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Temperature/pH/Enzyme Triple-Responsive Cationic Protein/ PAA-b-PNIPAAm Nanogels for Controlled Anticancer Drug and Photosensitizer Delivery Against Multidrug Resistant Breast Cancer Cells Trong-Ming Don, Kun-Ying Lu, Li-Jie Lin, Chun-Hua Hsu, Jui-Yu Wu, and Fwu-Long Mi Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00737 • Publication Date (Web): 23 Oct 2017 Downloaded from http://pubs.acs.org on October 25, 2017

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Molecular Pharmaceutics

1

Temperature/pH/Enzyme Triple-Responsive Cationic Protein/PAA-b-PNIPAAm

2

Nanogels for Controlled Anticancer Drug and Photosensitizer Delivery

3

Against Multidrug Resistant Breast Cancer Cells

4 5 6

Trong-ming Don1‡, Kun-Ying Lu2,3‡, Li-Jie Lin1,

7

Chun-Hua Hsu4, Jui-Yu Wu,3,5 Fwu-Long Mi3,5,6*

8 9

1.

10 11

Department of Chemical and Materials Engineering, Tamkang University, New Taipei City 25137, Taiwan.

2.

Graduate Institute of Biomedical Materials and Tissue Engineering, College of

12

Biomedical Engineering, Taipei Medical University, Taipei City 11031, Taiwan,

13

R.O.C.

14

3.

15 16

University, Taipei 11031, Taiwan 4.

17 18

21

Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan.

5.

19 20

Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical

Department of Biochemistry and Molecular Cell Biology, School of medicine, Taipei Medical University, Taipei 11031, Taiwan

6.

Graduate Institute of Nanomedicine and Medical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei 11031, Taiwan

22 23 24

*Corresponding author

25

Fwu-Long Mi, PhD

26

Professor

27

Department of Biochemistry and Molecular Cell Biology,

28

School of medicine, Taipei Medical University

29

Taipei City, Taiwan 110

30

Fax: 886-2-2735-6689

31

E-mail: [email protected]

32

* To whom correspondence should be addressed: [email protected] (Dr. F. L. Mi)

33

‡ These authors contributed equally to this work.

34

1

ACS Paragon Plus Environment


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ABSTRACT

36

The tumor microenvironments are often acidic and overexpress specific enzymes. In

37

this work, we synthesized a poly(AA-b-NIPAAm) copolymer (PAA-b-PNIPAAm)

38

using a reversible addition-fragmentation chain transfer (RAFT) polymerization

39

method. PAA-b-PNIPAAm and a cationic protein (protamine) were self-assembled

40

into nanogels, which effectively reduced the cytotoxicity of protamine. The

41

protamine/PAA-b-PNIPAAm nanogels were responsive to the stimuli including

42

temperature, pH and enzyme due to disaggregation of PAA-b-PNIPAAm, change in

43

random coil/α-helix conformation of protamine, and enzymatic hydrolysis of the

44

protein. Changing the pH from 7.4 to a lowered pHe (6.5-5.0) resulted in an increase

45

in mean particle size and smartly converted surface charge from negative to positive.

46

The cationic nanogels easily passed through the cell membrane and enhanced

47

intracellular localization and accumulation of doxorubicin-loaded nanogels in

48

multidrug resistant MCF-7/ADR breast cancer cells. Cold shock treatment triggered

49

rapid intracellular release of doxorubicin against P-glycoprotein (Pgp)-mediated drug

50

efflux, showing significantly improved anticancer efficacy as compared with free

51

DOX. Furthermore, the nanogels were able to carry a rose bengal photosensitizer and

52

caused significant damage to the multidrug resistant cancer cells under irradiation.

53

The cationic nanogels with stimuli-responsive properties show promise as drug carrier

54

for chemotherapy and photodynamic therapy (PDT) against cancers.

55

Keywords: N-isopropylacrylamide, protamine, nanogels, pH-responsive, thermo-responsive,

56

enzymatic digestion

57

2


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58

■ INTRODUCTION

59

Despite a variety of anticancer drugs have been developed, cancer therapy remains

60

one of the greatest challenges in modern medicine because chemoresistance becomes

61

a major problem that limits treatment with a success. Tumor-specific stimuli can

62

trigger drug release from nanocarriers to overcome this limitation. The temperature of

63

tumor microenvironment can be easily adjusted and controlled by applying an

64

external heat source or cold shock while pH changes and enzymatic digestion can

65

spontaneously occur in cancer cells or tumors.1-3 Polymeric micelles and nanoparticles

66

are easily modified with stimuli-responsive properties, allowing drug release

67

controlled by temperature, pH, enzymes and other biological active molecules.4-7

68

Poly(N-isopropylacrylamide) (PNIPAAm) is a thermo-responsive polymer that

69

exhibits a conformational change at a lower critical solution temperature (LCST)

70

around 32°C. Especially, PNIPAAm-based drug delivery systems with thermo- and/or

71

pH-responsive properties have been developed by different methods.8-13 Our

72

previously prepared nanogels based on poly(AA-co-NIPAAm) copolymers have

73

temperature and/or pH -responsive properties.14-16 However, the application of the

74

poly(AA-co-NIPAAm) nanogels for anticancer drug delivery was limited due to its

75

high solubility in physiological saline, leading to fast release of loaded drugs from the

76

carriers during circulating in blood. Our previous study also prepared protamine-based

77

nanoparticles that have enzyme-responsive and charge conversion properties.17

78

Protamine is a cationic polypeptide which is rich in arginine and basic amino acids.

79

The guanidino group of arginine residues in protamine has a pKa value near 13, thus

80

protamine is usually positively charged at most pH levels. Protamine has been used as

81

a cell-penetrating peptide which can effectively carry drugs, nucleic acids (DNA,

82

siRNA and miRNA) and proteins into cells.18,19 It can be digested by a matrix serine

83

protease (MSP) trypsin which is overexpressed in some cancer tissues, which has 3



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been found to be associated with tumor growth, invasion, and metastasis.20,21 In

this

work,

we

synthesized

a

poly(AA-b-NIPAAm)

copolymer

86

(PAA-b-PNIPAAm), via a reversible addition-fragmentation chain transfer (RAFT)

87

polymerization method. PAA-b-PNIPAAm greatly reduced the cytotoxicity of

88

protamine after formation of protamine/PAA-b-PNIPAAm complex nanogels. The

89

pH/thermo/enzyme-responsive properties were evaluated in vitro. The nanogels were

90

negatively charged at pH 7.4 (blood circulation) and positively charged at 6.0 and 5.0

91

(tumor microenvironments). The nanogel was also sensitive to trypsin, an enzyme

92

expressed in several human cancer cells and tumors, and was responsive to cold shock

93

treatments which could be used in cryotherapy to trigger doxorubicin (DOX) release

94

in acid environments (endosomes/lysosomes) for intracellular anticancer drug

95

delivery. Furthermore, the nanogels loaded with a photosensitizer for in vitro

96

photodynamic therapy (PDT) against DOX-resistant MCF-7/ADR breast cancer cells

97

were investigated.

98

■ EXPERIMENTAL SECTION

99

Materials and Reagents. N-isopropyl acrylamide (NIPAAm) and acrylic acid

100

(AA) were purchased from Acros Organics, Belgium. NIPAAm was purified by its

101

dissolution in hexane at 50 °C and then fitered to remove the impurity. It underwent

102

recrystalization from the filtrate as placed in freezer, followed by filtration to remove

103

the solvent. AA was purified by distilliation. Only the distillate in the middle stage

104

was collected. 2,2′-Azobisisobutyronitrile (AIBN) was supplied from UniRegion

105

Bio-Tech, Taiwan. It was purified by recrystallization in its methanol solution at -20

106

°C.

