Tumor Microenvironmental pH and Enzyme Dual Responsive Polymer

Subscriber access provided by UNIV OF BARCELONA

Article

Tumor Microenvironmental pH and Enzyme Dual Responsive PolymerLiposomes for Synergistic Treatment of Cancer Immuno-Chemotherapy Ya Liu, Zeng-Ying Qiao, Pei-Pei Yang, Hao Wang, and Xiguang Chen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01510 • Publication Date (Web): 08 Jan 2019 Downloaded from http://pubs.acs.org on January 9, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39 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

Biomacromolecules

1

Tumor Microenvironmental pH and Enzyme Dual Responsive

2

Polymer-Liposomes for Synergistic Treatment of Cancer

3

Immuno-Chemotherapy

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Ya Liu*, Zeng-Ying Qiao, Pei-Pei Yang, Hao Wang and Xi-Guang Chen* Ya Liu and Prof. Xi-Guang Chen College of Marine Life Science Ocean University of China No. 5 Yushan Road, Qingdao (China) Tel/Fax.: 86-0532-82032586; E-mail: [email protected] (Y. Liu); [email protected] (X. G. Chen) Pei-Pei Yang, Zeng-Ying Qiao and Prof. Hao Wang CAS Center for Excellence in Nanoscience CAS Laboratory for Biomedical Effects of Nanomaterials and Nanosafety National Center for Nanoscience and Technology (NCNST) No. 11 Beiyitiao, Zhongguancun, Beijing (China)

1

ACS Paragon Plus Environment

Biomacromolecules 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

20

Abstract

21

Despite recent advances in tumor treatment through cancer immunotherapy, the

22

efficacy of this approach remains to be improved. Looking forward to high rates of

23

objective clinical response, cancer immunotherapy combined with chemotherapy has

24

gained increasing attention recently. Here, we constructed liposomes with matrix

25

metalloproteinases (MMPs) responsive moiety and PD-L1 inhibitor conjugate

26

combine with low dose chemotherapy to achieve enhanced anti-tumor efficacy. Upon

27

introduction of the pH-responsive polymer to LPDp, the co-assembly could be almost

28

stable in physiological condition and tumor microenvironment and release the loaded

29

cargos at the lysosome. MMP-2 enzyme extracellularly secreted by the B16F10 cells

30

could cleave the crosslinker and liberate the PD-L1 inhibitor effectively disrupting the

31

PD-1/PD-L1interaction in vitro. Low dose DOX encapsulated in the LPDp was

32

capable of sensitizing B16F10 cells to CTLs by inducing overexpression of M6PR on

33

tumor cell membranes. In comparison with free PD-L1 inhibitor, LPDp improved the

34

biodistribution and on-demand release of the peptide inhibitor in tumor regions

35

following administration. LPDp achieved the optimal tumor suppression efficiency (～

36

78.7%), which demonstrated the significantly enhanced antitumor effect (P < 0.01)

37

than that of LPp (～57.5%) as well as that of LD (< 40%), attributing to synergistic

38

contribution from the substantial increase in M6PR expression on tumor cells and the

39

blockade of immune checkpoints. This strategy provides a strong rationale for

40

combining standard-of-care chemotherapy with relative non-toxic and high specific

41

immunotherapy.

42

2


Page 2 of 39

Page 3 of 39 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

Biomacromolecules

43

1. Introduction

44

Cancer immunotherapy by training or stimulating the inherent immunological

45

systems of the body to attack tumor cells, has been progressing rapidly and shown

46

tremendous promises as a powerful cancer treatment strategy1-3. Several

47

well-established cancer immunotherapeutic approaches such as interleukin-2, immune

48

checkpoint inhibitors (CTLA-4 and PD-1 receptors), chimeric antigen receptor T-cell

49

immunotherapy (CAR-T) have improved the survival in cancer patients

50

the exciting clinical results of immunotherapy for the treatment of cancer, the efficacy

51

of the approach remained to be improved

52

from immunotherapy with most patients being insensitive with no or low responses 8, 9.

53

Challenges of cancer immunotherapy included the ability of vaccines to generate

54

effective immune responses with the presence of numerous immunosuppressive

55

factors10,

56

parenchyma

57

immunization correctly

58

especially combination therapy, call for urgent in-depth investigations.

59

11,

6, 7.

4, 5.

Despite

Only a subset of patients can benefit

the ability of cytotoxic T lymphocytes (CTLs) to infiltrate tumor 12,

recognize tumor-associated antigen, and choose antigen for 13, 14.

Strategies to improving the immune response rate,

Chemotherapy was usually associated with immunosuppressive effects and not 14-16.

60

previously considered as an attractive adjunct to cancer immunotherapy

Recent

61

data suggested that prior chemotherapy enhanced the therapeutic outcome of

62

immunotherapy and further reversed chemoresistance after prolonged chemotherapy

63

17-19.

