Preparation of Magadiite-Sodium Alginate Drug Carrier Composite by Pickering-Emulsion-Templated-Encapsulation Method and Its Properties of Sustained Release Mechanism by Baker–Lonsdale and Korsmeyer–Peppas Model

In this study, the nanohybrid drug carrier were synthesized by Pickering emulsion-templated encapsulation (PETE) method to control the sustained-released properties of the nanohybrid drug carrier; magadiite-cetyltriphenyl phosphonium bromide (MAG-CTPB-KH550) and sodium alginate (NaC6H7O6) was dissolved in the aqueous phase but metronidazole (C6H9N3O3) was dissolved in the ethyl acetate (CH3COOC2H5) of the oil phase; both the oil phase and the aqueous phase were mixed and dispersed to prepared organically-modified magadiite-sodium alginate (MAG–CTPB–KH550/SA) nanohybrid drug carrier. X-ray diffraction (XRD), Flourier transform infrared spectrometry (FTIR) and scanning electron microscopy (SEM) results were shown that the most of Sodium alginate (SA) were encapsulated into the MAG–CTPB–KH550 but a few of SA were intercalated into the inner space layers of MAG–CTPB–KH550, metronidazole was combined with carrier materials through physical apparent adsorption, ion exchange and electrostatic interaction. In vitro result, it was showed that the slow release was shown less than 10% content of Sodium alginate; whereas, it was reduced the initial release percentage of Metronidazole but it was extended the sustained-released time. To reach at equilibrium, the sustained-released effects of the drug carrier were prepared with 10% of Sodium Alginate for 32 h and the maximum cumulative release percentage was 93.42% for 24 h. First order model, Baker–Lonsdale model and Korsmeyer–Peppas model were fitted to study the slow-release mechanism; the correlation coefficients (R2) of the three models were found over 0.9; thus, it was well described the release kinetics behavior of drug carrier composites. The slow-release mechanisms of the drug carrier were performed swelling and dissolving but the barrier effects of the lamina that were reduced the dissolution percentage of metronidazole.


Introduction
Nowadays, controlled release drug carriers have attracted more and more researchers' because of their advantages, such as safety, low dosage, biocompatibility, and low toxic side effects via decreasing the drug release rate thereby, which have great practical significance for treating patients [1,2]. Traditional drugs had burst release after administration, which lead to side effects and low efficiency-thus to solve these problems, many drug delivery and controlled release systems had been employed such as Liposomal drug delivery systems [3], Biocompatible PLGA Microspheres [4], Alginate/chitosan microparticles [5], threedimensional poly (lactic-co-glycolic acid)/silica colloidal crystal drug delivery system [6], Mesoporous Silica Nanoparticles [7]. Metronidazole was used as antibacterial and antiprotozoal medication, generally arranged in the treatment of anti-anaerobic infections, follicle worms, and acne diseases, usually administered orally, and can be rapidly absorbed in gastric after administration [8,9]. Long-term and excessive using this drug leads to drug resistance and side effects, therefore, it is necessary to develop controlled release drug carriers to reduce the drug dose at an appropriate level. Sodium alginate has become a promising polysaccharide for drug carriers [10].
Alginate have been widely used as nanoparticles or microparticles for oral delivery of insulin [11][12][13], hemoglobin [14], probiotics [15,16], and cells [17] owing to its high biocompatibility and biodegradability [18]. The drugloaded microspheres prepared by Sodium alginate alone have poor mechanical properties and drug loss during microsphere preparation, resulting in the uncontrollable release of the active pharmaceutical ingredient [19][20][21]. Many researchers had combined sodium alginate with other nanomaterials or polymers to modifying the structure of microspheres and improve the controlled release performance. Bardajee et al. [22] had prepared Thermo/pH/magnetictriple sensitive nanogel comprised of SA and magnetic graphene oxide for anticancer drug delivery. Xie et al. [23] had prepared pH-sensitive Carboxymethyl chitosan sodium salt (CMCS)/ hydrogel drug carrier. SA was also used to prepare sustained drug release system with montmorillonite [24][25][26]. However, due to the low ion exchange capacity of montmorillonite, the prepared drug carrier was not ideal in sustained release time and drug loading performance. So a two-dimensional layered silicate magadiite was developed in this study, which has special properties such as easily ion exchangeable hydrated sodium cations [27], active Si-OH groups on the layer, significant surface area associated with a negative charge, high ion exchange capacity (about 220 meq/100 g) [28], excellent adsorption capacity and none toxic to the human body [29]. Magadiite used as drug carrier has been reported [29,30] and it can also be used as solid particle emulsifier in Pickering Emulsion-Templated Encapsulation.
Many methods were used in drug carrier preparation, such as solvent evaporation method [31], Spontaneous emulsification/solvent diffusion method [32], Biocompatible crosslinked polymer preparation method [33] and Pickering Emulsion-Templated Encapsulation method. Emulsion methods have the advantages of high encapsulation efficiency and superior storage stability [34]. But the application of emulsification-volatilization methods to prepare drug carriers has been limited because the emulsifier added must be a food-grade material and will not interfere with the performance of the drug. Pickering emulsion is made by amphiphilic solid particles which has a suitable particle size and surface wettability rather than organic surfaceactive agent. The mass fraction of active agents required for stable Pickering emulsion is obviously less than that of conventional emulsions, so Pickering emulsion was more environmentally friendly and cost saving [35][36][37]. Pickering Emulsion-Templated Encapsulation had been applied in the field of drug carrier [38][39][40].

