Nanomedicine Formulations for Respiratory Infections by Inhalation Delivery: Covid-19 and Beyond

Abstract

For respiratory infections treatment and prevention, we analyze for the first time the possibility of providing a broad range medication based on metallic nanoparticles colloids (NpC) delivery by controlled aerosol inhalation. (i) Based on in-vitro data combined with aerosol deposition characteristics in the respiratory system, we calculate the required effective formulations, dosages and delivery parameters for an aerosol inhalation treatment. The goal is to achieve an effective NpC inhibitory concentration (IC) in the target airway surface liquid (ASL); (ii) We evaluate the clinical safety of such dosages, drawing on information from animal testing data and regulatory limits in the USA for such nanoparticles aerosol inhalation safety. Our analysis indicates a wide range of potentially safe and effective dosages that can be clinically explored, targeting the upper respiratory and bronchial tree system. Similar dosages can also provide antibacterial effectiveness for prophylactic treatment in hospital intensive care units to lower the risk of ventilator-associated pneumonia (VAP).

Our calculations are phenomenological, independent of mechanisms. Nevertheless, we highlight a mechanism of action by which any suitably designed NpC, with nanoparticles sized 2–10 nm and having a large negative zeta-potential, preferentially bind to viruses with predominantly positively-charged spike proteins. These will be ineffective against viruses with predominantly negatively-charged spike proteins. Accordingly, the popular silver metal base for NpC serves just as a construction ingredient, and other metal or metal-oxides which can serve to construct the noted nanoparticle properties would be similarly effective.

We suggest that inhalation delivery of the proposed antiviral formulations could be applied as a first-line intervention while respiratory infections are primarily localized to the upper respiratory system and bronchial tree.

Full text

Medical Hypotheses 159 (2022) 110753
Available online 3 January 2022
0306-9877/© 2022 The Author. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Nanomedicine formulations for respiratory infections by inhalation
delivery: Covid-19 and beyond
Oron Zachar
Yamor Technologies Ltd., 23 Mahane Yosef, Tel Aviv 6515325, Israel
ARTICLE INFO
Keywords:
Covid-19
Nanomedicine
Pneumonia
VAP
Respiratory infections
Viruses
ABSTRACT
For respiratory infections treatment and prevention, we analyze for the rst time the possibility of providing a
broad range medication based on metallic nanoparticles colloids (NpC) delivery by controlled aerosol inhalation.
(i) Based on in-vitro data combined with aerosol deposition characteristics in the respiratory system, we calculate
the required effective formulations, dosages and delivery parameters for an aerosol inhalation treatment. The
goal is to achieve an effective NpC inhibitory concentration (IC) in the target airway surface liquid (ASL); (ii) We
evaluate the clinical safety of such dosages, drawing on information from animal testing data and regulatory
limits in the USA for such nanoparticles aerosol inhalation safety. Our analysis indicates a wide range of
potentially safe and effective dosages that can be clinically explored, targeting the upper respiratory and
bronchial tree system. Similar dosages can also provide antibacterial effectiveness for prophylactic treatment in
hospital intensive care units to lower the risk of ventilator-associated pneumonia (VAP).
Our calculations are phenomenological, independent of mechanisms. Nevertheless, we highlight a mechanism
of action by which any suitably designed NpC, with nanoparticles sized 2–10 nm and having a large negative
zeta-potential, preferentially bind to viruses with predominantly positively-charged spike proteins. These will be
ineffective against viruses with predominantly negatively-charged spike proteins. Accordingly, the popular silver
metal base for NpC serves just as a construction ingredient, and other metal or metal-oxides which can serve to
construct the noted nanoparticle properties would be similarly effective.
We suggest that inhalation delivery of the proposed antiviral formulations could be applied as a rst-line
intervention while respiratory infections are primarily localized to the upper respiratory system and bronchial
tree.
Introduction
Whether it is the seasonal u, a sore throat, or early-stage Covid-19
symptoms, presently in the 21st century, there is still no home treatment
medication that is easily accessible and available in pharmacies world-
wide. The impact of this sorry state of medicine amounted to a global
disaster under Covid-19. Moreover, even in the absence of novel epi-
demics, each year the morbidity and economic cost of respiratory in-
fections are huge. The typical research tendency in novel
pharmaceuticals is to develop sophisticated, expensive, patent-
protected, highly potent medications for severely ill patients. In
contrast, when it comes to public health and pandemic prevention, it
may be more impactful to develop a broad-range, mild, cheap, and easily
accessible early-stage medications for home treatment, with low risk of
side effects and wide availability potential, to prevent deterioration mild
patients at home to the severe hospitalized state. From the pitiful present
venture point of anti-viral respiratory infections medication, there may
be a benet, humbly and open-mindedly, to revisit some neglected po-
tential options for advancement.
We argue that good candidates for such desired medication can be
found in a well-dened formulation of nanoparticle colloids for inha-
lation delivery. The antibacterial and antiviral potential of nanoparticle
colloids)NpC(has been extensively demonstrated in in-vitro and animal
testing [1–6]. Anti-microbial applications of NpC for wound care are
approved by the FDA. Unfortunately, a senseless and uncontrolled
“alternative medicine” practice of “colloidal silver” ingestion led the
pharmacological and academic establishment to widely disregard the
potential application of metal-based NpC as antimicrobial agents in
contexts other than wound care. In particular, there has been no
rigorous analysis of the potential use of inhalation-delivered NpC to
prevent or treat respiratory infections.
