amino acids for the development of

Chitosan functionalization with a series of sulfur-containing a-amino acids for the development of drug-binding abilities Reena Tondwal, Man Singh School of Chemical Sciences, Central University of Gujarat, Gandhinagar 382030, India Correspondence to: M. Singh (E - mail: [email protected]) and R. Tondwal (E - mail: [email protected])

Chitosan (Chi; 0.5 g) in 69.66 mM aqueous acetic acid was mixed with 312.4 mM methionine (methi) at 0.01 mL/s to disperse and cause optimum collisions for supporting condensation reactions through ANH2 of Chi and ACOOH groups of methi. The functionalized chitosan (f-Chi) product with methi developed an amide bond, which was represented as methi-functionalized chitosan [Chi–NHAC(@O)–methi]. Both the 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and Dean–Stark methods were followed for Chi functionalization. Sulfonation with chlorosulfonic acid in a dimethylformamide medium was conducted at 90 8C and 750 rpm with an approximately 72% yield. The Chi–NHAC(@O)–methi was characterized by 1H-NMR spectroscopy and Fourier transform infrared stretching frequencies. The onset temperature of 280 8C recorded by thermogravimetric analysis/differential scanning calorimetry analysis, confirmed the high stability of the covalent bonds in Chi–NHAC(@O)–methi. The synthesis was repeated with other series members of sulfur (S) atoms containing a-amino acids: homocysteine, ethionine, and propionine. The shielding of terminal ACH3 was enhanced on elongation of the terminal alkyl chain in the case of propionine. The peak for the ANH2 of Chi at a d value of 4.73 ppm shifted to 5.36 ppm in Chi–NHAC(@O)–methi because of the involvement of ANH2 in ANHAC(@O)A. Theoretically, the value of ANH2 of Chi was 5.11 ppm, with a difference of 0.38 ppm as compared to the experimentally determined value C 2017 Wiley Periodicals, Inc. J. of 4.73 ppm. Additionally, a new peak at a d value of 3.26 ppm also confirmed Chi functionalization. V ABSTRACT:

Appl. Polym. Sci. 2017, 135, 46000.

KEYWORDS: biodegradable; differential scanning calorimetry (DSC); polysaccharides; spectroscopy; thermogravimetric analysis (TGA)

Received 31 August 2017; accepted 2 November 2017 DOI: 10.1002/app.46000 INTRODUCTION

Functional methods for enhancing the abilities of macromolecules have been in high demand because of their targeted and specific applications, either for scavenging free radicals or for binding and releasing specified drugs.1–3 This does not mean that functionalization needs a host–guest chemistry to facilitate combinatorial symbiosis or science because functionalization is the function that is being performed by the functional molecules. The functionalization seems to be a specified new bond formation between the parent and other molecules that can assist binding, release, and steric activities over the drug by functional molecules with specified cavities, void spaces, and molecular motions.4 Thus, the new bond, which is formed on functionalization, should be strong enough to hold and carry forward for transportation of the drug species. Therefore, the selection of the parent molecule becomes the most critical choice for the functionalization process because competent functional groups are needed to support the chemical process to chemically or electronically establish a link between the parent and the additives.

Thereby, we chose Chi material because it has several AOH and ANH2 functional groups in its structure; these supported an electronic binding route with sulfur-containing a-amino acids (aAs) to functionalize Chi in a suitable environment in our case. Chi is a linear cationic heteropolymer or aminoglucopyran composed of repeating units (N-acetyl glucosamine) and glucosamine residues with 1,4-b linkages obtained from the deacetylation of chitin, which is the second most abundant natural biopolymer and is mainly derived from exoskeletons of crustaceans and also the cell walls of fungi and insects.5–9 Chitin is a natural polymer that naturally contains acetyl group and develops a highly stable product, where the acetyl group hinders their functionalization activities. Therefore, to take chitin to a functionalization level, deacetylation is chemically conducted to free the bound acetyl ANHA to ANH2. The free ANH2 is substantially and actively involved in the chemical process of functionalization with adequate capability for developing a covalent bond with functionalizing agents such as methionine (methi). The product (Chi) was procured from Sigma with 75–85% deacetylation. This caution was used in our study so that we could chemically bind a larger number of aAs to enhance Chi’s

C 2017 Wiley Periodicals, Inc. V

46000 (1 of 14)

J. APPL. POLYM. SCI. , DOI: 10.1002/APP.46000

ARTICLE

WILEYONLINELIBRARY.COM/APP

drug-binding ability, induce an adequate steric effect, and cause chemical units with van der Waals forces. Because the pure Chi gains an optimized structure with a comparatively higher potential energy (En) as the Lennard–Jones potential and because such an optimized structure of Chi could not accommodate the drug molecule to be bound with Chi, the purpose of Chi functionalization was to induce the interacting sites and methi, along with a series of aAs, to induce adequate branching to develop steric effects. Here, methi, homocysteine, ethionine, and propionine molecules were chemically bound with Chi through covalent amide bonds. These branching units induced adequate molecular motion, such as rotational, translational, and vibrational motions, to approach and entangle the drug so that the drug came into contact with the interacting units; this developed the drug-binding activities, which assisted with the transportation of the drug. Most interestingly, the hydrophobicity, which is a deciding factor in the binding and release of drugs, was also structurally constitutionalized in our approach through an increase in the number of ACH2A (methylene) groups in the aAs. The synthetic conditions were chosen in a manner so that the active methylene groups did not participate in the reaction and remained safer to constitute an element of hydrophobicity. Fourier transform infrared (FTIR) spectroscopy10 and 1H-NMR11 for acetylated and deacetylated Chi distinguished the spectroscopic features of the deacetylated Chi from that of the acetylated Chi. In view of the rapidly growing interest in the research of Chi, its chemical modification was done because, in its native form, its solubility in most of aqueous or other biocompatible media is negligible because of embedded functional groups. The functionalization of Chi with S atom containing molecules (thiolation) and the immersion of sulfate groups (sulfation) were chosen in several chemical modifications, such as quaternization, sulfation, thiolation, alkylation, acylation, oligomerization, carboxyalkylation, hydroxyalkylation, phosphorylation, graft copolymerization, and enzymatic and other reactions because of specific applications in anticholesterol,12,13 anticoagulant,14–16 antitumor,17 cardiovascular disease4,18,19 and drug delivery.2–4 These studies have been reported in a most scattered and piecemeal manner, and nothing can be generalized about a common principle with respect to the methodology of functionalization and the strength of the covalent bonds formed on functionalization. A sole method for the functionalization of a less functional molecule is to have the highest stability of the developed bonds because the product is supposed to be exposed to a variety of experimental conditions to make the best use of the said product. Therefore, contrary to the reported studies, we initiated its functionalization with a series of aA, where similar studies were also conducted with other series of amino acids for more and more suitable binding sites. Thereby, we performed the functionalization of Chi with aAs containing S atoms (methi). As an essential amino acid, methi has an S atom in its fundamental structure, which imparts additional properties because of its electronic configuration; this helps in voluminous biological activities and also in drug vehicles. Many drugs themselves contain S atoms in their structures. Thus, our studies would be most suitable for the binding

