Biomimetic synthesized nanoporous silica@poly(ethyleneimine)s xerogel as drug carrier: Characteristics and controlled release effect

International Journal of Pharmaceutics 467 (2014) 9–18

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Biomimetic synthesized nanoporous silica@poly(ethyleneimine)s xerogel as drug carrier: Characteristics and controlled release effect Jing Li, Lu Xu, Hongzhuo Liu, Yan Wang, Qifang Wang, Hongtao Chen, Weisan Pan, Sanming Li * School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 28 December 2013 Received in revised form 4 March 2014 Accepted 23 March 2014 Available online 24 March 2014

The purpose of this study was to prepare and characterize nanoporous silica@poly(ethyleneimine)s (NS@P) xerogel and methanol modified NS@P xerogel synthesized with biomimetic method, and investigate controlled release behavior of propranolol hydrochloride (PNH) loaded carrier materials in vitro and in vivo. Preparation was conducted at ambient conditions, and NS@P xerogel as well as PNH loaded NS@P xerogel were characterized using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and differential scanning calorimeter (DSC). Investigations on morphology and porous characteristics of NS@P xerogel and methanol modified NS@P xerogel were evaluated with scanning electron microscopy (SEM), transmission electron microscopy (TEM) and nitrogen adsorption. The results showed that the order of morphology compactness was NS@P xerogel > 25%NS@P xerogel > 75%NS@P xerogel because PEIs scaffold ability for silica condensation and forming hydrogen bond weakened with increasing volume ratio of methanol modification. Moreover, SBET decreased and uniformity of pore size distribution was interrupted after methanol modification. PNH loaded carrier materials displayed controlled release, and release effect was related with pore size of materials and PEIs scaffold ability. In vivo pharmacokinetic study demonstrated that release of PNH was delayed due to the PNH incorporated inside carrier materials and controlled release effect was in accordance with in vitro results. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Silica@poly(ethyleneimine)s Controlled release Biomimetic synthesis Characteristics In vivo

1. Introduction In the last years, many efforts have been devoted to the development of new formulations that control rate and period of drug delivery. Controlled drug delivery systems (CDDS) designed for long-term administration keep the drug level in the blood constant for a long time between the desired maximum and minimum value. It is generally accepted that traditional carriers currently employed are either natural or synthetic polymers (such as microcapsules, lipoproteins, liposomes), however an increasing number of studies is addressed to the development of nanoporous silica materials as alternative support (Ghedini et al., 2010). An important advantage of nanoporous silica materials is their relatively easy synthesis and excellent biocompatibility (Verraedt et al., 2010). In the area of controlled release, various applications of nanoporous silica materials have been described (Li et al., 2006; Qu et al., 2006). Nanoporous silica materials, defined as one kind of

* Corresponding author at: Wenhua RD 103, 110016, Shenyang, China. Tel.: +86 24 23986258. E-mail address: [email protected] (S. Li). http://dx.doi.org/10.1016/j.ijpharm.2014.03.045 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.

inorganic materials within nano pore size (1–100 nm), possess not only common characteristics of being drug carriers like non-toxic, good biocompatibility, drug loading ability, but also unique advantages over other organic materials, such as tunable particle size, stable structure, double functionality of internal and external surface area, etc. (Slowing et al., 2008). In nature, beautiful silica skeletons in biosilicas can be found in sponges and diatoms, and this biological silica formation is called biosilicification (Jin and Yuan, 2005a). Silica materials synthesized with biosilicification method have hierarchical structures and multiple morphologies with nanopores due to the abundant groups of Si

O

Si (Jaganathan and Godin, 2012). It is reported that biomimetic synthesized nanoporous silica materials can be realized by adding additives, such as polypeptides, polysaccharides, peptides, polyamines (Patwardhan, 2011). As a kind of widely used polyamines, poly(ethyleneimine)s (PEIs) can be assembled and modified to form superhydrophobic multilayered surfaces and used to form hybrid inorganic materials/carbon nanotubes (Shen et al., 2009; Zhou et al., 2012), and this function is similar to Pluronic F127 (Huang et al., 2012). It is known that PEIs can be divided into two types: branched PEIs prepared from the ringopening polymerization of cyclic ethyleneimine and linear PEIs

