Photocrosslinkable biodegradable responsive hydrogels as drug delivery systems

International Journal of Biological Macromolecules 49 (2011) 948–954

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Photocrosslinkable biodegradable responsive hydrogels as drug delivery systems J.F. Almeida a,b , P. Ferreira a,c,∗ , A. Lopes b , M.H. Gil a a

Department of Chemical Engineering, University of Coimbra, Rua Sílvio Lima, Polo II, 3030-790 Coimbra, Portugal Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127, 2781-901 Oeiras, Portugal c Department of Health Sciences, Portuguese Catholic University, Estrada da Circunvalac¸ão, 3504-505 Viseu, Portugal b

a r t i c l e

i n f o

Article history: Received 24 June 2011 Received in revised form 28 July 2011 Accepted 11 August 2011 Available online 19 August 2011 Keywords: Responsive hydrogel Dextran Photocrosslinking Drug delivery

a b s t r a c t Recently, controlled release from biocompatible materials has received much attention for biomedical applications. Due to their biocompatibility and biodegradability, glucopyranosides such as dextran appear as promising polymeric materials if one is able to regulate their rheological properties and the encapsulation/release efficiency. In this work graft polymer hydrogels from dextran and N-isopropylacrylamide (NIPAAm) were prepared and characterized. Dextran molecules were modified with 2-isocyanatoethylmethacrylate (IEMA) in order to obtain a polymer with carbon double bonds. Urethane linkages resulted from the reaction between hydroxyl groups (OH) of the dextran and isocyanate groups (NCO) of the IEMA. The obtained polymer was then crosslinked by UV irradiation in the presence of the photoinitiating agent Irgacure 2959 by CIBA® . The drug Ondansetron® was entrapped in the final system and its release profile was determined at 25 and 37 ◦ C. The characterization of the materials was accomplished by: ATR-FTIR (Attenuated Total ReflectanceFourier Transform Infrared) spectroscopy, elemental analysis, lower critical solution temperature (LCST) determination, swelling behaviour evaluation, determination of surface energy by contact angle measurement and drug delivery profile studies. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Hydrogels are materials that, when placed in water absorb and retain large amounts of the liquid without dissolving in the solution [1]. In the polymeric structure of hydrogels the hydrophilic parts of gels tend to be highly hydrated in the aqueous environment triggering the big water uptake that characterizes these structures [2]. Because of their properties, namely hydrophilicity and biocompatibility, hydrogels have been a subject of interest in different areas especially the biomaterials area [3,4]. One of the topics being explored is the different response of hydrogels to external stimuli, namely pH, and temperature (or both) and the implication of their use in biomedicine [5,6]. Recently, UV radiation has been used in different areas, such as sterilization, waste water treatment, and inks [7,8]. It has also been used largely in the biomaterials area, namely in the immobilization of cells and for the preparation of drug delivery systems [9–11]. In the biomaterials area, one of the main applications of the UV radiation is the preparation of photopolymerized materials. UV

∗ Corresponding author at: Department of Chemical Engineering, University of Coimbra, Rua Sílvio Lima, Polo II, 3030-790 Coimbra, Portugal. Tel.: +351 239798758; fax: +351 239798703. E-mail addresses: [email protected], [email protected] (P. Ferreira). 0141-8130/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2011.08.010

irradiation allows the polymerization in situ, producing materials with the desired texture and elasticity depending on their application, like matrices for bone implants [12]. Dextran is a polysaccharide produced mainly by bacteria, consisting essentially of ␣-1,6 linked d-glucopyranese residues [13]. This natural polymer has been reported as highly relevant in the biomedical field [14] and has been applied as plasma expander [15] and in the development of drug delivery systems [16], hydrogels [17] and wound dressings [18] among others. Poly(N-isopropylacrylamide) (PNIPAAm) has attracted much attention recently, especially due to its thermosensitive behaviour at temperatures close to physiological temperature [19], namely, the presence of the so called Lower Critical Solution Temperature (LCST) [20] a thermal transition occurring at 32.5 ◦ C. This property is mainly due to changes in the balance between hydrophilic and hydrophobic forces with the surrounding medium molecules and the break of hydrogen bonds between PNIPAAm and water molecules [21]. Due to this characteristic, PNIPAAm has been used in several applications such as drug delivery systems, biosensors [22], immunoassays [23], cell attachment/detachment [24] and even chromatography [25]. In this paper, we wish to report the synthesis of a responsive hydrogel based on dextran. This polymer was firstly modified with 2-isocyanatoethylmethacrylate. A photocrosslinkable polymer was obtained. After this chemical modification, N-isopropylacrylamide

