bioRxiv preprint doi: https://doi.org/10.1101/2024.09.06.611461; this version posted March 11, 2025.

The copyright holder for this preprint

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Intranasal dantrolene nanoparticles inhibit lipopoly-

saccharide-induced helplessness and anxiety be-
havior in mice
Jia Liu, MD, PhD1,2#, Yan Lu, MD1,3#, Piplu Bhuiyan MD1, Jacob Gruttner1, Lauren St. Louis1, Yutong
Yi1, Ge Liang, MD1, Huafeng Wei, MD, PhD1

1 Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
19104, U.S.A.
2 Department of Anesthesiology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, 26600, P. R. China
3 Department of Anesthesiology, Shanghai Key Laboratory of Maternal Fetal Medicine, Shanghai Institute of Maternal-Fetal Medi-

cine and Gynecologic Oncology, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University, Shanghai
200092, China
#Equal contributing authors

Correspondence should be addressed to Huafeng Wei, MD, PhD ([email protected])

Abstract: This study investigates the therapeutic effectiveness of intranasal dantrolene nanoparticles pretreatment to
inhibit lipopolysaccharide (LPS)-induced pathological inflammation and synapse destruction and depressive and anxiety
behavior in mice. B6SJLF1/J adult mice were pretreated with intranasal dantrolene nanoparticles (dantrolene: 5mg/kg),
daily, Monday to Friday, 5 days per week, for 4 weeks. Then, mice were treated with intraperitoneal injection of LPS (5mg/kg)
for one time. Behavioral tests for depression and anxiety were performed 24 hours after a one-time LPS injection.
Biomarkers for pyroptosis-related inflammation cytokines (IL-1β and IL-18) in blood and brains were measured using
enzyme-linked immunosorbent assay (ELISA) and immunoblotting, respectively. The changes of primary proteins
activation inflammatory pyroptosis (NLRP3: NLR family pyrin domain containing 3, Caspase-1, N-GSDMD: N terminal
protein gasdermin D) and synapse proteins (PSD-95 and synpatin-1) in brains were measured using immunoblotting.
Intranasal dantrolene nanoparticles robustly inhibited LPS-induced depression and anxiety behavior. Intranasal dantrolene
nanoparticles significantly inhibited LPS-induced pathological elevation of IL-1β and IL-18 in the blood and brain and
inhibited LPS induced activation of pyroptosis. Intranasal dantrolene nanoparticles significantly ameliorated decrease of
PSD-95 and synpatin-1 proteins in brains. Thus, intranasal dantrolene nanoparticles has demonstrated neuroprotection
against inflammation mediated depression and anxiety behaviors and should be studied furthermore as a future effective
drug treatment of major depression disorder or anxiety psychiatric disorder.

Pharmacotherapies for anxiety disorders also include drugs

Introduction that interact with GABA neurotransmission systems13. Unfor-
Three hundred million people worldwide suffer from major tunately, antidepressants have significant limitations, includ-
depressive disorder (MDD)1. MDD is a chronic and recurrent ing slow onset of action, high rates of nonresponse, and acute
disease affecting about 20% of the population and the leading worsening of anxiety14. Benzodiazepines are not recom-
cause of suicide2. MDD imposes a tremendous psychological mended for long-term use in some anxiety disorders, due to
burden, as well as significant social repercussions and contri- concerns about their potential for abuse, tolerance, and with-
butions to other disabilities3. The expected direct and indirect drawal, and they are ineffective in some anxiety spectrum dis-
costs of MDD are up to $6 trillion in the USA alone4. An esti- orders15. Treatment of MDD is prone to a high risk of resistance
mated 4% of the global population currently experience an (up to 30% of patients are unresponsive to the first treatment)
anxiety disorder. In 2019, 301 million people in the world had and relapse (up to 8%)3,10. Treatment-resistant anxiety with re-
an anxiety disorder, with a lifetime prevalence of approxi- mission rates may be as low as 25–35%, and relapse rates post
mately 34 percent5. Major depression disorder and anxiety dis- remission may be 30% after 10 years16. Thus, there is an urgent
orders were approximately twice as prevalent among need to develop novel approaches to treat depression and
women6,7, with overall age-specific rates remaining relatively anxiety, especially treatment-resistant depression, or anxiety,
stable or increasing across the lifespan8. In 2021, an estimated with minimal side effects or organ toxicity.
5.8 million (9.4%) children and adolescents were impacted by
anxiety9. Depression and anxiety often co-exist and are the Increasing studies indicated that glutamate neurotrans-
most common psychiatric diseases, which are chronic diseases mission plays a critical role in mood function and its imbalance
with unclear mechanisms of pathology10. may cause psychiatric disorders17. Glutamate is linked to the
development of anxiety disorders through its regulation of
Classical treatment of depression or anxiety psychiatric neuropeptides, fear extinction, and stress response. Gluta-
disorders includes atypical antipsychotics that modulate dopa- mate is also important in synaptic and neural plasticity related
mine and serotonin neurotransmission11,12. to psychiatric disorders. Ketamine, an N-methyl-d-aspartate

Liu and Lu et al. 2025 (preprint) 1

 bioRxiv preprint doi: https://doi.org/10.1101/2024.09.06.611461; this version posted March 11, 2025. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Intranasal dantrolene nanoparticles treat depression and anxiety

