Multilayer encapsulated mesoporous silica

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And the nanoparticles possessed good stability and strong mucosa adhesive ability. We believe ... lysine) (PLL) on funorescein loaded hollow mesoporous silica.

Materials Science and Engineering C 47 (2015) 313–324

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Multilayer encapsulated mesoporous silica nanospheres as an oral sustained drug delivery system for the poorly water-soluble drug felodipine Liang Hu a, Hongrui Sun b, Qinfu Zhao a, Ning Han a, Ling Bai a, Ying Wang a, Tongying Jiang a, Siling Wang a,⁎ a b

Department of Pharmaceutics, Shenyang Pharmaceutical University, P.O. Box 32, Liaoning Province, Shenyang 110016, PR China English Teaching Department, School of Basic Courses, Shenyang Pharmaceutical University, Wenhua Road 103, Shenyang 110016, PR China

a r t i c l e

i n f o

Article history: Received 24 March 2014 Received in revised form 27 September 2014 Accepted 21 October 2014 Available online 24 October 2014 Keywords: Mesoporous silica nanospheres Layer-by-layer Sustained release Poorly water-soluble drug Felodipine

a b s t r a c t We used a combination of mesoporous silica nanospheres (MSN) and layer-by-layer (LBL) self-assembly technology to establish a new oral sustained drug delivery system for the poorly water-soluble drug felodipine. Firstly, the model drug was loaded into MSN, and then the loaded MSN were repeatedly encapsulated by chitosan (CHI) and acacia (ACA) via LBL self-assembly method. The structural features of the samples were studied using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and nitrogen adsorption. The encapsulating process was monitored by zeta-potential and surface tension measurements. The physical state of the drug in the samples was characterized by differential scanning calorimetry (DSC) and X-ray diffractometry (XRD). The influence of the multilayer with different number of layers on the drug release rate was studied using thermal gravimetric analysis (TGA) and surface tension measurement. The swelling effect and the structure changes of the multilayer were investigated to explore the relationship between the drug release behavior and the state of the multilayer under different pH conditions. The stability and mucosa adhesive ability of the prepared nanoparticles were also explored. After multilayer coating, the drug release rate was effectively controlled. The differences in drug release behavior under different pH conditions could be attributed to the different states of the multilayer. And the nanoparticles possessed good stability and strong mucosa adhesive ability. We believe that this combination offers a simple strategy for regulating the release rate of poorly water-soluble drugs and extends the pharmaceutical applications of inorganic materials and polymers. © 2014 Elsevier B.V. All rights reserved.

1. Introduction It is commonly recognized that oral drug delivery is widely accepted and convenient administration route. For oral administration, a drug must first be dissolved in gastrointestinal fluids before adsorption. Since the dissolution is a rate-limiting step in drug absorption, poorly water-soluble drugs with low dissolution rate have low drug bioavailability [1,2]. Meanwhile, to achieve essential plasma drug concentration, the dosage must be raised. And the raised dosage will make the drug molecules easier to aggregate in gastrointestinal tract, which may also influence the drug dissolution. Nowadays, nearly 40% of commercially available drugs or new drug candidates under development are poorly water-soluble [3]. It is a great challenge for pharmaceutical researchers to improve the dissolution rate of poorly water-soluble drugs. Many strategies have been developed to solve this problem, such as nanotechnology and the use of solid dispersions, but the resulting formulations often suffer from poor stability. ⁎ Corresponding author at: Shenyang Pharmaceutical University, No.103, Wenhua Road, Shenyang 110016, PR China. E-mail address: [email protected] (S. Wang).

http://dx.doi.org/10.1016/j.msec.2014.10.067 0928-4931/© 2014 Elsevier B.V. All rights reserved.

In the past decade, silica-based mesoporous materials have attracted the attention of pharmaceutical researchers around the world. As we know, silica-based mesoporous materials have many unique properties, such as their non-toxic nature, large specific surface area and total pore volume, tunable pore size, as well as being chemically inert and having easily modified surface properties [4–9]. Various types of such materials have been developed into drug delivery systems [10,11]. It is generally considered that these materials are very promising to solve the problems mentioned above. When loaded using silica-based mesoporous materials, the drug molecules can be separated on the surface of these materials and so the intermolecular interactions of the crystal structure are prevented, which is the main reason why slow dissolution kinetics can be avoided [12–14]. In addition, the nanoscale drug particles in a higher free energy state are confined to the rigid porous structure, making the drug nanoparticles more stable [13]. Therefore, employing silica-based mesoporous materials as drug carriers will increase the dissolution rate and the stability of poorly water-soluble drugs. In addition, the increased delivery rate provides a foundation for further study and investigation of the application of poorly water-soluble drugs to provide controlled or sustained delivery.

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Fig. 1. Schematic representation of Fel loading into MSN and CHI/ACA multilayer encapsulation for drug release.

