Repair and reconstruction of a resected tumor defect using a composite of tissue flap-nanotherapeutic-silk fibroin and chitosan scaffold

Abstract

A multifaceted strategy using a composite of anti-cancer nanotherapeutic and natural biomaterials silk fibroin (SF) and chitosan (CS) blend scaffolds was investigated for the treatment of a tissue defect post-tumor resection by providing local release of the therapeutic and filling of the defect site with the regenerative bioscaffolds. The scaffold-emodin nanoparticle composites were fabricated and characterized for drug entrapment and release, mechanical strength, and efficacy against GILM2 breast cancer cells in vitro and in vivo in a rat tumor model. Emodin nanoparticles were embedded in SF and SFCS scaffolds and the amount of emodin entrapment was a function of the scaffold composition and emodin loading concentration. In vitro, there was a burst release of emodin from all scaffolds during the first 2 days though it was detected even after 24 days. Increase in emodin concentration in the scaffolds decreased the overall elastic modulus and ultimate tensile strength of the scaffolds. After 6 weeks of in vivo implantation, the cell density (p < 0.05) and percent degradation (p < 0.01) within the remodeled no emodin SFCS scaffold was significantly higher than the emodin loaded SFCS scaffolds, although there was no significant difference in the amount of collagen deposition in the regenerated SFCS scaffold. The presence and release of emodin from the SFCS scaffolds inhibited the integration of SFCS into the adjacent tumor due to the formation of an interfacial barrier of connective tissue that was lacking in emodin-free SFCS scaffolds. While no significant difference in tumor size was observed between the in vivo tested groups, tumors treated with emodin loaded SFCS scaffolds had decreased presence and size and similar regeneration of new tissue as compared to no emodin SFCS scaffolds.

Figure 1

(a) A schematic of the cross-section of silk fibroin and chitosan blend (SFCS)–emodin nanoparticle–latissimus dorsi muscle (LDM) flap composite applied at the tumor resected breast tissue site for the delivery of a nanotherapeutic to treat residual tumor overlaying the normal breast tissue and reconstruct the defect site with the SFCS scaffold via the process of tissue regeneration. (b) SFCS scaffold (25:75 blend) loaded with emodin in a circular petri dish mold (average diameter: 2.6 ± 0.1 cm and thickness: 1.3 ± 0.04 mm). (c) At the time of treatment of the tumor resected breast tissue site, emodin-loaded SFCS scaffold was sutured on the underside of the LDM flap. (d) Wound closures after scaffold–nanoparticle–flap composite implantation at the tumor bearing breast tissue site

Figure 2

The size of the emodin-loaded liposomes was measured using dynamic light scattering. (a) The volume based size distribution of the emodin-loaded liposomes without sonication results in higher frequency of particles in >360 nm range. (b) Sonication of liposomal emodin after 5 min of sonication results in higher frequency of nanoparticles ranging in size from 30 to 45 nm. (c) Mean diameter of liposomal emodin after sonication of 5 min decreased and the size was not affected by further exposure to sonication up to 60 min

Figure 3

Emodin loading efficiency within the liposomes and embedding within the SF and SFCS scaffold blend formulations. (a) Entrapment efficiency of emodin within the DMPC liposomes increased after sonication of 5 min and remained at 100% efficiency with increased sonication time n = 3, *p < 0.01 vs. all other sonication times. (b) Absolute emodin entrapment within the liposomal nanoparticles embedded in SF and SFCS scaffold blends (n = 3). *p < 0.001 vs. SF, 4.0 mg emodin, ^φ p < 0.001 vs. SF, 2.8 mg emodin, ^α p < 0.05 vs. SF, 1.7 mg emodin, ^β p < 0.001 vs. SF, 2.8 mg emodin, ^# p < 0.001 vs. SF, 4.0 mg, ^† p < 0.05 vs. 75:25 SFCS, 4.0 mg emodin, ^‡ p < 0.05 vs. 50:50 SFCS, 4.0 mg emodin, ^θ p < 0.01 vs. 25:75 SFCS, 4.0 mg emodin, emodin. (c) Percent entrapment of emodin within the liposomal nanoparticles embedded in SF and SFCS scaffold blends n = 3, *p < 0.05, **p < 0.01 vs. SF, 1.7 mg emodin, ^‡ p < 0.001 vs. SF, 2.8 mg emodin, ^θ p < 0.001 vs. SF, 4.0 mg emodin. All data reported as mean ± SEM

