Study on the effect of tunicate cellulose nanocrystals in the preparation of sodium alginate-based enteric capsule

In this work, tunicate cellulose nanocrystals (tCNCs) were extracted from the tunicate by bleaching and acid hydrolysis. They were used as filler in the preparation of sodium alginate-based enteric capsules. The addition of tCNCs with a high aspect ratio (65) rendered the enteric capsule excellent physical properties. Compared with the control sample, when the addition of tCNCs were 10% (wt), the water contact angle of the capsule was enhanced by 46.0%, the opacity was increased by 356.8%, the maximum tensile stress was increased by 142.6%, the modulus of elasticity was increased by 240.3%, and the elongation at break was increased by 133.8%. In the in vitro degradation experiments, the capsule hardly degraded in the gastric environment (pH 1.2), while in the intestinal environment (pH 6.8), the degradation became slower with the increase of tCNC content, which was consistent with the properties of the enteric capsule. This research developed a new direction for the application of tCNCs in the pharmaceutical material productions.


Introduction
In the food and pharmaceutical industries, encapsulation (Samakradhamrongthai et al. 2019) technology has been widely used. The capsules are less likely to be broken down by saliva in the mouth than tablets (Fathi et al. 2018;Grill et al. 2020;Yan et al. 2020), providing good protection from damage and protecting the digestive and respiratory organs. Generally, according to the dissolution location, the capsules can be divided into two categories, the gastric capsule and the enteric capsule. Unlike the gastric capsule, the enteric capsule does not disintegrate in the lower pH environment, i.e., the acidic stomach. Upon reaching the small intestine, the enteric capsule starts to disintegrate and releases the drug in the neutral pH environment. This could protect the stomach from drug-induced irritation (Al-Gousous and Langguth 2015).
Materials with good physical properties and biocompatibility are used to produce enteric capsules to maintain the stability of the medicine during the storage (Kathpalia et al. 2014). Currently in the market, the raw material for capsule manufacturing is mainly gelatin (derived from the skin and bone of animals such as pigs and cows). In addition, gelatin is prone to cross-linking (Digenis et al. 1994) and the high moisture content of the capsules leads to instability of the filled hygroscopic drugs (Duconseille et al. 2015). Recently, some non-gelatin-based capsules attracted interest in the pharmaceutical material industry. The most common alternatives are hydroxypropyl methylcellulose (HPMC) (Fu et al. 2020), pullulan (Ding et al. 2020) and starch-based capsules Ji et al. 2017). They serve as good substitutes for gelatin, but have some limitations since they are not pH-dependent and unstable in the enteral therapy. Sodium alginate (SA) is widely used in food, pharmaceutical, and bioengineering fields owing to its non-toxicity, stability, biocompatibility, and biodegradability (Abbasiliasi et al. 2019;Fayaz et al. 2009). Thus, SA has the potential to be used in the capsule production and could be an alternative of the traditional gelatin capsules. However, the mechanical properties of SA are weak, which makes it difficult to form capsule in the molding process.
The tunicates cellulose nanocrystals (tCNCs) are extracted from the mantle of tunicate (a kind of marine animal). Tunicate cellulose is a linear polymer with many D-glucopyranose rings linked by b-(1,4) glycosidic bonds. Recently, our research group has found that the crystallinity index (CrI) and aspect ratio of tCNCs is 93.9% and 65.0, respectively. However, the CrI and aspect ratio of the softwood nanocellulose are 70.9% and 10.5, respectively. These high CrI and aspect ratio render tCNCs excellent physical strength (Cheng et al. 2020). Therefore, from this point of view, tCNCs might be used as an enforcement agent in the SA capsule formation to increase the physical strength (Liu et al. 2021b(Liu et al. , 2021c. In this work, we applied tCNCs in the SA-based capsule using glycerol as a plasticizer. The prepared capsules were analyzed by mechanical characterizations, Fourier-transform infrared spectroscopy (FT-IR), and scanning electron microscope (SEM). The results showed that tCNCs were uniformly embedded into the SA matrix to form a rigid capsule. With the addition of tCNCs, the hydrophobic property, opacity and tensile properties were significantly improved in the resulted capsule. In addition, the capsule was relatively stable in an acidic medium but degraded well in neutral medium. Therefore, the tCNC-SA capsule is expected to load gastric irritant drugs as enteric capsules. As far as we know, this is the first research on the effect of tCNCs in the enteric capsule formation. This research would provide potential use of tCNCs in the pharmaceutical material industry.