We substantiate the potential of antimicrobial NpC formulations,
E-mail address: oron@yamor-tech.com.
Contents lists available at ScienceDirect
Medical Hypotheses
journal homepage: www.elsevier.com/locate/mehy
https://doi.org/10.1016/j.mehy.2021.110753
Received 29 June 2021; Received in revised form 2 November 2021; Accepted 17 November 2021

Medical Hypotheses 159 (2022) 110753
2
delivered by inhalation, to minimize the aggravation of respiratory
system infections. We evaluate both (A) viral respiratory infections
(including with SARS-CoV-2, the causative agent of COVID-19), and (B)
bacterial infections, particularly ventilator-associated pneumonia (VAP)
in intensive care unit (ICU) patients. Indeed, the most reliable experi-
ence concerning the treatment of respiratory infections has been gained
in the context ICU-acquired VAP [7]. There are unique factors to
consider for optimal treatment of the lungs. These include aerosol
characteristics, breathing patterns, geometrical factors (lung
morphology), disease state, pharmacokinetics (including lung clearance
and mucus transport). Both the total pulmonary drug dose and the
regional deposition distribution of the lung-deposited aerosol are critical
factors for the clinical success of an inhalation therapy [8].
The pathogenesis of respiratory infections begins mildly in the
nasopharynx or upper bronchial tree portions of the respiratory system
[9,10]. Aggravation occurs once the pathogens and associated inam-
mation migrate to lower portions of the respiratory system [11]. Greater
risk and aggravation of the disease state are associated with an increased
microbial load in the upper bronchial tree. Hence, a desirable clinical
endpoint for the proposed inhaled NpC treatment is the inhibition of
microbial load in the upper sections of the bronchial tree. Such an
endpoint implies treatment when most patients are still at home with
mild symptoms.
We lay the groundwork for future clinical evaluations of inhaled NpC
by determining: (i) the NpC material composition required for effective
antiviral activity; (ii) the effective inhibitory concentration (IC) required
in target respiratory tissues; (iii) the dosage required for practical
inhalation delivery of metal-based NpC antiviral and antibacterial for-
mulations, and (iv) the safe dosage range for clinical evaluation.
Conceptual approach
We follow the principles of antibiotics inhalation delivery [7,12], as
illustrated in Fig. 1. To calculate the dose delivered externally with an
aerosol nebulizer, one needs to consider the various losses that occur on
route to the target bronchial tree organ and sensitivity to the aerosol
properties that determine the deposition fraction.
Our dose calculation is based on the following results (each one
derived in dedicated sections):
•Nanoparticles: The antiviral potency of metal nanoparticles is highly
size-dependent, with optimal size being in the range of 3–7 nm.
Polymer capping agents (PVP, PVA) reduce the antiviral effective-
ness of NpC and better be avoided.
•Inhibitory concentration (IC): The target organ is the airway surface
liquid (ASL). For antiviral applications, the nanoparticles minimal
microbicidal IC (MMC) is about 10 µg/mL.
•Tissue deposition fraction (TDF): During oral breathing of 5 µm
aerosol droplets, NpC deposit onto the pharynx (30%), bronchial tree
(30%), and alveoli (25%).
•Inhalation timing losses (ITL): inhalation duration is about a third of
the breathing cycle. Thus, using a continuous aerosol source, the
aerosol uncontrollably cumulates in the oral cavity during 2/3 of the
breathing cycle. Hence, we assume only about 50% of the nebulized
substance is actually inhaled.
The required delivery dose is calculated by cumulating the effects of
each stage between the aerosolizing nebulizer device and nal target
tissue deposition and dilution into the local airway liquid. The mass (µg)
Fig. 1. Mechanisms by which the dose of an antimicrobial agent inserted into a nebulizer differs from the dose delivered to the target airway location (based on [7]),
with a focus on the fraction deposited in the bronchial tree.
Table 1
Dose calculations for aerosol droplets of various sizes to treat different target tissues.
Target
tissue
Droplet size
(µm)
IC In target tissue
(µg/mL)
MV Mucosal
Volume (mL)
TDF Tissue Deposit
Fraction
CC Colloid Concentration
(µg/mL)
Inhaled dose
(mL)
AD Aerosolized Dose
(mL)
(i) Bronchial
tree
5 10 1 0.25 50 1 2
(ii) Bronchial
tree
5 40 1 0.25 120 2 4
(iii) Bronchial
tree
3 25 1 0.1 120 2.75 5.5
(iv) Alveoli 3 10 10 0.33 120 2.75 5.5
O. Zachar

Medical Hypotheses 159 (2022) 110753
3
of nanoparticles deposited to the target organ location (e.g., bronchial
tree) is distributed into a liquid volume comprising the tissue airway
surface liquid (ASL) plus the deposited solution liquid volume. Equation-
(1) delineates the calculation of required NpC aerosolized dose (AD)
volume that needs to be put into the nebulizer device to achieve the
desired NpC inhibitory concentration (IC) in the target organ location:
AD =MV
TDF ×ITL (IC
CC −IC)(1)
where AD (in mL) is a function of the target inhibitory concentration (IC,
in µg/mL), mucosal volume (MV, in mL), tissue deposition fraction
(TDF), inhalation time losses fraction (ITL), and colloid concentration
(CC, in µg/mL).
