International Journal of Pharmaceutics 640 (2023) 123035

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Aqueous cannabidiol β-cyclodextrin complexed polymeric micelle nasal

spray to attenuate in vitro and ex vivo SARS-CoV-2-induced cytokine storms
Narumon Changsan a, Somchai Sawatdee b, Roongnapa Suedee c,
Charisopon Chunhachaichana d, Teerapol Srichana d, *
a
College of Pharmacy, Rangsit University, Pathumtani 12000, Thailand
b
Drug and Cosmetics Excellence Center and School of Pharmacy, Walailak University, Thasala, Nakhon Si Thammarat 80161, Thailand
c
Molecular Recognition Materials Research Unit, Nanotec-PSU Center of Excellence on Drug Delivery System Department of Pharmaceutical Chemistry, Faculty of
Pharmaceutical Sciences, Prince of Songkla University Hat Yai, Songkhla 90112, Thailand
d
Drug Delivery System Excellence Center, Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai,
Songkhla 90112, Thailand

A R T I C L E I N F O A B S T R A C T

Keywords: Cannabidiol (CBD) has a number of biological effects by acting on the cannabinoid receptors CB1 and CB2. CBD
Cannabidiol may be involved in anti-inflammatory processes via CB1 and CB2 receptors, resulting in a decrease of pro-
β-Cyclodextrin inflammatory cytokines. However, CBD’s poor aqueous solubility is a major issue in pharmaceutical applica­
Polymeric micelles
tions. The aim of the present study was to develop and evaluate a CBD nasal spray solution. A water-soluble CBD
Nasal spray solution
SARS-CoV-2
was prepared by complexation with β-cyclodextrin (β-CD) at a stoichiometric ratio of 1:1 and forming polymeric
Pro-inflammatory cytokine micelles using poloxamer 407. The mixture was then lyophilized and characterized using FT-IR, DSC, and TGA.
CBD-β-CD complex-polymeric micelles were formulated for nasal spray drug delivery. The physicochemical
properties of the CBD-β-CD complex-polymeric micelle nasal spray solution (CBD-β-CDPM-NS) were assessed.
The results showed that the CBD content in the CBD-β-CD complex polymeric micelle powder was 102.1 ± 0.5%
labeled claim. The CBD-β-CDPM-NS was a clear colorless isotonic solution. The particle size, zeta potential, pH
value, and viscosity were 111.9 ± 0.7 nm, 0.8 ± 0.1 mV, 6.02 ± 0.02, and 12.04 ± 2.64 cP, respectively. This
formulation was stable over six months at ambient temperature. The CBD from CBD-β-CDPM-NS rapidly released
to 100% within 1 min. Ex vivo permeation studies of CBD-β-CDPM-NS through porcine nasal mucosa revealed a
permeation rate of 4.8 μg/cm2/min, which indicated that CBD was effective in penetrating nasal epithelial cells.
CBD-β-CDPM-NS was tested for its efficacy and safety in terms of cytokine production from nasal immune cells
and toxicity to nasal epithelial cells. The CBD-β-CDPM-NS was not toxic to nasal epithelial at the concentration of
CBD equivalent to 3.125–50 μg/mL. When the formulation was subjected to bioactivity testing against monocyte-
like macrophage cells, it proved that the CBD-β-CDPM-NS has the potential to inhibit inflammatory cytokines.
CBD-β-CDPM-NS demonstrated the formulation’s ability to reduce the cytokine produced by S-RBD stimulation
in ex vivo porcine nasal mucosa in both preventative and therapeutic modes.

Abbreviations: β-CD, beta-cyclodextrin; γ-CD, gamma-cyclodextrin; ACE2, angiotensin-converting enzyme 2; CB1, endo-cannabinoid receptors 1; CB2, endo-
cannabinoid receptors 2; CBD, cannabidiol; CBD-β-CD, cannabidiol and beta cyclodextrin complex; CBD-β-CDPM, cannabidiol and beta cyclodextrin complex
polymeric micelles; CBD-β-CDPM-NS, cannabidiol and beta cyclodextrin complex polymeric micelle nasal spray solution; CD, cyclodextrin; COVID-19, Corona virus
disease-19; DM-β-CD, dimethyl beta cyclodextrin; DMEM, Dulbecco’s Modified Eagle Medium; DSC, differential scanning calorimetry; ELISA, enzyme-linked
immunosorbent assay; FBS, fetal bovine serum; FT-IR, Fourier transform infrared spectroscopy; HEC, hydroxyethyl cellulose; HP-β-CD, hydroxypropyl beta cyclo­
dextrin; HPLC, high-performance liquid chromatography; IL-1β, interleukin-1β; IL-18, interleukin-18; IL-6, interleukin-6; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-
diphenyl-tetrazolium bromide; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; S-RBD, recombinant spike receptor binding domain of SARS-CoV-
2; TGA, thermogravimetric analysis; TNF-α, tumor necrosis factor-α.
* Corresponding author.
E-mail address: teerapol.s@psu.ac.th (T. Srichana).