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formed from the hydrolysis of linear polyoxazoline. Branched chain architecture of PEIs suppresses the crystallinity, resulting in amorphous in the bulk state and freely soluble in water. On the contrary, linear PEIs exhibit very interesting crystalline phases (Yuan and Jin, 2005a). It is reported that branched PEIs can be remained in synthesized nanoporous silica materials when being applied in drug delivery system due to its nontoxicity at low molecular weight (<25 kDa) (Xia et al., 2009). Biomimetic synthesized nanoporous silica materials using branched PEIs as the template have characteristics as follows: (1) biomimetic synthesis processes are carried out at ambient conditions (normal temperature, normal pressure, static state without stirring), which save much source and energy (Patwardhan, 2011); (2) if the template is not removed, surface modification of nanoporous silica materials can be realized ascribed to hydrogen bond formed by branched PEIs and silanol groups. It has been reported that by choosing different solvents, especially methanol, (Jin and Yuan, 2005b; Yuan and Jin, 2005b) utilized during synthesis or modifying liner PEIs with a number of groups that can be connected (Jin, 2003; Jin and Yuan, 2006), morphology of nanoporous silica materials synthesized using liner PEIs as the template can be changed. However, whether silica shapes and structures will change as well when using branched PEIs remains unknown. It is widely known that sol–gel process involves the manufacture of inorganic matrixes through the formation of a colloidal suspension, which is called sol. Wet gel forms a globally connected solid matrix after gelation, and dry gel state forms after drying, which is named xerogel (Ahola et al., 2000). Silica xerogel is a potential biomaterial to be used as matrix materials (QuintanarGuerrero et al., 2009), and drug molecules impregnated into the sol state would be located within silica xerogel network. Sol–gel processed silica xerogel has been investigated as a carrier material in CDDS (Ahola et al., 2000) due to its biodegradability, high drug loading efficiency, more importantly, its low processing temperature, which allows in situ incorporation of drug molecules into silica matrix (Quintanar-Guerrero et al., 2009). Nifedipine (Maver et al., 2007), heparin (Ahola et al., 2001) and toremifene citrate (Ahola et al., 2000) have been used as model drugs to study their controlled release effect. As biomimetic synthesis of nanoporous silica materials using branched PEIs as the template can be formed with sol–gel process, it is undoubtedly reasonable to prepare nanoporous silica@PEIs (NS@P) xerogel as a carrier in CDDS. Water soluble drug propranolol hydrochloride (PNH, see Fig. 1) is a kind of b receptor blocker widely used in the treatment of hypertension, congestive heart failure and arrhythmia (Routledge and Shand, 1979). In practical administration of PNH tablet or injection, each dosage can be varied from 10 mg to 200 mg determined by patient’s condition and patient need to take medicine frequently due to its low half-life (2.0–3.0 h) (Prichard and Gillam, 1969).

Therefore, it is important to apply proper technique and materials to realize controlled release of PNH. The aim of present work was to systemically investigate a novel nanoporous silica xerogel as drug carrier to load PNH in order to control its release. Innovatively, branched PEIs with low molecular weight (<25 kDa) was applied as the template to biomimetic synthesize NS@P xerogel. Verification and physical state of NS@P xerogel, NS@P xerogel without template (NS xerogel) and PNH loaded NS@P xerogel were characterized using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and differential scanning calorimeter (DSC). Inspired by the scientific work that methanol modification of liner PEIs can lead to multiple morphologies of silica materials, methanol modification of NS@P xerogel was obtained by changing volume ratio of methanol (25%, 75%) in PEIs aggregate solution, which were called 25%NS@P xerogel and 75%NS@P xerogel, respectively. Their morphology and porous structure were studied using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and surface area as well as pore size distribution was also evaluated. PNH was loaded into carriers using in situ drug loading method, and in vitro release behavior and in vivo pharmacokinetic studies were carried out to confirm controlled release effect. The present study will help in the design of novel nanoporous silica materials applied in CDDS. 2. Materials and methods 2.1. Materials Tetramethoxysilane (TMOS) was purchased from Aladdin (Shanghai, China), branched poly(ethyleneimine)s (PEIs) with weight-average molecular weight of 20,000 was kindly donated by Qianglong new chemical materials (Wuhan, China). All other chemical were of reagent grade and deionized water was prepared by ion exchange. 2.2. Preparation of NS@P xerogel and methanol modified NS@P xerogel Briefly, NS@P xerogel was synthesized after accomplishing three main steps. Firstly, PEIs aggregate solution was prepared by mixing PEIs with aqueous solution to get the final PEIs concentration of 1.0 wt%. Then 1 ml PEIs aggregate solution was added into 2 ml mixed solution of silica source (TMOS) and absolute ethyl alcohol with volume ratio of 1:1, and left the system at ambient condition statically until the formation of light blue wet gel. Finally, drying wet gel at 40  C vacuum drying oven until xerogel state was formed to accomplish the synthesis of NS@P xerogel. Methanol modified NS@P xerogel was synthesized simply by changing the first preparation step. In detail, methanol modified PEIs aggregate solution was prepared by mixing PEIs with methanol aqueous solution to get the final PEIs concentration of 1.0 wt%. 25%NS@P xerogel and 75%NS@P xerogel were two kinds of methanol modified NS@P xerogel prepared using 25% methanol aqueous solution (methanol:aqueous = 1:3, v/v) and 75% methanol aqueous solution (methanol:aqueous = 3:1, v/v) to mix with PEIs to prepare PEIs aggregate solution. Moreover, nanoporous silica xerogel (NS xerogel) was obtained after removing the template of NS@P xerogel with heat treatment at 550  C for 6 h. 2.3. PNH loading procedure

Fig. 1. Chemical structure of model drug, PNH.

