Accepted Manuscript Title: Facile synthesis of functionalized ionic surfactant templated mesoporous silica for incorporation of poorly water-soluble drug Author: Jing Li Lu Xu Baixue Yang Hongyu Wang Zhihong Bao Weisan Pan Sanming Li PII: DOI: Reference:

S0378-5173(15)30033-8 http://dx.doi.org/doi:10.1016/j.ijpharm.2015.07.014 IJP 15014

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

9-4-2015 27-5-2015 6-7-2015

Please cite this article as: Li, Jing, Xu, Lu, Yang, Baixue, Wang, Hongyu, Bao, Zhihong, Pan, Weisan, Li, Sanming, Facile synthesis of functionalized ionic surfactant templated mesoporous silica for incorporation of poorly water-soluble drug.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2015.07.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Elsevier Editorial System(tm) for International Journal of Pharmaceutics Manuscript Draft Manuscript Number: IJP-D-15-00679R1 Title: Facile synthesis of functionalized ionic surfactant templated mesoporous silica for incorporation of poorly water-soluble drug Article Type: Research Paper Section/Category: Pharmaceutical Nanotechnology Keywords: mesoporous silica; ionic surfactant template; amine modification; carboxylic modification; drug incorporation Corresponding Author: Dr. Sanming Li, Corresponding Author's Institution: Shenyang Pharmaceutical University First Author: Jing Li Order of Authors: Jing Li; Lu Xu; Baixue Yang; Hongyu Wang; Zhihong Bao; Weisan Pan; Sanming Li Abstract: The present paper reported amino group functionalized anionic surfactant templated mesoporous silica (Amino-AMS) for loading and release of poorly water-soluble drug indomethacin (IMC) and carboxyl group functionalized cationic surfactant templated mesoporous silica (CarboxylCMS) for loading and release of poorly water-soluble drug famotidine (FMT). Herein, Amino-AMS and Carboxyl-CMS were facilely synthesized using co-condensation method through two types of silane coupling agent. Amino-AMS was spherical nanoparticles, and Carboxyl-CMS was well-formed spherical nanosphere with a thin layer presented at the edge. Drug loading capacity was obviously enhanced when using Amino-AMS and Carboxyl-CMS as drug carriers due to the stronger hydrogen bonding force formed between surface modified carrier and drug. Amino-AMS and Carboxyl-CMS had the ability to transform crystalline state of loaded drug from crystalline phase to amorphous phase. Therefore, IMC loaded Amino-AMS presented obviously faster release than IMC because amorphous phase of IMC favored its dissolution. The application of asymmetric membrane capsule delayed FMT release significantly, and Carboxyl-CMS favored sustained release of FMT due to its long mesoporous channels and strong interaction formed between its carboxyl group and amino group of FMT.

Cover Letter

Dear editor, We would like to submit the enclosed manuscript entitled “Facile synthesis of functionalized ionic surfactant templated mesoporous silica for incorporation of poorly water-soluble drug”, which we wish to be considered for publication as an original article in International journal of pharmaceutics. It should be declared that the work described in this submission has not been published previously and it is not under consideration for publication elsewhere. Moreover, publication of this manuscript is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright-holder. The present paper reported fundamental and applied research on functional surface modified mesoporous silica, having particular relevance to pharmaceutical field, which is within the scope of International journal of pharmaceutics. Surface modified mesoporous silica has great value to be studied as drug carrier. In our paper, we apply co-condensation method via two types of silane coupling agent to accomplish amino group and carboxyl group modification. For amino group functionalized mesoporous silica, anionic surfactant C16-L-alanine was used as template. While for carboxyl group functionalized

mesoporous

silica,

cationic

surfactant

cetyl trimethyl ammonium bromide (CTAB) was taken as template. Formation mechanism of these two as-synthesized materials was further described to illustrate how

the ionic colloid template induced co-condensation of silica, which may provide useful hint for related investigations of surface modified mesoporous silica. After surface modification, amino group functionalized anionic surfactant templated mesoporous silica (Amino-AMS) improved indomethacin loading capacity and its release, while carboxyl group functionalized cationic surfactant templated mesoporous silica (Carboxyl-CMS) enhanced famotidine loading and prolonged famotidine release. The discussion for these obvious functions were conduced, demonstrating that surface modified ionic surfactant templated mesoporous silica had important advantages to be applied in delivering poorly water-soluble drugs to favor formulation design. Overall, we believe that this work has significant value for the development of mesoporous silica materials and its application in pharmaceutical field. All authors have seen the manuscript and approved to submit to your journal.

Thank you very much for your attention and consideration. Best regards, Sanming Li School of Pharmacy,, Shenyang Pharmaceutical University, Wenhua RD 103, 110016, Shenyang, China. Tel.: +86-24-23986258;

E-mail address: [email protected]

*Author Checklist

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*Response to Reviewers

Responses to the opinions of reviewers Thank you so much for reviewers’ valuable opinions and considerations on the manuscript. Here we make responses one by one in details. Reviewers' comments and our answers: Question (1): The graphic abstract is not meaningful and appealing in its current form. Answer (1): Thank you for this comment and we have remade our graphic abstract in order to make it meaningful and appealing, hoping that the revised graphic abstract can meet your requirement.