J.F. Almeida et al. / International Journal of Biological Macromolecules 49 (2011) 948–954

was added to the mixture as well as the photoinitiating agent Irgacure® 2959. The choice of this photoinitiator was based on previous experiments that proved that this compound was well tolerated over a wide range of cell types and chemical concentrations [26]. Finally, after 5 min of UV irradiation the films were obtained. The drug Ondansetron® (an antiemetic used to treat nausea and vomiting, frequently following chemotherapy) was entrapped in the final system and its release profile was determined at two different temperature values: 25 and 37 ◦ C. 2. Experimental procedure 2.1. Materials ¯ w = 5, 000, 000–40, 000, 000 Da), 2-isocyanatoethyl Dextran (M methacrylate (IEMA) and N-isopropylacrylamide (NIPAAm; purity of 97%) were purchased from Sigma/Aldrich Chemical Company (Spain) and used with no further treatment. Dimethyl sulfoxide (DMSO) was also purchased from the same company but was dried previous to use. Calcium chloride (purity of 97%) was obtained from Riedel-de-Haën (USA). Irgacure® 2959 (Ir2959, purity of 97–99.5%) was gently supplied by CIBA (Ciba Specialty Chemicals, Basel, Switzerland) and was used as supplied. The drug Ondansetron was gently supplied by Dr. Armando Alcobia from Hospital Garcia de Orta, Portugal. 2.2. Dextran modification and responsive hydrogels preparation Dextran containing urethane groups was prepared by modifying its hydroxyl groups with IEMA. Two different degrees of modification were experimented: 25 and 33% using DMSO as solvent. For that purpose, 1 g of dextran was solubilised in 25 mL of DMSO by stirring the components in a conventional two neck round-bottomed glass flask in the absence of air (under a nitrogen atmosphere), and using a drying tube containing a desiccant agent (calcium chloride). The reaction vessel was placed in a water bath at the temperature of 60 ◦ C. After complete solubilisation, IEMA was added to the mixture in a molar proportion of NCO:OH of 25 and 33% for 24 h. After this period of time, the photoinitiator was added to the solution in a percentage of 4% of the number of moles of the IEMA. The mixture was kept under the initial conditions until complete solubilisation of Ir2959. Finally 1 g of NIPAAm was added to the solution which was kept stirring until this compound was completely solubilised. The resultant solution was removed from the water bath and was irradiated for the period of 5 min by using a UV lamp (Model UVGL-48, Multiband UV, from Mineral light® Lamp). After this period of time, two different films were obtained. After the reactions, each sample was washed with water. 2.3. ATR-FTIR analyses The ATR-FTIR technique was used to confirm the reaction between the dextran and IEMA (by observing the resultant urethane as well as the carbon double bonds bands) and also to verify the disappearance of the carbon double bands after photocrosslinking. All these analysis were performed on a Magma-IRTM Spectrometer 750 from Nicolet Instrument Corp., equipped with a Golden Gate Single Reflection Diamond ATR. Spectra were recorded on an average of 128 scans at a resolution of 4 cm−1 . 2.4. Elemental analyses This technique was used to determine the percentage weight of nitrogen (N) in the dextran at two stages. The first analysis was performed after reaction of this polymer with IEMA. The second

949

H3C O

N

N

N CH3 Fig. 1. Chemical structure of Ondansetron® .

analysis was performed to the final films obtained by crosslinking this modified dextran with NIPAAm. All samples were freeze-dried previously to analyses. The tests were performed on a Fisions Instruments EA-1108. 2.5. Lower critical temperature determination The lower critical solution temperature (LCST) was evaluated following the transmittance of the samples at 500 nm in a Jasco V-530 Spectrophotometer heating the solutions in a thermostatic bath from 25 ◦ C to 40 ◦ C in steps of 1 ◦ C. Transmittance values were measured for three samples of each film at all values of temperature after stabilizing the samples for 30 min. LCST was inferred from the inflexion point of the transmittance vs. temperature curve. 2.6. Water sorption and water releasing capacity Three samples of each crosslinked film were previously dried until constant weight at 40 ◦ C under vacuum conditions. The weight of the dried samples was obtained (Wd ). These samples were then placed in a container with a saturated solution of pentahydrated copper sulphate (CuSO4 ·5H2 O; ≈85% RH) and were weighted at different times until a maximum weight was achieved (Ws ). The swelling ratio was evaluated by using Eq. (1): Swelling ratio (%) =

