Composite of Functional Mesoporous Silica and DNA: An Enzyme

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Composite of Functional Mesoporous Silica and DNA: An Enzyme-Responsive Controlled Release Drug Carrier System Guilong Zhang, Minglei Yang, Dongqing Cai, Kang Zheng, Xin Zhang, Lifang Wu, and Zhengyan Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 12 May 2014 Downloaded from http://pubs.acs.org on May 16, 2014

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ACS Applied Materials & Interfaces

1

Composite of Functional Mesoporous Silica and DNA: An

2

Enzyme-Responsive Controlled Release Drug Carrier System

3

Guilong Zhang†,‡,¶, Minglei Yang†,‡, Dongqing Cai‡, Kang Zheng||, Xin Zhang§,

4

LifangWu‡,*, Zhengyan Wu‡,*

5

‡

6

Chinese Academy of Sciences, Hefei 230031, People’s Republic of China

7

¶

8

China

9

§

Key Laboratory of Ion Beam Bioengineering, Hefei Institutes of Physical Science,

University of Science and Technology of China, Hefei 230026, People’s Republic of

School of Life Sciences, Anhui Agricultural University, Hefei 230036, People’s

10

Republic of China

11

||

12

Academy of Sciences, Hefei 230031, People’s Republic of China

Key Laboratory of Materials Physics, Institute of Solid State Physics, Chinese

13 14

ABSTRACT: An efficient enzyme-responsive controlled release carrier system was

15

successfully fabricated using single-stranded

16

mesoporous silica nanoparticles. Mesoporous silica nanoparticles were initially

17

fabricated

18

cetyltrimethylammonium chloride (CTAC) solution, and the surface of nanoparticles

19

could be encapsulated with single-stranded DNA. This nanomaterial has excellent

20

bioactivity and its hydrolysate can not cause damage for normal cell, thus the

21

biocompatibility of the nanomaterial is improved. In addition, this nanomaterial

through

hydrolysis

of

DNA

tetraethyl

encapsulated functional

orthosilicate

1

ACS Paragon Plus Environment

(TEOS)

in


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showed an excellent drug release performance when loaded with drugs, which would

23

be helpful to increase the treatment efficiency and decrease side effects of drugs.

24

KEYWORD: mesoporous silica, DNA, enzyme-responsive, controlled release.

25 26

INTRODUCTION

27

Recently, controlled release drug carrier systems have attracted wide interest because

28

they can maintain the valid drug concentrations and improve the therapeutic efficacy.1-5

29

Because of nontoxicity, high biocompatibility, chemically stable porous nanostructure

30

and outstanding drug delivery performance,6-10 mesoporous silica nanoparticles

31

(MSNs) have been used as the most promising carrier materials for a controlled release

32

system (CRS). Some CRSs based on MSNs have been described,11-15 and controlled

33

release performances of these systems have also received more and more attention.

34

A number of CRSs with excellent controlled release performances have been

35

developed successfully as carriers of nanomedicines, and the prevailing ones are

36

usually fabricated using organic substances as the coating,16 and their organic coating

37

materials under enzyme catalysis could be decomposed into small molecules in body. It

38

is well known that lysosomes can secrete a variety of enzymes to resist the intrusion of

39

foreign substances into normal tissue through enzyme catalysis.17-18 When organic

40

substances-coated nanomedicine is injected into living body, the nanomedicine can be

41

delivered to lysosomes through endocytosis and then the organic substances will be

42

degraded through the enzyme catalysis, thus the drugs in CRS are released. Currently,

43

some organic-based delivery systems (PLA, PEG, liposome etc.) have been approved 2


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through FDA. However, some organic CRSs such as polymer still have potential risks

45

because the decomposed small organic molecules are usually harmful to living body.

46

Recently, some CRSs have been fabricated using DNA modified nanoparticle.19-24

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However, the majority of these DNA need to be modified with organic substance,

48

which would result in the same problem. To avoid the problem, it is necessary to

49

develop a new coating material. In this work, we chose DNA of food plant, a natural

50

and safe biomolecule, as the coating of nanomedicine, which makes CRS possess

51

higher biosafety and biocompatibility compared with organic substances.