By adopting in situ drug inclusion method, desired amount of drug was impregnated into NS@P xerogel, which was easy and efficient with no need to calculate drug loading amount as well as drug loading efficiency. Herein, a certain amount of PNH was dissolved in deionized water and 0.6 ml drug solution containing 10 mg PNH was added into gelling solution that consisted of TMOS

J. Li et al. / International Journal of Pharmaceutics 467 (2014) 9–18

absolute ethyl alcohol solution (1.6 ml, volume ratio of TMOS to absolute ethyl alcohol was 1:1) and PEIs aggregate solution (0.8 ml). During this process, drug solution should be added dispersedly into gelling solution in order to minimize variations from sample to sample. Then left the system at ambient condition statically until the formation of light blue wet gel and dried wet gel at 40  C vacuum drying oven until xerogel state was formed. For schematic diagram of drug loading process, please refer to Fig. 2. It should be noted that the holes of 24-well plates were of the same shape and volume content (3.0 ml), leading to no variation of samples when conducting in vitro experiment. Additionally, PNH loaded methanol modified NS@P xerogel was designed by alternating volume content of methanol (25%, 75%) in PEIs aggregate solution, which were called PNH loaded 25%NS@P xerogel and PNH loaded 75%NS@P xerogel, respectively. 2.4. Verification and physical state of materials as well as drug loaded carrier materials It should be noted that verification and physical state of 25% NS@P xerogel and 75%NS@P xerogel were consistent with that of NS@P xerogel since methanol modification just changed solvent constitution. Therefore, FT-IR, XRD and DSC results of NS@P xerogel could be also considered as the results of 25%NS@P xerogel and 75%NS@P xerogel. 2.4.1. FT-IR Fourier transform infrared spectroscopy (FT-IR, Spectrum 1000, Perkin Elmer, USA) spectra of samples were obtained over the spectral region 400–4000 cm
1. Samples were prepared by gently and respectively grounding NS@P xerogel, NS xerogel, PNH loaded NS@P xerogel and PNH with KBr. 2.4.2. XRD and DSC XRD patterns of NS@P xerogel, NS xerogel, PNH loaded NS@P xerogel and PNH were collected using an X-ray diffractometer (X’pert PRO, PANalytical B.V., The Netherlands) equipped with a Cu Ka target. The X-rays were generated at 30 mA and 30 kV with a Ni filtered Cu Ka line as the source of radiation. Data were obtained from 5  to 40  (diffraction angle 2u) at a step size of 0.02 and a scanning speed of 4 /min radiation. Thermal analysis was conducted mainly by utilizing differential scanning calorimeter (DSC, Q1000, TA Instrument, USA). Samples were placed in pierced aluminum pans and heated from 0  C to 300  C at a scanning rate of 10  C/min under nitrogen protection.

11

2.5. Morphology, porous structure and porous characteristics of carrier materials 2.5.1. SEM SEM was obtained with SURA 35 field emission scanning electron microscope (ZEISS, Germany) to analyze surface morphology of NS@P xerogel. Samples (NS@P xerogel, 25%NS@P xerogel and 75%NS@P xerogel) were mounted onto metal stubs using double-sided adhesive tape and sputtered with a thin layer of gold under vacuum. 2.5.2. TEM The porous structures of carrier materials (NS@P xerogel, 25% NS@P xerogel and 75%NS@P xerogel) were characterized using a Tecnai G2 F30 TEM instrument (FEI, The Netherlands) operated at 200 kV. Before examination, samples were dispersed in deionized water through sonication and subsequently deposited on carboncoated copper grids. 2.5.3. Surface area and pore size distribution The pore characteristics of NS@P xerogel, 25%NS@P xerogel and 75%NS@P xerogel were studied by determining the nitrogen adsorption and desorption using V-Sorb 2800P (app-one, China) at
196  C. The specific surface area, SBET, was evaluated from nitrogen adsorption data over the relative pressure range from 0.05 to 0.2 using the Brunauer–Emmett–Teller (BET) method. The total pore volume, Vt, was determined from the amount adsorbed at a relative pressure of 0.99. Pore size distributions (PSDs) were determined from adsorption branches of isotherms using the Barrett–Joyner–Halenda (BJH) method. The BJH pore diameter, WBJH, is defined as the maximum on the pore size distribution curve. All samples were degassed at 50  C for 12 h prior to analysis to remove adsorbed water. 2.6. In vitro dissolution Drug release experiment was carried out using USP II paddle method (100 rpm, 37  C, and 900 ml dissolution medium) with a ZRD6-B dissolution tester (Shanghai Huanghai Medicament Test Instrument Factory, China). PNH loaded carrier materials (NS@P, 25%NS@P, 75%NS@P) were exposed to enzyme-free simulated intestinal fluid (pH 6.8). At predetermined time intervals, 5 ml samples were withdrawn from the release medium and then an equivalent amount of fresh medium was added to maintain a constant dissolution volume. Samples administered through 0.45 mm microporous membrane were analyzed using UV-1750 (Shimadzu, Japan) at the wavelength of 289 nm. 2.7. In vivo pharmacokinetics

Fig. 2. Schematic diagram of in situ drug inclusion method. Drug solution was dispersedly added into gelling solution in holes of 24-well plates with injector.

