Starch-based nanocapsules fabricated through layer-by-layer assembly for oral delivery of protein to lower gastrointestinal tract

Accepted Manuscript Title: Starch-based nanocapsules fabricated through layer-by-layer assembly for oral delivery of protein to lower gastrointestinal tract Authors: Yiping Zhang, Chengdeng Chi, Xiaoyi Huang, Qin Zou, Xiaoxi Li, Ling Chen PII: DOI: Reference:

S0144-8617(17)30489-7 http://dx.doi.org/doi:10.1016/j.carbpol.2017.04.090 CARP 12278

To appear in: Received date: Revised date: Accepted date:

6-2-2017 25-4-2017 27-4-2017

Please cite this article as: Zhang, Yiping., Chi, Chengdeng., Huang, Xiaoyi., Zou, Qin., Li, Xiaoxi., & Chen, Ling., Starch-based nanocapsules fabricated through layer-bylayer assembly for oral delivery of protein to lower gastrointestinal tract.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.04.090 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Starch-based nanocapsules fabricated through layer-by-layer assembly for oral delivery of protein to lower gastrointestinal tract

Yiping Zhang1, Chengdeng Chi1, Xiaoyi Huang1,2, Qin Zou1, Xiaoxi Li1*, Ling Chen1*

1

Ministry of Education Engineering Research Center of Starch and Protein Processing,

Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China 2

Department of Chemistry, Materials, and Chemical Engineering “G.Natta”, Politecnico di

Milano, Milan 20131, Italy

* Corresponding Authors:

Xiaoxi Li，Ling Chen

Fax: +86 20 8711 3252 E-mail: [email protected]；[email protected]

Highlights ► CMS/QAS colloidal nanocapsules were fabricated through layer by layer assembly. ► Higher DS and lower Mw of CMS improve the encapsulating efficiency of BSA. ► CMS with lower DS or Mw benefit to nanocapsules with compact core-shell structure. ► Nanocapsules fabricated by CMS with lower DS and Mw showed colon-specific release. ► Nanocapsules structural changes in simulated GIT were modulated by DS and Mw of CMS.

Abstract: Anionic carboxymethyl starch (CMS) and cationic quaternary ammonium starch (QAS), were used to fabricate nanocapsules through electrostatic layer by layer (LbL) alternate deposition onto colloidal BSA particles. An ideal starch-based colloidal nanocapsule was achieved by adjusting the degree of substitution and molar mass Mw of CMS. The nanocapsules fabricated by lower DS or Mw of CMS possessed more compact and stable core-shell structure, which favored the BSA delivery from the upper gastrointestinal tract (GIT) to the colon. In particular, CMS/QAS nanocapsules constructed by CMS with lower DS and Mw showed better colon-specific delivery and release performance in simulated GIT fluid after 7 days’ storage in different kinds of beverage (33.04%-46.35% in upper GIT, 52.70%-64.97% in colon, respectively). These findings demonstrated that CMS/QAS nanocapsules constructed by CMS with lower DS and Mw can be further exploited as a potential oral delivery system for protein to colon.

Abbreviations CMS, Anionic carboxymethyl starch; QAS, cationic quaternary ammonium; DS, starchdegree of substitution; Mw, molar mass; BSA, bovine serum albumin; GIT, gastrointestinal tract; SAXS, small angle X-ray scattering; ITC, isothermal titration calorimetry; TEM, transmission electron microscope; SGF, simulated gastric fluid; SIF, simulated intestinal fluid; SCF, simulated colonic fluid; CTA, 3-chloro-2-hydroxy-propyl-trimethyl ammounium chloride; CA, chloroacetic acid; LbL, layer-by-layer.

Keywords: carboxymethyl starch; quaternary ammonium starch; layer-by-layer assembly nanocapsules; oral delivery; protein; colon targeting

Introduction In recent years there has been an increasing interest in the bioactive ingredients delivery through a specific and controlled manner in order to obtain optimal physiological benefits when foods are fortified with bioactive ingredients for health promotion or disease prevention. Bioactive polypeptides and proteins are one of the important nutraceuticals for their physiological effects but these polypeptides/proteines suffered from poor stability and lower bioavailability when orally administered due to the biological barriers associated with the changes of pH and proteolytic enzymes in the gastrointestinal tract (GIT). Thus, an ideal oral delivery system is needed for bioactive polypeptides and proteins, by which these bioactive ingredients could be sustainedly released in specific location of the lower GIT-colon, because the colon offers an appealing site with nearly neutral pH and lower proteoloytic enzyme activity. Recent developments about varieties of formulation strategies have been attempted, such as the used of protease inhibitors (Bernkop-Schnurch & Scerbe-Saiko, 1998), mucoadhesive and permeation enhancer (Situ, Li, Liu & Chen, 2015), as well as macrophage activity suppressors (Jalalipour, Najafabadi, Gilani, Esmaily & Tajerzadeh, 2008), to protect the bioactive polypeptides and proteins from degradation and denaturation. Besides, intelligent or environmentally responsive biomaterials, such as bacteria-sensitive, pH-responsive, pressure-sensitive, and time-responsive biopolymers have been extensively exploited as oral carriers for the bioavailability improvement of the bioactive ingredients (George & Abraham, 2007; Jain & Jain, 2008; Situ, Li, Liu & Chen, 2015). Particularly, due to their low-toxicity, safety, as well as good biocompatibility, natural and modified polysaccharides, such as alginate (George & Abraham, 2007), guar gum (Seeli & Prabaharan, 2016), chitosan (Luo, Teng, Li & Wang, 2015), and inulin (Chaturvedi, Ganguly, Nadagouda &

