pH Variation as a Simple and Selective Pathway for Obtaining

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

pH Variation as a Simple and Selective Pathway for Obtaining Nanoparticle or Nanocapsule Polysaccharides Chutamart Pitakchatwong, and Suwabun Chirachanchai Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03443 • Publication Date (Web): 03 Dec 2018 Downloaded from http://pubs.acs.org on December 5, 2018

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pH Variation as a Simple and Selective Pathway for Obtaining Nanoparticle or Nanocapsule Polysaccharides Chutamart Pitakchatwong,†, ‡ Suwabun Chirachanchai*,†,‡ †The

Petroleum and Petrochemical College, Chulalongkorn University, Bangkok 10330,

Thailand ‡Center

of Excellence on Petrochemical and Materials Technology, Chulalongkorn University,

Bangkok 10330, Thailand *E-mail:

[email protected]

KEYWORDS: Polysaccharide, chitosan, alginate, pH-tunable morphology, emulsion system

ABSTRACT: The fabrication of polysaccharides to be nanoparticles or nanocapsules is quite specific due to various parameters and factors. The present work demonstrates a simple pathway to selectively prepare the ionic polysaccharide flakes to be nanoparticles or nanocapsules. The systematic studies on the model cases of cationic polysaccharide (i.e. chitosan) and anionic polysaccharide (i.e. alginate) confirm that pKa is the key point to tune the polysaccharides to be nanoparticles or nanocapsules. When the ionic polysaccharides were in an oil/water emulsion system, the pH close to pKa leads to the densely packed polysaccharide chains under the hydrogen bond networks, and as a result the crosslink occurs all through the chains to be nanoparticles. On the other hand, when pH was adjusted to the lower or higher than pKa depending on the types of ionic polysaccharide, the polysaccharide chains are under charge-charge repulsive force, resulting in the alignment of polysaccharide chains to be hollow nanospheres, and at that time the

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crosslink initiates the formation of nanocapsules. The present work, for the first time, clarifies that pH variation is the key to selectively prepare nanoparticles or nanocapsules, and this is important for delivery systems, coatings, sensors, etc. Polymeric nanospheres either nanoparticles or nanocapsules have been variously proposed as potential materials for advanced applications such as controlled release system, coatings, sensors, and more.1-2 Basically, when polymer chains contain hydrophobic and hydrophilic pendants, the self-assembly to reduce surface tension leads to the nanoparticles.3 In this case, the polymer chains have to be functionalized with hydrophobic and hydrophilic groups so that the differences in hydrophobicity and hydrophilicity initiate the nanospherical morphologies, socalled core-corona nanoparticles.4-5 Block copolymers also provide good models to show how the differences in hydrophobicity or charges lead to the development of nanospheres.6 In fact, when those block copolymers demonstrate responsive functions, external stimuli can be the key factor in selectively preparing nanoparticles or nanocapsules.7-8 The use of an oil/water (o/w) emulsion system in combination with crosslinkers is also reported to the development of nanospheres.9-10 In this case, the emulsion system is important as it controls the assembly of polymer chains before crosslinking in the later step. For example, when the polymer chains are soluble in water or oil and form layers on the surface of water or oil droplets, the crosslink brings in the nanocapsules; whereas, if the polymer chains are phase separated, the crosslink among the chains results in nanoparticles. Currently, polymeric nanocapsules are accepted as the ideal morphology for delivery systems because of their high surface area, with the role of polymeric surface either to protect the incorporated species or to fine-tune the release mechanism.11-13 Approaches to obtain polymeric nanocapsules include molecular self-assembly,14-15 sacrificial core-template,16-17 the emulsion