The in vivo experiment was performed in accordance with the Ethical Guidelines for Investigations in Laboratory Animals and was approved by the Ethics Review Committee for Animal Experimentation of Shenyang Pharmaceutical University (Shenyang, China). Male Wistar rats weighing 200  20 g were randomly divided into four groups (A group, PNH loaded NS@P xerogel; B group, PNH loaded 25%NS@P xerogel, C group, PNH loaded 75%NS@P xerogel; D group, PNH commercial tablet) comprising three animals in each group and fasted for 12 h but allowed to free access to water prior to the experiment. The four types of PNH aqueous suspension at a dose of 5 mg/body weight, including aqueous suspension of PNH loaded NS@P xerogel, PNH loaded 25%NS@P xerogel, PNH loaded 75%NS@P xerogel and PNH commercial tablet, were orally administered (intragastric administration) to corresponding four groups of rats. Blood samples (0.5 ml) of each animal were sampled via the suborbital vein at 0,

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0.083, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12, 14, 16 h after administration. All the blood samples were immediately centrifuged at 5000 rpm for 10 min to collect plasma, and the obtained plasma was then stored at
20  C until analysis. Plasma samples were processed as follows: a 200 ml plasma sample was mixed with 20 ml of an internal standard (diltiazem hydrochloride) solution (50 mg/ml) and 200 ml 0.03 mol/l NaOH and vortexed for 1 min. Then 800 ml diethyl ether was added and the mixture was vortexed for 8 min. After centrifugation at 5000 rpm for 5 min, the above organic layer was transferred into another clean tube and 100 ml 0.02 mol/l HCl was added. The mixture sample was vortexed for 8 min and then centrifuged at 5000 rpm for 5 min. The supernatant of each sample (20 ml) was subjected to HPLC analysis. 3. Results and discussions 3.1. Preparation process and mechanism Visually can be seen, formation of NS@P xerogel went through different states, from liquid mixture waiting for reaction, to light blue sols, to semi-solid gels, and finally to solid dry gels, which can be called NS@P sol, NS@P gel, NS@P xerogel, respectively. It is known that the formation mechanisms of various silica materials are hydrolysis and deposition of silicate to form siloxane. Moreover, there are a large number of silanol groups on the surface of silica materials that contribute to be active sites for interacting with newcomers, such as drug molecules and silicon coupling agents, to realize drug loading and further surface modification (Barbe et al., 2004). NS@P xerogel was obtained due to

NH

sites from PEIs existing on the surface of precipitates to promote hydrolysis and condensation process. Preparation of NS@P xerogel using branched PEIs, a kind of soft template owing to its solution state, could be accomplished at ambient condition spontaneously and fast due to three functions of PEIs, which were scaffold, template and catalyst, respectively. The functionality of scaffold was to highly concentrate silica source and form hydrogen bond between PEIs and Si

OH or PEIs and other groups. PEIs catalyze the silica formation due to the alternating presence of protonated and nonprotonated amine groups in the polyamine chains, which allows hydrogen bond formation with the oxygen adjacent to silicon in the precursor and thus facilitate

Si

O

Si

bond formation (Patel et al., 2009). Template functionality of PEIs is to direct formation shape of silica materials. In addition, experiments showed that the formation rate of NS@P gel was largely dependent on total volume of NS@P sol as well as temperature. When using TMOS, consumption of time needed for preparing NS@P gel was about 40 min for total volume of 6 ml NS@P sol, while more than 24 h for 30 ml NS@P sol at ambient room temperature. However, if 30 ml NS@P sol was originally remained at 80  C water base, only several hours was enough for NS@P sol to completely transform to gel state. 3.2. FT-IR After removing the soft template (PEIs) by heat treatment (sintered at 550  C for at least 6 h), characteristic peaks belonging to silica xerogel (Fig. 3A,a), including Si

O

Si bending vibration at 466.4 cm
1, Si

O

Si symmetric stretching vibration at 804.2 cm
1, Si

O

Si antisymmetric stretching vibration at 1101.7 cm
1, H

O

H bending vibration at 1629.9 cm
1 and O

H of Si

OH antisymmetric stretching vibration at 3430.7 cm
1, can be observed. However, if template remained, extra peaks at 2853.7 cm
1 and 2924.6 cm
1 belonging to C

H stretching vibration of PEIs and the amide peak at 1637.0 cm
1 due to vibration of the chain of PEIs (Zhao and Su, 2012), were clearly

