Sex Differences Revealed in a Mouse CFA Inflammation Model with

Macrophage Targeted Nanotheranostics

Lu Liu1,2, Huseyin Karagoz3, Michele Herneisey1,2, Fatih Zor3, Takaaki Komatsu4, Shannon

Loftus1,2, Bratislav M. Janjic,5 Vijay S. Gorantla3*, Jelena M. Janjic1,2,*

1 Graduate School of Pharmaceutical Sciences, School of Pharmacy, Duquesne University,

Pittsburgh, PA, USA

2 Chronic Pain Research Consortium, Duquesne University, Pittsburgh, PA, US

3 Department of Surgery, Institute of Regenerative Medicine, Wake Forest University School of

Medicine, Winston-Salem, North Carolina, United States

4 Department of Pharmacology, Daiichi University of Pharmacy, Fukuoka, Japan

5 NRG Oncology Foundation Inc., University of Pittsburgh, Pittsburgh PA, United States

CO-CORRESPONDING AUTHORS:

*Jelena M. Janjic, PhD

Graduate School of Pharmaceutical Sciences,

Duquesne University

600 Forbes Avenue, 415 Mellon Hall

Pittsburgh, PA 15282, USA

412-396-6369 (Phone)

janjicj@duq.edu (Email)

*Vijay S. Gorantla, MD, PhD, MMM

Department of Surgery

Institute of Regenerative Medicine, Wake Forest University School of Medicine

391 Technology Way

Suite 333 Richard H. Dean Biomedical Building

Winston- Salem, NC 27101, USA

336-713-1494 (Phone)

vgorantl@wakehealth.edu (Email)

Keywords:

Pain nanomedicine, macrophages, sex differences, inflammatory pain, nanotheranostic

Abstract

Monocyte derived macrophages (MDMs) infiltrate sites of infection or injury and

upregulate cyclooxygenase-2 (COX-2), an enzyme that stimulates prostaglandin-E2 (PgE2).

Nanotheranostics combine therapeutic and diagnostic agents into a single nanosystem. In previous

studies, we demonstrated that a nanotheranostic strategy, based on theranostic nanoemulsions (NE)

loaded with a COX-2 inhibitor (celecoxib, CXB) and equipped with near-infrared fluorescent

(NIRF) reporters, can specifically target circulating monocytes and MDMs. The anti-inflammatory

and anti-nociceptive effects of such cell-specific COX-2 inhibition lasted several days following

Complete Freund’s Adjuvant (CFA) or nerve injury in male mice. The overall goal of this study

was to investigate the extended (up to 40 days) impact of MDM-targeted COX-2 inhibition and

any sex-based differences in treatment response; both of which remain unknown. Our study also

evaluates the feasibility and efficacy of a preclinical nanotheranostic strategy for mechanistic

investigation of the impact of such sex differences on clinical outcomes.

Methods: CFA was administered into the right hind paws of male and female mice. All

mice received a single intravenous dose of NIRF labeled CXB loaded NE twelve hours prior to

CFA injection. In vivo whole body NIRF imaging and mechanical hypersensitivity assays were

performed sequentially and ex vivo NIRF imaging and immunohistopathology of foot pad tissues

were performed at the end point of 40 days.

Results: Targeted COX-2 inhibition of MDMs in male and female mice successfully

improved mechanical hypersensitivity after CFA injury. However, we observed distinct sex-

specific differences in the intensity or longevity of the nociceptive responses. In males, a single

dose of CXB-NE administered via tail vein injection produced significant improved mechanical

hypersensitivity for 32 days as compared to the drug free NE (DF-NE) (untreated) control group.

In females, CXB-NE produced similar, though less prominent and shorter-lived effects, lasting up

to 11 days. NIRF imaging confirmed that CXB-NE can be detected up to day 40 in the CFA

injected foot pad tissues of both sexes. There were distinct signal distribution trends between males

and females, suggesting differences in macrophage infiltration dynamics between the sexes. This

may also relate to differences in macrophage turnover rate between the sexes, a possibility that

requires further investigation in this model.

Conclusions: For the first time, this study provides unique insight into MDM dynamics

and the early as well as longer-term targeted effects and efficacy of a clinically translatable

nanotheranostic agent on MDM mediated inflammation. Our data supports the potential of

nanotheranostics as presented in elucidating the kinetics, dynamics and sex-based differences in

the adaptive or innate immune responses to inflammatory triggers. Taken together, our study

findings lead us closer to true personalized, sex-specific pain nanomedicine for a wide range of

inflammatory diseases.

Introduction

Monocyte-derived macrophages (MDMs) and other immune cells infiltrate sites of injury and

produce numerous pro-inflammatory mediators (chemokines, cytokines, prostaglandins) that can

sensitize neurons and induce pain [1]. Macrophages, as drivers of inflammation, are implicated in

many diseases that lead to chronic pain, from rheumatoid arthritis (RA) [2, 3], osteoarthritis

(OA)[4], and inflammatory bowel disease (IBD) [5] to trauma [6]. In RA and OA, MDMs

upregulate cytokine production and trigger T cell proliferation, which results in joint destruction

and pain sensitization [3, 7].

An enzyme of particular interest in macrophage-driven inflammation and associated pain is

cyclooxygenase-2 (COX-2), the primary enzyme driving the production of prostaglandin-E2

(PgE2) [8-10]. In fact, macrophages expressing high levels of COX-2 have been shown to

exacerbate the inflammation driven pathology in osteoarthritis [11]. Nonsteroidal anti-

inflammatory drugs (NSAIDs) have been the standard of care alternatives to opioids to treat pain

and inflammation in RA, OA, and many other diseases [12]. NSAIDs such as celecoxib (CXB)

are potent inhibitors of COX-2 in immune cells (such as macrophages), which are critical drivers

of the innate or adaptive responses to inflammation.

The importance of COX-2 in macrophage mediated inflammatory pain is supported by studies

using macrophage COX-2 knockout mice. These studies showed reduced hyperalgesia post-CFA

injection compared to wild type mice and reduced response to pretreatment with celecoxib, a COX-

2 inhibitor [13]. COX-2 knockout mice also fail to develop collagen-induced arthritis (CIA)

compared to equally susceptible wild type and COX-1 knockout mice [14]. Recent studies have

shown that sex differences exist in COX-2 activity. Female COX-2 knockout mice exhibited

reduced edema compared to wild type mice when subjected to complete Freund’s adjuvant (CFA)

injection, while male COX-2 knockout mice showed no significant reduction in edema [15]. Sex

differences are also seen in PgE2 production; macrophages from female mice subjected to trauma

produced significantly more PgE2 compared to male mice experiencing the same trauma [16].

Additionally, there are sex differences in macrophage activity, including higher activation and

phagocytic activity in females [6].

These studies and our earlier work [10, 17] confirm that the macrophage enzyme COX-2 is a

critical mediator of pain in inflammatory disease, making it an attractive therapeutic target.

Nevertheless, the mechanisms underlying sex differences in clinical outcomes after COX-2

inhibition [18] are still poorly understood. We posit that the primary reason for this lies in

variability in target cell effects following systemic dosing (oral or parenteral) of the drug in

experimental models. One approach to this problem is delivery of NSAIDs directly to the immune

cell mediators of inflammation, such as macrophages, using nanotheranostics. Nanotheranostics

provide temporally and spatially controlled drug delivery to macrophages for prolonged

intracellular drug exposure and simultaneous, longitudinal imaging of these cells to monitor for

drug efficacy and potentially toxicity.

Here, we propose a novel nanotheranostic approach that could help provide preliminary

insights into mechanistic elucidation of sex-intrinsic differences in macrophage migration

dynamics and trafficking patterns to sites of inflammation, which can lead to advances in sex-

specific, personalized pain nanomedicine. Our group has recently developed such a

nanotheranostic approach, where COX-2 inhibitors were directly delivered to macrophages for

both control and imaging of inflammation in multiple animal models using complex fluorescently

labeled perfluorocarbon (PFC) nanoemulsions (NEs) as nanotheranostic formulations [17, 19, 20].

Macrophages as nanotheranostic targets have been investigated recently in rodent models of

inflammatory diseases and cancer [21-23]. In contrast to these studies, presented work for the first

time utilizes nanotheranostics in the context of identifying sex differences in inflammatory

diseases by directly targeting macrophages. Furthermore, we also show that nanotheranostic

formulations as presented can provide extended pain relief, bringing us closer to future

personalized pain nanomedicine.

Nanotheranostics internalized by MDMs provide a unique cell specific signature for in vivo

tracking using near-infrared fluorescence (NIRF), positron emission tomography (PET), or

magnetic resonance imaging (MRI). NEs are small-size (<500 nm) oil-in-water emulsion droplets

that are ideally suited for nanotheranostics, due to their surface-area-to-volume ratio that allows

for effective drug loading, sustained release, and the ability to functionalize the surface with

targeting ligands and imaging moieties (targeting agents, metal chelates, dyes, etc.) [24].

Therefore theranostic NEs can serve as unique tools for studying the effects of NSAIDs on

macrophage function in vivo and ex vivo. In the presented work, we used our established COX-2

inhibiting macrophage targeting nanotheranostic technology [17, 19] to investigate the effects of

extended COX-2 inhibition in mouse models of inflammatory pain. Extensive analyses were

performed in both males and females.

Presented study builds upon our earlier findings which demonstrated that NIRF-labeled

celecoxib NEs (CXB-NEs) injected 12 hours prior to inflammatory insult are taken up by MDMs

and found to associate specifically with COX-2-expressing macrophages in inflamed tissues [19].

Furthermore, COX-2 inhibition in macrophages via CXB-NE caused changes in macrophage

infiltration patterns as compared to drug-free NE (DF-NE) treated animals [19]. In an experimental

neuronal injury model, we also found that CXB-NE treatment decreased macrophage infiltration

at the site of injury and caused dramatic reduction in mechanical hypersensitivity [17]. In these

studies, only male animals, mice [19] and rats[17], were investigated, and we monitored NE-

labeled macrophages for a limited time (4 days) post-injury and treatment.

To the best of our knowledge, this is the first study to show that a single dose of CXB delivered

as an anti-inflammatory nanotheranostic formulation directly to macrophages can provide

extended pain relief as well as allow elucidation of sex differences in pain behavior and

inflammatory pain responses.

Materials and Methods

Animals, male and female C57BL/6 mice (2-3 month old), strain code: 027 were purchased

from Charles River Laboratories for imaging studies performed at Duquesne University under an

approved IACUC protocol. Pathogen-free adult male and female C57BL/6 mice (CLEA, Tokyo,

Japan) were used in the von Frey behavioral study. The experiments were performed with the

approval of the committee of Animal Experiments in Daiichi College of Pharmaceutical Sciences,

and complied with the recommendation of the International Association for the Study of Pain [25].

The mice (18-25g) were maintained in a controlled 12 hour light-dark cycle with food and water

ad libitum. Animals were used only once. CXB-NE and DF-NE carrying a NIRF reporter such as

DiR'; DiIC18(7) (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide) from

Invitrogen P12731 were manufactured following earlier established protocols [17, 26]. Complete

Freund’s Adjuvant (CFA), Inject, Thermo Scientific Product No. 77140.

Preparation of NEs

The NEs used in this study (celecoxib loaded, CXB-NE and drug-free, DF-NE) were produced

following earlier reported protocols in Janjic et al [17, 19]. Briefly, NEs were prepared by

incorporating the near-infrared fluorescent dye, DiR, DiIC18(7) (1,1'-Dioctadecyl-3,3,3',3'-

Tetramethylindotricarbocyanine Iodide) (5 mM) with/without celecoxib (2.5 mg/ml) into a

hydrocarbon oil. After adding perfluorocarbon and surfactants, the mixture was processed on a

microfluidizer (Microfluidics, M110S) following earlier established methods [17].