https://doi.org/10.1016/j.ijpharm.2023.123035
Received 11 December 2022; Received in revised form 23 April 2023; Accepted 5 May 2023
Available online 12 May 2023
0378-5173/© 2023 Elsevier B.V. All rights reserved.
N. Changsan et al.
1. Introduction
Coronavirus disease 2019 (COVID-19) is an acute respiratory disease
caused by the severe acute respiratory syndrome coronavirus-2 (SARS-
CoV-2) (Anil et al., 2021). Clinical manifestation might range from mild
to severe, or it can be asymptomatic. COVID-19 can cause pneumonia,
acute respiratory distress syndrome, and multiorgan failure in severe
cases.
Respiratory distress syndrome, the major cause of COVID-19 death,
is characterized by the release of pro-inflammatory cytokine storms,
such as interleukin 6 (IL-6) and interleukin 1β (IL-1β) as well as inter­
leukin 18 (IL-18). In critically ill patients, antiviral medications may be
insufficient to control the cytokine storm and respiratory distress. It is
consequently critical to develop novel therapeutic alternatives that can
decrease the cytokine storm to reduce the adverse outcome (Esposito
et al., 2020; Ramasamy and Subbian, 2019).
Cannabidiol (CBD) is a non-psychoactive phytocannabinoid synthe­
sized by Cannabis sativa (Vallée, 2022; Esposito et al., 2020). CBD is
regarded as one of the most fascinating emerging molecules in the
discipline of pharmacology since it has many therapeutic effects related
to its anticonvulsant, sedative, hypnotic, antipsychotic, anticancer, anti-
inflammatory, and neuroprotective properties (Esposito et al., 2020).
CB1 and CB2 endocannabinoid receptors are involved in anti-
inflammatory activities. CBD, as an agonist of the CB2 receptor, has a
wide spectrum of immunomodulatory and anti-inflammatory proper­
ties, and it can reduce the unregulated cytokine production that causes
acute lung injury. CBD has recently been considered as a possible
medication in the treatment of SARS-CoV-2 because it possesses anti-
inflammatory and immune-suppressive properties in pre-clinical
COVID-19 models. This compound has the potential to reduce the
release of pro-inflammatory cytokines, which are responsible for
inflammation during SARS-CoV-2 infection (Vallée, 2022). In addition
to anti-inflammatory effects, CBD has the ability to reduce the severity
and progression of the disease by down-regulating the expression of
angiotensin-converting enzyme 2 (ACE2) and transmembrane serine
protease 2, which are crucial viral gateways for SARS-CoV-2 cellular
invasion, thereby limiting SARS-CoV-2 entry into vulnerable hosts.
Furthermore, CBD, as a peroxisome proliferator-activated receptor-γ
agonist, has a direct antiviral effect and is a regulator of fibroblast/
myofibroblast activation and can limit the formation of pulmonary
fibrosis, thereby improving lung function in recovered patients (Holst
et al., 2022; Esposito et al., 2020; Janecki et al., 2022).
The nasal route can be employed for local or systemic treatment. The
nasal cavity is advantageous for systemic absorption due to its high
vascularization, wide surface area-to-volume ratio, and additional
benefits over alternative routes (Pires, et al., 2022). However, intranasal
delivery also has its limitations since only a small volume (maximum
150–200 μL in humans) can be applied, therefore potent drugs are
required. In addition, it is complicated developing drugs with poor water
solubility at high potency without using significant amounts of poten­
tially harmful cosolvents or surfactants. CBD is classified as bio­
pharmaceutics classification system Class II with very poor water
solubility with a solubility of 0.1 μg/mL in water (Amanat et al., 2020),
35 mg/mL in ethanol (Cayman Chemical, 2015), and a bioavailability of
around 6% (Koch et al., 2020). This restricts its application as a nasal
solution (Li et al., 2021).
CBD must be in a solution form at the site of absorption; conse­
quently, CBD in the form of better aqueous solubility may result in
greater CBD absorption. Particularly, a more water-soluble CBD would
have a wider range of applications in aqueous formulations like solu­
tions, ocular drops, injections, and aerosols. CBD nasal spray was
developed in various research projects and employed for various ap­
plications including anti-inflammatory and epilepsy treatments (Paudel
et al., 2010; Preve Ceutical Medical Inc, 2022; Patent WO2017/
208072A2). CBD intranasal administration has rapid absorption
compared to oral delivery (Polidoro et al., 2022).
2
International Journal of Pharmaceutics 640 (2023) 123035
Improved water solubility using inclusion complexes with cyclo­
dextrins (CDs) has been widely reported (Carneiro et al., 2019;
Hădărugă et al., 2019; Fenyvesi et al., 2020 Păduraru et al., 2022).
Several research studies reported CBD complexed with α-CD, γ-CD,
β-CD, HP-β-CD, and DM-β-CD (Lv et al., 2019; Li et al., 2021; Nivor­
ozhkin, 2019; Ramalho et al., 2021; Mannila et al., 2007; Hatziagapiou
et al., 2022). CBD/DM-β-CD and CBD/β-CD displayed strong in­
teractions (Li et al., 2021). The water solubility and dissolution rates of
the CBD/DM-β-CD and CBD/β-CD complexes were 614-fold and 17-fold,
respectively (Li et al., 2021). The bioavailability and pharmacokinetic
parameters of CBD/β-CD inclusion complexes showed that the CBD
plasma concentration was higher than orally administered ethanolic
CBD (Ramalho et al., 2021).
In this study, we developed a nasal aqueous solution of CBD by
complexing with β-CD to increase water solubility and then formed
polymeric micelles with poloxamer. Poloxamer refers to nonionic tri­
block copolymers formed by polar (polyethylene oxide) and non-polar
(polypropylene oxide) blocks (Kabanov, et al., 2002). Poloxamer 407
has a molecular weight of approximately 12,600 and is highly soluble in
water (Nagy et al., 2020; Kabanov, et al., 2002). Poloxamer 407 is
widely used in pharmaceuticals as a solubilizer, release modifier, and
suspending agent (Rowe et al., 2009). Furthermore, poloxamer 407 has
thermo-reversible and bioadhesive properties (Dumortier et al., 2006).
Poloxamer in aqueous solutions has an amphiphilic nature that leads to
aggregation of their molecules to form polymeric micelles with a hy­
drophobic interior composed of polypropylene oxide blocks and a hy­
drophilic exterior composed of polyethylene oxide blocks (Kabanov,
et al., 2002). Poloxamer forms polymeric micelles by self-assembly of
amphiphilic block copolymers in aqueous solutions (Ghezzi et al., 2021).
In a diluted aqueous solution, amphiphilic molecules exist as amphi­
philes. The critical micelle concentration of poloxamer 407 is 2.8 × 10− 6
M (~0.0342 mg/mL) and the hydrophilic-lipophilic balance value is 22
(Suksiriworapong et al., 2015; Nagy et al., 2020). Drugs can be encap­
sulated in the polymeric micelles during their formation depending on
the method used for the preparation and physicochemical characteris­
tics of the drug (Ghezzi et al., 2021).
The developed CBD-β-cyclodextrin complexed polymeric micelle
nasal spray formulation was tested for nasal cell cytotoxicity and the
ability to suppress pro-inflammatory cytokines produced from recom­
binant spike receptor binding domain (S-RBD) of SARS-CoV-2 induced
monocyte-like macrophages and ex vivo porcine mucosa explant tissue.
2. Material and methods
2.1. Materials
Cannabidiol was provided gratis by Quantum Biotech Co., Ltd.
(Pathum Thani, Thailand) with 99% purity. β-cyclodextrin (Cavamax
W7 FG) was purchased from Wacker Chemical Corporation, Michigan,
USA. Poloxamer 407 (Kolliphor P407) was purchased from BASF Can­
ada Inc., Mississauga, Canada. Hydroxyethyl cellulose (HEC) was ob­
tained from Chemipan Corporation Co., Ltd., Bangkok, Thailand).
Glycerin was procured from Chanjao Longevity Co., Ltd, Bangkok,
Thailand. Sodium citrate was purchased from Krungthepchemi Co., Ltd.,
Bangkok, Thailand. Citric acid monohydrate was obtained from RFCL
Limited, New Delhi, India. Benzalkonium chloride was obtained from
Sigma-Aldrich, Co., Darmstadt, Germany. Absolute ethanol and aceto­
nitrile were analytical grade and purchased from RCI Labscan, Bangkok,
Thailand.
2.2. Preparation of solid-state CBD-CD inclusion complexed polymeric
micelles (CBD-β-CDPM)
CBD 7.9 g (25 mmol) was dissolved in 78.0 g of the warm ethanol at
50 ◦ C and stirred with a magnetic stirrer (C-MAG HS 7, IKA® Works Co.
Ltd, Bangkok, Thailand.) until a clear solution was obtained that
N. Changsan et al. International Journal of Pharmaceutics 640 (2023) 123035

