Drug transport in stimuli responsive acrylic and methacrylic interpenetrating polymer networks

The interpenetrating polymer networks (IPNs) are recently gaining attention as sustained drug delivery systems because they could ensure a proper combination of functionality and network density to control the drug release profiles. This study aims to reveal how the functionality of two IPNs based on polyacrylamide and respectively poly(acrylic acid) (PAA) and poly(methacrylic acid) (PMAA) influences their smart behavior as well as their properties as delivery systems of the cationic drug verapamil hydrochloride (VPM). The “extra” α-methyl group of PMAA results into a loss of the temperature sensitivity in the studied region and changes the pH responsivity of the PMAA/PAAM IPNs as compared to the PAA/PAAM IPNs. Moreover, the VPM diffusion in both IPNs depends on their composition due to the change in their functionality as well as of their network density. The “extra” α-methyl group of PMAA defines its enhanced hydrophobicity and hence influences the VPM diffusion mechanism. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 134, 45380.


INTRODUCTION
In the recent years polymer based vehicles for modified drug release are an object of intensive work because of their many advantages as smart drug delivery systems. The drug delivery potential of a polymeric material depends on the drug release rate as well as on the extent of drug release/unloading. These two parameters are controlled by the drug solubility in the release media as well as by the drug diffusion in the polymer matrix. They could be additionally tuned via change in temperature and pH of the media when stimuli responsive drug delivery systems are used. Thus, smart polymer materials are increasingly gaining attention and their potential in the area of modified drug delivery is gradually being revealed although it is still not fully exploited at the moment.
It is well known from the literature that polymeric systems based on poly(acrylic acid) (PAA) and polyacrylamide (PAAM) are simultaneosly pH 1 and temperature responsive (exhibiting upper critical solution temperature, UCST, type behaviour 2,3 ). The presence of pendant COOH groups in PAA defines their pH-sensitivity: at pH > pKa PAA~4 .5 4 these groups are deprotonated, which results into their mutual electrostatic repulsion and increased swelling ability of the polymeric material. Hence, such materials could ensure an enhanced drug diffusion towards the release media as the pH changes from acidic, e.g. stomach-like conditions, to neutral, i.e. intestine like conditions. For example, this effect was utilized when developing a controlled release system for the antihypertensive drug losartan potassium 4 . With time, more and more carboxylic groups from PAA became deprotonated which resulted into a gradual increase in the polymer network swelling and thus to a prolonged drug release up to 34 hours. 4 The temperature sensitivity of PAA/PAAM based materials is due to the hydrogen bonding between their side chain pendant groups: -COOH and -NH2. At temperatures below UCST, the hydrogen bonds formed between these groups result into the collapse of the polymeric material. With increasing temperature, >UCST, the hydrogen bonds start to disrupt which increases the swelling of the material. Katono et al. 5,6 have studied in details the interpenetrating polymer networks (IPNs) of poly(acrylamide-co-butyl methacrylate) and PAA and revealed their potential as delivery systems for ketoprofen. At temperature above their UCST, these IPNs are in the so called "on" state (increased swelling ability) and thus start to release the loaded ketoprofen 5 . With decreasing temperature below UCST, a transition to almost completely "off" state is observed 6 . Similar "on-off" behaviour is also reported by Wang et al. 7 , who have utilized sequential IPNs from poly(acrylic acid)-graft-βcyclodextrin (PAAc-g-β-CD) and polyacrylamide (PAAM) for the controlled release of ibuprofen. The cyclic change of temperature from 25°C to 37°C, at constant pH, resulted into a pulsatile (modulated) drug release, following non-Fickian release mechanism of the ibuprofen. Thus, the polymer vehicle's smart behavior and its ability to change its structure and properties according to external stimuli, such as pH, temperature, ionic strength, etc., are lying behind the change of the mode of the drug transport into the polymer matrix and thus the drug release profile is controlled.
In a previous study, we have revealed the potential of IPNs of PAA and PAAM for the sustained release of verapamil hydrochloride (VPM) 8 . The tuning of the IPNs composition through varying the PAA/PAAM ratio allowed for modifying the VPM release profile. The best sustained release profile was observed for the IPN with the highest PAAM content: no initial burst effect occurred and ~ 90% of the drug VPM was released for over 25 hours. We have widen up our study by the synthesis of similar IPNs comprising the same side groups but replacing the poly(acrylic acid) (PAA) with poly(methacrylic acid ) (PMAA) and applied these new systems for the VPM sustained release 9 . The replacement of PAA with PMAA has changed the swelling ability of the IPNs due to the enhanced hydrophobicity of the latter, which has influenced the VPM release profile 8,9 . In this study we aim to directly compare both IPNs -PAA/PAAM and PMAA/PAAM and to reveal how the "small" change in the structure of one of the monomers would change their smart behavior. To this purpose the pH and the temperature responsiveness of both IPNs was studied. Moreover, as the "extra" α-CH3 group was expected to influence also the transport of the loaded cationic drug, the transport characteristics of the cationic drug VPM within both IPNs was compared.
Thus, the aim of the study is to reveal how the "extra" α-CH3 group in PMAA changed the smart behavior of and the drug (VPM) transport in PMAA/PAAM IPNs as compared to PAA/PAAM IPNs.