(NMDA) glutamate receptor antagonist, was approved in 1970 In this study, we investigated the therapeutic effective-
by the Food and Drug Administration (FDA) for use in children ness and the potential mechanisms of intranasal dantrolene
and adults as an anesthetic. Recent studies indicate that keta- nanoparticles on depression and anxiety behavior associated
mine is effective in treating MDD, especially treatment-re- with pathological inflammation in adult mice. We induced in-
sistant depression (TRD)18,19. Ketamine is also effective in treat- flammation by intraperitoneal injection of LPS. Our results
ing anxiety disorders, even those resistant to traditional treat- demonstrated that intranasal dantrolene nanoparticles for
ments9,20. While traditional antidepressants can take weeks to twelve consecutive weeks robustly inhibited LPS-induced de-
months to have an effect, ketamine has rapid effects on mood pression and anxiety behaviors in adult mice. These beneficial
and suicidality, with mood changes reported as early as the effects of intranasal dantrolene nanoparticles treatment were
first 4 hours after treatment21. As an antagonist of NMDAR, a associated with its robust and significant inhibition of LPS-in-
primary glutamate receptor on the plasma membrane that duced pathological elevation of pyroptosis related inflamma-
causes Ca2+ influx into the cytosol from extracellular space, ket- tion cytokine of IL-1β and IL-18 in blood and brains and loss of
amine has become a safe and broadly effective drug to treat synapse proteins (PSD-95 and synpatin-1) in brains. This study
depression or anxiety disorders, so is the intranasal esketa- provides proof of concept that intranasal dantrolene nanopar-
mine22. This indicates that glutamate-mediated excitotoxicity ticles may be an effective treatment for depression and anxiety
and associated disruption of intracellular Ca2+ homeostasis psychiatric disorders, potentially by inhibiting pathological in-
play a key role in the pathology of depression and anxiety dis- flammation and synapse destruction in CNS.
orders.
Although molecular mechanisms are unclear, increasing
evidence suggests that upstream disruption of intracellular
Materials and Methods
Animals
Ca2+ homeostasis and associated downstream inflammation
All procedures were approved by the Institutional Animal
and synapse dysfunction play critical roles in MDD patholo-
Care and Use Committee (IACUC) at the University of Pennsyl-
gies23-25, as well as anxiety disorder26,27. The overactivation of
vania. All B6SJLF1/J adult mice were obtained from Jackson Lab.
ryanodine receptors (RyRs) in MDD and associated excessive
Mice were divided into different cages according to age and
Ca2+ release from endoplasmic reticulum (ER) results in deple-
gender, with no more than five mice per cage. Both male and
tion of ER Ca2+ and pathological elevation of cytosol and mito-
female mice were used in this study.
chondrial Ca2+ concentrations, detrimental to synapse function
and cell survival25,28,29. RyRs overactivation have also been Experimental treatment groups
shown to increase anxious behavior28. Upstream Ca2+ dysregu- As demonstrated in supplemental figure 1, adult male and
lation results in mitochondria dysfunction30, mitochondrial and female mice (5-10 months old) were randomly divided into dif-
cellular oxidative stress10,31, activation of inflammasomes, and ferent experimental groups. The mice were pretreated with ei-
cell or neuron death by pyroptosis32-35. This results in the re- ther intranasal dantrolene nanoparticles (IN Dan, 5mg/kg) or
lease of inflammatory cytokines (IL-1β and IL-18) and patho- vehicle (Veh), once a day, 5X/week (Monday through Friday).
logical inflammation-mediated cell death by pyroptosis34,36. Control groups received no pretreatment. Fresh dantrolene
Pathological cytokines, especially pyroptosis-related IL-18 play nanoparticles were made every time before administration. In-
important roles in psychiatric disorders37,38. Gut dysbiosis and tranasal dantrolene nanoparticles stock solution was made at
associated inflammation also contribute to pathology in de- 5 mg/ml. The Ryanodex formulation vehicle was made fresh
pression and anxiety behaviors39,40. Thus, a drug that inhibits and contained all inactive ingredients in Ryanodex51,52. At the
upstream Ca2+ dysregulation and then downstream pathologi- end of the 4-week pretreatment period, mice were then
cal inflammation and programmed cell death by pyroptosis treated with one-time intraperitoneal injection of lipopolysac-
and synapse destruction is expected to treat MDD and anxiety charide (LPS, 5 mg/kg), once. Mice in the control group, with-
disorder effectively. out pretreatment, were treated with LPS (LPS, 5mg/kg) once
as well. The sham control group received no pretreatment or
Dantrolene, a RyRs antagonist, is a US Food and Drug Ad- treatment. At 24 hours following the one-time LPS treatment,
ministration-approved drug for the treatment of malignant hy- behavioral tests for depression or anxiety behaviors were per-
perthermia, muscle spasms, and neuroleptic syndrome, with formed on all mice, with the following order of least to most
tolerable side effects and occasional liver toxicity at high stressful tests: OPT, EPMT, TST, and FST. Thereafter, all mice
doses41. Dantrolene, with its ability to inhibit the common up- were euthanized, and the blood or brain were harvested for
stream critical Ca2+ dysregulation, is neuroprotective against examination of inflammation and programmed cell death by
many neurodegenerative diseases, including cerebral ische- pyroptosis and synapse destruction in blood and brains.
mia42,43, Huntington’s disease44, spinocerebellar ataxia45, amy-
otrophic lateral sclerosis46, and seizures47,48. Dantrolene ro- Intranasal dantrolene nanoparticle or vehicle administra-
bustly inhibited up to 78% of NMDA-induced elevation of cyto- tion
solic Ca2+ concentration in cerebral cortical neurons 49, suggest- Dantrolene (Sigma, St Louis, MO) was dissolved in the
ing it may have great potential to treat major depression dis- Ryanodex Formulation Vehicle (RFV: 125 mg mannitol, 25 mg
order with a similar molecular mechanism of ketamine50, polysorbate 80, 4mg povidone K12 in 5mL of sterile water and
which needs to be studies urgently. pH adjusted to 10.3), similar to our previous publications52,53.
For intranasal administration, the final concentration of