Aminabhavi, 2013), have drawn a great attention in developing oral bioactive ingredients delivery systems. Polyelectrolyte Micro- and Nanocapsules, which were fabricated through layer-by-layer (LbL) electrostatically alternate assembly of positively and oppositely charged biomacromolecules, are promising candidates for bioactive molecules controlled release system and have been successfully used for constructing ideal architectures in catalysis, biotechnology, etc (Diez-Pascual & Wong, 2010). The polyelectrolyte complexes could protect the bioactivities of the ingredients by maintaining theirs stable state under harsh gastrointestinal tract (GIT) environment (Park, Akiyama, Yamasaki & Kataoka, 2007; Patel & Velikov, 2011). Polyelectrolyte micro/nano-capsules can be obtained by electrostatically alternate assembly of natural or modified charged polysaccharides deposited onto colloidal particles surface. Hyaluronan, alginate, pectin, chitosan sulphate, chondroitin sulphate and dextran sulphate were widely employed for constructing LbL layers with negatively charged groups, while chitosan and cationic starch were frequently used as oppositely charged polymer species (Diez-Pascual & Shuttleworth, 2014; Li, Norde & Kleijn, 2012; Tapia-Hernandez et al., 2015). The micro/nano-capsules fabricated by chitosan and sodium alginate have demonstrated a high ability of proteins encapsulation and loading capacity, which guaranteed the entrapped proteins sustainedly release from the micro/nano-capsules in liquid environment (Haidar, Hamdy & Tabrizian, 2008; Ye, Wang, Liu, Tong, Ren & Zeng, 2006). In addition, the release of protein was remarkably controlled by the pH and calcium ions of the environment (Ye, Wang, Liu, Tong, Ren & Zeng, 2006). The multiple electrostatic interactions in layers with or against each other can modulate the stability and the controlled release properties of the fabricated micro/nano-capsules. The electrostatic interactions in layers are controlled by parameters such as

pH (Shiratori & Rubner, 2000), temperature (Tan, McMurdo, Pan & Van Patten, 2003), enzymes (Karamitros, Yashchenok, Mohwald, Skirtach & Konrad, 2013) and ionic strength (McAloney, Sinyor, Dudnik & Goh, 2001). Therefore, to control the release of proteins and polypeptides through oral delivery, a systematic design strategy for the preparation of LbL assembly materials must overcome the multiple biological barriers associated with the changes of pH, ionic concentration and enzymes in the gastrointestinal environment, and it is also important that the interactions between the assembly materials of micro/nano-capsules should have suitable responsiveness to these environmental changes mentioned above. However, there is a relatively poor understanding of how polyelectrolytes micro/nano-capsules behave within the human gastrointestinal tract (GIT), and how the polyelectrolyte biopolymers transform their physicochemical properties and influence the bioactive ingredient release from the ingested micro/nano-capsules. Starch, is an important polysaccharide extracted from higher plants which has been extensively employed in food, ranging from additives, stabilizers to packing materials, and also in pharmaceutics owing to its biodegradable and biocompatibility (Singh, Kaur & McCarthy, 2007). Our previous research have developed the starch-based microparticles for oral colon-specific polypeptide delivery with starch film coating. The engineered microparticles showed a great potential for the accurate delivery of bioactive polypeptides and protein to the colon (Situ, Chen, Wang & Li, 2014). Furthermore, a new resistant starch−glycoprotein complex based bioadhesive drug delivery system was also developed for oral colon-targeted delivery of bioactive ingredients, and it was exciting to find that the oral bioavailability of bioactive proteins and peptides were significantly enhanced (Situ, Li, Liu & Chen, 2015). In addition to starch film coating on the

surface of the solid microparticles, colloidal micro/nano-capsules fabricated through layer-by-layer assembly could regulate the release of bioactive ingredients in the colloidal or liquid systems (Zhang, Pan, Chen, Liu, Tao & Tian, 2017), which is benefit for the improvement of the stability and bioavailability of the bioactive ingredients for liquid foods such as solutions, emulsions, or gels. Therefore, starch-based LbL assembly colloidal micro/nano-capsules can be considered as a candidate for the oral delivery of functional proteins and peptides in liquid food systems. However, how to alter starch-based LbL assembly colloidal micro/nano-capsule responsiveness to environmental changes and unravel the mechanisms when these micro/nano-capsules transport across the GIT barriers are essential for oral delivery of the bioactive polypeptide and protein ingredients in liquid food systems. In the present study, two water-soluble polyelectrolytes, including anionic carboxymethyl starch (CMS) and cationic quaternary ammonium starch (QAS), were synthesized and used to construct nanocapsules through electrostatic LbL alternate deposition onto colloidal bovine serum albumin (BSA) particles which was used as a model protein. The effects of molar mass, the degree of substitution and electrostatic interactions between the two starch-based polyelectrolytes on the nanocapsule structures were investigated by small angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC) and transmission electron microscope (TEM). The in vitro release profile of BSA from this starch-based colloidal nanocapsules was carried out by mimicking the different pH and enzymes in gastric fluid (SGF), intestinal fluid (SIF) and colon fluid (SCF) respectively. Considering that the nanocapsules are generally added to the beverages and stored under suitable environment in food industry, the engineered starch-based nanocapsules were also added to the sprite (pH 3.02), apple vinegar (pH 3.71) and minute maid (pH 4.35), respectively, prior to the

investigation of BSA release in the simulated GIT conditions.