In order to improve patient compliance, drug efficacy and commercial value, it is better that the drug delivery rate is effectively controlled and, so, we focused on a recently developed nano/micro encapsulation technique, layer-by-layer (LBL) self-assembly technology. Using this technique, different particles may be repeatedly coated with oppositely charged polyelectrolytes. When placed in a polyelectrolyte solution with a relatively high concentration, charged particles can produce excess polyelectrolyte adsorption, which leads to surface charge neutralization and resaturation, and, eventually, charge reversal. The surface charge alternations will cause the positive and negative polyelectrolytes to assemble continuously, thereby resulting in the desired number of encapsulation layers [15]. By using such a simple procedure, drug crystals biological macromolecules and some drug/inorganic hybrids can be successfully encapsulated, resulting in desirable release features. Jasaswini Tripathy et al. have encapsulated bovine serum albumin (BSA) using sodium carboxymethyl cellulose (CMC) and poly(allylamine hydrochloride) (PAH) through LBL self-assembly. And the release profile showed a sustained release pattern up to 7 h [16]. When doxorubicin loaded porous CaCO3 was coated by CHI/ALG multilayer, a sustained drug release pattern was also achieved [17]. Yufang Zhu et al. have encapsulated cytosine-phosphodiester-guanine olifodeoxynucleotide (CpG ODN) and enzyme degradable poly(L -

lysine) (PLL) on funorescein loaded hollow mesoporous silica (HMS) through LBL technology, and an enzyme-triggered drug and gene codelivery system was achieved [18], while Yang et al. have used two polyelectrolytes pairs, PAH/PSS and ALG/CHI, to encapsulate drug-loaded mesoporous silica nanotubes, and obtained two different kinds of pH-controlled drug delivery systems [19]. As known, above the isoelectric point of SiOH (pH 2–3), the surface of mesoporous silica materials is negatively charged, which will favor the LBL self-assembly coating of polyelectrolytes [20]. CHI (pKa around 6.3 [21]) and acacia (ACA, negatively charged above pH 2.2 [22]), are natural macromolecular materials, and both exhibit good biocompatibility and are readily obtainable. Therefore, we chose this new polyelectrolyte pair to produce the LBL multilayer assembly. At pH 5.0, the positively charged CHI is well adsorbed on the negatively charged MSN, followed by the adsorption of the negatively charged ACA. Thus, polyelectrolyte multilayer can be easily coated on the surface of MSN and, so, we expected to be able to regulate the drug release rate by adjusting the number of layers. Here, we have proposed a combination of silica-based mesoporous material, employed as drug carriers, and the polyelectrolyte multilayer film, coated by the LBL technique, to regulate the release rate of a poorly water-soluble drug and, eventually, obtain an oral sustained drug delivery system. Hence we synthesized mesoporous silica nanospheres

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315

Fig. 2. SEM and TEM images of MSN (A, B), Fel-MSN (C, D) and Fel-MSN/(CHI/ACA)5 (E, F).

(MSN) and chose felodipine (Fel), a BCS class II drug (poor water solubility and high biomembrane permeability [23]), as a model drug to be loaded into the pores of MSN. Then, the polyelectrolyte multilayer shell, composed of CHI and ACA, was adsorbed on the drug loaded MSN according to the LBL self-assembly method. The whole process was summarized in Fig. 1. We believe that this combination will offer a simple strategy for regulating the delivery rate of poorly watersoluble drugs and extend the pharmaceutical applications of inorganic materials and polymers.

Table 1 Characteristics of drug carriers before and after drug uptake and after LBL encapsulation. Sample

Dap (nm)

SbBET (m2/g)

Vtc (cm3/g)

Loading content (%)

MSN Fel-MSN Fel-MSN/(CHI/ACA)1 Fel-MSN/(CHI/ACA)3 Fel-MSN/(CHI/ACA)5

2.4, 3.8

1051.82 521.21 407.40 314.86 168.43

0.75 0.38 0.31 0.26 0.25

18.31 17.53 16.72 16.24

Dpa is the pore diameter calculated by the BJH method on the branches of the nitrogen desorption isotherms. SBET b is the BET surface area calculated using experimental points at a relative pressure of P/P0 = 0.05 ~ 0.25. Vtc is the total pore volume determined at a relative pressure of 0.9814.

2. Materials and methods 2.1. Materials Tetraethyl orthosilicate (TEOS), hexadecyl trimethyl ammonium bromide (CTAB), anhydrous ethanol (99.7%, AR), methanol dried (≧ 99.5%, AR), ACA (≧ 99%) and sucrose were obtained from Tianjin Bodi Chemical Holding Co., Ltd. Ethyl ether (≧ 99.0%, AR) was produced by Tianjin Kaixin Chemical Industrial Co., Ltd. CHI (DAC ≧ 95%) was obtained from Haidebei Biotechnology (Jinan, China). Raw Fel was purchased from Changzhou Ruiming Pharmaceutical Co., Ltd. Fluorescein isothiocyanate (FITC) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Paraformaldehyde was purchased from Sigma-Aldrich. Cryoembedding media (OCT) and Triton × 100 were purchased from Shenyang Baoxin Co., Ltd. Rhodamine phalloidin was purchased from Shanghai Xinrui Co., Ltd. HOE33342 was purchased from Beijing Biyuntian Co., Ltd. Commercial available felodipine rapid-release tablets (5 mg) were produced by Beijing Union Pharm. Commercial available felodipine sustained release tablets (Plendil, 5 mg) were produced by AstraZeneca Pharmaceutical Co., Ltd. All other chemicals were commercially available and were used as purchased without any further purification. Double distilled water was used in all experiments.