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technique,18-19 and others. It should be noted that there are some difficulties involved with the preparations. For example, the pathway via self-assembly needs specific conditions in order to control the size, porosity, and thickness of the shell.18,20 On the other hand, the process via sacrificial core-templates has to be carried out using a multi-step process, which is time consuming, and includes a harsh treatment to remove the solid core.21-23 For polysaccharides, various preparation techniques for nanoparticles have been reported.24-26 In the past, our group showed that the conjugation of chitosan (CS) with polyethylene glycol hydrophilic chains and a phthalimido group induced chitosan nanoparticles. The conjugation can be done in water due to the fact that chitosan forms a complex with conjugating additives (i.e. N-hydroxylsuccinimide (NHS) and hydroxybenzyltriazole (HOBt)), and at that time the conjugating reaction with water soluble conjugating agents effectively develops chitosan chains conjugated with the functional groups. In this case, the conditions generally favor the development of nanoparticles rather than nanocapsules.27 The question is how to prepare the polysaccharides in the form of nanocapsules. In fact, in utilizing the pathway of o/w emulsion, the key factors are: (i) the stability of the polymer at the water-oil interface, (ii) the addition of a cross-linking agent and (iii) the removal of the oil core. Therefore, it is expected that the key factor in selectively obtaining nanoparticles or nanocapsules is to control polysaccharide chains either in the form of the dense aggregation, or in the form of the thin layer at oil/water interface before crosslinking step. As the ionic polysaccharides, e.g. chitosan (CS) and alginate (AG), are soluble in aqueous by protonating or deprotonating the amino and carboxylic acid functional groups to eliminate the hydrogen bond, they are considered to be pH responsive polymers. Because of this, varying pH is expected to be a simple, effective, and efficient way to selectively achieve the nanoparticles or nanocapsules.


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The present work, therefore, demonstrates the selective nanosphere formation via emulsion process based on model cases of ionic polysaccharides, i.e. CS and AG. In addition, as polysaccharides are accepted for their bio-related properties, the method chosen for selectively preparing nanoparticles and/or nanocapsules is also meaningful for the biomedical applications, surface coatings, sensors, etc.28-29

EXPERIMENTAL SECTION Materials. The CS (90% DD, Mw of 7.0 x 105) was supplied by Seafresh Chitosan (Lab) Co., Thailand. Alginate was purchased from Carlo Erba reagent, France. Hydrochloric acid (HCl, 37%), toluene, hexane, and sodium hydroxide (NaOH, 99%) were bought from RCI Labscan, Thailand. 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and dicyclohexylcarbodiimide (DCC) were purchased from TCI, Japan. N-hydroxysuccinimide (NHS) was purchased from Merck, Germany. Acetic acid was purchased from Univar, Australia. Glutaraldehyde (50% aqueous solution) was supplied from Acros organics, Belgium. Polyethylene glycol sorbitan monolaurate (Tween 80) was purchased from Sigma Aldrich, USA. Dialysis was performed using a cellulose membrane with a molecular weight cutoff of 12,000 g/mol. All chemicals were analytical grade and used without any purification. Purification of CS. CS (1% wt) was purified by dissolution in acetic acid (1% v/v). After that the solution was centrifuged for 20 min at 12,000 rpm and the supernatant was collected, following by filtration through cellulose membrane (Millipore sigma syringe filter with mixed cellulose esters membrane, pore size 0.45µm). The product was dialyzed until pH 7 and lyophilized to obtain pure CS.


