Synthesis and Characterization of Polyhydroxyalkanoate Organo

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Synthesis and Characterization of Polyhydroxyalkanoate Organo/Hydrogels Xu Zhang, Zihua Li, Xuemei Che, Linping Yu, Wangyue Jia, Rui SHEN, Jin-Chun Chen, Yi-Ming Ma, and Guo-Qiang Chen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00479 • Publication Date (Web): 16 May 2019 Downloaded from http://pubs.acs.org on May 17, 2019

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Biomacromolecules

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Synthesis and Characterization of Polyhydroxyalkanoate Organo/Hydrogels

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Xu Zhang1, Zihua Li2, Xuemei Che1,3, Linping Yu1, Wangyue Jia1, Rui

3

Shen1, Jinchun Chen1, Yiming Ma*1, Guo-Qiang Chen*1,3,4

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1 Center of Synthetic and Systems Biology, School of Life Sciences,

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Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing

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100084, China

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2 School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084,

8

China

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3 Center for Nano-and MicroMechanics, Tsinghua University, Beijing 100084,

10

China

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4 MOE Key Lab for Industrial Biocatalysis, Dept of Chemical Engineering,

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Tsinghua University, Beijing 100084, China

13 14

Corresponding author: Guo-Qiang Chen (Chen GQ)

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School of Life Science, Tsinghua University, Beijing 100084, China

1 ACS Paragon Plus Environment

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+86-10-62783844,

Fax:

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Tel:

+86-10-62794217,

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[email protected]

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Co-corresponding author: Yiming Ma,

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School of Life Science, Tsinghua University, Beijing 100084, China

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E-mail: [email protected].

e-mail:

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ABSTRACT: Synthetic organogels/hydrogels are attracting growing interests

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due to their potential applications in biomedical fields, organic electronics and

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photovoltaics. Photo-gelation methods for synthesis of organogels/hydrogels

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have shown particularly promising because of the high efficiency and simple

26

synthetic

27

polyhydroxyalkanoates

28

photo-crosslinking

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poly[(R)-3-hydroxyundecanoate-co-(R)-3-hydroxy-10-undecenoate] (PHU10U)

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with polyethylene glycol dithiol (PDT) as a photo-crosslinker. The PHU10U

31

was synthesized by an engineered Pseudomonas entomophila and

32

characterized via FTIR, 1H NMR and 13C NMR. With decreasing molar ratio

33

of PHU10U to PDT, both the swelling ratio and pore size were decreased.

34

Meanwhile, increasing densities of the gel networks resulted in a higher

procedures.

This

study

(PHA)-based using

synthesized

new

biodegradable

organogels/hydrogels

unsaturated

PHA

via

UV

copolymer


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Biomacromolecules

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compressive modulus. Cell cytotoxicity studies based on the CCK-8 assay on

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both the PHU10U precursor and PHU10U/PDT hydrogels showed that the

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novel PHA-based biodegradable acting as hydrogels possess good

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biocompatibility.

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KEYWORDS: PHB; Organogel; Hydrogel; Polyhydroxyalkanoates; PHA;

40

Photopolymerization

41 42 43 44

INTRODUCTION

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Gels are semi-solid polymers with special three-dimensional networks

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composed of several components such as macromolecules, polymeric

47

monomers, and a large amount of solvents.1-3 Gels can be divided into

48

hydrophobic

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applications include controlled-release matrices,4-6 tissue scaffolds,7-9

50

wound-healing adhesives,10-12 environmental protection,13,14 photo and

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electronically active soft materials,15-17 et al. Biomedical hydrogels have been

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successfully explored due to their hydrophilicity and biocompatibility.18, 19

organogels

and

hydrophilic

hydrogels.4

Their

potential



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However, very few biomedical applications of organogels have been reported

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due to their lack of biocompatibility.20, 21

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Based on the nature of the gelling molecules, organogels can be further

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subdivided into polymeric macromolecules22, 23 or low molecular weight

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(LMW) organogelators,24-26 which immobilize the organic solvents by forming

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a network of either crosslinked or entangled chains for chemical and physical

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gels, respectively. The easy self-assembly of LMW organogelators were

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formed by weak inter-chain interactions such as electrostatic, hydrogen

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bonding and van der Waals forces when immersed in organic solvent.27

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Various LMW organogelators of diverse molecular structures have been

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studied.28 However, it remains less frequent to develop gelators via chemical

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crosslinking. Thus, it is also difficult to study the structure−property

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relationships.3

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Organogels can be used as drug and vaccine delivery matrices via

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transdermal, oral, ophthalmic and parenteral administrations.4, 5, 20, 29

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Furthermore, organogels are moisture-sensitive allowing skin penetration,

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their organic nature also resists microbial contamination, indicating that

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organogels may be a candidate for drug delivery systems.30, 31 Therefore, it

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is necessary to understand the structure−property relationship of organogels.


