Fabrication of injectable and superelastic nanofiber rectangle matrices ("peanuts") and their potential applications in hemostasis

Abstract

Uncontrolled hemorrhage, which typically involves the torso and/or limb junctional zones, remains a great challenge in the prehospital setting. Here, we for the first time report an injectable and superelastic nanofiber rectangle matrix ("peanut") fabricated by a combination of electrospinning, gas foaming, hydrogel coating and crosslinking techniques. The compressed nanofiber peanut is capable of re-expanding to its original shape in atmosphere, water and blood within 10 s. Such nanofiber peanuts exhibit greater capacity of water/blood absorption compared to current commercial products and high efficacy in whole blood clotting assay, in particular for thrombin-immobilized samples. These nanofiber peanuts are capable of being packed into a syringe for injection. Further in vivo tests indicated the effectiveness of nanofiber peanuts for hemostasis in a porcine liver injury model. This new class of nanofiber-based materials may hold great promise for hemostatic applications.

Keywords: Electrospinning; Hemostasis; Injectable; Nanofiber rectangle matrix; Superelastic.

Figure 1

Schematic showing the fabrication and the use of injectable and shape memorable nanofiber peanuts. (A) Illustration of the process of fabrication of injectable and shape memorable nanofiber peanuts. (B) Illustration of the use of injectable and shape memorable nanofiber peanuts. Left: nanofiber pellets delivery through a cannula or syringe. Right: The expansion of nanofiber peanuts upon contact with blood, the blood plasma absorption, and the tamponade effect control bleeding.

Figure 2

Fabrication and cauterization of superelastic nanofiber peanuts. (A) The schematic shows the 3D structure of expanded PCL nanofiber peanuts. The red arrow indicates the direction of fiber alignment. (B) Photographs showing the morphology of PCL nanofiber mats before and after expansion. (C) Photograph showing a pile of PCL nanofiber peanuts. (D) The thickness distribution of PCL nanofiber peanuts. (E) The cross-section structures (Y-Z, X-Z, X-Y planes) of PCL nanofiber mats before and after expansion. (F) Morphological characterization of PCL nanofiber peanuts and gelatin-coated PCL nanofiber peanuts. Top row: photographs of PCL nanofiber peanuts before and after gelatin coating. Bottom row: SEM images showing the cross-sectional morphologies of PCL nanofiber peanuts before and after gelatin coating. (G) A series of photographs suggesting the compressed nanofiber peanuts can re-expand. (H) Cyclic compressive test of 0.5% gelatin-coated PCL nanofiber peanuts (5 cycles). In order to accommodate the testing equipment, the nanofiber peanuts cut in 2 cm thick were used for this test. Insets: the corresponding photographs of nanofiber peanuts at different stages during the compressive test.

Figure 3

Photographs showing compressed nanofiber pellets after putting into blood for different times. (A) t = 0 s. (B) t = 1 s. (C) t = 9 s. (D) t = 17 s.

Figure 4

Photographs illustrating the packing and injection of nanofiber peanuts. (A) Expanded nanofiber peanuts. (B) PVP nanofiber threads made by rolling the fine strips. (C) Packed nanofiber peanuts. (D) Packed nanofiber peanuts loaded in the syringe. (E) Inject packed nanofiber peanuts into water for different times.

Figure 5

The water uptake of hemostatic materials. (A) Photographs showing 0.5% gelatin-coated PCL nanofiber peanuts, commercial Gauze, Gelfoam^®, and Surgicel^® after putting in water. (B) The rate of water uptake of 0.5% gelatin-coated PCL nanofiber peanuts, commercial Gauze, Gelfoam^®, and Surgicel^®. (C) The amount of water absorbed of PCL nanofiber peanuts and 0.1%, 0.5% and 1% gelatin-coated PCL nanofiber peanuts. *p<0.05, **p<0.01. (D) Gelatin content in 0.1%, 0.5% and 1% gelatin-coated PCL nanofiber peanuts.

Figure 6

In vitro hemostatic efficacy test. (A) Photographs showing the hemostatic materials before and after blood absorption. (B) The blood absorption of 0.5% gelatin-coated PCL nanofiber peanuts, commercial Gauze, Gelfoam^®, and Surgicel^{®. **}p < 0.01. (C) The whole blood clotting assay of Surgicel^®, Gauze, Gelfoam^®, 0.5% gelatin coated PCL nanofiber peanuts, and thrombin-immobilized, 0.5% gelatin coated PCL nanofiber peanuts and the pH value of each group after whole blood clotting assay. (D) The hemoglobin binding efficiency of Surgicel^®, Gauze, Gelfoam^®, 0.5% gelatin coated PCL nanofiber peanuts, and thrombin-immobilized, 0.5% gelatin coated PCL nanofiber peanuts. (E) The blood clotting time of Surgicel^®, Gauze, Gelfoam^®, 0.5% gelatin coated PCL nanofiber peanuts, and thrombin-immobilized, 0.5% gelatin coated PCL nanofiber peanuts. (F) The blood protein absorption of Surgicel^®, Gauze, Gelfoam^®, 0.5% gelatin coated PCL nanofiber peanuts, and thrombin-immobilized, 0.5% gelatin coated PCL nanofiber peanuts. ^*p < 0.05, ^**p < 0.01.

Figure 7

The platelet adhesion assay of Surgicel®, commercial Gauze, Gelfoam®, 0.5% gelatin-coated PCL nanofiber peanuts, and thrombin-immobilized, 0.5% gelatin-coated PCL nanofiber peanuts. (A) SEM images showing platelet adhesion and activation. (B) Immunostaining of adhered platelets with CD41 (a marker for surface bound platelets) and CD62P (a marker for activated platelets).

Figure 8

Hemostatic efficacy test in vivo. (A) In vivo test procedure. (I) A pile of 0.5% gelatin-coated nanofiber peanuts. (II) Exposure of injury site (Grade V hepatic dome laceration) 1 h after treatment with nanofiber peanuts. Cotton lap pads have been removed, showing 0.5% gelatin-coated PCL nanofiber peanuts still packed into the injury defect. (III) Close-up of injury site. Forceps is beginning to extract the peanuts. (IV) Nanofiber peanuts after removal from wound. (B) SEM images showing the protein absorption and platelet adhesion on the outer surface and inner surface of 0.5% gelatin-coated PCL nanofiber peanuts after removal from wound. Insets: the corresponding highly magnified images. Black arrows indicate platelet adhesion on the nanofiber peanuts. (C) H&E staining of the wound. Green dots indicate the wound edge.

Figure 9

The schematic illustrating the working mechanism of superelastic property after compression. (A) PCL nanofiber peanuts. The PCL nanofiber peanuts failed to recover the shape completely after compression under 70% compressive strain. (B) 0.5% gelatin-coated PCL nanofiber peanuts. The 0.5% gelatin-coated PCL nanofiber peanuts were able to fully recover after compression under 70% compressive strain.