Inflammasome modulation with P2X7 inhibitor A438079-loaded dressings for diabetic wound healing

Abstract

The inflammasome is a multiprotein complex critical for the innate immune response to injury. Inflammasome activation initiates healthy wound healing, but comorbidities with poor healing, including diabetes, exhibit pathologic, sustained activation with delayed resolution that prevents healing progression. In prior work, we reported the allosteric P2X7 antagonist A438079 inhibits extracellular ATP-evoked NLRP3 signaling by preventing ion flux, mitochondrial reactive oxygen species generation, NLRP3 assembly, mature IL-1β release, and pyroptosis. However, the short half-life in vivo limits clinical translation of this promising molecule. Here, we develop a controlled release scaffold to deliver A438079 as an inflammasome-modulating wound dressing for applications in poorly healing wounds. We fabricated and characterized tunable thickness, long-lasting silk fibroin dressings and evaluated A438079 loading and release kinetics. We characterized A438079-loaded silk dressings in vitro by measuring IL-1β release and inflammasome assembly by perinuclear ASC speck formation. We further evaluated the performance of A438079-loaded silk dressings in a full-thickness model of wound healing in genetically diabetic mice and observed acceleration of wound closure by 10 days post-wounding with reduced levels of IL-1β at the wound edge. This work provides a proof-of-principle for translating pharmacologic inhibition of ATP-induced inflammation in diabetic wounds and represents a novel approach to therapeutically targeting a dysregulated mechanism in diabetic wound impairment.

Full text

Inflammasome modulation with
P2X7 inhibitor A438079-loaded
dressings for diabetic
wound healing
Jordan R. Yaron
1,2
*, Selin Bakkaloglu
1
, Nicole A. Grigaitis
1,3
,
Farhan H. Babur
1
, Sophia Macko
1
, Samantha Rhodes
1
,
Solenne Norvor-Davis
1
and Kaushal Rege
1,2,3,4
1
Center for Biomaterials Innovation and Translation, The Biodesign Institute, Arizona State University,
Tempe, AZ, United States,
2
School for Engineering of Matter, Transport & Energy, Arizona State
University, Tempe, AZ, United States,
3
Biological Design Graduate Program, Arizona State University,
Tempe, AZ, United States,
4
Chemical Engineering, Arizona State University, Tempe, AZ, United States
The inflammasome is a multiprotein complex critical for the innate immune
response to injury. Inflammasome activation initiates healthy wound healing, but
comorbidities with poor healing, including diabetes, exhibit pathologic, sustained
activation with delayed resolution that prevents healing progression. In prior
work, we reported the allosteric P2X7 antagonist A438079 inhibits extracellular
ATP-evoked NLRP3 signaling by preventing ion flux, mitochondrial reactive
oxygen species generation, NLRP3 assembly, mature IL-1brelease, and
pyroptosis. However, the short half-life in vivo limits clinical translation of this
promising molecule. Here, we develop a controlled release scaffold to deliver
A438079 as an inflammasome-modulating wound dressing for applications in
poorly healing wounds. We fabricated and characterized tunable thickness, long-
lasting silk fibroin dressings and evaluated A438079 loading and release kinetics.
We characterized A438079-loaded silk dressings in vitro by measuring IL-1b
release and inflammasome assembly by perinuclear ASC speck formation. We
further evaluated the performance of A438079-loaded silk dressings in a full-
thickness model of wound healing in genetically diabetic mice and observed
acceleration of wound closure by 10 days post-wounding with reduced levels of
IL-1bat the wound edge. This work provides a proof-of-principle for translating
pharmacologic inhibition of ATP-induced inflammation in diabetic wounds and
represents a novel approach to therapeutically targeting a dysregulated
mechanism in diabetic wound impairment.
KEYWORDS
wound dressing, silk fibroin, inflammasome, small molecule, drug delivery,
wound healing
Frontiers in Immunology frontiersin.org01
OPEN ACCESS
EDITED BY
Ronghua Yang,
Guangzhou First People’s Hospital, China
REVIEWED BY
Nik Theodoros Georgopoulos,
Sheffield Hallam University, United Kingdom
Weichang Li,
Sun Yat-sen University, China
*CORRESPONDENCE
Jordan R. Yaron
jyaron@asu.edu
RECEIVED 17 November 2023
ACCEPTED 29 January 2024
PUBLISHED 15 February 2024
CITATION
Yaron JR, Bakkaloglu S, Grigaitis NA,
Babur FH, Macko S, Rhodes S, Norvor-Davis S
and Rege K (2024) Inflammasome modulation
with P2X7 inhibitor A438079-loaded
dressings for diabetic wound healing.
Front. Immunol. 15:1340405.
doi: 10.3389/fimmu.2024.1340405
COPYRIGHT
© 2024 Yaron, Bakkaloglu, Grigaitis, Babur,
Macko, Rhodes, Norvor-Davis and Rege. This is
an open-access article distributed under the
terms of the Creative Commons Attribution
License (CC BY). The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the
copyright owner(s) are credited and that the
original publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or reproduction
is permitted which does not comply with
these terms.