W − W  s d Wd

× 100

(1)

2.7. Determination of water contact angle and surface energy It is widely recognized that surface energy is an important parameter affecting polymers adhesion, material wettability and even biocompatibility [27]. During this work, water contact angles as well as surface energies were determined for the crosslinked films. These parameters were evaluated by static contact angle () measurements in a DAS 1 from Kruss in order to compare them with the ones obtained from literature for skin. All the tests were performed on the air-facing surfaces of the samples with four liquids: water, formamide, ethylene glycol and propylene glycol using the sessile drop method. Nine measurements on different points were performed to calculate the mean static contact angle  and its standard deviation. The dispersive SD and polar SP components of the surface energy were determined according to the Owens–Wendt–Rabel and Kaelble relationship. 2.8. Release pattern A drug delivery system was prepared using the photocrosslinked films previously obtained. The chosen drug was Ondansetron® (Fig. 1), which is an antiemetic as formerly described. Three films samples were immersed in 5 mL of a drug solution (4 mg/mL) for 24 h. After this time, the remaining concentration of the solution was measured by UV–vis spectroscopy, using a Jasco V-530 spectrophotometer. The absorbed drug concentration was

950

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CH3 H2C

C C

O

CH2

O

OCH2CH2N

O

C

O

O

IEMA

OH

CH2 Dextran:IEMA

O OH

OH

O

OH

O C N CH2CH2O

OH Dextran

H2C

C

C O

H

CH

H2C C

O

CH3

NH

H3C

CH CH3

UV 5 min.

NIPAAm

Irgacure 2959 HO

CH2CH2O

O

CH3

C

C

OH

CH3 UV

O

CH2 O OH HO

O

OH

CH2CH2O

O

CH3

C

C

OH

CH3

O C N CH2CH2O

Benzoyl radical

Alkyl radical

C O

H CH2

C CH2 CH CH3

Dextran:IEMA:PNIPAAm

H3 C

C O NH CH3

Fig. 2. Schematic representation of the chemical reactions involved in the preparation of the films.

then evaluated as the difference between the initial and the final drug concentration in the solution. The quantification of the amount of drug released as a function of time was performed using the same spectrophotometer. The films were introduced in a dialysis membrane (to avoid any piece of the film interfering in the reading) and then inserted in erlemeyers containing 100 mL of physiological serum. Each assay was carried out at 25 and 37 ◦ C. Absorbance values were obtained at 312 nm (maximum of the drug absorption spectra), at predetermined time intervals, until a maximum of 196 h.

DEX:IEMA33% respectively. Afterwards, NIPAAm was mixed with the modified dextran and the mixture was then photocrosslinked under UV irradiation using Ir2959 as a photoinitiator. This compound is a ␣-hydroxy alkylphenone, a Type I photoinitiator, which, when exposed to UV radiation, undergoes a photofragmentation process (␣ or ␤ cleavage) originating a benzoyl radical and an alkyl radical (one with a hydroxyl and the other an isopropanol radical [28]. From these two, the benzoyl radical is the main responsible for the initiation of the polymerization [29]. The scheme representing the reaction steps involved in the preparation of the films is presented in Fig. 2.

3. Results and discussion 3.2. ATR-FTIR analysis 3.1. Synthesis The preparation of the crosslinked films was conducted trough two successive steps. The first one consisted on the insertion of carbon–carbon double bonds onto the dextran molecule. This chemical modification was achieved by reaction of dextran hydroxyl groups with the isocyanate groups of IEMA with the consequent formation of urethane groups. Two different degrees of modification were tested (25 and 33%) and therefore two different modified dextrans were obtained: DEX:IEMA25% and

The ATR-FTIR technique was used to confirm the presence of the urethane groups on the modified dextran as well as their disapearance after UV irradiation. Therefore, both the original (Fig. 3A) as well as the modified dextran (Fig. 3B) were analysed. Since the FTIR bands were similar for DEX:IEMA25% and DEX:IEMA33%, only the spectrum of the first is presented. The FTIR spectrum of dextran showed the following relevant bands: ∼3300 cm−1 (O–H stretching vibrations), ∼2922 cm−1 (C–H stretching), ∼1640 cm−1 (O–H bonding), 1150 cm−1 (C–O–C

