Degradation of chitosan hydrogel dispersed in dilute carboxylic acids by solution plasma and evaluation of anticancer activity of degraded products

Chitosan was obtained from the deacetylation of chitin, which was prepared from the shells of Metapenaeus dobsoni shrimp, provided by Surapon Foods Public. Glacial acetic acid (CH₃COOH, 99.9%), hydrochloric acid (HCl, 37.0%), sodium chloride (NaCl, 99.0%), and sodium hydroxide (NaOH, 97.0%) pellets were purchased from RCI Labscan. A 50% (w/v) NaOH solution was supplied by the Chemical Enterprise. Oxalic acid dihydrate [(COOH)₂ 2H₂O, 98.0%] and succinic acid [C₂H₄(COOH)₂, 99.5%] were purchased from Ajax Finechem. Malonic acid [CH₂(COOH)₂, 99.0%], glutaric acid [C₃H₆(COOH)₂, 99.0%], adipic acid [C₄H₈(COOH)₂, 99.0%], pimelic acid [C₅H₁₀(COOH)₂, 98.0%], and azelaic acid [C₇H₁₄(COOH)₂, 98.0%] were supplied by Sigma-Aldrich. Citric acid [C₃H₅O(COOH)₃, 99.5–102.0%] was purchased from Loba Chemie. Sodium borohydride (NaBH₄) and potassium bromide (KBr) were obtained from Carlo Erba Reagents and Wako, respectively.

M. dobsoni shrimp shells were washed and dried under sunlight before grinding into small flakes. The flakes of dried shrimp shell were demineralized by soaking in a 1 M HCl solution for 24 h. The demineralization step to remove calcium carbonate in shrimp shells was repeated by adding a fresh 1 M HCl solution and soaking for another 24 h before the flakes were removed and washed with distilled water until neutral. Subsequently, the demineralized shrimp shell flakes were deproteinized by immersion in a 4% (w/v) NaOH solution at 80 °C with vigorous stirring for 4 h. The flakes were filtered and washed with distilled water until the eluent reached a pH of 7.0. Then, the deproteinized product, known as chitin, was obtained. A 50% (w/v) NaOH solution containing 0.5% (w/v) NaBH₄ was added to the chitin flakes before the suspension was heated in an autoclave at 110 °C for 75 min. After filtration, the solid fraction from the deacetylation process was washed with distilled water until the pH became neutral. The deacetylation process was repeated twice to achieve the required degree of deacetylation (%DD) of chitosan (i.e., 90%), calculated by following an equation of Sabnis and Block.⁴⁹⁾ The solid-to-liquid ratio used in all processes was $1:10$. After each process, water in the product was removed by drying in an oven at 60 °C before continuing further steps or use.

Chitosan solution was prepared by dissolving chitosan flakes (2 g) into 200 ml of 2% (v/v) acetic acid solution. The obtained chitosan solution was then poured dropwise into 400 ml of 5% (w/v) NaOH solution with vigorous stirring.⁴⁸⁾ The gel-like precipitate was dialyzed against distilled water in a dialysis tube with a molecular weight cutoff of 12,132 Da (Sigma-Aldrich) until neutral to attain chitosan hydrogel.

A suspension of chitosan hydrogel for the degradation by the SP treatment was prepared by adding 6 g of chitosan hydrogel (having dry weight ∼0.1 g) to the SP reactor and then 50 ml of dilute acid solution was added to the suspension with an acid-to-chitosan mole ratio of $1:8$ or equivalent to 1.55 mM. The suspension was stirred at room temperature (30 °C) for 5 min. The SP reactor was made of a glass beaker having two tungsten electrodes (purity 99.9%, Nilaco) with a diameter of 1 mm inserted in it. The SP system used in this study has been described in previous works^40,42,46) and is schematically shown in Fig. 2. The operation parameters of the typical voltage, pulse frequency, pulse width, and electrode distance were 1.6 kV, 23 kHz, 2 µs, and 0.75 mm, respectively. The reaction temperature during the plasma operation was approximately 60–70 °C.