and release of those drugs because of the compatibility between the S atom of the drug and the S atom of the drug vehicles. The purpose of binding the aAs with parent Chi was to develop molecular factors that could interact with the drug at the cost of certain frictional and release mechanisms, which were the same on withdrawing the branching interactions of the f-Chi with the drug. Thus, the series members, with increasing length of the alkyl chain, developed an adequate amount of friccohesity20 for optimum drug-binding forces or physicochemical activities at infinitesimal mode. Similarly, the variable length of the series members also became an asset by allowing the contact of the drug with the medium and by undergoing steric activities to release the drug. Hence, the series of aAs (homocysteine, methi, ethionine, and propionine) as functionalizing agents was scientifically selected to investigate the effect of the terminal end bound to S atoms because the terminal end seemed to be responsible for the initiation of the pattern of molecular motion by causing a steric effect; this was an essential component for its use in the in binding and release of the drug. The S atom in the chain of the chosen aAs with different electronic configurations hindered the induction effect with respect to the length of alkyl chain, for example, of ethionine and propionine. Such electronic chemistry of the selected series of aAs induced weaker van der Waals forces required for binding the drug. The aAs used in our study, due to the generation of weak electrostatic poles [ANHAC(@O)A $ A1NH@C(@O)2A] derived from methi-functionalized chitosan [Chi–NHAC(@O)–methi], caused optimum binding of the drug, which was ideally needed for drug binding and safer transportation to the wanted sites as drug vehicles in the human body. The sole method of Chi functionalization was to develop a stable, chemically active structure with sufficient binding forces in terms of introducing chemically active sites in the resulting structure. The efficiency of f-Chi depends on the surface area, bulk density, hardness, pH, particle density, particle size, and surface chemistry.21 In this respect, the ANH2 of Chi was used to bind methi, as shown in Figure 1. Therefore, the f-Chi with a covalent bond was highly stable; this was also shown by thermogravimetric analysis (TGA). The stability of the drug vehicle is an essential requirement so that when binding or releasing the drug, it does not induce the structural transition in the drug or alter drug structure in any way. Additionally, our purpose for functionalizing Chi was to induce biocompatible bonding with adequate electronic configurations with highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) models to assist in binding, transport, and release of the drug to the desired sites. Several researchers have reported the Chi functionalization with cysteine,22 lysine,23 glutamic acid,24 and others, but no series of aAs with a systematic approach to explain the chain length of terminal alkyl chains attached to S atoms has been reported. Because the terminal alkyl chain performs a critical role in controlling and driving a drug to a chemically active electron cloud of the amide bond, in light of such novelty, our work not only advanced the mechanism of Chi functionalization with suitable binding agents for an adequate binding ability but also pioneered the new mechanism through the variation of the chemical environment of the terminal alkyl chain

46000 (2 of 14)


ARTICLE


Figure 1. Flow chart of the functionalization of Chi.

responsible for causing steric effects. However, research to further investigate the effect of Chi functionalized homocysteine, methi, and ethionine for drug release is being undertaken in our laboratory. Apart from the thiolation, including sulfur, carboxyl, amino, and terminal alkyl chain groups, which was introduced into the backbone of Chi, other functional groups (e.g., sulfate groups) were also introduced into the Chi structure with the help of chlorosulfonic acid (CSA) on reaction with AOH of Chi. Dimethylformamide (DMF) and CSA were also chosen; this became the most critical and efficient green route for using Cl2, H1, and OH2 to develop a sulfonated functionalized chitosan (f-Chi) with ASO3Na.25,26 Thus, the functional process is

industrially most desirable, and several molecules with different structures have been used for functionalization with several additives. We also tried our best to find relevant literature but found none on the theme, which we evolved by developing aAs linked to Chi with controlled hydrophobicity and steric effects. Thereby, our research method is a new breakthrough; it not only develops a new sulfonated functionalized product but also a new workable research methodology that is reproducible and does not produce any polluting byproduct except H2O. Therefore, our research approach could be noted not only as green chemistry but also as sufficiently functional green chemistry with the bifunctionalization of Chi. The sulfate groups have

46000 (3 of 14)


ARTICLE


750 rpm at room temperature (RT) for 10–20 min. Chi developed a stable emulsion in the 69.66 mM aqueous acetic acid. Similarly, methi and EDC (1:1 ratio) were dissolved in 10 mL of H2O to form a homogeneous solution. Because methi had amino and carboxylic functional groups, these groups assisted in its dissolution in H2O. Methi and EDC aqueous solution was added dropwise at 0.01 mL/s in 1% Chi aqueous acetic acid with continuous stirring at 750 rpm at RT. The stirring was continued for further 72 h. After 72 h of stirring, a 1 M NaOH aqueous solution was added dropwise to the reaction mixture. A cloudy precipitate was obtained by the addition of few drops of 1 M aqueous NaOH. The precipitate was centrifuged, and the residue was washed with water and pure methanol to neutralize the pH. The light brown product was dried at 50 8C in vacuo and stored in sample vial until further use. In the Dean–Stark method, Chi and methi in a 1:1 ratio were dispersed in toluene with continuous stirring at 750 rpm at 90–100 8C for 24 h into a 50 mL round bottom (RB) flask. The condenser-fitted Dean–Stark apparatus was fitted with this RB flask, and the apparatus was also filled with toluene. After 24 h, the round bottom (RB) flask was taken to RT, and similarly, the product was centrifuged with water and pure methanol, and the light brown product was stored in sample vial until use after drying at 50 8C in vacuo.

Figure 2. Flow chart of the sulfonation of f-Chi.

been proven to play a leading role in the biological activities of sulfated polysaccharides. So, naturally nonsulfated polysaccharides including Chi have also exhibited biological activities after sulfation and have wide uses in medicine and pharmacy in tracking disease.4,14,27–29 Our methodology could be extended to several other additives for efficient drug binding and release for other similar molecules in place of Chi, such as cellulose, starch, dendrimers, tween, graphene, and curcumin. With the consideration of existing research activities in the area of functionalization of Chi, our selection of the aA series as functionalizing agents opens a new window for the synthesis of safer and optimized drug vehicles.