64

anticancer effects and promote an immunogenic tumor phenotype. Several reports

Chemotherapeutic drugs combined with immunotherapy may result in enhanced

3



Page 4 of 39

65

indicated low dose chemotherapeutic agents were capable of improving the

66

permeability of tumor cell membranes to granzyme B (GranB) via upregulation of

67

mannose–6–phosphate receptor (M6PR) on tumor cells20,

68

mediator of cell apoptosis by CTLs in cell-mediated immune response22, 23. Hence,

69

noncytotoxic concentrations of antitumor agents were able to increase the

70

susceptibility of tumor cells to CTLs24-26.

21.

GranB was a critical

71

We rationalized that combination of an immunotherapy agent with a

72

chemotherapeutics agent can be enabled using nanoscale vehicles for a maximal

73

antitumor outcome27,

74

kinase-inhibiting supramolecular therapeutics to inhibit tumor growth effectively and

75

prolong survival in tumor-bearing mice in various tumor model systems29. Current

76

cancer immunotherapies employing nanoparticles have paid close attention to the

77

immune checkpoint pathways for the activation of tumor-targeting CTLs

78

al. have combined checkpoint inhibitor aPD1 and 1-MT with nanoplatform and have

79

achieved great success in tumor immunotherapy32. While nanoparticles have recently

80

been used as vaccines to induce an enhanced immune reaction against the cancer

81

cells17, 33, combination regimens of immunotherapy with nanoparticles encapsulated

82

low dose chemotherapeutic agents in cancer are yet to be studied.

28.

Sengupta et al. have combined checkpoint inhibitors with

30, 31.

Gu et

83

Herein, we attempted to build up polymer-liposomes (LPDp) grafted with

84

anti-PD-L1 peptide34 and encapsulated low dose doxorubicin (DOX) for combination

85

of cancer immunotherapy and chemotherapy (Scheme 1). The pH-responsive polymer

86

chains contained anchoring moiety for tight attachment onto the liposome membrane 4


Page 5 of 39 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

Biomacromolecules

87

surface (LP) and pH-sensitive moiety with capability of converting to protonated state

88

at acidic pH, accompanying the dynamic disordering of membrane. The mixing ratios

89

of lipid compositions with pH responders could be fine-tuned to optimize the

90

responsive sensitivity and specificity of LP. Low dose DOX was encapsulated in the

91

LPD with expect of sensitizing tumor cells to CTLs with negligible systemic side

92

effects. Hydrolysis resistant D-peptide (NYSKPTDRQYHF) as an antagonist to target

93

the PD-1/PD-L1 pathway was introduced into LPp via matrix metalloproteinases

94

(MMP) degradable cross-linkers (GPLGVRG)35, 36 to disrupt the immune-suppressing

95

pathway (Scheme 2). The bio-distribution of polymer-liposomes system in vivo was

96

evaluated via near-infrared imaging. Subsequently, LPDp was used as tumor

97

treatment in B16F10 mouse melanoma model and induced robust immune responses

98

with satisfactory therapeutic effects. We expect the MMPs/pH dual responsive

99

polymer-liposomes to provide an innovative strategy for combination of

100

immunotherapy and chemotherapy and facilitate the clinical treatment of melanoma.

101

2. Materials and methods

102

2.1.Materials

103

L-α-Phosphatidylcholine(PC),1,2-dioleoyl-3-trimethylammonium-propane(DOTAP

104

),cholesterol(CH),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyet

105

hyleneglycol)-2000](DSPE-PEG),and1,2-distearoyl-sn-glycero-3-phosphoethanolami

106

ne-N-[maleimide(polyethyleneglycol)-2000](DSPE-PEG-mal) were purchased from

107

Avanti Polar Lipids. Dulbecco’s Modified Eagle Medium (DMEM), penicillin,

108

streptomycin, phosphate-buffered saline (PBS), fetal bovine serum (FBS) and trypsin 5



Page 6 of 39

109

were obtained from HyClone/Thermo fisher (Beijing, China). B16F10 cell lines were

110

purchased from Cell Culture Center of Institute of Basic Medical Sciences, Chinese

111

Academy

112

N-hydroxysuccinimide (NHS), Hochest 33342 and Cy5 were purchased from

113

Sigma-Aldrich

114

3-(dibutylamino)-1-propylamine

115

glycol)diacrylate (average Mn 2000, PEGDA2000) were purchased from Aldrich

116

Chemical Corporation. Peptides (>90%) were prepared using standard Fmoc

117

solid-phase peptide synthesis methods and were purified by reverse-phase

118

high-performance liquid chromatography. Cell counting kit assay (CCK-8) was

119

obtained from Beyotime institute of biotechnology (Shanghai, China). 1,

120

6-Hexanediol diacrylate (HDDA), 3-(Dibutylamino)-1-propylamine (DBPA), and

121

dodecylamine were purchased from Aldrich Chemical Corporation. Methyl PEG-NH2

122

2000 was purchased from Seebio Biotech, Inc. C57BL/6 mice (B6, H-2b) (Female, 16

123

± 2 g) were purchased from Department of Laboratory Animal Science, Peking

124

University Health Science Center, and all animal experiments were conducted by its

125

center. All the other solvents used in this research were all of analytic grade and were

126

used without further purification.