TYPE Brief Research Report
PUBLISHED 15 February 2024
DOI 10.3389/fimmu.2024.1340405

1 Introduction
Chronic cutaneous wounds are a major medical burden. More
than 6 million chronic wound cases amount to a cost of over $20
billion per year in healthcare costs in the USA - nearly 5% of the
total cost of Medicare and Medicaid (1). Acute wound healing
proceeds along a spectrum of continuous and overlapping phases of
(i) hemostasis, (ii) inflammation, (iii) proliferation and (iv)
remodeling (2–4). Delay in the onset or resolution of any stage
leads to impaired healing and complex comorbidities such as
diabetes are commonly associated with poor healing. Diabetic
patients have a 15% lifetime risk for chronic foot ulcers, which
remain the primary cause for amputation and result in significant
negative emotional, physical, and financial costs (5). The 5-year
survival for lower limb amputations due to diabetic wounds is only
23%, approximately the same as the mortality rate seen for all
cancers (6). Selective targeting of dysregulated repair mechanisms
in diabetic wounds may provide an effective approach to reverting
impaired healing back to a healthy, acute healing state and is an
active field of investigation (7).
While inflammation is known to be important for initiating
reparative events in healing wounds (8), diabetic wounds are
characterized by dysregulated and sustained inflammation (9).
The inflammasome is a multi-protein platform that drives
inflammatory responses in a wide and growing array of infected
and sterile pathologies (10). The NLRP3 inflammasome is the most
widely studied of the various inflammasome platforms in part due
to its role in the greatest number of identified pathologies and is
most dominantly active in myeloid-lineage immune cells (11),
though other cells have been shown to exhibit NLRP3 pathway
activity (12–15). The NLRP3 inflammasome can be regarded as a
central nexus to cellular stressors upon myeloid cells and, once
licensed for activation by a priming signal such as interleukin (IL)-
1aor lipopolysaccharide (LPS), is activated by diverse stimuli such
as ion flux induced by extracellular damage associated molecular
patterns (DAMPs), frustrated phagocytosis of crystalline structures,
lysosomal instability, endocytic pathway disruption, and others (10,
16). While the NLRP3 inflammasome pathway has been shown to
be an important early player in healthy wound healing, its sustained
activation has been implicated in impaired wound healing in
diabetes (17).
Detection of extracellular ATP by the purinergic receptor P2X7
is a canonical initiator of NLRP3 inflammasome activity (18).
Targeting extracellular ATP-induced inflammasome activation
has been reported to improve diabetic wound impairment. Mirza
and colleagues were the first to report a role for the NLRP3
inflammasome in diabetic wound impairment using genetically
diabetic mice (19). Using glyburide, an inhibitor of K
ATP
potassium channel activity and extracellular ATP-induced NLRP3
activation, they showed an improvement in wound closure and a
reduction in ongoing inflammation at 10 days post-wounding.
Subsequently, Bitto and colleagues showed improved wound
closure in diabetic mice with the P2X7 receptor antagonist
Brilliant Blue G and the direct NLRP3 inhibitor Bay 11-7082 (20).
In both studies, the inhibitors required multiple administrations
owing to the short half-life of these molecules. Repeated
administration of drugs to wounds can cause secondary injury,
thus prolonging healing and increasing the risk of additional
complications (21,22). Further, multiple administrations of drugs
or dressings reduce patient compliance to complex wound care
regimens (23). Thus, dressings which are capable of sustained
delivery of drugs may enable improved clinical care for wound
care patients (24).
We have previously characterized the in vitro activity of
A438079, a potent small molecule inhibitor of P2X7 (25), and
reported that its use for P2X7 inhibition results in suppression of
both potassium and calcium fluxes and abrogation of mROS
generation thereby potently suppressing NLRP3 inflammasome
activation in macrophages in culture (26). Here, we develop a
biomaterial-based platform to act as a depot for sustained
delivery of A438079 in vivo and evaluate its efficacy in a model of
impaired diabetic wound healing. Prior reports indicate A438079
has a low bioavailability (19%), high plasma protein binding (84%),
and short half-life (1.02 hours) (27). Because inflammasome activity
persists for at least 3-6 days in diabetic wounds, repeated
administration or sustained release of inflammasome-modulating
drugs is necessitated for therapeutic augmentation. We describe the
development and characterization of a wound dressing fabricated
from purified silk fibroin, a naturally occurring biopolymer with
high biocompatibility and amenability to physicochemical
modification, that can be loaded with variable amounts of
A438079 and exhibit sustained delivery kinetics compatible with
modulation of early-stage over-activation of inflammasome activity
in diabetic wounds. We first test our all-in-one drug-loaded
dressings in vitro using mouse macrophages and then evaluate
efficacy in a wound healing model in vivo in diabetic mice. To our
knowledge, this is the first demonstration of a therapeutic topical
dressing platform to target P2X7-mediated inflammation in
diabetic wounds and presents a novel approach to therapeutically
modulate impaired wound healing in different pathologies.
