The release of ibuprofen sodium salt from permanently porous poly(hydroxyethyl methacrylate-co-trimethylolpropane trimethacrylate) resins

Microporous and Mesoporous Materials 217 (2015) 133e140

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The release of ibuprofen sodium salt from permanently porous poly(hydroxyethyl methacrylate-co-trimethylolpropane trimethacrylate) resins Agnieszka Kierys a, *, Marta Grochowicz b, Patrycja Kosik a a b

Maria Curie-Sklodowska University, Faculty of Chemistry, Department of Adsorption, M. Curie-Sklodowska Sq. 3, 20-031 Lublin, Poland Maria Curie-Sklodowska University, Faculty of Chemistry, Department of Polymer Chemistry, 33 Gliniana Str, 20-614 Lublin, Poland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 February 2015 Received in revised form 3 May 2015 Accepted 9 June 2015 Available online 19 June 2015

This study describes a newly synthesized crosslinked polymer poly(TRIM) (trimethylolpropane trimethacrylate, TRIM) and copolymers poly(HEMA-co-TRIM) (2-hydroxyethyl methacrylate, HEMA) as well as polymeredrug conjugates based on them. The resins were produced in the form of permanently porous beads via suspension-emulsion polymerization. The set of polymeredrug conjugates was prepared by loading ibuprofen sodium salt (IBNa) into polymer matrices by their swelling. Our study involves the characterization of polymers and polymeredrug conjugates, studies on the swelling of resins in the selected solvents as well as characteristics of drug release from conjugates. The conducted analyses reveal that the internal structure, total porosity and swelling characteristics of resins strongly depend on the presence of 2-hydroxyethyl methacrylate. The release profiles of ibuprofen sodium salt from the polymer
drug conjugates demonstrate that there are differences in the rate and efficiency of drug desorption from conjugates. © 2015 Published by Elsevier Inc.

Keywords: Porous polymer particles Swelling Drug release Ibuprofen sodium salt (IBNa) 2-hydroxyethyl methacrylate (HEMA)

1. Introduction The preparation of the crosslinked polymeric microspheres of desired functionality attracts a lot of attention due to the wide range of their possible applications [1e5] as well as the intrinsic interest. The polymer materials play a crucial role as a specific organic support in different multicomponent organic-inorganic composites [6e13]. Thus, we wish to report on investigations concerning a series of newly synthesized crosslinked resins and polymeredrug conjugates based on them. Macroporous homopolymer made of 1,1,1-trimethylolpropane trimethacrylate (TRIM) is characterized by permanent pore structure and significant mechanical rigidity. The fairly hydrophobic character of the polymer results in its relatively low tendency to swell in different solvents, especially of hydrophilic character, and as a consequence, it is only slightly sensitive to the surrounding solvent [2,14e17]. The presence of unpolymerized methacrylate groups makes it possible to easily obtain the desirable physical and

* Corresponding author. Tel.: þ48 81 537 5563; fax: þ48 81 533 33 48. E-mail addresses: [email protected] (A. Kierys), mgrochowicz@ umcs.pl (M. Grochowicz), [email protected] (P. Kosik). http://dx.doi.org/10.1016/j.micromeso.2015.06.009 1387-1811/© 2015 Published by Elsevier Inc.

chemical properties of original poly(TRIM) polymer by grafting them with different functional monomers [4,18,19]. Simultaneously, by using TRIM as a multifunctional crosslinking agent and employing the suspension polymerization a wide range of copolymers of desired chemical character were fabricated and intensively investigated [3,20e22]. In addition, due to a large variation of the polymerization parameters, it is possible to perform engineering in a wide range of properties of the TRIM based copolymers, such as their morphology, swelling ratio or porosity. For these reasons, the poly(TRIM) matrix was employed as an efficient organic support for the polymeresilica nanocomposites [9]. The usefulness of the polymer materials in a variety of medical and biological applications is associated with their biocompatibility and non-toxicity. Numerous studies have confirmed that it is possible to form biocompatible and a versatile polymeric materials for biomedical applications with the use of hydrophilic monomer 2-hydroxyethyl methacrylate (HEMA) [23e25]. The number of poly(HEMA) applications is continuously increasing, especially in such fields as ophthalmology [26,27], vehicles for drugs delivery and plastic surgery [28e35]. Moreover, the poly(HEMA) based materials were also proposed for numerous other applications, among which the following are worth mentioning: the preparation

