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pH responsive graft copolymers of chitosan
Accepted Manuscript Title: pH Responsive Graft Copolymers of Chitosan Author: Elvan Yilmaz Zulal Yalinca Kovan Ibrahim Yahya Uliana Sirotina PII: DOI: Reference:
S0141-8130(15)30007-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.10.003 BIOMAC 5417
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
11-6-2015 27-8-2015 1-10-2015
Please cite this article as: E. Yilmaz, Z. Yalinca, K.I. Yahya, U. Sirotina, pH Responsive Graft Copolymers of Chitosan, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Manuscript
pH Responsive Graft Copolymers of Chitosan
Elvan Yilmaza,*, ZulalYalincaa, KovanIbrahim Yahyaa,b, UlianaSirotina
a
a
Department of Chemistry, Faculty of Sciences, University of Duhok, Iraq
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b
ip t
Department of Chemistry, Eastern Mediterranean University, Famagusta, North Cyprus,
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ABSTRACT
Grafting suitable polymers onto chitosan can produce cationic or polyampholyte
an
polymers or hydrogels that are potential smart biomedical materials. Chitosan-graft-[poly(diethylamino)ethyl methacrylate] has been prepared in three different physical
M
forms as linear free chains in solution, chemical gels crosslinked with glutaraldehyde, and poly(diethylamino)ethyl methacrylate]
grafted onto chitosan tripolyphosphate
d
gel beads. In addition to chemical structure, the graft copolymers were characterized
Ac ce pt e
with respect to their dissolution and swelling behavior in aqueous solution. It has been established that solubility of the products is controlled by the grafting yield. While pH sensitive polymers, which collapse at a given pH value are obtained at lower grafting yields, hydrogels form at higher grafting yields with pH responsive swelling behavior. Glutaraldehydecrosslinkedchitosan-graft-[poly(diethylamino)ethyl methacrylate] gels and chitosantripolyphosphate gel beads grafted with poly[(diethylamino)ethyl methacrylate] exhibit pH sensitive swelling with highest equilibrium swelling capacity at pH=1.2. Keywords: Chitosan, polymethacrylate, environment responsive polymer. *Corresponding
author:
Tel.:
+903926301086,
E-mail
address:
[email protected]
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1. Introduction Polymersthat are classified aspH responsive materials contain ionizable groups, which respond to the pH of the environment. A polymer containing basic groups such as amine (-NH2), imidazole or pyridinegroup becomes protonated at low
ip t
pH in which the H+ ion concentration is high and hence is solubilized. At high pH, it becomes neutralized and undergoes a transition to the collapsed state or precipitates
cr
out of solution. Hence a phase change is observed.A polymer containing acidic
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groups such as carboxylic acid group obeys the same principle but behaves in the opposite way to a basic polymer. It exists in solution under basic conditions and
an
collapses in acidic medium.pH responsive hydrogels,on the other hand, respond to pH changes by undergoing volume transition according to the pH of the environment.
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Polymers and hydrogels with pH responsiveness are drawing increasing attention especially for targeted drug delivery as the pH of the tissues and cells show variations
d
depending on their type and function. For example, in the GI tract pH of the stomach
Ac ce pt e
is 1.0-3.0, pH of the duodenum is 4.8-8.2, the pH of the colon is 7.0-7.5 1. Furthermore, lysosomes inside the cells have a pH around 5.0. In some cases,pH of tumor cells may change between 5.7-7.8 2. These propertiescan be used to induce pH sensitive drug delivery.1, 2, 3.
Chitosan is a biobased polymer derived from the natural polymer chitin which
is a polysaccharide of β (1—>4) linked N-acetylglucose units shown in Fig. 1 (a). Chitosan is the partially deacetylated form of chitin.Itis a copolymer of β (1—>4) linked N-acetylglucose units and β (1—>4) linked glucosamine units as illustrated in Fig. 1 (b). Degree of deacetylation (DD) is one critical characteristic of chitosan, which affects its physicochemical responses and biological activity.
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The importance of these polymers for biomedical applications comes primarily from their biodegradability, biocompatibility and relative non toxicity. Theirversatility in terms of chemical and physicochemical properties add to their value. They are polyfunctional polymers with ease of modification,chelation/complexation capability,
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and ability to form chemically and physically crosslinked networks leading to gels, films, fibers, microspheres andnanospheres. They are biologically active polymers
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bearing mucoadhesivity, antibacterial activity, hypocholesterolemic activityin
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addition to acting as permeation enhancer, wound healing accelerator, having stimulatory effects on immune cells and local cell proliferation and integration of the
an
implanted material with the host tissue4.
Grafting is one polymer modification technique in which a polymer is linked
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to the backbone of a parent polymer, the substrate, by chemical linkages. Itis a preferred method of polymer modification for biomedical applications for several
d
reasons.It allows surface modification, as well asalteration of the bulk properties
Ac ce pt e
5,6.Furthermore,the shape and morphology of the polymer chain is known to affect the biological responses produced. As graft copolymers are branched polymers they exhibit different mechanical properties, and solution properties than their linear counterparts offering diversity in choices for given applications 6. Chitosan
was
demonstrated
to
be
a
suitable
substrate
for
graft
copolymerization of synthetic polymers for pH sensitive polymer and hydrogel design as it is a multi functional polymer with one primary alcohol on C-6, one secondary alcohol on C-3, and one amine or acetamide on C-2. It is soluble in aqueous acid solutions; a suitable medium for redox initiation. Grafting suitable polymers onto chitosan can produce cationic or polyampholyte polymers as potential drug delivery or controlled release agents. This has been thoroughly studied using redox
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initiation7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19. Less frequent examples of photo or radiation induced graftingare also available20, 21, 22, 23,24, 25, 26, 27. Newer strategies such as ATRP, RAFT and/or click chemistry gave graft copolymers of chitosan with well-defined architecture28, 29,
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30.Cerium(IV) ammonium nitrate (CAN) and potassium persulphate (KPS) or
ammonium per sulphate (APS) are commonly used to initiate grafting onto chitosan.
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Generally accepted mechanisms 31that proceed either via direct oxidation or
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complex formation are illustrated in Fig. 2(a), (b) and (c). Similar mechanisms to those of Ce4+ initiated mechanism have been proposed for grafting onto chitosan by
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potassium per sulphate initiation8, 32. It is clear that the possibility of obtaining a
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mixture of products is always available independent of the type of the initiator used. However, cleaner products are obtained as potassium salt is water soluble and there is
d
no complex formation mechanism involved as in the case of Ce4+ initiation. One
Ac ce pt e
characteristic of grafting onto chitosan by redox initiation is chain scission as a result of oxidation 32. According to our observations, in some cases depending on the type, an electron donating monomers such as 4-vinyl pyridine,and concentration of the monomer,and the pH of the medium the chitosan--Ce4+ complex formed does not dissociate completely 7. Usually a yellow colored product is obtained which is insoluble in common solvents. Hence products contaminated with Ce4+ are obtained which is a serious drawback for biomedical applications. This article describes graft copolymerization of (2-diethylamino) ethylmethacrylate), DEAEM, onto chitosan under homogeneous and heterogeneous conditions using potassium
persulphate
initiator.Chitosan-graft-poly((2-
diethylamino)ethylmethacrylate) has been prepared in three different physical forms as linear free chains in solution, chemical gels crosslinked with glutaraldehyde, and 4
Page 4 of 33
by grafting poly((2-diethylamino)ethylmethacrylate) onto chitosan tripolyphosphate gel beads to explore the limits of chitosan/DEAEM/KPS grafting system.The chemical structure has been characterized by FTIR and NMR spectroscopies. The effect of the grafting yield, the chemical structure and the physical form on the pH
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responsive swelling and dissolution behavior has been investigated.
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2. Materials and methods
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2.1. Materials
Chitosan (CHI, medium molecular weight, Aldrich, Germany, molar mass g/mol, and degree of deacetylation of 85%), 2-diethyl amino)ethyl
methacrylate
(DEAEM,
Aldrich,
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4.0x105
Germany),
Glutaraldehyde
(GA,
Aldrich,
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Germany),potassium persulfate (KPS, Aldrich, Germany), acetic acid (Riedel-de Häen, Germany), ethanol (Riedel-de Häen, Germany), acetone (Riedel-de Häen,
d
Germany), pentasodiumtripolyphosphate (TPP), 1,2-dichloroethane (Sigma-Aldrich,
Ac ce pt e
Germany) were used without any further purification.