2. Materials and methods 2.1 Materials Cassava starch was purchased from Huachen starch & sugar Co., Ltd. (Shijiazhuang, Hebei province, China). Pullulanase, pancreatin and bovine serum albumin (BSA) were purchased from Boao Biotechnology Co., Ltd. (Shanghai, China). 3-chloro-2-hydroxy-propyl-trimethyl ammounium chloride (CTA), chloroacetic acid (CA), ethanol, acetic acid and isopropanol were obtained from Damao Chemical Co. Ltd. (Tianjin, China). A bicinchoninic acid (BCA) protein assay kit was supplied by Newprobe Bioscience & Technology Co., Ltd. (Beijing, China). 2.2 Synthesis of anionic CMS and cationic QAS 2.2.1. Synthesis of CMS Cassava starch (20 g, dry basis) was added into a three-neck round bottom flask, sodium hydroxide (8.08 g) as well as 95% ethanol (40 mL) was added with continuous stirring (300 rpm) until sodium hydroxide was completely dissolved. Chloroacetic acid (CA) was added to the mixture and the reaction was kept at 55 °C for 4 h. At the end of the reaction, acetic acid was added to neutralize the un-reacted sodium hydroxide, and the slurry was filtered and washed with 95% ethanol until no chloride ions can be detected in the ethanol layer by reacting with AgNO3. The obtained CMS was dried at 40 °C. CMS with different degree of substitution (DS) of carboxymethyl was prepared by adjusting the amount of CA introduced to the reaction. DS of CMS was determined by a titration method (Volkert, Loth, Lazik & Engelhardt, 2004). In addition, CMS was debranched with pullulanase to obtain anionic polyelectrolytes with different molar mass (Mw)

according to our previous research (Zhang, Chen, Zhao & Li, 2013). 2.2.2. Synthesis of QAS Cassava starch (20 g, dry basis) was dispersed in 50 mL distilled water in a three-neck round bottom flask, then 2% sodium hydroxide and CTA (the weight ratio of cassava starch to CTA was 0.2−0.4) were slowly added to prepare QAS with different DS. The reaction was kept at 30 °C for 3 h, and then washed with 95% ethanol until no chloride ions can be detected in the ethanol layer by reacting with AgNO3. The obtained QAS was dried at 40 °C. QAS with different Mw was obtained by treating QAS with pullulanase for appropriate time, and the DS of CTA was determined according to the previous study (Radosta, Vorwerg, Ebert, Begli, Grülc & Wastyn, 2004). 2.3 Characterization of CMS and QAS Fourier transform infrared spectroscopy (FTIR) analysis was performed using an FTIR spectrometer (Tensor 37, Bruker Instrument Co., Germany) at wavelength between 400 and 4000 cm-1 with 4 cm-1 resolution. The Weight average molecular weight (Mw) of CMS and QAS were determined by gel permeation chromatography (GPC) (Waters, USA) equipped with three chromatographic columns (Styragel HR 3, Styragel HMW 6E, and Styragel HMW 7, Waters, USA) and simultaneously coupled with a multi-angle laser-light scattering detector (DAWN HELEOS MALS, Wyatt Technology Corp.) and a refractive index (RI) detector (Optilab rEx, Wyatt Technology Corp.) The mobile phase was DMSO with LiBr (50 mmol/L) filtered through a 0.22 m PTFE filter. All the samples was shaken at 60 °C for 12 h to ensure completely dissoved in the mobile phase and followed by filtered using a 5 μm membrane filter (Millipore Co., USA) before use (Zhang, Chen, Zhao & Li, 2013). Data was processed by Astra V software (Wyatt Technology Corp., USA).

The ζ-potential of CMS and QAS was measured by a Zetasizer® Nano-ZS (Malvern instruments, Malvern, UK) equipped with a 4 mW helium/neon laser at a wavelength output of 633 nm and a backscattering angle of 173° at 25 °C. CMS and QAS were diluted with phosphate-buffered saline (PBS) with different pH values (pH 1.2, 2.9, 3.5, 4.2, 5.0, 6.8 and 7.2) and placed in an electrophoretic cell for ζ-potential analysis. Each measurement was performed in triplicate. 2.4 Isothermal titration calorimetry (ITC) analysis The interaction between CMS and QAS was determined by measuring the heat changes occurred during titration using a Nano-ITC (TA Inc., America). CMS and QAS were dissolved in phosphate buffer saline (PBS) (0.01 M, pH 2.9) respectively. After equilibrated at 25 °C, the solutions were degassed under vacuum prior to titration. 50 μL of solution with different Mw of CMS (8 mg/mL) was titrated into the sample cell which contained 300 μL of QAS (Mw= 7.407×103 g/mol, DS=0.261) solution (18 mg/mL) every 300 seconds with a total of 20 injections at 25 °C. PBS (0.01 M, pH 2.9) was used as a reference and injected into the reference cell. The resulting titration curves were modeled and analyzed using the Launch Nano software. The thermodynamic values of association constant (Ka), enthalpy of binding (∆H), and entropy of binding (∆S) were obtained from the model. The change in free energy (∆G) during the titration was calculated using the Gibbs free energy equation, ∆G = ∆H - T∆S, where T is the absolute temperature in Kelvin. 2.5 Loading of BSA into the CMS/QAS/CMS nanocapsules by LbL Assembly About 0.3 g of BSA was completely dissolved in 50 mL PBS (pH 2.9, 0.4 M) to obtain positively charged polyelectrolyte solution; then CMS (3 g, db) was suspended in 200 mL PBS (pH 2.9, 0.4 M) to obtain negatively charged polyelectrolyte solution and added dropwise to the BSA

solution. The mixed solution was incubated at 25 °C under gentle stirring for 8 h. After centrifugation at 8000 rpm for 10 min, the precipitate was washed with distilled water in order to eliminate free polyelectrolytes adhered to the surface of the BSA/CMS complexes. The precipitate was suspended in 50 mL PBS (pH 5.0, 0.4 M) with high speed agitation (5.4 krpm) coupled with homogenization at 100 bar followed by 300 bar in order to well disperse the BSA/CMS complexes in the solution. Positively charged QAS solution (0.6 g dissolved in 50 mL of 0.4 M PBS, pH 5.0) was subsequently added into the BSA/CMS complex solution and the mixture was incubated at 25°C under gentle stirring for 8 h to obtain the BSA/CMS/QAS complexes. The BSA/CMS/QAS complex solution was centrifuged at 8000 rpm for 10 min and the precipitate was washed again to remove free QAS. The precipitate collected was re-suspended in 50 mL PBS (pH 2.9, 0.4M), and then 0.6 g CMS dissolved in 50 mL 0.4 M PBS (pH 2.9) was added to the BSA/CMS/QAS complex solution. After incubated at 25 °C for 8 h, the solution was centrifuged and washed as before to obtain the BSA-loaded CMS/QAS/CMS nanocapsules. The BSA encapsulation efficiency (EE) of the engineered nanocapsules was determined by quantifying the total amount of free BSA present in the collected supernatant after centrifugation by using a BCA kit at the wavelength of 562 nm. The EE of the nanocapsules was calculated using Eq. (1) (Haidar, Hamdy & Tabrizian, 2008): EE =