Sulfonation of f-Chi For the preparation of the sulfonated product, Chi– NHAC(@O)–methi was added to a sulfating complex consisting of DMF and CSA in a 1:1.5 ratio. This mixture was stirred at 90 8C for 4 h, and acetone was added to precipitate the product to salt out the precipitate as the acetone developed a stronger affinity toward H2O. The precipitate was redissolved in ice water, and the pH was adjusted to 9 by the addition of 3 M NaOH. The solution was centrifuged and washed with water and finally with acetone, and the sulfonated product was stored in a sample vial until use after drying in vacuo (Figure 3).

EXPERIMENTAL

Materials Chitosan (Chi; degree of deacetylation 5 75–85%, molecular weight 5 50–190 kDa), L-methi, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCHCl; Sigma-Aldrich), acetic acid, DMF, toluene, methanol, acetone (SRL, India), and chorosulfonic acid; (CSA; Qualikems, India) were used without further purification and were stored overnight in a vacuum desiccator filled with P2O5 until use. Functionalization of Chi The functionalization of Chi was materialized by two separate synthetic methods, namely, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and Dean–Stark (Figure 2). In the EDC method, the 1% Chi was dispersed in 69.66 mM aqueous acetic acid solution with continuous magnetic stirring at

Figure 3. Structural orientation of Chi and methi for conducting a condensation reaction.

46000 (4 of 14)


ARTICLE


framework of H2O and A1NH3, Chi seemed to disperse, as there was no structural resistance of H2O implied in Figure 4. The purpose was to activate the ANH2 group of Chi in a safer manner, and hence, on heating and stirring, aqueous CH3COOH also disrupted their hydrogen bonding so that the molecules could move ahead in the monodisperse mode with sufficient kinetic energy and react with each other. CH3COOH and H2O work on the basis of the Lewis conjugate acid–base concept, noted as follows: CH3 COOH ! CH3 COO2 1H1 Figure 4. Chi dispersion mechanism in aqueous acetic acid for an adequate orientation.

or H2 O1H1 ! H3 O1

Characterization of aA–f-Chi and Sulfonated f-Chi We recorded the FTIR spectra for f-Chi by two different methods and for sulfonated f-Chi with a PerkinElmer 65 series FTIR spectrophotometer by pelletizing a 2.0 mg sample with KBr. 1HNMR were recorded with a Bruker 500-MHz Ultra shield plus instrument with CD3COOD1D2O (E-Merck, 99.9%) as a solvent and TMS as an internal reference. The TGA study was performed with an Exstar TG/DTA 7300 analyzer from 50 to 600 8C under a nitrogen atmosphere at a rate of 100 mL/min with a slow ramp rate of 10 8C/min. RESULTS AND DISCUSSION

Chemistry and Mechanism In general, the molecules, which contained the ANH2 and AOH (alcoholic) groups in their structures, which were soluble in H2O because of hydrogen bonding, but Chi, contrary to holding the previous groups, was not dispersed in H2O alone in the absence of acetic acid (CH3COOH). This proved that the Chi structure had ANH2 and AOH groups in an embedded state; they could not establish the chemical link with the H2O alone and needed a comparatively protic solvent, such as CH3COOH, to activate the ANH2 of Chi. Similarly, because of a dipolar H2O structure, the hydrogen bonding was comparatively stronger and was not disrupted by Chi. Thus, we used the excellent monopoles of CH3COO2 and H1 to be added to H2O before the addition of Chi. CH3COO2 and H1 approached the oppositely charged H1 and O22 atoms of H2O, and this process weakened the H2O. Thus, here, the role of the H1 was significant in the activation of the ANH2 of Chi rather than disrupting the water structure. Once hydrogen bonding was broken, the H2O became free. This free H2O engaged the CH3COO2 released by the CH3COOH. Hence, aqueous CH3COOH was found to be an efficient solvent compared to hydrogen-bond breakers such as urea. Thus, this proved that an H1-releasing medium was needed to support and facilitate the functionalization of Chi. In such merit, the urea was found unfit as it could not release the H1. Probably, in light of the previous information, the CH3COOH as a disrupting agent disrupted the hydrogen-bonded H2O to form H3O1, which was not only kinetically active but also disrupted the hydrogenbonded H2O. The H1 could also approach the ANH2 of Chi and accept the lone-pair electrons of the ANH2 group to form A1NH3, which seemed to activate the ANH2 group to participate in the functionalization with aAs. In such a structural

or H3 O1 ! H2 O1H1

The H2O acts as a moderator to monitor the population of H1 obtained from the CH3COOH by forming H3O1 and then to release the H1. Thus, the combination of CH3COOH with H2O worked as a most optimized combinatorial model to initiate activation and orientation and destabilize the ANH2 of Chi to release H1; this was needed to complete the condensation reaction with methi. Of course, urea is a well-known H2O hydrogen breaker, but it also develops active chemical species; this could interfere with the condensation process, so the use of urea was avoided. However, the use of urea along with thiourea for the functionalization of Chi is being pursued in the laboratory: NH2 ACð@OÞANH2 $1 NH2 @CðAO2 ÞANH2 $ NH2 ACðAO2 Þ@1 NH2

It is surprising that researchers working in the area of the functionalization of Chi in aqueous media have used 174.68 mM CH3COOH. However, our critical research methodology has reduced the previous stoichiometric ratio of aqueous CH3COOH solution used as a dispersing medium. As compared to the 1% Chi in 174.68 mM aqueous CH3COOH, that is, the 1:1 ratio used by other researchers, we used 1% Chi in a 69.66 mM aqueous CH3COOH, that is, a 1:0.4 ratio, with an encouraging yield. This dynamic proved that CH3COOH was used to merely activate the ANH2 of Chi moderated through H3O1 formation in a safer manner because H1 ions were released on CH3COOH dissociation and could not directly approach the embedded ANH2 of Chi but were quickly attracted by the lone-pair electrons of the H2O. Hence, the 1:1 ratio of Chi to CH3COOH seemed invalid compared to the 1:0.4 ratio used in our research. The purpose was to functionalize a larger amount of Chi with a lower amount of CH3COOH to perform the reaction with greener solvents such as H2O. Therefore, our research methodology for using 69.66 mM aqueous CH3COOH solution was more effective with the avoidance of solute–solute interactions; this also inhibited the Chi interactions with CH3COOH, which affected the functionalization process; such interactions decreased the dispersion. Furthermore, 69.66 mM CH3COOH was reduced by the addition of a 312.4 mM methi aqueous solution, which further diluted the 69.66 mM CH3COOH. It was experimentally found to be most economic and chemically effective because of the higher

46000 (5 of 14)


ARTICLE


Figure 5. ANH2 of Chi and OH of methi at geometrical alignment per the Lennard–Jones potential for the reaction between them supported by HOMO and LUMO sites of methi.