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ethanol for three times on the 0.22 μm filter membrane. Finally, the white sample was dried at 60 °C for 24 h to remove the organic solvent completely. The products, named Fel-MSN, were passed through the 80-mesh sieve and placed in a vacuum dryer for further use. 2.4. Polyelectrolyte multilayer coating

Fig. 3. Zeta-potentials and surface tensions as a function of number of layers on Fel-MSN alternately encapsulated by CHI and ACA.

2.2. Preparation of mesoporous silica nanospheres The synthesis process of MSN was referred to the reference reported by Du et al. [24] with some modification. Briefly, 1.6 g CTAB was firstly added into the mixture of 200 ml double distilled water and 60 ml anhydrous ethanol, stirring until clear at room temperature. Then 1.6 ml ammonia and 40 ml ethyl ether were added into the solution. When a homogeneous solution obtained, 5 ml TEOS was added dropwise into the solution under vigorous stirring. After continuous stirring for 4 h at room temperature, a white precipitate was obtained. The resulting white precipitate was further homogenized using an ATS AH100D homogenizer (ATS Engineer Inc., China) at 800 bar for 20 cycles to reduce the aggregation of nanoparticles. Then separated by centrifugation, the products were washed by double distilled water and anhydrous alcohol for three times, respectively, and dried at 60 °C for 6 h. At last, the products were calcined in air at 550 °C for 6 h with the heating rate of 2 °C/ min to remove the template and other organic components.

2.3. Drug loading Fel was selected as a model drug. First, Fel was dissolved in anhydrous ethanol in a sealed glass bottle (20 mg/ml), and then certain amount of the fabricated MSN was added into the solution with the drug/silica mass ratio of 1:3. After ultrasonicated for 20 min and gently stirred at room temperature over night, the mixture was dried in air at 60 °C. In order to remove the drug adsorbed on the outer surface on MSN, the products were washed using elution method by anhydrous

CHI/ACA multilayer coating through LBL self-assembly was accomplished as follows. Before coating, 400 mg Fel-MSN were dispersed in 20 ml 0.5 M NaCl solution and the pH value was adjusted to 5.0 using acetic acid and NaOH. To deposit the first layer, an equal volume of the CHI solution (2 mg/ml in 0.5 M NaCl, pH 5.0) was added to the suspension of Fel-MSN and shaken at 80 rpm for 1 h. The resulting particles were treated with three repeated circles of centrifugation (3000 rpm, 3 min)/washing/redispersion with 0.5 M NaCl solution (pH 5.0). The oppositely charged ACA (20 ml, 2 mg/ml in 0.5 M NaCl, pH 5.0) was adsorbed through the same process. The deposition procedure of the CHI/ACA bilayer repeated until desired bilayers of CHI/ACA were obtained. The encapsulated Fel-MSN were centrifuged and washed with double distilled water for 3 times and then dried in air at 60 °C for 6 h. The samples obtained were passed through an 80-mesh sieve and referred to as Fel-MSN/(CHI/ACA)n (n = the number of CHI/ACA bilayers). 2.5. Morphology and structure characterization The morphology of the prepared samples was obtained by using SUPRA-35 field emission SEM (ZEISS, Germany). Before analyzing, samples were attached on the double side adhesive carbon tape, which was pasted on an aluminum stub, and then gold-coated under vacuum condition. The porous structure of the samples was obtained by using a TEM (Tecnai G2 F30, FEI, The Netherlands). Prior to analysis, samples were dispersed into double distilled water through sonication and then adsorbed on carbon-plated copper grids. The pore size, specific surface area and total pore volume of the samples were analyzed using an adsorption analyzer (Vsob-2800P, China). Prior to analysis, all the samples were degassed under vacuum at 60 °C for 6 h. The Brunauer–Emmett–Teller (BET) surface areas were determined using experimental points at a relative pressure of P/P0 = 0.05–0.25. The pore size distributions (DBJH) were computed out from the adsorption branch of isotherms by the Barrett-Joyner-Halenda (BJH) method. The total pore volumes were estimated from N2 amount adsorbed at a relative pressure of 0.9814. 2.6. The monitoring of the encapsulation process The encapsulation process was monitored using the zeta-potential analyzer and the surface tension measurement.

Fig. 4. XRD patterns (A) and DSC curves (B) of Fel, MSN, Fel-MSN, Fel-MSN/(CHI/ACA)1, Fel-MSN/(CHI/ACA)3, Fel-MSN/(CHI/ACA)5.

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Fig. 5. Dissolution profiles of Fel, Fel-MSN, Fel-MSN/(CHI/ACA)1, Fel-MSN/(CHI/ACA)3 and Fel-MSN/(CHI/ACA)5 in 0.3% SDS solution.

The zeta-potential of each layer adsorbed on the drug-loaded MSN dispersed in 20 ml 0.5 M NaCl solution (pH 5.0) during the encapsulation process was determined on a Zeta-Potential/Particle Sizer NICOMP 380 ZLS and the value was the average of three consecutive measurements. The surface tension measurement was carried out by du nouy ring method using Automatic Surface & Interface Tensiometer Model A201 (Kino, USA) when each layer encapsulated Fel-MSN were redispersed in 20 ml 0.5 M NaCl solution (pH 5.0). And the value was the average of three consecutive measurements.