Question (2): Since famotidine is susceptible to acid degradation, there is worry that it may interfere with the accurate measurement in the release study in 0.1 N HCl. Answer (2):

This consideration is very meaningful and we have carefully thought

about it. After reading the literature “Rania H. Fahmy *, Mohammed A. Kassem, Enhancement of famotidine dissolution rate through liquisolid tablets formulation: In vitro

and

in

vivo

evaluation,

European

Journal

of

Pharmaceutics

and

Biopharmaceutics 69 (2008) 993-1003”, we can know that “famotidine undergoes minimal first-pass metabolism and its oral bioavailability in man has been reported to be low and variable, ranging from 40% to 50% due to its poor aqueous solubility, high polarity, and gastric degradation” and their in vitro release work was conducted using 0.1 N HCl similarly as our method. This may suggest that gastric degradation does not interfere with in vitro dissolution test measurement. In addition, there are also reports (please see our reference (Tang et al., 2006)) to investigate in vitro release of famotidine loaded mesoporous silica using simulated gastric fluid. Therefore, we can do not worry about the accurate measurement of in vitro study in 0.1 N HCl. We sincerely hope to get your understanding and are pleased to have discussion with you.

1

Question (3): Provide more detailed description on in vitro characterization, e.g. TEM. Answer (3): Thank you for this suggestion and we have revised by adding more detailed description on in vitro characterization, including ① 2.3.1. FTIR， adding “Samples were milled and mixed with 100-fold amount of dried KBr in an agate mortar and pestle. KBr disks were prepared with a compression force of 10 tons using a 13-mm-diameter round flat face punch.” ② 2.3.2. TEM, adding some content and revise to “The mesoporous structure of Amino-AMS and Carboxyl-CMS was characterized using a Tecnai G2 F30 TEM instrument (FEI, The Netherlands) operated at 200 kV with the software package for automated electron tomography. Before examination, sample was dispersed in ethanol through sonication and subsequently deposited on carbon-coated copper grids with porous carbon films.” ③2.3.3. Surface area and pore volume, adding “To remove adsorbed water, Amino-AMS and Carboxyl-CMS were degassed at 120℃ for 15min before analysis.” Please check and we hope that the revised part can get your acceptance.

Question (4):

Try to use photographs in the same magnification for both silica

particles in Fig. 3; please use the same y-axis scale in Fig. 5. Answer (4):

Thank you for reminding these details. After revising, photographs in

the same magnification for both silica particles were presented in Fig. 3. In Fig. 5, the y-axis scale is the same, which is “Intensity (a.u.)”. After considering your question, maybe you refer to Fig. 4 because the y-axis scales in Fig. 4 are not the same. Therefore, we revised Fig. 4 and we feel thankful that you can tell us if this revision is 2

not your aim. Question 5: Are there data on in vitro release of drug-loaded non-modified mesoporous silica? Answer 5:

Yes, we have data on in vitro release of drug loaded non-modified

mesoporous silica and added in revised manuscript. Related content was added to “2.6. In vitro release of drug loaded surface modified IMS” and related discussion has been added in “3.4. In vitro release”. Thank you for reminding us this revision to improve this paper.

Thank you again for all these careful opinions and valuable suggestions and help us to improve this paper. We sincerely hope the revised paper can meet your requirement.

Best wishes. Your sincerely Sanming Li

3

*Graphical Abstract (for review)

Functionalized ionic surfactant templated mesoporous silica (IMS): synthesize using co-condensation method and apply as drug carrier

Amino group functionalized anionic surfactant templated mesoporous silica

Carboxyl group functionalized cationic surfactant templated mesoporous silica

Synthesize with APTES as silane coupling agent

Synthesize with APTTES as silane coupling agent

Improved loading and increased release of indomethacin

Enhanced loading and controlled release of famotidine

*Manuscript Click here to view linked References

1

Facile synthesis of functionalized ionic surfactant templated mesoporous silica for

2

incorporation of poorly water-soluble drug

3

Jing Li, Lu Xu, Baixue Yang, Hongyu Wang, Zhihong Bao, Weisan Pan and Sanming

4

Li*

5

School of Pharmacy, Shenyang Pharmaceutical University, Wenhua RD 103, 110016,

6

China

7

*Corresponding author：

8

Sanming Li:

Wenhua RD 103, 110016, Shenyang, China.

9

Tel.: +86-24-23986258.

10

E-mail address: [email protected]

11

Abstract

12

The present paper reported amino group functionalized anionic surfactant

13

templated mesoporous silica (Amino-AMS) for loading and release of poorly

14

water-soluble drug indomethacin (IMC) and carboxyl group functionalized cationic

15

surfactant templated mesoporous silica (Carboxyl-CMS) for loading and release of

16

poorly

17

Carboxyl-CMS were facilely synthesized using co-condensation method through two

18

types of silane coupling agent. Amino-AMS was spherical nanoparticles, and

19

Carboxyl-CMS was well-formed spherical nanosphere with a thin layer presented at

20

the edge. Drug loading capacity was obviously enhanced when using Amino-AMS

21

and Carboxyl-CMS as drug carriers due to the stronger hydrogen bonding force

water-soluble

drug

famotidine

1

(FMT).

Herein,

Amino-AMS

and

22

formed between surface modified carrier and drug. Amino-AMS and Carboxyl-CMS

23

had the ability to transform crystalline state of loaded drug from crystalline phase to

24

amorphous phase. Therefore, IMC loaded Amino-AMS presented obviously faster

25

release than IMC because amorphous phase of IMC favored its dissolution. The

26

application of asymmetric membrane capsule delayed FMT release significantly, and

27

Carboxyl-CMS favored sustained release of FMT due to its long mesoporous channels

28

and strong interaction formed between its carboxyl group and amino group of FMT.

