(194z) Fluorescent Tagging of Interleukin-4 for Visualizing in-Vivo Release from Coated Implantable Polypropylene Mesh for Correlation of Release Patterns to Downstream Outcomes

Introduction: Polypropylene
mesh is commonly used for the repair of tissue in the gynecologic conditions
pelvic organ prolapse and stress urinary incontinence, as well as for hernia
repair. Unfortunately, polypropylene mesh has been associated with complications
including mesh exposure through neighboring tissues, pain, and infection. In
women who have had pelvic mesh-related complications and have had surgical
removal of their mesh, it has been shown that the mesh-tissue-interface is
characterized by an abundance of macrophages exhibiting a pro-inflammatory
phenotype and that this characteristic response is seen even years after
initial mesh placement.¹ Macrophages, however, are plastic cell
types that display a variety of phenotypes (Figure 1) along a spectrum
of M1 pro-inflammatory vs M2 pro-remodeling/anti-inflammatory extremes.² 
It has been postulated that modulation of macrophage phenotype during initial
stages of healing could prevent chronic inflammation, improving downstream
outcomes and mesh incorporation. Previous research in our lab has shown that a
novel IL-4 eluting coating for polypropylene mesh initially polarizes
macrophages to the pro-remodeling/anti-inflammatory phenotype, which results in
a mitigation of the foreign body reaction downstream.³ However,
timing and duration of the in-vivo immunomodulatory release of IL-4 (and its
effect on macrophage phenotype transition) is important for timely shift to
anti-inflammatory phenotypes and eventual resolution of inflammation. Thus far,
we have characterized the in-vitro release profile of IL-4 from our coated mesh.
What we have found is that, regardless of how much IL-4 is loaded into our
coating, release starts immediately upon placement into aqueous solution and
plateaus at around two weeks. However, nothing is known about this release
profile in-vivo. If in-vivo release profiles were known, these profiles could
be manipulated (e.g., IL-4 release could be delayed, lengthened, shortened,
etc.) and then correlated to downstream outcomes (i.e., success or failure of
an implant as determined by tissue integration months after implantation) in
order to better understand mechanistically how early events in-vivo (e.g.,
early host macrophage polarization) impact downstream outcomes. To monitor the
in-vivo release of IL-4 in a spatial and temporal way, we aim to use
fluorescently tagged IL-4 to monitor the location and persistence of the
fluorescent signal with daily live-animal in-vivo imaging in animals implanted
with our coated mesh. In-vivo release profiles can then be correlated to
downstream outcomes, providing for a tunable system.

Methods: Recombinant
murine interleukin-4 (Peprotech, Rocky Hill, NJ) was reconstituted according to
manufacturer’s instructions. Using the Alexa Fluor® 594 Microscale Protein
Labeling Kit (Molecular Probes, Eugene, OR), 100μg reconstituted IL-4 was
subjected to a fluorescent labeling reaction. In brief, the Alexa Fluor® 594
dye was conjugated to a succinimidyl ester that reacts with primary amine
groups in the IL-4 protein. A gel-resin spin filter was used to separate
unreacted dye from protein-dye conjugates. To assess successful labeling of
IL-4, HPLC with a size exclusion column was
used. In order to provide the most physiologically relevant in-vivo release
profile, it is important that the fluorescently tagged modified protein
maintain appropriate bioactivity. IL-4 should polarize macrophages to a
pro-remodeling phenotype with increased arginase-1 production; therefore,
tagged IL-4 vs untagged IL-4 was supplemented into the media of naïve
macrophages for 48 hours in culture. Macrophages were fixed and stained for
arginase-1 using immunofluorescence, images were acquired, and intensity of
staining was analyzed for 5000 cells per group using Cell Profiler Image
Analysis Software (Broad Institute, Cambridge, MA).  Finally, tagged IL-4 was
loaded into a dermatan sulfate-chitosan layer by layer (LbL) coating of
Gynemesh (Ethicon, Inc., Somerville, NJ) polypropylene mesh using a protocol
previously established in our lab.³ Briefly, in this process, uncharged
polypropylene meshes are subjected to radio frequency glow discharge in the
presence of maleic anhydride monomers. Hydrolysis of the grafted maleic
anhydride molecule results in a negatively charged surface. After negative charge
acquisition, the modified polypropylene mesh is subjected to alternating
submersions in liquid polymer solutions of opposite charges. Chitosan was
chosen as the polycation and dermatan sulfate was chosen as the polyanion. To
load fluorescently tagged IL-4 protein, dermatan sulfate polymer solution was
incubated overnight with cytokine prior to coating. After coating, confocal
fluorescence microscopy was used to visualize tagged IL-4 incorporation into
the mesh coating. Proof of concept work for in-vivo visualization was completed
using an IVIS 200 in-vivo imaging platform.

Results: At
an HPLC detection wavelength of 280nm, untagged IL-4 eluted off the column at
12.9 minutes, while tagged IL-4 eluted at 10.0 minutes, suggesting the
successful conjugation of the dye to IL-4 (resulting in a larger molecule). The
in-vitro culture assay (Figure 2) showed that tagged (Figure 2d)
and untagged (Figure 2c) IL-4 supplementation produced equivalent levels
of increased arginase-1 (Figure 2e) when compared to macrophages that
were cultured in media without supplementation (Figure 2b) or an isotype
control (Figure 2a), suggesting that the fluorescent tag does not affect
the bioactivity of the IL-4 protein. Confocal imaging of mesh coated with
untagged IL-4 (Figure 3a), mesh void of all coatings (Figure 3b),
and mesh coated with fluorescently tagged IL-4 (Figure 3c), shows that
there is a uniform signal in the red channel only for mesh that is coated with
fluorescently tagged IL-4, indicating that the tagged protein is able to be
incorporated into the LbL dermatan sulfate-chitosan coating. An IVIS 200
in-vivo imaging platform with fluorescence imaging capabilities will be used
for daily monitoring of signal degradation in-vivo. Proof of concept work has
shown that our tagged coated mesh can be visualized within the system, and our
chosen method of sterilization, ethylene oxide gas, does not interfere with the
bioactivity of IL-4 or the ability of the tag to fluoresce (Figure 4). Next
steps include implanting the fluorescent mesh in-vivo, monitoring fluorescent
signal loss and location over time, and correlating downstream histologic and
biochemical outcomes.

Conclusions: Murine
IL-4 protein was able to be fluorescently tagged, while maintaining appropriate
bioactivity, and was successfully incorporated into a layer by layer mesh
coating. In-vivo implantation of this tagged mesh will allow daily live-animal
imaging of the same animal until loss of signal so that release profiles can be
manipulated and then correlated to downstream outcomes.

References: 

1. A. L. Nolfi, et al., AJOG, 215: 206.e1-8, 2016.

2. F. O. Martinez and S. Gordon, F1000Prime
Reports, 6:13, 2014.

3. D. Hachim et al.,
Biomaterials 112: 95e107, 2017.