Diffusion Coefficients of Timolol Maleate in Polymeric Membranes

Article pubs.acs.org/jced

Diffusion Coefficients of Timolol Maleate in Polymeric Membranes Based on Methacrylate Hydrogels Andreia S. G. Silva† and M. N. Coelho Pinheiro*,†,‡ †

Departamento de Engenharia Química e Biológica, Instituto Superior de Engenharia, Instituto Politécnico de Coimbra, Rua Pedro Nunes, Quinta da Nora, 3030-199 Coimbra, Portugal ‡ Centro de Estudos de Fenómenos de Transporte, Faculdade de Engenharia, Universidade do Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal ABSTRACT: Prediction accuracy from models which adequately describe drug release from polymeric matrices depends on the availability of good estimations/measurements for drug diffusion coefficients. Lenses obtained from membranes with 2-hydroxyethyl methacrylate (HEMA) as the main component were used in this study. Two hydrogels were prepared by thermal copolymerization: HEMA with methacrylic acid (MAA, hydrophilic monomer) and HEMA with methyl methacrylate (MMA, hydrophobic monomer). Timolol maleate was incorporated into the polymeric matrices by soaking or by addition to the polymerization mixture (occlusion). An experimental methodology with continuous monitoring of drug concentration in the release medium was implemented, thereby avoiding time-consuming sequential manual sampling. Considering diffusional mass transport as the controlling mechanism, the value of timolol maleate diffusivity when impregnated by occlusion was (1.74 ± 0.14) × 10−12 m2 s−1 for lenses with MAA, and (1.66 ± 0.14) × 10−12 m2 s−1 for lenses containing MMA. When the drug was added by soaking, the value of diffusivity was (1.49 ± 0.39) × 10−12 m2 s−1 for lenses containing MAA and (1.11 ± 0.26) × 10−12 m2 s−1 for lenses with MMA. The hydrogel with greater hydrophilic characteristics presented higher values of drug diffusion coefficients than the more hydrophobic, an observation which was valid for both methods used to incorporate the drug. However, the increase is more significant (by about 34%) when timolol maleate is added to the polymeric matrix by soaking. enhance its drug load capacity.10−12 MAA is a hydrophilic molecule that is ionized at pH values above 5.513 and MMA is a hydrophobic molecule. Ionic interactions and hydrogen bonds with timolol maleate occur in both monomers. At the same time, some efforts have been made in order to extend drug release duration, and new approaches have been developed: dispersion of nanoparticles encapsulating the drug in the polymeric matrix,2,7,14 addition of a biodegradable layer structure in contact lenses,3 and incorporation of diffusion barriers containing vitamin E.4−6 In parallel with the development of ophthalmic drug release systems that are effective and comfortable and can remain in the eye for a long period, some efforts have been made to model drug delivery from contact lens into the tear films and subsequent cornea absorption. Models do not replace clinical studies but help to design drug delivery systems to achieve desired therapeutic dosages. However, there are no global mathematical models covering all of the possible chemical and physical processes that can occur.

1. INTRODUCTION Drug effectiveness in combating disease depends on drug concentration at the intended location for an adequate period of time. In ophthalmic applications, eye drops are commonly used as the drug delivery system. They present low bioavailability because precorneal factors are responsible for losing about 95% of the drug applied.1 Soft contact lenses have recently been presented as a good vehicle to deliver drugs to the eyes.2−7 Glaucoma affects millions of people and is responsible for elevated intraocular pressures that causes serious damages to the eye with loss of vision and can even lead to blindness if not treated. Timolol is a very common drug used in glaucoma treatment, approved by the Food and Drug Administration (FDA) in 1979. Therapeutic contact lenses which deliver glaucoma medications have been the subject of recently developed research studies.2,5 Several hydrogels have been used for preparing potential therapeutic lenses. Poly(hydroxyethyl methacrylate) (polyHEMA) hydrogels have been widely studied as delivery support but its drug uptake is generally not sufficient for most purposes.8,9 Copolymers with small amounts of methacrylic acid (MAA) and methyl methacrylate (MMA) are used to © 2013 American Chemical Society