29 30

Keywords: mesoporous silica; ionic surfactant template; amine modification;

31

carboxylic modification; drug incorporation

32 33 34

1. Introduction Mesoporous silica materials are of special interest due to their common

35

properties

(facile

multifunctionalization,

36

biodegradability), unique features (high surface area, large pore volume, tunable pore

37

structures and excellent physicochemical stability), and potential applications in the

38

fields of catalysis, sensing, optically active materials, and biomaterials in drug

39

delivery (Xie et al., 2013). Commonly, mesoporous silica materials could be

40

fabricated by using cationic, non-ionic and anionic surfactants as templates to obtain

41

ordered mesostructures and well controlled morphologies, pore sizes, and porosities.

42

Among various mesoporous silica, anionic surfactant templated mesoporous silica 2

excellent

biocompatibility

and

43

(AMS) has attracted great attention and opens up an extensive area for research due to

44

its outstanding characteristics including the variety of the mesophases that can be

45

easily controlled and extremely the regular arrangement of the functional groups

46

introduced by the co-structure directing agent (CSDA) (Han and Che, 2013). Cationic

47

surfactant templated mesoporous silica (CMS) has also been widely reported to

48

prepare mesoporous silica with multiply structures and morphologies (Hu et al., 2012).

49

However, surface functionalized CMS synthesized using CSDA has rarely been

50

reported.

51

Hybrid mesoporous silica materials with organic functionalization on the exterior

52

and/or interior surfaces are of great interest due to their potential new applications in

53

adsorption, drug delivery, separation, catalysis and nanotechnology. The synthesis of

54

functionalized mesoporous silica materials based on organosilanes can be commonly

55

achieved by any of the following three methods: co-condensation (one-pot synthesis),

56

grafting (post-synthesis modification), and imprint coating method (Slowing et al.,

57

2008). Co-condensation and post-grafting are the two commonly available options.

58

The former designates the simultaneous hydrolysis and condensation of silica and

59

organic silane in one-pot, while the latter refers to the grafting of organic functional

60

species on the surface after the synthesis of mesoporous silica matrix (Xu et al., 2013).

61

Co-condensation method is relatively superior because of its relatively uniform

62

distribution of the organic groups and higher loading of organic groups without

63

closing the mesopores (Macquarrie and Jackson, 1997). To the best of our knowledge, 3

64

amino group functionalized mesoporous silica materials are well known for their use

65

in base-catalyzed reactions, further post-synthesis functionalization, waste-water

66

treatment, immobilization of enzymes, and drug delivery (Acosta et al., 2004; Lei et

67

al., 2002; Reynhardt et al., 2005). Carboxyl group functionalized mesoporous silica

68

has also been reported in drug delivery application (Tang et al., 2006). Therefore,

69

amino group and carboxyl group functionalized mesoporous silica materials have

70

potential value to be investigated and applied in drug delivery.

71

Significant efforts have been done to tackle the formulation challenges of

72

poorly water-soluble drugs, such as micronization, lipid-based formulations, solid

73

dispersion, co-solvents and complexation with cyclodextrin (Dressman and Reppas,

74

2007; Vogt et al., 2008). Among these, nanoparticle productions, which consist of raw

75

drug particles and a certain amount of surface active agents with a mean particle size

76

in the nanometer range of 10 and 1000 nm, have been emerged in the last decades and

77

developed as an alternative approach for the formulation of poorly soluble drugs

78

(Keck and Müller, 2006; Müller et al., 2001). Solubility and dissolution velocity of

79

poorly soluble drugs can be increased due to the reduced particle size according to the

80

Noyes-Whitney and Ostwald-Freundlich equation, thus tackling the above stated

81

formulation challenges (Kesisoglou et al., 2007). Unexpectedly, nanoparticles have

82

the drawback of instability caused by nucleation and particle growth, leading to the

83

demand for more effective stabilizer or matrix for nanoparticle formulations (Van

84

Eerdenbrugh et al., 2008). Due to unique properties, including stable material matrix, 4

85

large surface area and large pore volume, ordered mesoporous silica nanoparticles are

86

very effective in making up this drawback (Xu et al., 2013). Therefore, surface

87

functionalized ionic surfactant templated mesoporous silica (IMS) studied in this

88

work can also be chosen to load poorly-water soluble drugs for the purpose of

89

improving their solubility and dissolution.

90

Herein we report amino group functionalized AMS (Amino-AMS) and carboxyl

91

group functionalized CMS (Carboxyl-CMS) synthesized based on co-condensation

92

method using ionic surfactant (C16-L-alanine; cetyl trimethyl ammonium bromide,

93

CTAB), CSDA (3-aminopropyltriethoxysilane, APTES; 3-N-aminopropyl-tartaric

94

acid triethoxysilane, APTTES) and inorganic source (Tetraethoxysilane, TEOS). In

95

light of the wide application of mesoporous silica as drug carriers and unique

96

properties of surface modified IMS, attempts have been made to load poorly

97

water-soluble drug indomethacin (IMC, Fig. 1A) into Amino-AMS and famotidine

98

(FMT, Fig. 1B) into Carboxyl-CMS based on the following reasons. (1) Since IMC is

99

an acidic non-steroidal anti-inflammatory drug that may cause irritation of the

100

gastrointestinal mucosa (Tzankov et al., 2013), incorporation of IMC into AMS can

101

reduce side effect of causing local irritation due to the direct contact of free carboxyl

102

group (Shrivastava et al., 2003; Tzankov et al., 2013). (2) FMT is a histamine

103

H2-receptor antagonist mainly used to treat peptic ulcers and gastroesophageal reflux.