Results and analysis
Table 1 illustrates examples antiviral formulations delivered by the
oral inhalation of aerosol droplets, as calculated from the above
Equation-1. Using a typical continuous nebulizer (ITL =0.5 assumed)
with 5 µm droplets size, a nebulized dose of 4 mL is required to effec-
tively deposit the IC of 40 µg/mL in the bronchial tree. Inhaled antibi-
otics dosages for chronic airway infections are commonly 2–5 mL [13].
Hence, we expect inhalation of nebulized similar size dosage of antiviral
solutions to be well-tolerated by home users. For preferential deposition
in the alveoli, smaller aerosol droplets of size 3 µm are required (see
discussion in section 3.6.2).
As common in the practice of antibiotic inhalation treatments, a
target IC inhaled antimicrobial dose should be some signicant multiple
of the theoretical minimum IC (MIC) and also the minimum bactericidal
concentration (MBC). Since the antiviral minimum mucrobicidal con-
centration (MMC) for NpC is about 10 µg/mL, a recommended treatment
target IC may be 40 µg/mL, as illustrated in example (ii) in table-1, for
targeting the bronchial tree.
Target IC determination for antiviral applications
Antiviral effect is obtained only by nanoparticle of size <10 nm (see
section 3.2 below). The capping molecule used for the manufacture of
nanoparticle affects antiviral potency. The popular PVP cap appears to
inhibit antiviral effectiveness signicantly. Therefore, in analyzing the
published data, we limit our consideration to experiments involving
nanoparticles of size <15 nm and to non-PVP-stabilized NpC. The
minimum inhibitory concentration (MIC) is one at which occurs tran-
sition from microbial no-growth to growth. The Minimum Microbicidal
Concentration (MMC) is the least amount of antibiotic required to kill a
microbial organism (e.g., a 50% reduction after 1 h incubation), where
always MIC <MMC. In practice, published data is given at a few discrete
concentration points, from which the MIC or MMC needs to be deduced
somehow by interpolation and often not stated by the authors them-
selves (see an example below for the TGEV coronavirus). The evidence
summarized in Table-2 indicates that an IC of at least 10 µg/mL is
desirable at the target respiratory system location. We contend that the
optimal size of nanoparticles for antiviral effectiveness is 3–7 nm, not
the 10 nm diameter used in all the referenced experiments. Hence, there
is potential for better efcacy and lower MIC if an optimal NpC are used.
Relation to the mechanism of action
Our core calculations are phenomenological, independent of mech-
anisms. Yet, an understanding of mechanisms may guide future treat-
ment optimization and alternatives. There are multiple ways for bacteria
to interact with silver NpC components (both the nanoparticles and Ag
+
ions), as discussed by others [18]. Viruses are simpler. Based on the
broad-range effectiveness of silver NpC with diverse capping surface
molecules against many different viruses, we argue that the underlying
mechanism of action of NpC on viruses must be non-specic, simple, and
robust. The prominent material properties that affect the NpC antiviral
binding action are the nanoparticle size and surface electric zeta po-
tential, with all remaining aspects of the chemical composition being
secondary. Thus, we estimate that silver is not essential in itself other
than as a manufacturing basis for nanoparticles with appropriate phys-
ical properties. Other NpC with these properties, such as NpC based on
metal-oxides (e.g., zinc oxides, titanium oxides) would be effective as
well (Iron is not recommended, since iron promotes bacterial biolm
growth).
Antiviral mechanism of action – Nanoparticle size
The importance of nanoparticle size is much greater for antiviral
compared with antibacterial properties, and we contend that this is
related to the mechanism of action. The antiviral effect arises predom-
inantly from attachment of nanoparticles to the virus. We posit that
antiviral effectiveness is limited to nanoparticles of size <10 nm. This
universality arises from the rather uniform geometric scales of respira-
tory infections viruses (e.g., inuenza and coronavirus), all of which
have diameters of about 100 nm, the distance between neighboring
spike glycoproteins being 10–20 nm, and glycoprotein length of about
15 nm. Thus, based on geometrical limitations alone, for a nanoparticle
to interact effectively with a glycoprotein site, its diameter must be
around 10 nm or less. Virus functioning becomes disturbed only when
the virus is sufciently covered by attached nanoparticles [19].
Fig. 2. HIV-1 (a/b) with/without NpC treatment. (c) Size distribution of silver nanoparticles bound to HIV-1 collected from all tested preparations seems to peak at a
nanoparticle size of about 4 nm (adapted from [14]).
O. Zachar

Medical Hypotheses 159 (2022) 110753
4
We nd supporting evidence for the above argument from experi-
ments and direct imaging of nanoparticles binding to human immuno-
deciency virus (HIV) [14], whose size is 120 nm, with ~ 22 nm spacing
between the glycoprotein knobs. Interestingly, the observed sizes of
nanoparticles bound to HIV (see Fig. 2) are exclusively within 1–10 nm,
with peak virus attachment effectiveness for nanoparticle sizes in the
range of 3–7 nm. No nanoparticles of diameter greater than 10 nm were
observed to interact with the virus, even though about 40% of the
overall nanoparticle population in the sample was beyond this size range
[14].