317

Fig. 7. Surface tensions of MSN/(CHI/ACA)1, MSN/(CHI/ACA)3 and MSN/(CHI/ACA)5.

using Cu Kα radiation as the X-ray source. The X-rays were generated at 30 mA and 30 kV. Data were obtained from 5° to 40° (diffraction angle 2θ) at a step size of 0.02° and a scanning speed of 4°/min radiation. DSC was performed using a differential scanning calorimeter ((MettlerToledo, Switzerland)). Samples were put into the aluminum pans and the thermal analysis was conducted from 25 °C to 180 °C at a rate of 5 °C/min under a nitrogen flow.

2.9. In vitro drug release study 2.7. Drug loading analysis The actual drug loading of Fel-MSN and the encapsulated Fel-MSN was ascertained by extracting an accurately weighed amount of Felloaded composites with ethanol, followed by the determination of drug content using ultraviolet (UV) spectroscopy at a wavelength of 362 nm (UV-2000, Unico, USA). The standard curve was linear over the concentration range of 0.5–15 μg/ml. All the measurements were carried out in triplicate. 2.8. Physical state characterization by DSC and XRD The physical state of Fel in mesoporous silica was examined using DSC and XRD. XRD patterns of the samples were collected using an Xray diffractometer (X'pert PRO, PANalytical B.V., The Netherlands)

The dissolution behavior of samples was studied by a USP II paddle method in a dissolution apparatus (ZRS-8G, Tianjin Tianda Tianfa Technology Co., Ltd.). The paddle speed was 50 rpm and the volume of the dissolution medium, keeping the temperature at 37 ± 0.5 °C, was 1000 ml. 0.3% (w/v) sodium dodecyl sulfate (SDS) solution was used as the dissolution medium. At appropriate sampling times, 5 ml samples were withdrawn and replaced by equal volume of fresh medium instantly. Each sample was filtered through a 0.22 μm membrane filter and tested by UV spectrophotometry (UV-2000, Unico, USA) at 362 nm. Each sample was equivalent to 5 mg Fel and all the dissolution studies were carried out in triplicate.

2.10. TGA analysis To investigate the relationship between the drug release rate and the encapsulating amount, the TGA analysis of blank MSN, MSN/ (CHI/ACA)3 and MSN/(CHI/ACA)5 was carried out by TGA-50 instrument (Shimadzu, Japan) from 50 to 550 °C at the heating rate of 10 °C/min.

2.11. The influence of the multilayer on the drug release

Fig. 6. TGA patterns of MSN, MSN/(CHI/ACA)3 and MSN/(CHI/ACA)5.

To explore the changes of the polyelectrolyte multilayer during the drug release, while excluding the interferences of the drug and SDS on the measurements, we dispersed the encapsulated blank MSN in water and measured the changes of the surface tension with time. Briefly, MSN/(CHI/ACA)1, MSN/(CHI/ACA)3 and MSN/(CHI/ACA)5 were placed in 25 ml double distilled water and the systems were maintained at ambient temperature with 150 rpm stirring. The surface tensions of the systems were measured at appropriate intervals. All measurements were carried out in triplicate.

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Fig. 8. Dissolution profiles (A) and particle size changes (B) of Fel-MSN/(CHI/ACA)5, and schematic representation of the drug release mechanism (C) under different pH conditions.

2.12. In vitro pH-response study In order to study the pH effect, the dissolution profile of Fel-MSN/ (CHI/ACA)5 was studied in enzyme-free simulated gastric fluid (SGF, pH 1.2), enzyme-free phosphate buffer fluid (pH 4.5) and enzymefree simulated intestinal fluid (SIF, pH 6.8) with 0.3% (w/v) SDS. At appropriate sampling times, 5 ml samples were withdrawn and replaced by equal volume of fresh medium instantly. Each sample was filtered through a 0.22 μm membrane filter and tested by UV spectrophotometry (UV-2000, Unico, USA) at 362 nm. Each sample was equivalent to 5 mg Fel and all the dissolution studies were carried out in triplicate. 2.13. Swelling characteristics of Fel-MSN/(CHI/ACA)5

at 60 °C for 6 h. The specific surface area of the dried contents was then measured using an adsorption analyzer as mentioned above.

2.15. Accelerated stability study The accelerated stability studies were done on three different temperatures (60, 100 and 120 °C) to determine the elimination rate constant and expiry date accordingly. Fel-MSN/(CHI/ACA)5 capsules were prepared. And the capsules were packaged as commercially available preparations. The packaged capsules were placed in the oven at according temperature. At proper time intervals, the drug content was measured according to the method mentioned in Section 2.7. And the

To understand the relationship between the drug release behaviors and the states of the multilayer, the swelling of the multilayer was further studied under different pH conditions. We dispersed a selected amount of Fel-MSN/(CHI/ACA)5 in buffer solutions of pH 1.2, pH 4.5 and pH 6.8. Following a 2 hour incubation, the particle sizes under the three pH conditions were measured using a Zeta-Potential/Particle Sizer NICOMP 380 ZLS and the value was the average of three consecutive measurements. 2.14. Surface changing characteristics of MSN/(CHI/ACA)5 In order to investigate the surface structure changes of the multilayer coated MSN under different pH values during the drug release period, the specific surface areas were measured using an adsorption analyzer (Vsob-2800P, China). Briefly, four 50 ml EP tubes were prepared under each pH condition (pH 1.2, pH 4.5 and pH 6.8) and then each EP tube was loaded with 60 mg of MSN/(CHI/ACA)5 and 30 ml buffer solution. Then, after shaking for a selected time at 100 rpm, and 37 °C in a shaking incubator, one tube was taken out and the contents were dried

Fig. 9. Specific surface area changes at different sampling times under different pH conditions.