2.2.Preparation ofCHI-graft-PDEAEM under homogeneous conditions A 25 mL sample of CHIsolution of concentration 1% (w/v) prepared in 1% (v/v)
acetic acid solution was placed in a two-neck reaction vessel. A required amount of KPS, and monomer (2-diethylamino)ethyl methacrylate, DEAEM, were then added into CHI solution respectively under nitrogen atmosphere and at 70 °C. The reaction was carried out for 4 hours under vigorous magnetic stirring at 1200 rpm. Then, the product was precipitated in acetone and was dried at 50 °C overnight. Preparation conditions of all CHI-graft-PDEAEM prepared under homogeneous conditions are given in Table 1. The products were dialyzed against distilled water for 24 hours
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using a dialysis membrane of 6000-8000 MWCO to remove any unreacted monomer, initiator or any oligomers that may have been formed. After dialysis the products were cleaned by soxhlet extraction using 1,2-dichloroethane as the solvent to obtain
2.3. Preparation of GA crosslinked CHI-graft-PDEAEMgels
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pure graft copolymers free from any homopolymer.
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The gelation time for CHI and CHI-graft-PDEAEM samples dissolved in
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acetic acid (pH=3.0) and pH=1.0 (HCl/KCl buffer) was measured by placing 4.0 mL solution in a glass test tube. The solution was stirred by magnetic stirring. Gelation
an
was taken as complete when the magnet stops turning and the product in the tube does not flow when the tube is flipped. The preparation conditions for the GA
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crosslinkedCHI-graft-PDEAEM gels are shown in Table 2.
Preparation of CHI-TPP Beads and CHI-TPP-graft-PDEAEM
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2.4.
Ac ce pt e
A CHI solution of concentration 2% (w/v) was prepared in 1% (v/v) acetic
acid. The solution was added dropwise into 5% (w/v) TPP solution dissolved in distilled water [33]. CHI-TPP beads formed instantaneously upon coagulation at room temperature under magnetic stirring of 20 rpm. They were dried in the oven at 50°C overnight. Then a sample of CHI-TPP beads crosslinked or non crosslinked with GA weighing 0.25 g was placed and 25 mL 1% acetic acid solution. The monomer, 0.50mL, mixed with 1.0 mL ethanol was added into the flask containing 0.25g CHITPP beads and 0.1250 g KPS initiator in 25 mL acetic acid and grafting was carried under nitrogen atmosphere.
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2.5.
Fourier transform infrared (FTIR) analysis
The FTIR spectra of tested samples were recorded with KBr pellets on a Perkin Elmer Spectrum 65 FTIR spectrometer.
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2.6. C-13 NMR analysis C-13 NMR analysis of the samples was carried out at METU-Central Lab using a
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Bruker Super Conducting FT NMR Spectrometer Avance TM 300 MHz WB at
Gravimetric analysis
an
2.7.
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ODTÜ (METU) Merkez Laboratory in Ankara.
The grafting yield, homopolymer yield and crosslinking degree were calculated by the
𝑚 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 𝑎𝑓𝑡𝑒𝑟 𝑑𝑖𝑎𝑙𝑦𝑠𝑖𝑠
𝑚 𝑔𝑟𝑎𝑓𝑡𝑒𝑑 𝑐𝑖𝑡𝑜𝑠𝑎𝑛 −𝑚 𝑐𝑖𝑡𝑜𝑠𝑎𝑛 𝑚 𝑐𝑖𝑡𝑜𝑠𝑎𝑛
𝑥 100
𝑥 100
Ac ce pt e
𝐺𝑟𝑎𝑓𝑡𝑖𝑛𝑔 % =
𝑚 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 𝑎𝑓𝑡𝑒𝑟 𝑔𝑟𝑎𝑓𝑡𝑖𝑛𝑔 −𝑚 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 𝑎𝑓𝑡𝑒𝑟 𝑑𝑖𝑎𝑙𝑦𝑠𝑖𝑠
d
𝐻𝑜𝑚𝑜𝑝𝑜𝑙𝑦𝑚𝑒𝑟 % =
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following equations.
𝐶𝑟𝑜𝑠𝑠𝑙𝑖𝑛𝑘𝑖𝑛𝑔 % =
2.8.
𝑚 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 𝑎𝑓𝑡𝑒𝑟 𝑔𝑒𝑙𝑎𝑡𝑖𝑜𝑛 − 𝑚 𝑐𝑖𝑡𝑜𝑠𝑎𝑛 𝑥 100 𝑚 𝑐𝑖𝑡𝑜𝑠𝑎𝑛
% Swelling capacity determination The swelling and dissolution behaviors were investigated in terms of time of
swelling pH. The swelling properties of samples were studied by immersing the samples in a solution at 37°C. They were removed from the solution at given time intervals, blotted to get rid of excess water on the surface and were then weighed.
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2.9. % Oscillating swelling capacity determination Beads (approximately 0.05 g) were immersed and kept in the buffer solution (pH=7) for 1 hour. Then, the swollen gels were weighed after blotting with a filter paper to remove the surface water and then were immersed in pH=1.0 for 1 hour. The
ip t
% oscillating swelling of the beads was determined. Three consecutive treatments
SEM analysis
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2.10.
cr
were performed. The same procedure was carried out at pH=1.0 and pH=11.0.
SEM analysis of the samples was carried out at Cyprus International University,
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3. Results and Discussion
an
Nicosiausing a JEOL/JSM-6610LVF scanning electron microscope.
PDEAEM was grafted onto CHI under homogenous and heterogeneous
d
conditions by using potassium persulphate as the initiator to obtain pH responsive
Ac ce pt e
copolymers. A similar approach was taken for surface modification of CHI-TPP gel beads. CHI-TPP beads were prepared by coagulating CHI in acetic acid solution in aqueous TPP solution. Then they were grafted by PDEAEM by redox initiation.
3.1. Preparation of CHI-graft-PDEAEM and CHI-TPP-graft-PDEAEMgel beads The grafting conditions were studied withseveral different amounts of
DEAEM (0.25mL, 0.50mL, 0.75 mL) under homogenous conditions as shown in Table 3. The grafting yield increases with increasing monomer concentration up to 0.5 mLmonomer. The maximum grafting percentage of PDEAEM onto CHI was found to be 361% under homogeneous conditions using 0.5 – 0.75 mL of monomer with 1.00 g CHI dissolved in 1.0mL of 1.0% (w/v) acetic acid solution by using 0.125
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Page 8 of 33
g KPS, at 70◦C and 4 hour reaction time.At 1.00 mL, the grafting yield decreases due to
the
formation
of
oligomers
and
some
homopolymer.
Low
percent
homopolymerization (% H) values have been obtained showing that oligomers were formed, which were lost into the dialysis solution, rather than high polymers.
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The results of surface modification of physical gel beads of CHI-TPP by PDEAEM grafting are shown in Table 3. Grafting yield (%G) values changing between 31 and
cr
54 were obtained under the conditions given. These values are not very different from
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each other as several factors such as the bead size, degree of crosslinking and extent
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of swelling would affect the grafting yield.
3.2. Preparation of GA crosslinked PEAEM CHI gels
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The grafted product which was soluble in acidic medium was sample CHI-graftPDEAEM(294) with %G=294. Therefore, GA crosslinking was carried out on this
d
product to prepare chemically crosslinked PEAEM/CHI gels. The gelation times of
Ac ce pt e
the samples aregiven in Table 4.When gelation behaviorsof CHI are compared in acetic acid and HCl solution, it can be followed from Table 4that complete gel formation is not detectable for CHIatpH=1.00 containing with 1.0 % GA solution. Also, the gelation times for CHI are higher in hydrochloric acidsolution than in acetic acid solution. This behavior can be explained by considering the acidities of the solutions and the nature of the crosslinking reaction. Since at pH=1.0, the amines are protonated at a higher fraction that at pH=3.0 in 1.0% acetic acid solution,imine formation reaction is less probable due to the absence of free amine groups available for reaction. For CHI solution in 1.0 % acetic acid % crosslinking has been calculated as 15.4% and gelation time was measured as 9 minutes using 4% GA. On the other hand CHI solution in pH=1.0 % crosslinking percentage was obtained as 14.3% with
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Page 9 of 33
the gelation time 22 minutes under the same conditions. Longer gelation times have been recorded for the grafted products compared to CHI in pH = 1 solution. Since DEAEM bears tertiary amine groups, crosslinking reaction with GA is not expected from those functional groups. Crosslinking occurs only due to the imine formation
ip t
between the free amine groups of CHI and the aldehyde functionalities of GA. The crosslinking percentage for the grafted product was obtained as 15.4% prepared in pH
cr
=1 solution containing 4% GA. This value is very close to that obtained for CHI
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under the same conditions. This shows us that during the grafting process, monomer molecules cannot approach CHI chains from the amine groups. One reason is
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thatamine groups are either protonated or occupied by inter/intra molecular hydrogen bonding. The second one is that due to the bulky nature of the monomers C-6 of CHI
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is more available for the grafting reaction with the monomer molecules. Also, due to the branched nature of grafted chains the solution viscosity decreases increasing the
d
gelation time.It was not possible to obtain gels of CHI-graft-PDEAEM in acetic acid
Ac ce pt e
solution, due to the low viscosity of the solution prepared.
