Low-intensity focused ultrasound-activated piezoelectric gel bandage for diabetic wound repair and neuropathic pain relief

Domain: biology
Published: 2026-03-19
Editorial outcome: major_revision

Abstract
Nociceptive hypersensitivity and prolonged wound healing due to the interaction of peripheral
neuropathy and local immune disorders are key issues that need to be addressed in diabetic foot
ulcer (DFU) regenerative repair. Here, we present a low intensity focused ultrasound (LIFU)-
activated piezoelectric gel bandage for DFU wound repair and neuropathic pain relief. Acting as
an artificial “skin”, this self-powered, functionalized bandage not only temporarily shields tissue
from external environment, but also serves as a drug reservoir that can in situ release nitric oxide
upon LIFU to promote macrophage polarization, generate piezoelectric current to desensitize
TRPV1 nociceptor for on demand neuropathic pain relief and produce reactive oxygen species to
eliminate pathogens in male rodents. More importantly, this regimen can promote CGRP
neuropeptides release from sensory neurons to invigorate M2-like macrophages-mediated
protective cutaneous immunity. This LIFU-activated immune-nociceptor modulation strategy is
potentially applicable to other non-healing tissue regeneration.

Introduction
Diabetic foot ulcers (DFU) are one of the most devastating complications of advanced diabetes
mellitus, with high disability and mortality rates, imposing heavy clinical and economic burdens
on society1. More than half of DFUs become infected, with about 20% of moderate to severe
infections leading to amputation, and the mortality after amputation exceeds 70% within 5 years
for all diabetic patients2,3. Long-term poor glycemic control in diabetic patients not only creates
persistent local inflammatory microenvironment that leads to immune disorders and vasculopathy,
but also causes peripheral neuropathy that results in abnormal foot sensation and protective
function deficiency4,5. These factors work together to make DFU highly susceptible to infection
and prolonged healing when exposed to adverse external factors. Current treatment of DFU mainly
focuses on systemic glycaemic control, microvascular recanalization, infection prevention and
local wound care, focus solely on healing effect but meet with rather limited success6–8. Thus,
developing comprehensive therapeutic regimens to simultaneously remodel immune homeostasis
and relieve peripheral nerve perception becomes indispensable for DFU management.

Diabetic wound microenvironment, as a local factor that participates in the pathogenesis and
continues to influence wound repair process with the support of systemic factors, consists mainly
of fibroblasts, immune cells, endothelial cells and various cytokines9,10. Of these, the immune
components play a key role in coordinating tissue healing. During chronic inflammation in
diabetes, macrophages are recruited to trauma site and release cytokines to modulate the immune
microenvironment11. Notably, macrophages are highly plastic and can be categorized into pro-
inflammatory state (M1-like macrophages) and anti-inflammatory state (M2-like macrophages)
depending on their response to different stimuli and functional phenotypes. M1-like macrophages,
activated by Th1 cytokines (e.g., γ-interferon, tumor necrosis factor-α) and exogenous danger
signals, exhibits strong cytotoxicity and pro-inflammatory properties, whereas M2-like
macrophages mainly exert anti-inflammatory and pro-cell proliferation effects12. Besides, studies
have shown that macrophages are involved in regeneration process of damaged peripheral nerves.
Modulation of macrophage polarization is expected to reverse the local inflammatory
microenvironment and support peripheral nerve repair to promote diabetic wound recovery13,14.

In addition to dysregulated immune microenvironment, abnormal pain perception caused by
peripheral neuropathy remains a non-neglectable problem affecting diabetic wound healing and
even patients' life quality15. Considering that the expression, transport and phosphorylation status
of ion channels within sensory neurons directly affect functional abnormalities, targeting
regulation of specific ion channels may remodel diabetic peripheral nerve perception. Transient
receptor potential vanilloid-1 (TRPV1), a receptor highly expressed in dorsal root ganglia (DRG)
neurons, is closely involved in pain process16. TRPV1 can be activated by deleterious chemical,
mechanical and thermal insults, triggering calcium influx, and, in turn, a cascade of events linked
to neurogenic inflammation and nociception17,18. Growing studies have validated long-term
analgesic efficacy of TRPV1 agonists or antagonists in diverse preclinical chronic pain models19,20.
However, currently available selective TRPV1 agonists such as capsaicin (Cap) have limited
clinical efficacy due to its local irritation such as stinging, burning, and erythema. Therefore,
enormous efforts have been devoted to developing efficacious TRPV1 agonist without the adverse
effects associated with Cap application.

Local electrical stimulation has been proven to successfully stimulate receptor internalization,
macrophage polarization and tissue regeneration; however, the application of implanted electrodes
remains restrictive due to side effects such as invasiveness, infection, and electrode breakage. A
self-powered therapeutic strategy based on physical principles thus offers an ideal solution21–23.
Barium titanate (BaTiO , BT), a representative piezoelectric material, converts acoustic
3
mechanical energy into electrical energy, enabling ultrasound (US)-driven wireless stimulation;
meanwhile, the piezoelectric catalytic process produces reactive oxygen species (ROS) to
participate in the wound healing process24. These materials exhibit micro-pressure-induced
polarization, dynamic built-in electric fields, and excellent biocompatibility25,26. Studies confirm
that US-triggered piezoelectric BT systems significantly shorten the healing cycle of bacterially
infected wounds and improve immune homeostasis27,28. Critically, the ROS generated by
piezoelectric catalysis need to be properly regulated to coordinate its dual role of antibacterial and
healing29–31. Low-intensity focused ultrasound (LIFU), with its precise energy focusing capability,
avoids tissue damage and discomfort caused by excessive energy (e.g., significant suppression of
acute pain perception in the human dorsal anterior cingulate) while enhancing therapeutic
efficiency32. Therefore, the synergistic interaction between LIFU and piezoelectric materials
presents a promising strategy for achieving enhanced therapeutic outcomes.

Herein, we introduce a LIFU-activated piezoelectric gel (BT-dNO@Gel) bandage with immune
homeostasis modulation and neuropathic pain relief functions for comprehensive repair of diabetic
wounds. This self-powered system was composed of polyethyleneimine (PEI) and polyvinyl
alcohol (PVA) cross-linked hydrogel encapsulating piezoelectric BT nanoparticles with surface-
coated nitric oxide (NO) donor capable of sequentially releasing NO, generating ROS and
outputting localized direct current in response to LIFU stimulation. Firstly, the use of non-invasive
LIFU as a switch for precise spatiotemporal control avoided local damage or discomfort caused
by implanted electrodes or excessive incident energy. Secondly, the outer hydrogel layer possessed
favorable biocompatibility, adhesion, and mechanical flexibility to conformably fill complex
wound areas and effectively transmit US and electrical currents into deep regions. Finally, the
subsequent on-demand released NO synergistically with free radicals and piezoelectric currents
generated by BT mechanical deformation accelerates wound healing process through combating
pathogen infection, polarizing macrophages to M2-like phenotype, desensitizing TRPV1
nociceptor and releasing calcitonin gene related peptide (CGRP). This LIFU-mediated immune-
nociceptor modulation strategy not only present a valuable approach for diabetic wound care, but
also hold great potential for other chronic or acute wounds.

Results
Preparation and characterization of the BT-dNO@Gel bandage
The BT-dNO nanoparticles (NPs) with both piezoelectric and NO release properties were firstly
prepared by stepwise surface modification processes and NO donor loading (Fig. 1a). Since bare
BT NPs are prone to agglomeration, hydroxylation modification (BT-OH) and polydopamine
surface coating (BT-OH-pDA) were employed to enhance their hydrophilicity, conductivity and
biocompatibility. The typical peak at 1430 cm-1 in the Fourier transform infrared (FTIR) spectra
and the typical peak of C=C stretching of the benzene ring at 1565 cm-1 in the Raman spectra
confirmed the existence of OH and pDA, respectively (Supplementary Fig. 1a, b). Notably, since
the surface modification processes may form a covering layer on the BT-OH surface through π-π
stacking or hydrogen bonding interactions, this can interfere with the vibration freedom of free
hydroxyl groups, leading to a weakened or broadened 1430 cm-¹ peak that merges into the baseline
noise. X-ray photoelectron spectroscopy (XPS) and UV-vis spectra confirmed the successful
loading of NO donor (N-diazeniumdiolates, NONOate) (Fig. 1b, c). Thermogravimetric analysis
(TGA) demonstrated that the mass contents of NONOate was 3.62 wt%, which was in agreement
with the loading content (3.95 ± 0.14%) determined by the absorbance of NONOate at 253 nm in
UV-vis spectra (Fig. 1d). Transmission electron microscopy (TEM) images and energy dispersive
spectroscopy (EDS) elemental mapping displayed that BT-dNO NPs were polygonal-like shape
with a thin polymer layer on the surface and a uniform distribution of Ba, Ti, N, O and C elements
(Fig. 1e and Supplementary Fig. 2a, b). The average hydrated particle size of BT-dNO was 80.49
± 12.11 nm with the zeta potential of -17.9 ± 0.55 mV measured by dynamic light scattering (DLS)
(Fig. 1f and Supplementary Fig. 3). Furthermore, the synthesized BT-dNO NPs possessed high
colloidal stability, as observed by well dispersed in various solutions within a week, which may
be attributed to their negative charge and the anti-fouling effect of the deprotonated hydroxyl and
pDA coatings (Fig. 1g).

To immobilize the BT-dNO NPs on wound surface for long-lasting efficacy, PEI/PVA gel with
good acoustic permeability and adhesion property were designed as the bandage substrate (Fig.
1a). Briefly, BT-dNO NPs were added into phenylboronic-acid-functionalized branched PEI
(PBA-PEI) to form the precursor solution, followed by mixed with PVA solution to experience a
solution-gel transition under imine, boron ester and hydrogen bonding interactions without the
need for additional stimuli (Fig. 1h). The characteristic UV-vis absorption peak of PEI-PBA at 230
nm as well as FTIR absorption peak of C=N bond at 1645 cm-1 and B-O-C bond at 1371 cm-1
confirmed the reaction of amino group with aldehyde group and the formation of boron ester bond
(Supplementary Fig. 4a, b). Dynamic rheological tests showed the storage modulus (G′) was much
higher than the loss modulus (G″) values within 30 min with a frequency range of 0.1-100% Hz,
indicating the successful formation of BT-dNO@Gel network (Supplementary Fig. 5a, b).
Biological cryo-scanning electron microscopy (Cryo-SEM) presented a typical porous micro-
structure of BT-dNO@Gel, which may contribute to exudate absorption and oxygen diffusion (Fig.
1i). As demonstrated by confocal imaging, the red Cy3 signals from BT-dNO NPs displayed a
uniform distribution pattern in the FITC labeled gel, further indicating the satisfactory
encapsulation (Fig. 1j).

