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Controlled release studies through chitosan-based hydrogel synthesized at different polymerization stages
Accepted Manuscript Controlled release studies through chitosan-based hydrogel synthesized at different polymerization stages
Siti Zalifah Md Rasib, Hazizan Md Akil, Abbas Khan, Zuratul Ain Abd Hamid PII: DOI: Reference:
S0141-8130(18)35460-6 https://doi.org/10.1016/j.ijbiomac.2019.01.190 BIOMAC 11623
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
12 October 2018 2 January 2019 28 January 2019
Please cite this article as: S.Z.M. Rasib, H.M. Akil, A. Khan, et al., Controlled release studies through chitosan-based hydrogel synthesized at different polymerization stages, International Journal of Biological Macromolecules, https://doi.org/10.1016/ j.ijbiomac.2019.01.190
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ACCEPTED MANUSCRIPT Controlled Release Studies through Chitosan-based Hydrogel Synthesized at Different Polymerization Stages
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School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia, Engineering
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Department of Chemistry, Abdul Wali Khan University Mardan, 23200 Pakistan
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Campus, 14300 Nibong Tebal, Penang, Malaysia
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Siti Zalifah Md Rasib1, Hazizan Md Akil*1, Abbas Khan2 and Zuratul Ain Abd Hamid1
*Corresponding author:
Email: [email protected] Tel: +604 599 6161 Fax: +604599 6907
ACCEPTED MANUSCRIPT Abstract
An
earlier
study
showed
that
the
behaviour
of
chitosan-poly(methacrylic
acid-co-N-
isopropylacrylamide) [chitosan-p(MAA-co-NIPAM)] hydrogels synthesized at different reaction times are affected with regard to their pH and temperature sensitivities. The study was continued in this paper
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to identify the effects of different reaction times on the degradation, efficiency of rifampicin (Rif)
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loading and the Rif release profile under two different pH conditions (acidic and basic). The results that
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were obtained showed that the hydrogel had a faster degradation rate in the acidic condition than in the
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basic condition, where there was a loss of approximately 50% and 20%, respectively in its original weight within two weeks. The Rif loading efficiency was within 50% and the drug release was
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controlled by characteristics that were developed beyond the polymerization stages of the synthesis. Therefore, the reaction time for the synthesis of the hydrogel can be considered as a way to control the
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behaviour of the hydrogel as well as to modify the drug release profile in the chitosan-p(MAA-co-
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NIPAM) hydrogel.
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1. Introduction
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Keywords: Chitosan; hydrogel; drug delivery
Hydrogels have been classified as polymeric materials since the early 1900's [1]. A hydrogel with a 3D crosslink-network consisting of hydrophilic groups is able to retain a certain amount of water despite swelling [2]. The advantages of this hydrogel are that improvements can be made to its reactions by modifying it to be more sensitive to the environment either by physical or chemical crosslinking methods or both. Hence, this makes the hydrogels tremendously important in a wide variety of applications in the biotechnology, biomedical, pharmaceutical, and other related fields such as for wound dressings [3, 4], contact lenses [5, 6], and drug delivery systems [7, 8]. A unique property of
ACCEPTED MANUSCRIPT hydrogels is their capability to retain a certain amount of water. This enables them to resemble actual tissue cells, thus making them a very good biocompatibility material. Many significant contributions have been made to utilise the potential of hydrogels in drug delivery since they offer a suitable timecontrolled delivery, whereby the amount of drug released can be controlled according to physiological needs. Drugs can be delivered through the activation of stimuli-responsive hydrogels to pH, magnetism,
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ultrasound, temperature changes, electrical effects, and irradiation [9].
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Chitosan-p(MAA-co-NIPAM) hydrogels were synthesised by combining two stimuli, namely, pH
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and temperature, to enable the hydrogels to be more sensitive to various pH levels and to be continuously activated at body temperature. The emulsion polymerisation technique was applied to
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crosslink all the three polymers and monomers. The excellent behaviour of the p(MAA-co-NIPAM) hydrogel network in drug delivery has been well established by several researchers such as Constantin,
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Bucatariu, Harabagiu, Popescu, Ascenzi and Fundueanu [10] for self-regulated drug delivery; Sousa,
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Prior-Cabanillas, Quijada-Garrido and Barrales-Rienda [11] in slab-form hydrogels; Brazel and Peppas
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[12] for pulsatile local delivery; and Wu and Lee [13] for general drug delivery or diagnostic applications. A hydrogel network gains an extra advantage when chitosan is introduced into the network
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drug delivery [14].
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for improvements to the biodegradable and biocompatible characteristics, especially for site-targeting
A study was done on monomer conversion against polymerization time during the synthesis of the hydrogels, where the polymerization rate was different based on different parameters [15]. Various types of monomers were considered during the synthesis, where it was shown that each monomer had a different polymerization rate. Therefore, the core or shell of the hydrogel that was produced could be controlled to potentially affect the drug release mechanism. Not only that, the desired hydrophiliclipophilic properties could also be achieved by an accurate selection of the polymerization time [16].
ACCEPTED MANUSCRIPT The previous study was based on the application of different polymerization times on the chitosanp(MAA-co-NIPAM) hydrogels during the synthesis process in order to evaluate the formation of the hydrogels since the hydrogels consisted of many types of monomers and polymers. The study found that the hydrogels that were synthesized over a prolonged reaction time were continuously cross-linked, and the outer surface of the hydrogels was covered mostly by PNIPAM. The swelling of the hydrogels was
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restricted since the crosslink density kept increasing in the network and an intermediate reaction time
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was preferable, based on the sensitivity of the hydrogels to swelling in response to the pH and
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temperature [17]. Although prolonging the polymerization would lead to the completion of the
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polymerization process, the hydrogels produced had a high molecular weight and became more rigid
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[18, 19].
The hypothesis of this study was that changes in the network for a prolonged reaction time also
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affect the behaviour and the profile of drug release. Shi, Alves and Mano [20] also reported that
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additional chitosan in a hydrogel potentially lowers the release profile since chitosan also degrades with
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time. Further information on the degradation effects of the hydrogel on the drug release profile should also be investigated. The correlation between the structure and the swelling behaviour for different
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reaction times during the synthesis on the drug release behaviour should also be investigated to evaluate
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the potential of the hydrogel as a drug carrier. Therefore, this study continued with the previous work by covering the degradation of the hydrogel against time at a certain pH level, drug loading efficiency, kinetics, and drug release profile. Rifampicin was used as a model drug and the pH environment was varied between pH 7.4 (simulated intestinal fluid, blood) and 1.68 (simulated gastric fluid), whereas the temperature was kept constant at the temperature of the human body (37 °C).
