complete because the affinity of
the methacrylic acid mers for divalent ions is larger than that for
monovalent ones. In sum, the formation of a zinc/methacrylic
acid/imidazol coordination complex notably enhances the
stability of the zinc bonds to the network. This finding is in
agreement with previous papers that reported that polymers
4
7
(Figure 4). pHEMA-ZnMA hydrogels prepared without 1VI or
2
4VI (formulations A, B, G, and H) attained swelling values of
70-76% (Table 2), which are in the typical range value of
Figure 4. Swelling profiles of the pHEMA-ZnMA hydrogels (codes as
in Table 1) in water. Continuous and dotted lines correspond to
nonimprinted and ACT-imprinted networks.
2
1
Figure 3. H NMR spectra of urocanic acid in DMSO-d and 4VI in
CDCl .
3
6
Table 2. Percentage of Zinc Ions That Remain in the Hydrogels after the Cleaning Step, And Physical Properties of the Networks.
Mean Values and, In Parentheses, Standard Deviations
formulation
remaining Zn (%)
transmittance at 600 nm (%)
T
g
(ꢀC)
degree of swelling (%)
Dk (barrer)
0
85
76
70
68
64
29
29
73
77
22
30
110
66.0 (3.2)
73.0 (0.8)
70.6 (1.5)
42.8 (5.1)
55.5 (0.1)
52.7 (0.6)
53.4 (1.1)
77.5 (1.7)
75.2 (4.3)
55.5 (0.4)
61.0 (1.2)
12.4 (1.1)
12.6 (1.5)
17.7 (7.6)
9.2 (0.4)
A
B
C
D
E
F
G
H
I
22.6 (7.1)
11.3 (4.8)
38.7 (6.6)
43.7 (7.4)
73.6 (4.5)
71.9 (1.1)
10.0 (1.9)
13.9 (1.5)
76.9 (5.1)
90.8 (4.5)
109
115
126
132
125
125
110
109
116
123
12.8 (2.3)
14.8 (0.3)
13.4 (0.4)
18.0 (3.3)
24.8 (0.2)
17.2 (4.9)
15.6 (1.5)
J
7
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Biomacromolecules
ARTICLE
pHEMA hydrogels. Copolymerization with HEAA did not
change the degree of swelling. In contrast, those hydrogels
containing 1VI or 4VI exhibited a remarkably lower capability
to sorb water (Table 2). The synthesis in the presence of ACT
attenuated to a certain extent the decrease in the degree of
swelling; the D, F, and J imprinted hydrogels swelled more in
water than the corresponding C, E, and I nonimprinted net-
works.
Oxygen Permeability and Cytocompatibility. Hydrogels
intended as components of SCLs should have enough oxygen
permeability for preventing corneal hypoxia and edema. As
expected, the higher the degree of swelling, the greater the
oxygen permeability of the hydrogels was (Table 2). This means
that the hydrogels containing 1VI or 4VI are less gas-permeable
than the other hydrogels. Nevertheless, the differences are not
too large, and the values are in the range of those recorded for
50,51
Furthermore, 1VI and 4VI make the networks more rigid, as
indicated by the increase in the glass-transition temperature, Tg,
of the hydrogels prepared with these monomers (Table 2). The
presence of ZnMA did not alter the T of the pHEMA
commercially available SCLs.
Cytocompatibility studies were carried out according to the
direct contact test of the ISO 10993-5:1999 standard for the
simultaneous evaluation of the effect of the polymer network and
the leached substances, such as unreacted monomers. Fibroblast
showed an excellent viability (96-100%) when cultured on any
2
g
hydrogels, which is around 110 ꢀC (Table 2). It has been
4
9
reported that poly(1VI) has a T around 175 ꢀC. The DSC
g
scans of the hydrogels containing 1VI or 4VI showed only one T ,
hydrogel prepared (Figure 5), indicating that ZnMA , 1VI, and
g
2
at an intermediate temperature between the T of pHEMA and
that of poly(1VI), indicating that the monomers are miscible and
4VI do not deteriorate the cytocompatibility of the pHEMA
hydrogels and that no toxic substances are leaked from the
networks.
g
confirming that the vinylimidazole monomers are efficiently
39,46,49
copolymerized with the methacrylate ones.
Macromole-
ACT Loading and Release. Although both ACT and ETOX
exhibit similar ability to inhibit human CA isoform II and are
cular interactions between the carbonyl group of ethyl metha-
crylate and the imidazol fragments have been previously shown
52
active at the nanomolar range, relevant physicochemical differ-
ences between both molecules can be highlighted. CAIs bind in
the active site of the enzyme in deprotonated state, coordinating
to zinc, whereas the basic amino acids serve as proton acceptors.