3.3. FTIR analysis
In the FTIR spectrum of CHI shown in Fig.4 (a) characteristic amide band I
and II at 1649 cm-1, the C-H bending vibrations 1400-1500 cm-1 region, the -CH3 bending at 1380 cm-1 and the pyranose C-O-C and C-OH stretching vibrations of CHI in the region 1100-900 cm-1 are observable. The DEAEM spectrum in Fig.4 (b) shows adsorption band at 2974 cm-1 and 2935 cm-1 band which belong to C-H stretching . The band at 2811 cm-1 belong to H-C=O band, and 1721 cm-1 belong to C=O stretching. The band at 1639 cm-1 belong to C=C stretching, whereas 1456 cm-1 belong to C-H bending an 1382 cm-1 refer to C-H rocking .The peak at 1297 cm-1
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Page 10 of 33
belong to C-H wag.In the spectrum of the CHI-graft-PDEAEMin Fig.4 (c) the peak 1736 cm-1 refer to C=O stretching and the peak at 1643 cm-1 belong to the amide C=O of CHI. The shift from 1659 cm-1 to 1643 cm-1 is an indication of grafting of the monomer onto CHI from the amide nitrogen. The band at 1456 cm-1 and 1402 cm-1
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refers to C-H bending, and the peak at 1115 cm-1 refer to C-O-C of CHI.The FTIR spectrum of CHI-TPP-graft-PDEAEM beads is shown in Fig.4(d), the bands in the
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range 1024-1063 cm-1 belongs to C6-O of CHI TPP and the band at 1216 cm-1
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belongs to the P-O. It can be observed that absorption bands at 1727 cm-1 or 1731 cm-1 which belongs to C=O stretching which have been taken as evidence of
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3.5. C-13 NMR analysis
an
formation of grafting with DEAEM.
C-13 NMR spectrum of CHI, CHI-graft-PDEAEM, CHI-TPP-graft-PDEAEM is
d
shown in Fig.5 (a), (b) and (c) respectively.In the spectrum of CHI, the signals at
Ac ce pt e
169.7 ppm and 19.1 belong to the carbonyl carbon and the methyl group of the acetamide groups respectively. The signals at 100.7 ppm, 78.4 ppm, 70.9 ppm, 56.7 ppm, 53.1 ppm are assigned to the ring carbons: C-1, C-4, C-3,5, C-6 and C-2 respectively.
In the spectrum of CHI-graft-PDEAEM, the signal at 168.0 ppm belong to the
carbonyl carbon (C-1) of CHI and the intensity of this peak increased due to the grafting of DEAEM.
The signal at 17.3 ppm belongs to the methyl group of
acetamide of CHI and methyl group of DEAEM. The signal at 98.6 ppm is assigned to the C-1. The signals at 77.7 ppm 75.3 ppm, 74.7 ppm and 67.9 ppm are assigned to belong to CH2OH of DEAEM. The signals at 63.0 ppm, 59.4 ppm, 55.7 ppm,
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Page 11 of 33
54.6ppm are belong to are assigned to the ring carbons: C-4, C-3,5, C-6 and C-2 respectively. In the spectrum of CHI-TPP-graft-PDEAEM, the signal at 173.6 ppm belong to carbonyl ester bond. The chemical shifts at 168.6 ppm and 11.0 ppm belong to the
ip t
carbonyl carbon and the methyl group of the acetamide groups of CHI respectively. The signals at 98.5 ppm, 77.8 ppm, 70.1 ppm, 61.8 ppm and 54.7 ppm are assigned to
cr
the ring carbons: C-1, C-4, C-3,5, C-6 and C-2 respectively. The new signal appear at
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17.3ppm which, belongs to-CH2 of DEAEM. The signal at 39.6 ppm belongs to CH2NH2 of DEAEM.The signals at 49.9 ppm and 67.8 ppm belongs to CH2OH of
Scanning electron microscopy (SEM) analysis
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3.6.
an
DEAEM.
SEM micrographs of the CHI gel by 1% GA, CHI* gel by 1% GA, CHI* gel by 4%
d
GA, CHI*-graft-PDEAEM(294) by 1% GA , CHI-TPP beads and CHI-TPP-graft-
Ac ce pt e
PDEAEMare given in Fig.6 (a), (b), (c), (d), (e) and (f) respectively. The SEM images of gels of chitosan (Fig.6(a) and 6 (b)) show that smooth surfaceshave been obtained by crosslinking with 1% GA.Increasing GA% results in more roughness of the gel surface ((Fig. 6 (c)).The surface morphology of CHI*-graft-PDEAEM(294) ((Fig. 6(d)) revealed that grafting causeda less smooth surface. When the surface of CHITPP bead alone (Fig. 6 (e))is compared to that of CHI-TPP-graft-PDEAEM (Fig. 6(f)), the presence of the grafted polymer on the bead surface can easily be identified. 3.7. Swelling properties of products CHI graft PDEAEM gels exhibits improved pH responsive swelling when compared to pure CHIgels. Under acidic conditions CHI-GA gel exhibits an equilibrium swelling of187% within 6 hours while CHI-graft gel has an equilibrium swelling of
12
Page 12 of 33
276%.Similarly at pH=7.0 and 11.0 the GA-crosslinkedCHI gelshow an equilibrium swelling value of 130% and 163% respectively while the grafted product swells by 405% and 570% at pH=7.0 pH=11.0 respectively. The equilibrium % swelling value in basic conditions competes with the value in acidic conditions. This behavior could
ip t
be explained by basic and acidic hydrolysis of imine bonds giving rise to less crosslinked networks.The swelling behavior of CHI-TPP beads was followed in
cr
pH=1.0, 7.0 and 11.0. CHI-TPP swells with an equilibrium swelling capacity
us
%8800andthe PDEAEM grafted CHI-TPP beads have a swelling capacity of %5700 at pH=1.0. The reason why CHI-TPP beads have higher swelling capacity than CHI-
an
TPP-graft-PDEAEM beads can be attributed to screening effect of ethyl groups in between protonated tertiary amine groups on PDEAEM. In pH=7.00 and pH=11.0
%470.
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both CHI-TPP beads and CHI-TPP-graft-PDEAEMbeads have swelling capacity
d
Oscillating swelling behavior of CHI-TPP beads and CHI-TPP-graft-PDEAEMbeads
Ac ce pt e
was studied at pH=7.00/pH=1 and pH=11.00/pH=1.00. The oscillating swelling behavior at pH=7.00/pH=1 is shown in Fig7.When the beads are initially swollen in pH=7.0, the swelling capacity increases to 2470% for CHI-TPP beads and 650%CHITPP-graft-PDEAEMbeads following immersion in pH=1.0 as shown in Fig. 7.The same trend with increasing % swelling values is observed inrepeated steps. Similar behavior is observed for oscillating swelling for CHI-TPP-graft-PDEAEMbeads for consecutive immersion in pH=11.0 and pH=1.00.
4. Conclusions
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Page 13 of 33
Chi/DEAEM/KPS system is a versatile combination to obtain grafted chitosans by redox initiation under both homogeneous and heterogeneous conditions. Products whose solubility in aqueous acidic solution is controlled by the grafting yield are obtained under homogeneous reaction conditions. The graft copolymer sample, which
ip t
is soluble in aqueous acidic solution can becrosslinked by glutaraldehyde at pH=1 to obtain chemical gels with improved pH sensitive swelling capacity compared to
cr
glutaraldehydecrosslinked pure chitosan gels. Chitosan-TPP gel beads provide a
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suitable matrix for surface modification via graft copolymerization of DEAEM. In pH=1 solution, chitosan-TPP-graft-PDEAEM gel beads swell to perform as
an
superabsorbent gels.The grafted beads perform as pH responsive superabsorbent 3D matrices upon swelling in pH=7.00/pH=1.00 solutions in consecutive repeated steps.