Colloidal characterization

Dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, UK) was used to measure

droplet size and zeta potential at 25 °C. NE was dispersed in deionized water at a 1:80 dilution.

Size and zeta potential were recorded on the same sample in a cuvette or standard zeta potential

cell, respectively. Each sample was analyzed three times with at least 12 runs each. Example

measurements for DF-NE and CXB-NE size distribution and zeta potential are shown in Figure

S1A-D.

Enzyme-Linked Immunosorbent Assay

RAW 264.7 murine macrophages (ATCC) were plated at 300,000 cells/2ml/well in 6 well

plates for 24 hours. Cells were then exposed to the indicated concentrations of NE or drug

dispersed in full media for 24 hours. The cells were then washed and exposed to serum-free media

with or without 500 ng/ml LPS. After 18 hours, the cell culture supernatant was collected and

centrifuged at 2000 rpm 4°C for two minutes to remove any cell debris. PgE2 was measured in

these samples using the Prostaglandin E2 ELISA Kit – Monoclonal (Cayman Chemical, Ann

Arbor USA) according to the manufacturer’s instructions. CXB-NE inhibition of COX-2 in

macrophages is shown in Figure S2A-B.

Mechanical Hypersensitivity Assessments

Groups of male and female mice (n=5), weighing 18-25g, were treated with 200l of CXB-

NE, DF-NE or free drug solution (CXB dissolved in DMSO and saline mixture at 437.6µM) via

tail vein injection. Twelve hours later each mouse received 50l of CFA via intraplantar [i.pl]

injection into the footpad of the right hind paw. The achieved dose for CXB was 25µg/injection,

or approximately 1 mg/kg. Von Frey filaments were used to measure withdrawal threshold to

mechanical stimulation to the plantar surface of each hind paw up to day 40 post CFA induction

at distinct time points: days 0, 1, 3, 6, 11, 12, 18, 24, 32 and 40. [27]. Briefly, treated and control

mice were placed individually in a plastic cage (10 cm×10 cm×14 cm) with a wire mesh bottom.

After mice had adapted to the testing environment for 60 min, a von Frey filament with 0.4 g

bending force was pressed perpendicularly against the mid-plantar surface of the hind paw from

below the mesh floor and held for 3-5 s with the filament slightly buckled. Lifting of the paw was

recorded as a positive response. Stimulation of the same intensity (0.4 g filament) was applied to

the point of bending ten times to the plantar surface of the hind paw of each mouse at intervals of

5 s. Behavioral testing was performed between 10 am and 4 pm. Testing was performed for two

days before the start of the experiment to acclimatize the mice to testing procedures. Investigators

were blinded to treatment interventions during pain behavior tests. All data analyses and statistical

evaluations were performed by an independent operator.

Near-Infrared Fluorescence Imaging

NIRF whole body imaging (WBI) was conducted in male and female mice with group size

n=6. Animals were treated via tail vein injection with average dose of 200l of NIRF labeled

CXB-NE or DF-NE 12h prior to 50l i.pl CFA injection into the footpad of the right hind paw.

Whole body NIRF images were acquired at day 1, 3, 6, 9, 12, 18, 24, 32, and 40 post CFA injection

on Pearl® Trilogy Small Animal Imaging System. WBI imaging parameters were: 800nm channel

resolution 255 µm; Focus offset No. 2 [28-31]. Images were analyzed and signal quantified using

software Image Studio Lite. A rectangular box (area size 960) was placed on the back/shoulder

region of each animal across the study, we assigned this selected dark shape as background: signal

0.000. Region of interest (ROIs) were drawn around each CFA-induced footpad and its

contralateral footpad as control from toe to heel. Then, we recorded the signals from each footpad

as our raw data (example shown in Figure S5). The animal was anesthetized with 1.5-2.5 %

isoflurane/oxygen gas mixture inhalation. Then, the animal was placed on the pre-warmed to 37°C

imaging chamber bed at prone position. We anchored the animal’s face on the inhale nuzzle where

they will continuously receive anesthesia. The animal’s hind footpads were placed on each side of

the tail, and the palm of hind paws were facing up. This allows the camera to acquire a clean shot

of a picture representing the accumulation of NEs’ NIRF signal of the inflamed footpad (hind

right) and of the control footpad (hind left).

Euthanasia and Organ distribution

The mice were euthanized under anesthesia immediately after the final imaging time point with

a 0.5ml intraperitoneal injection of euthasol (pentobarbital sodium and phenytoin sodium solution,

Virbac AH, Inc., Fort Worth, TX). The animals were immediately perfused with 20ml of PBS

followed by 20ml of 4% paraformaldehyde in 1X PBS solution, administered into the left ventricle

of the heart, resulting in whole body fixation. We dissected select internal organs and quantified

fluorescence in each organ by drawing regions of interest (ROI) around each organ and

normalizing the total fluorescence to the organ weight [19]. In the biodistribution study (the ex

vivo imaging), all male animals were evaluated using the OdysseyCLx imaging system. The

imaging parameters were: 800nm channel Intensity at 0.5; Focus 1.0. All the female animals were

evaluated using the Pearl Trilogy Small Animal Imaging System. The imaging parameters were:

800nm channel Resolution 85 µm; Focus offset No. 2.

Immunofluorescence and Histopathology Analyses

Male and female mice were sacrificed after 40 days post CFA injection, and both of the right

and left paws were harvested. Tissues were prepared for histology with hematoxylin and eosin

(H&E) and immunostaining. The excised tissue and organ samples were fixed in 4% PFA solution,

embedded into paraffin, and cut into 10 μm sections using microtome (Leica, Buffalo Grove, IL).

The sections were stained with H&E according to standard protocols. For immunostaining; non-

specific binding was blocked with Dako serum free protein for 15 min at room temperature. Rat

anti-mouse CD68 (Bio-Rad, Hercules, CA) (1/200 dilution in DPBS/0.1% BSA/0.05% Tween-20)

and goat anti-mouse COX-2 antibodies (Novus, Centennial, CO) (at 1/50 dilution in PBS/0.1%

BSA/0.05% Tween-20) were added overnight at 4 °C followed by a secondary antibody, anti-rat-

Alexa 488 and anti-goat- Alexa 594 (Invitrogen, Grand Island, NY) (1/300 dilution in PBS/0.1%

BSA/0.05% Tween-20) for 1 h at RT. After washing in DPBS/0.05% Tween-20, coverslips were

mounted using Diamond anti-fading medium (Invitrogen, Grand Island, NY).

Immunofluorescence staining was monitored using an Olympus BX63 fluorescence microscope

with a multispectral camera by CRI (Perkin Elmer) and Nuance software.

Statistical Analyses

Imaging and behavior tests data was analyzed and plotted using Graph Pad Prism 8 software.

Data represents the mean ± SD, n = 5-6. Differences in treatment effects between groups for pain

behavior analyses were determined using two-way ANOVA and Tuckey’s multiple comparison

analyses between treatments (CXB-NE) and controls (DF-NE, free drug CXB, no treatment – CFA

alone and saline alone). Statistical significance was determined using the Holm-Sidak method,

with alpha=0.05. Each treatment was compared to controls at each time point was analyzed

individually, without assuming a consistent standard deviation across groups over time. The

statistical analyses results are provided in the Supplemental Information (Table S1 for males, Table

S2 for females).

Results

CXB-NE not free-drug inhibits COX-2 in macrophages and provides long term pain

control in male and female mice

Macrophage targeted NEs (CXB-NE) are designed for extended COX-2 inhibition resulting in

prolonged suppression of PgE2 release from macrophages at the site of injury leading to

mechanical hypersensitivity reduction [17]. Due to the major contribution of COX-2 in

macrophages in CFA induced pain, we hypothesized that CXB-NE can induce prolonged pain

relief using the same strategy in mice. To test this hypothesis, 12 hours prior to CFA injection,

groups of male and female mice were treated with a single dose of NIRF labeled CXB-NE, drug

free NE (DF-NE) or free celecoxib (CXB) solution, and followed for 40 days using von Frey

behavior assay. CXB-NE, DF-NE or free drug (CXB) solution were administered via tail vein

injection in both males and females. Overall, the CXB dose was kept at 1mg/kg across all groups

receiving drug treatment (CXB-NE or free drug) and the volume of injections (NE or free drug

solution) was kept constant across treatment groups. In all behavior analyses the statistical

significance was determined using two-way ANOVA with Tukey’s multiple comparison methods,

where withdrawals were compared for each treatment at each time point of follow up for both

males and females. All pain behavior studies were performed using blinded operators to the

treatment (CXB-NE or DF-NE).

Using von Frey assay we found persistent and statistically significant reduction in mechanical

hypersensitivity with CXB-NE over DF-NE and drug solution (CXB) controls (Figure 1A-D). In

male mice apparent pain control persists for up to 32 days (Figure 1A), while it lasts up to 11 days

in females (Figure 1B). Importantly, extended pain behavior change in both males and females is

achieved only when CXB is delivered in a NE as CXB-NE and directly to macrophages (Figure

1C-D). Both male and female mice when treated with matched low concentration of CXB

delivered as non-targeted, free drug solution showed no improvement in mechanical

hypersensitivity for up to 6 days (Figure S3 C-D). This means effectiveness of the low dose of

CXB requires targeted delivery to macrophages via NE, which is in agreement with our earlier

findings in rat injury models [17].

Macrophage targeted COX-2 inhibition with CXB-NE demonstrates sex-specific

differences

During the course of this study we observed significant differences in males and females in

response to CXB-NE treatment over controls (free drug and DF-NE). In male mice, the effects of

CXB-NE on improved mechanical hypersensitivity lasted up to 18 days post CFA induction versus

DF-NE control (Figure1A). However, there was an observable trend towards improvement in pain

behavior that persisted on days 24 and 32 post CFA induction, yet not statistically significant

(Figure 1A). To evaluate the antinociceptive effects more closely we ran two-way ANOVA for

each time point (statistical analyses results are provided in the Supplemental information, Tables

S1-2). A highly significant (p<0.001) reduction was observed in number of withdrawals when

male mice were treated with CXB-NE over free drug (CXB) at days 1 and day 32 (Figure 1C). In

female mice, we observed statistically significant differences in mechanical hypersensitivity

between CXB-NE treatment group and DF-NE treatment group up to day 6 post CFA induction

(Figure 1B). There was an obvious trend between the 2 groups on day 11 and day 12 post CFA

induction, yet not statistically significant (Figure 1B). When CXB-NE effects on females were

compared to free drug (CXB) treated animals, the mechanical hypersensitivity improvement is

sustained until Day 11 (Figure 1D). At early stages of post-CFA induced inflammation, up to day

6, both males and females responded to CXB-NE treatment over free drug and DF-NE as controls

demonstrated by significantly reduced mechanical hypersensitivity (Figure S3A-D). Importantly,

the CFA induced mechanical hypersensitivity both in males (Figure 1G) and females (Figure 1H)

as compared to the saline group. Under these conditions, as shown in Figure S4, no significant

difference in withdrawal counts between males and females treated with either CFA alone or saline

via i.pl injection was observed. Further, the withdrawal counts remained the same between males

and females treated with DF-NE control (Figure 1E). These findings suggest that a robust response

was obtained by CFA injection and this response was similar between males and females in this

experiment. However, when mechanical hypersensitivity was monitored for males and females

treated with drug loaded NE (CXB-NE) over the full course of the 40-day study we found highly

significant differences (Figure 1F). This finding warrants further investigation as it indicates that

the sex difference is exclusively tied to targeted delivery of CXB to macrophages and not found

in control treatment groups. The sex differences in response to treatment did not seem to be

confounded by differences in response to either CFA or saline injection (Figure 1G-H). Based on

these findings we concluded that COX-2 inhibition via targeted NE can enable extended pain relief

in animal models with marked sex-differences (Figure 1). Further we also showed that targeted

delivery is necessary to achieve pain relief with COX-2 inhibitor at a very low dose.