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After the degradation of chitosan hydrogel by the SP treatment, the suspension was centrifuged to collect the remaining solid residue assigned as the solid fraction, which was water-insoluble chitosan that was then dried in an oven at 60 °C. The supernatant was further freeze-dried and assigned as the solution fraction, which should contain water-soluble COS. All fractions were weighed and the percentage yield of each fraction was calculated using the following equation:

$$\begin{equation} \text{Yield}\ (\%) = \frac{W_{i}}{W_{0}}\times 100, \end{equation} \tag{ 1 }$$

where W₀ is the initial dry weight of chitosan in the system and W_i is the dry weight of the degraded chitosan fraction, while i refers to either the solid fraction or solution fraction.

The crystallinity of native chitosan and chitosan hydrogel before and after the SP treatment was characterized by using a wide-angle X-ray diffraction (WAXD) analyzer (Rigaku SmartLab) operated in a continuous mode with a scan speed of 5° min⁻¹ and a scattering angle (2θ) from 5 to 60° using Cu Kα as an X-ray source.

The morphology of the chitosan flakes and dried chitosan hydrogel was examined by using field-emission scanning electron microscopy (FE-SEM; JEOL JSM-7610F).

The changes in the average molecular weight and the molecular weight distribution of chitosan during the degradation via the SP treatment were determined using gel permeation chromatography (GPC; Shimadzu CTO-10A) apparatus equipped with a refractive index (RI) detector. An ultrahydrogel linear column (Waters 600E) for separation of the molecular weight in the range of 1.0 × 10³ to 2.0 × 10⁷ Da was connected in series with a guard column equipped in an oven at the operating temperature of 40 °C. The mobile phase was a 0.5 M acetate buffer solution at pH 4.0 and the flow rate was set at 0.6 ml·min⁻¹. The injection volume of a sample was 40 µl, comprising 3 mg·ml⁻¹ chitosan in the acetate buffer solution. Pullulans with molecular weight in the range of 2.17 × 10³ to 8.05 × 10⁵ Da were used as standard samples.

The investigation of any changes in the chemical structures of chitosan after the degradation via the SP treatment was carried out by using Fourier transform infrared (FT-IR) spectrometry (Thermo Fisher Scientific Nicolet iS5). FT-IR spectra with wavenumbers from 4000 to 400 cm⁻¹ were taken on KBr pellets at a resolution of 4 cm⁻¹ using 64 scans with correction for atmospheric carbon dioxide (CO₂).

2.6.1. Cell culture.

2.6.2. Cytotoxicity assay.

A test on the in vitro cytotoxicity of the chitosan sample was performed by MTT assay against both cancer and normal cells. Firstly, cells were seeded into 96-well microliter plates (10⁴ cells/well) and incubated in a 5% CO₂ humidified incubator at 37 °C for 24 h. After that, different concentrations of chitosan samples, diluted with sterile water, were added into the wells to make the desired final concentration (0.1 to 5 mg·ml⁻¹) and further incubated in an incubator at 37 °C and 5% CO₂. After 24 h incubation, the medium was discarded and replaced with phosphate buffer saline solution (PBS) containing 0.05% (w/v) of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution with further incubation for 3 h. Then, the solution was discarded and DMSO was added to dissolve the formed formazan crystals and the absorbance (A) of each well was measured at 570 nm using a microplate reader (Thermo Fisher Scientific Varioskan™ Flash Multimode Reader). The cell viability was calculated using the following equation:

$$\begin{equation} \text{Cell viability}\ (\%) = 100 \times \left|\frac{A_{\text{treated}} - A_{\text{blank}}}{A_{\text{untreated}} - A_{\text{blank}}}\right|. \end{equation} \tag{ 2 }$$

The percentage cell viability was plotted versus the concentration of the COS sample to determine the mean growth-inhibitory concentration (IC₅₀) value. All experiments were repeated five times. Doxorubicin was used as a positive control.