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of the hydrophilic modified poly(vinylidene fluoride) PVDF membranes by the in situ polymerization and micro-phase separation route [36]; the stimuli-responsive membranes [37]; the solid plastic poly(HEMA) doped with trivalent neodymium; and erbium [38]. Such favourable reports on the excellent properties of systems consisting of poly(HEMA) encouraged us to incorporate the HEMA monomer in the polymer matrix cross-linked with TRIM agent. The tremendous advantages of such poly(HEMA) based materials crosslinked with TRIM were noticed [39] and gained special attention in the preparation of the molecularly imprinted polymers (MIP) for molecular recognition [40]; quaternized copolymers exhibiting antibacterial activity against Escherichia coli and Staphylococcus aureus [41]; materials with the enhanced resistance to protein adsorption and cell adhesion with potential for artificial cornea applications [27]; and vehicles for immobilization and capsulation of 5-fluorouracil, an antimetabolic drug commonly used in cancer chemotherapy [42]. All the aforementioned poly(HEMA-co-TRIM) based materials were synthesized by bulk or UV polymerization and occurred in the form of blocks or films. The present article describes use of suspension-emulsion polymerization method [9,22,43] in producing permanently porous beads of poly(TRIM) and poly(HEMA-co-TRIM). In our approach, the investigation of the selected porous poly(TRIM) and poly(HEMAco-TRIM) beads is an introduction to preparing the polymeredrug conjugates. In this study, ibuprofen sodium salt (IBNa) was chosen as a model carboxylic drug. The salt form of the drug was chosen because it is known that, in the dissociation state, it generally exhibits higher physical stability and solubility than the corresponding unionized form [44,45]. The set of polymeredrug conjugates was prepared by loading ibuprofen sodium salt (IBNa) into polymer matrices by their swelling. The study involved the characterization of polymers and polymeredrug conjugates, the studies on the swelling of polymers in the selected solvents, and the discussion of the drug desorption from conjugates. The conducted studies (the low temperature N2 sorption together with spectroscopic techniques) provide insight into the changes and rearrangement of the internal structure of the poly(TRIM) and poly(HEMA-co-TRIM) as a result of the increase of the ratio of the HEMA to TRIM and after the introduction of drug molecules.

azobisisobutyronitrile was used as a polymerization initiator in the amount of 1%, based on the total weight of monomers. Polymerization was carried out in 80  C during 18 h; subsequently, the obtained beads were filtered, extracted with acetone in Soxhlet apparatus in order to remove any unreacted monomers and dried. The fabricated poly(TRIM) resin was denoted as HT01 and poly(HEMA-co-TRIM) copolymers with molar ratio of HEMA:TRIM 1:1 and 2:1 as HT11 and HT21, respectively. Dry resins were hermetically sealed, stored at ambient conditions and used as polymer matrices for loading of drug. 2.3. Preparation of polymeredrug conjugates A set of polymeredrug conjugates was prepared by loading of ibuprofen sodium salt (IBNa) into polymer matrices (HT01, HT11 and HT21) by swelling method. Firstly, drug solution was prepared by dissolving IBNa in anhydrous ethanol (EtOH, 34 mg/ml). Next, freshly prepared alcoholic solution was added drop by drop to polymer beads. The amount of the IBNa solution was adjusted so that it was fully absorbed during the polymer swelling. Afterwards, the polymer beads swollen in IBNa were immediately sealed hermetically and left for 3 h at room temperature. Subsequently, the solid product was dried at 50  C under vacuum for 6 h. It is clear that after the solvent evaporation, conjugates also retain the form of pellets. The polymeredrug conjugates were labelled as the HT01-IBNa, HT11-IBNa and HT21-IBNa, respectively. The final loading efficiency of the drug was estimated to be 51 mg/g for HT01-IBNa and 55 mg/g for HT11-IBNa and HT21-IBNa, taking into account the mass of the total carrier system. 2.4. Desorption of ibuprofen salt

2. Experimental section

The ibuprofen salt desorption experiments were performed under stirring at 280 rpm at 37 ± 0.1  C in a thermostated bath. Desorption profiles were obtained by soaking 200 mg of the polymeredrug conjugates in 50 ml of phosphate buffer solution (pH 7.4). At predetermined time intervals, 5 ml of release fluid was taken out for analysis and immediately replenished with fresh dissolution medium. The amount of the desorbed drug was determined by using a UV/Vis spectrophotometer (Varian Cary 100 Bio) at the wavelength of 222 nm. The final ibuprofen salt concentrations were corrected with respect to the dilution procedure.

2.1. Materials

2.5. Characterization methods

2-hydroxyethyl methacrylate (HEMA), trimethylolpropane trimethacrylate (TRIM), a,a’-azobisisobutyronitrile (AIBN), sodium dodecyl sulfate (SDS) and ibuprofen sodium salt (IBNa) were obtained from Sigma Aldrich. Solvents were purchased from POCh (Poland), and disodium hydrogen phosphate and sodium dihydrogen phosphate were obtained from Chempur (Poland). All reagents were analytical grade and used as received.