Received: April 16, 2013 Accepted: July 9, 2013 Published: July 18, 2013 2280

dx.doi.org/10.1021/je400377m | J. Chem. Eng. Data 2013, 58, 2280−2289

Journal of Chemical & Engineering Data

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Siepmann and Siepmann15 recently presented a survey of available models for diffusion controlled drug delivery systems with a useful classification scheme according to inner device structure, initial drug content and geometry. They present appropriate mathematical equations used to quantify drug release in the different schemes considered, with drug diffusion coefficient within polymeric matrix as the parameter. The availability of good estimations/measurements of model parameters dictates the prediction accuracy. The transport model for timolol release from p-HEMA contact lenses presented by Li and Chauhan16 represented a first effort in modeling drug release from contact lenses followed by cornea uptake. Fickian diffusion of drug in the lens was assumed, and the model also considers mass transfer enhancement by convection in the postlens tear film resulting from the flow induced by contact lens displacement during blinking. However, solute transport through hydrogels is a complex phenomenon depending on several factors such as the degree of swelling and solute interaction with the gel, and thus the assumption of Fickian diffusion through the contact lens is not appropriate.15,17 The literature frequently reports the use of empirical or semiempirical models which are not sufficient to predict exact drug behavior. For detailed and realistic predictions it is necessary to identify the most important transport mechanisms and take them into account during modeling, sometimes introducing significant complexity.15,17 Mathematical models accurately predicting drug release rates and drug mass transfer in the delivery system are very useful, as the needed number of experiments is greatly reduced and there is a better understanding of the physical mechanisms of drug transport. Model accuracy is conditioned by good estimates and measurements of parameters used as intake information. The drug diffusion coefficient in the delivery system is one of the most important parameters, and this is usually obtained by kinetic experiments of drug release. 1.1. Theory for Drug Diffusion Coefficient Determination. Several physical and chemical phenomena occur during drug release from delivery systems: solvent diffusion toward the inner structure of the system, drug diffusion through the polymeric matrix, adsorption/desorption processes, polymer swelling and erosion, among others. When the volume of a polymer changes to accommodate penetrant molecule build up internal stresses resulting in conformational modifications of polymeric chains, and polymer relaxation occurs with this structural change. When drug diffusion must be considered simultaneously, it makes drug release from polymeric matrices a complex phenomenon. Frequently three classes are distinguished according the relative magnitude between polymer relaxation and solute diffusion:18−21 (i) Case I or Fickian diffusion when solute diffusion is the dominant step (ii) Case II, another extreme case where polymer relaxation determines the process (iii) Case III or non-Fickian diffusion (also designated as anomalous diffusion) in which the superposition of diffusion and polymer relaxation must be used to describe the mass transfer process The simple equations used to describe Fickian diffusion and case II are expressed in terms of a single parameter: solute

diffusion coefficient and a zero-order kinetic constant for sink conditions in the release medium. In the light of the power law, a semiempirical model to describe drug release from polymeric matrixes, introduced by Peppas and co-workers in 198322 Mt = kt n M∞

(1)

where Mt and M∞ denote the total amount of drug released during the period of time t and after infinite time, respectively, k is a kinetic constant characteristic of drug/polymer system, and n is an exponent characteristic of the drug release mechanism. In eq 1, for thin polymeric films, n = 0.5 when Fickian diffusion is the dominant mechanism and n = 1 for case II, being drug release velocity time independent. In anomalous diffusion the process cannot be completely explained by a Fickian approach indicating relaxation in the polymeric network influences mass transfer. Although a universal approach does not exist, some authors mention that introducing concentration-dependent diffusion coefficients is not enough to describe the mass transfer process. For anomalous diffusion, two or more parameters are needed in the equations. In this case an interaction between diffusion and relaxation effects exists, and n from eq 1 takes an intermediate value between 0.5 and 1, for thin polymeric films. Taking logarithms in both terms of eq 1, the equation is linearized. Plotting log10(Mt/M∞) versus log10(t) from data obtained in kinetic experiments for drug release, the slope of the correlated straight lines could be used to determine exponent n. This parameter is an important indicator of the mechanisms controlling drug release from polymeric delivery systems. Diffusional mass transport of drugs in hydrophilic polymeric matrices is often the controlling mechanism, and Fick’s second law of diffusion can be used to describe the drug release process. For the geometry used in the present study (thin discs) the lateral surface of the delivery system is negligible and a one-dimensional (axial) nonsteady diffusion problem may be considered with Fick’s second law written as ∂CA ∂ 2C = D 2A ∂t ∂x

(2)

where CA is the drug concentration in the polymeric matrix, D is the drug diffusion coefficient, x represents the axial direction, and t is time. In order to quantify drug release the differential equation must be solved together with initial and boundary conditions. Considering that the diffusing specie is uniformly distributed through polymeric matrix, the initial condition may be written as

CA(x , t = 0) = CAi

(3)

where CAi stands for initial drug concentration in the hydrogel. For a well-stirred release medium, homogeneous hydrodynamic flow conditions at the delivery system boundary are achieved and considering a uniform structure throughout the polymer matrix, a symmetric drug concentration distribution (around the central plane of the membrane, x = 0) within the hydrogel is expected, and ∂CA (x = 0, t ) = 0 ∂x 2281

(4)



