Biodegradable and thermosensitive micelles inhibit ischemia-induced postoperative peritoneal adhesion

Abstract

Ischemia-induced adhesion is very common after surgery, and leads to severe abdominal adhesions. Unfortunately, many existing barrier agents used for adhesion prevention have only limited success. The objective of this study is to evaluate the efficacy of biodegradable and thermosensitive poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-PEG-PCL) micelles for the prevention of postoperative ischemia-induced adhesion. We found that the synthesized PCL-PEG-PCL copolymer could self-assemble in an aqueous solution to form micelles with a mean size of 40.1 ± 2.7 nm at 10°C, and the self-assembled micelles could instantly turn into a nonflowing gel at body temperature. In vitro cytotoxicity tests suggested that the copolymer showed little toxicity on NIH-3T3 cells even at amounts up to 1,000 μg/mL. In the in vivo test, the postsurgical ischemic-induced peritoneal adhesion model was established and then treated with the biodegradable and thermosensitive micelles. In the control group (n=12), all animals developed adhesions (mean score, 3.58 ± 0.51), whereas three rats in the micelles-treated group (n=12) did not develop any adhesions (mean score, 0.67 ± 0.78; P<0.001, Mann-Whitney U-test). Both hematoxylin and eosin and Masson trichrome staining of the ischemic tissues indicated that the micelles demonstrated excellent therapeutic effects on ischemia-induced adhesion. On Day 7 after micelle treatment, a layer of neo-mesothelial cells emerged on the injured tissues, which confirmed the antiadhesion effect of the micelles. The thermosensitive micelles had no significant side effects in the in vivo experiments. These results suggested that biodegradable and thermosensitive PCL-PEG-PCL micelles could serve as a potential barrier agent to reduce the severity of and even prevent the formation of ischemia-induced adhesions.

Keywords: poly(ε-caprolactone)–poly(ethylene glycol)–poly(ε-caprolactone) (PCL–PEG–PCL); postsurgical adhesion; surgical complications, barrier agent.

Figure 1

Characterization of PCL-PEG-PCL micelles. Notes: (A) Particle size of micelles at 10°C. (B) Zeta potential of micelles at 10°C. Abbreviation: PCL–PEG–PCL, poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone).

Figure 2

Thermosensitivity of prepared PCL–PEG–PCL micelles. Notes: (A) Sol–gel phase transition of the thermosensitive micelles as a function of temperature. (B) Particle size of micelles at different temperatures. Abbreviation: PCL–PEG–PCL, poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone).

Figure 3

Cytotoxicity of the micelles in vitro using MTT assay. Abbreviation: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

Figure 4

Establishment and treatment of ischemic-induced postoperative peritoneal adhesion model in sprague Dawley rats. Notes: (A) Normal saline-treated group as control group. (B) PCL–PEG–PCL micelles-treated group. Abbreviation: PCL–PEG–PCL, poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone).

Figure 5

Evaluation of ischemia-induced adhesion prevention on postoperative Day 7. Notes: (A) Adhesions were observed in control group, which scored 4. (B) No adhesion was observed in micelles-treated group, which scored 0.

Figure 6

Histopathological analyses of tissues from control group and micelles-treated group. Notes: (A) Adhesion tissues from control group stained with H&E (100×). (B) Adhesion tissues from control group stained with Masson (100×). (C) Tissues from micelles-treated group stained with H&E (100×). (D) Tissues from micelles-treated group stained with Masson (100×). (E) The appearance of neo-mesothelial cells in tissues stained with H&E on Day 7 after micelles treatment (400×). (F) The appearance of neo-mesothelial cells in tissues stained with Masson on Day 7 after micelles treatment (400×). Black arrows in each slide showed the position of adhesion or mesothelial cells in each figure. Abbreviations: H&E, hematoxylin and eosin; Me, mesothelial cells; SK, abdominal wall skeletal muscle; SS, silk suture.

Figure 7

SEM images of neo-mesothelial cells on Day 7 after micelle treatment. Notes: Images are taken at 2,000× (A), and 10,000× magnification (B). Inset shown in A is magnified in B. Abbreviation: SEM, scanning electron microscope.

Figure 8

(A) Body weight, (B) food intake, and (C) water intake of mice in each group after surgery.

Figure 9

Photographs of mouse major organs after administration of micelles (200×) on Day 7 (A–J).