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Novel scaffolds of gelators to solve the poor biocompatibility in biomedical

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application.4

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Polyhydroxyalkanoates, short as PHA, are a family of biopolyesters

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accumulated

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biocompatible with favorable thermal and mechanical properties for medical

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usages.37, 38 Functional PHA synthesized by engineered bacteria have

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shown potentials because of possibility to widen their properties.39-47

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Langlois et al. reported a variety of modified PHA with improving the

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properties of polyesters,48, 49 such as unsaturated PHA grafted with PEG,50

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terminal

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poly(oxyethylene-co-oxypropylene)s,52 these modified PHA show different

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physical properties such as enhanced hydrophilicity or amphiphilicity

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compared with original hydrophobic PHA. Recently, Nomura et al.53, 54

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prepared

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chain-length

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poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxy-10-undecenoate] (PHBU) from

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engineered E. coli LSBJ for crosslinking with the pentaerythritol tetrakis

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(3-mercaptopropionate) (PETMP) via thiol-ene click chemistry. An increase in

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tensile strength with not significant cytotoxicity toward human cells was

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observed on these modified PHA materials, resulting in a material with

by

various

acid,51

carboxylic

a

series

bacteria.32-36

of

unsaturated

(MCL)

PHA

PHA

are

and

biodegradable

and

amino-terminated

short-chain-length

(SCL)–medium

copolymer,

namely,



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physical properties and biocompatibility closer to those for known soft tissue

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replacement.53 Furthermore, Adler et al observed organogelation with

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thermo-reversibility

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poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) thermo-dissolved in

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toluene.55 However, no crosslinked organogel between unsaturated PHA and

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PEG-dithiol terminated macromers has been reported.

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This study aimed to develop first PHA gel (organogels/hydrogels depending

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on solvents) via photo-crosslinking based on thiol−ene click reaction (Figure

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1), and investigated the relationship between the structure and the property.

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Scaffolds with the gel incorporating the advantages of PHA were developed to

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address biocompatibility of most organogel/hydrogel.

during

the

cooling

process

of

103 104

Figure 1. Synthesis of functional PHA and its organogels prepared via

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photo-crosslink. 6 ACS Paragon Plus Environment

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EXPERIMENTAL SECTION

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2.1 Materials

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The

109


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was synthesized by Pseudomonas entomophila (P. entomophila) LAC23, a

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mutant of P. entomophila L48 deleted with β-oxidation related genes fadB,

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fadA, and PSEEN 0664.56 10-Undecenoic acid (>98%) and 1-undecanol

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(>99%) were purchased from TCI Co. (Shanghai, China). PEG-dithiol

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(PDT, >95%, Mn=2000, PDI=1.05, Ponsure, Co. China) was the crosslinker.

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Chloroform (A.R.), tetrahydrofuran (THF, A.R.) and ethanol (A.R.) were

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obtained

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2,2-Dimethoxy-2-phenylacetophenone (DMPA, >98%) was purchased from

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Energy Chemical Co. China. Human Embryonic Kidney 293T (HEK 293T)

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cells containing red fluorescent protein (HEK 293T-RFP cell) were kindly

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provided by Dr. Qiong Wu in School of Life Sciences, Tsinghua University,

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China.

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2.2 Synthesis of PHU10U alkene precursor

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An engineered strain P. entomophila LAC23 developed by this lab was used

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in this study to produce PHU10U based on a cultivation procedure modified

MCL

from

unsaturated

Beijing

Chemical

copolymer

Works

(China).



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from a previously reported method.56 In brief, a 100 mL Erlenmeyer flask with

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20 mL of Luria-Bertani (LB) medium containing 10 g L−1 tryptone, 5 g L−1

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yeast extract, and 10 g L−1 NaCl, was inoculated with P. entomophila LAC23.

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Subsequently, 5% (v/v) seed cultures were inoculated into 4YLB medium

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containing 12 g L-1 tryptone and 24 g L-1 yeast extract, which was co-cultured

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with 2 g L-1 1-undecanol and 2 g L-1 10-undecenoic acid. After cultivated at

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30°C for 48 h in a rotary shaker set at 200 rpm, cells were collected by

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centrifugation at 8000 rpm for 20 min under room temperature, and the

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supernatant was discarded. The collected strains were washed with absolute

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ethanol to remove residual carbon sources, and then collected again by

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centrifugation under the same conditions, and washed with deionized water.