The morphology and internal structure of organic matrices and polymeredrug conjugates were examined using scanning electron microscope (FEI Quanta 3D FEG) working at 30 kV. The ability of the studied polymer and copolymers to swell in selected solutions i.e. toluene, anhydrous ethanol and anhydrous ethanol solution of ibuprofen sodium salt (34 mg/ml) was tested using Nikon SMZ1 500 stereoscopic optical microscope with NIS-elements software and automatic time-lapse imaging on the statistical sample of about 100 objects. The measurements were conducted using polymer beads fraction with diameter range from 100 to 200 mm. The diameter of the polymer beads was measured in the dry state (before immersion in the solution) and in the state of the maximum swelling, just before the solvent started to evaporate. The Vs, denoted as the volume of material beads after swelling, and the Vd, denoted as the volume of the dry beads of resin, were calculated from the measured average diameters of the polymer beads. Subsequently, the swellability coefficients B expressed as B ¼ [(Vs-Vd) * 100%]/Vd were determined in the aforementioned solution [48]. Parameters characterizing the porosity of polymers and conjugates (the specific surface area, SBET, the total pore volume, Vp, and

2.2. Preparation of porous polymer resins The poly(TRIM) and poly(HEMA-co-TRIM) resins were prepared in the form of porous beads via suspension-emulsion polymerization [21,43,46,47]. Polymerization experiments were performed in 0.25% w/w aqueous solution of sodium dodecyl sulfate, using toluene as the pore forming agent. The homopolymer poly(TRIM) was prepared only with trimethylolpropane trimethacrylate monomer, whereas poly(HEMA-co-TRIM) copolymers were obtained with the functional 2-hydroxyethyl methacrylate and TRIM in the molar ratio of HEMA:TRIM 1:1 and 2:1. The volume ratio of monomers to toluene was 1/1.5 [21,22,43]. In all cases, a,a’-

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the pore size distribution, PSD) were determined by nitrogen adsorption at
196  C using a Micromeritics ASAP 2420 analyzer. Prior to the experiment, all of the investigated samples were dried overnight at 60  C under vacuum. The pore size distributions (PSDs) were determined from both the adsorption and desorption branch of the nitrogen isotherm using the BarretteJoynereHalenda (BJH) procedure [49]. The presence of the drug in conjugates was verified by Raman spectroscopy. Raman spectra were collected using Raman microscope inVia Reflex from Renishaw (UK) which used a chargecoupled device (CCD) detector with a spectral resolution of 1 cm
1 for detection. Exciting radiation at 514 nm was provided by an Arþ laser at the cross-section of dried samples. The ATR-FTIR spectra were recorded on a Tensor 27 (Bruker) spectrometer equipped with a diamond crystal over the 4000e600 cm
1 range at the resolution of 4 cm
1 and maximum source aperture. Interferograms of 50 scans collected at room temperature were averaged for each spectrum. 3. Results and discussion 3.1. Analysis of texture and morphology The pore structure of the poly(TRIM) and poly(HEMA-co-TRIM) beads in the dry state was probed using the low-temperature nitrogen sorption porosimetry. The N2 sorption isotherms of HT01 depicted in Fig. 1a are of type IV, which is typical for mesoporous materials [50]. The adsorption and desorption branches of these isotherms do not coincide, which causes arising of a hysteresis loop of type H2, extended along the pressure axis. Such hysteresis loop is ascribed to materials of the highly interconnected pore network which consists of free volumes with alternating enlargements and constrictions [50]. Therefore, it may be expected that mesopores are heterogeneous in size in the investigated HT01 homopolymer. Suspension-emulsion polymerization of TRIM and HEMA comonomers with different molar ratio makes it possible to form porous resins in the shape of tiny beads. The increase of the content of the hydrophilic monomer strongly influences the internal pore network of resins. As a consequence, the shape of the nitrogen adsorption/desorption isotherms of copolymers substantially changes and the value of N2 adsorption decreases in comparison to the HT01 homopolymer (Fig. 1a). It should be noted that the HT21 resin displays an incomplete type II isotherm and a type H3 hysteresis loop. This type of isotherm is characteristic for non-porous or macroporous adsorbents, whereas the H3 type of hysteresis may be explained as a consequence of the presence of slit-shaped pores. It should also be emphasized that adsorption and desorption branches of the isotherm do not overlap in the lowest pressure range for all investigated resins. The irreversibility of gas adsorption may indicate some kinetics restrictions of the desorption, as well as