Consequently, we estimate that experiments in the literature, pre-
dominantly performed with larger NpC, have skewed IC values too high,
since their effectiveness arises wholly from the margins of the distri-
bution of particles with sizes <10 nm. The best we can do, with the
presently available, is to focus on analyzing published data only from
experiments conducted with ~ 10 nm NpC size. However, there is a need
to perform experiments with purposefully produced NpC nanoparticle
sizes peak distribution in the range of 3–7 nm.
Antiviral mechanism of action –The electrostatic potential of nanoparticles
Nanoparticles are composites, having a metal core and an envelope
capping material (Fig. 3). In a liquid environment, nanoparticles have a
surface electric potential, called the zeta potential, the magnitude of
which is dependent on solution pH. For a typical silver NpC, the zeta
potential is negative.
Unlike bacteria, we conjecture that the broad-range antiviral effec-
tiveness of NpC is a consequence of primarily electrostatic interactions.
The spike proteins of many human affecting viruses (including inuenza
and coronaviruses) are positively charged, likely promoting their bind-
ing to the predominantly negative surface charge of the host cell re-
ceptors (such as ACE2) [20]. Correspondingly, the highly negative
surface zeta-potential of the nanoparticles (which is required to keep
them in colloidal form) leads the nanoparticles to bind selectively to the
spike proteins of viruses and thereby neutralize their receptor binding
afnity. Hence, nanoparticles with a zeta potential stronger than −20
mV or −30 mV are preferred. The silver as a particular atom is unim-
portant in itself, other than as a manufacturing method for generating a
stable, charged, composite nanoparticle colloid. It follows that any
colloid of nanoparticles whose size distribution is predominantly in the
2–10 nm range and that possess a highly negative zeta-potential strength
of more than −20 mV would work as effectively as silver NpC. Corre-
spondingly, viruses whose spike protein binding sites are less positively
charged will be less affected by the NpC.
Generally, the chemistry of solutions is such that the zeta potential
becomes more negative with increasing pH. Changing pH may even
cross the zeta potential between negative and positive values. In
humans, normal pH values of the bronchial tree mucus are in the range
6.9–9.0 [22]. Therefore, NpC should preferably have a pH of around
7–7.5 in order that their zeta potential not overly degrade in the mucosal
environment.
Coronavirus evidence
Transmissible gastroenteritis virus (TGEV), a porcine coronavirus,
causes very high mortality in piglets. Fig. 4 presents the results of testing
the effect of NpC on coronavirus TGEV [17]. With nanoparticles of non-
optimal size 10–20 nm, tested for 1 h incubation, there is a signicant
inhibitory (>50% reduction) effect at a 12.5 µg/mL concentration and
what seems like near static (no growth) at a 3 µg/mL concentration.
Hence we would conclude from this experiment the values of MIC =3
µg/mL , and MMC =12 µg/mL.
Material properties
Distinguishing ionic vs nanoparticle colloids
It is essential to distinguish between ionic solution (recognized by
clear water-like color) and nanoparticle colloids (identied by a visibly
yellow-brownish color). Ionic silver solution consists of positively
charged silver ions dissolved in water, whereas silver NpC consists of
negatively charged nano-sized chunks of elemental silver, size 1–100
nm. Both ionic silver and silver NpC manifest antibacterial properties. In
contrast, regarding antiviral properties, it has been argued that colloidal
particles are signicantly more potent than ionic silver [23].
Signicance of selected stabilization coating of the nanoparticles
Prior testing results suggest that the capping agent material can
affect antiviral effectiveness. For example, it appears that PVP-capped
NpC had practically no antiviral effect on the coronavirus TGEV,
whereas a non-PVP NpC exhibited strong antiviral effectiveness [17]. A
similar result was obtained for HIV, where a ten-fold greater
Fig. 3. (a) A silver nanocrystal core, the PVA capping, and a full snapshot of an equilibrated PVA-stabilized nanoparticle in aqueous media [21]. (b) The zeta
potential of a colloidal nanoparticle is the potential (here negative) at the slip plane surface.
Fig. 4. The coronavirus TGEV (MOI 0.5) was incubated with the indicated
concentrations of silver-based nanoparticles (AgNP) at 37
◦C for 1 h in DMEM.
The AgNP were of size <20 nm (adapted from [17]).
O. Zachar

Medical Hypotheses 159 (2022) 110753
5
concentration was needed for a PVP-coated NpC sample to achieve the
same antiviral effect as a non-PVP NpC [13]. Overall, it appears that a
thinner capping layer may have a less deleterious effect on antiviral
potency.