Liu and Lu et al. 2025 (preprint) 2

 bioRxiv preprint doi: https://doi.org/10.1101/2024.09.06.611461; this version posted March 11, 2025. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Intranasal dantrolene nanoparticles treat depression and anxiety

dantrolene was 5 mg/mL as we have previously described. wall. The locomotor activity was recorded for six minutes with
Mice were held and fixed by the scruff of their necks with one a camera above the apparatus under dim light. The anxiety be-
hand. With the other hand, mice were given a total of havior of each mouse was measured using the total distance
1μL/gram of body weight of dantrolene nanoparticles or RFV. traveled, center entries, immobility time, and time spent in the
Accordingly, a mouse weighing 20 g would be given 20μL of central zone. After each test, the apparatus was cleaned with
solution, which is equal to a dantrolene dose at dose at 5mg/kg. 75% ethanol. Any-maze software (Stoelting USA) was used to
The solution was slowly delivered directly into the mouse’s analyze the collected data. The lesser the central zone distance
nose. Care was taken to ensure that mice were minimally and mean speed, the increased the anxiety behavior. The more
stressed, and that the solution remained in the nasal cavity and immobile time, the more severe the anxiety behavior.
did not enter the stomach or lungs.
Elevated plus maze test (EPMT)
Forced swimming test (FST) Anxiety was also assessed using EPMT, as described pre-
Depression behavior was assessed using the FST, as de- viously, although with some modification57. Mice were placed
scribed previously with some modification54. The mice from in the center of an elevated plus-maze (arms are 33 cm × 5 cm,
each group were settled in a clear glass tank of 25 cm height, with 25 cm tall walls on the closed arms) under dim lighting
10 cm diameter, 15 cm water depth, and (23 ± 2) °C water tem- and their behavior was videotaped for 5 minutes. We used
perature. The test's total period was 6 minutes, 2 minutes for Any-maze software (Stoelting USA) to analyze the collected
adaptation, and the total immobility time was recorded in the data. The time spent in the closed and open arms, as well as
next 4 minutes. Immobility occurred when the mice discontin- the number of explorations of open arms, was measured and
ued floundering on the surface of the water, instead appearing recorded. The more time spent in closed arms, the more se-
as floating in an equilibrium state. The greater the immobility vere the anxiety behavior.
time, the more severe the depression behavior.
Euthanasia and tissue collection
Tail suspension test (TST) Mice were euthanized after the completion of all behav-
Depression behavior was assessed using the tail suspen- ioral tests. As described previously52, mice were deeply anes-
sion test described previously, with modifications55. We use thetized with 2–4% isoflurane delivered through a nose cone,
specially manufactured tail suspension boxes, made of cartons with the concentration was adjusted according to response to
with the dimensions (42 cm height X 18 cm width X 30 cm a toe pinch. The skin of each mouse was prepared, and an in-
depth). To prevent animals from observing or interacting with cision was made to open the chest and expose the heart. Blood
each other, each mouse was suspended within its three-walled was collected from the heart for the serum study using a sy-
rectangular box. Each day, animals were acclimated to the ringe equipped with a 27G needle. The blood was centrifuged
testing room for at least 1 hour before the test. Each mouse at 1400 rpm at 4◦C for 30 min, and the supernatant was col-
was suspended in the middle of the box. The width and depth lected and frozen at –80◦C. The mice were euthanized by trans-
of the box were sufficiently sized to prevent the mouse from cardial perfusion and exsanguination was conducted with cold
contacting the walls. In this setting, the approximate distance phosphate-buffered saline.
between the mouse's nose and the apparatus floor was 20-25
cm. A plastic suspension bar (50 cm. height X 40 cm. depth), Measurements of serum concentration of IL-1β and IL-18
was used to suspend the tail of each mouse and positioned on We measured the serum IL-1β and IL-18 cytokines using
the top of the box. At the bottom of each box, we placed a an ELISA kit, following company instructions, and as we de-
paper towel to collect feces or urine from the animals. A dark scribed previously for measurement of S100β, with some mod-
grey box, for albino animals, and a white colored box, for mice ification58,59. All reagents and samples (the supernatant of the
of other coat colors, were used. Before evaluation, the tail of blood) were thawed to room temperature (18 - 25 °C) before
the mouse was securely adhered to the suspension bar, which use. It is recommended that all standards and samples be run
was able to withstand the mouse’s weight. A video camera was at least in duplicate. Add 100 µL of each standard and sample
placed in position and started recording the TST session. The into appropriate wells. Cover wells and incubate for 2.5 hours
total duration of the test was 6 minutes. The paper towel was at room temperature or overnight at 4 °C with gentle shaking.
replaced after each trial. After all sessions were finished, Any- Discard the solution and wash it 4 times with 1X Wash Solution.
maze software (Stoelting, USA) was used to analyze the col- Wash by filling each well with Wash Buffer (300 µL) using a
lected data. multi-channel Pipette or auto washer. Complete removal of
During the behavioral analysis, the mobility time of each liquid at each step is essential to reliable performance. After
mouse was measured and subtracted from 360 seconds, pro- the last wash, remove any remaining Wash Buffer by aspirating
ducing the immobility time. The investigator was blinded to or decanting. Invert the plate and blot it against clean paper
the animal groups. The greater immobility time, the more se- towels. Add 100 µL of 1x prepared Detection Antibody to each
vere the depressive behavior. well. Cover wells and incubate for 1 hour at room temperature
with gentle shaking. Discard the solution. Repeat the wash pro-
Open field test (OFT) cedure as in step 3. Add 100 µL of prepared Streptavidin solu-
The OFT was performed as previously described to evaluate tion to each well. Cover wells and incubate for 45 minutes at
anxiety behavior as described56. Each mouse was placed indi- room temperature with gentle shaking. Discard the solution.
vidually into the OFT apparatus (44 × 44 × 44 cm3), facing the Repeat the wash as in step 3. Add 100 µL of TMB One-Step

