Biogenic Polyphosphate Nanoparticles from Synechococcus sp. PCC

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Bioactive Constituents, Metabolites, and Functions

Biogenic Polyphosphate Nanoparticles from Synechococcus sp. PCC 7002 Exhibit Intestinal Protective Potential in Human Intestinal Epithelial Cells In Vitro and Murine Small Intestine Ex Vivo Guangxin Feng, Yinong Feng, Tengjiao Guo, Yisheng Yang, Wei Guo, Min Huang, Haohao Wu, and Ming-Yong Zeng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03381 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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Journal of Agricultural and Food Chemistry

Biogenic Polyphosphate Nanoparticles from Synechococcus sp. PCC 7002 Exhibit Intestinal Protective Potential in Human Intestinal Epithelial Cells In Vitro and Murine Small Intestine Ex Vivo

Guangxin Feng, Yinong Feng, Tengjiao Guo, Yisheng Yang, Wei Guo, Min Huang, Haohao Wu*, Mingyong Zeng*

a

College of Food Science and Engineering, Ocean University of China, 5 Yushan

Road, Qingdao, Shandong Province, 266003, China

* Authors to whom correspondence should be addressed; E-mails: [email protected] (Wu, H.); [email protected] (Zeng, M.); Tel. & Fax: +86-532-8203-2400 (Wu, H.); +86-532-8203-2783 (Zeng, M.).

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ACS Paragon Plus Environment


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ABSTRACT

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Polyphosphates are one of the active compounds from probiotics to maintain gut

3

health. The current research extracted and purified intact biogenic polyphosphate

4

nanoparticles (BPNPs) from Synechococcus sp. PCC 7002 cells. BPNPs were

5

near-spherical anionic particles (56.9 ± 15.1 nm) mainly composed by calcium and

6

magnesium salt of polyphosphate, and were colloidally stable at near-neutral and

7

alkaline pH. BPNPs survived gastrointestinal digestion in mice, and could be

8

absorbed

9

dose-dependently increased the tightness of intercellular tight junction and the

10

expression of claudin-4, occludin, zonula occludens-1 and heat shock protein 27 in

11

Caco-2 cell monolayers. BPNPs also effectively attenuated H2O2-induced cell death,

12

plasma membrane impairment and intracellular superoxide production in NCM460

13

cells. In addition, they conferred resistance to H2O2-induced barrier disruption in

14

freshly excised mouse small intestine. Our results suggest that BPNPs are a promising

15

postbiotic nanomaterial with potential applications in gut health maintenance.

16

KEYWORDS: biogenic polyphosphate nanoparticles, Synechococcus sp. PCC 7002,

17

intestinal permeability, cytoprotective activities, Caco-2 cell monolayer

and

transported

by

polarized

Caco-2

2


cell

monolayers.

They

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INTRODUCTION

19

Inorganic polyphosphates (polyP) with the general formula Mn+2PnO3n+1 are linear

20

polymers containing tens to hundreds of orthophosphate residues linked by

21

high-energy phosphoanhydride bonds.1 PolyP are extremely ancient biopolymers,

22

probably even predating life itself, and are ubiquitously conserved in all extant forms

23

of life, from prokaryotes to mammals.2 These biopolymers have been found to mainly

24

accumulate in acidocalcisomes of living cells as polyP particles, the size of which

25

depends on microorganism itself and living environment.3-7 In biological systems,

26

polyP act as various roles like a reservoir of phosphate, an alternative energy supply, a

27

chelator of metals, a buffer against alkali, a regulator of responses to stress, blood

28

clotting and bone and teeth regeneration.8

29

Besides its fundamental role in digestion and absorption of nutrients, gut also act as

30

the first-line defender against food-borne harmful substances and microorganisms.9,10

31

Probiotics are living microorganisms that confer a health benefit on the host, and they

32

produce some active compounds (so-called postbiotics) to improve intestinal barrier

33

function.11 Postbiotics could potentially be a safer alternative to living bacteria

34

especially during intestinal inflammation when probiotics have been found to exert

35

detrimental effects.12 PolyP have been well documented as a postbiotic.13-16

36

Production of polyP by heterotrophic microorganisms usually requires expensive

37

energy and carbon sources like glucose and propionate.17 Marine cyanobacteria,

38

which live on natural sunlight and CO2 in marine water, store large amounts of 3



39

phosphorus in the form of polyP to overcome the frequent phosphorus limitation in

40

the ocean, and are thus promising “photo-bioreactors” to produce polyP in an energy

41

and fresh water saving way.18

42

Synechococcus sp. PCC 7002 is a unicellular marine cyanobacterium strain with

43

many excellent features for biotechnological and industrial applications. This

44

cyanobacterium is capable of living photoautotrophically, mixotrophically and

45

heterotrophically, and can survive a wide spectrum of salt concentrations and

46

temperatures.19 Synechococcus sp. PCC 7002 makes use of high-light irradiation

47

efficiently, and propagates very fast with a doubling time shorter than 4 h.20 We

48

previously found that polyP are accumulated in this strain as intracellular

49

near-spherical particles smaller than 100 nm.21 Nano-sized materials have been

50

increasingly used in food and medical fields to improve bioavailability of nutrients

51

and drugs.22 Therefore, Synechococcus sp. PCC 7002 is an excellent host for

52

production of nano-sized polyP particles.

