Tunicate were purchased from Weihai Sea Food Market in Shandong, China. Treasure blue edible pigment was purchased from Wilton Industries, Inc. Dialysis bag was purchased from MYM biological technology Co., Ltd. Phenyl isocyanate was purchased from Xiya Reagent Co., Ltd. KBr, KOH, phosphate buffered solution (PBS), NaClO2, H2SO4, CH3OH, acetic acid, glycerol, tetrahydrofuran, pyridine and sodium alginate (SA) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. All the chemicals were used in this study without further purification.
2.2.1 Preparation of tCNC
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℃ for 6 hours, and the bleach solution was changed every 2 hours. 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 ℃. 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 12000-14000 until the pH of the solution became neutral. After dialysis, the suspension was sonicated for 30 min and designated as tCNC.
2.2.2 Gel permeation chromatography (GPC) analysis of tCNC and SA
The gel permeation chromatography (GPC) (Waters, Polymer Standards Service, Milford, USA) was used to determine the weighted average molecular weight (Mw) and degree of polymerization (DP) of tCNC and SA. Prior to the analysis, the tCNC and SA were derivatized by phenyl isocyanate based on a literature method (Hubbell and Ragauskas 2010). The tCNC/SA (15 mg) was weighed and placed in a test tube equipped with a micro-stirring bar. Then anhydrous pyridine (4.00 mL) and phenyl isocyanate (0.50 mL) were added and the tubes were capped with rubber film and reacted in an oil bath (DF101, Shanghai Qiuzuo Scientific Instrument Co., Ltd, China) at 70 ℃ for 72 h. And then 1.00 mL of CH3OH was added to quench the reaction. The reaction solution was added dropwisely to a volume ratio of 7:3 CH3OH/H2O solution (100.0 mL) to precipitate the reaction product. The solids were collected by filtration and then washed with methanol/water (1×50.0 mL) followed by water (2×50.0 mL). The derivatized tCNC/SA was dried under vacuum at 40 ℃ for 12 h. The derivatized tCNC and SA were then dissolved in tetrahydrofuran (2 mg/mL) for the GPC analysis. The DP is calculated by dividing the Mw of tCNC and SA by 519 g/mol and 436 g/mol, which are the molecular weights of the derivatized cellulose repeating units and SA repeating units, respectively.
2.2.3 Transmission electron microscopy (TEM) analysis of tCNC
The morphology of tCNC was studied by TEM (TECNAI G2 F20, FEI Co., USA). One drop of tCNC 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.
2.2.4 Preparation of tCNC-SA film
In order to perform the physical and mechanical properties testing of the tCNC-SA capsule, the tCNC-SA was firstly form a film since it has flat shape which is convenient for testing. The tCNC-SA film was formed as following: SA (3g) was firstly dissolved in DI water (100 mL) at 70°C in the water shaking bath (SY-2230, Crystal Instruments, USA). Then the glycerol (0.75 mL) was dropwisely added in the SA solution. After that, the tCNC was added to the solution in amounts of 0%, 1%, 3%, 5% and 10% of the total dry weight mass, which were marked as F0, F1, F2, F3 and F4, respectively. The tCNC was 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 mins prior to the viscosity measurement. The viscosity of the tCNC-SA gel was determined by a viscometer (NDJ-5S, Shanghai Xingliang Optical Instrument Co., Ltd, China). In the meanwhile, the resulted gel was poured into a 90 mm diameter Petri dish and dried at 45℃ for 24 h to form the tCNC-SA film. The preparation process is shown in Fig. 1.
2.2.5 Mechanical properties measurement
The mechanical properties (tensile strength, modulus of elasticity, elongation) of the tCNC-SA film were measured by universal material testing machine (Instron 3365, Instron Corporation, USA). The tCNC-SA film was cut into long strips of 62 mm × 20 mm and the film thickness was measured by vernier calipers (Baigong 0-150mm, 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 bended and twisted to visually observe its flexibility.
2.2.6 Opacity measurement
The opacity of the tCNC-SA film was determined by its UV absorbance based on a literature method (Abbasiliasi et al. 2019). Briefly, the tCNC-SA film was cut into squares with the size of 20 mm × 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 equation (1) (Siripatrawan and Harte 2010):
Where A is the UV absorbance of the film at 600 nm, t is the film thickness, mm.
2.2.7 FT-IR analysis
The SA, glycerol, tCNC, and tCNC-SA film samples were placed on a Fourier infrared spectrometer (VERTEX 70, Germany Brooke 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.
2.2.8 X-ray diffraction analysis
The X-ray diffraction (XRD) was used to analyze the crystallinity of the tCNC and tCNC-SA film samples. The scanning speed was set to 2°/min and scanned in the range of 2θ = 5°~45° with a voltage of 40 kV and a current of 40 mA.
According to the study of French (French 2014), the tCNC was judged to be cellulose Iβ. The crystallinity index (CrI) was calculated according to Segal’s equation (2) (Segal et al. 1959; Yousefhashemi et al. 2019):
Where I200 is the diffraction intensity (2θ = 22.5°) of the (200) lattice plane in type I cellulose, and Iam is the diffraction intensity of the non-crystalline region at the minimum in the intensity near 18°.
2.2.9 Preparation of tCNC-SA capsule
The tCNC-SA capsules were prepared to observe the morphological changes in the in vitro degradation experiments. Basically, the tCNC-SA capsule and the tCNC-SA film shared the same chemical compositions. Their differences were on the formation process: the tCNC-SA capsule was dried in a mold while the tCNC-SA film was dried in a Petri dish.
The capsules were prepared following a reference method (Abbasiliasi et al. 2019). Preheat the stainless steel capsule mold at 70℃ oven. Repeat the experimental step 2.2.4 to prepare tCNC-SA gel with different tCNC ratios, and 1% (wt) of treasure blue edible pigment was also added in the mixture. The formed gel was stirred well and was poured into the preheated mold. Finally, the mold was dried in 45℃ oven for 4h. The amounts of 100 capsules were prepared by adding different proportions of tCNC film (F0-F4). The following equation (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.
2.2.10 Characterization of the tCNC-SA capsule
The capsule characterization includes the appearance, the dry loss and water contact angle testing. The appearance of the tCNC-SA capsule was characterized by visual observation, and the dry loss was tested according to US Pharmacopoeia (USP) (Pharmacopeial 2016) method. The cap and body of the capsule were separated and weighed. After that, they were dried at 105℃ oven for 6 hours and weighed the dry weight. The following equation (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 following: at ambient temperature, a certain size of capsule (20 mm × 20 mm) was cut and fixed on a carrier table, and the water of 5 µL 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.
2.2.11 Scanning electron microscopy (SEM) analysis
The morphology of the 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.
2.2.12 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 the 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 step, 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℃). 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℃ water shaking bath. The undissolved solid samples were collected through filteration at different time intervals (0 min, 15 min, 30min 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 the 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℃. During this process, the undissolved capsules were collected and the weight losses were also calculated, as described in the first step.