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Entrapment of bacterial cellulose nanocrystals stabilized Pickering emulsions droplets in alginate beads for hydrophobic drug delivery
Colloids and Surfaces B: Biointerfaces 177 (2019) 112–120
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Entrapment of bacterial cellulose nanocrystals stabilized Pickering emulsions droplets in alginate beads for hydrophobic drug delivery
T
Huiqiong Yana,b, Xiuqiong Chena,b, Meixi Fengb, Zaifeng Shib, Wei Zhangb, Yue Wangb, ⁎ Chaoran Keb, Qiang Lina,b, a Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou, 571158, China b Key Laboratory of Water Pollution Treatment & Resource Reuse of Hainan Province, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou, 571158, Hainan, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacterial cellulose nanocrystals Pickering emulsions Alginate composite beads Interfacial assembly Encapsulation Sustained release
In this work, the interfacial assembly of amphiphilic bacterial cellulose nanocrystals (BCNs) by Pickering emulsion method was proposed to improve the compatibility between the alginate and hydrophobic drug. BCNs prepared by sulfuric acid hydrolysis of biosynthesized bacterial cellulose was used as the particulate emulsifiers, whereas the model drug, alfacalcidol, dissolved in CH2Cl2 was used as the oil phase. The oil-in-water Pickering emulsions were prepared by ultrasonic dispersion method and then they were well dispersed in alginate solution. Ultimately, the drug-loaded alginate composite beads were successfully fabricated by external gelation. The characterization results revealed that BCNs possessed good colloidal property and could form flocculated fibril network, which was beneficial to stabilize Pickering emulsions. The irreversible adsorption of BCNs at the oilwater interface could make the Pickering emulsions preserve the droplets against coalescence and Ostwald ripening when they were dispersed in alginate solution. The interfacial assembly of amphiphilic BCNs and the hydrogel shells of the alginate composite beads formed by external gelation achieved the loading and sustained release of alfacalcidol. The release curves were well fitted by Korsmeyer Peppas model and the release mechanism of alfacalcidol from the composite beads was attributed to non-Fickian transport. In addition, the resultant alginate composite beads exhibited low cytotoxicity and good capabilities for osteoblast differentiation.
1. Introduction Over the last decade, much more attentions have been paid to alginate hydrogel beads for their extensive applications in food industry, drug delivery and tissue engineering [1–3]. Since alginate has unique advantages such as aqueous-solubility, renewability, non-toxicity, biocompatibility, biodegradability and non-immunogenicity [4,5], it is suitable to encapsulate large molecules or hydrophilic materials at high efficiencies [6]. Various cargos including living cells, protein drugs, enzymes, food ingredients and catalysts have successfully been encapsulated in alginate hydrogel beads [7]. However, when it comes to hydrophobic drugs or compounds, the loading capacity is low because of the poor affinity of alginate for the hydrophobic drugs or compounds, resulting from the large amounts of free hydroxyl and carboxyl groups along alginate backbone [8]. This inherent drawback of alginate was commonly overcome by chemical modification involving the
introduction of hydrophobic groups onto its hydrophilic backbone, blending with other polymers and incorporating clays [9,10]. To date, the encapsulation of Pickering emulsions in alginate hydrogel beads to improve the loading capacity of hydrophobic drugs is rarely reported, especially when using bacterial cellulose nanocrystals (BCNs) as the Pickering emulsifiers. BCNs are prepared by acid hydrolysis of bacterial cellulose to remove amorphous regions to obtain rod-like crystalline forms of cellulose, which are high modulus and tensile strength, environmentally friendly, nontoxic, edible, degradable and biocompatible [11,12]. Especially, BCNs prepared by sulfuric acid hydrolysis can be endowed with anionic sulfate half-ester groups on their surface, which could be further oxidized by hydrogen peroxide, thereby resulting in the uniform aqueous dispersion [13,14]. BCNs are considered to be amphiphilic overall owing to the presence of high density of hydroxyl groups on the surface [15], while the hydrophobic interactions come from the
⁎ Corresponding author at: Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou, 571158, China. E-mail addresses: [email protected], [email protected] (Q. Lin).
https://doi.org/10.1016/j.colsurfb.2019.01.057 Received 29 July 2018; Received in revised form 8 January 2019; Accepted 26 January 2019 Available online 29 January 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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composite beads were also investigated. As far as we know, this new drug-loading technique with high efficiency, sustained-releasing performance and good bioactivity via the Pickering emulsion interfacial assembly of amphiphilic BCNs in bio-pharmaceutical field has not been reported.
crystalline organization along with extensive hydrogen bonding of polymer chains [16]. Therefore, their amphiphilic properties could be applied to stabilize surfactant-free Pickering emulsions. The self-assembly of BCNs at liquid–liquid interfaces has been well studied, which could be applied to immobilize hydrophobic drugs or compounds. In this case, BCNs spontaneously localize at the interface to minimize the helmholtz free energy [17–19]. Pickering emulsions involve the irreversible adsorption of solid colloidal particles at the oil-water interface and stabilize the emulsion droplets against coalescence by forming a mechanically robust monolayer [11]. The irreversible adsorption requires a much higher energy to remove the colloidal particles from the oil–water interface, making Pickering emulsions exhibit peculiar advantages over conventional emulsions in terms of outstanding stability against coalescence and Ostwald ripening [20]. In contrast to conventional emulsions stabilized by surfactants, Pickering emulsions have a number of merits, such as more robust formulations, reduced foaming problems and lower toxicity [21], thus being widely used in biomedical, health and cosmetic fields where the use of surfactants is undesirable [19]. It is reported that inorganic or organic particles of silica, clay, calcium carbonate, hematite, polystyrene, and microgels, ranging in size from nanometers to micrometers, could all stabilize Pickering emulsions [11]. But the increasing legal and consumer requirements of Pickering emulsions such as non-toxicity, biocompatibility and high ecological accept-ability make BCNs become the ideal candidate for Pickering emulsifiers. In comparison to other solid lipid particles, the highly enhanced emulsions’ stabilization against coalescence and Oswald ripening by BCNs makes Pickering emulsions be able to conserve the droplets under high concentration of dispersed phase. Alfacalcidol, a kind of hydrophobic drug, is an analogue of vitamin D used for supplementation in humans, which is of great importance in health and disease prevention [2]. It is considered to be a more useful form of vitamin D supplementation, mostly due to much longer half-life and lower kidney load [22]. It is well known that the success of a medical treatment depends not only on the pharmacodynamic activity of a drug, but also on the availability of the active agent at the site of action in the human body. Although alfacalcidol could be loaded in CH2Cl2/water Pickering emulsions stabilized by BCNs with long-term stability towards coalescence, the emulsion droplets may not withstand the influences of physiological environment, owing to the possible change of size and surface properties of BCNs in physiological medium [20]. Several methods could be applied to ensure mechanical robustness and stability of the emulsions, including gel trapping technique, locking particles together by polyelectrolyte and chemical cross-linking of individual building blocks [21]. Among these methods, the gel trapping technique by calcium alginate seems to be simple and feasible. As previously mentioned, the drug-loaded Pickering emulsions could preserve the droplets when they are dispersed in bulk alginate solution, which could be crosslinked by multivalent cations, such as Ca2+, to form hydrogel beads by chelation [23,24]. Therefore, alginate hydrogel beads with excellent properties in combination with the Pickering emulsions may be a promising delivery vehicle to protect alfacalcidol from harmful conditions (such as immune responses) and to restrict its release in a timely manner, thus improve its bioavailability [25–27]. In this study, we combined the high encapsulating performance of alginate hydrogel beads with the ideal emulsifying property of BCNs to develop a new formulation with high loading capacity of hydrophobic drugs. The loading of hydrophobic alfacalcidol in alginate beads was achieved via the emulsification of eco-friendly Pickering emulsions and the immobilization of them within hydrogel matrix of alginate by external gelation as shown in Scheme 1. After removing CH2Cl2, hydrophobic alfacalcidol and BCNs could be deposited into the network of alginate hydrogel, similar to common emulsion solvent evaporation method [28]. The physicochemical properties and emulsifying performance of BCNs were assessed, and the morphology, encapsulation efficiency, release performance and cytocompatibility of alginate
2. Experimental 2.1. Materials Bacterial cellulose was produced by Acetobacter xylinum (CGMCC5173) obtained from China General Microbiological Culture Collection Center according to previous method [13]. Sodium alginate (MW = 465 kDa, G/M = 1.5), alfacalcidol, sulfuric acid, hydrogen peroxide, CH2Cl2, CaCl2 and other reagents were purchased from Sigma-Aldrich (New York, USA). They were analytical grade and were used without further purification. 2.2. Synthesis of BCNs In this work, acid hydrolysis of BC was performed to synthesize BCNs according to previous method with some modifications [29,30]. About 10 g of BC was dispersed in 200 mL of 50 wt% sulfuric acid under vigorous mechanical stirring. The hydrolysis was performed at 45 °C for 3 h, and the mixture was diluted five-fold to quench the hydrolysis reaction. Then 20 mL of 30 wt% aqueous hydrogen peroxide was added to bleach and further oxidize BCNs. Afterwards, the resultant suspension was centrifuged at 9000 rpm for 15 min to separate the crystals, which were washed and treated ultrasonically for 20 min to eliminate excess acid. At last, the precipitate was further dialyzed against deionized water for 7 d using a dialyzing membrane with a molecular weight cutoff of 3500 to remove residual sulfuric acid as well as other low-molecular weight impurities. The resultant BCNs suspension was stored in a refrigerator for further use. Simultaneously, a specified amount of BCNs aqueous suspensions was lyophilized to calculate BCNs content in the suspensions and for further characterization. Based on the weight of BC, the yield of BCNs was 62%. 2.3. Characterization of BCNs The micro-morphology of dry BCNs was observed by using a Hitachi S-3000N (Japan) scanning electron microscope after fixing the samples on a brass holder and coating them with gold. And the structure of BCNs was observed by a JEM 2100 TEM (JEOL Co., Japan) at an acceleration voltage of 200 kV. Transmission electron microscope (TEM) images of samples were obtained by placing a few drops of the aqueous dispersion of the samples on a carbon-coated copper grid, and evaporating the solvent prior to observation. The molecular weight of the BCNs was determined using the Mark–Houwink equation according to previous report [31]. The structure and crystallinity of BCNs were examined by using a Bruker T27 FT-IR spectrophotometer (Germany) and a Bruker AXS/D8 advance X-ray diffractometer system (Germany) with Cu-Kα radiation (λ = 0.154 nm). The freeze-dried samples were mixed with KBr compressed into semitransparent KBr pellets before the FT-IR measurement. The XRD measurement was operated at 40 kV and 100 mA in a step scan mode, which performed over a 2θ range of 5-60° with the scanning speed of 0.025°/s. The crystallinity index (CrI) of BCNs was then calculated by using the following empirical equation [32,33].