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Preparation of Glutaraldehyde Saturated Toluene (GA). GA was prepared according to the method reported elsewhere.30 Briefly, glutaraldehyde (4 mL) and toluene (4 mL) was mixed in the round bottom and stirred for 1 h and was kept at 4 °C for 24 h. The upper part was GA. Estimation of the GA content in toluene was carried out by 1H-NMR spectroscopy. The upper part was dried under vacuum to remove toluene, followed by adding deuterium oxide (D2O). 1,4-Dioxane (10 µL) was used as external standard for quantitative measurement of GA concentrations which was 6.8 %wt in toluene (3.41x10-4 mol/mL). Nanocapsules Formation. The preparation of chitosan nanostructures was based on the crosslink among CS chain with GA at the interface of emulsion which was prepared from oil in water miniemulsion. CS (1% wt, 0.05 g) was dissolved in acetic acid (difference pH 2, 3, 4, 5 and 6, 5 mL), following by adding tween 80 (19 mg). Toluene (0.5 g) containing GA (30 µL, 3 % molar ratio of amino and hydroxyl groups of CS) and hexadecane (0.013 g) was mixed with CS aqueous solution. Emulsification was performed by using a BRANSON Sonifier Generator Model 450-D, Inc., (1/2 in. probe, pulse 1 s, pause 1 s, 80% amplitude) for 4 min in ice bath. The capsule formation with crosslinking agent was conducted at room temperature for 24 h. After that, the product was purified by removing toluene, following by dialysis (Spectra/Por/Biotech dialysis membrane, regenerated cellulose (RC) membrane tubing, MWCO 8-10 kDa, Flat width 16 mm, diameter 10 mm) to remove excess amount of GA and tween 80. The products were purified by washing them several times with acetic acid (1% v/v). After washing, CS was converted to nanospheres (the term includes both nanocapsules and nanoparticles) about 55±5%. In addition, CS nanospheres with an increase in size were synthesized by changing surfactant from tween 80


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to sodium dodecyl sulfate (SDS, 2mg/mL). To differentiate the morphology, the products were stained with fluorescein isothiocyanate (FITC) dye and analyzed by TEM and confocal microscopy. AG nanospheres were prepared in similar, except the pH variation was changed to 4, 5, 7, and 9. The products were purified by evaporating toluene, dialyzing and adjusting pH to 9 to remove pristine AG. GA in Oil/Water (o/w) Emulsion Evaluated by NMR Technique. Five different pHs of D2O (2 mL) were adjusted to 2, 3, 4, 5, and 6 using deuterium hydrochloride solution (DCl) and sodium deuteroxide solution (NaOD), and mixed with toluene-d8 (0.1 mL) containing GA 10 µL. A sonication probe (BRANSON Sonifier Generator Model 450-D, Inc., 1/2 in. probe, pulse 1 s, pause 1 s) was placed at the interface between D2O/toluened8 phase, after sonication, the solution was left for 24 hour. The samples were centrifuged to separate two phases (D2O and toluene-d8) which the upper part was toluene-d8 after that this part was characterized by NMR technique. Preparation of CS Nanosphere Formation in o/w Emulsion with EDC/NHS as Crosslinking Agent. A CS toluene/water emulsion (1% wt toluene, 10% wt water) was prepared by mixing two phases of toluene and water phase consisting of tween 80 (3 mM), succinic acid (0.74% wt), EDC, and NHS as cross-linking agent in water phase at pH 3. The mixture was emulsified by ultra-sonication for 24 hours. To study the effect of phase of cross-linking agent, the emulsion was purified by removing toluene and followed by dialysis (Spectra/Por/Biotech dialysis membrane, regenerated cellulose (RC) membrane tubing, MWCO 8-10 kDa, flat width 16 mm, diameter 10 mm). The unreacted CS was removed by acetic acid (1% v/v). The morphology of the sample was observed by TEM.