53

The present study extracted, purified and systematically characterized polyP

54

nanoparticles from Synechococcus sp. PCC 7002, here called biogenic polyP

55

nanoparticles (BPNPs). Considering the potential application of BPNPs in food and

56

medical fields, we investigated their colloidal stability under various pH and ionic

57

strength conditions, gastrointestinal digestibility in mice, and absorption and

58

transportation in polarized Caco-2 cell monolayers. The intestinal protective potential

4


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of BPNPs was also evaluated in Caco-2 cell monolayers, NCM460 cells and ex vivo

60

intestinal loops.

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EXPERIMENTAL SECTION

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Chemicals. Dihydroethidine (DHE), methylthiazolyldiphenyl-tetrazolium bromide

63

(MTT), dimethylsulfoxide (DMSO), FITC-conjugated dextran (FD-4; average

64

molecular weight 4000 Da), radioimmunoprecipitation assay (RIPA) lysis buffer [50

65

mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100，1% sodium deoxycholate, and

66

0.1% SDS], 4’,6-diamidino-2-phenylindole (DAPI), lactate dehydrogenase (LDH)

67

based cytotoxicity detection kit (4744934001), phenylmethanesulfonyl fluoride

68

(PMSF), bovine serum albumin (BSA) and TBST [10 mM tris (pH 7.5), 150 mM

69

NaCl, and 0.05% Tween 20] were provided by Sigma-Aldrich Co. (Shanghai, China).

70

RPMI 1640 medium and Dulbecco’s modified Eagle’s medium (DMEM) were

71

purchased from Yisheng Biotechnology Co., Ltd. (Shanghai, China). Fetal bovine

72

serum (FBS), 1×penicillin-streptomycin-glutamine, TrypLE™ Express Enzyme, BCA

73

Protein Assay Kit (23227), Dulbecco’s phosphate-buffered saline (D-PBS) and

74

Hank’s balanced salt solution without phenol red (HBSS) were obtained from

75

ThermoFisher Scientific (San Jose, CA, USA). Hydrogen peroxide (H2O2) was

76

acquired from Guoyao Company (Shanghai, China). The primary antibodies claudin-4

77

(ab15104), heat shock protein 27 (Hsp-27) (ab5579), occludin (ab167161), zonula

78

occludens-1 (ZO-1) (ab59720) and secondary anti-rabbit IgG (ab6721) and

79

anti-mouse IgG (ab6728) antibodies were purchased from Abcam (Cambridge, MA). 5



80

The primary antibody β-actin (R1011) was purchased from Sigma-Aldrich Co.

81

(Shanghai, China). Other reagents used were of analytical grade and commercially

82

available.

83

Production and Characterization of BPNPs. BPNPs were produced by

84

Synechococcus sp. PCC 7002. The pre-cultured cyanobacteria were inoculated into

85

5-L serum bottles containing sterilized liquid standard medium A.23 The incubation

86

was carried out at 30 °C under continuous illumination with cool white fluorescent

87

lights (100 µmol photons m−2s−1) and air bubbling with 1% CO2 in air for 9 days.

88

For fluorescence observation of intracellular BPNPs, Synechococcus sp. PCC 7002

89

cells were fixed with 4% formaldehyde for 20 min at room temperature,

90

permeabilized with 0.3% Triton X-100 for 5 min, and stained with 50 µg/mL DAPI at

91

room temperature for 30 min. The DAPI-stained samples were mounted on glass

92

slides and viewed on an inverted fluorescence microscope (MF52, Mshot Co.,

93

Guangzhou, China) using a personalized filter set with a bandpass excitation

94

maximum at 350 nm and a 500 nm long pass emission filter.

95

For transmission electron microscopic (TEM) observation of intracellular BPNPs,

96

Synechococcus sp. PCC 7002 cells were fixed with 2.5% glutaraldehyde in 0.1 M

97

phosphate buffer solution (pH 7.4) for 2 h at room temperature and in a 4 °C

98

refrigerator for another 12 h. After glutaraldehyde fixation, the cells were immersed in

99

1% OsO4 for 30 min. After ultra-thin sectioning, the algal cells were observed under a

100

JEM-1200 TEM operating at 80 kV. 6


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BPNPs were extracted from algal cells according to Patrick et al. with some

102

modifications.24 Briefly, cells were harvested by centrifugation at 5000 g for 5 min

103

and resuspended in five volumes of HEPES buffer (pH 7). The suspension was heated

104

in a boiling water bath for 15 min before being cooled immediately in an ice-water

105

bath. After centrifugation at 6000g for 10 min, the supernatant was used as the crude

106

extract.