52

Here,

we

described

an

enzyme-responsive

CRS

using

53

(3-Aminopropyl)triethoxysilane (APTES) modified mesoporous silica nanoparticles

54

(AMSNs) encapsulated with the DNA from natural food plant as the carrier. Currently,

55

some reports on gene therapy indicated that DNA could possess excellent the

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safety.25,26 DNA of food plant especially has excellent bioactivity and its hydrolysate

57

can not cause damage for normal cell, thus the biocompatibility of nanomedicine can be

58

improved. In this work, it is first time that enzyme-responsive CRS was fabricated

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through the nanoparticle modified with single-stranded DNA of food plant. The

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modification of APTES makes MSNs aminated and the surface of nanopaticles

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positively charged because of the amino protonation in simulated body fluid (SBF),

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which is beneficial for making negatively charged DNA encapsulate AMSNs through

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electrostatic interactions. As depicted in Scheme 1, once mesopores in AMSNs were

64

loaded with drugs, the curly DNA molecules on the surface of nanoparticles could

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encapsulate the nanoparticles, acting as gates of the mesopores to keep the drugs 3



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enclosed. When DNase I enzyme was added into SBF, the DNA was hydrolyzed to

67

plenty of tiny fragments, and obviously the gates started to open and drugs could be

68

released from the mesopores. Moreover, in comparison with the reported CRSs, this

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system may possess higher biocompatibility and lower side effect.

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EXPERIMENTAL SECTION

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Materials: All chemical reagents were used as received without further purification.

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Triethanolamine

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(3-Aminopropyl)triethoxysilane (APTES) were purchased from Sinopharm Co.

74

(Shanghai, China). Colchicine (COLC) and tetraethyl orthosilicate (TEOS) were

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received from Aladdin Co. (Shanghai, China). These chemical reagents were all of

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analytical grade. In addition, RNAprep Pure Plant kit was obtained from Tiangen Co.

77

(Beijing China), RNaseA and LA Taq DNA polymerase were purchased from Takara

78

Co. (Japan). Nanoclay hydrophilic bentonite was received from Sigma-Aldrich Co.

79

(USA). Potassium acetate and GelRed were purchased from Sangon Biotech Co.

80

(Shanghai, China). M-MLV Reverse Transcriptase was obtained from Promega Co.

81

(Madison, USA). These reagents were all of bioreagent grade.

82

Synthesis of MSN: MSN was synthesized using a soft template method.27 Briefly,

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CTAC (2.5 g) was added into ethanol solution (10 mL ethanol in 76 mL deionized

84

water) under magnetic stirring at 60 oC. After 30 min, TEA (2 mL) was added into the

85

resulted solution and stirred continuously for 2 h. Then, TEOS (1 mL) was added into

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the mixture solution dropwise and stirred continuously for 12h, and the product was

(TEA),

cetyltrimethylammonium

chloride

4


(CTAC)

and

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centrifuged under 15000 rpm for 20 min. Afterwards, the soft template was removed

88

through reflux twice in acetone for 24 h at 60 oC.

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Amine modified MSNs: MSNs were added into the mixture of toluene and APTES to

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aminate the silica surface. The detailed procedures are as follows: MSNs (50 mg) was

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dispersed into 20 mL toluene under magnetic stirring, and then 50 µL of APTES was

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added into the resulted solution under nitrogen and stirred for 24 h. Finally, the

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aminated nanoparticles were centrifuged and washed with ethanol and deionized water

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for three to five times.

95

DNA extraction: To extract the genome DNA, leaves of tomato (Solanum

96

lycopersicum) and wheat (Triticum aestivum) (approximately 5g) were ground into

97

powder by mortar in liquid nitrogen respectively. Then the powder was gently

98

dispersed in extraction buffer containing 100 mM Tris-HCl (pH 8.0), 20mM EDTA

99

(pH 8.0), 1.5 M NaCl, 10% (W/W) CTAB, 0.5% (W/W) bentonite, 2% (W/W) PVP-10,

100

and 0.2% (W/W) 2-mercaptoethnol at 65 oC for 30 min, and then 34% (V/V) potassium

101

acetate (5M, pH 4.8) was added to extraction buffer in ice bath for 20 min. Afterwards,

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the following steps of chloroform washing, DNA precipitation and washing, and

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RNaseA treatment were carried out as previously described.28

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To obtain 1000 bp DNA fragments, the total RNA was extracted from the flowers of

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Pistacia chinensis using RNAiso plus. The cDNAs obtained from RNA using the