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elimination rate and expiry date were calculated using Arrhenius approach [25,26]. 2.16. Mucosa adhesive ability in rat intestine The encapsulated nanospheres were labeled by FITC through the condensation between the isothiocyanate groups of FITC and the NH2 groups of CHI. Briefly, 5 mg of MSN/(CHI/ACA)5 was added into 10 ml of 0.5 mg/ml FITC solution. The suspension was stirred at room temperature overnight, and the FITC labeled encapsulated MSN were collected by centrifugation. After being washed with water for three times, the FITC labeled nanoparticles were redispersed in 10 ml water and kept in dark place for further use. This study was carried out in 8 male rats (about 200 g) divided into two groups. Each rat in the control group was given 0.5 ml of 0.5 mg/ml FITC solution through an intragastric tube after overnight fasting. Meanwhile, each rat in the dosing group was given 0.5 ml of the FITC labeled nanoparticle suspension. After appropriate time intervals, rats were sacrificed, and different intestinal segments were selected and removed. The selected duodenum, jejunum and ileum segments were immersed in 10% paraformaldehyde solution and 30% sucrose solution at 0 °C for two days, respectively. Then the samples were frozen in cryoembedding media for subsequent cryostat sectioning at 20 μm (CM3050S, Leica). The sections were applied to glass slides, immersed in TPBS (0.15 ml Triton × 100 in 5 ml PBS) for 4 min and rinsed with PBS for three times. Then, the cytomembrane was stained with rhodamine phalloidin, and the nucleus was counterstained with HOE33342. Afterwards, the sections were observed by confocal laser scanning microscopy (CLSM, FluoView™ FV1000, Olympus, Japan).

319

Table 2 Elimination rate constant values at different temperatures. Temperature

333 K

373 K

393 K

k × 104/h−1

5.481

172.7

322.4

that the mesopores of the MSN were radially arranged from the center to the surface, which should favor drug loading and release. As shown in Table 1, the synthesized MSN had a pore size distribution at 2.4 nm and 3.8 nm, a high specific surface area of 1051.82 m2/g and a large total pore volume of 0.75 cm3/g. All the features mentioned above confirmed that the synthesized MSN had the potential to be used as excellent drug carriers. After drug loading and polyelectrolyte multilayer coating, as the SEM images (Fig. 2C and E) showed, the nanospheres remained almost monodispersed. Compared with Fel-MSN, as shown in Fig. 2D, the edge of which looked very smooth, we could clearly see that a thin layer was present at the edge of the nanospheres as the arrows show in Fig. 2F, which was likely due to the polyelectrolyte multilayer coating on Fel-MSN. Moreover, after drug loading and polyelectrolyte encapsulation, the specific surface area and total pore volume, as summarized in Table 1, were both reduced sharply. All these findings confirmed the success of drug loading and polyelectrolyte encapsulation. 3.2. Felodipine loading and characteristics

In order to verify the practical application value of the prepared formulations, the drug release patterns of the commercial available felodipine rapid-release and sustained release tablets were also studied in 0.3% SDS solution. And the method was the same as Section 2.8.

Fel was loaded according to the solvent deposition method, and then washed with ethanol to remove the adsorbed drug on the surface of the carriers so as to expose the silanol groups for the subsequent coating process. The total process was performed as Fig. 1 represented. UV measurement, as shown in Table 1, confirmed that the drug loading of Fel could reach 18.31%. After the encapsulation process, it could be seen that only a little drug loaded in MSN was lost, which might be attributed to the particularly poor solubility of Fel and the protection of the polyelectrolyte multilayer. The high drug loading is necessary for the preparation of sustained drug release formulations.

3. Results and discussion

3.3. The monitoring of the encapsulation process

3.1. Morphology and structure of the samples

In order to control the drug release rate, CHI and ACA were repeatedly coated on Fel-MSN using the LBL self-assembly method. The entire process was monitored by measuring the zeta-potential as shown in Fig. 3. We produced the polyelectrolyte encapsulation at pH 5.0, because at this pH the surface of MSN is negatively charged [20], while CHI and ACA were also highly charged [21,22]. The zeta-potential of bare Fel-

2.17. Drug release comparison with commercial available tablet

As shown in Fig. 2A, the well-formed spherical MSN were nearly monodispersed with a diameter of about 300 nm. Some large honeycombed mesopores were distributed on the surface of the MSN, which was caused by the gasification of ethyl ether during the synthesis process [24]. From the TEM images of the MSN (Fig. 2B) we could see

Fig. 10. The variation of lg (drug content) vs. time (hours) at different temperatures.

Fig. 11. The variation of lg k vs. 1/T for the Arrhenius approach.