CrI =
I200 − Iam × 100% I200
(1)
where I200 is the intensity value of crystalline cellulose, and Iam is the intensity value of amorphous region material (2θ = 18°). 13 C solid state NMR experiment was performed at 25 °C using a Bruker AVANCE400 WB spectrometer at resonance frequencies of 113
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Scheme 1. Schematic illustration of the preparation of the drug-loaded alginate composite beads by entrapping BCNs stabilized Pickering emulsions droplets in alginate beads.
100.6 MHz for 13C nuclei. The 13C CP/MAS NMR spectrum was recorded under observation conditions, a CP contact time of 3 ms, a repetition time of 5 s, a spinning speed of 7 KHz and the numbers of scans were 4 k. In addition, the size and zeta potential of BCNs were further determined by DLS with a Malvern Nano-ZS90 Zetasizer (UK) at a scattering angle of 90° at 25 °C, employing an (He-Ne) argon laser (λ = 633 nm). Refractive indices of 1.47 for BCNs and 1.33 for deionized water were used. In addition, the viscosity value of deionized water was 0.894 mPa s at 25 °C. The size of the 0.9 wt% BCNs suspensions was obtained from the Cumulant analysis of its correlation function by using the CONTIN analysis. The zeta potential of 0.9 wt% BCNs suspensions was calculated from the electrophoretic mobility (u) using the Smoluchowski relationship (ζ = ηu/ε) under the assumption that κα ≪1, where η is the solution viscosity, ε is the dielectric constant of the medium, and κ and α are the Debye−Hückel parameter and the particle radius, respectively. Zeta potentials were averaged over 20 runs. The viscosity of the 0.9 wt% BCNs suspensions was measured at 25 °C using a TA DHR Rheometer (USA).
solution were collected with a Leica DMRX optical microscope (Switzerland) equipped with a high performance digital camera. The emulsion droplets were placed directly onto a glass microscope slide and viewed under 10× and 40× magnification.
2.5. Fabrication of drug-loaded alginate composite beads The fabrication of drug-loaded alginate composite beads, abbreviated as SA/BCNs CBs, was illustrated in Scheme 1. Briefly, the above alginate solution containing drug-loaded Pickering emulsion droplets was dripped into 0.25 M CaCl2 solution. 30 min later, the drug-loaded SA/BCNs CBs were formed and washed with distilled water, and then vacuum dried at 60 °C. The alginate composite beads encapsulating the drug-loaded Pickering emulsions stabilized by 0.3, 0.6 and 0.9 wt% BCNs were respectively abbreviated as SA/BCNs-0.3 CBs, SA/BCNs-0.6 CBs and SA/BCNs-0.9 CBs. For comparison, the similar drug-loaded alginate beads, abbreviated as SA beads, with the absence of BCNs were also prepared. The surface morphology of the composite beads was examined using a Hitachi S-3000N (Japan) scanning electron microscope after fixing the samples on a brass holder and coating them with gold.
2.4. Preparation and characterization of drug-loaded Pickering emulsions CH2Cl2, as the least toxic of the simple chloro-hydrocarbons with high dissolving ability, was chosen to disperse alfacalcidol, which was used as the oil phase in the preparation of Pickering emulsions. The drug-loaded Pickering emulsion with an oil/water ratio of 1:9 (v/v) was prepared by ultrasonic dispersion with a JY92-II DN ultrasonic device (China). Actually, 1 mL of CH2Cl2 solution dissolving 50 mg alfacalcidol was added to 9 mL of BCNs suspension with various concentrations (0.3, 0.6 and 0.9 wt%) to form the emulsions with the aid of ultrasonication. After the CH2Cl2 and BCNs were respectively stained with Nile red and fluorescent brightener, the microstructure of Pickering emulsion droplets was observed by using a reconstructive Nikon Ti-S fluorescent microscope (Japan). The imaging of emulsion droplets was performed with the fluorescence successively excited at 492 nm and 408 nm. Meanwhile, the size of the emulsion droplets was measured by laser light diffraction using a Malvern Mastersizer 2000 apparatus (UK) equipped with a He–Ne laser operating at 633 nm. The average droplet diameter was taken to be the volume-weighted average diameter (d4/3) from triplicate measurements, which could be calculated by the following equation [18].
d4/3 =
∑ ni × di4 ∑ ni × di3
2.6. Loading and in vitro release of alfacalcidol The encapsulation of alfacalcidol into the alginate hydrogel matrix was ultimately achieved by external gelation [34]. The residual alfacalcidol in the gelling solution and washing solution was in the supernatant which could be extracted by chloroform. The encapsulation efficiency (EE) could be calculated using the following equation.
Encapsulation Efficiency (EE ) total alfacalcidol − residual alfacalcidol = × 100% total alfacalcidol
(3)
The release of alfacalcidol from the composite beads was performed in pH 7.4 PBS. 20 mg of the dried composite beads was immersed into 50 mL PBS in centrifuge tube. 40 mL of the solution was replaced with the same volume of fresh PBS by centrifugation at different time intervals, which could avoid the influences of the saturated solutions. The released alfacalcidol in the supernatant for each time interval was extracted by 40 mL chloroform. The chloroform extract was placed in an ice water bath to avoid the volatility of chloroform that would affect alfacalcidol concentration. The amount of alfacalcidol in chloroform was measured by using a Shimadzu UV-1800 (Shimadzu, Kyoto, Japan) UV–vis spectrophotometer at 265 nm with a calibration curve prepared by standard concentration of alfacalcidol in chloroform ranging from 10 to 50 μg/mL [2]. The drug release procedure was performed in triplicate to calculate the standard deviation.
(2)
where ni is the number of the droplets with the diameter di. Subsequently, 0.8 g of sodium alginate was dissolved in 50 mL water with vigorous stirring for 4 h to form a homogenous solution. Then 10 mL of the drug-loaded Pickering emulsion was well dispersed in 40 mL of 2% (w/v) alginate solution under gentle mechanical stirring. The optical micrographs of drug-loaded Pickering emulsions in alginate 114
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of short and rod-like microfibrils of BCNs, attributed to the removal of the amorphous components and the cleavage of the crystalline microfibrils. BCNs revealed very thin layers in their SEM image and bundles or ribbons in their TEM image, indicating the presence of interfibrillar hydrogen bonds. According to the Mark-Houwink equation, the molecular weight of the BCNs was 41.7 kDa. FT-IR analysis is a useful method for elucidating the specific functional groups or chemical bonds in BCNs. The FT-IR spectra of BC and BCNs revealed the similar profiles as shown in Fig. 2a. However, BCNs showed the additional weak peaks at 1732.65 and 862.11 cm−1 respectively for the bending vibration of −COOH and stretching vibration of CeO [18], indicating the further oxidation of pyranose ring by hydrogen peroxide. Additionally, the peak of hydroxyl stretching at 3350.45 cm−1 in the spectrum of BC became sharp and made a blue shift to 3411.17 cm−1 in the spectrum of BCNs. The results indicated that sulfuric acid hydrolysis could destroy the original intramolecular hydrogen bonding of BC [3], promoting the cleavage of the glycosidic bonds. The change of crystal structure of BCNs during the hydrolysis process was also analyzed by XRD. As shown in Fig. 2b, both of the BC and BCNs exhibited three crystalline peaks at 2θ = 14.7°, 16.8° and 22.7° corresponding to the (-110), (110) and (200) crystal planes, as well as an amorphous background at about 2θ = 18°, indicating the typical cellulose I structure, consistent with the result reported by Moriana et al. [35]. But the intensity of the BCNs diffraction peaks significantly increased in contrast to that of BC, confirming the removal of the amorphous components during the hydrolysis process. According to Eq. (1), the CrI of BC and BCNs were 75.1% and 89.6%, which was close to that reported in previous study for BC produced from pretreatment WWCJ medium (75.42%) [36]. 13C CP/ MAS solid-state NMR experiments are actually the combination of cross polarization, magic angle spinning and high power decoupling. Fig. 2c showed the representative 13C CP/MAS spectrum of solid BCNs. And the 13 C peaks of BCNs had been marked in 13C CP/MAS spectrum. BCNs have board resonances that extend over chemical shift ranges of δ = 110˜100, 92˜79 and 57˜67 ppm attributed to the C1, C4, and C6 signals, respectively. To note, the 13C peaks ranging from 86 to 79 ppm assigned to the C4 signals of amorphous cellulose disappeared, indicating the high crystallinity for BCNs, which was consistent with the XRD results. Besides, the size, zeta potential and viscosity of BCNs at the concentration of 0.9 wt % were measured by DLS and rheometer. BCNs exhibited a relatively narrow size distribution nearly involving one range, and their average size was 259.6 nm with the PDI of 0.26 (Fig. 2d). The relatively narrow size distribution and low average size possibly resulted from the elimination of amorphous components and the cleavage of the glycosidic bonds, which was in accordance with FTIR and XRD analysis. Additionally, from Fig. 2e, BCNs showed negative zeta potentials for the presence of carboxyl groups on the pyranose ring. To note, BCNs exhibited relatively high zeta potential at about -34.8 mV, which could be well-dispersed in aqueous solution because of the strong electrostatic repulsion forces [3]. Furthermore, the BCNs
