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Supplementary Information

Self-assembled Peptidic Nanomillipede to Fabricate Tuneable Hybrid Hydrogel

Jianhui Liu a, Rong Ni a, b and Ying Chau*, a

a Department of Chemical and Biological Engineering, the Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kongb Institute for Advanced Study, the Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong

Corresponding Author* Email: keychau@ust.hk (Y.C.)

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2019

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Table of Contents

1 Supplementary Figures.................................................................................3

2 Supplementary Tables ................................................................................34

3 Experimental Procedures ...........................................................................35

4 References ....................................................................................................42

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1 Supplementary Figures

Fig. S1 Molecular structure of (a) nNSAP and (b) cNSAP. Hydrophobic and hydrophilic residues are marked with yellow and blue colour, respectively. The peptide sequence was designed based on a well-characterized amyloid-inspired β-sheet forming self-assembling peptide. cNSAP contains thiol (SH) group at the N-terminus of nNSAP. The hydrophobic intermolecular interactions, pi-pi stacking, and hydrogen bonding contribute to the self-assembly process. [1]

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Fig. S2 Molecular structure of: (a) snNSAP and (b) scNSAP. Hydrophobic and hydrophilic residues are marked with yellow and blue colour, respectively. Compared with the self-assembling peptide, the hydrophobic residues within the scramble peptide are much more evenly distributed to significantly diminish the hydrophobic driving force.

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Fig. S3 MALDI mass spectroscopy of (a) nNSAP: Theoretical MW 1891; Measured MW 1891. ([M – H + Na+] = 1913) (b) cNSAP: Theoretical MW 1994; Measured MW 1994. ([M – H + K+] = 2032) (c) snNSAP: Theoretical MW 1891; Measured MW 1891. ([M – H + Na+] = 1913) (d) scNSAP: Theoretical MW 1994; Measured MW 1994. ([M – H + Na+] = 2016).

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Fig. S4 Concentration dependent ATR-FTIR spectrum of self-assembling peptide (48 h incubation): (a) nNSAP and (b) cNSAP. The absorbance intensity peak near 1624 cm-1 (amide Ⅰ stretch peak) and the shoulder near 1695 cm-1 indicate the well-defined antiparallel β-sheet architecture.

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Fig. S5 Concentration dependent ATR-FTIR spectrum of scrambled self-assembling peptide (48 h incubation): (a) snNSAP and (b) scNSAP. The peak at 1640 cm-1 within the amide Ⅰ region indicates random coil architecture, where peptides are oriented randomly and incorporate no ordered component.

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Fig. S6 Concentration dependent CD spectrum of self-assembling peptide (48h incubation): (a) nNSAP and (b) cNSAP. The positive peaks around 198 nm and the negative peaks around 218 nm exist for both nNSAP and cNSAP, which indicate well-defined β-sheet architecture.

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Fig. S7 Concentration dependent CD spectrum of scrambled self-assembling peptide (48h incubation): (a) snNSAP and (b) scNSAP. The negative peak near 200 nm indicates random coil structure, where peptides are oriented randomly and incorporate no well-defined ordered component.

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Fig. S8 AFM images of freshly prepared nanomillipedes. (scale bar: 200nm) The peptide solution was diluted 100 times for sample preparation.

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Fig. S9 TEM images of mature nanomillipede (low magnification, scale bar: 200nm).

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Fig. S10 ATR-FTIR spectrum of peptidic nanomillipede.

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Fig. S11 CD spectrum of peptidic nanomillipede.

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Fig. S12 ATR-FTIR spectrum of scrambled SAP mixture.

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Fig. S13 CD spectrum of scrambled SAP mixture.

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Fig. S14 Tryptophan fluorescence measurement with different incubation time (a) peptidic nanomillipede and (b) scrambled SAP mixture.

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Fig. S15 Tryptophan fluorescence measurement with different incubation time (a) peptidic nanomillipede and (b) scrambled SAP mixture.

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Fig. S16 The incubation of ultrasound-treated (30-min) nanomillipede in viscous buffer for (a) 0h (b) 12h and (c) 48h before TEM characterization. Scale bar: 100 nm. The peptide solution was diluted 1000 times for TEM sample preparation.