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the presence of irregularities and constriction in the polymer pore system. Moreover, according to the IUPAC, this phenomenon may be associated with the swelling of a non-rigid porous structure or with the irreversible uptake of molecules in pores (or through pore entrances) of about the same width as that of the adsorbate molecule [50]. The distribution of the pore size in the resins was computed from both the adsorption and desorption branches of the isotherm (Fig. 1b and c). In the case when only if the BJH pore size distributions are calculated from the desorption branch of the isotherm, the PSD is of bimodal character (Fig. 1c), while the PSD curves calculated from the adsorption branches (Fig. 1b) exhibit the broad distributions, and the peak centred at 3.8 nm is absent from the all curves. Similar findings were presented before for permanently porous polymer resins derived from multifunctional (meth)acrylate monomers [2,14]. Flodin and co-workers have noted that the size of smaller pores of diameter about 4 nm (determined from N2 sorption data) is independent both of TRIM and initiator concentrations, while the size of large pores (probed via Hg intrusion) is clearly dependent on monomer concentration. These observations have been attributed to a very regular structure of the polymer on the micro level scale. In conclusion, they stated that the type of the used solvent as well as the monomer to the solvent ratio are the most important factors, since they affect the pore structure [14]. On the other hand, according Sherrington et al. the pore size distributions of almost all TRIM-based resins cover a broad range from micro-through meso-to macropores [2]. Notwithstanding, the BJH pore size distributions calculated from the desorption branch of the isotherm always exhibit a sharp, often large maximum in the PSD curve. This may lead to the erroneous conclusion that a very narrow pore size distribution is present for such resins. Therefore, it has been postulated that the porosity data should be computed from the adsorption branches of N2 isotherms, since an artefact (which was attributed to a percolation process) arises in the pore size distributions if it is derived from the corresponding desorption data [2]. However, the pore size distribution of TRIM-based copolymers derived from the inverse size exclusion chromatography was also found to be of bimodal character. This technique, which makes it possible to determine the porous properties of the beads in a swollen state, exhibits the presence of two maxima on the PSD curve. They have been assigned to free volumes of micro and meso size present in the internal pore network of studied resins [21]. Moreover, the use of positron annihilation lifetime spectroscopy (PALS) to characterize the structural properties [51,52] also confirmed the complex pore structure of the poly(TRIM) resin in the dry state. Mesopores of diameter above 3 nm and much smaller pores of diameter below 1 nm are present in the homopolymer [9]. Hence, it can be assumed that the internal structure of the crosslinked poly(TRIM) resin is of a complex character, both in the dry and swollen state. The conclusions are consistent with the

Fig. 1. The low-temperature nitrogen adsorption/desorption isotherms of the polymer HT01 and copolymers HT11 and HT21 (a) and the pore size distributions determined by applying the BJH method to the adsorption (b) and desorption isotherm (c) under study.

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proposed mechanism of formation of the polymer porous network [31,53,54]. The mechanism is based on the assumption that formation process of the porous polymer network consists of two major steps. Firstly, primary particles are generated through crosslinking between vinyl groups in a monomer and then they agglomerate via intermolecular crosslinking into larger clusters called microspheres. The increase of the number of clusters in the synthesis system results in the polymer phase becoming continuous. It is well known that the final structure of the polymer network, and hence the pore network, depends to a large extent on the features of monomers and the property of solvent used in the polymerization [55]. The phase separation is observed at the high conversion of monomers to polymer only if the monomers and the porogen exhibit a good compatibility. This creates a complex internal network of highly interconnected individual nuclei and larger microspheres. As a result, the polymer resin is characterized by the high specific surface area and the pore size distribution with a maximum in the region of micro-to mesopores. Otherwise, the synthesized polymer has a broad pore size distribution through the mesopore range and into the macroporous regime. According to Flodin, TRIM molecule has a rigid and a compact tetrahedral structure [14]. Thus, the intramolecular cyclization is unlikely to occur [14]. Instead, it is more probable that the multilevel internal structure of the poly(TRIM) resin is formed by crosslinking of pendant vinyl groups on neighbouring monomers. The analysis of the shape of N2 adsorption/desorption isotherms for HT01 suggest the presence of pores between spherically shaped elements of a solid or pores in the shape of the ink-bottle which may be interconnected. Taking into account the proposed mechanism of formation of the polymer porous network, it may be assumed that the smaller mesopores at the PSD curve correspond to the pores between nuclei, whereas the bigger ones to those between larger structures-microspheres. On the basis of the experimental data presented in Table 1, it may be concluded that the copolymerization of TRIM with monofunctional HEMA monomer influences the size of larger mesopores. The increase of the ratio of the HEMA to TRIM shifts the pore size distribution towards macropores regime, as it is visible for HT21 copolymer, whose structure becomes almost macroporous. The decrease of the amount of the crosslinking agent strongly reduces the SBET of the final copolymers and, to a lesser extent, their pore volume. It is reasonable if the likely mechanism of formation of the porous network of poly(HEMA-co-TRIM) is taken into account. First of all, the porogenic solvent employed in this copolymerization reaction seems to exhibit poor compatibility with used hydrophilic monomer e 2-hydroxyethyl methacrylate. Therefore, a phase separation occurs at the lower conversion of monomers. This probably results in the formation of heterogeneous polymer network, consisting of unsaturated very fine units which are very tightly packed and form larger species, microspheres, which are irregular in size and in shape. It is highly probable that the coalescence of the tiny units leads to creation of very small, slit-shaped pores, or even closed ones which may be inaccessible to nitrogen molecules. On

