Electrospun mini-MiSp spidroin/poly(L-lactide-co-ε-caprolactone) nanofibrous scaffolds for ARPE-19 cells

Figures & data

Figure 1. Schematic illustration of the expression plasmid pET-SUMO-R2C.

Figure 2. Schematic illustration of the preparation of the electrospun R2C/PLCL scaffolds. (A) Preparation of the electrospinning solutions of R2C/PLCL. (B) Schematic illustration of the electrospinning process. HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; HVDC, high-voltage direct current.

Figure 3. Amino acid sequence of spidroin R2C. The spidroin R2C consists of a Rep2 domain and a CT domain. The DNA sequences of the Rep2 and CT domains were from A. ventricosus MiSp (GeneBank accession number AFV31615.1) and were translated into amino acid sequences via DNAMAN software. The amino acid sequences of Rep2 and CT domains were marked with green and blue shading, respectively.

Figure 4. SDS-PAGE analysis of the recombinant spidroin R2C. Lane M, Protein marker (C600525, Sangon, Shanghai, China); lane 1, lysates of uninduced cells; lane 2, lysates of induced cells; lane 3, supernatant of cell lysates; lane 4, pellet of cell lysates; lane 5, flow-through of the Ni-NTA column by passing the supernatants through the Ni-NTA column; lane 6, eluate of Ni-NTA column by using elution buffer; lane 7, fragments after SUMO protease digestion; lane 8, purified spidroin R2C. The target band of spidroin R2C is indicated by the black arrow.

Figure 5. Preparation and mechanical properties of the wet-spun fibers of R2C. (A) Continuous silk-like fibers formed in coagulation solution of 90% methanol. (B) Silk-like fibers more than 10 cm in length. (C) SEM micrography of the wet-spun fiber of R2C using the full backscattered electron detector. The scale bar is 4 μm. (D) Stress-strain curve of the wet-spun fibers of R2C.

Table 1. Mechanical properties of the wet-spun fibers of R2C.

Name	Diameter (μm)	Stress at break (MPa)	Strain at break (mm/mm)	Young’s modulus (GPa)	Toughness (MJ/m^3)
R2C	5.67 ± 0.72	106.68 ± 20.53	0.26 ± 0.07	3.51 ± 0.92	24.67 ± 9.51

Figure 6. Preparation of the electrospinning R2C/PLCL scaffolds and their effects on ARPE-19 cells. (A) SEM micrography of the electrospinning R2C/PLCL scaffolds using the full backscattered electron detector. The scale bar is 10 μm. (B) Fiber diameter distribution of the electrospinning R2C/PLCL scaffolds. (C) Volcano plot of differential gene expression after co-culture of ARPE19 cells with membranes. (D) Heatmap of differential gene expression with a threshold of a log₂ FoldChange more than 3 and a p adjust value less than 0.05. The volcano plot and heatmap were generated by the online tool of Majorbio Cloud Platform (https://cloud.majorbio.com/page/tools/).

Table 2. Up-regulated genes with a log₂ FoldChange more than 3.

Gene name	Gene description	Log2 FoldChange
TGFB2-OT1	TGFB2 overlapping transcript 1	24.218
KRTAP2-3	keratin associated protein 2-3	8.815
C3	complement C3	7.1
AMTN	amelotin	4.286
PDF	peptide deformylase (mitochondrial)	3.897
TAGLN	transgelin	3.739
SERPINE1	serpin family E member 1	3.618
IL32	interleukin 32	3.423
KRT33B	keratin 33B	3.324
KRT38	keratin 38	3.222
IER3	immediate early response 3	3.178
CCDC85B	coiled-coil domain containing 85B	3.151
UGT1A8	UDP glucuronosyltransferase family 1member A8	3.079
KRT32	keratin 32	3.01

Table 3. Down-regulated genes with a log₂ FoldChange more than 3.

Gene name	Gene description	Log2 FoldChange
HRNR	hornerin	−5.747
ZBTB20	zinc finger and BTB domain containing 20	−4.405
SLC5A3	solute carrier family 5 member 3	−3.817
FLG	filaggrin	−3.759
MYH15	myosin heavy chain 15	−3.704
CCDC144B	coiled-coil domain containing 144B (pseudogene)	−3.279
RAB11FIP1P1	RAB11 family interacting protein 1 pseudogene 1	−3.165

Data availability statement

The data that support the findings from this study are available from the corresponding author [EM], upon reasonable request.