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Fig. S17 SEM image of VS-Dex peptide hybrid hydrogel (Low resolution, scale bar: 50 µm). Components: VS-Dex (DM=8%, concentration=5 wt%, pH=7.4) and peptidic nanomillipede (total peptide concentration=10 mM, pH=7.4).

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Fig. S18 FTIR spectrum of freeze-dried hybrid hydrogel.

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Fig. S19 FTIR spectrum of (VS-Dex)-scrambled SAP mixture.

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Fig. S20 Dynamic strain sweep test. (ω = 10 rad/s) Yield point: γy= 30%.

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Fig. S21 Dynamic frequency sweep test. (strain γ = 2%)

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Fig. S22 Shear-thinning property. (γ = 2%)

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Fig. S23 Step-strain measurement. Alternative low strain (γ = 2%, normal state) and high strain (γ = 150%, disruption, much higher than γy) were applied to the hydrogel.

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Fig. S24 A possible mechanism proposed for the self-healing process.

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Fig. S25 Dynamic frequency sweep test of hybrid hydrogels. Strain γ = 2%.

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Fig. S26 Viscosity-frequency curve of hybrid hydrogels. Strain γ = 2%.

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Fig. S27 Cycling step-strain measurement for different hybrid hydrogels. Alternative low strain (γ = 2%, normal state) and high strain (γ = 150%, disruption, much higher than γy) were applied to the hydrogel.

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Fig. S28 Dynamic frequency sweep test of hybrid hydrogels with different value. Strain γ 𝑅𝑐

=2%.

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Fig. S29 1H NMR spectrum of VS modified dextran with different reaction time: (a) 30s (b) 60s (c) 120 s

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Fig. S30 Controlling DM of VS modified dextran by varying reaction time.

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2 Supplementary Tables

Tab. S1 Gelation condition examination of hybrid hydrogel. (−: No hydrogel formation within 48h. +: Hydrogel forms within 5 minutes.)

[a] 10 wt% unmodified dextran (2000 kDa) was added instead of VS-modified dextran. [b] 5 mM snNSAP and 5 mM scNSAP were added instead of 5 mM nNSAP and 5 mM cNSAP.

Experiment (Exp.)

1 2 3 4 5 6 7 8 [a] 9 10 11 [b]

VS-Dex (wt%) 10 % 10 % 10 % 10 % 10 % 5 % 0 0 10 % 10 % 10%

nNSAP (mM) 0.05 0.5 1 2 5 5 5 5 0 10 0

cNSAP (mM) 0.05 0.5 1 2 5 5 5 5 0 0 0

Result − − − + + + − − − − −

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3 Experimental Procedures

Materials

Fmoc-protected amino acids (Fmoc-Cys-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp-OH, Fmoc-Leu-

OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Gln-OH, Fmoc-Gly-OH, Fmoc-Ser-

OH, Fmoc-Pro-OH, Fmoc-Asp-OH) and rink amide resins were purchased from GL Biochem

(Shanghai) Corporation. Ltd. Fmoc-Hexacid-OH was purchased from Binhai Hanhong

Biochemical Co., LTD. (China). The rest of chemicals used were purchased from Sigma-

Aldrich (USA) without specific statement.

Characterization

Nuclear magnetic resonance (NMR) spectroscopy

The 1H NMR spectrometry was performed on 300 MHz High Resolution NMR spectrometer

Varian Mercury 300 VX (Varian, USA) using D2O (99.9 atom % D) as the solvent.

Mass spectrometry (MS)

The molecular weight (MW) of peptide was determined by MALDI TOF/TOF

ultrafleXtremeTM Mass Spectrometer (Bruker Corporation, USA). The sample volume is 100

µL and the concentration is about 100 µM.