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3.4. Physical state characterization by XRD and DSC

Table 3 Calculated stability data at 25 °C. Temperature/°C 25

lg k −5.357

k × 106/h−1

t0.9/year

4.381

0.1054/k 2.75

MSN was −21.52 mV. After the first CHI layer encapsulation, the zetapotential remained negative, but increased by about 14 mV compared with bare Fel-MSN. As the first ACA was deposited, the zeta-potential value decreased again to about 30 mV. As the CHI/ACA polyelectrolyte encapsulation continued, the zeta-potential changed regularly. The apparent zeta-potential variation indicated that the polycationic CHI and polyanionic ACA were able to be deposited on Fel-MSN by the LBL self-assembly method [17,19]. In addition, the encapsulation process was also monitored by the surface tension measurement. It is reported that CHI has little effect on the surface tension of water [27,28]. But ACA is a widely used surfactant and has a stronger ability to reduce the surface tension of water [29, 30]. As shown in Fig. 3, the surface tension only decreased slightly after the first CHI encapsulation, which might be explained by the slight adsorption of hydrophobic sequences of CHI molecules, supplied by the slight dissolution of the outmost CHI layer when redispersed, on the air/solution interface [27,28]. And after the first ACA deposited, the surface tension value presented a larger decline, which should be attributed to the greater ability of ACA molecules, supplied by the slight dissolution of the outmost ACA layer, to reduce the surface tension. As the encapsulation process continuing, the surface tension value showed an apparent variation, which might help to demonstrate the successful LBL self-assembly encapsulation of CHI and ACA. So, zeta-potential and surface tension measurements, together with the TEM images and the dramatic decrease in SBET and Vpor, clearly demonstrated the successful encapsulation of the CHI/ACA multilayer by the LBL self-assembly method.

The XRD patterns of the drug loaded samples were used to determine whether a crystalline Fel phase could be detected. As shown in Fig. 4A, the diffraction pattern of pure Fel was highly crystalline in nature as indicated by the numerous peaks. However, no crystalline Fel was detected in Fel-MSN. The absence of distinctive peaks indicated that the Fel loaded into the pores of MSN was in a noncrystalline state [31–33]. On the other hand, the XRD pattern of MSN was amorphous [14]. Moreover, the diffraction profiles of the encapsulated samples (Fel-MSN/(CHI/ACA)1, Fel-MSN/(CHI/ACA)3 and Fel-MSN/(CHI/ACA)5) still showed no peaks. Therefore, Fel maintained the noncrystalline state in the pores of MSN throughout the encapsulation of CHI/ACA. The physical state of Fel in mesoporous silica was also examined by DSC analysis. When in its crystalline state in the pores, the amount of drug would be detected and estimated from the melting point depression using DSC. If the drug in the pores is in a noncrystalline form, no melting point depression would be detected. As shown in Fig. 4B, the DSC curve of pure Fel exhibited a single endothermic peak at 143.7 °C, corresponding to its intrinsic melting points. However, no melting peak of Fel was distinguished in the DSC curve obtained for Fel-MSN. The absence of phase transition owing to Fel in the DSC analysis is evidence that Fel in the pores of MSN is in a noncrystalline state. In addition, after CHI/ACA encapsulation, still no melting peak of Fel could be found. So it was indicated that the recrystallization of Fel did not occur throughout the encapsulating process, and the drug in the pores was still in the noncrystalline form. And the results of the DSC analysis confirm those obtained from the XRD study. 3.5. In vitro drug release study Fig. 5 showed the dissolution profiles of pure Fel, Fel-MSN, Fel-MSN/ (CHI/ACA)1, Fel-MSN/(CHI/ACA)3 and Fel-MSN/(CHI/ACA)5 in 0.3% SDS solution. Compared with pure Fel, Fel-MSN showed a much faster release behavior. The drug release within 2 h could reach more than 90%. The immediate dissolution profile of Fel-MSN could be explained

Fig. 12. CLSM pictures of duodenum, jejunum and ileum from control group and dosing group after 45 min.

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Fig. 13. CLSM pictures of duodenum, jejunum and ileum from control group and dosing group after 6 h.

by the dispersing effect of MSN with a particularly high surface area, the inhibition of drug crystal growth by the rigid porous structure and, of course, the hydrophilicity of mesoporous silica [12–14,34]. In addition, the encapsulated Fel-MSN exhibited a more sustained release pattern, and the release rate decreased as the number of bilayers increasing. As for Fel-MSN/(CHI/ACA)5, less than 30% of Fel could be released within 2 h, and the drug release rate was effectively slowed down during 24 h. The sustained release of Fel from the encapsulated Fel-MSN must be due to the blocking effect of the multilayer encapsulated outside Fel-MSN. 3.6. TGA analysis Fig. 6 showed the TGA image of the blank MSN and the encapsulated blank MSN with different number of bilayers. For the blank MSN, the line was almost horizontal, which suggested that there was no organic residue in the blank MSN. The lines for MSN/(CHI/ACA)3 and MSN/ (CHI/ACA)5 were declining as the temperature was increasing, which should be owed to the mass loss of the organic multilayer. And for MSN/(CHI/ACA)5, the mass loss was 17.82%, larger than that for MSN/ (CHI/ACA)3 (8.55%). This indicated that more amount of polyelectrolyte was adsorbed on the surface of MSN as the increase of the number of bilayers, which would show a stronger blocking effect during the drug release. Meanwhile, the decreased specific surface area and total pore volume, as shown in Table 1, also proved this point. Therefore, the drug release rate could be slowed down after multilayer encapsulation, and be finely regulated by changing the number of layers. 3.7. The changes of the multilayer during the drug release To explore the changes of the polyelectrolyte multilayer during the drug release, while excluding the interferences of the drug and SDS on the measurement, we dispersed the encapsulated blank MSN with