ACCEPTED MANUSCRIPT
2. Materials and Methods 2.1
Materials
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N-isopropylacrylamide (NIPAM), N, N-Methylenebisacrylamide (MBA) (purity > 95.0%) and
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chitosan (Mw ≈ 700-1000 kDa, degree of deacetylation ≈ 90.0%) were purchased from Zhejiang
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Golden-Shell Pharmaceutical Co., Ltd (Zhejiang, China), Span™ 80 (Croda International Plc., UK), and acetic acid (≥ 99.85%) from Sigma–Aldrich. NIPAM was purified by recrystallization from a toluene/n-
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hexane (1:3) mixture. Methacrylic acid (MAA) (purity > 95.0%) was further purified by distillation
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under reduced pressure, while all the other chemicals were used as received and without further purification. Deionised distilled water (DDH2O) was used for all the reactions and solution preparations,
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and the hydrogels were purified by using the membrane filter, Spectra/Por® molecularporous membrane
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tubing, which was obtained from Spectrum Laboratories, Inc., Rancho Dominguez, CA, USA; cut-off
Preparation of chitosan-p(MAA-co-NIPAM) hydrogels
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2.2
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12,000–14,000.
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Emulsion polymerization by different reaction time was applied to produced chitosan-p(MAA-coNIPAM) hydrogel. Method of synthesis has already been described in the previous paper [21] by varying time to stop the polymerization reaction to 30 minutes (RT30), 60 minutes (RT60), 120 minutes (RT120), and 180 minutes (RT180). The copolymer hydrogels that were obtained were then purified by centrifugation (Sorvall RC-6 Plus superspeed centrifuge, Thermo Electron Co., Waltham, MA, USA) and decantation, and were washed with water. The resultant hydrogels were purified by dialysis through a Spectra/Por molecular porous membrane tubing using distilled water with a pH of 5±0.3 at room
ACCEPTED MANUSCRIPT temperature (25 ℃) that was frequently changed for 1 week. The purified hydrogels were freeze–dried for 48 hours with a LABCONCO freeze dry system after freezing overnight at -40 ℃.
2.3
In vitro degradation analysis
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Freeze-dried hydrogels was weight 5 mg for three sample for each reaction time hydrogel. The
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hydrogels then were incubated in 3 ml PBS at 37 °C for 24 hours with pH 1.68 and 7.5 respectively.
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Then, the hydrogel was centrifuged, supernatant was removed and wet hydrogel were weighted (W0).
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The hydrogels were incubated again at the same condition (3 ml PBS with pH 1.68 and 7.5 at 37 °C). The weights of wet hydrogels were measured in certain days (W1). The final weight ratios were
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Determination of Rifampicin Encapsulation Efficiency
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2.4
(1)
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W – W0 Wet mass % 1 x 100 W0
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calculated by equation 1.
The encapsulation efficiency (EE) of hydrogels were determined as follows. The 10 mM Rif drug stock
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solution was prepared by dissolved the drug in methanol solution. 60 mg of freeze dried hydrogels were immersed in 0.4 ml Rif from the stock solution and stirred slowly (100rpm/min) for various time under room temperature. Then, 2.6 ml of distilled water was added into the solution and stirred for 5 seconds. After the loading procedure, the suspensions were centrifuged (3300 Micro Centrifuge, Kubota, Japan) at 13000 rpm for 20 min and the Rif that remained in the supernatant phase was diluted 10 times to determine by using a UV-Vis. The DLC and EE percentage was calculated by equation 2.
ACCEPTED MANUSCRIPT M - M2 Drug Loading 1 x 100 M3
(2)
Where M1 is the initial amount of drug, M2 is the amount of Rif after loading (unloaded drug from the
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In Vitro Release Studies
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supernatant), and M3 is the amount of hydrogel.
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Drug release from hydrogel was studied using a dialysis method. The hydrogels loaded with Rif (10 mg/ml under different PBS pH 1.68 and 7.4) were added in a dialysis bag (MWCO 10 kDa; Thermo
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Fisher Scientific) and suspended in 500 mL of PBS (pH 7.4 and 1.68). The dissolution medium was
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stirred at 200 rpm in a laboratory shaker and maintained the temperature at 37oC. The sample (5 mL) was periodically removed and the same volume of fresh PBS buffer at the same temperature was added
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immediately to maintain constant release volume. These experiments were performed three times. The
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amount of released Rif was analyzed with a UV-Vis near-infrared spectrophotometer (UV–Vis 3600,
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Shimadzu) with wavelength 476 nm.
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The mechanism of Rifampicin release from the chitosan-p(MAA-co-NIPAM) hydrogels was investigated using Korsmeyer-Peppas model [22]
M t / M Kt n
(1)
where M t is the mass of drug released at time t, M is the total mass of released drug, K is a kinetic constant, and n is the diffusional exponent. Graph is plotted as log cumulative percent drug release against log time. Drug transport was classified based on the n value as shown in the Table 1 [23].
ACCEPTED MANUSCRIPT Table 1: Ritger-Peppas diffusion exponent and drug release mechanism for spherical devices. Drug release mechanism Fickian diffusion
0.43 < n < 0.85
Anomolus (non-Fickian) transport
0.85
Polymer swelling
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0.43
In vitro degradation analysis
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3.1
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3. Results and Discussion
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The freeze-dried hydrogels were immersed in different pH solutions for 16 days to observe the hydrolysis reactions in both basic and acidic mediums. The results for the 16 days’ hydrolysis in an
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acidic solution as shown in Figure 1 revealed that the hydrogels had been reduced to more than 50% of
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its original wet mass. From the similar composition and the hydrogel produced at the end of synthesis as shown in previous report by Rasib et al (2018) , PNIPAM content is more pronounced in the hydrogel
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composition and there is a higher presence of the chitosan content in hydrogel RT30, the degradation
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varies widely in hydrogel RT30 as compared to the other hydrogels. It was found that the high content of chitosan was the reason for hydrogel RT30 to lose 50% of its wet mass on day 6, at a rate that is
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faster than the other hydrogels.
ACCEPTED MANUSCRIPT 110
RT30 RT60 RT120 RT180
100
80 70
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60
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Wet Mass (%)
90
40 30 2
4
6
8
10
12
14
16
18
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0
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50
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Time (days)
Fig. 1. Degradation profiles of different reaction time Chitosan-p(MAA-co-NIPAM) hydrogels in acidic
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solution pH 1.68 by wet mass loss.
The hydrolysis results for the other hydrogels showed that there was a severe reduction in
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hydrogel RT60 and RT120 at the earlier immersion days but an increase of weight was observed in the
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initial immersion of RT180 hydrogel. Based on the hydrodynamic diameter in the acidic solution from the Rasib et al (2018), hydrogel RT180 had a slightly lesser swelling as compared to hydrogel RT120.
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The degree of crosslinking hindered the amount of water that goes into the network but with the solubility of the chitosan, it was able to hold a reasonable amount of water although the network had shrunk as a result of PNIPAM’s hydrophobicity, hence lowering the hydrolysis rate. While, the other hydrogels smaller amount of PNIPAM shrink lesser and more water was able to penetrate into the network and resulted in a cleavage of the chitosan.
There is a continuous weight loss on the following day but an increase in the weight is observed on the 8th day. This condition is caused by the cleavage of chitosan that has opened up a larger space in
ACCEPTED MANUSCRIPT the network. This is clearly proven by the slight increase of the weight in hydrogel RT30 in which the chitosan that covered the surface of the hydrogel has opened more space in the network. Weight gain can also be seen in hydrogels RT60 and RT120 when the reaction time is extended. However, a decline in the hydrogel RT180’s weight is observed, which is due to the higher crosslink factor. The weight for hydrogel RT30 began to slow down after the 8th day. Hence, can be deduce that when there is a higher
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chitosan content on the surface of the hydrogel, the hydrolysis process becomes easier and the number
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of chitosan glycosidic linkages hydrolysed faster after immerse for many days. Thus, impedes the
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weight loss of the hydrogel.