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To investigate the effect of crosslinker, CS toluene/water emulsion was prepared similarly but using dodecanedioic acid and DCC conjugating agent in toluene phase. Quantitative Analysis of Free Amino Groups of CS Nanospheres. A simple method for quantitative analysis of free amino groups of chitosan based on the ninhydrin reaction was carried out as follows. Ninhydrin (2 g) was dissolved in dimethylsulfoxide (DMSO, 75 mL) followed by adding acetate buffer (4 M, 25 mL) to adjust pH to 5.2. The reagent was stored in a dark bottle before using. The CS nanosphere solution (solid content 5 mg/mL, 500 µL) was mixed with ninhydrin reagent (375 µL) and heated in the oven at 80 C for 30 min. The solution was cooled and mixed with ethanol (50 %v/v, 500 µL). The solution was observed by UV/VIS spectrophotometer. The glycine solution was used as a standard. Characterization. The FTIR spectra were recorded using a Bruker ALPHA Fourier transform infrared spectrophotometer (ATR), and 1H NMR spectra were obtained from an Ultrashield 500 Plus Bruker spectrometer (500 MHz). Chemical shifts are reported in delta (δ) units, expressed in parts per million (ppm) downfield from tetramethylsilane (D2O, 1H: 4.79 ppm, Toluene d8, 1H: 2.08, 6.97, 7.01 ppm). The hydrodynamic diameter, size distribution, and zeta potential of polymeric nanospheres were measured using a Malvern Zetasizer Nano ZS. All samples had a final concentration of 0.5 mg/mL. Data from three individual measurements were recorded with at least 100 runs at room temperature. The average hydrodynamic diameter was determined using the Stokes−Einstein equation. The morphologies of all nanospheres were observed by an H7650 Hitachi transmission electron microscope at an acceleration voltage of 100 kV. The samples for TEM measurement were prepared by placing a drop of the sample solution on the


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surface of Formvar carbon film-coated copper grids. The morphologies of polymeric nanostructures were characterized with a Park Systems atomic force microscope (AFM, Park XE-100). The surface morphologies of samples were acquired in non-contact mode. The samples were prepared by dropping the colloid solution onto glass slide and drying at room temperature for 48 h before measurements. To confirm the morphologies of CS nanostructures, the FITClabeled CS nanostructures with different pH (pH 3 and pH 5) were studied by using the confocal laser scanning microscope (CLSM) (ZEISS LSM 800, Carl Zeiss Microscopy). The UV/Vis spectra were recorded with an Agilent Technologies Cary 5000 UV/Vis NIR spectrometer. The pH values were monitored using a FiveEasy Plus, Mettler Toledo pH meter.

RESULTS AND DISCUSSION CS nanosphere formation. Scheme 1 demonstrates the preparation steps of CS in an o/w emulsion system. The CS (1 w/w %) was mixed with surfactant, i.e. tween 80 (3 mM), in water (continuous phase of CS) and toluene phase (dispersed phase for CS) containing glutaraldehyde (GA, 0.74 wt %), the crosslink in the final step results in the development of nanospheres. Scheme 1. Schematic for the steps of preparation of CS nanosphere in o/w emulsion system.


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To ensure the stability of the droplets in the emulsion process for the generation of nanostructures, the optimized processing conditions and parameters were investigated (see Supporting information, Figure S1-S3). As CS has its pKa at 6.4,31 the CS solutions were prepared in the range of pH 2-6 so that CS is completely soluble in an aqueous system. At this step, the o/w emulsion systems containing CS solutions were crosslinked and the products were observed using TEM. It is clear that in the case of pH 6 (Figure 1 (a)), the sizes of the products are about 30-110 nm (Figure 1 (b)). For pH 5 (Figure 1 (c)), the particle size is about 50 nm (Figure 1 (d)). In fact, the products from both conditions are tightly packed in the form of nanoparticles, as suggested by TEM images. The pH effect was studied. The CS nanoparticles are as small as 40 nm when the pH was at 4 (Figure 1 (e), (f)). At this condition, the nanoparticles are less aggregated. When the pH was as low as 2 or 3, the TEM images of the products clearly show nanocapsules morphology (Figure 1 (g), (i)), with an average size of only about 35 nm. In fact, as both nanoparticles and nanocapsules are in the nanosphere morphology, the SEM images can clearly confirm the appearances (Figure S4).


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Figure 1. Transmission electron micrographs including the schematic of CS nanoparticles and nanocapsules and the size distribution at various pHs; pH 6 (a and b), pH 5 (c and d), pH 4 (e and f), pH 3 (g and h), and pH 2 (i and j).