107

For purification of BPNPs, the crude extract was applied to a Milli-Q

108

water-equilibrated Sephadex G-100 column (2.5×70 cm). The column was eluted with

109

Milli-Q water at a flow rate of 1 mL/min. The eluate was monitored at 220 nm with a

110

HD-3 UV detector (Shanghai HuXi Analysis Instrument Factory Co., Ltd., Shanghai,

111

China). All peaks were collected manually and assayed for DAPI fluorescence and

112

light scattering count rates using a Hitachi F-4600 fluorescence spectrophotometer

113

(Hitachi Co. Ltd., Japan) and a Zetasizer Nano ZS (Malvern Instruments, Herrenberg,

114

UK), respectively. The fraction containing BPNPs was concentrated by using 3-kDa

115

ultrafiltering centrifuge tubes (Merck Millipore, Shanghai, China), and was then used

116

for further characterization as well as cellular and ex vivo studies.

117

For TEM observation, the sample solution was dropped onto a carbon-coated

118

copper grid, allowed to air-dry, and then examined using a JEM-2100Plus device at

119

200 kV. The energy-dispersive X-ray (EDX) mapping analysis was carried out on a

120

FEI-Tecnai G2 TF20 to analyze the elemental composition of BPNPs. For scanning

121

electron microscopic (SEM) examination, the lyophilized sample of BPNPs was 7



122

mounted on an aluminum stub, gold-sputtered, and viewed in a TESCAN VEGA3

123

device at 20.0 kV. Dynamic light scattering and nanoparticle tracking analysis were

124

analyzed on a Zetaview Laser Scattering Video Microscope (Particle Metrix GmbH,

125

Microtrac, Meerbusch, Germany). The ζ-potential measurements were performed

126

using a Malvern Nano ZS instrument. For an absolute quantification, BPNPs were

127

fully hydrolyzed in 2 N HCl at 95 °C for 40 min before orthophosphate was

128

determined with a molybdenum blue method.13

129

Cellular Experiments. The human colon carcinoma cell line Caco-2 and the

130

normal human colon epithelial cell line NCM460 were obtained from the Cell Bank of

131

the Chinese Academy of Sciences (Shanghai, China), and were routinely maintained

132

in DMEM and RPMI 1640 medium, respectively, supplemented with 10% FBS and

133

100 U/mL penicillin-streptomycin-glutamine at 37 °C in a humidified atmosphere

134

containing 5% CO2. Caco-2 cells were used at passage levels of 55 to 65, and

135

NCM460 cells were used at passage levels of 5 to 10. BPNPs were used at doses from

136

10 to 75 µg P/mL, based on the results of Segawa et al. (2011) and Sakatani et al.

137

(2016).13,14

138

Caco-2 cells were seeded in 12-well transwell plates (1.12 cm2 polycarbonate

139

membrane, 0.4 µm pore size, Corning, Shanghai, China) at a density of 2×105

140

cells/well. By measuring LDH release, transepithelial electrical resistance (TER), and

141

Western blotting, the effects of BPNPs on plasma membranes, epithelial permeability,

142

and tight junction protein expression were investigated, respectively, in Caco-2 cell 8


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monolayers at 12 d post-confluence, when the cell monolayers reached a plateau of

144

the TER around 1000–1200 Ω.cm2. The LDH activity in apical medium was

145

determined with an LDH-based cytotoxicity detection kit according to the

146

manufacturer’s instructions. The TER measurements were performed on a

147

Millicell-ERS system (Millipore, Bedford, MA, USA).

148

According to a previously described protocol,25 the uptake and transport of BPNPs

149

were measured in polarized Caco-2 cell monolayers at 21 d old, when the monolayers

150

have become fully differentiated regarding the expression of many transport proteins

151

and brush border hydrolases. Briefly, cell monolayers were washed with pre-warmed

152

HBSS at 37 °C for 15 min before their TER values were measured. After the

153

treatments with BPNPs, TER values were measured again to ensure the integrity of

154

the monolayer, and culture medium from both sides was collected for determining

155

DAPI fluorescence. The cell monolayers were fixed with 2.5% glutaraldehyde,

156

post-fixed with 1% OsO4, thin sectioned, and observed under a JEM-1200 TEM at 80

157

kV.

158

NCM460 cells were seeded into 96-well plates at a density of 1×105 cells/well and

159

cultured for 24 h before being treated with BPNPs or H2O2. To evaluate cellular

160

toxicity, the culture supernatant and the cells were assayed for LDH release and MTT

161

reduction, respectively. The LDH activity was measured using an LDH-based

162

cytotoxicity detection kit according to the manufacturer’s instructions. To assay MTT

163

reduction, the cells were washed with DPBS for three times, before being incubated 9



164

with 0.5 mg/mL MTT in fresh medium for 4 h, and the resulted formazan crystals

165

were dissolved with DMSO, followed by measurement of the absorbance at 570 nm

166

on a microplate reader (BioTek Instruments, Winooski, VT, USA). To determine the

167

level of intracellular superoxide radical, NCM460 cells were incubated with 10 µM

168

DHE in HBSS for 1 h, washed with HBSS twice, and read in a Fluorescence

169

Multi-Detection Reader (BioTek Instruments, Winooski, VT, USA) at 488 nm

170

excitation wavelength with a 610-nm emission filter.