107

protamine, doxorubicin (DOX) and rose bengal (RB) were all purchased from

108

Sigma–Aldrich (Louis, MO, USA). All other chemicals were at least analytical grade

109

and used without further purfication.

2-(Dodecylsulfanylthiocarbonylsulfanyl)-2-methylpropionic

4


acid

(DMP),

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110

RAFT Polymerization of PAA-b-PNIPAAm Block Copolymer. A living free

111

radical polymerization method, RAFT, was adopted to synthesize PAA-b-PNIPAAm

112

block copolymer following previous studies with a slight modification (Schilli et al.,

113

2004; Kulkarni et al., 2006). In the first stage, AA was dissolved in 20 mL methanol

114

and placed in a reaction vessel equipped with a condenser and the addition funnel.

115

The reactor was purged with nitrogen and heaed to 75 °C. The DMP as chain transfer

116

agent for the RAFT polymerization and the AIBN as an initiator, both pre-dissolved

117

in methanol, were added into the reactor to start polymerization. The molar feed ratio

118

of AA: DMP: AIBN was 77.45: 1.0: 0.1. The reaction was continued for 1.5 h. To

119

remove the un-reacted monomer and initiator, the reactor was cooled down by

120

immersion in the ice-water bath. Cold ether was then added with 20-time volume of

121

methanol to precipitate the reaction product, i.e. PAA-CTA, as the new macro-chain

122

transfer agent. The precipitate was separated by centrifugation at 4 °C for 15 min. It

123

was dissolved in methanol again and repeated the procedure of precipitation in ether

124

and centrifugation. Finally, it was dried at 80 °C to obtain the yellowish PAA-CTA.

125

In the second stage, NIPAAm was also dissolved in methanol and added with the

126

PAA-CTA and AIBN followed by the same reaction procedure in the first-stage

127

RAFT polymerization. Yet, the reaction time was increased to 3 h. The molar feed

128

ratio of NIPAAm: PAA-CTA: AIBN was 160: 1: 0.1. The reaction is shown in the

129

following Fig. 1A.

130

Chemical structure of the synthesized PAA-b-PNIPAAm block copolymer was

131

analyzed by Fourier transform infrared (FTIR) spectrometer (iS10, ThermoFisher,

132

USA), nulcear magnetic resonance (NMR) spectrometer (DMX-600, Bruker,

133

Germany) and gel permeation chromatography (GPC, Jasco PU-2080 plus pump and

134

Shodex RI-101 detector, Japan). To obtain the FTIR spectrum, dried sample was

135

ground with KBr powder and the mixture was then pressed into transparency disk. It 5



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136

was scanned 32 times from 4000 to 400 cm-1 with a resolution of 4 cm-1 in the IR

137

transmittance mode. Characteristic absorption peaks for the PAA-b-PNIPAAm (cm-1):

138

3435-2500 ((C=O)-OH), 3354 (NH), 2976 and 2877 (-CH3), 2937 and 1459 (>CH2),

139

1732 (C=O), 1652 (C=O, amide I), 1548 (NH, amide II), 1388 and 1368 (CH3,

140

isopropyl group), 1073 (C=S). For NMR analysis, PAA-b-PNIPAAm was dissolved

141

in deuterated DMSO. Its NMR spectrum was then obtained for analysis.

142

Characteristic resonance peaks (ppm): 0.95 (-CH3 in the terminal DMP), 1.10-1.45

143

(>CH2 in the DMP, -CH3 in the isopropyl groups of both DMP and PNIPAAm),

144

1.50-2.15 (>CH2 in the PAA and PNIPAAm blocks), 2.30-2.55 (>CH- in the PAA and

145

PNIPAAm blocks), 3.85 (>CH- in the isopropyl groups of PNIPAAm).

146

Preparation of Protamine/PAA-b-PNIPAAm Nanogel. PAA-b-PNIPAAm

147

nanogels were prepared by directly heating the PAA-b-PNIPAAm aqueous solution (1

148

mg/mL, 25 mL) at 37 °C above its LCST for 10 mins. On the other hand,

149

protamine/PAA-b-PNIPAAm nanogels were prepared via a polyelectrolyte complex

150

method by mixing the PAA-b-PNIPAAm(aq) (1 mg/mL) with the protamine(aq) (2

151

mg/mL) at different weight ratios and at different temperatures (25 °C and 37 °C)

152

using a pipette (Table 1). The surface morphology and particle size of nanogels in

153

their dry state were observed by using a transmission electron microscope (TEM,

154

H-7650, Hitachi, Japan). Dilute nanogel solutions were applied to carbon-coated

155

copper grids and then dried. Measurements of particle size and surface charge of

156

PAA-b-PNIPAAm and protamine/PAA-b-PNIPAAm nanogels in solutions were

157

performed using a Zetasizer Nano (Malvern, UK). The FTIR spectra were also

158

recorded to determine the interaction between protamine and PAA-b-PNIPAAm.

159

LCST of PAA-b-PNIPAAm Solutions. A UV/Visible spectrophotometer

160

(ThermoFisher, Helios α, USA) equipped with a temperature controller was used to

161

investigate the lower critical solution temperature (LCST) of the nanogel solutions at 6


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162

different pH values. The copolymer solutions were prepared at a concentration of 1

163

mg/mL and their transmittances were recorded at the wavelength of 500 nm at

164

temperatures ranging from 10 °C to 60 °C. Values for the LCST were then determined

165

at the differential peak temperatures of transmittance curves. The LCST values were

166

also determined by differential scanning calorimeter (Diamond DSC, Perkin Elmer,

167

USA) at a heating rate of 1 °C/min.

168

Characterization of pH/Thermo/Enzyme-Responsive Properties. In order to

169

investigate the pH/thermo-responsive properties of the PAA-b-PNIPAAm and

170

protamine/PAA-b-PNIPAAm, both nanogels(aq) in distilled water were separately

171

added into several dissolution mediums of pH 5.0 (acetate buffer), 6.0 (HCl +

172

phosphate buffer) and 7.4 (phosphate-buffered saline, PBS) at different volume ratios.

173

Two different temperatures at 4 °C and 37 °C were tested. A series of experiments to

174

characterize the fluorescence recovery, optical transmittance and the change in

175

particle size distribution were then performed at the selected pH values and

176

temperatures. Procedures for measuring optical transmittance and particle size

177

distribution were described in the previous section. For the experiment of

178

fluorescence recovery, 5-aminofluorescein-labeled PAA-b-PNIPAAm was prepared

179

as described in the following. 5-aminofluorescein (1.5 mg) was first dissolved in

180

DMSO, which together with EDC(3.0 mg)/NHS(4.5 mg) were added into 5.0 mL of

181

PAA-b-PNIPAAm solution (5.0 mg) at pH 4.8. The coupling reaction was allowed to

182

continue for 8 h at 35 °C. The 5-aminofluorescein-labeled PAA-b-PNIPAAm was

183

purified by dialysis against DI water in the dark. The fluorescence spectra of

184

5-aminofluorescein-labeled PAA-b-PNIPAAm and protamine/PAA-b-PNIPAAm

185

nanogels at different pH values and temperatures were recorded using a fluorescence

186

spectrophotometer equipped with a temperature-controllable circulating water bath

187

(Hitachi,

F-7000,

Japan)

to

examine

assembly-disassembly

7


of

the


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188

protamine/PAA-b-PNIPAAm

189

disassembly behavior, the nanogels were added into a phosphate buffer (pH 6.5)

190

containing trypsin at a concentration of 0.2 mg/mL. At different time intervals, the

191

optical transmittance was measured by using the above mentioned method.