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After drying under lyophilization, PHA were purified via Soxhlet extraction

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followed by ethanol precipitation. Soxhlet extractions were performed in 120

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mL chloroform for 4 h, and then the solution was evaporated under ventilated

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conditions for no less than 5 h. The resulting film was dissolved in a minimal

140

amount of chloroform, and precipitated by a 10-fold volume of ethanol. The

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final white PHA powder was obtained after the freeze drying process, and

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stored at room temperature protected from light until the organogel fabrication

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started.

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2.3 Preparation of photo-polymerized PHU10U/PDT organogels


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“Thiol-ene” reaction, known as click chemistry, was used for the synthesis of

146

PHU10U/PEG organogels.57, 58 Briefly, PHA solution was prepared by

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dissolving 0.100 g PHU10U (0.32 mmol of alkene) in 3 ml anhydrous

148

tetrahydrofuran (THF) in a cylindrical glass bottle at 40℃ under agitation for

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30 min. After complete dissolution, 2 mg photoinitiator DMPA was added

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corresponding to the different molar ratios of cross-linking PDT, including

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equivalents of 0.125, 0.250, 0.375, and 0.5 (0.04, 0.08, 0.12 and 0.16 moles

152

of alkene, respectively) was added. Unless stated, all organogels were made

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at a concentration of 2.5% (w/v). The mixtures were poured into the molds

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(diameter: 2 cm) and exposed to UV irradiation with ultraviolet light (6.9 mW

155

cm−2, 360–480 nm). The gelation time was determined by the vial tilting

156

method. All studies above were repeated for three times. After the gelation, all

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organogel samples were immersed in chloroform for 2 h to remove unreacted

158

substances including photo-initiator. Subsequently, oragnogels without

159

chloroform were placed in large amount of 75% ethanol for at least 48 h,

160

followed by changing 75% ethanol after additional 8 h. After removed 75%

161

ethanol, all organogels were soaked in deionized water for an additional 48 h,

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with deionized water changed every 24 h. The resulting PHU10U oragnogel

163

was lyophilized for 48 h before further characterizations and cytotoxicity

164

studies.



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2.4 Characterizations of PHU10U

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2.4.1 Nuclear Magnetic Resonance (NMR) Characterization

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5-10 mg Purified PHU10U was dissolved in 500 μl deuterated chloroform

168

(CDCl3).1H NMR and

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Bruker 400 MHz spectrometer or a Bruker 800 MHz spectrometer. Its

170

chemical shifts were reported relative to residual protonated solvent.

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Additionally, the ratio of unsaturated bonds was determined by 1H NMR

172

spectra, after being processed by TOSPIN v1.3 from Bruker BioSpin, the ratio

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of the alkene proton signal at 5.78 ppm to the polymer backbone stereocenter

174

at 5.17 ppm was taken as the alkenyl ratio of the copolymer.

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2.4.2 Gel Permeation Chromatography (GPC) Characterization

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GPC was performed as previous described for Mw study.44 In brief, the

177

molecular weights (Mw) and polydispersity of samples were detected by a

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GPC containing Shimadazu LC-20A instrument equipped with a RID-10A

179

refractive index detector and a Shimadzu GPC-804C column using an elution

180

liquid with chromatographically pure chloroform at a flow rate of 1 ml min-1 at

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40°C. 30 μl Sample solutions were prepared at a concentration of 1 mg ml-1 ,

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and undissolved impurities were filtered by 0.22 μm nylon-microporous

183

membrane (JingLong, China). To calculate the molecular weights and

13C

NMR spectra of the PHU10U were obtained on a


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polydispersity index of the samples, a standard calibration curve was

185

established based on polystyrene standards with different number-average

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molecular weights (3 × 103, 5 × 103, 3 × 104, 5 × 104, 1.5 × 105, and 3 × 105;

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Sigma-Aldrich, USA), respectively.

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2.5 Physical characterizations of PHU10U/PDT gels

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2.5.1 Fourier transform infrared (FTIR) spectroscopy

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In order to characterize the insoluble PHU10U/PDT organogels, Fourier

191

transform infrared (FTIR) spectroscopy was examined in an attenuated total

192

reflectance (ATR) mode on a Bruker VERTEX70 FT-IR spectrometer (Horiba,

193

Germany). Data collected were analyzed using OPUS 7.0 software, each

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spectrum consisted of 128 scans from 4000 cm-1 to 500 cm-1 plotted in a

195

transmittance mode. Subsequently, data were re-plotted using Originpro 8 to

196

generate high quality images.