Anti-bacterial applications
For bacterial infections, the literature suggests the presence of a
poorly differentiated mixed effect arising from both silver ions and
nanoparticles [18]. A major problem in deriving clear numerical con-
clusions from the published literature is that the IC, commonly stated in
weight fraction units (µg/mL), does not take into account the sensitivity
to nanoparticle size. For the same weight fraction (µg/mL) the number
density (N/mL) of smaller nanoparticles is higher than larger nano-
particles. Since higher number density (number of nanoparticles per
volume) increases the probability of nanoparticle–pathogen
interactions, we expect smaller nanoparticles to exhibit greater antimi-
crobial effectiveness for same weight fraction (µg/mL) than larger
nanoparticles (as has been manifested experimentally [24]). Confusion
arises from published studies using different-sized nanoparticles from
one another while expressing the results in weight fraction µg/mL. The
variance in the reported outcomes between publications is an artifact of
the different nanoparticle sizes used in the various protocols, obscuring
the consistency of NpC effects. Considering the antibacterial properties
of silver NpC (e.g., on Pseudomonas aeruginosa) [24], the typical IC
values (~7 µg/mL) of small nanoparticles (~7 nm) are similar to those
of the typical antiviral IC, according to our analysis.
Dose calculation
Antimicrobial drugs exhibit concentration-dependent efcacy.
Therefore, ensuring an appropriate concentration in the relevant body
uid is essential. For inhaled antimicrobials, the applicable body uid
for drug concentration purposes is the airway surface liquid (ASL) [12],
also referred to as epithelial lining uid (ELF). The delivered dose is
deposited on and diluted by the ASL. We argue that dosage planning,
correction, and controlled verication can be achieved by examining the
trachea or primary bronchi (G1). Deposition models indicate that the
trachea and G1 bronchi can serve as lower/upper bounds for all of the
concentration estimators for all rst 10 generations of the bronchial tree
(see Fig. 5). Therefore, BAL sampling of tracheal & G1 bronchi NpC
concentration is a well-dened and measurable verication method of
target IC in the whole bronchial tree organ.
Theoretically, to calculate the dose that needs to be deposited to
achieve the target IC requires knowing the volume of the ASL. Unlike the
predictability of blood volume, the ASL volume may have signicant
inter-personal variability. Such variability may result from individual
disease states (e.g., pneumonia, healthy) or lifestyles (e.g., smoking).
The ASL concentration of an individual patient can be veried by
bronchoalveolar lavage (BAL) sampling.
Concentration in airway surface liquid (ASL)
A dose calculation yields a range of possibilities rather than a xed
number. In clinical practice with antibiotics, the indicated dosage for
achieving a signicant inhibitory concentration (IC) is a substantial
Fig. 5. Local concentration of inhaled aerosol in different generations of the
bronchial tree, where the trachea is generation 0. Adapted from [12].
Fig. 6. Deposition fractions in primary respiratory system structures when breathing at rest. Adapted from [25].
O. Zachar

Medical Hypotheses 159 (2022) 110753
6
multiple of the MIC within wide clinician-determined margins limited
by the bounds of safety. It is common for antibiotics inhalation to target
IC that can be ten times (10x) the MIC. Similar considerations apply to
our nanoparticles dose calculation.
The bronchial tree’s rst 10 generations surface area is about 1 m
2
(10,000 cm
2
). The ASL layer thickness ranges from about 6 µm in the
trachea to 3 µm in the 10th generation of the bronchial tree [12],
amounting to a combined mucosal volume in the top half of the bron-
chial tree to be about 1 mL in a healthy adult. The surface area of the
alveoli is about 100 m
2
, with mucosal thickness of about 0.07 µm,
resulting in a total ASL volume of 7–10 mL. Therefore, the more realistic
target treatment is disinfection of the upper bronchial tree at early stages
before the infection spreads deep into the lungs. The disease state
signicantly affects antimicrobial inhalation delivery. Increased mucus
production dilutes the drug concentration in the ASL, compared with
healthy adults. Fortunately, for inhalation of NpC formulations, our
theoretically computed MIC or MMC are more than 40 times smaller
than the estimated safety limit (see section 3.7 below).
Deposition fraction in target tissue
The deposition fraction factor depends on the:
i. Target tissue location: extra-thoracic, trachea-bronchial (TB)
tree, or pulmonary (alveoli)
ii. Mode of inhalation: nasal or oral
iii. Size of the aerosol droplets.
There is a signicant difference in tracheal-bronchial (TB) tree
deposition between nasal and oral breathing (Fig. 6). Peak nasal
breathing TB deposition is only about 10%, whereas oral breathing TB
deposition is about 30%. Hence, we recommend oral inhalation.
Depending on aerosol droplet size, the deposition fraction is different
in each tissue region. As illustrated in Fig. 6: (a) the peak bronchial tree
deposition fraction (of about 30%) is obtained with aerosol droplets of
diameter ~ 6
μ
m, whereas (b) peak alveoli deposition (of about 30%) is
with aerosol droplets of diameter ~ 3
μ
m. For 5 µm droplets, typical in
mass-produced devices presently on the market, the bronchial tree
deposition fraction is ~ 25% and the alveoli deposition fraction is also
~ 25%.
Inhalation timing losses
Inhalation represents only about a third of the duration of a complete
breathing cycle. Consequently, when aerosols are delivered continu-
ously, during 2/3 of the time the aerosol is cumulated in a cloud within
the oral cavity until the next inhalation begins. Hence, we postulate a
lower-bound working assumption that only about 50% of the aero-
solized dose is considered to have been effectively inhaled. This would
be common for home users utilizing standard commercial medicament
aerosol devices available in pharmacies. By contrast, using a breath-
actuated nebulizer would correspondingly lower wastage of silver NcP
colloid, changing the associated dosage instructions.
Lung clearance
The lungs have innate mechanisms to remove deposited particles.