Table 1 2.4. Assay of CBD in the CBD-β-CDPM-NS formulation

Composition of the CBD-β-CD complexed polymeric micelle (CBD-β-CDPM)
formulation. High-performance liquid chromatography (HPLC) was used to
Ingredients Amount (g) Function analyze the CBD content in the CBD-β-CDPM powder and CBD-β-CDPM-
Cannabidiol 7.9 Active ingredient
NS formulation. The HPLC system included the CBM-20A system
β-Cyclodextrin 28.4 Complexing agent controller (Shimadzu Corporation, Tokyo, Japan), LC-30AD pump, SIL-
Poloxamer 407 9.9 Solubilizing agent 30AC autosampler, SPD-M20A PDA detector, and CTO-20AC column
Ethanol 78.0 Solvent of CBD oven. A Shim-pack GIS C18 (150 mm × 4.6 mm, 3 μm) from Shimadzu
Purified water 875.8 Vehicle
(Shimadzu Corporation, Tokyo, Japan) at 15 ◦ C was used to perform the
Total weight 1000
separations. A degassed mixture of 70% acetonitrile and 30% ultrapure
Note: The molar ratio of cannabidiol, β-cyclodextrin, and poloxamer 407 was water made up the mobile phase. The sample or standard CBD substance
approximately 1:1:0.03. was dissolved in the mobile phase. The injection volume and flow rate
were 10 μL and 1 mL/min, respectively. A spectrophotometer was used
to monitor the separation at 207 nm. LC Solution software (Shimadzu
Table 2
Composition of the CBD-β-CDPM nasal spray solution.
Corporation, Tokyo, Japan) was used to process the acquired data. The
validation parameters included precision, accuracy, specificity, limit of
Ingredients Amount (g) Function
quantitation, limit of detection, linearity, and robustness. There was no
CBD-β-CDPM powder 0.30* Active ingredient matrix interference of either CBD-β-CDPM powder or CBD-β-CDPM-NS
Hydroxyethyl cellulose (HEC) 0.05 Mucoadhesive polymer in the chromatographic analysis.
Citric acid monohydrate 0.20 Buffering agent
Sodium citrate 0.28 Buffering agent
Glycerin 2.15 Humectant 2.5. Characterization of dried CBD-β-CDPM powder
Ethanol (99.8%) 2.00 Permeation enhancer
Benzalkonium chloride 0.02 Preservative The moisture content of the dried CBD-β-CDPM powder was
Sterile water for injection 95.00 Vehicle
analyzed using thermogravimetric analysis (TGA). Samples were accu­
*
Note: The 0.30 g of CBD-β-CDPM powder is equivalent to 0.05 g of pure CBD. rately weighed and subjected to heating at a constant rate of 10 ◦ C/min
from 30 to 150 ◦ C under a nitrogen purge using a thermogravimetric
resulted in an alcoholic CBD solution. Poloxamer 407, 9.9 g (0.79 mmol) analyzer (Model TGA 7 PerkinElmer, Inc., USA).
and β-CD 28.4 g (25 mmol) were dispersed in separate vessels in 400 mL All samples including CBD, β-CD, poloxamer 407, CBD-β-CD, CBD-
and 476 mL, respectively, of water at 70 ◦ C by slow addition and stirring β-CDPM powder, and the physical mixing of CBD, β-CD, and poloxamer
with a high shear mixer (IKA RW 20, Co. Ltd, Bangkok, Thailand) at 407 were subjected to differential scanning calorimetry (DSC). DSC
1000 rpm for at least 60 min or until a clear solution was formed. The analysis was carried out on a differential scanning calorimeter (DSC 800,
aqueous β-CD solution and aqueous poloxamer 407 solution were then Perkin Elmer Inc., USA). Each sample (5–10 mg) was heated at a rate of
combined to create a homogenous mixture. The aqueous mixture of 10 ◦ C/min from 30 to 300 ◦ C under a flowing argon atmosphere (flow
β-CD and poloxamer 407 was then mixed with CBD ethanolic solution rate: 20 mL/min) in an aluminum pan.
and the mixture was stirred at a low shear rate (500 rpm) for at least 30 All samples including CBD, β-CD, poloxamer 407, CBD-β-CD, and
min or until a uniform dispersion was created. As a consequence, 1000 g CBD-β-CDPM powder were determined by Fourier transform infrared
of the combination were produced. (FT-IR) spectroscopy. The sample powder (1 mg) was mixed with KBr in
The mixture solution was freeze-dried to produce a water-soluble a 1:100 ratio following by compression to obtain 2 mm transparent disc.
CBD-β-CDPM using the Martin Christ Model Delta 2–24 LSC plus FT-IR spectra between 4000 and 400 cm− 1 were determined after an
freeze-dryer (Osterode am Harz, Germany) using the following condi­ accumulation of 16 scans using a Spectrum One (Perkin Elmer, Inc.,
tions: pre-freeze temperature at − 40 ◦ C for 24 h; primary drying at USA).
− 10 ◦ C for 24 h at a chamber pressure of 0.633 mbar; and secondary
drying at 10 ◦ C for 4 h and 20 ◦ C for 20 h. The schematic diagram of 2.6. Characterization of CBD-β-CDPM-NS
CBD-β-CDPM preparation is illustrated in Supplementary Fig. S1, and
the ingredients of the CBD-β-CDPM are listed in Table 1. CBD-β-CDPM was dispersed in a purified water at a concentration of
0.3% w/v which was equal to the concentration of CBD-β-CDPM in the
2.3. Preparation of the nasal spray solution containing CBD-β-CD CBD-β-CDPM-NS formulation. The particle size distribution and zeta
complexed polymeric micelles (CBD-β-CDPM-NS) potential were then determined by a Zetasizer (Nano ZS ZEN3600,
Malvern Instruments Ltd., Worcestershire, UK). CBD-β-CDPM-NS was
All ingredients for the nasal spray formulation containing (CBD- also analyzed without dilution. The sample measurements were per­
β-CDPM) are provided in Table 2. For each 100 mL of nasal solution, 20 formed at 25 ◦ C. The average of ten measurements at an angle of 90◦ was
mL of sterile water for injection and 2 g of ethanol were combined. Then, used to calculate the particle size and polydispersity index of the
0.3 g of the dried CBD-β-CDPM powder (equivalent to CBD 50 mg) was investigated formulations.
gradually added during constant stirring. After all excipients were dis­ The viscosity of CBD-β-CDPM-NS was analyzed using a Modular
solved, sodium citrate, benzalkonium chloride, and citric acid mono­ Advanced Rheometer System (HAAKE MARS 60; Thermo Fisher Scien­
hydrate were added. Glycerin was then added in the last step. HEC was tific, Bremen, Germany). The rheometer used a 60 mm diameter parallel
disseminated in sterile water for injection and then homogeneously plate at a gap of 0.5 mm with a Peltier temperature control system. After
combined with the CBD-β-CDPM solution to obtain the nasal spray so­ loading a 3 g sample onto the bottom plate, the upper plate was moved
lution The resultant nasal spray formulation was kept at controlled room to the designed gap. The flow experiments were conducted at a constant
temperature for subsequent analysis. As a control, a formulation without 25 ◦ C with a shear rate of 1 to 1000 s− 1. A frequency sweep was con­
CBD-β-CDPM was prepared. Also, CBD-β-CD was prepared similarly to ducted in a range of 0.1 Hz to 100 kHz. Each batch of tests was per­
the above method without adding poloxamer 407 to compare the formed in triplicate.
interaction or characteristics of CBD and β-CD without surfactant The surface tension of CBD-β-CDPM-NS formulation was measured
interference. by the Du Noüy ring method. The Du Noüy ring is an automated oper­
ation to determine the surface tension. The tensiometer used in this
experiment was a model DY-300 (Kyowa Interface Science Co., Ltd.,