Preparation of tCNCs
The tunicate specimens were slit with a knife to remove the internal organs. The residual tunicate mantles were washed thoroughly with deionized water (DI water). After that, the mantles were soaked in a 5% (w/v) KOH solution for 10 h. Then, 300 mL 2% sodium chlorite solution and 5 mL of anhydrous acetic acid were mixed properly to make bleaching solution. The mantles were washed 3 times with DI water to neutral, and bleached with 300 mL bleaching solution at 70°C for 6 h, and the bleach solution was changed every 2 h. At last, they were washed thoroughly and cut into small pieces, which were designated as tunicate cellulose.
The tunicate cellulose was further hydrolyzed by sulfuric acid following a literature method (Tang et al. 2014). The tunicate cellulose was weighed 3 g in 105 mL 64% (v/v) sulfuric acid solution and stirred continuously for 2 h at 45°C. The reaction was terminated by adding excess (10 times) DI water, and the precipitate was collected by centrifugation at 8000 r/min for 30 min. The precipitate was dialyzed in a dialysis bag with a cut-off molecular weight of 12,000-14,000 until the pH of the solution became neutral. After dialysis, the suspension was sonicated for 30 min and designated as tCNCs.
b-D-mannuronic(M)/a-L-guluronic analysis(G) analysis of SA The M/G ratio of SA was determined by infrared spectroscopy through a literature method (Filippov and Kohn 1974). Briefly, the SA and KBr were pressed in the ratio of 1:200 and placed on a Fourier infrared spectrometer (VERTEX 70, Germany Bruker Technology Co., Ltd, Germany) to determine the infrared spectrum. The resolution was set to 4 cm -1 and the samples were scanned 32 times in the range of 4000 to 400 cm -1 . The absorbance ratio of wave numbers 1290/1320 is designated as M/G ratio, which is 1.02 in this study.
Gel permeation chromatography (GPC) analysis of tCNCs and SA Weighted average molecular weight (Mw) and number-average molecular weight (Mn) of tCNCs and SA, and the degree of polymerization of tCNCs were determined by GPC. Prior to the GPC analysis, tCNCs were firstly derivatized by phenyl isocyanate in the anhydrous pyridine following a literature method (Hubbell and Ragauskas 2010) The derivatized tCNCs were then dissolved in THF for GPC analysis containing ultraviolet (UV) detector (Agilent 1200 series, Agilent Technologies, Santa Clara, USA). The sample was filtered through a 0.45 lm membrane filter prior to injection. 20 lL of the sample was automatically injected. GPC analyses were carried out using a UV detector on a 4-column sequence of Styragel columns (HR0.5, HR2, HR4 and HR6, Waters Corporation, Milford, USA) at 1.00 mL/min flow rate. Polystyrene standards were used for calibration. WinGPC Unity software (Version 7.2.1, Polymer Standards Service USA, Inc.) was used to collect data and determine number average molecular weight (Mn) and weight average molecular weight (Mw) of the tricarbanilated tCNCs. The weightaverage degree of polymerization (DPw) for the tCNCs was obtained by dividing Mw by 519 g/mol, the molecular weight of the tricarbanilated cellulose repeat unit.
The Mn and Mw determination of SA was carried out in a GPC system equipped with a refractive index (RI) detector (Agilent 1200 series, Agilent Technologies, Santa Clara, USA) on a 3-column sequence of Ultrahydrogel columns (120, 250 and 500, Waters Corporation, Milford, USA). The RI detector was set at 35°C. The mobile phase was an alkaline sodium hydroxide/acetate solution (0.2 M sodium hydroxide and 0.1 M sodium acetate, pH 12-13) and the flow rate was 0.5 mL/min. The SA dissolved in mobile phase (1 mg/mL) and the solution was then filtered with a 0.2 lm filter. The filtered sample (25 lL) was injected into the GPC column system for analysis. The pullulan standard samples were used as narrow calibration standards. The WinGPC Unity software was also applied to determine the Mn and Mw of SA.