Fig. 3. FT-IR spectra of A including a, NS xerogel; b, NS@P xerogel; B including c, PNH; d, PNH loaded NS@P xerogel.

detected (Fig. 3A,b), which sufficiently illustrated the accurate preparation of NS@P xerogel. Physical state of PNH loaded NS@P xerogel was illustrated in Fig. 3B. PNH showed a strong hydroxyl band at 1456.4 cm
1, which corresponded to hydroxyl group presented in PNH. After loading PNH, the band assigned to OH bending vibration belonging to PNH was observed at 1463.6 cm
1, which showed a slight shift from 1456.4 cm
1 due to the hydrogen bonding of the silanol groups to the hydroxyl group of PNH. Moreover, the amide peak at 1637.0 cm
1 belonging to PEIs was not shown after loading PNH, indicating that hydrogen bonding was also formed between the amide groups of PEIs and the hydroxyl groups of PNH. 3.3. XRD and DSC results As shown in Fig. 4A, no crystalline PEIs was observed in the NS@P xerogel sample, indicating the amorphous state of PEIs. In addition, NS xerogel was amorphous single-phase materials due to the presence of a broad band between 15 and 30 2u (Radin et al., 2002). The XRD pattern of the PNH loaded NS@P xerogel was recorded to determine whether a crystalline PNH phase could be detected. As shown in Fig. 4B, the diffraction pattern of pure PNH was highly crystalline in nature as indicated by the numerous peaks. However, no crystalline PNH was detected in PNH loaded NS@P xerogel, indicating that the PNH was loaded into the pores of NS@P xerogel in a noncrystalline state (Salonen et al., 2005; Kapoor et al., 2009). Crystalline state can be also demonstrated by conducting DSC experiment. DSC thermogram of NS@P xerogel showed endothermic phenomena but not obvious endothermic peaks (Fig. 5A,a), indicating that branched PEIs known as amorphous polyamine,

J. Li et al. / International Journal of Pharmaceutics 467 (2014) 9–18

Fig. 4. XRD patterns of A including a, NS xerogel; b, NS@P xerogel; B including c, PNH; d, PNH loaded NS@P xerogel.

was adsorbed onto silica. While after PEIs was removed by heat treatment at 550  C for 6 h, thermogram turned out to be almost plat (see Fig. 5A,b). It can be concluded that NS xerogel was amorphous, which was in agreement with XRD result. As crystalline silica is known to cause a rapid influx of inflammatory cells, increase collagen deposition in lungs, and change histological state of pulmonary lymph nodes (Kortesuo et al., 2000), NS@P xerogel and NS xerogel were safe to be drug carriers due to amorphous state of NS xerogel and non-toxicity of branched PEIs with weight-average molecular weight smaller than 25 kDa (Xia et al., 2009). As shown in Fig. 5B, the DSC curve of PNH exhibited a single endothermic peak at 163.74  C, which corresponded to its intrinsic melting points. However, no melting peak of PNH was identified in the DSC curves obtained from PNH loaded NS@P xerogel. The absence of phase transitions owing to PNH in the DSC analysis was evidence that PNH was in a noncrystalline state. 3.4. SEM The morphology of NS@P xerogel was mainly analyzed by SEM (see Fig. 6). It was obvious that methanol used with different volume ratios (25%, 75%) in PEIs aggregate solution influenced morphology delicately. It has been reported that multiple morphologies of silica can be directed by liner PEIs through facile tuning of the media in which the PEIs pre-organize. The silica shapes and structures ranged from flattened silica particles, cotton-like spheres with nanofibers to monolith without shapes

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Fig. 5. DSC thermogram of A including a, NS@P xerogel; b, NS xerogel; B including c, PNH; d, PNH loaded NS@P xerogel.

after methanol modification with methanol volume contents of 0, 25, 75%, respectively (Jin and Yuan, 2005b). However, no multiple morphologies of carrier materials (NS@P xerogel, 25%NS@P xerogel and 75%NS@P xerogel) could be observed according to SEM images because the PEIs used to form NS@P xerogel was branched and amorphous, which had no ability of forming crystalline state to prominently direct various silica shapes and structures during silica formation. Interestingly, differences of morphology of carrier materials synthesized using branched PEIs could be also observed. It can be concluded that morphology compactness increased with decreasing volume content of methanol. Possibly, this might be largely dependent on the polarity of PEIs aggregate solution. Since polarity of water was much stronger than methanol, the order of polarity of PEIs aggregate solution was aqueous solution > 25% methanol aqueous solution > 75% methanol aqueous solution. Therefore, branched PEIs exposed to higher polarity of system, which was the lower volume ratio of methanol, turned out to possess stronger functionality of scaffold to accelerate silica condensation and form hydrogen bond between PEIs and Si

OH so morphology was more compactness. On the contrary, the higher volume ratio of methanol attributed to lower polarity of PEIs aggregate solution, resulting in weaker scaffold ability and so morphology of obtained carrier materials was looser. In the process of forming carrier materials, we also discovered that the higher volume ratio of organic solvent, the faster reaction rate. Possible

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Fig. 6. SEM photographs of A, NS@P xerogel; B, 25%NS@P xerogel; C, 75%NS@P xerogel.