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PDEAEM grafted chitosan gelshave a potential to find a place in drug delivery as well as novel bio-based adsorbents following further in-vitro and in-vivo detailed
Ac ce pt e
d
investigationand biological analysis.
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Page 14 of 33
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[11] S.B. Lee, D.I. Ha, S.K. Cho, S.J. Kim, Y.M. Lee , Appl. Polym. Sci. 92(2004) 2612-2620.
[12]G.R. Mahdavinia, M.J. Zohuriaan-Mehr, A.Pourjavadi , Polym. Adv. Technol. 15(2004) 173-180.
[13] H.Caner, E. Yilmaz, O.Yimaz , Carbohydr. Polym. 69(2007) 318-325. [14] E.Yilmaz, T. Adali, O. Yilmaz, M.Bengisu , React. Funct. Polym.67(2007) 1018. [15] T. Adali, E.Yilmaz , Carbohydr. Polym.77(2009) 136-141. [16]P.J. Lv, Y.Z. Bin, Y.Q. Li, R. Chen, X. Wang, B.Y. Zhao ,Polymer. 50(2009)
15
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5675-5680. [17] C. Spagnol, F.H.A. Rodrigues, A.G.B. Pereira, A.R. Fajardo, A.F. Rubira, E.C.Muniz , Carbohydr. Polym. 87(2012) 2038-2045. [18] G.Y. Li, L. Guo, Q.W. Wen, T.Zhang, Int. J. Biol. Macromol. 55 (2013) 69-
ip t
74. [19] T.Adali ,Bio-Medical Materials and Engineering. 23 (2013) 349-359.
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[20] K.Zhang, Z.K. Wang, Y.L. Li, Z.Q. Jiang, Q.L. Hu, M.Y. Liu, Q.X.
us
Zhao, Carbohydr. Polym. 92(2013) 662-667.
[21] P.Mukhopadhyay, K. Sarkar, S. Bhattacharya, A. Bhattacharyya, R. Mishra,
an
P.P. Kundu, Carbohydr. Polym. 112(2014) 627-637.
74(2005) 26-30.
M
[22] H. Cai, Z.P. Zhang, P.C. Sun, B.L. He, X.X.Zhu , Radiat . Phys. Chem.
[23] M.F.A. Taleb, Polym. Bull. 61(2008) 341-351.
d
[24] I.M. El-Sherbiny, H.D.C. Smyth , Carbohydr. Polym. 81(2010) 652-659.
Ac ce pt e
[25] I.M. El-Sherbiny, H.D.C. Smyth , Carbohydr. Res. 345 (2010)2004-2012. [26] S. Saber-Samandari, M. Gazi, E.Yilmaz , Polym. Bull. 68(2012) 1623-1639. [27] S. Saber-Samandari, O. Yilmaz, E. Yilmaz , J. Macromol. Sci. A. 49(2012) 591-598.
[28] A.S.Carreira, F. Goncalves, P.V. Mendonca, M.H. Gil, J.F.J.Coelho , Carbohydr. Polym. 80(2010) 618-630.
[29] W.Z. Yuan, Z.D. Zhao, S.Y. Gu, T.B. Ren, J.Ren , Mater.Lett. 65(2011) 793796. [30] K.Zhang, Z.K. Wang, Y.L. Li, Z.Q. Jiang, Q.L. Hu, M.Y. Liu, Q.X. Zhao, Carbohydr. Polym. 92(2013) 662-667.
16
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[31] R. Jayakumar, M.Prabaharan, R.L. Reis, J.F. Mano , Carbohydr. Polym. 62 (2005) 142-158. [32] S.C. Hsu, T.M. Don , W.Y. Chiu , Polym. Degrad. Stab. 75 (2002) 73-83. [33] Z. Yalinca , E. Yilmaz , F. T. Bullici , J. Appl. Polym. Sci. 125(2012) 1493–
Ac ce pt e
d
M
an
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cr
ip t
1505.
17
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List of Tables Table 1. Preparation conditions of all CHI-graft-PDEAEM. Table 2. The preparation conditions of GA crosslinked CHI-graft-PDEAEM gels.
ip t
Table 3. The preparation conditions of GA crosslinked and non crosslinkedCHI-TPPgraft-PDEAEM beads.
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Table 4. Gelation time determination of GA crosslinkedCHI and GA crosslinked
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CHI-graft-PEAEM gels.
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List of Figures
Fig. 1. Chemical structure of (a) chitin and (b) chitosan.
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Fig. 2. Grafting mechanism onto chitosan by (a) direct oxidation by Ce4+, (b) complex formation by Ce4+, (c) persulphate initiation.
d
Fig. 3. Chemical structure of DEAEM.
Ac ce pt e
Fig. 4. FTIR spectra of (a) CHI, (b) DEAEM, (c) CHI-graft-PDEAEM, (d)CHI-TPPgraft-PDEAEM beads.
Fig. 5. C-13NMR spectra of (a) CHI, (b) CHI-graft-PDEAEM, (c)CHI-TPP-graftPDEAEM.
Fig. 6. SEM micrographs of the a) GA(1)-CHI, b) GA (1)-CHI* c) GA (4)-CHI* d) GA (4)-CHI*-graft-PDEAEM(294) (e) CHI-TPP beads and (f)CHI-TPP-graftPDEAEM.
Fig. 7. The comparison of oscillating swelling behavior of CHI-TPP and CHI-TPPgraft-PDEAEM between pH=7.0 and pH=1.0.
18
Page 18 of 33
Table 1
DEAEM (mL)
T (°C)
Time (hr)
KPS (g)
H%
CHI-graft-PDEAEM(294)
0.25
70
4
0.1250
3.37
294
CHI-graft-PDEAEM(361)
0.50
70
4
0.1250
4.95
361
CHI-graft-PDEAEM(356)
0.75
70
4
0.1250
3.26
356
CHI-graft-PDEAEM(221)
1.00
70
4
0.1250
1.69
221
G%
Ac ce p
te
d
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an
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ip t
Sample ID
cr
Table 1. Preparation conditions of all CHI-graft-PDEAEM.
Page 19 of 33
Table 2
Table 2. The preparation conditions of GA crosslinked CHI-graft-PDEAEM gels. Volume (µL) of GA
GA (1)-CHI-graft-PDEAEM(294)
3960
40
GA (2)-CHI-graft-PDEAEM(294)
3920
80
GA (3)-CHI-graft-PDEAEM(294)
3880
GA (4)-CHI-graft-PDEAEM(294)
3840
ip t
Volume (µL) of CHI
cr
120 160
Ac ce p
te
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an
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Sample ID
Page 20 of 33
Table 3
Table 3. The preparation conditions of GA crosslinked and non crosslinked CHI-TPP-graft-
GA, mL
Bead Size,
G%
CHI-TPP-graft-PDEAEM(31)
0.50
-
710
31
GA(0.1)- CHI-TPP-graft-PDEAEM(49)
0.50
0.1
710
49
GA(0.3)- CHI-TPP-graft-PDEAEM(43)
0.50
0.3
710
43
GA(0.5)- CHI-TPP-graft-PDEAEM(48)
0.50
0.5
520
48
GA(1.0)- CHI-TPP-graft-PDEAEM(54)
0.50
1.0
212
54
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Ac ce p
te
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an
Sample ID
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DEAEM, mL
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PDEAEM beads.
Page 21 of 33
Table 4
Table 4. Gelation time determination of GA crosslinked CHI and GA crosslinked CHI-graftPEAEM gels.
CHI
CHI*
CHI*-graft-PDEAEM(294)
dissolved in acetic acid
dissolved in pH=1.0
dissolved in pH=1.0
cr
GA%
ip t
Gelation Time (minutes)
35 26
190
3
17
43
4
9
not detectable.