171

Western Blotting Analysis. Caco-2 cell monolayers were lysed in RIPA buffer

172

supplemented with 1 mM PMSF at 4 °C for 30 min. The cell suspension was

173

centrifuged at 13,000 r/min for 15 min at 4 °C, and the supernatant was harvested as

174

whole cell lysate. The protein concentration in whole cell lysate was measured with a

175

BCA Protein Assay Kit according to the manufacturer’s instructions. Samples were

176

boiled in a loading buffer, separated by SDS-PAGE gel and then transferred to PVDF

177

membranes. After being blocked with 5% BSA (5 g BSA power in 100 mL of

178

1×TBST) at room temperature for 1h, the membranes were incubated with

179

anti-Claudin-4 (1:200 dilution), anti-Hsp-27 (1:1000 dilution), anti-Occludin (1:300

180

dilution), anti-ZO-1 (1:200 dilution), or anti-β-actin (1:5000 dilution) overnight at

181

4 °C. Immunolabeled proteins were incubated with HRP-conjugated secondary

182

antibodies (1:8000 dilution) at room temperature for 1 h, washed with TBST for three

183

times, visualized by ECL luminescence reagent, and photographed on a Tannon 5200

184

Multi image analyzer (Tanon Science & Technology Co., Ltd., Shanghai, China). 10


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Animal Experiments. Male C57BL/6 mice (7-8 weeks old) were provided by

186

Lukang Pharmaceutical Co. (Shandong, China) and housed in an air-conditioned

187

animal facility (20–24°C, 55%–65% humidity) with a 12-h/12-h light/dark cycle. All

188

experiments were performed ethically in accordance with the principles in the

189

National Institutes of Health (NIH) Guide for the Care and Use of Laboratory

190

Animals and were approved by the Committee on the Ethics of Animal Experiments

191

of Ocean University of China.

192

BPNPs (150 µg P/mL in 200 µL PBS) were orally administered to overnight

193

fasted mice (n = 4 per group, a total of 16 animals) by gavage to investigate whether

194

they could survive gastrointestinal digestion. At 2 h and 4 h post gavage, mice were

195

sacrificed, and their stomach, small intestine and large intestine were excised. The

196

content of each gastrointestinal section was flushed out with 5 mL warm (35°C)

197

physiological saline. The amounts of polyP in the gastrointestinal contents were

198

measured by the DAPI fluorescence.

199

For ex vivo intestinal loop studies, mice were sacrificed and the small intestine

200

was removed beginning at the ligament of Treitz, followed by being divided into

201

about 6-cm lengths and washed with sterile PBS. The small intestine segments were

202

filled with 10%-FBS supplemented RPMI 1640 medium with or without containing 3

203

mM H2O2 and/or 75 µg P/mL BPNPs, followed by ligation of each end with silk

204

sutures, and were then incubated in culture dishes filled with 5 mL 10%-FBS

205

supplemented RPMI 1640 media at 37 °C in a 5% CO2 incubator for 2 h. After 11



206

replacement of the luminal media with 10%-FBS supplemented RPMI 1640 media

207

containing 100 µM FD-4, the loops continued being cultured and samples from the

208

media outside bathing loops were taken at 10, 20, 30 and 40 min to determine

209

transmural flux of FD-4 by reading in a Fluorescence Multi-Detection Reader

210

(BioTek Instruments, Winooski, VT, USA) at 490-nm excitation wavelength with a

211

520-nm emission filter.

212

Statistical Analysis. Quantity One 4.6.2 software (Bio-Rad, Hercules,USA) was

213

used for band analysis. Statistical analyses were done using OriginPro 2016 software

214

(OriginLab Co., Northampton, USA). Data were expressed as means ± standard

215

deviations. The data were analyzed using Student's t-test for the assays. P < 0.05 was

216

considered as statistically significant.

217

RESULTS AND DISCUSSION

218

Preparation and Characterization of BPNPs. Synechococcus sp. PCC 7002

219

(Figure 1a) contained near spherical particles with diameters typically smaller than

220

100 nm under TEM in Figure 1b, and the EDX spectra of the red circle region in

221

Figure S1a reveals strong signals of phosphorus and oxygen (Figure S1b). DAPI has

222

been frequently utilized to characterize polyP synthesis within living cells due to its

223

distinguishable green and blue fluorescence when binding to polyP and nucleic acids,

224

respectively.26 As shown in Figure 1c, the DAPI-stained Synechococcus sp. PCC 7002

225

cells exhibited both green and blue fluorescence under light microscope. These results

12


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suggest that Synechococcus sp. PCC 7002 can synthesize polyP, which seem to be

227

present particulately at the nano level within cells.