Transmission electron microscopy (TEM) analysis of tCNCs
The morphology of tCNCs were studied by TEM (TECNAI G2 F20, FEI Co., USA). One drop of tCNCs suspension (1 wt%) was deposited on the surface of a copper grid covered with a porous carbon film. The grids were then allowed to float in a 2 wt% uranyl acetate solution for 3 min to stain the samples. Finally, the samples were dried at room temperature for 24 h and then tested by TEM with a resolution of 0.2 nm at an accelerating voltage of 100 kV.

Preparation of tCNC-SA film
In order to perform physical and mechanical property tests on tCNC-SA capsules, tCNC-SA film was firstly formed since it was convenient for testing. The tCNC-SA film was formed as following: SA (3 g) was firstly dissolved in DI water (100 mL) at 70°C in the water shaking bath (SY-2230, Crystal Instruments, USA). The glycerol (0.72 mL) was dropwisely added in the SA solution as plasticizer. During the formation of tCNC-SA film, the intercalation of glycerol could significantly decrease the interactions of the polymers of tCNCs and SA (Sothornvit and Krochta 2001). Thus, it can be used as plasticizer in tCNC-SA film. According to the study of Shimokawa et al. (2018), the optimal percentage of plasticizer addition is 30%, which is equivalent to 0.72 mL glycerol in this study. In addition, when the amount of tCNCs exceeded 10% in the tCNC-SA mixture, the agglomeration of tCNCs rendered tCNC-SA film inhomogeneous and decreased its physical strength. Similar observation was also reported in the previous literature (Merayo et al. 2017). Thus, in tCNC-SA film formations, the addition amounts of tCNCs in tCNC-SA mixture varied in different weight proportions, i.e. 0%, 1%, 3%, 5%, and 10%, which were marked as samples F0, F1, F2, F3 and F4, respectively. The tCNCs were dispersed evenly in the SA solution by ultrasound and the tCNC-SA gel was formed. After that, the resulted gel was degased by water vacuum pump for 30 min prior to the viscosity measurement. Rotor No. 4 was selected and the viscosity of tCNC-SA gel was measured at 12 rpm/min with a viscometer (NDJ-5S, Lichenkeyi Co., Ltd.) at 25°C. In the meanwhile, the resulted gel was poured into a 90 mm diameter Petri dish and dried at 45°C for 24 h to form the tCNC-SA film. The preparation process is shown in Fig. 1.

Mechanical properties measurement
The mechanical properties (tensile strength, modulus of elasticity, elongation) of tCNC-SA film were measured by a universal material testing machine (Instron 3365, Instron Corporation, USA). The tCNC-SA film was cut into long strips of 62 mm 9 20 mm and the film thickness was measured by vernier calipers (Baigong 0-150 mm, Shanghai Shenhan Measuring Tools Co., Ltd, China). The clamping distance was 25 mm and the stretching speed was 10 mm/min. Three parallel samples were measured and the average value was adopted. In addition, the tCNC-SA films (F0 and F3) were bent and twisted to visually observe their flexibility.

Opacity measurement
The opacity of tCNC-SA film was determined by its UV absorbance based on a literature method (Abbasiliasi et al. 2019). Briefly, tCNC-SA film was cut into squares with the size of 20 mm 9 20 mm. The UV absorbances of these samples were recorded at 600 nm by UV spectrophotometer (Agilent 8453, Agilent Technologies Co. Ltd, USA) to calculate the opacity with the following Eq. (1) (Siripatrawan and Harte 2010): where A is the UV absorbance of the film at 600 nm, t is the film thickness, mm.

FT-IR analysis
The glycerol, tCNCs, and tCNC-SA film samples were placed on a Fourier infrared spectrometer to determine the infrared spectrum. The resolution was set to 4 cm -1 and the samples were scanned 32 times in the range of 4000 to 400 cm -1 .

X-ray diffraction analysis
The X-ray diffraction (XRD) was used to analyze the crystallinity of tCNCs and tCNC-SA film samples. The scanning speed was set to 2°/min and scanned in the range of 2h = 5°* 45°with a voltage of 40 kV and a current of 40 mA. According to the study of French (French 2014), the tCNCs were judged to be cellulose I b . The crystallinity index (CrI) was calculated according to Segal's Eq. (2) (Segal et al. 1959;Yousefhashemi et al. 2019): where I 200 is the diffraction intensity (2h = 22.5°) of the (200) lattice plane in type I cellulose, and I am is the diffraction intensity of the non-crystalline region at the minimum in the intensity near 18°.