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2.5.2 Morphology of the PHU10U/PDT gels

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To analyze the microstructure of photo-crosslinked PHU10U/PDT organogels,

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FEI Quanta 200 scanning electron microscopy (FEI, America) was used to

200

visually observe interior morphology. The swollen gel samples, having

201

reached their maximum swelling ratio in deionized water at room temperature,

202

were quickly frozen in liquid nitrogen and then lyophilized. Before observation, 11 ACS Paragon Plus Environment


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all of the freeze-dried gel samples were coated with a layer of gold and the

204

images were taken at an accelerated voltage of 10 kV.

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2.5.3 Swelling measurements in THF and deionized water

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The dried photo-crosslinked PHU10U/PDT gels were weighted (weighted as

207

W0) and soaked in either anhydrous THF or deionized water at room

208

temperature. At regular intervals, the samples were taken out and then

209

weighted (weighted as Wt) after removing surface liquid. The swelling ratio (Q)

210

was calculated as followed (1):7

211

Q = (Wt-W0)/W0×100%

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All swelling ratio results were obtained from triplicate measurements and data

213

are expressed as means ± standard deviations (SD).

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2.5.4 PHU10U/PDT gel fractions

215

To determine the extent of the cross-linked network within PHA gels, the gel

216

fraction of each sample was measured after incubation in anhydrous THF.

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Dried PHU10U/PDT samples were swollen for 72 h in anhydrous THF at 25°C,

218

with the solvent changed for 4-6 times every 24 hours. The final swollen gels

219

were freeze-dried and then weighted. The gel fraction (cross-linking density)

220

(Gf) was calculated as followed (2):7

(1)


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Gf = mf/m0×100%

(2)

222

where mf is the dry gel sample weight after anhydrous THF extraction, and m0

223

is the initial weight of the sample.

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All gel fraction results were obtained from triplicate measurements and data

225

are expressed as means ± standard deviations (SD).

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2.5.5 Mechanical property studies

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The compressive mechanical test of the PHU10U/PDT gels was conducted

228

with a 4466 electro-mechanical universal testing instrument (Instron, Norwood,

229

MA). All of the cylindrical hydrogel samples were10 mm in diameter and 5 mm

230

in thickness. Each of the PHU10U/PDT hydrogel was placed in the center of

231

the bottom plate and compressed perpendicularly by the upper plate at room

232

temperature until the hydrogel was fractured. A strain rate (compression) of

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0.1 mm min−1 was employed. The compressive modulus and maximum strain

234

were calculated from the stress–strain curves, which was re-plotted using

235

Originpro 8 to generate high quality images.

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2.6 Cytotoxicity of PHU10U/PDT hydrogels

237

2.6.1 Cell culture



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HEK 293T-RFP cells were cultured at 37℃ in 5% CO2 in Dulbecco’s modified

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Eagle medium (DMEM) (Gibco, USA) containing 10% fetal bovine serum

240

(FBS) (Gibco, USA), 100 units ml-1 penicillin (Biodee, China) and 0.1 mg ml-1

241

streptomycin (Biodee, China). The medium was changed every 2 days. Cells

242

were grown to a minimum of 70% confluence before harvesting.

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2.6.2 Cytotoxicity of PHU10U and PHU10U/PDT hydrogels

244

To evaluate the cytotoxicity of PHU10U/PDT hydrogels towards human

245

embryonic kidney 293T (HEK 293T) cells containing red fluorescent protein

246

(HEK 293T-RFP cell), CCK-8 assay was performed in this study. Briefly, the

247

lyophilized PHU10U/PDT as organogels were completely immersed in 75%

248

alcohol for 12 h, an excessive PBS was used to replace the 75% alcohol

249

solution for at least 24 h and changed every 8 h. After totally removed PBS,

250

the PHA xerogels (without any solvent) were sterilized for 15 min. The

251

sterilized

252

cross-linkages and native PHU10U were incubated in culture medium for 24 h

253

at 37℃, after which the materials were removed and the extraction medium

254

was obtained.

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Initially, HEK 293T-RFP cells with an appropriate cell density (3000 cells well-1)

256

were seeded onto 96-well plates and incubated over a time range of 24–96 h

257

to investigate the effect of incubation time on cell viability. Before seeding, the

photo-crosslinked

PHU10U/PDT

xerogels

with

different


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plates were sterilized with 75% ethanol for 12 h and then washed with PBS for

259

3 times. Cells were cultured in fresh prepared medium without treating any

260

material, which were used as the blank control.