Thus, we need to examine whether the nanoparticles reside long enough
in respiratory system surface tissue to fulll their antiviral potential. For
indicative reference, we consider the behavior of inhaled antibiotics
(tested in ventilated ICU patients) [26,27]. We note that inhalation-
deposited antibiotics reside for an effective peak duration of about
2–3 h in the lungs. The NpC experimental data we analyzed were
commonly for 1 h treatments (e.g., for the coronavirus presented in
Fig. 4). Hence, experience with inhaled antibiotics suggests that also the
inhaled NpC will be retained in lung surface tissue for long enough to
exhibit antiviral potency.
Clinical safety
Clinical safety evaluations are performed in the context of an esti-
mated treatment dose and schedule. To be specic, we analyze the
example outlined in table-1(ii): 2 mL of NpC with a concentration of 120
μ
g are inhaled (i.e., a dose of 240
μ
g per treatment), taken two times per
day, which amounts to a daily deposition of about 480
μ
g , taken over
ve days (similar to an antibiotics regimen).
The human body has mechanisms for the disposal of silver. The
natural median daily intake of silver from food and drinks has been
reported to be up to 80
μ
g/day [28]. Therefore, our above scenario of a
recommended treatment regimen (deposition of about 480
μ
g) is a sig-
nicant increase over normal daily dietary intake for a short acute
treatment duration of a few days. When addressing safety or toxicity, the
professional guidelines distinguish between acute exposure of <14 days
and prolonged (repeated dose) or chronic exposure of more than 14
days. Only acute (<14 days) exposure is relevant in our context, but we
shall discuss both.
USA occupational guidelines and exposure limits for airborne silver
dust are all dened on a mass basis. The Occupational Safety and Health
Administration (OSHA) in the USA adopted a threshold-limit value time-
weighted average (TLV-TWA) chronic exposure over a 40-hr week of
100
μ
g/m
3
(0.1
μ
g/L) of metallic silver dust. Under a regular breathing
rate of 6–8 L/min, the resulting silver nanoparticle inhalation of 36–42
μ
g/h is considered a safe work environment. It amounts to daily, 8 h,
inhaling ~ 300
μ
g of silver nanoparticles on a prolonged routine basis.
Thus, the treatment regimen stipulated here, of 240
μ
g dose given two
times per day, is close to the safety bounds for acceptable chronic intake
in the work environment in the USA.
However, chronic work environment exposure is not the appropriate
context for acute drug toxicity evaluation. We should focus on the
assessment of short-term inhalation. It has been shown that even after
90 days of continuous inhalation exposure to high doses of silver
nanoparticles (total of 1,143
μ
g/day of silver – about three times (3x)
our realistic treatment regimen example – the accumulated tissue levels
recover to normal within about 12 weeks from the end of treatment
(Fig. 7). This provides a rm indication of the safety and tolerance of
short-term silver NpC inhalation exposure in that it does not lead to any
lasting accumulation in body tissues.
A test closest to our envisioned treatment protocol was performed on
rats for 10 days, 4 h/day, at a concentration of 3,300
μ
g/m
3
(3.3
μ
g/L)
using an aerosol of 5 nm silver nanoparticles, resulting in minimal
pulmonary toxicity or inammation [31]. Human breathing at an
average rate of 6 L/min translates to a daily inhaled dosage of silver
nanoparticle aerosol of 4,700
μ
g/day. This study indicates that, for acute
short-term treatment of <10 days, even an inhalation intake of 4,000
Fig. 7. Tissue silver concentrations in female rats exposed to high levels of
AgNP (381
μ
g/m
3
silver nanoparticles of ~ 15 nm diameter, for 6 h/day, which
amounts to an inhalation dosage of 1,143
μ
g/day of silver) in a 90-day inha-
lation study, followed by a 12-week recovery period. Adapted from [29,30].
O. Zachar

Medical Hypotheses 159 (2022) 110753
7
μ
g/day, which is about 10 times our realistic treatment regimen
example, would induce no adverse reaction or a lasting residue.
Discussion
Though marred by the charlatan claims of unprofessional “alterna-
tive medicine” products, there is well-established scientic research on
the antibacterial and antiviral properties of silver NpC. However, the
potential applications for respiratory infections treatment have never
been adequately explored. The surveyed literature indicates that silver
nanoparticles colloids (NpC) of diameter 3–7 nm can be highly effective
in suppressing many viral pathogens at an inhibitory concentration (IC)
of 10 µg/mL (Table 2). For a treatment via inhalation delivery of silver
NpC (Table 1), inhalation of 1 mL of NpC at a concentration of 120
μ
g
can achieve a signicant IC of 40
μ
g (i.e., 4x of MMC) in the bronchial
tree airway surface liquid (ASL). With common market continuous
nebulizers, one would need to nebulize a dose of 240
μ
g (because of 50%
inhalation time losses). A twice daily treatment protocol corresponds to
a total daily deposition of 240
μ
g. The available safety information in-
dicates that such doses and regimens are well within the safe range and
enable the safe delivery of even ten times (10x) the IC noted in Table 1.
We estimate that these formulations can effectively prevent and treat
any early-stage respiratory viral infection, including infection with
SARS-CoV-2.