dissociation. In Chi–NHAC(@O)–methi, the S atom stopped the development of the induction effect by terminal alkyl groups because of its electron cloud, which did not allow the passage of terminal ACH3 electron to reach the ACH2A bypassing S atom, which was placed before the terminal ACH3 in case of methi, ACH2CH3 in the case of ethionine, and ACH2CH2CH3 in case of propionine. Similar repulsion was caused by ANH2 because (as shown in the circled A region of Figure 5) in Chi–NHAC(@O)–methi could hybridize the electron as a single integrated cloud; this could have also been a reason for the stability of the newly formed ANHAC(@O)A amide bond, as it could repel out the electronic character. Structurally, Chi–NHAC(@O)–methi seemed to have substantially effective HOMO and LUMO sites with separate electron densities and electronic potentials (in electron volts). Such differences in the electronic energy caused a spontaneity that led to the induction of molecular motions, which were responsible for binding the drug and for functionalization process. Figure 5 electronically illustrates the LUMO and HOMO populations having unequal sets of shared pairs and nonshared pairs of electrons. Because the collisions are the most important requirement for orienting the reacting species with an adequate involvement of electronic charges, the stirring made Chi, methi, and other molecules monodisperse when their cohesive forces were weakened. Thus, an adequate 750 rpm was obtained when several samples were run, but the 750 rpm produced the reaction orientation with the maximum yield. We conducted the two parallel synthetic methods, namely, EDC and Dean–Stark, by transforming the ANH2 groups into ANHAC(@O)A amide groups to assess the mechanism and experimental conditions. In the Dean–Stark method, additional heating at 90 8C for 24 h was maintained without EDC, and the ANHAC(@O)A formation was a condensation reaction, which evolved too much water. As per Lechatelier law, the water molecules, which were formed on condensation, were accumulated at the head with the overhead pressure of the apparatus. Such water (vapor) pressure of the released H2O as an overhead could reverse the reaction, so to prevent an accumulation of water the Dean– Stark method was used. In this method, the H2O vapor has the

highest affinity toward toluene to form an azeotrope. Because the electronic profile of toluene due to hyperconjugation constitutes the dipolar structure where the water dipole is easily attracted and forms an azeotrope that facilitates the condensation reaction between Chi and methi, toluene is a carcinogenic volatile organic compound, and its vapor seriously affects the laboratory environment. Therefore, research work to substitute the toluene is being pursued with safer chemical substances, such as a series of alcohols and acetone to quickly use the water. So, on the utilization of water vapors, the reaction moved forward for a maximum yield. If H2O is not evacuated, there is no space to further accommodate the released H2O. Hence, there is a possibility of moving the reaction with kb, as shown in eq. (1). Here, we developed the collisions by providing a high thermal energy, which activated Chi and methi to reach to a Lennard–Jones potential distance so that the electron cloud of interacting atoms was exchanged to develop new chemical bonds. When kf > 1, a temperature of 90 8C was maintained:

kf 5

½Chi2NH2Cð5OÞ2methi ½H2 O ½Chi2NH2 ½HOOC2methi

(1)

The conditions for the reaction proceeded as under kf > 0 (0– 0.9) or kf > 1 (1–9) for better yield, and kf < 0 did not yield the product. Because the H2O was used in azeotrope formation and no amount was left unused so as per Lechatelier’s law, the reaction moved forward. Thus, the evacuation of H2O quickened the reaction, and hence, a stronger azeotrope forming the nature of the media affected the rate of the reaction. So, when the H2O was removed, the final equation was as follows: kf 5

½Chi2NH2Cð5OÞ2methi ½Chi2NH2 ½HOOC2methi

(2)

Equation (2) depicts that Chi–NH2 and HOOC–methi favored a forward reaction, as the H2O was not in the denominator term. In this process, the concentration of [Chi–NH2] and [HOOC–methi] decreased, although the amount of product [Chi–NHAC(@O)–methi] increased when kf > 1. Contrary to the Dean–Stark method, the EDC method seems to be a green chemistry route. In general, the functionalization has become an inevitable condition for developing safer, stable drug vehicles. In this regard, we successfully attained the functionalization of Chi through a simple and green solvent, such as H2O, and acetic acid with an emphasis on electrophilic and nucleophilic conditions at RT. Of course, toluene was also used as a medium to disperse Chi, but the functionalization reaction was conducted at 90 8C. Therefore, over toluene as the medium, Chi was dispersed in diluted acetic acid, and methi was dissolved in H2O. In this case, the reaction was conducted at RT on with a green solvent, such as H2O, and acetic acid. In case of the EDC method, the H2O and acetic acid not only acted as ANH2 group activating agents through a simple hydronium-ion formation mechanism but also moderated the H1 activities; this

46000 (6 of 14)


ARTICLE


Scheme 1. Synthesis scheme for Chi–NHAC(@O)–methi.

reduced the relaxation time of the water as a medium. However, in contrast to the aforementioned model of reacting and interacting steps, the toluene used in the Dean–Stark method initiated a secondary chemical process, which was a azeotrope

formation, where the reacting mixture, apart from a functionalization reaction, also underwent a secondary chemical mechanism for azeotrope formation, which could affect the functionalization process in terms of the activation energy.

Scheme 2. Synthesis scheme for sulfonated f-Chi.

46000 (7 of 14)


ARTICLE


Figure 6. 1H-NMR of (a) Chi, (b) Chi–NHAC(@O)–methi via the Dean–Stark method, (c) Chi–NHAC(@O)–methi via the EDC method, (d) sulfonated f-Chi via the Dean–Stark method, and (e) sulfonated f-Chi via the EDC method.

Therefore, the EDC method seemed superior over the Dean– Stark method mechanism-wise along with the moderation of H1 by H3O1 formation. Hence, in the EDC method shown in Scheme 1, EDC had a better proton-attracting ability because it had three N atoms each with one unshared electron pair. So, the H1 ions were needed in larger numbers to optimize EDC. The low relaxation time of EDC implied that the H1 ions remained bound with this for a shorter time, so we could monitor the release of H1. Thus, the H1 ions were furnished by ANH2 groups of Chi and also from the ACOOH of methi, and the release of H1 also followed from the CH3COOH. Such a situation created an additional spontaneity of EDC for H1. EDC seemed to dissociate the H1 from methi and ACOO2 attacks on the carbon of AN@C@NH1A (EDC). Thus, the H1 of ANHþ 3 (Chi) catalyzed by EDC and ANH2 was activated to establish ANHAC(@O)A amide bonds by attacking AC@O of the amino acid–EDC complex. Thereby, the additional stability of the biocompatible ANHAC(@O)A peptide bond made it a drug vehicle without changing its structure. The chemistry of Chi–NHAC(@O)–methi seemed most workable because of the ASA atom and ANH2, which were helpful for encapsulation, and the drug remained operational. Because it seemed that methi could have developed multiple units with Chi that could cause workable void spaces on the surface of