127

2.2. Synthesis and characterization of polymer-liposomes

128

2.2.1. Synthesis of functional peptide

of

Medical

(Shang,

Sciences

China).

(Beijing,

1,

China). Irinotecan

6-Hexanediol

(DBPA),

dodecylamine

Hydrochloride,

diacrylate and

(HDDA),

poly(ethylene

129

Peptide (CPLGVRGK-GGG-NYSKPTDRQYHF) was obtained using standard

130

Fmoc solid-phase peptide synthesis methods. Cys-Pro-Leu-Gly-Val-Arg-Gly-Lys 6


Page 7 of 39 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

Biomacromolecules

131

(SH-CPLGVRGK) was metalloproteinase-2 (MMP-2) substrate sequence and the

132

D-peptide Asn-Tyr-Ser-Lys-Pro-Thr-Asp-Arg-Gln-Tyr-His-Phe (NYSKPTDRQYHF)

133

performed an inhibitor targeting programmed death ligand 1 (PD-L1).

134

2.2.2. Responsive cleavage of functional peptide by MMP-2

135

The enzyme mixture was prepared: (2 mg mL-1) peptide, (15 ng mL-1) MMP-2 in

136

buffer (20 mM Tris∙HCl/50 mM NaCl/100 mM CaCl2/0.05% Brij 35, pH 7.4) with a

137

total volume of 0.5 ml. The reaction was followed at room temperature for 1 h. The

138

original peptide and after exposition for 1 h to gelatinase were analysed by HPLC

139

(acetonitrile (0.1% TFA) /water (0.1% TFA) linear gradient from 5%/95% to

140

60%/40% in 30 min flux 1 mL/min, 220 nm detection).

141

2.2.3. Preparation of pH-responsive polymers

142

The pH-responsive polymers were prepared by Michael addition. HDDA, DBPA

143

and mPEG-NH2 2000 were dissolved into DMSO and then bubbled with N2 for 15

144

min under stirring, then heated at 50 °C for 7 days in dark to obtain P1. Then

145

dodecylamine and P1 were added into DMSO and bubbled with N2 for 15 min under

146

stirring. The mixture was allowed to react for 5 days at 50 °C and dialyzed against

147

deionized water (MWCO: 3,500 Da). 1H NMR spectra (400 MHz) of the copolymers

148

were recorded on a Bruker ARX 400 MHz spectrometer.

149

2.2.4. pH titration of copolymers

150

The graft copolymers (P1) were dissolved in HCl solution with a final

151

concentration of 1.0 mg mL-1. The pH was adjusted to be 10.0 with a 0.1 M NaOH

152

solution at an increment of 10 µl. The NaCl aqueous solution was acted as control 7



153

group. The exact pH increases of the solution were monitored with a pH meter at

154

room temperature.

155

2.2.5. Preparation of polymer-liposomes complexes

156

Liposomes (L) were prepared by a thin lipid film hydration method followed by

157

extrusion. Briefly, PC: CH: DOTAP: DSPE-PEG: DSPE-PEG-NHS=62 : 30 : 5.5 : 2 :

158

0.5 (mol ratio) were dissolved in a chloroform/methanol mixture (4 : 1) in a

159

round-bottom flask. The pH-responsive polymers dissolved in methanol were added

160

to engineer polymer-liposomes (LP). CPLGVRGK-GGG-NYSKPTDRQYHF peptide

161

(1mg mL-1) was added to the liposomes (LPp) with gentle stirring for 12 h. DOX was

162

encapsulated in the complexes (LPDp) via an active transmembrane pH gradient

163

method. Size and surface charge of liposomes were measured by using Zetasizer

164

Nano ZS (Malvern Instruments Ltd).

165

2.2.6. Morphology study of polymer-liposomes

166

The morphology of polymer-liposomes obtained by hydration with PBS (pH 7.4) or

167

acetate buffer (pH 5.0) was observed by transmission electron microscopy (TEM,

168

Tecnai G2 F20 U-TWIN). The liposomes sample was stained by uranium acetate for

169

30 s and washed by distilled water twice. The stability of all kinds of

170

polymer-liposomes was investigated by storing at 37 °C for a period of 2 weeks

171

separately. The morphological changes of them were evaluated.