𝐵𝑆𝐴𝑡𝑜𝑡𝑎𝑙 − 𝐵𝑆𝐴𝑠𝑢𝑝𝑒𝑟 𝐵𝑆𝐴𝑡𝑜𝑡𝑎𝑙

(1)

Where BSAtotal is the initial total amount of BSA added; BSAsuper is the amount of free BSA present in the supernatant. 2.6 Characterization of the engineered nanocapsules Nano-scale structure of the nanocapsules was investigated by small angle X-ray scattering

(SAXS) technique with SAXSess (Anton-Paar, Graz, Austria) as reported by our previous study (Zhu, Li, Chen & Li, 2013). The nanocapsules were well suspended in PBS (0.4 M) at different pH conditions (pH 1.2, 6.8 and 7.2) with concentration of 10 mg/mL. The samples were filled into a capillary of 1 mm diameter and 0.01 mm wall thickness, and placed in a TCS 120 temperature-controlled sample holder unit (Anton Paar) at 37 oC. The housing of SAXS was vacuumized and the samples were measured at 50 mA and 40 kV with a PW3830 X-ray generator (PANalytical) at the wavelength of 0.1542 nm. The exposure time under X-ray lasted for 10 min, and the data which was recorded in an image plate was collected by the IP Reader software using a PerkinElmer Storage Phosphor System. All collected data were analyzed by SAXSquant 2D software and then normalized, smeared using SAXSquant 3.0 software for further analysis. The morphologies of the nanocapsules were investigated by using a JEM-1010 transmission electron microscope (TEM). The BSA-loaded CMS/QAS/CMS nanocapsules suspended in PBS with different pH values (pH 1.2, 6.8 and 7.2) was dropped onto the copper mesh and stained with 2% phosphotungstic acid. After drying at 30 °C for 1 h, the samples were observed by TEM. 2.7 In vitro BSA release The release of BSA from the nanocapsules was performed with a dissolution rate test apparatus (RCZ-8B, Tianda Tianfa Co., Ltd., Tianjin, China) according to our previous study (Pu, Chen, Li, Xie, Yu & Li, 2011). In order to simulate the bioactive ingredients transition in human gastrointestinal tract, the nanocapsules were incubated in simulated gastric fluid (SGF) for 2 h, followed by incubation in simulated intestinal fluid (SIF) for another 4 h and additional 26 h incubation in simulated colonic fluid (SCF). All in vitro measurements were kept at 37 oC with gentle stirring (100 rpm). The SGF (pH 1.2) consisted of 0.2 g NaCl, 7.0 mL HCl and 3.2 g pepsin;

the SIF (pH 6.8) comprised 6.8 g KH2PO4, 190 mL NaOH (0.2 M) and 10.0 g pancreatin; the SCF (pH 7.2) was composed of PBS (0.1 M). Each 5 mL of sample was collected at pre-set time points to determine BSA release content in vitro. The release profile of BSA from the nanocapsules which were added to real liquid food system (e.g. beverage) and stored for one week was also investigated. 3. Results and discussion 3.1 Characterization of CMS and QAS 3.1.1. FTIR spectroscopy CMS and QAS with different DS were synthesized by adjusting the weight ratio of CA or CTA to starch. The introduction of CA and CTA to native cassava starch was confirmed by FTIR. Spectra of native starch and modified starches are shown in Fig. S1. For native starch, the absorption bands at 1652 and 3400 cm-1 is assigned to the stretching mode of the –OH. An intense absorption at 1156 and 2929 cm-1 is due to C-O stretching and C-H stretching, respectively. Bands appeared at 1000 and 1160 cm-1 are attributed to the C-O bond signals, and the most intense belongs to glycoside linkage C-O-C stretching vibrations (Kizil, Irudayaraj & Seetharaman, 2002). The successful synthesis of CMS was confirmed by the appearance of new absorption bands at 1610 and 1420 cm-1, which could be assigned to carboxyl stretching vibration (Stojanovic, Jeremic & Jovanovic, 2000). The FTIR spectrum of QAS showed new bands at 1485 and 1385 cm-1 belong to the C-N and and C-H stretching respectively in trimethyl ammonium (CH3)3N+- (Pigorsch, 2009). The peak intensity at 3400 cm-1 became weaker due to the introduction of (CH3)3N+- to the starch backbone. The CMS and QAS with different DS were synthesized and their Mw was regulated by debranching enzyme (pullulanase) treatment. As shown in Table S1, CMS and QAS with different DS and Mw were obtained, the DS ranged from 0.041 to 0.138 for CMS and from 0.261 to 0.283 for