228

Synechococcus sp. PCC 7002 cells were boiled to obtain the crude extract (Figure

229

1d). After Sephadex G-100 gel filtration (Figure 1e), two fractions were obtained with

230

peak I and peak II showing light scattering count rates of 208 ± 3.9 and 6 ± 1.2 kcps,

231

respectively. As tested by blue dextran, peak I is the exclusion peak, and is a

232

particle-containing fraction considering its intense light scattering signal. The peak I

233

fraction also displayed an intense DAPI fluorescence, and gave a great deal of green

234

particles under fluorescence microscope (Figure 1f). These results suggest that BPNPs

235

in the crude extract were eluted in the peak I fraction.

236

Figure 2 depicts the detailed analysis of BPNPs in the peak I fraction. TEM

237

analysis reveals monodispersed spherical particles with an average size of 56.9 ±

238

15.1 nm (Figures 2a and 2b), and whole area EDX analysis revealed prominent

239

signals for sodium, potassium, magnesium, calcium, oxygen, phosphorus, carbon and

240

copper (Figure 2c). The EDX mapping analysis of a single particle show that

241

phosphorus, oxygen, calcium, and magnesium distributed uniformly in the nano-size

242

sphere, with non-spherical distribution observed for sodium and potassium (Figures

243

2d and 2e). Additionally, the linear EDX scanning profiles of BPNPs revealed little

244

signal for nitrogen and sulfur (Figure 2f), so nitrogen- and sulfur-containing organic

245

compounds like proteins and nucleic acids seem to be absent in BPNPs. These results

13



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further confirm the presence of BPNPs in the peak I fraction, and demonstrated that

247

BPNPs were mainly composed by calcium and magnesium salt of polyP.

248

The results of dynamic light scattering in Figure 2g revealed the intensity-weighted

249

hydrodynamic diameters (HDD) of BPNPs to be around 100 nm. Nanoparticle

250

tracking analysis in Movie S1 shows BPNPs were mostly monodispersed with

251

aggregated particles sporadically observed. The ζ-potential of BPNPs was around

252

-28.9 ± 1.8 mV (Figure 2h), indicating the strongly anionic properties of BPNPs. The

253

high-magnification SEM analysis of lyophilized samples confirmed a spherical

254

morphology of BPNPs with an average diameter less than 100 nm (Figure 2i).

255

PolyP granules are abundantly found in many bacterial and cyanobacterial strains

256

such as Lactobacillus casei, Propionibacterium shermanii, Nostoc pruniforme and

257

Plectonema

258

calcium/magnesium at submicrometer scale with diameters of several hundreds of

259

nanometers.27-30 In this study, we observed rather smaller polyP granules at the

260

nanoscale in Synechococcus sp. PCC 7002 cells, which could be explained by the fact

261

that Synechococcus sp. PCC 7002 is a tiny cyanobacterium with an average cell

262

diameter of only 750 nm.31 We also observed the prominent presence of calcium and

263

magnesium in BPNPs, confirming that the intracellular polyP granules in

264

Synechococcus sp. PCC 7002 cells are calcium/magnesium precipitates of polyP.

265

PolyP are usually extracted from biological cells under acid or alkaline conditions or

266

with EDTA at near neutral pH.32 Here, polyP nanoparticles from Synechococcus sp.

boryanum,

and

are typically the

precipitates

14


of

polyP

and

Page 15 of 41


267

PCC 7002 cells were obtained intact in a green way by using hot water extraction and

268

Sephadex G-100 gel filtration.

269

Effects of pH and Ionic Strength on the Colloidal Stability of BPNPs.

270

Considering their potential postbiotic applications in food and medical fields, BPNPs

271

may encounter complicated delivery and biological circumstances with various pH

272

and ionic strengths, which usually determine a nanoparticle’s colloidal behavior. As

273

shown in Figure 3a, BPNPs exhibited negative ζ-potential in the pH range of 2-10,

274

and this could be explained by the strongly acidic hydrogen at each residue of

275

phosphate (pKa of 1 to 2) within the chain of long-chain polyP.33 BPNPs showed

276

remarkably higher negative ζ-potential in the presence of 10 mM NaCl (low ionic

277

strength) than 100 mM NaCl (medium ionic strength, like those found in

278

gastrointestinal fluids) at all pH values (Figure 3a), which is associated with the

279

compression of electrical double layer or an increased electrostatic shielding effect by

280

the increased counter-ions with the increase in ionic strength. In the presence of both

281

10 mM and 100 mM NaCl, the ζ-potential of BPNPs gradually declined with pH

282

decreasing from 6 to 2, with no significant change observed at the pH range of 6-10

283

(Figure 3a). The reduction of pH in the system below 4 caused remarkable rise in

284

conductivity (Figure 3b).

285

Long-chain polyP have two weakly acidic hydrogens at the ends of the chain, and

286

their pKa values were around 6 or 4 in the presence of Na+ or Mg2+, respectively.34

287

The acid-induced reduction of ζ-potential of BPNPs below pH 6 (Figure 3a) might be 15



288

the result of deionization of the terminal phosphate of polyP. Mg2+ and Ca2+ bound to

289

the terminal phosphate of polyP might disassociate from BPNPs below pH 4,34 which

290

is probably the reason for an acid-induced increase in conductivity in Figure 3b.