Liu and Lu et al. 2025 (preprint) 3

 bioRxiv preprint doi: https://doi.org/10.1101/2024.09.06.611461; this version posted March 11, 2025. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Intranasal dantrolene nanoparticles treat depression and anxiety

Substrate Reagent (Item H) to each well. Cover wells and incu- (186.6 vs. 101.5). In contrast, the intranasal dantrolene nano-
bate for 30 minutes at room temperature in the dark with gen- particles vehicle control did not demonstrate same inhibitory
tle shaking. Add 50 µL of Stop Solution (Item I) to each well. effects on helplessness behaviors. The elevated plus maze test
Read absorbance at 450 nm immediately. Mouse IL-1 beta (EPMT) and open field test (OPT) are commonly used to evalu-
ELISA Kit and Mouse IL18 ELISA Kit come from the company ate anxiety related behaviors in mice and test drug efficacy to
SIGMA(RAB0275-1KT/RAB08100-1KT). treat anxiety behaviors27,63,64. In both EPMT (Fig. 1C) and OFT
(Fig. 1D), LPS induced significantly increased time stay in closed
Immunoblotting (Western Blot) arm in EPMT (anxiety behaviors) by 46% (162 vs. 237) and im-
As we described previously60, total brain tissues were ex- mobile time in OPF by 130% (82 vs. 189) respectively. Intrana-
tracted by homogenization using cold RIPA buffer (#9806S, Cell sal dantrolene nanoparticle pretreatment robustly inhibited
Technology, USA) supplemented with protease inhibitor cock- the increased time stay in closed arm in EPMT by 29% (237 vs.
tails (P8340 Roche). The brain homogenates were rocked at 169) and immobile time in OPT by 40% (189 vs. 114). In con-
4°C for 90 minutes and then centrifuged at 14,000 rpm (Brush- trast, the intranasal dantrolene nanoparticles vehicle control
less Microcentrifuge, Denville 260D) for 20 minutes at 4°C to did not demonstrate same inhibitory effects on depression be-
remove cell debris. After collecting the supernatant, the pro- haviors.
tein concentration was measured using a BCA protein assay kit In the open field test, the less central zone distance mice
(Pierce, Rockford, IL 61101 USA). Briefly, equal amounts of pro- traveled and less moving speed, the more severe the anxiety
tein (20µg/lane) were loaded onto 4-20% gel electrophoresis behavior (supplemental figure 2). Compared to control with-
of mini-protein TGX precast (Cat. #4561094, BIO-RAD) and out treatment, one-time LPS treatment significantly decreased
transferred to polyvinylidene difluoride (PVDF) membranes mean speed (anxiety behavior) but not traveled zone distance.
(Immobilon-P, MERK Millipore, Ireland) using wet electro- Intranasal dantrolene nanoparticles trended to inhibit LPS in-
transfer system (BIO-RAD, USA). Following the transfer mem- duced decrease of travel zone distance and mean speed (Sup-
brane blocking with 5% BSA (Sigma-Aldrich) for 1 hour, then plemental figure 2).
the PVDF membrane was incubated with primary antibodies
overnight at 4 °C including NLRP3, cleaved caspase-1, GSDMD-
NT, IL-1β, IL-18, PSD95, Synapsin-1, GAPDH (Table 1). Then, the
membranes were incubated with a secondary antibody includ-
ing anti-Mouse IgG1 HRP-linked; Anti-rabbit IgG, HRP-linked
that was conjugated to horseradish peroxidase and washed
with Tris-buffered saline containing 0.2% Tween-20 (TBST).
Following that, TBST was used for washing the membranes
three time for 10 minutes. After incubation with secondary an-
tibodies, protein bands were visualized using ECL Western
Blotting Detection Reagents (Cytiva, amersham, UK) and quan-
tified for band intensity using ImageJ software (National Insti-
tutes of health, Bethesda, MD, USA), by two persons blinded
to treatments and averaged for each mouse.