2 Materials and methods
2.1 Generation and characterization of silk
fibroin films
Silk fibroin was obtained by degumming Bombyx mori silkworm
cocoons as previously described (28). Briefly, raw cocoons were cut
into small sections and washed thoroughly in nanopure water
(resistivity 18.2 MW-cm). Degumming was performed by boiling
washed cocoon pieces in a solution of Na
2
CO
3
in nanopure water
(0.005 g/mL, 0.5% w/v) for 30 minutes with slow stirring. Degummed
material was washed three times in nanopure water for 15-20 minutes
in each wash, pulled into thin sections, and dried overnight at room
temperature. The dried silk fibroin material was dissolved in 9.4M
lithium bromide at 60°C (2 g silk fibroin per 10 mL solution) with
stirring by adding the silk piece wise. Once dissolved, the solution was
continuously stirred for an additional 4 hours at 60°C. Dissolved silk
solution was clarified through cotton filter paper, transferred to 3.5
kDa dialysis membranes, and dialyzed against nanopure water for 72
hours at 4°C. Concentration and weight percentage of resultant silk
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fibroin solution was determined by back-calculation of the mass of
dried, known volume of silk in a pre-weighed weigh boat. Purified silk
composition was characterized by FT-IR on a Thermo Nicolet
6700 spectrometer.
Films were cast in a 100 mm petri dish by dispensing 4 or 8 mL
of an 8% w/v solution followed by overnight solvent evaporation
at room temperature. Rheometry of 10x15-mm films was
performed by Dynamic Mechanical Analysis on a Discovery HR
30 rheometer (TA Instruments) with a frequency sweep from 1 Hz
to 10 Hz and a strain of 0.1% to measure storage and loss moduli.
Round films were produced by using a 6-mm hollow punch tool.
Films were made insoluble by placing in an autoclavable pouch and
autoclaving in a 20-minute steam/heat cycle. Films were measured
using a micrometer-type caliper (Rexbeti) to confirm thickness
prior to use.
2.2 Scanning electron microscopy
Silk films were sputter coated with 1.5-2nm gold coating on a
Cressington 108 sputter coater (exposure 120s with an argon gas
environment). High-resolution field emission SEM was performed
on an Auriga (Zeiss) scanning electron microscope equipped with a
high-resolution Gemini Field Emission-SEM column and Schottky
thermal field emitter operating at 5.00 kV HV and a monopole
magnetic immersion final lens. Images were collected en face and in
cross-section after cutting the film with a scalpel.
2.3 Film swelling and passive
degradation studies
Autoclaved films were placed in 1X PBS and incubated for up to
40 days at room temperature. Film thickness was measured with a
micrometer-type caliper (Rexbeti) and degradation was determined
by protein quantity in solution at 40 days using a BCA protein
assay (Pierce).
2.4 A438079 molecular docking
A438079 was docked to the published structure for P2X7 in the
closed, apo state (PDB 5U1L) (29) using Webina, a webserver for
Autodock Vina (30). Docking results were visualized in Chimera X
version 1.3 (31). Results were validated against prior docking
reported by Allsopp et al. (32).
2.5 A438079 spectroscopy analysis
A438079 was purchased from Santa Cruz Biotechnology and
dissolved in a solution of 50% DMSO in nanopure water to produce a
25 mM stock solution. Aliquots were prepared and stored at -20°C.
Two-fold serial dilutions from 2.5 mM to 0.04 mM were generated in
1X PBS and dispensed in 50 µL volumes in a UV-transparent 96-well
plate. Absorbance spectroscopy was performed on a Biotek Synergy
H2 plate reader from 200-300 nm with a 2 nm resolution. Diagnostic
peaks were observed at 226 nm and 258 nm.
2.6 Loading and release studies
Insolubilized silk films were incubated in solutions of 1.25 mM
A438079 for 24 hours. The amount of the small molecule inhibitor
drug loaded was evaluated by absorbance spectroscopy of the post-
loaded solution versus solution that was not incubated in the
presence of a film. Release studies were performed by incubating
a loaded film in 1X PBS at room temperature with shaking. At
regular intervals, the PBS soaking solution was removed and
analyzed by absorbance spectroscopy. The films were placed in
fresh solutions of PBS to generate continuous sink conditions.
Cumulative release was calculated and fit to a two phase burst
release model in GraphPad Prism with the “two phase association”
equation (33):
SpanFast = (Plateau −Y0)*PercentFast*:01
SpanSlow = (Plateau −Y0)*(100 −PercentFast)*:01
Y = Y0 + SpanFast*(1 −exp( −KFast*X)) + SpanSlow*(1 −exp( −KSlow*X))
(1)
2.7 Cell culture
J774DUAL cells (Invivogen) were cultured in DMEM with 10%
FBS containing 1% penicillin/streptomycin and additional
supplementation of selective antibiotics according to
manufacturer’s procedure (5 µg/mL Blasticidin, 100 µg/mL
Zeocin®). Cells were fed twice a week and passaged by cell
scraping. Cell viability during passaging was evaluated by Trypan
Blue staining using an EVE™Plus Automated Cell Counter
(NanoEnTek) to ensure 95% or greater viability prior to use.