Considering that gel bandages are vulnerable to deformity by mechanical external forces, the
viscosity and self-healing capacity of BT-dNO@Gel were first tested. Both G′ and G″ values were
relatively stable in the strain range from 0.1% to 100%, indicating that the BT-dNO@Gel were in
the linear viscoelastic region with favorable viscosity (Supplementary Fig. 6a, b). Macroscopic
healing tests using rhodamine B (red) and methylene blue (blue) staining exhibited that two
separate gels in contact with each other were able to heal rapidly without any external interference,
and the healed gel could be lifted by its own weight without cracks (Fig. 1k). Further continuous-
step strain test yielded the same results. When a high dynamic strain (500%) exceeding the critical
value was applied, the value of G′ was dropped from ~100 Pa to ~10 Pa and lower than that of G″,
whereas once adopting a lower strain (1%), the values of G′ and G′′ can immediately recovered
almost to the original values even after four alternating cycles, demonstrating the autonomous
healing behavior of the BT-dNO@Gel (Fig. 1k). An ideal wound dressing should be able to absorb
tissue exudates and establish a moist local micro environment conducive to wound repair. As
expected, the BT-dNO@Gel displayed a time-dependent swelling behavior in PBS at 37 °C with
a swelling rate of about 90% in 1 h and reached an equilibrium swelling state in ~2 h, indicating
that the prepared bandage is sufficient to effectively absorb wound exudate (Fig. 1l). The
hemostatic potential of BT-dNO@Gel was further investigated. In both the rat tail hemorrhage
model and liver hemorrhage model, BT-dNO@Gel treatment reduced rat blood loss to a great
extent, which can be attributed to strong adhesion between gel bandage and tissues (Fig. 1m and
Supplementary Fig. 7). In addition, BT-dNO@Gel degraded almost completely in physiological
media within about 10 days (Supplementary Fig. 8). The above results indicated the successful
fabrication of BT-dNO@Gel bandage with tight adhesion, high self-healing, strong swelling and
excellent hemostatic capacity, which is well suited for extracorporeal wound dressing applications.

BT-dNO@Gel bandage functionality in response to LIFU
Under LIFU irradiation, the outer NONOate of the BT-dNO decomposes to release NO, and the
inner BT core would produce an unbalanced charge on their surface and undergo cascading redox
reaction process, which leads to the generation of ROS and a localized electric field (Fig. 2a). The
LIFU-responsive NO production profile of the BT-dNO@Gel was first evaluated under
physiological conditions using classical Griess assay. NONOate is an exogenous NO donor and
free NONOate displayed a concentration-, LIFU intensity-, and irradiation time-dependent NO
release features (Supplementary Fig. 9a-c). Subsequent grafting NONOate onto the surface of BT
NPs also produced a suitable concentration of NO that is beneficial for wound healing, and the
NO-responsive release property remained undiminished after further encapsulation into the
PEI/PVA gel (Fig. 3b). The NO production yield was not only positively linear with the
concentration of BT-dNO in the gel, but also strongly correlated with LIFU intensity or duration
(Fig. 2c, d and Supplementary Fig. 10).

Then, the piezoelectric catalytic performance of the BT-dNO@Gel was systematically
estimated. The X-ray diffractometer (XRD) pattern of BT-dNO showed a peak splitting occurred
around 2θ = 45°, resulting in the separated (002) and (200) reflections, verifying the preservation
of tetragonality to generate piezoelectricity (Fig. 2e). A typical butterfly amplitude curve with a
ramp voltage starting from -10 to +10 V observed in piezoforce microscopy (PFM) suggested a
constantly varied strain induced by the applied external electric field (Fig. 2f, g and Supplementary
Fig. 11a, b). Besides, on the phase chart, there was an ~180° phase switch in the local circuit of
piezoelectric hysteresis, exhibiting the desirable piezoelectric efficacy of BT-dNO. The LIFU-
response output voltage of BT-dNO@Gel was measured using a high-precision benchtop digital
multimeter. The BT NPs hardly produced any voltage in the absence of LIFU (Fig. 2h). In contrast,
the output voltage of either BT or BT-dNO NPs was markedly increased under LIFU stimulation
at 1.5 W cm-2, indicating that surface modification and drug loading process did not affect the
piezoelectric properties of BT NPs. More importantly, the voltage transmission capability of BT-
dNO NPs was amplified to hundreds of millivolts after encapsulation in gel substrate, suggesting
that higher electrical signal outputs could be obtained from BT-dNO@Gel at the same LIFU
parameter. In line with the results of the piezoelectric voltage output, the real-time piezoelectric
current generated by the gel bandage under LIFU stimulation showed that BT-dNO generated a
peak current density of 0.12 ± 0.04 μA cm-², especially after encapsulated in gel matrix, the current
transmission capacity of BT-dNO nanoparticles was amplified about 10 times (Fig. 2i). The built-
in electric field of BT-dNO NPs under LIFU stimulation enables piezoelectric separation of
electrons and holes to catalyze the ROS generation. The rhodamine B degradation assay confirmed
the ROS generation capacity of BT-dNO@Gel under LIFU activation, and the ROS yield was
positively correlated with BT-dNO concentration, LIFU power and duration (Fig. 2j and
Supplementary Fig. 12a-c). Electron spin resonance (ESR) was adopted to systematically identify
and quantify the ROS types produced by BT-dNO@Gel upon LIFU using 5,5-dimethyl-1-
pyrroline-N-oxide (DMPO) spin trapping agent. Characteristic 1:2:2:1 and 1:1:1:1 quartet signals
were observed in BT-dNO and BT-dNO@Gel groups under LIFU irradiation, confirming the
generation of hydroxyl radicals (·OH) and superoxide anions (·O -), respectively (Fig. 2k, l). The
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relative contributions of each ROS were quantified by signal deconvolution, revealing ·OH as the
predominant species. These results suggested that LIFU can be used as a switch to
spatiotemporally control NO release, direct current generation and ROS production from BT-
dNO@Gel bandage, thus facilitating subsequent biological applications.

In vitro pro-repair, anti-inflammatory and antibacterial performance of BT-dNO@Gel
bandage
Prior to cellular experiments, the cytotoxicity of BT-dNO@Gel was first assessed. CCK-8 assay
showed negligible effect of BT-dNO (less than 0.3 mg mL-1) and PEI/PVA gel (less than 3 mg
mL-1) on HUVEC viability (Supplementary Fig. 13a, b). Meanwhile, hemolysis test exhibited that
the blood cell hemolysis rate of BT-dNO was less than 5% within the safe range (less than 0.3 mg
mL-1), suggesting its favorable hemocompatibility (Supplementary Fig. 14 and Supplementary
Table 1). Consistent with this, live/dead staining and relative quantitative analysis revealed that
either LIFU alone or BT-dNO@Gel combined with LIFU did not affect the cellular activity of
HUVECs (Fig. 3a, b). Cell migration is essential for wound healing. The scratch assay showed
that the combination of LIFU and BT@Gel or BT-dNO@Gel significantly promoted the migration
of HUVECs compared with LIFU irradiation alone, and in particular, the 48 h migration rate of
HUVECs reached 84.7% after BT-dNO@Gel+LIFU treatment (about 2.5 times higher than the
control group) (Fig. 3c, d). Similar phenomena were observed in keratinocyte (HaCat cell line)
and fibroblast (HSF cell line), confirming the high biocompatibility and robust pro-cell migration
ability of BT-dNO@Gel+LIFU treatment regimen (Supplementary Fig. 15a-h). To elucidate the
mechanism of the pro-healing effect, we first measured the NO release kinetics at the cellular level.
Griess assay displayed that both BT-dNO and BT-dNO@Gel liberated NO in situ under LIFU
activation. Under preset conditions, the maximum instantaneous concentration of NO released
from BT-dNO@Gel was about 0.2 μM, which is considered beneficial concentration range for
wound healing (Supplementary Fig. 16a, b). It is noteworthy that a small amount of NO was still
detectable after the removal of LIFU, which may be due to the slow spontaneous release of NO
from the NONOate donors. Consistent with our conjecture, blocking NO signaling with NS-2028,
a soluble guanylate cyclase (sGC) inhibitor, significantly decreased the cell migration rate of
HUVECs, indicating the released NO was the main contributor of LIFU-activated BT-dNO@Gel
bandage to promoting cell migration (Supplementary Fig. 17a-e). Moreover, tube formation results
revealed that BT-dNO@Gel+LIFU treatment achieved greater total tube length and more tube
meshes than BT@Gel+LIFU treatment (Fig. 3e-g). This satisfactory healing efficacy may be
attributed to the released NO and piezoelectric current from BT-dNO@Gel under LIFU activation
that stimulated the growth and migration of endothelial cells.

Given the pivotal role of macrophages in orchestrating the inflammatory phase of wound healing,
we assessed the ability of our piezoelectric gel bandage to promote the anti-inflammatory M2-like
macrophage polarization essential for resolution of inflammation and tissue repair. Expressions of
the inflammatory marker CD86 and the anti-inflammatory marker CD206 in macrophages
(RAW264.7) were assessed using cellular immunofluorescence (IF) staining and flow cytometry.
IF staining showed that BT-dNO@Gel+LIFU concurrently downregulated lipopolysaccharides
(LPS) and interferon-γ (IFN-γ) - induced M1-like macrophages while upregulating M2-like
macrophages (Supplementary Fig. 18a, b), suggesting that BT-dNO@Gel+LIFU could encourage
macrophages’ repolarization from M1- to M2-like phenotype. Similar findings were verified in
flow cytometry, where BT-dNO@Gel+LIFU treatment significantly increased the M2/M1 ratio
by about 6-fold compared to the M1 control, underscoring the anti-inflammatory properties of
piezoelectric gel bandage (Fig. 3h, i).

Infection is one of the major obstacles to diabetic wound healing, dressings with antimicrobial
activity would be helpful in impeding pathogen invasion and boosting wound recovery. The
bactericidal effect of BT-dNO@Gel bandage was tested in Gram-positive bacteria represented by
S. aureus and Gram-negative bacteria represented by E. coli. As shown by the SEM, bacteria in
the control group exhibited an intact morphology with a smooth surface, while partial cytoplasmic
efflux from bacterial membrane damage was seen in the Gel and BT@Gel groups (Fig. 3j).
Notably, significant morphological disruption was observed in the BT@Gel+LIFU and BT-
dNO@Gel+LIFU groups, with the bacterial membrane severely wrinkled and disrupted, causing
inner contents leakage. This confirmed the antibacterial efficacy of LIFU-activated BT-
dNO@Gel. Several studies have shown that ROS can imbalance the antioxidant system of bacteria,
thereby inhibiting their growth and activity33,34. The fluorescent probe O11 is a common tool to
visualize bacterial ROS generation, and strong ROS green fluorescence was detected in BT@Gel
and BT-dNO@Gel-treated S. aureus after LIFU irradiation (Fig. 3k, l). Then, the antimicrobial
properties of BT-dNO@Gel against S. aureus was evaluated using the typical spread plate method.
Compared to LIFU alone, which had no apparent effect on bacterial growth, the blank gel treatment
inhibited bacterial viability to a certain extent (Fig. 3m, n). This is in line with previous studies
that cationic polyamine PEI can interact with microbial membranes to cause membrane
disruption35. Both BT@Gel and BT-dNO@Gel treatment dramatically reduced S. aureus survival
rate in the presence of LIFU, as manifested by substantial protein leakage from the bacteria, which
was also confirmed by the bacterial live/dead staining (Fig. 3o, p and Supplementary Fig. 19).
Similar phenomenon was observed in E. coli (Supplementary Fig. 20a-h). To confirm whether
ROS at this bactericidal concentration would cause damage to normal cells, we assessed the
production of ROS in HUVECs, which was consistent with the previously non-toxic results. Piezo-
catalyzed ROS production within HUVECs was lower than that of positive control (Rosup
concentration < 50 μg mL-1) and progressively decreased over time, which was within the safe
threshold range of cell viability (Supplementary Fig. 21a-f). The LIFU-triggered ROS generation
from BT-dNO@Gel indeed affected bacteria survival but not HUVECs, probably due to the fact
that the respiratory chain of eukaryotic cells is located in intracellular mitochondria and their
superior ROS-scavenging systems, which is more resistant to external ROS interference than
bacteria36–38. Overall, the above data indicated that LIFU-activated BT-dNO@Gel bandage
possessed excellent pro-cell migration, angiogenic, macrophage polarization and bactericidal
capacities in vitro.