Whole body NIRF confirms localization of CXB-NE and DF-NE at the site of CFA

injection in males and females

High dose of NIRF labeled NEs was used to achieve maximal labeling of macrophages upon

i.v. injection produce strong NIRF signal affected region (CFA injection site) as the region of

interests (ROI). We found that NIRF fluorescent signal in affected paws, which we hypothesized

based on earlier studies that corresponds to macrophage infiltration levels, persists for 40 days post

NE injection in both males and females. NIRF whole body images were collected at distinct time

points: days 1, 3, 6, 9, 12, 15, 18, 24, 32, and 40 post-CFA induction in males and females injected

with DF-NE or CXB-NE. Whole body NIRF images of DF-NE treated male (Figure 2A) and

female (Figure 2B) mice and CXB-NE treated males (Figure 2C) and females (Figure 2D) at days

1 and 40, showing earliest and latest time-points. Additional time points collected representative

images for all groups are shown in supplemental information (Figure S6). The persistence of NIRF

signal, which correlated to the presence of CXB-NE or DF-NE in affected ROIs, at the site of CFA

injection, was further confirmed by immunofluorescence of excised inflamed tissues at day 40

(Figure 6, below). To the best of our knowledge, this is the first time NIRF labeled macrophage

targeted NEs were used to follow these cells beyond initial acute inflammation and 40 days post

CFA insult. Because of the extended follow up duration and single injection used, we want to

emphasize that NIRF signal distribution patterns must be interpreted cautiously. We observed no

statistically significant differences in NIRF signal in CFA injected hind paws in males (Figure 2E)

and females (Figure 2F) between CXB-NE and DF-NE treatment groups. However, we observed

interesting differences in distribution of NIRF signal in animals treated with CXB-NE and DF-NE

in both males and females (Figure 2E-F). There was a statistically higher signal in male animals

treated with DF-NE at later time points (Days 24, 32 and 40), Figure 2G. This sex difference in

NIRF signal at CFA treated paw not observed in CXB-NE treated animals across all time points

(Days 1-40), Figure 2H.

Furthermore, in order to study the correlation between live NIRF imaging of infiltrating

macrophages and CFA-induced inflammation, we plotted WBI data at different time points up to

40 days in both male and female animals administered CXB-NE and DF-NE. To control for inter-

subject variability, the fluorescence signal in the injured (CFA) paw at each specific time point

was divided by the fluorescence signal in the injured paw prior to CFA injection (CFA/pre-injury).

The control (non-injured) footpads were also evaluated using this approach. Female mice treated

with either DF-NE or CXB-NE showed statistically significant differences on days 1, 3, and 6

post-CFA injection when comparing the CFA/pre-injury ratio to the control/pre-injury ratio

(Figure 3A-B). In the DF-NE treated male group, statistically significant differences were also

observed on days 1, 3, and 6 post CFA injection when comparing the CFA/pre-injury ratio to the

control/pre-injury ratio (Figure 3C). However, in the CXB-NE treated male group, the ratio of

CFA/pre-injury only showed a statistically significant difference on day 6 post CFA injection

(Figure 3D). These data demonstrated that CFA injection induced severe inflammation at early

time points (days 1, 3, and 6 post injection), which confirms the findings in previous reports [32,

33]. Further, the results in Figure 3D suggested that in male mice, treatment with CXB-NE reduces

macrophage infiltration in the injured (CFA) footpad.

Ex vivo NIRF enables quantitative evaluation of tissue distribution of CXB-NE and DF-

NE in recipient animals

Ex vivo NIRF imaging was used to quantify the percent injected dose of NE per gram of organ

weight (%ID/g) in male and female mice administered CXB-NE or DF-NE (males, n=6/group;

females, n=3/group). We demonstrated that the %ID/g in the heart, lung, liver, spleen, kidneys,

and CFA injected leg muscle was not statistically significantly different (p-value<0.05) between

the CXB-NE and the DF-NE treatments for both male and female mice (Figure 4A-B).

Representative fluorescent images of biodistribution in the studied organs are shown in Figure 4C.

The biodistribution study reconfirmed our earlier findings that the liver and spleen accumulate NE

the most compared to the other studied non-target organs [19]. These results (Figure 4) were

expected because macrophages are present in large quantities in the liver and spleen [34, 35].

Though we did not observe a significant difference in the biodistribution between CXB-NE and

DF-NE (for both male and female mice), there may still be a biological difference [36]. For

example, the percentage of inflammatory (M1) macrophages present in the liver and spleen may

have decreased in response to treatment with CXB-NE. Previous work has demonstrated that

treatment with CXB-NE decreases the percentage of M1 macrophages present at the site of injury

[10]. This may be the case in off target organs as well.

Immunohistopathology and Immunofluorescence evaluation at end-point reveals

differential inflammatory patterns in hind paws of males and females

Male and female mouse hind paws were analyzed by routine histopathology (H&E) and

immunofluorescence staining for macrophage specific markers (CD68 and COX-2) at 40 days

following CFA injection. We chose coronal sections as they allow superior spatial interpretation

and anatomical localization of inflammatory patterns within the hind paw and assessment of

variations in degree of immune cell infiltration after CFA injury. Significant inflammation as

manifested by diffuse inflammatory cell infiltration is seen in the deep dermis in both female

(Figure 5 [DF-NE (Panel A)], and male controls (Figure 5 [DF-NE (Panel C)], In contrast to control

subjects, where there was minimal difference in degree of inflammation, at 40 days, there is

improvement of inflammation in both sexes, as manifested by resolution of infiltrates (Figure 5

[CXB-NE (Panels B and D)]. Male mice demonstrated more significant resolution of inflammation

as compared to female mice with CXB-NE treatment. This effect was particularly prominent in

the periosteal and synovial zones as well as in the deep dermis.

Immunofluorescent staining (Figure 6) for CD68 expression showed strong positivity in

female control mice as compared to male mice (DF-NE). No significant differences were observed

in the levels of COX-2 expression in controls. However, CXB-NE treatment substantially

suppressed COX-2 expression in both sexes. The merged panels in both male and female CXB-

NE treated mice on Figure 6 demonstrate that there is a persistence of CD68 positive macrophages

in the hind paws at 40 days but COX-2 expression is very low or negligible in both sexes. DAPI

nuclear counterstaining with NE confirmed the intracellular (peri-nuclear/intracytoplasmic)

localization and persistence of NE in macrophages in both DF-NE and CXB-NE groups at 40 days

after CFA injection. Figure 7 shows anatomical context to better localize the inflammation which

is marked in the periosteal zone of the metatarsals and in the synovial sheaths of the flexor

digitorum brevis tendons. There is absent or negligible inflammation in the plantar interossei

(interposed between the tendons and the metatarsals.

Discussion

Monocytes and macrophages are attractive targets for both diagnosis and treatment of

inflammation as they are shown to directly contribute to pathology of multiple inflammatory

diseases such as osteoarthritis, rheumatoid arthritis and inflammatory bowel disease [30-32]. In

prior studies we demonstrated that CXB-NEs when delivered intravenously in rat chronic

constriction injury (CCI) model, are taken up by macrophages changing their infiltrating patterns

as compared to DF-NE [33]. We also showed that both CXB-NE and DF-NE co-localize with

CD68 positive cells (macrophages) and COX-2 in tissues excised from male mice hind paws

treated with CFA at day 4 post-induction [3]. Here we extended our imaging studies to 40 days at

selected time points after CFA injection in both male and female mice between CXB-NE and DF-

NE groups.

Our study confirms that administration of a single dose of CXB-NE significantly reduces CFA-

induced mechanical hypersensitivity in both male and female mice for longer than one week

(Figure 1A, B). However, this pain reduction was more significant in male mice at later time points

(Figure 1D). CXB-NE provided extended improvement in pain behavior in males (32 days, Figure

1A) versus females (11 days, Figure 1B), which may be dependent on targeted delivery of CXB

directly to macrophages expressing COX-2 enzyme. This is likely due to extended inhibition of

PgE2 release from these cells achieved by prolonged COX-2 exposure to the inhibitor. This effect

is achieved by internalized CXB-NE droplets serving as drug depot inside macrophages.

Importantly, CXB administered as a free drug showed no effect in either males or females

when administered at matched concentration to that delivered with CXB-NE (Figure 1C-D). This

finding strongly suggests that effectiveness of CXB in inflammatory pain can be dramatically

improved by incorporation into macrophage targeted NEs. CFA treated paw NIRF signal in female

and male mice when administered CXB NE or DFNE, persists up to day 40, Figure 2, while it

changes in intensity over time. This finding indicates that targeted delivery of CXB directly to

sites of inflammation can be monitored over long period of time using NIRF imaging, where NIRF

signal from NE may be used to infer levels of drug accumulation. The value of these findings is to

demonstrate the potential for using nanotheranostics, such as the NEs evaluated on this study, as

tools for studying macrophage turnover and life-span in chronic inflammation. Further, these data

indicate there are macrophage infiltration sex-differences in males and females that warrant further

investigation in preclinical models. For example, we found that fluorescent ratio of CFA/control

decreases to 1.0 in female mice (Figure 2D) at the same time point (11 days) when hyperalgesia

begins to show less of significant effect of CXB-NE treated mice over controls (free drug and/or

DF-NE) in female mice (Figure 1B). This was not observed in male mice. The data indicates there

are differences in response to COX-2 inhibition in males and females that may correspond to

changes in NE labeled macrophage infiltration levels between the sexes. Immunofluorescence

staining (Figure 6) on hind paw samples from Day 40, revealed significantly more CD68 positive

macrophages containing phagocytosed NE, in DF-NE treated male mice as compared to females.

Tissue counterstaining with DAPI confirmed the intracytoplasmic location of NE in both DF and

CXB-NE groups. This finding correlated with a statistically significant increase in NIRF signal

intensity in males versus female DF-NE treated mice (Figure 2G). On H&E (Figure 6), we

observed increased gross infiltration in inflammatory cells at 40 days after CFA in both male and

female mice receiving DF-NE. Despite the visible decrease infiltration at day 40 in both male and

female mice receiving CXB-NE, the clearance of infiltrates was much more marked in male mice

(Figure 6B [upper and lower panels, d,e,f]).

However, we must acknowledge limitations in approach or analyses in this study that

prevented us from making more substantive or empirical assumptions: All mice received only a

single injection of NE and a dose-response study was not performed. Thus, it may be hypothesized

that newly generated monocytes that differentiated into MDMs and infiltrated to the sites of

inflammation late after the DF or CXB-NE injection may not have retained the fluorescent label.