Materials
Magadiite was prepared in the laboratory according to the referenced literature [41], Cetyltriphenyl phosphonium bromide was involved of analytical-grade reagent, which was purchasedfrom Zhejiang Fenghong Clay Chemical Co., Ltd, China; Metronidazole was provided by Aladdin Ltd; SA and ethyl acetate were provided by Tianjin Fuchen Chemical Reagent Factory, China; Other chemicals of reagent grade were involved in all analytical-grade reagents that was purchased from Guangzhou Qianhui Company, China.

Preparation of Hydrophobic Organic-Magadiite (Org-MAG)
Magadiite and Cetyltriphenyl phosphonium bromide with a set mass ratio (1:2) were mixed with deionized water, then magnetically stirred at 60 °C for 6 h, and then wash several times until the AgNO 3 test was negative. Separating the solid with filtration and dried at 80 °C for 12 h.

Preparation of Org-MAG/SA/Metronidazole
30 mg metronidazole which was used as a simulated drug and a certain amount of Org-MAG were dispersed into 500 mL ethyl acetate solution as the oil phase, ultrasound treated for 10 min and then stirred. A certain amount of SA was dissolved in 500 mL of deionized water as the water phase. Finally, the ethyl acetate dispersion of Org-MAG and the sodium alginate solution were mixed and stirred for 1 h, formed a stable milky white Pickering emulsion. Thereafter, the ethyl acetate and the water were volatilized to obtain Org-MAG/SA/metronidazole drug carrier, which was collected as powders. The preparation process of drug carrier was shown in Fig. 1 and the content of SA and Org-MAG in different sample was shown in Table 1.

Metronidazole Standard Concentration Curve Drawing
UV/vis spectrophotometer was used to analyze the solution concentration and the optimum wavelength was 321 nm by wavelength scanning test. The concentration of metronidazole in solution was calculated by using the measured standard concentration curve. The linear equations of metronidazole in HCl solution of pH = 4.0 were: y = 0.03387x + 0.04483, R 2 = 0.999.

X-Ray Powder Diffraction
X-ray powder diffraction (Model D8 ADVANCE, Bruker AXS, Karlsruhe, Germany) patterns were recorded in the 2θ range of 2-10˚ at a scanning rate 6°/min and scanning step 0.02 o using monochromatic Cu-Kα radiation with the tube voltage and the tube current was 40 kV and 40 mA.

Fourier Transform Infrared Spectra
The spectral scanning rate of Fourier transform infrared spectra (NEXUS Model 670 Fourier, Thermo Nicolet Corporation, Waltham MA, USA) was 400 ~ 4000 cm −1 , the diameter of the sample was about 1 cm and the resolution ratio was 2 cm −1 .

Scanning Electron Microscopy
SEM analyses (Nova Nano SEM 430, FEI, Hillsboro, OR, USA) were used to observe the surface morphology of the drug carriers, the operating voltage was 10-20 kV.