192

Doxorubicin

Loading

nanogels.

and

To

Page 8 of 37

Release.

investigate

Both

the

enzyme-triggered

PAA-b-PNIPAAm

and

193

protamine/PAA-b-PNIPAAm nanogels loaded with doxorubicin (DOX) were

194

prepared to determine their drug release behaviors. DOX (25 mg) was first dissolved

195

in PAA-b-PNIPAAm aqueous solution (1 mg/mL, 25 mL) at 10 °C, and then the

196

premixed DOX/PAA-b-PNIPAAm solutions were heated to 37 °C with and without

197

the addition of protamine solution (protamine to PAA-b-PNIPAAm weight ratio= 2:9)

198

to

199

protamine/PAA-b-PNIPAAm. After centrifugation, drug loading efficiency of the

200

nanogel was determined by measuring the optical transmittance at 480 nm in the

201

supernatant using a Perkin Elmer EnSpire 2300 multimode plate reader (USA). The

202

amount of free doxorubicin was thus determined from the calibration curve. To

203

examine the drug release behavior, the DOX-loaded nanogel was then transferred to

204

each of three dialysis tubes against different dialysis solutions (45 mL) of pH 5.0, 6.0

205

and 7.4, which were placed in a beaker and shaken at 37 °C, 100 rpm. Drug release

206

studies were also performed at pH 6.5 in the presence of enzyme (0.2 mg mL-1 trypsin)

207

and its inhibitor. At specific time intervals, the dialysate (0.5 mL) was withdrawn and

208

replaced with fresh dissolution medium, and the collected dialysate was used to

209

determine the doxorubicin release percentage colorimetrically using the multimode

210

plate reader.

obtain

the

DOX-loaded

nanogels

of

PAA-b-PNIPAAm

and

211

In Vitro Cytotoxicity of DOX-loaded Nanogels. MCF-7 and MCF-7/ADR

212

(doxorubicin-resistant) breast cancer cells were seeded in 96-well plates at a density

213

of 1×104 cells per well. After 24 h of incubation, the cells were exposed to 8


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214

DOX-loaded protamine/PAA-b-PNIPAAm nanogels by replacing the cell culture

215

medium with serum-free medium containing the nanogels. After 2 h of incubation at

216

37 °C (pH 6.5), the cells were washed and treated with a low-temperature treatment

217

for 15 min at 4 °C (a cold shock treatment). The cells were further cultured for 24 h

218

and MTT assay was used to determine the dose-dependent cytotoxicity (0.9, 1.8, 9.0

219

µg/mL doxorubicin equivalent) caused by the nanogels after reading the optical

220

density at 570 nm on a microplate reader (Model 3550, Bio-Rad, USA).

221

Cellular Uptake of Doxorubicin-Loaded Nanogels. To investigate cellar uptake

222

of the nanogels, PAA-b-PNIPAAm was labeled with a green fluorescence

223

5-aminofluorescein and the fluorescent protamine/ PAA-b-PNIPAAm nanogels were

224

prepared according to the previously described procedure. The cells were then

225

incubated with the fluorescent nanogels for 2 h at pH 6.5. After incubation, the cells

226

were washed with PBS, and then DAPI staining and paraformaldehyde fixation were

227

performed either immediately or after a cold shock treatment. The cells were

228

visualized by a confocal microscope (TCS SP5, Leica Microsystems, Wetzlar,

229

Germany). Densitometric analysis of fluorescence intensity was performed using the

230

Image J software (National Institute of Health, Bethesda, MD, USA).

231

Photodynamic Treatments. MCF-7 and MCF-7/ADR cells were cultured

232

according to the above-mentioned procedure. Rose bengal-loaded nanogels were

233

prepared by the method similar to that for preparation of DOX-loaded nanogels in

234

section 2.7, except for dissolving a photosensitizer (2 mg/mL rose bengal) in the

235

PAA-b-PNIPAAm aqueous solution. Rose bengal loading efficiency was determined

236

by measuring the amount of free rose bengal in the supernatant at 546 nm in the

237

supernatant using a Perkin Elmer EnSpire 2300 multimode plate reader (USA). The

238

cells were incubated with the rose bengal-loaded nanogels (8.0 µg/mL rose bengal

239

equivalent) for 4 h. After replacing the culture medium, the plates (w/ and w/o a cold 9



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240

shock treatments) were immediately exposed to a green light LED (550 nm, 50

241

mW/cm2) for 8 min. The cells were cultured for an additional 24 h and the

242

photocytotoxicity was evaluated using the above-mentioned MTT assay.

243

Statistical Analysis. Statistical analyses were conducted using Student’s t-test with

244

replicate measurements for data (n= 5). Differences with a P value < 0.01 were regarded

245

to be significant.

246

RESULTS AND DISCUSSION

247

Characterization of PAA-b-PNIPAAm Block Copolymer. A living free radical

248

polymerization method, RAFT, was adopted to synthesize PAA-b-PNIPAAm block

249

copolymer following previous studies with a slight modification (Figure 1A).15

250

Because of the presence of both PAA block and PNIPAAm block in the same

251

polymer chain, this block copolymer is therefore sensitive to the environmental

252

changes of acidity and tmperature. After reaction of 1.5 h for synthesizing the PAA

253

block in the first stage and another 3 h for the further growth of PNIPAAm block,

254

NMR analysis was carried out not only to confirm the chemical structure bout also to

255

determine the chain length (degree of polymerization, DP) of each block. The most

256

distinct absorption peaks of the PAA-b-PNIPAAm block copolymer were caused by

257

the isopropyl group in the PNIPAAm block including -CH3 at 1.20 ppm and >CH- at

258

3.85 ppm. The broad absorption peaks of the >CH2 and >CH- groups in the main

259

chain were at 1.50-2.15 and 2.30-2.55 ppm, respectively. The individual chain lengths

260

of the PAA and PNIPAAm blocks were thus calculated from the respective peak area

261

ratios of the characteristic absorption peaks of PAA segment and PNIPAAm segment

262

to the terminal DMP group. The results indicated that the DP values of the PAA and

263

the PNIPAAm blocks were 28 and 106, respectively. The corresponding AA

264

conversion in the first stage was 36%, whereas the NIPAAm conversion in the second

265

stage was 66%. The molecular weight of the PAA-b-PNIPAAm was thus 11480 and 10


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266

the PDI value measured from GPC was 1.23. This narrow molecular-weight

267

distribution is agreed to the living characteristics of the RAFT polymerization.

268

Chemical and Physical Properties of Nanogels. Fig. 1B shows the schematic

269

diagram for the preparation of protamine/PAA-b-PNIPAAm nanogels by mixing both

270

polymers that were dissolved in deionized (DI) water. The nanogels were prepared by

271

a method of thermo-induced aggregation in combination with polyelectrolyte complex.

272

Protamine was largely assembled with PAA-b-PNIPAAm via polyelectrolyte complex

273

on the outer layer of thermally aggregated PAA-b-PNIPAAm nanogels (Figure 1B).

274

The PAA-b-PNIPAAm aqueous solution (1 mg/mL) was transparent at room

275

temperature (25 °C), but formed thermally aggregated nanogels upon heating to 37 °C.

276

Yet, at room temperature, upon the addition of a small amount of aqueous protamine

277

into PAA-b-PNIPAAm solution (weight ratio of protamine to PAA-b-PNIPAAm=

278

1:45), protamine/PAA-b-PNIPAAm nanogels formed immediately. This is because of

279

the electrostatic attraction between the negative-charged PAA block and the

280

positive-charged protamine and probably also due to their hydrogen bonding.