291

We also observed significantly increased HDD (intensity weighted) and scattering

292

count rates of BPNPs with pH decreasing from 6 to 2 in the presence of both 10 mM

293

and 100 mM NaCl (Figures 3c and 3d). This should be owing to the increased

294

aggregation of BPNPs below pH 6, as evidenced by the elevated polydispersity

295

indexes (PDI) (Figure 3e) and the greater proportions of large particles (Figures 3f

296

and S2). Apparently, the acid-induced diminishing of ζ-potential below pH 6 impaired

297

the colloidal stability of BPNPs. In addition, Mg2+ and Ca2+ dissociated from the

298

terminal phosphate of polyP below pH 4 might facilitate aggregation of BPNPs by

299

forming salt bridges between particles.

300

Gastrointestinal Digestibility of BPNPs in Mice. The postbiotic application of

301

BPNPs greatly depends on whether polyP could survive the gastrointestinal digestion

302

thereby reaching the intestinal (especially colonic) epithelium. As shown in Figure 4a,

303

the oral administration of BPNPs significantly increased the polyP levels in stomach

304

(P < 0.05), small (P < 0.001) and large (P < 0.01) intestine at 2 h post gavage,

305

suggesting that oral ingestion of BPNPs effectively increased the intestinal

306

availability of postbiotic polyP to the hosts. However, after 4 h of the oral

307

administration of BPNPs, gastrointestinal polyP levels almost restored to basal values

308

(Figure 4b), possibly owning to the intestinal absorption or hydrolysis of polyP. In 16


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fact, very slow enzymic and acid hydrolysis of long-chain polyP (n ≥ 5) could be

310

expected in the gastrointestinal tract according to previous reports,32,35 so the

311

intraluminal polyP seemed to be readily absorbed by the host, rather than to be rapidly

312

hydrolyzed, during 2-4 h post gavage of BPNPs.

313

Uptake and Transport of BPNPs by Caco-2 Cell Monolayers. Polarized Caco-2

314

cell monolayers on permeable filters have become a popular gut epithelial model and

315

were used to investigate the intestinal uptake and transport of BPNPs (Figure 5a).

316

Transwells with 3-µm pore size filters are frequently employed to investigate the

317

transportation of nanoparticles; nevertheless, filters with a pore diameter of 3 µm have

318

been found to allow the cells to crawl through the pores to the opposite side of the

319

filter, resulting in a double monolayer.25 To establish a monolayer of polarized Caco-2

320

cells, we used 0.4-µm pore-size transwells in this study, and according to the results

321

of BPNP transportation in empty transwells (Figure S3), 0.4-µm pore-size filters did

322

not severely restrict the permeability of BPNPs.

323

Thin-section TEM revealed well-differentiated cells with densely packed

324

microvilli and tight junction strands (white arrow) (Figure 5b). The treatments with

325

BPNPs resulted the observation of well-dispersed electron opaque nanoparticles

326

between microvilli or within endosomes (Figures 5c and 5d), and EDX analysis of the

327

regions of microvilli and endosomes showed stronger phosphorus signal (Figure S4).

328

BPNPs thus seem to be taken up by intestinal epithelial cells via the endocytic

329

pathway, which is in line with the results of probiotic-derived polyP as reported by 17



330

Tanaka et al. (2015).16 BPNPs were monodispersed and intact between microvilli and

331

within endosomes, suggesting their good colloidal and chemical stabilities during

332

intestinal absorption.

333

According to the results of DAPI fluorescence in Figure 4e, apical BPNPs almost

334

disappeared after an incubation of 12 h, and meanwhile a steady rise in basolateral

335

DAPI fluorescence was observed (Figure 5f). It thus seems that BPNPs could be

336

transported across intestinal epithelial monolayers. However, according to the

337

fluorescence intensities in Figures 5e and 5f, the overwhelming majority of apical

338

BPNPs was absorbed by polarized Caco-2 cells within 12 h, with only a minor portion

339

of them being transported into the basolateral side, which indicates that most apical

340

BPNPs were retained within cells after an incubation of 12 h.

341

BPNPs Enhanced Intestinal Epithelial Barrier Function In Vitro. Epithelial cell

342

plasma membrane and intercellular tight junction (TJ) are crucial in the preservation

343

of intestinal barrier function, and their leaking or even slightly perturbing caused by

344

pathogenic bacteria or certain pathological conditions (e. g. hyperglycemia) can lead

345

to serious consequences, including intestinal inflammation and systemic infection

346

complications.36 To investigate the effects of BPNPs on plasma membranes of Caco-2

347

cell monolayers, the leakage of cytosolic LDH was tested. As shown in Figure 6a,

348

BPNPs lead to no elevated LDH release at the dosages of 10, 25 and 75 µg P/mL,

349

indicating that these dosages were safe to avoid membrane leakage in Caco-2 cell

350

monolayers. TER is an instantaneous measurement for the tightness degree of TJ 18


Page 18 of 41

Page 19 of 41


351

barrier. As shown in Figure 6b, BPNPs at 25 and 75 µg P/mL resulted significantly

352

higher TER in Caco-2 cell monolayers from 12 h and 24 h of incubation, respectively

353

(P < 0.05), suggesting that BPNPs reduced the TJ permeability dose-dependently. The

354

effects of BPNPs on the expression of typical TJ proteins in Caco-2 cell monolayers

355

were evaluated by Western blotting (Figures 6c and 6d). BPNPs at 25 and 75 µg P/mL

356

significantly increased the protein levels of claudin-4, occludin and ZO-1 during an

357

incubation of 24 h. These results suggest that BPNPs could enhance intestinal

358

epithelial barrier integrity by boosting the expression of TJ proteins.

