Structural and functional characterization of proteins adsorbed on hydrophilized polylactide-co-glycolide microfibers

Figures & data

Figure 1 Scanning electron micrographs of Pluronic^® F-108 blended and unblended PLGA microfibrous mesh. (A) Pure PLGA, (B) PLGA blended with 2.0% PF-108 (this is a representative micrograph of blended samples [PF-0.5 to PF-1.5] that are morphologically similar to those reported previously²⁰).

Abbreviations: PF, Pluronic^® F; PLGA, polylactide-co-glycolide.

Figure 2 Plot of bovine serum albumin released from PF-0.5 to PF-2.0 blended PLGA microfibrous meshes and pure PLGA microfibrous mesh (control) as a function of time (2, 8, and 24 hours).

Note: *P < 0.001 between unblended PLGA and PF-108 blended PLGA samples at the 2-hour time point.

Abbreviations: PF, Pluronic^® F; PLGA, polylactide-co-glycolide; BSA, bovine serum albumin.

Figure 3 Plot of lysozyme released from PF-0.5 to PF-2.0 blended PLGA microfibrous meshes and pure PLGA microfibrous mesh (control) as a function of time (2, 8, and 24 hours).

Note: *P < 0.001 between unblended PLGA and PF-108 blended PLGA samples at the 2-hour time point.

Abbreviations: PF, Pluronic^® F; PLGA, polylactide-co-glycolide.

Figure 4 Schematic of PF-108 conformation in/on PLGA microfiber surface. (A) Structure of PF-108 depicting ethylene oxide and propylene oxide domains. (B) Conformation of PF-108 before exposure of water to PF-108 blended PLGA microfiber surface. (C) Conformation of PF-108 after exposure of water to PF-108 blended PLGA microfiber surface. The ethylene oxide component of PF-108 takes on a mushroom-shaped conformation, whereas the propylene oxide component of PF-108 remains embedded in the PLGA microfiber. (D) Noninteracting mushroom-shaped conformations of ethylene oxide on PLGA microfiber surface at lower concentrations of PF-108. (E) Brush-shaped conformations of ethylene oxide on PLGA microfiber surface at higher concentrations of PF-108.

Abbreviations: PF, Pluronic^® F; PLGA, polylactide-co-glycolide; EO, ethylene oxide; PO, propylene oxide.

Figure 5 Schematic depicting the cross-sectional view of PF-108 conformation in/on PLGA microfiber surface. (A) Change in conformation of ethylene oxide units of PF-108 from mushroom-shaped to brush-shaped conformation with increase in surface density of PF-108 on PLGA microfiber surface. (B) Primary and secondary protein adsorption behavior on mushroom-shaped and brush-shaped ethylene oxide units.

Abbreviations: PF, Pluronic^® F; PLGA, polylactide-co-glycolide; EO, ethylene oxide; PO, propylene oxide.

Table 1 Secondary structure of lysozyme content as determined by experimental and deconvolution techniques

Secondary structure	Experimental (from PDB)	Deconvoluted
α-helices	38.87% ± 2.9%	31.08% ± 3%
β-sheets	10% ± 1.4%	20.17% ± 4.9%
Random chains	51.12% ± 3.27%	48.75% ± 4.61%

Abbreviation: PDB, Protein Data Bank.

Figure 6 Deconvolution spectra of lysozyme amid I peak (1600–1700 cm ⁻¹). (A) Complete Fourier transform infrared spectrum of lyophilized lysozyme powder. (B) Zoomed view of amid I peak (solid line in spectrum depicts the amid I region and the dotted lines depict deconvoluted Gaussian peaks of various secondary structures of lysozyme).

Table 2 Percentage α-helical content of adsorbed and released bovine serum albumin and lysozyme from PF-108 blended and unblended PLGA microfibers (percentage α-helical content of powdered bovine serum albumin and lysozyme were taken as controls)

Sample ID	Adsorbed BSA	Released BSA	Adsorbed lysozyme	Released lysozyme
PF-0.0	41.98 ± 2.1	39.92 ± 2.4	33.03 ± 2.2	32.73 ± 1.1
PF-0.5	37.37 ± 1.6*	36.83 ± 1.7	*37.69 ± 3.4	33.44 ± 1.6^$
PF-1.0	34.7 ± 1.2***	35.77 ± 2.2*	30.25 ± 1.5	31.85 ± 1.8
PF-1.5	42.85 ± 3.0	36.79 ± 2.4^##	31.17 ± 2.1	32.07 ± 2.1
PF-2.0	40.9 ± 2.3	36.36 ± 1.9^#	27.58 ± 2.6**	32.86 ± 1.9^$$
Powder BSA/Lys	32.6 ± 1.6		31.08 ± 2.5

Notes: PF-1.0, all adsorbed BSA samples showed improvement in α-helical content (PF-0.0 and PF-2.0, P < 0.001; PF-0.5 and PF-1.5, P < 0.01) as compared with powdered BSA; except for PF-1.0 and PF-2.0, all BSA samples released showed marginal improvement in α-helical content (PF-0.0, P < 0.001; PF-0.5; and PF-1.5, P < 0.05) as compared with powdered BSA; significance with respect to PF-0.0 in each column (excluding powder BSA/Lys),

* P < 0.05,

** P < 0.01,

*** P < 0.001; except for PF-0.5 (P < 0.001), all adsorbed and released lysozyme samples did not show any significant difference in α-helical content as compared with powder lysozyme; comparison between adsorbed and released BSA.

# P < 0.05,

## P < 0.001; comparison between adsorbed and released lysozyme.

$ P < 0.05,

$$ P < 0.01.

Abbreviations: BSA, bovine serum albumin; Lys, lysosome; PF, Pluronic^® F; PLGA, polylactide-co-glycolide.

Figure 7 Enzymatic activity of lysozyme (units/mL) released from 0.5%–2.0% PF-108 blended and unblended PLGA microfiber meshes.

Notes: *P < 0.001 (except PF-0.5, P < 0.01) between unblended PLGA and PF-108 blended PLGA samples at the 2-hour time point. ^#P < 0.001 (except PF-1.0 and PF-2.0, P < 0.01) between unblended PLGA and PF-108 blended PLGA samples at the 8-hour time point. ^§P < 0.01 between unblended PLGA and PF-108 blended PLGA samples at the 24-hour time point.

Abbreviations: PF, Pluronic^® F; PLGA, polylactide-co-glycolide.

VasitaRManiGAgrawalCMKattiDSSurface hydrophilization of electrospun PLGA micro-/nano-fibers by blending with Pluronic® F-108Polymer20105137063714

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