RESEARCH ARTICLE
www.advmat.de
Toward a Radically Simple Multi-Modal Nasal Spray for
Preventing Respiratory Infections
John Joseph, Helna Mary Baby, Joselyn Rojas Quintero, Devin Kenney,
Yohannes A Mebratu, Eshant Bhatia, Purna Shah, Kabir Swain, Dongtak Lee,
Shahdeep Kaur, Xiang-Ling Li, John Mwangi, Olivia Snapper, Remya Nair, Eli Agus,
Sruthi Ranganathan, Julian Kage, Jingjing Gao, James N Luo, Anthony Yu,
Dongsung Park, Florian Douam, Yohannes Tesfaigzi, Jeffrey M Karp, and Nitin Joshi*
Nasal sprays for pre-exposure prophylaxis against respiratory infections show
limited protection (20–70%), largely due to their single mechanism of
action—either neutralizing pathogens or blocking their entry at the
nasal lining, and a failure to maximize the capture of respiratory
droplets, allowing them to potentially rebound and reach deeper airways.
This report introduces the Pathogen Capture and Neutralizing Spray (PCANS),
which utilizes a multi-modal approach to enhance efficacy. PCANS coats
the nasal cavity, capturing large respiratory droplets from the air, and serving
as a physical barrier against a broad spectrum of viruses and bacteria, while
rapidly neutralizing them with over 99.99% effectiveness. The formulation
consists of excipients identified from the FDA’s Inactive Ingredient Database
and Generally Recognized as Safe list to maximize efficacy for each step in the
multi-modal approach. PCANS demonstrates nasal retention for up to 8 hours
in mice. In a severe Influenza A mouse model, a single pre-exposure dose
of PCANS leads to a >99.99% reduction in lung viral titer and ensures 100%
survival, compared to 0% in the control group. PCANS suppresses pathological
manifestations and offers protection for at least 4 hours. This data suggest
PCANS as a promising daily-use prophylactic against respiratory infections.
J. Joseph, H. M. Baby, P. Shah, K. Swain, D. Lee, S. Kaur, X.-L. Li,
J. Mwangi, O. Snapper, E. Agus, S. Ranganathan, J. Kage, J. Gao, A. Yu,
J. M Karp, N. Joshi
Center for Accelerated Medical Innovation
Department of Anesthesiology
Perioperative and Pain Medicine
Brigham and Women’s Hospital
Boston, MA 02115, USA
E-mail: [email protected]; [email protected]
J. Joseph, H. M. Baby, P. Shah, D. Lee, S. Kaur, X.-L. Li, J. Mwangi,
O. Snapper, E. Agus, S. Ranganathan, J. Kage, J. Gao, A. Yu, J. M Karp,
N. Joshi
Center for Nanomedicine
Department of Anesthesiology
Perioperative and Pain Medicine
Brigham and Women’s Hospital
Boston, MA 02115, USA
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adma.202406348
DOI: 10.1002/adma.202406348
- Introduction
Respiratory infections result in significant
morbidity and mortality worldwide. [1] The
past few decades have witnessed numer-
ous outbreaks, often leading to epidemics or
unanticipated pandemics such as COVID- - Although vaccines are available against
Influenza A virus (IAV), severe acute respi-
ratory syndrome coronavirus 2 (SARS-CoV-
2), respiratory syncytial virus (RSV) and
Streptococcus pneumoniae, the emergence
of mutants often reduces the efficacy of
vaccines. [2] Additionally, there are several
pathogens, including adenovirus, Klebsiella
pneumoniae, Staphylococcus aureus, and Es-
cherichia coli, which can cause severe res-
piratory diseases, but do not have clinically
available vaccines, as of now. In the face of
an unforeseen pandemic, the timeline for
developing vaccines targeting a pathogen
can range from 1 to 10 years, contingent
upon the specific nature of the pathogen.[3,4]
J. Joseph, J. R. Quintero, Y. A Mebratu, D. Lee, S. Kaur, R. Nair, J. Gao,
J. N Luo, A. Yu, Y. Tesfaigzi, J. M Karp, N. Joshi
Harvard Medical School
Boston, MA 02115, USA
E-mail: [email protected]
J. R. Quintero, Y. A Mebratu, Y. Tesfaigzi
Division of Pulmonology
Brigham and Women’s Hospital
Boston, MA 02115, USA
D. Kenney, F. Douam
National Emerging Infectious Diseases Laboratories
Department of Microbiology
Boston University
Chobanian & Avedisian School of Medicine
Boston, MA 02118, USA
E. Bhatia
Indian Institute of Technology
Mumbai 400076, India
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The rapid creation of efficacious COVID-19 vaccines stands as
an unparalleled scientific achievement. However, it took several
months for the vaccine to become available, during which nu-
merous hospitalizations and deaths were reported.[5] Addition-
ally, multiple obstacles, such as production complexities, vac-
cine nationalism, and the emergence of novel variants, collec-
tively posed major challenges around the world. A large percent-
age of the population did not consent to vaccination for vari-
ous reasons, which posed a significant hurdle in minimizing
the transmission of pathogens. Another concern pertaining to
vaccines is their partial mitigation of the pathogen burden,[6,7]
which implies that vaccinated people can still contract and dis-
seminate the infection, albeit at a reduced rate compared to
those who are unvaccinated. Thus, there is a critical need to
develop a pre-exposure prophylactic approach that can be eas-
ily and rapidly deployed either independently or in tandem
with vaccines, serving as the primary safeguard against cur-
rent and emerging respiratory pathogens. Such an approach
should efficiently reduce pathogen load, and be radically sim-
ple to scale up and manufacture to ensure widespread global
adoption.
Transmission of most respiratory pathogens predominantly
occurs through inhalation of contaminated respiratory droplets
and their subsequent deposition in the nasal cavity, which has an
entry checkpoint.[8] For instance, SARS-CoV-2 virus binds to the
angiotensin-converting enzyme 2 (ACE2) located in nasal epithe-
lial cells via its receptor-binding domain (RBD). The nasal cavity
is a primary target for SARS-CoV-2 infection due to high expres-
sion of ACE2, [9–11] which decreases towards the lower respiratory
tract.[12] The infection spreads to the deeper airways via virus-
laden extracellular vesicles secreted by infected cells in the nasal
cavity. [13] Similarly, bacteria, including S. pneumoniae and S. au-
reus adhere to nasal mucin via a specific adhesin receptor. [14,15]
Considering the vulnerability of nasal cavity and its critical role
in the transmission of respiratory pathogens, chemoprophylactic
nasal sprays have been developed to offer pre-exposure prophy-
laxis against respiratory infections. This approach utilizes chem-
ical agents, including small molecule drugs, antiseptics, or nitric
oxide, to deactivate the pathogen in the nasal cavity or a polymer
that acts as a physical barrier to prevent pathogen entry through
J. N Luo
Department of Surgery
Brigham and Women’s Hospital
Boston, MA 02115, USA
D. Park
Center for Functional Nanomaterials
Brookhaven National Laboratory
Upton, NY 11973, USA
J. M Karp
Harvard–Massachusetts Institute of Technology Division of Health
Sciences and Technology
Massachusetts Institute of Technology
Cambridge, MA 02139, USA
J. M Karp
Broad Institute
Cambridge, MA 02142, USA
J. M Karp
Harvard Stem Cell Institute
Cambridge, MA 02138, USA
the nasal lining.[16,17] Although multiple pre-exposure chemopro-
phylactic approaches have been previously developed,[18–20] they
have resulted in sub-optimal efficacy with only 20–60% protec-
tion achieved in pre-clinical and clinical studies. [21–23] We con-
tend that the sub-optimal clinical efficacy of previous chemo-
prophylactic nasal sprays can be attributed, at least in part, to
their dependence on a single mode of action, typically centered
around either pathogen neutralization or hindering pathogen en-
try through the nasal lining. Additionally, as pathogens are de-
posited in the nasal cavity through the impaction of large respi-
ratory droplets, [24] the potential for these droplets to bounce off
the cavity wall and reach the deeper airways has been overlooked
in previous approaches. A comprehensive prophylactic strategy
should prioritize the effective capture of pathogen-laden droplets,
preventing them from bouncing off—a critical consideration ne-
glected in prior efforts. Previous chemoprophylactic approaches
also target a limited type/class of pathogen,[21,25–28] which could
potentially compromise their effectiveness against newly emerg-
ing pathogens. Finally, many chemoprophylactic nasal sprays
face limitations for repeated/daily application due to toxicity
concerns.[29,30]
Herein, we report a Pathogen Capture and Neutralizing Spray
(PCANS), which, unlike previously developed chemoprophylac-
tic approaches, acts via a multi-modal approach that involves
three key steps (Figure 1). First, PCANS enhances the capture
of pathogen-laden respiratory droplets from inspired air by pre-
venting them from bouncing off the nasal lining. Second, PCANS
provides a physical barrier over nasal mucosa to intercept inva-
sion/colonization of different pathogens. Last, PCANS rapidly
neutralizes a wide range of pathogens. To ensure safety during
daily or repeated use and achieve design simplicity, PCANS was
meticulously designed as a “drug-free” formulation, utilizing ma-
terials from inactive ingredient database (IID) or generally recog-
nized as safe (GRAS) list of the Food and Drug Administration
(FDA) that are present as excipients in commercially available
nasal/topical formulations and are available commercially in tons
of quantities. Using a highly iterative approach, we performed
rigorous screening of these excipients, their different concentra-
tions and combinations to identify optimal agents and their con-
centrations that maximize the efficacy of each step of the multi-
modal approach, while maximizing the residence time of PCANS
in the nasal cavity. Consequently, the final formulation of PCANS
comprises a blend of multiple active agents, each contributing to
the overall effectiveness through a synergistic multi-modal ap-
proach. Given the diverse active agents in PCANS, we also con-
ducted comprehensive studies to ascertain the optimal concen-
tration for each agent, ensuring that their individual activities re-
mained unaltered. This meticulous investigation was imperative
to guarantee the efficacy at every stage of the multi-modal ap-
proach.
In vitro, PCANS demonstrated excellent physical barrier prop-
erty against multiple viruses and bacteria preventing their trans-
port by >99.99%. The unique composition of PCANS enabled
broad-spectrum neutralization activity against a range of viruses
(both enveloped and non-enveloped) and bacteria, resulting in99.99% reduction in the pathogen load. On the other hand,
iota/kappa carrageenan, the key component of a commercially
available chemoprophylactic nasal spray- AGOVIRAX, resulted
in only 90% reduction in the viral loads of pathogens includ-
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Figure 1. Pathogen Capture and Neutralizing Spray (PCANS) acts via a multi-modal approach against respiratory pathogens. An aqueous, “drug-free”
solution of PCANS, comprising mucoadhesive biopolymers, surfactants, and alcohol, is administered using a pocket-sized nasal spray device and
undergoes a phase transition to form a hydrogel layer over nasal mucosa. Surfactants in PCANS reduce interfacial tension of the nasal lining and increase
wettability to enhance the capture or reduce the bounce-off of pathogen-laden respiratory droplets from the inhaled air. PCANS layers as a physical barrier
preventing the transport of pathogens through the nasal lining. Finally, pathogens are neutralized by biopolymers and surfactants present in PCANS.
PCANS is cleared via the native mucosal clearance mechanism and is eliminated through the digestive route.
ing IAV and SARS-CoV-2 viruses. Coating a 3D-model of hu-
man nasal cavity with PCANS significantly increased the cap-
ture of large respiratory droplets, compared to an only mucus-
coated nasal cavity. Intranasal administration of PCANS-loaded
with a fluorescent dye resulted in at least 8 h of residence time
in the mouse nasal cavity, measured as the retention of fluo-
rescence signal over time. No discernible tissue inflammation
was observed in mouse nasal turbinate after repeated dosing of
PCANS, establishing its safety in mice. In a proof-of-concept in
vivo study performed in a mouse model of severe Influenza A
infection induced by a supra-lethal dose of PR8 virus (a mouse-
adapted H1N1 Influenza virus), PCANS showed superior pro-
phylactic effect compared to the efficacy reported for previous
approaches.[21] Pre-exposure prophylactic administration of a sin-
gle dose of PCANS was effective within 15 min, resulting in
99.99% reduction in lung viral titer, and 100% survival by day
10 as compared to 0% observed in the PBS-treated group. PCANS
also suppressed pathological manifestations, and offered protec-
tion for at least 4 h. Overall, PCANS holds promise as a pre-
exposure prophylactic approach to prevent current and emerging
respiratory infections. The straightforward and readily scalable
manufacturing process of PCANS, combined with its “drug-free”
composition and robust stability demonstrated in this study, posi-
tions it favorably for widespread adoption and global distribution.