3
N. Changsan et al.
Tokyo, Japan). Approximately 200 mL of CBD-β-CDPM-NS was placed in
a glass surface tension cup and loaded on the equipment. The ring was
cleaned after each measurement to ensure the residue was completely
removed. All measurements were performed in triplicate.
The osmolality of CBD-β-CDPM-NS was determined by an automatic
cryoscopic osmometer (Osmomat® 030, Gonotec, ELITechGroup Inc.,
USA). All measurements were performed in triplicate.
2.7. Stability study
To determine the stability under actual storage conditions in
Thailand, which is in a hot and humid zone, 20 mL of the CBD-β-CDPM-
NS formulation was placed in a polyethylene nasal spray bottle and kept
for six months at 30 ± 0.5 ◦ C and at a relative humidity of 75 ± 5%
(ASEAN Guideline on Stability of Drug Products, 2018). The content of
CBD in CBD-β-CDPM-NS and physical properties of the formulation
including appearance, pH, particle size, and zeta potential were
recorded.
2.8. In vitro drug release studies
In vitro dissolution of the nasal formulation in a nasal cavity was
developed according to the previous study (Inoure et al., 2022). The CBD
dissolution of CBD-β-CDPM-NS was evaluated. Phosphate-buffered sa­
line (PBS) pH 6.4 was used as an artificial nasal fluid (Washington et al.,
2000). The temperature of the medium was kept at 35 ◦ C. A quantity of
500 μL of CBD-β-CDPM-NS (equivalent to 50 μg × 5 doses of CBD) was
loaded into the simulated chamber with 10 mL dissolution medium.
After dosing, a 100 μL aliquot of medium was collected from the bottom
of the chamber at intervals of 0, 1, 2, 3, 4, 5, 7, 10, and 15 min and
replaced with 100 μL fresh medium. The amount of CBD dissolved was
analyzed by HPLC as described in Section 2.4.
2.9. Cell culture conditions for cell toxicity studies
The RAW 264.7 mouse monocyte/macrophage cell line (ATCC TIB-
71, USA) was grown in Dulbecco’s Modified Eagle Medium (DMEM,
Gibco®, USA) containing 10% fetal bovine serum (FBS, Gibco®, USA)
and 100 U/mL penicillin/streptomycin (Gibco®, USA). The media was
replenished every two days while the cells were cultured at 37 ◦ C in a 5%
CO2 incubator. The cells were collected by gentle rocking. A new cell
suspension was then prepared by adding fresh culture media, which was
then used for further incubation.
The RPMI 2650 human nasal septum epithelial cell line (ATCC: CCL-
30, Rockville, MD, USA) was cultured in Eagle’s Minimum Essential
Medium (EMEM, Gibco®, USA) supplemented with 10% fetal bovine
serum (FBS, Gibco®, USA), 100 U/mL penicillin/streptomycin (Gibco®,
USA). The cells were incubated at 37 ◦ C in a 5% CO2 incubator and the
media were changed every two days. They were harvested by gentle
rocking, followed by the addition of fresh culture medium to create a
new cell suspension for further incubation.
2.10. Cell proliferation and viability assay
A quantity of 100 μL of RAW 264.7 cells or RPMI 2650 at a con­
centration of 1 × 105 cell/mL was seeded in a 96-well plate with com­
plete medium. After 24 h of incubation, the culture plates were treated
with CBD-β-CDPM-NS at CBD concentrations equivalent to 3.12–50 µg/
mL in fresh media. Untreated cells were used as a negative control. After
the cells were exposed to the test samples for 24 h, cell viability was
measured using a solution (5 mg/mL) of 3-(4, 5-dimethylthiazol-2-yl)-
2,5-diphenyl-tetrazolium bromide (MTT). Briefly, the cells were
treated with 80 µL of fresh media and the MTT solution (20 µL), which
was then incubated at 37 ◦ C under 5% CO2 for 4 h. The media containing
MTT were then removed and dimethyl sulfoxide (100 µL) was added.
Absorbance was recorded using a microplate reader (Biohit 830,
4
International Journal of Pharmaceutics 640 (2023) 123035
Biohit®, Helsinki, Finland) at 570 nm. The percentage of cell prolifer­
ation was calculated and compared to the negative control.
2.11. In vitro CBD-β-CDPM-NS efficacy to reduce S-RBD-induced pro-
inflammatory cytokine production
This experiment was performed according to previous studies (Sri­
chana et al., 2022). The coding sequence for the S-RBD of SARS-CoV-2
(S-RBD, residue 317–539) was optimized for expression in mammalian
cells from the sequence obtained from the reference strain (Wuhan-Hu-
1) and cloned into pSecTag using restriction enzyme cloning (BsiWI and
XhoI) in-frame with the Igκ leader sequence at the N-terminus and the
Myc-His tag at the C-terminus. Recombinant tagged S-RBD protein was
produced according to a previously published protocol (Amanat et al.,
2020). Briefly, human embryonic kidney 293 T cells were maintained in
OptiMEM with 10% fetal bovine serum supplement and transfected with
pSecTag-SRBD using FuGENE transfection reagent according to the
manufacturer’s protocol. The cell media were then changed to OptiMEM
with no supplement at 6 h post-transfection. At 72 h post-transfection,
the supplement was conditioned with binding buffer (4X) (1.2 mM
NaCl, 200 mM NaH2PO4, 40 mM imidazole). The conditioned super­
natant was then mixed with Ni-NTA agarose resin (Qiagen) for 1 h. The
unbound proteins were washed off with a wash buffer (300 mM NaCl,
50 mM NaH2PO4, 20 mM imidazole). Recombinant S-RBD was then
eluted with an elution buffer (300 mM NaCl, 50 mM NaH2PO4, 250 mM
imidazole). The eluted fractions were pooled, concentrated and buffer-
exchanged into PBS using a 10 molecular weight cut-off centrifugal fil­
ter unit. The protein concentration was measured using the Bradford
method (Bradford, 1976). Recombinant S-RBD was stored at − 80 ◦ C in
small aliquots prior to use.
RAW 264.7 cells (100 μL) at a cell density of 105 cells/mL in com­
plete media were seeded into each well of a 96-well plate. The cells were
allowed to grow until 70–80% confluence for 2 h. Following incubation,
the cells were treated with S-RBD and CBD-β-CDPM-NS in different or­
ders as follows:
Treatment mode: Cells were stimulated with S-RBD protein (10 μg/
mL) and incubated at 37 ◦ C under 5% CO2 for 24 h before treatment with
CBD-β-CDPM-NS containing CBD equivalent to 50 μg/mL and incubated
at 37 ◦ C under 5% CO2 for 24 h.
Co-administration mode: Cells were stimulated with S-RBD protein
(10 μg/mL) and concurrently treated with CBD-β-CDPM-NS containing
CBD equivalent to 50 μg/mL for 24 h at 37 ◦ C in 5% CO2.
After 24 h of incubation, the cell supernatant of each experimental
mode was assayed for pro-inflammatory cytokine levels that included
TNF-α, IL-1β, or IL-6, which were generated in the cell supernatants
obtained from the experiment explained earlier. Measurements used a
rat TNF-α, IL-1β, or IL-6 Quantikine enzyme-linked immunosorbent
assay kit (R&D Systems, Inc., Minneapolis, MN, USA). Using the 96-well
plates included in the ELISA kit, 50 μL of TNF-α, IL-1β, or IL-6 diluent
was added to each well. The experimental cell supernatant in the
amount of 50 μL was added and incubated for 2 h at room temperature.
Each well was washed (5X) with a buffer solution followed by adding the
conjugate solution (100 μL) of either TNF-α, IL-1β, or IL-6 to each well
and incubated for another 2 h. Each well was then washed with a buffer
(5X) and 100 μL of substrate solution was added. The plates were
incubated for 30 min at room temperature followed by adding a stop
solution (100 μL). The absorbance of a consequent reaction was
measured at 450 nm with a microplate reader (Biohit 830, Biohit®,
Helsinki, Finland). The absorbance results were quantified using TNF-α,
IL-1β, and IL-6 standard curves.
2.12. Ex vivo studies on the activity of CBD-β-CDPM-NS on porcine nasal
epithelia mucosa
Pigs (22–23 weeks old, 80 kg) were dissected by the veterinary staff
of Charoen Pokphand (CP) Foods Public Company, Ltd, Thailand. To
N. Changsan et al.
Fig. 1. Schematic diagrams represent an overview of ex vivo experimental design.
maintain the tissue integrity, the nasal mucosa tissue was immediately
removed from the pig snout. The mucosa explants were prepared using
the procedure described by Tulinski et al. (2013), with slight modifi­
cations. In brief, isolated nasal mucosa explants were placed on sterile
gauze soaked in DMEM supplemented with 1 mg/mL streptomycin and
1000 U/mL penicillin. The air–liquid interface was kept open and the
cilia membrane exposed to the air. The process of explant preparation is
depicted in Fig. 1.
Porcine nasal mucosa explants were divided into pieces approxi­
mately 1 cm2 and placed into a well of a 24-well plate filled with DMEM
to the half-height of the explants. The ability of CBD-β-CDPM-NS to
suppress the cytokine provoked by the S-RBD protein was examined in
two experimental studies: prevention and treatment. The surface of a
porcine nasal mucosa explant was exposed to S-RBD protein 50 µL (10
µg/mL), which was then incubated for 6 h in a CO2 incubator to activate
the immune system and generate pro-inflammatory cytokines. In the
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International Journal of Pharmaceutics 640 (2023) 123035
prevention study, nasal mucosa explants were treated with 25 μL of
CBD-β-CDPM-NS for 30 min before S-RBD protein challenge. Following
6 h of S-RBD incubation in the treatment study, 25 μL of CBD-β-CDPM-
NS was applied to the explant surface and incubated for 30 min. After
the incubation time ended, the tissue was diced into small fractions
suspended in culture medium (DMEM) and transferred to a micro­
centrifuge tube, placed in an ice bath, and then subjected to a 10 min
sonication process. The generated cytokines (TNF-α, IL-1β, or IL-6) were
detected in the supernatant after centrifuging the tissue suspension at
5000 rpm for 5 min. The Quantikine® ELISA Kit (R&D Systems, Inc.,
Minneapolis, MN, USA) was used to measure the porcine TNF-α, IL-1β,
or IL-6 cytokine levels following the manufacturer’s instructions.
N. Changsan et al. International Journal of Pharmaceutics 640 (2023) 123035