106

M-MLV Reverse Transcriptase were used for polymerase chain reaction (PCR) as a

107

template, and then the PCR reaction (25 µL reaction volume containing template DNA,

108

dNTPs, MgCl2, Taq DNA polymerase, primer and PCR buffer) was carried out. The 5



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primer was designed to amplify 12-ox-phytodienoic acid reductase gene, which has the

110

length of approximately 1200 base pairs. Amplification was performed in a thermo

111

cycler. After programmed for an initial denaturation at 94 °C for 8 min, 94 °C for 30 sec

112

and 55 °C for 30 sec, 72 °C for 1 min for 35 cycles and a final auto extension step, the

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amplification products were analyzed by agarose (1.5%) and stained with GelRed.

114

Storage and release of drug: (a) AMSNs (10 mg) were dispersed in 5 mL of COLC

115

solution (10 mg/mL) by shaking at ambient temperature for 24 h, making COLC loaded

116

in pores of AMSNs. Then, 500 µL of DNA (1.5 mg/mL) was added into the mixture

117

solution and heated at 95 oC for 1 h to obtain flexible single strands. After that, urea (50

118

mg) was added into the resulted solution to prevent DNA single strands from forming

119

rigid double strands. The resulted system was shaken at an ice bath for 24 h to obtain

120

DNA-encapsulated AMSNs. (b) 10 mg of DNA-encapsulated AMSNs were dispersed

121

in 6 mL SBF by shaking for 24 h at 36.5 oC. The COLC concentration in the

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supernatant after centrifuging was determined using a UV-Vis spectrophotometer (UV

123

2550, Shimadzu Co., Japan) at wavelength of 244 or 350 nm to get the drug release

124

performance.

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Characterization: The morphology and microstructure were observed on a scanning

126

electron microscope (Sirion 200, FEI Co., USA) and an H-800 transmission electron

127

microscope (Hitachi Co., Japan). The particle size distribution was determined by a

128

dynamic light scattering (DLS) detector (Zetasizer 300, Malvern Co., UK). The pore

129

size distribution was measured using a porosimetry analyzer (Tristar II, 3020M,

6


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Micromeritics Co., USA). The structure and interaction were analyzed using a Fourier

131

transform infrared spectrometer (Bruker Co., Germany).

132

RESULTS AND DISCUSSION

133

MSN was synthesized by adding a mixture of TEOS and TEA to homogeneous CTAC

134

solutions at 60 oC, and the size of MSN depend on the adding amount of TEA. Scanning

135

electron microscope (SEM) images showed that MSNs had homogenous size of

136

approximately 100 nm (Figure 1a and b). In addition, the particle size distribution of

137

MSNs was shown in Figure 2a, and the same result was obtained (approximately 100

138

nm). Transmission electron microscope (TEM) images showed that MSN in the

139

solution possessed obvious mesopores and high dispersibility (Figure 1c and d). To

140

make MSN positively charged, APTES was used to aminate MSN, which could

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increase the zeta potential of nanoparticles because of the protonation of amino group

142

in simulated body fluid (SBF). It was shown that AMSN had more positive charges

143

than MSN (Figure 2b).

144

The nitrogen adsorption-desorption isotherm curves of MSNs and AMSNs and

145

their corresponding pore size distribution curves were shown in Figure 3. It was found

146

that the amounts of nitrogen absorption at 0.8-1.0 of P/P0 increased sharply, which is

147

probably because of the pores among the aggregated particles (Figure 3a). In addition,

148

the isotherm curves of MSNs at 0.15-0.6 of P/P0 exhibited that MSN possessed

149

ordered mesoporous structure and high porosity, and the pore size distribution curve

150

showed that pore sizes narrowly distributed over 2-4 nm (dominantly 2.9 nm) (inset of

151

Figure 3a). However, the isotherm curve of AMSNs indicated lower porosity and 7



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smaller pore size of AMSN compared with MSN (Figure 3b), because the amino

153

groups on the surface of AMSN decrease the porosity and pore size. COLC molecule

154

size was estimated through Chemdraw software to be around 1.34 nm (inset of Figure

155

3b). Therefore, although the pore size of AMSN was decreased to 2.0 nm, it was also

156

suitable for loading drug molecules .