A comparison of the morphology of native chitosan flakes and freeze-dried chitosan hydrogel was performed by using FE-SEM, and the FE-SEM images are shown in Fig. 3(a). The FE-SEM image revealed that the chitosan flakes were composed of well-packed multilayered sheets. After dissolving chitosan flakes in the acetic acid solution, followed by reprecipitation in the NaOH solution to form chitosan hydrogel, intra- and intermolecular hydrogen bonding interactions within the structure of native chitosan were disrupted. The dissolution and regeneration of chitosan hydrogel resulted in the loss of the former well-packed multilayered structure, leading to a more amorphous region in chitosan hydrogel compared with native chitosan.⁵⁰⁾

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Moreover, the change in the crystalline structure of native chitosan and chitosan hydrogel was examined by WAXD analysis, as shown in Fig. 3(b). The WAXD spectrum of native chitosan showed that the main characteristic peaks of native chitosan appeared at 2θ = 9.8 and 19.9°. As reported in the previous works,^51,52) the reflection of 2θ around 9–10.5° indicated that chitosan had a hydrate polymorph. In the hydrate tendon crystal, the chain conformation appeared as a 2-fold helix stabilized by a hydrogen bond (O3•••O5) with the orientation of O6. Hydrogen bonds also occurred between adjacent chains (via N2•••O6) to form a sheet structure. These sheets stacked together in a parallel fashion and could be stabilized by water molecules that presented inside the chitosan structure.⁵²⁾ Upon the transformation of native chitosan to hydrogel, the crystalline structure in native chitosan was changed. The WAXD result of chitosan hydrogel exhibited clearly broader peaks at 2θ = 9.8°. The characteristic peak at 2θ = 19.9° disappeared, while two new broad peaks at 2θ = 28.1 and 35.0° could be observed. The WAXD result suggested that the crystalline structure of native chitosan was destroyed. The peaks of 2θ at 28.1 and 35.0° indicated the formation of chitosan acetate salt as reported by Modrzejewska et al.⁵³⁾ According to this evidence, the chitosan hydrogel had lower crystallinity than native chitosan and should be more susceptible to degradation by the SP treatment.

The degradation of chitosan hydrogel dispersed in the dilute acetic acid by applying the SP treatment and the conventional heat treatment were conducted under the same condition. The chitosan hydrogel was found to disperse well in the dilute acetic acid solutions. It was found that the weight-average molecular weight (M_w) of chitosan hydrogel after SP treatment dramatically decreased, while the slight reduction of M_w for chitosan hydrogel by the conventional heating treatment occurred, as shown in Table I. Furthermore, the degradation of chitosan hydrogel dispersed in the dilute acetic acid by the SP treatment was performed for comparison with the degradation of chitosan solution dissolved in 1 M acetic acid by SP treatment, which has been studied in some previous works.^29,39,40) It was found that the M_w values of both types of chitosan samples were greatly reduced after the SP treatment. From GPC measurement, the degradation of chitosan hydrogel dispersed in the dilute acetic acid by applying the SP treatment for 60 min could reduce M_w for chitosan by approximately 98%. On the other hand, M_w for chitosan in the chitosan solution was decreased by 95% under the same condition. Therefore, it might be concluded that chitosan hydrogel dispersed in the dilute acetic acid was readily degraded by the SP treatment. Moreover, it also indicated that the SP treatment is an effective tool for promoting the degradation of chitosan to produce COS, even though only a dilute organic acid solution was present in the reaction.