the molecules. Similar chemistry worked in the case of homocysteine, ethionine, and propionine-functionalized chitosan (aA–f-Chi), and hence, the reaction was conducted in a similar manner. These parameters supported the activities of aA–f-Chi as superior and nondamaging to the structure of the drug, and such assets of the drug vehicles made the vehicles useful for several drugs. In sulfonation (Scheme 2), a white mist was formed on partial H1 and Cl2 release from both CSA and DMF. The sulfonation was materialized to make f-Chi with better dispersion properties for encapsulating the drug. Chi–NHAC(@O)–methi was stirred at 750 rpm for 4 h at 90 8C in DMF–CSA so that f-Chi was dispersed in the mixed acid medium, where the CSA initiated a reaction with alcoholic AOH of Chi–NHAC(@O)–methi. Because DMF and CSA had the most active chemical species, which quickly interacted and made the medium most receptive for the functionalization of Chi. So, in this context, the CSA route for Chi functionalization seemed faster with a better yield. After 4 h, a dark brown color appeared in the mixture. The DMF structure seemed highly active with a high kinetic energy to percolate any molecular cluster or chain spaces of Chi-type molecular structures without directly involving the chemical process. Thus, the electron clouds of Chi–NHAC(@O)–methi and the DMF–CSA complex simulated and led to interaction so

46000 (8 of 14)


ARTICLE


Table I. Chemical Shifts of aA–f-Chi Before sulfur (ppm)

After sulfur (ppm)

Sample

ACH(NH2)A

ANH2

ASH

ACH2A

ACH2A

ACH3

Chi–NHAC(@O)–methi

3.26

4.73

—

—

—

2.18

Chi–NHAC(@O)–homo

3.26

4.73

1.5

—

—

—

Chi–NHAC(@O)–ethio

3.26

4.73

—

2.48

—

1.25

Chi–NHAC(@O)–prop

3.26

4.73

—

2.44

1.38

0.90

that f-Chi was not permanently engaged with the complex, except for the attack of ASO3H of the complex, and DMF was released as such. Therefore, the combination of DMF and CSA seemed to develop the most functional interface executed in a most integrated and simultaneous manner for Chi functionalization. CSA was also selected as it was quickly dissociated with a high dissociation constant and released Cl2 and H1 species. The success of this reaction depended on the decomposition of CSA. At last, a larger amount of NaOH produced a larger amount of OH2; this sufficiently neutralized the H1 produced from the AOSO3H. The addition of aqueous NaOH was done for the neutralization of an acidic environment and the precipitation of the product, and the excess NaOH neutralized the excessively released Cl2 when present; this caused NaCl and also its OH2 to attack the H1 to form H2O. Structural Characterization of F-Chi and Sulfonated F-Chi NMR resolved the state of 1H of Chi–NHAC(@O)–methi and sulfonated f-Chi due to shielding and deshielding effects; Chi had peaks at d values of 1.99, 3.04, 3.5–3.9, and 4.5–4.73 ppm for acetyl H, H-2, H-3,4,5,6, and H-1 and solvent, respectively. The peak at a d value of 4.73 ppm for ANH2 attached with an a-C atom of amino acid is present in methi and in Chi– NHAC(@O)–methi separately because the ANHAC(@O)A amide group could not be coupled with the proton of ANH2 attached to the a-C atom of methi in Chi–NHAC(@O)–methi after Chi functionalization. Similarly, AOH of methi could not couple with the protons, as mentioned previously for methi alone. Therefore, the chemical shift of the proton of ANH2 of the a-C atom of methi remained ineffective in Chi– NHAC(@O)–methi and also for methi alone; this implied Chi functionalization. The peak at a d value of 3.26 ppm depicted a product structure, and the rest of the peaks were merged in the Chi peaks depicted in Figure 6. The NMR spectra of the Chi– NHAC(@O)–methi synthesized with EDC showed a highly intense peak at a d of 3.26 ppm. However, a similar peak also

Figure 7. Graph of chemical shifts (ds) and a series of aAs.

appeared in the Chi–NHAC(@O)–methi synthesized with the Dean–Stark method, and this peak, shown in Figure 6, proved that the maximum number of ANH2 (primary amino group) of the polymerized Chi were transformed into ANHAC(@O)A (secondary amino group) in case of the EDC method. Because the additional lone pair of nitrogen of EDC facilitated the reaction to occur quickly so the higher intensity with EDC could be valid. In both processes, the functionalization could not have transformed all of the ANH2 in the amide bond, but it seemed that the highest intensity of the product synthesized with EDC indicated a maximum methi population. In Chi, the two hydrogen atoms of ANH2 were in a coupling state, and on chemical engagement of ANH2 with methi, the ANHAC(@O)A group was formed with different chemical environments around the stretching atoms, such as H and O. In the ANHAC(@O)A group, hydrogen atoms were decoupled, so their chemical shift underwent a visible change from a d value of 4.73–5.36 ppm. In the series of aAs, the purpose of increasing the terminal alkyl chain was to increase the element of steric hindrance, but at the same time, the process of increasing the length of the terminal alkyl chain should not affect the binding strength of the ANHAC(@O)A (Table I). This was responsible for the establishment of the chemical linkage with Chi. Thus, we studied the chemical environment by studying the shielding and deshielding effects of H atoms around each carbon by 1H-NMR in Table I. For example, the high field d of 0.90 for terminal ACH3 of propionine predicted a high stability that could withstand the chemical changes caused by either chemical substances or variations in the temperature and pH during the drug-binding and release processes with aA–f-Chi. However, the shielding of terminal ACH3 was strengthened when the peak shifted to a higher field in propionine with maximum stability as per the Zeeman splitting effect and degenerating activities. The chemical shift data for 1 H placed at various positions of aA–f-Chi indicated a two-order process; this was distinguished vis-a-vis their positions before S and after S atoms. The chemical shift of the proton placed along with carbon at after an S atom split at a higher field because of the stability in propionine (Figure 7). The new peak at a d value of 1.2 ppm, the shift of the peak at 3.04–3.10 ppm for H-2, and the merging of the peaks shown in Figure 6 confirmed that Chi– NHAC(@O)–methi was converted into sulfonated f-Chi. All of these sciences based on the working molecular coordinates were fitted in our hypothesis so that a novel and safer drug-binding and release model was developed with aA–f-Chi. The split in the FTIR spectra implied a variable environment of its stretching atoms of specific [AOH, ANH2,

46000 (9 of 14)


ARTICLE


Figure 8. FTIR spectra of (a) Chi, (b) Chi–NHAC(@O)–methi via the Dean–Stark method, (c) Chi–NHC(@O)–methi via the EDC method, (d) sulfonated f-Chi via the Deana˜ Stark method, and (e) sulfonated f-Chi via the EDC method.