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determine the drug diffusion coefficient D in the polymeric device. 1.2. Kinetic Experiments of Drug Release. Recent developments of new pharmaceutical products, particularly with ophthalmic applications based in polymeric matrices as drug support, have been accompanied by extensive experimental studies in vitro and finally by clinical studies with in vivo experiments.26 In vitro studies are frequently done in order to obtain drug release profiles. This kind of study usually requires an extensive series of experiments to optimize existing drug delivery devices and to design new systems to achieve desired therapeutic dosages. To evaluate the effectiveness of a drug to combat an ocular disease it is necessary to characterize the drug release kinetic from the delivery system, in this case soft contact lenses. Several studies presented in the literature quantify the accumulated mass of drugs released into a medium, simulating the eye environment over time. Periodically, small aliquots are removed for drug quantification from the release medium in which the lens is immersed. Care is taken to maintain a constant volume, and the aliquot used for drug quantification is restituted to the release medium after being analyzed, or an equal volume of fresh medium is added. This sequential manual sampling is time-consuming and limited data is available from these experiments. For this reason the implementation of an experimental technique allowing continuous monitoring of the drug concentration in the release medium is attractive. In this study a recirculation loop of the release medium passing continuously through the analytical equipment for drug quantification was used in the kinetic experiments. The imposed recirculation flow rate was not high and does not significantly affect the hydrodynamic characteristics of the release medium. The delay in data caused by the recirculation loop was not significant because a small loop volume was used. After the start up, the proposed experiments did not need any human participation since the signal from the analytical equipment is continuously acquired and registered by a computer. The methodology used in this study avoids manual sampling and allows the collection of a large volume of experimental results, particularly at the beginning of the drug release process, which is essential for an accurate determination of diffusion coefficients. As a consequence of this simple kinetic experiment implementation, a significant number of similar experiments were performed and a statistical treatment was possible, which is useful for comparing diffusion coefficients for the delivery scenarios studied. It should be noted that individual dimension and weight characterizations of all of the lenses prepared were made, which also contributed to diffusion coefficient accuracy. There is a lack of essential parameters as drug diffusion coefficients in polymeric matrices to support accurate predictions from available drug delivery models. For this reason it is expected that this work will contribute to providing the scientific community working in therapeutic lens for glaucoma treatment with precise measurements of a parameter which dictates prediction accuracy.

which represents the symmetry condition. The other boundary condition states that drug concentration at the polymeric device surface is not time-dependent and is equal to CAs: CA(x = S , t ) = CAs

(5)

S represents half of the geometry thickness. With this simplification an analytical solution of eq 2 is possible, applying Laplace transforms or using dimensionless variables and solving the problem by the method of separation of variables23,24 CA(x , t ) − CAi 4 =1− CAs − CAi π

∞

∑ n=0

⎡ (2n + 1)πx ⎤ ( −1)n cos⎢ ⎥ ⎣ ⎦ 2n + 1 2S

⎡ (2n + 1)2 π 2Dt ⎤ exp⎢ − ⎥ ⎦ ⎣ 4S2

(6)

Equation 6 represents the distribution of drug concentration within the delivery system for each instant of time t. This equation was used also by Wall et al.25 to obtain solute diffusion coefficients from suspended porous discs in a large volume of solvent or solutions of different solute concentrations. The authors clearly presented the conditions for eq 6 to be valid and referred to the possibility of applying the methodology to polymeric materials. The drug flux at hydrogel surface (x = S ) leaving the delivery system at time t is obtained from JA = −D

∂CA (x = S , t ) ∂x

(7)

and from a material balance −

dm = JA dt

(8)

where m represents the total amount of diffusion specie within the hydrogel at instant t. A time integration of eq 8, with JA from eq 7 and CA replaced by eq 6, allows the computation of the total amount of drug released during the period of time t (Mt) as Mt =1− M∞

∞

∑ n=0

⎡ (2n + 1)2 π 2Dt ⎤ 8 exp ⎥ ⎢− ⎦ ⎣ (2n + 1)2 π 2 4S2 (9)

An equivalent equation can be expressed in terms of the complementary error function (ierfc) as ∞ ⎛ n S ⎞⎤ ⎛ Dt ⎞1/2 ⎡ 1 Mt ⎟⎥ = 2⎜ 2 ⎟ ⎢ 1/2 + 2 ∑ ( −1)n ierfc⎜ ⎝ S ⎠ ⎢⎣ π ⎝ 2 Dt ⎠⎥⎦ M∞ n=0

(10)

and taking the limit as t tends to zero, the following approximation of eq 10 might be applied for short times:23 ⎛ Dt ⎞1/2 Mt = 2⎜ 2 ⎟ ⎝ πS ⎠ M∞

(11)

This approximation of Fick’s second law solution applied to thin films with uniform initial drug concentration is valid for short times and represents a special case of the power law where the exponent n is equal to 0.5, indicating that diffusion is the controlling mechanism of drug release. From eq 11, the fraction of drug released is linearly dependent with the square root of time, and the drug release profiles obtained from kinetic experiments could be used to

2. MATERIALS AND METHODS 2.1. Materials. Two different compositions of polyHEMA based hydrogels were prepared: one with a small amount of MAA and another with a small amount of MMA. All of the 2282