To our knowledge, this is the first study to describe a multi-modal
chemoprophylactic nasal spray, as well as the first to demon-
strate broad-spectrum activity of a chemoprophylactic nasal spray
against both bacteria and viruses. Additionally, this nasal spray is
the first to achieve an 8-hour residence time in mouse nasal cav-
ity, and the first chemoprophylactic strategy to show 100% pro-
tection in animals within a pre-clinical model of respiratory virus
infection.
- Results
2.1. Leveraging Biopolymers to Restrict Pathogen Entry via
Formation of a Physical Barrier
We selected mucoadhesive biopolymers that are listed in the IID
or GRAS list of the FDA and are present as excipients in commer-
cially available nasal/topical formulations. Specifically, gellan,
pectin, hydroxypropyl methylcellulose (HPMC), carboxymethyl
cellulose sodium salt (CMC), carbopol, and xanthan gum were
selected. The biopolymers were screened for their ability to im-
part physical barrier property to PCANS. Since a metered spray
device would be used to administer PCANS, we first identified
sprayable concentration of each biopolymer by performing rhe-
ological measurements (Figure 2a–f). Dynamic viscosity curves
were generated using a rotational rheometer by varying shear
rates up to 40 s-1, which is within the lower limits of shear
rates encountered while dispensing formulations through a nasal
spray device. Concentrations that exhibited a viscosity of less
than 0.1 Pa.s were considered “sprayable”.[31,32] Next, we deter-
mined the mechanical strength of each biopolymer at the high-
est sprayable concentration before and after the addition of sim-
ulated nasal fluid (SNF). SNF was added to mimic the physio-
logical environment in the nasal cavity. Mechanical strength was
measured using a rotational rheometer and quantified as storage
modulus (G’), which represents the amount of structure present
in a material.[33] In the presence of SNF, gellan showed the high-
est G’ as compared to other biopolymers (Figure 2g), indicating
its superior mechanical strength. Gellan showed a 100-fold in-
crease in its G’ in the presence of SNF (Figure 2g), which is con-
sistent with its ability to undergo in situ gelation under phys-
iological conditions. Mono and divalent cations present in the
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Figure 2. Biopolymers were screened for physical barrier property against pathogen entry. Viscosity as a function of shear rate up to 40 s−1 at 25 °C for
different concentrations of a) gellan, b) pectin, c) hydroxy propyl methyl cellulose (HPMC), d) carboxymethylcellulose (CMC), e) Carbopol and f) xanthan
gum in water. The sprayable viscosity window is shown below the dashed line. g) Storage modulus (G’) of 0.4% (w/v) gellan, 2% (w/v) pectin, 0.5%
(w/v) HPMC, 0.5% (w/v) CMC, 0.2% (w/v) Carbopol, and 0.2% (w/v) xanthan gum, without and with simulated nasal fluid (SNF). Amplitude sweep
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SNF complex with glucuronic monomeric units of gellan to form
a crosslinked hydrogel. [34] Compared to gellan, other biopoly-
mers showed minimal or no increase in their storage modulus,
suggesting poor in situ gelation. To investigate physical barrier
property of biopolymers, a trans-membrane assay was devised
(Figure S1, Supporting Information), which involved evaluating
the transport of IAV through an SNF-coated cell strainer (pore
size ≈70 μm) or a cell strainer coated with simulated mucus/SNF
mixture or a biopolymer/SNF mixture. After 4 h, the viral titer in
the chamber below the strainer was quantified by performing a
plaque assay in Madin-Darby canine kidney (MDCK) host cells.
Consistent with its excellent mechanical strength, Gellan/SNF
reduced the transport of IAV particles by >4-log fold (99.99%)
as compared to only SNF-coated or mucus/SNF-coated strain-
ers (Figure 2h). Xanthan/SNF, CMC/SNF and HPMC/SNF also
significantly reduced the IAV transport, but not as efficiently as
gellan/SNF. Interestingly, despite significantly lower mechani-
cal strength of pectin/SNF as compared to gellan/SNF, it inter-
cepted the IAV transport with similar efficiency as gellan/SNF.
Carrageenan, a biopolymer used in previously reported and com-
mercially available chemoprophylactic nasal sprays,[35] was used
as a control and did not reduce IAV transport in the presence of
SNF.
Reduction in the transport of IAV particles by anionic biopoly-
mers could be a result of their physical barrier property and/or
electrostatic interactions between their negatively charged poly-
meric chains and the positively charged capsid of IAV. To decou-
ple the effects of physical barrier property and electrostatic inter-
actions, we studied the transport of a low molecular weight dye,
rhodamine B isothiocyanate, which does not exhibit electrostatic
interactions with any of the biopolymers evaluated, as confirmed
using an in silico study (Figure S2, Supporting Information). Gel-
lan/SNF resulted in 100% reduction in the transport of the dye,
confirming excellent physical barrier property (Figure 2i). In con-
trast, other biopolymers did not reduce the transport of the dye,
indicating their poor physical barrier property. This confirms that
the reduction in IAV transport by pectin was primarily mediated
via electrostatic interaction of pectin’s chains with the virus cap-
sid.
Next, we aimed to investigate the underlying mechanism re-
sponsible for the observed reduction in the transport of rho-
damine B isothiocyanate by the gellan/SNF system. Data from
the in silico study exploring the intermolecular interactions of
rhodamine B isothiocyanate with different biopolymers (Figure
S2 and Table S1, Supporting Information) indicated that the dye
interacts with gellan primarily through hydrogen bonds, similar
to its interactions with other biopolymers. Notably, rhodamine
B isothiocyanate does not display hydrophobic interactions with
gellan but does with certain other biopolymers. Therefore, the in-
teractions between the dye and gellan do not appear to be unique
and are unlikely to be the primary factor contributing to the ob-
served inhibition of the dye transport with gellan/SNF. Interest-
ingly, gellan/SNF reduced the transport of rhodamine B isothio-
cyanate in a concentration-dependent manner (Figure 2j), with
a minimum of 0.1% w/v gellan required. Higher polymer con-
centration in a gel can increase tortuosity. [36] As the concentra-
tion of the polymer increases, the network of cross-linked poly-
mer chains becomes denser. This increased tortuosity creates
more complex and convoluted diffusion pathways, thereby re-
ducing the effective diffusion coefficient. Previous studies have
shown that in systems with large pores, where the pore diameter
is approximately 30–40 times the size of the diffusing molecule,
geometric tortuosity is the primary factor influencing diffusive
transport.[37] In contrast, for pores with diameters barely 10 times
the molecular size, intermolecular interactions are more pro-
nounced. Given the small size of rhodamine B isothiocyanate
(≈2 nm) relative to the micron-sized pores in the gellan gel,[38]
the diffusion of the dye through gellan gel aligns with the for-
mer scenario, suggesting that the observed transport reduction
could be attributed to the increased tortuosity caused by the gel’s
cross-linking. Another physical factor responsible for the reduced
transport of the dye could be the mechanical strength of the gel,
measured as the storage modulus (G’), which increased with
the concentration of gellan (Figure S3, Supporting Information).
An increase in the mechanical strength of gels results in re-
duced swelling and hence reduces the diffusivity of molecules. [39]
Therefore, taken together, the primary drivers for the reduction
in dye transport by the gellan/SNF can be attributed to both in-
creased tortuosity and gel strength at specific gellan concentra-
tions.
Gellan/SNF also showed excellent physical barrier property
against bacteria. A 0.2% w/v concentration of gellan also reduced
the transport of E.coli bacteria by >8-log fold (100%) (Figure S4,
Supporting Information), suggesting it’s broad spectrum physi-
cal barrier property to limit the transport of both viruses and bac-
teria. Mucus, on the other hand, only showed a 1-log fold (90%)
reduction. To conclude, gellan at a concentration of 0.2% w/v and
above impeded the transport of rhodamine B dye, E. coli, and IAV
by 100%.
To ensure maximum coverage of the nasal cavity, we evalu-
ated the spray characteristics of gellan at a concentration of 0.2%
w/v or higher when sprayed using a nasal spray pump (Aptar).
measurements were performed at 37 °C by varying oscillatory strain between 0.005% to 10% at 1 Hz frequency. *P < 0.0001, P < 0.05. n.s., not
significant. h) Amount of Influenza A virus (IAV) that permeated within 4 h through a simulated nasal fluid (SNF)-coated cell strainer (pore size ≈70 μm)
or a cell strainer coated with simulated mucus/SNF mixture or a biopolymer/SNF mixture. Permeation of viral particles was quantified by evaluating the
viral titer in the chamber below the strainer using plaque assay performed in MDCK host cells. Results are expressed in plaque-forming units (PFU/mL).
P < 0.01, P < 0.05 compared to mucus/SNF, n.s, not significant. Percentage permeation of a fluorescent dye, rhodamine B isothiocyanate through
i) an SNF-coated cell strainer or a cell strainer coated with simulated mucus/SNF mixture or a biopolymer/SNF mixture. *P < 0.0001 compared to
mucus/SNF and j) an SNF-coated strainer or strainer coated with gellan/SNF at different concentrations of gellan. P < 0.0001 compared to 0.05%
w/v gellan/SNF. k) Percentage drip length of free brilliant green dye or mucoadhesive polymers mixed with brilliant green dye on sheep mucosal tissue.
Drip length from the spray area was measured as the distance traversed in 4 h by the biopolymer or free dye from the point of deposition. The percentage
drip length of each biopolymer was calculated with respect to the drip length of the free dye. *P < 0.0001 compared to free dye. For g and h, P-values
were determined using two-way ANOVA with Tukey’s multiple comparisons tests. For i-k, P-values were determined using one-way ANOVA with Tukey’s
post hoc analysis. Data in a-f are from a single experiment (experiment repeated three times). Data in g–k are means ± SD of technical repeats (n = 3,
each experiment performed at least twice).
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Plume geometry and spray coverage were measured with a high-
speed image acquisition system. Increasing gellan concentration
resulted in a significant reduction in the angle of emitted plume
of the spray (defined as “plume angle”) and coverage area (Figure
S5a–d, Supporting Information). Henceforth, we used 0.2% w/v
gellan due to its superior physical barrier property, plume angle,
and coverage area, as compared to other concentrations.
Next, we evaluated the retention ability of gellan and other
biopolymers at the mucosal tissue upon spraying. Mucosal re-
tention was measured as the drip length, defined as the distance
traversed in 4 h by the biopolymer from the point of deposition on
sheep’s intestinal mucosa placed vertically. To visualize dripping,
biopolymers were mixed with a brilliant green dye. The percent-
age drip length of each biopolymer was calculated with respect to
the drip length of the free dye. Gellan (0.2% w/v) demonstrated
excellent mucosal retention with zero drip length (Figure 2k;
Figure S6, Supporting Information). Other biopolymers, includ-
ing carrageenan, which was used as a control showed >95%
drip length, indicating poor mucosal retention. Gellan’s superior
mucosal retention is attributed to its ability to strongly entangle
with mucin glycoprotein in the mucosal tissue during the sol-gel
transition. [40]
2.2. Identifying Agents for Neutralizing a Broad-Spectrum of
Respiratory Pathogens
To impart PCANS a broad-spectrum pathogen neutralization
ability, we screened agents from three different classes of com-
pounds, including biopolymers, surfactants, and alcohols. We de-
fine “neutralization” as a process that impedes pathogen entry
into host cells by either destabilizing the pathogen cell mem-
brane or blocking the receptor-mediated binding/fusion of the
pathogen through chemical interactions. These compounds were
selected based on their previously reported ability to neutralize
different types of pathogens. [41–43] To maximize safety and trans-
latability of PCANS, we only selected agents that are listed in the
IID or GRAS list of the FDA and are present as excipients in com-
mercially available nasal/topical formulations (Figure 3a). We
first evaluated the neutralization ability of these agents against
viruses (Figure 3b–k). Neutralization was studied in vitro by in-
cubating each agent individually with either IAV or SARS-CoV-2
for 10 or 60 min, followed by 1-min centrifugation and subse-
quent infection of target cells with the supernatant evaluated us-
ing plaque forming or focus-forming assay. We chose IAV and
SARS-CoV-2 due to their high prevalence worldwide as respi-
ratory viruses and also due to a difference in their capsid pro-
teins and charge.[44,45] Biopolymers were evaluated at their high-
est sprayable concentration, except for gellan and carrageenan.