ANHAC(@O)A] groups. Thereby, the FTIR spectroscopy became a more informatory tool for predicting its functionality for encapsulation. The FTIR spectrum of the sample was recorded, and the stretching frequencies of the Chi– NHAC(@O)–methi molecules were compared with those of Chi and methi shown in Figure 8. The spatial arrangements of

the AOH and ANH2 groups were highly appreciable, as the AOH groups and ANH2 were placed in a different spatial location of Chi, and this caused different environmental effects on their stretching frequencies within 3300–3500 cm21. The stretching frequencies of AOH and ANH2 were weak and closely placed, and this implied that they were embedded in the

46000 (10 of 14)


ARTICLE


Chi monomer unit. The molecular weight of Chi was 150 kDa, where an infinite number of AOH and ANH2 groups could have been chemically entangled, and hence, they produced weak stretching frequencies. Therefore, the 3N-6 modes were normal modes of vibrations with En, which is the quadratic function of the normal coordinate because of electron–electron, electron– proton, and proton–proton interactions as per Born–Oppenheimer approximation. Hence, En was determined with eqs. (3) and (5): 1 (3) En 5h n1 m 2 But m5

1 2p

rffiffiffi j l

where m is the reduced mass and j is force constant. Therefore, the final equation is obtained when the stretching frequency (m) from eq. (4) is put into eq. (3): rffiffiffi 1 1 j (5) En 5h n1 2 2p l where n is the principal quantum number and h is planck’s constant, which is calculated as follows: l5

Figure 9. Graph of the OD and a series of aAs.

(4)

m1 m2 m1 1m2

Because there is a higher En, the stretching frequency shown in Figure 8(a) for AOH and ANH2 was very weak because of stronger binding, the stretching frequency of f-Chi as compared to that of pure Chi were further weakened; this implied an engagement of ANH2 of Chi with AOH of methi to form the ANHAC(@O)A amide group. In case of Chi, many AOH and ANH2 groups were unused, although they were embedded in the polymerized Chi units. However, in the case of f-Chi, the ANH2 group was used to form an amide group. Thus, the ANH2 stretching frequency was further reduced. Therefore, manifolds split from 3300–3500 cm21 were not produced except for the stretching frequency for the embedded AOH group [Figure 8(b)]. The Dean–Stark and EDC methods were applied for f-Chi preparation, Chi was dispersed in toluene in the case of the Dean–Stark method and dilute acetic acid in the case of EDC method. Toluene is an aprotic solvent, whereas the acetic acid is a protic solvent. In this context, Figure 8(c) predicts a reduction in the absorbance (A) at 3400 cm21, in contrast to Figure 8(a,b). This proved that CH3COOH in aqueous medium dissociated into CH3COO2 1 H1 in the case of the EDC method. EDC catalyzed the ACOOH groups of methi through the H1 ion; this recovered after amide-bond formation. Thus, there was an excess of H1 population because the H1 was highly active and acted as an electrophilic agent, and hence, it approached the embedded AOH to form A1OH2. Such structural species present in the sample reduced the UV absorption, as shown at 3400 cm21 in Figure 8(c). Figure 8(a–c) shows different stretching of the amino and amide group because of the chemically varied environment. Thus, the ANHAC(@O)A bond as compared to the ANH2 bond of Chi were present in their separate chemical environments and binding or En as per their electron configuration. The

aforementioned parameters affected the stability of the produced product. Therefore, there was a prominent link between the 1HNMR, FTIR spectroscopy, and TGA/differential scanning calorimetry (DSC) of the samples. At 2928.6 cm21, a new stretching frequency was generated for the ACH3 stretching of a terminal alkyl chain; this confirmed the structural confirmation of the Chi–NHAC(@O)–methi via the EDC and Dean–Stark methods. For both cases, the EDC and Dean–Stark methods, Figure 8 shows the characteristic peaks of amide groups at 1589 and 1581.8 cm21 in Chi– NHAC(@O)–methi. This showed that methi got attached to the ANH2 group of the Chi through amide bond. A shift in the peaks of Chi–NHAC(@O)–methi confirmed the sulfonated structures of f-Chi, as shown in Figure 8(d,e). The stretching frequencies of CAOAS and S@O were observed at 894.7 and 1252.2 cm21 in the case of Dean–Stark method and 815.07 and 1264.6 cm21 in the case of the EDC method, respectively; this demonstrated the sulfonated f-Chi (Figure 8(d,e)). Figure 9 shows that methi produced the least absorption because there was no electron cloud of the terminal ACH3, which strongly responded to the IR. Homocysteine, with H atoms attached to the S atoms, as compared to the SAC bond had an active electron cloud that substantially responded to the IR light. In ethionine, the induction effect was initiated, which absorbed the IR more than methi and less than homocysteine. The series of aAs seemed to develop the aA–f-Chi with a consideration of getting desired optical activity because aA–f-Chi was aimed to bind, transport, and release the drug. The drugs, because of their heteroatomic structure with a unique electronic configuration, showed an extraordinary optical activity, and as per photonics activities, the drug could undergo certain transitional changes during a binding chemical process. Thereby, the series members from methi with a 0.0133 optical density (OD) from 4000 to 3200 cm21, ethionine with a 0.1427 OD from 4000 to 3300 cm21, and homocysteine with a 0.2677 OD were used for aAs. Figure 9 shows that the ACOOH of the aAs as their functionalization unit along with the development of the optimum electrostatic poles needed for drug binding, whereas their alkyl chain with ACHACH2ACH2A length caused a stretching frequency that induced the steric effect to encircle the drug. However, the terminal part where AH, ACH3, and ACH2CH3 of homocysteine, methi, and ethionine, respectively,

46000 (11 of 14)


ARTICLE


were observed with effective OD. So, they structurally induced a favorable factor over the drug to ensure its proper binding and release in a different chemical environment. Thus, aA–f-Chi had many drug-binding chemical coordinates, which were compatible with drug-delivery science. The use of various functional groups and p conjugation with delocalized electron clouds seem safer where the positive charge AN1 of the aA–f-Chi could bind the drug with weak van der Waals forces. The distances between two HOMO models were the same in homocysteine, methi, and ethionine, so the electron voltage of these localized models with the same distance remained the same as per eq. (6): 12 r r6 (6) [ðrÞ5 4E 2 r r where r is the bond length, U is potential energy, E is the energy, and r is the intermolecular separation. Because the amide bond was responsible for binding the drug and its terminal alkyl chain was responsible for the encapsulationtype mechanism, the chemical work was done by amide bonding, and the mechanical work was rendered by the terminal alkyl chain to geometrically drive the drug toward chemically active sites to generate weaker van der Waals forces. Thus, this implied that the interacting sites of the drug should have approached the electrostatic sites of the amide bond of aA–f-Chi. Therefore, the distance allowed the binding interactions with the generation of adequate binding potential on undergoing geometrical chemical interfaces. Fundamentally, the OD was determined by r!r*, n!n*, and p!p* transitions at 0–280 nm, but our purpose was not to focus on the transition but to consider the steric motions of the f-Chi, where methi and others were involved in the steric hindrance; this caused their activities to facilitate the drug-binding and drugrelease activities. Therefore, our focus was on the kinetic energy in terms of the frequency of the functional groups such as ANH2, AOH, ANHAC(@O)A, and others to generate the m (stretching frequency) used to calculate the energy. Thus, the OD was determined in terms of the kinetic energy and not as the electron transition, as the purpose was to prevent the electron transitions; this could have induced unwanted chemical activities in the drug structure. Therefore, A was calculated with A 5 2 2 log10 %T (T is transmittance). In light of the previous discussion, the NMR and FTIR spectroscopy, both complementary to each other, substantially