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absorbent paper. In the beginning the procedure was repeated every 30 min, and then the time periods became longer until sample weight was constant. The equilibrium water content was determined for the two prepared hydrogels (poly(HEMA-coMAA) and poly(HEMA-co-MMA)), and five swelling experiments of each were carried out to calculate an average value and the associated standard deviation. 2.3. Release Medium Characterization. An aqueous solution of NaCl used as the release medium was prepared several times, with an average concentration of 9.03 mg mL−1 and a standard deviation of 0.02 mg mL−1. The solution pH was measured every time a new release medium was prepared. The average value obtained (without adjusting the pH) with the corresponding standard deviation was 5.60 ± 0.06. 2.4. Timolol Maleate Release Experiments. The drug release experiments were carried out in an experimental setup (schematically represented in Figure 1) allowing the monitoring of timolol concentration in the release medium.

monomers were purchased from Acros Organics and had a purity greater than 97 %. Ethylene glycol dimethacrylate (EGDMA) from Acros Organics was used as the cross-linking agent in the copolymerization. The reaction was initiated by adding α−α’azoisobutyronitrile (AIBN) purchased from Fluka. Timolol maleate salt (98 %) was used as the drug in the release process, purchased from Sigma-Aldrich. The release medium was prepared by dissolving sodium chloride (99.5% from Panreac) in distilled water to obtain a concentration of 9 mg mL−1. 2.2. Hydrogel and Sample Preparation and Characterization. An amount of the monomer (MAA or MMA) equivalent to 3 % (w/w) was dissolved in HEMA, together with EGDMA and AIBN (mole fraction of 0.5 % and 0.25%, relative to the monomer quantity, respectively) and mixed in a small beaker. When the mixture was completely homogenized this was divided into two equal portions: one used to prepare blank hydrogels and the other used to prepare hydrogels with the drug incorporated (0.4 %, w/w). The prepared solutions were immediately injected into molds. The molds were made of two glass plates firmly tightened together (to prevent solution leakage) and separated by a frame of a silicone membrane (0.5 mm thick). The inner surface of the glass (in contact with copolymers) was covered with a polypropylene sheet to facilitate hydrogel removal after polymerization. All of the molds were placed in an oven at 60 °C for 24 h, in order to guarantee the same polymerization conditions for all the hydrogels prepared. After removal from the molds, the hydrogel sheets were cut into small discs (18 mm diameter), a dimension similar to commercial contact lenses. To facilitate the cutting operation, the hydrogel sheets were placed in a humid atmosphere until they became soft. In order to characterize the lenses obtained, each of them was weighed and measured individually. Diameter and thickness measurements were performed in the four quadrants of each disc and a mean value was used to characterize the geometry. Knowing the average thickness value of each lens used in the experiments is essential when applying eq 11 to calculate the drug diffusion coefficient. All the lenses were stored in desiccators which were wrapped in aluminum foil for light protection. About ten lenses obtained from each hydrogel poly(HEMA-co-MAA) and poly(HEMA-coMMA) sheet were reserved to be saturated with timolol maleate by soaking. The lenses were immersed in 2.5 mL of an aqueous solution with 1 mg mL−1 of timolol maleate, in small vials protected from light with aluminum foil. The amount of timolol maleate in the initial solution and in the medium after 65 h and (140 to 165) h was determined by spectrophotometry. The drug absorbed by the lenses was calculated as the difference between the amount of timolol maleate in solution at the beginning and in the sample collected from the medium. Since the amount of drug absorbed after (140 to 165) h was similar to the amount predicted for each lens when the timolol maleate was occluded in the polymer solution, the absorption process was interrupted. After that the lenses were dried (for 24 h at 60 °C) and stored in desiccators wrapped in aluminum foil. The swelling kinetics was studied for lenses made from each prepared dry hydrogel. After being weighed the samples were placed in 5 mL of NaCl solution in vials which were immersed in a thermostatic bath maintained at (36 ± 0.1) °C. At different times the samples were weighed after careful wiping with

Figure 1. Experimental setup used in drug release experiments. 1, glass flask with release medium for drug delivery; 2, spectrophotometer with a flux cell; 3, peristaltic pump; 4, thermostatic bath; 5, magnetic stirrer.

Each experiment began by placing the lens in 50.00 mL of NaCl solution contained in a glass flask (1). The NaCl solution flowing in a closed circuit was pumped from the glass flask and forced to pass continuously through a quartz flux cell (the cuvette) inside the spectrophotometer (2) where the absorbance of the release medium was measured. A peristaltic pump (3) maintained a liquid flow rate of 0.186 ± 0.002 mL s−1 in a closed circuit during the experiment. The temperature of the release medium was nearly constant at 36 °C, which is human body temperature. To maintain the temperature in the solution inside the glass flask a stream of hot water, coming from a thermostatic bath (4) circulated through a flexible plastic tube placed around the flask. A magnetic stirrer (5) was used to promote agitation of the release medium in order to obtain a homogeneous solution. A spectrophotometer (Spectronic Unicam, Helios Gamma) is connected by an RS 232 cable to a computer with a data acquisition system. The absorbance value of the release medium was registered every 300 s for 24 h (or 48 h), the duration of a drug release experiment. Before extensive use of the procedure presented in the kinetic experiments of timolol maleate release, care was taken to compare data with that obtained from the traditional methodology used to quantify drug concentration in the release medium. Ten experiments with continuous monitoring of the release medium absorbance were carried out with lenses from both hydrogels with timolol maleate incorporated by occlusion. The evolution of the average timolol maleate concentration over time is represented in Figure 2a,b, together with the results 2283