Table 3 epithelium explant membrane. The diffusion cells were maintained in a

Initial physical properties and CBD content of CBD-β-CDPM powder and after circulating water bath at 100 rpm. Samples of 500 μL were collected at
reconstitution to CBD-β-CDPM-NS with stability results (n = 3–6). each time point of 0, 5, 10, 15, 20, 25, 30, 45, and 60 min and fresh PBS
Test Initial data Data after 6 months pH 6.4 was used to replace the equal amounts of sampling. The 500 μL
storage collected samples were diluted with 300 μL of mobile phase. The diluted
CBD-β-CDPM powder samples were filtered with a 0.22 μm nylon membrane filter before the
CBD content (%) 102.1 ± 0.5 100.7 ± 0.6 CBD analysis using HPLC described in Section 2.4. Following the
Appearance White to off-white off-white powder experiment, the leftover porcine nasal mucosa membranes were placed
powder
in centrifuge tubes with an additional 800 μL of mobile phase. The
Water content (%) 4.79 ± 0.3 4.40 ± 0.2
Particle size (Z-average, 101.4 ± 6.9 102.5 ± 7.3 samples were digested with a cell digestion machine (IKA, Bangkok,
nm) * Thailand) and filtered through a 0.22 μm nylon membrane. The super­
PDI 0.25 ± 0.01 0.31 ± 0.02 natant was then subjected to HPLC analysis to determine the CBD con­
Zeta potential (mV)* − 8.9 ± 0.4 − 10.5 ± 0.2 tent retained in the membrane. The experiment was done in quadruplet.
CBD-β-CDPM-NS
CBD content (%) 99.80 ± 1.3 98.80 ± 0.8
From the in vitro membrane permeation data, steady-state flux (Jss) (μg/
Appearance Clear colorless solution Clear colorless solution cm2/min) and the cumulative amount of CBD through the mucosa
pH 6.02 ± 0.02 6.12 ± 0.01 membranes (μg/cm2) were determined.
Osmolality (mOs/kg) 300 ± 0.8 302 ± 0.6
Particle size (Z-average, 111.9 ± 0.7 121.4 ± 1.1
2.14. Statistical analysis
nm)
PDI 0.15 ± 0.01 0.22 ± 0.02
Zeta potential (mV) 0.8 ± 0.1 1.0 ± 0.2 All results were reported as mean and standard deviations. Signifi­
Viscosity (mN s/m2 [cP]) 12.04 ± 2.64 15.20 ± 1.03 cant differences in the mean parameters were analyzed using one-way
All values are presented as mean ± SD. analysis of variance (ANOVA). The significance level was set at 0.05
*
After reconstitution with sterile water for injection. for all tests.

2.13. Ex vivo nasal epithelium permeation studies using Franz diffusion
cells
Porcine nasal epithelium explants were cut into 2 × 2 cm2 pieces and
CBD transport was determined through the membrane. The explants
were mounted between the donor and receptor chamber of the Franz
diffusion cells (Hanson Technology, Chatsworth, CA USA) with an
effective diffusion area of 1.76 cm2 and cell volume of 8 mL filled with
freshly prepared PBS pH 6.4. After an equilibrium time of 15 min at 35 ◦ C
± 0.5 ◦ C, 500 μL of CBD-β-CDPM-NS (equivalent to 250 μg CBD) were
loaded into the donor compartment through direct contact with nasal
3. Results and discussion
3.1. Validation results of CBD in the CBD-β-CDPM and CBD-β-CDPM-NS
The HPLC analysis was very specific to CBD. The retention time, limit
of CBD detection, and limit of quantitation were 3.7 min, 0.25 μg/mL,
and 1 μg/mL, respectively. The accuracy and precision of the analysis
were both 99% with a 1.2% relative standard deviation. The results of
the HPLC system were linear over a range of 10–50 μg/mL, and the
system was able to give consistent results for different operators.

Fig. 2. CBD release from CBD-β-CDPM powder (blue line) and CBD-β-CDPM-NS (black line). Drug release studies were performed by dialysis simulated condition in
the nasal cavity (PBS pH 6.4, 8 mL) under sink conditions while maintaining the temperature at 35 ± 0.5 ◦ C. The data presented is mean of drug release with
standard deviation of 3 replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

6
N. Changsan et al. International Journal of Pharmaceutics 640 (2023) 123035

Fig. 3. CBD from the CBD-β-CDPM-NS permeability through nasal porcine mucosa determined by Franz diffusion cells in PBS. (A) Steady-state flux across nasal
porcine mucosa. (B) Cumulative amount of CBD over time through nasal porcine mucosa. Data are expressed as mean ± SD (n = 4).

3.2. Development of CBD-β-CDPM
CBD complexation with CD was developed to improve water solu­
bility. β-CD has undergone extensive safety testing and has been
approved for use in pharmaceutical products (Loftsson and Brewster,
2010; Lv et al., 2019; Haimhoffer et al., 2019). Also, β-CD is safe for
human ingestion and application (Pereira et al., 2021; Poulson, et al.,
2022; European Medicines Agency, 2014). Furthermore, β-CD was
chosen in this study due to the similarity of its internal cavity size of
6.0–6.4 Å (Poulson, et al., 2022) and the size of the CBD molecule. From
the literature, the crystal structure of CBD was reported to be monoclinic
with dimensions of a = 10.617 Å, b = 10.649 Å, and c = 17.266 Å and β
= 95.30(4)◦ (Jones et al., 1977), and another study reported similar
dimensions of a = 10.4395 Å, b = 10.8739 Å, and c = 16.7853 Å and β =
95.448(1)◦ (Mayr et al., 2017). We confirmed the results of those studies
from our CBD crystallography as shown in the supplementary data files.
These findings indicated that a portion of the CBD molecule could be
inserted into the β-CD cavity of 6.0–6.4 Å. In addition, another group
reported that the β-CD cavity was 7.8 Å (Păduraru et al., 2022). β-CD has
hydrophilic properties with a solubility in 25 ◦ C water of 18 mg/mL
(Chatjigakis et al., 1992). Furthermore, as the temperature is increased,
β-CD becomes more soluble (Poulson, et al., 2022; Yong et al., 2008). For