IPNs' synthesis
The procedure to obtain IPNs of PAA/PAAM and PMAA/PAAM is described elsewhere 8,9 . Briefly, a two stage sequential method was applied. At the first stage, a single network (SN) of the polyacid was obtained via free crosslinking radical polymerization of the acidic monomer (AA or MAA), MBAA (4 mol.%) and PPS (0.1 mol.%). Each of thus prepared SN hydrogels was purified in distilled water to completely remove traces from the nonreacted chemicals (the residuals were checked by UV). Then, the 2 nd (PAAM) network was in situ synthesized into the 1 st SN. To this purpose, dry SNs were transferred into aqueous solutions with different AAM concentrations (from 1M to 5M), containing also MBAA (0.1 mol.%) and PPS (0.1 mol.%). Each of the synthesized IPN's was washed out with distilled water to completely remove traces from the non-reacted chemicals (the residuals were checked by UV). Following this procedure, IPNs with different composition, i.e. weight fraction of the polyacid, were obtained using respectively PAA (Table 1) and PMAA ( Table 2).
The exact IPNs' composition was determined by: (i) titration of the residual acidic monomer (respectively AA or MAA) in the waste waters obtained after the 1 st stage (polyacid SN purification) and (ii) determination of the non-reacted AAM in the waste waters obtained after purification of the IPNs by using UV method for AAM determination. More details for the synthetic procedure and the determination of the IPNs' composition are provided in the Supplementary info.

Temperature responsiveness
Measurements of the equilibrium swelling ratio (ESR) for both types IPNs were performed at different temperatures in the range 20-65°C. To this purpose, dry disk-shaped samples with diameter 4.5 mm were left to swell at certain temperature in distilled water. After reaching an equilibrium (typically for 7 hours, as detected by the lack of change in their weight after this period), their weight in the swollen state was measured. ESR at a defined temperature was calculated by using the equation: Here, 0 TC swollen m and dry m are respectively the weights of the swollen at certain temperature sample and in dry state. The data were averaged for at least three pieces.

pH responsiveness
Measurements were performed in a similar to the above described way but using different pH values in the range from 2 to 10 at temperature 24±1°С. Briefly, dry disk shaped samples with diameter 4.5 mm were swollen in buffer media with defined pH. After reaching equilibrium (typically for 24 hours) the mass of each piece in its swollen state was measured. ESR was calculated for each pH value using the equation: Here, pH swollen m and dry m are respectively the weights of the swollen piece at certain pH and when dry. Buffer solutions, used throughout the experiment, were prepared following the procedure described by Pourjavadi et al 10 .

Drug loading and release
The VPM loading and release were described elsewhere 8,9 . Briefly, dry disk shaped samples with diameter of 8 mm were immersed in water solution of VPM with concentration 100 mg/ml, at temperature 24°C±1°C. After swelling for 24 h, the drug loaded samples were drawn from the loading media, their surface was gently washed out with distilled water and the samples were left at room temperature to dry.
The drug release was perfomed by using a dissolution test apparatus (Erweka DT 600, Germany). The USP paddle method was applied. The test was carried out at a paddle rotation speed of 50 rpm, in experiments. To this purpose, the VPM loaded IPNs were first immersed in 0.1 mol L -1 HCl solution (pH 1.2) for 2 h and then transferred into phosphate buffer solution (pH 6.8) for up to 24 h. 5 ml aliquots of dissolution media were withdrawn at selected intervals up to 24 h. Each sample was filtered through a 0.45-mm membrane filter (Sartorius cellulose acetate filter, Germany). The quantity of the drug in the sample solution was determined by UV spectroscopy at 278±2 nm using a Hewlett-Packard 8452 A Diode Array spectrophotometer, USA. The cumulative percentage of the drug release was calculated and the average of six determinations was used in the data analysis.