different number of bilayers in water and measured the changes of the surface tension with time. As shown in Fig. 7, it was clearly seen that the surface tensions of the encapsulated samples with different number of bilayers gradually decreased with time, indicating that the multilayer could be dissolved gradually in the water. And the dissolved polymers, especially the ACA, which has a strong surface activity [29, 30], would adsorb on the air/solution interface to reduce the surface tension. Meanwhile, the surface tension showed a greater reduction as the number of bilayers increased, which suggested that larger amount of polymers was dissolved when the number of bilayers was higher. And it should be noticed that the declination speed of the surface tension accelerated as the number of bilayers increased, especially at the beginning of putting the encapsulated MSN in water, when there was no polymer in the water to affect the dissolution behavior and it was easier to compare the changes of the layer with different number of bilayers. Therefore, the faster declination speed as the number of bilayers increased might indicate that the outer layer was looser than the inner layer and, so, the outer layer was more easily to be dissolved to reduce the surface tension. So the initial burst release could be attributed to the easier dissolution of the outer layer. 3.8. In vitro pH-response study To further study the influence of pH on the dissolution behavior of Fel-MSN/(CHI/ACA)5, we tuned the above dissolution media to pH 1.2, pH 4.5 and pH 6.8. Fig. 8A showed that the drug release rate could be slowed down effectively in all three of these dissolution media. The drug release profiles all increased slowly throughout the whole release period and the final release ratios were all higher than 80%. However, it should be noted that the drug release rates at pH 1.2 and pH 6.8 were a little faster than that at pH 4.5. This release difference could be attributed to the influence of different pH values on the states of the multilayer coated on the drug loaded MSN.

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Fig. 14. CLSM pictures of duodenum, jejunum and ileum from control group and dosing group after 12 h.

3.9. Swelling characteristics of Fel-MSN/(CHI/ACA)5

3.10. Surface changing characteristics

To investigate the relationship between the drug release behavior and the states of the multilayer film, swelling or constriction, the coated nanospheres were kept at pH 1.2, pH 4.5 and pH 6.8. Fig. 8B presented a comparison of the particle size distributions of Fel-MSN/(CHI/ACA)5 under different pH values. Clearly, the particle size of Fel-MSN/(CHI/ ACA)5 under pH 1.2 and pH 6.8 was larger than that under pH 4.5, which suggested the greater swelling of the multilayer film under pH 1.2 and pH 6.8, and the constricted state under pH 4.5. The swelling would assist drug release from the pores of MSN and the constriction would provide greater resistance to drug release. This could be used to explain why the drug release rates were a little faster at pH 1.2 and pH 6.8 than at pH 4.5. Fig. 8C schematically represented the different states of the multilayer film and the different drug release mechanisms under different pH conditions. It has been reported that the pKa of ACA is about 2.2 [22] and the pKa of CHI is around 6.3 [21]. Therefore, the amino groups on CHI and the carboxyl groups on ACA will be well ionized at pH 4.5. Thus, CHI and ACA could be combined together tightly through electrostatic interaction in the multilayer, so the swelling of the multilayer was limited. At pH 6.8, the carboxyl groups on ACA were mainly in the form of COO−, while the amino groups of CHI were mostly unionized. At this time, in order to neutralize the excess COO−, positive ions, such as Na+, would diffuse into the multilayer, which could lead to swelling of the multilayer due to the osmolality difference [35–37]. Also, the silanol groups were negatively charged and they could cause repulsion with COO−. Therefore, the particle size was larger because of the swelling of the multilayer. As a result, the drug release rate was accelerated to some extent. When the pH value was 1.2, the amino groups on CHI were almost completely ionized and the carboxyl groups on ACA, together with the silanol groups on MSN, were mostly unionized. The swelling of the multilayer produced by the ionized NH3 + groups on CHI might accelerate the drug release to a certain extent, although the hydrogen bonds within the polyelectrolyte multilayer would hinder this [19].

To further investigate the drug release mechanism from the multilayer coated MSN, the structural changes in the multilayer coated blank MSN (named MSN/(CHI/ACA)5) were investigated under different pH conditions. As shown in Fig. 9, the specific surface area of MSN/ (CHI/ACA)5 changed with time at different pH values. At pH 1.2 and pH 6.8, there was an initial fall and then an increase with the time. The initial fall suggested that the swelling of the multilayer at the beginning did not expose the pores but blocked the pores because of the increased volume [13]. As the swelling continued, cracks appeared through the multilayer film and, so, some pores were exposed, increasing the specific surface areas. In addition, the swelling behavior would facilitate the partial dissolution of the multilayer, and the dissolution could be accelerated as the swelling increased, which would also increase the specific surface areas. At pH 4.5, the specific surface area did not change much because of the tight combination between the positively charged CHI and negatively charged ACA. The initial decrease might be attributed to the minor swelling caused by the stretching of the ionized polyelectrolyte molecules because of the repulsion between the same ionized groups on one molecule. Then, because the combination between the positively charged CHI and negatively charged ACA was tight, the gradual increase would mainly be caused by the slow dissolution of the multilayer. Overall, it could be seen that, despite the swelling and partial dissolution of the multilayer under different pH conditions, the pores were not completely exposed (the specific surface area of the blank MSN is 1051.82 m2/g), which implies that the drug release could be controlled effectively during the drug release period. No matter the state of the multilayer, swelling or closely combined, an effective blocking effect could be obtained by the LBL encapsulation method, and eventually produce a sustained drug release system. 3.11. Accelerated stability study Fig. 10 showed the variation of lg (drug content) as a function of time at three temperatures (333, 373 and 393 K). And the elimination

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Fig. 15. CLSM pictures of duodenum, jejunum and ileum from control group and dosing group after 24 h.

