Available online at www.sciencedirect.com
ScienceDirect Procedia Chemistry 19 (2016) 388 – 393
5th International Conference on Recent Advances in Materials, Minerals and Environment (RAMM) & 2nd International Postgraduate Conference on Materials, Mineral and Polymer (MAMIP), 4-6 August 2015
Effect of Different Composition on Particle Size Chitosan-PMAAPNIPAM Hydrogel SZM Rasiba, HM Akila* and AS Yahyab a
School of Materials and Mineral Resources Engineering, Engineering Campus, Universiti Sains Malaysia, 14300 Nibong Tebal Pulau Pinang b School of Civil Engineering, Engineering Campus, Universiti Sains Malaysia, 14300 Nibong Tebal Pulau Pinang
Abstract Chitosan-based (chitosan-poly(methacrylic acid)-poly(N-isopropylacrylamide) [Cs-PMAA-PNIPAM] copolymer hydrogels were synthesized by free radical emulsion polymerization to study the effect of different composition of monomer on hydrogel particle size. Chitosan usually applied for medical use such as drug delivery due to its biodegradability, bio-compatibility, and nontoxicity properties. Co-polymerized chitosan with MAA and NIPAM is an improvement of chitosan gel to be more responsive to the environment of human body included different pH, temperature, ionic strength, electric field, and enzyme activities. Small size of the particles is particularly important to ensure the particles reach the target site especially as a drug delivery. A full factorial experimental design (23) was employed to identify which factors influenced most on the particle size. The design considered three factors which is amount of MAA, NIPAM and N,N'-Methylenebisacrylamide (MBA) while particle size are chosen as the responses of the variation on each composition. Particle size distribution was measured by laser diffractionin wet condition. From the design of experiment, NIPAM shows the main factor affected the particle size. However combination of the others factors also contributed on the whole size of the hydrogel. © B.V. This is an open access article under the CC BY-NC-ND license © 2016 2016The TheAuthors. Authors.Published PublishedbybyElsevier Elsevier B.V. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia. Peer-review under responsibility of School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia Keywords: Chitosan; Hydrogel; Drug delivery; Particle size
1. Introduction Chitosan is much known especially in medical and pharmaceutical application. Its biocompatible properties allow * Corresponding author. Tel.: +6-045-996-161; fax: +6-045-941-011. E-mail address:
[email protected]
1876-6196 © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of School of Materials and Mineral Resources Engineering, Universiti Sains Malaysia doi:10.1016/j.proche.2016.03.028
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it usable in medical application such as topical ocular application1, implantation2or injection3. Chitosan also metabolised by certain human enzymes and this considered that chitosan is biodegradable4and also bioadhesive due to positive charges at physiological pH which increase retention at the site of application5. Others than that chitosan promote wound-healing especially in veterinary medicine6and has bacteriostatic effects with broader spectra of activity, a higher killing rate, and lower toxicity toward mammalian cells7. Since chitosan properties are excellent in swollen state, therefore chitosan in hydrogel form is selected for medical application and further research on chitosan hydrogel for multi-purpose application will be more advantages8. Its’ performance as a drug delivery give more attraction in medical field especially when chitosan become more responsive to the environment in human body. Copolymerized chitosan with MAA and NIPAM is one of alternative to enhance the pH and temperature responsive of the chitosan hydrogel. However, different composition of chitosan, MAA and NIPAM monomer during copolymerization result on different particle size of the hydrogel. Particle size is particularly important on biodistribution and circulation profile within the body9.Varied size of hydrogel carrier in body resulting in different blood circulation times. Once hydrogel is injected into circulation system, certain hydrogel usually small particle size below 10 nm will be removedand certain over than 10 nm will be penetrated into small capillaries within the body tissues10. Hydrogel with particle size with range from 50 to 500 nm is usually being used with prolong circulation time and suitable for drug delivery which take days and sometimes almost a month in circulation system. Hydrogel with larger than 4 μm which is the smallest diameter size of capillaries system will be captured and withheld in the lung. This will risk on human body11. However particle size of hydrogelin different part of human body is depend on its site target and also its application such as particle size of hydrogel for oral delivery can be larger compared to particle size for transdermal or blood by injection. Since Chitosan-PMAA-PNIPAM hydrogel function as a carrier in body system, it is particular to achieve particle size in range below 500 nm so that the application of this chitosan-PMAA-PNIPAM will be enhanced for any part of body. An experimental design is use to study the main factor affected the particle size and correlation each of the factors that contributed to variation of hydrogel size. Nomenclature CS-PMAA-PNIPAM Chitosan MAA NIPAM MBA DDH2O
Chitosan-poly(methacrylic acid)-poly(N-isopropylacrylamide) Chitosan Methacrylic acid N-isopropylacrylamide N,N'-Methylenebisacrylamide Deionised distilled water
2. Experimental 2.1. Materials N-isopropylacrylamide (NIPAM), N,N-Methylenebisacrylamide (MBA) (purity > 95.0%) and Chitosan (Mw ≈ 160,000 g/mol, degree of deacetylation ≈ 91.0%) were purchased from Aldrich (St. Louis, MO, USA), and all other chemicals were obtained from Acros (Geel, Belgium). NIPAM was purified by recrystallization from toluene/nhexane (1:3) mixture. Methacrylic acid (MAA) (purity > 95.0%) was further purified through distillation under reduced pressure, whereas all other chemicals were used as received and without further purification. Deionised distilled water (DDH2O) was used for all reactions, solution preparations, and hydrogel purified by using membrane filtere, which was obtained from a Milli-Q®system,Millipore (Bedford, MA, USA).