104

Prolong the working time in the stomach becomes an efficient method to improve

105

therapeutic effect of FMT (Mady et al., 2010). Since FMT has good solubility in 5

106

enzyme-free simulated gastric fluid (pH 1.0), it is not satisfied to apply

107

Carboxyl-CMS as drug carrier to investigate release behavior. Therefore, asymmetric

108

membrane capsule was introduced. The asymmetric membrane capsule is a controlled

109

delivery device which consists of a drug core surrounded by a membrane of an

110

asymmetric structure. Asymmetric membrane is semipermeable membrane with a

111

higher rate of water influx and allows the release of drugs with a lower osmotic

112

pressure or lower solubility (Wang et al., 2005). Similar to conventional telescoping

113

hard gelatin capsule, the asymmetric membrane capsule consists of a cap and a body

114

that fit into each other. The cap is shorter in length and has a slightly larger diameter

115

than the body which is longer and has a smaller diameter. In contrast to gelatin

116

capsules, the walls of asymmetric membrane capsules are made from a

117

water-insoluble polymer such as cellulose acetate (CA), ethylcellulose (EC), cellulose

118

acetate butyrate (CAB), and their mixtures. Thus, the capsule shell does not dissolve

119

to instantly release the drug filled in it. Instead, the drug is released over a prolonged

120

duration by diffusion through the capsule walls (Thombre et al., 1999). We believe

121

that this research will be of significant help in designing better oral drug delivery

122

systems using surface modified IMS as drug carrier.

123

2. Materials and methods

124

2.1. Materials

125

Tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) were

126

purchased from Aladdin (Shanghai, China). N, N '- dicyclohexyl carbon imine (DCC), 6

127

N-dimethyl aminopyridine (DMAP), palmitic acid and L-alanine methyl ester were

128

purchased from Chengdu Xiya Chemical Technology Co. Ltd.(Chengdu, China). All

129

other chemicals were of reagent grade and were used without further purification. Ion

130

exchange system was used for preparation of deionized water, which was used

131

throughout the experimental work.

132

2.2. Fabrication of surface modified IMS

133

2.2.1 Amino-AMS

134

Initially, C16-L-alanine was synthesized to be used as template for preparing

135

Amino-AMS. Typically, palmitic acid and DMAP were added into 100 ml

136

dichloromethane (molar ratio of composite was 10:1), then the mixture was stirred on

137

0 ℃ water bath for 1h. L-alanine methyl ester solution was made by mixing

138

triethylamine, 26 mmol L-alanine methyl ester and 50 ml dichloromethane and

139

stirring at ambient temperature for 40 min. Afterwards, DCC solution can be prepared

140

by dissolving 28.4 mmol DCC into 50 ml dichloromethane. When these reactions

141

accomplished, L-alanine methyl ester solution was added to the solution consisting of

142

palmitic acid and DMAP, then DCC solution was dropwise added to the above mixed

143

solution. Stirring was maintained at 0℃ water bath for 3 h, and then stirring was kept

144

overnight at ambient temperature. The obtained final solution was filtered and

145

successively washed with water, a saturated aqueous solution of NaCl, a saturated

146

aqueous solution of NaHCO3, HCl (1 M), and again water. The organic layer was

147

dried by using MgSO4 and filtrated. Crude product was obtained after the evaporation 7

148

of organic solvent. The final product (C16-L-alanine methyl ester) was purified by

149

crystallization using ethyl acetate/n-hexane (ratio 1/1, v/v) to yield a white solid.

150

C16-L-alanine was synthesized by hydrolyzing C16-L-alanine methyl ester, and this

151

process was stated as follows. A certain amount of methanol was utilized to dissolve

152

C16-L-alanine methyl ester under stirring on 0℃ water bath. Afterwards, 1 M NaOH

153

was added to the above mixture, and then the methanol remained in the mixture was

154

removed using reduced pressure distillation. White precipitate was separated out after

155

adjusting pH of the obtained mixture to 2-3 by 1 M HCl. Finally, filtered, water

156

washed, and dried the precipitates to get C16-L-alanine.

157

In a typical run, 0.33 g C16-L-alanine was dissolved in 10 ml deionized water, to

158

which 10 ml NaOH (0.1 M) and then 10 ml HCl (0.01 M) were added under stirring.

159

The mixture was stirred at ambient temperature for 1 h. Afterwards, a mixed solution

160

consisting of 0.24 ml APTES and 1.57 ml TEOS was added to the mixture under

161

vigorous stirring. The synthesized mixture was remained statically for 24 h, filtered,

162

centrifuged, water washed and dried. The as-synthesized sample was extracted by

163

reflux with an ethanolic solution of ethanolamine (17 vol.%) for 12 h at its boiling

164

temperature. Thus, Amino-AMS can be obtained through the ionic exchange between

165

protonated amino group and amine [15]. The anionic surfactant templated mesoporous

166

silica without modification (AMS) was prepared using the same procedure except

167

calcination method was used instead of reflux method to remove the template and

168

3-aminopropyl groups. 8

169

2.2.2. Carboxyl-CMS

170

For the first time, a new type of silane coupling agent named as APTTES was

171

synthesized. Briefly, 2 mmol L-tartaric acid was dissolved using 20 ml anhydrous

172

ethanol on 60℃ water bath,

173

stirring for more than 4 h, the precipitate named as APTTES was obtained by

174

centrifugation and drying. Carboxyl-CMS was prepared with CTAB as template. 1.6 g

175

CTAB was firstly added into the mixture of 200 ml double distilled water and 60 ml

176

anhydrous ethanol, stirring until clear at room temperature. 1.6 ml ammonia was

177

added into the solution then 1.0 g APTTES was added under stirring. When a

178

homogeneous solution obtained, 5 ml TEOS was added dropwise into the solution

179

under vigorous stirring. After continuous stirring for 4 h at room temperature, a white

180

precipitate was obtained. The resulting white precipitate was remained statically for

181

24 h, and separated by centrifugation, washed by double distilled water and anhydrous

182

alcohol for three times, and dried at 60℃ for 6 h. The template CTAB was removed

183

by reflux in a methanol solution of 0.01 M HCl for 12 h at its boiling temperature.