Figure 10. DSC of (a) Chi, (b) Chi–NHAC(@O)–methi, and (c) sulfonated f-Chi.

supported our series in favor of its ability to act as a drug vehicle with adequate binding forces and also release dynamics. Dynamic Studies by DSC and Thermogravimetry (TG) DSC curves were recorded in a nitrogen environment from ambient temperature to 600 8C and are shown in Figure 10. DSC specifically revealed the heat (q in milliwatts) induced transitions in the Chi, Chi–NHAC(@O)–methi, and sulfonated f-Chi structures. For Chi, the endothermic peak at 91.1 8C in the first range (50–250 8C) was associated with the loss of water, whereas the exothermic peak at 308.1 8C in the second range (50–550 8C) corresponded to the degradation and deacetylation of Chi. Figure 10 implies that the maximum heat flow of 6.09 mW in the case of the bare Chi, 0.18 mW in the case of Chi– NHAC(@O)–methi, and 0.71 mW in the case of the sulfonated f-Chi as Chi > Sulfonated f-Chi > Chi–NHAC(@O)–methi were noted. Interestingly, the sulfonated f-Chi had a higher q flow than the Chi–NHAC(@O)–methi by 0.53 mW and 5.5 8C. This proved that the sulfonated f-Chi further weakened the Chi– NHAC(@O)–methi structure by involving AOH in sulfonation. As for the Chi–NHAC(@O)–methi and sulfonated f-Chi, the endothermic peaks shifted to 115.4 and 100.3 8C, respectively, depending on the type of functionalization; this could have been due to the much stronger interactions between water and the functionalized groups of Chi. Both the sulfonated f-Chi and Chi–NHAC(@O)–methi had lower thermal stabilities than Chi because their decomposition peaks were 305.2 and 299.7 8C; these were lower than that of Chi (308.1 8C). These results were estimated because the degradation of the functionalized groups further induced the degradation of the whole material. All of the products were considered to be homogeneous during the thermal degradation because only one exothermic peak was found in the DSC curves (Figure 10). The TGA curves for Chi, Chi–NHAC(@O)–methi, and sulfonated f-Chi recorded in the nitrogen environment are shown in Figure 11. The temperature and weight loss (%) are given in Table II. When compared with Chi–NHAC(@O)–methi and sulfonated f-Chi, the Chi had a comparatively lower water evaporation activity and higher thermal degradation temperatures. Chi showed an onset temperature at 280 8C with a 52.2% weight loss in contrast to the 250 8C and 50.3% onset temperature and weight loss of Chi–NHAC(@O)–methi, respectively. This implied that the functionalization weakened the compactness of Chi and facilitated the drug-binding activities of Chi–NHAC(@O)–methi.

Figure 11. TG curves of (a) Chi, (b) Chi–NHAC(@O)–methi, and (c) sulfonated f-Chi.

46000 (12 of 14)


ARTICLE


Table II. Peak Temperatures and Weight Loss in DSC and TG during the Thermal Degradation of Chi, Chi–NHAC(@O)–Methi, and Sulfonated F-Chi First range (50–250 8C)

Second range (50–550 8C)

Sample

DSC minimum (8C)

Weight loss (%)

DSC maximum (8C)

Weight loss (%)

Chi

91.1

4.0

308.1

52.2

Chi–NHAC(@O)–methi

115.4

7.3

305.2

50.3

Sulfonated f-Chi

100.3

5.3

299.7

Interpretation

Water evaporation

Both the TGA and DSC data also implied the successful functionalization of Chi. The narrow gap between transitions indicated homogeneity in the Chi structure. The broad gap between the transitions indicated or supported the f-Chi because the two separate units were attached and expressed their nature toward heat response in Chi–NHAC(@O)–methi; this directly implied the stability of the final product. The sulfonation had homogenized structural activities, as indicated by Figure 11, as a comparatively narrow gap in the transitional structure. Figure 12 shows the relationship between the thermodynamic Gibbs free energy (DG) values of Chi, f-Chi, and sulfonated f-Chi. DG was calculated by the equation 2DG5nRT lnTs

Because Chi, f-Chi, and sulfonated f-Chi were polymers, n 5 1 or Ts was the transition temperature. Chi had the highest DG, as depicted in Figure 12; this value was 214193.26, compared to those of f-Chi (214169.84)and sulfonated f-Chi (214124.79). Because the chemistry and mechanism of the constitutional components of f-Chi, which were most adequately investigated in terms of their structure-determining tools, such as 1H-NMR, FTIR spectroscopy, TGA, and DSC, proved the smart and multifunctional activities of f-Chi, there was a wider scope to functionalize Chi with n-number functionalizing agents, where the void spaces could be the sole criteria with adequate steric effects and stability. In this context, f-Chi with fluorescent dyes, such as Rhodamine B, transitional metals, and lanthanides, especially with gadolinium (Gd31), and with a 17 electronic configuration, induced remarkable contrasting properties or solartrapping abilities, which could be useful in the development of remarkable photocatalytic and conducting thin films. Thus, in

Figure 12. Graph of the DG and Chi, Chi–NHAC(@O)–methi, and sulfonated f-Chi.