J. Li et al. / International Journal of Pharmaceutics 467 (2014) 9–18

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Fig. 8. Nitrogen adsorption/desorption isotherms of A, NS@P xerogel; B, 25%NS@P xerogel; C, 75%NS@P xerogel.

illustration for formation rate may be that the higher volume ratio of organic solvent, which had lower polarity and weaker scaffold functionality, required less time to form this looser state of carrier materials. 3.5. TEM TEM images (see Fig. 7) confirmed the existence of nanopores both on the surface and inside of carrier materials (NS@P xerogel, 25%NS@P xerogel and 75%NS@P xerogel), demonstrating their nanoporous structure. 3.6. Surface area and pore size distribution The nitrogen adsorption/desorption isotherms and pore size distribution of NS@P xerogel, 25%NS@P xerogel and 75%NS@P xerogel were presented in Figs. 8 and 9, and the values for SBET, Vt and WBJH were given in Table 1. As can be seen in Fig. 8, NS@P xerogel and 75%NS@P xerogel represented typical type-IV isotherms according to the IUPAC nomenclature with clear hysteresis

Fig. 7. TEM photographs of A, NS@P xerogel; B, 25%NS@P xerogel; C, 75%NS@P xerogel.

Fig. 9. Pore size distribution curves of A, NS@P xerogel; B, 25%NS@P xerogel; C, 75% NS@P xerogel.

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Table 1 Specific surface area, pore volume and pore diameter of samples. Sample

SBET (m2/g)

Vt (cm3/g)

WBJH (nm)

NS@P xerogel 25%NS@P xerogel 75%NS@P xerogel

788 405 588

0.13 0.13 0.05

3.6 2.8 23.6

loops which in the relative pressure scope from 0.4 to 0.8 and 0.8 to 1.0, respectively, indicating their mesoporous structure (Cao et al., 2009). On the contrary, nitrogen adsorption/desorption isotherm of 25%NS@P xerogel was a combination of type-I isotherm and type-IV isotherm, demonstrating that it had microporous and mesoporous structure. Pore size distribution curves (Fig. 9) of these three samples showed that NS@P xerogel and 75%NS@P xerogel were mesoporous carriers, and pores of 75%NS@P xerogel (23.6 nm) were quite larger than NS@P xerogel (3.6 nm). The larger pore size distribution of 75%NS@P xerogel compared with NS@P xerogel could explain its delay of a second steep increase of nitrogen uptake ascribed to the filling of mesopores due to capillary condensation. For 25%NS@P xerogel, it contained a large number of micropores and a small part of mesopores though the higher peak of micropore size distribution was not completely detected, and the mesopores of 25%NS@P xerogel (2.8 nm) was smaller than NS@P xerogel (3.6 nm). It should be noted that there were two peaks of pore size distribution for 25%NS@P xerogel and 75%NS@P xerogel while only one peak for NS@P xerogel, indicating that uniformity of pore size distribution was obviously interrupted after methanol modification for NS@P xerogel, which decreased SBET significantly. Moreover, lower volume ratio (25%) of methanol modification narrowed pore size and higher volume ratio (75%) of methanol modification enlarged pore size. The estimated reason was ascribed to scaffold functionality of PEIs when pre-organized in methanol, which was strong in 25% (v/v) methanol but weak in 75% (v/v) methanol according to SEM results.

Fig. 10. Release profiles of A, PNH loaded NS@P xerogel; B, PNH loaded 25%NS@P xerogel; C, PNH loaded 75%NS@P xerogel. Each data point represents the mean  S. D. of three determinations.

3.7. Drug loading and release discoveries It has been documented that the release mechanism of silicabased xerogel is diffusion and erosion (Ahola et al., 2000). The release profile of drug impregnated into silica xerogel depends on gel characteristics and chemical interactions between the gel network and impregnated drug (Wu et al., 2007). Drug release of PNH loaded carrier materials (NS@P xerogel, 25%NS@P xerogel and 75%NS@P xerogel) displayed distinct controlled-release due to the fact that model drug was sustained to release for more than 24 h with an initial burst release and a sustained release, as can be seen in Fig. 10. The initial burst release may be attributed to the presence of PNH impregnated on the external surface, which allowed a certain amount of drug to be released quickly into the release medium. Then the release rate slowed down due to the time required for erosion of xerogel and the amount of PNH impregnated at the internal of nanopores of carrier materials. In addition, it can be concluded that controlled release effect was improved with decreasing volume ratio of methanol. This