261

After incubations lasting 24, 48, and 96 hours, respectively, on the basic of

262

the manufacturer’s protocol, cell counting kit-8 solution was added to each

263

well, including both samples and controls. After incubation at 37°C for further

264

1 h, the solution absorbance of each well was measured at 450 nm with a

265

Thermo Scientific Varioskan Flash microplate reader (Thermo, America) to

266

evaluate the cell viability quantitatively. The relative cell proliferation (%) was

267

measured using the CCK-8 assay as described earlier, which was calculated

268

according to the equation (3):

269

Relative cell proliferation (%) =

270

[(ODsample−ODextraction solution blank) / (ODcontrol−ODmedium blank)] ×100%

271

(3)

272

where ODsample, ODextraction solution blank, ODcontrol and ODmedium blank

273

represent treated samples with various hydrogels extraction solutions, blank

274

test of the extraction solution (without cells), untreated blank control (cells with

275

normal cultured medium) and blank test of normal cultured medium (without

276

cell), respectively.



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To study cell morphology, the cultured process was conducted similar to the

278

CCK-8 assay with 8000 cells well-1 were seeded into the 24-well plates after

279

incubation at 37℃ with 5% CO2 for 96 h. Subsequently, the living cell

280

morphology and fluorescence images were taken using a Nikon Ti-E inverted

281

microscope (Nikon, Japan).

282

RESULTS AND DISCUSSION

283

3.1 Synthesis and characterization of PHU10U

284

The

285


286

(Figure 1), was synthesized using related carbon sources 1-undecanol and

287

10-undecenoic acid by engineered P. entomophila deleted with its

288

chromosomal β-oxidation related genes. This copolymer was designed and

289

synthesized with sufficient unsaturated monomers to form networked

290

organogels with adjustable degree of crosslinking under photo-crosslinking

291

reaction. The so-resulted PHA generated flexibility for preventing brittleness

292

while retaining sufficient stiffness. This is necessary to meet special

293

requirements of soft tissues such as cartilage or tendon. Based on our

294

previous results,59 MCL PHA copolymer consisting of approximately 50%

295

unsaturated monomers has balanced physical properties and adequate vinyl

296

groups for cross-linking purpose. A series PHU10U copolymers of different

microbial

polymer

containing

alkene

group

termed


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unsaturated ratios from 0-100% could be synthesized by adjusting the carbon

298

source ratio in the fed media. The chemical structure and unsaturated

299

monomer content of PHU10U was revealed by NMR spectroscopy (Figure 2).

300

Double bonds in PHU10U was confirmed by 1H NMR (Figure 2a) with

301

appearances of proton signals at δ = 5.70-5.90 ppm (—CH=CH2—, 2) and δ =

302

4.88-5.07 ppm (—CH=CH2—, 1). Chemical shifts at δ = 5.11-5.26 ppm

303

(—CH—, 9+9’) and δ = 2.41-2.66 ppm (—CH2—, 10+10’) were the typical

304

characteristic peaks of PHA structural backbone. Side chain signals of

305

PHU10U were also confirmed at δ = 0.83-0.95 ppm (—CH3, 1) representing

306

the characteristic hydrogen related to terminal saturated methyl group. On the

307

other hand, the unsaturated ratio of PHU10U of 58.8 mol% was determined

308

by 1HNMR spectra (Figure 2a), in that the ratio of the vinyl proton signal at

309

5.80 ppm to the polymer backbone stereocenter at 5.20 ppm was taken as the

310

unsaturated ratio of the copolymer. Accordingly, the presence of peaks at δ

311

=114.3 ppm (=CH2, 1) and δ =139.0 ppm (—CH=, 2) in 13C NMR spectrum

312

(Figure 2b) was attributed to carbon signals from the double bonds, and

313

peaks around δ =169.4 ppm (C=O, 11+11’) in 13C NMR could be related to

314

carbon signals from the carbonyl groups. All other carbon signals of PHU10U

315

were identified and labelled in the 13C NMR spectrum (Figure 2b). The

316

engineered microbial cells were able to grow to a cell dry weight (CDW) of

317

approximately 4.26 g L-1 containing approximately 45% PHU10U with the 17 ACS Paragon Plus Environment


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number average molecular weight (Mn) and polydispersity (PDI) of 80,665

319

and 1.57, respectively (Table 1).

320

3.2 Synthesis of PHU10U/PDT gels

321

Theoretically, UV radiation with a photo-initiator should allow PHU10U with

322

pendant vinyl moiety to proceed free radical crosslinking reactions.59 This

323

study first employed 2,2-dimethoxy-2-phenylacetophenone (DMPA) (e.g.

324

0.5wt%) as the photo-initiator. However, it could not form a networked gel

325

easily. A long time UV radiation resulted in an analogous gel which was easily

326

dissociated possibly due to the loose networked structure.60


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327 328

Figure 2. NMR spectra of PHU10U containing 58.8% vinyl group

329

(a) 1H NMR spectrum and (b) 13C NMR spectrum.