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951

Table 1 Results obtained for the % N for both systems before and after the grafting with PNIPAAm and photopolymerization. %N

Unmodified dextran DEX:IEMA25% DEX:IEMA33%

Before (grafting with PNIPAAm + UV irradiation)

After (grafting with PNIPAAm + UV irradiation)

0.000 2.365 3.659

0.000 6.874 8.589

topolymerization and the consequent introduction of PNIPAAm in the final structure of the gel. 3.3. Elemental analysis

Fig. 3. FTIR-ATR spectra of dextran; dextran modified with IEMA; NIPAAm; and the final gel after UV irradiation (DEX:IEMA:PNIPAAm).

stretching) and a sharp band at ∼1020 cm−1 (stretching vibration of the hydroxyl group) [30]. On the other hand, when analysing the spectrum of the modified dextran it was possible to verify a decrease of the band around ∼3300 cm−1 related with the decrease of the total number of –OH groups in the polysaccharide structure [31]. The urethane bands are also visible at 3378 cm−1 (N–H hydrogen bonded stretching) and at 1525 cm−1 (C–N stretching and N–H bending). Finally it was possible to verify the presence of the C C double bonds by the detection of the correspondent peak at 3010 cm−1 . Since the modified dextran was then photocrosslinked in the presence of NIPAAm, FTIR analysis was also performed for this compound (Fig. 3C). In this spectrum it is possible to see a sharp peak at ∼3280 cm−1 for N–H stretching vibration, a peak at 2960 cm−1 due to C–H stretching, and the typical amide I and II bands at ∼1650 cm−1 and 1550 cm−1 (due to C O stretching and N–H bending respectively). The peaks around ∼1430–1370 cm−1 are ascertain for CH3 deformation. Finally, in Fig. 3D, is represented the FTIR spectrum of the obtained films after UV crosslinking. When compared to Fig. 3B (modified dextran before UV irradiation) we can verify an increase in the peaks around 1650 and 1550 cm−1 related with the incorporation of PNIPAAm in the structure of the gel [32]. Also, it is possible to verify the decrease of the peak related with the C C bond at 3010 cm−1 , suggesting the success of the pho-

The elemental analysis was used to determine the success of both the substitution reaction and the photopolymerization by the detection and quantification of the % of nitrogen (N). For that purpose, a film containing only dextran in its composition was prepared and used as negative control. Elemental analysis was also used to characterize the DEX:IEMA25% and DEX:IEMA33%. Finally, the percentage of N in the films obtained from both modified dextrans grafted with PNIPAAm (after UV irradiation) was quantified. Table 1 shows the results obtained for all this quantifications. As expected, the film prepared from dextran did not present any N in its composition and the percentage value was 0%. During the first step of the reaction, chemical groups containing N were attached to dextran. Therefore, the monitorization of the total % of N present in the samples allows the determination of the degree of substitution. From Table 1 it is possible to verify an increase in the total percentage of this element presented in the samples when compared with pure dextran. This result suggests that the reaction of modification was well succeeded. We can also see that there is an increase in the final amount of N related with the increase of the degree of substitution. In Table 1 are also presented the percentages of nitrogen after UV crosslinking. These results were calculated as the difference between the final amount of Nitrogen and the amount that was present before the photopolymerization. Therefore, these values represent the amount of N present which is due only to the presence of PNIPAAm in the systems. It was then possible to verify an increase in the percentage of nitrogen in the gels suggesting that NIPAAm was successfully grafted onto dextran. When comparing the results obtained for the different degrees of substitution one can verify that when increasing the degree of substitution, it becomes evident an increase in the final amount of nitrogen introduced in the system, suggesting a higher concentration of PNIPAAm in the gel. This result may be explained by the fact that a higher degree of substitution means a higher concentration of C–C double bonds, the ones involved in the crosslinking with NIPAAm. The total percentage of copolymer introduced in each system was also calculated using Eq. (2): % copolymer =

Wp − W0 × 100 W0

(2)

where Wp is the weight of the gel after photopolymerization and W0 is the weight before the reaction. The results obtained indicated a total percentage of copolymer of 49% and 60% for the 25% degree of substitution gel and the 33% degree of substitution gel respectively. These results are in consonance with the ones obtained for the total percentage of nitrogen quantified and point out the success of the reaction and the increase of the PNIPAAm in each gel with the increase of the degree of substitution.