Figure 2 shows the hydrolysis profile of hydrogels in the basic solution with a pH of 7.4 for 16
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days. It was observed that the same pattern also applied to the hydrogels were immersed in the acidic solution, where there was a weight gain on the initial day, followed by a reduction of weight after 4 to 5
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days, a rise again and eventually dropped until day 16.
However, a difference was observed in
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hydrogel RT180, where there was no weight gain in the early days after the immersion was done.
ACCEPTED MANUSCRIPT 130
RT30 RT60 RT120 RT180
120
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100
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90
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Wet Mass (%)
110
70 0
2
4
6
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80
8
10
12
14
16
18
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Time (days)
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Fig.2. Degradation profiles of Chitosan-p(MAA-co-NIPAM) hydrogels in basic solution pH 7.4 by wet
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mass loss.
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At pH 7.4, the chitosan ions is less than the carboxyl ions from PMAA. The ionization of PMAA causes the hydrogel to swell and hold water for a certain period of time. In this case, the hydrolysed
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chitosan is slower as compared to the chitosan hydrolysed in an acidic solution and the swelling of the
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hydrogel is more dominant than its degradation. However, the hydrophobicity property of hydrogel RT180 with a higher content of PNIPAM causes it to be more dominant to hydrolyse. This case does not occur in hydrogel RT120 that contains lesser amount of PNIPAM where the hydrogel is more dominant to swell. Hydrogel RT180’s behaviour was classified by the researcher as steric shielding, meaning to say that as the chitosan begins to degrade, the PNIPAM chain will start to fold, thus shielding water from the network that consequently restrains the swelling of the hydrogel [24]. This test is repeated four times since this treatment is different from the other hydrogels, where the same result of a weight reduction is observed.
ACCEPTED MANUSCRIPT The weight loss of the hydrogel continued until the 16th day with an increment on day 4, a scenario that is similar to a hydrogel that is immersed in an acidic solution. In basic conditions, the cleavage of the chitosan is still present. Since chitosan is not soluble in that pH, its degradation rate is slower than in an acidic condition where in acidic condition there are more mechanisms that help in the
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acceleration of the degradation process. The same concept applies where the cleavage causes the
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hydrogel to have more space to expand, thus increasing its ability to hold more water from the
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environment. For hydrogels RT30 and RT60, the weight gains are higher than hydrogels RT120 and
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RT180. This is in line with the development of the hydrogel and its ability to absorb higher amounts of water as compared to RT120 and RT180, which are only limited to expand and the blockage from
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PNIPAM that tends to get rid of water at 37 °C. Overall, the remaining weight of the hydrogel is about 80%, while in acidic conditions it reaches to almost 40% after 16 days. This shows that the hydrogel can
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last longer in basic conditions but only less than two weeks in acidic conditions without any presence of
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Entrapment Efficiency of Rifampicin Drug
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3.2
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enzymes.
The percentage of entrapment efficiency (EE) of rifampicin (Rif) into different reaction times of
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polymerization on chitosan-p(MAA-co-NIPAM) hydrogels is shown in Figure 3. The EE for hydrogel RT120 was at a maximum level of 62% while for RT60, it was at a minimum level of 50%. It should be noted that with the two different degrees of EE, almost 1.5 mg to 2.0 mg of the rifampicin drug were loaded into the hydrogel from the full dosage of 3.28 mg. However, the results showed that the EE measured were not that much significant with the hydrogel that was polymerized at different reaction times.
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30
20
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Entrapment efficiency (%)
50
0 RT60
RT120
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RT30
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10
RT180
Time
In Vitro Drug Release
hydrogels
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Figure 3: Effect of varying reaction time on entrapment efficiency of chitosan-p(MAA-co-NIPAM)
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The different reaction times on polymerization chitosan-p(PMAA-co-NIPAM) hydrogels were
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investigated based on the time the Rif drug was released under different pH conditions (basic and acidic). The changes in the network that were caused by the swelling, collapsing and degrading processes controlled the diffusion of the embedded drug molecule within the hydrogel. The release profiles of the Rif drug from the hydrogel network for pH 7.4 are presented in Figure 4. Generally, the release of the Rif drug is below 25% in the first 72 hours, where there is an initial burst of release in the first hours followed by an almost constant rate of the drug release. The bursting effect is attributed to the surface layer of the hydrogel in which the drug is easily diffused out and also due to the collapsing action by PNIPAM in the network after the temperature rise to 37 °C [25]. Hydrogel RT30
ACCEPTED MANUSCRIPT demonstrated the highest bursting level while the bursting rates of the other three hydrogels became slower after four hours of the drug release. Due to the higher amount of release at the initial stage, hydrogel RT30 had the highest release of drug within 72 hours in the basic condition. From the composition with a higher level of chitosan content, the absence of crosslinkers within the chitosan that
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Cumulative Drug Release (%)
25
15 12
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20 15
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10 5 0 10
20
30
40
Time (hr)
50
60
70
9 6 3 0
0
1
2
3
4
5
Time (hr)
6
7
8
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0
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Cumulative Drug Release (%)
30
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RT30 RT60 RT120 RT180
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retain the chain from swelling continuously has allowed the hydrogel to release the drug more easily.
Figure 4: RIF release profile from different polymerization reaction time of Chitosan-p(MAA-co-
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NIPAM) hydrogels in basic condition pH 7.4 under constant temperature 37oC. Right graph: is a
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magnification of the rectangle region from left graph.
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The release of the Rif drug were subsequently followed by the hydrolysis results from hydrogels RT120 and RT180. Hydrogel RT180 releases the drug at a much slower as compared to RT120. Hypothetically, more drugs are supposed to be released with the effect of PNIPAM that collapses the hydrogel. However, in hydrogel RT180, the obstacle that prevents it from collapsing is due to the rigidity of the chitosan that is present in the network plus a high crosslinking within the structures, which controlled the hydrogel from bursting even further. For hydrogel RT120, since there was lesser crosslinking within the structures, it was able to release more drugs as compared to hydrogel RT180. Hydrogel with 60 minutes reaction showed the lowest drug release of about 4% within the 72 hours.
ACCEPTED MANUSCRIPT Although the swelling was higher at this pH level, the swelling also counteracted with the less shrinkage from PNIPAM. Hence, the hydrogel experienced a burst from the surface layer and a small amount of the drug is released as a result of the shrinkage and swelling.
From Figure 5(a), it can be seen that the maximum amount of the Rif released from chitosan-
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p(MAA-co-NIPAM) hydrogel in the presence of an acidic medium within the span of 72 hours is 25%.