2.8 IL-1bELISA analyses
On day 1, J774DUAL cells were seeded at 10
5
cells/well in a tissue
culture-treated 96-well plate and incubated overnight at 37°C/5%
CO
2
in a humidified incubator in complete DMEM without selective
antibiotics. On the same day, an ELISA plate (R&D systems, DY008)
was coated with capture antibody to IL-1b(R&D Systems, DY401)
according to manufacturer’s instructions, sealed with plate film, and
incubated at room temperature overnight. On day 2, cells were either
left untreated or primed with 1 µg/mL E. coli LPS (O111:B4; “first
signal”)in100µLfinal volume fresh DMEM (no selective antibiotics)
and returned to the incubator for 4 hours. Concurrently, A438079-
loaded films were incubated in 220 µL fresh DMEM (10% FBS, 1%
pen/strep, no selective antibiotics) at room temperature in a 1.5 mL
microcentrifuge tube. ELISA plates were washed and blocked during
the priming period according to manufacturer’s protocol. During the
final 30 minutes of priming (3.5 hours after initial treatment with
LPS), 50% (i.e., 50 µL) of the incubation medium was replaced with
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either fresh DMEM or film-conditioned medium. The plate was
returned to the incubator for the final 30 minutes of priming. To
stimulate inflammasome activation (“second signal”), cells were
treated with 3 mM final concentration of extracellular ATP (3 µL
of a 100 mM stock solution prepared in fresh DMEM mixed into 100
µL final culture volume) and returned to the incubator for 45
minutes. After 45 minutes, complete (100 µL) cell supernatants
were collected, transferred to the blocked ELISA plate along with
recombinant IL-1bstandards, sealed with plate film, and incubated
overnight at 4°C. On day 3, the ELISA plate was washed, probed with
detection antibodies, and developed according to manufacturer’s
procedure. The developed ELISA plate was read on a Biotek
Synergy H2 plate reader at 450 nm with background subtraction at
540 nm. Data analysis using 4-parameter logarithmic regression was
performed in GraphPad Prism.
2.9 Immunofluorescence microscopy
Cells were treated as in Section 2.8 above for ELISA analyses. After
45 minutes of ATP treatment, the culture medium was aspirated, and
cells were fixed in 2% formaldehyde (freshly prepared from
paraformaldehyde in 1X PBS) for 20 minutes at room temperature.
Cells were permeabilized with 1X TBS containing 0.2% Tween-20
(TBST) for 20 minutes at room temperature and blocked in 5% BSA
prepared in 1X TBST for 1 hour at room temperature. Cells were
incubated overnight at 4°C with rabbit polyclonal antibody to ASC
(AL177, 1:200 dilution, Adipogen) in 5% BSA/TBST. The following day,
cells were washed with TBST and incubated with CF568-conjugated
donkey anti-rabbit secondary antibody (1:500 dilution, Biotium) and
iFluor 488-conjugated Phalloidin (1:1000 dilution, AAT Bioquest) for 2
hours at room temperature protected from light. Cells were washed with
TBST and incubated for 30 minutes with 10 µg/mL DAPI (Abcam) in
1X PBS at room temperature protected from light. Cells were washed
with PBS and submerged in 250 µL Fluoromount G (Thermo-Fisher)
andstoredat4°Cprotectedfromlightuntilimaging.Cellswereimaged
on a Nikon AXR confocal (22.1 µm pinhole) mounted to a Ti2 base
with a Plan Apo 60× Oil NA 1.42 objective lens using 408, 488, and 561
nm laser lines paired to DAPI (429-474 nm), AF488 (503-541 nm), and
AF568 (571-625 nm) emission windows, respectively, with at
1024x1024 resolution setting with 2X frame averaging. Images were
collected with NIS-Elements AR software (ver. 5.41.01 build 1709) and
analyzed using FIJI/ImageJ (ver. 2.14.0/1.54f).
2.10 In vivo wound healing studies
All animal procedures were approved by the Institutional Animal
Care and Use Committee of Arizona State University under protocol
#21-1830R. Mice were purchased from the Jackson Laboratory and
kept on a standard 12/12 light-dark cycle in specific pathogen-free
housing conditions and given food and water ad libitum. Full-
thickness wound healing was performed in male and female 12-
week old obese, diabetic “db/db”mice (BKS.Cg-Dock7
m
+/+ Lepr
db
/J;
JAX strain code 000642) as previously described (34,35). Briefly,
non-fasting blood glucose was measured within one week of surgery
by saphenous vein collection and confirmed to be >400 mg/dL using
a glucometer. Mice were anesthetized and a 1x1-inch midline
intrascapular area between the base of the neck and apex of the
spine was shaved and sterilized with successive washes with alcohol
and chlorhexidine gluconate solution. A 6-mm full-thickness biopsy
punch was performed and wounds were either treated directly with
20 µL saline, 100 µM A438079 in 20 µL saline, empty silk film, or silk
film loaded with A438079. Treatments were applied a single time
during the study. A silicone splint (14mm OD x 7mm ID x 0.5mm
thick, Grace Biolabs) covered with Tegaderm occlusive dressings
(3M) were affixed using cyanoacrylate glue (Krazy Glue) and six
interrupted sutures (4-0 black Ethilon monofilament with a FS-2
reverse cutting needle; Ethicon, Inc.). Mice were returned to single
housed cages to prevent removal of splints by cage mates and
monitored daily until euthanasia at 10 days post-wounding.