172

2.2.7. Turbidity study

173

The respective amount of pH-responsive polymer (0, 0.5, 1, 1.5 mol%) was added

174

to the lipid mixture before formation of the lipid film. Then the lipid film was hydrated 8


Page 8 of 39

Page 9 of 39 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

Biomacromolecules

175

with different buffer (pH 7.4, 6.8, 5.5). And the changes of turbidity were determined

176

by OD600 nm using multifunctional microplate reader (Tecan infinite M200).

177

2.3 Hemolysis Assay

178

For hemolysis analysis, LPDp was dispersed in PBS and incubated with the

179

erythrocyte stock dispersions to make the final concentration of LPDp at 0.125 to 2

180

mg/ml. Incubation was carried out at 37 °C for 2 h. Then reaction mixtures were

181

centrifugated at 800rpm for 5 min to remove intact erythrocytes. The supernatants

182

were dissolved in a hydrochloric acid andethanol mixture, following an additional

183

centrifugation (800 rpm for 3 min). PBS solution was used as negative control (0%

184

lysis), and distilled water was used as positive control (100% lysis). The absorbance

185

of the supernatant was determined at 545 nm by an ultraviolet spectrophotometer. The

186

hemolysis rate (HR %) was calculated by Eq. (1): Dt ― Dnc

187

HR%=Dpc ― Dnc × 100

(1)

188

Where Dt, Dnc, and Dpc were the absorbances of the tested sample, the negative

189

control, and the positive control, respectively. The experiments were run in triplicate.

190

2.4. Loading efficiency of DOX

191

DOX was encapsulated in the polymer-liposomes via an active transmembrane pH

192

gradient method as described. The encapsulation efficiency (EE%) and loading

193

capacity (LC%) of DOX was calculated by Eq. (2) and Eq. (3), respectively.

194

EE %=(Drugt  Drug f ) Drugt × 100

(2)

195

LC %=(Drugt  Drug f ) Lt × 100

(3)

9



Page 10 of 39

196

Where Drugt is the total amount of Drug; Drugf is the free amount of Drug in

197

supernate; and Lt is the total weight of polymer-liposomes.

198

2.5. Drug leakage

199

DOX leakage was investigated to evaluate the leakage of drug from

200

polymer-liposomes in the physiological environment at 37 °C for a period of 2 weeks.

201

Various polymer-liposomes (5 ml) were placed into a cellulose membrane dialysis

202

tube (molecular weight cutoff = 2000 Da), respectively. The amount of DOX released

203

in the medium was assayed at 480 nm.

204

2.6. pH-responsive drug release

205

LPDp or LD (5 mL) was placed into a cellulose membrane dialysis tube

206

(molecular weight cutoff = 8000-10,000). The tube was then introduced into 15 mL

207

PBS (pH 7.4), PBS (pH 6.8) or acetate buffer (pH 5.5) and stirred at 100 rpm at 37 °C.

208

At predetermined time intervals (between 0.25 h and 24 h), whole medium was

209

removed and replaced by fresh release medium to maintain a sink conditions. The

210

amount of DOX released in the medium was assayed at 480 nm. All procedures were

211

carried out in the dark to protect DOX from light. The accumulative drug release

212

percentage (Q%) of drug in acetate buffer was calculated by Eq. (4) and all

213

experiments were carried out in triplicate:

214

Q%=(C n  V 

i

n 1 0

C i  Vi ) (Drugt  EE %)× 100

(4)

215

Where Cn was the Drug concentration at Tn, V was the total volume of release medium,

216

Vi was the sampling volume at Ti, Ci was the Drug concentration at Ti (both V0 and C0

217

were equal to zero), Drugt was the total amount of Drug used for preparation of the 10


Page 11 of 39 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

Biomacromolecules

218

original mixture, EE% was the encapsulation efficiency of drug loaded into the

219

liposomal systems.

220

2.7. 50% inhibitory concentration (IC50) of DOX

221

B16F10 cells were cultured in DMEM medium supplemented and were kept in an

222

atmosphere of 5% CO2 and 95% air at 37 °C. Considering the toxicity of DOX (0.05

223

µg mL-1 to 25.6 µg mL-1), the cell viability was investigated by the CCK-8 assay. The

224

percentage of viability was expressed as relative growth rate (RGR %) by Eq. (5):

225

Tests were performed in quadruplicate for each sample.

226

Dt

RGR%=DC × 100%

(5)

227

Where Dt and Dc were the absorbances of the tested sample and the negative control.

228

The IC50 was defined as the concentration of DOX produced 50 per cent relative

229

growth rate.