Repairing efficacy of BT-dNO@Gel bandage on diabetic wound
Biosafety is a prerequisite for clinical application and translation, the local and systemic toxicity
of BT-dNO@Gel plus LIFU were assessed before in vivo treatment. Healthy rats were subjected
to PBS or BT-dNO@Gel plus LIFU treatment, respectively. H&E staining of local skin tissues
collected on day 14 after treatment showed no significant difference between the two groups in
terms of either cellular morphology and structure or inflammatory cell infiltration, suggesting the
as-prepared bandage had no toxicity to normal skin (Supplementary Fig. 22a). Meanwhile, analysis
of blood specimens from rats in the middle and late stages of treatment revealed that the blood
cells and liver & kidney function indicators of treated rats were all within normal levels
(Supplementary Fig. 22b). Consistently, H&E staining of major organs after treatment also
revealed no abnormalities (Supplementary Fig. 22c). The above results indicated that the BT-
dNO@Gel+LIFU treatment regimen had satisfactory biocompatibility with high translational
potential.

Skin wounds can self-heal within a few days under normal physiological conditions. In diabetic
wounds, however, the sustained inflammatory microenvironment caused by chronic
hyperglycemia presents a significant challenge to normal physiological healing 39. Encouraged by
the favorable efficacy in vitro, the repair effect of BT-dNO@Gel was next evaluated in a full-
thickness skin wound model of streptozotocin (STZ)-induced diabetic rats (Fig. 4a). Successful
modeling was defined as random blood glucose concentration >16.7 mmol L-1 measured by blood
sampled from the tail vein after one week. Two weeks after injection, a 2 cm full-thickness skin
wound was created on the back of the rats using a sterile perforator. Diabetic rats with back wounds
were randomly divided into 6 groups: (1) control, (2) LIFU, (3) Gel, (4) BT@Gel, (5)
BT@Gel+LIFU and (6) BT-dNO@Gel+LIFU and treated every other day (dose of BT was 0.3 mg
mL-1, dose of BT-dNO was 0.3 mg mL-1, LIFU parameters were 1.0 MHz, 1.5 W cm-2, 50% duty
cycle and 120 s duration). During the observation period, there were no significant differences in
body weight of the rats in each group, and the blood glucose level of all diabetic rats remained
consistently >16.7 mmol L-1 (Supplementary Fig. 23a, b). Recognizing that the therapeutic efficacy
of BT-dNO@Gel+LIFU treatment involve the regulation of NO release, whose overproduction
would interfere with wound healing, we first investigated the kinetics of NO release within the
wound skin tissue following treatment. BT-dNO@Gel bandage coated on diabetic wounds rapidly
released NO under LIFU stimulation, reaching a peak concentration of about 0.25 μM, and then
gradually decreased and maintained at a relative low level, which is considered beneficial for
wound healing (Supplementary Fig. 24a, b). It is noteworthy that a small amount of NO was still
detectable after the removal of LIFU, which may be due to the slow spontaneous release of NO
from the NONOate donors. Digital photos and quantitative analysis of the wound sites showed
that all the gel containing groups facilitated healing process to some extent compared to the control
group with significantly delayed wound healing, especially in BT-dNO@Gel+LIFU treatment
group, which may be attributed to well-adherent hydrogels providing a suitable matrix-like
environment for tissue regeneration (Fig. 4b, c). Notably, the wound closure rate of BT@Gel with
LIFU was significantly higher than that without LIFU, suggesting a pro-healing effect mediated
by piezoelectric stimulation. Consistent with expectations, the BT-dNO@Gel+LIFU group
achieved optimal wound repair, with a 93.15% wound closure rate compared to the control.
Moreover, this treatment regimen exhibited an obviously faster healing rate than recombinant
bovine basic fibroblast growth factor (rb-bFGF) dressing, which is commercially available for
wound repair, in promoting diabetic wound healing (Supplementary Fig. 25a-d).

Consistent with accelerated macroscopic closure, H&E staining revealed significant histological
improvements in BT-dNO@Gel+LIFU-treated wounds, as displayed by abundant neoplastic
granulation tissue in the wound center with fewer inflammatory cells and increased epidermal
viability compared to untreated diabetic controls (Fig. 4d, g, h). Moreover, more collagen
deposition and highly mature and oriented collagen structures, which are essential for cell
proliferation, differentiation and wound healing, were observed in the BT-dNO@Gel+LIFU group
compared to the other groups, showing better extracellular matrix (ECM) remodeling (Fig. 4e, i
and Supplementary Fig. 26). The co-analysis of K14-labeled keratinocytes, vimentin-positive
fibroblasts, and CD31-marked endothelial cells provides critical insights into the epidermal-
dermal-vascular tri-lineage synergy during skin regeneration40–42. Dual IF staining for K14 and
Ki67 revealed a significantly expanded zone of proliferating basal keratinocytes (K14+Ki67+) at
the wound edge after BT-dNO@Gel+LIFU treatment, directly supporting the accelerated re-
epithelialization observed in H&E (Supplementary Fig. 27a, b). Similarly, dual IF staining with
vimentin and Ki67 showed an obviously increased percentage of proliferating fibroblasts after BT-
dNO@Gel+LIFU treatment, which is essential for ECM production (Supplementary Fig. 27c, d).
In parallel, there was a noticeably higher density of CD31+α-SMA+ vessels in BT-
dNO@Gel+LIFU group, along with an increased proportion of proliferating endothelial cells
(CD31+Ki67+), suggesting the active neovascularization and maturation (Fig. 4f, j-l). This
phenomenon was further verified in enzyme-linked immunosorbent assay (ELISA), where the BT-
dNO@Gel+LIFU group secreted the highest amount of epidermal growth factor (EGF) and
vascular endothelial growth factor (VEGF) compared to the other groups (Fig. 4m, n). These
results demonstrated that BT-dNO@Gel bandage combined with LIFU can significantly
accelerated diabetic wound healing by promoting re-epithelialization, collagen deposition and
amplified angiogenesis.

Inflammation is crucial in wound repair process and reducing inflammation level will help the
wound move into the proliferative phase43. Macrophages, as essential immune cells in the
regulation of inflammation during wound repair, are highly plastic and capable of phagocytizing
pathogens to promote wound healing44. To elucidate the therapeutic mechanism of LIFU-activated
BT-dNO@Gel bandage, we assessed the macrophage status in diabetic wound microenvironment.
The in vivo M2/M1 macrophage ratio was confirmed by IF staining for CD206 and CD86.
Compared to other groups, higher populations of M2-like macrophages and lower populations of
M1-like macrophages were observed in BT@Gel+LIFU and BT-dNO@Gel+LIFU groups on both
day 7 and day 14, which resulted in the greater M2/M1 ratio, indicating decreased proinflammatory
and increased anti-inflammatory capacities (Supplementary Fig. 28a-c and Fig. 4o, p). Notably,
BT-dNO@Gel+LIFU treatment exhibited the highest anti-inflammatory efficacy, which can be
attributed to the synergistic action of released NO and the piezoelectric current triggered by LIFU.
To gain mechanistic insights, we employed a mouse model as a well-established system for
analyzing immune cell responses and profiling macrophage activation following the treatments.
M2-like macrophages (CD11b+ F4/80+ CD206+) and M1-like macrophages (CD11b+ F4/80+
CD86+) were quantified by flow cytometry, and the results were in agreement with the IF trends
(Supplementary Fig. 29). In addition, compared to the persistent inflammatory responses in the
control, LIFU, Gel and BT@Gel groups, the expression of pro-inflammatory cytokines including
tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) at the wound site decreased dramatically
after BT-dNO@Gel+LIFU treatment, while the expression of anti-inflammatory cytokine
interleukin-10 (IL-10) increased substantially (Supplementary Fig. 28d-f and Fig. 4q-s). Taken
together, these findings conclusively showed that BT-dNO@Gel+LIFU treatment could
effectively regulate local immune-inflammation homeostasis to accelerate diabetic wound healing.

Antibacterial efficacy of BT-dNO@Gel bandage against infected diabetic wounds
Inspired by the commendable antibacterial effect of LIFU-activated BT-dNO@Gel bandage in
vitro, we further investigated its efficacy against rat diabetic infected wounds. Diabetic rat model
with full-thickness wound was constructed as mentioned before. For the S. aureus infection wound
model, a suspension of S. aureus (150 μL, 108 CFU mL-1) was first dropped onto the skin wound
site and bacterial infection was confirmed 24 h later by standard swab culture and solid agar plate
counting (Fig. 5a and Supplementary Fig. 30). The infected diabetic rats were randomly divided
into 6 groups and treated every other day according to the aforementioned regimens. Digital photos
of the infected wounds were recorded and corresponding wound areas for each group were
calculated. The findings revealed that groups containing gel all promoted wound healing to some
extent, especially BT-dNO@Gel+LIFU treatment achieved a 90.16% wound healing rate on day
14 (Fig. 5b-d). Meanwhile, sample fluid from the wound edges of each group was collected on
days 7 and 14 after different interventions. Despite the fact that obvious bacterial colonies were
observed in all groups on day 7, it was evident that colony counts in BT@Gel+LIFU and BT-
dNO@Gel+LIFU groups were slightly less than those in other groups, which could be attributed
to bactericidal effect of ROS generated by LIFU-activated piezoelectric catalytic reaction (Fig. 5e,
f). Notably, the antibacterial effect of BT@Gel+LIFU and BT-dNO@Gel+LIFU groups was
particularly excellent by day 14, with only a trace of bacterial growth. Recognizing that the
efficacy of antibacterial treatment with piezoelectric gel bandage lies in the regulation of ROS
generation, whose overproduction leads to oxidative stress. Therefore, we investigated the kinetics
of ROS release in wound skin tissue after treatment. DHE staining showed that low levels of ROS
fluorescence were detected in rat skin tissues after BT-dNO@Gel+LIFU treatment
(Supplementary Fig. 31a-d). Over time, the ROS fluorescence was gradually absorbed and
degraded, decreasing in concentration after exerting an effective antibacterial effect and thus
progressing towards the direction of favorable wound healing. Consistent with the antibacterial
results, wounds treated with BT-dNO@Gel+LIFU had narrower granulation tissue width and
thicker regenerated epithelial tissue (Fig. 5g, i, j). Meanwhile, BT-dNO@Gel+LIFU-treated
wounds had thicker collagen bundles with better alignment, orientation and collagen deposition
density than other groups (Fig. 5h, k).