VPM transport in IPNs
The drug transport in both types of IPNs was studied by applying the Korsmeyer-Peppas model. The Korsmeyer-Peppas Model was developed especially for modeling the release of a drug molecule from a polymeric matrix, such as a hydrogel. This semi-empirical model assumes an exponential relation of the drug release and the elapsed time 11 . Thus, the type of the drug transport could be evaluated by applying the equation: where Mt stays for the amount of the drug released at time t; М  is the amount of the drug that should be released at infinite time, t=∞; kKP, is the rate constant, and n is the diffusion exponent, which value depends on the sample geometry and reveals the mode of the drug diffusion. This equation is valid when the release is one-dimensional and the sample width-to-thickness ratio is at least 10 12 . The relation between n values and the drug diffusion mode is presented in Table 3 13 .
From the diffusion exponent values, taking into account the cylindrical geometry of our IPNs samples, one could obtain the diffusion coefficient of the drug (VPM), using the equation: where KKP is the same from equation (3), π is the Ludolphine number (π=3.14), l is the sample height (in meters) and n is the diffusion exponent from equation (3).

6
The dependence of 0 TC ESR on temperature for IPN PAA/PAAM is presented in Figure 1. All studied IPNs PAA/PAAM are temperature sensitive and exhibit UCST as demonstrated by their ESR increase with temperature.
It is known from the literature, that for any system (copolymers, blends, IPNs, etc.), consisting of PAA and PAAM, an UCST is observed due to the hydrogen bond formation between PAA's carboxylic groups and PAAM's amide groups 2 (Scheme 1A). At temperature below the UCST, PAA preferentially forms interpolymer hydrogen bonds with PAAM, and the IPN PAA/PAAM hydrogels shrink due to the chain-chain zipper effect 14 . When the temperature becomes higher than UCST, the interpolymer hydrogen bonds between PAA and PAAM are disrupted and the IPN hydrogels swell through the relaxation of both polymer networks' chains. Therefore, the IPNs swell to higher ESR at temperatures above the UCST due to the disruption of the PAA/PAAM hydrogen bonds (Scheme 1B).
The IPNs with comparable content of PAA and PAAM, e.g. PA41 and PA28, show an abrupt change in their ESR when temperature increases ( Figure 1). This could be explained by the higher number of hydrogen bonds between PAA and PAAM upon which disruption the ESR increases stronger. On the contrary, when PAAM prevails (e.g. in IPN 19 and IPN22), the number of interpolymer hydrogen bonds decreases and the ESR increases continuously with temperature ( Figure 1). Similar behaviour we have observed also for the microgels of PAA/PAAM IPN 15 and were also reported by Katono et al. 5,6 . Thus, the number of the hydrogen bonds between PAA and PAAM below UCST defines the "strength" of the temperature response of the IPNs PAA/PAAM.
From Figure 1, it could be also concluded that the higher PAA content results into higher ESR of the IPNs PAA/PAAM. This trend is expected as PAA is charged at neutral pH (polyanion) and the repulsive forces between its COOanions keep the IPN network expanded when the PAA content is higher.
When the number of COO --containing monomeric units decreases, i.e. PAA content decreases, the swelling ratio of the IPNs is also expected to decrease.
The UCST values for the studied IPNs PAA/PAAM were determined as the inflection point of the respective experimental curve ( Figure 1) and the obtained values are presented as a function of the IPNs' composition in Figure 2. There, it could be seen that UCST decreases as the PAA content in the IPN PAA/PAAM increases. This is related, as discussed above, to the increased swelling ability of the IPNs PAA/PAAM in water as the PAA content increases. The PAA interaction with water is enhanced either through an increased number of hydrogen bonds between the PAA and water molecules or by the enhanced COOsolvation by water molecules, which together with the repulsive forces between adjacent COOresults into higher swelling ability of the IPNs.
In summary, the temperature responsiveness of the IPNs PAA/PAAM is defined by the balance between polymer-polymer vs. polymer-solvent (water) interactions. As the IPNs' composition varies, this balance changes and the UCST increases from 38 to 47 o C ( Figure 2). The IPNs' composition defines the number of the hydrogen bonds formed between PAA and PAAM in the IPN. That is why, as the ratio between both components declines from ~1:1, the ESR dependence on temperature changes from discontinuous to continuous.
If one compares the ESR increase under both temperature and pH increase, it could be concluded that the IPNs PAA/PAAM are more sensitive towards changes in pH rather than in temperature as their ESR changes stronger when pH increases. This strong pH dependence of the ESR of the studied IPNs PAA/PAAM means that these materials are good candidates for drug delivery applications. The IPNs PAA/PAAM could be appropriate for oral drug delivery as they will prevent the drug release in the stomach (low pH, low ESR, shrunk IPNs) and will preferably release the drug in intestine-like conditions (neutral pH, 3 to 5 times increase in ESR as compared to the acidic pH), i.e. they could define a targeted release of the loaded drug in the intestines.
It was expected that IPNs of PMAA/PAAM will show pH responsiveness similar to the IPNs of PAA/PAAM as they also possess COOH groups. Figure  Thus, it could be concluded that the "extra" α-CH3 group in PMAA defined a continuous ESR increases as pH increases in contrast to the discontinuous way of ESR increase observed for PAA/PAAM IPNs.