305 155
M
2
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Complete gel formation
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1
4
Ac ce p
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22
20
Page 22 of 33
Ac ce p
te
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*dissolved in pH=1.0
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Figure1 (a)
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Figure1 (b)
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Figure 2 (a)
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Figure 2 (b)
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Figure 2 (c)
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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Figure 7
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S0141-8130(15)30007-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.10.003 BIOMAC 5417
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
11-6-2015 27-8-2015 1-10-2015
Please cite this article as: E. Yilmaz, Z. Yalinca, K.I. Yahya, U. Sirotina, pH Responsive Graft Copolymers of Chitosan, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Manuscript
pH Responsive Graft Copolymers of Chitosan
Elvan Yilmaza,*, ZulalYalincaa, KovanIbrahim Yahyaa,b, UlianaSirotina
a
a
Department of Chemistry, Faculty of Sciences, University of Duhok, Iraq
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b
ip t
Department of Chemistry, Eastern Mediterranean University, Famagusta, North Cyprus,
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ABSTRACT
Grafting suitable polymers onto chitosan can produce cationic or polyampholyte
an
polymers or hydrogels that are potential smart biomedical materials. Chitosan-graft-[poly(diethylamino)ethyl methacrylate] has been prepared in three different physical
M
forms as linear free chains in solution, chemical gels crosslinked with glutaraldehyde, and poly(diethylamino)ethyl methacrylate]
grafted onto chitosan tripolyphosphate
d
gel beads. In addition to chemical structure, the graft copolymers were characterized
Ac ce pt e
with respect to their dissolution and swelling behavior in aqueous solution. It has been established that solubility of the products is controlled by the grafting yield. While pH sensitive polymers, which collapse at a given pH value are obtained at lower grafting yields, hydrogels form at higher grafting yields with pH responsive swelling behavior. Glutaraldehydecrosslinkedchitosan-graft-[poly(diethylamino)ethyl methacrylate] gels and chitosantripolyphosphate gel beads grafted with poly[(diethylamino)ethyl methacrylate] exhibit pH sensitive swelling with highest equilibrium swelling capacity at pH=1.2. Keywords: Chitosan, polymethacrylate, environment responsive polymer. *Corresponding
author:
Tel.:
+903926301086,
address:
[email protected]
1
Page 1 of 33
1. Introduction Polymersthat are classified aspH responsive materials contain ionizable groups, which respond to the pH of the environment. A polymer containing basic groups such as amine (-NH2), imidazole or pyridinegroup becomes protonated at low
ip t
pH in which the H+ ion concentration is high and hence is solubilized. At high pH, it becomes neutralized and undergoes a transition to the collapsed state or precipitates
cr
out of solution. Hence a phase change is observed.A polymer containing acidic
us
groups such as carboxylic acid group obeys the same principle but behaves in the opposite way to a basic polymer. It exists in solution under basic conditions and
an
collapses in acidic medium.pH responsive hydrogels,on the other hand, respond to pH changes by undergoing volume transition according to the pH of the environment.
M
Polymers and hydrogels with pH responsiveness are drawing increasing attention especially for targeted drug delivery as the pH of the tissues and cells show variations
d
depending on their type and function. For example, in the GI tract pH of the stomach
Ac ce pt e
is 1.0-3.0, pH of the duodenum is 4.8-8.2, the pH of the colon is 7.0-7.5 1. Furthermore, lysosomes inside the cells have a pH around 5.0. In some cases,pH of tumor cells may change between 5.7-7.8 2. These propertiescan be used to induce pH sensitive drug delivery.1, 2, 3.
Chitosan is a biobased polymer derived from the natural polymer chitin which
is a polysaccharide of β (1—>4) linked N-acetylglucose units shown in Fig. 1 (a). Chitosan is the partially deacetylated form of chitin.Itis a copolymer of β (1—>4) linked N-acetylglucose units and β (1—>4) linked glucosamine units as illustrated in Fig. 1 (b). Degree of deacetylation (DD) is one critical characteristic of chitosan, which affects its physicochemical responses and biological activity.
2
Page 2 of 33
The importance of these polymers for biomedical applications comes primarily from their biodegradability, biocompatibility and relative non toxicity. Theirversatility in terms of chemical and physicochemical properties add to their value. They are polyfunctional polymers with ease of modification,chelation/complexation capability,
ip t
and ability to form chemically and physically crosslinked networks leading to gels, films, fibers, microspheres andnanospheres. They are biologically active polymers
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bearing mucoadhesivity, antibacterial activity, hypocholesterolemic activityin
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addition to acting as permeation enhancer, wound healing accelerator, having stimulatory effects on immune cells and local cell proliferation and integration of the
an
implanted material with the host tissue4.
Grafting is one polymer modification technique in which a polymer is linked
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to the backbone of a parent polymer, the substrate, by chemical linkages. Itis a preferred method of polymer modification for biomedical applications for several
d
reasons.It allows surface modification, as well asalteration of the bulk properties
Ac ce pt e
5,6.Furthermore,the shape and morphology of the polymer chain is known to affect the biological responses produced. As graft copolymers are branched polymers they exhibit different mechanical properties, and solution properties than their linear counterparts offering diversity in choices for given applications 6. Chitosan
was
demonstrated
to
be
a
suitable
substrate
for
graft
copolymerization of synthetic polymers for pH sensitive polymer and hydrogel design as it is a multi functional polymer with one primary alcohol on C-6, one secondary alcohol on C-3, and one amine or acetamide on C-2. It is soluble in aqueous acid solutions; a suitable medium for redox initiation. Grafting suitable polymers onto chitosan can produce cationic or polyampholyte polymers as potential drug delivery or controlled release agents. This has been thoroughly studied using redox
3
Page 3 of 33
initiation7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19. Less frequent examples of photo or radiation induced graftingare also available20, 21, 22, 23,24, 25, 26, 27. Newer strategies such as ATRP, RAFT and/or click chemistry gave graft copolymers of chitosan with well-defined architecture28, 29,
ip t
30.Cerium(IV) ammonium nitrate (CAN) and potassium persulphate (KPS) or
ammonium per sulphate (APS) are commonly used to initiate grafting onto chitosan.
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Generally accepted mechanisms 31that proceed either via direct oxidation or
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complex formation are illustrated in Fig. 2(a), (b) and (c). Similar mechanisms to those of Ce4+ initiated mechanism have been proposed for grafting onto chitosan by
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potassium per sulphate initiation8, 32. It is clear that the possibility of obtaining a
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mixture of products is always available independent of the type of the initiator used. However, cleaner products are obtained as potassium salt is water soluble and there is
d
no complex formation mechanism involved as in the case of Ce4+ initiation. One
Ac ce pt e
characteristic of grafting onto chitosan by redox initiation is chain scission as a result of oxidation 32. According to our observations, in some cases depending on the type, an electron donating monomers such as 4-vinyl pyridine,and concentration of the monomer,and the pH of the medium the chitosan--Ce4+ complex formed does not dissociate completely 7. Usually a yellow colored product is obtained which is insoluble in common solvents. Hence products contaminated with Ce4+ are obtained which is a serious drawback for biomedical applications. This article describes graft copolymerization of (2-diethylamino) ethylmethacrylate), DEAEM, onto chitosan under homogeneous and heterogeneous conditions using potassium
persulphate
initiator.Chitosan-graft-poly((2-
diethylamino)ethylmethacrylate) has been prepared in three different physical forms as linear free chains in solution, chemical gels crosslinked with glutaraldehyde, and 4
Page 4 of 33
by grafting poly((2-diethylamino)ethylmethacrylate) onto chitosan tripolyphosphate gel beads to explore the limits of chitosan/DEAEM/KPS grafting system.The chemical structure has been characterized by FTIR and NMR spectroscopies. The effect of the grafting yield, the chemical structure and the physical form on the pH
ip t
responsive swelling and dissolution behavior has been investigated.
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2. Materials and methods
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2.1. Materials
Chitosan (CHI, medium molecular weight, Aldrich, Germany, molar mass g/mol, and degree of deacetylation of 85%), 2-diethyl amino)ethyl
methacrylate
(DEAEM,
Aldrich,
an
4.0x105
Germany),
Glutaraldehyde
(GA,
Aldrich,
M
Germany),potassium persulfate (KPS, Aldrich, Germany), acetic acid (Riedel-de Häen, Germany), ethanol (Riedel-de Häen, Germany), acetone (Riedel-de Häen,
d
Germany), pentasodiumtripolyphosphate (TPP), 1,2-dichloroethane (Sigma-Aldrich,
Ac ce pt e
Germany) were used without any further purification.
2.2.Preparation ofCHI-graft-PDEAEM under homogeneous conditions A 25 mL sample of CHIsolution of concentration 1% (w/v) prepared in 1% (v/v)
acetic acid solution was placed in a two-neck reaction vessel. A required amount of KPS, and monomer (2-diethylamino)ethyl methacrylate, DEAEM, were then added into CHI solution respectively under nitrogen atmosphere and at 70 °C. The reaction was carried out for 4 hours under vigorous magnetic stirring at 1200 rpm. Then, the product was precipitated in acetone and was dried at 50 °C overnight. Preparation conditions of all CHI-graft-PDEAEM prepared under homogeneous conditions are given in Table 1. The products were dialyzed against distilled water for 24 hours
5
Page 5 of 33
using a dialysis membrane of 6000-8000 MWCO to remove any unreacted monomer, initiator or any oligomers that may have been formed. After dialysis the products were cleaned by soxhlet extraction using 1,2-dichloroethane as the solvent to obtain
2.3. Preparation of GA crosslinked CHI-graft-PDEAEMgels
ip t
pure graft copolymers free from any homopolymer.