the other hand, large mesopores and even macropores are created between microspheres. This results in the formation of copolymer beads with inhomogeneous, almost macroporous, internal pore network, and as a consequence, the lower specific surface area. SEM micrographs in Fig. 2aec reveal that the investigated resins possess permanent well-developed porous structure in the dry state. Regardless of this, changes in the internal topography are observed with the increase of the HEMA content in their copolymer network. It may be observed with the increase of the HEMA content, the internal structure of resins is becoming more loosely packed (Fig. 2c). Although all newly synthesized resins are spherical in shape, they are not entirely uniform in size (Fig. 1aec see in the Supplementary Data). The size distribution of raw polymer beads (before their fractionation) in a dry state was estimated with the use of an image analysis program over the statistical sample of about 100 objects from the optical pictures. The most uniform in size are beads of the HT01 which are 155 ± 35 mm in diameter. The copolymerization of the TRIM with monofunctional HEMA monomer influences not only the internal pore structure but also the size of the obtained copolymer beads. The HT11 beads are, on the average, twice the size in the diameter, 330 ± 105 mm, and are also much less homogeneous in size. The further increase of HEMA content results in the decrease of diameter of HT21 beads which reaches 205 ± 85 mm. The presented results indicate that the internal structure of the HEMA-enriched resins differ from each other and from the HT01. Therefore, the FTIR spectra were measured for all the investigated porous resins. Fig. 3 presents the ATR-FTIR spectra of the polymeric materials. Since the chemical structure of monomers TRIM and HEMA are similar, their FTIR spectra are also almost the same. Nevertheless, some small differences in the spectra of HT01 and poly(HEMA-co-TRIM) copolymers are visible and testify to the fact that HEMA units are present in the copolymer networks. In each spectrum of copolymers, bands at about 1140 cm
1, arising from the vibration of the CeOeC group, are observed. However, this band is shifted to a higher wave numbers when HEMA is incorporated into polymer network, and for HT21 its maximum is at 1147 cm
1, whereas in HT01, it is at 1139 cm
1. Moreover, with the increase of HEMA to TRIM ratio, the increase of the intensity of the band at 3527 cm
1, attributed to the eOH group vibration present in HEMA units, is visible. Additionally, peaks at 1638 cm
1 presented in each spectrum indicate that unreacted C]C double bonds are still present in the structure of the prepared materials. However, this peak is the most intense for HT01 polymer, which may testify to the fact that the number of unreacted double bonds in its structure is higher in comparison to copolymers. On the basis these results, conversion of double bonds in the synthesized microspheres was calculated. Intensities of peaks responsible for stretching vibrations of the C]C (1638 cm
1) group before and after polymerization were compared. As an internal standard the intensity of carbonyl group absorption band (1723 cm
1) was used. The degree of conversion (DC) was calculated by the following equation:

Table 1 The parameters characterizing the porosity of the samples obtained from N2 adsorption/desorption at
196  C: the specific surface area, SBET, the total pore volume, Vp and the pore diameter at the peak of PSD, Dp, derived from the desorption branch of N2 isotherm. Sample

Mass ratio HEMA:TRIM

SBET (m2/g)

Vp (cm3/g)

Dp1 (nm)

Dp2 (nm)

HT01 HT01-IBNa HT11 HT11-IBNa HT21 HT21-IBNa

0

536 449 391 390 219 172

0.78 0.76 0.73 1.05 0.60 0.56

3.8 3.8 3.8 3.8 3.8 3.8

15.3 15.1 24.0 32.0 33.5 34.2

0.38 0.77

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Fig. 2. SEM micrographs of the polymer HT01 (a) and copolymers HT11 (b) and HT21 (c) (see other magnification in the Supplementary Data).

Fig. 3. FTIR spectra of the copolymers HT21 (a) and HT11 (b), as well as the polymer HT01 (c).

0



B DC ¼ 100%
@



IC¼C IC¼O IC¼C IC¼O

1

 polymer  100%C A

Table 2 The swellability coefficient (B) obtained from the measurements taken with the use of optical microscope. Sample

monomer

The calculated values of DC for HT01, HT11 and HT21 materials are: 72%, 78% and 90%, respectively. Thus, it is seen that use of monovinyl monomer HEMA increases the degree of double bonds conversion [21].

3.2. Swelling characteristics of resins Since the investigated resins possess a permanent porous structure, it seems very interesting to see if the alcoholic solution of the drug (the alcoholic solution of ibuprofen salt 34 mg/ml) easily penetrates their pore network. In addition to the results, the ability to swell of the investigated resins in toluene and anhydrous ethanol is presented for comparative purposes. The swelling process of investigated polymer matrices occurred rapidly because the developed and permanent pore network gives the solvent molecules easy access to the beads' interior. Therefore, the swelling process was monitored with Nikon SMZ 1500 optical microscope and time-lapse imaging. This equipment makes it possible to monitor the very high rate of changes during the swelling. Fig. 2 in the Supplementary Data shows the population of the beads in both the dry and wetted state (the state of the maximum swelling). The values of swellability coefficient (B) computed from the images of samples are collected in Table 2. The ability to swell of studied materials differs significantly between each other and strongly depends on the solvent type. As it is seen, the volume of polymer HT01 beads increases tremendously after its immersion both in toluene and in anhydrous ethanol, as well as, to a lesser extent, in the anhydrous ethanol solution of ibuprofen sodium salt. By contrast, HEMA-rich copolymer HT21 swells much less in all applied solvents, with the lowest value of its swellability coefficients found in toluene. This confirms that HT21 is