Tryptophan fluorescence measurement

The fluorescence emission spectrum was measured at 25 ℃ by microplate reading. 100 µL

peptide solution was added to the 96-well black microplate (Greiner Bio-One International

GmbH, Germany) and loaded to microplate reader (Varioskan LUX Multimode Microplate

Reader, Thermo Fisher Scientific, USA). The excitation wavelength was set to be 280 nm and

the emission wavelength range was set to be 300 – 550 nm. The step of scanning was set to be

1 nm.

Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy

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The secondary structure of peptide was characterized by ATR-FTIR spectroscopy. The ATR

accessory (GladiATR Vision, PIKE Technologies, USA) was connected to FTIR spectrometer

(Vertex 70 Hyperion 1000, Bruker Corporation, USA). For the peptide solution sample, 15 µL

solution was dropped on the surface of diamond crystal and then dried with air purge until a

uniform semi-transparent film was formed, while the buffer background was subtracted before

the ATR-FTIR characterization. For the hybrid hydrogel sample, the freeze-drying process was

carried out overnight and 20 mg sample was positioned on the diamond crystal, while the air

background was subtracted before the ATR-FTIR characterization. The resolution of spectrum

was set to be 4 cm-1 within 4000 – 400 cm-1. The spectra were collected with an average of 128

scans.

Circular dichroism (CD) spectroscopy

The secondary structure of peptide was characterized with circular dichroism (CD)

spectrophotometer (J-815 circular dichroism spectrophotometer, JASCO, Japan). 25 µL sample

was loaded carefully to o-shaped CD cuvette (UV quartz, 0.1 mm light path, FireflySci, USA).

The scan was carried out at room temperature after subtracting the buffer background. The

spectrum was collected within the range of 300 nm – 190 nm with an average of 3 scans. The

step of scan and scan speed were set to be 0.5 nm and 100 nm/s, respectively.

Transmission electron microscopy (TEM)

The microscopic morphology was characterized by transmission electron microscope (JEM-

2100 transmission electron microscope, JEOL Ltd., Japan) using an acceleration voltage of 200

kV. The surface of TEM grids (400 mesh, copper supported carbon grids, Ted Pella Inc., USA)

were cleaned using plasma cleaner (Harrick Scientific Products Inc., USA) for 2 min before

sample preparation. 6 µL solution was dropped on the surface of a parafilm, after which the

droplet was covered by the carbon surface of the TEM grid. The excess sample was removed

with filter paper after 2 min. Then 6 µL negative staining solution (2 wt% uranyl acetate) was

dropped on the surface of the parafilm, followed by covering the carbon surface of TEM grid

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on the droplet. The excess negative staining solution was removed with filter paper after 2 min.

The TEM grid was left overnight for complete desiccation before imaging. In this work, the

sample was diluted 1000 times before sample preparation.

Atomic force microscopy (AFM)

The microscopic morphology of peptide was imaged by AFM (Dimension 3100, Veeco

Instruments Inc., USA). 20 µL of sample was applied to freshly cleaned mica (Ted Pella Inc.,

USA) and incubated for 2 min, followed by wicking off the excess sample solution with filter

paper and rinsing the surface with distilled water. The treated AFM mica was left overnight for

complete desiccation before imaging. The scan rate was set to be 0.5 Hz. In this work, the

sample was diluted 100 times before AFM sample preparation.

Scanning electron microscopy (SEM)

The microscopic morphology of hydrogel was characterized by SEM (JSM-6390, JEOL Ltd.,

Japan) at an accelerating voltage of 20 kV. 20 µL hydrogel precursor was dropped on the

surface of double-sided carbon conductive adhesive tapes (Agar Scientific Ltd., UK), followed

by affixing the tape to an aluminium SEM stage. The sample was left overnight for complete

gelation. The SEM stage was then shock-frozen with liquid nitrogen for 30 min and freeze-

dried overnight. The sample was then coated with a thin layer of gold nanoparticles with SEM

sputter coater (Scancoat Six, Edwards, UK) for 15 min before imaging.