QAS, the Mw ranged from 2.345×107 g/mol to 4.373×106 g/mol for CMS, and from 1.344×104 g/mol to 7.407×103 g/mol for QAS, respectively. 3.1.2. ζ-potential measurement The surface charge of polyelectrolytes is one of the most important parameters in controlling the fabrication of micro/nano-capsules through electrostatically LbL assembly and can remarkably influence on the physiochemical characteristics of the engineered micro/nano-capsules (Feng, Wu, Wang & Liu, 2016). ζ-potential measurements were carried out to characterize the surface charge of CMS and QAS at different pH conditions in order to understand the interaction behavior between CMS and QAS (Fig. 1). As showed in Fig. 1(a1) and Fig. 1(a2), the ζ-potential of CMS was pH dependent, which was nearly neutral at pH 1.2 and became negative when pH was higher than 1.2. This observation can be explained as the carboxyl groups is protonated at pH 1.2 and deprotonated at pH > 1.2 and resulted in negatively charged of CMS. When the pH was increased from 2.9 to 7.2, the ζ-potential of CMS constantly increased because of the gradual deprotonation of carboxyl groups. It was noticed that CMS (Mw= 2.345×107 g/mol) with higher DS possessed a considerably higher absolute value of ζ-potential because of more carboxyl groups on CMS molecules. The ζ-potential of CMS (DS=0.041) with different Mw also increased when the pH was increased but changed slightly as the Mw variation. Similarly, ζ-potential of QAS at different pH conditions were measured and the results were shown in Fig. 1(b1) and Fig. 1(b2). All QAS with different DS and Mw displayed positive charge at different pH due to the presence of positively charged quaternary ammonium groups on the QAS molecules. As expected, QAS showed the most strongly positive ζ-potential at the lowest pH condition (pH 1.2) because the increase of the concentration of H+ induced higher conductivity of

the solution. And then the ζ-potential reduced substantially as the pH increased because the quaternary ammonium is difficult to ionization with the H+ ions decreasing. QAS (Mw=7.407×103 g/mol) with higher DS showed slightly stronger ζ-potential due to more positively charged quaternary ammonium groups on the QAS molecules. However, similar to CMS, Mw did not display significant influence on the ζ-potential of QAS (DS=0.261) at different pH conditions. Negatively charged CMS and positively charged QAS were then used as oppositely charged polyelectrolytes to fabricate nanocapsules. The structural properties of the engineered nanocapsules could be strongly affected by the structure of the CMS and QAS. Since DS and Mw did not significantly induce the variation of the ζ-potential of QAS within a wide range of pH, QAS with the DS of 0.261 and Mw of 7.407×103 g/mol was selected as a positively polyelectrolytes for fabricating the nanocapsules. The effects of DS and Mw of CMS on the structural properties of the nanocapsules constructed by CMS and QAS were investigated. 3.3. Interaction between CMS and QAS The integrated enthalpy of ITC titration curves for the binding of CMS to QAS showed in Fig. 2. It indicated that the titration of QAS with CMS triggered an exothermic binding event, which was ascribed to the electrostatic interaction between carboxyl and quaternary ammonium groups. The exothermic enthalpy declined as the injection amount of CMS increased, suggesting the saturation interaction between the quaternary ammonium groups of QAS and carboxyl groups of CMS. The thermodynamic parameters of QAS titrated with CMS with various DS and Mw were presented in Table 1. The negative values of free energy (∆G) suggested that the interaction between QAS and CMS was exothermic and spontaneous. It is notable that the ∆G became more negative as the DS of CMS increased and the Mw of CMS decreased, which was in line with the variation of binding

enthalpies (∆H) and the binding affinity (Ka) (i.e. more negative ∆H and high Ka), suggesting that the titration of QAS with CMS was more favoured with high DS and low Mw. CMS with higher DS was more negatively charged, which facilitated stronger electrostatic interactions between QAS and CMS molecules. The less favourable binding of CMS to QAS at higher Mw was probably due to the presence of steric hindrance which prevented the electrostatic interaction between CMS and QAS molecules. Therefore, CMS with higher DS and lower Mw may benefit to fabricate the nanocapsules with compactness structural properties. 3.4. Loading of BSA into CMS/QAS/CMS nanocapsules BSA was loaded into the nanocapsules through the alternated deposition of cationic QAS and anionic CMS onto the surface of colloidal BSA driven by electrostatic interactions, as schematically presented in Fig. S2. BSA (pI ~4.7) was positively charged while CMS was negatively charged at pH=2.9, so that the addition of CMS to BSA would lead to the formation of stable BSA/CMS complexes via strong electrostatic interactions. Nanocapsules with multilayers possessing different surface charge and composition were then prepared through the alternate stacking of positively charged QAS and negatively charged CMS to the BSA/CMS complexes. The EE of BSA shown in Fig. S3(a) indicated that nanocapsules fabricated by CMS with higher DS displayed the higher encapsulation efficiency. The highest EE of BSA was achieved (45.52%) when the DS of CMS was 0.138. This was reasonable since higher DS resulted in stronger electrostatic interaction between CMS and BSA. In addition, loading BSA into the nanocapsules was also promoted by CMS with lower Mw (Fig. S3(b)), while the lowest Mw of 4.373×106 g/mol displayed the increased encapsulation efficiency of 23.48%, which could be attributed to the fact that CMS with lower Mw displayed weaker steric hindrance, so that the carboxyl groups of CMS

was more accessible to BSA, leading to stronger electrostatic interactions between CMS and BSA and ultimately more BSA loaded into the nanocapsules. Therefore, higher DS and lower Mw of CMS may help to fabricate nanocapsules with higher EE of BSA. 3.5. Nanoscale structure of nanocapsules SAXS is considered as a useful technique to characterize the nano-scale structure of nanomicelles or nanocapsules (Zhu, Li, Chen & Li, 2013). The fractal structure, which is determined from α according to the power-law equation: I ~ q-α (I is the SAXS scattering intensity and α was calculated from the slope of regression line of double logarithmic SAXS curves), was used to characterize the compactness of the scattering objects. In addition, sample with higher α indicated higher compactness of the scattering objects (Zhang, Li, Liu, Xie & Chen, 2013). The double-logarithmic SAXS patterns of the nanocapsules at different pH, presented in Fig. 3, showed that the engineered nanocapsules possessed certain fractal structures only at pH 1.2 rather than at pH 6.8 or 7.2. The engineered nanocapsules displayed more compact fractal structure with α of 1.259 when DS of CMS (Mw=2.345×107 g/mol) was 0.041, while displayed loose fractal structure when DS increased to 0.067 and 0.138 (Fig. 3). It was thus concluded that CMS with lower DS had advantages in engineering nanocapsules with more compact structures. It indicated that DS of CMS is an important factor influencing the nano-scale structure of the nanocapsules. Despite that CMS with lower DS could promote the compactness of the nanocapsules at pH=1.2 (the pH of the simulated gastric fluid), the structure of the nanocapsules with lower DS still loose at pH=6.8, which is not helpful for the protection of proteins and peptides in the upper gastrointestinal tract. To further investigate the effect of the structure change of nanocapsules on its control release

