Device-based local delivery of siRNA against mammalian target of rapamycin (mTOR) in a murine subcutaneous implant model to inhibit fibrous encapsulation

Abstract

Fibrous encapsulation of surgically implanted devices is associated with elevated proliferation and activation of fibroblasts in tissues surrounding these implants, frequently causing foreign body complications. Here we test the hypothesis that inhibition of the expression of mammalian target of rapamycin (mTOR) in fibroblasts can mitigate the soft tissue implant foreign body response by suppressing fibrotic responses around implants. In this study, mTOR was knocked down using small interfering RNA (siRNA) conjugated with branched polyethylenimine (bPEI) in fibroblastic lineage cells in serum-based cell culture as shown by both gene and protein analysis. This mTOR knock-down led to an inhibition in fibroblast proliferation by 70% and simultaneous down-regulation in the expression of type I collagen in fibroblasts in vitro. These siRNA/bPEI complexes were released from poly(ethylene glycol) (PEG)-based hydrogel coatings surrounding model polymer implants in a subcutaneous rodent model in vivo. No significant reduction in fibrous capsule thickness and mTOR expression in the foreign body capsules were observed. The siRNA inefficacy in this in vivo implant model was attributed to siRNA dosing limitations in the gel delivery system, and lack of targeting ability of the siRNA complex specifically to fibroblasts. While in vitro data supported mTOR knock-down in fibroblast cultures, in vivo siRNA delivery must be further improved to produce clinically relevant effects on fibrotic encapsulation around implants.

Figure 1

Gel migration and cytotoxicity assays of mTOR siRNA/bPEI complexes. (a) Gel migration of mTOR siRNA/bPEI complexes at different NP ratios (–40). (b) Cell cytotoxicity of mTOR siRNA/bPEI complexes in 3T3 fibroblast in serum-based media. Assay was performed 24 hours-post transfection. (*p=0.0004, compared with cultures without treatment).

Figure 2

RNA message knock-down in 3T3 fibroblast cultures in serum-based media. (a) Western blot analysis for mTOR expression after mTOR siRNA/bPEI exposure. Cellular mRNA levels of (b) mTOR and (c) COL1A1 in cells treated with non-targeting siRNA (control) and mTOR siRNA/bPEI complexes were analyzed by RT-PCR. Cyclophilin B (housekeeping gene) mRNA was used as a control.

Figure 3

Microscopic images of fibroblast serum-based cultures (a) treated with non-targeting siRNA, (b) mTOR siRNA at NP ratio 20, and (c) cell proliferation in the siRNA-treated cells (NP 20, *p = 0.028), scale bar = 250μm. Images were taken 5 days after siRNA transfection. Numbers of cells treated with non-targeting siRNA was normalized to 100%.

Figure 4

Release of siRNA/bPEI complex from PEG-based hydrogels in vitro. (a) Cumulative siRNA release profiles in PBS media sink conditions. NP ratio of the siRNA/bPEI complex was 20. Release profiles in Formulation 1 (PEGDM: 4.25mg, DTT: 0.09mg) and Formulation 2 (PEGDM: 8.5mg, DTT: 0.18mg) are indicated by the solid line and dash-dotted line, respectively. (b) Western blot analysis for mTOR expression in fibroblasts after incubating cells with hydrogel-released siRNA/bPEI complexes. Cell lysis was harvested after three-day culture in this media. Numbers of days shown reflect the hydrogel release time prior to media collection and cell culture.

Figure 5

Comparison of in vivo collagen capsule thickness for murine sub-dermal hydrogel-coated implants explanted after two weeks for (a) negative control, no siRNA loaded, (b) 2μg mTOR siRNA loaded, (c) 10μg mTOR siRNA loaded and (d) positive control, 10 μg TGF-β siRNA loaded. Localization of fibrous capsule is marked by white arrows (scale bar = 100μm). (e) Summary of collagen capsule thickness data. P values for each individual group vs. negative control without siRNA treatment are: 0.3112 (2μg mTOR siRNA), 0.3954 (10μg mTOR siRNA), and 0.7045 (10μg TGF-β siRNA).

Figure 6

Immunostains of mTOR protein expression in foreign body capsules from murine histological sections. Tissue samples surrounding implants (I) were harvested from mice two-week post-implantation. Immunohistochemical staining for mTOR in foreign body capsules around filter paper from (a) negative control group (no siRNA loaded), (b) 2μg mTOR siRNA-treated group, and (c) 10μg mTOR siRNA-treated group. Sections were stained with mTOR antibody, and counterstaining was done with hematoxylin. These treatments stain target proteins red and cell nuclei dark (blue). Arrows denote fibroblasts (scale bar = 125μm).