Preparation of tCNC-SA capsule
The tCNC-SA capsules were prepared to observe the morphological changes in the in vitro degradation experiments. Basically, tCNC-SA capsule and tCNC-SA film shared the same chemical compositions. Their differences were in the formation process: the tCNC-SA capsule was dried in a mold while tCNC-SA film was dried in a Petri dish. The capsules were prepared following a reference method (Abbasiliasi et al. 2019). The stainless-steel capsule mold was preheated in a 70°C oven. The experimental step of preparation of tCNC-SA film was repeated to prepare tCNC-SA gel with different tCNCs ratios. In the meanwhile, the treasure blue edible pigment (1 wt%) was also added to the mixture. The formed gel was stirred well and was poured into the preheated mold. Finally, the molds were dried in a 45°C oven for 4 h. The amounts of 100 capsules were prepared by adding different proportions of tCNC film (F0-F4). The following Eq. (3) is used to calculate the capsule formation rate (C). Finally, the dried capsules were removed from the mold for subsequent characterization.
where N1 is the number of capsules formed, and N2 is the number of capsules made.

Characterization of the tCNC-SA capsule
The capsule characterization includes the appearance, the dry loss and water contact angle (WCA) testing. The appearance of tCNC-SA capsule was characterized by visual observation, and the dry loss was tested according to US Pharmacopoeia (USP) (Pharmacopeial 2017) method. The cap and body of the capsule were separated and weighed. After that, they were dried at 105°C ovens for 6 h and weighed the dry weight. The following Eq. (4) was used to calculate the dry loss.
where m1 is the mass of the sample before drying, and m2 is the mass of the sample after drying. The capsule appearance includes surface smoothness, color uniformity, shape deformation and odor. The water contact angle testing of the capsules was described as follows: at ambient temperature, a certain size of the capsule (20 mm 9 20 mm) was cut and fixed on a carrier table, and the water of 5 lL was dropped onto the capsule surface. The WCA was measured by goniometer (DSA 30, Kruss, Germany) to characterize the film hydrophobicity. Five points were tested for each sample and the average value was adopted.

Scanning electron microscopy (SEM) analysis
The morphology of tCNC-SA capsule was measured by SEM (Nova Nano SEM 230, FEI Co., USA). The treated sample was gold sprayed (100 s) by vacuum ion sputter (JFC-1600, JEOL Ltd, Japan), and the morphology of the sample was observed.

In vitro degradation experiments of tCNC-SA capsule
In order to protect the gastric mucosa from the irritation caused by the drug components, the capsules should not disintegrate and release drugs under acidic conditions. Therefore, in this research, the degradation study of tCNC-SA was divided into two steps. The influences of tCNC loading and pH of PBS buffer solution on the capsule degradation were studied in the first and second steps, respectively.
The first step focused on the effect of tCNC loading on capsule degradation time in a simulated human intestinal fluid of pH 6.8 and temperature (37°C). The optimized tCNC loading was based on the degradation time (60 min). In practice, each tCNC-SA capsule (F0, F1, F2, F3, F4) was weighed into glass test tubes, and 7 mL of pH 6.8 PBS was added separately and dissolved in a 37°C water shaking bath. The undissolved solid samples were collected through filteration at different time intervals (0 min, 15 min, 30 min and 60 min). The sample weight loss during the dissolution was calculated by the ratio of the initial sample weight (o.d. weight) and the o.d. weight of the sample in a certain time interval.
In the second step, the optimized tCNC loading was applied in tCNC-SA capsule formation. These capsules were used to study the pH effect on their degradation. Two pHs, 1.2 and 6.8 were chosen to simulate the human gastric and intestinal fluid environment, respectively (Ilgin et al. 2020). In this step, the capsules were dissolved in two PBS (pH 1.2 and pH 6.8) at 37°C. During this process, the undissolved capsules were collected and the weight losses were also calculated, as described in the first step.

GPC analysis of SA and tCNCs
The Mw and Mn of tricarbanilated tCNCs and SA, and the weight-average degree of polymerization (DPw) of tCNCs are listed in Table 1. The Mw and Mn of tricarbanilated tCNCs are 2.49 9 10 6 g/mol, and 1.28 9 10 6 g/mol, respectively. The DPw of tCNCs is 4798. The Mw of SA is 1.32 9 10 5 g/mol and Mn of SA is 7.89 9 10 4 g/mol.