Fig. 11. Plasma concentration–time profiles of A, aqueous suspension of PNH loaded NS@P xerogel; B, aqueous suspension of PNH loaded 25%NS@P xerogel; C aqueous suspension of PNH loaded 75%NS@P xerogel; D aqueous suspension of PNH commercial tablet. Each data point represents the mean  S.D. of three determinations.

phenomenon can be explained in two aspects, including pore size and PEIs scaffold ability. It is widely accepted that fast drug release can be achieved by enlarging pore size of mesoporous carrier (Horcajada et al., 2004; Mellaerts et al., 2008; Yang et al., 2008). Because 25%NS@P xerogel contained a large number of micropores and a small part of mesopores, it should not be considered as a simple mesoporous carrier material. For two mesoporous carrier materials, NS@P xerogel and 75%NS@P xerogel, mesopore size of 75%NS@P xerogel (23.6 nm) was quite larger than

Table 2 Pharmacokinetic parameters of PNH after oral administration of aqueous suspension of PNH loaded drug carrier materials and PNH commercial tablet. A group: PNH loaded NS@P xerogel; B group: PNH loaded 25%NS@P xerogel; C group: PNH loaded 75%NS@P xerogel; D group: PNH commercial tablet. Each value represents the mean  S.D. (n = 3). Pharmacokinetic parameter

A

B

C

D

Cmax (mg/l) Tmax (h) AUC0 ! 16 h (mg h/l) MRT (h) T1/2 (h)

294.033  0.287 0.083  0.000 2010.772  92.200 7.041  0.123 6.364  2.123

361.991 151.340 0.500  0.250 1816.853  820.174 4.806  0.544 5.264  1.994

402.615  71.602 0.417  0.144 1580.610  129.338 3.580  0.308 2.694  0.427

262.814  55.345 0.583  0.144 1499.250  260.416 4.717  0.316 9.469  6.154

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the former (3.6 nm). Therefore, 75%NS@P xerogel exhibited a lower steric diffusion hindrance caused by pore size and drug molecules had a greater chance of escaping from mesopores and diffusing into the release medium. In another aspect, PEIs functioned as the template in NS@P xerogel, influenced drug release due to its methanol modification. According to SEM studies, we got to know that lower volume ratio of methanol in PEIs aggregate solution leaded to stronger scaffold functionality of PEIs to accelerate silica condensation and increase hydrogen bonding force formed between PEIs and Si-OH as well as PEIs and hydroxyl group of PNH, resulting in more compactness of NS@P xerogel. Therefore, (1) PNH could release faster from 25%NS@P xerogel compared to NS@P xerogel, though pore size of NS@P xerogel was litter larger; (2) drug release from 75%NS@P xerogel was faster compared to 25% NS@P xerogel and NS@P xerogel. These explanations also indicated that rate of dissolution and drug release was reduced with increasing the water to alkoxide ratio, which was in accordance with published literature (Barbe et al., 2004). Moreover, it demonstrated that drug release can be controlled to reach appropriate and desired level simply by changing volume ratio of methanol in PEIs aggregate solution because methanol modification influenced pore size and PEIs scaffold ability. This is an obvious novel and meaningful technique to design a drug carrier with regular changeable controlled release of PNH. 3.8. In vivo pharmacokinetic studies The primary aim of in vivo pharmacokinetic studies of drug loaded carrier materials (NS@P xerogel, 25%NS@P xerogel and 75% NS@P xerogel) without adding other excipients was to further investigate and confirm their controlled release effect. The plasma concentration–time profiles and the main pharmacokinetic parameters of PNH resulted from the oral administration in Wistar rats were presented in Fig. 11 and Table 2, respectively. Interestingly, there showed two peaks for A group, B group and C group, while one common peak for the control group (D group) in plasma concentration–time profiles. It is well accepted that the presence of two peaks in plasma concentration–time profile is mainly related to several factors, including enterohepatic circulation or gastrointestinal circulation of drug, reabsorption of active metabolite of drug, special formulations or more than one drug for administration (Wei et al., 2005). Taking into the control group for consideration, only one peak was observed for every animal, excluding the factors of enterohepatic circulation or gastrointestinal circulation of drug and reabsorption of active metabolite of drug. Moreover, two peaks showed in the plasma concentration– time profile for each animal in experimental groups (A group, B group, C group) obviously confirmed that two peaks were attributed to the factor of formulations, which was the factor of carrier materials in this study. As carrier materials for loading PNH possessed nanoporous structure, the second peak of experimental groups was ascribed to the release of PNH located at the internal of nanopores while the first peak was largely due to the release of PNH on the surface or located at the external of nanopores. It can be clearly seen that all the second peaks of experimental groups showed up later than the peak of control group, demonstrating that PNH located at the internal of nanopores delayed the release, which could further illustrate the ability of carrier materials to control drug release. Additionally, the showing time of second peak slowed down and MRT increased with decreasing volume ratio of methanol modified PNH loaded carrier materials, reflecting the controlled release effect of carrier materials increased with decreasing volume ratio of methanol modification, which was quite in agreement with in vitro results. The Cmax and AUC0 ! 16 h values of the experimental groups were greater than the control group, indicating that carrier materials in our investigation could