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Lenses without timolol maleate incorporated (blank lenses) were also used in the experiments to quantify any possible substances extracted from the hydrogels, such as unreacted monomers, initiator or cross-linker that could interfere in the absorbance at 292 nm. An average absorbance value of all the experiments performed with the same kind of blank lenses, poly(HEMA-co-MAA) or poly(HEMA-co-MMA), was calculated every 300 s. The average profile obtained with blank lenses was used to correct the data acquired in the experiments conducted to evaluate the drug release from lenses with timolol maleate added by occlusion. The correction was not applied to data obtained from lenses with timolol maleate added by soaking because lenses were immersed in the drug solution for several days and possible unreacted substances were released. Instead, the correction was introduced in the absorbance value measured in the sample collected from the medium at the end of drug absorption process.

3. RESULTS AND DISCUSSION When a hydrogel is immersed in a solvent such as an aqueous NaCl solution, it starts to diffuse through the polymeric network by osmosis (a slow process) followed by a progressive swelling of the polymer mass. The polymeric chains respond with elastic retractive forces resisting the expansion strain, and the swelling equilibrium is reached when osmotic forces equal the viscoelastic restoring forces.27 The amount of water uptake in the hydrogel can be computed using eq 12 w − w0 water content (%) = w0 (12)

Figure 2. Evolution of release medium concentration in timolol maleate over time during kinetics experiments performed with continuous monitoring and periodic sampling for (a) poly(HEMAco-MAA) lenses and (b) poly(HEMA-co-MMA) lenses with timolol maleate loaded by occlusion.

where w is the weight of the hydrogel sample at time t and w0 is the initial weight of the hydrogel sample (when it is dry). Figure 3 presents the dynamic swelling behavior of poly(HEMA-co-MAA) and poly(HEMA-co-MMA) hydrogels

obtained from experiments performed in similar conditions with periodic sampling of the release medium. The samples were collected every 45 min over 9 h and after an absorbance measurement they were restituted to the release medium in order to prevent volume changes. Figure 2a,b shows that the results obtained from the two experimental techniques are almost coincident, the proposed technique being much more advantageous as the volume of data obtained is greater with no need of human participation. The evolution of timolol maleate concentration in the release medium over time is obtained using the absorbance data recorded during the experiment and the calibration curve obtained previously. The absorbance is linear with timolol maleate concentration in the range (0 to 0.03) mg mL−1, and the calibration curve was obtained for the wavelength of maximum absorbance of 292 nm (y = 22.650 x + 5.2596 × 10−4, where y is the absorbance and x is the timolol maleate concentration in mg mL−1). Several release experiments (8 to 11) with lenses obtained from the same sheet of polymer were performed for the purpose of applying a statistical analysis to the obtained values of diffusivity and to check data reproducibility. Lenses cut from the hydrogel sheets of poly(HEMA-co-MAA) and poly(HEMAco-MMA) with the drug incorporated before polymerization were used in experiments carried out in similar conditions. With the objective of studying the effect of adding the timolol maleate by soaking, the experiments were repeated with the poly(HEMA-co-MAA) and poly(HEMA-co-MMA) lenses after being saturated in a drug solution.

Figure 3. Swelling behavior of poly(HEMA-co-MAA) and poly(HEMA-co-MMA) lenses immersed in NaCl solution (pH 5.60 ± 0.06) at 36 °C ± 0.1 °C.

samples, at 36 °C ± 0.1 °C. The swelling equilibrium appears to be reached approximately 2 h after sample immersion in the NaCl solution. The maximum water content obtained for poly(HEMA-co-MAA) hydrogel was 46.6 % ± 0.3 %, slightly higher than the 43.8 % ± 0.2 % correspondent value for poly(HEMA-co-MMA) hydrogel. Although the percentage of hydrophilic monomer (MAA) in the hydrogel prepared is not high (3 %, w/w), the increased water uptake may be due to the greater number of hydrogen bonds formed. The water uptake rates were calculated from swelling data for both hydrogels. A value of (0.725 ± 0.026) mg mg−1 h−1 was 2284



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Table 1. Characteristics of the Lenses (With Different Compositions and Drug Incorporation Methods) Used in the Experiments diameter/mm poly(HEMA-co-MAA)

blank drug occluded drug absorbed blank drug occluded drug absorbed

poly(HEMA-co-MMA)