281

However, the hydrodynamic particle size (volume-averaged) increased substantially

282

from 92.5±3.5 nm to 601.1±23.2 nm when increasing the weight ratio from 1:45 to

283

1:25. Moreover, the nanogels became unstable during 24 h of storage and then

284

precipitated, when they were prepared at weight ratios of protamine to

285

PAA-b-PNIPAAm higher than 1:25. As shown in Table 1, increasing the weight ratio

286

of protamine to PAA-b-PNIPAAm from 1:45 to 1:25 leads to the formation of nearly

287

neutral nanogels (–20.60±0.78 mV vs. –0.07±0.10 mV), which decreases the

288

repulsion among individual nanogels. The results indicate that the complex nanogels

289

of protamine/PAA-b-PNIPAAm prepared at room temperature can’t be used as a drug

290

delivery carrier.

291 11



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292 293

Page 12 of 37

Table 1. Average size and zeta potential of protamine/PAA-b-PNIPAAm nanogels at different weight ratios of protamine to PAA-b-PNIPAAm Volume ratio1

Weight ratio

Average size

2

PDI

Zeta potential

(nm)

PAA-b-PNIPAAm 25°C 37°C

(mV)

33.91±1.76 34.71±2.39

0.175 0.034

0.24±0.61 –0.13±0.36

protamine/PAA-b-PNIPAAm 25°C 1:90 1:50

1:45 1:25

92.52±3.46 601.10±23.20

0.110 0.116

–20.60±0.78 –0.07±0.10

1:9 2:9 3:9

2:9 4:9 6:9

142.23±0.71 137.27±1.05 201.57±3.00

0.103 0.020 0.046

9.97±0.96 –0.01±0.17 –0.14±0.17

37°C

294 295

1

The volume ratio of the added protamine(aq) solution (2 mg/mL) to the PAA-bPNIPAAm(aq) solution (1 mg/mL).

296

2

The weight ratio of the protamine to the PAA-b-PNIPAAm block copolymer.

297 298

Interestingly, when a protamine solution (2 mg/mL, 1 mL) was added into a

299

thermally aggregated PAA-b-PNIPAAm (1 mg/mL, 9 mL) at 37 °C, a highly cloudy

300

and stable suspension of protamine/PAA-b-PNIPAAm was obtained. The average

301

particle size and zeta potential of the nanogels with different compositions are

302

summarized

303

protamine-to-PAA-b-PNIPAAm weight ratio of 2:9, which have a zeta potential of

304

9.97±0.96 mV, revealing that the nanogels were covered with the positively charged

305

protamine. The nanogels were very stable and the average particle size wasn’t

306

obviously

307

PAA-b-PNIPAAm nanogels under heating at 37 °C (T > LCST), the positively

308

charged protamine are attracted to the negatively charged PAA block (Figure 1B),

309

leading to the formation of protamine/PAA-b-PNIPAAm polyelectrolyte complex

310

layer on the outer layer of PAA-b-PNIPAAm nanogels (Figure 1B).

in

Table

changed

1.

for

14

The

nanogels

days.

By

can

be

pre-forming

12


prepared

thermally

at

a

high

aggregated

Page 13 of 37

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(A)

311 312 (B)

313 314

Figure 1. (A) Schematic diagram for the synthesis of PAA-b-PNIPAAm copolymer,

315

(B) schematic diagram for the preparation of protamine/PAA-b-PNIPAAm nanogels.

316

Protamine/PAA-b-PNIPAAm

317

aggregation/polyelectrolyte complex method. Changing the pH from systemic

318

circulation (pH 7.4) to tumor microenvironment (pH 6.5) resulted in smartly

319

converting surface charge from negative to positive. The cationic nanogels easily

320

passed through the cell membrane and enhanced intracellular accumulation of

321

DOX-loaded nanogels. Cold shock treatment triggered rapid intracellular release of a

nanogels

were

prepared

13


by

a

thermal


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

322

large amount of doxorubicin or a photosensitizer rose bengal against multidrug

323

resistant MCF-7/ADR breast cancer cells.

324

TEM imagine shows the protamine/PAA-b-PNIPAAm nanogels that were prepared

325

by the thermal aggregation/polyelectrolyte complex method (Figure 2A-a). The

326

average size of the protamine/PAA-b-PNIPAAm nanogels is larger than that of

327

PAA-b-PNIPAAm nanogels (Figure 2A-b). The FT-IR spectra of protamine,

328

PAA-b-PNIPAAm, and protamine/PAA-b-PNIPAAm nanogels (weight ratio= 2:9)

329

are shown in Figure 2B. The bands at 1655 cm-1 and 1540 cm-1 are assigned to the

330

stretching of amide I (>C=O) and amide II (>N-H) of the peptide bonds in protamine,

331

respectively. The PAA-b-PNIPAAm copolymer shows several characteristic peaks

332

assigned to the absorption bands of carboxylic acid (1732 cm-1), amide group (1652

333

cm-1 and 1548 cm-1), and isopropyl group (1388 cm-1 and 1368 cm-1). Obviously, the

334

amide and guanidino absorption bands of protamine are overlapped with the

335

absorption of PAA-b-PNIPAAm. Still, the spectrum of protamine/PAA-b-PNIPAAm

336

nanogels shows a shoulder at about 1715 cm-1, revealing the formation of molecular

337

interactions between protamine and PAA-b-PNIPAAm. (A)

(B)

338 339

Figure 2. (A) TEM micrographs of PAA-b-PNIPAAm and protamine/PAA-b-

340

PNIPAAm nanogels, (B) FT-IR spectra of PAA-b-PNIPAAm, protamine, and

341

protamine/PAA-b-PNIPAAm nanogels, 14


Page 14 of 37

Page 15 of 37

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342

Thermo- and pH-Induced Assembly and Disassembly of Nanogels. The phase

343

transition temperatures of PNIPAAm-based materials can be determined by DSC

344

analysis.15, 22 Figure 3A shows the DSC curves of the swollen PAA, PNIPAAm, and

345

PAA-b-PNIPAAm copolymer in the distilled water. The phase transition temperature

346

(LCST) of the empty PNIPAAm is found at 31.5 °C, which is agreed to the literature

347

value. After copolymerization with the PAA, the prepared PAA-b-PNIPAAm (about

348

pH 4.2 when dissolved in distilled water) has a lower LCST value than the empty

349

PNIPAAm. At this low pH value, most carboxylic acid groups are in their neutral

350

form (-COOH) since the pKa of the PAA is about 4.75. The result suggests that

351

hydrogen bonding might occur between the amide groups of PNIPAAm and the

352

carboxylic acid groups of PAA, thus decreasing the LCST value.

353

The transition behaviors of various nanogels were studied by the studies of

354

temperature-induced fluorescence quenching and protein conformational changes.

355

Figure 3B and 3C show the fluorescence quenching and recovery of fluorescein

356

amine-labeled PAA-b-PNIPAAm after assembly and disassembly of the nanogels.

