-
Supporting Information
Self-assembling peptide hydrogels functionalized with LN- and
BDNF- mimicking epitopes synergistically
enhance peripheral nerve regeneration
Shuhui Yang1*, Chong Wang2*, Jinjin Zhu3*, Changfeng Lu2,4,
Haitao Li2, Fuyu Chen2, Jiaju Lu1, Zhe Zhang1,
Xiaoqing Yan1,5, He Zhao1, Xiaodan Sun1, Lingyun Zhao1, Jing
Liang6, Yu Wang2, Jiang Peng2, and Xiumei
Wang1
1. State Key Laboratory of New Ceramics and Fine Processing, Key
Laboratory of Advanced Materials of
Ministry of Education, School of Materials Science and
Engineering, Tsinghua University, Beijing 100084,
China
2. Institute of Orthopedics, Chinese PLA General Hospital,
Beijing 100853, China; Co-innovation Center of
Neuroregeneration, Nantong University, Nantong, Jiangsu Province
226007, China
3. Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital,
Zhejiang University School of Medicine
& Key Laboratory of Musculoskeletal System Degeneration and
Regeneration Translational Research of
Zhejiang, Hangzhou 310016, China
4. Department of Orthopaedics and Trauma, Peking University
People’s Hospital, Beijing 100191, China
5. School of Clinical Medicine, Tsinghua University, Beijing
100084, China
6. Department of Pediatrics, Tianjin Hospital, Tianjin
University, No. 406 Jiefang Nan Road, Tianjin 300211,
China
* Contributed equally to this work
Corresponding author: Xiumei Wang, E-mail:
[email protected], Phone: 86-10-62782966. Fax:
86-10-62771160. Jiang Peng, E-mail: [email protected]. Yu
Wang, E-mail:
[email protected].
mailto:[email protected]:[email protected]:[email protected]
-
Methods
Cell proliferation assay
The proliferation of RSCs on different hydrogels was evaluated.
50 μL of peptide solution was loaded
directly into the wells of a 96-well plate, and then 100 μL of
culture medium was gently added onto the peptide
solution. After incubation for 15 min at 37 °C, the medium was
removed and changed at least twice to equilibrate
the hydrogels. RSCs were seeded on the surface of the hydrogel
at a density of 8 × 103 cells/well (n = 4), and
the medium was changed with fresh medium every 2 days. Cell
proliferation in different groups was assessed
using Cell Counting Kit-8 (CCK-8, DOJINDO, Tokyo, Japan). After
culture for 1, 4, and 7 days, the samples
were incubated with 10% CCK-8 working solution in cell culture
medium for 3 h at 37 °C. Then, 100 μL of the
supernatant was transferred to a new 96-well plate, and the
absorbance was measured at 450 nm using an EnSpire
Multimode Plate Reader (PerkinElmer, USA).
-
Table S1. Primer sequences used for quantitative RT-PCR
Gene Primers (F=forward, R=reverse)
GAPDH F: ATGATTCTACCCACGGCAAG
R: CTGGAAGATGGTGATGGGTT
NGF F: CGCTCTCCTTCACAGAGTTTT
R: GACATTACGCTATGCACCTCAGA
BDNF F: TCTACGAGACCAAGTGTAATCCCA
R: CTTATGAACCGCCAGCCAAT
CNTF F: ATGGCTTTCGCAGAGCAAAC
R: CAACGATCAGTGCTTGCCAC
IGF-2 F: GAACAACAATAGCCGCCCAAACTC
R: CATGTTCTGTTCCTCTCCTTGGGT