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J.F. Almeida et al. / International Journal of Biological Macromolecules 49 (2011) 948–954 Table 3 Results obtained for the exponent of the water release mechanism and the correlation coefficient for both systems at 25 ◦ C and 37 ◦ C. System

DEX:IEMA25%:PNIPAAm DEX:IEMA33%:PNIPAAm

Fig. 4. Transmittance values obtained with temperature for dextran, PNIPAAM and the gels obtained with both modification degrees (25 and 33%).

3.4. LCST determination Fig. 4 shows the optical transmittance curves for dextran, PNIPAAm and both films prepared: DEX:IEMA25%:PNIPAAm and DEX:IEMA33%:PNIPAAm. The values represent the mean value of transmittance at each measurement point. Standard deviations are not represented because of their very small value (between 0.02 and 0.05). As it can be seen in Fig. 4, a sharp decrease in optical transmittance of all systems containing PNIPAAm occurred at a certain temperature, i.e., all systems but dextran presented a temperature sensitive behaviour (with a correspondent value of LCST). The lowest value was obtained for pure PNIPAAm, ca. 32–33 ◦ C, in accordance with literature values for this property [33]. For all the other gel systems the LCST, ca. 33–34 ◦ C, presented a small increase when compared with PNIPAAm, mainly due to the presence of the hydrophilic part of dextran in the gels. Other modification studies [34] showed more significant increases of the LCST than the ones obtained during this study (36 ◦ C). This difference in results is mainly related to the use of maleic acid by the authors, which resulted in a higher hydrophilicity of the system. In this case, the system is more hydrophobic due to two co-related main factors: (a) the incorporation of a more hydrophobic molecule (IEMA) in the system and (b) the decrease of total hydroxyl groups remaining in the dextran molecule after modification with the IEMA. 3.5. Swelling capacity The swelling ratio is a good indicator of the hydrophilicity/hydrophobicity of the final gel and is of crucial importance for any material to be applied as biomaterial. For these systems it was possible to verify that swelling equilibrium was achieved within a week of incubation in a water saturated atmosphere. The values obtained for this parameter are presented in Table 2. When observing these results we can conclude that both gels are hydrogels independently of the test temperature. However, signif-

Table 2 Swelling capacity results determined for three samples of each film at two different temperature values (25 and 37 ◦ C). System

DEX:IEMA25% DEX:IEMA33%

Swelling capacity (%) 25 ◦ C

37 ◦ C

180.7 ± 4.5 205.3 ± 4.7

61.2 ± 3.1 50.8 ± 3.0

25 ◦ C

37 ◦ C

n

R2

n

R2

0.500 0.515

0.994 0.991

0.623 0.642

0.961 0.945

icant differences associated with the composition and temperature are easily detected. At 25 ◦ C the gel containing a higher degree of substitution was the one presenting the highest swelling value. This suggests that hydrophilicity increases with the incorporation of PNIPAAm within the dextran structure. Contrarily, at 37 ◦ C, the swelling capacity is lower for the system with higher PNIPAAm content. These two markedly different behaviours can be fairly explained by the presence of PNIPAAm in the gels and the fact that, at 37 ◦ C, the systems were above the LCTS for this material. In fact, at this temperature there is an increase of the intramolecular interactions involving PNIPAAm segments on the gel which precludes water from the precipitated/collapsed segments of PNIPAAm leading to the observed differences [6]. This also elucidates why the swelling of the gels decreases with temperature regardless of the substitution degree. The rate of water release from the gels was also evaluated. Release kinetics was determined using Eq. (3) [35]: Mt = kt n M∞

(3)