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CS nanospheres with an increase in sizes were synthesized to clarify the differences between nanocapsules and nanoparticles (pHs 3 and 5, respectively) by changing the surfactants from tween 80 to sodium dodecyl sulphate (SDS, 2mg/mL). The products were stained with fluorescein isothiocyanate (FITC) dye based on the reaction between isothiocyanate groups of FITC and amino groups of CS. As shown in Figure 2A, at pH 3, the CS nanocapsules were obtained as confirmed by the clear contrast between the inner part and the edges (Figure 2A (a)). The collapse of nanocapsules at the edge during the drying process for TEM was also observed. In contrast, at pH 5, CS nanoparticles were achieved as seen from the solid color of the nanoparticles and their shape are retained even in the dried state (Figure 2A (b)). In addition, AFM was also used to confirm the differences between nanocapsules and nanoparticles. The AFM image clearly shows the nanocapsule morphology at pH 3 with the hollow core in threedimensional phase mode. The result obtained is in the good agreement with that revealed by TEM (Figure 2A (b)). The condition at pH 5 leads to the CS nanoparticles (Figure 2B (b)). Moreover, as the CS nanospheres were labeled with FITC, confocal laser scanning microscope (CLSM) was applied to examine the different morphologies. The micrograph in Figure 2A (c) clearly shows the hollow core of CS nanocapsule while the high fluorescence intensity strongly appeared at the edge of nanocapsules at pH 3. When pH was 5, the fluorescence intensity was observed in the form of solid nanoparticles without showing any contrast between core and shell. The abovementioned results strongly support the different morphologies of CS nanospheres by simply varying the pH.


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Figure 2. CS nanostructures prepared by using SDS as surfactant, (A) CS nanocapsules at pH 3, and (B) CS nanoparticles at pH 5 for (a) TEM images, (b) AFM images in phase mode, (c) Confocal microscopy images.

Characterization of crosslinking of CS nanospheres. A question arises about how changing the pH leads to different nanostructures. Figure 3A shows a curve fitting FTIR spectrum of the product obtained from pH 2. The absorption bands at 890-1160 cm-1 show the characteristic peak of a pyranose ring of CS. There are new peaks at 1614 cm-1 and 1063 cm-1, which correspond to C=N (Schiff base linkage) and C-O-C (hemi-acetal bond), respectively.32-34 Here, the crosslink related to the pH was quantitatively analyzed by taking the ratio between absorption bands of Schiff base at 1614 cm-1 and hemi-acetal bond at 1063 cm-1, regarding the internal standard at 1152 cm-1 (pyranose ring) (Figure 3B). It is clear that the ratio was the least at pH 2 and increased 12 ACS Paragon Plus Environment

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with increasing the pH. This might be due to at low pH, the amino groups were protonated to obstruct the Schiff base formation. It is important to note that the ratio between absorption bands of hemi-acetal bond at 1063 cm-1 and CS at 1152 cm-1 were similar at all pHs. This reflects that pH variations hardly affect the hemi-acetal formation. However, to further confirm the above discussion, a general method, i.e. ninhydrin assay, to quantify the free primary amino groups of CS was carried out. The CS nanoparticles and nanocapsules obtained from various pH were dispersed in Ninhydrin reagent in pH 5.2. It was found that the solution developed the blue color depending on the number of free amino groups. The plots of glycine at various concentrations were used as reference standard (Figure S5 (a)). The amount of free amino groups was the highest at pHs 2 and 3 and became less when the pH was increased to 4-6 (Figure S5 (b)). In other words, the pH plays an important role to form nanoparticles or nanocapsules under the crosslink network with GA.