Dynamic mechanical analysis (DMA)

The mechanical properties of hydrogels were studied by DMA with rheometer (ARES-3, TA

Instruments, USA). 50 µL hydrogel sample (pie-like sample, diameter 8mm, thickness 1mm)

was transferred to an 8mm parallel plate transducer and the gap between plates was set to be 1

mm. Hybrid hydrogel components contain VS-Dex (DM=8%, concentration=5 wt%, pH=7.4)

and peptidic nanomillipede (total peptide concentration=10 mM, pH=7.4) except specification.

Each experiment was repeated at least 3 times until stable data was obtained. For shear-thinning

property, we define viscosity attenuation factor as Equation S1:

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(S1)∆𝑁𝜂 =

𝜂𝜔 = 0.1 𝑟𝑎𝑑/𝑠

𝜂𝜔 = 100 𝑟𝑎𝑑/𝑠

Quantitative analysis of peptidic nanomillipedes

The number and length of peptidic nanomillipedes of each sample were analysed using

Gwyddion 2.50 software.

Vinyl sulfone modified dextran synthesis

Dextran is a branched polysaccharide composed of repeating glucose molecules, [2] which is

widely used in biomedical and pharmaceutical engineering due to satisfying biocompatibility

and biodegradation properties. [3] The hydroxyl group (OH) of dextran was modified by the

nucleophilic conjugation addition with divinyl sulfone (DVS) under alkaline conditions. [4] This

reaction mechanism can be controlled by increasing DVS to OH molecular ratio and optimizing

reaction conditions (pH and time), thereby generating VS modified dextran backbone. This

reaction is highly pH dependent, which was terminated by adjusting the pH from alkaline

conditions to acid conditions. The degree of modification (DM) was controlled by varying

reaction time, which is defined as Equation S2: [5]

(S2)𝐷𝑀 =

𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑉𝑆 𝑀𝑜𝑑𝑖𝑓𝑖𝑒𝑑 𝑂𝐻 𝐺𝑟𝑜𝑢𝑝𝑇𝑜𝑡𝑎𝑙 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑅𝑒𝑝𝑒𝑎𝑡𝑖𝑛𝑔 𝑈𝑛𝑖𝑡𝑠

5 g Dextran ((MW 2000 kDa) was dissolved in 100 mL H2O, followed by rigorous vortex,

magnetic stirring and centrifugation to form homogenous solution. 1 M NaOH was then used

to adjust the solution to alkaline condition (pH=12.3, [OH−]=0.02 M). Divinyl sulfone (DVS)

was dropped instantly into vigorously stirring alkaline polymer solution at a molar ratio of 1.2

times the hydroxyl groups of dextran. After certain reaction time, the reaction was terminated

by adding 1 M HCl instantly to adjust the pH to 5. The modified dextran was then transferred

to dialysis bag (Spectra/Por Dialysis Membrane, MWCO:8000, Spectrum Labs, USA) against

ultrapure water for further one-week dialysis purification and 3-day freeze-drying. DM was

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calculated from 1H NMR spectra by comparing the integral of signals at δ=6.9 and at δ=6.4

(Fig. S29), while DM and reaction time indicate strong linear relationship (Fig. S30). VS

modified dextran with certain DM was then used for further hybrid hydrogel preparation.

Peptide synthesis and purification

All peptides synthesis was carried out using microwave-assisted peptide synthesizer (Biotage,

Sweden), which is based on Fmoc solid phase peptide synthesis. The resin was transferred to

peptide synthesis vessel (P140025M, Kemtech Inc., USA) after the synthesis, followed by the

cleavage cocktail (95 v% trifluoroacetic acid, 5 v% triethylsilane) treatment for 2 hours. The

filtrate was then mixed with 4 ℃ cold ether to form precipitation, which was further washed by

cold ether for 3 times. The peptide crude was transferred to vacuum desiccator for 0.5 h for

further solvent evaporation.

Peptide crude was dissolved in acetonitrile (ACN) aqueous solution, followed by centrifugation

and supernatant collection. It was further purified using high performance liquid

chromatography (HPLC, Waters Corporation, USA) on HPLC column (Vydac C18 column,

19×250 mm, Hichrom Limited., UK). Gradient was set to be 2 %/min and flow rate was set to

be 10 ml/min. The ACN was then removed by rotate evaporation, followed by liquid nitrogen

shock-frozen and lyophilization. The molecular weight identification of purified peptide was

done by mass spectrometry.