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The percentage of drug release is expected to be higher in acidic solutions as a result of the hydrolysis
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and swelling factor; however the results show that the percentage is not that significantly different from
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the amount of drug released in a basic solution. By comparing both profiles of the acidic and basic conditions in Figure 5(b), it is evident that there was a quicker burst release in the acidic medium. This
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result supported the chitosan release studied by Manca, Loy, Zaru, Fadda and Antimisiaris [26], where it was observed that there was a higher burst release at pH 4.40 when compared with the release profile at
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pH 7.40, which is explained by the higher solubility of chitosan in an acidic media.
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For the same reason, hydrogel RT30 demonstrated the highest burst release among all the hydrogels due to the higher chitosan content in the network. Chitosan is hygroscopic in nature, having a
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great capability to form hydrogen bonding (formed with both hydroxyl (-OH) and amino (-NH2) groups)
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with water, which contributes to its swelling ability. Moreover, chitosan possesses properties that allow the acid solution to penetrate inside its polymer structure. These two factors were not present in the basic condition [27]. The amount of the drug release was followed by hydrogels RT180, RT60 and RT120 respectively. The burst release for hydrogel RT180 was higher due to the high PNIPAM content in the network where the hydrogel had shrunk within the initial hours of the release. A higher crosslink within the network controlled the drug release by restricting the network from swelling even further and over collapsing. A smaller amount of PNIPAM content will induce a lesser amount of hydrogel shrinkage and as a result, hydrogel RT120 released small amount of drug in the initial hours. Hence, a
ACCEPTED MANUSCRIPT lower crosslink has allowed a balance in the shrinking and swelling of the network in the drug release. As opposed to hydrogels RT180 and RT120, hydrogel RT60 tended to swell and was able to release the
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drug easily. A less PNIPAM content and a lower crosslink allowed the drug to be released faster.
Figure 5: (a) RIF release profile from different polymerization reaction time of Chitosan-p(MAA-coNIPAM) hydrogels in acidic medium pH 1.68 under constant temperature 37oC, (b) A combination release profile in acidic medium with pH 1.68 (red and hollow symbol) and pH 7.4 (black and solid symbol) represented faster release in acidic medium
ACCEPTED MANUSCRIPT Since the hydrogel structure consists of chitosan, the chitosan chain has the potential to produce cleavage that will affect the percentage of the drug release. Referring to Figure 1 and Figure 2, the hydrogel loses 10% to 20% of its mass in an acidic medium and less than 5% in a basic medium within a span of 72 hours. Therefore, the percentage of drug release is also contributed by the cleavage although it is difficult to specifically determine its effect on the percentage of the drug release.
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Unfortunately, this study could not be continued for further observation on the degradation effect of the
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drug release due to the limited diffusion of drug produced by the membrane dialysis [28]. The drug
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moved passively from the inner dialysis bag to a release medium and the concentration equilibrium was
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easily reached although a small amount of the drug had been successfully diffused out. Thus, an assumption can be made that the percentage of drug release will increase synchronise to the reduction
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hydrolysis reduction. The mechanism of drug release will explain further correlation of drug release to kinetic release of chitosan-p(MAA-co-NIPAM) hydrogel. Korsemeyer-Peppas model was applied to
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study the mechanism of drug release. The corresponding n values represent as diffusional exponent
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which explained the diffusion mechanism of the hydrogel [29]. The investigation was applied in both
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environment, acidic and basic as given in Table 2 based on plot of log M t / M versus log t shown in
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Figure 6.
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The values of n were in range 0.54 to 0.70 in acidic environment while 0.58 to 0.95 in basic environment for different reaction time of hydrogel. Based on the drug release mechanism by Peppas, this indicated that the hydrogel is non fickian (anamolous) drug release kinetics. Drug was released by overlapping both diffusion and hydrogel relaxation in controlling water uptake and drug release [22, 30, 31]. This matched with the explanation before on the drug release behaviour which the drug release tend to diffused once immersed in the release medium and was controlled by the swelling behaviour in releasing the drug. Although there are unfavourable amount of soluble polymer in the structure of RT30,
ACCEPTED MANUSCRIPT the network tend to shift from from non-Fickian in acidic medium to polymer swelling in basic medium
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due to the osmotic pressure [32].
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Figure 6: Korsmeyer-Peppas model of Rifampicin release in a) acidic environment, b) basic
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environment from chitosan-p(MAA-co-NIPAM) hydrogel.
Sample code RT30 RT60 RT120 RT180
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Table 2: Release kinetic studies of sphere hydrogel according to Korsemeyer-Peppas model. Acid R2 0.7840 0.7193 0.8594 0.7479
n 0.70 0.54 0.58 0.61
Basic R2 0.9144 0.8202 0.8610 0.8586
n 0.95 0.58 0.68 0.67
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4.
Conclusion
As a conclusion, the hydrolysis factor is less affected to the drug release within the first 72 hours.
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Based on the swelling, shrinkage, crosslinking and the hydrolysis behaviours presented within the
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periods, it can be predicted that the drug was released continuously once the hydrolysis took over, in
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which the hydrogel continued to shrink and is controlled with the swelling according to the PNIPAM and hydrophilic polymer (chitosan and PMAA) contents in the hydrogel network. A fast reaction time
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within 30 minutes showed a fast release and the Rif drug release can be controlled by the hydrogel through prolonging the reaction time to 180 minutes, which in this case, less than 20% of drug was
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released within the 72 hours. Chitosan also plays an important part in the continuous shrinkage control
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by PNIPAM and at the same time reducing the rate of the drug release. The drug also was release by
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combination diffusion and controlled by the swelling behaviour of the hydrogel.
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Acknowledgements
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This research was financially supported by the Ministry of Higher Education Malaysia [FRGS-
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6071325 and FRGS-6071337] and School of Materials and Mineral Resources Engineering, Engineering Campus Universiti Sains Malaysia for facilities and technical support.
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ACCEPTED MANUSCRIPT hydrochloride (DIL. HCl) into poly [(N-isopropylacrylamide)-co-(methacrylic acid)] hydrogels and its release behaviour from hydrogel slabs, J Control Release 102(3) (2005) 595-606. [12] C.S. Brazel, N.A. Peppas, Pulsatile local delivery of thrombolytic and antithrombotic agents using poly (N-isopropylacrylamide-co-methacrylic acid) hydrogels, J Control Release 39(1) (1996) 5764.
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copolymer micelles: molecular architectures and camptothecin drug release, Colloid Surface B:
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chitosan, PLGA and chitosan-coated PLGA microparticles, Colloid Surface B: Biointerfaces 67(2) (2008) 166-170.
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Potential Drug Release and Tissue Engineering Systems - Synthesis, Characterization, Swelling Kinetics and Mechanism, Journal of Physical Chemistry & Biophysics 7(3) (2017). [28] S. Hua, Comparison of in vitro dialysis release methods of loperamide-encapsulated liposomal gel for topical drug delivery, Int J Nanomed 9 (2014) 735. [29] G. Arora, K. Malik, I. Singh, Formulation and evaluation of mucoadhesive matrix tablets of taro gum: Optimization using response surface methodology, Polimery w medycynie 41(2) (2011).