Gellan was evaluated at 0.2% w/v due to its superior physical
barrier property compared to 0.1% w/v concentration and su-
perior spray pattern compared to 0.4% w/v concentration. Car-
rageenan, used as a control, was evaluated at 0.16% w/v, as this
concentration is present in a commercially available chemopro-
phylactic nasal spray.[46,47] Surfactants and alcohols were evalu-
ated at the highest concentration previously used in humans via
nasal route.[48,49] Compared to carrageenan, pectin exhibited su-
perior neutralization of IAV, regardless of the incubation time,
and demonstrated a 4-log fold (99.99%) reduction in viral titer in
the host cells in comparison to PBS (Figure 3b). Ten min of in-
cubation with carbopol did not reduce the IAV titer, but a 4-log
fold (99.99%) reduction was observed with 60 min of incubation.
Gellan exhibited similar neutralization of IAV as carrageenan, re-
sulting in only a 1-log fold (90%) reduction in viral load in the host
cells. For SARS-CoV-2, both pectin and carrageenan showed less
than a 1-log fold decrease in viral load in the host cells (Figure 3g).
Gellan showed a 4-log fold (99.99%) reduction in the viral titer,
but only with 1 h incubation time. Among surfactants, tween 80
and benzalkonium chloride (BKC) showed a 1-log log fold reduc-
tion in IAV titer in the host cells, regardless of the incubation
time (Figure 3c). Rapid neutralization of SARS-CoV-2 was ob-
served with BKC, resulting in a 5-log fold (>99.99%) reduction
in viral load in the host (Figure 3h). Alcohols did not neutralize
SARS-CoV-2, and minimum neutralization was observed for IAV,
resulting in less than 1-log fold (90%) reduction in viral load for
chlorobutanol and phenethyl alcohol (PEA) (Figure 3d,i). Overall,
this extensive screening identified pectin and BKC as the most
effective agents for rapid neutralization of IAV and SARS-CoV-2,
respectively. Neutralization ability of pectin and BKC was found
to be dose-dependent (Figure 3e,j). Minimum concentrations of
0.75% w/v and 0.01% w/v were required for pectin and BKC, re-
spectively, to achieve >4-log fold (>99.99%) reduction in the viral
load with 10 min of incubation time.
To elucidate the viral neutralization mechanism of pectin and
BKC, we performed in silico modeling to determine their bind-
ing affinity with the receptor binding domains (RBD) of IAV
and SARS-CoV-2, respectively. For IAV, anionic pectin targets
RBD at the distal part of hemagglutinin, which is positively
charged, thus averting the virus entry into the host cell (Figure 3l).
Compared to the host ligand sialic acid present in mucosal ep-
ithelia, pectin showed stronger binding to RBD through dis-
tant hydrogen bonding with Se228, Ser186, and Thr187 and hy-
drophobic linkage with Ser227 and Glu190 (Figure S7, Support-
ing Information). BKC was found to exhibit hydrophobic inter-
actions with the ACE2 binding motif of spike protein of SARS-
CoV-2 (Figure 3m). BKC also showed hydrophobic interactions
with Phe23 and Phe26 in membrane helices via pi-pi stacking
(Figure 3m), which can distort the helical conformation of adja-
cent helices, as aromatic stacking of Phe23 and Phe26 is a pre-
requisite to stabilizing helix-helix interface of the envelope trans-
membrane protein. BKC fits into the pentameric ion channels
at the N terminus of the transmembrane domain through in-
teraction with Thr11 and potentially blocks the influx/efflux of
ions (Figure 3n). To determine the role of electrostatic interaction
in pectin- and BKC-mediated neutralization of IAV and SARS-
CoV-2, respectively, we performed a neutralization assay by pre-
treating pectin and BKC with counter ions to offset the charge. As
anticipated, anionic pectin in the presence of positively charged
polyethyleneimine lost its neutralization activity and failed to
show a significant reduction in the viral load compared to PBS
(Figure 3f). Likewise, the pretreatment of BKC with negatively
charged bovine serum albumin diminished the ability of BKC to
reduce the SARS-CoV-2 titer in the host cells (Figure 3k).
Next, we investigated whether ionic interactions between an-
ionic gellan or pectin with cationic BKC, when present to-
gether in a formulation, would impact the neutralization abil-
ity of pectin or BKC. Notably, the neutralization efficiency of
pectin (0.75% w/v) against IAV remained conserved even with a
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dose-dependent increase in BKC up to a concentration of 0.1%
w/v (Figure 3o). Neutralization efficiency of BKC (0.01% w/v)
against SARS-CoV-2 was not impacted by gellan or pectin at 0.2%
w/v or 0.75% w/v concentrations, respectively, but reduced at
higher concentrations (Figure 3p,q). These results further under-
score that the concentration of each agent is critical for efficient
neutralization.
Finally, we also screened surfactants and alcohols to assess
their neutralization ability against bacteria, including E. coli and
K. pneumoniae. Neutralization was determined by measuring the
bactericidal activity. Each agent was individually incubated with
either E. coli or K. pneumoniae for 30 or 60 min, followed by 1-
min centrifugation, and then evaluating the bacterial load in the
supernatant using a colony-forming assay. BKC was more effec-
tive than non-ionic surfactants, resulting in a 4-log fold (99.99%)
and 7-log fold (99.99%) reduction in colony-forming units (CFU)
of E. coli and K. pneumoniae, respectively, with an incubation
time of 30 min (Figure 3r,t). Previously reported molecular mod-
eling studies have shown that BKC incorporates into the bacte-
rial membrane, resulting in its destabilization.[50] This involves
BKC’s attachment to the bacterial cell surface followed by its
quick integration into the lipid bilayer, where it stays for at least
several nanoseconds, likely causing membrane destabilization.
Alcohols had a negligible bactericidal effect over the exposure
periods of 30 or 60 min (Figure 3s,u). Altogether, our data on
physical barrier property, spray pattern, mucosal retention, and
neutralization indicate gellan, pectin, and BKC as the three crit-
ical components to formulate PCANS. However, we also incor-
porated phenethyl alcohol (PEA), as it is commonly added as a
stabilizer to nasal formulations to prevent the growth of gram-
negative bacteria, [48] thereby ensuring a long shelf-life.
2.3. Utilizing Surfactants to Promote the Capture of Respiratory
Droplets
Pulmonary surfactant layers the alveolar epithelium to en-
hance wettability and trap airborne particles.[51] We adopted
this biomimetic approach to capture pathogen-laden respiratory
droplets in the nasal cavity. Specifically, we identified surfactants
to reduce interfacial tension of PCANS and reduce the bounce
off/escape of respiratory droplets. We evaluated surfactants listed
in the IID list, including Tween-20, Tween-80, and BKC. Screen-
ing was performed using a twin impinger, which is a glass appa-
ratus that can be used to assess the deposition of aerosolized par-
ticles in different regions of the respiratory tract (Figure 4a).[52,53]
Simulated mucus or a biopolymer mixture of gellan (0.2% w/v)
and pectin (0.75% w/v) without or with different concentrations
of surfactants was sprayed into the SNF-coated oropharyngeal re-
gion of the impinger (Figure 4a). Droplets with mass medial aero-
dynamic diameter >5 μm and laden with rhodamine B-loaded
liposomes (size ≈400 nm) were generated using a jet nebulizer
to mimic pathogen-laden large respiratory droplets. Droplet cap-
ture was determined by quantifying the fluorescence intensity of
rhodamine B in the biopolymer/surfactant mixture or the mucus
layer. Biopolymer mixture without any surfactant showed simi-
lar fluorescence intensity as mucus (Figure 4b). Combining the
biopolymer mixture with Tween-80 or Tween-20 at a concentra-
tion higher than 0.005% w/v or with BKC at a concentration
higher than 0.01% w/v resulted in a significant increase in the
fluorescence intensity as compared to mucus or only biopoly-
mer mixture, suggesting increased capture of droplets due to
surfactants. Compared to Tween-20, BKC and Tween-80 resulted
in a significantly higher fold increase in the fluorescence inten-
sity when added to the biopolymer mixture at a concentration
of 0.05% w/v or higher (Figure 4b). At 0.05% w/v concentration,
both BKC and Tween-80 containing biopolymer mixtures showed
similar fluorescence intensity, which was 4-fold higher than the
fluorescence intensity of mucus or biopolymer mixture without a
surfactant. Since 0.01% w/v is the most commonly used concen-
tration of BKC in commercially available nasal formulations,[54,55]
and also showed excellent neutralization activity against SARS-
CoV-2, we decided to use this concentration in PCANS, even
though BKC didn’t increase the capture of respiratory droplets
at this concentration.
To impart respiratory droplet-capturing ability, we decided to
proceed with Tween-80 and determined its safe concentration
that would not compromise the permeability or metabolic activ-
ity of nasal epithelium. To that end, we performed an in vitro
Figure 3. Biopolymers, surfactants and alcohols were screened for neutralization of different respiratory pathogens. a) Table summarizes different
components and their concentrations to determine the neutralization ability against respiratory pathogens. Each component was individually evaluated
for its pathogen neutralization potential. IAV and SARS-CoV-2 viral loads in the host cells after 10 or 60 min incubation of the virus with b, g) different
biopolymers, c, h) different surfactants, and d, i) different alcohols. Viable viral titer was quantified using plaque assay in MDCK host cells for IAV and
focus-forming assay in Vero E6 cells for SARS-CoV-2 virus. Results are expressed in plaque-forming units (PFU/mL) or focus-forming units (FFU/mL).
P < 0.0001, P < 0.001, P < 0.01, P < 0.05 compared to 10 minutes of incubation with PBS. n.s, not significant. Viral loads in the host cells
after 10 min incubation of e) IAV and j) SARS-CoV-2 with different concentrations of pectin and BKC, respectively. P < 0.01, P < 0.05 compared to
PBS. Viral loads in the host cells after 10 min incubation of f) IAV and k) SARS-CoV-2 with pectin (0.75% w/v) + polyethylenimine and BKC (0.01% w/v) +
bovine serum albumin, respectively. P<0.01 compared to PBS. n.s, not significant. l) Pectin (yellow) binds to the receptor binding site of IAV (purple)
at the distal part of hemagglutinin monomer (colored in purple) through hydrophobic interactions with Ser227 and Glu190, and hydrogen bonding with
Ser228, Ser186, and Thr187. Blue and red dots in hydrogen bonding maps represent carbon and oxygen atoms, respectively. m) Chemical interaction of
BKC (green) with ACE2 binding motif (red) in the spike protein of SARS-CoV-2. Interaction map reveals the hydrogen bonding of BKC with Tyr505 and
Gly496. n) Aromatic pi-pi interaction of BKC (green) with Phe23 (purple) in the transmembrane domain and with Thr11 (brown) membrane helices.
Interaction analysis shows 10 hydrophobic bonds with Phe23 and 8 hydrophobic bonds with Phe26. o) Viral load in host cells after 10 min incubation
of IAV with pectin (0.75% w/v) in the presence of different concentrations of BKC. *P < 0.0001 compared to PBS. Viral load in the host cells after
10 min incubation of SARS-CoV-2 with BKC (0.01% w/v) in the presence of different concentrations of p) gellan and q) pectin. P < 0.0001 compared
to PBS. Effect of surfactants (r, t) and alcohols (s, u) against gram-negative bacteria E. coli and K. pneumoniae using colony-forming unit (CFU) plate
count method after 30 and 60 minutes of exposure. Viable bacterial colonies are expressed in CFU/mL. P<0.05 compared to PBS. For b–d, P values
were determined using two-way ANOVA with Tukey’s post hoc analysis. For e, f, j, k, o–q, P values were determined using one-way ANOVA. Data in b-k
and o-u are presented as Means ± S.D of technical repeats (n = 3, each experiment performed at least twice). Ser, serine; Thr, threonine; Glu, glutamic
acid; Phe, phenylalanine; Tyr, tyrosine; Gly, glycine.