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Figure 3. (A) FTIR spectra of (a) CS nanocapsules at pH 2, (b) pristine CS, and (B) semiquantitative FTIR of CS nanoparticles/nanocapsules at various pHs; (●) the integral ratio of 1614/1152, and (○) the integral ratio of 1063/1152. 14 ACS Paragon Plus Environment

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The question is how pH plays the role to form nanoparticles or nanocapsules. It is known that when pH is below pKa, CS is positively charged, and the chains are repulsive to each other, leading to a decrease in packing structures. At that time, the crosslink might be formed along the chains, which were under the electrostatic repulsion of CS chain, resulting in the development of nanocapsules. The repulsive effect was further confirmed by the charge density based on the zeta-potential (Figure 4). At pH 2-3, the amino groups were completely protonated and at this time the zeta-potential is as high as 60 mV. When the pH was further increased to 4, 5 and 6, the zeta-potential is decreased to 30 mV, 24 mV and 14 mV, respectively. As shown in Figure 4, tween 80, and toluene/water emulsion containing tween 80 maintain their zeta-potential even when the pH was varied. However, toluene/water emulsion containing tween 80 and CS shows the changes in electrostatic charges depending on the pH. This means that the zeta-potential of the emulsion is directly related to the electrostatic charges of CS.

Figure 4. Zeta potential at various pHs of (●) CS, (○) tween 80, (▲) toluene/water emulsion containing tween 80, and (∆) toluene/water emulsion containing tween 80 and CS. Data presented as mean (± SD) were obtained from three independent experiments.


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Investigation of the effect of types of cross-linking agent and phase of the crosslink on the morphology of CS nanosphere. It is clear that the surface charge of CS is the key parameter that controls the morphology of CS nanospheres to be either nanoparticles or nanocapsules. It comes to the point how we confirm the pH effect on nanoparticles or nanocapsules formation. In order to clarify this, the experiments were done as follows. As the first condition, CS in pH 2-3 was prepared in toluene/water emulsion system. The dicarboxylic acid (succinic acid), EDC, NHS crosslink system were used. At that time, the nanocapsules were obtained (Figure 5 (a)). This implies that the crosslink at the interface of toluene/water, where CS layers were existed, was effective (Scheme 2A (a)). The second condition of using GA as the crosslinker was also studied. It is known that GA is also a pHdependent molecule35 and prefers to be in toluene phase rather than in water at pH 2-3 (Figure S6). Therefore, CS was in the interface (pH 2-3) while the GA crosslinker was in toluene phase, only nanocapsules were obtained. This reflects that CS chains were not in the toluene phase but rather than at the interface (Scheme 2A (b)).

Figure 5. TEM micrographs of CS emulsion at pH 3; after crosslink by (a) succinic acid, and (b) dodecanedioic acid.


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When pH was increased to as high as pH 5, the CS tends to pack with each other by the inter- and intra-molecular hydrogen bond36 and at the same time most of GA molecules can be transferred to the water phase (Figure S6). This brought the crosslink of CS in water phase to be nanoparticles with only few amounts of GA (Scheme 2B). Another condition by using dodecanedioic acid, which prefers to be in toluene was considered. It is expected that this condition will bring CS nanocapsules when pH as low as two as shown in Figure 5 (b) (Scheme 2A (c)). As a result, different types of cross-linking agents did not exhibit any significant effect on CS nanosphere formation. This emphasizes that the main factor in fine-tuning the nanoparticles and/or nanocapsules is the pH of the aqueous solution of CS, rather than type or phase of the crosslinker. Scheme 2. Proposed mechanisms for the formation of CS nanospheres through o/w emulsion with different pH levels (A) pH 2-3, and (B) pH 4-6 and different crosslinker (a) dicarboxylic acid, (b) GA, and (c) dodecanedioic acid.

Selective Nanospheres formation for Anionic polysaccharide. If pH is the key factor in controlling CS morphology, it is natural to see similar tunable morphology for other ionic polysaccharides, as illustrated in Scheme 2. Here, the anionic polysaccharide, alginate (AG), was studied. It should be noted that the pKa of AG is around 4 which means that


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when pH is higher than 4, carboxyl groups of AG are deprotonated, giving negative charges. As seen in the case of CS, AG shows different morphologies depending on pHs (Figure 6). At pH 4, the nanoparticles are confirmed. When pH was increased from 5 to 9, the nanocapsules were mainly observed. The results confirmed that pH is the factor to finetune the nanosphere of pH-responsive polysaccharides (e.g. CS, and AG) in o/w emulsion.