357

Converting of PAA-b-PNIPAAm from solution into nanogels due to the increase of

358

temperature from 25 °C to 37 °C was monitored by the reduction in fluorescence due

359

to self-quenching of fluorescein amine conjugated with PAA-b-PNIPAAm (78.2% of

360

original intensity), indicating thermal-induced aggregation of the copolymer at a

361

temperature higher than its LCST. Adding protamine to the PAA-b-PNIPAAm

362

nanogels at a weight ratio of 2:9 further quenched the fluorescence (65.9% of original

363

intensity), suggesting that PAA-b-PNIPAAm was even more aggregated with the

364

assistance of protamine (Figure 3B). Unexpectedly, the quenching effect decreased

365

with increasing the protamine-to-PAA-b-PNIPAAm weight ratio from 2:9 to 4:9 and

366

6:9, resulting in the recovery of the fluorescence intensity from 65.9% to 82.7% and

367

84.5% of original intensity. The fluorescence change can be correlated to the decrease 15



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Page 16 of 37

368

of zeta potential when increasing the weight ratio from 2:9 to 4:9 and even 6:9 (Table

369

1). This might be due to the decrease in thermal-induced aggregation of

370

PAA-b-PNIPAAm copolymer due to the protamine-involved electrostatic attraction

371

and possible hydrogen bonding as well. The existent ionized PAA block is assumed to

372

be fixed by the cationized protamine, thus the chain mobility of PAA-b-PNIPAAm

373

copolymer is further limited, leading to the unapparent fluorescence quenching. The

374

results suggest that the excessive increase in the number of protamine molecules

375

(positively

376

PAA-b-PNIPAAm, consequently leading to an increase of the fluorescence intensity

377

and decrease of the zeta potential. So, the optimal condition for preparing the complex

378

nanogels should be at a protamine-to-PAA-b-PNIPAAm weight ratio of 2:9.

379

Decreasing the temperature from 37 °C to 25 °C and 4 °C caused a fluorescence

380

recovery of the protamine/PAA-b-PNIPAAm nanogels (increases from 65.9% of

381

original intensity at 37 °C to 80.4% at 25 °C and 97.1% at 4 °C) (Figure 3C),

382

indicating the disassembly of the nanogels under a low-temperature condition.

charged)

may

disturb

the

thermal-induced

aggregation

of

383

Temperature-Induced Protein Conformational Changes. To evaluate if the

384

conformation of protamine could be affected by PAA-b-PNIPAAm after association

385

into

386


387

Formation of the protamine/PAA-b-PNIPAAm nanocomplex was indicated by the

388

shift of a negative band at 198 nm and the decrease of its ellipticity along with the

389

disappearance of a positive band near 217 nm.23 Figure 3D shows the CD spectra of

390

protamine and protamine/PAA-b-PNIPAAm nanogels at different temperatures. A

391

random coil-like conformation is characterized by a negative peak at 198 nm and a

392

positive band at 217 nm in the CD spectrum of only protamine molecules.24 The

393

conformation was found to change from a random coil to α-helix after the formation

polyelectrolyte

complex,

we

measured

the

CD

spectra

of

nanocomplex formed at different temperatures.

16


Page 17 of 37

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394

of protamine/PAA-b-PNIPAAm nanocomplex. α-helix signal (a negative band at 208

395

nm and a positive band at 220 nm) was observed in the CD spectra for the complex

396

nanogel formed at 37 °C (Figure 3D). However, when decreasing the temperature

397

from 37 °C to 25 °C and 4 °C, the negative peak at 208 nm was blue-shifted to 206

398

nm and 205 nm, and a positive peak appeared at around 217 nm and 215 nm,

399

revealing that the conformation of protamine changed due to the disassembly of the

400

nanogels.

401 (A)

402

(B)

(C)

(D)

403 404

Figure 3. (A) DSC curves of PAA, PNIPAAm, and PAA-b-PNIPAAm copolymer, (B)

405

fluorescence quenching of fluorescein amine-labeled PAA-b-PNIPAAm after addition

406

of protamine; the insert shows the fluorescence remaining and quenching ratios

407

calculated from the intensity of the fluorescein amine-labeled PAA-b-PNIPAAm and

408

the fluorescent nanogels self-assembled at protamine-to-PAA-b-PNIPAAm weight

409

ratio

of

2:9,

4:9

and

6:9,

(C)

fluorescence

recovery

17


of

fluorescent


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 37

410

protamine/PAA-b-PNIPAAm nanogels at different temperatures; the insert shows the

411

fluorescence remaing and quenching ratios calculated from the intensity of the

412

fluorescein amine-labeled PAA-b-PNIPAAm and the fluorescent nanogels at 37 °C,

413

25 °C and 4 °C, (D) CD spectra of protamine and the protamine/PAA-b-PNIPAAm

414

nanogels at different temperatures

415

Phase

Transition

Behavior.

Decrease

in

optical

transmittance

of

416

PAA-b-PNIPAAm solution caused by thermal and pH-induced aggregation was

417

investigated to observe its phase transition behavior. The temperature responsive

418

properties of PAA-b-PNIPAAm copolymer are easily affected by changes in the

419

solution pH value because the acid groups in the PAA block could undergo ionization

420

when the external pH is raised above its pKa (=4.75). The LCST of the

421

PAA-b-PNIPAAm copolymer solution was determined by optical transmittance in

422

various buffer solutions of pH 5.0, 6.0 and 7.4 (Figure 4A) and compared to the

423

protamine/PAA-b-PNIPAAm nanogels at the same pH values (Figure 4B).

424

PAA-b-PNIPAAm copolymer did not display LCST behavior in phosphate buffers at

425

pH 6.0 and 7.4 up to 60 °C. This is because the PAA block was highly ionized in

426

these buffers, leading to a great increase in electrostatic repulsion and hydrophilic

427

property for the copolymer. Yet, by decreasing the pH value to 5.0, close to the pKa

428

of PAA block, it was expected that the phase transition would become apparent and

429

the copolymer would self-assemble into nanogels, because the ionization extent of the

430

PAA block would be much lower. Indeed, as shown in Figure 4A, a rapid decline in

431

the optical transmittance upon heating was observed, and the LCST determined from

432

the differential peak temperature was 32.4 °C (Figure 4A). Interestingly, phase

433

transition could be observed even in the buffers of pH 6.0 and 7.4 when

434

PAA-b-PNIPAAm and protamine assembled together to form complex nanogels. The

435

LCST values of the protamine/PAA-b-PNIPAAm nanogels in pH 6.0 and 7.4 buffers 18


Page 19 of 37

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436

were 22.8 and 25.4 °C, respectively (Figure 4B), which were lower than the LCST of

437

PNIPAAm. The protamine was bound with the ionized PAA-b-PNIPAAm in pH 6.0

438

and 7.4 buffers via the electrostatic interactions formed between the oppositely

439

charged protamine and PAA-b-PNIPAAm. Consequently, the phase transition

440

appeared in the transmittance-temperature curve with a lower LCST due to the

441

increased hydrophobicity and thermal-induced aggregation of PAA-b-PNIPAAm in

442

the complex nanogels (Figure 4B). In pH 5.0 buffer, the formation of

443


444

PAA-b-PNIPAAm

445

transformation of PNIPAAm segments. Therefore, the decline curves are not as sharp

446

as that of PAA-b-PNIPAAm copolymer (Figure 4A). It has to point out that after the

447

rapid decline of transmittance due to the aggregation, there was a slight increase in the

448

transmittance with temperature in the pH 5 solution. It is suspected that, in the pH 5.0

449

buffer, the PAA-b-PNIPAAm aggregates fixed by the protamine/PAA-b-PNIPAAm

450

complex layer that was formed on the outer layer of the nanogels was not as stable as

451

those in the pH 6.0 and 7.4 buffers at 37 °C, which is advantageous for pH-triggered

452

drug release in endosomes or lysosomes.

along

complex with

the

reduced decrease

the of

chain

mobility

of

hydrophilic-to-hydrophobic

453

We next examined the pH- and temperature-dependent size changes of

454

protamine/PAA-b-PNIPAAm nanogels at various buffer solutions by dynamic light

455

scattering (DLS). As shown in Figure 4C, the hydrodynamic diameter of the nanogel