17.86 17.09 16.81 16.75 17.00 16.89

Table 2. Drug Load for the Lenses Used in the Experiments

poly(HEMA-coMAA) poly(HEMA-coMMA)

occlusion soaking occlusion soaking

mass of drug/mass of lens/mg g−1

± ± ± ±

4.47 5.61 ± 0.12 5.29 5.60 ± 0.15

0.66 0.83 0.70 0.83

0.01 0.02 0.02 0.02

0.24 0.10 0.10 0.06 0.11 0.15

thickness/mm 0.53 0.52 0.54 0.52 0.52 0.53

± ± ± ± ± ±

0.01 0.01 0.01 0.00 0.01 0.01

mass/mg 164.7 147.1 148.8 143.2 132.0 148.5

± ± ± ± ± ±

4.4 1.7 4.1 2.1 3.2 2.2

As can be seen from Figures 4 and 5 the amount of timolol maleate released from the polymeric matrix is smaller when the drug was added by soaking, indicating a stronger bonding drug/ polymer or less solvent accessibility to drug sites due to structural changes in polymeric chains. The smaller initial delivery rates of timolol maleate when it is added by soaking is also the consequence of a lower affinity of the drug to the release medium. The percentages of timolol maleate loaded into the polymeric matrices released into the NaCl solution (maintained at 36 °C) for the different situations were calculated and are indicated in Table 3 with 95 % confidence intervals. As can be seen from Table 3, the percentage of timolol maleate released into the medium during experiments is significantly smaller when the drug was incorporated into the hydrogel by soaking. This happens even when the initial mass of drug added per mass of lens is slightly higher than the drug added to polymeric mixture before polymerization (see Table 2). The inclusion of a hydrophilic modifier (the monomer MAA) in the hydrogel composition results in almost 6 % more timolol maleate released when it was occluded in the polymeric matrix, but it seems to have no influence when the drug is added by soaking. In order to evaluate whether diffusion of timolol maleate in polymeric matrices is the controlling release mechanism, the exponent n from the power law (eq 1) was obtained from the several sets of data. As stated earlier, the correlation between log10(Mt/M∞) and log10(t) will be linear with slope equal to n. For the sake of illustration, Figure 6 shows this kind of representation together with the best linear fit obtained for one of the release experiments performed with a poly(HEMA-co-MAA) lens where the timolol maleate was incorporated by occlusion. A very good correlation was obtained for all the experiments performed. Table 4 summarizes the average values obtained (with 95 % confidence intervals) in the experiments with both poly(HEMA-co-MAA) and poly(HEMA-co-MMA) lenses having timolol maleate incorporated by occlusion and soaking. As can be seen from Table 4 the average values of the exponent are essentially the same for all conditions used and nearly equal to 0.6. This indicates that the diffusion of timolol maleate is probably influenced by polymer swelling, but in a small extent. Because only the initial time of release was considered (corresponding to a Mt/M∞ ratio less than 0.6) the swelling of polymeric matrices was considerable as discussed before, the first hour of immersion in the release medium having more than 93 % of the total water uptake. As the diffusional mass transport of timolol maleate was essentially the controlling mechanism, Fick’s second law can be used to describe the drug release process. Consequently, at the first stages of the delivery process, eq 11 is applicable and can be used to calculate timolol maleate diffusion coefficient. The

obtained for the initial water uptake rate per unit mass of polymer for the poly(HEMA-co-MAA) lenses, while the correspondent value was slightly smaller for the poly(HEMAco-MMA) samples, (0.691 ± 0.031) mg mg−1 h−1. The lower initial capacity of poly(HEMA-co-MMA) polymer to absorb water reflects the more hydrophobic characteristics of monomer MMA. After 1 h the water uptake rates are already very small (less than 0.035 mg mg−1 h−1) for both hydrogels, indicating that further swelling is no longer significant. As was noted earlier, one of the advantages of the approach used in this study is the dimension characterization of the individual prepared samples. Each lens was also weighed after having been prepared and dried. Table 1 summarizes the dimensions and weight of the samples used in the experimental work, presenting the average values with 95 % confidence intervals. The lenses with drug incorporated by occlusion were obtained by dispersing an amount of timolol maleate in the monomers solution before polymerization. Assuming uniform distribution in the polymeric matrix, it is possible to calculate the drug load in each lens. The average mass of drug added per lens and the mass ratio of timolol maleate incorporated by occlusion and soaking into the lenses used in the experimental study is presented in Table 2; when it is applicable a 95 % confidence interval is indicated.

mass of drug per lens/mg

± ± ± ± ± ±

Drug release experiments conducted in similar conditions with hydrogel samples of different compositions with timolol maleate incorporated in different ways allowed several sets of data to be collected. Figures 4 and 5 show the release profiles of timolol maleate from poly(HEMA-co-MAA) and poly(HEMAco-MMA) lenses, respectively. In each figure the results obtained for both methods used to incorporate the drug into the polymeric matrices are presented: occlusion and soaking. Not all data have been shown in Figures 4 and 5, to facilitate the visualization of the error bars representing the standard deviation for the set of (8 to 11) similar experiments performed. Although the data presented correspond to a period of 24 h, the majority of the experiments performed had a duration of 48 h. Care was taken to verify if the mass of drug in the release medium during the following 24 h was maintained and a variation of less than 5 % was observed. 2285



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Figure 4. Timolol maleate release profile in NaCl solution (pH 5.60 ± 0.06) from poly(HEMA-co-MAA) lenses at 36 °C. The drug was loaded into the polymeric matrix by occlusion and soaking.