Wound area (planimetry) after splint removal was documented by
digital photography using a mobile phone and analyzed with
calibration in ImageJ/FIJI.
2.11 Immunohistochemistry
Wound tissues were collected at day 10 post-wounding and
fixed in 10% neutral-buffered formalin. Tissues were dehydrated
through graded alcohol into xylene followed by paraffin perfusion.
Tissues were embedded into paraffin blocks and 5-6 µm sections
were captured onto positively charged glass slides. Sections were
rehydrated and epitope retrieval was performed by incubation
in sodium citrate buffer (10 mM, pH 6.0 with 0.05% Tween-20)
at 60°C overnight. Sections were blocked with 5% bovine serum
albumin (BSA) in TBS/0.1% Tween-20 for 1 hour at room
temperature and incubated overnight with goat anti-mouse IL-1b
primary antibody (R&D Systems, AF-401-NA, 1:100) in 5% BSA in
TBS/0.1% Tween-20 at 4°C overnight. Sections were washed with
TBS/0.1% Tween-20 and peroxide quenching was performed with
3% hydrogen peroxide in PBS for 15 minutes at room temperature
followed by thorough washing. Sections were incubated in
horseradish peroxidase (HRP)-conjugated donkey anti-goat
secondary antibody (Jackson Immunoresearch #705-035-147,
1:500) for 2 hours at room temperature followed by thorough
washing. Sections were developed with ImmPACT DAB substrate
(Vector Labs #SK-4105) for 5 minutes, counterstained with
hematoxylin (Gill No. 2, #GHS232, Sigma Aldrich), dehydrated,
and mounted with CytoSeal XYL (Thermo Fisher). Slides were
scanned at 40X magnification on an Olympus VS200 Slide Scanner
and quantified using QuPath software v0.4.3 (36).
3 Results
3.1 Fabrication and characterization of
insoluble, tunable thickness silk fibroin
film dressings
We sought to generate a tunable thickness, insoluble silk fibroin
film as a long-lasting wound dressing matrix. We enriched silk fibroin
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from Bombyx mori silkworm cocoons and characterized the
polypeptide by FT-IR spectroscopy (Figure 1A) with expected
amide I peak at 1650, amide II peak at 1520, and amide III peak at
1240 cm
-1
(37). We cast 100-mm diameter sheets of silk fibroin in
petri dishes and generated films by solvent evaporation at room
temperature overnight. Using a hollow punch tool, we fabricated 6-
mm diameter wound dressings (Figure 1B), which we made insoluble
by autoclaving (38). Autoclaving is thought to increase the beta sheet
content in silk fibroin, which makes them resistant to dissolution in
aqueous solutions (39). We performed scanning electron microscopy
(SEM; Figure 1C) on insolubilized films and observed that while the
exposed surfaces of the films were relatively smooth and without
notable features, the cross-section of the films indicated a highly
complex network structure. Dynamic mechanical analysis of the films
(Figure 1D) indicated that the films exhibited durable elastic
properties with high storage and loss moduli of ~3300 MPa and
~200 MPa, respectively, with a low tan(d) of ~0.06, which indicated
elastic nature of these films. Film thickness was tuned by modulating
the amount of silk solution used to cast the initial film sheet, with 4
mL of 8 w/v% silk solution in a 100-mm petri dish resulting in films
of ~60-µm in thickness, and 8 mL of 8 w/v% silk solution resulting in
films of ~120-µm thickness (Figure 1E). We evaluated the passive
degradation of the films in PBS over a period for 40 days at room
temperature and found that 60-µm films cast from 4 mL solutions did
not exhibit any swelling behavior (-2% swelling ratio), while 120-µm
films cast from 8 mL solutions exhibited minor swelling behavior to
~140-µm (16.7% swelling ratio). In both cases, BCA protein assay of
the incubation solution indicated no presence of dissolved protein
(data not shown) and thus no passive degradation over 40 days.
Taken together, we generated insoluble and durable, tunable
thickness silk fibroin films appropriate for wound dressing
applications (40,41).