184

The cationic surfactant templated mesoporous silica without modification (CMS) was

185

prepared using the same procedure except no APTTES was added.

186

2.3. Characterization of Amino-AMS and Carboxyl-CMS

187

2.3.1. FTIR

then 0.5 ml APTES was added under stirring. After

188

Fourier transform infrared spectroscopy (FT-IR, Spectrum 1000, Perkin Elmer,

189

USA) spectra of samples (C16-L-alanine, Amino-AMS, APTTES and Carboxyl-CMS) 9

190

were recorded from 400 to 4000 cm-1 in transmittance mode with a resolution of 1

191

cm-1. Samples were milled and mixed with 100-fold amount of dried KBr in an agate

192

mortar and pestle. KBr disks were prepared with a compression force of 10 tons using

193

a 13-mm-diameter round flat face punch.

194

2.3.2. TEM

195

The mesoporous structure of Amino-AMS and Carboxyl-CMS was characterized

196

using a Tecnai G2 F30 TEM instrument (FEI, The Netherlands) operated at 200 kV

197

with the software package for automated electron tomography. Before examination,

198

sample was dispersed in ethanol through sonication and subsequently deposited on

199

carbon-coated copper grids with porous carbon films.

200

2.3.3. Surface area and pore volume

201

The surface area and pore volume of Amino-AMS and Carboxyl-CMS were

202

studied by determining the nitrogen adsorption and desorption using a SA3100

203

surface area and pore size analyzer (Beckman Coulter, USA). To remove adsorbed

204

water, Amino-AMS and Carboxyl-CMS were degassed at 120℃ for 15min before

205

analysis. The specific surface area (SBET) was evaluated from nitrogen adsorption data

206

over the relative pressure range from 0.05 to 0.2 using the Brunauer–Emmett–Teller

207

(BET) method. Pore size distributions (PSDs) were determined from adsorption

208

branches of isotherms using the Barrett–Joyner–Halenda (BJH) method. The total

209

pore volume (Vt) was determined from the amount adsorbed at a relative pressure of

210

0.99. 10

211

2.3.4. Functional group content analysis

212

For Amino-AMS, amino group content was determined using acid-base titration

213

method. 0.1 g Amino-AMS was added to 4.0 ml 0.01 M HCl standard solution and

214

stirred homogeneously. Centrifuged the mixture and washed the precipitate using

215

distilled water, then collected the supernatant and the distilled water. A certain

216

amount of phenolphthalein solution was added to the supernatant, and 0.01 M NaOH

217

was used to titrate until the color changed from colorless to pink and this conversion

218

can keep for 30 seconds. Amino group content was calculated according to the

219

volume of NaOH (V) used in titration process. Amino group content mmol/g 

0.01 ∗ 4.0  V 0.1

220

Similarly, 0.1 g Carboxyl-CMS was dissolved in 10 ml distilled water and stirred

221

homogeneously. Centrifuged the mixture and washed the precipitate using distilled

222

water, then collected the supernatant and the distilled water. A certain amount of

223

phenolphthalein solution was added to the supernatant, and 0.01 M NaOH was used to

224

titrate until the color changed from colorless to pink and this conversion can keep for

225

30 seconds. Amino group content was calculated according to the volume of NaOH

226

(V) used in titration process. Carboxyl group content mmol/g 

227

2.4. Drug loading

228

2.4.1. IMC loaded Amino-AMS

11

0.01 ∗ V 0.1

229

The solvent deposition method, which involved the soaking equilibrium and then

230

solvent evaporation, was applied to load IMC into Amino-AMS. In detail, IMC was

231

dissolved in acetone to obtain a high concentrated solution (15 mg/ml) then this

232

solution (equivalent to 30 mg IMC) was mixed with certain amount of Amino-AMS

233

to obtain samples with the drug-silica carrier ratio of 1:3 (w/w). After gentle stirring

234

for 24 h, the solvent was allowed to evaporate and then the precipitated powder was

235

washed with acetone to remove the drugs on the surface of carrier. Finally, the

236

obtained powder was dried under vacuum drying. Drug loading capacity was

237

measured by taking an accurately weighed quantity of IMC loaded Amino-AMS, then

238

extracting the loaded IMC completely using methanol under ultrasound, and finally

239

measuring IMC content with ultraviolet spectroscopy (UV-1750, Shimadzu, Japan) at

240

a wavelength of 318 nm.

241

2.4.2. FMT loaded Carboxyl-CMS

242

FMT was dissolved in ethanol to obtain a high concentrated solution (10 mg/ml)

243

then 1 ml FMT ethanol solution was mixed with 30 mg Carboxyl-CMS to obtain

244

samples with the drug-silica carrier ratio of 1:3 (w/w). After gentle stirring for 24 h,

245

the solvent was allowed to evaporate under vacuum drying and the precipitated

246

powder was washed with ethanol to remove the drugs on the surface of carrier. Finally,

247

the obtained powder was dried under vacuum drying. Drug loading capacity was

248

analyzed by taking an accurately weighed quantity of FMT loaded Carboxyl-CMS,

249

then extracting the loaded FMT completely using 0.1 M HCl under ultrasound, and 12

250

finally measuring FMT content with ultraviolet spectroscopy (UV-1750, Shimadzu,

251

Japan) at a wavelength of 267 nm.