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2.2. Hydrogel synthesis All the hydrogels were synthesized by the free-radical copolymerization of chitosan, MAA, NIPAM and MBA, using ammonium persulfate (APS) as an initiator. Briefly, approximately different amount of chitosan (0.3 g), MAA, and NIPAM were dissolved in 100 mL doubly distilled and deionized water, under constant stirring for approximately 18–20 h, in a three-neck round-bottom flask equipped with an N2 gas inlet and condenser. After 20 h of stirring under N2 purging at 30 °C, MBA (which was used as the crosslinker) was added to the reaction flask. The temperature of the reaction mixture was slowly raised to 70 °C under N2 purging along with continuous stirring and gradual heating in a silicon-filled oil bath. After 30 minutes of constant heating at 70 °C, 5 mL of APS (0.05 M) were added to the reaction mixture in order to start the polymerization reaction. Several minutes after the addition of the initiator, the color of the reaction mixture turned milky. The polymerization reaction was allowed to proceed for 5 hours at constant stirring under N2 purging at 70 °C. The obtained copolymer hydrogels were then purified by centrifugation (Sorvall RC-6 Plus supers peed centrifuge, Thermo Electron Co., Waltham, MA, USA) and decantation, and were then washed with water. Next, each resultant hydrogel was further purified by dialysis for 1 week (Spectra/Por molecular porous membrane tubing from Spectrum Laboratories, Inc., Rancho Dominguez, CA,USA; cut-off 12,000–14,000; the same tubing was used below) against a frequently changed dilute HCl solution in water, with a pH 5±0.3 at room temperature (25 °C). 2.3. Characterization Since particle size is in range 0.3 to 1µm, therefore particle size is measured by using laser diffraction (Mastersizer 3000) to optimized the suitable composition of the hydrogel. 2.4. Experimental Design In this study, a two-level 23 factorial experiment design was employed to identify which factors influenced most on the particle size of the hydrogel form. The factor is based on different amount of MAA, NIPAM and MBAwhich has two levels (‘low’ -1 and ‘high’ 1) with total of 16 runs in replicate were performed by using MINITAB 16 software. All the factors are defined in Table 1 as follow. Table 1. Real values of coded levels. Factor
Code
MAA NIPAM MBA
3.
A B C
Unit g/100 mL g/100 mL mg/100 mL
Level -1 1 1 10
1 3 3 30
Results and discussion
Based on 16 runs of different composition generated by MINITAB software on Table 2, synthesis hydrogel has been done and particle size measured by using laser diffraction Mastersize 3000 is shown. Particle size presented is in range from 27 µm to 1410 µm which particle size for experimental run 4 is the smallest and particle size for experimental run 14 is the biggest particle size. Based on the composition of run 4, less amount of crosslinker (MBA) is used during the polymerization. Therefore less crosslink density produce within the structure. Crosslink tied amide group from chitosan with another amide group of chitosan result mobility of chain increase and hence larger particles size formed12. The particle size also changed from the smallest amount to the highest amount of monomer feed. This shows that varied the composition of monomer contributed on different particle size of the hydrogel.