7
N. Changsan et al.
Fig. 4. The cytotoxicity profiles of the CBD-β-CDPM, CBD-β-CDPM-NS with CBD concentrations equivalent to 3.125–50 μg/mL, and nasal spray blank formulation
against the human nasal septum epithelial cell line (RPMI 2650) (A), and mouse monocyte/macrophage cell line (RAW264.7) (B). Data are expressed as mean ± SD
(n = 4).
this reason, the manufacturing process in this study was performed at
70 ◦ C. After preparation, CBD-β-CDPM was lyophilized to produce a dry
powder. This technique provided the amorphous formulation of CBD as
reported in a previous study (Koch, et al., 2020). This current method is
similar to the method by Koch et al., (2020) except CBD was dissolved in
ethanol before adding it to the CD solution, which resulted in a higher
amount of CBD loaded into the CD molecules.
The CBD encapsulated within the cavity of β-CD to form a 1:2
host–guest inclusion complex is described in the literature (Lv et al.,
2019). Another consistent study prepared CBD as a β-CD inclusion
complex. The molar stoichiometry of the CBD and β-CD complex was 1:2
by the precipitation method in which only 7 mg of β-CD was required to
complex 1 mg of CBD (Mannila et al., 2007). However, Li et al. (2022)
reported CBD was inclusion complexed with hydroxypropyl-β-CD at a
ratio of 1:1.
In this study, CBD was formulated with β-CD in a 1:1 ratio and
poloxamer to improve the aqueous solubility and bioavailability of the
poorly soluble CBD. The CBD-β-CDPM solution was freeze-dried into
powder and reconstituted as an active ingredient in a nasal spray
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International Journal of Pharmaceutics 640 (2023) 123035
formulation. The developed water-soluble CBD-β-CDPM is white to an
off-white amorphous powder at room temperature after the lyophiliza­
tion process, and the powder is free flowing with a high bulk volume.
The yield of water-soluble CBD-β-CDPM was 90.9% (42.0 g) of the total
weight of the solid ingredients (46.2 g) in the formulation. The TGA
analysis showed moisture content of the water-soluble CBD-β-CDPM was
4.8 ± 0.3% w/w. After reconstituting the CBD-β-CDPM with water for
injection, a clear colorless solution that easily dissolved was obtained.
The solubility of CBD-β-CD was 0.4 μg/mL, which is 4 times higher than
pure CBD. When CBD-β-CD was formulated with poloxamer 407, its
solubility at 25 ◦ C was 2.5 mg/mL, which is equal to 427.5 μg/mL of
CBD (theoretically, 46.2 g of CBD-β-CDPM powder contains 7.9 g of
CBD). Thus, the aqueous solubility of CBD-β-CDPM was 4275 times
higher than the solubility of pure CBD at 0.1 μg/mL (Amanat et al.,
2020), which is beneficial for developing aqueous nasal spray solutions.
3.3. Characterization of CBD-β-CDPM
A previously published thermal analysis reported that poloxamer
N. Changsan et al.
Fig. 5. In vitro results of cytokine levels: (A) TNF-α, (B) IL-1β, and (C) IL-6 produced from macrophage-like cells (RAW264.7) after exposure to S-RBD, culture
medium (negative control), CBD-β-CDPM-NS, S-RBD following with CBD-β-CDPM-NS 24 h later (treatment), and co-administration of S-RBD and CBD-β-CDPM-NS.
Data are expressed as mean ± SD (n = 3).
407 had the lowest melting point of 59.49 ◦ C (Emam et al., 2021) fol­
lowed by CBD at 67.5 ◦ C (Stinchcomb et al., 2004), whereas β-CD melted
at 267–269 ◦ C (Thurein et al., 2018). However, the broad endothermic
profile of β-CD showed a peak at around 100–150 ◦ C, which corresponds
to the loss of crystalized water molecules from the β-CD cavity. Several
other researchers reported results such as a centered peak at 145 ◦ C
(Marangoci et al., 2019), a range of 34–155 ◦ C (peak ~ 125 ◦ C) (Júnior
et al., 2019), and a peak at 113.2 ◦ C (David et al., 2019). In this study,
the thermograms of the raw materials of pure compound poloxamer
407, CBD, and β-CD had melting peaks at 53.9, 64.2, and 122.9 ◦ C,
respectively. The thermogram of the physical mixture showed an
absence of the characteristic melting peaks of poloxamer 407 and β-CD
(Supplementary Fig. S2). The melting point of CBD shifted to a lower
melting point temperature of 60 ◦ C. This indicated that the physical
mixture formed a complex with β-CD during the heating phase that
resulted in a much lower melting point than the original melting point of
pure β-CD caused by eutectic phenomena. Also, the melting point of CBD
shifted to a lower value in the mixture of the three components with an
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International Journal of Pharmaceutics 640 (2023) 123035
absence of the poloxamer 407 peak. This indicated that the three mol­
ecules could interact even in the physical mixture during the heating
stage, and that the lowest melting components of poloxamer 407 and
CBD could act as a cosolvent system while forming a complex structure
of those three components.
When the CBD complexed with β-CD (CBD-β-CD), it was found that
part of the CBD structure was possibly inserted into the CD cavity. The
small CBD peak at 60 ◦ C was found to have a much smaller peak area
together with an amorphous halo from 100 to 120 ◦ C that was possibly
caused from free CBD that did not form the inclusion complex with β-CD
(Fig. S2).
The DSC thermogram showed that the CBD-β-CDPM is an amorphous
solid, while the DSC thermogram did not show the response peak of any
compounds. Hence the mixture was completely amorphous. It can be
postulated that the mixture is readily soluble in water without free CBD
remaining (Fig. S2). When CBD and poloxamer 407 were prepared as a
mixture, there was no crystallinity observed in the thermogram, which
indicated only an amorphous phase in the mixture.
N. Changsan et al.
Fig. 6. Ex vivo results of cytokine levels: (A) TNF-α, (B) IL-1β, and (C) IL-6 produced from porcine nasal mucosa explant after exposure to S-RBD, culture medium
(negative control), CBD-β-CDPM-NS, S-RBD 6 h following with CBD-β-CDPM-NS for 30 min (treatment), and CBD-β-CDPM-NS for 30 min following with S-RBD for 6 h
(prevention). Data are expressed as mean ± SD (n = 3).
The FT-IR spectra of CBD, poloxamer 407, β-CD, CBD-β-CD and CBD-
β-CDPM are shown in Supplementary Fig. S3. The potential interactions
between CBD and β-CD were investigated by FT-IR analysis by
comparing the respective absorption spectra, considering specific func­
tional groups of the CBD, poloxamer 407 and β-CD alone, and complexes
of CBD-β-CD. The potential interactions of poloxamer 407 in the com­
plexes system were also investigated. If CBD forms the inclusion com­
plexes with β-CD, the characteristic peaks of CBD probably shift,
decrease, or disappear (Li et al., 2021). The characteristic bands at
2925–2856 cm− 1 (C–H stretching) and at ~ 3408 cm− 1 (O–H stretching)
were found in CBD and β-CD alone and also in the complex of CBC-β-CD
and CBD-β-CDPM. The CBD spectrum showed characteristic bands at
1623–1583 cm− 1 (cyclic alkene C = C stretching) that were not found in
the β-CD and poloxamer 407. Only C–O stretching of aliphatic ether was
found in β-CD at the wave number 1158 cm− 1 and also in the poloxamer
407 that usually occurs in the range 1260–1000 cm− 1 (Pavia et al.,
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International Journal of Pharmaceutics 640 (2023) 123035
2001). The characteristic IR bands of β-CD and CBD were found in the
complex of CBD-β-CD and peak intensity decreased compared to raw
compounds, which confirmed that molecular interaction between CBD
and β-CD occurred.
From the FT-IR results, CBD, β-CD and poloxamer did not chemically
interact with one another as all fingerprints of each component appeared
in the spectra. Possible structures of CBD-β-CDPM could be a mixture of:
(A) the CBD inclusion complex with β-CD at a ratio of 1:1; (B) CBD
without complex encapsulation in the core of poloxamer; (C) CBD-β-CD
complex may encapsulate in the core of the poloxamer but might be
difficult to occur due to hydrophilicity molecules; and (D) CBD-β-CD
complex inserted between hydrophilic parts of poloxamer micelles
(Supplementary Fig. S4).
N. Changsan et al.
Fig. 7. Schematic diagram of CBD-β-CDPM-NS as a potential agent to reduce cytokine storm caused by SARS-CoV-2 infection.
3.4. Development and stability of CBD-β-CDPM-NS
CBD-β-CDPM was used as an active ingredient along with a buffering