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Figure 4. PCANS enhances the capture of respiratory droplet-mimicking aerosol and exhibits prolonged nasal residence time in mice. a) Experimental
design for measuring the capture of respiratory droplet-mimicking aerosol. A twin impinger was used to simulate the aerodynamics of the human
respiratory tract. Mucus or gellan (0.02% w/v) and pectin (0.75% w/v) solution (G+P), without or with different concentrations of Tween-80, Tween-20
or BKC was coated on the inner surface of the throat region of the impinger using a nasal spray device. Droplets with mass medial aerodynamic diameter5 μm and laden with rhodamine B-loaded liposomes (size ≈400 nm) were generated using a jet nebulizer and administered into the impinger under
vacuum (15 L min−1 ). Droplet capture was determined by quantifying the fluorescence intensity of rhodamine B in the biopolymer/surfactant mixture or
mucus layer. b) Fold increase in fluorescence intensity with respect to mucus. *P < 0.0001, P < 0.05 compared to mucus. c) Transepithelial electrical
resistance (TEER) across the human nasal epithelial cell (RPMI-2650)-based monolayer at different time points after treatment with only medium or
medium containing Triton-X (0.1% w/v) or different concentrations of Tween-80. Surfactant-containing medium was replaced at 4 h with fresh medium
to examine impedance recovery. P < 0.0001, P < 0.001 compared to untreated control. n.s, not significant. d) Experimental design for measuring
the capture of respiratory droplet-mimicking aerosol using a 3D human nasal cavity model (Koken cast). The inner surface of the nasal cavity was coated
with mucus, G+P solution or PCANS (the final formulation) using a nasal spray device. The throat part of the model was coupled to a vacuum pump for
simulating the respiratory airflow (15 L min−1 ). Nostrils were then exposed to nebulized rhodamine B-loaded liposomes for 1 minute. Droplet capture
was determined by quantifying the fluorescence intensity of rhodamine B in the nasal cavity. e) Fold increase in fluorescence intensity with respect to
mucus. P < 0.01 compared to mucus. n.s, not significant. f) Experimental outline for the evaluation of the nasal residence time of PCANS in mice.
C57BL/6 mice were intranasally administered with 10 μL of free DiR or DiR-loaded PCANS (PCANS/DiR) into each nostril. Mice were euthanized at
different time points over 24 h, and nasal cavity was harvested and imaged using an in vivo imaging system (IVIS). g) Representative images of the
nasal cavity excised at different time points. h) Quantification of fluorescence intensity in the nasal cavity at different time points. i) Fold change in total
flux at 8 h in the nasal cavity relative to G+P. P < 0.05, compared to G+P. n.s, not significant. j) Experimental design to assess the biocompatibility of
PCANS in mouse nasal cavity. 10 μL PCANS or PBS was administered into each nostril of C57BL/6 mice once daily for 14 consecutive days. Animals
were euthanized on day 15 and nasal cavity was analyzed histologically. k) Representative images of H&E-stained sections of nasal turbinate from mice
captured using a 4X objective. Insets represent healthy olfactory epithelium (i) and (iii), and lamina propria (ii) and (iv) captured at 20X objective. For
b, e and i, P values were determined by one-way ANOVA using Tukey’s post hoc analysis. For b, concentrations for each surfactant were compared
individually. For c, P values were determined by two-way ANOVA with Tukey’s multiple comparison test. Data in b,c, and e are presented as Means ± S.D
of biological repeats (n = 3, each experiment performed at least twice). Data in h and i are presented as Means ± SEM (n = 5 mice/group).
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assay evaluating the transepithelial electrical resistance (TEER)
across the human nasal epithelial cell (RPMI-2650)-based mono-
layer upon treatment with different concentrations of tween-80.
A transient dip of less than 15% in TEER was observed in the
monolayer immediately after the addition of Tween-80, irrespec-
tive of the concentrations evaluated in this study. However, the
TEER reversed rapidly to the original value in less than 1 h af-
ter replacing tween-80-containing medium with fresh medium
(Figure 4c). The drop in the TEER for Tween-80 was signifi-
cantly less compared to Triton-X (negative control), which re-
sulted in a permanent change in the TEER. Second, we evalu-
ated the effect of different concentrations of Tween-80 on the
metabolic activity of RPMI-2650 cells upon 24 or 48 h of incuba-
tion. Cells incubated with 0.01% or 0.05% w/v tween-80 showed
similar metabolic activity as cells incubated in medium. How-
ever, Tween-80 (0.5% w/v) resulted in a significant reduction in
the metabolic activity of RPMI cells (Figure S8, Supporting In-
formation). Thus, we decided to use 0.05% w/v as the final con-
centration of Tween-80 in PCANS. Overall, based on our data
for physical barrier property, spray pattern, mucosal retention,
neutralization, droplet capture, and nasal epithelial cell toxicity,
we decided on gellan, pectin, BKC, PEA, and Tween-80 as the fi-
nal components for PCANS, and validated the respiratory droplet
capturing ability of the final formulation using a 3D- model of
human nasal cavity (Koken cast) with the anatomical intricacies
(Figure 4d).[56] Consistent with the twin impinger results, there
was no significant difference in the fluorescence intensity be-
tween gellan and pectin mixture, and mucus (Figure 4e). PCANS,
on the other hand, showed a 2-fold higher fluorescence com-
pared to mucus, suggesting the potential of PCANS to increase
the capture of pathogen-laden respiratory droplets from inhaled
air.
2.4. Prolonged Nasal Retention of PCANS and Safety Upon
Repeated Administration
Next, we evaluated the retention of PCANS in the nasal
cavity of mice (Figure 4f). PCANS (10 μL) mixed with
a fluorescent dye – (DiIC18(7) (1,1′-Dioctadecyl-3,3,3′,3′-
Tetramethylindotricarbocyanine Iodide) (DiR) was administered
into both nostrils of C57/BL6 mice. Free DiR was used as a
control. Mice were euthanized at different time points over 24 h,
and nasal cavity was harvested and imaged using an in vivo
imaging system (IVIS) to quantify the fluorescence signal from
DiR. Free DiR resulted in negligible fluorescence signal, even
at 15 min after administration, suggesting its rapid clearance
(Figure 4g,h). Interestingly, mice administered with DiR-loaded
PCANS showed significant fluorescence for up to 8 h, sug-
gesting prolonged nasal retention of PCANS (Figure 4g,h). We
hypothesized that prolonged retention of PCANS is attributed to
the presence of surfactants, including Tween-80 and BKC, which
have previously been shown to reduce cilia beat frequency in the
nasal cavity. [57] To test our hypothesis, we compared nasal reten-
tion of DiR-loaded mixture of gellan and pectin without or with
tween-80 or BKC. The addition of both BKC or tween-80 signifi-
cantly enhanced the nasal retention of gellan and pectin mixture
at 8 h post-nasal administration, as evident from the fluorescent
signal of DiR in the nasal cavity (Figure 4i). However, tween-80
resulted in significantly higher nasal retention than BKC. The
ability of tween-80 to enhance the nasal retention of gellan and
pectin mixture was found to be concentration-dependent (Figure
S9, Supporting Information). However, considering irreversible
nasal epithelial permeabilization and cytotoxicity at 0.5% w/v or
higher concentration of tween-80, we maintained 0.05% w/v in
PCANS for further experiments. Notably, nasal administration
of DiR-loaded PCANS only showed fluorescence signals in the
nasal cavity and stomach, suggesting no systemic absorption.
PCANS was fully cleared at 24 h (Figure 4g,h;Figure S10,
Supporting Information), resulting in negligible fluorescence
signal in both the nasal cavity and the stomach. To confirm
safety of PCANS, we performed a repeat-dose toxicity study in
healthy mice intranasally administered with PCANS or PBS
once daily for 14 consecutive days (Figure 4j). Hematoxylin and
eosin (H&E) stained sections of nasal cavity from both PBS or
PCANS-administered mice did not show any inflammation or
other gross evidence of toxicity, as evident by a defined lamina
propria (Figure 4k). This connotes the safety of PCANS for daily
administration.
2.5. Broad-Spectrum Activity, Spray Characteristics, and Shelf
Stability of PCANS
Having identified the final components of PCANS, along with
their optimal concentrations, we sought to demonstrate the
physical barrier property and neutralization ability of PCANS
against a broad spectrum of respiratory pathogens, including en-
veloped viruses (IAV, SARS-CoV-2, RSV), a non-enveloped virus
(adenovirus), and bacteria (E. coli and K. pneumoniae). Physi-
cal barrier property was evaluated by assessing the transport of
pathogens through an SNF-coated cell strainer or a cell strainer
coated with simulated mucus/SNF mixture or PCANS/SNF mix-
ture. PCANS/SNF prevented the transport of all the pathogens
by >4-log fold (>99.99%) (Figure 5a-f), suggesting its broad-
spectrum physical barrier property. For all pathogens, except
RSV, mucus/SNF mixture showed significantly less prevention
of pathogen transport compared to PCANS/SNF. PCANS also ef-
ficiently neutralized all the tested pathogens within 10 or 30 min
of incubation time, resulting in >3-log fold (>99.9%) reduction
in pathogen load (Figure 5g–l). Excitingly, PCNAS demonstrated
remarkable efficacy in co-neutralizing both bacterial and viral
pathogens (Figure S11, Supporting Information), achieving >4-
fold (>99.99%) reduction in the load of E. coli and SARS-CoV-
2, used as proof-of-concept pathogens in this experiment. This
finding is clinically significant, as respiratory droplets can carry
both bacterial and viral pathogens, highlighting PCNAS’s poten-
tial in providing comprehensive protection against respiratory in-
fections. We also evaluated the spray characteristics of PCANS
sprayed through a standard and commercially used VP3 multi-
dose nasal spray pump (Aptar, USA). The droplet distribution
data showed that 10% of PCANS droplets had size >10 μm, and
90% had size <200 μm (Figure 5m), which is desirable to maxi-
mize the deposition in nasal cavity, while minimizing deposition
into deep lungs. PCANS resulted in a wide plume angle within
the ideal range of 35–55°, an ovality close to 1, covering a circu-
lar area of up to 8%, which is in line with the commercial nasal
sprays (Figure 5m).[58,59]
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Figure 5. PCANS exhibits broad-spectrum physical barrier property with pathogen neutralization, and is sprayable and shelf-stable. a–d) Amount of
different viruses that permeated within 4 h through a simulated nasal fluid (SNF)-coated cell strainer or a cell strainer coated with simulated mucus/SNF
mixture or PCANS/SNF mixture. Virus permeation was quantified by plaque assay in MDCK cells (IAV), Vero E6 cells (SARS-CoV-2), and Hep-2 cells (RSV
and adenovirus). P < 0.0001, P < 0.001, P < 0.01, P < 0.05. n.s, not significant. Amount of e) E. coli and f) K. pneumoniae that permeated
within 4 h through an SNF-coated cell strainer or a cell strainer coated with simulated mucus/SNF mixture or PCANS/SNF mixture. Bacterial permeation
was quantified using a CFU plate count method. P < 0.0001, P < 0.001, *P < 0.01. n.s, not significant. g–j) Viral titer for IAV, SARS-CoV-2,
adenovirus, and RSV after treatment with PBS or PCANS. IAV and SARS-CoV-2 were incubated with PCANS for 10 min, while adenovirus and RSV were
treated for 30 min. Anti-bacterial activity of PCANS against k) E. coli and l) K. pneumoniae using CFU plate count method after 30 and 10 min incubation,
respectively. *P < 0.001, P < 0.01, *P < 0.05. m) Spray characteristics of PCANS. The droplet size distribution of PCANS was analyzed using a
laser diffraction system. Representative images of single time delay plume angle and ovality ratio, captured using a high-speed digital camera and laser
light sheet. n) Experimental design to assess the stability of PCANS in accelerated temperature conditions (40 °C). PCANS was stored in glass amber
bottles. Aliquots were taken at different time points to investigate spray characteristics and pathogen neutralization efficacy. o) Plume angle, p) ovality,
q) mean droplet diameter and r) spray deposition area over a period of 60 days. P < 0.001, P < 0.01 compared to day 0, n.s, not significant. s)
Percent reduction in the viral load of IAV and SARS-CoV-2 in their respective host cells after 10 min incubation with PCANS aliquoted at different time
points in the stability study. P < 0.05 compared to day 0. n.s, not significant. For a–f and o–s, P values were determined using one-way ANOVA with
Tukey’s post-hoc analysis. For g–l, P values were determined using a two-tailed t-test. Data are presented as Means ± SD of biological repeats (n = 3,
each experiment performed at least twice).