2.7. Cell viability of SA/BCNs CBs Cytotoxicity of the SA/BCNs CBs was assessed by the use of mouse osteoblastic MC3T3-E1 cells which were cultured with α-MEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin under the condition of 37 °C, 5% CO2, 95% air and 100% relative humidity, and the medium was changed every 2 days. The sterilized SA/BCNs CBs with serial concentrations were blended with cell suspension at a cell density of 5 × 104 cells/cm2, which were placed into 24-well culture plates. The cell counting kit-8 (CCK-8) assay was used to evaluate cell viability on the SA/BCNs CBs, and the obtained optical density was proportional to the number of living cells. After 2 days and 7 days incubation, the culture medium was replaced by 500 μL fresh culture medium with 50 μL CCK-8 solution for further 4 h incubation at 37 ℃. 100 μL of the solution from each well was transferred to new 96well cell culture plates and the optical density (OD) of each well was read using a microplate reader machine (Bio-rad X-mark, America) at a wavelength of 450 nm. Simultaneously, the cells cultured on the tissue culture polystyrene (TCP) was served as the blank control group. Additionally, the culture medium containing 90% LG-DMEM, 10% fetal bovine serum, 1% penicillin/streptomycin, 10 mmol/L β-glycerophosphate, 0.1 μmol/L dexamethasone and 50 μg/mL ascorbic acid used as an osteogenic medium to achieve the cell differentiation under the condition of 37 °C, 5% CO2, 95% air and 100% relative humidity, and the medium was changed every 3 days. The cell differentiation of MC3T3-E1 cells on the SA/BCNs CBs was determined by alkaline phosphatase (ALP) expression using a QuantiChrom ALP kit. After 10 days incubation, the SA/BCNs CBs with cells were rinsed in PBS and then lysed in a 0.2% (w/w) Triton X-100 in PBS under the ice-bath environment for 30 min. After high speed centrifugation at 12,000 rpm for 5 min at 4 °C to discard cell debris, 50 μL of supernatant was transferred to a new 96-well plate for ALP determination. Moreover, MC3T3-E1 cells cultured on the TCP was served as the blank control group. And the relative ALP activities for all samples were standardized by the blank control group. 2.8. Statistical analysis All data presented as means ± standard deviation. Single-factor ANOVA was performed by SPSS software to analyze the variables, and a value of p < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Characterization of BCNs It is reported that sulfuric acid hydrolysis can not only induce the removal of the amorphous components of BCNs but also endow BCNs with anionic sulfate half-ester groups [29,33]. As shown in Fig. 1, the sulfuric acid hydrolysis of bacterial cellulose resulted in the formation
Fig. 1. Scanning electron microscope (SEM) image (a) and transmission electron microscope (TEM) image (b) of BCNs. 115
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Fig. 2. (a) FT-IR spectra and (b) X-ray diffractograms of BC and BCNs. (c) and (f) viscosity of 0.9 wt% BCNs suspensions.
13
C CP/MAS spectrum of solid BCNs. (d) Size distribution, (e) zeta potential distribution
Fig. S1. Under bright field, the emulsion droplets exhibited good spherical shape, indicating the good emulsifying performance of BCNs. Nevertheless, under dark field, the emulsion droplets appeared green for the Nile red excitation at 492 nm whereas the emulsion droplets appeared blue for the fluorescent brightener excitation at 408 nm. Since oil-soluble Nile red only existed in oil phase and appeared green for the excitation at 492 nm, emulsion type was confirmed to be oil-in-water type [11]. Additionally, it is observed that BCNs were adsorbed at the oil-water interface with few emerging in aqueous phase, indicating that BCNs could form stable three-dimensional flocculated fibril network via the coverage of themselves at the oil-water interface to resist coalescence [38]. The droplet diameter distribution of drug-loaded Pickering emulsions stabilized by BCNs with various concentrations is presented in Fig. S2. As d4/3 diameter is more sensitive to the presence of large droplets, it is regarded as the average droplet diameter in the experiment. Based on Eq. (2), d4/3 diameters of the drug-loaded Pickering emulsions
suspensions exhibited shear-thinning behavior with three regions against the shear rate, as shown in Fig. 2f. Similar viscosity behavior has also been reported in previous work [37]. The viscosity variation of the BCNs suspensions depended on the three-dimensional flocculated fibril network formed by hydrogen bonding interaction between BCNs and water molecules [38]. The results indicated that BCNs possessed good colloidal property and could form flocculated fibril network, which was beneficial to stabilize Pickering emulsions. 3.2. Characterization of drug-loaded Pickering emulsions Due to the amphiphilic property of cellulose chains [16], BCNs could emulsify CH2Cl2 solution of alfacalcidol to form the Pickering emulsions via ultrasonic dispersion method. The microstructure of emulsion droplets was observed by fluorescent microscope after fluorescence staining. The fluorescent images of the dyed Pickering emulsions stabilized by BCNs at a concentration of 0.9 wt % are shown in 116
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Fig. 3. Optical micrographs of drug-loaded Pickering emulsions stabilized by various concentration of BCNs in alginate solution: (a) 0.3 wt %, (b) 0.6 wt % and (c) 0.9 wt %.
rough and filiform surface morphology with some gaps. In addition, as observed in their cross-sections (Fig. 4c and f), the SA/BCNs CBs exhibited more pore structures than SA beads, further confirming the high encapsulation efficiency of CH2Cl2 solution of alfacalcidol. On the basis of the hydrogel shells formed by external gelation and the emulsification of amphiphilic BCNs, alfacalcidol was encapsulated inside the composite beads in the course of the preparation of the composite beads. According to Eq. (3), the EEs of SA beads, SA/BCNs0.3 CBs, SA/BCNs-0.6 CBs and SA/BCNs-0.9 CBs were respectively 54.2%, 80.3%, 89.6% and 91.4% as shown in Table 2, revealing good drug-loading capacity. It was worth noting that the EEs of 89.6% for SA/BCNs-0.6 CBs and 91.4% for SA/BCNs-0.9 CBs were not much difference, probably attributed to the sufficient coverage of BCNs at the oil-water interface during the preparation of the drug-loaded Pickering emulsions when BCNs concentration reached up to 0.6 wt%. The excess BCNs may have little effect on the drug-loading capacity.
stabilized by BCNs with the concentrations of 0.3, 0.6 and 0.9 wt% were respectively 18.5, 14.6 and 8.8 μm. It was obvious that the BCNs concentration had a distinct influence on the size of the emulsion droplets. With the increase of BCNs concentration, the size of the emulsion droplets decreased and became uniform, similar to previous report for the stabilization of Pickering emulsions [39]. The variation of emulsion droplets diameter with different concentrations of BCNs was attributed to the coverage of BCNs at the oil-water interface, which protected the emulsion droplets against coalescence by forming a flocculated fibril network [38]. It was the irreversible adsorption demanding a much higher energy to remove the BCNs from the oil–water interface that made the Pickering emulsions preserve the droplets against coalescence and Ostwald ripening when they were dispersed in alginate solution [20]. The optical micrographs of drug-loaded Pickering emulsions stabilized by different concentrations of BCNs in alginate solution are shown in Fig. 3. When the BCNs concentration was low, the formed flocculated fibril network was weak. The weak flocculated fibril network easily led to a certain degree of coalescence [19], thereby resulting in the big emulsion droplets, as shown in Fig. 3a. With increasing BCNs concentration, more and more BCNs were absorbed at the oil-water interface, which could cover a much larger area to reduce the size of emulsion droplets and prevent their coalescence [40]. Consequently, the droplets of the emulsions became small and uniform (Fig. 3b and c) when the BCNs concentration increased.
3.4. In vitro release studies The release of alfacalcidol was achieved through its diffusion out of the swollen beads. The alfacalcidol release profiles of the composite beads are presented in Fig. 5. All of the release curves showed a rapid release in the initial stage (0–2000 min) and then a slow release in the following stage. The alfacalcidol accumulation on the surface of the composite beads resulting from the poor compatibility between alginate solution and CH2Cl2 solution of alfacalcidol and the extensive water uptake properties of the alginate hydrogel could have been responsible for the initially rapid release [43]. The coverage of BCNs on the surface of alfacalcidol after the evaporation of CH2Cl2 (Scheme 1), which probably formed a mechanically robust layer, and hydrogel shells of the composite beads formed by external gelation may account for the slow release of alfacalcidol [11]. In addition, the electrostatic forces and intermolecular hydrogen bonds formed between BCNs and alginate may also retard the release of alfacalcidol [44,45]. It was worth noting that the release process had lasted for more than 6600 min, indicating good sustained release performance. In actual drug therapy, the prolongation of release time is beneficial to improve the drug efficacy and drug utilization [46]. Additionally, SA/BCNs CBs exhibited a better sustained release performance over SA beads due to the barrier of BCNs within the polymeric matrix and the sufficient coverage of BCNs on the surface of alfacalcidol. With the increase of BCNs concentration, the sustained release performance improved. To further investigate the release mechanisms, the release profiles of the alfacalcidol up to 60% were fitted using the following common kinetic models [47–49].