To set the scale of potential benets, we ash out the remarkable
results of an Inuenza H3N2 in-vivo experiment done on mice (one of
the very few in vivo animal experiments in the NpC literature),
comparing the effectiveness of intranasal administration of NpC to that
of Tamiu (Oseltamivir) [15]. Tamiu is claimed to reduce the number
of patients with severe u complications, such as pneumonia (by 44%)
or hospitalization (by 63%). As shown in Fig. 8 below, it appears that
potentially NpC can be as effective as Tamiu. A NpC u treatment, if
effective as in the mice model, could cost <1/10 of Tamiu, with lower
side effects risk, be available essentially OTC (as market availability of
NpC already is) and be manufactured locally in any country. However,
no investment in clinical human trials was ever done to investigate the
possibility. We can only speculate that this is due to conventional
pharma companies’ lack of nancial incentives to make the required
investment in development and regulatory procedures. One of the main
goals of this paper is to motivate, guide, and advocate for future in-
vestment in the clinical and regulatory development of NpC treatment
formulations.
For bacterial infections, particularly in the context of preventing
ICU-acquired VAP, the same formulations are expected to be applicable.
An additional risk-reduction benet of silver NpC inhalation treatment
for ventilated patients is the possibility of suppression of biolm for-
mation inside the endotracheal or tracheostomy tube.
Funding
This research received no external funding.
Declaration of Competing Interest
The authors declare the following nancial interests/personal re-
lationships which may be considered as potential competing interests:
Yamor Technologies promotes a drug development program based on
the framework presented in this article.
Acknowledgements
We thank Alex Singer for editorial assistance in the manuscript’s
preparation.
Consent statement/Ethical approval
Not required.
References
[1] Aderibigbe BA. Metal-based nanoparticles for the treatment of infectious diseases.
Available from Molecules [Internet]. 2017;22(8):1370. http://www.mdpi.com/
1420-3049/22/8/1370.
[2] Nakamura S, Sato M, Sato Y, et al. Synthesis and application of silver nanoparticles
(Ag NPs) for the prevention of infection in healthcare workers. Available from: Int
J Mol Sci [Internet] 2019;20(15):3620. https://www.mdpi.com/1422-0067/20/1
5/3620.
[3] Siadati SA, Afzali M, Sayadi M. Could silver nano-particles control the 2019-nCoV
virus?; An urgent glance to the past. Available from: Chem Rev Lett [Internet]
2020;3(1):9–11. http://www.chemrevlett.com/article_105425.html.
[4] Galdiero S, Falanga A, Vitiello M, Cantisani M, Marra V, Galdiero M. Silver
nanoparticles as potential antiviral agents. Available from Molecules [Internet]
2011;16(10):8894–918. http://www.mdpi.com/1420-3049/16/10/8894.
[5] Balavandy SK, Shameli K, Biak DRBA, Abidin ZZ. Stirring time effect of silver
nanoparticles prepared in glutathione mediated by green method. Chem Cent J
2014;8(1):11.
[6] Haggag E, Elshamy A, Rabeh M, et al. Antiviral potential of green synthesized silver
nanoparticles of Lampranthus coccineus and Malephora lutea. Available from: Int J
Nanomed [Internet] 2019;14:6217–29. https://www.dovepress.com/antiviral-pote
ntial-of-green-synthesized-silver-nanoparticles-of-lampr-peer-reviewed-article-IJN.
[7] Rouby JJ, Bouhemad B, Monsel A, Brisson H, Arbelot C, Lu Q. Aerosolized
antibiotics for ventilator-associated pneumonia: Lessons from experimental studies.
Anesthesiology 2012;117(6):1364–80.
[8] Darquenne C, Fleming JS, Katz I, et al. Bridging the gap between science and
clinical efcacy: Physiology, imaging, and modeling of aerosols in the lung.
J Aerosol Med Pulm Drug Deliv 2016;29(2):107–26.
[9] Behrens G, Stoll M. In: Pathogenesis and immunology [Internet]. Paris, France:
Flying Publisher; 2006. p. 92–109 [cited 2020 Nov 30]. Available from:.
[10] Garland JS. Ventilator-associated pneumonia in neonates: An update. Available
from Neoreviews [Internet] 2014;15(6):e225–35. https://neoreviews.aappublicati
ons.org/content/15/6/e225.
[11] Manjarrez-Zavala EM, Patricia D, Horacio L, Ocadiz-Delgado R, Cabello-Gutierrez
C. Pathogenesis of viral respiratory infection [Internet]. In: Respiratory Disease and
Infection - A New Insight. Mahboub BH (Ed.), InTech (2013) [cited 2020 Nov 30].
Available from: https://doi.org/10.5772/54287.
[12] Hasan MA, Lange CF. Estimating in vivo airway surface liquid concentration in
trials of inhaled antibiotics. J. Aerosol Med 2007;20(3):282–93.
[13] Ringshausen FC, Chalmers JD, Pletz MW. In: Anti-infectives and the Lung.
European Respiratory Society; 2017. p. 57–79. https://doi.org/10.1183/
2312508X.10004616.
Table 2
Inhibitory concentration (IC) for non-PVP stabilized nanoparticle colloids (NpC)
with diameter ≤10 nm.