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Shelf-stability is a key attribute governing the translational po-
tential of formulations. We tested shelf-stability of PCANS over
60 days at 40 °C temperature, as per the International Confer-
ence on Harmonization (ICH) guidelines for stability testing un-
der accelerated storage conditions (Figure 5n). Over a period of 60
days, we observed no substantial variations in the spray character-
istics, including plume angle, ovality, coverage area, and droplet
size distribution (Figure 5o–r; Figure S12, Supporting Informa-
tion). PCANS also displayed no changes in its neuralization ac-
tivity over 60 days, resulting in >99.99% reduction in Influenza
A and SARS-CoV-2 viral loads in the host cells upon 10 min of
incubation (Figure 5s). Interestingly, in a separate stability study,
we found that PEA’s presence in PCANS is critical for minimiz-
ing bacterial growth after the formulation was challenged with
E.coli (Figure S13, Supporting Information). This is consistent
with previous reports,[48] which have shown that PEA prevents
bacterial growth in nasal sprays. Collectively, these data confirm
the shelf-stability of PCANS.
2.6. PCANS Exhibits Prophylactic Activity In Vivo
Next, in a proof-of-concept study, we investigated the prophylac-
tic efficacy of PCANS against respiratory infection in vivo. PR8,
a mouse-adapted strain of H1N1 Influenza virus, was used to in-
duce infection. PR8 is a highly virulent strain that induces severe
respiratory infection in mice,[60] and can be lethal at a dose of
10 PFU. [61] In vitro assay revealed excellent potency of PCANS to
neutralize 106 PFU of PR8 within 10 min of incubation, resulting
in >5-log fold (>99.99%) reduction of the viral load in host cells
(Figure S14, Supporting Information). To demonstrate efficacy in
vivo, PCANS or PBS (10 μL) was administered prophylactically
to both the nostrils of healthy mice on day 0 (Figure 6a). Fifteen
minutes later, animals were challenged intranasally with a free-
flowing PR8 solution (10 μL/nostril), exposing mice to a total dose
of 250 PFU, which has been previously used by other groups. [62,63]
Intranasal instillation of 10 μL of a fluorescent dye (DiR) solution
into each nostril of mice showed a stronger fluorescent signal in
the lungs compared to the nasal cavity (Figure S15, Supporting
Information) at 2 h post-administration. This confirms that the
liquid reached the lungs and was not primarily retained in the
nasal cavity, suggesting that the viral suspension administered
as a liquid bolus also reached the deep lungs.
Remarkably, all mice in the PCANS-treated group survived for
at least 10 days after the infection, whereas the PBS-treated group
showed 100% lethality by day 8 (Figure 6b). Over 10 days, no dis-
cernible change was observed in the body weight of the PCANS-
treated animals, while significant weight loss was observed for
PBS-treated ones after 3 days post-infection (Figure 6c). PCANS
also curtailed the lung viral titer to undetectable levels on days
2 and 4 post-infection, resulting in >5-log fold (>99.99%) re-
duction compared to PBS-treated mice (Figure 6d, e). Com-
pared to healthy mice, mice infected with PR8 and treated with
PBS showed significant differences in the levels of inflamma-
tory cells, including leukocytes, neutrophils, lymphocytes, and
macrophages in bronchoalveolar lavage (BAL) fluid (Figure 6f–i).
Prophylactic treatment of mice with PCANS restored the levels of
inflammatory cells in BAL fluid to normal. Additionally, cytokine
profile from lung homogenate showed a significant reduction of
IL-6 and TNF-a levels in PCANS-treated mice, as compared to
the PBS-treated group (Figure 6j–l). No reduction was, however,
observed in the levels of IL-1𝛽. Histological examination of lung
sections revealed a substantial reduction in leukocyte infiltrates
in PCANS-treated mice, as compared to the PBS-treated group,
which showed an abundant presence of bronchial and alveolar
infiltrates (Figure 6m). Overall, compared to PBS-treated mice,
we observed a significant reduction in pulmonary inflammation
score for PCANS-treated group (Figure S16, Supporting Infor-
mation). An escalated dose challenge was performed to deter-
mine the potency of PCANS to neutralize a higher viral load of
PR8 (500 PFU). Compared to the PBS-treated group, prophylac-
tic treatment with PCANS significantly improved survival and
body weight and reduced lung viral titer on days 2 and 4 post-
infection (Figure S17, Supporting Information). PCANS also pro-
tected mice against the PR8 challenge performed after 2 and 4 h
of prophylactic treatment, as evident from significant reduction
observed in lung viral titer on day 2, as compared to the PBS-
treated group (Figure 6n–p). Specifically, average reductions of
60% and 78% in lung viral titer were observed for 2 and 4 h
challenge groups, respectively; however, there was no statistically
significant difference between PCANS-mediated reductions ob-
served in animals challenged after 2 versus 4 h. These data clearly
indicate the potential of nasally administered PCANS to protect
against respiratory infection in mice for at least 4 h.
- Discussion
We report PCANS – a radically simple and multi-modal pre-
exposure prophylactic nasal spray to offer protection against res-
piratory pathogens. Unlike vaccines, which are pathogen-specific
and exhibit reduced efficacy as the pathogen mutates, [64] PCANS
has the potential to offer broad spectrum protection against a
wide range of pathogens. In a proof-of-concept study performed
in mice, a single intranasal dose of PCANS was effective against
supra-lethal dosages of a highly virulent mouse-adapted strain
of H1N1 Influenza virus (PR8), and efficiently reduced the lung
viral titer providing protection for at least 4 hours. This under-
scores the potential utility of PCANS as an additional layer of
protection in conjunction with vaccines to minimize pathogen
load, considering that vaccines alone often achieve only partial re-
duction. For example, in a clinical study, participants vaccinated
with BNT162b2 and mRNA-1273 had only 40 percent less de-
tectable virus compared to those who were unvaccinated when
infected. [65]
To our knowledge, PCANS is the first multi-modal chemo-
prophylactic approach, equipped with three critical attributes
that involve 1) capturing pathogen-laden respiratory droplets in
the nasal cavity, 2) preventing pathogen transport via nasal lining
and 3) neutralizing a broad spectrum of pathogens. This is the
first report of a chemoprophylactic nasal spray that exhibits neu-
tralization activity against a broad spectrum of pathogens, includ-
ing both bacteria and viruses (enveloped and non-enveloped),
and the first that has shown >4 log-fold reduction in pathogen
load across multiple bacteria and viruses. Previously devel-
oped nasal sprays have demonstrated limited effectiveness with
only a 1–2.5-log fold reduction in pathogen load. For instance,
compared to xylitol-based formulations pHOXWELL and Xlear,
which reduced viral titer of SARS-CoV-2 by 2 and 2.5 log fold,
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Figure 6. Pre-exposure prophylactic treatment with intranasal PCANS reduces respiratory infection in mice. a) Experimental outline for the prophylac-
tic efficacy study. C57B/6 mice received a single dose (10 μL) of PCANS or PBS before 15 minutes of intranasal inoculation with 250 PFU Influenza
A/PR/8/34. One cohort of animals was followed for body weight changes and survival for a period of 10 days. Animals from a second cohort were eutha-
nized on day 2 or 4 after infection to enumerate lung viral titer, inflammatory cell count in bronchoalveolar lavage (BAL) fluid, and inflammatory cytokine
levels in lung homogenate. Hematoxylin and eosin (H&E) stained lung tissue sections from animals euthanized were assessed for inflammation. b)
Survival and c) body weight change of mice over a period of 10 days post-infection. P = 0.0007 compared to the PBS-treated group for Kaplan-Meier
survival curve. * P < 0.01 compared to PBS-treated group for body weight change curves. d) Viral titer from lung homogenate of mice and e) percentage
reduction in viral load in the lungs on day 2 and 4 post-infection, as quantified by plaque assay performed in MDCK cells. P = 0.001. f–i) Inflammatory
cell count in BAL on day 2 and 4 after infection. *P < 0.0001, *P < 0.01. Levels of j) IL-6, k) TNF- 𝛼 and l) IL-1𝛽 in lung tissues. P < 0.0001,
P = 0.005. n.s, non-significant, n.d, not detected. m) Representative images of H&E-stained lung tissue sections of virus-challenged mice that were
prophylactically treated with PBS or PCANS. Histology images were captured using 10X and 40X objectives. Scale bar: 100 μm. High-magnified insets
depict the difference in the extent of inflammatory infiltrates. Scale bar: 20 μm. n) Experimental outline to evaluate time-dependent nasal protection by
PCANS. Mice received a single dose of PCANS at 2 or 4 h prior to intranasal inoculation with 100 PFU influenza A/PR/8/34. Animals were euthanized
on day 2 post-infection to enumerate lung viral titer o) Viral titer quantified from lung homogenate and p) percentage reduction in viral load in the lungs
on day 2 post-infection for animals challenged after 2 or 4 h after prophylactic treatment. P < 0.01, *P < 0.05, n.s., not significant. For b, P values
were determined using the Gehan-Breslow-Wilcoxon test. For c, P values were determined using one-way ANOVA with Brown-Forsythe. For d and f–l,
P values were determined using two-way ANOVA with Tukey’s post hoc analysis. For o, P values were determined using one-way ANOVA with Tukey’s
post hoc analysis. n = 6 mice/group for b. Data in c are presented as Means ± SEM (n = 6 mice/group). Data in d–l are presented as Means ± SEM (n
= 4 mice/group). Data in o, p are presented as Means ± SEM (n = 6 mice/group).
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respectively,[23,66] PCANS demonstrated a remarkable 5 log fold
reduction. In our study, iota + kappa carrageenan, the key com-
ponent of a commercially available nasal spray- AGOVIRAX®,
resulted in only 1-log fold reduction in the viral loads of both
IAV and SARS-CoV-2 within 10 min, while PCANS showed >4
log fold reduction for both IAV and SARS-CoV-2 within 10 min.
The superior prophylactic effect of PCANS was also demon-
strated in vivo. Compared to a previous study,[21] where multi-
ple dosages of iota + kappa carrageenan showed 70% survival
of mice in a PR8 model of infection, a single dose of our for-
mulation showed 100% survival of mice. To our knowledge, this
is the first chemoprophylactic strategy to show 100% protection
within a pre-clinical model of respiratory virus infections. Addi-
tionally, we also demonstrated >99.99% reduction in lung viral
titer. On the other hand, iota + kappa carrageenan only showed
75% reduction.[21] PCANS also exhibits an unprecedented resi-
dence time of 8 h in the mouse nasal cavity and provides protec-
tion for at least 4 h. To our knowledge, this is the longest nasal res-
idence time that has been reported for nasal sprays in mice.
The interaction of PCANS with mucosal tissue and its prolonged
retention time involve three key mechanisms: (1) mucoadhe-
sive forces between the biopolymers (gellan and pectin) and the
mucin proteins in the mucosal layer, [67,68] (2) gelation of PCANS,
facilitated by cross-linking of gellan in the presence of physio-
logical electrolytes in nasal fluid, which intercalates with mu-
copolysaccharides to form a hydrogel mesh, preventing nasal
dripping, and (3) the presence of tween-80, which has been previ-
ously shown to slow mucociliary clearance by reducing cilia beat
frequency,[69] thereby enhancing the nasal retention of PCANS.
Such a long nasal residence time would potentially minimize
dosage frequency in future clinical studies, offering an advan-
tage over previously developed chemoprophylactic approaches,
including SaNOtize, which require 3–6 doses per day. [20,29] Fi-
nally, to our knowledge, this is the first nasal formulation that can
effectively trap large respiratory droplets from the air we breathe
in, which would be essential for maximizing its preventive effec-
tiveness in future clinical studies.