Interestingly however, we saw persistence of NE in macrophages even at 40 days in both male and

female DF-NE mice (Figure 6, DAPI+NE merge). In contrast, very little NE was present in

macrophages in the CXB-NE treated male and female mice at day 40. We are currently

investigating if the drug loading affects the pharmacokinetics of the NE. However, the marked

resolution of inflammation at day 40 as seen on H&E in both male and female (male > female)

mice treated with CXB-NE suggest that the anti-inflammatory effects of CXB may persist long

beyond the life-span, turnover and removal of CXB-NE laden macrophages. A significant

limitation of this study is that histologic analyses were done only at end point and

immunohistochemistry for macrophage specific markers was not performed. Other limitations of

this work include (1) different groups of animals were used to perform the pain and the NIRF

imaging studies; (2) only one type of pain assay was used. To the best of our knowledge, the

presented exploratory, predominantly observational work is the only example of investigating the

extended impact (up to 40 days) on pain and inflammation with COX-2 inhibition in MDMs

through targeted drug delivery. Further, this is the first, albeit proof-of-concept study where sex

differences in macrophage specific COX-2 inhibition were ever investigated with a

nanotheranostic approach. We envision future work to include sequential imaging, advanced

immunohistopathologic analyses, and additional pain behavior assays beginning at earlier time

points and coinciding with crucial time points in the inflammation response to injury. Further

studies in large animal models may be necessary to validate biological sex differences in

inflammatory pain.

Conclusion

Our study highlights the potential of nanotheranostics as tools to investigate the complex

biology and sex-specific nuances in responses to inflammation and pain. Our results in male and

female mice highlight the value of a cell-targeted nanotheranostic approach for in-depth

investigation of macrophage behavior in inflammatory diseases. This technology can be used to

improve our understanding of the mechanism behind sex differences in macrophage biology,

inflammation and response to commonly used drugs such as NSAIDs. A multipronged approach

including behavioral testing, in vivo real time cell tracking, and ex vivo histological analyses such

as presented here is critical to improve our understanding of the processes that drive such

inflammation. Our work provides key foundational insights into development of better

personalized pain management approaches for patients.

Acknowledgements

Author contributions: JM Janjic designed the overall approach for inflammatory pain and

targeted inflammation imaging with nanotheranostics, designed COX-2 inhibiting nanoemulsions,

analyzed pain behavior data and edited the manuscript. L. Liu produced all nanoemulsions used in

this study, managed mouse imaging and pain behavior studies, collected tissues for ex vivo

analyses and performed critical imaging experiments. Immunofluorescence and H&E analyses

were performed by H. Karagoz and F. Zor and these data were processed and interpreted by V.S.

Gorantla. L. Liu and M. Herneisey performed WBI in male and female mice, collected and

analyzed the data, and contributed to drafting the manuscript. Pain behavior studies were

performed by T. Komatsu. B.M. Janjic assisted in statistical analyses. S. Loftus performed in vitro

studies on nanotheranostics.

Drs. Jelena M. Janjic and Vijay S. Gorantla serve as co-corresponding authors. They jointly

edited and approved the manuscript. The authors would like to thank Denise Butler-Buccilli,

Duquesne University, for her support in animal studies. This work was in part supported by

Duquesne University start-up research funds, NIDA award number R21DA039621-02, and DSF

Charitable Foundation.

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Figures

Figure 1. Mechanical hypersensitivity monitoring for 40 days with von Frey in male and

female mice following tail vein injection with 200 µL of CXB-NE or DF-NE 12h prior to i.pl

injection with CFA. A) Mechanical hypersensitivity responses in male mice, show significant

difference between mice administered CXB-NE versus DF-NE (control, drug free NE) for 18 days

and trend continued up to 32 days post injection; B) Mechanical hypersensitivity responses in

female mice show significant difference between mice administered CXB-NE over DF-NE for 6

days; C) CXB-NE show marked improvement in mechanical hypersensitivity in males at days 1

and 32 as compared to free drug (CXB) solution control at matched low dose injected i.v. There is

no significant difference between DF-NE and free drug control as both do not produce any pain

relief. D) CXB-NE results in statistically significant improvement in pain behavior in females at

days 1 and 11 over free drug (CXB) control. E) Male and female mice administered DF-NE reveal

no statistical significance in mechanical hypersensitivity during the course of the experiment

except at day 12 post CFA injection (*p<0.05). F) Male and female mice reveal significant

differences between footpad withdrawal responses. There is a marked difference during the course

of the experiment for 32 days showing that CXB-NE results in greater improvement in mechanical

hypersensitivity in males than females. G) Mechanical hypersensitivity responses show significant

difference for male mice administered CFA i.p. injection vs. saline i.p. injection in right hind

footpads. H) Mechanical hypersensitivity responses show significant difference for female mice

administered CFA i.p. injection vs. saline i.p. injection in right hind footpads. GraphPad Prism 8

was used for statistical analyses and graphical representation. Data represents average ± SEM, n =

5 across all treatment groups.

Figure 2. NIRF whole body imaging of CXB-NE and DF-NE NEs accumulation at the site of

inflammation following CFA injection in males and females. The NEs were injected 12h prior

to the CFA induction at the right hind paw in male and female mice. Whole body NIRF images

were collected post-CFA induction at distinct time points: days 1, 3, 6, 9, 12, 15, 18, 24, 32, and

40. A-B) Representative NIRF images showing the accumulation of DF-NE (drug free NE) at the

site of inflammation (hind right paw) at days 1 and 40 post-CFA induction in males (A) and

females (B). C-D) Accumulation of CXB-NE (drug free NE) at the site of inflammation (hind right

paw) at days 1 and 40 post-CFA injection in males (C) and females (D); E-H) The ratio of

quantified fluorescence of inflamed hind right paw footpad is calculated to that of the control

(contralateral, left paw) footpad in both between male and females at days 1, 3, 6, 9, 12, 15, 18,

24, 32, and 40 post-CFA. E) DF-NE and CXB-NE associated signal distribution in males over 40

days follow up. CXB-NE treated animals show decreasing signal over time, though not statistically

different from DF-NE treatments. This indicate the CXB-NE may impact macrophage infiltration

dynamics in males. F) Comparison of signal distribution in DF-NE and CXB-NE treated female

mice shows no statistical significance between the treatment groups. G) DF-NE accumulation at

the site of CFA insult differs between males and females with a lower overall trend of accumulation

in females. There is a lower (statistically significant) signal in females versus males at later time

points (Days 24, 32 (p<0.05) and day 40 (p<0.001). H) CXB-NE shows no significant difference

in accumulation patterns between males and females over 40 days of follow up. GraphPad Prism

8 was used for statistical analyses and graphical representation. Data represents average ± SEM, n

= 5 across all treatment g roups. Two-way ANOVA was used to establish statistical significance

between all groups.

Figure 3. Whole Body NIRF of CXB-NE and DF-NE at the ratio of CFA footpad/ itself pre-

injury in males and females. A) In DF-NE treated female group, the ratio of CFA/pre-injury

showed statistically significant difference at day 1, 3, and 6 post CFA injection compare to the

ratio of control/pre-injury; B) In CXB-NE treated female group, the ratio of CFA/pre-injury

showed statistically significant difference at day 1, 3, and 6 post CFA injection compare to the

ratio of control/pre-injury; C) In DF-NE treated male group, the ratio of CFA/pre-injury showed

statistically significant difference at day 1, 3, and 6 post CFA injection compare to the ratio of

control/pre-injury; D) In CXB-NE treated male group, the ratio of CFA/pre-injury only showed

statistically significant difference at day 6 post CFA injection compare to the ratio of control/pre-

injury (Figure 3D).

Figure 4. Biodistribution of theranostic NEs. A. Representative fluorescence image

quantification of indicated organs showing NE biodistribution at 40 days post CFA-induced

inflammation in male animals ex vivo as evaluated by Odyssey CLx imaging system. B.

Representative fluorescence image quantification of indicated organs showing NE biodistribution

at 40 days post CFA-induced inflammation in female animals ex vivo as evaluated by Pearl Trilogy

Small Animal Imaging System. C. Representative fluorescent images of biodistribution in the

organs.

Figure 5. H&E staining of inflamed paw sections collected at end-point from male and female

mice treated with CXB-NE or DF-NE. A) Coronal Section of Female Mouse Hind Paw at 40

days following CFA Injection: Panel A shows a coronal section of the mid foot in a female

mouse receiving DF-NE injection. Significant inflammatory infiltration (white arrowheads) is

seen in the periosteal zone around the metatarsals (4th metatarsal is shown in [a]), soft tissue

(including epidermo-dermal structures, interfaces and adnexa in [b]) and the synovial sheaths of

the flexor tendons (the flexor digitorum brevis tendons and sheath are shown for the 2nd metatarsal

in [c]) Panel B shows a coronal section of the mid foot in a female mouse receiving CXB-NE

injection. There is only partial improvement or resolution of inflammation (white arrows) as

manifested by infiltration in the periosteal zone around the metatarsals (2nd metatarsal is shown in

[d]), soft tissue (including epidermo-dermal structures, interfaces and adnexa in [e]) and the

synovial sheaths of the flexor tendons (the flexor digitorum brevis tendons and sheath are shown

for the 3rd metatarsal in [f]); B) Coronal Section of Male Mouse Hind Paw at 40 days following

CFA Injection: Panel C shows a coronal section of the mid foot in a male mouse receiving DF-

NE injection. Significant inflammatory infiltration (white arrowheads) is seen in the periosteal

zone around the metatarsals (2nd metatarsal is shown in [a]), soft tissue (including epidermo-dermal

structures, interfaces and adnexa in [b]) and the synovial sheaths of the flexor tendons (the flexor

digitorum brevis tendons and sheath are shown for the 4th metatarsal in [c]) Panel D shows a

coronal section of the mid foot in a male mouse receiving CXB-NE injection. Compared to that in

the female mouse hind paw, there is marked improvement or resolution of inflammation (white

arrows) as shown by discernible reduction in infiltration in the periosteal zone around the

metatarsals (4th metatarsal is shown in [d]), soft tissue (including epidermo-dermal structures,

interfaces and adnexa in [e]) and the synovial sheaths of the flexor tendons (the flexor digitorum

brevis tendons and sheath are shown for the 1st metatarsal in [f])

Figure 6. Representative images of immunofluorescence staining of excised tissues from

CFA-treated footpad in males and females on day 40 post CFA induction. Sections were

stained with Rat anti mouse-CD68 (selective macrophage marker, green) and Goat anti mouse-

COX-2 (COX-2, red). DF-NE and CXB-NE shown as purple. The merged panel shows the co-

localization of CXB-NE or DF-NE with COX-2 expressing macrophages, confirming the

persistence of both NEs in affected tissues at day 40 post-CFA injection. The lowermost panel

shows DAPI nuclear counterstaining co-localized with NE in both DF-NE and CXB-NE groups,

confirming the intracellular (peri-nuclear) localization of NE inside macrophages (CD68 positive or

negative)

Figure 7. Coronal section of the mouse hind paw showing contextual anatomical localization of

CFA-induced inflammation. A coronal section of the mid foot is shown with the arrow

demonstrating the location of CFA injection on the plantar aspect of the sole of the hind paw. The

metatarsals of the big toe (m1), and other toes are labeled (m2, m3, m4, m5). The flexor digitorum

brevis (FDB) tendons are shown for each toe (t1 for great toe, and t2, t3, t4, t5 for other toes). The

FDB arises from the tendon of plantaris as three slender muscles which pass over into long tendons.

Each tendon, at the base of the first phalanx, divides into two and insert on the proximal end of the

second phalanx of the second, third and fourth toes. The plantar interossei for the 2nd, 3rd and 4th

toe are shown (i2, i3 [2nd digit]; i4, i5 [3rd digit]; i6, i7 [4th digit]). These are small slender muscles

on the volar surface of the metatarsals. There are two interossei for 2nd, 3rd and 4th metatarsals,

and one each for the great toe and little toe.