3.3. Morphology and encapsulation efficiency of drug-loaded composite beads As the alginate hydrogel beads were prepared by external gelation through dripping an alginate solution into a CaCl2 solution, the gelling occurred only on the surface of alginate droplets to form the hydrogel shells [41]. It was the hydrogel shells that could immobilize the drugloaded Pickering emulsions in alginate solution, thus achieving the loading and sustained release of alfacalcidol (Scheme 1). The digital photographs of the wet SA beads and SA/BCNs CBs as well as the SEM images of the dried composite beads are shown in Fig. 4. Both of the wet SA beads and SA/BCNs CBs exhibited uniformly spherical shapes (Fig. 4a and d). However, volume shrinkage occurred when water was evaporated form the wet composite beads during the drying process, resulting in the formation of rough and collapsed surface morphology (Fig. 4b and e). It was observed that the surface of the SA beads was relatively smooth over that of the SA/BCNs CBs, which should be ascribed to the barrier properties of BCNs [42]. The SA beads with low amount of CH2Cl2 solution could possess a uniform rate of water loss on their surface, generating relatively smooth surface morphology [9]. The poor compatibility of alginate solution and CH2Cl2 solution of alfacalcidol led to the low encapsulation efficiency. On the contrary, the emulsification of CH2Cl2 solution of alfacalcidol by interfacial assembly of amphiphilic BCNs could effectively improve the compatibility between alginate solution and CH2Cl2 solution of alfacalcidol, thereby enhancing the encapsulation efficiency. The evaporation of CH2Cl2 during the drying process and the well-dispersion of BCNs resulted in
1
A = kt 2
Higuchi model
ln(1 − A) = −kt A = kt n
Korsmeyer Peppas model
ln(1 − A) = −at b 117
First order kinetic model
Weibull model
(4) (5) (6) (7)
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Fig. 4. (a) Digital photographs of SA beads, (b) SEM images of SA beads’ surface and (c) SEM images of SA beads’ cross-section. (d) Digital photographs of SA/BCNs CBs, (e) SEM images of SA/BCNs CBs’ surface and (f) SEM images of SA/BCNs CBs’ cross-section.
Table 2 Encapsulation efficiency (EE) and fitting results of release data by Korsmeyer Peppas model. Formulation
EEa
SA beads SA/BCNs-0.3 CBs SA/BCNs-0.6 CBs SA/BCNs-0.9 CBs
54.2 80.3 89.6 91.4
a
where A is the fractional release of drug in time t, K is a constant incorporating structural and geometrical characteristics of the delivery system, n is the diffusion exponent characteristic of the release mechanism, a and b are constants. The correlation coefficients (R2) of fitting release data to different models are shown in Table 1. By contrast, the correlation coefficients (R2) of fitting release data obtained from Korsmeyer Peppas model were higher than that from other models, indicating that the release curves
Higuchi First order Korsmeyer Peppas Weibull
SA/BCNs-0.6 CBs
SA/BCNs-0.9 CBs
0.9772 0.9691 0.9951 0.9906
0.9678 0.9839 0.9987 0.9917
0.9806 0.9311 0.9965 0.9895
0.9864 0.8831 0.9935 0.9888
0.0065 0.0031 0.0045 0.0052
0.7297 0.6854 0.6497 0.6320
Non-Fickian Non-Fickian Non-Fickian Non-Fickian
Values for EE expressed as the mean ± the standard deviation.
The cytotoxicity of the SA/BCNs CBs was evaluated on MC3T3-E1 cells by CCK-8 assay using Osteoblastic MC3T3-E1 cells. As shown in Fig. 6a, the MC3T3-E1 cells with various concentration of SA/BCNs CBs exhibited good proliferation in comparison to the blank control, indicating that the MC3T3-E1 cells were viable and proliferate well on the SA/BCNs CBs, which was attributed to the good cytocompatibility of SA/BCNs CBs. In particular, when the concentrations of SA/BCNs CBs were 20 mg/mL and 40 mg/mL, the OD values obtained after 2 days and 7 days incubation were significantly different, which was significantly higher than that of the blank control. But the high concentration of SA/BCNs CBs (80 mg/mL) resulted in the reduction of proliferation activity of the MC3T3-E1 cells. The results implied that low concentration of SA/BCNs CBs (20˜40 mg/mL) could facilitate the proliferation of MC3T3-E1 cells remarkably, but the too high concentration of SA/BCNs CBs may inhibit the viability of cells. Subsequently, the osteoblast differentiation of MC3T3-E1 cells after 10 days
Correlation coefficient(R2) SA/BCNs-0.3 CBs
Mechanism
3.5. Cytocompatibility of SA/BCNs CBs
Table 1 Correlation coefficients (R2) of release data fitted to different models.
SA beads
5.1% 3.6% 2.7% 2.8%
n
were well fitted by the Korsmeyer Peppas model. The fitting results of release data by Korsmeyer Peppas model are presented in Table 2. It is reported that the release exponent, n, indicates the mechanism of drug release. For spherical alginate beads, when n ≤ 0.43, the release mechanism is attributed to Fickian diffusion; when 0.43 < n < 0.85, the release mechanism is attributed to non-Fickian transport; when n ≥ 0.85, the release mechanism is attributed to a zero order release mechanism [50]. Since the n values of all formulations were in the range from 0.43 to 0.85, the release mechanism of alfacalcidol from the composite beads was attributed to non-Fickian transport, which implied that water migration into the beads and diffusion of the drug through continuously swelling beads were carried out simultaneously [45].
Fig. 5. In vitro release profiles of alfacalcidol from SA beads, SA/BCNs-0.3 CBs, SA/BCNs-0.6 CBs and SA/BCNs-0.9 CBs in pH 7.4 PBS (error bars represent the standard deviation of three replicates).
Model
± ± ± ±
k (min−n)
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Fig. 6. (a) CCK-8 assay for proliferation of MC3T3-E1 cells cultured on the SA/BCNs CBs after 2 days and 7 days incubation. (b) Relative ALP activity of MC3T3-E1 cells on the SA/BCNs CBs after 10 days incubation. * denotes p < 0.05.
Natural Science Foundation of China (21566009).
incubation was determined using ALP activity as ALP enzyme was a vital biomarker for indicating early osteoblast differentiation [51]. As shown in Fig. 6b, the relative ALP activity of MC3T3-E1 cells exceeded 1.0 with the concentration of SA/BCNs CBs ranging from 20 mg/mL to 80 mg/mL. Especially, higher activity was obviously found in the presence of relatively lower concentration (20–40 mg/mL) of SA/BCNs CBs as compared to the control. The results indicated that low concentration of SA/BCNs CBs may have good biocompatibility to MC3T3-E1 cells, which could effectively facilitate cells proliferation and osteoblast differentiation. On the basis of good merits, such as drug-loading capacity, release performance and biocompatibility, SA/BCNs CBs may have broad range of applications in food, medicine, and pharmaceutics, where they are used as delivery systems for encapsulation, protection, and controlled release of hydrophobic compounds.
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4. Conclusions In summary, the loading and sustained release of alfacalcidol for the composite beads was achieved by the interfacial assembly of amphiphilic BCNs in oil-in-water Pickering emulsions followed by external gelation. The characterization of BCNs revealed that the sulfuric acid hydrolysis induced the removal of the amorphous components and the cleavage of the crystalline microfibrils, making BCNs exhibit rod-like shape, high crystallinity and good colloidal property. The coverage of BCNs at the oil-water interface that could form the stable three-dimensional flocculated fibril network by hydrogen bonding interaction was beneficial to preserve the droplets of the drug-loaded Pickering emulsions against coalescence and Ostwald ripening in alginate solution. The emulsifying performance of BCNs at the oil-water interface that improved the compatibility between alginate and alfacalcidol and the hydrogel shells of the composite beads formed by external gelation should be responsible for the loading and sustained release of alfacalcidol. The composite beads exhibited good sustained release performance with the release mechanism of non-Fickian transport, which was helpful to improve the drug efficacy and drug utilization. Additionally, the composite beads exhibited low cytotoxicity and good capabilities for osteoblast differentiation. This novel drug-loading technique with high efficiency and sustained-releasing performance combined biocompatible BCNs with natural alginate by eco-friendly Pickering emulsion method, exhibiting great potential in bio-medicine and bio-pharmaceutical. Acknowledgments We gratefully acknowledge the financial support from the Natural Science Foundation of Hainan Province (218QN233) and the National 119
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Entrapment of bacterial cellulose nanocrystals stabilized Pickering emulsions droplets in alginate beads for hydrophobic drug delivery
T
Huiqiong Yana,b, Xiuqiong Chena,b, Meixi Fengb, Zaifeng Shib, Wei Zhangb, Yue Wangb, ⁎ Chaoran Keb, Qiang Lina,b, a Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou, 571158, China b Key Laboratory of Water Pollution Treatment & Resource Reuse of Hainan Province, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou, 571158, Hainan, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacterial cellulose nanocrystals Pickering emulsions Alginate composite beads Interfacial assembly Encapsulation Sustained release
In this work, the interfacial assembly of amphiphilic bacterial cellulose nanocrystals (BCNs) by Pickering emulsion method was proposed to improve the compatibility between the alginate and hydrophobic drug. BCNs prepared by sulfuric acid hydrolysis of biosynthesized bacterial cellulose was used as the particulate emulsifiers, whereas the model drug, alfacalcidol, dissolved in CH2Cl2 was used as the oil phase. The oil-in-water Pickering emulsions were prepared by ultrasonic dispersion method and then they were well dispersed in alginate solution. Ultimately, the drug-loaded alginate composite beads were successfully fabricated by external gelation. The characterization results revealed that BCNs possessed good colloidal property and could form flocculated fibril network, which was beneficial to stabilize Pickering emulsions. The irreversible adsorption of BCNs at the oilwater interface could make the Pickering emulsions preserve the droplets against coalescence and Ostwald ripening when they were dispersed in alginate solution. The interfacial assembly of amphiphilic BCNs and the hydrogel shells of the alginate composite beads formed by external gelation achieved the loading and sustained release of alfacalcidol. The release curves were well fitted by Korsmeyer Peppas model and the release mechanism of alfacalcidol from the composite beads was attributed to non-Fickian transport. In addition, the resultant alginate composite beads exhibited low cytotoxicity and good capabilities for osteoblast differentiation.