Org-MAG/SA Drug Carrier Simulated Sustained Release In Vitro
Metronidazole can be absorbed quickly and completely in gastric after oral administration and will be widely distributed in tissues and body fluids. In order to simulate the gastric juice environment, the drug carrier samples were subjected to in vitro simulated sustained release experiments under HCl solution of pH = 4 at 37 °C. The amount of drug carrier loaded with metronidazole (1 g) was put into a pretreated dialysis bag in a 100 mL slow-release medium and stirred. When the metronidazole drug was released for 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 36 and 48 h, the amount of 5.0 mL upper clear liquid was taken out and concentration of metronidazole was tested using UV-vis spectrophotometer (at 321 nm). In order to maintain the consistency of the sustained release conditions, the amount of 5 mL release medium solution (at 37 °C) was poured back into the drug release system. The corresponding time concentration is calculated as follows: The cumulative release amount for the corresponding time is calculated as follows: (1) C n = X n The cumulative release percentage of metronidazole in the corresponding time is calculated as follows where n represents the number of times the buffer solution medium was taken out; C n represents the cumulative release concentration of metronidazole when taking the buffer solution n times, mg/mL, and X n represents the concentration of solution corresponding to the absorbance when taking the buffer solution n times, mg/mL; C i represents the concentration of the replacement liquid taken at the i time, mg/mL; V 0 and V e respectively represent the total volume of the initial sustained-release medium and the replacement volume of the sustained-release medium, mL; Q is drug loading capacity (mg/g) and Q n is the cumulative release amount before the n-th replacement of buffer solution (mg/g); M represents the cumulative release percentage of metronidazole, %. (2)

Scanning Electron Microscopy (SEM) Analysis
The SEM images of the drug carriers with different content of SA were shown in Fig. 2. It can be seen from Fig. 2a that MAG has a layered structure with rosette morphology. Figure 2c showed the microscopic morphology of S1, a portion of the metronidazole molecule can be observed as white small particles irregularly distributed between the layers of Org-MAG. Some of the SA molecule can only be observed on a few surfaces of Org-MAG layer as the content of SA was limited to 5%. As the content of SA increased (Fig. 2d), the boundary of the MAG interlayer became vague, while the surface morphology of the materials became flatter, a portion of metronidazole molecules were intercalated between the layers and another portion were attached to the surface in a block shape, which may be caused by random sorting of the MAG layers. When the content of SA was 25%, as shown in Fig. 2e, blocks with different angularities can be observed, which was due to the SA attaching to the interlayer space of MAG, making the surface smooth, and a similar morphology was observed with SA content of 50% (Fig. 2f). In these two samples, metronidazole could not be observed on the surface of drug carrier, indicating that metronidazole was covered by SA. In Fig. 2h, the SA content was 75% and the surface of the drug carrier became flat. Because SA can form a film, which can wrap MAG more tightly, and when the content of SA increases to a certain extent, SA was completely wrapped MAG, as is shown in Fig. 2 (S6). It can be inferred that when the content of SA was overweighed, particles size distribution could be uniform, while SA formed a film, wrapping MAG and metronidazole, contributing to the decline of the maximum cumulative release. With the appropriate ratio of SA and Org-MAG, the particle size distribution might not be uniform to some extent due to the random sorting of MAG layers, while it could increase the release of the drug.