252

2.5. Conversion of drug crystalline state

253

X-ray diffraction (XRD) is a common method to test whether a crystalline drug

254

phase can be detected. Herein, IMC and FMT crystalline state were measured using

255

XRD (X'pert PRO, PANalytical B.V., The Netherlands). XRD patterns of samples

256

were generated at 30 mA and 30 kV with a Ni filtered CuKa line as the source of

257

radiation. Data were obtained from 5° to 40° (diffraction angle 2θ).

258

2.6. In vitro release of drug loaded surface modified IMS

259

In vitro dissolution experiment was carried out using USP paddle method (50

260

rpm, 37℃) with a ZRD6-B dissolution tester (Shanghai Huanghai Medicament Test

261

Instrument Factory, China). IMC, IMC loaded AMS and IMC loaded Amino-AMS

262

were respectively exposed to enzyme-free simulated intestinal fluid (pH 6.8). At

263

predetermined time intervals, 5 ml samples were withdrawn from the release medium

264

and then an equivalent amount of fresh medium was added to maintain a constant

265

dissolution volume. Samples administered through 0.45 µm microporous membrane

266

were analyzed using UV-1750 (Shimadzu, Japan) at the wavelength of 320 nm.

267

In the present study, asymmetric membrane capsule was made by loading

268

capsule material liquid into empty capsule cap and body (1#). The capsule material

269

liquid consisted of CA, acetone, polyethylene glycol (PEG 400) and diethyl phthalate

270

(DEP). Asymmetric membrane was formed after evaporating organic solvent at 4℃ 13

271

refrigerator. Then the asymmetric membrane capsule cap and body were obtained by

272

stripping off from empty capsule cap and body. In vitro dissolution experiment of

273

FMT was carried out using USP paddle method (50 rpm, 37℃) with a ZRD6-B

274

dissolution tester (Shanghai Huanghai Medicament Test Instrument Factory, China).

275

Asymmetric membrane capsule containing FMT, asymmetric membrane capsule

276

containing FMT loaded CMS, and asymmetric membrane capsule containing FMT

277

loaded Carboxyl-CMS were respectively exposed to enzyme-free simulated gastric

278

fluid (pH 1.0). At predetermined time intervals, 5 ml samples were withdrawn from

279

the release medium and then an equivalent amount of fresh medium was added to

280

maintain a constant dissolution volume. Samples administered through 0.45 µm

281

microporous membrane were analyzed using UV-1750（Shimadzu, Japan）at the

282

wavelength of 267 nm.

283

3. Results and Discussion

284

3.1. Formation mechanism of Amino-AMS and Carboxyl-CMS

285

In the present work, Amino-AMS and Carboxyl-CMS were synthesized using

286

co-condensation method via two types of silane coupling agent, which was the

287

relatively superior method to realize surface modification of mesoporous silica

288

(Macquarrie and Jackson, 1997). Table 1 presented the synthetic strategy for

289

Amino-AMS

290

organic/inorganic interface chemistry is constructed with synthesis route of (S-M+I-).

291

S- , M+ and I- stand for anionic surfactant, APTES with cationic amino groups, and

and

Carboxyl-CMS.

In

14

our synthesis

of

Amino-AMS,

the

292

TEOS with negative charges, respectively. Upon the addition of APTES and TEOS,

293

the negatively charged headgroups of the C16-L-alanine interact electrostatically with

294

the positively charged ammonium sites of the APTES (Qiu and Che, 2011). This

295

electrostatic repulsion induces the co-condensation of silica source TEOS and silane

296

coupling agent APTES. As for Carboxyl-CMS, it is the first time to apply a new

297

silane coupling agent (APTTES) to realize the formation of carboxyl group

298

functionalized mesoporous silica. The organic/inorganic interface chemistry of

299

Carboxyl-CMS is constructed with synthesis route of (M-S+ I-), in which M- stands for

300

APTTES with negative carboxyl groups, S+ stands for cationic surfactant and I- stands

301

for TEOS with negative charges. Upon the addition of APTTES and TEOS, the

302

positively charged headgroups of the CTAB interact electrostatically with the

303

negatively charged carboxyl groups of the APTTES and also negatively charged

304

silicon hydroxyl groups from hydrolyzed TEOS, thus inducing the co-condensation of

305

silica source TEOS and silane coupling agent APTTES. CMS can be formed simply

306

by not adding APTTES because the positively charged headgroups of the CTAB can

307

interact electrostatically with negatively charged silicon hydroxyl groups from

308

hydrolyzed TEOS. The clear description of formation mechanism of Amino-AMS and

309

Carboxyl-CMS can help to have a better understanding of their synthesized process

310

and provide useful hint for further related investigations.