property, CMS (DS 0.041) with different molar mass was used to construct the LbL assembly nanocapsules. From Fig 3, when the Mw of CMS was larger than 4.373×106 g/mol the engineered nanocapsules possessed certain fractal structures only at pH 1.2 rather than at pH 6.8 or 7.2, which could be related to the protonation of CMS in the outer layer of nanocapsules at pH = 1.2. The carboxylate groups (–COONa) of the outer layer are converted to carboxyl groups (–COOH), thus reducing the solubility of the CMS and limiting the gastric fluid penetration into the nanocapsules and making the nanocapsules compact. At higher pH (pH 6.8 of SIF and pH 7.2 of SCF), the carboxyl groups are deprotonated and ionized, thus favoring hydration, swelling of nanocapsules, thus leading to loose structure of the nanocapsules. However, nanocapsules fabricated by CMS with Mw of 4.373×106 g/mol also possessed fractal structures at pH 6.8 and 7.2, indicating the formation of more compact structures compared with that of nanocapsules prepared from CMS with higher Mw. This could also be confirmed by the increased α value of the nanocapsules fabricated by CMS with lower Mw, revealing enhanced compactness. The compact structures of this nanocapsule at pH 6.8 could limit the release of the entrapped protein and be helpful for the protection of the protein in the SIF. Thus, the nanocapsules, fabricated by CMS with lower DS and Mw (DS= 0.041, Mw=4.373×106 g/mol) and QAS (DS= 0.261, Mw=7.407×103 g/mol), had more compact structure, which could protect the proteins form degradation in the upper gastrointestinal tract and benefit to target to colon with sustained release behaviour. 3.6. Morphology of nanocapsules The TEM images of the nanocapsules at different pH conditions are showed in Fig. 4. The nanocapsules were orderly assembled by charged polyelectrolytes alternately deposited onto the BSA surface and showed a core-shell structure. When the nanocapsules were incubated in PBS at

pH of 1.2, the outer CMS layer was protonated and reduced the solubility of the CMS, limiting the PBS penetration and the swelling of the nanocapsules and then leading to smaller capsule size of the nanocapsules. Therefore, the nanocapsules at pH 1.2 remained clearly spherical structure (Fig. 4, left panel). At higher pH of 6.8, the carboxyl groups are deprotonated and ionized, thus favouring the outer CMS layer hydration and the PBS penetration into the nanocapsules, leading to the swelling and larger size of nanocapsules. At pH 6.8, CMS was negatively charged and interacted with QAS so that the nanocapsules remained integral in most of the case (Fig. 4, middle panel). As pH further increased to 7.2, CMS and QAS became soluble so that the CMS and QAS layers were detached from the nanocapsules, leading to small particle sizes. (Fig. 4, right panel). From Fig. 4(a), the nanocapsules assembled by CMS with lower DS showed more swelling and larger size compared with the nanocapsules assembled by the CMS with higher DS. CMS with lower DS could assemble with QAS to form nanocapsules with more compact nano-scale structure and more tolerated to slightly acidic pH conditions (Fig. 3). The nanocapsules assembled by CMS with DS of 0.041 showed a relative intact core-shell structure even in the SCF at pH 7.2. However, as the DS of CMS increased, some of the shell dissociated from capsules at pH 6.8 and 7.2 owing to the looser structure of the nanocapsules and easy solubilisation of the CMS/QAS layer. CMS with low Mw easily interacted with QAS (Fig. 2 and Table 1), and the engineered nanocapsules displayed more compact nano-scale structures (Fig. 3) with higher EE of BSA (Fig. S3). As shown in Fig. 4(b), nanocapsules fabricated by CMS with the higher Mw more easily swelled so that fragments detached from the nanocapsules and disintegrated nanocapsules were detected as the pH increased. From the results of Fig. 3, the interaction between CMS and QAS enhanced as the Mw of the CMS decreased when the nanocapusles transferred from SGF to the SIF

and then to SCF. Thus, when the Mw of CMS was 4.373×106 g/mol, the nanocapsules were more compact and resistant to pH changes resulting intact structure at pH 6.8 and 7.2. As discussed above, we proposed BSA-loaded nanocapsules assembled by CMS with the lower DS and Mw would undergo structural conversion when transited through human gastrointestinal tract, as schematically presented in Fig. 5. At pH 1.2, the protonation of CMS in the outer layer of nanocapsules reduced the solubility and limited the nanocapsules swelling or collapse, thus keeping the intact core-shell structure of the nanocapsules. At higher pH (pH 6.8), the carboxyl groups of CMS are deprotonated and ionized, favouring the shell layer solubilisation which would promote the BSA release. Meanwhile, the enhanced interaction between BSA, CMS and QAS favoured a relative compact and stable structure (the fractal structures showed in the left of Fig. 3), which hindered BSA release from the nanocapusles. However, the shell layer of the nanocapusles could be degraded by the pancreatic α-amylase when the nanocapsules were transferred to the SIF which could trigger the BSA release in SIF. When the nanocapsules transited through the upper gastrointestinal tract and reached colon (pH 7.2), the decreased α value of the nanocapsules (the left in Fig. 3) made the engineered nanocapsules remain its stability and sustainedly released BSA from the nanocapsules. 3.7. In vitro release of BSA 3.7.1. Effect of DS and Mw of CMS on BSA release For verification the release mechanism of the nanocapsules, the release behaviour of BSA from nanocapsules constructed by CMS with different DS and Mw and QAS in simulated human gastrointestinal tract was investigated. As displayed in Fig. 6, the BSA release from the nanocapsules can be regulated and controlled by altering the DS and Mw of the CMS layer.