X-Ray Diffraction (XRD)
The XRD patterns of MAG, SA, Org-MAG, and Org-MAG/SA were shown in Fig. 3. Comparing MAG (Fig. 3a) and Org-MAG (Fig. 3b) spectra, the XRD curve of MAG had an obvious 001 characteristic diffraction peak at 2θ = 5.48°, and the corresponding layer spacing was 1.61 nm. After the modification of CTPB, the XRD patterns of Org-MAG changed significantly: 001 characteristic diffraction peak moved to 2θ = 2.65°, and the corresponding layer spacing increased to 3.33 nm. The original diffraction peak at 2θ = 5.48° basically disappeared, while a weak diffraction peak appeared at 2θ = 4.62°, in which the layer spacing was 1.91 nm, almost identical to that of pure MAG. From the results above, it could be referred that part of the organic modifier could intercalate into the layers of MAG, expending the interlayer structure space. The XRD diagram of Org-MAG/SA was shown in Fig. 3c; it can be seen that the characteristic diffraction peak at 2θ = 2.65° on Org-MAG (Fig. 3b) didn't change, but the intensity increased significantly, and the peak at 2θ = 4.62° on Org-MAG (Fig. 3c) transferred to 2θ = 5.40°, the interlayer space of Org-MAG/SA was roughly same to Org-MAG. From Fig. 3d, it could be seen that there was no characteristic diffraction peak in the measurement range of SA. The result indicated that the addition of SA didn't change the structure of Org-MAG. Although the ordered structure was destroyed to a certain extent, its overall crystal and layered structure were not destroyed. This is consistent with the phenomenon observed in SEM images. Figure 4 exhibits the chemical structure of Org-MAG, SA, and Org-MAG/SA/metronidazole which refers to the FTIR spectrum. In Fig. 4b the absorption peaks at 3451 cm −1 , 1643 cm −1 , 1420 cm −1 , 1322 cm −1 , and 1137 cm −1 were assigned to the stretching vibration of -OH, C=C, C=OH, C-O-C and C-O, respectively [42]. Figure 4d was the FTIR spectrum of Org-MAG/SA/metronidazole. Compared with the FTIR spectra of Org-MAG showed in Fig. 4c, it was found that the characteristic absorption peaks at 2923 cm −1 and 2861 cm −1 caused by organic modifiers intercalated into the MAG interlayer disappeared, which indicated that due to the preparation of drug carrier, the characteristic absorption peaks at 2923 cm −1 and 2861 cm −1 had disappeared. The organic modifier was exfoliated from the interlayer of MAG, while metronidazole entered the MAG interlayer and was coated by the MAG interlayer, which was corresponding to XRD pattern mentioned above [43,44]. Compared with the standard FTIR spectra of metronidazole, the characteristic absorption peak between the wave number of 1500-400 cm −1 has a large multi-peak superposition, which also indicated that metronidazole was encapsulated inside the Org-MAG/SA/metronidazole [45]. The expansion vibration absorption peak of the C-N single bond was between the wave number of 1300-900 cm −1 , the absorption peak intensity of the C-N group was reduced since the oxygen atom in the molecule of metronidazole and was close to the -C-N group; thus, that the intensity of the absorption peak of the C-N group was small. Therefore, several stretching  -h), the SA contents were 0% (S0), 5% (S1), 10% (S2), 15% (S3), 25% (S4), 50% (S5), 75% (S6) vibration absorption peaks of C-N bond at the wave number of 1300-900 cm −1 will be combined into a single absorption peak with very small intensity and large width.

Sodium Alginate/Magadiite Drug Release In Vitro
The cumulative release percentage for different content of SA were shown in Fig. 5 and the cumulative release percentage were shown in Table 2, which showed that the final release percentage of Org-MAG was very low, indicating that most of the drugs hadn't released. The main reason was the adsorption effect of MAG layers [46]. When a certain amount of SA was added, the cumulative release percentage was increasing continuously until the content of SA was 10%, reaching the maximum value of 93.42%. It can be seen that the addition of SA can significantly increase the release percentage of the drug, the low release percentage of Org-MAG was due to the fact that the drug in the Org-MAG interlayer was obstructed and adsorbed by the layer so that it cannot be released. Meanwhile the addition of SA expands the interlayer spacing of MAG, a good agreement with the XRD results of Fig. 3, reduces the interaction between drug molecules and MAG layer. When the content of SA was excessive, as shown by electron microscope ( Fig. 2 (g, h)), the layer space of MAG was completely encapsulated by SA, which leads to the impossibility of releasing the drugs in MAG layer. Table 3 showed time acquired for the release percentage reach to 90% of the balance release percentage, and it can be seen that with the increasing of SA content, the drug sustained-released time has been extended (the time of S1, S2 and S3 were over 20 h), and when the SA content was 10%, the release took the longest time to reach balance. As the SA content continuously increased, the time acquired to reach 90% of the drug balance release percentage became shorter. The reason was that appropriate amount of SA can encapsulate a part of the drug, when the drug released in-vitro, the drug at the edge of the MAG layer released first, but the drug between the layers cannot released immediately due to the blocking effect of SA macromolecules and MAG layer, resulting in effectively extending sustained-released time. When SA was further increased, the layer space of MAG was completely encapsulated by SA, as shown by electron microscopy (in Fig. 2), so during the in-vitro release, only drugs adsorbed outside the MAG layer can be released in an even higher percentage, but the drug between the MAG layers coated by SA was difficult to release, because SA was difficult to dissolve in acid solution. In general, the release of a biodegradable polymer-loaded drug in vitro was controlled by three mechanisms: diffusion of drug molecules, biodegradation of high molecular polymers, and swelling or dissolution of high molecular polymers [47]. The sustained release steps of drug carriers could be generally divided into two stages: first, the drug attached to the surface of the drug carrier diffuses in the buffer medium, and secondly, the polymer swells and dissolves in the solution. The sustained release figure reveals that the release rate of different drug carriers was fast within 0 ~ 4 h and slow down after 4 h.