agent, preservative, and ethanol as a permeation enhancer (Bhise et al.,
2008) to formulate a nasal spray solution. The formulation for CBD-
β-CDPM nasal spray (CBD-β-CDPM-NS) was a clear solution.
HEC (0.05 %w/w) was used as a mucoadhesive polymer in this
experiment due to its adhesiveness to the nasal epithelial cells (Cho
et al., 2021). In addition to the good mucoadhesive properties, Hansen
et al. reported that HEC enhanced the transport of drug molecules across
the nasal epithelium (Hansen et al., 2015). A preliminary study of HEC
concentration at varying concentrations of 0.05, 0.1, 0.15, 0.2, and
0.25% w/w had no effect on surface tension but had a significant impact
on viscosity. The viscosity of HEC ranged from 6.2 cP at 0.05% (w/v) to
82.1 cP at 0.25% (w/v) (Supplementary Fig. S5). On the basis of these
results, the lowest concentration of HEC with the lowest viscosity was
chosen for the formulation for easy release from the container.
The average size of the CBD-β-CDPM before freeze-drying was 101.4
± 6.9 d.nm and its polydispersity index was 0.249 ± 0.013 (n = 6)
(Table 3). The literature reported that polymeric micelles of pure
poloxamer 407 showed a particle size under 100 nm (Bahman et al.,
2019). The particle size of CBD-β-CDPM was larger than empty polox­
amer micelles, which was likely due to the insertion of CBD or CBD-β-CD
in the core of the micelles. The apparent zeta potential of the CBD-
β-CDPM before freeze-drying was − 8.9 ± 0.4 mV. This result indicated
the micelles were negatively charged, which was similar to the results of
− 13.57 mV in a previous report on polymeric micelles of gambogic acid
(Saxena and Hussain, 2012). Both the polypropylene oxide and poly­
ethylene oxide segments in poloxamer 407 are nonionic; therefore, the
addition of CBD and β-CD affected the surface charge of the polymeric
micelles.
After storage of CBD-β-CDPM for six months, reconstitution of the
powder with sterile water for injection resulted in a particle size of CBD-
β-CDPM of 102.5 ± 7.3 d.nm and a zeta potential of − 10.5 ± 0.2 mV.
The size of the CBD-β-CDPM was reduced with a lower negative charge.
This was possibly due to stabilization of the size and charge by
11
International Journal of Pharmaceutics 640 (2023) 123035
equilibrium moisture during storage.
The particle size of CBD-β-CDPM-NS initially was 111.9 ± 0.7 d.nm
and the zeta potential was 0.8 ± 0.1 mV. The particle size of CBD-
β-CDPM-NS after storage at room temperature for six months increased
to 121.4 ± 1.1 d.nm with a small change in the surface charge. The CBD-
β-CD complex polymer micelle enlargement indicated some degree of
instability although no precipitation or aggregation occurred, and the
surface charge increased to nearly zero. This result was possibly caused
by the buffering agent and preservative in the nasal spray formulation
that affected the counter ions of the colloidal system. In addition, the
environmental change of the colloidal system may lead to slightly larger
sizes and structures of the micelles (Perumal et al., 2022).
The osmolarity of this formulation was 300 ± 0.8 mOsm/kg in the
range of 285–310, which indicated an isotonic solution suitable for nasal
preparation (USP43; Ehrick et al., 2013; Barros et al., 2022). The vis­
cosity of the nasal spray formulation was 12.04 ± 2.64 cP at the shear
rate of 50 s− 1. The rheogram of the CBD-β-CDPM-NS showed a shear
thinning system, which was typical of pseudoplastic behavior (Supple­
mentary Fig. S6). As the shear rate increased, the viscosity decreased.
The local pH value inside the nasal cavity directly influences the rate and
extent of drug absorption. It has been suggested that the optimum pH
value of nasal spray should range from 4.5 to 6.5 (Dhakar et al., 2011) or
5.5 to 6.5, which is close to the physiological pH (Barros et al., 2022).
The pH value of the nasal spray formulation in this current study was
6.02 ± 0.02, which is suitable for nasal epithelial cells. The surface
tension was 34.5 mN/m, which was lower than sterile water (72.8 mN/
m at 20 ◦ C) (Sinko, 2011). This result indicated that poloxamer 407
reduced the surface tension and formed complex polymeric micelles in
the bulk solution.
The assay results of CBD are shown in Table 3. The CBD content in
the CBD-β-CDPM was 102.1 ± 0.5% of the theoretical value (found 8.06
g in the theoretical CBD content of 7.9 g in a total solid content of 46.2
g). When CBD-β-CDPM powder was formulated as a nasal spray solution
(CBD-β-CDPM-NS), the assay content was 99.8 ± 1.3% of the theoretical
content.
The chemical and physical properties, which included CBD content,
N. Changsan et al.
appearance, pH, osmolality, particle size, and zeta potential, of the CBD-
β-CDPM powder and CBD-β-CDPM-NS did not significantly change (p-
value < 0.5). These results confirmed that the product was stable in
storage up to six months at room temperature.
3.5. CBD release profile from CBD-β-CDPM-NS and permeation through
nasal epithelium
The in vitro CBD release from polymeric micelles of CBD-β-CDPM-NS
in simulated nasal cavity conditions through the dialysis membrane is
shown in Fig. 2. CBD release from CBD-β-CDPM-NS was 104 ± 0.26%,
while CBD release from CBD-β-CDPM was 91.94 ± 0.15% in 15 min. The
CBD in the nasal spray (CBD-β-CDPM-NS) was 100% released within the
first minute because the CBD molecules were complexed with β-CD in
the form of micelle and colloidal systems that were easily released from
the complexed state. In contrast to the CBD-β-CDPM powder, CBD
release was slower than CBD-β-CDPM-NS.
The CBD permeation rate and amount of CBD per area of the mem­
brane from CBD-β-CDPM-NS through the porcine nasal mucosa are
shown in Fig. 3 (A) and (B). The steady-state flux (Jss) of CBD over
excised porcine nasal mucosa was 4.8 μg/cm2/min, and the rapid ab­
sorption of CBD from CBD-β-CDPM-NS increased after CBD-β-CDPM-NS
was administered to the diffusion cells. After 15 min the CBD permeated
the receptor cells at a lower permeation rate. The results showed that the
permeation rate of CBD was enhanced by increased aqueous solubility of
CBD (Ghezzi et al., 2021). However, CBD remained in the nasal
epithelium and in the donor phase. The CBD retained in the nasal
epithelial explants and the permeation across the mucosa correlated
with the ex vivo CBD-β-CDPM-NS as described in Section 3.6.
3.6. Cell viability and in vitro efficacy of CBD-β-CDPM-NS
The cytotoxicity profiles of the CBD-β-CDPM, CBD-β-CDPM-NS, and
nasal spray blank formulation against the human nasal septum epithelial
cell line (RPMI 2650) and mouse monocyte/macrophage cell line
(RAW264.7) are presented in Fig. 4A and Fig. 4B, respectively. CBD-
β-CDPM and CBD-β-CDPM-NS with CBD concentrations equivalent to
3.125–50 μg/mL were used to challenge the cell line. The results showed
that all concentrations of CBD-β-CDPM, CBD-β-CDPM-NS, and the nasal
spray blank formulation were safe for both RPMI 2650 and RAW264.7
cells with nearly 100% viability.
The SARS-CoV-2 virus’s recombinant S-RBD is a viral protein that
binds to the host cell’s ACE2 receptor and enters the cell, causing
pathological changes in lung injury and inflammatory responses that
lead to excessive production of pro-inflammatory cytokines, inflamma­
tion, and tissue injury (Montazersaheb et al., 2022). These immune re­
actions might indicate the onset of a cytokine storm with severe
clinicopathological consequences and severe COVID-19 disease (Mah­
mudpour et al., 2020). Stimulation of RAW264.7 macrophage-like cells
by S-RBD as a COVID-19 surrogate was employed to determine the ef­
ficacy of CBD-β-CDPM-NS formulation to inhibit excessive pro-
inflammatory cytokines, such as TNF-α, IL-1β, and IL-6. Stimulation
resulted in the release of extraordinarily high levels of TNF-α, IL-1β, and
IL-6, which were 539.18 ± 5.22 pg/mL (Fig. 5A), 273.38 ± 4.61 pg/mL
(Fig. 5B), and 1666.36 ± 5.96 pg/mL (Fig. 5C), respectively. The CBD-
β-CDPM-NS formulation, on the other hand, did not trigger RAW264.7
cells to release any pro-inflammatory cytokines because the levels
detected were very low and equivalent to the levels detected in the
untreated control group (3rd column in Fig. 5 A–C). This indicated that
the CBD-β-CDPM-NS formulation did not cause injury to the cells.
The capacity of the CBD-β-CDPM-NS formulation to suppress the