230

2.8.M6PR expression

231

The effect of DOX on mannose 6-phosphate receptor (M6PR) expression was

232

determined by immunofluorescence. B16F10 cells were incubated with 1 mL of

233

freshly prepared LPDp suspension (DOX concentration from 0.25 µg mL-1 to 1.0 µg

234

mL-1). After 12 h incubation, B16F10 cells were fixed with 4% cold

235

paraformaldehyde for 15 min, washed three times with PBS, blocked with 10% BSA

236

for 1 h at room temperature, and then incubated with primary mouse monoclonal

237

M6PR antibody (1:100, Ab8093; Abcam, Cambridge, MA, USA) overnight at 4 ºC.

238

Cells were washed three times and incubated with FITC-conjugated rabbit anti-mouse

239

secondary antibody (1:1000, zsBio, Beijing, China) for 1h at room temperature. 11



240

Nuclei were stained with 4’, 6-diaminoindole (DAPI) (S36938, Invitrogen) for 10 min

241

after washing three times with PBS. Cell membranes were labeled with commercially

242

available membrane tracker (DiI, red color). Cells were observed using a confocal

243

microscope (LSM710, Carl Zeiss, Germany). The cell resuspension was finally

244

subjected to flow cytometry (BD Calibur) and quantitatively analyzed M6PR

245

expression in the different treatment groups with CellQuest software through

246

fluorescence channel 1 (FL1)．

247

For western blotting, protein lysates from cells were extracted in TNE lysis buffer

248

(0.5% NP-40, 10 mM Tris, 150 mM NaCl, 1mM EDTA and protease inhibitors).

249

Total protein (50 mg) was separated by 8% gel and transferred to a PVDF membrane

250

and treated with primary mouse monoclonal M6PR antibody (1:100, Ab8093; Abcam,

251

Cambridge, MA, USA), and then immunoblotted with peroxidase-conjugated

252

secondary antibody (1:5000, zsBio, Beijing, China).

253

2.9. Blockage of PD-1/PD-L1 interaction

254

Since the polymer-liposomes relied on MMP-2 to degrade the linker for anti-PD-L1

255

peptide, MMP-2 concentration in the original cell media of B16F10 cells was

256

measured using a MMP-2 ELISA Kit. The Responsive cleavage of functional peptide

257

linker by MMP-2 secreted by B16F10 cells after 24 h culture was evaluated using

258

HPLC. After treatment with IFN-γ (20 ng/mL) to upregulate the expression of PD-L1

259

in vitro, B16F10 cells harvested in a logarithmic growth phase were seeded on

260

glass-bottomed dishes. After 24 h, a commercial Cy3-labeled recombinant mouse

261

PD1 protein (ab216248) was added to the cells. Meanwhile, the culture medium was 12


Page 12 of 39

Page 13 of 39 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

Biomacromolecules

262

replaced by serum-free medium containing LPp, LP, or D-peptide. After incubation

263

at 37 °C for 4 h, the cells were washed three times with PBS and stained with 10 μg

264

mL−1 Hoechst 33342 for 10 min, then viewed by confocal laser scanning microscope.

265

To test the blocking efficiency, we conducted flow cytometry experiments.

266

2.10. Apoptosis analysis

267

The apoptosis of B16F10 cells was further determined by fluorescence-activated

268

cell sorting (FACS) using annexin V-FITC/propidium iodide (PI) double staining

269

after treatment with PBS, GranB, LPDp, or LPDp ＋ GranB for 24 h. Briefly, the

270

treated cells were trypsinized and collected by centrifugation for 5 min. Then the cells

271

were washed and resuspended with 100 μL of PBS. Finally, the cells were first

272

stained with annexin V-FITC (25 μg/mL) for 15 min on ice and then incubated with

273

propidium iodide (50 μg/mL) for 5 min before FACS analysis.

274

2.11. In vivo imaging

275

All animal experiments were performed complying with the NIH guidelines for the

276

care and use of laboratory animals and according to the protocol approved by the

277

Institutional Animal Care. 5×106/mL-1 B16F10 cells, collected in serum free DMEM

278

medium (200 μL), were subcutaneously (s. c.) injected into the right lateral hind hip

279

of the female C57BL/6 mice. The tumor volume was calculated by Eq (6): Tests were

280

performed in quadruplicate for each sample.

281 282 283

V = AB2/2

(6)

Where A was the maximum diameter, B was the minimum diameter. When tumors had developed to approximate 150 - 200 mm3, the mice were 13



Page 14 of 39

284

intravenously injected Cy5 labeled LP or LPp (200 μL 1mg mL-1). Near-infrared

285

imaging was carried out after 4 h, 8 h, and 24 h using a Maestro in vivo spectrum

286

imaging system (excitation filter, 649 nm; emission filter, 670 nm). Mice treated with

287

intravascular injection of PBS were acted as control group. Then animals were

288

sacrificed and tumor, heart, liver, spleen, lung and kidney were excised for flourecsent

289

intensity measurement.