330

Previous study demonstrated that the photo-crosslinked reactivity of the

331

pendant unsaturated PHA depends on the polymer properties and gelation 19 ACS Paragon Plus Environment


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time.59 Although unsaturated PHA has possibility to form an analogous gel

333

film by itself, it is difficult to form 3D organogels or hydrogels itself. To

334

synthesize PHU10U 3D gels, a cross-linker polyethylene glycol dithiol (PDT)

335

was added to facilitate photo-crosslinking of PHU10U (Figure 1), together with

336

0.1wt% DMPA serving as the photo-initiator to trigger cross-linking in an

337

organic solvent under 5 min UV irradiation (Scheme 1). PDT with Mn=2000

338

and PDI=1.05 was used as cross-linker to form PHU10U/PDT solution

339

(Scheme 1a), various transparent PHU10U/PDT gels with different molar

340

ratios of PDT were generated under UV radiation, respectively (Scheme 1b).

341

While with a Mn of 8000 for PDT, PHU10U/PDT could only form a gel after the

342

gelation time increased to 30 min. In this case, the resulted gels were loose

343

and quickly disappeared in THF or H2O.

344 345

Scheme 1. Thiol-ene cross-linking of PHU10U/PDT organogels.

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(a) PHU10U/PDT and DMPA mixture before photo-crosslinking reaction (b) a

347

PHU10U/PDT transparent gel obtained after 5 min of UV irradiation. 20 ACS Paragon Plus Environment

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Properties of PHU10U/PDT gels including the interior morphology, swelling

349

behavior in THF or water, and compressive stress were investigated. They

350

were found to depend on the PHU10U/PDT molar feed ratio (Table 1).

351

Table 1. Gel fraction (cross-linking density) of PHU10U/PDT gels related to

352

ratios of double bonds and thio-groups Sample

Mn

PDI

Molar ratio of >C=C< and -SH

PHU10U

80665

1.57

100: 0

0

PDT

2000

1.05

0: 100

0

1: 0.25

25

1: 0.50

47

1: 0.75

73

1: 1

98

Gf (%)

PHU10U/PDT1 PHU10U/PDT2 PHU10U/PDT3 PHU10U/PDT4 353

Gf: Gel fraction (cross-linking density). Five minutes UV radiation (6.9 mW cm−2, 360–480

354

nm) to PHU10U/PDT dissolved in THF at room temperature.



Page 22 of 58

355

3.3 Characterization of organogels

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ATR-FTIR spectra were used to confirm the successful photo-crosslinking

357

between PHU10U and PDT (Figure 3A). The spectra clearly illustrated the

358

gels of different PHU10U/PDT molar ratios in comparison to the PHU10U

359

precursor (Figure 3A). Absorption peaks located at around 2921, 2851, and

360

1721 cm−1 represent unique PHA characteristic peaks corresponding to the

361

C-H stretching vibrations of –CH3, –CH2 and ester carbonyl (>C=O) in the

362

PHU10U polymer molecules, respectively. Furthermore, Typical peaks at

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3078 and 1641 cm−1 represent strong −CH=CH2 and C=C stretching vibration

364

in PHU10U, respectively. After photo-crosslinking, all IR spectra of the

365

samples appeared only slightly different from each other due to different molar

366

ratios of the crosslinked PDT. A decreasing vinyl signal in the FT-IR spectrum

367

at 3078 cm-1 (Figure 3B) and 1641 cm−1 (Figure 3C) were considered as an

368

evidence for the conversion of the vinyl group to the thio-ether product. With

369

increasing molar ratio of PDT, characteristic IR absorption peaks of CH=CH2

370

at 3078 cm-1 tended to decrease and disappeared when the PHU10U/PDT

371

molar feed ratio was 1:0.5 (Figure 3B). Meanwhile, absorption peaks at 1641

372

cm-1 representing C=C were also reduced with increasing molar ratios of

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PDT, IR spectra of all gel samples generated a new and strong peak at 1115

374

cm-1 (Figure 3A b-e) compared to IR data of PHU10U (Figure 3Aa), indicating


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the stretching vibration of the ether group C-O-C from PDT. All the IR data

376

indicated that PHU10U and PDT had been successfully photo-crosslinked to

377

form gels.

378 379

Figure 3. FTIR spectra of (A) PHU10U and PHU10U/PDT gels with different

380

molar ratio of PDT, (B and C) Enlargements of (A) at peaks ranging from

381

3040-3110 cm-1 to 1620-1660 cm-1. (a) PHU10U (b-e) PHU10U/PDT gels at

382

different molar ratios of PHU10U to PDT (b) 1: 0.125 (c) 1: 0.25 (d) 1: 0.375 (e)

383

1: 0.5.