S100 F: GTTGCCCTCATTGATGTCT
R: CTGCTCTTTGATTTCCTCC
MBP F: TCTGGCAAGGACTCACACAC
R: AAATCTGCTGAGGGACAGGC
NCAM F: TTCAGTGACGACAGTTCGGAGC
R: TGCGAAGACCTTGAGGTGGAT
PMP22 F: TGTACCACATCCGCCTTGG
R: GAGCTGGCAGAAGAACAGGAAC
NRP2 F: CTTGCTCCCTCTTTGCTG
R: TTCCTTGTGGTGTCTTCTG
VEGF F: ACCATGCCAAGTGGTGAAGT
R: GGGCTTCATCATTGCAGCAG
P0 F: AAGTCTATGGTGCTGTGGGC
R: CCCATACCTAGTGGGCACTTT
-
Table S2. Physicochemical properties of self-assembling backbone
and functional motifs
Sequences Net charge at pH=7 Hydrophilic residue ratio
Description
(RADA)4 0.0 50% Backbone
G2-IKVAV 1.0 14% LA motif
G2-RGIDKRHWNSQ 2.1 54% BDNF motif
G2-IKVAV-G2-RGIDKRHWNSQ 3.1 40% Dual-motif
-
Table S3. Estimated secondary structure fractions of different
peptides
Peptides Secondary structure fractions (%)*
H (r) H (d) S (r) S (d) Turn Unrd
RAD 4.8 0.0 32.3 14.1 15.4 33.4
RAD-IKV 8.5 11.4 0.0 8.4 24.9 46.9
RAD-RGI 4.8 1.9 0.0 6.1 32.2 55.0
RAD-IKV-GG-RGI 3.7 6.3 11.4 9.2 26.4 43.1
RAD/IKV 6.5 0.0 36.4 15.8 12.3 29.0
RAD/RGI 1.9 2.7 33.8 14.1 15.9 31.7
RAD/IKV-GG-RGI 2.5 0.0 28.6 11.7 20.4 36.9
RAD/IKV/RGI 2.1 0.1 32.8 13.1 19.4 32.4
*Abbreviations: H (r), regular α-helix; H (d), distorted
α-helix; S (r), regular β-strand; S (d), distorted β-strand;
Turn, β-turn structure; Unrd, unordered structure. Total Sheet
content = S (r) + S (d).
-
Table S4. Nanofiber width from molecular models and AFM
measurements
Peptides Wthe* Wadj** Wmea
RAD 5.9 nm 12.1 nm 15.4 ± 2.1 nm
RAD-IKV 8.3 nm 14.5 nm -***
RAD-RGI 10.5 nm 16.7 nm -
RAD-IKV-GG-RGI 13.0 nm 19.2 nm -
RAD/IKV 10.7 nm 16.9 nm 18.1 ± 1.9 nm
RAD/RGI 15.1 nm 21.3 nm 23.0 ± 1.5 nm
RAD/IKV-GG-RGI 20.1 nm 26.3 nm 29.6 ± 2.2 nm
RAD/IKV/RGI 15.1 nm 21.3 nm 21.6 ± 0.9 nm
*According to the molecular model proposed in Figure 1A.
**Wthe: theoretical fiber width; Wmea: measured fiber width by
AFM; Wadj: adjusted theoretical fiber width
considering the size effect of the AFM probe tip based on the
estimation 𝑊𝑎𝑑𝑗 = 𝑊𝑡ℎ𝑒 + 2 (2𝑅𝑡𝐻 − 𝐻2)1/2,
Rt = AFM tip size (~ 10 nm), H = sample height (~ 0.5 nm) [1,
2].
***No nanofiber formation was observed.
-
Figure S1. Analytical HPLC trace of RAD peptide.
Figure S2. The electrospray ionization mass spectrometry
(ESI-MS) of RAD peptide.
10.12
8'
11.47
2'
12.24
8'
13.95
0'
0
200
400
600
800
1000
1200
1400
1600
mV
2 4 6 8 10 12 14 16 18 20 22 24 min
299.0 839.4 1379.8 1920.2 2460.6 3001.0
Mass (m/z)
0
5.3E+4
0
10
20
30
40
50
60
70
80
90
100
% In
ten
sity
Voyager Spec #1=>MC[BP = 1713.2, 53175]
1713.24
-
Figure S3. Analytical HPLC trace of RAD-IKV peptide.
Figure S4. The electrospray ionization mass spectrometry
(ESI-MS) of RAD-IKV peptide.
7.9
72
8.1
87
9.0
38
10.2
00
12.6
33
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
699.0 1359.4 2019.8 2680.2 3340.6 4001.0
Mass (m/z)
0
2.4E+4
0
10
20
30
40
50
60
70
80
90
100
% In
ten
sity
Voyager Spec #1[BP = 2338.6, 24086]
2338.56
2361.60
-
Figure S5. Analytical HPLC trace of RAD-RGI peptide.