456

suspension would change accordingly with different temperature and pH values. At

457

37 °C; the PAA-b-PNIPAAm nanogel was very stable at pH 5.0 but was easily

458

dissolved at pH 6.0 and 7.4 as indicated by its much smaller particle sizes (less than 2

459

nm) (Figure 4C-a). On the other hand, the protamine/PAA-b-PNIPAAm complex

460

nanogel shows a completely different pH-responsive property compared with its

461

PAA-b-PNIPAAm counterpart. First, the protamine/PAA-b-PNIPAAm nanogels had 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

462

larger particle size than the PAA-b-PNIPAAm alone at pH 5.0 and 37 °C. Moreover,

463

the protamine/PAA-b-PNIPAAm nanogels were very stable even at pH 6.0 and 7.4

464

(Figure 4C-b), in contrast to the rapid dissolution of PAA-b-PNIPAAm nanogels. As

465

explained previously, the hydrophilic portion of ionized PAA block can bind with

466

protamine to form electrostatic complex layers, and the PNIPAAm block can form

467

self-aggregated domain caused by raising the temperature above the LCST. In

468

addition, increasing the pH value resulted in a slight decrease in particle size of

469

protamine/PAA-b-PNIPAAm nanogels as shown in Figure 4C-b. This is because of

470

the higher ionization extents of the PAA block at higher pH values (such as pH 7.4),

471

leading to the stronger electrostatic interactions and thus the smaller particle sizes.

472

The protamine/PAA-b-PNIPAAm nanogels showed a pH-responsive size-tunable

473

property at 25 °C (Figure 4C-c) that were similar to those at 37 °C (Figure 4C-b). It

474

was worth noting that the zeta potential changed from -2.7 mV to 11.5 mV while the

475

average size change from 153.5 nm to 244.9 nm for pH decline 7.4 to 5.0 at 37 °C,

476

indicating that the nanogels may have pH-responsive and charge-conversion

477

properties (Figure 4D). These properties can be attributed to the conformational

478

change of protamine in the nanogels at different pH values, allowing the shielding and

479

stretching out the arginine-rich domain.17 As the temperature was decreased to 4 °C,

480

the nanogels completely dissolved in all pH buffer solutions (pH 5.0, 6.0 and 7.4),

481

thus the size distributions were not measurable (Figure 4C-d). Almost no signal

482

corresponding to the size distribution of the nanogels was detectable at this low

483

temperature. The hydrophilic effect was greatly increased on decreasing the

484

temperature to 4 °C, thus weakening the inter/intramolecular hydrogen bonding and

485

causing a disintegration in the aggregates of the PNIPAAm segments. The

486

protamine/PAA-b-PNIPAAm nanogels were disassembled and then were completely

487

dissolved in the medium at 4 °C regardless of pH values. Although the 20


Page 20 of 37

Page 21 of 37

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488

PAA-b-PNIPAAm was ionized at pH 7.4 to a large extent, the electrostatic force

489

between the PAA-b-PNIPAAm and protamine at such a low temperature (4 °C) did

490

not provide the nanogels with sufficient binding strength for self-aggregation,

491

consequently leading to the complete dissolution of the protamine/PAA-b-PNIPAAm

492

nanogels. Because a low temperature treatment at 4 °C caused a more pronounced

493

temperature-responsive behavior compared to the treatments at 25 °C, we selected 4

494

°C for the cold treatment in the following studies. (A)

(B)

495 (C) (a)

(b)

(d)

(c)

496

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 37

(D)

497 498

Figure 4. Optical transmittance of PAA-b-PNIPAAm (A) and protamine/

499

PAA-b-PNIPAAm nanogels (B) at various buffer solutions of pH 5.0, 6.0 and 7.4, (C)

500

hydrodynamic diameters of PAA-b-PNIPAAm and protamine/PAA-b-PNIPAAm

501

nanogels at different temperatures (4 °C, 25 °C and 37 °C) and various buffer

502

solutions of pH 5.0, 6.0 and 7.4, (D) pH-dependent zeta potential change at 37 °C

503

Thermo- and pH-Responsive Drug Release. The doxorubicin encapsulation

504

efficiency of PAA-b-PNIPAAm and protamine/PAA-b-PNIPAAm nanogels were

505

42.4% and 54.9%, respectively. Figure 5A shows the pH-responsive drug release

506

behaviors

507

protamine/PAA-b-PNIPAAm nanogels. The nanogels prepared by self-aggregation of

508

PAA-b-PNIPAAm alone rapidly released doxorubicin in PBS (pH 7.4), revealing that

509

the nanogels were not able to prevent a quick release of the drug from the vesicles

510

under normal physiological conditions (normal tissues) or in blood circulation system.

511

By contrast, protamine/PAA-b-PNIPAAm nanogels demonstrated a distinct

512

pH-responsive property for controlled doxorubicin release. To simulate the conditions

513

of nanoparticles in blood circulation (pH 7.4) and subsequently transported to late

of

doxorubicin

from

the

PAA-b-PNIPAAm

22


and

Page 23 of 37

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514

endosomes and lysosomes of cancer cells (pH 5.0) at different temperatures,

515

doxorubicin release was examined by changing the pH of the release medium. While

516

keeping the temperature at 37 °C, doxorubicin release from the nanogels was faster at

517

pH 5.0 (80% after 24 h of release), compared to the slower drug release at pH 7.4

518

(31.1%). As previously studied in Figure 4B, the nanogels has high stability at pH 7.4

519

and the release of doxorubicin was slow and consecutive, but the nanogels were

520

disassembled in acidic environment because the PAA blocks were at a lower level of

521

ionization, thus doxorubicin release from the nanogels was faster at pH 5.0 due to the

522

collapse of the nanogels and the weakened electrostatic forces between the positively

523

charged doxorubicin and protamine, with the negatively charged PAA-b-PNIPAAm.

524

To simulate the conditions of the low temperature treatment (cryotherapy),

525

doxorubicin release was examined by changing the temperature of the release medium

526

(Figure 5B). It was worth noting that a burst release of doxorubicin from the

527

protamine/PAA-b-PNIPAAm nanogels could be found when a low-temperature (cold

528

shock) treatment was performed. The cloudy nanogel suspension became clear when

529

the temperature drop to a low level at 4 °C. The accumulative release reached more

530

than 92.5% of the loaded doxorubicin from the nanogels placed in the medium with a

531

pH changing from 7.4 to 5.0, followed by a 15 min of cold shock treatment. On the

532

other hand, the nanogels released a smaller amount of doxorubicin during the same

533

time period if the low-temperature treatment was not performed. The total amount of

534

doxorubicin released from the nanogels was determined to be 57.6% upon changing

535

the pH from 7.4 to 5.0 w/o cold shock treatment, yet was still higher than the

536

doxorubicin release from the nanogels w/o changing the pH and temperature (31.2%

537

at pH 7.4, 37 °C). The PAA-b-PNIPAAm demonstrated greater hydrophilic properties

538

at

539

protamine/PAA-b-PNIPAAm nanogels were rapidly disassembled and became

4

°C

when

compared

to

that

at

37°C,

23


consequently,

the


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 37

540

completely dissolved at 4 °C. The results suggest that the nanogels are indeed

541

pH-responsive and thermo-responsive at the same time. At pH 5.0 or at temperatures

542

below 4 °C, the nanogels were in a disassembled conformation, which obviously

543

accelerated doxorubicin release from the nanogels. These dual-responsive nanogels

544

thus will be able to deliver doxorubicin to cancer cells, in which the acidic

545

endosome/lysosome environment can trigger the drug release. Furthermore, this

546

nanogel is applicable to temperature-controlled drug release which can be associated

547

with cryotherapy.