Statistical Analysis
All data were represented as mean ± standard deviations
(Means±SD). Statistical analyses were employed with
GraphPad Prism (Version 9.3.1, CA, USA). Comparisons of more
than two groups were conducted by one-way ANOVA with
Tukey’s multiple comparison test. The N values in each group
represent the number of mice. P<0.05 was considered statisti-
cally significant.
Results
Intranasal dantrolene nanoparticle pretreatment signifi-
cantly inhibited LPS-induced helplessness and anxiety be-
haviors Figure 1. Intranasal dantrolene nanoparticles significantly reduced
The FST and TST tests are commonly used to evaluate LPS-induced acute helplessness and anxiety-related behaviors.
helplessness or depression behaviors in mice and to test drug’s Adult B6SJLF1/J mice were treated with intranasal dantrolene nanopar-
ticles (IN-DAN) or vehicle (VEH) for 28 days, followed by a single i.p.
efficacy versus side effects27,61,62. In both FST (Fig. 1A) and TST injection of lipopolysaccharide (LPS) (IN-DAN+LPS). Control mice ei-
(Fig. 1B), LPS induced significantly increased immobile times ther had no treatment (No Tx), only LPS (LPS), or VEH+LPS. The
(helplessness or depression behaviors) by 89% (60.1 vs. 113.3) forced swim test (FST, A), tail suspension test (TST, B), elevated plus
maze (EPMT, C) and open field test (OFT, D) were performed 24 hours
and 90% (98.1 vs. 186.6) respectively. Intranasal dantrolene
after the LPS injection. Increased mobility time indicates helplessness
nanoparticle pretreatment robustly inhibited the increased or anxiety-related behaviors (A, B, D). Increased time in the closed arm
immobile time in FST by 41% (113.3 vs. 66.9) and in TST by 46% indicates anxiety (C). N=18-19 mice, Mean±95%CI, One-Way ANOVA
followed by Tukey post hoc test. *P<0.05, **P<0.01, ****P<0.0001

Liu and Lu et al. 2025 (preprint) 4

 bioRxiv preprint doi: https://doi.org/10.1101/2024.09.06.611461; this version posted March 11, 2025. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Intranasal dantrolene nanoparticles treat depression and anxiety

Intranasal dantrolene nanoparticle pretreatment signifi-

cantly inhibited LPS-induced helplessness and anxiety be-
haviors primarily in female mice.
We further analyze the effects of sex on neuroprotective
effects of intranasal dantrolene nanoparticles on helplessness
and anxiety behavior. As demonstrated in Fig. 2, intranasal
dantrolene nanoparticles but not its vehicle control primarily
inhibited helpless behavior determined by both FST (Fig. 2A)
and TST (Fig. 2B) in female but not male mice. For anxiety
behavior, although intranasal dantrolene nanoparticle but not
vehicle control inhibited LPS induced anxiety behavior
determined by EPMT (Fig. 2C) in male mice, the inhibitory
effects of dantrolene on anxiety behaviors were most
significant in female mice determined by both EPMT (Fig. 2C)
and OFT (Fig. 2D).

Figure 3. Intranasal Dantrolene Significantly Inhibited lipopoly-

.saccharide-induced pathological elevation of IL-18 and IL-1β cy-
tokines in the blood and brains. B6SJLF1/J adult mice were pre-
Figure 2. Intranasal dantrolene nanoparticles reduced LPS-induced treated with intranasal dantrolene nanoparticles DAN, 5mg/kg) or ve-
acute helplessness and anxiety-related behaviors more in female hicle (Veh) control daily, Monday to Friday, for continuous 4 weeks.
than male mice. The sex of the adult B6SJLF1/J mice, pretreated with Mice were then treated with one-time IP injection of lipopolysaccharide
intranasal dantrolene nanoparticles (IN-DAN) or vehicle (VEH) for 28 (LPS, 5 mg/kg). Mice in control (No Tx) group received no treatments.
days, followed by a single i.p. injection of LPS (IN-DAN+LPS), was exam- IL1β in blood (A) and brain (C, E) and IL18 in blood (B) and brains (D,
ined. No treatment controls (No Tx), LPS only (LPS), or VEH+LPS. The F) were measured using ELISA assay kit (A, B) or immunoblotting (C-
forced swimming test (FST, A), tail suspension test (TST, B), elevated F)., N=18-20 mice for blood measurement (A, B) and N=4 brains for
plus maze (EPMT, C), and open field test (OFT, D) were performed 24 immunoblotting (C-F), Means±95% CI, One-Way ANOVA followed by
hours after the LPS injection. N=8-10 mice, Mean±95%CI, Two-Way Tukey post hoc test. **P<0.01, ****P<0.0001 respectively.
ANOVA followed by Tukey post hoc test. *P<0.05,**P<0.01, ****P<0.0001

Intranasal dantrolene nanoparticles inhibited LPS-induced

Intranasal dantrolene nanoparticles significantly inhibited programmed cell death by pyroptosis in mice brains.
LPS-induced pathological elevation of pyroptosis-related
Compared to control mice without treatment, LPS
inflammation cytokines in blood and brains.
treatment increased the proteins levels of pyroptosis pathway
Compared to control without treatment, IP injection of LPS
(5mg/kg) for one time significantly increased blood activation (NLRP3, caspase-1 and N terminal GSDMD) in mice
concentrations of IL-1β by 147% (Fig. 3A, 136 vs. 336) and IL- brains. Intranasal dantrolene nanoparticles significantly
18 by 67% (Fig. 3B, 233 vs. 390). Intranasal dantrolene inhibited increase of Caspase-1 (P20), a biomarker of
programmed cell death by pyroptosis (Fig. 4 C, D,
nanoparticles but not its vehicle control significantly inhibited
supplemental figure 6), and trended to inhibit LPS-induced
the increased IL-1β by 46% (336 vs. 183) and elevated IL-18 by
23% (Fig. 3B, 390 vs. 299). Furthermore, intranasal dantrolene elevation of pyroptosis pathway activation proteins (Figure 4,
nanoparticles significantly inhibited LPS-induced elevation of A and B, supplemental figure 5, NLRP-3 and Fig. 4 E and F,
IL-1β and IL-18 proteins in both blood (Fig. 3 A, B) and in brains supplemental figure 7, N terminal GSDMD (GSDMD-NT))
(Fig. 3 C, D, supplemental figure 3 and 4).