Drug transport in IPNs PAA/PAAM and PMAA/PAAM
In the previous sections it was demonstrated that the replacement of PAA with PMAA into their IPNs with PAAM resulted into: (i) a loss of the temperature responsiveness in the studied temperature region and (ii) a change in the mode of the pH responsiveness from discontinuous to continuous one.
Here, the transport mode of the model cationic drug (VPM) in both "acidic" IPNs, differing in their hydrophobicity and smart behavior, will be compared. To this purpose, the Korsmeyer-Peppas model The dependence of n on the IPNs PAA/PAAM composition is presented in Figure 6A. As it was mentioned above, n values could be used to evaluate the mode of the drug diffusion into the polymer system (Table 3). According to Figure 6A, the VPM diffusion in both SNs PAA and PAAM is Anomalous diffusion means that the rate of the drug diffusion is comparable to the rate of the polymer chains relaxation, i.e. Rdrug ≈ Rrelax (Table 3) When one compare PA19 to the other IPNs samples (PA62 to PA23), the latter all exhibit Fickian diffusion of VPM ( Figure 6A). In addition, a gradual decrease in their n values is observed as the PAA content increases. Similar is the UCST dependence on the PAA content -it decreases as the PAA content increases (Figure 2). In fact, the UCST for PA41 and PA28 is ~37 o C, which is the temperature at which the VPM release was carried out, i.e. the temperature at which the drug diffusion is evaluated. At the UCST the hydrogen bonds between PAA and PAAM start to disrupt, which means that at this temperature most of PAA's carboxylic groups become free from the PAA-PAAM interaction and thus available for interaction with the VPM molecules. The interaction between VPM and PAA was revealed by IR spectroscopy (see the Supplementary info) to be mainly electrostatic and also via hydrogen bonds. This is in agreement by the reported by other authors ionic interactions between PAA containing IPNs 18 and cationic drugs.
Thus, it could be summarized that the diffusion of VPM in IPNs PAA/PAAM as PAA content increases is slowed down as compared to the polymer chain relaxation rates due to: (i) the enhanced interaction between the drug (VPM) and PAA as well as to (ii) the gradual change of the inner structure of the IPN hydrogels as the IPN's composition changes.
The slowdown of the VPM in the IPN as the PAA content increases is better illustrated in Figure 6B, where the dependence of the VPM's diffusion coefficient (D) on the IPN's composition is presented.
As PAA content in the IPNs increases, the diffusion coefficient of VPM gradually decreases, i.e. the VPM molecules diffuse slower and slower ( Figure 6B). This is related to the enhanced interaction between the cationic drug VPM and the acidic component of the IPN (PAA) as the content of the latter increases.
It should be mentioned that in this study we have preferably studied the VPM transport in IPNs with low PAA content because according to our previous investigations 8 , exactly these IPNs' compositions were better performing as VPM sustained release systems, PA19 being the best one among them.
This fact was explained there by the optimal combination of functionality (in terms of PAA content) and network density (in terms of PAA/PAAM ratio, defining the interlacing and hydrogen bond formation between them). Thus, for higher PAA content (when it was in an excess to PAAM), the VPM release was far from 100% -between 40 and 70% depending on the exact PAA amount 8    The results obtained within the current study confirm and also explain the mechanism behind the best performing systems for sustained VPM release from both IPNs of PAA/PAAM and PMAA/PAAM.
If one compares the best performing PAA/PAAM IPN and PMAA/PAAM IPN, it could be concluded that the anomalous diffusion of VPM in PA19 combined with its "smart" characteristics define better performance as VPM extended release vehicle as compared to PMA20.

CONCLUSIONS
The drug diffusion into IPNs of PAAM with poly (acrylic acid) and poly(methacrylic acid) is governed by the IPNs components nature as well as by the IPNs' composition. The replacement of the hydrophilic PAA with the more hydrophobic PMAA resulted into a loss of the IPNs' temperature sensitivity in the studied temperature region, change of the pH responsiveness mode and also into a loss of a clear diffusion exponent dependence on the IPNs' composition. Thus, the appropriate choice of the IPNs' components and composition could be used to finely tune their structure, properties and hence their characteristics as drug delivery systems.