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3.4 Morphology and gel fraction 23 ACS Paragon Plus Environment


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385

The interior morphology of a gel is vital for biomedical application as it

386

associates with nutrients or small molecules transports and communication of

387

biological moieties that are significant for potential gel tissue scaffolds.61 To

388

understand

389

cross-sections of the gels were studied under scanning electron microscopy

390

(SEM). Generally, all the freeze-dried gels displayed porous network

391

structures (Figure 4). The average pore size of these honeycomb-like porous

392

structures decreased with increasing amounts of PDT as a cross-linker. Molar

393

ratios of PHU10U to PDT were found to control the pore size of hydrogels. For

394

example, PHU10U/PDT-1 hydrogel with the largest molar ratio of PHU10U to

395

PDT showed the largest pore size (Figure 4a), with an average diameter of

396

34.8 μ m. While PHU10U/PDT-4 hydrogel with the smallest molar ratio of

397

PHU10U to PDT with 1: 0.25, possessed the smallest pore size with an

398

average diameter of 8.0 μm (Figure 4d). The interior morphological data are

399

consistent with the effect of the different ratios of PHU10U to PDT on

400

compressive moduli data discussed above (Figure 4).

401

In addition, this study suggested that the pore size of PHA gels was correlated

402

to the actual cross-linking density (Figure 4 and Table 1). For example, with a

403

gel fraction 98%, this gel was found to have the smallest pore size. When the

the

structure–function

relationship

of

PHU10U/PDT

gels,


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404

gel fraction was only 25% which was the smallest cross-linking density, the

405

pore size was the largest.

406 407 408

Figure 4. SEM images of PHU10U/PDT gels at different molar ratios of PHU10U to PDT (a) 1: 0.125 (b) 1: 0.25 (c) 1: 0.375 (d) 1: 0.5.

409

3.5 Swelling performance of PHU10U/PDT gels

410

Generally, organogels or hydrogels can demonstrate swelling ability, which is

411

very important for biomedical applications as drug delivery carriers and

412

implant scaffolds due to their elastic physical and chemical properties

413

including external and internal diffusion property.62 The swelling kinetics of

414

PHU10U/PDT xerogels in water (Figure 5A) and THF (Figure 5B) were

415

investigated for 30 h. All the PHU10U/PDT gels in distilled water generally

416

showed a low swelling capacity because of its relatively hydrophobic



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417

backbone of PHU10U, the swelling equilibrium ratio in distilled water showed

418

that the equilibrated swelling ratios at room temperature depended on the

419

molar ratio of PHU10U to PDT as hydrogels (Figure 5A). All PHU10U/PDT

420

hydrogels swelled rapidly in distilled water during the first 8 h, followed by

421

slow expansion (Figure 5Aa-d). All hydrogels reached their swelling

422

equilibrium within approximately 24 h, with swelling values ranging from 80%

423

to 160% (Figure 5C). The PHU10U/PDT hydrogels with a molar ratio of

424

PHU10U to PDT at 1: 0.125 showed the highest swelling equilibrium value

425

(160%). As the molar ratio of PHU10U to PDT decreased from 1:0.125 to

426

1:0.5, the hydrogel swelling equilibrium declined due to the increasing

427

cross-linking density. Moreover, with increasing swelling equilibrium values,

428

PHU10U/PDT became more and more transparent. Interestingly, the relatively

429

high cross-linking density of PHU10U/PDT hydrogels (Figure 5Ac and d)

430

reached equilibrated swelling status within 6 h, quicker than the other two

431

studies (Figure 5Aa and b), implying that the incorporation of PDT segment

432

into the hydrophobic PHU10U enhanced the hydrophilicity, thus increasing the

433

swelling of the PHU10U hydrogels with higher PDT ratio. In other words, the

434

increasing ratios of hydrophilic PDT enlarged the swelling capacity of

435

PHU10U/PDT hydrogels in distilled water partially.


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PHU10U/PDT demonstrated organogel properties with different molar ratios

437

of PHU10U to PDT when placed in THF at room temperature up to 30 h

438

(Figure 5B). Generally, all PHU10U/PDT organogels had similar swelling

439

performances in THF solvent: an initially sharp increase of swelling ratio

440

during the first 2 h, followed by a rather slow increase. The fast swelling

441

kinetics and high organic solvent retention capability of these organogels are

442

attributed to their hydrophobic and cross-linking natures. For example, a

443

PHU10U/PDT organogel with a molar ratio of PHU10U/PDT of 1:0.125

444

presented the highest equilibrium swelling ratio in THF up to approximately

445

6000% (Figure 5E). Even more, when a PHU10U/PDT xerogel with a molar

446

ratio of PHU10U/PDT of 1:0.125 was immersed in THF, it became opaque

447

and small (Figure 5D). After reaching the swelling equilibrium, this organogel

448

became transparent with enough solvent in its larger expanded size

449

compared to the initial xerogel (Figure 5E). The PHU10U/PDT organogel with

450

a molar ratio of 1:0.5 was revealed to have the smallest swelling ratio of

451

around 700%, while the swelling ratios of all PHU10U/PDT organogels in THF

452

were much higher than that of PHU10U/PDT hydrogels in distilled water. The

453

effect of PHU10U/PDT molar ratio on swelling ratio in THF was also found to

454

be consistent with that in the distilled water. This trend could also be observed