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increase drug absorption ability because PNH was loaded into drug carrier materials in noncrystalline form (Liu et al., 2012). 4. Conclusions NS@P xerogel, 25% NS@P xerogel and 75% NS@P xerogel have been successfully synthesized using biomimetic synthesis method and the formation process included three states, which were sol, semi-solid gels and dry gels, respectively. Characteristic analysis of NS@P xerogel and NS xerogel revealed that PEIs and NS xerogel were amorphous. After methanol modification for NS@P xerogel, morphology compactness of NS@P xerogel increased with decreasing volume ratio of methanol in PEIs aggregate solution. Moreover, SBET decreased and uniformity of pore size distribution was interrupted, resulting in smaller nanopores of 25%NS@P xerogel and quite larger nanopores of 75%NS@P xerogel compared to NS@P xerogel. After impregnating PNH into carrier materials by in situ drug inclusion method, in vitro drug release experiment and in vivo pharmacokinetic studies showed that NS@P xerogel and methanol modified NS@P xerogel had tremendous application in CDDS as evidenced by the fact that model drug was controlled to release for more than 24 h in vitro and release of drug loaded at the internal of nanopores was delayed in vivo. Both pore size and PEIs scaffold ability influenced controlled release effect. Release rate was faster for 75%NS@P xerogel compared to NS@P xerogel due to its larger pore size. Furthermore, PNH could release from 25%NS@P xerogel faster than NS@P xerogel though pore size of NS@P xerogel was litter larger because PEIs scaffold ability decreased with increasing volume ratio of methanol modification. Therefore, it is flexible to control release effect simply by tuning methanol volume content in PEIs aggregate solution prior to the formation of PNH loaded carrier materials. Nanoporous silica xerogel synthesized with biomimetic method could broaden the application of silica materials used as carrier materials in CDDS. This research is a significant contribution to the design of nanoporous silica materials as drug carrier and flexibly regulating of controlled release effect. References Ahola, M., Kortesuo, P., Kangasniemi, I., Kiesvaara, J., Yli-Urpo, A., 2000. Silica xerogel carrier material for controlled release of toremifene citrate. International Journal of Pharmaceutics 195, 219–227. Ahola, M.S., Säilynoja, E.S., Raitavuo, M.H., Vaahtio, M.M., Salonen, J.I., Yli-Urpo, A.U., 2001. In vitro release of heparin from silica xerogels. Biomaterials 22, 2163– 2170. Barbe, C., Bartlett, J., Kong, L., Finnie, K., Lin, H.Q., Larkin, M., Calleja, S., Bush, A., Calleja, G., 2004. Silica particles: a novel drug–delivery system. Advanced Materials 16, 1959–1966. Cao, L., Man, T., Kruk, M., 2009. Synthesis of ultra-large-pore SBA-15 silica with twodimensional hexagonal structure using triisopropylbenzene as micelle expander. Chemistry of Materials 21, 1144–1153. Ghedini, E., Signoretto, M., Pinna, F., Crocellà, V., Bertinetti, L., Cerrato, G., 2010. Controlled release of metoprolol tartrate from nanoporous silica matrices. Microporous and Mesoporous Materials 132, 258–267. Horcajada, P., Ramila, A., Perez-Pariente, J., Vallet-Regı, M., 2004. Influence of pore size of MCM-41 matrices on drug delivery rate. Microporous and Mesoporous Materials 68, 105–109. Huang, C., Tang, Z., Zhou, Y., Zhou, X., Jin, Y., Li, D., Yang, Y., Zhou, S., 2012. Magnetic micelles as a potential platform for dual targeted drug delivery in cancer therapy. International Journal of Pharmaceutics 429, 113–122. Jaganathan, H., Godin, B., 2012. Biocompatibility assessment of Si-based nano-and micro-particles. Advanced Drug Delivery Reviews 64, 1800–1819. Jin, R.-H., 2003. Colloidal crystalline polymer generated in situ from growing star poly (oxazolines). Journal of Materials Chemistry 13, 672–675. Jin, R.-H., Yuan, J.-J., 2005a. Synthesis of poly (ethyleneimine)s–silica hybrid particles with complex shapes and hierarchical structures. Chemical Communications 1399–1401. Jin, R.-H., Yuan, J.-J., 2005b. Simple synthesis of hierarchically structured silicas by poly (ethyleneimine) aggregates pre-organized by media modulation. Macromolecular Chemistry and Physics 206, 2160–2170. Jin, R.-H., Yuan, J.-J., 2006. Shaped silicas transcribed from aggregates of four-armed star polyethyleneimine with a benzene core. Chemistry of Materials 18, 3390– 3396.

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