Figure 6. TEM micrographs of AG at; (a) pH 4, (b) pH 5, (c) pH 7, and (d) pH 9.

CONCLUSIONS The present work demonstrates an approach to fine tune the morphology of pHresponsive polysaccharides through model cases of cationic chitosan and anionic alginate. In an oil/water emulsion system, by setting the chitosan in the pH below its pKa, the electrostatic repulsion caused the chitosan to align on the interphase of toluene and aqueous phases. The crosslink in the next step led to the formation of nanocapsules. A similar result was observed in the case of alginate when the pH was above its pKa. The


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as-desired polysaccharide morphologies at a nano-scale level under the simple pH variations are meaningful for developing nanocarriers for versatile applications. ASSOCIATED CONTENT Supporting information Materials, synthesis, and characterization of optimum condition of CS nanostructure formation. This material is available free of charge via the internet at http://pubs/acs/org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS The authors would like to express their appreciation for the useful discussion and suggestions from Professor Katharina Landfester, and Associate Professor Daniel Crespy, Max Plank Institute for Polymer Research, Germany. Appreciation is also expressed for the Royal Golden Jubilee, Thailand Research Fund (PHD/0124/2557) for the research funds. This work was fully supported by PTT Public Company Limited under the PTT NSTDA Chair Professor Scholarship No. FDA-CO-2558-1309-TH Green and Sustainable Polymers: A Challenge for Renewable Resource-Rich Thailand. The author would like to thank the Center of Excellence on Petrochemical and Materials Technology, Chulalongkorn University for the additional support.


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“for Table of Contents use only”


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Scheme 1. Schematic for the steps of preparation of CS nanosphere in o/w emulsion system. 276x84mm (300 x 300 DPI)


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Figure 1. Transmission electron micrographs including the schematic of CS nanoparticles and nanocapsules and the size distribution at various pHs; pH 6 (a and b), pH 5 (c and d), pH 4 (e and f), pH 3 (g and h), and pH 2 (i and j). 210x442mm (300 x 300 DPI)


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Figure 2. CS nanostructures prepared by using SDS as surfactant, (A) CS nanocapsules at pH 3, and (B) CS nanoparticles at pH 5 for (a) TEM images, (b) AFM images in phase mode, (c) Confocal microscopy images. 264x165mm (300 x 300 DPI)


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Figure 3. (A) FTIR spectra of (a) CS nanocapsules at pH 2, (b) pristine CS, and (B) semi-quantitative FTIR of CS nanoparticles/nanocapsules at various pHs; (●) the integral ratio of 1614/1152, and (○) the integral ratio of 1063/1152. 322x832mm (300 x 300 DPI)


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Figure 4. Zeta potential at various pHs of (●) CS, (○) tween 80, (▲) toluene/water emulsion containing tween 80, and (∆) toluene/water emulsion containing tween 80 and CS. Data presented as mean (± SD) were obtained from three independent experiments. 205x124mm (300 x 300 DPI)


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Figure 5. TEM micrographs of CS emulsion at pH 3; after crosslink by (a) succinic acid, and (b) dodecanedioic acid. 235x125mm (300 x 300 DPI)


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Scheme 2. Proposed mechanisms for the formation of CS nanospheres through o/w emulsion with different pH levels (A) pH 2-3, and (B) pH 4-6 and different crosslinker (a) dicarboxylic acid, (b) GA, and (c) dodecanedioic acid.


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Figure 6. TEM micrographs of AG at; (a) pH 4, (b) pH 5, (c) pH 7, and (d) pH 9. 177x193mm (300 x 300 DPI)


pH Variation as a Simple and Selective Pathway for Obtaining

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