Considering that high bacterial burden would worsen inflammatory response and exacerbate
immune disorders at wound site, we wondered whether bacterial infections affect the regulation
capacity of BT-dNO@Gel+LIFU on local inflammatory microenvironment. IF staining showed
that even in infected diabetic wound model, BT-dNO@Gel+LIFU treatment could still promote
macrophage polarization toward the M2-like phenotype, especially in the early stages of wound
repair, as verified by the higher CD206 expression and lower CD86 expression at the wound site
(Fig. 5l, m). Unsurprisingly, inflammatory indicators gradually recovered as the immune disorder
resolved, with a decrease in pro-inflammatory cytokines (e.g. TNF-α and IL-6) and an increase in
anti-inflammatory factors (e.g. IL-10) (Fig. 5n-p). Similar results were observed in E. coli-infected
diabetic wound, where BT-dNO@Gel+LIFU treatment significantly promoted refractory wound
regenerative repair by reducing bacterial load and modulating inflammatory microenvironment
(Supplementary Fig. 32a-p). Collectively, our results validated the antibacterial repair
effectiveness of LIFU-activated BT-dNO@Gel in diabetic wounds infected with S. aureus and E.
coli.

Antinociceptive efficacy of BT-dNO@Gel bandage under LIFU
Interactions between the local inflammatory microenvironment of diabetic wounds and resident
immune cells can alter nociceptive messaging through releasing proinflammatory cytokines and
chemokines45. To test the antinociceptive effect of BT-dNO@Gel upon LIFU irradiation, a
diabetic neuropathic pain (DNP) model was constructed (Fig. 6a). Naive rats of same weight were
used as negative controls without receiving any treatment. Von Frey filament testing and hot plate
assay were applied to test mechanical and thermal pain behavior, respectively. Compare to naive
rat, DNP rat exhibited significantly lower pain thresholds, as displayed by decreased mechanical
paw withdraw threshold (PWT) and thermal paw withdrawal latency (PWL) (Fig. 6b, c). The
successfully modeled DNP rats were then randomly divided into six groups and received different
treatments according to the aforementioned regimens. Digital photos of DFU and corresponding
quantitative analysis of wound area revealed that the BT-dNO@Gel+LIFU treatment group
achieved the best wound healing rate than other groups on day 14 (Supplementary Fig. 33a-c). In
addition, compared with the PBS group, BT-dNO@Gel+LIFU therapy significantly increased the
pain threshold in DNP rats, manifested by an increase in both 50% PWT and PWL (Fig. 6d, e).
More importantly, the pain thresholds of the diabetic rats did not decrease during the observation
period, suggesting that our LIFU-mediated immune-nociceptor modulation strategy had a durable
analgesic effect. In contrast, the pain threshold of non-diabetic wound rats was recovered rapidly
by day 14 regardless of treatment, which was consistent with their uncompromised healing
capacity (Supplementary Fig. 34a-c).

Next, we wonder whether the BT-dNO@Gel plus LIFU exerts antinociceptive effects in vivo
via targeting TRPV1, a critical pain nociceptor overexpressed in DRG sensory neurons. Cap, a
selective TRPV1 channel agonist, was introduced as a positive control. High concentrations of
Cap induce TRPV1 activation and Ca2+ influx, which causes receptor desensitization of TRPV1
ion channels46,47. TRPV1 receptor desensitization then decreases TRPV1 response to
inflammatory mediators or endogenous ligands, and in turn reduces TRPV1-dependent
hyperalgesia (Fig. 6f). As expected, Cap (1 μM) treatment markedly promoted TRPV1 expression
in expression
in DRG neurons, which was instead diminished with prolonged incubation time (Fig. 6g). Similar
phenomenon and more pronounced TRPV1 inactivation were observed after BT-dNO@Gel plus
LIFU treatment. Meanwhile, intracellular Ca2+ levels were measured in DRG neurons using
calcium imaging techniques. Compared to the control group with no significant change in Ca2+
signal, both Cap and BT-dNO@Gel plus LIFU treatment induced a dramatic increase in
intracellular Ca2+ fluorescence signal, which then diminished at about 30 min (Fig. 6h). The above
results suggested that the antinociceptive efficacy of BT-dNO@Gel plus LIFU strategy may be
due to impaired Ca2+ influx resulting from TRPV1 receptor desensitization.

To further validate the mechanism of BT-dNO@Gel plus LIFU-induced TRPV1 desensitization
at organism, L4/L5 DRG and spinal cord of rats were collected 24 h after intervention. Na 1.8 is
v
a voltage-gated sodium channel highly expressed in DRG nociceptive neurons. As a functional
and structural marker of nociceptors, Na 1.8 expression is tightly linked to neuronal excitability
v
and pathological pain states, making it a robust complementary target for assessing sensory neuron
activity48. In the rat DNP model, the co-upregulation of Na 1.8 and TRPV1 expression reflects
v
neuronal response mechanisms to persistent noxious stimuli. After BT-dNO@Gel+LIFU
intervention, the observed reduction in activity of Na 1.8+TRPV1+ neurons demonstrates a
v
significant correlation with the attenuation of mechanical allodynia, providing critical insights into
their role in mediating pathological pain (Fig. 6i-k). Given that the glial region is involved in
neuronal signaling, particularly the transmission of nociceptive message, we further examined the
activation of astrocytes (GFAP+) and microglia (Iba-1+) in spinal dorsal horn glial region. In line
with previous results, BT-dNO@Gel+LIFU treatment markedly inhibited the activation of
microglia and astrocytes in DNP rats (Fig. 6l-n). Endogenous opioid pathway and α -adrenergic
2
receptor pathway are also common potential pain targets49,50. To ascertain whether the analgesic
effect of BT-dNO@Gel+LIFU depends on these pathways, we examined the effects of the
nonselective opioid receptor antagonist Naloxone and the α -adrenergic receptor antagonist
2
Yohimbine on BT-dNO@Gel+LIFU-induced analgesia through the Von Frey filament testing and
hot plate assay. As shown in Supplementary Fig. 35, intraperitoneal administration of Naloxone
or Yohimbine did not attenuate the analgesic potency, suggesting that BT-dNO@Gel+LIFU exert
antinociceptive effects independently of the function of opioid receptors and α -adrenergic
2
receptors. Collectively, the above results suggested that our piezoelectric gel bandage hinders Ca2+
influx by desensitizing TRPV1 nociceptor under LIFU activation, thereby attenuating nociceptive
messaging-related neuronal excitability for effective neuropathic pain relief.

BT-dNO@Gel bandage orchestrated neuro-immune crosstalk
To comprehensively explore transcriptional dynamics after LIFU plus BT-dNO@Gel bandage
treatment, we collected total RNA from skin wounds of well-established DNP rats and analyzed
the transcriptome landscape. Principal component analysis (PCA) demonstrated clear separation
among the naive, control and BT-dNO@Gel+LIFU treatment groups, confirming distinct global
transcriptomic signatures (Fig. 7a). The Venn diagram showed that 565 differentially expressed
genes (DEGs) were shared among the three groups (Fig. 7b). Hierarchical clustering of DEGs
showed significant differences between the naive group and control group, highlighting the
extensive transcriptional reprogramming induced by diabetes and injury (Supplementary Fig. 36a,
b). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment
analyses revealed that genes altered by diabetes and ulcers were significantly enriched not only in
glucose and lipid metabolism pathways, but primarily in pathways related to inflammation and
oxidative stress, ECM remodeling, and cell differentiation, migration and response to stimulus
(Supplementary Fig. 36c, d). This profile reflects a molecular landscape skewed towards chronic
inflammation, compromised tissue regeneration and hyperalgesia, characteristic of the non-healing
diabetic wound. Crucially, comparison of BT-dNO@Gel+LIFU group and control group identified
2942 significant DEGs (1292 upregulated and 1650 downregulated), demonstrating potent ability
of piezoelectric gel bandage to modulate the wound transcriptome (Fig. 7c). KEGG and GO
analyses revealed that BT-dNO@Gel+LIFU treatment significantly reversed diabetes-induced
expression patterns dysregulation, as verified by shifts in key transcriptional pathways including
inflammation-immune response regulation, ECM remodeling, cell surface receptor signaling
pathway and cytokine-cytokine receptor interaction (Fig. 7d, e). Furthermore, targeted analysis
displayed that genes encoding immune and inflammatory regulation, tissue repair, and pain
regulation in BT-dNO@Gel+LIFU group were shifted significantly towards the naive profile
compared to the control group, which was in accordance with the alleviation of pain sensitivity
symptoms and remodeling of immune homeostasis (Fig. 7f).

The transcriptome atlas outcomes impelled us to further explore the mechanism of neuro-
immune interplay in DFU wound regeneration. Recent studies revealed that the sensory nerves
located within cutaneous tissue may signal dermal immune cells through the neuropeptide CGRP
during the healing process51. Given the importance of peptidergic sensory neurons in mediating
neuro-immune communication during tissue healing, we evaluated the expression of CGRP
neuropeptide after treatment. RT-qPCR results showed that BT-dNO@Gel+LIFU treatment up-
regulated Calca expression by 3.4 times in DRG neurons, which was consistent with the ELISA
assay that BT-dNO@Gel+LIFU treatment increased soluble CGRP release in DRGs (Fig. 7g, h).
Further multiplex IF co-staining for CGRP with definitive neuronal markers Na 1.8 and M2-like
v
macrophage markers CD206 revealed that the CGRP signal occurred specifically in Na 1.8
v
cutaneous nociceptors, and its spatial association with CD206+ macrophages suggested a neuro-
immune axis driving wound repair (Fig. 7i). Quantitative analysis showed that compared with the
control group, BT-dNO@Gel+LIFU treatment significantly promoted CGRP release and
subsequent polarization of CD206+ macrophages, while the polarization process of macrophages
toward anti-inflammatory phenotype could be blocked by CGRP receptor antagonist (CGRP8-37),
which did not affect the release of soluble CGRP in skin tissue (Fig. 7j-l and Supplementary Fig.
37). To directly validate the correlation between CGRP signaling and macrophage phenotype, we
assessed the polarization of RAW264.7 macrophages in vitro, a widely used and well-established
model for studying macrophage activation. Expressions of the inflammatory marker CD86 and the
anti-inflammatory marker CD206 in macrophages were assessed using flow cytometry. The results
showed that LPS and IFN-γ treatment significantly increased the proportion of M1-like
macrophages, while IL-4 treatment increased the proportion of M2-like macrophages (Fig. 7m).
Compared with the M1 positive control group, fewer CD86-labeled M1-like macrophages and
more CD206-labeled M2-like macrophages were detected in the α-CGRP group. After blocking
CGRP signaling, the proportion of CD206-marked macrophages decreased, which directly
confirmed the role of CGRP in inducing macrophage phenotypic transformation and anti-
inflammatory regulation. The above findings confirmed that the LIFU-mediated immune-
nociceptor modulation strategy can orchestrate subcutaneous sensory nerve and M2-like
macrophages interactions to achieve wound regenerative repair and analgesia by invigorating
protective cutaneous immunity.