where Mt is the amount of water release at a given t time, M∞ is the total amount of water used in the preparation of gels, k is the kinetic constant, t is time and n the exponent that characterizes the mechanism of the release. Table 3 shows the results obtained for the exponent and the correlation coefficient (R2 ) for both systems at 25 ◦ C and 37 ◦ C. From Table 3 we can see that all values for n, at 25 ◦ C, are near or equal to 0.5, suggesting a Fickian mechanism for the release of water from the systems. This suggests that the release mechanism is mainly diffusional meaning that is the difference between the concentrations inside and outside the gels that controls water release. With the increase of the temperature, the values of the exponent change to the interval between 0.5 and 1.0, meaning that the release pattern of water depends not only on the difference of concentrations inside and outside the gels but also on the relaxation and degradation of the polymeric chains. The difference between these results may be justified by the precipitation of PNIPAAm chains due to the increase of temperature and therefore the overcome of the LCTS. 3.6. Determination of water contact angle and surface energy Water contact angles were measured for both systems with different substitution degrees and at 25 and 37 ◦ C. Values for the water contact angle, at 25 ◦ C, varied between 68.9◦ and 61.5◦ for 25% and 33% degree of substitution respectively and between 117.5◦ and 132.5◦ for the same systems at 37 ◦ C. These results uphold the conclusions taken from the swelling capacity, e.g. all systems presented high hydrophilicity levels depending on the degree of substitution and also depending on the temperature of the assay. We verified that the system with higher degree of substitution presented a more accentuated temperature depending behaviour which is consistent with the higher concentration of PNIPAAm in the final structure of the material. Surface energy is an important parameter affecting polymers adhesion, material wettability and even biocompatibility [27]. The

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953

Table 4 Surface energy values and correspondent polar (SP ) and dispersive (SD ) components. Substrate

Surface energies (mN/m) 25 ◦ C

Skin DEX:IEMA25%:PNIPAAm DEX:IEMA33%:PNIPAAm

37 ◦ C

s

SP

SD

s

SP

SD

38–56 29.8 31.2

– 20.1 23.2

– 9.7 8.1

38–56 25.1 24.8

– 14.2 12.7

– 10.9 12.1

surface energy values for each prepared film were also determined according to the Owens–Wendt–Rabel and Kaelble relationship. The main purpose of this study was to determine if the crosslinked films would become adherent when placed over skin. As determined by other authors, the surface energy of skin varies between 38 and 56 mN/m depending on its temperature and relative humidity [36]. According to a fundamental thermodynamic principle, films will adhere to skin if their surface energy is equal or lower than these values. The measured values for surface energy at 25 and 37 ◦ C are presented in Table 4. The obtained results showed that values of surface energy determined for both systems were similar and that independently of temperature, both systems presented a lower surface energy value than skin. These values suggest that adhesive forces between the gel and the skin will prevail against the intermolecular forces of the gel, resulting in the adherence of the films to the epidermis. However, it is noticeable that at 37 ◦ C the values of the dispersive components are higher than at 25 ◦ C. This fact is related with the thermal behaviour induced by PNIPAAm and suggests an increasing in the hydrophobicity related with the precipitation/aggregation of PNIPAAm. Nevertheless, at any of the tested temperatures the surface energies of the photocrosslinked films are lower than the ones mentioned for skin. For that reason, it is possible to suggest that when the films are placed over the epidermis surface, they would not slough off easily since adhesion forces will be considerable. 3.7. Release pattern Release studies were performed by using UV spectroscopy. The yield of drug released by each system was evaluated by using a validated calibration curve at 312 nm. The total amount of drug absorbed, by each system, was determined by UV–vis spectroscopy measuring the absorbance of the drug solution before and after the introduction of each gel in it. The essays were carried out at two different temperatures below and above LCST (25 ◦ C and 37 ◦ C respectively). We verified that the total amount of drug absorbed by the system varied from 1.215 mg/g polymer to 1.896 mg/g polymer at 25 ◦ C for the DEX:IEMA25%:PNIPAAm and DEX:IEMA33%:PNIPAAm systems respectively. At 37 ◦ C, the obtained values were 1.058 mg/g polymer for DEX:IEMA25%:PNIPAAm and 0.558 for DEX:IEMA33%:PNIPAAm. The observed differences can be related with the different behaviour of each system regarding the swelling properties that have been described previously in this paper. At each time, the amount of drug released was expressed as a percentage of the initial amount entrapped in the gel. Release kinetics was determined using Eq. (3). Fig. 5 shows the release pattern for both systems at 25 ◦ C and ◦ 37 C. The results represent the mean value of three samples. Standard deviation values were calculated but were all below 0.1. Therefore, they are not represented in order to simplify the results interpretation. As an overall conclusion it is visible that drug release is significantly higher at 25 ◦ C. In fact these results are consistent with the ones already registered for swelling. It is also possible to observe

Fig. 5. Release pattern of Ondansetron® for both systems (DEX:IEMA25%:PNIPAAm and DEX:IEMA33%:PNIPAAm) at temperatures values of 25 and 37 ◦ C. Table 5 Results obtained for the release exponent, the kinetic constant and the correlation coefficient. System

