Delivery of a transforming growth factor β-1 plasmid to mesenchymal stem cells via cationized Pleurotus eryngii polysaccharide nanoparticles

Figures & data

Figure 1 The process of polysaccharide of Pleurotus eryngii extraction and purification.

Figure 2 The process of spermine grafting to the polysaccharide (dextran is used as an example).

Notes: The reductive amination method is used to produce the cationized polysaccharide: the polysaccharide is first oxidized by KIO₄; after the addition of spermine, NaBH₄ is used to reduce the reactants.

Figure 3 The process of cationized Pleurotus eryngii polysaccharide (CPEPS) preparation.

Table 1 Amino group content and cross-linking of CPEPS

Polycation	Nitrogena (μmol/mg)	Primary aminesb (μmol/mg)	Sperminec (μmol/mg)	Secondary aminesd (μmol/mg)	Secondary to primary amine ratio	Cross-linked sperminee (%)
CPEPS	3.92 ± 0.23	1.24 ± 0.09	0.98 ± 0.06	2.94 ± 0.18	2.40 ± 0.30	0

Notes:

a Determined by elemental analysis (n = 3);

b determined by the trinitrobenzene sulfonic acid method (n = 3);

c calculated from elemental analysis (total nitrogen divided by 4);

d theoretical secondary amine content (in case no cross-linking occurred: spermine content multiplied by 3);

e calculated for secondary to primary amine ratio ≥ 3.

Abbreviation: CPEPS, cationized Pleurotus eryngii polysaccharide.

Figure 4 Fourier transform infrared spectra of polysaccharide of Pleurotus eryngii (PEPS) and cationized P. eryngii polysaccharide (CPEPS).

Figure 5 Agarose gel electrophoresis of the CPEPS-pTGF-β1 nanoparticles.

Notes: Well 1, free pTGF-β1; wells 2–7, CPEPS-pTGF-β1 nanoparticles with various weight ratios of CPEPS/pTGF-β1 (from left to right: 5:1, 10:1, 20:1, 30:1, 60:1, and 80:1).

Abbreviations: CPEPS, cationized Pleurotus eryngii polysaccharide; pTGF-β1, plasmid encoding transforming growth factor beta-1.

Figure 6 The zeta potentials of free pTGF-β1, CPEPS, and CPEPS combined with different amounts of pTGF-β1 to yield the following CPEPS/pTGF-β1 weight ratios: 10:1, 20:1, and 30:1.

Note: The values are the means ± standard error of the mean of three experiments.

Abbreviations: TGF-β1, transforming growth factor beta-1; pTGF-β1, plasmid encoding TGF-β1; CPEPS, cationized P. eryngii polysaccharide.

Table 2 The size distribution of nanoparticles with CPEPS/pTGF- β1 weight ratios of 10:1, 20:1, and 30:1

CPEPS/pTGF-β1 weight ratios	10:1	20:1	30:1
Particle size (means ± SEM, nm)	248.3 ± 7.4	80.8 ± 6.8	151.1 ± 7.2

Abbreviations: CPEPS, cationized Pleurotus eryngii polysaccharide; pTGF-β1, plasmid encoding transforming growth factor beta-1; SEM, standard error of the mean.

Figure 7 Particle size distribution and morphology. (A) The particle size distribution of the CPEPS/pTGF-β1 nanoparticles with a weight ratio of 20:1; (B) transmission electron microscopy image of the CPEPS/pTGF-β1 nanoparticles with a weight ratio of 20:1.

Abbreviations: CPEPS, cationized Pleurotus eryngii polysaccharide; pTGF-β1, plasmid encoding transforming growth factor beta-1.

Figure 8 Cytotoxicity assay. From left to right: Lipofectamine™ 2000; polyethylenimine (25 kDa); free plasmid; CPEPS-pTGF-β1 nanoparticles with CPEPS/pTGF-β1 weight ratios of 10:1, 20:1, and 30:1.

Note: The values are the means ± standard error of the mean of three experiments.

Abbreviations: CPEPS, cationized Pleurotus eryngii polysaccharide; PEI, polyethylenimine; pTGF-β1, plasmid encoding transforming growth factor beta-1.

Figure 9 Transmission electron microscopy (TEM) images of MSCs before and after transfection. (A) TEM image of mesenchymal stem cells before transfection; (B–D) TEM images of mesenchymal stem cells after transfection by CPEPS-pTGF- β1 nanoparticles with CPEPS/pTGF-β1 weight ratios of 10:1, 20:1, and 30:1, respectively.

Abbreviations: CPEPS, cationized Pleurotus eryngii polysaccharide; pTGF-β1, plasmid encoding transforming growth factor beta-1.

Figure 10 TGF-β1 protein released into the medium from mesenchymal stem cells 72 hours after treatment with CPEPS-pTGF-β1 nanoparticles. (A) TGF-β1 protein concentrations in the medium with various weight ratios of CPEPS to pTGF-β1: 10:1 (first bar), 20:1 (second bar), and 30:1 (third bar), as well as in free plasmid DNA (fourth bar). (B) A comparison of TGF-β1 expression levels: free plasmid DNA (first bar); Lipofectamine™ 2000 (second bar); polyethylenimine (25 kDa, third bar); and CPEPS-pTGF-β1 nanoparticles with a CPEPS/pTGF-β1 weight ratio of 20:1 (fourth bar).

Note: The values are the means ± standard error of the mean of three experiments.

Abbreviations: TGF-β1, transforming growth factor beta-1; pTGF-β1, plasmid encoding TGF-β1; CPEPS, cationized Pleurotus eryngii polysaccharide; PEI, polyethylenimine.

Figure 11 Messenger RNA expression of TGF-β1 in mesenchymal stem cells (MSCs).

Notes: Well 1, MSCs transfected with free pTGF-β1; well 2, MSCs transfected with Lipofectamine™ 2000/pTGF-β1; well 3, MSCs transfected with CPEPS-pTGF- β1 nanoparticles.

Abbreviations: TGF-β1, transforming growth factor beta-1; CPEPS, cationized Pleurotus eryngii polysaccharide; pTGF-β1, plasmid encoding TGF-β1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 12 Flow cytometry of propidium iodide-stained mesenchymal stem cells (MSCs). (A) MSCs treated with plasmid TGF-β1; (B) MSCs treated with Lipofectamine™ 2000/pTGF-β1; (C) MSCs treated with CPEPS-pTGF-β1 nanoparticles; (D) MSCs treated with medium containing pTGF-β1 protein.

Abbreviations: TGF-β1, transforming growth factor beta-1; pTGF-β1, plasmid encoding TGF-β1; CPEPS, cationized Pleurotus eryngii polysaccharide.