Methods
Animals, cell lines and bacterial strains
All animal studies comply with policies of the National Ministry of Health and obtained approval
from the Laboratory Animal Center of Shanghai Tenth People’s Hospital (SHDSYY-2023-4674).
Male Sprague-Dawley (SD) rats (170-200 g) and male BALB/c mice (6-8 weeks) were ordered
from Salccas Biotech Co., Ltd. All animal experiments were conducted exclusively with male
animals. This approach was taken to eliminate the potential confounding effect of estrogen on
wound healing and glycemic control in the diabetic model. Animals were housed in a specific
pathogen-free environment under a controlled conditions (20 ± 3 °C, about 40-70% humidity and
12 h light/dark cycle). Cell lines (HUVECs (CRL-4053), HSF (CRL‑4066), HaCat (CRL‑4048)
and RAW264.7 (TIB-71)) and bacterial strains (S. aureus (6538) and E. coli (8739)) were obtained
originally from American Type Culture Collection (ATCC). Cells were cultured in a humidified
incubator containing 5% CO at 37°C using specific medium (Dulbecco's Modified Eagle
2
Medium (DMEM, Gibcom) supplemented with 1% penicillin-streptomycin (Gibco) and 10% fetal
bovine serum (FBS, Gibco)). Cells were routinely tested for mycoplasma contamination every
two months. S. aureus and E. coli were suspended in Luria-Bertani medium (Solarbio) and cultured
in oscillation incubator at 37 °C. Bacteria were harvested during the exponential growth phase and
adjusted to 1 × 108 CFU mL-¹ prior to use.

Materials
Barium titanate (BT, 99.9%), NONOate (98%), polyethyleneimine (PEI, 99%) and polyvinyl
alcohol (PVA, 99%) were obtained from Rhawn. Dopamine (98%) and rhodamine B (≥ 99%) were
purchased from Macklin. Phenylboronic-acid (PBA, 98%) and 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDC, 99%) were gained from Bidepharm. FITC (≥ 90%) and
cyanine3 (Cy3, ≥ 95%) were purchased from BioFroxx GmbH. Methylene blue (≥ 70%), Capsaicin
(Cap, ≥ 95%), Naloxone (≥ 97%) and Yohimbine (≥ 99%) were gained from Aladdin. Calcium ion
indicator (Fluo-4 AM), Griess reagent kit and DCRFH-DA were gained from Beyotime.
Streptozotocin (STZ, ≥ 98%) was purchased from MaPokang Biotech Co., Ltd. Rat VEGF and EGF
ELISA kits were acquired from Enzyme-linked Biotech Co., Ltd. Rat TNF-α, IL-10, IL-6 and
CGRP ELISA kits were acquired from Multi Sciences Biotech Co., Ltd. α-CGRP (99.8%) and rat
CGRP-(8-37) (99%) were purchased from MedChemExpress.

Synthesis and characterization of BT-dNO
In detail, BT (1 g) was refluxed in hydrogen peroxide (H O ) aqueous solution (50 mL, 30 wt%)
2 2
at 105 °C under stirring. The hydroxylated modified BT (BT-OH) was cooled to room temperature
(RT), then collected after centrifugation (7200 × g, 5 min), washing (three times with deionized
water) and drying (60 °C in a vacuum oven for overnight). BT-OH (12.5 mg) was dissolved in Tris
buffer at pH 8.5 (25 mL, 10 mM) and homogenized using an ultrasonicator. Fresh dopamine
(2.5 mg) was rapidly added to the solution to initiate oxidative polymerization with a following
overnight agitation at RT. Polydopamine-coated BT-OH (BT-OH-pDA) was then collected by
centrifugation (8900 × g, 10 min). Finally, NONOate aqueous solution (4 mL, 1 mg mL-1) was
dropwise added to the BT-OH-pDA solution (8 mL, 1 mg mL-1) to obtain BT-dNO through π–π
attraction. The loading content of NONOate on BT-dNO was determined by UV-vis spectrum at
253 nm and by TGA (NETZSCH STA 449 F5). During TGA, the sample heated from RT to 600 °C
at a rate of 5 °C min-¹ in a nitrogen environment.

Preparation and characterization of BT-dNO@Gel bandage
PEI (1 g) was dissolved in methanol (5 mL), followed by adjustment of the pH of the PEI solution
to 5.0-6.0 with HCl (1 M). PBA powder (1 g) was dissolved in DMF (5 mL). EDC (2 g) was then
added to the PBA solution, followed by the prepared diluted PEI solution. The pH value was
maintained within 5.0 to 6.0 throughout the 3 h reaction process. The product was dialyzed against
deionized water for 3 days by changing deionized water three times daily. Finally, the PEI-PBA
concentrated solution was collected and vacuum freeze-dried for reserve. BT-dNO solution (0.3
mL, 2 mg mL-1) was thoroughly mixed with PEI-PBA solution (1 mL, 40 mg mL-1), followed by
the addition of PVA aqueous solution (1.3 mL, 40 mg mL-1) to form a BT-dNO@Gel complex.
The biological cryo-SEM (FEI Quanta 450) was used to examine the surface and section
morphology of the BT-dNO@Gel bandage. The rheological properties were evaluated using a
Thermo Haake Rheostress rheometer (RS6000, Thermo Scientific).

Characterization
Chemical structures were characterized by XPS (Thermo Escalab 250Xi) and raman microscope
(Renishaw inVia). UV-vis spectra and FT-IR spectra were recorded using UV-vis-NIR
spectrophotometer (PE Lambda 950) and Nicolet FT-IR IS 10 spectrometer, respectively. TEM
(FEI Tecnai G2 F30) was used to analyze sample morphology and elemental composition. Particle
size as well as zeta potential were detected by a laser particle analyzer (Zetasizer Nano ZS90).
XRD analysis was performed using a Rigaku D/MAX-2200 PC XRD diffractometer with Cu Kα
radiation (λ = 1.5406 Å). The piezoelectric output was measured under an electrochemical analysis
instrument (CHI660E). PFM measurement was conducted by atomic force microscope (AFM,
Bruker Dimension ICON) equipped with a ferroelectric test module. BT-dNO samples were
dispersed onto a conductive Pt-coated silicon substrate. A Cr/Au-coated conductive probe was
used in contact mode. During imaging, a 3 V AC excitation voltage was applied to the tip, with
the drive frequency tuned to the contact-resonance range (250-400 kHz). Amplitude and phase
images were recorded simultaneously for each scanned area.

Swelling and degradation assays
Hydrogel samples were freeze-dried and recorded for initial dry weights (W ), then immersed in
0
PBS solution (5 mL, 37°C) simulating physiological conditions. The hydrogel was removed at the
specified time point and gently blotted with filter paper to remove surface moisture. For the
swelling test, the hydrogels were weighed as W , and the swelling ratio was determined according
ts
to: swelling ratio (%) = (W -W ) / W × 100%. For the degradation test, the hydrogel samples
ts 0 0
were lyophilized and weighed as W . The degradation percent of the hydrogel was computed by
td
using the formula: degradation percent (%) = (W -W ) / W × 100%.
0 td td

Hemostatic assay
For rat tail amputation model, after anesthesia with isoflurane, the rats were secured to a corkboard.
Approximately 50% of the tail was surgically removed using sterile scissors. Following tail
transection, BT-dNO@Gel (500 μL) was applied to the wound. Blood loss was continuously
monitored during hemostasis, while wounds without any treatment were used as controls. For liver
hemorrhaging model, rats were anesthetized with isoflurane and the liver was exposed via
abdominal incision. After gently removing excess surface fluid, mouse liver was positioned on a
pre-weighed filter paper. An incision of 5 mm was made in the liver using micro scissors to induce
bleeding, followed by immediate application of 500 μL BT-dNO@Gel. After complete
hemostasis, bleeding time and the increase in filter paper weight were recorded and compared with
the control group.

NO release and piezoelectrocatalytic properties
The NO release performance of BT-dNO was assessed using the Griess assay. The release profiles
under various conditions were determined by recording characteristic absorbance (450 nm) using
a UV-vis-NIR spectrophotometer and a microplate reader (Thermo Multiskan FC). NO level was
determined based on a standard calibration curve generated using NaNO , with untreated deionized
2
water serving as the control. Rhodamine B degradation and ESR were used to characterize ROS
generation of the gel bandage, and DCFH-DA was used as an indicator of ROS at the cellular
level. BT-dNO (5 mg) was dispersed in aqueous rhodamine B solution (5 mL, 10 mg L-¹) and
stirred for 1 h to establish adsorption-desorption equilibrium. Following various treatments, the
samples were centrifuged to collect transparent supernatants, and the residual rhodamine B was
measured by UV-vis-NIR spectroscopy. Bruker A300-10/12 ESR spectrometer further detected
different types of ROS. DMPO (Sigma) served as the spin-trapping reagent for ·OH and ·O -,
2
and the resulting signals were recorded using an ESR spectrometer at ambient temperature.

Cytotoxicity evaluation
Cytotoxicity and cell proliferation assay were assessed using CCK-8 assay (Dojindo). HUVECs,
HSF and HaCat cells were pre-seeded into 96-well plates (5 × 10³ cells per well) and cultured
overnight. BT-dNO solutions and gel leachates at varying concentrations were added and
incubated for another 24 h. After adding CCK-8 reagent to each well, cell viability was assessed
by measuring absorbance at 450 nm using a microplate reader. Moreover, cells were subjected to
different treatments for 2 days, and their viability was gauged using a Calcein-AM/PI Double
Staining Kit (Dojindo), followed by observation using confocal laser scanning microscopy
(CarlZeiss LSM900). Fresh red blood cells (RBCs) were collected from healthy male SD rats and
suspended in PBS for the hemolysis assay. The RBC suspension was incubated with BT-dNO
solutions at different concentrations for 2 h at 37 °C. Hemolysis was quantified by measuring the
supernatant absorbance at 570 nm and calculated using: Hemolysis (%) = (OD - OD )
sample negative
/ (OD - OD ) × 100%. The negative control and positive control were deionized water
positive negative
and PBS, respectively.

Scratch assay
HUVEC, HSF and HaCat cells were seeded into 6-well plates (5 × 105 cells per well) and cultured
until confluent. Use a sterile micropipette tip to create a scratch in the culture dish, then rinse to
remove suspended cells and debris. Subsequently, the medium was replaced according to the
groups and subjected to various treatments. Images were captured by a microscope at different
culture time (0, 24, and 48 h). Migration rate was determined according to the following formula:
cell migration (%) = (S - S) / S × 100%. S and S denote the scratch area at 0 h and the
0 t 0 0 t
predetermined time, respectively.

Tube formation assay
Matrigel was thawed overnight in an ice bath (4 °C). Diluted Matrigel (200 μL) was pre-added to
24-well plate to allow gelation. HUVECs (5 × 104 cells per well) were then evenly seeded onto the
polymerized Matrigel and incubated with different materials for 24 h. After incubation, the cells
were stained with 0.5 µM Calcein-AM dye and observed under a fluorescence microscope. The
junctions, total tube length and meshes were quantified using ImageJ software (version 2.1.0).