ACCEPTED MANUSCRIPT [30] F. Ullah, F. Javed, M. Othman, A. Khan, R. Gul, Z. Ahmad, H. Md. Akil, Synthesis and Functionalization of Chitosan built Hydrogel with Induced Hydrophilicity for Extended Release of Sparingly Soluble Drugs, J Biomat Sci-Polym E 29(4) (2017) 376-396. [31] M. Stanojević, M.K. Krušić, J. Filipović, J. Parojčić, M. Stupar, An Investigation into the Influence of Hydrogel Composition on Swelling Behavior and Drug Release from Poly(Acrylamide-co-
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Itaconic Acid) Hydrogels in Various Media, Drug Deliv 13(1) (2006) 1-7.
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[32] M.A. Malana, R. Zohra, The release behavior and kinetic evaluation of tramadol HCl from
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chemically cross linked Ter polymeric hydrogels, DARU J Pharm Sci 21(1) (2013) 10.
ACCEPTED MANUSCRIPT Highlights 1. Changes in the hydrogel network for different polymerization stages affect the behaviour and the profile of drug release. 2. Prolong the reaction time, chitosan, poly(methacrylic acid) (PMAA), and Poly(Nisopropylacrylamide) (PNIPAM) were continuously crosslinked and slowing the hydrolysis rate
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3. Chitosan plays an important part in the continuous shrinkage of PNIPAM consequently allowed
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the hydrogel keep releasing the drug.
Siti Zalifah Md Rasib, Hazizan Md Akil, Abbas Khan, Zuratul Ain Abd Hamid PII: DOI: Reference:
S0141-8130(18)35460-6 https://doi.org/10.1016/j.ijbiomac.2019.01.190 BIOMAC 11623
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
12 October 2018 2 January 2019 28 January 2019
Please cite this article as: S.Z.M. Rasib, H.M. Akil, A. Khan, et al., Controlled release studies through chitosan-based hydrogel synthesized at different polymerization stages, International Journal of Biological Macromolecules, https://doi.org/10.1016/ j.ijbiomac.2019.01.190
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ACCEPTED MANUSCRIPT Controlled Release Studies through Chitosan-based Hydrogel Synthesized at Different Polymerization Stages
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School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia, Engineering
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Department of Chemistry, Abdul Wali Khan University Mardan, 23200 Pakistan
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2
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Campus, 14300 Nibong Tebal, Penang, Malaysia
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1
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Siti Zalifah Md Rasib1, Hazizan Md Akil*1, Abbas Khan2 and Zuratul Ain Abd Hamid1
*Corresponding author:
Email: [email protected] Tel: +604 599 6161 Fax: +604599 6907
ACCEPTED MANUSCRIPT Abstract
An
earlier
study
showed
that
the
behaviour
of
chitosan-poly(methacrylic
acid-co-N-
isopropylacrylamide) [chitosan-p(MAA-co-NIPAM)] hydrogels synthesized at different reaction times are affected with regard to their pH and temperature sensitivities. The study was continued in this paper
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to identify the effects of different reaction times on the degradation, efficiency of rifampicin (Rif)
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loading and the Rif release profile under two different pH conditions (acidic and basic). The results that
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were obtained showed that the hydrogel had a faster degradation rate in the acidic condition than in the
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basic condition, where there was a loss of approximately 50% and 20%, respectively in its original weight within two weeks. The Rif loading efficiency was within 50% and the drug release was
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controlled by characteristics that were developed beyond the polymerization stages of the synthesis. Therefore, the reaction time for the synthesis of the hydrogel can be considered as a way to control the
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behaviour of the hydrogel as well as to modify the drug release profile in the chitosan-p(MAA-co-
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NIPAM) hydrogel.
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1. Introduction
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Keywords: Chitosan; hydrogel; drug delivery
Hydrogels have been classified as polymeric materials since the early 1900's [1]. A hydrogel with a 3D crosslink-network consisting of hydrophilic groups is able to retain a certain amount of water despite swelling [2]. The advantages of this hydrogel are that improvements can be made to its reactions by modifying it to be more sensitive to the environment either by physical or chemical crosslinking methods or both. Hence, this makes the hydrogels tremendously important in a wide variety of applications in the biotechnology, biomedical, pharmaceutical, and other related fields such as for wound dressings [3, 4], contact lenses [5, 6], and drug delivery systems [7, 8]. A unique property of
ACCEPTED MANUSCRIPT hydrogels is their capability to retain a certain amount of water. This enables them to resemble actual tissue cells, thus making them a very good biocompatibility material. Many significant contributions have been made to utilise the potential of hydrogels in drug delivery since they offer a suitable timecontrolled delivery, whereby the amount of drug released can be controlled according to physiological needs. Drugs can be delivered through the activation of stimuli-responsive hydrogels to pH, magnetism,
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ultrasound, temperature changes, electrical effects, and irradiation [9].
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Chitosan-p(MAA-co-NIPAM) hydrogels were synthesised by combining two stimuli, namely, pH
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and temperature, to enable the hydrogels to be more sensitive to various pH levels and to be continuously activated at body temperature. The emulsion polymerisation technique was applied to
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crosslink all the three polymers and monomers. The excellent behaviour of the p(MAA-co-NIPAM) hydrogel network in drug delivery has been well established by several researchers such as Constantin,
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Bucatariu, Harabagiu, Popescu, Ascenzi and Fundueanu [10] for self-regulated drug delivery; Sousa,
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Prior-Cabanillas, Quijada-Garrido and Barrales-Rienda [11] in slab-form hydrogels; Brazel and Peppas
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[12] for pulsatile local delivery; and Wu and Lee [13] for general drug delivery or diagnostic applications. A hydrogel network gains an extra advantage when chitosan is introduced into the network
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drug delivery [14].
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for improvements to the biodegradable and biocompatible characteristics, especially for site-targeting
A study was done on monomer conversion against polymerization time during the synthesis of the hydrogels, where the polymerization rate was different based on different parameters [15]. Various types of monomers were considered during the synthesis, where it was shown that each monomer had a different polymerization rate. Therefore, the core or shell of the hydrogel that was produced could be controlled to potentially affect the drug release mechanism. Not only that, the desired hydrophiliclipophilic properties could also be achieved by an accurate selection of the polymerization time [16].
ACCEPTED MANUSCRIPT The previous study was based on the application of different polymerization times on the chitosanp(MAA-co-NIPAM) hydrogels during the synthesis process in order to evaluate the formation of the hydrogels since the hydrogels consisted of many types of monomers and polymers. The study found that the hydrogels that were synthesized over a prolonged reaction time were continuously cross-linked, and the outer surface of the hydrogels was covered mostly by PNIPAM. The swelling of the hydrogels was
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restricted since the crosslink density kept increasing in the network and an intermediate reaction time
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was preferable, based on the sensitivity of the hydrogels to swelling in response to the pH and
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temperature [17]. Although prolonging the polymerization would lead to the completion of the
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polymerization process, the hydrogels produced had a high molecular weight and became more rigid
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[18, 19].