49.7 Thermal degradation

light of the peculiar features of the structure–activity relationship and structure–friccohesity relationship, our research work has attained high academic and industrial significance. It altogether opens a new window for the development smart materials on choosing the structure of the functionalizing agent with variable molecular weights of Chi. CONCLUSIONS 1

H-NMR and FTIR spectroscopy both confirmed Chi functionalization on the formation of highly stable covalent bonding between the ANH2 of Chi and the ACOOH groups of methi. The variations in the chemical environments at the terminal end of the aAs constitutionally controlled the molecular motions as per the chemical environment of aA–f-Chi. The terminal group remained free because it did not participate in the amide-bond formation in contrast to ACOOH, which took part in the condensation reaction depicted by 1H-NMR and FTIR spectroscopy. The sulfonation additionally sensitized the Chi– NHAC(@O)–methi; this could have been favorable for its dispersion and binding with the varieties of drugs. DSC of Chi, fChi, and sulfonated f-Chi all showed exothermic processes. Comparatively, Chi showed more exothermic processes. Therefore, f-Chi did not undergo many exothermic processes, and this would be favorable for its use as a good drug vehicle. TGA for Chi and f-Chi also showed visible changes in the transition at approximately 250–310 8C. We observed that a 49.70% weight loss occurred with sulfonation. In general, 52.5, 50.30, and 49.70% weight losses for Chi, f-Chi, and sulfonated f-Chi, receptively, implied higher stabilities. In all of the cases, the parent structure of Chi remained intact because there were no drastic changes in the FTIR spectroscopy, TGA, or 1H-NMR except for the assignation of AOH and ANH2 of Chi. The chemical involvement of AOH and ANH2 was evidenced from reasonable changes in the FTIR spectra, 1H-NMR, and TGA. The lone pair of electrons of N of ANHAC(@O)A derived from f-Chi was attracted by C1 (electron-deficient C) and deshielded the H of ANHAC(@O)A; this resulted in a chemical shift of 5.36 ppm. ANHAC1(@O2)A was formed as a highly electronegative O atom and attracted the shared electrons of ANHA(C@O)A to form C1. Probably, the S atom in these aA did not support the shielding or deshielding of H either of the terminal ACH3 or ACH2 as well, as it was placed just after the terminal ACH3 with increasing alkyl chains attached to S atom in other amino acids of the chosen series. Our research methodology could also support Chi functionalization with a series of a, b, and g amino acids.

46000 (13 of 14)


ARTICLE


ACKNOWLEDGMENTS

The authors are thankful to the Central University of Gujarat (Gandhinagar, India) for its infrastructural support and the University Grants Commission (New Delhi, India) for its financial support (RGNF F. no. F1-17.1/2015-16/RGNF-SC-HAR-12768).

13. Sugano, M.; Fujikawa, T.; Hiratsuji, Y.; Nakashima, K.; Fukuda, N.; Hasegawa, Y. Am. J. Clin. Nutr. 1980, 33, 787. 14. Liu, W.; Zhang, J.; Cheng, N.; Cao, Z.; Yao, K. J. Appl. Polym. Sci. 2004, 94, 53. 15. Wang, Z. M.; Xiao, K. J.; Li, L.; Wu, J. Y. Cellulose 2010, 17, 953. 16. Vongchan, P.; Sajomsang, W.; Subyen, D.; Kongtawelerta, P. Carbohydr. Res. 2002, 337, 1239.

REFERENCES

1. Feng, T.; Gong, J.; Du, Y.; Huang, Z. J. Appl. Polym. Sci. 2009, 111, 545.

17. Yu, M.; Ji, Y.; Qi, Z.; Cui, D.; Xin, G.; Wang, B.; Cao, Y.; Wanga, D. Saudi Pharm. J. 2017, 25, 464.

2. Elbarbary, A. M.; El-Sawy, N. M.; Hegazy, E. S. A. J. Appl. Polym. Sci. 2015, 132, DOI: 10.1002/app.42105.

18. Hu, X.; Zhang, Y.; Zhou, H.; Wan, H. J. Appl. Polym. Sci. 2015, 132, DOI: 10.1002/app.42623.

3. Tiew, S. X.; Misran, M. J. Appl. Polym. Sci. 2017, 134, DOI: 10.1002/app.45273.

19. Avramoff, A.; Khan, W.; Mizrahi, B.; Domb, A. J. J. Appl. Polym. Sci. 2012, 126, E196.

4. Majee, S. B.; Avlani, D.; Biswas, G. R. Afr. J. Pharm. Pharmacol. 2017, 11, 68. 5. Mati-Baou che, N.; Hele`ne De Baynast.; Vial, C.; Audonnet, F.; Sun, S.; Petit, E.; Pennec, F.; Prevot, V.; Michaud, P. J. Appl. Polym. Sci. 2015, 132, DOI: 10.1002/app.41257.

20. Singh, M.; Singh, S.; I.; Asiri, A. M. J. Mol. Liq. 2017, 244, 7.

6. Cobos, M.; Gonzalez, B.; Fernandez, M. J.; Fernandez, M. D. J. Appl. Polym. Sci. 2017, 134, DOI: 10.1002/app.45092.

23. Gomez, M. D.; Esparza, M. H.; Trevino, F. A. R.; Reyes, R. C. Macromol. Symp. 2003, 197, 277.

7. Abd E-Ghaffar, M. A.; Hashem, M. S. J. Appl. Polym. Sci. 2009, 112, 805.

24. Ghaffar, M. A. A.; Hashem, M. S. Carbohydr. Polym. 2010, 81, 507.

8. Santhosh, S.; Mathew, P. T. J. Appl. Polym. Sci. 2008, 107, 280.

25. Huang, R.; Du, Y.; Yang, J.; Fan, L. Carbohydr. Res. 2003, 338, 483.

9. Xiao, W.; Wu, T.; Peng, J.; Bai, Y.; Li, J.; Lai, G.; Wu, Y.; Dai, L. J. Appl. Polym. Sci. 2013, 128, 1193. 10. Kumirska, J.; Czerwicka, M.; Kaczynski, Z.; Bychowska, A.; Brzozowski, K.; Thoming, J.; Stepnowski, P. Mar. Drugs 2010, 8, 1567.

21. Singh, K.; Bharose, R.; Singh, V. K.; Verma, S. K. Ind. Eng. Chem. Res. 2011, 50, 10074. 22. Ding, Y.; Gu, G.; Xia, X. H.; Huo, Q. J. Mater. Chem. 2009, 19, 795.

26. Zhang, K.; Helm, J.; Peschel, D.; Gruner, M.; Groth, T.; Fischer, S. Polymer 2010, 51, 4698. 27. Nishimura, S.; Kai, H.; Shinada, K.; Yoshida, T.; Tokura, S.; Kurita, K. Carbohydr. Res. 1998, 306, 427.

11. Queiroz, M. F.; Melo, K. R. T.; Sabry, D. A.; Sassaki, G. L.; Rocha, H. A. O. Mar. Drugs 2015, 13, 141.

28. Ruocco, N.; Costantini, S.; Guariniello, S.; Costantini, M. Molecules 2016, 21, 551.

12. Jennings, C. D.; Boleyn, K.; Bridges, S. R.; Wood, P. J.; Anderson, J. W. Proc. Soc. Exp. Biol. Med. 1988, 189, 13.

29. Jain, A.; Gulbake, A.; Shilpi, S.; Jain, S. K. Crit. Rev. Ther. Drug Carrier Syst. 2013, 30, 91.

46000 (14 of 14)