TEM analysis of tCNCs
As shown in the TEM image (Fig. 2), the length and width of tCNCs were measured as 1300 nm and 20 nm, respectively. Then the aspect ratio was calculated as 65, which is in the range of previous report (Peng et al. 2011).

Formation of tCNC-SA film/capsule
During tCNC-SA film/capsule formation, the hydrogen bonding is formed between the hydroxyl groups in SA and the hydroxyl groups in tCNCs, which forms a network structure (Fig. 3a). The glycerol is inserted between the molecular chains of SA and tCNCs as a plasticizer. The intermolecular forces between the cellulose and SA molecular chains can be weakened by the glycerol, and the distance between them is increased (Xu et al. 2012). As a result, the possibility of movement between SA and tCNCs are increased, and the entanglement between molecular chains is reduced, resulting in increased plasticity of the composites.
The viscosity of tCNC-SA gel is demonstrated in Fig. 3b. As tCNC content increases, the viscosity of tCNC-SA film gradually increases from 1.28 Pa s to 1.69 Pa s, which is 31.32% higher than that of blank sample F0. This phenomenon is attributed to the hydrogen bonding between tCNCs and SA (Salas et al. 2014).

Mechanical property analysis of tCNC-SA film
Five stress-strain curves of tCNC-SA films are shown in Fig. 4a. There is basically no residual strain after the fracture, and the section is perpendicular to the direction of stress, indicating the film has a certain degree of rigidity and toughness (Liu et al. 2021a;Revin et al. 2019). As the content of tCNCs increases Table 1 The molecular weight analysis of tCNCs and SA