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The gelation time for CHI and CHI-graft-PDEAEM samples dissolved in
us
acetic acid (pH=3.0) and pH=1.0 (HCl/KCl buffer) was measured by placing 4.0 mL solution in a glass test tube. The solution was stirred by magnetic stirring. Gelation
an
was taken as complete when the magnet stops turning and the product in the tube does not flow when the tube is flipped. The preparation conditions for the GA
M
crosslinkedCHI-graft-PDEAEM gels are shown in Table 2.
Preparation of CHI-TPP Beads and CHI-TPP-graft-PDEAEM
d
2.4.
Ac ce pt e
A CHI solution of concentration 2% (w/v) was prepared in 1% (v/v) acetic
acid. The solution was added dropwise into 5% (w/v) TPP solution dissolved in distilled water [33]. CHI-TPP beads formed instantaneously upon coagulation at room temperature under magnetic stirring of 20 rpm. They were dried in the oven at 50°C overnight. Then a sample of CHI-TPP beads crosslinked or non crosslinked with GA weighing 0.25 g was placed and 25 mL 1% acetic acid solution. The monomer, 0.50mL, mixed with 1.0 mL ethanol was added into the flask containing 0.25g CHITPP beads and 0.1250 g KPS initiator in 25 mL acetic acid and grafting was carried under nitrogen atmosphere.
6
Page 6 of 33
2.5.
Fourier transform infrared (FTIR) analysis
The FTIR spectra of tested samples were recorded with KBr pellets on a Perkin Elmer Spectrum 65 FTIR spectrometer.
ip t
2.6. C-13 NMR analysis C-13 NMR analysis of the samples was carried out at METU-Central Lab using a
cr
Bruker Super Conducting FT NMR Spectrometer Avance TM 300 MHz WB at
Gravimetric analysis
an
2.7.
us
ODTÜ (METU) Merkez Laboratory in Ankara.
The grafting yield, homopolymer yield and crosslinking degree were calculated by the
𝑚 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 𝑎𝑓𝑡𝑒𝑟 𝑑𝑖𝑎𝑙𝑦𝑠𝑖𝑠
𝑚 𝑔𝑟𝑎𝑓𝑡𝑒𝑑 𝑐𝑖𝑡𝑜𝑠𝑎𝑛 −𝑚 𝑐𝑖𝑡𝑜𝑠𝑎𝑛 𝑚 𝑐𝑖𝑡𝑜𝑠𝑎𝑛
𝑥 100
𝑥 100
Ac ce pt e
𝐺𝑟𝑎𝑓𝑡𝑖𝑛𝑔 % =
𝑚 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 𝑎𝑓𝑡𝑒𝑟 𝑔𝑟𝑎𝑓𝑡𝑖𝑛𝑔 −𝑚 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 𝑎𝑓𝑡𝑒𝑟 𝑑𝑖𝑎𝑙𝑦𝑠𝑖𝑠
d
𝐻𝑜𝑚𝑜𝑝𝑜𝑙𝑦𝑚𝑒𝑟 % =
M
following equations.
𝐶𝑟𝑜𝑠𝑠𝑙𝑖𝑛𝑘𝑖𝑛𝑔 % =
2.8.
𝑚 𝑝𝑟𝑜𝑑𝑢𝑐𝑡 𝑎𝑓𝑡𝑒𝑟 𝑔𝑒𝑙𝑎𝑡𝑖𝑜𝑛 − 𝑚 𝑐𝑖𝑡𝑜𝑠𝑎𝑛 𝑥 100 𝑚 𝑐𝑖𝑡𝑜𝑠𝑎𝑛
% Swelling capacity determination The swelling and dissolution behaviors were investigated in terms of time of
swelling pH. The swelling properties of samples were studied by immersing the samples in a solution at 37°C. They were removed from the solution at given time intervals, blotted to get rid of excess water on the surface and were then weighed.
7
Page 7 of 33
2.9. % Oscillating swelling capacity determination Beads (approximately 0.05 g) were immersed and kept in the buffer solution (pH=7) for 1 hour. Then, the swollen gels were weighed after blotting with a filter paper to remove the surface water and then were immersed in pH=1.0 for 1 hour. The
ip t
% oscillating swelling of the beads was determined. Three consecutive treatments
SEM analysis
us
2.10.
cr
were performed. The same procedure was carried out at pH=1.0 and pH=11.0.
SEM analysis of the samples was carried out at Cyprus International University,
M
3. Results and Discussion
an
Nicosiausing a JEOL/JSM-6610LVF scanning electron microscope.
PDEAEM was grafted onto CHI under homogenous and heterogeneous
d
conditions by using potassium persulphate as the initiator to obtain pH responsive
Ac ce pt e
copolymers. A similar approach was taken for surface modification of CHI-TPP gel beads. CHI-TPP beads were prepared by coagulating CHI in acetic acid solution in aqueous TPP solution. Then they were grafted by PDEAEM by redox initiation.
3.1. Preparation of CHI-graft-PDEAEM and CHI-TPP-graft-PDEAEMgel beads The grafting conditions were studied withseveral different amounts of
DEAEM (0.25mL, 0.50mL, 0.75 mL) under homogenous conditions as shown in Table 3. The grafting yield increases with increasing monomer concentration up to 0.5 mLmonomer. The maximum grafting percentage of PDEAEM onto CHI was found to be 361% under homogeneous conditions using 0.5 – 0.75 mL of monomer with 1.00 g CHI dissolved in 1.0mL of 1.0% (w/v) acetic acid solution by using 0.125
8
Page 8 of 33
g KPS, at 70◦C and 4 hour reaction time.At 1.00 mL, the grafting yield decreases due to
the
formation
of
oligomers
and
some
homopolymer.
Low
percent
homopolymerization (% H) values have been obtained showing that oligomers were formed, which were lost into the dialysis solution, rather than high polymers.
ip t
The results of surface modification of physical gel beads of CHI-TPP by PDEAEM grafting are shown in Table 3. Grafting yield (%G) values changing between 31 and
cr
54 were obtained under the conditions given. These values are not very different from
us
each other as several factors such as the bead size, degree of crosslinking and extent
an
of swelling would affect the grafting yield.
3.2. Preparation of GA crosslinked PEAEM CHI gels
M
The grafted product which was soluble in acidic medium was sample CHI-graftPDEAEM(294) with %G=294. Therefore, GA crosslinking was carried out on this
d
product to prepare chemically crosslinked PEAEM/CHI gels. The gelation times of
Ac ce pt e
the samples aregiven in Table 4.When gelation behaviorsof CHI are compared in acetic acid and HCl solution, it can be followed from Table 4that complete gel formation is not detectable for CHIatpH=1.00 containing with 1.0 % GA solution. Also, the gelation times for CHI are higher in hydrochloric acidsolution than in acetic acid solution. This behavior can be explained by considering the acidities of the solutions and the nature of the crosslinking reaction. Since at pH=1.0, the amines are protonated at a higher fraction that at pH=3.0 in 1.0% acetic acid solution,imine formation reaction is less probable due to the absence of free amine groups available for reaction. For CHI solution in 1.0 % acetic acid % crosslinking has been calculated as 15.4% and gelation time was measured as 9 minutes using 4% GA. On the other hand CHI solution in pH=1.0 % crosslinking percentage was obtained as 14.3% with
9
Page 9 of 33
the gelation time 22 minutes under the same conditions. Longer gelation times have been recorded for the grafted products compared to CHI in pH = 1 solution. Since DEAEM bears tertiary amine groups, crosslinking reaction with GA is not expected from those functional groups. Crosslinking occurs only due to the imine formation
ip t
between the free amine groups of CHI and the aldehyde functionalities of GA. The crosslinking percentage for the grafted product was obtained as 15.4% prepared in pH
cr
=1 solution containing 4% GA. This value is very close to that obtained for CHI
us
under the same conditions. This shows us that during the grafting process, monomer molecules cannot approach CHI chains from the amine groups. One reason is
an
thatamine groups are either protonated or occupied by inter/intra molecular hydrogen bonding. The second one is that due to the bulky nature of the monomers C-6 of CHI
M
is more available for the grafting reaction with the monomer molecules. Also, due to the branched nature of grafted chains the solution viscosity decreases increasing the
d
gelation time.It was not possible to obtain gels of CHI-graft-PDEAEM in acetic acid
Ac ce pt e
solution, due to the low viscosity of the solution prepared.