Figure 5. Timolol maleate release profile in NaCl solution (pH 5.60 ± 0.06) from poly(HEMA-co-MMA) lenses at 36 °C. The drug was loaded into the polymeric matrix by occlusion and soaking.

Table 3. Percentages of the Initial Timolol Maleate Load into the Lenses That Were Released during the Experiments percentage of initial timolol maleate load released poly(HEMA-co-MAA) poly(HEMA-co-MMA)

occlusion

soaking

76.6 % ± 1.7 % 70.8 % ± 1.7 %

49.0 % ± 1.7 % 48.7 % ± 3.5 %

representation of Mt/M∞ versus t1/2, from data until Mt/M∞ = 0.6, gives a very good linear correlation for all experiments performed. An example of such representation is in Figure 7 that corresponds to a release experiment performed with a lens made of poly(HEMA-co-MAA) with timolol maleate incorporated in the polymeric matrix by occlusion. Ten more experiments were carried out using lenses with similar characteristics. A graphical representation of the diffusion coefficient values obtained is presented in Figure 8 using a box and whisker plot. This type of plot was selected because it combines a display of all the calculated values with a statistical summary, which is

Figure 6. log10(Mt/M∞) versus log10(t) obtained from data until Mt/ M∞ = 0.6 for a release experiment performed with a poly(HEMA-coMAA) lens with timolol maleate loaded into the polymeric matrix by occlusion.

useful for comparing diffusion coefficients for the delivery scenarios studied. The central box corresponds to the range of values between the upper and lower quartiles (75 % to 25 % 2286



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Table 4. Average Values of Power Law Exponent n Obtained from All Data Acquired during the Initial Period of Experiments

interquartile range and inside the upper quartile plus 1.5 times the interquartile range. For this reason, all values were considered when the average values of timolol maleate diffusion coefficients were computed (× in Figure 8). The values obtained for the four delivery scenarios studied experimentally are indicated with the correspondent 95 % confidence intervals in Table 5.

exponent n value poly(HEMA-coMAA) poly(HEMA-coMMA)


0.60 0.64 0.58 0.61

± ± ± ±

0.02 0.05 0.02 0.04

correlation coefficient range 0.9820 0.9912 0.9909 0.9961

to to to to

0.9991 0.9981 0.9997 0.9993

Table 5. Average Values of Timolol Maleate Diffusion Coefficient for the Delivery Scenarios Used with Indication of the Correlation Coefficient Range for the Least Square Lines Obtained diffusion coefficient, 1012, D/m2 s−1 value poly(HEMA-coMAA) poly(HEMA-coMMA)


1.74 1.49 1.66 1.11

± ± ± ±

0.14 0.39 0.14 0.26

correlation coefficient range 0.9916 0.9888 0.9913 0.9947

to to to to

0.9986 0.9990 0.9995 0.9995

The method used to incorporate timolol maleate into the polymeric matrices (occlusion or soaking) has a significant influence later in the release process, shown in Figures 4 and 5, and now corroborated by the diffusion coefficients obtained in both scenarios for the hydrogels used. When the drug was incorporated by occlusion in the hydrogels the value of the timolol maleate diffusion coefficient was higher than when the soaking method was applied to incorporate the drug. The increase was about 17 % for poly(HEMA-co-MAA) lenses and 49 % for poly(HEMA-co-MMA) lenses, indicating that drug release will be easier when it is added before polymerization. When the drug is added to the monomers solution, the interactions between their chains promoted by the cross-linker during the polymerization occur with the timolol maleate present. A complex drug-monomer group is formed and the resulting polymer network structure seems to facilitate drug diffusion when it is immersed in a release medium. Some authors6,28,29 refer to structural differences in polymeric matrices in the cases where chain networks are originated in the absence of a drug. Alvarez-Lorenzo and co-workers6 studied

Figure 7. Mt/M∞ versus t1/2 obtained from data until Mt/M∞ = 0.6 for a release experiment performed with a poly(HEMA-co-MAA) lens with timolol maleate loaded into the polymeric matrix by occlusion.

percentile) and the middle line represents the median (50 % percentile). The vertical lines (the whiskers) extend from the minimum to the maximum value after excluding the outliers. A total of 25 % of the diffusivity values are distributed between the top of the central box to the maximum value and 75 % of the values are between the top of the central box and the minimum value. Information about the distribution of diffusion coefficients values in its domain is available from this kind of graphical representation. From Figure 8 it is clear that the dispersion of the diffusion coefficient values calculated is greater when the timolol maleate was added to the lens by soaking. Although there was a high dispersion of results no mild outlier was observed. All diffusion coefficients values calculated from experiments (symbols in Figure 8) were inside the lower quartile minus 1.5 times the

Figure 8. Box and whisker representation of timolol maleate diffusion coefficient in poly(HEMA-co-MAA) and poly(HEMA-co-MMA) lenses when the drug was loaded into the polymeric matrix by occlusion and soaking. Symbols correspond to experimental values and × represents the average value. 2287