HT01 HT21

Swellability coefficient B (%) Toluene

Anhydrous ethanol

Anhydrous Ethanol þ IBNa

79 23

80 43

55 35

a highly crosslinked copolymer because it is generally known that highly crosslinked polymers have a low tendency to swell even in very good solvents. It is associated with lower elasticity of the network resulting from the crosslinking of the polymer chains [56]. A higher value of swellability coefficient in polar anhydrous ethanol is related to the chemical structure of the studied copolymer. The presence of polar functional groups is responsible for the higher affinity for swelling in polar solvents. Thus, the presence of HEMA functional groups in the HT21 copolymers enhances network interactions with solvents and causes the copolymer to expand more easily after its immersion in EtOH. Simultaneously, it should be taken into account that HT21 copolymer exhibits a much smaller specific surface area and the total pore volume. Hence, its ability to uptake the solvent is much smaller. The great ability to swell of poly(TRIM) is understandable if its high specific surface area and pore volume in dry state is taken into account (Table 1). Moreover, the presence of unreacted double bonds in HT01 polymer network together with its high B value indicate that the degree of crosslinking is lower, compared to the copolymers. Therefore, after the immersion of HT01 in the solvent, its molecules fill in the free volumes and also solvate and easily penetrate polymer network which is elastic to some extent. Following on from Table 2, the ability to swell of the investigated matrices in alcoholic solution of drug is much lower in comparison to the pure alcohol. This effect may be related to the clogging up of the pore system of the transport character by drug molecules which are larger than solvent molecules. This prevents further diffusion of

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solvent and drug molecules into the matrix and, consequently, significantly limits the expanding of the polymer matrices. Additionally, the volume of HT01 and HT21 beads in a dry state, following the evaporation of the solvent from matrices (after their swelling in anhydrous ethanol and alcoholic solution of drug, respectively) was estimated. It is interesting that the diameter of dry resin beads reaches almost the value observed before the immersion in EtOH. Unlike with swelling in EtOH, swelling in drug solution and the subsequent evaporation of the solvent from matrices cause both HT01 and HT21 beads to maintain the enlarged size. They are greater up to 25 and 7.5% of the initial volume beads, respectively. The extended volume of polymer and copolymer beads following the solvent evaporation indicates that the drug molecules to a large extent prevent the network from collapsing. Similar mechanisms were also observed in other systems presented before [13,57]. Therefore, it may be assumed that drug molecules are spread over the entire HT01 network, and, in the case of HT21, the ibuprofen sodium salt locates mainly in the interior of large mesopores. 3.3. Physicochemical characterization of ibuprofen-loaded conjugates The presented findings show the significant differences both in the internal pore network and the chemical character between the investigated cross-linked resins. Therefore, each of the newly synthesized resins was employed and tested as a matrix in the synthesis of the polymeredrug conjugates. The proposed idea of the controlled release from the polymer microspheres is not new, as it was reported in the 1960 [29], but it still arouses great interest [13,57]. To verify the presence of the drug in the polymereIBNa conjugates after the evaporation of the solvent, the Raman spectra were measured for all the investigated samples. The representative Raman spectra were investigated in the wavenumber range 1550e1630 cm
1, where no polymer peaks are present, so that ibuprofen sodium salt loading within polymer beads can be demonstrated. As it is shown in Fig. 4, in this region, the Raman spectrum of free IBNa exhibits two characteristic sharp peaks at 1576 and 1614 cm
1 which are generally assigned to vibrations of C]C and CeH bonds of the IBNa aryl ring [58,59]. In the same figure, there are also typical spectra reported, which are taken from the cross-section of the representative collection of beads of the copolymer HT21 and the HT21-IBNa copolymeredrug conjugate. The characteristic Raman bands of the drug are clearly visible in the HT21-IBNa spectrum. This confirms the successful introduction of

Fig. 4. Raman spectra of free ibuprofen sodium salt (a), the HT21-IBNa conjugate (b), and the pure polymer HT21 (c) recorded on dried samples.