1. Introduction Over the last decade, much more attentions have been paid to alginate hydrogel beads for their extensive applications in food industry, drug delivery and tissue engineering [1–3]. Since alginate has unique advantages such as aqueous-solubility, renewability, non-toxicity, biocompatibility, biodegradability and non-immunogenicity [4,5], it is suitable to encapsulate large molecules or hydrophilic materials at high efficiencies [6]. Various cargos including living cells, protein drugs, enzymes, food ingredients and catalysts have successfully been encapsulated in alginate hydrogel beads [7]. However, when it comes to hydrophobic drugs or compounds, the loading capacity is low because of the poor affinity of alginate for the hydrophobic drugs or compounds, resulting from the large amounts of free hydroxyl and carboxyl groups along alginate backbone [8]. This inherent drawback of alginate was commonly overcome by chemical modification involving the
introduction of hydrophobic groups onto its hydrophilic backbone, blending with other polymers and incorporating clays [9,10]. To date, the encapsulation of Pickering emulsions in alginate hydrogel beads to improve the loading capacity of hydrophobic drugs is rarely reported, especially when using bacterial cellulose nanocrystals (BCNs) as the Pickering emulsifiers. BCNs are prepared by acid hydrolysis of bacterial cellulose to remove amorphous regions to obtain rod-like crystalline forms of cellulose, which are high modulus and tensile strength, environmentally friendly, nontoxic, edible, degradable and biocompatible [11,12]. Especially, BCNs prepared by sulfuric acid hydrolysis can be endowed with anionic sulfate half-ester groups on their surface, which could be further oxidized by hydrogen peroxide, thereby resulting in the uniform aqueous dispersion [13,14]. BCNs are considered to be amphiphilic overall owing to the presence of high density of hydroxyl groups on the surface [15], while the hydrophobic interactions come from the
⁎ Corresponding author at: Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou, 571158, China. E-mail addresses: [email protected], [email protected] (Q. Lin).
https://doi.org/10.1016/j.colsurfb.2019.01.057 Received 29 July 2018; Received in revised form 8 January 2019; Accepted 26 January 2019 Available online 29 January 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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composite beads were also investigated. As far as we know, this new drug-loading technique with high efficiency, sustained-releasing performance and good bioactivity via the Pickering emulsion interfacial assembly of amphiphilic BCNs in bio-pharmaceutical field has not been reported.
crystalline organization along with extensive hydrogen bonding of polymer chains [16]. Therefore, their amphiphilic properties could be applied to stabilize surfactant-free Pickering emulsions. The self-assembly of BCNs at liquid–liquid interfaces has been well studied, which could be applied to immobilize hydrophobic drugs or compounds. In this case, BCNs spontaneously localize at the interface to minimize the helmholtz free energy [17–19]. Pickering emulsions involve the irreversible adsorption of solid colloidal particles at the oil-water interface and stabilize the emulsion droplets against coalescence by forming a mechanically robust monolayer [11]. The irreversible adsorption requires a much higher energy to remove the colloidal particles from the oil–water interface, making Pickering emulsions exhibit peculiar advantages over conventional emulsions in terms of outstanding stability against coalescence and Ostwald ripening [20]. In contrast to conventional emulsions stabilized by surfactants, Pickering emulsions have a number of merits, such as more robust formulations, reduced foaming problems and lower toxicity [21], thus being widely used in biomedical, health and cosmetic fields where the use of surfactants is undesirable [19]. It is reported that inorganic or organic particles of silica, clay, calcium carbonate, hematite, polystyrene, and microgels, ranging in size from nanometers to micrometers, could all stabilize Pickering emulsions [11]. But the increasing legal and consumer requirements of Pickering emulsions such as non-toxicity, biocompatibility and high ecological accept-ability make BCNs become the ideal candidate for Pickering emulsifiers. In comparison to other solid lipid particles, the highly enhanced emulsions’ stabilization against coalescence and Oswald ripening by BCNs makes Pickering emulsions be able to conserve the droplets under high concentration of dispersed phase. Alfacalcidol, a kind of hydrophobic drug, is an analogue of vitamin D used for supplementation in humans, which is of great importance in health and disease prevention [2]. It is considered to be a more useful form of vitamin D supplementation, mostly due to much longer half-life and lower kidney load [22]. It is well known that the success of a medical treatment depends not only on the pharmacodynamic activity of a drug, but also on the availability of the active agent at the site of action in the human body. Although alfacalcidol could be loaded in CH2Cl2/water Pickering emulsions stabilized by BCNs with long-term stability towards coalescence, the emulsion droplets may not withstand the influences of physiological environment, owing to the possible change of size and surface properties of BCNs in physiological medium [20]. Several methods could be applied to ensure mechanical robustness and stability of the emulsions, including gel trapping technique, locking particles together by polyelectrolyte and chemical cross-linking of individual building blocks [21]. Among these methods, the gel trapping technique by calcium alginate seems to be simple and feasible. As previously mentioned, the drug-loaded Pickering emulsions could preserve the droplets when they are dispersed in bulk alginate solution, which could be crosslinked by multivalent cations, such as Ca2+, to form hydrogel beads by chelation [23,24]. Therefore, alginate hydrogel beads with excellent properties in combination with the Pickering emulsions may be a promising delivery vehicle to protect alfacalcidol from harmful conditions (such as immune responses) and to restrict its release in a timely manner, thus improve its bioavailability [25–27]. In this study, we combined the high encapsulating performance of alginate hydrogel beads with the ideal emulsifying property of BCNs to develop a new formulation with high loading capacity of hydrophobic drugs. The loading of hydrophobic alfacalcidol in alginate beads was achieved via the emulsification of eco-friendly Pickering emulsions and the immobilization of them within hydrogel matrix of alginate by external gelation as shown in Scheme 1. After removing CH2Cl2, hydrophobic alfacalcidol and BCNs could be deposited into the network of alginate hydrogel, similar to common emulsion solvent evaporation method [28]. The physicochemical properties and emulsifying performance of BCNs were assessed, and the morphology, encapsulation efficiency, release performance and cytocompatibility of alginate
2. Experimental 2.1. Materials Bacterial cellulose was produced by Acetobacter xylinum (CGMCC5173) obtained from China General Microbiological Culture Collection Center according to previous method [13]. Sodium alginate (MW = 465 kDa, G/M = 1.5), alfacalcidol, sulfuric acid, hydrogen peroxide, CH2Cl2, CaCl2 and other reagents were purchased from Sigma-Aldrich (New York, USA). They were analytical grade and were used without further purification. 2.2. Synthesis of BCNs In this work, acid hydrolysis of BC was performed to synthesize BCNs according to previous method with some modifications [29,30]. About 10 g of BC was dispersed in 200 mL of 50 wt% sulfuric acid under vigorous mechanical stirring. The hydrolysis was performed at 45 °C for 3 h, and the mixture was diluted five-fold to quench the hydrolysis reaction. Then 20 mL of 30 wt% aqueous hydrogen peroxide was added to bleach and further oxidize BCNs. Afterwards, the resultant suspension was centrifuged at 9000 rpm for 15 min to separate the crystals, which were washed and treated ultrasonically for 20 min to eliminate excess acid. At last, the precipitate was further dialyzed against deionized water for 7 d using a dialyzing membrane with a molecular weight cutoff of 3500 to remove residual sulfuric acid as well as other low-molecular weight impurities. The resultant BCNs suspension was stored in a refrigerator for further use. Simultaneously, a specified amount of BCNs aqueous suspensions was lyophilized to calculate BCNs content in the suspensions and for further characterization. Based on the weight of BC, the yield of BCNs was 62%. 2.3. Characterization of BCNs The micro-morphology of dry BCNs was observed by using a Hitachi S-3000N (Japan) scanning electron microscope after fixing the samples on a brass holder and coating them with gold. And the structure of BCNs was observed by a JEM 2100 TEM (JEOL Co., Japan) at an acceleration voltage of 200 kV. Transmission electron microscope (TEM) images of samples were obtained by placing a few drops of the aqueous dispersion of the samples on a carbon-coated copper grid, and evaporating the solvent prior to observation. The molecular weight of the BCNs was determined using the Mark–Houwink equation according to previous report [31]. The structure and crystallinity of BCNs were examined by using a Bruker T27 FT-IR spectrophotometer (Germany) and a Bruker AXS/D8 advance X-ray diffractometer system (Germany) with Cu-Kα radiation (λ = 0.154 nm). The freeze-dried samples were mixed with KBr compressed into semitransparent KBr pellets before the FT-IR measurement. The XRD measurement was operated at 40 kV and 100 mA in a step scan mode, which performed over a 2θ range of 5-60° with the scanning speed of 0.025°/s. The crystallinity index (CrI) of BCNs was then calculated by using the following empirical equation [32,33].
CrI =
I200 − Iam × 100% I200
(1)
where I200 is the intensity value of crystalline cellulose, and Iam is the intensity value of amorphous region material (2θ = 18°). 13 C solid state NMR experiment was performed at 25 °C using a Bruker AVANCE400 WB spectrometer at resonance frequencies of 113
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Scheme 1. Schematic illustration of the preparation of the drug-loaded alginate composite beads by entrapping BCNs stabilized Pickering emulsions droplets in alginate beads.
100.6 MHz for 13C nuclei. The 13C CP/MAS NMR spectrum was recorded under observation conditions, a CP contact time of 3 ms, a repetition time of 5 s, a spinning speed of 7 KHz and the numbers of scans were 4 k. In addition, the size and zeta potential of BCNs were further determined by DLS with a Malvern Nano-ZS90 Zetasizer (UK) at a scattering angle of 90° at 25 °C, employing an (He-Ne) argon laser (λ = 633 nm). Refractive indices of 1.47 for BCNs and 1.33 for deionized water were used. In addition, the viscosity value of deionized water was 0.894 mPa s at 25 °C. The size of the 0.9 wt% BCNs suspensions was obtained from the Cumulant analysis of its correlation function by using the CONTIN analysis. The zeta potential of 0.9 wt% BCNs suspensions was calculated from the electrophoretic mobility (u) using the Smoluchowski relationship (ζ = ηu/ε) under the assumption that κα ≪1, where η is the solution viscosity, ε is the dielectric constant of the medium, and κ and α are the Debye−Hückel parameter and the particle radius, respectively. Zeta potentials were averaged over 20 runs. The viscosity of the 0.9 wt% BCNs suspensions was measured at 25 °C using a TA DHR Rheometer (USA).
solution were collected with a Leica DMRX optical microscope (Switzerland) equipped with a high performance digital camera. The emulsion droplets were placed directly onto a glass microscope slide and viewed under 10× and 40× magnification.