Figure 4 shows flow charts giving a comparison of the experimental procedures for the degradation of chitosan solution and chitosan hydrogel to produce COS by SP treatment employed in this study and other previous works.^19,29) At a high concentration of acetic acid (pH ≈ 4.0), chitosan can dissolve well because most NH₂ groups at the C2 position in the structure of chitosan are protonated by protons from the dissociation of acetic acid and become soluble under an acidic environment.³⁶⁾ However, at a very low concentration (pH ≈ 6.5) of acetic acid, only a small number of NH₂ groups in chitosan can be protonated due to the lack of protons and consequently, the chitosan hydrogel still remains as a suspension in the dilute acetic acid solution. Accordingly, the water-soluble degraded product of COS obtained from the SP treatment of the chitosan hydrogel dispersed in the dilute acetic acid could be easily separated from the remaining solid fractions by using simple centrifugation. The COS dissolving in the solution was collected as a supernatant, while the solid fraction that contained water-insoluble, high-molecular-weight chitosan was separated as a precipitate after centrifugation. On the other hand, the degraded product from the degradation of the chitosan solution in the presence of a high concentration of acetic acid by the SP treatment required neutralization by using NaOH in order to separate the water-insoluble, high-molecular-weight chitosan from the water-soluble, low-molecular-weight product that contained COS. Furthermore, the addition of a non-solvent such as ethanol or acetone was necessary for separation of the COS products from the solution as a precipitate. According to the flow charts in Fig. 4, the production of COS from chitosan hydrogel was simpler and less time-consuming than that obtained from chitosan solution.

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In addition to acetic acid, various types of other carboxylic acids were also used to disperse chitosan hydrogel in order to study their influence on the degradation of chitosan hydrogel by applying the SP treatment. The various types of other carboxylic acids including chemical structures, acid dissociation constants,⁵⁴⁾ number of carboxylic groups, and number of methylene groups on the backbone are shown in Table II. According to GPC analysis, the results on molecular weight reduction as a function of the SP treatment time are shown in Table III. M_w for the original chitosan hydrogel before the SP treatment was 4.8 × 10⁵ Da. M_w was found to decrease for all types of carboxylic acids with increasing SP treatment time. For the addition of oxalic acid, the dramatic reduction of M_w was observed and the scale of molecular weight reduction was comparable to that obtained from acetic acid. M_w for the degraded products obtained from the systems containing acetic acid and oxalic acid was approximately 6 × 10³ Da after 60 min of SP treatment. It was expected that the degraded products would contain COS having M_w less than 5 kDa³⁰⁾ as a major component. In the systems with the addition of malonic acid, succinic acid, glutaric acid, and pimelic acid, the SP treatment could reduce M_w for the chitosan hydrogel from 4.8 × 10⁵ Da to the range of 7.1 × 10³–1.3 × 10⁴ Da, while by the addition of azelaic acid and citric acid, M_w for the degraded chitosan was equal to approximately 2 × 10⁴ Da after the SP treatment for 60 min.

The rate constant of the degradation reaction of chitosan hydrogel was analyzed on the basis of the following linear relationship between inverse molecular weight (1/M_w) and reaction time (t) in previous reports:^29,55)

$$\begin{equation} \frac{1}{M_{t}} = \left(\frac{1}{M_{0}}\right) + \left(\frac{kt}{M}\right) = \left(\frac{1}{M_{0}}\right) + k't, \end{equation} \tag{ 3 }$$

where M_t is M_w for the sample at the reaction time (t), M₀ is the initial M_w for the sample, M is the molecular weight of the monomer, k (min⁻¹) or k' (mol g⁻¹ min⁻¹) is the rate constant, and t is the reaction time. The k values are shown in Table IV. The k value obtained by the system with the addition of oxalic acid was equal to 3.67 × 10⁻⁴ min⁻¹, which was slightly higher than that for the addition of acetic acid (k = 3.48 × 10⁻⁴ min⁻¹). Meanwhile, the addition of the other dicarboxylic acids led to lower k values than that obtained by the system with the addition of acetic acid. The obtained k values for the systems using dicarboxylic acids ranged from 1.36 × 10⁻⁴ to 3.67 × 10⁻⁴ min⁻¹, which seemed to vary, depending on the number of methylene groups on the backbone. For the tricarboxylic acid, citric acid, the k value was the lowest among all the studied carboxylic acids. Nevertheless, the k values obtained in this study from the degradation of chitosan hydrogel dispersed in dilute carboxylic acids by applying the SP plasma treatment were comparable to those obtained from the degradation of chitosan solution by using concentrated H₂O₂ or using various types of energy with the k values ranging from 10⁻⁴ to 10⁻² min⁻¹.^32,39,55,56)