drug into the polymer network. Moreover, it clearly appears that the position of peaks of salt entrapped into polymer beads does not change. As sodium salt cannot form intermolecular hydrogen bonds [58], the encapsulation process does not affect the molecular form of the drug. Raman spectra for HT01, HT11, HT01-IBNa and HT11IBNa are not shown since they are almost identical with those presented here. The drug conjugates preserve the spherical shape of the matrices beads since they are crosslinked systems, which are insoluble in the applied drug solution. The SEM images of drugloaded conjugates (Fig. 5aec) confirm changes in their internal topography after the precipitation of ibuprofen sodium salt molecules into the pore network of the polymer matrices. The interior of the conjugates is more compact and the fine grained structure is barely visible for the HT01-IBNa and HT11-IBNa (Fig. 5a and b). However, the tiny microgel nanoparticles are distinct in the HT21IBNa (Fig. 5c). The immersion of the resins in the alcoholic ibuprofen solution clearly indicates that it is an efficient method to introduce drug molecules into the polymer beads (Fig. 4). A similar technique of drug introduction into the porous polymer beads was proposed earlier [13,57,60]. There, it was shown that the precipitation of drug molecules within the polymer matrix results in the deformation and reconstruction of the internal pore network [13,57]. Therefore, the nitrogen sorption results for the investigated polymeredrug conjugates illustrated in Fig. 3 (Supplementary Data) and Table 1 are very surprising. The measured N2 adsorptionedesorption isotherms are almost identical in shape to those of pure polymer matrix; additionally, the adsorption values are similar for each of the matrices before drug loading, with the exception of HT11-IBNa conjugate. The HT11 matrix behaves similarly to commercially available polymer matrix [13,57]. Following the precipitation of the ibuprofen salt molecules, the changes in the hysteresis shape and in the PSD are clearly visible. Simultaneously, from the analysis of the parameters characterizing the porosity (see Table 1) it may be concluded that the specific surface area is unchanged whereas the total pore volume and the pores' size of the HT11-IBNa conjugate are significantly higher, in comparison to those in the pure HT11 matrix. This suggests the loosening of the internal structure of the HT11 during the drug solution uptake. In contrast, the parameters characterizing the porosity (see Table 1) of other conjugates diminish slightly due to the presence of drug molecules. In addition, the pore size distributions are almost unchanged. A possible explanation may be that the loading of the ibuprofen sodium salt is too small to create any significant changes in the internal pore network. However, the results previously presented clearly exhibit significant changes both in the porosity parameters and in the internal structure of conjugates even at a level of low drug loading [13,57]. On the other hand, the investigated matrices possess large mesopores. Therefore, even if drug is precipitated in their interiors, the changes in the shape of adsorption/desorption isotherms might not be seen. Therefore, the characterization of polymeredrug conjugates whose internal structure has been found to be of a complex character with the use of a conventional nitrogen sorption method seems to be inappropriate and involves certain difficulties. The presented micrographs along with the swelling experiments indicate that the internal structure and chemical nature of the polymer matrices differ significantly and greatly affect the embedding of drug molecules. Although the drug molecules were successfully introduced into the pore network of resins, they are located differently. The HT01 resin exhibits the highest SBET and pore volume. Following the immersion of HT01 in the drug solution, it easily expands and adequately deforms, which facilitates the uptake of the molecules of both the solvent and the large drug molecules. Moreover, the entry of drug into the interior of matrix is

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Fig. 5. SEM images of the beads interior of drug-loaded conjugates HT01 (a), HT11 (b), HT21 (c).

not limited by the interaction of matrix with drug molecules. Therefore, it is likely that the drug molecules may deeply penetrate the HT01 matrix. Thus, it may be presumed that ibuprofen sodium salt occupies mainly the free volumes between nuclei, as well as mesopores of HT01. However, it is also probable that, to some extent, it penetrates the primary particles of the polymer matrices in the form of the molecular dispersion. The copolymerization of TRIM with HEMA monomer strongly influences the chemical nature of the resulting resins. Moreover, the high HEMA content causes the copolymer's internal network to be almost macroporous, and, as a result, it exhibits a lower specific surface area and pore volume. Thus, it may be presumed that these factors reduce the effectiveness of matrix network penetration by drug molecules. Unlike with HT01, the chemical nature of poly(HEMA-co-TRIM) favours the interaction with drug molecules and, additionally, limits the deep penetration into the copolymer network. The swelling experiment in the drug solution is consistent with the presented hypothesis. Therefore, it is highly probable that the drug molecules are mostly located in the large free volumes of the polymer network, on the surface of microspheres, and also occupy the external surface of the beads.

3.4. Ibuprofen release To compare the influence of different polymer matrices on the drug desorption rate, ibuprofen sodium salt release studies for all investigated conjugates were carried out in the phosphate buffer solution at 37  C, which was used as a dissolution medium. The drug release profiles are provided in Fig. 6. Following from these release curves, the ibuprofen salt is most effectively leached out from the HT21-IBNa conjugate. Simultaneously, the burst release is observed and almost 60% of the drug is released within the first

Fig. 6. Ibuprofen sodium salt release from conjugates measured in the phosphate buffer solution at 37  C.