T = 25 ◦ C n

DEX:IEMA25%: PNIPAAm DEX:IEMA33%: PNIPAAm

T = 37 ◦ C n

k(h )

2

k(hn )

R2

R

n

0.516 0.0734

0.985

0.555 0.0369

0.989

0.525 0.0647

0.986

0.587 0.0283

0.996

that, at 25 ◦ C, and after 196 h, the amount of Ondansetron® released was nearly 100% of the amount initially entrapped. The results also showed that the gel with the highest degree of substitution was the one presenting a slightly lower drug release rate. This result suggests that the total concentration of PNIPAAm in the films influences the release of the entrapped drug. This effect may be due to the increase in the crosslinking degree of the gel, causing a higher retention of drug inside the gel matrix. Nonetheless, the differences are not very significant in these systems. The influence of the concentration of PNIPAAm in the release of the drug is more easily noticed when analysing the curves for the temperature of 37 ◦ C. In this case, it was verified that the system containing higher concentration of PNIPAAm presented a lower amount of drug release. This result is in conformity with the values registered for swelling and described previously. The release exponent (R2 ), the kinetic constant (k) and the correlation coefficient (n) were calculated using Eq. (3) and the obtained results are presented in Table 5. The release exponents’ values were close to 0.5 for systems at 25 ◦ C, suggesting a Fickian mechanism for the release of Ondansetron® . This means that the diffusion of the drug molecules is the major phenomenon involved in the release. We can also see that the systems kept at 37 ◦ C presented higher exponents values and that which suggests a non-Fickian release pattern, indicating some influence of the polymer relaxation process. These results are consistent with the ones obtained for the swelling capacity, where the results for 37 ◦ C showed also the influence of the precipitated polymer in this property. 4. Conclusions Throughout this work dextran was modified with IEMA and finally photocrosslinked in the presence of NIPAAm and Ir2959

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under UV radiation. Both dextran’s modification and crosslinking was confirmed by ATR-FTIR technique. At the end of these processes insoluble films were obtained. Two different substitution degrees were tested: 25 and 33%. It was possible to verify that different substitution degrees lead to different amounts of PNIPAAm grafted in dextran and consequently to different systems properties, including LSCT. Ultimately, this allows the fine tuning of the LCST according with the specific needs of the practical application. Swelling behaviour of both systems was also influenced by PNIPAAm concentration and consequently by the tested temperatures (25 and 37 ◦ C). Surface energy results showed that, even though the values were different depending on the temperature, all systems presented lower surface energies than skin, suggesting a good adhesion between each film and epidermis. The drug release studies showed that temperature has a significant influence on the amount of drug that diffuses from the films. In fact, release patterns showed that, when films are kept at 25 ◦ C, the amount of drug released is higher than at 37 ◦ C. PNIPAAM concentration proved to be another important parameter influencing drug release. However, this fact was more noticeable at 37 ◦ C, when a higher concentration of PNIPAAm corresponded to a lower concentration of drug release. These differences suggest that the reaction time for film preparation and the temperature of operation altogether can be controlled to tune the kinetics of drug release. Acknowledgements The authors would like to thank Fundac¸ão para a Ciência e Tecnologia for the financial support to J.F. Almeida (SFRH/BD/19707/2004). They would also like to thank Dr. Armando Alcobia from Hospital Garcia de Orta for gently supplying the drug Ondansetron® . References [1] W.E. Hennink, C.F. van Nostrum, Adv. Drug Deliv. Rev. 54 (2002) 13–36. [2] T. Coviello, P. Matricardi, C. Marianecci, F. Alhaique, J. Control. Release 119 (2007) 5–24. [3] A.S. Hoffman, Adv. Drug Deliv. Rev. 43 (2002) 3–12.