311

3.2. Characterization of Amino-AMS and Carboxyl-CMS

312

3.2.1. FTIR 15

313

The successful synthesized C16-L-alanine and APTTES were verified with FTIR

314

analysis (Fig. 2). The FTIR spectrogram of C16-L-alanine showed characteristic peaks

315

at 1718 and 1692 cm-1, which assigned to the carbonyl groups of the carboxylic acid

316

and amide (Hu et al., 2011a). Additionally, the stretching peak of –NH at 3315.1 cm -1

317

and two bands assigned to CH2 stretching at 2918.2 and 2849.7 cm-1 due to hexadecyl

318

group (Song et al., 2005) further demonstrated the formation of C16-L-alanine. With

319

the synthesis of anionic surfactant C16-L-alanine as template, Amino-AMS was

320

prepared evidenced by characteristic peaks of silica (including Si-O-Si bending

321

vibration at 461.7 cm-1 and Si-O-Si antisymmetric stretching vibration at 1070.5 cm-1)

322

and amino modification (including –NH2 bending vibration at 1490.1 cm-1, –NH2

323

antisymmetric stretching vibration at 1571.3 cm-1 and N-H stretching vibration at

324

3362.1 cm-1). As for APTTES, carbonyl group stretching vibration of amide was

325

observed at 1632.7 cm-1, indicating the successful connection of APTES with

326

L-tartaric acid. Moreover, the existence of carbonyl group stretching vibration of

327

carboxylic acid at 1697.0 cm-1 and out-of-plane bending vibration of carboxylic

328

hydroxyl at 943.2 cm-1 proved that only one carboxyl group of L-tartaric acid

329

functioned with amino group of APTES, and the unbounded carboxylic acid can be

330

used to conduct carboxylic modification. The Si-O-C antisymmetric stretching

331

vibration shown at 1092.2 and 1150.4 cm-1 together with no appearance of Si-O-Si

332

peaks illustrated that the APTES used in the synthesis did not hydrolyze in the

333

reaction. On the basis of the template APTTES, Carboxyl-CMS was synthesized 16

334

evidenced by characteristic peaks of silica (including Si-O-Si bending vibration at

335

464.8 cm-1, Si-O-Si antisymmetric stretching vibration at 1071.0 cm-1, and O-H of

336

Si-OH antisymmetric stretching vibration at 3424.9 cm-1) and carboxylic modification

337

(including carbonyl group stretching vibration of carboxylic acid at 1699.4 cm-1 and

338

out-of-plane bending vibration of carboxylic hydroxyl at 958.3 cm-1).

339

3.2.2. TEM

340

As can be seen in Fig. 3, Amino-AMS exhibited spherical nanoparticles with

341

mesopores. The well-formed spherical Carboxyl-CMS was nearly monodispersed

342

with a diameter of about 300 nm and a thin layer was present at the edge of the

343

nanosphere. A large number of long mesoporous channels can be observed, especially

344

obvious in the thin layer. Since the functional group (3-N-aminopropyl-tartaric acid

345

group) of APTTES was longer than that of TEOS (oxyethyl group), condensation rate

346

of APTTES was slower than TEOS, leading to the formation of nanosphere core due

347

to fast condensation reaction and a thin layer originating from slow condensation

348

reaction. This result reflected the co-structure directing function of APTTES as CSDA

349

significantly, further confirming the function of silane coupling agent as CSDA in our

350

co-condensation method to synthesize surface modified IMS.

351

3.2.3. Surface area and pore volume

352

Nitrogen adsorption/desorption isotherms and pore size distribution curves of

353

Amino-AMS and Carboxyl-CMS were presented in Fig. 4, and calculated parameters

354

were shown in Table 2. The nitrogen adsorption/desorption isotherms of Amino-AMS 17

355

and Carboxyl-CMS were type IV isotherm with a hysteresis loop according to the

356

IUPAC classification. Their SBET and Vt were not quite large compared with reported

357

mesoporous silica materials [5] due to surface modification.

358

3.2.4. Surface modification content and drug loading capacity

359

Acid-base titration, a simple and efficient method, was used to determine surface

360

modification content. As can be seen in Table 2, amino modification content of

361

Amino-AMS was 0.154 mmol/g, and carboxylic modification content of

362

Carboxyl-CMS was 0.081 mmol/g, which also confirmed the successful surface

363

modification in addition to FTIR analysis. According to Table 3, IMC loading

364

capacity of Amino-AMS (31.82%) was significantly higher than that of AMS

365

(23.65%) and FMT loading capacity of Carboxyl-CMS (25.61%) was also higher than

366

that of CMS (21.58%). The results revealed that the surface modification obviously

367

enhanced drug loading capacity possibly due to the stronger hydrogen bonding force

368

formed between surface modified carrier and drug (amino group of Amino-AMS with

369

carboxylic acid moiety of IMC molecule, carboxyl group of Carboxyl-CMS with

370

amino group of FMT).

371

3.3. Conversion of drug crystalline state

372

According to Fig. 5A, the diffraction pattern of pure IMC was highly crystalline

373

in nature as indicated by the numerous peaks. However, no crystalline IMC was

374

detected in IMC loaded Amino-AMS (Hu et al., 2011b). It was assumed that the

375

limited mesopores and mesostructure of Amino-AMS prevented the crystallization of 18

376

IMC due to the space confinement (Madieh et al., 2007; Van Speybroeck et al., 2009).

377

Before being loaded, FMT was crystalline state evidenced by multiply classic peaks

378

of XRD pattern, then crystalline state converted to amorphous (Fig. 5B) because the

379

framework of mesoporous channels limited living space of FMT in Carboxyl-CMS

380

and prevented its crystalline formation, thus increasing FMT stability in

381

Carboxyl-CMS.