Macrophage polarization analysis
RAW264.7 (1 × 105 cells per well) were seeded in confocal dishes and cultured until confluent.
The M1-like phenotype was induced by co-incubation with LPS (100 ng mL-1, ABclonal,
RPM0001) and IFN-γ (20 ng mL-1, ABclonal, RP01070), while M2-like phenotype was induced
by IL-4 (20 ng mL-1, ABclonal, RP01161) stimuli. IF staining (Leica SP8 LSCM) and flow
cytometry (BD LSRFortessa™ X-20) were performed to evaluate macrophage polarization status.
Antibodies used in this assay included anti-CD86 (diluted at 1: 400, Abcam, ab119857, clone no.
GL-1), anti-CD206 (diluted at 1: 500, Abcam, ab64693), Alexa Fluor® 488-conjugated secondary
antibody (diluted at 1: 800, Abcam, ab150157), Alexa Fluor® 594-conjugated secondary antibody
(diluted at 1: 800, Abcam, ab150080), APC anti-CD86 (0.2 μg per test, Biolegend, 159215, clone
no. A17199A), PE anti-CD206 (0.2 μg per test, Biolegend, 141705, clone no. C068C2).
For flow cytometry analysis, the harvested tissue was first digested into a single-cell suspension
using hyaluronidase/collagenase II. Antibodies used for staining included: APC-Cy7 anti-CD45
(Biolegend, 103115, clone no. 30-F11), APC anti-F4/80 (Biolegend, 157305, clone no. QA17A29),
FITC anti-CD11b (Biolegend, 101205, clone no. M1/70), PE-Cy7 anti-CD86 (Biolegend, 105013,
clone no. GL-1), PE anti-CD206 (Biolegend, 141705, clone no. C068C2). Each antibody was
diluted to a final working concentration of 0.2-0.5 μg per test. Data were collected and analyzed
using flow cytometer (BD LSRFortessa™ X-20) and FlowJo software (10.6.2).

In vitro antibacterial assessment
SEM (Sigma 300) was used to observe the bacteria (S. aureus and E. coli) morphology after
different treatments. The ROS level of bacteria was assessed using the bacterial ROS detection kit
(BestBio) and imaged under an upright microscope (Leica DM6 B). Spread plate assay was
adopted to evaluate the antibacterial effect. In brief, prepared bacterial suspensions (15 μL, 107
mL-1) were added to the pre-treated 24-well cell plate and received different group treatments.
Bacterial suspensions were harvested with sterile PBS (1 mL) and diluted approximately 800-fold.
Bacterial survival was determined by enumerating CFUs after 18 h of incubation on agar plates.
All experiments were conducted in triplicate, and survival rates were calculated as: survival rate
(%) = (C / C ) × 100%. C and C denote colony numbers in control and treated groups, respectively.
0 0
In addition, bacterial suspensions subjected to different treatments were passed through 0.22 μm
filters, and the concentration of bacterial protein leakage was calculated by BCA protein
quantification kit (Beyotime). To evaluate bactericidal efficacy, each sample was tested using a
viability/cytotoxicity assay kit (L6060, UElandy) and observed under an upright microscope
(Leica DM6 B, Germany). The bacterial reduction rate (%) was calculated as: reduction rate (%)
= (N / N ) × 100%. N and N denote the total and the dead bacterial count, respectively.
0 0

Study in diabetic wound model
For establishment of the diabetic models, animals were fasted for more than 12 h prior to STZ
administration. The rat diabetes model was induced by a single i.p. injection of STZ (50 mg kg-¹),
while the mouse diabetes model was established through five consecutive i.p. injections of STZ
(50 mg/kg). Blood glucose levels in all animals were measured twice a week using a glucometer
(Accu-Chek®) for 4 weeks after STZ injection. Successful modeling of diabetic animals was
defined as randomized glucose >16.7 mmol L-1 for two consecutive readings. After modeling, the
diabetic animals were anesthetized (1% pentobarbital sodium) and a back full-thickness skin
wound (2 cm diameter for rats and 8 mm diameter for mice) was created using a round punch
under aseptic conditions. The successfully modeled animals were randomly divided into six
groups: Control (PBS), LIFU, Gel, BT@Gel, BT@Gel+LIFU and BT-dNO@Gel+LIFU (n = 5
rats per group). The dose of BT or BT-dNO was 0.3 mg mL-1, LIFU parameters were 1.5 W cm-2,
50% duty cycle, 1.0 MHz and 120 s duration, and freshly prepared gel bandage was replaced every
other day to ensure sustained bioactive ion release. Wound images were acquired at predetermined
time points to evaluate the healing process. The wound closure rate was calculated based on the
following formula: wound closure (%) = (A - A) / A × 100%. A and A denote the original
0 t 0 0 t
wound area and the wound area at different time point, respectively.

Study in rat diabetic infected wound model
For diabetic rat with infected wound model, after experiencing the above STZ-induced diabetes
and establishing the back skin defect, the S. aureus or E. coli suspension (150 μL, 1 × 108 CFU
mL-1) was spread onto the wounds. Wound infection was confirmed 24 h after inoculation by
routine swab culture followed by spread plate analysis on solid agar. The successfully modeled
rats were than randomly allocated into groups based on the aforementioned grouping (n = 5 rats
per group). Wound healing was monitored by photographing and measuring wound areas at
predetermined time points, while bacterial infection was assessed by swab culture and spread plate
methods on days 7 and 14.

Study in rat DNP model
For DNP model, the diabetic rats were first constructed according to the aforementioned method,
and the blood glucose status and behavioral changes were monitored. One month after injection,
rats with blood glucose levels >16.7 mmol L-1 and 50% PWT decreased more than 50% from
baseline indicated successful establishment of the DNP model. The DNP rats were anesthetized
(1% pentobarbital sodium) and a right foot full-thickness skin wound (8 mm diameter) was created
to establish the DFU model on day 0. The successfully modeled DNP combined with DFU rats
were randomly divided into six groups to receive the different treatments described above (n = 5
rats per group). The wound healing process and behavioral changes were monitored during
treatment.

Behavior tests
Mechanical nociception was determined using the Von Frey filaments by measuring paw
withdrawal responses to stimulation on the plantar surface of rat hind paw. Prior to routine testing,
each rat received a 30 min adaptation period on pain test mesh rack covered with Plexiglas
chambers. Adaptation was confirmed when the rat entered a quiet state with reduced voluntary
activities. Von Frey filaments of increasing force (1.4-26 g) were applied vertically to the central
plantar surface of the hind paw for 6-8 s. Paw withdrawal or licking was considered a positive
response, proceeding to the next lower-force filament, whereas failure to respond led to use the
next higher-force filament. The testing sequence was continued until four measurements were
collected after the first behavioral change, or until four successive negative responses or five
successive positive responses occurred. The 50% PWT (denoted in grams, g) was determined by
the up-down method of Dixon. Hot nociception was evaluated using hot plate test, specifically by
timing the latency of rapid thumping of the hind paw, hind paw-licking or jumping. Rats were
placed on the plate at RT for 30 min prior to formal testing. Subsequently, they were transferred
to a hot plate heated to 55 °C for observation. Measurements were conducted three times with a
15 min interval between each session and a 30 s cut-off time is set to avoid foot burns.

Isolation, cultivation and experimentation of DRG neurons
DRG neurons were isolated from rat embryos on day 18 of embryonic development and
immediately plated into cold DMEM medium. Then DRGs were then washed with DMEM
medium and digested with an enzyme mixture solution (DNase Ⅰ, trypsin and collagenase) at 37°C
for 20 min until cell detachment. Next, the cell suspension was filtered with cell strainer (100 μm)
and purified with Percoll. DRG neurons were resuspended in Neurobasal medium with 2% B-27
(Gibco) and cultured in an incubator.
For IF staining of TRPV1, DRGs were pre-seeded in confocal dishes (1 × 105 cells per well) and
subjected to different treatments. After PBS washing, the neurons were fixed with 4%
polyformaldehyde (PFA) at RT (15 min), and incubated in QuickBlock™ Blocking Buffer
(Beyotime, P0260) for 15 min. After triple washing by PBS, the specimens were incubated with
anti-TRPV1 primary antibody (diluted at 1:200, ABclonal, A8564) overnight at 4 °C, and then
incubated with Alexa Fluor® 488-conjugated secondary antibody (diluted at 1:800, Abcam,
ab150077) for 1 h at RT. Before observed under CLSM, the nuclei were stained with DAPI.
For calcium imaging, DRG neurons were inoculated into confocal dishes (1 × 105 cells per well)
and cultured until cell attachment. Fluo-4 AM (4 μM) was added to the serum-free DMEM medium
and co-incubated with neurons in the dark at 37 °C for 30 min. Neurons were then washed and
treated with Cap or BT-dNO@Gel in culture media. LIFU stimulation was carried out at 1.5 W cm-
2 for 30 s. The fluorescence images were captured using CLSM.

Histological analysis
To assess wound closure status, epidermal regeneration, immune infiltration and analgesia
mechanism, the rats skin tissues, L4/L5 DRGs and spinal cord dorsal horn were harvested and
fixed in 4% PFA for 20 min-1 h, followed by dehydration and paraffin embedding. Tissue sections
of 5 μm thickness were cut using a microtome for histological analysis. Re-epithelialization and
collagen deposition were evaluated by H&E staining, Masson’s trichrome staining and
immunohistochemistry staining of collagen I (dilution at 1: 500, Abcam, ab270993, clone no.
EPR24331-53) and collagen III (dilution at 1: 200, Abcam, ab7778). ROS levels within wound
tissue were assessed using DHE dye (Beyotime, S0064S). For IF staining, tissue sections
underwent a 90 min blocking step followed by overnight incubation with the primary antibody at
4°C and a 2 h incubation with the secondary antibody at RT. Prior to imaging (Leica DM3000 and
Zeiss Axio Imager Z1), cell nuclei were stained with DAPI. Primary antibodies involved in
experiment include Ki67 (dilution at 1: 500, Abcam, ab15580), CD31 (dilution at 1: 100, Abcam,
ab222783, clone no. EPR17260-263), α-SMA (dilution at 1: 500, Bioss, bsm-33187M, clone no.
3F9), K14 (dilution at 1: 100, Thermo Fisher Scientific, MA5-11599, clone no. LL002), vimentin
(dilution at 1: 1000, ABclonal, A19607, clone no. ARC0086), CD68 (dilution at 1:100, Abcam,
ab283654, clone no. EPR23917-164), Arg1 (dilution at 1: 200, CST, #93668, clone no. D4E3M),
CD206 (dilution at 1: 500, Abcam, ab64693), CD86 (dilution at 1: 200, ABclonal, A16805),
Na 1.8 (dilution at 1: 2000, Abcam, ab307817, clone no. EPR25132-222), TRPV1(dilution at 1:
v
200, ABclonal, A8564), GFAP (dilution at 1: 500, CST, #3670, clone no. GA5), Iba-1 (dilution at
1: 200, CST, #17198, clone no. E4O4W) and CGRP (dilution at 1: 200, CST, #14959, clone no.
D5R8F).

Transcriptome sequencing
Total RNA was isolated from wound tissues using TRIzol reagent at the end of treatment. RNA
quality and quantification were tested using Agilent 2100 Bioanalyzer and ND-2000 NanoDrop
spectrophotometer. Library construction and sequencing were performed on an Illumina HiSeq
2000 system. Differential expression analysis among the naive, control and BT-dNO@Gel+LIFU
groups was analyzed using DESeq2 (v1.4.5) with a significance threshold of Q value ≤ 0.05.
KEGG and GO enrichment analysis of DEGs were conducted using Phyper based on the
hypergeometric test, and statistical significance was corrected by Bonferroni adjustment with a Q
value threshold of ≤ 0.05.