The hypothesis of this study was that changes in the network for a prolonged reaction time also
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affect the behaviour and the profile of drug release. Shi, Alves and Mano [20] also reported that
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additional chitosan in a hydrogel potentially lowers the release profile since chitosan also degrades with
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time. Further information on the degradation effects of the hydrogel on the drug release profile should also be investigated. The correlation between the structure and the swelling behaviour for different
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reaction times during the synthesis on the drug release behaviour should also be investigated to evaluate
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the potential of the hydrogel as a drug carrier. Therefore, this study continued with the previous work by covering the degradation of the hydrogel against time at a certain pH level, drug loading efficiency, kinetics, and drug release profile. Rifampicin was used as a model drug and the pH environment was varied between pH 7.4 (simulated intestinal fluid, blood) and 1.68 (simulated gastric fluid), whereas the temperature was kept constant at the temperature of the human body (37 °C).
ACCEPTED MANUSCRIPT
2. Materials and Methods 2.1
Materials
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N-isopropylacrylamide (NIPAM), N, N-Methylenebisacrylamide (MBA) (purity > 95.0%) and
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chitosan (Mw ≈ 700-1000 kDa, degree of deacetylation ≈ 90.0%) were purchased from Zhejiang
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Golden-Shell Pharmaceutical Co., Ltd (Zhejiang, China), Span™ 80 (Croda International Plc., UK), and acetic acid (≥ 99.85%) from Sigma–Aldrich. NIPAM was purified by recrystallization from a toluene/n-
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hexane (1:3) mixture. Methacrylic acid (MAA) (purity > 95.0%) was further purified by distillation
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under reduced pressure, while all the other chemicals were used as received and without further purification. Deionised distilled water (DDH2O) was used for all the reactions and solution preparations,
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and the hydrogels were purified by using the membrane filter, Spectra/Por® molecularporous membrane
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tubing, which was obtained from Spectrum Laboratories, Inc., Rancho Dominguez, CA, USA; cut-off
Preparation of chitosan-p(MAA-co-NIPAM) hydrogels
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2.2
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12,000–14,000.
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Emulsion polymerization by different reaction time was applied to produced chitosan-p(MAA-coNIPAM) hydrogel. Method of synthesis has already been described in the previous paper [21] by varying time to stop the polymerization reaction to 30 minutes (RT30), 60 minutes (RT60), 120 minutes (RT120), and 180 minutes (RT180). The copolymer hydrogels that were obtained were then purified by centrifugation (Sorvall RC-6 Plus superspeed centrifuge, Thermo Electron Co., Waltham, MA, USA) and decantation, and were washed with water. The resultant hydrogels were purified by dialysis through a Spectra/Por molecular porous membrane tubing using distilled water with a pH of 5±0.3 at room
ACCEPTED MANUSCRIPT temperature (25 ℃) that was frequently changed for 1 week. The purified hydrogels were freeze–dried for 48 hours with a LABCONCO freeze dry system after freezing overnight at -40 ℃.
2.3
In vitro degradation analysis
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Freeze-dried hydrogels was weight 5 mg for three sample for each reaction time hydrogel. The
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hydrogels then were incubated in 3 ml PBS at 37 °C for 24 hours with pH 1.68 and 7.5 respectively.
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Then, the hydrogel was centrifuged, supernatant was removed and wet hydrogel were weighted (W0).
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The hydrogels were incubated again at the same condition (3 ml PBS with pH 1.68 and 7.5 at 37 °C). The weights of wet hydrogels were measured in certain days (W1). The final weight ratios were
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Determination of Rifampicin Encapsulation Efficiency
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2.4
(1)
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W – W0 Wet mass % 1 x 100 W0
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calculated by equation 1.
The encapsulation efficiency (EE) of hydrogels were determined as follows. The 10 mM Rif drug stock
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solution was prepared by dissolved the drug in methanol solution. 60 mg of freeze dried hydrogels were immersed in 0.4 ml Rif from the stock solution and stirred slowly (100rpm/min) for various time under room temperature. Then, 2.6 ml of distilled water was added into the solution and stirred for 5 seconds. After the loading procedure, the suspensions were centrifuged (3300 Micro Centrifuge, Kubota, Japan) at 13000 rpm for 20 min and the Rif that remained in the supernatant phase was diluted 10 times to determine by using a UV-Vis. The DLC and EE percentage was calculated by equation 2.
ACCEPTED MANUSCRIPT M - M2 Drug Loading 1 x 100 M3
(2)
Where M1 is the initial amount of drug, M2 is the amount of Rif after loading (unloaded drug from the
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In Vitro Release Studies
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2.5
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supernatant), and M3 is the amount of hydrogel.
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Drug release from hydrogel was studied using a dialysis method. The hydrogels loaded with Rif (10 mg/ml under different PBS pH 1.68 and 7.4) were added in a dialysis bag (MWCO 10 kDa; Thermo
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Fisher Scientific) and suspended in 500 mL of PBS (pH 7.4 and 1.68). The dissolution medium was
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stirred at 200 rpm in a laboratory shaker and maintained the temperature at 37oC. The sample (5 mL) was periodically removed and the same volume of fresh PBS buffer at the same temperature was added
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immediately to maintain constant release volume. These experiments were performed three times. The
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amount of released Rif was analyzed with a UV-Vis near-infrared spectrophotometer (UV–Vis 3600,
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Shimadzu) with wavelength 476 nm.
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The mechanism of Rifampicin release from the chitosan-p(MAA-co-NIPAM) hydrogels was investigated using Korsmeyer-Peppas model [22]
M t / M Kt n
(1)
where M t is the mass of drug released at time t, M is the total mass of released drug, K is a kinetic constant, and n is the diffusional exponent. Graph is plotted as log cumulative percent drug release against log time. Drug transport was classified based on the n value as shown in the Table 1 [23].
ACCEPTED MANUSCRIPT Table 1: Ritger-Peppas diffusion exponent and drug release mechanism for spherical devices. Drug release mechanism Fickian diffusion
0.43 < n < 0.85
Anomolus (non-Fickian) transport
0.85
Polymer swelling
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0.43
In vitro degradation analysis
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3.1
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3. Results and Discussion
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The freeze-dried hydrogels were immersed in different pH solutions for 16 days to observe the hydrolysis reactions in both basic and acidic mediums. The results for the 16 days’ hydrolysis in an
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acidic solution as shown in Figure 1 revealed that the hydrogels had been reduced to more than 50% of
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its original wet mass. From the similar composition and the hydrogel produced at the end of synthesis as shown in previous report by Rasib et al (2018) , PNIPAM content is more pronounced in the hydrogel
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composition and there is a higher presence of the chitosan content in hydrogel RT30, the degradation
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varies widely in hydrogel RT30 as compared to the other hydrogels. It was found that the high content of chitosan was the reason for hydrogel RT30 to lose 50% of its wet mass on day 6, at a rate that is
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faster than the other hydrogels.
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RT30 RT60 RT120 RT180
100
80 70
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60
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Wet Mass (%)
90
40 30 2
4
6
8
10
12
14
16
18
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0
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50
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Time (days)
Fig. 1. Degradation profiles of different reaction time Chitosan-p(MAA-co-NIPAM) hydrogels in acidic
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solution pH 1.68 by wet mass loss.
The hydrolysis results for the other hydrogels showed that there was a severe reduction in
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hydrogel RT60 and RT120 at the earlier immersion days but an increase of weight was observed in the
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initial immersion of RT180 hydrogel. Based on the hydrodynamic diameter in the acidic solution from the Rasib et al (2018), hydrogel RT180 had a slightly lesser swelling as compared to hydrogel RT120.