290

2.12. In vivo combined therapeutic study

291

2.12.1. Tumor treatment

292

When the tumors reached sizes of 50 –100 mm3, at which time the mice (in groups

293

of six each) received different treatments: (a)LPDp, (b)LPp, (c)LD (1 mg/kg in

294

DOX), (d)free DOX (10 mg/kg in DOX), or (e) PBS. The drugs were intravenously

295

administered to mice on every second day (day 1, 3, 5, 7, 9, 11 and 13). DOX

296

concentration in normal tissues was measured after 24 h injection using HPLC.

297

During the process of the treatment, the tumor volumes and body weight were

298

measured every two days. After in vivo anti-tumor treatment, mice were sacrificed

299

and blood, tumor, heart, liver, spleen, lung, kidney were collected for blood

300

biochemistry and histological analysis. The tumor inhibition ratio (IR%) was

301

calculated by Eq. (7): Tests were performed in quadruplicate for each sample.

302

IR%= (Vc  Vt ) Vc 100

(7)

303

Where the Vc was the average tumor volume of control group after treated with

304

intravascular injection of PBS for 17 d, and the Vt was the average tumor volumes of

305

treatments with LPDp, LPp, LD, or free DOX, respectively. 14


Page 15 of 39 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

306

Biomacromolecules

2.12.2. Immunohistochemical staining

307

M6PR expression in tumor tissues was detected by Immunohistochemistry. Tumors

308

were fixed in 4% paraformaldehyde for 4 h at 4 °C and cut into 6 μm sections for

309

histological analysis. Briefly, sections were deparaffinized, hydrated, and subjected to

310

proteinase K treatment for antigen retrieval. Endogenous peroxidase was blocked by

311

3% hydrogen peroxide/methanol. The slides were blocked with normal goat serum

312

and then incubated with corresponding primary antibody (1:50, Abcam, USA)

313

overnight at 4 °C, respectively. After washing with PBS, the sections were incubated

314

with HRP-conjugated Goat Anti-mouse IgG (1:500, Abcam, USA) and DAB was

315

used for color development. The obtained experiment slices were observed by optical

316

microscopy. All measurements were carried out using Image J.

317

2.12.3. The intratumoral CD8+ T cells

318

The tumor tissues were harvested and the cells separated from tumor tissues were

319

stained with PE-CD8a antibody for 20 min on ice at dark. The cells were washed 3

320

times with PBS buffer, centrifuged and re-suspended in PBS buffer. Cell resuspension

321

was finally subjected to flow cytometry (BD Calibur) and analyzed with CellQuest

322

software.

323

The frozen tumor tissue sections of 10 mm thickness were prepared. CD8 positive

324

cells in tumor sections were stained using anti-rat CD8 antibody (1:500 dilution,

325

eBioscience) and Cy3-anti-rat IgG (1:500 dilution, Jackson immune research) as a

326

secondary antibody (Red). Finally, the sections were washed three times with PBS

327

and the nuclei were labeled with DAPI. The apoptosis in tumors was detected by 15



328

the TUNEL assay. Sections were then washed, covered with coverslip and observed

329

using a LSM710 confocal microscope.

330

3. Results and discussions

331

3.1. Characterization of polymer-liposomes complexes

332

The pH-sensitive copolymer P1 was synthesized by Michael addition of

333

hydrophobic monomer HDDA, pH-sensitive monomer DBPA and hydrophilic

334

PEG-NH2 (Supporting Information, Figure S1). For effective insertion into

335

membrane of liposome, dodecylamine was conjugated onto the both ends of P1,

336

gaining copolymer P2. The 1H NMR spectrum of P1 and P2 was shown in

337

Figure S2 and Figure S3, respectively (Supporting Information). The pH

338

sensitivity of P2 was evaluated by a pH titration method. After adding 0.1 M

339

NaOH, a high buffer capacity of copolymers was observed in the range of pH

340

4.0–9.0 compared to NaCl solution, which was in accordance with other

341

copolymers with diethyl amino groups (Figure S4, Supporting Information).

342

PC: CH: DSPE-PEG: DOTAP: DSPE-PEG-MAL= 62: 30: 2: 5.5: 0.5(mol

343

ratio) were dissolved in a chloroform/methanol mixture (4: 1) to prepare

344

liposomes (L)

345

mol%) dissolved in methanol was added to the lipid mixture before formation

346

of the lipid film. The lipid film was hydrated with phosphate buffered saline

347

(PBS, pH 7.4) following evaporating the organic solvents. DOX was

348

encapsulated in LP using the active transmembrane pH gradient method

349

(LPD).The peptide (p) (CPLGVRGK-GGG-NYSKPTDRQYHF) was coupled

37, 38.

For polymer-liposomes (LP) preparetion, P2 (0–2.5

16


Page 16 of 39

Page 17 of 39 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

Biomacromolecules

350

to

the

maleimide

group

of

DSPE-PEG2000-MAL

351

Responsiveness and selective cleavage site of the linker p in the presence of

352

MMP-2 were validated by HPLC (Figures S5, Supporting Information). DOX

353

was encapsulated in L to prepare LD. Schematic representation of the

354

component details can be found in the Supporting Information (Figure S6).