548

Trypsin has also been confirmed to be expressed in various adenocarcinoma

549

tissues where pH levels are in the range of 6.5 to 6.8, which is able to digest

550

protamine via hydrolysis of peptides on the C-terminal side of arginine residues.25,26

551

Figure

552

protamine/PAA-b-PNIPAAm nanogels. Doxorubicin release in the medium w/ trypsin

553

was faster than that w/o trypsin at pH 6.5. Without digestion by trypsin, only 46.9%

554

doxorubicin was released within 24 h, however, the doxorubicin release percentage

555

considerably increased due to enzymatic digestion of protamine, and more than 88.6%

556

of DOX was released in the presence of trypsin. The enzyme-triggered drug release

557

could be inhibited by trypsin inhibitor (58.9% of DOX release), implying that the

558

trypsin-involved drug release could be due to enzymatic digestion-induced

559

disassembly of the nanogels. The transmittance of the protamine/PAA-b-PNIPAAm

560

nanogels with (w/) and without (w/o) trypsin was monitored to investigate the

561

enzyme-triggered disassembly of the protamine/PAA-b-PNIPAAm nanogels (Figure

562

5D). The nanogels have good stability in PBS w/o digestion by trypsin, showing no

563

appreciable change in size distribution; on the contrary, the optical ＋ rapidly

564

increases under tryptic digestion, suggesting that the disassembly of nanogels was

565

caused by enzymatic digestion.

5C

shows

enzyme-responsive

release

of

24


doxorubicin

from

the

Page 25 of 37

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(B)

(A)

566 (C)

(D)

567 568

Figure 5. (A) pH- (pH 5.0, 6.0 and 7.4) and (B) thermo-responsive (changed from 37

569

°C to 4°C) drug release behavior of doxorubicin from the PAA-b-PNIPAAm and

570


571

protamine/PAA-b-PNIPAAm nanogels in the medium w/ and w/o trypsin (0.2 mg/mL)

572

at pH 6.5, (D) optical transmittance of the protamine/ PAA-b-PNIPAAm nanogels w/

573

and w/o trypsin (0.2 mg/mL) at pH 6.5.

nanogels,

(C)

Doxorubicin

release

from

574

Intracellular Drug Delivery. Microenvironment of solid tumors is known to be

575

weakly acidic. Under hypoxic culture condition, the pH can even decrease to 6.1.27

576

Yang reported the charge conversion of photo- and pH-responsive polypeptides for

577

enhanced and targeted cancer therapy. The polypeptides were less positively charged

578

(1.2 mV) at physiological condition (pH 7.4) and became more positively charged

579

(5.5 mV) at the pH near tumor microenvironments (pH 6.0).28 As shown in Figure 4D,

580

the zeta potential was converted from -2.7 mV to 3.1 mV when pH changed from 7.4

581

to 6.5, revealing that the protamine/PAA-b-PNIPAAm nanogels became cationic 25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

acidic

conditions.

To

further

examine

intracellular

Page 26 of 37

582

under

distribution

of

583

doxorubicin-loaded nanogels, PAA-b-PNIPAAm was labeled with fluorescein amine

584

and then was used to prepare fluorescent protamine/PAA-b-PNIPAAm nanogels.

585

Figure 6A shows the cellular uptake and intracellular distribution of the fluorescein

586

amine-labeled and doxorubicin-loaded nanogels in MCF-7 breast cancer cells. Free

587

DOX penetrate rapidly into nuclei and the nuclei exhibited red fluorescence. The cells

588

incubated with protamine/PAA-b-PNIPAAm nanogels at pH 6.5 for 2 h showed a

589

green fluorescence in cytoplasm with their nuclei stained with doxorubicin (red

590

fluorescence) (Figure 6A), revealing that the nanogels were efficiently internalized by

591

MCF-7 cells and doxorubicin were delivered into nuclei. The effective uptake of the

592

nanogels by the cancer cells is possibly due to the reason that the nanogels are

593

positively charged at pH 6.5, which may improve endocytic uptakes of the nanogels

594

through binding to the negatively charged plasma membrane.

595

However, fluorescence of free DOX wasn’t observed in the multidrug resistant

596

MCF-7/ADR cells, indicating rapid DOX efflux from the drug-resistant cancer cells.

597

The cancer cells treated with DOX-loaded protamine/PAA-b-PNIPAAm nanogels

598

demonstrated weaker intracellular fluorescence intensity of DOX (red fluorescence)

599

because self-quenching of DOX fluorescence in the well-organized nanoparticles

600

(Figure 6B). Next, MCF-7 and MCF-7/ADR cells were treated by a cold shock at 4

601

°C. MCF-7 cells subjected to a cold-shock treatment demonstrated a similar

602

intracellular fluorescence intensity compared to the cells incubated at a normal

603

condition (Figure 6A). In contrast, MCF-7/ADR cells treated with DOX-loaded

604

protamine/PAA-b-PNIPAAm nanogels demonstrated much stronger intracellular

605

DOX fluorescence intensity than their free DOX-treated counterparts (Figure 6B).

606

The relative DOX fluorescence intensity in the nanogels-treated MCF-7/ADR cells

607

was shown to be much stronger than their counterparts treated with free DOX, 26


Page 27 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


608

revealing that the nanogels can overcome the cellular barrier and doxorubicin can

609

enter into the cancer cells by the nanogel delivery system (Figure 6C). These results

610

revealed that the nanogels may be internalized by the cells and then releasing

611

doxorubicin into cytosol. The nanogels tend to escape from endosome for releasing

612

doxorubicin in cytoplasm, causing a stronger red fluorescence in the cells under cold

613

shock treatment. (A)

614 615 (B)

616

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(C)

617 618

Figure 6. Cellular uptake and intracellular distribution of the fluorescein

619

amine-labeled and DOX-loaded protamine/PAA-b-PNIPAAm nanogels in MCF-7 (A)

620

and MCF-7/ADR (B) breast cancer cells, (C) the relative DOX fluorescence intensity

621

in MCF-7/ADR breast cancer cells treated with free DOX and DOX-loaded

622

protamine/PAA-b-PNIPAAm nanogels w/ and w/o cold treatment at 4 °C for 15

623

minutes. Each data is represented as mean ± SD (n = 3); * indicates p < 0.01.

624

Cytotoxicity. Figure S1 shows that PAA-b-PNIPAAm significantly reduces the

625

cytotoxicity of protamine. The empty nanogels didn’t cause noticeable cellular death

626

in MCF-7 cells while free doxorubicin and the doxorubicin-loaded nanogels caused

627

remarkable cytotoxicity towards the cancer cells. As shown in Figure 7A, free DOX

628

exhibited greater cytotoxicity in MCF-7 cells than DOX-loaded nanogels (5.1% vs.

629

24.3% cell viability and 39.5% vs. 65.8% cell viability for the cells treated with 10.0

630

µg/mL and 1.0 µg/mL equivalent free RB and RB-loaded nanogels) (p < 0.01), which

631

is consistent with the higher cellular uptake of free DOX (Figure 6A). Free DOX

632

rapidly diffused into nuclei and effectively inhibited the replication of DNA. However,

633

doxorubicin was gradually released from the protamine/PAA-b-PNIPAAm nanogels

634

that were taken up by MCF-7 cells via endocytosis, causing a lower cytotoxicity in the 28


Page 28 of 37

Page 29 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


635

cancer cells than free DOX. Because PAA-b-PNIPAAm nanogels readily dissolved in

636

culture medium (pH near 7.4) before cellular uptake and then released DOX quickly

637

(Figure 5A), its cytotoxicity is similar to that of free DOX.