From Fig. 6(a), the nanocapsules fabricated by CMS with different DS and QAS shown different release properties (shown in table S2). There was about 18.86% to 32.42% of the BSA released in the initial 2 h, and 27.34% to 36.76% in the following 4 h in SIF and a sustained release of 40.25% to 44.39% BSA in SCF. The BSA release in the simulated upper GIT decreased as the DS of the outer CMS layer decreased. The nanocapsules assembled by CMS with DS of 0.041 showed less release percentage with 55.61% of BSA in the SGF and SIF and more in SCF with 44.39% (p<0.05). Because the outer CMS layer is protonated at pH 1.2 and the nanocapsules showed more compact fractal structure when DS of CMS was 0.041, thus leading to less BSA release in SGF. When the nanocapsules were transferred to the SIF the outer CMS layer is deprotonated and ionized and the shell was degraded by the pancreatic α-amylase in SIF, resulting the increase of BSA release. It can be concluded that CMS with lower DS would be benefit to construct LbL assembled nanocapsules for delivering BSA to lower GIT. The Mw of CMS also affected the release of BSA from the nanocapsules. As indicated in Fig. 6(b), when the Mw of CMS was 2.345×107 g/mol, the release percentage was up to 18.86% within 2 h, whereas the one with Mw of 4.373×106 g/mol was reduced to 15.56%. The nanocapsules fabricated by CMS with lower Mw showed declined release of BSA, which could be ascribed to the more compact and stable core-shell structure of nanocapsules in the simulated GIT as discussed above. When the nanocapsules were transferred to the SIF, the nanocapusles assembled by the CMS with lower Mw of 4.373×106 g/mol still possessed the compact fractal structure which can inhibit the further swelling of the nanocapsules and the degradation of the shell layer by the pancreatic α-amylase, resulting in the limited BSA release. About 31.25% of the BSA released from the nanocapsules and 53.19% of BSA passed through the upper GIT and sustainedly released up to 26 h

in SCF. Therefore, a desirable nanocapsules with colon-specific delivery and sustained release behaviour could be achieved by adjusting the DS and Mw of the outer CMS layer. 3.7.2. BSA release after storage in beverage As discussed above, the BSA-loaded nanocapsule delivery system constructed with CMS with lower DS and Mw showed lower BSA release in the upper GIT. CMS with lower DS and Mw assembled more compact core-shell structure with QAS which can inhibit the swelling and degradation of the nanocapsules and therefore limited BSA release in SGF and SIF. Thus, CMS with DS of 0.041 and Mw of 4.373×106g/mol was considered as a desirable candidate for constructing a BSA-loaded nanocapsule delivery system with sustained release performance. To mimic the different condition of nanocapsules applied in liquid food systems, BSA-loaded nanocapsules were respectively added to the sprite (pH 3.02), apple vinegar (pH 3.71) and minute maid (pH 4.35) and then stored for 7 days. The in vitro BSA release from the nanocapsules in liquid food systems was investigated as previously described. The leakage percentage of BSA in beverage was shown in Table 2, which indicated that the leakage percentage of BSA increased as the storage time prolonged. Nanocapsules in sprite displayed the highest leakage of BSA (up to 8.71%) after storage, followed by apple vinegar (5.16%) and minute maid (1.30%), indicating that the beverage environment influenced the BSA release from the nanocapsules. The BSA release from the nanocapsules pre-stored in beverage for 7 days were presented in Fig. 6(c). All of the samples showed limited BSA release in upper gastrointestinal tract and colon targeting release. Based on the data shown in Table S3, it can be seen that the nanocapsules pre-stored in minute maid showed the minimum BSA release (33.04%) within the first 6 h in the upper GIT, followed by those pre-stored in apple vinegar (43.93%) and sprite (46.35%) (p<0.05).

However, all of these nanocapsules exhibited sustained release of 52.70% to 64.97% of BSA in SCF up to 26 h. The difference in BSA release from nanocapsules pre-stored in beverage may caused by the changes in the interaction between the CMS and QAS and different fractal structure of nanocapsules at different pH conditions. Based on the above release tests, it was concluded that CMS with lower DS and Mw may represent a most ideal potential candidate anionic polyelectrolyte in constructing nanocapsules as oral colon-specific sustained release delivery system for the proteins and/or polypeptides.

4. Conclusions Nanocapsules were fabricated by the negtively chaged CMS and the positively charged QAS through alternate layer-by-layer deposition onto the surface of BSA colloidal particles. The surface charged of CMS was pH dependent and had a great influence on the properties of the nanocapsules. Nanocapsules displayed more compact and stable core-shell structure when fabricated by CMS with lower DS and lower Mw during the transport from the upper GIT to the lower GIT because of the enhanced electrostatic interaction between CMS and QAS, which were confirmed by SAXS and TEM. In vitro BSA release experiments indicated that the nanocapsules constructed by CMS with lower DS and Mw of CMS displayed better colon-specific delivery and release performance with 33.04% BSA released in the simulated upper GIT and 64.97% BSA sustainedly released in SCF. Therefore, an ideal nanocapsule for bioactive ingredient delivery system targeting to the colon can be obtained by adjusting DS and Mw of CMS. These findings suggested that this kind of nanocapsules fabricated by CMS and QAS has a great potential to be exploited as tunable nanocarriers for bioactive polypeptides and/or proteins oral delivery.