2.5. Fabrication of drug-loaded alginate composite beads The fabrication of drug-loaded alginate composite beads, abbreviated as SA/BCNs CBs, was illustrated in Scheme 1. Briefly, the above alginate solution containing drug-loaded Pickering emulsion droplets was dripped into 0.25 M CaCl2 solution. 30 min later, the drug-loaded SA/BCNs CBs were formed and washed with distilled water, and then vacuum dried at 60 °C. The alginate composite beads encapsulating the drug-loaded Pickering emulsions stabilized by 0.3, 0.6 and 0.9 wt% BCNs were respectively abbreviated as SA/BCNs-0.3 CBs, SA/BCNs-0.6 CBs and SA/BCNs-0.9 CBs. For comparison, the similar drug-loaded alginate beads, abbreviated as SA beads, with the absence of BCNs were also prepared. The surface morphology of the composite beads was examined using a Hitachi S-3000N (Japan) scanning electron microscope after fixing the samples on a brass holder and coating them with gold.
2.4. Preparation and characterization of drug-loaded Pickering emulsions CH2Cl2, as the least toxic of the simple chloro-hydrocarbons with high dissolving ability, was chosen to disperse alfacalcidol, which was used as the oil phase in the preparation of Pickering emulsions. The drug-loaded Pickering emulsion with an oil/water ratio of 1:9 (v/v) was prepared by ultrasonic dispersion with a JY92-II DN ultrasonic device (China). Actually, 1 mL of CH2Cl2 solution dissolving 50 mg alfacalcidol was added to 9 mL of BCNs suspension with various concentrations (0.3, 0.6 and 0.9 wt%) to form the emulsions with the aid of ultrasonication. After the CH2Cl2 and BCNs were respectively stained with Nile red and fluorescent brightener, the microstructure of Pickering emulsion droplets was observed by using a reconstructive Nikon Ti-S fluorescent microscope (Japan). The imaging of emulsion droplets was performed with the fluorescence successively excited at 492 nm and 408 nm. Meanwhile, the size of the emulsion droplets was measured by laser light diffraction using a Malvern Mastersizer 2000 apparatus (UK) equipped with a He–Ne laser operating at 633 nm. The average droplet diameter was taken to be the volume-weighted average diameter (d4/3) from triplicate measurements, which could be calculated by the following equation [18].
d4/3 =
∑ ni × di4 ∑ ni × di3
2.6. Loading and in vitro release of alfacalcidol The encapsulation of alfacalcidol into the alginate hydrogel matrix was ultimately achieved by external gelation [34]. The residual alfacalcidol in the gelling solution and washing solution was in the supernatant which could be extracted by chloroform. The encapsulation efficiency (EE) could be calculated using the following equation.
Encapsulation Efficiency (EE ) total alfacalcidol − residual alfacalcidol = × 100% total alfacalcidol
(3)
The release of alfacalcidol from the composite beads was performed in pH 7.4 PBS. 20 mg of the dried composite beads was immersed into 50 mL PBS in centrifuge tube. 40 mL of the solution was replaced with the same volume of fresh PBS by centrifugation at different time intervals, which could avoid the influences of the saturated solutions. The released alfacalcidol in the supernatant for each time interval was extracted by 40 mL chloroform. The chloroform extract was placed in an ice water bath to avoid the volatility of chloroform that would affect alfacalcidol concentration. The amount of alfacalcidol in chloroform was measured by using a Shimadzu UV-1800 (Shimadzu, Kyoto, Japan) UV–vis spectrophotometer at 265 nm with a calibration curve prepared by standard concentration of alfacalcidol in chloroform ranging from 10 to 50 μg/mL [2]. The drug release procedure was performed in triplicate to calculate the standard deviation.
(2)
where ni is the number of the droplets with the diameter di. Subsequently, 0.8 g of sodium alginate was dissolved in 50 mL water with vigorous stirring for 4 h to form a homogenous solution. Then 10 mL of the drug-loaded Pickering emulsion was well dispersed in 40 mL of 2% (w/v) alginate solution under gentle mechanical stirring. The optical micrographs of drug-loaded Pickering emulsions in alginate 114
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of short and rod-like microfibrils of BCNs, attributed to the removal of the amorphous components and the cleavage of the crystalline microfibrils. BCNs revealed very thin layers in their SEM image and bundles or ribbons in their TEM image, indicating the presence of interfibrillar hydrogen bonds. According to the Mark-Houwink equation, the molecular weight of the BCNs was 41.7 kDa. FT-IR analysis is a useful method for elucidating the specific functional groups or chemical bonds in BCNs. The FT-IR spectra of BC and BCNs revealed the similar profiles as shown in Fig. 2a. However, BCNs showed the additional weak peaks at 1732.65 and 862.11 cm−1 respectively for the bending vibration of −COOH and stretching vibration of CeO [18], indicating the further oxidation of pyranose ring by hydrogen peroxide. Additionally, the peak of hydroxyl stretching at 3350.45 cm−1 in the spectrum of BC became sharp and made a blue shift to 3411.17 cm−1 in the spectrum of BCNs. The results indicated that sulfuric acid hydrolysis could destroy the original intramolecular hydrogen bonding of BC [3], promoting the cleavage of the glycosidic bonds. The change of crystal structure of BCNs during the hydrolysis process was also analyzed by XRD. As shown in Fig. 2b, both of the BC and BCNs exhibited three crystalline peaks at 2θ = 14.7°, 16.8° and 22.7° corresponding to the (-110), (110) and (200) crystal planes, as well as an amorphous background at about 2θ = 18°, indicating the typical cellulose I structure, consistent with the result reported by Moriana et al. [35]. But the intensity of the BCNs diffraction peaks significantly increased in contrast to that of BC, confirming the removal of the amorphous components during the hydrolysis process. According to Eq. (1), the CrI of BC and BCNs were 75.1% and 89.6%, which was close to that reported in previous study for BC produced from pretreatment WWCJ medium (75.42%) [36]. 13C CP/ MAS solid-state NMR experiments are actually the combination of cross polarization, magic angle spinning and high power decoupling. Fig. 2c showed the representative 13C CP/MAS spectrum of solid BCNs. And the 13 C peaks of BCNs had been marked in 13C CP/MAS spectrum. BCNs have board resonances that extend over chemical shift ranges of δ = 110˜100, 92˜79 and 57˜67 ppm attributed to the C1, C4, and C6 signals, respectively. To note, the 13C peaks ranging from 86 to 79 ppm assigned to the C4 signals of amorphous cellulose disappeared, indicating the high crystallinity for BCNs, which was consistent with the XRD results. Besides, the size, zeta potential and viscosity of BCNs at the concentration of 0.9 wt % were measured by DLS and rheometer. BCNs exhibited a relatively narrow size distribution nearly involving one range, and their average size was 259.6 nm with the PDI of 0.26 (Fig. 2d). The relatively narrow size distribution and low average size possibly resulted from the elimination of amorphous components and the cleavage of the glycosidic bonds, which was in accordance with FTIR and XRD analysis. Additionally, from Fig. 2e, BCNs showed negative zeta potentials for the presence of carboxyl groups on the pyranose ring. To note, BCNs exhibited relatively high zeta potential at about -34.8 mV, which could be well-dispersed in aqueous solution because of the strong electrostatic repulsion forces [3]. Furthermore, the BCNs
2.7. Cell viability of SA/BCNs CBs Cytotoxicity of the SA/BCNs CBs was assessed by the use of mouse osteoblastic MC3T3-E1 cells which were cultured with α-MEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin under the condition of 37 °C, 5% CO2, 95% air and 100% relative humidity, and the medium was changed every 2 days. The sterilized SA/BCNs CBs with serial concentrations were blended with cell suspension at a cell density of 5 × 104 cells/cm2, which were placed into 24-well culture plates. The cell counting kit-8 (CCK-8) assay was used to evaluate cell viability on the SA/BCNs CBs, and the obtained optical density was proportional to the number of living cells. After 2 days and 7 days incubation, the culture medium was replaced by 500 μL fresh culture medium with 50 μL CCK-8 solution for further 4 h incubation at 37 ℃. 100 μL of the solution from each well was transferred to new 96well cell culture plates and the optical density (OD) of each well was read using a microplate reader machine (Bio-rad X-mark, America) at a wavelength of 450 nm. Simultaneously, the cells cultured on the tissue culture polystyrene (TCP) was served as the blank control group. Additionally, the culture medium containing 90% LG-DMEM, 10% fetal bovine serum, 1% penicillin/streptomycin, 10 mmol/L β-glycerophosphate, 0.1 μmol/L dexamethasone and 50 μg/mL ascorbic acid used as an osteogenic medium to achieve the cell differentiation under the condition of 37 °C, 5% CO2, 95% air and 100% relative humidity, and the medium was changed every 3 days. The cell differentiation of MC3T3-E1 cells on the SA/BCNs CBs was determined by alkaline phosphatase (ALP) expression using a QuantiChrom ALP kit. After 10 days incubation, the SA/BCNs CBs with cells were rinsed in PBS and then lysed in a 0.2% (w/w) Triton X-100 in PBS under the ice-bath environment for 30 min. After high speed centrifugation at 12,000 rpm for 5 min at 4 °C to discard cell debris, 50 μL of supernatant was transferred to a new 96-well plate for ALP determination. Moreover, MC3T3-E1 cells cultured on the TCP was served as the blank control group. And the relative ALP activities for all samples were standardized by the blank control group. 2.8. Statistical analysis All data presented as means ± standard deviation. Single-factor ANOVA was performed by SPSS software to analyze the variables, and a value of p < 0.05 was considered statistically significant. 3. Results and discussion 3.1. Characterization of BCNs It is reported that sulfuric acid hydrolysis can not only induce the removal of the amorphous components of BCNs but also endow BCNs with anionic sulfate half-ester groups [29,33]. As shown in Fig. 1, the sulfuric acid hydrolysis of bacterial cellulose resulted in the formation
Fig. 1. Scanning electron microscope (SEM) image (a) and transmission electron microscope (TEM) image (b) of BCNs. 115
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Fig. 2. (a) FT-IR spectra and (b) X-ray diffractograms of BC and BCNs. (c) and (f) viscosity of 0.9 wt% BCNs suspensions.