For further investigation, the chemical structures of chitosan hydrogel before and after degradation by applying the SP treatment in the presence of various types of dilute carboxylic acids were examined by using FT-IR, and the FT-IR results are shown in Fig. 5. The absorption peaks of chitosan hydrogel before the SP treatment at the wavenumber of approximately 3450 cm⁻¹ were assigned to the N–H and hydrogen bonded O–H stretching vibration and the peaks at the wavenumbers of 1650 and 1600 cm⁻¹ were referred to the C=O stretching of the amide bond and the N–H bending vibrations of the secondary amide, respectively.^6,15,57,58) The peaks at around 1420 and 1375 cm⁻¹ were attributed to –CH₂– bending with the orientation of the primary hydroxyl group in the polysaccharides and methyl group.^59,60) The peak at 1070 cm⁻¹ was assigned to the stretching of C–O–C.⁶⁾ According to the FT-IR results, the overall chemical structures of the degraded chitosan hydrogel dispersed in various dilute carboxylic acids did not change after the SP treatment. For the degraded chitosan hydrogel obtained from the system with the addition of acetic acid, strong absorption at 1650 cm⁻¹ was observed. This might indicate the ionic interaction between the protonated amino groups of chitosan and the carboxylate anion (–COO⁻) of the carboxylic acid.⁶¹⁾ Normally, protons (H⁺) from the dissociation of acetic acid (CH₃COOH) can protonate amino groups (–NH₂) of chitosan to form ammonium groups (–NH₃⁺).⁵⁰⁾ Then, acetate anions (CH₃COO⁻), which have relatively small size and low steric hindrance, should be able to penetrate into the structure of chitosan and undergo ionic interaction with –NH₃⁺. After that, these interactions could lead to an electrostatic repulsion between chitosan chains.¹⁹⁾ Consequently, the structure of chitosan may freely expand and distribute in the solution, as demonstrated later in Fig. 7(a), leading to an increasing probability that radicals generated by plasma discharge could penetrate to the β-(1,4)-glycosidic linkages of chitosan and greatly promote the M_w reduction of chitosan hydrogel. For the addition of oxalic acid, the FT-IR spectra of the degraded product showed that the absorption at 1650 cm⁻¹ became sharp and stronger than that of the degraded product obtained from the system with the addition of acetic acid. In the case of oxalic acid, which is a dicarboxylic acid with no methylene group, it was reported that its dissociation could be influenced by the phenomenon called the mesomeric effect.⁶²⁾ The mesomeric effect might lead to intramolecular hydrogen bonding between the first dissociated carboxylic group and the OH part of the undissociated group in the oxalic acid, which bears a partial positive charge. Similar to acetic acid, oxalic acid could interact with chitosan, but the interaction might be slightly stronger, leading to a slightly higher rate of degradation than that of acetic acid. Meanwhile, the FT-IR spectra of the degraded product from the system with the addition of other dicarboxylic acids and the tricarboxylic acid exhibited the characteristic peak at 1600 cm⁻¹, which slightly shifted to lower wavenumbers, and the absorption at 1650 cm⁻¹ became obvious but had lower intensity than that obtained by the addition of oxalic acid. According to the pK_a values in Table II, all dicarboxylic acids and the tricarboxylic acid are able to dissociate in the water and can lead to the protonation of –NH₂ in chitosan, similar to acetic acid and oxalic acid. The result might indicate that there were ionic interactions between the –NH₃⁺ groups of chitosan and the –COO⁻ groups of the carboxylic acids. However, since dicarboxylic acid and tricarboxylic acid contain two and three groups of –COO⁻, respectively, they could undergo ionic crosslinking or complexation with some adjacent chitosan chains, as reported in some previous studies.^63–65) These ionic crosslinking or complexation might be the reason for the slower M_w reduction of chitosan hydrogel. Moreover, the result showed that the rate of the degradation of chitosan depended on the number of methylene groups in the structures of dicarboxylic acid and tricarboxylic acid.