SUPPLEMENTAL INFORMATION

Sex Differences Revealed in a Mouse CFA Inflammation Model with

Macrophage Targeted Nanotheranostics

Lu Liu1,2, Huseyin Karagoz3, Michele Herneisey1,2, Fatih Zor3, Takaaki Komatsu4, Shannon

Loftus1,2, Bratislav M. Janjic,5 Vijay S. Gorantla3*, Jelena M. Janjic1,2,*

1 Graduate School of Pharmaceutical Sciences, School of Pharmacy, Duquesne University,

Pittsburgh, PA, USA

2 Chronic Pain Research Consortium, Duquesne University, Pittsburgh, PA, US 4 Department of Surgery, Institute of Regenerative Medicine, Wake Forest University School of

Medicine, Winston-Salem, North Carolina, United States

3 Department of Pharmacology, Daiichi University of Pharmacy, Fukuoka, Japan

5 NRG Oncology Foundation Inc., Pittsburgh, PA, United States

CO-CORRESPONDING AUTHORS:

*Jelena M. Janjic, PhD

Graduate School of Pharmaceutical Sciences,

Duquesne University

600 Forbes Avenue, 415 Mellon Hall

Pittsburgh, PA 15282, USA

412-396-6369 (Phone)

janjicj@duq.edu (Email)

*Vijay S. Gorantla, MD, PhD, MMM

Department of Surgery

Institute of Regenerative Medicine, Wake Forest University School of Medicine

391 Technology Way

Suite 333 Richard H. Dean Biomedical Building

Winston- Salem, NC 27101, USA

336-713-1494 (Phone)

vgorantl@wakehealth.edu (Email)

Methods

Preparation of nanoemulsions

The nanoemulsions used in this study (celecoxib loaded, CXB-NE and drug-free, DF-NE) were

produced following earlier reported protocols in Janjic et al[1, 2]. Briefly, nanoemulsions were

prepared by incorporating the near-infrared fluorescent dye, DiR, DiIC18(7) (1,1'-Dioctadecyl-

3,3,3',3'-Tetramethylindotricarbocyanine Iodide) (5 mM) with/without celecoxib (2.5 mg/mL) into

a hydrocarbon oil. After adding perfluorocarbon and surfactants, the mixture was processed on a

microfluidizer (Microfluidics, M110S) following earlier established methods.[1]

Colloidal characterization

Dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, UK) was used to measure

droplet size and zeta potential at 25 °C. Nanoemulsion was dispersed in deionized water at a 1:80

dilution. Size and zeta potential were recorded on the same sample in a cuvette or standard zeta

potential cell, respectively. Each sample was analyzed three times with at least 12 runs each.

PgE2 ELISA

RAW 264.7 murine macrophages (ATCC) were plated at 300,000 cells/2mL/well in 6 well plates

for 24 hours. Cells were then exposed to the indicated concentrations of nanoemulsion or drug

dispersed in full media for 24 hours. The cells were then washed and exposed to serum-free media

with or without 500 ng/mL LPS. After 18 hours, the cell culture supernatant was collected and

centrifuged at 2000 rpm 4°C for two minutes to remove any cell debris. PgE2 was measured in

these samples using the Prostaglandin E2 ELISA Kit – Monoclonal (Cayman Chemical, Ann

Arbor USA) according to the manufacturer’s instructions.

WBI image acquisition

Pearl Trilogy Small Animal Imaging System, was used to evaluate the whole body live imaging

(WBI) of an individual mouse with different treatment and at different time points until the end of

the study (40 days post-CFA induction). The animal was anesthetized with 1.5-2.5 %

isoflurane/oxygen gas mixture inhalation. Then, the animal was placed on the pre-warmed to 37°C

imaging chamber bed at prone position. We anchored the animal’s face on the inhale nuzzle where

they will continuously receive anesthesia. The animal’s hind footpads were placed on each side of

the tail, and the palm of hind paws were facing up. This allows the camera to acquire a clean shot

of a picture representing the accumulation of nanoemulsions’ NIRF signal of the inflamed footpad

(hind right) and of the control footpad (hind left).

Results

Fig. S1. Comparison of size and zeta potential

distributions of the nanoemulsions used in WBI. A.

Size distribution for CXBNE (green) vs DFNE

(blue) 1 day after production and B. zeta potential

distribution. C. Size distribution for CXBNE after 1

day (blue) and 6 months (green) and D. Size distribution for DFNE after 1 day (blue) and 6

months (green).

Figure S2. PgE2 ELISA Assay. RAW 264.7 macrophage cells were cultured with increasing concentrations of CXB-

NE, DF-NE or free drug (CXB) for 24 h prior LPS exposure for 3h and supernatant collection. A.PgE2 production in

inhibition from cells treated with a 5-fold dilution dose curve of CXB-NE or DF-NE. B. PgE2 production in cells

treated with a 5-fold dilution curve of CXB-NE or CXB dissolved in DMSO. Error bars represent standard deviation

from triplicate culture aliquots.

Figure S3. Reanalysis of von Frey results in male and females. First six days post CFA injection mechanical

hypersensitivity measurements shows significant pain behavior improvement both in males and females with CXB-

NE over controls (CXB or DF-NE).

Figure S4. Reanalysis of von Frey results in male and females. A) Mechanical hypersensitivity monitoring for 40

days after CFA i.pl injection in male and female mice. There is no significant difference observed in responses to

CFA (B) or saline intraplantar injection between sexes.

Figure S5. A representation on how we collect the data from whole body

imaging (male, DF-NE on day 1 post CFA induction). Images were

analyzed and signal quantified using software Image Studio Lite. A

rectangular box (area size 960) was placed on the back/shoulder region

of each animal across the study, we assigned this selected dark shape as

background: signal 0.000. Region of interest (ROIs) were drawn around

each CFA-induced footpad and its contralateral footpad as control from

toe to heel. Then, we recorded the signals from each footpad as our raw data.

Figure S6. NIRF whole body live images showing the accumulation of nanoemulsion at the site of inflammation (hind

right footpad) at different time points (Days 1, 3, 6, 9, 12, 18, 24, 32, and 40 post CFA-induction). The treatment

(CXB-NE or DF-NE) were injected i.v. via tail vein injection prior to the CFA induction at the right hind paw (50µ

intraplantar). A) Representative NIRF images showing the accumulation of DF-NE at the site of inflammation in male

mice group from day 1 to day 40 post-CFA induction. B) Representative NIRF images showing the accumulation of CXB-NE at the site of inflammation in male mice group from day 1 to day 40 post-CFA induction. C) Representative

NIRF images showing the accumulation of DF-NE at the site of inflammation in female mice group from day 1 to day

40 post-CFA induction. D) Representative NIRF images showing the accumulation of CXB-NE at the site of

inflammation in female mice group from day 1 to day 40 post-CFA induction.

References:

1. Janjic, J.M., et al., Low-dose NSAIDs reduce pain via macrophage targeted nanoemulsion

delivery to neuroinflammation of the sciatic nerve in rat. J Neuroimmunol, 2018. 318: p.

72-79.

2. Patel, S.K., et al., Theranostic nanoemulsions for macrophage COX-2 inhibition in a

murine inflammation model. Clin Immunol, 2015. 160(1): p. 59-70.

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A B C D E F G H I

Von Frey ASSAY in MALE MICE

Table Analyzed Males - vonFrey

Two-way ANOVA OrdinaryAlpha 0.05

Source of Variation % of total variati P value P value summarSignificant?Interaction 12.97 < 0.0001 **** YesRow Factor 31.84 < 0.0001 **** YesColumn Factor 35.89 < 0.0001 **** Yes

ANOVA table SS DF MS F (DFn, DFd) P valueInteraction 267.3 36 7.426 F (36, 200) = 3.7P < 0.0001Row Factor 656.4 9 72.93 F (9, 200) = 36.6P < 0.0001Column Factor 739.9 4 185 F (4, 200) = 92.9P < 0.0001Residual 398.1 200 1.99

Within each row, compare columns (simple effects within rows)

Number of families 10 Number of missing values 0Number of comparisons per family 10Alpha 0.05

Tukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary1 Saline vs. CXB-NE (CFA) 0.5 -1.956 to 2.956 No ns Saline vs. CFA -4 -6.456 to -1.544 Yes *** Saline vs. CXB (CFA) -3.3 -5.756 to -0.8437 Yes ** Saline vs. DF-NE (CFA) -3.8 -6.256 to -1.344 Yes *** CXB-NE (CFA) vs. CFA -4.5 -6.956 to -2.044 Yes **** CXB-NE (CFA) vs. CXB (CFA) -3.8 -6.256 to -1.344 Yes *** CXB-NE (CFA) vs. DF-NE (CFA) -4.3 -6.756 to -1.844 Yes **** CFA vs. CXB (CFA) 0.7 -1.756 to 3.156 No ns free drug innefective - no change vs untreated (CFA alone) CFA vs. DF-NE (CFA) 0.2 -2.256 to 2.656 No ns drug-free nanoemulsion ineffective CXB (CFA) vs. DF-NE (CFA) -0.5 -2.956 to 1.956 No ns

3 Saline vs. CXB-NE (CFA) 0 -2.456 to 2.456 No ns Saline vs. CFA -4.6 -7.056 to -2.144 Yes **** Saline vs. CXB (CFA) -3.9 -6.356 to -1.444 Yes *** Saline vs. DF-NE (CFA) -4.3 -6.756 to -1.844 Yes **** CXB-NE (CFA) vs. CFA -4.6 -7.056 to -2.144 Yes **** CXB-NE (CFA) vs. CXB (CFA) -3.9 -6.356 to -1.444 Yes *** CXB-NE (CFA) vs. DF-NE (CFA) -4.3 -6.756 to -1.844 Yes **** CFA vs. CXB (CFA) 0.7 -1.756 to 3.156 No ns CFA vs. DF-NE (CFA) 0.3 -2.156 to 2.756 No ns CXB (CFA) vs. DF-NE (CFA) -0.4 -2.856 to 2.056 No ns

6 Saline vs. CXB-NE (CFA) 0 -2.456 to 2.456 No ns Saline vs. CFA -3.8 -6.256 to -1.344 Yes *** Saline vs. CXB (CFA) -6 -8.456 to -3.544 Yes **** Saline vs. DF-NE (CFA) -5.4 -7.856 to -2.944 Yes **** CXB-NE (CFA) vs. CFA -3.8 -6.256 to -1.344 Yes *** CXB-NE (CFA) vs. CXB (CFA) -6 -8.456 to -3.544 Yes **** CXB-NE (CFA) vs. DF-NE (CFA) -5.4 -7.856 to -2.944 Yes **** CFA vs. CXB (CFA) -2.2 -4.656 to 0.2563 No ns CFA vs. DF-NE (CFA) -1.6 -4.056 to 0.8563 No ns CXB (CFA) vs. DF-NE (CFA) 0.6 -1.856 to 3.056 No ns

11 Saline vs. CXB-NE (CFA) 0 -2.456 to 2.456 No ns Saline vs. CFA -4.3 -6.756 to -1.844 Yes **** Saline vs. CXB (CFA) -3.5 -5.956 to -1.044 Yes ** Saline vs. DF-NE (CFA) -3.5 -5.956 to -1.044 Yes ** CXB-NE (CFA) vs. CFA -4.3 -6.756 to -1.844 Yes **** CXB-NE (CFA) vs. CXB (CFA) -3.5 -5.956 to -1.044 Yes ** CXB-NE (CFA) vs. DF-NE (CFA) -3.5 -5.956 to -1.044 Yes ** CFA vs. CXB (CFA) 0.8 -1.656 to 3.256 No ns CFA vs. DF-NE (CFA) 0.8 -1.656 to 3.256 No ns CXB (CFA) vs. DF-NE (CFA) 0 -2.456 to 2.456 No ns