Sample
Mw (g/mol) Mn (g/mol) DPw tCNCs 2.49 9 10 6* 1.28 9 10 6* 4798 SA 1.32 9 10 5 7.89 9 10 4 -* The value refers to the tricarbanilated tCNCs   240.3% when it contains 10% of tCNCs (sample F4). Generally, the higher the modulus of elasticity, the more rigid the sample is and the less likely to be deformed. The improvements of the tensile stress and elastic modulus of tCNC-SA film are due to the inherent advantages of high aspect ratio and high elastic modulus of tCNCs, which shows excellent reinforcement in the polymer matrix (Iwamoto et al. 2009;Xu et al. 2021). When tCNCs were applied in the capsule formation, it has a certain degree of resistance to compression, which is conducive to alleviating the extrusion of the capsule during production, handling and storage. When tCNCs were added at 1% (sample F1), the elongation of the film before fracture is increased by 100% compared to F0, as shown in the Fig. 4c. In a previous study by our group, it was found that tCNCs had significant high aspect ratio of 65, which renders it forming mesh-like structure in the composite. The high aspect ratio of tCNCs could enhance the binding sites between tCNCs and SA (Boufi et al. 2016;Ming et al. 2021), forming interweaving network in the film. That might be the reason that the increase tCNCs content in the film could improve the elongation, as shown in Fig. 4c. It should be noted that, when the adding amounts increased from the 1% (sample F1) to 10% (sample F4), the film elongation rate was increased from 100 to 144% when compared with the contrast F0 sample. The results indicate the small quantity of tCNCs could significantly increase the mechanical strength of tCNC-SA. Further addition of tCNCs have limit strength enhancement for the film. Furthermore, the addition of tCNCs could also improve the film flexibility, as shown in Fig. 4 (d, e, f, g, h and i). The film made from the pure SA (sample F0) was rigid. Upon the bending and twisting force, it recovered to the original form rapidly. In contrast, tCNCs enhanced film (sample F3) is soft and flexible. The flexibility and softness is important for the capsules since these properties could reduce the irritation to the mucosa during the deglutition (Kathpalia et al. 2014).
The elastic modulus and elongation rate of the enteric capsule are compared with the previous report, as shown in Table 2. In current work, the elasticity modulus is much higher than that of HPMC/ Fatty acids (Jimenez et al. 2010) and the elongation at break is slightly higher than that of highly carboxymethylated starch HCMS/Glycerol (Jimenez et al. 2010;Kim et al. 2002). This indicates the mechanical properties of the capsule prepared in this study are superior or comparable to the reported data.
Physical properties of tCNC-SA capsules Physical properties of the capsule mainly include appearance, opacity, testing of WCA and the SEM observations. Table 3 summarizes the appearance, dry loss and formation rate data of capsules with different addition of tCNCs. Without the addition of tCNCs, the capsule formation rate was 0 (sample F0) since there is no complete tCNC-SA capsule formed in the mold. From the visual observation, the formed capsules (F1 to F4) have smooth surface with no cracks, no air bubbles and no odor.
As the amount of tCNCs increases, the capsule formation rate gradually increases. The capsule formation rate reaches 92% when the amount of tCNCs increases to 10%. TCNCs have a high aspect ratio and could form hydrogen bonds with the SA, resulting in improved physical strength and flexibility of tCNC-SA film, eventually leading to an increase of capsule formation rate. The dry loss of F0 was only 8.61% and reached a maximum of 15.45% (sample F4) with the 10 wt% of tCNCs added. This is due to the hydrogen bonding between tCNCs and SA, which results in the absorption of water (Gilormini and  Verdu 2018) in the prepared film and therefore the dry loss increases. It can be visually observed in Fig. 5a, c that the opacity of tCNC-SA capsule increases with the increase of tCNC content. The F0 is a SA film with an opacity of 0.44 and high transparency. When 1% tCNCs and 10% tCNCs were added (Sample F1 and F4), the increases of opacity are 15.9% and 356.8%, respectively. This is because the tCNCs are partially agglomerated in the film and forms a self-assembled network, which hinders the passage of light through the film (Qian et al. 2010). Since all the film thickness are similar as discussed in the sample preparation, the film opacity is directly related to the film UV absorbance, as indicated in Eq. 1. According to the opacity data in Table 3, tCNC-SA film opacities increase with the increase of tCNCs content, which means that the film material has the potential to act as a UV absorber, giving it light sheltering abilities. Therefore, the adding of tCNCs in the capsule could reduce the amount of shading agent added during the preparation of capsules.
The WCA is used to determine the resistance of tCNC-SA capsule surface to liquid water, which can reflect the hydrophobicity/hydrophilicity of the sample surface (Rhim 2011). The pure SA film (sample F0) has poor water resistance, and the addition of tCNCs can improve this hydrophobicity, as shown in Fig. 5b. There is no tCNCs in the F0 sample, which has a WCA of 36.5°, and the water diffuses randomly in all directions. As tCNC content increases, the WCA of the film becomes enlarged, which also leads to a more circular water permeation shape. This indicates that the addition of tCNCs make the propagation of water resistance more uniform. When the addition of tCNCs was 10%, the water contact angle of the film surface was 53.3°, which increased by 46.0% compared with 0% tCNC addition sample. A strong hydrogen bonding can occur between the hydroxyl group on tunicate nanocellulose and the hydroxyl groups in SA. Thus, improving the bonding of these two substrates and inhibiting the water absorption of the sample. After wetting, water molecules cannot break through these strong hydrogen bonds, so it plays   (Abdollahi et al. 2013). The ratios of tCNCs are different, and the surface structure of the tCNC-SA capsule is also different. The surface and cross-section of the tCNC-SA films were characterized using SEM to investigate the morphology of tCNCs in the SA substrate film, as shown in Fig. 6. From the surface diagram (a-e), it can be observed that tCNCs with high aspect ratio exists in the film in the form of thin strips. From F0 to F4, tCNCs are added more and more, the surface of the film becomes rougher and even some agglomeration occurs. In the cross-sectional view (Fig. 6h-j), it is observed that tCNCs are uniformly distributed in the film in the form of network, which could enhance the physical properties of the film. This is the reason why the mechanical properties of tCNC-SA films become stronger as the amount of tCNCs increases.
Chemical characterization of tCNC-SA film Figure 7a represents the FT-IR spectra of tCNCs, glycerol, SA, F0, F1, F2, F3 and F4. No chemical reaction occurred during the preparation of tCNC-SA film, so no new characteristic peaks appear in F0, F1, F2, F3 and F4. And the transmittance of F0, F1, F2, F3 and F4 in the IR spectrum increases with the increase of tCNC content. The absorption peaks at 3415 cm -1 , 3342 cm -1 , 1638 cm -1 and 1413 cm -1 are attributed to the O-H stretching vibration. The absorption peaks at 2920 cm -1 and 2904 cm -1 are attributed to the C-H stretching vibration. The absorption peak at 1620 cm -1 is attributed to C = C stretching vibration. The two absorption peaks at 1299 cm -1 and 1089 cm -1 are caused by C-O stretching vibration, and the absorption peak at 817 cm -1 is attributed to the C = C bending vibration (Saravanakumar et al. 2020). Two characteristic peaks of glycerol at 2937 cm -1 and 2879 cm -1 belong to CH 2 asymmetric and symmetric stretching vibration, respectively. The characteristic peaks of tCNCs, SA, and glycerol interact with each other, which results in peak overlaps in two large ranges (3778 cm -1 -2437 cm -1 and 1856 cm -1 -1016 cm -1 ) . Figure 7b shows the XRD spectrum of the crystal structure of tCNCs and tCNC-SA films (samples F0 to F4). It can be seen that the diffraction characteristics of the films (F1, F2, F3 and F4) are essentially the same as those of tCNCs, which is a typical cellulose I-type structure with three typical lattice surfaces with intensities of 2h = 14.66°(1-10), 16.58°(110) and 22.76°(200), respectively (Cheng et al. 2020;Narita et al. 2005 The addition of tCNCs inhibited the degradation of the capsule as analyzed in Fig. 8a, b. F1, F2, F3 and F4 disintegrate at 15 min without affecting the release of the drug, which is in accordance with the US Pharmacopoeia requirements on disintegration time of hard capsules (Pharmacopeial 2017). F1 degrades completely within 60 min, F2, F3 and F4 cannot be completely degraded within 60 min, especially F4, where the mass loss value is about 50% at 60 min. This is due to the fact that tCNCs and SA are crosslinked by hydrogen bonds and form a mesh structure, which hinder the disintegration of the capsules. Therefore, with the increase of tCNC content, the mass loss is smaller.
In vitro degradation of tCNC-SA capsule in different pH environments Figure 9a shows the in vitro degradation mass loss and degradation photos of F2 at different pH environments (pH 1.2 and pH 6.8). The mass loss of the tCNC-SA capsule in pH 6.8 increases continuously with time and  finally reached 67.3% at 60 min. In contrast, in the solution with pH = 1.2, the mass loss of the tCNC-SA capsule is 8.2% after 30 min, and then the mass is stabilized (Fig. 9b). The SA contains a large amount of -COO -. Under acidic conditions, it is turned into -COOH. Thus, the ionization and the hydrophilicity of SA decrease, leading to the contraction of molecular chains (Hua et al. 2010). Therefore, the prepared tCNC-SA capsule hardly degrades in acidic conditions. The capsule in pH 6.8 is softened at 15 min and cannot maintain their original shape, and most of them degrade at 60 min. Based on these observations, the tCNC-SA capsule can be applied as enteric-soluble capsule, which is loaded with drugs that are irritating to the stomach. As a result, the tCNC-SA capsule can be digested and absorbed in the intestine.