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Figure 6 shows the WAXD patterns of chitosan hydrogel before the SP treatment and the degraded chitosan hydrogel after applying the SP treatment in the presence of various types of carboxylic acid solutions at a concentration of 1.55 mM. The presence of acetic acid during the SP treatment resulted in a degraded product with an amorphous structure and, in the case of oxalic acid, very low intensity of the peaks at 2θ = 19.6 and 22.2° was observed. This may imply that the destruction of the crystalline structure of chitosan was facilitated by the presence of acetic acid and oxalic acid during the SP treatment. Meanwhile, the addition of other dicarboxylic acids and the tricarboxylic acid gave peaks with relatively stronger intensity at 2θ = 19.6 and 22.2°. According to a previous study, the broad peak at around 2θ = 19 and 23° might be ascribed to the diffraction of the plane of the crystal region in the chitosan carboxylate.⁶⁶⁾ The chitosan carboxylate was induced by the formation of ionic interactions between the –NH₃⁺ groups of chitosan and –COO⁻ groups of the carboxylic acid. In the case of the dicarboxylic acids and tricarboxylic acid, they could undergo ionic crosslinking or complexation with some adjacent chitosan chains, leading to stabilization of the crystal structure of chitosan and, consequently, these two peaks could be clearly observed. In particular, the relatively high crystallinity of the degraded chitosan products from the systems with the addition of azelaic acid and citric acid was evidence that indicated some extent of crosslinking within the structure of degraded chitosan. Therefore, the WAXD results were consistent with the FT-IR results and could also be used to explain the reason that different rates of degradation were obtained when the chitosan hydrogel dispersed in the different carboxylic acids was degraded by SP treatment.

A schematic drawing of a possible degradation mechanism of chitosan hydrogel dispersed in various carboxylic acid solutions by applying SP treatment is shown in Fig. 7(b). The reaction solution containing only molecules of H₂O, carboxylic acids, and chitosan was subjected to SP treatment. The major component in the system was H₂O molecules. The plasma discharge can result in the excitation and ionization of H₂O molecules.⁶⁷⁾ Electrons are emitted as a result of ionization (i.e., H₂O → H₂O⁺ + e⁻) and then continuously collide with the surrounding H₂O molecules (i.e., e⁻ + H₂O → OH + ⁻H) to produce radicals. In addition, the excitation of H₂O molecules also directly leads to the formation of radicals (i.e., H₂O^* → OH + H). Both the ionization and excitation of H₂O molecules by SP treatment rapidly produce OH, which is necessary for the degradation of chitosan.^60,68) The generated OH could lead to the chain scission of the chitosan at β-1,4-glycosidic linkage which have been reported in the previous studies.^29,55,60) The breakdown of the β-1,4-glycosidic linkage leads to the reduction of the molecular weight of chitosan.⁶⁰⁾ Moreover, the electron from the plasma discharge can also directly collide with a chitosan molecule, leading to the chain scission.