The “drug-free” nature of PCANS is favorable for the regula-
tory process, which could be tedious for chemoprophylactic ap-
proaches based on investigational new drugs such as IgM-14. [70]
Also, since all the components used in PCANS are commercially
available off-the-shelf and require simple mixing without chem-
ical modifications, our approach is amenable to scale-up and
large-scale manufacturing. PCANS is also safe for daily admin-
istration, as demonstrated in mice, which is a significant advan-
tage over previously developed povidone iodine-based anti-viral
nasal sprays, [19,30] which are associated with iodine burns, thy-
roid toxicity, and disruption of the mucosal barrier, constrain-
ing repeated administration. Similarly, frequent use of a nitric
oxide (NO)-inducing nasal spray (SaNOtize), which has shown
potential in post-exposure prophylaxis of SARS-CoV-2 infection,
can result in elevated Th2 cytokines, which mediate autoimmune
disorders.[71] In addition, excessive NO can cause tissue dam-
age and cell death.[29] The incidence of such adverse effects with
PCANS is likely to be low, as the formulation is devoid of im-
munomodulatory molecules such as NO and steroids.
Our study has several strengths. First, the components consti-
tuting PCANS were identified via rigorous in vitro and in vivo
screenings of excipients from the IID and GRAS list of the FDA,
and their different concentrations and combinations. These ex-
tensive screening experiments were aimed to optimize the key
parameters, including sprayability, mucoadhesiveness, capture
of respiratory droplets, physical barrier property, broad spectrum
pathogen neutralization activity, and nasal residence time. Our
comprehensive investigations allowed us to construct an un-
precedented, multi-modal chemoprophylactic strategy. Second,
to evaluate the respiratory droplet capturing ability of PCANS,
we used a 3D- model of human nasal cavity (Koken cast), which
has been previously used for in vitro evaluation of nasal drug
delivery, as it replicates all the anatomical intricacies of human
nasal cavity. [56] We measured capture efficiency by comparing
the fold increase in rhodamine B fluorescence intensity in the
PCANS-coated nasal cavity compared to the one coated with mu-
cus. This provided a robust and reproducible measurement. We
also attempted to measure droplet capture efficiency by counting
the droplets that passed through the model. To achieve this, we
connected the nasal cavity cast to an aerosol monitoring system.
However, the readings were inconsistent due to technical issues
like ambient dust inflow through gaps in the nebulizer-nasal cav-
ity connector and non-specific droplet deposition in the connec-
tor, leading to high variability. Third, physical barrier property
of different biopolymers was evaluated by two complementary
techniques – quantification of viral and bacterial transport using
plaque-forming and colony forming assays as well as quantifica-
tion of the transport of small molecule dye using fluorescence
spectroscopy. Fourth, we demonstrated broad spectrum physical
barrier property and neutralization ability of PCANS in five dif-
ferent pathogens – three enveloped viruses (IAV, SARS-CoV-2,
RSV), one non-enveloped virus (adenovirus), and two bacteria (E.
coli and K. pneumoniae). Lastly, to demonstrate prophylactic effi-
cacy of PCANS in vivo, we used a highly virulent mouse-adapted
strain of H1N1 Influenza virus (PR8) that induces severe respi-
ratory infections in mice. [60] Prophylactic efficacy of PCANS was
demonstrated against three different dosages of the virus, which
were 10–50 times higher than the previously established lethal
dose for PR8 in mice.[61] Due to its prolonged nasal residence
time, PCANS was effective for at least 4 h after nasal administra-
tion.
Our study also has certain limitations, and there are additional
questions that need to be answered. First, for the in vivo efficacy
study, we instilled the viral suspension intranasally as a liquid bo-
lus. Although aerosol administration of virus more closely reca-
pitulates real-life exposure in humans, intranasal inoculation as a
liquid bolus is a widely accepted standard in mouse models. [72,73]
Studies have shown that both methods result in comparable lev-
els of morbidity, mortality, and viral titers in lung and nasal tis-
sues for different influenza viruses as well as SARS-CoV-2.[74,75]
Thus, our choice of intranasal inoculation is scientifically justi-
fied. Excitingly, with intranasal inoculation—where the virus’s
contact time with PCANS is limited to just a few seconds—we
observed a greater than 99.99% reduction in lung viral titers
and improved animal survival for 250 PFU dosage of PR8 virus.
This underscores the significance of our findings. However, the
500 PFU group showed higher viral titer on day 4 and an early
mortality compared to 250 PFU. We believe that reduced effi-
cacy at 500 PFU might be partly, if not entirely, attributed to
the limited contact time of virus particles with PCANS. Inhala-
tion administration of the virus would deposit pathogen-laden
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respiratory droplets on the PCANS layer, providing significant
contact time between the pathogen and PCANS, potentially en-
hancing its prophylactic efficacy. Similar to the virus inoculation,
we also instilled PCANS as a liquid bolus intranasally, while in
humans PCANS would be sprayed using a metered dose spray
pump, which is technically difficult to recapitulate in mice due to
significant difference in their respiratory tract anatomy as com-
pared to humans. To overcome these limitations, future studies
should evaluate the efficacy of PCANS in large animal models,
for example non-human primates (NHPs) that allow the admin-
istration of PCANS as a nasal spray and can be subsequently chal-
lenged with virus-laden respiratory droplets as an aerosol. Sec-
ond, compared to >99.99% reduction in lung viral titer achieved
by PCANS for PR8 challenge after 15 min, a 78% reduction was
observed when the challenge was performed after 4 h. This can
be largely explained by clearance of PCANS in the nasal cav-
ity, resulting in significantly less formulation available to cap-
ture/neutralize the virus at 4 h as compared to 15 min. The res-
idence time study with DiR-loaded PCANS showed ∼50% clear-
ance of PCANS in the first 4 h. This was also the reason for
not evaluating the prophylactic efficacy of PCANS beyond 4 h in
this study. Since mucociliary clearance in NHPs and humans is
slower than mice, [76] future efficacy studies in large animal mod-
els are warranted to accurately determine the local pharmacoki-
netics of PCANS and duration of protection following a single
administration. Notably, the 78% reduction in lung viral titer, as
observed in our study after 4 h of prophylactic treatment is still
an improvement over 71% reduction observed with an inhalable
formulation – spherical hydrogel inhalation for enhanced lung
defense (SHIELD), developed recently as a prophylactic approach
against SARS-CoV-2.[77] Finally, although we demonstrated broad
spectrum activity of PCANS against multiple pathogens in vitro,
our proof-of-concept in vivo efficacy study only focused on PR8.
Future studies should also evaluate the prophylactic efficacy of
PCANS against other pathogens to confirm the broad-spectrum
activity in vivo, and this should be ideally performed in NHPs
to mimic the virus challenge and PCANS administration in
humans. - Conclusion
PCANS presents a promising chemoprophylactic approach
against respiratory infections. Besides its potential to act as a
first line of defense against respiratory pathogens and emerging
variants for which there are no vaccines available, our approach
could also be potentially used as an added layer of protection with
existing vaccines. Given its broad-spectrum prophylactic activity
and shelf stability, we anticipate PCANS holds the potential for
global distribution, especially in countries with low vaccination
rates against respiratory pathogens. Alongside, the benefits of
PCANS can also be extended to immunocompromised patients,
high-risk individuals with co-morbidities, and vaccine-hesitant
populations. Its pocket-sized spray format allows for easy porta-
bility, making it convenient to carry during social gatherings and
travel. With these significant benefits, we believe PCANS will ex-
perience rapid widespread adoption, enhancing the accessibility
of respiratory infection prevention. By enabling people to breathe
clean and minimizing the transmission of respiratory infections,
PCANS could potentially play a pivotal role in safeguarding pub-
lic health worldwide. - Experimental Section
Preparation of Biopolymer Solutions and PCANS: Biopolymer solutions
were prepared by the addition of the biopolymer (0.2 to 2% w/v) to ul-
trapure deionized sterile water (Invitrogen). The solution was then mixed
to attain a homogenous mixture with slight heating at 60 °C. Biopoly-
mers including gellan (Gelzan), pectin, carboxymethylcellulose (CMC), hy-
droxypropyl methylcellulose (HPMC), carrageenan, xanthan gum, and Car-
bopol were purchased from Sigma Aldrich. To prepare PCANS, 0.4% w/v
gellan and 1.5% w/v pectin solutions were mixed in a ratio of 1:1, followed
by the addition of tween-80 (Sigma Aldrich) to obtain a final concentra-
tion of 0.05% w/v. The solution was then supplemented with benzalko-
nium chloride (BKC) (Sigma Aldrich) and subjected to immediate mixing
by pipetting several times to yield 0.01% w/v in the solution. Finally, 0.25%
w/v phenethyl alcohol (Sigma Aldrich) was added, and the pH of the so-
lution was adjusted to 5.5. For cell culture experiments and in vivo effi-
cacy study, the individual components of PCANS were sterile filtered using
0.2 μm PVDF syringe filters (EMD Millipore) and combined as described
above.
Preparation of Simulated Nasal Fluid (SNF) and Simulated Mucus: SNF
was prepared by dissolving 1.32 g sodium chloride (150 mM), 447 mg
potassium chloride (39.9 mM), and 88.5 mg calcium chloride (5.3 mM) in
150 mL ultrapure deionized sterile water and filtered using 0.2 μm filter.[78]
The healthy simulated mucus was formulated by dissolving 0.6 mg mucin
from porcine stomach Type II (Sigma Aldrich), 0.8 mg mucin from porcine
stomach Type III (Sigma Aldrich), 0.32 mg bovine serum albumin (Sigma
Aldrich) in 10 mL ultrapure deionized water containing 20 mM HEPES
buffer and 38 mM sodium chloride solution. [79] The mixture was stirred
vigorously under slight heating to attain a homogenous solution.
Rheological Measurements: Dynamic viscosity behavior of biopolymer
solutions was evaluated using a rotational rheometer (Discovery HR-2,
TA Instruments) using a 40 mm diameter cone with a geometry angle of
1 0 . Samples were subjected to a linear shear rate ramp up to 40 s−1 at
25° C to mimic the strain encountered by the formulation when actuated
through the nozzle of the spray device. The viscosity of the biopolymer
solution was measured during the upward ramp in triplicates. The sol-gel
transition of biopolymer solutions with and without the presence of SNF
was evaluated by rotational rheology. The mechanical strength in terms of
storage modulus was assessed by applying amplitude sweep with a varying
oscillatory strain at 1 Hz at 37° C.
Ex Vivo Mucosal Retention Study: Tissue harvested from sheep was cut
open to expose the mucosal surface and trimmed down to 75×26 mm.
Mucosal tissue was then mounted on a glass slide facing upwards and
positioned at 45 0 to align it with the spray actuation angle. The tissue was
initially moistened with SNF using a generic nasal spray device, and ex-
cessive fluid was removed with sterile wipes. Brilliant green dye (Sigma
Aldrich) loaded polymeric solution was sprayed, keeping the spray nozzle
tip at a distance of 5 cm from the slide surface. The slides were examined
for runoff/drip after 4 h of spraying. The distance traveled by the poly-
mer solution down the glass slide from the bottom end of formulation
deposited on mucosal tissue was measured as drip length. Drip length of
free dye was considered 100%.
Cell Culture: Madin-Darby canine kidney cells (ATCC®) were cultured
in T-175 flasks (CELLTREAT) at 37 °C and 5% CO 2 in DMEM (Gibco) sup-
plemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-
(streptomycin (Invitrogen). Hep2 cells and Vero E6 cells (ATCC®) were
cultured in T-75 flasks at 37 °C and 5% CO 2 in EMEM supplemented with
10% FBS and 1% penicillin-streptomycin. Human nasal epithelial cells
(ATCC®) were cultured in T-175 flasks at 37 °C and 5% CO 2 in EMEM
supplemented with 10% FBS and 1% penicillin-streptomycin.