Acknowledgments This work is supported by the National Natural Science Foundation of China (NSFC) (31271824), the National Key Research and Development Program of China(2016YFD0400401-3), NSFC-Guangdong Joint Foundation Key Project (U1501214), YangFan Innovative and Entepreneurial Research Team Project (No. 2014YT02S029), the Science and Technology Program of Guangzhou (201607010109), the Innovative Projects for Universities in Guangdong Province (2015KTSCX006) and the Fundamental Research Funds for the Central Universities (2015ZZ106).

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5

5

（ a1）

（ a2）

pH 2.9

0

4.2

3.5

5.0

6.8

7.2

pH 2.9

Zeta potential(mv)

Zeta potential(mv)

1.2 -5

-10

-15

5.0

6.8

7.2

1.2

-5 7

Mw=2.345×10 g/mol

DS=0.138 DS=0.067 DS=0.041

-20

4.2

3.5

0

7

Mw=1.990×10 g/mol 6

Mw=4.373×10 g/mol -10

-25

24 18

DS=0.261 DS=0.265 DS=0.283

(b1)

21

4

1.344×10 g/mol 3 8.779×10 g/mol 3 7.407×10 g/mol

(b2) 15

15

zeta-potential(mv)

zeta-potential(mv)

18

12 9 6

12

9

6

3

3 0 1.2

0

2.9

4.2

3.5

pH

5.0

6.8

7.2

1.2

2.9

3.5

4.2

5.0

6.8

7.2

pH

Fig. 1 ζ-potential of CMS and QAS under different pH conditions. (a1) CMS with different DS (Mw= 2.345×107 g/mol); (a2) CMS with different Mw (DS=0.041); (b1) QAS with different DS (Mw=7.407×103 g/mol); (b2) QAS with different Mw (DS=0.261)

0

Enthalpy(KJ/mol)

Enthalpy(KJ/mol)

(b)

(a)

0

-5

DS=0.041 DS=0.067 DS=0.138

-10

-15

-5

-10

-15

0

5

10

15

20

6

4.373×10 g/mol 7 1.990×10 g/mol 7 2.345×10 g/mol 0

5

10

15

20

Injection number

Injection number

Fig. 2 The integrated enthalpy the titration of CMS with different (a) DS and (b) Mw into QAS (Mw= 7.407×103 g/mol, DS=0.261).

pH=6.8 pH=7.2

0.1

1

-1

q(nm )

7

pH=6.8 pH=7.2

0.1

-1

q(nm )

1

pH=1.2 pH=6.8 pH=7.2

-1

q(nm )

1

6

α=1.612

7

0.1

1

-1

q(nm )

Mw=1.990×10 g/mol

α=1.561 Relative Intensity(a.u.)

Relative Intensity(a.u.)

pH=1.2

pH=7.2

0.1

1

-1

pH=1.2

pH=6.8

q(nm )

Mw=2.345×10 g/mol α=1.259

α=1.259

Relative Intensity(a.u.)

pH=7.2

pH=1.2

Mw=4.373×10 g/mol pH=1.2

Relative Intensity(a.u.)

pH=1.2 pH=6.8

0.1

α=1.120

Relative Intensity(a.u.)

Relative Intensity(a.u.)

α=0.791

DS=0.041

DS=0.067

DS=0.138

α=1.058 pH=6.8 α=0.976

0.1

pH=7.2

-1

1

q(nm )

Fig. 3 SAXS analysis of the fractal structures of the nanocapsules fabricated by QAS (DS= 0.261, Mw=7.407×103 g/mol) and CMS (Mw=2.345×107 g/mol) with different DS or CMS (DS=0.041) with different Mw at different pH.

Fig. 4 TEM images of the nanocapsules at different pH conditions. (a) Nanocapsules fabricated by CMS (Mw = 2.345×107) with different DS; (b) Nanocapsules fabricated by CMS (DS=0.041) with different Mw

Fig. 5 Schematic diagram of nanocapsule changes transiting through human gastrointestinal tract.

100

SGF

SGF

SIF

SCF Cumalative release(%)

Cumalative release(%)

80

100

(a) 60

40

DS=0.138 DS=0.067 DS=0.041

20

SIF

SCF

80

(b)

60

40 7

Mw=2.345×10 g/mol 7

Mw=1.990×10 g/mol

20

6

Mw=4.373×10 g/mol

0

0

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

Time(h)

Time(h)

SGF

100

SIF

SCF

Cumalative release(%)

80

(c)

60

40

Minute maid Apple vinegar Sprite

20

0 0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

Time(h)

Fig. 6 In vitro BSA release from nanocapsules assembled by (a) CMS (Mw = 2.345×107 g/mol) with different DS, (b) CMS (DS=0.041) with different Mw, and (c) CMS (DS= 0.041, Mw of 4.373×106g/mol) pre-stored 7 days in different beverage.

Table 1 Thermodynamic parameters of QAS titrated with CMS with various DS and Mw. Mw (g/mol)

4.373×106

1.990×107

2.345×107

2.345×107

2.345×107

DS

DS=0.041

DS=0.041

DS=0.041

DS=0.067

DS=0.138

Ka(M-1)

6.129×103

2.610×103

1.001×101

1.581×105

1.040×106

△H(kJ/mol)

-1983

-1682

-553.6

-778.9

-1020.3

△G(kJ/mol)

-41.9

-36.19

-11.68

-30.2

-35.25

Table 2 The BSA leakage properties of the nanocapsules in different beverage leakage percentage% time

Sprite

Apple vinegar

Minute maid

(pH 3.02)

(pH 3.71)

(pH 4.35)

1d

1.42±0.13c

0.98±0.08c

0.65±0.09c

4d

5.03±0.07b

2.39±0.12b

0.94±0.06b

7d

8.71±0.14a

5.16±0.15a

1.30±0.08a

Values followed by the same letters within a column are not significantly different (n = 3, P < 0.05)