The degraded chitosan hydrogel obtained by SP treatment in the presence of various dilute carboxylic acids for 60 min was centrifuged in order to separate the degraded products into water-soluble and water-insoluble degraded products. The solid fraction consisted of water-insoluble chitosan, while the liquid fraction contained the water-soluble COS. Further purification after the centrifugation was unnecessary because a very low concentration of carboxylic acids was used in the reaction. Moreover, carboxylic acids are generally found in many natural products and are safe for use in food and drug production.⁶⁹⁾ After centrifugation, in most cases of the studied carboxylic acids, the production yield of water-soluble COS was in the range of 50 to 70% with the exception of azelaic acid and citric acid, where the production yields of water-soluble COS were 40% and less than 5%, respectively. The production yield in this study was comparable to that obtained by different methods in other previous studies, as shown in supplementary data (Table III). For further investigation of the molecular weight reduction, M_w, number-average molecular weight (M_n), and PDI of the water-soluble COS were determined by GPC, as shown in Table IV. On the basis of M_n, the major oligosaccharides in the water-soluble COS could be estimated for each carboxylic acid, as shown in Table V. Degradation of chitosan hydrogel dispersed in the dilute solution of citric acid could produce COS with the major oligosaccharide of 10-mers, while the addition of acetic acid, oxalic acid, and adipic acid resulted in the COS containing mainly oligosaccharides of 12-mers. The addition of malonic acid, glutaric acid, and azelaic acid could give the major oligosaccharides of 13-mers and the COS with the major oligosaccharide of 14-mers could be produced by the addition of succinic acid and pimelic acid. Therefore, it could be concluded that the degradation of chitosan hydrogel dispersed in the dilute solution of various carboxylic acids by applying SP treatment could produce COS with a DP ranging from 10 to 14, which are difficult to produce by enzymatic degradation.⁶⁾ Besides, since the carboxylate anions still remained in the degraded products, they should undergo ionic interactions with the COS leading to the formation of COS-carboxylates.

To evaluate the anticancer activity and to determine the concentrations of the COS-carboxylate samples that could kill 50% of the cell population (IC₅₀), H460 and MRC-5 were used as representatives of the cancer cell line and the normal cell line, respectively. The COS-carboxylate samples were tested at final concentrations ranging from 1 to 5 mg·ml⁻¹. IC₅₀ of the COS-carboxylate and the normal COS samples against cancer cells (H460) and normal cells (MRC-5) are shown in Table V. The morphology of H460 cells untreated and treated with the COS-carboxylate samples at a concentration of 5 mg·ml⁻¹ is also shown in the online supplementary data at http://stacks.iop.org/JJAP/57/0102B5/mmedia (Fig. S1). The normal COS was obtained by following the protocol as reported in a previous work,¹⁹⁾ using SP treatment of chitosan solution. For comparing the COS-carboxylate and the COS, the result suggested that the presence of carboxylate in the structure of the COS could enhance the anticancer activity. Among the studied COS-carboxylate samples, it was found that the COS-carboxylate samples with the majority of 14-mers, which were obtained from the degradation of chitosan hydrogel dispersed in succinic acid and pimelic acid, exhibited the best inhibitory effect on the growth of H460 cells (IC₅₀ = 1.6 mg·ml⁻¹). For cytotoxicity against MRC-5 cells, the COS-acetate and COS-succinate samples had the highest IC₅₀ value at 5 mg·ml⁻¹. Moreover, the selectivity index (SI) of the COS-carboxylate and COS samples could be determined by the IC₅₀ ratio of a compound tested against cancer cells and normal cells. SI implies the difference in the cytotoxic activity of the tested sample against cancer and normal cells.^70,71) Therefore, a high SI value indicates a higher selectivity for cytotoxic activity against H460 cells than MRC-5 cells. Among the studied COS-carboxylate samples, the SI of the COS-succinate had the highest value of 3.1. The COS-succinate was also found to have better selectivity to cancer cells than COS. Since the last decade, succinate has been extensively studied for the treatment of cancer, because it was found to be a key metabolic factor in the cancer-immune and may lead to cancer immunotherapies.^72,73) Previous studies reported that vitamin E succinate showed an inhibitory effect on cancer cells and tumors.^74,75) Therefore, the combination of COS and succinate, which exhibited better selectivity against cancer cells, could be recommended for further development as a potential anticancer agent. In addition, according to Table VI, the cell viability obtained for the COS-succinate was comparable to that of the COS prepared by enzymatic degradation. From this evidence, it was proved that SP treatment can be used to reduce the molecular weight of chitosan in order to obtain COS without changing the biological properties such as anticancer activity, which naturally belongs to COS.