Figure 4

Representative curves for the emodin release from liposomal nanoparticle and SF based scaffolds. (a) Percent emodin released from SF and 25:75 SFCS scaffolds for emodin loading concentration of 4.0 mg shows higher amounts of emodin released from SF scaffolds as compared to the SFCS scaffolds (n = 3). (b) Comparison of percent emodin released from 25:75 SFCS with three different emodin loading concentrations (n = 3). All data reported as mean ± SEM

Figure 5

Mechanical properties of SF and SFCS scaffolds with and without emodin loading for the three concentrations were determined using uniaxial tensile testing. (a) Representative stress vs. strain curves for SF scaffolds (no emodin), 25:75 SFCS scaffolds (no emodin), and 25:75 SFCS scaffolds, 1.7 mg emodin are shown. (b) Comparison of the elastic modulus of SF and SFCS scaffolds with and without emodin nanoparticle loading (n = 6). ^‡ p < 0.001 vs. SF no emodin control, *p < 0.05 vs. 75:25 SFCS no emodin control, ^α p < 0.01 vs. 50:50 SFCS no emodin control, ^β p < 0.05 and ^ββ p < 0.001 vs. 25:75 SFCS no emodin control. (c) Comparison of ultimate tensile strength (UTS) of SF and SFCS scaffolds with and without emodin nanoparticle loading (n = 6). ^‡ p < 0.001 vs. SF no emodin control, *p < 0.05 and **p < 0.01 vs. 50:50 SFCS no emodin control, ^φ p < 0.05 vs. 75:25 SFCS no emodin control, ^β p < 0.01 and ^ββ p < 0.001 vs. 25:75 SFCS no emodin control, ^# p < 0.01 vs. SF (4.0 mg emodin). All data reported as mean ± SEM

Figure 6

(a) Cell viability of GILM2 breast cancer cells measured using the MTT assay after 4 days of exposure to liposomal emodin nanoparticles. The increase in the emodin concentration decreased the number of viable cells (n = 3), ^# p < 0.001 vs. control, ^‡ p < 0.05 vs. 20 μM emodin concentration. (b) Comparison of GILM2 breast cancer cell viability after exposure to liposomal emodin 25:75 SFCS, 4.0 mg emodin scaffolds with no emodin controls with and without the 25:75 SFCS scaffold (n = 3), *p < 0.01 vs. control, ^† p < 0.05 vs. SFCS only. All data reported as mean ± SEM

Figure 7

Histological microsections of the reconstructed GILM2 breast tumor site after 6 weeks of implantation of 25:75 SFCS-emodin nanoparticle-muscle flap composite stained with either H&E or Movat stains. (a) A Movat stained cross-section of the breast tumor site reconstructed with the LD muscle tissue flap only. (b) A H&E stained cross-section of the tumor site reconstructed with the SFCS scaffold shows the muscle flap components (skin, connective tissue, muscle) interfaced and well-integrated with the partially degraded emodin-loaded SFCS scaffold that contains the interspersed fibrils embedded in the regenerated tissue. (c) A H&E stained cross-section of tumor scaffold interface that was seamlessly integrated when reconstructed with SFCS scaffold-LDM flap composite with no emodin loading. (d) A Movat stained cross-section of remodeled emodin-loaded SFCS scaffold interfaced with a tumor via layers of connective tissue. (e) A Movat stained cross-section showing the blood vessels (see arrows) formed at the interface of the muscle and the emodin loaded SFCS scaffold. (f) A Movat stained cross-section of the SFCS scaffold showing partial degradation and remodeling of the scaffold. The remainder scaffold fibrils (red) are surrounded by collagen (yellow) deposited in the regenerated tissue

Figure 8

The degradation of SFCS scaffold was quantified using image processing in Image J software. An example of the (a) original H&E image and (b) the image after processing shows the area covered by non-degraded scaffold