455

in the mechanical studies with the increased crosslinking densities of the

456

PHU10U/PDT hydrogel networks as described below. 27 ACS Paragon Plus Environment


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457 458

Figure 5. Swelling kinetics of photo-crosslinked PHU10U/PDT.

459

(A) hydrogels (B) organogels (C) Optical images of PHU10U/PDT hydrogels

460

with different molar ratio of PDT after reaching their swelling equilibrium (D-E)

461

Typical optical image of PHU10U/PDT organogels at a ratio of PHU10U to

462

PDT of 1: 0.125 in THF (D) Xerogel to (E) organogel which reached its

463

swelling equilibrium (a-d) PHU10U/PDT organogels and hydrogels at different

464

molar ratios of PHU10U to PDT: (a) 1: 0.125 (b) 1: 0.25 (c) 1: 0.375 (d) 1: 0.5.

465

3.6 Mechanical property of PHU10U/PDT hydrogels

466

Mechanical properties of PHU10U/PDT hydrogels are affected by the

467

cross-linking density, swelling ratio and rigidity of the polymer networks.

468

Typical stress-strain of PHU10U/PDT hydrogels with different molar ratio of

469

PHU10U to PDT could be illustrated (Figure 6). The hydrogel at molar ratio of


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Biomacromolecules

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PHU10U to PDT of 1: 0.125 showed its maximum fracture strain up to around

471

60%. After incorporating PDT with different molar ratios to these hydrogel

472

systems, the increased crosslinking density of PHU10U/PDT hydrogels led to

473

brittleness with their fraction strain declining from 60% to 42% (Figure 6a-d),

474

similar trend of swelling behavior for those hydrogels were observed in

475

distilled water. On the other hand, the qualitative compressive moduli based

476

on these stress-strain curves significantly increased after incorporation of PDT

477

with increased molar ratio in the PHU10U/PDT hydrogels, indicating that the

478

increased crosslinking densities of the PHU10U/PDT hydrogel networks with

479

the decreased molar ratio of PHU10U to PDT, resulting in rigidity and stiffness

480

for the hydrogels. Previous studies indicated that the crosslinking density of

481

these PHU10U/PDT hydrogels also influenced their swelling ratios, implying

482

that the swelling ratios of these hydrogels became more limited as the

483

crosslinking density increased. A similar trend among the crosslinking density,

484

swelling ratio and compressive moduli were reported in a relatively

485

hydrophobic hydrogels obtained from pseudo-poly (amino acid) and

486

polyethylene glycol diacrylate (PEGDA) precursors.63



Page 30 of 58

487 488

Figure 6. Stress–strain of PHU10U/PDT hydrogels.

489

(a-d) Different molar ratios of PHU10U/PDT (a) 1:0.125 (b) 1:0.25 (c) 1:0.375 (d)

490

1: 0.5.

491

3.7 Cytotoxicity of PHU10U and PHU10U/PDT hydrogels

492

As a tissue scaffold or an implant, its constitutive material should be

493

biocompatible without releasing toxic substances for adverse reactions, it

494

should also not lead to the malformation of cultured cells. This can be verified

495

via in vitro cytotoxic tests.8 In this study, the cytotoxicity of PHU10U and

496

PHU10U/PDT hydrogels were evaluated based on the CCK-8 assay.

497

Meanwhile, to solve the invisible problems in the CCK-8 assay, cell

498

morphology was characterized. The result of relative cell proliferation using

499

CCK-8 assay indicated that the cell viability had no significant decrease

500

compared to the blank control without any treatment in the first 24 h (Figure 7).

501

Namely, more than 95% cells incubated in different materials extraction media

502

were able to grow or proliferate. During the additional 48 h cultivation, 30 ACS Paragon Plus Environment

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503

PHU10U, PHU10U/PDT-1 (Figure 7a), PHU10U/PDT-2 (Figure 7b) and

504

PHU10U/PDT-3 (Figure 7c) were not statistically significantly different from

505

the blank control at p>0.05 (unpaired t test) for cell growth supports,

506

respectively, while PHU10U/PDT-4 showed a significant difference with the

507

blank control with p

Synthesis and Characterization of Polyhydroxyalkanoate Organo

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