382

3.4. In vitro release

383

Compared with IMC (Fig. 6A), IMC loaded AMS and IMC loaded Amino-AMS

384

presented obvious faster release through in vitro process and its cumulative release

385

was more than 90% at 60 min. It is widely known that several factors may contribute

386

to the dissolution enhancement, including the lack of crystalline form and presence of

387

amorphous form, the reduction in drug particle size to nanosize range and the

388

hydrophilic surface, etc. The formation of the highly ordered crystalline IMC can be

389

restricted by the confined space of the mesopores, thus retaining its amorphous form

390

(Hu et al., 2012). Therefore, after loading IMC into AMS or Amino-AMS, crystal

391

form of IMC had been successfully converted to amorphous state based on XRD

392

analysis, resulting in the increase of apparent solubility and dissolution of IMC, which

393

significantly favored IMC formulation design. It was worth noticing that in vitro

394

release of IMC loaded Amino-AMS was slower than that of IMC loaded AMS,

395

demonstrating that the stronger hydrogen bonding force formed between IMC and

396

Amino-AMS reduced IMC release rate. This decreased release phenomenon avoided 19

397

burst release that occurred to IMC loaded AMS, further confirming the superior

398

application of Amino-AMS for loading and release of IMC.

399

As can be seen in the small inserted image in the bottom right corner of Fig. 6B,

400

as-synthesized asymmetric membrane capsule was a transparent and glossy capsule.

401

The application of asymmetric membrane capsule significantly delayed FMT release

402

for more than 5 h in enzyme-free simulated gastric fluid because its capsule shell

403

synthesized using water-insoluble polymer CA prolonged drug release by diffusion

404

(Thombre et al., 1999). Additionally, the advantage of Carboxyl-CMS was also

405

obvious. Compared with asymmetric membrane capsule containing FMT, in vitro

406

release of asymmetric membrane capsule containing FMT loaded CMS and

407

asymmetric membrane capsule containing FMT loaded Carboxyl-CMS presented

408

slower release in the whole process and prolonged releasing time to 9 h, indicating

409

that CMS and Carboxyl-CMS controlled FMT release because of long mesoporous

410

channels. Moreover, FMT loaded Carboxyl-CMS displayed slower release than FMT

411

loaded CMS due to the stronger hydrogen bonding force formed between carboxyl

412

group of Carboxyl-CMS and amino group of FMT. It was undoubted that the

413

combined application of asymmetric membrane capsule and Carboxyl-CMS favored

414

FMT sustained release formulation design.

415

4. Conclusion

416

Surface modified IMS, including Amino-AMS and Carboxyl-CMS, were

417

successfully synthesized using co-condensation method via two types of silane 20

418

coupling agent. After surface modification, drug loading capacity was obviously

419

enhanced due to the stronger hydrogen bonding force formed between surface

420

modified carrier and drug. Furthermore, Amino-AMS and Carboxyl-CMS had the

421

ability to transform crystalline state of loaded drug from crystalline phase to

422

amorphous phase. Based on this transformation, IMC loaded Amino-AMS presented

423

obvious faster release than IMC because amorphous phase of IMC favored its

424

dissolution. The application of asymmetric membrane capsule delayed FMT release

425

significantly, and Carboxyl-CMS can also prolonged FMT release due to its

426

mesopores and strong interaction formed between its carboxyl group and amino

427

group of FMT. It was obvious that surface modified IMS had important advantages

428

to be applied in delivering poorly water-soluble drugs to favor formulation design.

429

Overall, we believe that this work has significant value for the development of

430

mesoporous silica materials and its application in pharmaceutical field.

431 432 433 434 435 436 437 438 21

439

References

440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479

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*Manuscript Click here to view linked References

Figure caption Fig. 1 Structural formula of IMC (A) and FMT (B). Fig. 2 FTIR spectra of A, C16-L-alanine and Amino-AMS; B, APTTES and Carboxyl-CMS. Fig. 3 TEM images of A, Amino-AMS; B, Carboxyl-CMS. Fig.4 Nitrogen adsorption/desorption isotherms and Pore size distribution curves of Amino-AMS and Carboxyl-CMS. Fig. 5 A, XRD patterns of IMC and IMC loaded Amino-AMS; B, XRD patterns of FMT and FMT loaded Carboxyl-CMS. Fig. 6 In vitro release of A, IMC, IMC loaded AMS and IMC loaded Amino-AMS; B, FMT stands for asymmetric membrane capsule containing FMT, FMT loaded CMS stands for asymmetric membrane capsule containing FMT loaded CMS, FMT loaded Carboxyl-CMS stands for asymmetric membrane capsule containing FMT loaded Carboxyl-CMS.

*Manuscript Click here to view linked References

Table headings Table 1 The synthetic strategy for Amino-AMS and Carboxyl-CMS. Table 2 Surface modification content (mmol/g), surface area (SBET), and pore volume (Vt) of Amino-AMS and Carboxyl-CMS. Table 3 Drug loading capacity (%) of IMC loaded Amino-AMS, IMC loaded AMS, FMT loaded Carboxyl-CMS, and FMT loaded CMS.

Figure(s)

Figure 1

A

B

Figure(s)

Figure 2

A

B

Figure(s)

Figure 3

A

B

Figure(s)

Figure 4

A

B

Figure(s)

Figure 5

A

B

Figure(s)

Figure 6

A

B

Table(s)

Table 1 Amino-AMS

Carboxyl-CMS

C16-L-alanine

CTAB

APTES

APTTES

Template

CSDA

Interaction

Table(s)

Table 2 Materials Surface modification content (mmol/g) SBET (m2/g) Vt (cm3/g)

Amino-AMS 0.154 190.74 0.3923

Carboxyl-CMS 0.081 460.46 0.3009

Table(s)

Table 3 Drug loaded materials Drug loading capacity (%)

IMC loaded Amino-AMS

IMC loaded AMS

FMT loaded Carboxyl-CMS

FMT loaded CMS

31.82

23.65

25.61

21.58