13
C CP/MAS spectrum of solid BCNs. (d) Size distribution, (e) zeta potential distribution
Fig. S1. Under bright field, the emulsion droplets exhibited good spherical shape, indicating the good emulsifying performance of BCNs. Nevertheless, under dark field, the emulsion droplets appeared green for the Nile red excitation at 492 nm whereas the emulsion droplets appeared blue for the fluorescent brightener excitation at 408 nm. Since oil-soluble Nile red only existed in oil phase and appeared green for the excitation at 492 nm, emulsion type was confirmed to be oil-in-water type [11]. Additionally, it is observed that BCNs were adsorbed at the oil-water interface with few emerging in aqueous phase, indicating that BCNs could form stable three-dimensional flocculated fibril network via the coverage of themselves at the oil-water interface to resist coalescence [38]. The droplet diameter distribution of drug-loaded Pickering emulsions stabilized by BCNs with various concentrations is presented in Fig. S2. As d4/3 diameter is more sensitive to the presence of large droplets, it is regarded as the average droplet diameter in the experiment. Based on Eq. (2), d4/3 diameters of the drug-loaded Pickering emulsions
suspensions exhibited shear-thinning behavior with three regions against the shear rate, as shown in Fig. 2f. Similar viscosity behavior has also been reported in previous work [37]. The viscosity variation of the BCNs suspensions depended on the three-dimensional flocculated fibril network formed by hydrogen bonding interaction between BCNs and water molecules [38]. The results indicated that BCNs possessed good colloidal property and could form flocculated fibril network, which was beneficial to stabilize Pickering emulsions. 3.2. Characterization of drug-loaded Pickering emulsions Due to the amphiphilic property of cellulose chains [16], BCNs could emulsify CH2Cl2 solution of alfacalcidol to form the Pickering emulsions via ultrasonic dispersion method. The microstructure of emulsion droplets was observed by fluorescent microscope after fluorescence staining. The fluorescent images of the dyed Pickering emulsions stabilized by BCNs at a concentration of 0.9 wt % are shown in 116
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Fig. 3. Optical micrographs of drug-loaded Pickering emulsions stabilized by various concentration of BCNs in alginate solution: (a) 0.3 wt %, (b) 0.6 wt % and (c) 0.9 wt %.
rough and filiform surface morphology with some gaps. In addition, as observed in their cross-sections (Fig. 4c and f), the SA/BCNs CBs exhibited more pore structures than SA beads, further confirming the high encapsulation efficiency of CH2Cl2 solution of alfacalcidol. On the basis of the hydrogel shells formed by external gelation and the emulsification of amphiphilic BCNs, alfacalcidol was encapsulated inside the composite beads in the course of the preparation of the composite beads. According to Eq. (3), the EEs of SA beads, SA/BCNs0.3 CBs, SA/BCNs-0.6 CBs and SA/BCNs-0.9 CBs were respectively 54.2%, 80.3%, 89.6% and 91.4% as shown in Table 2, revealing good drug-loading capacity. It was worth noting that the EEs of 89.6% for SA/BCNs-0.6 CBs and 91.4% for SA/BCNs-0.9 CBs were not much difference, probably attributed to the sufficient coverage of BCNs at the oil-water interface during the preparation of the drug-loaded Pickering emulsions when BCNs concentration reached up to 0.6 wt%. The excess BCNs may have little effect on the drug-loading capacity.
stabilized by BCNs with the concentrations of 0.3, 0.6 and 0.9 wt% were respectively 18.5, 14.6 and 8.8 μm. It was obvious that the BCNs concentration had a distinct influence on the size of the emulsion droplets. With the increase of BCNs concentration, the size of the emulsion droplets decreased and became uniform, similar to previous report for the stabilization of Pickering emulsions [39]. The variation of emulsion droplets diameter with different concentrations of BCNs was attributed to the coverage of BCNs at the oil-water interface, which protected the emulsion droplets against coalescence by forming a flocculated fibril network [38]. It was the irreversible adsorption demanding a much higher energy to remove the BCNs from the oil–water interface that made the Pickering emulsions preserve the droplets against coalescence and Ostwald ripening when they were dispersed in alginate solution [20]. The optical micrographs of drug-loaded Pickering emulsions stabilized by different concentrations of BCNs in alginate solution are shown in Fig. 3. When the BCNs concentration was low, the formed flocculated fibril network was weak. The weak flocculated fibril network easily led to a certain degree of coalescence [19], thereby resulting in the big emulsion droplets, as shown in Fig. 3a. With increasing BCNs concentration, more and more BCNs were absorbed at the oil-water interface, which could cover a much larger area to reduce the size of emulsion droplets and prevent their coalescence [40]. Consequently, the droplets of the emulsions became small and uniform (Fig. 3b and c) when the BCNs concentration increased.
3.4. In vitro release studies The release of alfacalcidol was achieved through its diffusion out of the swollen beads. The alfacalcidol release profiles of the composite beads are presented in Fig. 5. All of the release curves showed a rapid release in the initial stage (0–2000 min) and then a slow release in the following stage. The alfacalcidol accumulation on the surface of the composite beads resulting from the poor compatibility between alginate solution and CH2Cl2 solution of alfacalcidol and the extensive water uptake properties of the alginate hydrogel could have been responsible for the initially rapid release [43]. The coverage of BCNs on the surface of alfacalcidol after the evaporation of CH2Cl2 (Scheme 1), which probably formed a mechanically robust layer, and hydrogel shells of the composite beads formed by external gelation may account for the slow release of alfacalcidol [11]. In addition, the electrostatic forces and intermolecular hydrogen bonds formed between BCNs and alginate may also retard the release of alfacalcidol [44,45]. It was worth noting that the release process had lasted for more than 6600 min, indicating good sustained release performance. In actual drug therapy, the prolongation of release time is beneficial to improve the drug efficacy and drug utilization [46]. Additionally, SA/BCNs CBs exhibited a better sustained release performance over SA beads due to the barrier of BCNs within the polymeric matrix and the sufficient coverage of BCNs on the surface of alfacalcidol. With the increase of BCNs concentration, the sustained release performance improved. To further investigate the release mechanisms, the release profiles of the alfacalcidol up to 60% were fitted using the following common kinetic models [47–49].
3.3. Morphology and encapsulation efficiency of drug-loaded composite beads As the alginate hydrogel beads were prepared by external gelation through dripping an alginate solution into a CaCl2 solution, the gelling occurred only on the surface of alginate droplets to form the hydrogel shells [41]. It was the hydrogel shells that could immobilize the drugloaded Pickering emulsions in alginate solution, thus achieving the loading and sustained release of alfacalcidol (Scheme 1). The digital photographs of the wet SA beads and SA/BCNs CBs as well as the SEM images of the dried composite beads are shown in Fig. 4. Both of the wet SA beads and SA/BCNs CBs exhibited uniformly spherical shapes (Fig. 4a and d). However, volume shrinkage occurred when water was evaporated form the wet composite beads during the drying process, resulting in the formation of rough and collapsed surface morphology (Fig. 4b and e). It was observed that the surface of the SA beads was relatively smooth over that of the SA/BCNs CBs, which should be ascribed to the barrier properties of BCNs [42]. The SA beads with low amount of CH2Cl2 solution could possess a uniform rate of water loss on their surface, generating relatively smooth surface morphology [9]. The poor compatibility of alginate solution and CH2Cl2 solution of alfacalcidol led to the low encapsulation efficiency. On the contrary, the emulsification of CH2Cl2 solution of alfacalcidol by interfacial assembly of amphiphilic BCNs could effectively improve the compatibility between alginate solution and CH2Cl2 solution of alfacalcidol, thereby enhancing the encapsulation efficiency. The evaporation of CH2Cl2 during the drying process and the well-dispersion of BCNs resulted in
1
A = kt 2
Higuchi model
ln(1 − A) = −kt A = kt n
Korsmeyer Peppas model
ln(1 − A) = −at b 117
First order kinetic model
Weibull model
(4) (5) (6) (7)
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Fig. 4. (a) Digital photographs of SA beads, (b) SEM images of SA beads’ surface and (c) SEM images of SA beads’ cross-section. (d) Digital photographs of SA/BCNs CBs, (e) SEM images of SA/BCNs CBs’ surface and (f) SEM images of SA/BCNs CBs’ cross-section.
Table 2 Encapsulation efficiency (EE) and fitting results of release data by Korsmeyer Peppas model. Formulation
EEa
SA beads SA/BCNs-0.3 CBs SA/BCNs-0.6 CBs SA/BCNs-0.9 CBs
54.2 80.3 89.6 91.4
a
where A is the fractional release of drug in time t, K is a constant incorporating structural and geometrical characteristics of the delivery system, n is the diffusion exponent characteristic of the release mechanism, a and b are constants. The correlation coefficients (R2) of fitting release data to different models are shown in Table 1. By contrast, the correlation coefficients (R2) of fitting release data obtained from Korsmeyer Peppas model were higher than that from other models, indicating that the release curves
Higuchi First order Korsmeyer Peppas Weibull
SA/BCNs-0.6 CBs
SA/BCNs-0.9 CBs
0.9772 0.9691 0.9951 0.9906
0.9678 0.9839 0.9987 0.9917
0.9806 0.9311 0.9965 0.9895
0.9864 0.8831 0.9935 0.9888
0.0065 0.0031 0.0045 0.0052
0.7297 0.6854 0.6497 0.6320
Non-Fickian Non-Fickian Non-Fickian Non-Fickian
Values for EE expressed as the mean ± the standard deviation.