355

Transmission electron microscope (TEM) images of LP with the respective

356

amount of P2 (0–2.5 mol%) (FigureS7, Supporting Information). The

357

self-aggregations of P2 were detected visually with the amount of P2 up to 2

358

mol% and 2.5 mol%. P2 with the amount ≤ 1.5mol% was able to be entirely

359

incorporated into liposomes. The LP containing 1.5 mol% of P2 exhibited

360

hollow and spherical structures in PBS buffer solution (pH 7.4) (Figure1A).

361

The hydrodynamic diameter of all liposomal systems was around 60-70 nm

362

with PDI ≤ 0.2 and zate potential about 20 mV (Table S1, Supporting

363

Information).The LPp with 1.5 mol% of P2 was re-dissolved in acetate buffer

364

(pH 5.0) and incubated for 30 min. The bilayer membrane of LPp was

365

disrupted, which was clearly observed by TEM. The morphological change of

366

L (containing 0 mol% P2) was unobvious in the same condition. The turbidity

367

of LPp with 0–1.5 mol% of P2 was checked to evaluate the integrality of

368

liposome membrane by monitoring the optical density at 600 nm (OD600

369

(Figure1B). At pH 6.8, the OD600 nm of all liposomal systems was detected with

370

almost no change in comparison with that at pH 7.4. The turbidity of LPp with

371

1.5 mol% P2 at pH 5.5 reduced to 44.1% compared to that at pH 7.4. For L 17


to

form

LPDp.

nm)


372

without mixing of P2, that acidic condition caused no significant change of the

373

OD600

374

and tumor microenvironment and release the loaded cargos at the lysosome (pH

375

5–6), implying that the LPDp could be applied as pH-responsive drug carriers

376

for intracellular delivery.

nm.

Therefore, the LPDp were almost stable in physiological condition

377

Hemolysis rates of LPDp suspension are shown in Figure S8 (Supporting

378

Information). The HR % of LPDp at the highest concentration (2 mg/ml) was

379

below 3.5%, indicating that LPDp had good hemocompatibility.

380

3.2. Loading and release of DOX

381

The introduction of the P2 into polymer-liposomes

382

The encapsulation efficiencies of the DOX in polymer-liposomes were >60%, at

383

loading capacities of approximately 8–10% (Supporting Information, Table S1). The

384

DOX leakage from the delivery systems was quantitatively determined after storing

385

for 2 weeks at 37 °C. The amount of DOX leakage from the LD was found to be

386

about 18.7%. In contrast, only about 3.6% leakage was obtained from the LPD and

387

LPDp under the same conditions. These results indicate that the pH-responsive

388

polymers helped to stabilize the liposomes and reduce leakage of the payloads in the

389

physiological environment. Furthermore, morphological change after the storage of 2

390

weeks at 37 °C was observed using TEM to monitor the physical stability of the

391

particles. LD showed obvious fusion and severe aggregation (Figure 1C). For LPD

392

and LPDp in the identical conditions, only a slight aggregation could be detected.

393

The drug release profiles were monitored at different pH (7.4, 6.8, 5.0), and the 18


Page 18 of 39

Page 19 of 39 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

Biomacromolecules

394

amount of released DOX was quantified by HPLC. At pH 7.4 and pH 6.8, only 5%

395

and 10% of the loaded DOX was released from LD, respectively. In acetate buffer

396

(pH 5.0), higher solubility of DOX caused more drug release (about 34% for 24 h).

397

When LPDp was incubated at pH 5.0, the release of DOX was significantly

398

accelerated (>65%), which was attributed to the dissociation of the liposomes. The

399

results demonstrated that the pH-responsive drug release was in accordance with the

400

turbidity assay.

401

The pH-responsive polymer chains incorporated in LPD and LPDp contained

402

anchoring moiety for tight attachment onto the liposome membrane surface and made

403

LPD and LPDp more stable to reduce leakage of the payloads in physiological

404

environment. At acidic pH, the pH-sensitive moiety of the pH-responsive polymer

405

chains was capable of converting to protonated state accompanying the dynamic

406

disordering of membrane. Therefore, the pH-sensitive liposomes could reduce leakage

407

of DOX and protected the inner liposomal core in the physiological environment prior

408

to arrival at the targeted cells.

409

3.3. Up-regulation of the M6PR induced by low-dose DOX

410

We optimized the dosage of DOX by assessing the efficacy of up-regulating M6PR

411

on B16F10 cells. The IC50 of DOX was about 2.8 µg mL − 1(Figure S9, Supporting

412

Information) by evaluating cell viability of B16F10 cells and optimized the low dose

413

of DOX (< 2 µg mL-1) for further experiments with negligible systemic side effects.