production of pro-inflammatory cytokines as a result of S-RBD stimu­
lation was evaluated when CBD-β-CDPM-NS formulation was applied
after S-RBD stimulation (treatment mode) or concurrently with S-RBD
stimulation (co-administration mode). Upon comparison with the S-RBD
stimulation control group, all experiment modes demonstrated the
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International Journal of Pharmaceutics 640 (2023) 123035
capacity of the CBD-β-CDPM-NS formulation to significantly lower the
levels of TNF-α, IL-1β, and IL-6. However, it is apparent from comparing
the treatment mode and co-administration mode that the co-
administration mode demonstrated significantly greater efficacy than
the treatment mode. All measured cytokine levels generated when S-
RBD and CBD-β-CDPM-NS formulation were administered together were
extremely low and similar to the untreated control levels as depicted in
Fig. 5, A–C. These findings are in agreement with our previous reports
(Srichana et al., 2022), which showed that co-administration of a CBD
formulation and allergens had the greatest impact on reducing the level
of macrophage-produced cytokines. CBD binding to the CB2 receptor on
macrophages/monocyte resulted in a decrease in cytokine production in
RAW264.7 cells exposed to S-RBD (Perwee, 2008). Co-administration of
the CBD-β-CDPM-NS formulation and S-RBD would also function
through additional mechanisms in addition to the CBD-CB2 interaction
to lessen the cytokine release induced by S-RBD. This was suggested to
be the result of competing interactions between S-RBD and CBD with
macrophages/monocytes; however, CBD interactions with macro­
phages/monocytes did not lead the immune cells to release response
cytokines like S-RBD did. In consequence of this, macrophage/monocyte
production of cytokines decreased when CBD was used instead of S-RBD
in the interaction between macrophages/monocytes. According to the
findings, the combined effects of CBD-CB2 interaction and CBD
competitive contact with macrophages resulted in a very low cytokine
level that was identical to the untreated control.
3.7. Ex vivo efficacy of CBD-β-CDPM-NS with porcine nasal mucosa
explants
CBD-β-CDPM-NS efficacy was also evaluated in ex vivo porcine nasal
mucosa to demonstrate the formulation’s activity in living tissue. Since
the nasal cavity is the primary site of infection for the SARS-CoV-2 virus,
it stands to reason that the nasal cavity would be the target of preven­
tative and therapeutic treatments. Thus, the CBD-β-CDPM-NS adminis­
tered to porcine nasal mucosa explants was investigated in both
preventive and therapeutic models. In the preventative model, CBD-
β-CDPM-NS was administered 30 min before S-RBD protein exposure. In
the treatment model, explants were exposed to S-RBD for 6 h to induce
an immunological response, followed by treatment with CBD-β-CDPM-
NS to reduce cytokine production. Co-administration of CBD-β-CDPM-
NS and S-RBD was omitted from the experiments because it does not
reflect the actual use of CBD-β-CDPM-NS.
Ex vivo effectiveness in reducing cytokine production induced by S-
RBD correlated with the in vitro cell culture model (Section 3.6). In terms
of TNF-α, IL-1β, and IL-6 levels (Fig. 6) CBD-β-CDPM-NS did not cau­
se explant tissue to produce all the tested cytokines because the cytokine
levels found in the CBD-β-CDPM-NS exposed tissue were not signifi­
cantly different from the negative control tissue (p-value > 0.05). The
tissue stimulated by S-RBD produced a significant quantity of response
cytokines more than the negative control (p-value < 0.05). CBD-
β-CDPM-NS administration both before (prevention) and after (treat­
ment) S-RBD exposure significantly decreased the cytokine level relative
to the S-RBD exposure control tissue. The prevention condition showed
TNF-α values decreased from 44.59 ± 8.17 pg/g to 11.37 ± 6.34 pg/g,
IL-1β values decreased from 610.45 ± 37.65 pg/g to 405.05 ± 48.83 pg/
g, and IL-6 values decreased from 1655.30 ± 53.21 pg/g to 747.97 ±
143.60 pg/g, p-value < 0.05). In addition, the treatment condition can
decrease the cytokine to 15.38 ± 2.22 pg/g, 418.78 ± 53.78 pg/g and
843.34 ± 206.42 pg/g of TNF-α, IL-1β, and IL-6 respectively, p-value <
0.05). There were no significant differences in the reduction of produced
cytokines from explant tissue exposed to S-RBD between the prevention
and treatment models (p-value > 0.05). This demonstrated that CBD-
β-CDPM-NS was effective in alleviating the effect of S-RBD in stimu­
lating living tissue to produce response cytokines in both prevention and
treatment models, which thereby relieved the viral-induced inflamma­
tory reaction.
N. Changsan et al.
Furthermore, β-CD has been demonstrated to have antiviral activities
owing to its high cholesterol sequestering capacity. The depletion of
cholesterol and destruction of lipid rafts, the virus’s cholesterol-rich
membrane regions, occurs as a result of structural deformation of the
viral envelope (Braga, 2019). In addition, Lu et al. (2008) reported that
lipid rafts are crucial for SARS-CoV entry into cells (Lu et al., 2008).
Cyclodextrins can also reduce the amount of cholesterol in host cell
membranes and make them less susceptible to viral infection (Braga,
2019).
Furthermore, a nasal spray with a polysaccharide base, such as HEC,
can establish a physical barrier to limit virus-cell surface interaction by
trapping or binding to the virus for removal by mucociliary action,
thereby preventing viral proliferation and distribution in the airways
(Fais et al., 2022; Moakes et al., 2021).
In summary, CBD, β-CD, and poloxamer were successfully formu­
lated as water-soluble CBD-β-CD complexed-poloxamer micelles in a
molar ratio of approximately 1:1:0.03 as proposed in Fig. 7 and can be
used as an active ingredient in developing nasal spray formulations.
4. Conclusions
The CBD-β-CD complex-polymeric micelle nasal spray solution (CBD-
β-CDPM-NS) showed physicochemical characteristics suitable for
application as a nasal spray. CBD-β-CDPM-NS demonstrated its efficacy
and safety due to very low cytokine production from nasal immune cells
and very low toxicity to nasal epithelial cells. The formulations were
successful in reducing COVID-19/SARS-CoV-2-related inflammatory
reactions both in vitro and ex vivo. The findings of this study indicate that
CBD-β-CDPM-NS strongly inhibits the immunological response of
macrophage/monocytes induced by the SARS-CoV-2 spike protein RBD,
which may eventually lead to asymptomatic infection or symptom relief
once infection has commenced. The developed CBD-β-CDPM-NS may be
recommended as an early-stage prophylactic in SARS-CoV-2 infection
since it may limit viral proliferation and viral loads in the nasal cavity.
However, for an accurate conclusion, animal studies should be
performed.
CRediT authorship contribution statement
Narumon Changsan: Methodology, Investigation, Writing – orig­
inal draft, Writing – review & editing, Visualization. Somchai Sawat­
dee: Methodology, Investigation, Writing – original draft, Writing –
review & editing, Visualization. Roongnapa Suedee: Conceptualiza­
tion, Writing – original draft, Writing – review & editing. Charisopon
Chunhachaichana: Methodology, Investigation. Teerapol Srichana:
Conceptualization, Methodology, Writing – original draft, Writing –
review & editing, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
This research was supported by the National Science, Research and
Innovation Fund and Prince of Songkla University (PHA 64050875). The
authors would like to thank the Drug Delivery System Excellence Center,
Faculty of Pharmaceutical Sciences, Prince of Songkla University for
providing the facilities and the National Biotechnology Center (BIO­
TEC), NSTDA, Ministry of Higher Education Science Research and
13
International Journal of Pharmaceutics 640 (2023) 123035
Innovation, Thailand for providing the spike protein of SARS-CoV-2, as
well as Ms. Titpawan Nakpheng for technical support. The authors
would like to thank the veterinary staff of Charoen Pokphand, Foods
Public Company, Ltd, Thailand for nasal tissue preparation.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijpharm.2023.123035.
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