Virus NpC diameter distribution
peak (nm)
MMC (µg/
mL)
Reference
Inuenza A H3N2 9.5 <12.5 [15]
Monkeypox
(MPV)
10 <12.5 [16]
Coronavirus
TGEV
1
10 ~10 [17]
1
TGEV, transmissible gastroenteritis virus.
Fig. 8. In-vivo intranasal NpC administration protected mice from H3N2
infection. Adapted from [15].
O. Zachar

Medical Hypotheses 159 (2022) 110753
8
[14] Elechiguerra JL, Burt JL, Morones JR, et al. Interaction of silver nanoparticles with
HIV-1. Available from: J. Nanobiotechnology [Internet]. 2005;3(1):6. http://jnano
biotechnology.biomedcentral.com/articles/10.1186/1477-3155-3-6.
[15] Xiang D, Zheng C, Zheng Y, et al. Inhibition of A/Human/Hubei/3/2005 (H3N2)
inuenza virus infection by silver nanoparticles in vitro and in vivo. Int. J.
Nanomedicine [Internet]. 8(1), 4103 (2013). Available from: https://doi.org/
10.2147/IJN.S53622.
[16] Rogers JV, Parkinson CV, Choi YW, Speshock JL, Hussain SM. A preliminary
assessment of silver nanoparticle inhibition of Monkeypox virus plaque formation.
Nanoscale Res Lett 2008;3(4):129–33.
[17] Lv X, Wang P, Bai Ru, Cong Y, Suo S, Ren X, et al. Inhibitory effect of silver
nanomaterials on transmissible virus-induced host cell infections. Biomaterials
2014;35(13):4195–203.
[18] Dakal TC, Kumar A, Majumdar RS, Yadav V. Mechanistic basis of antimicrobial
actions of silver nanoparticles. Front Microbiol 2016;7:1831.
[19] Morris D, Ansar M, Speshock J, et al. Antiviral and immunomodulatory activity of
silver nanoparticles in experimental rsv infection. Available from: Viruses
[Internet] 2019;11(8). https://pubmed.ncbi.nlm.nih.gov/31398832/.
[20] Prabakaran P, Xiao X, Dimitrov DS. A model of the ACE2 structure and function as
a SARS-CoV receptor. Biochem Biophys Res Commun 2004;314(1):235–41.
[21] Kyrychenko A, Pasko DA, Kalugin ON. Poly(vinyl alcohol) as a water protecting
agent for silver nanoparticles: The role of polymer size and structure. Phys Chem
Chem Phys 2017;19(13):8742–56.
[22] Clary-Meinesz C, Mouroux J, Cosson J, Huitorel P, Blaive B. Inuence of external
pH on ciliary beat frequency in human bronchi and bronchioles. Eur Respir J 1998;
11(2):330–3.
[23] Lara HH, Ayala-Nu˜
nez NV, Ixtepan-Turrent L, Rodriguez-Padilla C. Mode of
antiviral action of silver nanoparticles against HIV-1. Available from:
J Nanobiotechnol [Internet] 2010;8(1):1. http://jnanobiotechnology.biomedcen
tral.com/articles/10.1186/1477-3155-8-1.
[24] Martínez-Casta˜
n´
on GA, Ni˜
no-Martínez N, Martínez-Gutierrez F, Martínez-
Mendoza JR, Ruiz F. Synthesis and antibacterial activity of silver nanoparticles
with different sizes. Available from: J Nanoparticle Res [Internet] 2008;10(8):
1343–8. https://link.springer.com/article/10.1007/s11051-008-9428-6.
[25] Cheng YS. Mechanisms of pharmaceutical aerosol deposition in the respiratory
tract. Available from AAPS PharmSciTech [Internet] 2014;15(3):630–40. https://li
nk.springer.com/article/10.1208/s12249-014-0092-0.
[26] Dhanani J, Fraser JF, Chan HK, Rello J, Cohen J, Roberts JA. Fundamentals of
aerosol therapy in critical care. Crit Care 2016;20(1):1–16.
[27] Lu Q, Girardi C, Zhang M, Bouhemad B, Louchahi K, Petitjean O, et al. Nebulized
and intravenous colistin in experimental pneumonia caused by Pseudomonas
aeruginosa. Intensive Care Med 2010;36(7):1147–55.
[28] WHO. Silver in drinking-water: Background document for development of WHO
Guidelines for Drinking-water Quality [Internet]. Geneva. Available from: https
://www.who.int/water_sanitation_health/dwq/chemicals/silver.pdf.
[29] Fewtrell L. Silver: Water disinfection and toxicity [Internet]. Available from: https
://www.who.int/water_sanitation_health/dwq/chemicals/Silver_water_disinfecti
on_toxicity_2014V2.pdf.
[30] Song KS, Sung JH, Ji JH, Lee JH, Lee JS, Ryu HR, et al. Recovery from silver-
nanoparticle-exposure-induced lung inammation and lung function changes in
Sprague Dawley rats. Nanotoxicology 2013;7(2):169–80.
[31] Stebounova LV, Adamcakova-Dodd A, Kim JS, Park H, O’Shaughnessy PT,
Grassian VH, et al. Nanosilver induces minimal lung toxicity or inammation in a
subacute murine inhalation model. Part Fibre Toxicol 2011;8(1). https://doi.org/
10.1186/1743-8977-8-5.
O. Zachar