Stability Enhancement of Freeze-Dried Gelatin/Alginate Coacervates for bFGF Delivery

Abstract

Chronic wound sites have elevated levels of proteolytic enzymes that negate the activity of topically applied growth factors. bFGF encapsulated in gelatin/alginate coacervates was protected from protease and showed better activity than bFGF in solution; however, its activity decreased with particle size and PDI increase after freeze-drying and rehydration. In this study, we aim to improve the stability of bFGF coacervates during freeze-drying to enable a topically applied growth factor delivery system for diabetic foot ulcer. Trehalose, mannitol, and Tween 80 at various concentrations were tested as cryoprotectant candidates. Trehalose improved the mechanical property of freeze-dried coacervates and physical properties after rehydration, resulting in stable size and PDI values. It also enhanced the bFGF activity in hyperglycemic human dermal fibroblasts with better cell viability, migration, and procollagen synthesis compared to the coacervates without trehalose. Hydrogen bonding interactions between trehalose and polymers probed by ATR-FTIR contribute to the stability of coacervates during freeze-drying. In conclusion, the freeze-dried gelatin/alginate coacervates encapsulating bFGF was effectively stabilized with trehalose, and the resulting coacervate composition is suggested as a potential therapeutic modality for chronic wounds including diabetic foot ulcer.

Keywords: coacervates; cryoprotectants; diabetic foot ulcer; fibroblast growth factor; freeze-drying.

Figure 1

Appearance of GA/SA coacervates acidified with citric acid before freeze-drying (F/D) and after rehydration of freeze-dried samples; (a) without cryoprotectant, (b) coacervates containing trehalose, mannitol, or Tween80 before freeze-drying, (c) after rehydration of freeze-dried coacervates containing trehalose, mannitol, or Tween80. Red circles show aggregated particles.

Figure 2

Effects of freeze-drying (F/D) on GA/SA coacervates acidified with acetic acid or citric acid; (a) particle size, (b) PDI before freeze drying and after rehydration of freeze-dried coacervates.

Figure 3

Effects of freeze-drying (F/D) on GA/SA coacervates acidified with citric acid without (w/o) or with trehalose, mannitol, and Tween 80; (a) particle size and (b) PDI value before freeze drying (F/D) and after rehydration of freeze-dried coacervates.

Figure 4

Microscope images of GA/SA coacervates acidified with citric acid without or with cryoprotectants (Left: Fluorescence images, Middle: Optical images, Right: Overlapped Fluorescence and Optical images): (a) before freeze-drying, (b) after rehydration of freeze-dried coacervates without cryoprotectant, (c–e) after rehydration of freeze-dried coacervates with trehalose (2%, w/v), with mannitol (2%, w/v), and with Tween 80 (2%, w/v). The scale bars represent 20 μm.

Figure 5

Microscope images of GA/SA coacervates acidified with acetic acid without or with cryoprotectants (Left: Fluorescence images, Middle: Optical images, Right: Overlapped Fluorescence and Optical images): (a) before freeze-drying, (b) after rehydration of freeze-dried coacervates without cryoprotectant, (c–e) after rehydration of freeze-dried coacervates with trehalose (2%, w/v), with mannitol (2%, w/v), and with Tween 80 (2%, w/v). The scale bars represent 20 μm.

Figure 6

Morphology and texture of freeze-dried GA/SA coacervates acidified with citric acid; (a) with various concentrations of cryoprotectants, (b) texture of freeze-dried coacervates with 2% (w/v) cryoprotectants.

Figure 7

SEM images of freeze-dried GA/SA coacervates acidified with acetic acid or citric acid, (a) without trehalose (AA-F/D, CA-F/D) and (b) with trehalose (2%, w/v) (AAT-F/D, CAT-F/D). Scale bars represent 100 μm, 10 μm, and 10 μm, for 110×, 400×, and 1000×, respectively.

Figure 8

ATR-FTIR spectra of trehalose, GA/SA/trehalose powder mixture and CAT-F/D.

Figure 9

bFGF ELISA results after rehydration of freeze-dried GA/SA coacervates acidified with acetic acid or citric acid, without trehalose (AA-F/D, CA-F/D) or with trehalose (2%, w/v) (AAT-F/D, CAT-F/D). * p < 0.05.

Figure 10

In vitro bFGF release profiles of freeze-dried GA/SA coacervates acidified with acetic acid or citric acid, without trehalose (AA-F/D, CA-F/D) or with trehalose (2%, w/v) (AAT-F/D, CAT-F/D), compared to the bFGF solution and freeze-dried physical mixture.

Figure 11

In vitro activity assay of various samples at 5 days (n = 3); (a) HDF viability after treatment with coacervates before (AA, CA) and after freeze-drying without trehalose (AA-F/D, CA-F/D), (b) freeze-dried samples with or without trehalose 2% (w/v). P represents the positive control HDF cultured in 5 mM glucose and 10% FBS. Other samples were all cultured in 25 mM glucose and 0.5% FBS. N represents the negative control HDF cultured in 25 mM glucose and 0.5% FBS without any treatment. **** p < 0.0001, *** p < 0.001, ** p < 0.01 * p < 0.05. Red arrows emphasize the changes after freeze-drying.

Figure 12

PICP ELISA assay of supernatants various samples collected at day 5. Treatment with freeze-dried samples without (AA-F/D, CA-F/D) or with trehalose 2% (w/v) (AAT-F/D, CAT-F/D). Samples were all cultured in 25 mM glucose and 0% FBS, except N, the negative control cultured in 25 mM glucose and 0% FBS without any treatment. **** p < 0.0001, ** p < 0.01 * p < 0.05.

Figure 13

In vitro HDF scratch wound assay results; (a) Representative bright field images at right after scratching (0 h) and 24 h after treatment with various samples (24 h), (b) Wound healing rates of various conditions relative to the Negative group (n = 3). **** p < 0.0001, *** p < 0.001, * p < 0.05.