414

M6PR on the surface of the B16F10 cells incubation with LPDp (DOX concentration

415

from 0.25 µg mL − 1 to 1 µg mL − 1) was comfirmed to be up-regulated obviously by 19



Page 20 of 39

416

immunofluorescence (Figure 2A). The expression of the M6PR was verified

417

quantitatively by flow cytometry. The M6PR expression improved with the DOX

418

concentration increase (Figure 2B). Compared with control group, about 6-fold higher

419

(P < 0.01) mean fluorescence intensity (32.12± 3.21) was detected in B16F10 cells

420

treated with LPDp with DOX concentration of 1 µg mL-1 (Figure S10, Supporting

421

Information). The expression of the M6PR was also verified with respect to protein

422

level (Figure 2C) by Western blotting. These results were consistent with reports that

423

DOX could induce overexpression of M6PR on tumor cell membranes.

424

3.4. Blockade of PD-1/PD-L1 interaction in vitro

425

Matrix metalloproteinases (MMPs), up-regulated in tumor regions

39,

have been 40.

426

extensively studied as target enzymes for drug-delivery systems design

427

concentration MMP-2 in B16F10 cell media was measured using ELISA Kit. As

428

demonstrated in Figure S11 (Supporting Information), MMP-2 was overexpressed in

429

B16F10 cells media (about 0.76 μgmL−1) after 12 h culture, which was about 50-fold

430

higher than the concentration we used to cleave the linker of anti-PD-L1 peptide in

431

section 2.2.2. When the incubation time is extended to 24 h, the MMP-2 concentration

432

was up to about 2-fold higher (P < 0.05) in cell culture supernatants. We confirmed

433

that amount of MMP-2 secreted by B16F10 cells in vitro was sufficient to release the

434

PD-L1 antagonist using HPLC (Figure S12, Supporting Information). As PD-L1 was

435

low-expressed during in vitro culturing

436

upregulated the PD-L1 on B16F10 cells and confirmed by confocal laser scanning

437

microscope and flow cytometry experiment (Figure 3A and Figure S13, Supporting

41, 42,

The

we used interferon-γ (IFN-γ) to

20


Page 21 of 39 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

Biomacromolecules

438

Information). In vitro PD-1/PD-L1 blockage experiments were conducted by treating

439

B16F10 cells with PBS, LP, LPp, or D-pep (0.1 mg mL−1 in peptide) in the presence

440

of Cy3-labeled recombinant mouse PD1 protein for 4 h at 37 °C. In comparison with

441

control group, LPp and D-pep exhibited high inhibitory effect with significant

442

fluorescence intensity decrease (p ＜ 0.01) as revealed in CLSM image (Figure 3B).

443

On the contrary, negligible blockage was detected when B16F10 cells treated with LP.

444

The blocking rate of PD-1/PD-L1 interaction was also assessed quantitatively by flow

445

cytometry (Figure 3C). It could be seen that LPp blocked PD1/PD-L1 interaction

446

more significantly (Plung>spleen>heart

499

concentration of DOX in liver (0.08 μg/ml) was about approximately 4-fold higher (P

500

< 0.05) than that in heart. The toxicity of the LPDp in vivo was further investigated

501

by blood and histological assays (Table S2 and Figure S18, Supporting Information).

5A),

was

indicating

evaluated

nontoxicity

by

of

using

the

HPLC,

(Supporting

low-dose

indicated

Information,

DOX

the Figure

encapsulated

order S17).

of The

502

The platelet counts, white blood cell and red blood cell of mice in LPDp treated

503

group showed no significant changes compared to that in control group. Moreover, 23



504

the levels of ALP, CR, BUN, ALT and AST in LPDp treated group were within the

505

normal ranges, indicating low toxicity to liver and kidney. No noticeable organ

506

damage was detected after pathological assay, which indicated low dose DOX showed

507

low toxicity to normal tissues.

508

The M6PR had been shown to play a role in endocytic uptake of GranB46, since

509

target cells overexpressing M6PR had an increased sensitivity to the cytotoxic effect

510

of CTLs47,

511

Immunohistochemistry (Figure 5B). The M6PR expression levels in the LPDp

512

treatment group were the highest (0.015), which demonstrated a far more significant

513

up-regulation (P < 0.01) than that of LPp and a slightly higher one compared to that

514

of LD (Figure S19, Supporting Information). The cell apoptosis in tumor tissue was

515

measured by TUNEL assay (Figure 5C). The fluorescent intensity in tumor tissue

516

sections indicated the order of LPDp>LPp >LD >PBS. Compared with LPp, about

517

2-fold higher (P

Tumor Microenvironmental pH and Enzyme Dual Responsive Polymer

Recommend Documents