638

In contrast, the DOX-loaded protamine/PAA-b-PNIPAAm nanogels demonstrated

639

significant inhibitory effect on MCF-7/ADR cells compared to its free doxorubicin

640

counterpart (Figure 7B). At the same doxorubicin equivalent, the cytotoxicity of

641

doxorubicin was improved by loading it in the complex nanogels (92.1% vs. 82.8%

642

cell viability for the cells treated with 10.0 µg/mL equivalent free RB and RB-loaded

643

nanogels) (p < 0.1). Doxorubicin is an anthracycline that poorly accumulates in

644

MCF-7/ADR cells because of the over-expression of multidrug efflux pumps in the

645

doxorubicin-resistant cells. Protamine is arginine-rich peptide, which can enhance the

646

cellular uptake and further endosomal escaping of nucleic acid. Doxorubicin-loaded

647

protamine/PAA-b-PNIPAAm nanogels exhibited superior accumulation of the drug in

648

MCF-7/ADR cells compared to free doxorubicin (Figure 6B), consequently may

649

enhance the cytotoxicity against the cancer cells.

650

Cell viability of MCF-7 and MCF-7/ADR cells treated with DOX-loaded

651

protamine/PAA-b-PNIPAAm nanogels followed by treatment of the cells at different

652

temperatures (4-37 °C) for 15 minutes are shown in Figure 7C. The chemotherapy

653

using DOX-loaded nanogels associated with a cold shock treatment (4 °C) induced a

654

higher cytotoxicity in MCF-7/ADR cells compared with isothermal (37 °C) treated

655

groups (63.4% vs. 82.3% and 81.8% cell viability for the cells treated with

656

DOX-loaded nanogels at 4 °C vs. at 25 °C and 37 °C) (p < 0.01). The higher

657

cytotoxicity

658

protamine/PAA-b-PNIPAAm nanogels rapidly disintegrate at 4 °C after endocytosis,

659

facilitating a rapid release of a large amount of anticancer drugs.29 The excess

660

doxorubicin released in cytoplasm that was not readily effluxed from the

in

the

cells

under

a

cold

shock

29


treatment

is

because


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

661

drug-resistant cancer cells caused more notable cellular death.

662

We further examine photocytotoxicity of rose bengal (RB)-loaded nanogels

663

against MCF-7/ADR cells when it was combined with cold shock treatment. To

664

evaluate whether the RB-loaded nanogels can affect the proliferation of MCF-7/ADR

665

cells upon green light excitation, the cancer cells internalized with the empty and

666

RB-loaded nanogels were photo-irradiated using green LED light (550 nm),

667

respectively (Figure 7D). The MCF-7/ADR cells internalized with free RB or empty

668

nanogels did not show noticeable cellular death upon photo irradiation while the

669

RB-loaded nanogels caused obvious phototoxicity towards the cancer cells. (A)

(B)

670 671 (C)

(D)

672 673

Figure 7. (A) Dose-dependent inhibition of cell proliferation in MCF-7 (A) and

674

MCF-7/ADR (B) breast cancer cells by DOX-loaded protamine/PAA-b-PNIPAAm

675

nanogels (w/o cold shock treatment), (C) inhibition of cell proliferation in

676

MCF-7/ADR breast cancer cells by DOX-loaded protamine/PAA-b-PNIPAAm 30


Page 30 of 37

Page 31 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


677

nanogels (10.0 µg/mL DOX equivalent; w/ cold shock treatment at different

678

temperatures), (D) the photodynamic treatments of MCF-7/ADR cells with rose

679

bengal (RB)-loaded protamine/PAA-b-PNIPAAm nanogels (8.0 µg/mL RB

680

equivalent; w/ cold shock treatment at 4 °C) under irradiated with 550 nm using a

681

high-power green light-emitting diode (LED) array. The data are shown as mean ± SD

682

(n = 5); * indicates P < 0.05 and ** indicates P < 0.01.

683

The RB encapsulation efficiency of protamine/PAA-b-PNIPAAm nanogels was

684

52.3%. Free RB induced lower cytotoxicity in MCF-7/ADR cells upon photo

685

irradiation as compared with the RB-loaded nanogels. Under exposure to green LED

686

light, the phototoxicity of RB was improved by loading it into the nanogels (82.3% vs.

687

39.3% cell viability for photodynamic treatment with 8.0 µg/mL equivalent free RB

688

and RB-loaded nanogels) (p < 0.01) (Figure 7D). Rose bengal is a photosensitizer that

689

poorly accumulates in cancer cells because it is difficult to penetrate through cell

690

membranes. Furthermore, RB can be effluxed from MCF-7/ADR cells, resulting in a

691

reduced photocytotoxicity. Rose bengal loaded in protamine/PAA-b-PNIPAAm

692

nanogels exhibited superior accumulation in MCF-7/ADR cells compared to its free

693

form, consequently the cytotoxicity against the cancer cells was enhanced upon photo

694

irradiation. However, without photo irradiation, free RB and the RB-loaded nanogels

695

exhibited negligible cytotoxicity in MCF-7/ADR cells, indicating that, without the

696

photodynamic treatment, the empty and rose bengal-loaded nanogels were safe and

697

did not show phototoxicity.

698

■ CONCLUSION

699

The triple-stimuli-responsive nanogels were successfully prepared via electrostatic

700

interactions

701


702

PAA-b-PNIPAAm aggregates remarkably increased the stability of the nanogels that

between

PAA-b-PNIPAAm complex

and

layer

protamine. on

the

31


Formation

thermally

of

induced


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

703

were well dispersed in PBS. The nanogels were disassembled and then released

704

doxorubicin quickly by decreasing temperature, reducing pH, and treating with

705

enzyme. After loading doxorubicin or rose bengal in the nanogels, an improvement in

706

inhibition efficiencies, caused by chemical therapeutic or photodynamic effects, was

707

observed in doxorubicin-resistant MCF-7/ADR cancer cells.

708

■ ACKNOWLEDGMENTS

709

The authors gratefully acknowledge the financial support provided by the Ministry of

710

Science and Technology, Taiwan, ROC (MOST 101-2221-E-038-016-MY3).

711

SUPPORTING INFORMATION

712

Cell viability: protamine and empty nanogels

713 714

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36


Page 36 of 37

Page 37 of 37

Table of Contents (TOC)

+

H 2N

C-terminal domain (arginine-rich)

NH

N H

m

O

O

O

2

11 O

PAA-b-PNIPAAm copolymer PAA-b-PNIPAAm nanogel

PAA block

N H

Systemic circulation (pH 7.4)

OH

polyelectrolyte complex (PEC)

37°C

-

O

cationic protein (protamine)

thermo-induced aggregation

HN

O H N N H

2

2 O

NH2

NH

O

H N

n

NH2

NH

O

+

H 2N

+

H 2N

NH2

+

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


doxorubicin

Protamine/PAA-b-PNIPAAm nanogel

PNIPAAm block conformational change

Tumor environment (pH 6.3)

Cold shock (4°C)

endocytosis

photodynamic therapy I O

chemotherapy Burst intracellular release

I O

O O

I

O

OH

OH

I Cl

COO

Cl

Cl

charge conversion

OH

ROS and 1O2

O

O

OH

doxorubicin

H O

O

Cl

rose bengal

+

N H3

Overcome drug resistance ACS Paragon Plus Environment

OH

PNIPAAm Nanogels for Controlled Anti - ACS Publications - American

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