Liu and Lu et al. 2025 (preprint) 5

 bioRxiv preprint doi: https://doi.org/10.1101/2024.09.06.611461; this version posted March 11, 2025. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Intranasal dantrolene nanoparticles treat depression and anxiety

Figure 5. Intranasal dantrolene nanoparticles significantly inhib-

ited lipopolysaccharide-induced synapse protein loss in brains.
B6SJLF1/J adult mice were pretreated with intranasal dantrolene na-
noparticles (Dan, 5mg/kg) or vehicle (Veh) daily, Monday to Friday, for
continuous 4 weeks. Mice were then treated with one-time IP injection
of lipopolysaccharide (LPS, 5 mg/kg). Mice in control (No Tx) group
received no treatments. Western blot measured changes of synapse
proteins of PSD-95 (A, C) and Synapsin-1 (B, D) in the brains. GAPDH
as loading control. N=4 different mice in each group, Means±95%CI,
One-Way ANOVA followed by Tukey post hoc test. *P<0.05, **P<0.01,
***P<0.001, P<0.0001 respectively.

Discussion
Figure 4. Effects of intranasal dantrolene nanoparticles on lipo- There is an urgent need for new therapeutic interventions
polysaccharide-induced programed cell death by pyroptosis in to prevent or treat MDD3,65 and anxiety5 psychiatric disorders,
the brains. B6SJLF1/J adult mice (5-10 months old) were pretreated
with intranasal dantrolene nanoparticles (Dan, 5mg/kg) or vehicle especially those drug-resistant and recurrent depression and
anxiety5,16,66. This study has demonstrated that intranasal
(Veh) daily, Monday to Friday, for continuous 4 weeks. Mice were then
treated with one-time IP injection of lipopolysaccharide (LPS, 5 dantrolene nanoparticles are capable of ameliorating
mg/kg). Mice in control (No Tx) group received no treatments. Repre-
helplessness and anxiety behaviors in adult mice, associated
sentative western blot (A, C, E) and statistical analysis (B, D, F) deter-
with its ability to inhibit pathological elevation of pyroptosis
mined the protein levels of critical regulatory proteins of pyroptosis
related cytokines (IL-1β and IL-18) in blood, pyroptosis
pathway (A, B, NLRP3; C, D, Active caspase-1 (P-20), E, F, N terminal
GSDMD (GSDMD-NT) in brains. N=4 different mice brain in each biomarker caspase-1 (P20) and synapse proteins loss in brains.
group. GAPDH as loading control. Means±95%CI, One-Way ANOVA
followed by Tukey post hoc test. * P<0.05.
Considering that upstream Ca2+ dysregulation plays an
important role in the pathology of depression and anxiety,
evidenced by the effectiveness of ketamine and esketamine in
Intranasal dantrolene nanoparticles significantly inhibited treating depression and anxiety resistant to traditional drug
LPS-induced synapse protein loss in mice brains. treatments14,16, a new class of drugs, such as dantrolene, could
Compared to control without treatment, LPS significantly be developed with a mechanism to correct the disruption of
decreased the synapse proteins of PSD-95 by 45% (1.322 vs. upstream Ca2+ dysregulation and associated downstream
0.726, Fig. 5, A, C, supplemental figure 8) and synapsin-1 by 37% pathological inflammation and pyroptosis in psychiatric
(0.925 vs. 0.579, Fig. 5, B, D, supplemental figure 9) in mice disorders.
brains. Intranasal dantrolene nanoparticles but not vehicle
control pretreatment for 4 weeks significantly inhibited the Increasing studies suggests that inflammation is an
pathologically reduction of synapse proteins of PSD-95 by 77% important pathology causing MDD and anxiety and could be a
(0.726 vs. 1.286, Fig. 5 A, C, supplemental figure 8) and target for effective treatments67-70. Intracellular Ca2+
synpasin-1 by 76% (0.579 vs. 1.021, Fig. 5 B and D, dysregulation has been proposed as the upstream cause of
supplemental figure 9). downstream mitochondria dysfunction, oxidative stress, and
inflammation in MDD25,71,72 and anxiety38,73. Inhibition of
upstream Ca2+ dysregulation through inhibiting over-activation
of RyRs has been shown to suppress inflammation in Gulf War
illness characterized by depression and dementia28. Although
dantrolene has been shown to inhibit pathological
inflammation with increased cytokines in diabetes74,75,