To explore the stability of the prepared nanoparticles, the acceleration test was carried out. Fig. 12 showed the CLSM pictures of different sections after 45 min. The cytomembrane and the nuclei were dyed red and blue, respectively. And FITC presented a green fluorescence. The colorful image was formed by the superposition of the former three. For the control

group, we could see green fluorescence in duodenum. And for the dosing group, green fluorescence could been clearly seen in jejunum and ileum. So FITC solution was delivered into the intestinal tract. And FITC labeled nanoparticles were also translated into the intestinal tract. After 6 h, as shown in Fig. 13, no green fluorescence could been seen in any section for the control group, suggesting FITC solution was easily removed. However, for the dosing group, green fluorescence could be clearly seen in duodenum, jejunum and ileum, indicating that compared with FITC solution, FITC labeled nanoparticles had a stronger mucosa adhesive ability. After 12 h, as shown in Fig. 14, for the dosing group, we could still see light green fluorescence in ileum, showing the strong mucosa adhesive ability of the nanoparticles. And after 24 h, as shown in Fig. 15, FITC labeled nanoparticles were completely removed in the three sections of intestinal.

Fig. 16. Dissolution profiles of commercial available Fel rapid-release tablet and Fel-MSN in 0.3% SDS.

Fig. 17. Dissolution profiles of commercial available Fel sustained release tablet, Fel-MSN/ (CHI/ACA)3 and Fel-MSN/(CHI/ACA)5 in 0.3% SDS solution.

rate constants were determined using the slope of Fig. 10. Table 2 demonstrated these elimination rate constant values. Using these values, the Arrhenius plot of the fuel elimination reaction was shown in Fig. 11. Therefore, the elimination rate constant and the shelf life t0.9 could be calculated and shown in Table 3. The preparation was considered to have good stability at room temperature. 3.12. Mucosa adhesive ability in rat intestine

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3.13. Drug release comparison with commercial available tablets In order to verify the practical application value of the prepared formulation, the drug release patterns of the commercial available felodipine rapid-release and sustained release tablets were also investigated for comparison. Compared with the commercial available rapidrelease felodipine tablet, as shown in Fig. 16, the dissolution rate of felodipine from Fel-MSN was faster. The dispersion effect of MSN promoted the dissolution of felodipine. And the increased dissolution rate provided a foundation for the regulation of drug release rate. Compared with commercial available sustained release tablet, as shown in Fig. 17, the drug release pattern of Fel-MSN/(CHI/ACA)3 presented a clear burst release. But the burst release could be suppressed effectively by increasing the number of bilayers of CHI/ACA. And it showed a good sustained drug release pattern when 5 bilayers of CHI/ACA were encapsulated. Although the drug release from Fel-MSN/(CHI/ACA)5 was not so complete as that from the commercial available tablet, it could still reach more than 85%, which would not affect its practical application. In addition, we believe that the ideal drug release pattern can be obtained through further adjustment of the number of layers. 4. Conclusion In summary, we have successfully developed an effective way to control the release rate of a poorly water-soluble drug. Mesoporous silica nanospheres were synthesized and used as drug carriers for felodipine. To slow down the drug release rate, a new CHI/ACA polyelectrolyte multilayer pair was coated on the drug loaded MSN using LBL self-assembly technology. A sustained drug delivery system for felodipine was successfully obtained, although the drug release behavior was slightly affected by the state of the encapsulated multilayer under different pH conditions. Thus, we believe that this simple method will have potential applications in controlling the drug delivery rate of poorly water-soluble drugs. In addition, this combination of silicabased mesoporous materials and the LBL self-assemble technology will extend the pharmaceutical applications of inorganic materials and organic polymers. Acknowledgments This work was supported by National Basic Research Program of China (973 Program) (No. 2015CB932100) and National Natural Science Foundation of China (No. 81473165). References [1] C.A. Lipinski, et al., Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings, Adv. Drug Deliv. Rev. 23 (1997) 3–25. [2] C.A. Lipinski, Poor aqueous solubility-an industry wide problem in ADME screening, Am. Pharm. Rev. 5 (2002) 82–85. [3] Y. Liu, et al., Mechanism of dissolution enhancement and bioavailability of poorly water soluble celecoxib by preparing stable amorphous nanoparticles, J. Pharm. Pharm. Sci. 13 (2010) 589–606. [4] Y. Liu, et al., Steam-stable aluminosilicate mesostructures assembled from zeolite type Y seeds, J. Am. Chem. Soc. 122 (2000) 8791–8792. [5] Y. Liu, et al., Steam‐stable MSU‐S aluminosilicate mesostructures assembled from zeolite ZSM‐5 and zeolite beta seeds, Angew. Chem. Int. Ed. 40 (2001) 1255–1258.

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