12 Saline vs. CXB-NE (CFA) 0 -2.456 to 2.456 No ns Saline vs. CFA -6.4 -8.856 to -3.944 Yes **** Saline vs. CXB (CFA) -6.6 -9.056 to -4.144 Yes **** Saline vs. DF-NE (CFA) -4.4 -6.856 to -1.944 Yes **** CXB-NE (CFA) vs. CFA -6.4 -8.856 to -3.944 Yes **** CXB-NE (CFA) vs. CXB (CFA) -6.6 -9.056 to -4.144 Yes ****

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A B C D E F G H I CXB-NE (CFA) vs. DF-NE (CFA) -4.4 -6.856 to -1.944 Yes **** CFA vs. CXB (CFA) -0.2 -2.656 to 2.256 No ns CFA vs. DF-NE (CFA) 2 -0.4563 to 4.456 No ns CXB (CFA) vs. DF-NE (CFA) 2.2 -0.2563 to 4.656 No ns

18 Saline vs. CXB-NE (CFA) 0 -2.456 to 2.456 No ns Saline vs. CFA -7.5 -9.956 to -5.044 Yes **** Saline vs. CXB (CFA) -5.2 -7.656 to -2.744 Yes **** Saline vs. DF-NE (CFA) -4.6 -7.056 to -2.144 Yes **** CXB-NE (CFA) vs. CFA -7.5 -9.956 to -5.044 Yes **** CXB-NE (CFA) vs. CXB (CFA) -5.2 -7.656 to -2.744 Yes **** CXB-NE (CFA) vs. DF-NE (CFA) -4.6 -7.056 to -2.144 Yes **** CFA vs. CXB (CFA) 2.3 -0.1563 to 4.756 No ns CFA vs. DF-NE (CFA) 2.9 0.4437 to 5.356 Yes * CXB (CFA) vs. DF-NE (CFA) 0.6 -1.856 to 3.056 No ns

24 Saline vs. CXB-NE (CFA) 0 -2.456 to 2.456 No ns Saline vs. CFA -3.7 -6.156 to -1.244 Yes *** Saline vs. CXB (CFA) -2.2 -4.656 to 0.2563 No ns Saline vs. DF-NE (CFA) -2.2 -4.656 to 0.2563 No ns CXB-NE (CFA) vs. CFA -3.7 -6.156 to -1.244 Yes *** CXB-NE (CFA) vs. CXB (CFA) -2.2 -4.656 to 0.2563 No ns CXB-NE (CFA) vs. DF-NE (CFA) -2.2 -4.656 to 0.2563 No ns CFA vs. CXB (CFA) 1.5 -0.9563 to 3.956 No ns CFA vs. DF-NE (CFA) 1.5 -0.9563 to 3.956 No ns CXB (CFA) vs. DF-NE (CFA) 0 -2.456 to 2.456 No ns

32 Saline vs. CXB-NE (CFA) 0 -2.456 to 2.456 No ns Saline vs. CFA -3.4 -5.856 to -0.9437 Yes ** Saline vs. CXB (CFA) -2.2 -4.656 to 0.2563 No ns Saline vs. DF-NE (CFA) -2.6 -5.056 to -0.1437 Yes * CXB-NE (CFA) vs. CFA -3.4 -5.856 to -0.9437 Yes ** CXB-NE (CFA) vs. CXB (CFA) -2.2 -4.656 to 0.2563 No ns CXB-NE (CFA) vs. DF-NE (CFA) -2.6 -5.056 to -0.1437 Yes * CFA vs. CXB (CFA) 1.2 -1.256 to 3.656 No ns CFA vs. DF-NE (CFA) 0.8 -1.656 to 3.256 No ns CXB (CFA) vs. DF-NE (CFA) -0.4 -2.856 to 2.056 No ns

40 Saline vs. CXB-NE (CFA) 0 -2.456 to 2.456 No ns Saline vs. CFA -1.4 -3.856 to 1.056 No ns Saline vs. CXB (CFA) 0 -2.456 to 2.456 No ns Saline vs. DF-NE (CFA) -0.6 -3.056 to 1.856 No ns CXB-NE (CFA) vs. CFA -1.4 -3.856 to 1.056 No ns CXB-NE (CFA) vs. CXB (CFA) 0 -2.456 to 2.456 No ns CXB-NE (CFA) vs. DF-NE (CFA) -0.6 -3.056 to 1.856 No ns CFA vs. CXB (CFA) 1.4 -1.056 to 3.856 No ns CFA vs. DF-NE (CFA) 0.8 -1.656 to 3.256 No ns CXB (CFA) vs. DF-NE (CFA) -0.6 -3.056 to 1.856 No ns

Test details Mean 1 Mean 2 Mean Diff. SE of diff. N1 N2 q DF

0 Saline vs. CXB-NE (CFA) 0 0 0 0.8923 5 5 0 200 Saline vs. CFA 0 0 0 0.8923 5 5 0 200 Saline vs. CXB (CFA) 0 0 0 0.8923 5 5 0 200 Saline vs. DF-NE (CFA) 0 0 0 0.8923 5 5 0 200 CXB-NE (CFA) vs. CFA 0 0 0 0.8923 5 5 0 200 CXB-NE (CFA) vs. CXB (CFA) 0 0 0 0.8923 5 5 0 200 CXB-NE (CFA) vs. DF-NE (CFA) 0 0 0 0.8923 5 5 0 200 CFA vs. CXB (CFA) 0 0 0 0.8923 5 5 0 200 CFA vs. DF-NE (CFA) 0 0 0 0.8923 5 5 0 200 CXB (CFA) vs. DF-NE (CFA) 0 0 0 0.8923 5 5 0 200

1 Saline vs. CXB-NE (CFA) 2 1.5 0.5 0.8923 5 5 0.7925 200 Saline vs. CFA 2 6 -4 0.8923 5 5 6.34 200 Saline vs. CXB (CFA) 2 5.3 -3.3 0.8923 5 5 5.23 200 Saline vs. DF-NE (CFA) 2 5.8 -3.8 0.8923 5 5 6.023 200 CXB-NE (CFA) vs. CFA 1.5 6 -4.5 0.8923 5 5 7.132 200 CXB-NE (CFA) vs. CXB (CFA) 1.5 5.3 -3.8 0.8923 5 5 6.023 200 CXB-NE (CFA) vs. DF-NE (CFA) 1.5 5.8 -4.3 0.8923 5 5 6.815 200 CFA vs. CXB (CFA) 6 5.3 0.7 0.8923 5 5 1.109 200 CFA vs. DF-NE (CFA) 6 5.8 0.2 0.8923 5 5 0.317 200 CXB (CFA) vs. DF-NE (CFA) 5.3 5.8 -0.5 0.8923 5 5 0.7925 200

3 Saline vs. CXB-NE (CFA) 3.4 3.4 0 0.8923 5 5 0 200

165166167168169170171172173174175176177178179180181182183184185186187188189190191192193194195196197198199200201202203204205206207208209210211212213214215216217218219220221222223224225226227228229230231232233234235236237238239240241242243244245246

A B C D E F G H I Saline vs. CFA 3.4 8 -4.6 0.8923 5 5 7.291 200 Saline vs. CXB (CFA) 3.4 7.3 -3.9 0.8923 5 5 6.181 200 Saline vs. DF-NE (CFA) 3.4 7.7 -4.3 0.8923 5 5 6.815 200 CXB-NE (CFA) vs. CFA 3.4 8 -4.6 0.8923 5 5 7.291 200 CXB-NE (CFA) vs. CXB (CFA) 3.4 7.3 -3.9 0.8923 5 5 6.181 200 CXB-NE (CFA) vs. DF-NE (CFA) 3.4 7.7 -4.3 0.8923 5 5 6.815 200 CFA vs. CXB (CFA) 8 7.3 0.7 0.8923 5 5 1.109 200 CFA vs. DF-NE (CFA) 8 7.7 0.3 0.8923 5 5 0.4755 200 CXB (CFA) vs. DF-NE (CFA) 7.3 7.7 -0.4 0.8923 5 5 0.634 200

6 Saline vs. CXB-NE (CFA) 2.6 2.6 0 0.8923 5 5 0 200 Saline vs. CFA 2.6 6.4 -3.8 0.8923 5 5 6.023 200 Saline vs. CXB (CFA) 2.6 8.6 -6 0.8923 5 5 9.51 200 Saline vs. DF-NE (CFA) 2.6 8 -5.4 0.8923 5 5 8.559 200 CXB-NE (CFA) vs. CFA 2.6 6.4 -3.8 0.8923 5 5 6.023 200 CXB-NE (CFA) vs. CXB (CFA) 2.6 8.6 -6 0.8923 5 5 9.51 200 CXB-NE (CFA) vs. DF-NE (CFA) 2.6 8 -5.4 0.8923 5 5 8.559 200 CFA vs. CXB (CFA) 6.4 8.6 -2.2 0.8923 5 5 3.487 200 CFA vs. DF-NE (CFA) 6.4 8 -1.6 0.8923 5 5 2.536 200 CXB (CFA) vs. DF-NE (CFA) 8.6 8 0.6 0.8923 5 5 0.951 200

11 Saline vs. CXB-NE (CFA) 2.3 2.3 0 0.8923 5 5 0 200 Saline vs. CFA 2.3 6.6 -4.3 0.8923 5 5 6.815 200 Saline vs. CXB (CFA) 2.3 5.8 -3.5 0.8923 5 5 5.547 200 Saline vs. DF-NE (CFA) 2.3 5.8 -3.5 0.8923 5 5 5.547 200 CXB-NE (CFA) vs. CFA 2.3 6.6 -4.3 0.8923 5 5 6.815 200 CXB-NE (CFA) vs. CXB (CFA) 2.3 5.8 -3.5 0.8923 5 5 5.547 200 CXB-NE (CFA) vs. DF-NE (CFA) 2.3 5.8 -3.5 0.8923 5 5 5.547 200 CFA vs. CXB (CFA) 6.6 5.8 0.8 0.8923 5 5 1.268 200 CFA vs. DF-NE (CFA) 6.6 5.8 0.8 0.8923 5 5 1.268 200 CXB (CFA) vs. DF-NE (CFA) 5.8 5.8 0 0.8923 5 5 0 200

12 Saline vs. CXB-NE (CFA) 1.6 1.6 0 0.8923 5 5 0 200 Saline vs. CFA 1.6 8 -6.4 0.8923 5 5 10.14 200 Saline vs. CXB (CFA) 1.6 8.2 -6.6 0.8923 5 5 10.46 200 Saline vs. DF-NE (CFA) 1.6 6 -4.4 0.8923 5 5 6.974 200 CXB-NE (CFA) vs. CFA 1.6 8 -6.4 0.8923 5 5 10.14 200 CXB-NE (CFA) vs. CXB (CFA) 1.6 8.2 -6.6 0.8923 5 5 10.46 200 CXB-NE (CFA) vs. DF-NE (CFA) 1.6 6 -4.4 0.8923 5 5 6.974 200 CFA vs. CXB (CFA) 8 8.2 -0.2 0.8923 5 5 0.317 200 CFA vs. DF-NE (CFA) 8 6 2 0.8923 5 5 3.17 200 CXB (CFA) vs. DF-NE (CFA) 8.2 6 2.2 0.8923 5 5 3.487 200