Figure S6. The electrospray ionization mass spectrometry
(ESI-MS) of RAD-RGI peptide.
7.7
84'
8.3
92'
8.9
23'
-200
-100
0
100
200
300
400
500
600
700
800
900
1000
mV
2 4 6 8 10 12 14 16 18 min
+Q1: 4 MCA scans from Sample 136 (04010019507-3-1 B Q SE) of
04010019507.wiff (Turbo Spray), Centroided Max. 6.1e7 cps.
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600
1700 1800 1900 2000m/z, Da
0.0
5.0e6
1.0e7
1.5e7
2.0e7
2.5e7
3.0e7
3.5e7
4.0e7
4.5e7
5.0e7
5.5e7
6.0e7
In
te
ns
ity
, c
ps
802.3
1069.8
642.1
1604.0
535.1 798.1665.5 820.2 1064.1638.6 946.9 1093.9385.3
1283.4565.5414.1 697.9 1661.51632.2 1718.01420.8 1924.91503.91214.5
1302.9322.3
-
Figure S7. Analytical HPLC trace of RAD-IKV-GG-RGI peptide.
Figure S8. The electrospray ionization mass spectrometry
(ESI-MS) of RAD-IKV-GG-RGI peptide.
7.1
52
'
7.4
48
'
0
200
400
600
800
1000
1200
1400
1600
mV
1 2 3 4 5 6 7 8 9 10 11 12 13 min
699.0 1759.4 2819.8 3880.2 4940.6 6001.0
Mass (m/z)
0
2.6E+4
0
10
20
30
40
50
60
70
80
90
100
% In
ten
sity
Voyager Spec #1[BP = 3830.9, 26112]
3830.87
-
Figure S9. The size of pores in different self-assembling
peptide hydrogels. (A) Typical distribution of pore
sizes in different groups. (B) Analysis of the mean diameter of
pores. Values are represented as mean ± SD.
Statistical analysis was carried out using one-way analysis of
variance (ANOVA), followed by Tukey’s post hoc
test (n = 3).
-
Figure S10. The average G’ and G’’ of self-assembling peptide
hydrogels. The average G’ values were
respectively 2942.6 ± 250.0 Pa, 4600.0 ± 101.4 Pa, 2629.7 ±
675.1 Pa, 3246.3 ± 426.6 Pa, and 2594.8 ± 502.9
Pa. The average G’’ values were respectively 406.4 ± 51.4 Pa,
606.4 ± 116.7 Pa, 298.1 ± 64.7 Pa, 386.9 ± 39.0
Pa, and 340.2 ± 35.4 Pa. Values are represented as mean ± SD.
Statistical analysis was carried out using one-
way analysis of variance (ANOVA), followed by Tukey’s post hoc
test. *P < 0.05, versus the G’ of RAD/IKV
hydrogel. #P < 0.05, versus the G’’ of RAD/IKV hydrogel (n =
3).
-
Figure S11. The proliferation of RSCs on different peptide
hydrogels after 1, 4, and 7 days. Values are
represented as mean ± SD. Statistical analysis was carried out
using one-way analysis of variance (ANOVA),
followed by Tukey’s post hoc test (equal variances) or Dunnett’s
T3 post hoc test (unequal variances). *P < 0.05,
and **P < 0.01 (n = 4).
-
Figure S12. Nerve graft preparation (A), implantation (B) and
harvest of the nerve grafts at 7 days
postoperatively (C) of the different groups.
-
Figure S13. Scheme of transverse section of regenerated nerve at
12 weeks after surgery.
References
[1] Hong Y, Legge RL, Zhang S, Chen P. Effect of amino acid
sequence and pH on nanofiber formation of self-
assembling peptides EAK16-II and EAK16-IV. Biomacromolecules.
2003; 4: 1433-42.
[2] Horii A, Wang X, Gelain F, Zhang S. Biological designer
self-assembling peptide nanofiber scaffolds
significantly enhance osteoblast proliferation, differentiation
and 3-D migration. PLoS One. 2007; 2: e190.
