Micelle-Coated, Hierarchically Structured Nanofibers with Dual-Release Capability for Accelerated Wound Healing and Infection Control

Abstract

Tailoring nanofibrous matrices-a material with much promise for wound healing applications-to simultaneously mitigate bacterial colonization and stimulate wound closure of infected wounds is highly desirable. To that end, a dual-releasing, multiscale system of biodegradable electrospun nanofibers coated with biocompatible micellar nanocarriers is reported. For wound healing, transforming growth factor-β1 is incorporated into polycaprolactone/collagen (PCL/Coll) nanofibers via electrospinning and the myofibroblastic differentiation of human dermal fibroblasts is locally stimulated. To prevent infection, biocompatible nanocarriers of polypeptide-based block copolymer micelles are deposited onto the surfaces of PCL/Coll nanofibers using tannic acid as a binding partner. Micelle-modified fibrous scaffolds are favorable for wound healing, not only supporting the attachment and spreading of fibroblasts comparable to those on noncoated nanofibers, but also significantly enhancing fibroblast migration. Micellar coatings can be loaded with gentamicin or clindamycin and exhibit antibacterial activity as measured by Petrifilm and zone of inhibition assays as well as time-dependent reduction of cellular counts of Staphylococcus aureus cultures. Moreover, delivery time of antibiotic dosage is tunable through the application of a novel modular approach. Altogether, this system holds great promise as an infection-mitigating, cell-stimulating, biodegradable skin graft for wound management and tissue engineering.

Keywords: dual drug release; infection prevention; layer-by-layer; nanofibrous matrices; wound healing.

Figure 1.

Schematic representation of a modular system to simultaneously stimulate wound healing and mitigate infection. TGF-β1 was incorporated into PCL/Coll nanofibers to stimulate fibroblast-to-myofibroblast differentiation. Micellar nanocarriers were deposited on the surface of PCL/Coll nanofibers and loaded with antibiotics to prevent infection.

Figure 2.

PCL/Coll nanofibers with TGF-β1 support the attachment and fibroblast-to-myofibroblast differentiation of NHDFs. Immunofluorescent staining of focal adhesion protein vinculin (green) and nuclei with DAPI (blue) (A,B), and immunofluorescent staining of FAK (green) (C,D), and α-SMA (red) (E, F) after 7 days culture of 2×10⁴ cells/sample. (G) Cell migration tracks, with individual cell tracks displayed in different colors. Each track start was equalized to the center of the plot. Plot is shown from −150 to 150 μm. (H) Mean displacement for the NHDFs (n=10) tracked at 60-min lapse interval. (I) Quantification of α-SMA positive cells normalized against the cell nuclei stained with DAPI. Data presented as mean ± SD, n=3, p-values are calculated using an unpaired student t-test, **p <0.01.

Figure 3.

Surface coverage of BCM on PCL/Coll NFs depends on deposition time while average micellar size does not. Morphology of PCL/Coll NFs after 1.5 bilayers of BCM/TA deposited for 5 min (A) or 30 min (B). Surface coverage and average micellar size as a function of deposition time (C). Data presented as mean ± SD, n=2.

Figure 4.

Bare and BCM/TA-coated PCL/Coll NFs support the growth and attachment of NHDFs. Attachment of NHDFs as imaged by SEM imaging after 24 h culture of 5.0×10⁴ cells (A). (B) Live/dead staining of NHDFs after 24 h culture of 5.0×10⁴ cells/sample. Immunofluorescent staining of focal adhesion protein vinculin (green) and nuclei with DAPI (blue) (C), and FAK (green) (D) with DAPI (blue) after 48 h culture of 2×10⁴ cells/sample.

Figure 5.

BCM/TA coatings enhance NHDFs migration rate and proliferation on PCL/Coll NFs. (A) NHDFs (2×10⁵ per matrix) were seeded on either bare or BCM/TA coated PCL/Coll NFs with an insert in the middle. After 24 h, the insert was removed to generate a 0.9-mm wound gap. Cells were allowed to migrate into the wound gap, and visualized after 24 and 72 h using methylene blue staining. (B) Quantification of the distance between the front lines of migrating NDHFs. Data presented as mean ± SD, n=3, p-values are calculated using an unpaired student t-test, *p <0.05.

Figure 6.

BCM/TA coatings on Si wafers can be loaded with gentamicin and prevent bacterial growth. (A) 3.5 bilayer PVP/TA or BCM/TA coatings loaded and unloaded with gentamicin were inoculated with 10³, 10⁵, 10⁷ CFU cm⁻² of S. aureus ATCC 12600 and the growth of bacteria enumerated using Petrifilm plates (B).

Figure 7.

BCM/TA coatings on PCL/Coll NFs inhibit growth of S. aureus on agar plates and in TSB solution. ZOI diameter in mm (A) and images of ZOIs from bare (B) and BCM/TA-coated (C) PCL/Coll NFs loaded with clindamycin. ZOIs for BCM/TA-coated PCL/Coll NFs loaded with clindamycin are shown for freshly prepared samples (red) as well as samples after 20-weeks of storage at 5 °C (blue). OD of bacterial culture (D) and CFU per mL (E) over time show that clindamycin-loaded BCM/TA coatings on NFs significantly reduce OD and CFU. Data presented as mean ± SD, n=12 for A, n = 2 for D and E, p-values are calculated using an unpaired student t-test, *p <0.02.

Figure 8.

Highly tunable inhibition of S. aureus from clindamycin-loaded coatings on PCL/Coll NFs via a modular approach. (A) Images of zones of inhibition from clindamycin-loaded coatings on NFs with 0, 2 and 4 spacer layers at time points, 15 min, 1 and 3 h. (B) Quantification of zones of inhibition from clindamycin-loaded coatings on NFs with 0, 2 and 4 spacer layers. Data presented as mean ± SD, n=2.