Liu and Lu et al. 2025 (preprint) 6

 bioRxiv preprint doi: https://doi.org/10.1101/2024.09.06.611461; this version posted March 11, 2025. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Intranasal dantrolene nanoparticles treat depression and anxiety

sepsis76,77 and in COVID-19 infection78,79, its effects to inhibit dantrolene nanoparticles suggested that dantrolene may
Ca2+ dysregulation and associated inflammation for the inhibit critical pathology and etiology contributing depression
treatment of depression and anxiety behaviors has not yet and anxiety behaviors. Although the exact mechanism
been investigated until now and studied herewith. unclears, we propose that dantrolene inhibited the upper
stream critical Ca2+ dysregulation and associated inflammation
Several animal models to establish depression and and associated programmed neuron death by pyroptosis and
anxiety behaviors have been suggested based on the proposed synapse protein loss.
neurobiology mechanisms, including altered
neurotransmission, HPA axis abnormalities involved in chronic Intranasal administration of drugs, especially in a
stress, inflammation, reduced neuroplasticity, and network nanoparticle formulation, significantly promotes drugs that
dysfunction80-82. Induction of inflammation in animals has been bypass the blood-brain barrier (BBB) and penetrate the CNS,
increasingly used to establish depression and anxiety animal with reduced peripheral toxicity94-96. Our recent research work
models, especially using LPS to induce inflammation, indicated that intranasal dantrolene nanoparticles achieved
depression, and anxiety behaviors to test drug treatment higher therapeutic efficacy in the brain compared to oral and
efficacy37,71,83-86. The common approach of LPS administration subcutaneous forms administration, with minimal or no side
is intraperitoneal injection because of its easiness and effects after chronic use52,53,97. Intranasal dantrolene
feasibility. The dose and duration of IP injection of LPS to nanoparticles significantly ameliorated memory loss in adult
establish depression or anxiety behaviors has been varied but 5XFAD mice as a disease-modifying drug52. Increasing studies
appears to be dose dependent. The high dose of LPS at 5 mg/kg, suggest ryanodine receptor overactivation and associated Ca2+
administered once, seems to be dependable in inducing dysregulation is an upstream critical pathology leading to
inflammation and establishing depression behavior71. Our multiple downstream pathologies including mitochondria
study confirmed the efficiency of a one-time administration of dysfunction98, oxidative stresses99, pathological
IP LPS (5 mg/kg) to induce inflammation and depression and inflammation , and neuron damage by pyroptosis101.
100

anxiety behavior in adult mice consistently. Accordingly, the Eventually, these pathologies result in depression and/or
LPS-induced inflammation-mediated depression behaviors are anxiety psychiatric disorders34,70,102. This study demonstrated
adequate to evaluate the therapeutic effects of dantrolene to that inhibition of RyRs overactivation by dantrolene inhibited
treat depression. Our results demonstrated robust inhibition pathological inflammation and synapse proteins destruction
of LPS-induced inflammation and associated helplessness and and ameliorates depression and anxiety behavior robustly.
anxiety behaviors by intranasal dantrolene nanoparticles This study further strengthens the indication that upstream
pretreatment in adult mice. In addition with the protective RyRs overactivation and Ca2+ dysregulation and associated
effects of dantrolene against memory loss in various AD animal downstream pathological inflammation and synapse
models52,87-89, intranasal dantrolene nanoparticles also destruction play important roles in depression and anxiety
significantly inhibited helplessness and anxiety behaviors, and psychiatric disorders. Like esketamine22,103, the intranasal
associated inflammation and pyroptosis, suggesting that dantrolene nanoparticles could become an effective disease-
pathological inflammation and pyroptosis play a critical role in modifying drug treatment for both depression and anxiety
both cognitive dysfunction and psychiatric disorders. Drugs psychiatric disorders and need to be investigated in future
that target inflammation pathology should be developed to clinical studies.
treat both memory loss and psychiatric disorders in AD
patients. Nevertheless, intranasal dantrolene nanoparticles Since depression and anxiety psychiatric disorders are
significantly inhibited LPS-induced pathological elevation of chronic diseases, they require long-term drug treatments.
pyroptosis related inflammation cytokines (IL-1β and IL-18) in Accordingly, proposed drugs must limit side effects or organ
blood, and ameliorated both depression and anxiety behavior toxicity with chronic treatment. The major advantage of using
in adult mice, suggesting that intranasal dantrolene intranasal dantrolene nanoparticles, in comparison to
nanoparticles could be developed as an effective drug to treat commonly used oral or intravenous approaches, is that it
depression and/or anxiety in major depression disorder (MDD) significantly increases the brain/blood concentration ratio of
patients potentially by its inhibition of inflammation and dantrolene52,53, especially in aged mice97. This promotes its
associated programmed cell death by pyroptosis. CNS therapeutic effects, while minimizing its side effects or
organ toxicity52. Our previous study demonstrated no side
MDD affect female twice more than male patients90,91. effects on nose structure and smell function52,104, liver
The proposed mechanisms for this sex difference include the structure52 and function, or muscle function51,52,87 after up to
effects of estrogen92, serotonin and tryptophan metablism93, 10 months of chronic treatment in adult 5XFAD mice52,
different reactions to stresses91 etc. An important finding in suggesting that intranasal dantrolene nanoparticles are safe to
this study is that intranasal dantrolene nanoparticles primarily be used chronically in animals. Chronic use of dantrolene in
be effective to ameliorate helplessness and anxiety behaviors patients’ needs to be investigated further.
primarily in female than in male mice. Our result is consistent
with MDD prevalence, more in female than male mice. Also, This study has the following limitations: 1). The mice age
the more significant inhibition of inflammation associated is from 5 to 10 months old. Although they are considered adult
helplessness and anxiety behavior in female mice by intranasal mice, future studies need to focus on different age group,