The cytotoxicity of the SA/BCNs CBs was evaluated on MC3T3-E1 cells by CCK-8 assay using Osteoblastic MC3T3-E1 cells. As shown in Fig. 6a, the MC3T3-E1 cells with various concentration of SA/BCNs CBs exhibited good proliferation in comparison to the blank control, indicating that the MC3T3-E1 cells were viable and proliferate well on the SA/BCNs CBs, which was attributed to the good cytocompatibility of SA/BCNs CBs. In particular, when the concentrations of SA/BCNs CBs were 20 mg/mL and 40 mg/mL, the OD values obtained after 2 days and 7 days incubation were significantly different, which was significantly higher than that of the blank control. But the high concentration of SA/BCNs CBs (80 mg/mL) resulted in the reduction of proliferation activity of the MC3T3-E1 cells. The results implied that low concentration of SA/BCNs CBs (20˜40 mg/mL) could facilitate the proliferation of MC3T3-E1 cells remarkably, but the too high concentration of SA/BCNs CBs may inhibit the viability of cells. Subsequently, the osteoblast differentiation of MC3T3-E1 cells after 10 days
Correlation coefficient(R2) SA/BCNs-0.3 CBs
Mechanism
3.5. Cytocompatibility of SA/BCNs CBs
Table 1 Correlation coefficients (R2) of release data fitted to different models.
SA beads
5.1% 3.6% 2.7% 2.8%
n
were well fitted by the Korsmeyer Peppas model. The fitting results of release data by Korsmeyer Peppas model are presented in Table 2. It is reported that the release exponent, n, indicates the mechanism of drug release. For spherical alginate beads, when n ≤ 0.43, the release mechanism is attributed to Fickian diffusion; when 0.43 < n < 0.85, the release mechanism is attributed to non-Fickian transport; when n ≥ 0.85, the release mechanism is attributed to a zero order release mechanism [50]. Since the n values of all formulations were in the range from 0.43 to 0.85, the release mechanism of alfacalcidol from the composite beads was attributed to non-Fickian transport, which implied that water migration into the beads and diffusion of the drug through continuously swelling beads were carried out simultaneously [45].
Fig. 5. In vitro release profiles of alfacalcidol from SA beads, SA/BCNs-0.3 CBs, SA/BCNs-0.6 CBs and SA/BCNs-0.9 CBs in pH 7.4 PBS (error bars represent the standard deviation of three replicates).
Model
± ± ± ±
k (min−n)
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Fig. 6. (a) CCK-8 assay for proliferation of MC3T3-E1 cells cultured on the SA/BCNs CBs after 2 days and 7 days incubation. (b) Relative ALP activity of MC3T3-E1 cells on the SA/BCNs CBs after 10 days incubation. * denotes p < 0.05.
Natural Science Foundation of China (21566009).
incubation was determined using ALP activity as ALP enzyme was a vital biomarker for indicating early osteoblast differentiation [51]. As shown in Fig. 6b, the relative ALP activity of MC3T3-E1 cells exceeded 1.0 with the concentration of SA/BCNs CBs ranging from 20 mg/mL to 80 mg/mL. Especially, higher activity was obviously found in the presence of relatively lower concentration (20–40 mg/mL) of SA/BCNs CBs as compared to the control. The results indicated that low concentration of SA/BCNs CBs may have good biocompatibility to MC3T3-E1 cells, which could effectively facilitate cells proliferation and osteoblast differentiation. On the basis of good merits, such as drug-loading capacity, release performance and biocompatibility, SA/BCNs CBs may have broad range of applications in food, medicine, and pharmaceutics, where they are used as delivery systems for encapsulation, protection, and controlled release of hydrophobic compounds.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.01.057. References [1] G. Lawrie, I. Keen, B. Drew, A. Chandler-Temple, L. Rintoul, P. Fredericks, L. Grøndahl, Interactions between alginate and chitosan biopolymers characterized using FTIR and XPS, Biomacromolecules 8 (2007) 2533–2541. [2] Q. Li, C.G. Liu, Z.H. Huang, F.F. Xue, Preparation and characterization of nanoparticles based on hydrophobic alginate derivative as carriers for sustained release of vitamin D3, J. Agric. Food Chem. 59 (2011) 1962–1967. [3] H. Yan, X. Chen, J. Li, Y. Feng, Z. Shi, X. Wang, Q. Lin, Synthesis of alginate derivative via the Ugi reaction and its characterization, Carbohydr. Polym. 136 (2016) 757–763. [4] J.M. Yang, N.C. Wang, H.C. Chiu, Preparation and characterization of poly(vinyl alcohol)/sodium alginate blended membrane for alkaline solid polymer electrolytes membrane, J. Membr. Sci. 457 (2014) 139–148. [5] Y. Yue, J. Han, G. Han, A.D. French, Y. Qi, Q. Wu, Cellulose nanofibers reinforced sodium alginate-polyvinyl alcohol hydrogels: core-shell structure formation and property characterization, Carbohydr. Polym. 147 (2016) 155–164. [6] E.S. Chan, Preparation of Ca-alginate beads containing high oil content: influence of process variables on encapsulation efficiency and bead properties, Carbohydr. Polym. 84 (2011) 1267–1275. [7] J.P. Paques, E. Linden, C.J.M. van Rijn, L.M.C. Sagis, Alginate submicron beads prepared through w/o emulsification and gelation with CaCl2 nanoparticles, Food Hydrocolloids 31 (2013) 428–434. [8] J.S. Yang, Y.J. Xie, W. He, Research progress on chemical modification of alginate: a review, Carbohydr. Polym. 84 (2011) 33–39. [9] H. Yan, X. Chen, Y. Feng, F. Xiang, J. Li, Z. Shi, X. Wang, Q. Lin, Modification of montmorillonite by ball milling method for immobilization and delivery of acetamiprid based on alginate/exfoliated montmorillonite nanocomposite, Polym. Bull. 73 (2016) 1185–1206. [10] L.Q. Yang, B.F. Zhang, L.Q. Wen, Q.Y. Liang, L.M. Zhang, Amphiphilic cholesteryl grafted sodium alginate derivative: synthesis and self-assembly in aqueous solution, Carbohydr. Polym. 68 (2007) 218–225. [11] P. Paximada, E. Tsouko, N. Kopsahelis, A.A. Koutinas, I. Mandala, Bacterial cellulose as stabilizer of o/w emulsions, Food Hydrocolloids 53 (2016) 225–232. [12] Y. Habibi, L.A. Lucia, O.J. Rojas, Cellulose nanocrystals: chemistry, self-assembly, and applications, Chem. Rev. 110 (2010) 3479–3500. [13] Z. Hu, S. Ballinger, R. Pelton, E.D. Cranston, Surfactant-enhanced cellulose nanocrystal Pickering emulsions, J. Colloid Interface Sci. 439 (2015) 139–148. [14] L. Zhong, S. Fu, X. Peng, H. Zhan, R. Sun, Colloidal stability of negatively charged cellulose nanocrystalline in aqueous systems, Carbohydr. Polym. 90 (2012) 644–649. [15] T.A. Dankovich, D.G. Gray, Contact angle measurements on smooth nanocrystalline cellulose (I) thin films, J. Adhes. Sci. Technol. 25 (2011) 699–708. [16] B. Lindman, G. Karlström, L. Stigsson, On the mechanism of dissolution of cellulose, J. Mol. Liq. 156 (2010) 76–81. [17] J.J. Blaker, K.Y. Lee, X. Li, A. Menner, A. Bismarck, Renewable nanocomposite polymer foams synthesized from Pickering emulsion templates, Green Chem. 11 (2009) 1321–1326. [18] Y. Jia, X. Zhai, W. Fu, Y. Liu, F. Li, C. Zhong, Surfactant-free emulsions stabilized by tempo-oxidized bacterial cellulose, Carbohydr. Polym. 151 (2016) 907–915.
4. Conclusions In summary, the loading and sustained release of alfacalcidol for the composite beads was achieved by the interfacial assembly of amphiphilic BCNs in oil-in-water Pickering emulsions followed by external gelation. The characterization of BCNs revealed that the sulfuric acid hydrolysis induced the removal of the amorphous components and the cleavage of the crystalline microfibrils, making BCNs exhibit rod-like shape, high crystallinity and good colloidal property. The coverage of BCNs at the oil-water interface that could form the stable three-dimensional flocculated fibril network by hydrogen bonding interaction was beneficial to preserve the droplets of the drug-loaded Pickering emulsions against coalescence and Ostwald ripening in alginate solution. The emulsifying performance of BCNs at the oil-water interface that improved the compatibility between alginate and alfacalcidol and the hydrogel shells of the composite beads formed by external gelation should be responsible for the loading and sustained release of alfacalcidol. The composite beads exhibited good sustained release performance with the release mechanism of non-Fickian transport, which was helpful to improve the drug efficacy and drug utilization. Additionally, the composite beads exhibited low cytotoxicity and good capabilities for osteoblast differentiation. This novel drug-loading technique with high efficiency and sustained-releasing performance combined biocompatible BCNs with natural alginate by eco-friendly Pickering emulsion method, exhibiting great potential in bio-medicine and bio-pharmaceutical. Acknowledgments We gratefully acknowledge the financial support from the Natural Science Foundation of Hainan Province (218QN233) and the National 119
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