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The degree of crosslinking hindered the amount of water that goes into the network but with the solubility of the chitosan, it was able to hold a reasonable amount of water although the network had shrunk as a result of PNIPAM’s hydrophobicity, hence lowering the hydrolysis rate. While, the other hydrogels smaller amount of PNIPAM shrink lesser and more water was able to penetrate into the network and resulted in a cleavage of the chitosan.
There is a continuous weight loss on the following day but an increase in the weight is observed on the 8th day. This condition is caused by the cleavage of chitosan that has opened up a larger space in
ACCEPTED MANUSCRIPT the network. This is clearly proven by the slight increase of the weight in hydrogel RT30 in which the chitosan that covered the surface of the hydrogel has opened more space in the network. Weight gain can also be seen in hydrogels RT60 and RT120 when the reaction time is extended. However, a decline in the hydrogel RT180’s weight is observed, which is due to the higher crosslink factor. The weight for hydrogel RT30 began to slow down after the 8th day. Hence, can be deduce that when there is a higher
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chitosan content on the surface of the hydrogel, the hydrolysis process becomes easier and the number
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of chitosan glycosidic linkages hydrolysed faster after immerse for many days. Thus, impedes the
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weight loss of the hydrogel.
Figure 2 shows the hydrolysis profile of hydrogels in the basic solution with a pH of 7.4 for 16
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days. It was observed that the same pattern also applied to the hydrogels were immersed in the acidic solution, where there was a weight gain on the initial day, followed by a reduction of weight after 4 to 5
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days, a rise again and eventually dropped until day 16.
However, a difference was observed in
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hydrogel RT180, where there was no weight gain in the early days after the immersion was done.
ACCEPTED MANUSCRIPT 130
RT30 RT60 RT120 RT180
120
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100
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90
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Wet Mass (%)
110
70 0
2
4
6
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80
8
10
12
14
16
18
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Time (days)
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Fig.2. Degradation profiles of Chitosan-p(MAA-co-NIPAM) hydrogels in basic solution pH 7.4 by wet
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mass loss.
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At pH 7.4, the chitosan ions is less than the carboxyl ions from PMAA. The ionization of PMAA causes the hydrogel to swell and hold water for a certain period of time. In this case, the hydrolysed
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chitosan is slower as compared to the chitosan hydrolysed in an acidic solution and the swelling of the
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hydrogel is more dominant than its degradation. However, the hydrophobicity property of hydrogel RT180 with a higher content of PNIPAM causes it to be more dominant to hydrolyse. This case does not occur in hydrogel RT120 that contains lesser amount of PNIPAM where the hydrogel is more dominant to swell. Hydrogel RT180’s behaviour was classified by the researcher as steric shielding, meaning to say that as the chitosan begins to degrade, the PNIPAM chain will start to fold, thus shielding water from the network that consequently restrains the swelling of the hydrogel [24]. This test is repeated four times since this treatment is different from the other hydrogels, where the same result of a weight reduction is observed.
ACCEPTED MANUSCRIPT The weight loss of the hydrogel continued until the 16th day with an increment on day 4, a scenario that is similar to a hydrogel that is immersed in an acidic solution. In basic conditions, the cleavage of the chitosan is still present. Since chitosan is not soluble in that pH, its degradation rate is slower than in an acidic condition where in acidic condition there are more mechanisms that help in the
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acceleration of the degradation process. The same concept applies where the cleavage causes the
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hydrogel to have more space to expand, thus increasing its ability to hold more water from the
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environment. For hydrogels RT30 and RT60, the weight gains are higher than hydrogels RT120 and
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RT180. This is in line with the development of the hydrogel and its ability to absorb higher amounts of water as compared to RT120 and RT180, which are only limited to expand and the blockage from
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PNIPAM that tends to get rid of water at 37 °C. Overall, the remaining weight of the hydrogel is about 80%, while in acidic conditions it reaches to almost 40% after 16 days. This shows that the hydrogel can
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last longer in basic conditions but only less than two weeks in acidic conditions without any presence of
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Entrapment Efficiency of Rifampicin Drug
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3.2
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enzymes.
The percentage of entrapment efficiency (EE) of rifampicin (Rif) into different reaction times of
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polymerization on chitosan-p(MAA-co-NIPAM) hydrogels is shown in Figure 3. The EE for hydrogel RT120 was at a maximum level of 62% while for RT60, it was at a minimum level of 50%. It should be noted that with the two different degrees of EE, almost 1.5 mg to 2.0 mg of the rifampicin drug were loaded into the hydrogel from the full dosage of 3.28 mg. However, the results showed that the EE measured were not that much significant with the hydrogel that was polymerized at different reaction times.
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30
20
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Entrapment efficiency (%)
50
0 RT60
RT120
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RT30
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10
RT180
Time
In Vitro Drug Release
hydrogels
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3.3
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Figure 3: Effect of varying reaction time on entrapment efficiency of chitosan-p(MAA-co-NIPAM)
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The different reaction times on polymerization chitosan-p(PMAA-co-NIPAM) hydrogels were
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investigated based on the time the Rif drug was released under different pH conditions (basic and acidic). The changes in the network that were caused by the swelling, collapsing and degrading processes controlled the diffusion of the embedded drug molecule within the hydrogel. The release profiles of the Rif drug from the hydrogel network for pH 7.4 are presented in Figure 4. Generally, the release of the Rif drug is below 25% in the first 72 hours, where there is an initial burst of release in the first hours followed by an almost constant rate of the drug release. The bursting effect is attributed to the surface layer of the hydrogel in which the drug is easily diffused out and also due to the collapsing action by PNIPAM in the network after the temperature rise to 37 °C [25]. Hydrogel RT30
ACCEPTED MANUSCRIPT demonstrated the highest bursting level while the bursting rates of the other three hydrogels became slower after four hours of the drug release. Due to the higher amount of release at the initial stage, hydrogel RT30 had the highest release of drug within 72 hours in the basic condition. From the composition with a higher level of chitosan content, the absence of crosslinkers within the chitosan that
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Cumulative Drug Release (%)
25
15 12
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20 15
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10 5 0 10
20
30
40
Time (hr)
50
60
70
9 6 3 0
0
1
2
3
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Time (hr)
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7
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Cumulative Drug Release (%)
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RT30 RT60 RT120 RT180
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retain the chain from swelling continuously has allowed the hydrogel to release the drug more easily.
Figure 4: RIF release profile from different polymerization reaction time of Chitosan-p(MAA-co-
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NIPAM) hydrogels in basic condition pH 7.4 under constant temperature 37oC. Right graph: is a
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magnification of the rectangle region from left graph.
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The release of the Rif drug were subsequently followed by the hydrolysis results from hydrogels RT120 and RT180. Hydrogel RT180 releases the drug at a much slower as compared to RT120. Hypothetically, more drugs are supposed to be released with the effect of PNIPAM that collapses the hydrogel. However, in hydrogel RT180, the obstacle that prevents it from collapsing is due to the rigidity of the chitosan that is present in the network plus a high crosslinking within the structures, which controlled the hydrogel from bursting even further. For hydrogel RT120, since there was lesser crosslinking within the structures, it was able to release more drugs as compared to hydrogel RT180. Hydrogel with 60 minutes reaction showed the lowest drug release of about 4% within the 72 hours.