Production of NanoLuc Luciferase Expressing Recombinant SARS-CoV-2:
All replication-competent SARS-CoV-2 experiments were performed in a
BSL-3 facility at the Boston University National Emerging Infectious Dis-
eases Laboratories. A recombinant SARS-CoV-2 virus expressing a Neon-
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Green fluorescent protein (rSARS-CoV-2 mNG) was generously provided
by the Laboratory of Pei-Yong Shei. [80] To propagate the virus, 1 × 10 7 Vero
E6 cells were seeded in a T-175 flask one day prior to propagation. The next
day, 10 μL of rSARS-CoV-2 mNG virus stock was diluted in 10 mL of Opti-
MEM, added to cells, and then incubated for 1 h at 37 °C. After incubation,
15 mL of DMEM containing 10% FBS and 1% penicillin/streptomycin was
added to cells. The next morning, media was removed, cells were washed
with 1X PBS and 25 mL of fresh DMEM containing 2% FBS was added.
Virus was incubated for an additional 48 h. The supernatant was collected
at 72 h, filtered through a 0.22 μm filter, and stored at −80 °C. The vi-
ral stock was thawed and concentrated by ultracentrifugation (Beckman
Coulter Optima L-100k; SW32 Ti rotor) on a 20% sucrose cushion (Sigma-
Aldrich) at 25000 x g for 2 h at 4 °C. Media and sucrose were then dis-
carded, pellets were dried for 5 min at room temperature, and viral pellets
were resuspended in 100 μL of cold 1X PBS at 4 °C overnight. The next day,
concentrated virus was combined, aliquoted and stored at −80 °C.
In Vitro Physical Barrier Assay: A 70 μm pore size mesh cell strainer
was coated with 15 μL of mucus, or a biopolymer solution, or PCANS. The
formulation was spread evenly using a sterile stainless-steel spatula with a
tapered end. To facilitate in situ gelation, 15 μL of SNF was added, covering
the entire surface of the strainer. The strainer was placed in a 6-well plate
containing 0.9 mL of serum-free DMEM (for virus/bacteria penetration)
or ultrapure deionized water (for rhodamine B isothiocyanate penetration)
in each well, and 0.1 mL of diluted virus (≈1 × 10 5 PFU mL−1 )/bacteria
(1 × 10 7 CFU mL−1 ) stock or rhodamine B isothiocyanate (1 mg mL−1 )
was added to the upper compartment of the strainer. After 4 h of incu-
bation at 37 °C, medium or deionized water from the bottom reservoir
was retrieved, and the viral titer permeated through the hydrogel layer was
quantified using plaque assay for IAV performed in MDCK cells, crystal
violet staining for RSV performed in Hep-2 cells, immunostaining for ade-
novirus performed in Vero E6 cells, focus forming assay for SARS-CoV-2 in
Vero E6 cells, and colony forming unit (CFU) plate count method for bacte-
ria, as described in the following sections. The permeation of dye through
biopolymer solution/mucus was quantified by measuring the fluorescence
intensity using a microplate reader.
In Vitro Neutralization Assay with Influenza A: Neutralization activity
of different excipients and PCANS was evaluated by plaque assay. MDCK
cells were seeded at a density of 2–3 million cells per well in a 6-well
plate and then incubated at 37 °C to achieve ≈80–90% confluency one
day before infection. On the day of infection, 50 μL of HKx31 Influenza A
virus (H3N2, 5 × 10 4 –1 × 10 5 PFU mL−1 ) (BEI Resources) in infection
media (serum-free DMEM containing 3 mg mL−1 TPCK-trypsin) was pre-
treated with 50 μL of PCANS, biopolymer solution, surfactant solution,
alcohol solution or PBS. Samples were vortexed for 10 seconds and in-
cubated at 37 °C for 10 or 60 min. After incubation, samples were cen-
trifuged for 1 min at 1000 RPM, and the supernatant was subjected to a
10-fold serial dilution until eighth dilution using infection medium. MDCK
cells were then exposed to pre-treated virus dilutions for 1 h. After infec-
tion, an overlay growth medium containing 2X DMEM with 2% agarose
(50:50) was poured onto the top of the cell monolayer and incubated for
72 h. The overlay was removed, and cells were then fixed using 1 mL
of 10% formalin and left for 1 h at room temperature, followed by the
addition of 1% crystal violet for 5–15 min. Wells were washed with wa-
ter and left to dry out and PFUs were counted to determine the viral
titer.
In Vitro Neutralization Assay with SARS-CoV-2: The day prior to infec-
tion experiment, 8 × 10 4 Vero E6 cells/well were plated in a 24-well plate.
To perform neutralization assay, 50 μL of PCANS, biopolymer solution,
surfactant solution, alcohol solution or PBS was mixed with 8 × 10 4 PFU
of SARS-CoV-2 mNG in 50 μL of infection media (OptiMEM (Gibco) con-
taining 3 mg mL−1 TPCK-trypsin), vortexed and centrifuged briefly prior
to incubation at 37 °C for 10 or 60 min. After incubation, samples were
centrifuged for 1 min at 1000 RPM and serially diluted 10-fold until eighth
dilution with infection medium. Of each dilution, 200 μL was then plated
into a 24-well plate and incubated for 1 h at 37 °C prior addition of 800 μL
of 1.2% Avicel (Dupont). Following a 24 h incubation period at 37 °C, Avi-
cel was removed, cells were washed with 1X PBS and fixed for 3 h with
10% neutral buffered formalin. Focal forming units (FFU) per mL were de-
termined by counting NeonGreen expressing foci using an Evos M5000
fluorescent microscope (Thermo Scientific).
In Vitro Neutralization Assay with Adenovirus and Respiratory Syncytial
Virus: The broad-spectrum neutralization potency of PCANS was evalu-
ated against adenovirus type 5 (ADV-5, ATCC, VR-2554) and respiratory
syncytial virus strain A2 (RSV-A2, ATCC, VR-1540) using plaque assay.
Briefly, the day prior to the infection, 1 × 10 5 Vero E6 cells/well or 1.5 × 10 5
Hep-2 cells/ well were plated in a 24-well plate for ADV-5 and RSV-A2, re-
spectively. On the day of infection, 50 μL of PCANS was mixed with 50 μL
of virus (1 × 10 6 PFU/mL of ADV-5 and 2 × 10 6 PFU/mL of RSV-A2) in the
infection media and incubated at 37 °C for 30 min. The pre-treated mix-
ture was 10-fold serially diluted in infection media after the incubation.
Cells were washed with serum-free media before infection and 200 μL of
each dilution was transferred to the cells for a 1 h incubation prior to the
addition of a 1 mL overlay medium containing methylcellulose. Following
a 72 h incubation, the overlay layer was removed, and cells were fixed us-
ing 10% formalin with subsequent immunostaining for Vero E6 cells and
crystal violet staining for Hep-2 cells. Plaques were counted using a plaque
reader (Bioreader-600-Va).
In Vitro Neutralization Assay with Bacteria: The neutralization potency
of components and PCANS was studied against gram-negative bacteria
including E. coli and K. pneumoniae. An overnight culture of bacteria was
prepared in 5 mL tryptic soy broth (TSB, Sigma Aldrich) media. On the day
of the experiment, bacteria suspension was adjusted to obtain an OD600nm
= 0.2, which corresponds to 10 8 CFU/mL. A 50 μL of bacterial suspension
in TSB media was incubated with 50 μL of PCANS, biopolymer solution,
surfactant solution or alcohol solution at 37 °C for 10 or 60 min. After incu-
bation, the sample/bacteria mixture was 10-fold serially diluted in 1X PBS,
and 10 μL of each dilution was plated onto pre-poured LB (Luria Broth,
HiMedia Laboratories Pvt Ltd) agar plates followed by an incubation of
16–18 h at 37 °C, 5% CO 2 . The plates were then counted for CFUs.
In Vitro Assay for Co-Neutralization of Bacteria and Virus: An overnight
culture of E. coli was prepared in tryptic soy broth (TSB, Sigma Aldrich)
media, and the bacterial suspension was adjusted to obtain a titer cor-
responding to 10 9 CFU/mL. A 10 μL bacterial suspension was added to
1 mL of PCANS at 37 °C for 60 mins. Following the incubation, a 100 μL
PCANS/bacteria mixture underwent a 10-fold serial dilution in 1x PBS, and
a CFU assay was performed. The remaining PCNAS/bacteria mixture was
syringe filtered using a 0.22 μm filter to remove the E. coli, and neutraliza-
tion of SARS-CoV-2 was performed as described above.
Molecular Docking Simulation for Rhodamine B Isothiocyanate: Molec-
ular docking simulations were conducted using Biovia Discovery Stu-
dio 2021 Client and PyRx (version 0.8) software to investigate the bind-
ing affinities and interaction types between rhodamine B isothiocyanate
and various biopolymers. Initially, the 3D structures of rhodamine B
isothiocyanate (CID: 44 134 929), gellan gum (SID: 443 546 310), pectin
(CID: 854), HPMC (CID: 57 503 849), CMC (CID: 24 749), xanthan gum
(CID: 47 933), Carbopol (CID: 91 824 753), and 𝜅-carrageenan (SID:
481 108 992) was obtained in.sdf format from the PubChem database.
These structures were imported into PyRx, wherethe OpenBabel toolkit
was employed for energy minimization and geometric confirmation. The
molecules were then converted to.pdbqt format and categorized either
as macromolecules (rhodamine B isothiocyanate) or as ligands (biopoly-
mers). Docking analyses were carried out using AutoDock Vina within
PyRx, employing a grid box method with dimensions of 25 × 25 × 25 Å. Af-
ter the docking process, the binding free energies (expressed in kcal/mol)
of the rhodamine B isothiocyanate-biopolymer complexes were evaluated.
These docked complex files were subsequently imported into Biovia Dis-
covery Studio to analyze various interaction types, including hydrogen
bonds, carbon-hydrogen bonds, and hydrophobic interactions between
the dye molecule and biopolymers.
In Silico Modeling of Viral Protein Interactions Neutralizing Agents: In-
silico binding analysis was conducted using AutoDock Vina (https://
vina.scripps.edu/). The receptor binding domains of influenza hemag-
glutinin (PDBID: 4WE4), SARS-CoV-2 spike receptor-binding domain
(PDBID:6M0J) and Envelope Protein Transmembrane Domain (PDBID:
7K3G) were sourced from the Protein Data Bank (PDB) available at RCSB
(https://www.rcsb.org/). To prepare for docking simulations, molecular
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files of the ligands were first generated using Marvin Sketch and then con-
verted into PDB format using PyMOL for 3D structural visualization. The
protein and ligand files were converted into AutoDock-readable formats
(.pdbqt) to facilitate docking simulations. The simulations were performed
with the receptor binding domains of the proteins to predict the most fa-
vorable binding conformations. AutoDock Vina and PyMOL were used to
visualize and analyze these conformations. The binding energies associ-
ated with the predicted conformations were reported. Furthermore, to gain
insights into the potential interactions between protein and ligand, LigPlot
(https://www.ebi.ac.uk/thornton-srv/software/LigPlus/) software was uti-
lized. This tool provided a detailed visual representation of the molecular
interactions, highlighting the key residues involved in binding.
TEER Assay and In Vitro Cytotoxicity of tween-80: RPMI 2650 cells were
seeded on the apical part of Transwell inserts (6.5 mm polyester mem-
brane ≈ 0.4 μm pore size, Corning) at a density of 1.5 × 10 5 cells/cm2
in 0.1 mL EMEM. The basolateral compartment of the insert was filled
with 0.6 mL EMEM media supplemented with 10% FBS followed by in-
cubation at 37 °C. On day 4, the medium was removed from the top
of the inserts, and media volume in the bottom well was reduced to
200 μL. Every 2 days the medium was changed, and TEER was measured.
An epithelial volt ohmmeter (World Precision Instrument) was used to
measure the impedance. Until the monolayer formed with a constant
impedance around 12 Ω, cells were grown with an air-liquid interface. On
day 12, TEER was measured prior to the treatment of cells with surfac-
tants. 200 μL of medium containing Triton X-100 (0.1% w/v) or tween-
80 at different concentrations was added to the insert. Plate was incu-
bated at 37 °C for 4 h. After incubation, wells were replenished with fresh
medium, and TEER was measured after 4, 5, 12, and 24 h. The cytotoxic
effect of tween-80 at different concentrations was also studied on RPMI
2650 cells. Briefly, 20000 cells/well were seeded in a 96-well plate and in-
cubated at 37 °C overnight to achieve 70–80% confluency. Tween-80 (0.01,
0.05, and 0.5% w/v) solution in 0.2 mL EMEM media was added to the
wells, followed by an incubation for 24 and 48 h. The metabolic activity of
RPMI 2650 cells was measured using an XTT (2,3-bis(2-methoxy-4-nitro-5-
sulfophenyl)−2H-tetrazolium-5-carboxanilide) assay kit (ATCC®) accord-
ing to the manufacturer’s protocol.