3.3. FTIR analysis
In the FTIR spectrum of CHI shown in Fig.4 (a) characteristic amide band I
and II at 1649 cm-1, the C-H bending vibrations 1400-1500 cm-1 region, the -CH3 bending at 1380 cm-1 and the pyranose C-O-C and C-OH stretching vibrations of CHI in the region 1100-900 cm-1 are observable. The DEAEM spectrum in Fig.4 (b) shows adsorption band at 2974 cm-1 and 2935 cm-1 band which belong to C-H stretching . The band at 2811 cm-1 belong to H-C=O band, and 1721 cm-1 belong to C=O stretching. The band at 1639 cm-1 belong to C=C stretching, whereas 1456 cm-1 belong to C-H bending an 1382 cm-1 refer to C-H rocking .The peak at 1297 cm-1
10
Page 10 of 33
belong to C-H wag.In the spectrum of the CHI-graft-PDEAEMin Fig.4 (c) the peak 1736 cm-1 refer to C=O stretching and the peak at 1643 cm-1 belong to the amide C=O of CHI. The shift from 1659 cm-1 to 1643 cm-1 is an indication of grafting of the monomer onto CHI from the amide nitrogen. The band at 1456 cm-1 and 1402 cm-1
ip t
refers to C-H bending, and the peak at 1115 cm-1 refer to C-O-C of CHI.The FTIR spectrum of CHI-TPP-graft-PDEAEM beads is shown in Fig.4(d), the bands in the
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range 1024-1063 cm-1 belongs to C6-O of CHI TPP and the band at 1216 cm-1
us
belongs to the P-O. It can be observed that absorption bands at 1727 cm-1 or 1731 cm-1 which belongs to C=O stretching which have been taken as evidence of
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3.5. C-13 NMR analysis
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formation of grafting with DEAEM.
C-13 NMR spectrum of CHI, CHI-graft-PDEAEM, CHI-TPP-graft-PDEAEM is
d
shown in Fig.5 (a), (b) and (c) respectively.In the spectrum of CHI, the signals at
Ac ce pt e
169.7 ppm and 19.1 belong to the carbonyl carbon and the methyl group of the acetamide groups respectively. The signals at 100.7 ppm, 78.4 ppm, 70.9 ppm, 56.7 ppm, 53.1 ppm are assigned to the ring carbons: C-1, C-4, C-3,5, C-6 and C-2 respectively.
In the spectrum of CHI-graft-PDEAEM, the signal at 168.0 ppm belong to the
carbonyl carbon (C-1) of CHI and the intensity of this peak increased due to the grafting of DEAEM.
The signal at 17.3 ppm belongs to the methyl group of
acetamide of CHI and methyl group of DEAEM. The signal at 98.6 ppm is assigned to the C-1. The signals at 77.7 ppm 75.3 ppm, 74.7 ppm and 67.9 ppm are assigned to belong to CH2OH of DEAEM. The signals at 63.0 ppm, 59.4 ppm, 55.7 ppm,
11
Page 11 of 33
54.6ppm are belong to are assigned to the ring carbons: C-4, C-3,5, C-6 and C-2 respectively. In the spectrum of CHI-TPP-graft-PDEAEM, the signal at 173.6 ppm belong to carbonyl ester bond. The chemical shifts at 168.6 ppm and 11.0 ppm belong to the
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carbonyl carbon and the methyl group of the acetamide groups of CHI respectively. The signals at 98.5 ppm, 77.8 ppm, 70.1 ppm, 61.8 ppm and 54.7 ppm are assigned to
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the ring carbons: C-1, C-4, C-3,5, C-6 and C-2 respectively. The new signal appear at
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17.3ppm which, belongs to-CH2 of DEAEM. The signal at 39.6 ppm belongs to CH2NH2 of DEAEM.The signals at 49.9 ppm and 67.8 ppm belongs to CH2OH of
Scanning electron microscopy (SEM) analysis
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3.6.
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DEAEM.
SEM micrographs of the CHI gel by 1% GA, CHI* gel by 1% GA, CHI* gel by 4%
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GA, CHI*-graft-PDEAEM(294) by 1% GA , CHI-TPP beads and CHI-TPP-graft-
Ac ce pt e
PDEAEMare given in Fig.6 (a), (b), (c), (d), (e) and (f) respectively. The SEM images of gels of chitosan (Fig.6(a) and 6 (b)) show that smooth surfaceshave been obtained by crosslinking with 1% GA.Increasing GA% results in more roughness of the gel surface ((Fig. 6 (c)).The surface morphology of CHI*-graft-PDEAEM(294) ((Fig. 6(d)) revealed that grafting causeda less smooth surface. When the surface of CHITPP bead alone (Fig. 6 (e))is compared to that of CHI-TPP-graft-PDEAEM (Fig. 6(f)), the presence of the grafted polymer on the bead surface can easily be identified. 3.7. Swelling properties of products CHI graft PDEAEM gels exhibits improved pH responsive swelling when compared to pure CHIgels. Under acidic conditions CHI-GA gel exhibits an equilibrium swelling of187% within 6 hours while CHI-graft gel has an equilibrium swelling of
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Page 12 of 33
276%.Similarly at pH=7.0 and 11.0 the GA-crosslinkedCHI gelshow an equilibrium swelling value of 130% and 163% respectively while the grafted product swells by 405% and 570% at pH=7.0 pH=11.0 respectively. The equilibrium % swelling value in basic conditions competes with the value in acidic conditions. This behavior could
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be explained by basic and acidic hydrolysis of imine bonds giving rise to less crosslinked networks.The swelling behavior of CHI-TPP beads was followed in
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pH=1.0, 7.0 and 11.0. CHI-TPP swells with an equilibrium swelling capacity
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%8800andthe PDEAEM grafted CHI-TPP beads have a swelling capacity of %5700 at pH=1.0. The reason why CHI-TPP beads have higher swelling capacity than CHI-
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TPP-graft-PDEAEM beads can be attributed to screening effect of ethyl groups in between protonated tertiary amine groups on PDEAEM. In pH=7.00 and pH=11.0
%470.
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both CHI-TPP beads and CHI-TPP-graft-PDEAEMbeads have swelling capacity
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Oscillating swelling behavior of CHI-TPP beads and CHI-TPP-graft-PDEAEMbeads
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was studied at pH=7.00/pH=1 and pH=11.00/pH=1.00. The oscillating swelling behavior at pH=7.00/pH=1 is shown in Fig7.When the beads are initially swollen in pH=7.0, the swelling capacity increases to 2470% for CHI-TPP beads and 650%CHITPP-graft-PDEAEMbeads following immersion in pH=1.0 as shown in Fig. 7.The same trend with increasing % swelling values is observed inrepeated steps. Similar behavior is observed for oscillating swelling for CHI-TPP-graft-PDEAEMbeads for consecutive immersion in pH=11.0 and pH=1.00.
4. Conclusions
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Page 13 of 33
Chi/DEAEM/KPS system is a versatile combination to obtain grafted chitosans by redox initiation under both homogeneous and heterogeneous conditions. Products whose solubility in aqueous acidic solution is controlled by the grafting yield are obtained under homogeneous reaction conditions. The graft copolymer sample, which
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is soluble in aqueous acidic solution can becrosslinked by glutaraldehyde at pH=1 to obtain chemical gels with improved pH sensitive swelling capacity compared to
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glutaraldehydecrosslinked pure chitosan gels. Chitosan-TPP gel beads provide a
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suitable matrix for surface modification via graft copolymerization of DEAEM. In pH=1 solution, chitosan-TPP-graft-PDEAEM gel beads swell to perform as
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superabsorbent gels.The grafted beads perform as pH responsive superabsorbent 3D matrices upon swelling in pH=7.00/pH=1.00 solutions in consecutive repeated steps.
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PDEAEM grafted chitosan gelshave a potential to find a place in drug delivery as well as novel bio-based adsorbents following further in-vitro and in-vivo detailed
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investigationand biological analysis.
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References [1] D. Schmaljohann, Adv. Drug Deliv. Rev. 58(2006) 1655-1670. [2] S. Kim, J.H.Kim, O. Jeon, I.C. Kwon, K. Park, Eur. J. Pharm. Biopharm.71(2009) 420-430.
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[3] R. Rayakumar, Int. J. Biol. Macromol. 74(2015) 240-262. [4] E. Yilmaz, Adv. Exp. Med. Biol. 553 (2004) 59-68.