3.2 Characterization and drug release
characterization of P2X7 receptor
inhibitor A438079
A438079 is a water-insoluble, selective antagonist of the P2X7
purinergic receptor that allosterically binds in a region of the ion-
permeating channel in the left flipper of the extracellular domain in
close proximity to the previously described residues F88, F92, T94,
F95, and F103 (Figure 2A)(32,42). This allosteric site is ideal to
facilitate druggability of the P2X7 receptor, as allosteric sites are
commonly more selective, allow lower target-based toxicity,
fewer side effects, and accessible physicochemical properties
(43,44). We previously investigated the inhibitory function of
A438079 and reported a potent ability to ameliorate extracellular
ATP-evoked potassium and calcium flux and subsequent
inflammasome activation in mouse macrophages (26). We
performed UV-VIS spectrophotometric analysis of A438079 in
PBS (Figure 2B) and identified two diagnostic absorption peaks at
226 nm and 258 nm which exhibit linear response from 40 µM to
2500 µM (Figure 2C). We passively loaded 60-µm and 120-µm films
with A438079 overnight in a 1.25 mM loading solution in PBS and
measured release over 72 hours into PBS. Kinetics analysis indicated
a release profile fitting to a two-phase burst release model (Equation
1), with release amount proportional to film thickness (Figure 2D).
A concentration of approximately 100 µM and 200 µM in 100 µL
PBS was measured in the first 6 hours from 60-µm and 120-µm
films, respectively. Release continued at ~40 µM and ~60 µM per
day for the first 2 days for 60-µm and 120-µm films, slowing to ~20
µM and ~30 µM by the third day, respectively. We observed that
prolonging the drug loading time from 1 days to 3 days increased
the amount of released drug, indicating a one-day load does not
saturate loading capacity of 60-µm films (Supplementary Figure S1).
Films were stored dry in plastic bags after loading and kept at room
temperature for up to 1 week prior to use. Thus, insoluble silk
fibroin wound dressings were loaded with A438079 in a film-
tunable and loading-tunable manner with differential release
kinetics into aqueous solution.
3.3 In vitro evaluation of A438079-
loaded films
We next sought to determine whether A438079-loaded films
would exhibit therapeutic properties in an in vitro system of
inflammation. We used the mouse macrophage cell line,
J774DUAL (derivative of J774A.1), to investigate inflammasome
signaling. We first performed ELISA to measure production and
release of IL-1b,akeyinflammatory cytokine and secreted
inflammasome mediator (Figure 3A). J774DUAL cells primed
with LPS for 4 hours with E. coli LPS and stimulated for 45
minutes with 3 mM extracellular ATP to activate P2X7 receptor
signaling robustly secreted IL-1b. When cells were treated with
conditioned medium exposed to A438079-loaded silk films, near-
complete inhibition of IL-1bsecretion was observed. We
next evaluated whether this inhibition of IL-1bsecretion was
due to inhibition of inflammasome activity. We performed
immunofluorescence of J774DUAL cells primed with LPS and
stimulated with 3 mM extracellular ATP to evaluate the
formation of perinuclear ASC specks, a canonical indicator of
inflammasome assembly (Figures 3B,C). Without inhibitor
treatment, approximately 6% of the population exhibited
perinuclear ASC specks. We note these levels of ASC speck
positivity are in agreement with prior work by us (26) and others
(45,46), but are likely an underestimation due to repeated wash
steps during immunofluorescence processing. When treated with
conditioned medium exposed to A438079-loaded silk films, <0.5%
of the population exhibited ASC specks. Thus, A438079-loaded silk
films robustly inhibit inflammasome assembly and activity when
evaluated with mouse macrophages in vitro.
3.4 In vivo evaluation of A438079-
loaded films
We generated full-thickness 6-mm biopsy punch wounds in the
dorsum of genetically diabetic db/db mice (Figure 4). Wounds were
treated at the time of wounding either with saline or saline solution
containing 100 µM A438079 without silk film (“direct A438079;”
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A
B
DE
C
FIGURE 1
Fabrication and characterization of insoluble, tunable silk fibroin wound dressings. (A) FT-IR spectra of purified silk fibroin with key peaks indicated
for amide I (1650 cm
-1
), amide II (1520 cm
-1
), and amide III (1240 cm
-1
). (B) Representative 6-mm diameter silk wound dressing. (C) SEM of top
surface (“top”) and cross-section (“cross”) of insoluble silk dressing at 10,000x. Scale bar = 1 µm. (D) DMA results for Storage Modulus, Loss Modulus,
and Tan(d) of insoluble silk dressing. (E) 40-day swelling study of 4 mL (60-µm thick) and 8 mL (120-µm thick) cast silk dressings. Results are shown
as mean ± standard error. Statistics are performed by two-way ANOVA with Fisher’s LSD. *p<0.05.
A
B DC
FIGURE 2
Characterization of A438079 cargo and drug delivery kinetics. (A) Chemical structure of A438079 and illustration of molecular docking region in the
P2X7 structure in top and side view. ATP-binding pocket residues are colored green, while A438079-binding residues are colored red. One
monomeric unit of P2X7 is colored cyan. (B) Spectrophotometric analysis of A438079 with diagnostic wavelengths indicated at 226 nm and 258 nm.