15 min. The initial very fast release significantly decreases in the case of the HT01-IBNa. However, the maximum of ~98% of salt is desorbed only from the HT21-IBNa conjugate whereas the increase of the crosslinking component decreases the maximum amount of released drug within 30 h. The differences in the rate of ibuprofen salt desorption from conjugates are caused both by their chemical character and the porosity. The HT01-IBNa conjugate possesses permanent and highly porous structure, which makes it possible to easily introduce solvent molecules into its large free volumes. However, poor wettability of HT01 and, as a consequence, its poor swelling in aqueous solution are caused mainly by its chemical character but also by its crosslinked structure. Thus, after the initial fast desorption, very slow and almost linear release of deeply trapped drug is observed. Contrary to the presented conjugate based on the HT01 matrix, the HT11-IBNa and HT21-IBNa conjugates are more hydrophilic due to the HEMA content. Therefore, it may be presumed that the poly(HEMA-co-TRIM) matrix is easily wetted in the used dissolution medium. Moreover, as noted above, the drug molecules are mostly located in the large free volumes of the polymer network. Thus, the placement of the conjugates in the buffer solution causes the fast desorption of drug, which is directly related to the solubility of the drug, located on the most external part of copolymer matrix. Nevertheless, the studied kinetics of ibuprofen salt release from drug-loaded copolymers also testifies to the fact that the copolymer network is able to swell in the solution medium permitting drug release. 4. Conclusions The present article has demonstrated the permanently porous poly(HEMA-co-TRIM) and poly(TRIM) resins prepared in the form of beads via suspension-emulsion polymerization as well as polymereibuprofen sodium salt conjugates based on them and their drug release kinetics. In the course of study, the complex internal structure of the polymer and copolymers was revealed. It was proved that the increase of the ratio of the monofunctional HEMA monomer to TRIM results in the formation of the copolymer spherical beads with inhomogeneous, almost macroporous, internal pore network and with the lower specific surface area. It was presented that the immersion of investigated materials in the alcoholic solution of ibuprofen sodium salt causes their easy and rapid swelling, which results in an increase of the polymer beads size even after the solvent evaporates. The Raman spectra of the investigated materials confirm the successful introduction of drug into the polymer and copolymers network. SEM micrographs and N2 adsorption experiment have revealed that the internal network is reorganized after the introduction of the drug molecules into the organic carrier. As demonstrated above, the differences in the

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internal structure of conjugates after drug embedding as well as in the rate of drug desorption strongly depend on the chemical character and the porosity of the initial polymer and copolymer. It was proved that the higher the content of hydrophilic monomer is, the ibuprofen sodium salt is more effectively leached out from the conjugate. Acknowledgments Authors would like to express their gratitude to Professor Jacek Goworek for his support and remarks. The research was carried out with the equipment purchased thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (contract no. POIG.02.01.00-06-024/09 Center of Functional Nanomaterials). The authors thank Dr. Michał Rawski from Center of Functional Nanomaterials for SEM measurements. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.micromeso.2015.06.009. References [1] D.C. Wu, F. Xu, B. Sun, R.W. Fu, H.K. He, K. Matyjaszewski, Chem. Rev. 112 (2012) 3959e4015. [2] T. Rohr, S. Knaus, H. Gruber, D.C. Sherrington, Macromolecules 35 (2002) 97e105. [3] M. Grochowicz, B. Gawdzik, React. Funct. Polym. 71 (2011) 625e633. [4] P.K. Dhal, S. Vidyasankar, F.H. Arnold, Chem. Mater. 7 (1995) 154e162. sek, D. Hora k, [5] J. Koubkov a, P. Müller, H. Hlídkov a, Z. Plichta, V. Proks, B. Vojte New. Biotechnol. 31 (2014) 482e491. [6] Z.M. Chen, S.J. Li, F.F. Xue, G.N. Sun, C.G. Luo, J.F. Chen, Q. Xu, Colloid Surf. A 355 (2010) 45e52. [7] I. Halasz, A. Kierys, J. Goworek, H.M. Liu, R.E. Patterson, J. Phys. Chem. C 115 (2011) 24788e24799. [8] R. Zaleski, A. Kierys, M. Dziadosz, J. Goworek, I. Halasz, Rsc Adv. 2 (2012) 3729e3734. [9] R. Zaleski, A. Kierys, M. Grochowicz, M. Dziadosz, J. Goworek, J. Colloid Interf. Sci. 358 (2011) 268e276. [10] Z.Q. Wang, J.Q. Guan, S.J. Wu, C. Xu, Y.Y. Ma, J.H. Lei, Q.B. Kan, Mater. Lett. 64 (2010) 1325e1327. [11] A. Kierys, R. Zaleski, W. Buda, S. Pikus, M. Dziadosz, J. Goworek, Colloid Polym. Sci. 291 (2013) 1463e1470. [12] W. Buda, B. Czech, Water Sci. Technol. 68 (2013) 1322e1328. [13] A. Kierys, M. Rawski, J. Goworek, Micropor. Mesopor. Mat. 193 (2014) 40e46. [14] J.E. Rosenberg, P. Flodin, Macromolecules 19 (1986) 1543e1546. [15] A. Schmid, M. Walenius, P. Flodin, J. Appl. Polym. Sci. 45 (1992) 1995e2004. [16] B.N. Kolarz, D. Jermakowicz-Bartkowiak, A. Trochimczuk, Eur. Polym. J. 34 (1998) 1191e1197. [17] B.N. Kolarz, D. Bartkowiak, A.W. Trochimczuk, W. Apostoluk, B. Pawlow, React. Funct. Polym. 36 (1998) 185e195.

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