Conclusions
In this study, tCNCs enhanced SA-based capsules were prepared. Due to the formation of hydrogen bonds between tCNCs and SA, the mechanical properties, water contact angle, opacity of tCNC-SA capsules are significantly improved. When tCNCs loading was 10% (w/w), the water contact angle of the capsules increased by 46.0%, opacity increased by 356.8%, tensile stress increased by 142.6%, elastic modulus increased by 240.3%, and elongation increased by 137.0%, respectively.
During the in vitro disintegration experiment, the conversion of -COOin SA to -COOH under acidic conditions leads to decrease of capsule ionization. As a result, tCNC-SA capsules barely disintegrated under acidic conditions (pH 1.2), while they could disintegrate at a pH of 6.8 in 15 min, which indicates the excellent pH responsibilities.
Therefore, the prepared tCNC-SA composite is expected to be used as enteric capsules, which lays the foundation for the application of tCNCs in pharmaceutical material production.
Author contribution DX: Conceptualization, methodology, validation, formal analysis, investigation. YC: Conceptualization, methodology, validation, formal analysis, investigation. SW: Resources, data curation, writing-original draft. QZ and AKM: Methodology, validation, formal analysis, investigation. DN: Writing-review and editing, visualization. FH: Supervision, writing-review and editing, project administration, funding acquisition. Fig. 9 a Degradation of F2 tCNC-SA capsules at pH 1.2 and pH 6.8, b weight loss of F2 tCNC-SA capsules at pH 1.2 and pH 6.8 Declarations The authors report no declarations of interest.