ACCEPTED MANUSCRIPT Although the swelling was higher at this pH level, the swelling also counteracted with the less shrinkage from PNIPAM. Hence, the hydrogel experienced a burst from the surface layer and a small amount of the drug is released as a result of the shrinkage and swelling.
From Figure 5(a), it can be seen that the maximum amount of the Rif released from chitosan-
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p(MAA-co-NIPAM) hydrogel in the presence of an acidic medium within the span of 72 hours is 25%.
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The percentage of drug release is expected to be higher in acidic solutions as a result of the hydrolysis
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and swelling factor; however the results show that the percentage is not that significantly different from
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the amount of drug released in a basic solution. By comparing both profiles of the acidic and basic conditions in Figure 5(b), it is evident that there was a quicker burst release in the acidic medium. This
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result supported the chitosan release studied by Manca, Loy, Zaru, Fadda and Antimisiaris [26], where it was observed that there was a higher burst release at pH 4.40 when compared with the release profile at
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pH 7.40, which is explained by the higher solubility of chitosan in an acidic media.
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For the same reason, hydrogel RT30 demonstrated the highest burst release among all the hydrogels due to the higher chitosan content in the network. Chitosan is hygroscopic in nature, having a
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great capability to form hydrogen bonding (formed with both hydroxyl (-OH) and amino (-NH2) groups)
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with water, which contributes to its swelling ability. Moreover, chitosan possesses properties that allow the acid solution to penetrate inside its polymer structure. These two factors were not present in the basic condition [27]. The amount of the drug release was followed by hydrogels RT180, RT60 and RT120 respectively. The burst release for hydrogel RT180 was higher due to the high PNIPAM content in the network where the hydrogel had shrunk within the initial hours of the release. A higher crosslink within the network controlled the drug release by restricting the network from swelling even further and over collapsing. A smaller amount of PNIPAM content will induce a lesser amount of hydrogel shrinkage and as a result, hydrogel RT120 released small amount of drug in the initial hours. Hence, a
ACCEPTED MANUSCRIPT lower crosslink has allowed a balance in the shrinking and swelling of the network in the drug release. As opposed to hydrogels RT180 and RT120, hydrogel RT60 tended to swell and was able to release the
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drug easily. A less PNIPAM content and a lower crosslink allowed the drug to be released faster.
Figure 5: (a) RIF release profile from different polymerization reaction time of Chitosan-p(MAA-coNIPAM) hydrogels in acidic medium pH 1.68 under constant temperature 37oC, (b) A combination release profile in acidic medium with pH 1.68 (red and hollow symbol) and pH 7.4 (black and solid symbol) represented faster release in acidic medium
ACCEPTED MANUSCRIPT Since the hydrogel structure consists of chitosan, the chitosan chain has the potential to produce cleavage that will affect the percentage of the drug release. Referring to Figure 1 and Figure 2, the hydrogel loses 10% to 20% of its mass in an acidic medium and less than 5% in a basic medium within a span of 72 hours. Therefore, the percentage of drug release is also contributed by the cleavage although it is difficult to specifically determine its effect on the percentage of the drug release.
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Unfortunately, this study could not be continued for further observation on the degradation effect of the
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drug release due to the limited diffusion of drug produced by the membrane dialysis [28]. The drug
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moved passively from the inner dialysis bag to a release medium and the concentration equilibrium was
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easily reached although a small amount of the drug had been successfully diffused out. Thus, an assumption can be made that the percentage of drug release will increase synchronise to the reduction
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hydrolysis reduction. The mechanism of drug release will explain further correlation of drug release to kinetic release of chitosan-p(MAA-co-NIPAM) hydrogel. Korsemeyer-Peppas model was applied to
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study the mechanism of drug release. The corresponding n values represent as diffusional exponent
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which explained the diffusion mechanism of the hydrogel [29]. The investigation was applied in both
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environment, acidic and basic as given in Table 2 based on plot of log M t / M versus log t shown in
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Figure 6.
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The values of n were in range 0.54 to 0.70 in acidic environment while 0.58 to 0.95 in basic environment for different reaction time of hydrogel. Based on the drug release mechanism by Peppas, this indicated that the hydrogel is non fickian (anamolous) drug release kinetics. Drug was released by overlapping both diffusion and hydrogel relaxation in controlling water uptake and drug release [22, 30, 31]. This matched with the explanation before on the drug release behaviour which the drug release tend to diffused once immersed in the release medium and was controlled by the swelling behaviour in releasing the drug. Although there are unfavourable amount of soluble polymer in the structure of RT30,
ACCEPTED MANUSCRIPT the network tend to shift from from non-Fickian in acidic medium to polymer swelling in basic medium
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due to the osmotic pressure [32].
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Figure 6: Korsmeyer-Peppas model of Rifampicin release in a) acidic environment, b) basic
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environment from chitosan-p(MAA-co-NIPAM) hydrogel.
Sample code RT30 RT60 RT120 RT180
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Table 2: Release kinetic studies of sphere hydrogel according to Korsemeyer-Peppas model. Acid R2 0.7840 0.7193 0.8594 0.7479
n 0.70 0.54 0.58 0.61
Basic R2 0.9144 0.8202 0.8610 0.8586
n 0.95 0.58 0.68 0.67
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4.
Conclusion
As a conclusion, the hydrolysis factor is less affected to the drug release within the first 72 hours.
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Based on the swelling, shrinkage, crosslinking and the hydrolysis behaviours presented within the
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periods, it can be predicted that the drug was released continuously once the hydrolysis took over, in
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which the hydrogel continued to shrink and is controlled with the swelling according to the PNIPAM and hydrophilic polymer (chitosan and PMAA) contents in the hydrogel network. A fast reaction time
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within 30 minutes showed a fast release and the Rif drug release can be controlled by the hydrogel through prolonging the reaction time to 180 minutes, which in this case, less than 20% of drug was
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released within the 72 hours. Chitosan also plays an important part in the continuous shrinkage control
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by PNIPAM and at the same time reducing the rate of the drug release. The drug also was release by
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combination diffusion and controlled by the swelling behaviour of the hydrogel.
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Acknowledgements
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This research was financially supported by the Ministry of Higher Education Malaysia [FRGS-
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6071325 and FRGS-6071337] and School of Materials and Mineral Resources Engineering, Engineering Campus Universiti Sains Malaysia for facilities and technical support.
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ACCEPTED MANUSCRIPT Highlights 1. Changes in the hydrogel network for different polymerization stages affect the behaviour and the profile of drug release. 2. Prolong the reaction time, chitosan, poly(methacrylic acid) (PMAA), and Poly(Nisopropylacrylamide) (PNIPAM) were continuously crosslinked and slowing the hydrolysis rate
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3. Chitosan plays an important part in the continuous shrinkage of PNIPAM consequently allowed
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the hydrogel keep releasing the drug.