157

Colchicine (COLC), a drug used in cancer treatment, was selected as a model drug to

158

be loaded in AMSN, and DNA molecules could act as the “guard” to control the release

159

of drugs from the pores of AMSN. Drug-loading capacity of AMSN was determined

160

using a UV-Vis spectra. The absorption wavelength and corresponding calibration

161

curve of COLC were shown in Figure 2c and d. It was shown that AMSN possessed

162

high drug storage capacity (242 µg/mg) through UV-Vis analysis, which is helpful to

163

provide continuous treatment to disease tissue. The rigid duplex DNA was thermally

164

treated at 95 oC and then treated with urea to obtain the stable flexible single strands,

165

which is beneficial for encapsulating the AMSN. As was shown in Figure 4a,

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molecular weight of DNA could be verified through agarose gel electrophoresis

167

(AGE). Compared with sample III, Sample IV showed smaller molecular weight and

168

possessed flexible chains, which indicated that duplex DNA was successfully divided

169

into single strands. In order to obtain the interactions among AMSN, AMSN

170

encapsulated with DNA (DAMSN) and DAMSN after enzyme degradation, FTIR

171

analysis was carried out (Figure 4c). For AMSN, the peak at 1089 cm-1 assigns to Si-O

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stretching vibration. For DAMSN, the peak at 3430 cm-1 ascribes to the NH stretching

173

vibration of DNA, indicating the binding of nucleic acid bases onto the surface of 8


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AMSN. The peaks at 1667 and 1634 cm-1 are assigned to the C=O stretching vibration

175

of nucleotide. This splitting of the C=O stretching vibration peak is assigned to

176

polynucleotide double helix structure because of the antiparallel orientation of each

177

helix.29 In addition, the peaks at 1456 and 1472 cm-1 reflect the purine and pyrimidine

178

(DNA bases) ring modes.30 These results indicated that DNA successfully encapsulated

179

AMSN. The FTIR spectrum of DAMSN after enzyme degradation displayed that some

180

DNA characteristic peaks became weakened or even disappeared, indicating that the

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DNA coating on AMSN has been hydrolyzed by DNase I enzyme.

182

The hydrolysis of DNA coating was also investigated by agarose gel electrophoresis

183

(Figure 4b). Large electrophoresis spot could be seen clearly for both sample 1 and 3,

184

because DNA with high molecular weight was unable to penetrate the agarose gel.

185

Nevertheless, some DNA fragments could be found in SBF when DNase I was added

186

into the solution (sample 2 and 4 in Figure 4b). These results implied that the DNA

187

gates of AMSN could be opened effectively through the hydrolization effect of DNase

188

I.

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To investigate enzyme-responsive release performance of COLC, COLC-loaded

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DAMSNs (CDAMSNs) were placed in DNase I SBF at 36.5 oC. Figure 5a showed that

191

few drugs were released when AMSNs were encapsulated with DNA single strands,

192

which indicated that the gates of the mesopores were almost closed because of the

193

strong electrostatic interactions between the positive groups (NH3+) on the surface of

194

AMSN and the negative groups of single-stranded DNA. However, after about 2 hours

195

since the addition of DNase I, the COLC molecules began to be released in varying 9



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degrees, because the gates started to open resulting from the hydrolysis of DNA

197

strands. Additionally, the COLC release rate decreased with the increase of the

198

molecular weight of DNA, which is because DNA with higher molecular weight was

199

harder to hydrolyze as shown in Figure 4b (106 bp DNA was hydrolyzed faster

200

compared with 109 bp DNA). Interestingly, the release rate of COLC from CDAMSN

201

with DNA of 109 bp increased steadily and gradually, suggesting that this drug delivery

202

system might be long-acting and continuous, which is helpful to maintain the effective

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concentration of drugs in diseased tissue, enhance treatment efficiency, and decrease

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drug toxicity for normal cells.

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This enzyme-responsive nanomedicine was investigated further, and the release

206

behaviors of COLC in DAMSN before and after addition of DNase I were shown in

207

Figure 5b-d. The absorbance intensity of COLC before addition of DNase I was

208

monitored for 10 h, and the flat baselines showed that drug leakages from

209

nanomedicine were weak and could be negligible. After DNase I was added into

210

release system solution, the absorbance intensity of COLC increased rapidly, which

211

indicates that the hydrolysis of DNA through DNase I could open the gates of MSN.

212

The similar release behaviors appeared in all three DAMSNs (coated with 103, 106 or

213

109 bp DNA). These results demonstrated that CDAMSN was efficient for the storage

214

of drugs and could meanwhile release drugs after addition of DNase I.