18 Saline vs. CXB-NE (CFA) 1.8 1.8 0 0.8923 5 5 0 200 Saline vs. CFA 1.8 9.3 -7.5 0.8923 5 5 11.89 200 Saline vs. CXB (CFA) 1.8 7 -5.2 0.8923 5 5 8.242 200 Saline vs. DF-NE (CFA) 1.8 6.4 -4.6 0.8923 5 5 7.291 200 CXB-NE (CFA) vs. CFA 1.8 9.3 -7.5 0.8923 5 5 11.89 200 CXB-NE (CFA) vs. CXB (CFA) 1.8 7 -5.2 0.8923 5 5 8.242 200 CXB-NE (CFA) vs. DF-NE (CFA) 1.8 6.4 -4.6 0.8923 5 5 7.291 200 CFA vs. CXB (CFA) 9.3 7 2.3 0.8923 5 5 3.645 200 CFA vs. DF-NE (CFA) 9.3 6.4 2.9 0.8923 5 5 4.596 200 CXB (CFA) vs. DF-NE (CFA) 7 6.4 0.6 0.8923 5 5 0.951 200

24 Saline vs. CXB-NE (CFA) 2.8 2.8 0 0.8923 5 5 0 200 Saline vs. CFA 2.8 6.5 -3.7 0.8923 5 5 5.864 200 Saline vs. CXB (CFA) 2.8 5 -2.2 0.8923 5 5 3.487 200 Saline vs. DF-NE (CFA) 2.8 5 -2.2 0.8923 5 5 3.487 200 CXB-NE (CFA) vs. CFA 2.8 6.5 -3.7 0.8923 5 5 5.864 200 CXB-NE (CFA) vs. CXB (CFA) 2.8 5 -2.2 0.8923 5 5 3.487 200 CXB-NE (CFA) vs. DF-NE (CFA) 2.8 5 -2.2 0.8923 5 5 3.487 200 CFA vs. CXB (CFA) 6.5 5 1.5 0.8923 5 5 2.377 200 CFA vs. DF-NE (CFA) 6.5 5 1.5 0.8923 5 5 2.377 200 CXB (CFA) vs. DF-NE (CFA) 5 5 0 0.8923 5 5 0 200

32 Saline vs. CXB-NE (CFA) 1.4 1.4 0 0.8923 5 5 0 200 Saline vs. CFA 1.4 4.8 -3.4 0.8923 5 5 5.389 200 Saline vs. CXB (CFA) 1.4 3.6 -2.2 0.8923 5 5 3.487 200 Saline vs. DF-NE (CFA) 1.4 4 -2.6 0.8923 5 5 4.121 200 CXB-NE (CFA) vs. CFA 1.4 4.8 -3.4 0.8923 5 5 5.389 200 CXB-NE (CFA) vs. CXB (CFA) 1.4 3.6 -2.2 0.8923 5 5 3.487 200 CXB-NE (CFA) vs. DF-NE (CFA) 1.4 4 -2.6 0.8923 5 5 4.121 200 CFA vs. CXB (CFA) 4.8 3.6 1.2 0.8923 5 5 1.902 200 CFA vs. DF-NE (CFA) 4.8 4 0.8 0.8923 5 5 1.268 200 CXB (CFA) vs. DF-NE (CFA) 3.6 4 -0.4 0.8923 5 5 0.634 200

247248249250251252253254255256257

A B C D E F G H I40 Saline vs. CXB-NE (CFA) 4 4 0 0.8923 5 5 0 200 Saline vs. CFA 4 5.4 -1.4 0.8923 5 5 2.219 200 Saline vs. CXB (CFA) 4 4 0 0.8923 5 5 0 200 Saline vs. DF-NE (CFA) 4 4.6 -0.6 0.8923 5 5 0.951 200 CXB-NE (CFA) vs. CFA 4 5.4 -1.4 0.8923 5 5 2.219 200 CXB-NE (CFA) vs. CXB (CFA) 4 4 0 0.8923 5 5 0 200 CXB-NE (CFA) vs. DF-NE (CFA) 4 4.6 -0.6 0.8923 5 5 0.951 200 CFA vs. CXB (CFA) 5.4 4 1.4 0.8923 5 5 2.219 200 CFA vs. DF-NE (CFA) 5.4 4.6 0.8 0.8923 5 5 1.268 200 CXB (CFA) vs. DF-NE (CFA) 4 4.6 -0.6 0.8923 5 5 0.951 200

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A B C D E F G H IVon Frey ASSAY in FEMALE MICE

Two-way ANOVA OrdinaryAlpha 0.05

Source of Variation % of total varia P value P value summaSignificant?Interaction 11.46 < 0.0001 **** YesRow Factor 44.57 < 0.0001 **** YesColumn Factor 27.63 < 0.0001 **** Yes

ANOVA table SS DF MS F (DFn, DFd) P valueInteraction 247.3 36 6.87 F (36, 200) = 3.8P < 0.0001Row Factor 962.3 9 106.9 F (9, 200) = 60.6P < 0.0001Column Factor 596.5 4 149.1 F (4, 200) = 84.5P < 0.0001Residual 352.8 200 1.764

Number of missing values 0

Within each row, compare columns (simple effects within rows)

Number of families 10Number of comparisons per family 10Alpha 0.05

Tukey's multiple comparisons test Mean Diff. 95% CI of diff. Significant? Summary

Day 1Saline vs. CXB-NE (CFA) -1.6 -3.913 to 0.7125 No nsSaline vs. CFA -5.9 -8.213 to -3.587 Yes ****Saline vs. CXB (CFA) -4.2 -6.513 to -1.887 Yes ****Saline vs. DF-NE (CFA) -6 -8.313 to -3.687 Yes ****CXB-NE (CFA) vs. CFA -4.3 -6.613 to -1.987 Yes ****CXB-NE (CFA) vs. CXB (CFA) -2.6 -4.913 to -0.2875 Yes *CXB-NE (CFA) vs. DF-NE (CFA) -4.4 -6.713 to -2.087 Yes ****CFA vs. CXB (CFA) 1.7 -0.6125 to 4.013 No nsCFA vs. DF-NE (CFA) -0.1 -2.413 to 2.213 No nsCXB (CFA) vs. DF-NE (CFA) -1.8 -4.113 to 0.5125 No ns

Day 3Saline vs. CXB-NE (CFA) -1.4 -3.713 to 0.9125 No nsSaline vs. CFA -5 -7.313 to -2.687 Yes ****Saline vs. CXB (CFA) -5.2 -7.513 to -2.887 Yes ****Saline vs. DF-NE (CFA) -5 -7.313 to -2.687 Yes ****CXB-NE (CFA) vs. CFA -3.6 -5.913 to -1.287 Yes ***CXB-NE (CFA) vs. CXB (CFA) -3.8 -6.113 to -1.487 Yes ***CXB-NE (CFA) vs. DF-NE (CFA) -3.6 -5.913 to -1.287 Yes ***CFA vs. CXB (CFA) -0.2 -2.513 to 2.113 No nsCFA vs. DF-NE (CFA) 0 -2.313 to 2.313 No nsCXB (CFA) vs. DF-NE (CFA) 0.2 -2.113 to 2.513 No ns

Day 6Saline vs. CXB-NE (CFA) -3.3 -5.613 to -0.9875 Yes **Saline vs. CFA -5.9 -8.213 to -3.587 Yes ****Saline vs. CXB (CFA) -6.5 -8.813 to -4.187 Yes ****Saline vs. DF-NE (CFA) -6.9 -9.213 to -4.587 Yes ****CXB-NE (CFA) vs. CFA -2.6 -4.913 to -0.2875 Yes *CXB-NE (CFA) vs. CXB (CFA) -3.2 -5.513 to -0.8875 Yes **CXB-NE (CFA) vs. DF-NE (CFA) -3.6 -5.913 to -1.287 Yes ***CFA vs. CXB (CFA) -0.6 -2.913 to 1.713 No nsCFA vs. DF-NE (CFA) -1 -3.313 to 1.313 No nsCXB (CFA) vs. DF-NE (CFA) -0.4 -2.713 to 1.913 No ns

Day 11Saline vs. CXB-NE (CFA) -2.4 -4.713 to -0.08750 Yes *Saline vs. CFA -4.8 -7.113 to -2.487 Yes ****Saline vs. CXB (CFA) -5.2 -7.513 to -2.887 Yes ****Saline vs. DF-NE (CFA) -4.6 -6.913 to -2.287 Yes ****CXB-NE (CFA) vs. CFA -2.4 -4.713 to -0.08750 Yes *CXB-NE (CFA) vs. CXB (CFA) -2.8 -5.113 to -0.4875 Yes **CXB-NE (CFA) vs. DF-NE (CFA) -2.2 -4.513 to 0.1125 No nsCFA vs. CXB (CFA) -0.4 -2.713 to 1.913 No nsCFA vs. DF-NE (CFA) 0.2 -2.113 to 2.513 No nsCXB (CFA) vs. DF-NE (CFA) 0.6 -1.713 to 2.913 No ns

Day 12Saline vs. CXB-NE (CFA) -3.6 -5.913 to -1.287 Yes ***Saline vs. CFA -4.2 -6.513 to -1.887 Yes ****Saline vs. CXB (CFA) -4.2 -6.513 to -1.887 Yes ****Saline vs. DF-NE (CFA) -5.6 -7.913 to -3.287 Yes ****CXB-NE (CFA) vs. CFA -0.6 -2.913 to 1.713 No nsCXB-NE (CFA) vs. CXB (CFA) -0.6 -2.913 to 1.713 No nsCXB-NE (CFA) vs. DF-NE (CFA) -2 -4.313 to 0.3125 No nsCFA vs. CXB (CFA) 0 -2.313 to 2.313 No nsCFA vs. DF-NE (CFA) -1.4 -3.713 to 0.9125 No nsCXB (CFA) vs. DF-NE (CFA) -1.4 -3.713 to 0.9125 No ns

18Saline vs. CXB-NE (CFA) -4.2 -6.513 to -1.887 Yes ****Saline vs. CFA -5 -7.313 to -2.687 Yes ****Saline vs. CXB (CFA) -4.8 -7.113 to -2.487 Yes ****Saline vs. DF-NE (CFA) -3.4 -5.713 to -1.087 Yes ***CXB-NE (CFA) vs. CFA -0.8 -3.113 to 1.513 No ns

93949596979899

100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172173174175176177178179180181182183184

A B C D E F G H ICXB-NE (CFA) vs. CXB (CFA) -0.6 -2.913 to 1.713 No nsCXB-NE (CFA) vs. DF-NE (CFA) 0.8 -1.513 to 3.113 No nsCFA vs. CXB (CFA) 0.2 -2.113 to 2.513 No nsCFA vs. DF-NE (CFA) 1.6 -0.7125 to 3.913 No nsCXB (CFA) vs. DF-NE (CFA) 1.4 -0.9125 to 3.713 No ns

24Saline vs. CXB-NE (CFA) -4.1 -6.413 to -1.787 Yes ****Saline vs. CFA -4.7 -7.013 to -2.387 Yes ****Saline vs. CXB (CFA) -2.8 -5.113 to -0.4875 Yes **Saline vs. DF-NE (CFA) -3.7 -6.013 to -1.387 Yes ***CXB-NE (CFA) vs. CFA -0.6 -2.913 to 1.713 No nsCXB-NE (CFA) vs. CXB (CFA) 1.3 -1.013 to 3.613 No nsCXB-NE (CFA) vs. DF-NE (CFA) 0.4 -1.913 to 2.713 No nsCFA vs. CXB (CFA) 1.9 -0.4125 to 4.213 No nsCFA vs. DF-NE (CFA) 1 -1.313 to 3.313 No nsCXB (CFA) vs. DF-NE (CFA) -0.9 -3.213 to 1.413 No ns