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S.Z.M. Rasib et al. / Procedia Chemistry 19 (2016) 388 – 393 Table 2. Combinations of the experimental variables in experiment design. Run
Variables B -1 -1 1 1 -1 -1 1 1 -1 -1 1 1 -1 -1 1 1
A -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Response Particle Size (µm) 1010 672 793 38 145 976 138 73.1 1150 611 102 27.2 161 1410 130 71.5
C -1 -1 -1 -1 1 1 1 1 -1 -1 -1 -1 1 1 1 1
The interaction and main effect of particles size has been analysed with standardized effect of factor studied at alpha 0.05. Fig. 1 shows the normal plot of the standardized effect on the particles size. In general, the far point of pvalue from the straight line, the more significant the terms observed. From the point below, factor B (NIPAM) is the most significant factor which affected the particle size and followed with interaction factor A*C (MAA and MBA), A*B*C (MAA*NIPAM*MBA) and A*B (MAA*NIPAM). Even though factor A (MAA) and C (MBA) is not significant on particles size, both of the factor should be consider due to hierarchical model since interaction factor A*C (MAA and MBA) is significant. Both of the factors also acceptable to be used since this two factors is very important in producing the hydrogel. Normal Plot of the Standardized Effects (response is Particle Size, Alpha = 0.05) 99 95 AC
Percent
90
Factor A B C
80 70 60 50 40 30 20 10
Effect Type Not Significant Significant Name PMAA PNIPAM MBA
AB ABC B
5 1 -5.0
-2.5
0.0 Standardized Effect
2.5
5.0
Fig. 1. Normal plot of the standardized effect for MAA,NIPAM, MBA and interaction factors.
Table 3 shows significant value for the single and interaction factors. From the table, significant value is synchronise with normal plot of the standardized effect (Fig. 1). Factor B (NIPAM) shows very small p-value which is less to 3 decimal value, followed with interaction factor A (MAA) and C (MBA) with p-value 0.002.
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Table 3. Significant value and estimated effect for particle size. Variable
Particle Size
Constant A B C A*B A*C B*C A*B*C
p-value
Estimated Effect
0.771 0.000 0.156 0.032 0.002 0.813 0.027
31.2 -595.3 -162.3 -269.5 457.9 25.4 -281.3
Fig. 2 is the interaction plot for particle size between the three factors. It is clearly seen that there are no or very small interaction between factor B (NIPAM) and factor C (MBA). Based on interaction within factor A (MAA) and B (NIPAM), particle size is smaller by increased both amount of MAA and NIPAM. However, particles are still in smaller size even though small amount of MAA with same amount of NIPAM is used during the synthesis. This shows that less contribution of MAA on the particle size compared to the amount of NIPAM. While, the interaction between MAA and MBA, the smallest particle size can be achieve by decrease the amount of MAA and increased the amount of MBA. Therefore, the amount of MAA should be decreased while the amount of NIPAM and MBA should be increased to acquire very small particles of hydrogel.
Interaction Plot for Particle Size Fitted Means
1
3
10
30 1000
MAA
500
0 1000
NIPAM
MAA 1 3 NIPAM 1 3
500
0 MBA Fig. 2. The interaction plot for particle size between the three factors.
Table 4 shows an idea for linear and interaction effect of the parameter to the particle size. Correlation within the single factors and interaction factors can be generated by linear regression to determine the particle size in equation 1. This particle size model is accepted to be used based on the fit quality of R2 value which is 89.61%.
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S.Z.M. Rasib et al. / Procedia Chemistry 19 (2016) 388 – 393 Table 4. Significant value, estimated coefficient and estimated effect for particle size. Variable Constant A B C A*B A*C B*C A*B*C
Particle Size Estimated Coefficient 2748.56 -735.438 -616.188 -112.716 146.563 51.0288 29.4038 -14.0663
(1) 4.
Conclusion
Effect of different composition of MAA, NIPAM and MBA on particle size has been studied. Amount of NIPAM is the main influence on the particle size. Fewer amounts of MAA and increased amount of MBA is necessary to achieved very small particle size of Chitosan-PMAA-PNIPAM hydrogel. The smallest particle size for this model is 27.2 µm and this value can be less by optimization the amount of each factor based on the regression equation of the model. Acknowledgements The authors wish to acknowledge the USM for sponsoring this project under USM-FRGS-6071242 and the School of Materials and Mineral Resources Engineering, Engineering Campus, Universiti Sains Malaysia for their technical support. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
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