215

CONCLUSION

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In summary, an efficient enzyme-responsive controlled release carrier system was

217

successfully fabricated using a single-stranded DNA encapsulated AMSN. Based on 10


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the electrostatic interactions between flexible single-stranded DNA and AMSN, the

219

single-stranded DNA coating could act as gates for storage of drugs. These gates could

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be opened to release drugs through the enzyme hydrolysis effect of DNase I, and the

221

drug release performance could be controlled by the molecular weight of DNA,

222

therefore, this drug delivery system might make a long-acting and continuous effect of

223

drugs, which would be helpful to increase the treatment efficiency and decrease the side

224

effects of drugs.

225 226

AUTHOR INFORMATION

227

Corresponding authors.

228

*Tel.: +86-551-65595012, +86-551-65591413; Fax: +86-551-65591413. E-mail:

229

[email protected] (L.W.), [email protected] (Z.W.).

230

Notes

231

The authors declare no competing financial interest.

232

†

233

ACKNOWLEDGMENTS

234

The authors acknowledge financial support from the National Natural Science

235

Foundation of China (No. 10975154, No. 11075178) and Hefei Center of Physical

236

Science and Technology of China (No. 2012FXCX006).

G.Z. and M.Y. are co-first authors.

237 238

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Controlled-Release

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Nanocontainers. Nucleic Acids Res. 2011, 39, 1638.

DNA-Capped

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(23) He, D.; He, X.; Wang, K.; Chen, M.; Cao. J.; Zhao, Y. Reversible

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Stimuli-Responsive Controlled Release Using Mesoporous Silica Nanoparticles

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Functionalized with a Smart DNA Molecule-Gated Switch. J. Mater. Chem.

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2012, 22, 14715.

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(24) Chen, L.; Wen, Y.; Su, B.; Di, J.; Song, Y.; Jiang, L. Programmable DNA Switch for Bioresponsive Controlled Release. J. Mater. Chem. 2011, 21, 13811.

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Y. Combination Gene Therapy Targeting on Interleukin-1β and RANKL for Wear

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Debris-Induced Aseptic Loosening. Gene Ther. 2013, 20, 128.

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Therapy Clinical Trials Worldwide to 2012 – An Update. J. Gene Med. 2013, 15,

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Isolation from Pear Leaves Rich in Polyphenolic Compounds. Int. J. Hortic. Sci.

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Malhotra, B. D.; Chand, S. Application of Electrochemically Prepared

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Polypyrrole–Polyvinyl Sulphonate Films to DNA Biosensor. Biosens. Bioelectron.

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Radiotolerant Bacterium Pantoea Agglomerans by FT-IR Spectroscopy. Talanta

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323

Figure legend

324

Scheme 1. Schematic representation of drug release process of drug-loaded AMSN

325

encapsulated with DNA.

326 327

Figure 1. SEM (a, b) and TEM (c, d) images of mesoporous silica nanoparticles.

328 329

Figure 2. (a) Particle size distribution of MSN, (b) zeta potential of (A) MSN and (B)

330

AMSN, (c) UV-Vis absorption wavelength and (d) calibration curve of COLC.

331 332

Figure 3. N2 adsorption-desorption isotherms of (a) MSN and (b) AMSN (inset of (a):

333

pore size distribution of MSN, inset of (b): pore size distribution of AMSN, and

334

structure and size of COLC molecule).

335 336

Figure 4. (a) DNA electrophoresis images (Marker (M), 109 bp DNA (I), 106 bp DNA

337

(II), 2×103 bp DNA (III), 2×103 bp DNA after melting at 95 oC (IV)); (b) DNA

338

electrophoresis images (M: marker, samples 1 and 3: 109 and 106 bp DNA, samples 2

339

and 4: 109 and 106 bp DNA after DNase I degradation); (c) FTIR spectra of AMSN ,

340

DAMSN , and DAMSN after enzyme degradation.

341 342

Figure 5. Release behavior of COLC in AMSN encapsulated with DNA: (a) with and

343

without addition of DNase I; (b)-(d) before and after adding DNase I for 103 bp (b),

344

106 bp (c) and 109 bp (d) DNA. 17



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.

346 347 348

Scheme 1

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Composite of Functional Mesoporous Silica and DNA: An Enzyme

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