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The value obtained experimentally for the exponent of the power law was nearly 0.6 for all of the scenarios studied. This parameter is a characteristic of the drug release mechanism. The value indicates that the polymer swelling effect is small and Fickian diffusion was considered as the dominant mechanism. The percentage of the timolol maleate loaded into the polymeric matrices that was released during experiments was significantly higher (in all cases greater than 20 %) when timolol maleate was added to polymeric mixture before polymerization. Probably this observation is the result of structural differences in polymeric matrices when chain networks are originated in the presence of drug. From the timolol maleate diffusion coefficients obtained for the delivery scenarios studied it was possible to conclude that: (i) when the occlusion method is applied to incorporate the drug into the hydrogels the values are higher, indicating that the drug release will be easier; (ii) the effect of hydrogel composition in the timolol maleate diffusion coefficients seems to be more pronounced when the drug was added by soaking; and (iii) the poly(HEMA-co-MAA) polymer, with greater hydrophilic characteristics, presented higher values than poly(HEMA-co-MMA) lenses, which are more hydrophobic. The interactions between timolol maleate and the polymeric networks affect the diffusional mobility of drug molecules. For that reason, the experimentally determined diffusion coefficients are apparent quantities and constitute valuable parameters to obtain accurate predictions from available models for the scenarios studied. When different membrane compositions and other polymerization and release conditions are considered, extensive experimental determinations must be done in order to obtain new parameters. The experimental technique proposed represents an efficient and attractive way of doing that.

the influence of the polymer composition on the timolol maleate loading capability of weakly cross-linked HEMA hydrogels. They also evaluated the applicability of an imprinting technique to reload soft contact lenses for timolol maleate administration. The results obtained from the dynamic mechanical analysis performed with hydrogels containing MAA and MMA groups and synthesized with timolol maleate (imprinted lenses) and without timolol maleate (nonimprinted lenses) indicated structural modifications in the polymeric networks. The modification of inter and intrachain interactions observed suggested that when hydrogel polymerization occurs with timolol maleate the interactions and relative positions between MAA or MMA groups are affected. Probably these structural changes in polymeric chains referred by the authors result in a greater accessibility to drug sites and the diffusion coefficients are higher when the drug was occluded, as observed in the present study. From Figure 8 and Table 5 the effect of hydrogel composition in the timolol maleate diffusion coefficients is also evident. Poly(HEMA-co-MAA) lenses, with greater hydrophilic characteristics, presented higher values of timolol maleate diffusion coefficients than poly(HEMA-co-MMA) lenses, which are more hydrophobic. This observation is valid for both methods used to incorporate the drug, although the increase in the diffusion coefficient is more significant (about 34 %) when timolol maleate was loaded by soaking. The same conclusion was made by Garcı ́a et al.30 when they studied the influence of poly(HEMA-co-MAA) hydrogel composition (prepared by free-radical polymerization) in the swelling and the timolol maleate release at 37 °C. The authors stated that as the MAA content in the hydrogel increases the swelling and drug diffusion coefficients are higher due to the increase in carboxylic acid groups, ionized groups allowing the absorption of a great amount of water. According to Reinhart and Peppas31 larger swelling ratios of the polymeric matrices leads to higher drug diffusion coefficients in the polymeric matrices. Garcı ́a et al.30 obtained values of timolol maleate diffusion coefficients of (0.53 ± 0.02) × 10−11 m2 s−1 at the early stages of drug release at 37 °C from poly(HEMA) membranes. Although direct comparison is not possible because the hydrogel composition and the conditions used during polymerization and drug incorporation are different, the diffusion coefficients obtained in the present study are of the same order of magnitude.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +351 239790200 (ext. 308). Fax: +351 239790341. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Elsa A.N. Ribeiro for the kinetic experiments performed with periodic sampling to drug concentration measurement in the release medium. The authors would also like to thank Particles, Polymers and Biomaterials Technology group from Chemical Process Engineering and Forest Products Research Centre (CIEPQPF), Department of Chemical Engineering, Faculty of Sciences and Technology of University of Coimbra for their helpful suggestions in hydrogel manufacturing.

4. CONCLUSION This study presents an attractive experimental technique allowing continuous monitoring of drug concentration in the release medium during kinetic experiments which are usually performed for the development of new drug delivery devices based in polymeric supports. After an initial period the experimental methodology does not require any human presence. Volume changing of the release medium during drug concentration measurement which occurs in the traditional procedure used in drug kinetic experiments is also eliminated with the proposed experimental setup. The human error in drug concentration measurements is diminished and a large volume of experimental results will become available for an accurate determination of diffusion coefficients. A significant number (8 to 11) of similar kinetic experiments were performed to study the release of timolol maleate from poly(HEMA-co-MAA) and poly(HEMA-co-MMA) hydrogels when the drug was incorporated either by occlusion or soaking.

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REFERENCES

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Diffusion Coefficients of Timolol Maleate in Polymeric Membranes

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