359

Heat-shock proteins are a class of stress-induced chaperons that protect cells from

360

various stress conditions.37 A considerable body of evidence indicates that probiotics

361

can maintain physiological expression of inducible heat shock proteins in the

362

intestine.38-41 PolyP have been reported as the key probiotic-derived factor to induce

363

the expression of Hsp-27 in Caco-2 cells.13,42 In this study, the effects of BPNPs on

364

the expression of Hsp-27 in Caco-2 cell monolayers were also investigated by

365

Western blotting (Figures 6c and 6d). BPNPs increased the protein levels of Hsp-27 at

366

all dosages tested with a good dose dependence. Therefore, BPNPs might also protect

367

intestinal

368

pathophysiological stress by inducing the expression of heat shock proteins.

epithelial

barrier

function

from

environmental,

metabolic

or

369

Cytoprotective Effects of BPNPs against Oxidative Injury in NCM460 Cells.

370

Oxidative stress has been implicated to mediate mucosal injury and immune

371

activation in several gastrointestinal disorders (e.g. ulcerative colitis and Crohn’s 19



372

disease).43-45 In this study, we used a normal colon epithelial cell line NCM460, which

373

is much more sensitive to oxidative stress than the carcinoma cell line Caco-2, to

374

establish a model of oxidative stress-induced intestinal injury.46 As shown in Figure

375

7a, NCM460 cells underwent a 57% cell death following the treatment with 2 mM

376

H2O2 for 3 h, and this was dose-dependently attenuated by the 6-h preincubation with

377

BPNPs, which showed no cytotoxic effect at all doses used (Figure S5). As shown in

378

Figure 7b, the treatment with 500 µΜ H2O2 for 3 h caused a 72% rise in LDH release,

379

suggesting a severely impaired plasma membrane integrity by H2O2-induced oxidative

380

stress, and this was effectively prevented by the 6-h preincubation with BPNPs in a

381

dose-dependent manner. These results suggest the cytoprotective effects of BPNPs

382

against oxidative stress-induced intestinal injury.

383

Besides their activities to elicit the production of cytoprotective Hsp-27, polyP

384

themselves can function as inorganic protein-protective chaperones to confer

385

oxidative stress resistance to a variety of prokaryotic and eukaryotic cells.47 PolyP is

386

also well-known to reduce oxidative stress by sequestering redox-active transition

387

metals.48,49 In addition, the intracellular Mn2+-polyP complex has been demonstrated

388

to detoxify superoxide radicals.50,51 In this study, the H2O2-induced production of

389

superoxide radical was monitored by detecting DHE fluorescence (Figure 7c). The

390

treatment with 500 µΜ H2O2 for 3 h induced an 11% increase in DHE fluorescence (P

391

< 0.01), and this was completely attenuated by the preincubation with BPNPs at 75 µg

392

P/mL, indicating an ability of BPNPs to detoxify superoxide radicals. 20


Page 20 of 41

Page 21 of 41


393

BPNPs Protect Intestinal Tissues from Oxidative Stress in Ex Vivo

394

Preparation of Mice. The above-mentioned results show beneficial potential of

395

BPNPs in intestinal epithelial barrier function as well as oxidant-induced intestinal

396

injury. To further validate these in vitro results, ex vivo studies were carried out in

397

freshly excised mouse small intestine. As shown in Figure 8, an intraluminal exposure

398

to 3 mM H2O2 remarkably increased transmural FD-4 fluxes, suggesting an

399

oxidant-induced impairment of mucosal integrity, and this was significantly

400

attenuated by the co-exposure to 75 µg P/mL BPNPs. Apparently, BPNPs conferred

401

resistance to oxidant-induced barrier disruption in mouse intestinal tissues.

402

Importantly, the intestine loops treated with both H2O2 and BPNPs gave even lower

403

permeability of FD-4 than the untreated ones, which is in line with the results of

404

probiotic-derived long chain polyP in ex vivo models as reported by Segawa et al.

405

(2011),13 and this confirmed the intestinal barrier function-enhancing activity of

406

BPNPs in Figure 6b.

407

In conclusion, polyP nanoparticles from Synechococcus sp. PCC 7002 cells, i.e.