Values in the legend are given as [A438079] in mM. (C) Log-log concentration versus absorbance for A438079 at diagnostic wavelengths of 226 nm
and 258 nm demonstrating linear response with 95% confidence intervals and R
2
of regression indicated. (D) Release kinetics of A438079 over 72
hours at room temperature in 1X PBS from 60-µm thick and 120-µm thick films versus unloaded 60-µm thick films (control) with curve fit for two
phase burst release model results. R
2
given for model fit. Results are shown as mean ± standard error.
Yaron et al. 10.3389/fimmu.2024.1340405
Frontiers in Immunology frontiersin.org06

dosage selected according to release dynamics in vitro –Figure 2D)
or had empty silk films or silk films loaded with A438079 applied.
Planimetric analysis was performed at 10 days post-wounding,
similar to prior reports (19). We found that silk films alone and a
topical aqueous solution of A438079 partially, but non-significantly,
enhanced wound closure. By comparison, silk films loaded
with A438079 resulted in a 40% wound closure which was
statistically significant versus saline treated, empty film-treated,
and direct drug-treated wounds (Figures 4A,B). We performed
immunohistochemical staining for IL-1bas the most direct
downstream inflammatory mediator of inflammasome activation
(Figures 4C,D). Quantification of IL-1bpositive cells at the wound
margin indicated a statistically significant reduction in wounds
treated with silk films loaded with A438079 compared to saline or
empty silk film treated wounds. Comparison to direct A438079
treated wounds approached significance (p=0.21), suggesting even a
day 0 treatment with A438079 exerted some anti-inflammasome
activity over the course of diabetic healing. Thus, A438079-loaded
films, acting as a depot, promote enhanced and sustained anti-
inflammasome activity resulting in improved wound closure in
diabetic wounds.
4 Discussion
We describe a silk fibroin wound dressing loaded with
inflammasome-modulating cargo for use in diabetic wound
treatments. Silk fibroin has gained attention as a sustainable,
biocompatible, and physicochemically alterable biomaterial
amenable to fabrication into sutures, films, and particles for use
in wound care (47). By insolubilizing the films with autoclave
pretreatment, our films exhibit no loss of integrity by dissolution.
However, we cannot exclude the possibility of proteolytic
degradation in the wound exudate (48), which may explain the
A
B
C
FIGURE 3
In vitro evaluation of A438079-loaded films. (A) ELISA quantification of supernatant IL-1breleased from J774DUAL macrophages left untreated or
primed for 4 hours with 1 µg/mL LPS and stimulated for 45 minutes with 3 mM ATP. Cells were treated for the final 30 minutes of priming with
conditioned media exposed to A438079-loaded silk films for 4 hours. Results are given as mean ± standard error with statistics calculated by two-
way ANOVA and Fisher’s LSD. ****p<0.0001. (B) Quantification of perinuclear ASC specks from LPS-primed J774DUAL macrophages stimulated for
45 minutes with 3 mM ATP with or without 30 minutes pre-treatment with conditioned media exposed to A438079-loaded silk films. Results are
given as mean ± standard error with statistics calculated by one-way ANOVA with Fisher’s LSD. ***p<0.001. (C) Representative confocal micrographs
of J774DUAL macrophages immunostained for ASC (red) and stained for DNA with DAPI (blue) and F-actin with phalloidin (green). Perinuclear ASC
specks are indicated with white arrows and shown with enhanced visibility in zoomed fields. Scale bars represent 50 µm in the first two rows and 25
µm in the zoomed field.
Yaron et al. 10.3389/fimmu.2024.1340405
Frontiers in Immunology frontiersin.org07

modest and non-significantly enhanced closure by unloaded films
(Figure 4), as soluble silk fibroin products are known to exhibit
immune-stimulating and regenerative properties (49,50).
We also note a similar non-significant effect of topical direct
application of A438079 at the time of wounding, which may have
blunted initial responses to ATP released at the time of injury.
However, extracellular ATP may be released by post-injury death in
cells undergoing apoptosis via pannexin channels or necroptotic
rupture (51–53). Thus, the sustained delivery of A438079 in
silk films allows prolonged suppression of P2X7 receptor
activation and inflammasome activity, as demonstrated by our
immunohistochemical finding of reduced IL-1blevels at day 10.
This effect may be enhanced further by negative feedback, as
inhibition of P2X7-mediated pyroptotic cell death has been
shown to suppress subsequent extracellular ATP release (54) and
IL-1bsignaling positively regulates further expression of IL-1b(55).