[4] K. Ulbrich, V. Subr, P. Podperova, M. Buresova, J. Control. Release 34 (1995) 155–165. [5] L. Verestiuc, C. Ivanov, E. Barbu, J. Tsibouklis, Int. J. Pharm. 269 (2004) 185–194. [6] B. Jeong, S.W. Kim, Y.H. Bae, Adv. Drug Deliv. Rev. 54 (2002) 37–51. [7] E. Napadensky, Ink-Jet 3D printing of Photopolymers Materials: An Emerging Rapid prototyping Technology, Radtech Europe 2005 Conference & Exhibition, 2005. [8] M. Huang, Liquid photopolymer useful in fabricating printing plates which are resistant to solvent based ink, United States Patent 6555292, 2003. [9] G.M. Cruise, O.D. Hegre, F.V. Lamberti, S.R. Hager, R. Hill, D.S. Scharp, J.A. Hubbell, Cell Transplant. 8 (1999) 293–306. [10] Q. Li, J. Wang, S. Shahani, D.D. Sun, B. Sharma, J.H. Elisseeff, K.W. Leong, Biomaterials 27 (2006) 1027–1034. [11] K.T. Nguyen, J.L. West, Biomaterials 23 (2002) 4307–4314. [12] K.H. Kraus, C. Kirker-Head, Vet. Surg. 35 (2006) 232–242. [13] H.K. Can, B.K. Denizli, A. Günera, Z.M.O. Rzaev, Carbohyd. Polym. 59 (2005) 51–56. [14] J. Maia, L. Ferreira, R. Carvalho, M.A. Ramos, M.H. Gil, Polymer 46 (2005) 9604–9614. [15] R. McCahon, J. Hardman, Anaesth. Intensive Care 11 (2010) 75–77. [16] M.A. Casadei, F. Cerreto, S. Cesa, M. Giannuzzo, M. Feeney, C. Marianecci, P. Paolicelli, Int. J. Pharm. 325 (2006) 140–146. [17] L. Weng, A. Romanov, J. Rooney, W. Chen, Biomaterials 29 (2008) 3905–3913. [18] J.G. Clay, D. Zierold, K. Grayson, F.D. Battistella, J. Surg. Res. 155 (2009) 89–93. [19] A.S. Mathews, C.S. Ha, W.J. Cho, I. Kim, Drug Deliv. 13 (2006) 245–251. [20] R. Salgado-Rodriguez, A. Licea-Claverie, K.F. Arndt, Eur. Polym. J. 40 (2004) 1931–1946. [21] C.D. Vo, D. Kuckling, H.-J.P. Adler, M. Schönhoff, Colloid Polym. Sci. 280 (2002) 400–409. [22] T. Matsuda, S. Ohra, Langmuir 21 (2005) 9660–9665. [23] N. Malmstadt, S. Hoffman, P.S. Stayton, Lab Chip 4 (2004) 412–415. [24] M. Ebara, M. Yamato, M. Hirose, T. Aoyagi, A. Kikuchi, K. Sakai, T. Okano, Biomacromolecules 5 (2004) 505–510. [25] M. Zhou, A. Sivaramakrishnan, K. Ponnamperuma, W.-K. Low, C. Li, J.O. Liu, D.E. Bergbreiter, D. Romo, Org. Lett. 8 (2006) 5247–5250. [26] C.G. Williams, A.N. Malik, T.K. Kim, P.N. Manson, J.H. Elisseeff, Biomaterials 26 (2005) 1211–1218. [27] S.C.H. Kwok, J. Wang, P.K. Chu, Diam. Relat. Mater. 14 (2005) 78–85. [28] L. Zang, H. Zhang, J. Luo, J. Guo, J. Macromol. Sci. A 48 (2011) 332–338. [29] N.S. Allen, M.C. Marin, M. Edge, D.W. Davies, J. Garrett, F. Jones, S. Navaratnam, B.J. Parsons, J. Photochem. Photobiol. A 126 (1999) 135–149. [30] X. Li, W. Liu, G. Ye, B. Zhang, D. Zhu, K. Yao, Z. Liu, X. Sheng, Biomaterials 26 (2005) 7002–7011. [31] G. Ciardelli, V. Chiono, G. Vozzi, M. Pracella, A. Ahluwalia, N. Barbani, C. Cristallini, P. Giusti, Biomacromolecules 6 (2005) 1961–1976. [32] Y. Katsumoto, T. Tanaka, H. Sato, Y. Ozaki, J. Phys. Chem. A 106 (2002) 3429–3435. [33] D. Lu, Z. Liu, M. Zhang, X. Wang, Z. Liu, Biochem. Eng. J. 27 (2006) 336–343. [34] S.R.V. Tomme, W.E. Hennink, Expert Rev. Med. Devices 4 (2007) 147–164. [35] N.A. Peppas, Pharm. Acta Helv. 60 (1985) 110–111. [36] S. Venkatraman, R. Gale, Biomaterials 19 (1999) 1119–1136.