408

BPNPs, were obtained intact in a green way by using hot water extraction and

409

Sephadex G-100 gel filtration. BPNPs were near spherical anionic particles mainly

410

composed by calcium and magnesium salt of polyP. They were colloidally stable at

411

near-neutral and alkaline pH, and tended to aggregate at pH < 6. BPNPs could survive

412

gastrointestinal digestion and be absorbed in the intestine. They were capable of

413

enhancing

intestinal

epithelial

barrier

function

and

21


preventing

oxidative


414

stress-induced intestinal injury. BPNPs are thus an attractive candidate to maintain gut

415

health. However, further studies are needed to evaluate their postbiotic efficacy in

416

animal models.

417

418

Supporting Information

419

This material is available free of charge via the Internet at http://pubs.acs.org.

420

Supplementary Figure S1. (a) Representative TEM image of thin sections of

421

Synechococcus sp. PCC 7002 cells. (b) Energy dispersive X-ray analysis of the red

422

circle region.

423

Supplementary Figure S2. Volume-weighted lognormal size distributions versus pH at

424

100 mM NaCl.

425

Supplementary Figure S3. The transport of BPNPs in empty transwells with 0.4 and 3

426

µm pore sizes and expressed as DAPI fluorescence in the basolateral side. Data were

427

expressed as means ± standard deviations (n = 3).

428

Supplementary Figure S4. Energy dispersive X-ray spectra of thin sections of Caco-2

429

cells treated with or without BPNPs.

430

Supplementary Figure S5. Viabilities of NCM460 cells following an incubation with

431

0, 10, 25 and 75 µg P/mL of BPNPs for 6 h. (a) The relative cell viability (%) related

432

to control wells. (b) Photograph showing a typical MTT assay. Data were expressed

433

as means ± standard deviations (n = 3).

434

ASSOCIATED CONTENT

ACKNOWLEDGMENTS 22


Page 22 of 41

Page 23 of 41


435

This work was financially supported by the National Natural Science Foundation of

436

China (No. 31601406), the Natural Science Foundation of Shandong Province of

437

China (No. ZR2016CB30), and the Applied Basic Research Project of Qingdao of

438

China (No. 16-5-1-16-jch).

439

440

Conflicts of Interest There are no conflicts to declare.

441

442

Corresponding Authors

443

*Phone: +86-532-8203-2400. E-mail: [email protected].

444

*Phone: +86-532-8203-2783. E-mail: [email protected]

445

446

(1) Albi, T.; Serrano, A., Inorganic Polyphosphate in the Microbial World. Emerging

447

Roles for a Multifaceted Biopolymer. World J Microbiol Biotechnol. 2016, 32, 27.

448

(2) Rao, N. N.; Gómezgarcía, M. R.; Kornberg, A., Inorganic Polyphosphate:

449

Essential for Growth and Survival. Annu. Rev. Biochem. 2008, 78, 605-647.

450

(3) Docampo, R.; Souza, W. D.; Miranda, K.; Rohloff, P.; Moreno, S. N. J.,

451

Acidocalcisomes? Conserved from Bacteria to Man. Nat. Rev. Microbiol. 2005, 3,

452

251-261.

453

(4) Tillberg, J. E.; Rowley, J. R.; Barnard, T., X-Ray Microanalysis of Leakage from

454

Polyphosphate Granules in Scenedesmus. J. Ultrastruct. Res. 1980, 72, 316.

AUTHOR INFORMATION

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513.

Pathways

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Stress-Induced

Accumulations

594

30


of

Inorganic

Page 31 of 41


595

Figure captions

596

Figure 1. Preparation and characterization of BPNPs. (a) Appearance of the

597

Synechococcus sp. PCC 7002 culture. (b) Typical TEM image of thin sections of

598

Synechococcus sp. PCC 7002 cells. (c) Fluorescence microscope image of

599

Synechococcus sp. PCC 7002 cells stained with DAPI (×400). (d) Photograph of

600

crude extract from Synechococcus sp. PCC 7002. (e) Purification of crude extract by

601

Sephadex G-100 gel filtration. (f) Fluorescence microscope image of DAPI-stained

602

BPNPs (×400).

603

Figure 2. Detailed characterization of BPNPs. (a, b, d) TEM images. (c) EDX-plot. (e)

604

EDX elemental mapping. (f) linear EDX scanning. (g) Size distribution. (h)

605

ζ-potential. (i) SEM images.

606

Figure 3. Colloidal stability tests of BPNPs versus pH and ionic strength. (a)

607

ζ-potentials. (b) Conductivities. (c) Hydrodynamic diameters (HDD). (d) Scattering

608

count rates. (e) Polydispersity indexes (PDI). (f) Volume-weighted lognormal size

609

distributions at 10 mM NaCl. Data were expressed as means ± standard deviations (n

610

= 3).

611

Figure 4. Effects of the oral administration of BPNPs on gastrointestinal polyP levels

612

in mice at (a) 2 h and (b) 4 h post gavage. Data were expressed as means ± standard

613

deviations (n = 3). Statistical differences were determined by student's t-test (*p

Biogenic Polyphosphate Nanoparticles from Synechococcus sp. PCC

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