32Saline vs. CXB-NE (CFA) -3.6 -5.913 to -1.287 Yes ***Saline vs. CFA -3.3 -5.613 to -0.9875 Yes **Saline vs. CXB (CFA) -1.3 -3.613 to 1.013 No nsSaline vs. DF-NE (CFA) -2.6 -4.913 to -0.2875 Yes *CXB-NE (CFA) vs. CFA 0.3 -2.013 to 2.613 No nsCXB-NE (CFA) vs. CXB (CFA) 2.3 -0.01250 to 4.613 No nsCXB-NE (CFA) vs. DF-NE (CFA) 1 -1.313 to 3.313 No nsCFA vs. CXB (CFA) 2 -0.3125 to 4.313 No nsCFA vs. DF-NE (CFA) 0.7 -1.613 to 3.013 No nsCXB (CFA) vs. DF-NE (CFA) -1.3 -3.613 to 1.013 No ns

40Saline vs. CXB-NE (CFA) -2.5 -4.813 to -0.1875 Yes *Saline vs. CFA -3.6 -5.913 to -1.287 Yes ***Saline vs. CXB (CFA) -1.6 -3.913 to 0.7125 No nsSaline vs. DF-NE (CFA) -2.2 -4.513 to 0.1125 No nsCXB-NE (CFA) vs. CFA -1.1 -3.413 to 1.213 No nsCXB-NE (CFA) vs. CXB (CFA) 0.9 -1.413 to 3.213 No nsCXB-NE (CFA) vs. DF-NE (CFA) 0.3 -2.013 to 2.613 No nsCFA vs. CXB (CFA) 2 -0.3125 to 4.313 No nsCFA vs. DF-NE (CFA) 1.4 -0.9125 to 3.713 No nsCXB (CFA) vs. DF-NE (CFA) -0.6 -2.913 to 1.713 No ns

Test details Mean 1 Mean 2 Mean Diff. SE of diff. N1 N2 q DF

1Saline vs. CXB-NE (CFA) 1.8 3.4 -1.6 0.84 5 5 2.694 200Saline vs. CFA 1.8 7.7 -5.9 0.84 5 5 9.933 200Saline vs. CXB (CFA) 1.8 6 -4.2 0.84 5 5 7.071 200Saline vs. DF-NE (CFA) 1.8 7.8 -6 0.84 5 5 10.1 200CXB-NE (CFA) vs. CFA 3.4 7.7 -4.3 0.84 5 5 7.239 200CXB-NE (CFA) vs. CXB (CFA) 3.4 6 -2.6 0.84 5 5 4.377 200CXB-NE (CFA) vs. DF-NE (CFA) 3.4 7.8 -4.4 0.84 5 5 7.407 200CFA vs. CXB (CFA) 7.7 6 1.7 0.84 5 5 2.862 200CFA vs. DF-NE (CFA) 7.7 7.8 -0.1 0.84 5 5 0.1683 200CXB (CFA) vs. DF-NE (CFA) 6 7.8 -1.8 0.84 5 5 3.03 200

3Saline vs. CXB-NE (CFA) 3.8 5.2 -1.4 0.84 5 5 2.357 200Saline vs. CFA 3.8 8.8 -5 0.84 5 5 8.417 200Saline vs. CXB (CFA) 3.8 9 -5.2 0.84 5 5 8.754 200Saline vs. DF-NE (CFA) 3.8 8.8 -5 0.84 5 5 8.417 200CXB-NE (CFA) vs. CFA 5.2 8.8 -3.6 0.84 5 5 6.061 200CXB-NE (CFA) vs. CXB (CFA) 5.2 9 -3.8 0.84 5 5 6.397 200CXB-NE (CFA) vs. DF-NE (CFA) 5.2 8.8 -3.6 0.84 5 5 6.061 200CFA vs. CXB (CFA) 8.8 9 -0.2 0.84 5 5 0.3367 200CFA vs. DF-NE (CFA) 8.8 8.8 0 0.84 5 5 0 200CXB (CFA) vs. DF-NE (CFA) 9 8.8 0.2 0.84 5 5 0.3367 200

6Saline vs. CXB-NE (CFA) 2.3 5.6 -3.3 0.84 5 5 5.556 200Saline vs. CFA 2.3 8.2 -5.9 0.84 5 5 9.933 200Saline vs. CXB (CFA) 2.3 8.8 -6.5 0.84 5 5 10.94 200Saline vs. DF-NE (CFA) 2.3 9.2 -6.9 0.84 5 5 11.62 200CXB-NE (CFA) vs. CFA 5.6 8.2 -2.6 0.84 5 5 4.377 200CXB-NE (CFA) vs. CXB (CFA) 5.6 8.8 -3.2 0.84 5 5 5.387 200CXB-NE (CFA) vs. DF-NE (CFA) 5.6 9.2 -3.6 0.84 5 5 6.061 200CFA vs. CXB (CFA) 8.2 8.8 -0.6 0.84 5 5 1.01 200CFA vs. DF-NE (CFA) 8.2 9.2 -1 0.84 5 5 1.683 200CXB (CFA) vs. DF-NE (CFA) 8.8 9.2 -0.4 0.84 5 5 0.6734 200

11Saline vs. CXB-NE (CFA) 2 4.4 -2.4 0.84 5 5 4.04 200Saline vs. CFA 2 6.8 -4.8 0.84 5 5 8.081 200Saline vs. CXB (CFA) 2 7.2 -5.2 0.84 5 5 8.754 200Saline vs. DF-NE (CFA) 2 6.6 -4.6 0.84 5 5 7.744 200CXB-NE (CFA) vs. CFA 4.4 6.8 -2.4 0.84 5 5 4.04 200CXB-NE (CFA) vs. CXB (CFA) 4.4 7.2 -2.8 0.84 5 5 4.714 200CXB-NE (CFA) vs. DF-NE (CFA) 4.4 6.6 -2.2 0.84 5 5 3.704 200CFA vs. CXB (CFA) 6.8 7.2 -0.4 0.84 5 5 0.6734 200CFA vs. DF-NE (CFA) 6.8 6.6 0.2 0.84 5 5 0.3367 200

185186187188189190191192193194195196197198199200201202203204205206207208209210211212213214215216217218219220221222223224225226227228229230231232233234235236237238239240241242243244245

A B C D E F G H ICXB (CFA) vs. DF-NE (CFA) 7.2 6.6 0.6 0.84 5 5 1.01 200

12Saline vs. CXB-NE (CFA) 2.6 6.2 -3.6 0.84 5 5 6.061 200Saline vs. CFA 2.6 6.8 -4.2 0.84 5 5 7.071 200Saline vs. CXB (CFA) 2.6 6.8 -4.2 0.84 5 5 7.071 200Saline vs. DF-NE (CFA) 2.6 8.2 -5.6 0.84 5 5 9.428 200CXB-NE (CFA) vs. CFA 6.2 6.8 -0.6 0.84 5 5 1.01 200CXB-NE (CFA) vs. CXB (CFA) 6.2 6.8 -0.6 0.84 5 5 1.01 200CXB-NE (CFA) vs. DF-NE (CFA) 6.2 8.2 -2 0.84 5 5 3.367 200CFA vs. CXB (CFA) 6.8 6.8 0 0.84 5 5 0 200CFA vs. DF-NE (CFA) 6.8 8.2 -1.4 0.84 5 5 2.357 200CXB (CFA) vs. DF-NE (CFA) 6.8 8.2 -1.4 0.84 5 5 2.357 200

18Saline vs. CXB-NE (CFA) 3.4 7.6 -4.2 0.84 5 5 7.071 200Saline vs. CFA 3.4 8.4 -5 0.84 5 5 8.417 200Saline vs. CXB (CFA) 3.4 8.2 -4.8 0.84 5 5 8.081 200Saline vs. DF-NE (CFA) 3.4 6.8 -3.4 0.84 5 5 5.724 200CXB-NE (CFA) vs. CFA 7.6 8.4 -0.8 0.84 5 5 1.347 200CXB-NE (CFA) vs. CXB (CFA) 7.6 8.2 -0.6 0.84 5 5 1.01 200CXB-NE (CFA) vs. DF-NE (CFA) 7.6 6.8 0.8 0.84 5 5 1.347 200CFA vs. CXB (CFA) 8.4 8.2 0.2 0.84 5 5 0.3367 200CFA vs. DF-NE (CFA) 8.4 6.8 1.6 0.84 5 5 2.694 200CXB (CFA) vs. DF-NE (CFA) 8.2 6.8 1.4 0.84 5 5 2.357 200

24Saline vs. CXB-NE (CFA) 3.5 7.6 -4.1 0.84 5 5 6.902 200Saline vs. CFA 3.5 8.2 -4.7 0.84 5 5 7.912 200Saline vs. CXB (CFA) 3.5 6.3 -2.8 0.84 5 5 4.714 200Saline vs. DF-NE (CFA) 3.5 7.2 -3.7 0.84 5 5 6.229 200CXB-NE (CFA) vs. CFA 7.6 8.2 -0.6 0.84 5 5 1.01 200CXB-NE (CFA) vs. CXB (CFA) 7.6 6.3 1.3 0.84 5 5 2.189 200CXB-NE (CFA) vs. DF-NE (CFA) 7.6 7.2 0.4 0.84 5 5 0.6734 200CFA vs. CXB (CFA) 8.2 6.3 1.9 0.84 5 5 3.199 200CFA vs. DF-NE (CFA) 8.2 7.2 1 0.84 5 5 1.683 200CXB (CFA) vs. DF-NE (CFA) 6.3 7.2 -0.9 0.84 5 5 1.515 200

32Saline vs. CXB-NE (CFA) 3.7 7.3 -3.6 0.84 5 5 6.061 200Saline vs. CFA 3.7 7 -3.3 0.84 5 5 5.556 200Saline vs. CXB (CFA) 3.7 5 -1.3 0.84 5 5 2.189 200Saline vs. DF-NE (CFA) 3.7 6.3 -2.6 0.84 5 5 4.377 200CXB-NE (CFA) vs. CFA 7.3 7 0.3 0.84 5 5 0.505 200CXB-NE (CFA) vs. CXB (CFA) 7.3 5 2.3 0.84 5 5 3.872 200CXB-NE (CFA) vs. DF-NE (CFA) 7.3 6.3 1 0.84 5 5 1.683 200CFA vs. CXB (CFA) 7 5 2 0.84 5 5 3.367 200CFA vs. DF-NE (CFA) 7 6.3 0.7 0.84 5 5 1.178 200CXB (CFA) vs. DF-NE (CFA) 5 6.3 -1.3 0.84 5 5 2.189 200

40Saline vs. CXB-NE (CFA) 4.2 6.7 -2.5 0.84 5 5 4.209 200Saline vs. CFA 4.2 7.8 -3.6 0.84 5 5 6.061 200Saline vs. CXB (CFA) 4.2 5.8 -1.6 0.84 5 5 2.694 200Saline vs. DF-NE (CFA) 4.2 6.4 -2.2 0.84 5 5 3.704 200CXB-NE (CFA) vs. CFA 6.7 7.8 -1.1 0.84 5 5 1.852 200CXB-NE (CFA) vs. CXB (CFA) 6.7 5.8 0.9 0.84 5 5 1.515 200CXB-NE (CFA) vs. DF-NE (CFA) 6.7 6.4 0.3 0.84 5 5 0.505 200CFA vs. CXB (CFA) 7.8 5.8 2 0.84 5 5 3.367 200CFA vs. DF-NE (CFA) 7.8 6.4 1.4 0.84 5 5 2.357 200CXB (CFA) vs. DF-NE (CFA) 5.8 6.4 -0.6 0.84 5 5 1.01 200