Preparation of peptidic nanomillipede

Normal nanomillipede-forming self-assembling peptide (nNSAP) and cysteine-terminated

nanomillipede-forming self-assembling peptide (cNSAP) were synthesized following the

aforementioned peptide synthesis method. Then nNSAP and cNSAP were dissolved in HFIP at

the concentration of 10 mM to disrupt the preformed β-sheet peptide aggregates. After 2-hour

HFIP treatment, the solution was transferred to vacuum desiccator for solvent evaporation. The

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dried peptide was then dissolved in ultrapure water, followed by adjusting pH to 7.4 using 4-(2-

hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) stock solution (100 mM, pH=8.5) to

finalize 10 mM HEPES solution. The peptide co-assembly was then incubated in HEPES

solution for 48h to form mature thiol-containing peptidic nanomillipede crosslinker. The hybrid

hydrogel network formation is based on the Michael addition reaction between vinyl sulfone

(VS) group and thiol (SH) group. The final concentration of peptide is 10 mM, including nNSAP

and cNSAP. We define the ratio of cysteine-containing peptide within nanomillipede as

Equation S3:

(S3)𝑅𝑐 =

𝑛𝑐𝑁𝑆𝐴𝑃

𝑛𝑛𝑁𝑆𝐴𝑃 + 𝑛𝑐𝑁𝑆𝐴𝑃

Where and represent number of moles of cNSAP and nNSAP, respectively. This is 𝑛𝑐𝑁𝑆𝐴𝑃 𝑛𝑛𝑁𝑆𝐴𝑃

the parameter to characterize the crosslinking site density for nanomillipedes.

Tuning length of peptidic nanomillipede through ultrasound treatment

1 mL of aforementioned peptidic nanomillipede solution was prepared in 1.5 mL vial. Each

peptide sample was treated with an ice-bathed ultrasound cleaner (maximal power setting,

Powersonic P1100D-45, Crest Ultrasonics Corporation, USA) for certain time before TEM

sample preparation and hydrogel formation.

Preparation of hybrid hydrogel

VS modified dextran (DM=8%) was dissolved in preformed peptidic nanomillipede solution,

followed by rigorous stirring to form homogenous precursor solution. The precursor was then

transferred to predesigned mould which was placed in sealed constant humidity chamber. The

precursor was incubated (25 ) for 72h for complete gelation. For hybrid hydrogel (5 wt% VS-℃

Dex, DM=8%), the concentration of VS group is:

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𝑐𝑉𝑆 =

50162

× 8% 𝑚𝑜𝑙

1𝐿= 24.7 𝑚𝑀

This value is excess compared with nanomillipedes (10 mM) with different value, thereby 𝑅𝑐

indicating the crosslinking reaction is thiol group (nanomillipede)-limiting step, which is

consistent with the ‘length changing’-based mechanical tuning and value-based mechanical 𝑅𝑐

tuning.

Gelation condition examination

VS modified dextran (VS-Dex) (DM=8%) was added as the polymer backbone. The VS-Dex

was then mixed with preformed nanomillipedes to prepare 1 mL hybrid hydrogel precursor

solution. The gelation condition was determined by inverted vial (1.5 mL) test to examine

whether the gel-like material can self-support.

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4 References

[1]. R. Ni and Y. Chau, J. Am. Chem. Soc., 2014, 136, 17902–17905.

[2]. M. Yalpani and P. O. Hedman, Crit. Rev. Biotechnol., 1985, 3, 375–421.

[3]. Q. Cai, Y. Wan, J. Bei and S. Wang, Biomaterials, 2003, 24, 3555–3562.

[4]. E. J. Oh, K. Park, K. S. Kim, J. Kim, J. A. Yang, J. H. Kong, M. Y. Lee, A. S. Hoffman

and S. K. Hahn, J. Control. Release, 2010, 141, 2–12.

[5]. Y. Yu and Y. Chau, Biomacromolecules, 2012, 13, 937–942.