Liu and Lu et al. 2025 (preprint) 7

 bioRxiv preprint doi: https://doi.org/10.1101/2024.09.06.611461; this version posted March 11, 2025. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Intranasal dantrolene nanoparticles treat depression and anxiety

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 bioRxiv preprint doi: https://doi.org/10.1101/2024.09.06.611461; this version posted March 11, 2025. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Intranasal dantrolene nanoparticles treat depression and anxiety

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mood disorders: at the intersection of adrenal and gonadal hormones. This work was supported by grants to HW from the National Institute on
Horm Metab Res 2012; 44: 607-18 Aging (R01AG061447) and NIA R01 Supplemental (3R01AG061447-03S1). The
92. Iqbal J, Huang GD, Xue YX, Yang M, Jia XJ: Role of estrogen in sex differ- research was performed in the lab of Dr. Huafeng Wei and should be attributed
ences in memory, emotion and neuropsychiatric disorders. Mol Biol Rep to the Department of Anesthesiology, University of Pennsylvania. We appreci-
2024; 51: 415 ate the technical support from Rebecca Chae from Rowan University, New Jer-
93. Songtachalert T, Roomruangwong C, Carvalho AF, Bourin M, Maes M: sey, U.S.A and Kyulee Kim from UPENN, Pennsylvania, U.S.A.
Anxiety Disorders: Sex Differences in Serotonin and Tryptophan Metab-
olism. Curr Top Med Chem 2018; 18: 1704-1715 Author contributions
H.W. conceived and designed the study. J.L., Y.L, P.B. J.G, L.S.L, Y.Y., G.L,
H.W. conducted experiments, acquired and the data, J.L., Y.L, P.B. J.G, L.S.L,

Liu and Lu et al. 2025 (preprint) 10

 bioRxiv preprint doi: https://doi.org/10.1101/2024.09.06.611461; this version posted March 11, 2025. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Intranasal dantrolene nanoparticles treat depression and anxiety

Y.Y., G.L, H.W. analyzed data and contributed to the manuscript preparation. Alzheimer’s Disease” in multiple countries by The Trustees of the University of
H.W. wrote the manuscript. All the authors reviewed and approved the final Pennsylvania.
manuscript.

Competing interest statement

Drs. Huafeng Wei and Ge Liang are listed as inventors of patent applica-
tions entitled “Intranasal Administration of Dantrolene for Treatment of

Table 1

Cat. and Manufacture Dilution

Primary antibody
Anti- NLRP3 68102-1-Ig; Proteintech 1:2000
Anti-Human procaspase-1/ cleaved #2225S, Cell signaling Technol- 1:1000
caspase-1 p20 ogy,USA
Anti- Cleaved GSDMD #36425S; Cell signaling Technology, 1:1000
USA
Anti-IL-1β 31202S, Cell signaling Technology, 1:1000
USA
Anti-IL-18 10663-1-AP; Proteintech 1:3000
Anti-PSD95 #2507; Cell signaling Technology, 1:1000
USA
Anti-Synapsin-1 #5297; Cell signaling Technology, 1:1000
USA
GAPDH MA5–15738, Thermo Fisher Scien- 1:10,000
tific
Secondary antibody
Anti-Mouse IgG1; HRP -linked # PA1-74421, Thermo Fisher Scien- 1:10,000
tific
Anti-rabbit IgG, HRP-linked #7074; Cell signaling Technology, 1:1000
USA

Liu and Lu et al. 2025 (preprint) 11

 bioRxiv preprint doi: https://doi.org/10.1101/2024.09.06.611461; this version posted March 11, 2025. The copyright holder for this preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Intranasal dantrolene nanoparticles treat depression and anxiety

Supplemental figure 1. Experimental Design. Adult B6SJLF1/J mice were pretreated with intranasal dantrolene nanoparticles in Ryanodex
formulation (DNRF, 5mg/kg) or vehicle (Veh) for 4 weeks. Control group received no treatments. Mice were then treated with intraperitoneal (IP)
injection of lipopolysaccharide (LPS, 5mg/kg) for one time. Depression and anxiety behavior tests were performed at 24 hours after one-time IP
LPS injection. PreTX: Pretreatment, Tx: Treatment, IN: Intranasal, Dan: Dantrolene, OFT: Open Field Test, EPMT: Elevated Plus Maze Test, TST:
Tail Suspension Test, FST: Forced Swimming Test

Supplemental figure 2. Effects of intranasal dantrolene nanoparticles on lipopolysaccharide-induced anxiety behavior in adult mice.
B6SJLF1/J adult mice (5-10 months old) were pretreated with intranasal dantrolene nanoparticles (Dan, 5mg/kg) or vehicle (Veh) control daily,
Monday to Friday, for continuous 4 weeks. Mice were then treated with one-time IP injection of lipopolysaccharide (LPS, 5 mg/kg). Mice in control
(No Tx) group received no treatments. Open field test (OFT) was performed at 24 hours after one-time IP LPS injection. The less central zone
distance or less mean speed, the more anxiety behavior for OFT. N=18-20 mice. Means±95%CI, One-Way ANOVA followed by Tukey post hoc
test. ***P<0.001.

Liu and Lu et al. 2025 (preprint) 12