Real-time quantitative Polymerase Chain Reaction (RT-qPCR)
Total RNA was extracted with the Eastep Total RNA Extraction Kit (Promega). Complementary
DNA (cDNA) was synthesized from total RNA using the HiScript® II First Strand cDNA
Synthesis Kit (Vazyme Biotech Co., Ltd.). RT-qPCR analysis was performed with SYBR Green
PCR Master Mix (Biotechnology), and gene expression was normalized to Gapdh. Primers were
ordered from Sangon Biotech. The primer sequences for Calca and Gapdh used were as follows51:
Rat_Calca forward primer: 5’- GAAATGCCACCTTTTGACAGTG -3’,
Rat_Calca reverse primer: 5’- TGGATGCTCTCATCAGGACAG -3’,
Rat_Gapdh forward primer: 5’- AGGTCGGTGTGAACGGATTTG -3’,
Rat_Gapdh reverse primer: 5’- TGTAGACCATGTAGTTGAGGTCA -3’.

Statistical analysis
All experiments were performed with at least three biological replicates. Quantitative data were
presented as mean ± SD. Statistical analyses were performed using GraphPad Prism software
(version 8.2.1), IBM SPSS software (version 27.0.1.0), and OriginPro 2024 software. A two-tailed
unpaired Student’s t-test was applied for comparisons between two groups, whereas one-way
ANOVA with Tukey’s post hoc test was used for multiple-group comparisons. Statistical
differences were denoted as ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001 and
****P<0.0001.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this paper, the authors prepared a low intensity focused ultrasound (LIFU)-activated piezoelectric gel bandage for DFU wound repair and evaluated the therapeutic efficacy in a rat model of diabetic neuropathic pain. In general, the paper is well organized with good novelty. Some revisions are required as follows.\n1. BT-dNO@Gel designed in this study can produce both NO and ROS. In this regard, could the release of NO be precisely controlled because NO is short-lived radical demonstrating dual effect at different dose? At least, some discussion on this point is needed. Next, the NO can react with ROS to generate peroxynitrite (ONOO) with high level toxicity. Have the authors notice the production of ONOO and its effect on the wound healing?\n2. The authors mentioned that ROS produced by LIFU activated piezoelectric catalytic reaction has bactericidal effect, but excessive accumulation of ROS may lead to cell damage, oxidative stress and even delayed wound healing. Therefore, it is recommended to detect the production of ROS in vivo, and provide relevant discussion.\n3. Mechanistically, the relieved neuropathic pain is ascribed to the desensitizing of TRPV1 receptor by the BT-dNO@Gel. Relevant experimental results are required to verify this hypothesis Are there other possible mechanisms for pain relief that have not been considered?\n4.In the current version, the gene expression was evaluated through transcriptome analysis. It would be helpful to validate these changes by other experimental methods, such as qPCR or Western blot? In addition, are there experimental data to support the functional significance of these gene expression changes?", "reviewer_2": "This paper investigated the effects of low-intensity focused ultrasound (LIFU)-activated piezoelectric gel dressing (BT-dNO@Gel) on diabetic foot ulcer (DFU) treatment, exploring its impact on immune homeostasis and neuropathic pain relief. The paper presents a novel concept of using LIFU-activated piezoelectric gel dressing for DFU treatment. The experimental design is reasonable, and the result analysis is relatively sufficient. However, the presented work raises several critical concerns that need to be addressed before considering it for publication.\n(1) The introduction section provides a detailed introduction to the pathogenesis and treatment status of DFU, but there is insufficient elaboration on the current research status of the application of piezoelectric materials and LIFU in wound healing. It is recommended to supplement relevant content.\n(2) As the author stated, Under LIFU irradiation, the outer NONOate of the BT-dNO decomposes to release NO. However, NO, as a bioactive molecule, has various important roles and functions in the human body and organisms. The effects of different NO concentrations are very different. When NO concentration <1-30 nM, it could promote cell survival and proliferation, when >1000 nM, it could kill bacterial bioflms. The authors should add relevant experiments to reflect the specific concentration curve of NO released by this hydrogel under US irradiation.\n(3) Additionally, under LIFU irradiation, the inner BT core would produce an unbalanced charge on their surface and undergo cascading redox reaction process, which leads to the generation of ROS and a localized electric field. The types of ROS include various species such as superoxide anions (O2-), hydroxyl radicals (• OH), etc. There are differences in the bactericidal performance of different ROS. Please supplement Electron spin resonance to detect the various ROS produced by BT dNO.\n(4) BTdNO@Gel Bandages have good in vitro bactericidal ability. As far as we know, high concentrations of NO and ROS both contribute to the bactericidal effect. In this process, are the two synergistically antibacterial or do they rely solely on LIFU-triggered ROS generation?\n(5) BT@Gel and BT-dNO@Gel can lead to the rupture of bacterial film and the leakage of a large amount of protein, resulting in the death of bacteria. Please supplement the electron microscope images of bacteria to show the above process more directly.\n(6) As mentioned in the paper, infection is one of the major obstacles to wound healing in diabetes, and dressings with antibacterial activity will help block pathogen invasion and promote wound recovery, but the authors only demonstrated that this bandage has a satisfactory bactericidal effect against Staphylococcus aureus. The antibacterial effect of gram-negative bacteria is also crucial for diabetic wound healing, so please supplement the relevant experiments.\n(7) As shown in FIG. 6a-6d, there was a decrease in M1 macrophages and an increase in M2 macrophages in the BT-dNO@Gel+LIFU group. What is the mechanism for regulating the polarization of macrophages? Is it related to NO?", "reviewer_3": "Li and colleagues report the development of a piezoelectric gel bandage activated by low-intensity focused ultrasound to aid in diabetic wound repair and neuropathic pain relief.\nThe authors use a series of immunohistochemical, physiological and molecular biological assays to test the ability of this hydrogel formulation to heal diabetic wounds. This addresses a significant problem, since diabetic foot ulcers are notoriously painful and difficult to treat. Additionally, this formulation represents a multifunctional approach, incorporating strategies that have been developed elsewhere and now applied to diabetic neuropathy.\nHowever, there are several issues with the manuscript which curtailed my enthusiasm:\n1. Line 64 appears to imply the TRPV1 agonist capsaicin has abuse liability. Presumably this refers to a now-deleted mention of opioids, because there is no evidence I’m aware of that capsaicin or TRPV1 modulators in general exhibit abuse liability.\n2. The rationale for settling on electrical stimulation could be better articulated. For example, citing evidence that the approach has potential, talk about the potential mechanisms of action and then outline the current difficulties/limitations of this approach that their novel formulation will circumvent.\n3. The authors mention that the device liberates ROS without first mentioning the therapeutic potential of this approach. Fig. 1 appears to suggest the benefit of ROS is derived solely from being bactericidal, but ROS can be directly pro- and anti-inflammatory also, this should be addressed.\n4. Couldn’t the beneficial effects of the formulation also be mediated by NO-driven vasodilation? This doesn’t seem to have been considered.\n5. Fig. 1 is a graphical abstract – either include it as such, or move it to the end of the results section as a tentative schematic, or remove it.\n6. It was not clear if the durability of NO mobilization and/or NO release kinetics were tested in vivo. In other words, is there evidence that LIFU stimulation several days after application is still able to liberate physiologically relevant quantities of NO/ROS?\n7. The study would benefit from comparison to standard wound dressings/antibiotics, or standard systemic dosing with a nitric oxide donor, such as nitroglycerin or sodium nitroprusside.\n8. Fig. 3 seems to show NO release/ROS production in vitro over the course of 2-3 minutes. What evidence is there that LIFU can be used to generate sustained NO release/ROS in vivo? Presumably a shifting of macrophage phenotype requires more than a few minutes of enhanced ROS.\n9. Could the authors use L-NAME to block NO and test whether the effects on HUVEC proliferation/migration are blocked?\n10. Is >16.7mM on non-fasted rats a benchmark for diabetes following STZ that has precedent?\n11. The statistical tests, particularly in Fig. 5 (unpaired t-test), seems inappropriate. Two-way, repeated measures ANOVA would be more appropriate for Fig. 5D for example.\n12. The authors do not directly test the effect of this formulation on macrophages in vitro to establish if the beneficial effects on wound healing are a direct effect of the formulation on macrophage function. It’s possible there is an association here, but the chief driver could be the bactericidal activity which dampens innate inflammation and results in a less-pro-inflammatory microenvironment and fewer M1-like macrophages being present.\n13. Fig. 6 – requires additional evidence to localize the iNOS and Arg1 staining with a known macrophage marker (F4/80, CD68).\n14. Which cells are Ki67 positive? It would bolster the claims being made regarding growth factors if there was a concordant increase in proliferation of the target cell type (keratinocytes/vascular endothelial cells).\n15. Why wasn’t more testing of wound healing done in the hindpaw DFU model?\n16. In the pain behavior testing, there is no group where gel formulations are delivered to non-diabetic rats, where presumably there would be no effect on mechanical sensitivity.\n17. PC-12 cells are not an ideal model of sensory neurons in this context, especially when there may be effects on multiple sensory neuron channels that PC12 cells do not express, and the team show they can access lumbar DRG from rats for other downstream applications.\n18. Figure 8e doesn’t look like plasma membrane staining at all, which would be expected for a functional ion channel.\n19. Why is the effect size in the pain behavior testing very small – approximately 15% reversal versus naïve controls?\n20. The IHC evidence for activity of TRPV1 sensory neurons is generally weak. For example, the interpretation that a TRPV1+ cell is less likely to be Fos+ doesn’t necessarily mean that reduced TRPV1 activity is responsible for this.\n21. The TRPV1 staining pattern in DRGs is unconvincing, because it looks to be labeling most neurons rather than a subset of neurons, and there is an unexpected signal from what looks like nuclei as well.\n22. What was the skin RNA-seq compared against as control. This is important to explain clearly in order to know if the differentially-expressed genes can be ascribed to LIFU, ROS production, etc.\n23. Using PC12 cells to illustrate the promotion of CGRP expression/release is not optimal. The IHC for CGRP in Fig. 9 does not resemble cutaneous nerves, nor is there any co-staining with a neuronal marker (e.g. beta 3-tubulin, PGP9.5) to aid in identifying the cells responsible for the CGRP immunoreactivity.\n24. There are also no experiments attempting to interfere with CGRP signaling in macrophages to confirm that this signaling is necessary for the formulation’s beneficial effects.\n25. Fig. S18 – I struggle to interpret whether the immunostaining is specific or if there is a high degree of background.\nMinor points:\nThe wording of the introduction could benefit from editing to optimize grammar and word choices.\nMacrophages can adopt a stereotypical profile of M1 or M2 in vitro, but this is broadly considered to be an oversimplification when discussing the spectrum of macrophage function in vivo. Where possible, that terminology should be avoided, or at the very least acknowledge its limitations.\nThe transcript name for CGRP should be styled Calca (italicized) rather than \"CGRP.\"\nResults section, page 224: it was not clear why ROS green fluorescence should be expected. I think some explanation of the experimental design is missing in this part.\nBiosafety supplementary figures at the end does not form a logical narrative flow and should be moved closer to the initial characterization of the formulation.