Capture of Respiratory Droplets: The inner surface of a glass twin im-
pinger’s (Copley Scientific) oropharyngeal region (denoted by red arrows
in Figure 4a) was coated with SNF followed by spraying the gellan (0.2%
w/v) and pectin (0.75% w/v) mixture without or with different concentra-
tions of tween-20, tween-80 or BKC using a VP3 nasal spray pump (Aptar).
Droplets with mass median aerodynamic diameter >5 μm and laden with
rhodamine B-loaded liposomes (size ≈400 nm) were generated using a
jet nebulizer. Nebulized droplets were administered into the impinger un-
der vacuum at a flow rate of 15 L min−1 for 3 min. The gel was retrieved,
and fluorescence intensity was quantified at an excitation and emission
wavelength of 543 and 580 nm. Rhodamine B-loaded liposomes were syn-
thesized using the thin-film hydration method. [81,82] Briefly, the lipids,
DSPE-PEG (2000) amine (Avanti Polar lipids), cholesterol (Sigma) and
L-𝛼-phosphatidylcholine, hydrogenated (Soy) (HPC, Avanti Polar lipids)
were dissolved in chloroform to prepare a 10 mg mL−1 lipid stock solu-
tion in 1:1:3 molar ratio. A 2 mL of lipid stock solution was added to a
round-bottom flask containing 0.8 mL of rhodamine B isothiocyanate from
a 1 mg mL−1 stock. The organic solvent was then evaporated using a ro-
tary evaporator for 5 min to form a thin lipid layer. The lipid film was then
hydrated using 10 mL ultrapure water (Invitrogen) and silica glass beads
were added to the flask to suspend the lipid in the solution with vigorous
shaking using the rotary evaporator at 40 °C for 45 min. The hydrated lipid
suspension was sonicated (Probe sonicator) at 30% amplitude for 1 min
with a 2 sec pulse on and off condition. The size of liposomes was then
analyzed using a Zeta Analyzer (Malvern).
To emulate the capture of pathogen-laden droplets in the human nasal
cavity, a 3D transparent, silicone human nose model (Koken Co, Ltd) was
used. The anterior region of the Koken model was deposited with SNF
followed by the gellan (0.2% w/v) and pectin (0.75% w/v) mixture or
PCANS with a single actuation using a nasal spray pump (Aptar). Koken
model was connected to a vacuum pump at an air flow rate of 15 L min−1
and rhodamine B-loaded liposomes were then nebulized for 1 min. The
model was disassembled to retrieve the formulation and captured dye-
loaded droplets after nebulization. The capture of droplets was measured
by quantifying the fluorescence intensity at an excitation and emission
wavelength of 543 and 580 nm.
Spray Characterization: Multi-dose nasal spray vials were filled with
water or gellan solution or PCANS. The pump (140 μL) with an insertion
depth of 1.8 cm (Aptar) was used to study the spray characteristics in-
cluding plume geometry, spray plume, and droplet size distribution. Three
replicate measurements were performed for each sample. Plume geome-
try and spray pattern were measured using a Spray-View measurement
system (Proveris Scientific, Hudson, MA) at a distance of 30 mm from
the nozzle orifice of the actuator. This acquisition system employs a high-
speed digital camera and laser light sheet to capture images. Data were
analyzed using an image processing software, Viota. Actuation parameters
including velocity, acceleration and hold time, and settings for camera and
laser were kept identical across all the samples. Plume geometry measures
the angle of plume ejected from the nozzle orifice. Ovality and plume area
were evaluated to quantify the spray pattern of the samples. Ovality was
defined as the ratio of maximum to minimum cross-sectional diameter
of the spray plume. A uniform circular plume with an ovality close to 1
can be considered an optimal condition for nasal sprays. [83] Droplet size
analysis of samples was inspected using a Malvern Spraytec laser diffrac-
tion system. The FDA recommends reporting the measurements of size
distribution data at D(v,0.1), D(v,0.5), and D(v,0.9) thresholds which corre-
spond to the size of 10%, 50%, and 90% droplets by volume distribution,
respectively.[58] It was suggested to have droplet population with D(v,0.1)10 μm, D(v,0.5) between 30–70 μm and D(v,0.9) <200 μm. Droplet pop-
ulations smaller than 10 μm have a propensity to induce a non-targeted
deposition at the lungs, and droplets greater than 200 μm tend to drip/
run off the nasal cavity.[58]
Shelf-Stability Study: PCANS (15 mL) was filled in a sterile multi-dose
nasal sprays (Aptar) capped with the actuator. The nasal spray vials were
stored at an accelerated temperature condition (40° C). Aliquots were re-
trieved at different time points and evaluated for neutralization activity
against IAV and SARS-CoV-2 using plaque forming and focus forming as-
says, respectively, as described above. Aliquots were collected from three
different vials. Similarly, 5 mL aliquots were used to evaluate the spray
features, including spray pattern, plume geometry, and droplet size distri-
bution.
Mice: Animal experiments were conducted according to ethical guide-
lines approved by the Institutional Animal Care and Use Committee
(IACUC) of Brigham and Women’s Hospital. Experiments were conducted
in 6–8 weeks-old C57BL/6 mice (Jackson Laboratories, USA). Mice were
maintained under pathogen-free conditions and randomly assigned to var-
ious experiment groups, irrespective of gender. The group size of animals
in experiments was decided based on the minimum number of animals
required to attain a statistical significance of P<0.05 among different test
groups. For mouse model of influenza infection, experiments were con-
ducted in Biosafety Level 2 according to ethical guidelines approved by
the Institutional Animal Care and Use Committee (IACUC-A4752-01) of
Brigham and Women’s Hospital.
In Vivo Biodistribution and Nasal Retention: Nasal retention of the for-
mulation was performed in mice. Briefly, C57BL/6 mice were administered
with 10 μL per nostril of free DiR (Thermofisher) or PCANS mixed with
DiR at a final concentration of 10 μg mL−1 ). Mice were euthanized at dif-
ferent time points and nasal cavity was harvested and imaged using IVIS
(Bruker’s In-Vivo Xtreme optical and x-ray in vivo imaging system) at an
excitation and emission wavelength of 680/700 nm. Vital organs such as
lung, liver, spleen, kidney, and heart, were also imaged at 2 and 24 h time
points. To determine the mechanism of long residence time, animals were
intranasally instilled with DiR-mixed gellan (0.02% w/v) and pectin (0.75%
w/v) mixture without or with BKC and tween-80. After 8 h, animals were
euthanized to harvest and image the nasal cavity using Perkin Elmer IVIS
Lumina II and the total flux was expressed in (p/sec/m2 /sr).
In Vivo Prophylactic Activity of PCANS: Mice were intranasally instilled
with 10 μL PCANS or PBS into each nostril under brief anesthesia using
isoflurane. After 15 min, animals were challenged with 250 or 500 PFU
of PR8 intranasally. One cohort of animals was followed for body weight
Adv. Mater. 2024, 2406348 © 2024 Wiley-VCH GmbH2406348 (17 of 20)
www.advancedsciencenews.com www.advmat.de
changes and survival for a period of 10 days. Animals were euthanized
when the body weight was reduced to 20%. Animals from a second cohort
were euthanized either on day 2 or 4 after infection to enumerate lung
viral titer, inflammatory cell count in bronchoalveolar lavage (BAL) fluid,
and inflammatory cytokine levels in lung homogenate. BAL fluid was iso-
lated by gently instilling saline solution into bronchioles with a catheter
inserted through the trachea. The total cells and immune cell types from
the collected BAL fluid were quantified using Diff-quik kit as per man-
ufacturer’s protocol. For lung viral titer and cytokine profiling, left lung
was homogenized and centrifuged at 2000 g for 10 min at 4 °C to collect
the supernatant. The obtained supernatant was further used for down-
stream assays. Viral titer was enumerated using plaque assay with MDCK
cells, as detailed above. Cytokine profiling was performed using respective
ELISA kits of IL-6, TNF-a, and IL-1b (BioLegend) according to the manu-
facturer’s protocol. Histopathology of the right lung was determined using
hematoxylin and eosin staining, and inflammation scoring was performed
as reported previously.[84] To evaluate the time-dependent protection of
PCANS, animals were challenged with 100 PFU of PR-8 via intranasal route
after 2 or 4 h of PCANS or PBS treatment and euthanized on day 2 post-
infection to quantify lung viral titer using plaque assay.
Statistics: Statistical analysis and graphing were conducted using
Graphpad Prism. A one-way ANOVA with Tukey’s post hoc analysis was
used to compare multiple groups. Two-way ANOVA with Tukey’s multi-
ple comparison tests was used to analyze the data with two variables.
To evaluate the efficiency of PCANS, survival plots were generated using
the Kaplan-Meier survival curve, and the statistical significance of the re-
sults was analyzed using the Gehan-Breslow-Wilcoxon test. P values for
the body weight changes were determined using one-way ANOVA with
Brown-Forsythe post hoc analysis. A P-value of less than 0.05 was con-
sidered statistically significant.
Supporting Information
Supporting Information is available from the Wiley Online Library or from
the author.
Acknowledgements
The authors acknowledge the use of BioRender for creating schematic
illustrations - Fig. 1, Figs. 4a, 4f, 4j, Fig. 5n, and Figs. 6a, 6n. in
the manuscript. The authors acknowledge funding support from Gillian
Reny Stepping Strong Center for Trauma Innovation at the Brigham and
Women’s Hospital (to NJ and JMK), Department of Anesthesiology, Peri-
operative, and Pain Medicine at the Brigham and Women’s Hospital (to
NJ), Fulbright-Nehru Postdoctoral Fellowship (to JJ), and Boston Univer-
sity (to FD) and the Peter Paul Career Development Award (to FD). The
metered dose spray pumps were generously gifted by Aptar Inc. The au-
thors acknowledge Integrated BioTherapeutics (IBT) Bioservices for eval-
uating the neutralization activity of PCANS against RSV and adenovirus.
Conflict of Interest
J.J., H.M.B, Y.T., and J.M.K have one pending patent based on the PCANS
formulation described in this manuscript. N.J. and J.M.K are paid con-
sultants, scientific advisory board members, and hold equity in Akita Bio-
sciences, a company that has licensed IP generated by N.J. and J.MK. that
may benefit financially if the IP was further validated. The interests of N.J.
and J.MK. were reviewed and overseen by their institution in accordance
with its conflict of interest policies.
Author Contributions
J.J., H.M.B., and J.R.Q contributed equally to this work. J.J., H.M.B., J.R.Q.,
Y.T., J.M.K., N.J. performed conceptualization. J.J, H.M.B., J.R.Q., D.K.,
E.B., D.L., D.P. was performed data curation. J.J., H.M.B., J.R.Q., D.K.,
E.B., D.L., D.P. performed data analysis. J.M.K., N.J. performed funding
acquisition. J.J., H.M.B., J.R.Q., Y.M., E.B., P.S., K.S., O.S., R.N., E.A., S.R.,
J.K. performed investigation. J.J., H.M.B., J.R.Q., D.K., S.K., X.L.L., J.M.,
J.G., J.N.L, A.Y., F.D. performed methodology. Y.T., J.M.K., N.J. performed
project administration. F.D., Y.T., J.M.K., N.J. performed supervision. J.J.,
H.M.B., J.R.Q., J.M.K., N.J. performed validation. J.J., H.M.B., S.R., N.J.,
wrote manuscript – original draft: J.J., H.M.B., D.K., F.D., Y.T., J.M.K, N.J.
edited the manuscript.
Data Availability Statement
The data that support that findings of this study are available from the
corresponding author upon reasonable request.
Keywords
antibacterial, antiviral, broad spectrum protection, nasal prophylaxis,
nasal spray, pathogen capture, respiratory infections, virus neutralization
Received: May 3, 2024
Revised: August 18, 2024
Published online:
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