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[5] V. Pillay, A. Seedat, Y.E. Choonara, L.C. Toit, P. Kumar, V.M.K. Ndesendo,
[6] L.Y., Qiu, Y.H. Bae ,Pharm Res. 23(2006) 1-30.
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AAPS Pharmscitech. 14(2013) 692-711.
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[7] H. Caner, H. Hasipoglu, O. Yilmaz, E. Yilmaz, Eur. Polym. J. 34(1998) 493-497.
77(2000) 2314-2318.
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[8]A.M.K. Najjar, W. Yunus, M.B. Ahmad, M.Z.A. Rahman, J. Appl. Polym. Sci.
577-581.
d
[9] M.Yazdani-Pedram, C. Tapia, J. Retuert, J.L. Arias, Macromol. Biosci. 3(2003)
Ac ce pt e
[10]A. Pourjavadi, G.R. Mahdavinia, M.J. Zohuriaan-Mehr ,J. Appl. Polym. Sci. 90(2003) 3115-3121.
[11] S.B. Lee, D.I. Ha, S.K. Cho, S.J. Kim, Y.M. Lee , Appl. Polym. Sci. 92(2004) 2612-2620.
[12]G.R. Mahdavinia, M.J. Zohuriaan-Mehr, A.Pourjavadi , Polym. Adv. Technol. 15(2004) 173-180.
[13] H.Caner, E. Yilmaz, O.Yimaz , Carbohydr. Polym. 69(2007) 318-325. [14] E.Yilmaz, T. Adali, O. Yilmaz, M.Bengisu , React. Funct. Polym.67(2007) 1018. [15] T. Adali, E.Yilmaz , Carbohydr. Polym.77(2009) 136-141. [16]P.J. Lv, Y.Z. Bin, Y.Q. Li, R. Chen, X. Wang, B.Y. Zhao ,Polymer. 50(2009)
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5675-5680. [17] C. Spagnol, F.H.A. Rodrigues, A.G.B. Pereira, A.R. Fajardo, A.F. Rubira, E.C.Muniz , Carbohydr. Polym. 87(2012) 2038-2045. [18] G.Y. Li, L. Guo, Q.W. Wen, T.Zhang, Int. J. Biol. Macromol. 55 (2013) 69-
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74. [19] T.Adali ,Bio-Medical Materials and Engineering. 23 (2013) 349-359.
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[20] K.Zhang, Z.K. Wang, Y.L. Li, Z.Q. Jiang, Q.L. Hu, M.Y. Liu, Q.X.
us
Zhao, Carbohydr. Polym. 92(2013) 662-667.
[21] P.Mukhopadhyay, K. Sarkar, S. Bhattacharya, A. Bhattacharyya, R. Mishra,
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P.P. Kundu, Carbohydr. Polym. 112(2014) 627-637.
74(2005) 26-30.
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[22] H. Cai, Z.P. Zhang, P.C. Sun, B.L. He, X.X.Zhu , Radiat . Phys. Chem.
[23] M.F.A. Taleb, Polym. Bull. 61(2008) 341-351.
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[24] I.M. El-Sherbiny, H.D.C. Smyth , Carbohydr. Polym. 81(2010) 652-659.
Ac ce pt e
[25] I.M. El-Sherbiny, H.D.C. Smyth , Carbohydr. Res. 345 (2010)2004-2012. [26] S. Saber-Samandari, M. Gazi, E.Yilmaz , Polym. Bull. 68(2012) 1623-1639. [27] S. Saber-Samandari, O. Yilmaz, E. Yilmaz , J. Macromol. Sci. A. 49(2012) 591-598.
[28] A.S.Carreira, F. Goncalves, P.V. Mendonca, M.H. Gil, J.F.J.Coelho , Carbohydr. Polym. 80(2010) 618-630.
[29] W.Z. Yuan, Z.D. Zhao, S.Y. Gu, T.B. Ren, J.Ren , Mater.Lett. 65(2011) 793796. [30] K.Zhang, Z.K. Wang, Y.L. Li, Z.Q. Jiang, Q.L. Hu, M.Y. Liu, Q.X. Zhao, Carbohydr. Polym. 92(2013) 662-667.
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[31] R. Jayakumar, M.Prabaharan, R.L. Reis, J.F. Mano , Carbohydr. Polym. 62 (2005) 142-158. [32] S.C. Hsu, T.M. Don , W.Y. Chiu , Polym. Degrad. Stab. 75 (2002) 73-83. [33] Z. Yalinca , E. Yilmaz , F. T. Bullici , J. Appl. Polym. Sci. 125(2012) 1493–
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1505.
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List of Tables Table 1. Preparation conditions of all CHI-graft-PDEAEM. Table 2. The preparation conditions of GA crosslinked CHI-graft-PDEAEM gels.
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Table 3. The preparation conditions of GA crosslinked and non crosslinkedCHI-TPPgraft-PDEAEM beads.
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Table 4. Gelation time determination of GA crosslinkedCHI and GA crosslinked
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CHI-graft-PEAEM gels.
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List of Figures
Fig. 1. Chemical structure of (a) chitin and (b) chitosan.
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Fig. 2. Grafting mechanism onto chitosan by (a) direct oxidation by Ce4+, (b) complex formation by Ce4+, (c) persulphate initiation.
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Fig. 3. Chemical structure of DEAEM.
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Fig. 4. FTIR spectra of (a) CHI, (b) DEAEM, (c) CHI-graft-PDEAEM, (d)CHI-TPPgraft-PDEAEM beads.
Fig. 5. C-13NMR spectra of (a) CHI, (b) CHI-graft-PDEAEM, (c)CHI-TPP-graftPDEAEM.
Fig. 6. SEM micrographs of the a) GA(1)-CHI, b) GA (1)-CHI* c) GA (4)-CHI* d) GA (4)-CHI*-graft-PDEAEM(294) (e) CHI-TPP beads and (f)CHI-TPP-graftPDEAEM.
Fig. 7. The comparison of oscillating swelling behavior of CHI-TPP and CHI-TPPgraft-PDEAEM between pH=7.0 and pH=1.0.
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Table 1
DEAEM (mL)
T (°C)
Time (hr)
KPS (g)
H%
CHI-graft-PDEAEM(294)
0.25
70
4
0.1250
3.37
294
CHI-graft-PDEAEM(361)
0.50
70
4
0.1250
4.95
361
CHI-graft-PDEAEM(356)
0.75
70
4
0.1250
3.26
356
CHI-graft-PDEAEM(221)
1.00
70
4
0.1250
1.69
221
G%
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Sample ID
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Table 1. Preparation conditions of all CHI-graft-PDEAEM.
Page 19 of 33
Table 2
Table 2. The preparation conditions of GA crosslinked CHI-graft-PDEAEM gels. Volume (µL) of GA
GA (1)-CHI-graft-PDEAEM(294)
3960
40
GA (2)-CHI-graft-PDEAEM(294)
3920
80
GA (3)-CHI-graft-PDEAEM(294)
3880
GA (4)-CHI-graft-PDEAEM(294)
3840
ip t
Volume (µL) of CHI
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120 160
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Sample ID
Page 20 of 33
Table 3
Table 3. The preparation conditions of GA crosslinked and non crosslinked CHI-TPP-graft-
GA, mL
Bead Size,
G%
CHI-TPP-graft-PDEAEM(31)
0.50
-
710
31
GA(0.1)- CHI-TPP-graft-PDEAEM(49)
0.50
0.1
710
49
GA(0.3)- CHI-TPP-graft-PDEAEM(43)
0.50
0.3
710
43
GA(0.5)- CHI-TPP-graft-PDEAEM(48)
0.50
0.5
520
48
GA(1.0)- CHI-TPP-graft-PDEAEM(54)
0.50
1.0
212
54
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Sample ID
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DEAEM, mL
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PDEAEM beads.
Page 21 of 33
Table 4
Table 4. Gelation time determination of GA crosslinked CHI and GA crosslinked CHI-graftPEAEM gels.
CHI
CHI*
CHI*-graft-PDEAEM(294)
dissolved in acetic acid
dissolved in pH=1.0
dissolved in pH=1.0
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GA%
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Gelation Time (minutes)
35 26
190
3
17
43
4
9
not detectable.
305 155
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2
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Complete gel formation
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1
4
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22
20
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*dissolved in pH=1.0
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Figure1 (a)
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Figure1 (b)
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Figure 2 (a)
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Figure 2 (b)
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Figure 2 (c)
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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Figure 7
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