A438079 has previously been shown effective in several models
of inflammatory disease (56–62). However, despite potent effect, it
has been noted that clinical translation may be a challenge owing to
its short half-life of 40 minutes (63) to ~1 hour (25). Similarly, prior
work on delivering small molecule inflammasome modulators to
wounds also required repeated administration (19,20). Here, we
demonstrate that tunable wound dressings which may be stored dry
after drug loading are capable of releasing A438079 for several days
without repeated administration, providing an appropriate use-case
for clinical wound application. By comparison, hydrogel
formulations releasing modulators of inflammasome pathway
components pose potential challenges for shelf stability and end-
A
B
DC
FIGURE 4
In vivo evaluation of A438079-loaded films. (A) Planimetry at 10 days post-wounding of db/db mice treated with saline, A438079, silk films, or silk loaded
with A438079. Data are given as mean ± standard error and statistics are two-way ANOVA with Sidak multiple comparisons test *p<0.05, ***p<0.001.
(B) Representative wound images at day 0 and day 10 post-wounding with traces to illustrate degree of closure shown in gold (day 0) and blue (day 10)
with 1-cm scale bar. (C) Quantification of IL1bpositive (+ve) cells at the wound edge on day 10 with the indicated treatments. Data are given as mean ±
standard error and statistics are two-way ANOVA with Sidak multiple comparisons test *p<0.01, ***p<0.05. (D) Representative immunohistochemistry
micrographs of IL1bstaining on day 10 post-wound tissues for the indicated treatments. Red arrow terminates at the edge of the migrating epidermis.
Scale bars are 200 µm.
Yaron et al. 10.3389/fimmu.2024.1340405
Frontiers in Immunology frontiersin.org08

user complexity (64). Furthermore, whereas drug-loaded hydrogels
require costly reagents, recombinant proteins, and fabrication
methods, silk fibroin wound dressings are low cost, sustainable,
and can be produced in large-scale batches with minimal
difficulty (65).
Several groups have noted that the NLRP3 inflammasome is
critical for wound healing. Work by Weinheimer-Haus and
colleagues (66), and confirmed by Ito et al. (67), reported that
mice deficient in NLRP3 or caspase-1 exhibited reduced
epithelialization rates and angiogenesis versus wildtype mice. In
this respect, inhibition of inflammasomes may be detrimental to
healthy wound healing. By contrast, repeated observation that
several comorbidities characterized by delayed healing have been
associated with sustained inflammasome activation, including
diabetes (19), aging (68), and burns (69,70), underscores the
dichotomous role for this key innate immune pathway in health
and disease (71). For example, Tan et al. reported that disruption of
IL-1 signaling, using either genetic knockout of the IL-1 receptor or
introduction of a recombinant fusion matrix-binding IL-1 receptor
antagonist, can stimulate healing in diabetic mouse wounds (64).
Thus, for wounds in which inflammasome activity is dysregulated
(e.g., diabetic wounds), pharmacologic inhibition may be
appropriate. In effect, initial inflammasome signaling facilitates
early healing processes, but appropriate control is necessary to
negatively regulate the pathway and allow a pro-resolution
phenotype to begin. Diabetes and other comorbidities fail to
provide this negative regulation, and unchecked sustained activity
results in prolonged inflammation and an inability to shift towards
a pro-resolution phenotype. The question remains: why is
inflammasome activity needed for healthy wound healing, but
detrimental in diabetic wound healing? Future work will
investigate this dichotomous role for inflammasome signaling
with respect to targeted drug delivery.
Data availability statement
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.
Ethics statement
The animal study was approved by Institutional Animal Care
and Use Committee of Arizona State University. The study was
conducted in accordance with the local legislation and
institutional requirements.
Author contributions
JY: Conceptualization, Data curation, Formal analysis, Funding
acquisition, Investigation, Methodology, Project administration,
Resources, Supervision, Visualization, Writing –original draft,
Writing –review & editing. SB: Investigation, Methodology,
Writing –review & editing. NG: Investigation, Writing –review
& editing. FB: Investigation, Writing –review & editing. SM:
Investigation, Writing –review & editing. SR: Investigation,
Writing –review & editing. SN-D: Investigation, Writing –
review & editing. KR: Conceptualization, Funding acquisition,
Project administration, Resources, Supervision, Writing –review
& editing.
Funding
The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. This work
was supported by NIH K01EB031984 to JY. KR is grateful to NIH
(R01 AR074627) for partial support of this work.
Acknowledgments
The authors acknowledge the use of facilities within the Eyring
Materials Center at Arizona State University supported in part by
NNCI-ECCS-1542160 and would like to thank Sisouk Phrasavath
for technical assistance. The authors would also like to acknowledge
resources and support from the Biodesign Institute Advanced Light
Microscopy and from the Regenerative Medicine and Bioimaging
core facilities at Arizona State University.
Conflict of interest
JY is affiliated with Vivo Bioconsulting, LLC and Endotat
Biotechnologies, LLC. KR is affiliated with Synergyan, LLC and
Endotat Biotechnologies, LLC.
The remaining authors declare that the research was conducted
in the absence of any commercial or financial relationships that
could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online at:
https://www.frontiersin.org/articles/10.3389/fimmu.2024.1340405/
full#supplementary-material
Yaron et al. 10.3389/fimmu.2024.1340405
Frontiers in Immunology frontiersin.org09

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