49
by FTIR spectroscopy. However, we could not observe rele-
-
1
vant shifts in the carbonyl band at 1707 cm , probably because
of the relatively low proportion of 1VI and 4VI in the hydrogels
compared with HEMA. The FTIR spectra mainly showed the
characteristic bands of pHEMA (data not shown).
Therefore, the lower the pK , the more favorable the binding is.
a
ACT is more acidic (pK 7.4) and significantly less lipophilic
a
5
3
(
Log P = -0.26) than ETOX (pK 8.0; Log P = 2.01).
a
Compared with ETOX, ACT is more ionized, and its solubility
is greater at physiological pH 7.4. Although both CAIs obey
Lipinski “rule of 5” properties and show good membrane
permeability, ACT has seven hydrogen bond acceptors and three
hydrogen bond donors, whereas ETOX has just five hydrogen
bond acceptors and two hydrogen bond donors. Therefore, ACT
may be anchored to the active site of the enzyme through more
53
hydrogen bonds. Drug loading experiments were designed to
have the same number of molecules of ACT or ETOX in the
volume of medium in which the hydrogels were immersed.
Therefore, the first consequence of the physicochemical differ-
ences between ACT and ETOX was that the loading experiments
with the latter had to be carried out by immersion of the
hydrogels in an aqueous suspension because of the low solubility
of ETOX. Loading with ACT was carried out by immersion in 0.2
g/L solution.
Figure 5. Cell viability for different pHEMA-ZnMA hydrogels. Mean
values and standard deviations (n = 3).
2
Table 3. ACT Loaded, Network/Water Partition Coefficients, ACT Released in NaCl 0.9% Solution, and Release Rate Constants
a
Obtained after Fitting to the Square-Root Kinetics
(% h-
0.5
)
R
2
formulation
loading (mg/g)
K
N/W
ACT released at 48 h (mg/g)
K
H
0
1.22 (0.10)
1.50 (0.30)
1.18 (0.36)
1.48 (0.20)
1.74 (0.12)
3.37 (0.03)
3.16 (0.25)
1.51 (0.10)
1.38 (0.10)
3.15 (0.29)
3.28 (0.15)
5.40 (0.18)
6.67 (1.46)
5.11 (1.77)
6.63 (0.96)
7.79 (0.58)
16.40 (0.16)
15.35 (1.26)
6.52 (0.48)
5.90 (0.50)
15.29 (1.47)
15.64 (0.72)
1.17 (0.04)
0.87 (0.04)
0.74 (0.06)
0.92 (0.13)
1.16 (0.02)
1.64 (0.05)
1.87 (0.20)
0.68 (0.04)
0.64 (0.04)
1.61 (0.18)
2.46 (0.30)
24.20 (2.05)
10.48 (0.53)
11.67 (0.26)
10.56 (0.47)
14.07 (2.98)
6.86 (0.18)
8.49 (0.45)
8.61 (0.71)
8.99 (0.79)
7.31 (0.04)
13.04 (0.16)
0.994
0.962
0.967
0.991
0.946
0.995
0.995
0.948
0.955
0.994
0.993
A
B
C
D
E
F
G
H
I
J
a
Mean values and, in parentheses, standard deviations (n = 6).
7
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Biomacromolecules
ARTICLE
Copolymerization of HEMA with ZnMA solely or with
that resemble the functionalities of the components of the natural
receptor at the active site significantly decreased the ACT release
rate. Most hydrogels required 15 days to complete the release.
Hydrogels A and B showed a certain decrease in ACT release rate
compared with control hydrogels, which could be due to a slight
increase in the mesh size of the network owing the divalent Zn
ions connecting neighbor methacrylic acid mers. The release
profiles fitted quite well to the square root kinetics, and the
hydrogels E, F, and I showed the lowest release rate (Table 3).
Imprinted hydrogels (i.e, formulations B, D, F, H, and J) seem to
release ACT faster than the corresponding nonimprinted net-
works (Figure 6), although statistically significant differences
were recorded only for formulation F compared with E and for
formulation J compared with I (t test, R < 0.01). The reasons
behind this behavior are not clear but could be related to small
changes in the microstructure of the hydrogels caused by the
presence of ACT molecules during polymerization. The removal
of the template molecules may have opened paths into the
network for an easier entrance/exit of subsequent drug mole-
cules. In fact, the synthesis in the presence of template molecules
caused an increase in the degree of swelling of the imprinted
networks compared with the nonimprinted ones (Table 2).
ETOX Loading and Release. The loading of ETOX was
carried out by immersion of the hydrogels in aqueous suspen-
sions of the drug because of its limited solubility (21.38 mg/L).