Drug Release Kinetics Study
The study of drug release kinetics is helpful to understand the mechanism of drug release. The analysis of the drug delivery process of Org-MAG/SA drug carrier in vitro shows that the sustained release of Pickering emulsion drug carrier consists of diffusion in buffer medium, swelling and dissolution of the polymer, and interlayer diffusion. The drug molecules encapsulated by drug carriers may diffuse into the release medium through the swelling and dissolution of polymers, and the small interaction between drug molecules and layered lamellae and polymers will affect the release kinetics. Under the example of the sustained release of drug carrier, the kinetics fitting analysis of the sustained release process was carried out.
The sustained release of metronidazole of the drug carrier may undergo several steps, one is the diffusion of the drug in the interlayer; the second is the solid-liquid boundary of the drug between the MAG and the release medium, in which liquid film diffusion occurs, and the third stage is the concentration gradient diffusion of the drug in the release medium. The release behavior was studied using the Zero order model, the First order model, the Higuchi model, the release-time reciprocal model, the Baker-Lonsdale model, and the Korsmeyer-Peppas model [46,47]. Zero-order model expression: First order model expression: Baker-Lonsdale model expression: Korsmeyer-Peppas model expression: In the above expressions, Q t and Q ∞ represent the cumulative release amount and maximum cumulative release of metronidazole during t time respectively, mg/g; k 0 , k 1 , k 2 , and n represent the release constants in the corresponding kinetic model, respectively; For release time, h; C KP represent the constants in the corresponding kinetic model, respectively.
The relevant parameters for a linear fitting of the release kinetic model of S1, S2, and S3 are shown in Table 4. The fitting parameters of these three samples are similar. It can be seen from the data that the linear correlation coefficients of the first order model, Baker-Lonsdale and  Korsmeyer-Peppas models for drug sustained-release fitting are above 0.9. All three models can describe the sustained release process of drug carrier. Among the four models, First-order model relatively high linear correlation coefficient, indicating that it is prior to describing the release kinetic behavior of the composite drug carrier, while the zero-order model does not match.
According to the results of the drug release test, we selected S2 as the typical sample due to its highest cumulative release percentage. Figure 6 showed the fitting curves of different models for drug composite carriers and the fitting parameters of S2 under different release kinetic models were showed in Table 4. In the Korsmeyer-Peppas model, n is the diffusion index, and its value can be used to describe the drug release mechanism. The value of n is between 0.43 and 0.85, indicating that the sustained release mechanism of the composite carrier to metronidazole belongs to non-Fick diffusion, which is the synergistic effect of drug diffusion and matrix dissolution, which may be due to the barrier effect of the interlayer space of MAG and the swelling and dissolution of SA [48]. The release kinetics of the drug carrier also fits well with the First-order model, indicating that the sustained release of metronidazole is related to its concentration profile in the laminate structure of the carrier material [49][50][51], mainly due to the presence of layers in the MAG. The drug molecule is limited by the MAG interlayer space and the encapsulation of SA, and the MAG interlayer space has an inhibitory effect on the in vitro dissolution of metronidazole, thus causing a change in release percentage.

Conclusions
In this study, a serious of Org-MAG/SA drug carriers containing metronidazole were prepared through Pickering Emulsion-Templated Encapsulation and their controllable sustained release property were studied. XRD, FT-IR, and SEM analysis showed that the majority of SA encapsulated the Org-MAG, and a few SA was intercalated into the Org-MAG inner space layers. Metronidazole was combined with the carrier material through physical apparent adsorption, electrostatic action, and coating mechanism.
In vitro release test was conducted in the condition that the pH was determined to be 4 to simulate gastric the acid-base environment in which the metronidazole was released after oral administration. SA content has a significant influence on the sustained release of metronidazole, a small amount of SA (5%-10%) can braced layer the Org-MAG inner layer and formed a film to wrap the lamellar spacing, which can prevent metronidazole from completely stuck in the drug carrier, this helped to increase drug release amount and slow down release rate. But if the SA was excess (more than 15%), the loaded drug cannot go through the thick SA film and release. The Pickering drug carrier prepared by 10% SA reached a balance in over 32 h. Metronidazole which encapsulated by MAG and SA in drug carrier can maintained a relatively low release rate because of the drug carrier's mesoporous structures, the swelling and dissolution of SA and the barrier effect of the MAG layer [52].