\nThere needs to be more emphasis on the incidence of DFU infection in the introduction. It wasn’t clear before reading the results that this was part of the formulation’s intended activity.", "reviewer_4": "In the manuscript titled low intensity focused ultrasound-activated piezoelectric gel bandage for diabetic wound repair and neuropathic pain relief,' Li et al. report that the of BT-dNO@Gel_LIFU significantly promoted diabetic skin wound healing in a sterile or bacteria infection environment. The proposed mechanism involves macrophage polarisation, desensitisation of TRPV-1 nociceptors and the produce of ROS against invasive pathogens. Furthermore, the treatment may promote the release of CGRP from sensory neurons, which in turn contributes to macrophage polarisation. While the therapeutic efficacy of BT-dNO@Gel_LIFU in accelerating diabetic wound closure is acknowledged, the manuscript raised several major concerns regarding experimental design and data interpretation:\n1. In the most statistical analysis, the authors included more than two groups in the experiments but conducted two-tailed unpaired student’s t-test. This approach is not appropriate for analysing the data with more than two groups in the experimental design. The authors should reanalyse data by using a one-way ANOVA or two-way ANOVA with post-hoc tests for most of the figures to ensure proper statistical analysis and interpretation of the results.\n2. In figure 4, the authors investigated the effects of BT-dNO@Gel with LIFU only on HUVEC. However, in the proliferation phase of wound healing processes, keratinocytes and fibroblast are also critical for promoting skin wound closure. The authors should perform the migration assay and cytotoxicity assay on these two cell types to understand whether BT-dNO@Gel with LIFU could directly affect keratinocyte and fibroblast cell growth and proliferation. Additionally, given the gel was applied immediately right after the skin punch, the immune cell responses, which are important for skin wound healing in the early of phase, would be affected by BT-dNO@Gel and required for further investigation.\n3. In line 232-235, the authors stated that “Of note, the LIFU-triggered ROS generation from BT-dNO@Gel indeed affected bacteria survival but not HUVECs, probably due to the fact that the respiratory chain of eukaryotic cells is located in intracellular mitochondria, which is more resistant to external ROS interference than bacteria.” Please cite relevant references to justify their statement.\n4. In the method, the authors described using a single ip injection of STZ to induce diabetes in a rat model. Two weeks post-injection, they performed the skin injury procedure and harvested the tissue two weeks post the injury. This implies that tissues harvesting occurs four weeks after the single injection of STZ.\nSince that STZ induced diabetes and hyperglycaemia in rats may not be persistent for extended period, it is crucial for the authors to measure blood glucose levels weekly throughout the experimental period to confirm that hyperglycaemia persisted up to four weeks following the single injection of STZ. Additionally, the authors should clarify whether blood glucose levels were measured in a fasting or non-fasting state and provide these details in the method section to enhance the reproducibility and reliability of study.\n5. In the animal work, the authors conducted 120s (50 % duty circle) of LIFU at the tissue injury site where the BT-dNO@Gel was delivery every other day. It implies this regime only allowed the gel to generate the current for 120 seconds every 48 hours. This raises a concern that such LIFU regime may result in the production of ROS and the release of NO for only a brief two-minute period, potentially limiting the therapeutic effects. To address this, the authors should evaluate the extent of ROS and NO generation at the wound site several hours post LIFU applications. This would help confirm whether the therapeutic effect of BT-dNO@Gel is sustained or merely transient following stimulation.\n6. In line 260, the authors stated that “Digital photos and quantitative analysis of the wound sites showed that all the gel groups containing different formulations facilitated healing process to some extent compared to the control rats with significantly delayed wound healing, which can be attributed to well-adherent hydrogels providing a suitable matrix-like environment for fibroblast proliferation and epidermal regeneration (Fig. 5b-d).” However, there is no data or results to directly support that fibroblasts proliferation in vivo in this model. Please consider removing this statement or conduct relevant experiments, such us immunostaining, such as Ki67 and fibroblast markers, to support their points.\n7. In line 269, the authors described that “In contrast to the immature granulation tissue, local inflammatory cell infiltration and incomplete epidermal bridging in the control group, the gel-containing groups had abundant neoplastic granulation tissue in the wound center with fewer inflammatory cells and increased epidermal viability, especially in BT-dNO@Gel+LIFU group (Fig. 5e-g). However, the authors did not provide the data to support how they identified inflammatory cell infiltration into the tissue site. The authors should perform experiments, such as immunostaining of pro-inflammatory immune markers, to identify and evaluate the extent of pro-inflammatory cell infiltration to the skin wound.\n8. In figure 5, the excessive collagen disposition observed at the wound at the later timepoint (day 14) could suggest a contribute to scar formation and fibrosis, which are typically detrimental to skin wound healing. Could the authors clarify why they consider a larger collage area in the G6 groups at day 14 to be beneficial for skin wound healing? Additionally, it would be helpful to discuss whether the quality and organization of the collagen (e.g., alignment, type I vs. type III ratio) were assessed, as these factors are critical in determining whether collagen deposition supports regenerative healing or leads to fibrosis.\n9. In figure 6a-d, the authors demonstrated a reduced number of iNOS+ cells and an increased large number of Arginase+ cells in the BT-dNO@Gel+LIFU-treated skin wounds, suggesting that the treatment regulates macrophage polarisation. However, macrophages are not the sole source of iNOS and arginase-1. The authors should perform IHC co-staining of F480 (or any macrophage makers) with iNOS, as wells F480 with Arginase on the same tissue sections to confirm that the observed changes in iNOS and Argainse-1 expression occurs in macrophages rather than in other cell types. Besides, it is not annotated in the manuscript which part of the skin wound was represented in the in Figure 6a and c. Were those images taken from the granulation tissue or the edges of the wound? Additionally, since immune cells are not evenly distributed across the tissue injury site, the authors should capture multiple images from a single tissue section. Quantification and averaging the positive staining cells from these images provide a more accurate representation of the number of positive staining cells. Alternatively, using flow cytometry to evaluate immune cell population in the wound is encouraging. In the figure 6e, f and g, there are no details of antibodies of TNFa and IL-6, IL-10 include in this manuscript. And given that they are detected by using immunostaining, the data presented as the intensity by percentage is puzzling. Please add all details to clarify how the quantification of the staining was conducted. Moreover, given that the backgrounds of staining images of TNFa, IL6 and IL10 are very high and inconsistent among all images as well as the result obtained from the IHC is semi-quantitive which cannot represent the condition in the entire skin wound. Using ELISA to detect these cytokines are required. There are same issues and concerns found in Figure 7g, h, i, j, k and Figure 9.\n10. In figure 6k, it is not clear which cell type the ki67+ cells are. It could be immune cells, fibroblast, endothelial or keratinocytes. Since monocyte proliferation has been linked to chronic inflammation and delay skin wound healing in diabetic mice (PMID: 33443065), it is very crucial to perform co-staining of IHC to verify which cell types of these Ki67+ cells are. Lastly, in Figure 6h, the quantification of Ki67 should be presented as the percentage of Ki67+ cells out of the total cell number. It is not very common to quantify Ki67+ staining as the intensity. Could the authors clarify why they considered to present Ki67+ staining as the intensity?\n11. Could the authors clarify whether the rats in Figure 8 received skin punch? Please add the skin punch procedure in the timeline presented in Figure 8a\n12. In DNP model, mice received the treatment two weeks after the foot skin injury in which the skin wound was almost healing. Therefore, the BT-dNO@Gel_LIFU-indueced macrophage polarisation and CGRP release at the tissue injury observed in figure 8 might not contribute to wound regenerative repair. The authors should treat the skin wound right after the injury as they did in Figure 5 and observe macrophage polarisation, CGRP expression, and wound clousre to support their statement that “LIFU-mediated immune-nociceptor modulation strategy can orchestrate subcutaneous sensory nerve and M2 type macrophages interactions to achieve wound regenerative repair and analgesia.”\n13. In Figures 8d–f, the authors used the PC-12 cell line as an in vitro model to investigate the direct effects of treatments on nociceptors. They measured the TRPV-1 levels in PC-12 cells and observed that the treatment reduced TRPV-1 expression in the presence of capsaicin. However, since the PC-12 cell line lacks proper characterization as a nociceptor neuron, the response observed here might not fully replicate the phenotype or function of native nociceptors. To address issue, the authors should use primary neuron cells isolated from DRG and detect TRPV1 expression in nociceptors, which can be identified by using nociceptor markers (PMID:26053673), after the treatment.\n14. In the abstract, the authors state that piezoelectric current to desensitize TRPV1 nociceptor. However, in this manuscript, it is clearly showed that BT-dNO@Gel_LIFU induced the gel to generate ROS and NO. Since ROS and NO have been demonstrated to affect nociceptive pain (PMID: 17317010; PMID: 21723953), the effects of ROS and NO generated by the BT-dNO@Gel_LIFU on neurons should not be disregarded. The authors should investigate the role of these two factors on nociceptors. Additionally, if piezoelectric currents are indeed the primary factor causing TRPV1 nociceptor desensitization, the authors should measure the level of current generated by BT-dNO@Gel_LIFU to confirm whether it is sufficient to induce TRPV1 desensitization.\n15. In figure 9f, the authors should use co-staining to confirm CGRP expression on nociceptors and Arg1 on macrophages. Particularly, the CGRP expression is almost co-localisation with Arg1, implying that CGRP might be expressed by macrophages (PMID: 20141542). Besides, the data obtained by using immunostaining approach is semi-quantitive. The authors should use ELISA to detect CGRP levels in the injury sites or in the corresponding DRG to support their statement.\n16. The authors concluded that the BT-dNO@Gel_LIFU triggered CGRP release from nociceptors, therapy regulating macrophage polarisation tissue injury sites. However, based on their current data, BT-dNO@Gel_LIFU regulated directly affect HUVECs that have been demonstrated to affect macrophage activation (PMID: 23542777). Moreover, ROS and NO may also have impact on macrophage polarisation. To elucidate the critical role of CGRP, which might be upregulated by BT-dNO@Gel_LIFU, the authors should use genetic knockout (LysMCre-RAMP1floxed) animals or CGRP receptor antagonists (CGRP8-37, PMID: 1797334) on the skin wounds to confirm if BT-dNO@Gel_LIFU- promoted skin wound closure and M2 macrophage polarisation are disrupted.\nMinor comments\n1. In line 210, the authors stated that “ the blood cell hemolysis rate of BT-dNO was less than 5% within the safe range, suggesting its favorable hemocompatibility. Could the authors include the relevant reference to support this statement? Additionally, the bar graph in Figure S14 shows that 0.5mg/ml BT-dNO resulted in ~ 15% of hemolysis, which appears inconsistent with the statement in line 212 (5%). Could the authors verify the raw data and provide it in a table format instead of the bar graph to ensure accurate calculation of the percentage of homolysis?\n2. In Figure S1, authors claims that the typical peak of C-C benzene ring at 1430 and 1565 cm-1 in the Raman spectra confirmed the existence of OH and pDA, respectively (fig. S1a, b). However, the peak of C-C benzene ring at 1430 cm-1 is only observed in BT-OH, not in the sample of BT-OH-pDA or BT- dNO. Could the authors clarify how to interpret the data and add these details in the result section?\n3. In material and methods, line 560, how was PVA solution prepared?\n4. In the line 658. the authors should clarify whether they replaced the gel every other days.\n5. In figure 8b and c, should the label of x axis be “days after DNP”?", "reviewer_author_response": "The following part is the point-by-point responses to the reviewers:\nResponse to Reviewers 1\nReviewer #1 (Remarks to the Author):\nIn this paper, the authors prepared a low intensity focused ultrasound (LIFU)-activated piezoelectric gel bandage for DFU wound repair and evaluated the therapeutic efficacy in a rat model of diabetic neuropathic pain. In general, the paper is well organized with good novelty. Some revisions are required as follows."}, "author_response": ""}]

Decision letter