The suspensions contained the same number of molecules of
ETOX per liter as in the case of ACT. The hydrogels loaded less
ETOX than ACT, which may be due to the fact that the drug first
has to dissolve to be available to interact with the network.
Nevertheless, the hydrogels (even the control one) showed
remarkably high affinity for ETOX (network/water partition
coefficients above 36), which suggest unspecific hydrophobic
2
HEAA did not significantly modify the amount of ACT uptaken
by the hydrogels (Table 3). These hydrogels retained a small
proportion of the zinc ions incorporated during synthesis, and
thus no specific drug-network interactions could be established.
A slight improvement in the loading was recorded for formula-
tions having 1VI. In contrast, hydrogels bearing 4VI moieties
showed a remarkably greater ability (two-fold) to load ACT
(
Table 3). The network/water partition coefficient, KN/W, values
obtained for formulations E, F, I, and J almost triplicate the values
recorded for the other hydrogels. No further improvement was
achieved by adding ACT as template during polymerization. This
means that by using the functional monomers that mimic the best
components of the CA receptor, it is feasible to endow the
hydrogels with high affinity for ACT. Such a notable increase in
affinity was also evidenced in a better control of drug release
(
Figure 6). Control hydrogels (formulation 0) rapidly released
the ACT loaded. Copolymerization with functional monomers
adsorption to the pHEMA network. pHEMA-ZnMA hydrogels
2
containing 4VI were again those with greater ability to host
ETOX and exhibited 90% greater KN/W values than the other
hydrogels (Table 4), which indicates the contribution of specific
interactions with the biomimetic pockets. The hydrogels sus-
tained the release of ETOX for 2 weeks, after which the amount
released was still <50% (Figure 7). The release rate decreased
after 72 h, which could be due to the attainment of equilibrium
between the free drug in the medium and the drug bound to the
hydrogel due to the high drug-network affinity. It should be also
noticed that the volume of the medium, although enough to
dissolve the whole ETOX dose loaded by the hydrogels, was
Figure 6. ACT release profiles in 0.9% NaCl medium from pHEMA
hydrogels containing diverse functional comonomers. Continuous and
dotted lines correspond to nonimprinted and ACT-imprinted networks,
respectively. Codes as in Table 1. The insert shows data used for the
fitting to the square-root kinetics.
Table 4. ETOX Loaded, Network/Water Partition Coefficient, ETOX Released in NaCl 0.9% Solution, And Release Rate
a
Constants Obtained after Fitting to the Square-Root Kinetics
(% h-
0.5
)
R
2
formulation
loading (mg/g)
K
N/W
ETOX released at 48 h (mg/g)
K
H
0
0.99 (0.17)
0.91 (0.12)
0.80 (0.16)
0.81 (0.14)
1.01 (0.04)
1.51 (0.07)
1.50 (0.18)
1.00 (0.02)
0.95 (0.11)
1.55 (0.12)
1.71 (0.18)
45.70 (7.82)
42.11 (5.64)
36.86 (7.55)
37.38 (6.40)
46.73 (1.92)
70.36 (3.45)
69.84 (8.27)
46.26 (1.08)
44.04 (5.18)
72.32 (5.44)
79.38 (7.33)
0.43 (0.06)
0.34 (0.04)
0.28 (0.04)
0.28 (0.05)
0.42 (0.02)
0.35 (0.03)
0.36 (0.03)
0.42 (0.03)
0.36 (0.01)
0.35 (0.03)
0.48 (0.06)
7.03 (0.66)
7.03 (0.46)
6.70 (0.48)
7.29 (0.97)
7.86 (0.27)
4.81 (0.18)
4.98 (0.45)
8.53 (0.22)
7.61 (0.99)
4.02 (0.63)
4.92 (0.44)
0.969
0.957
0.945
0.926
0.954
0.954
0.907
0.971
0.956
0.921
0.962
A
B
C
D
E
F
G
H
I
J
a
Mean values and, in parentheses, standard deviations (n = 6).
7
07
dx.doi.org/10.1021/bm101562v |Biomacromolecules 2011, 12, 701–709

Biomacromolecules
ARTICLE
’
AUTHOR INFORMATION
Corresponding Author
*
Fax: 34-981547148. E-mail: carmen.alvarez.lorenzo@usc.es.
’
ACKNOWLEDGMENT
Work supported by MICINN (SAF2008-01679) and FEDER
Spain) and Funda c- ~a o para Ci ^e ncia e Tecnologia (FCT, Praxis
(
grant SFRH/BD/40947/2007, Portugal). We thank C. Fern ꢀa ndez
Masaguer and J. Gonz ꢀa lez Parga for their help with the
synthesis of 4VI and A. Rey-Rico for help with the cytocompat-
ibility tests.
’
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