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Formulations for COVID-19 Treatment via Silver Nanoparticles Inhalation

Delivery

Preprint · May 2020

DOI: 10.31219/osf.io/adnyb

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 Formulations for COVID-19 Treatment via Silver Nanoparticles
Inhalation Delivery
Oron Zachar, Ph.D
Yamor Technologies Ltd.
23 Hamelacha, Rosh Haayin 4809173, Israel
Email: oron@yamor-tech.com
Abstract
Objectives: For suppression of both viral and bacterial respiratory infections, we present candidate
formulations and treatment protocols based on nano-silver colloids (NAgC) by inhalation delivery.
Special evaluation is given to Corona virus. We are not aware of any published theoretical or clinical
research on medicinal inhalation of silver particles. In this article we lay the grounds for such clinical
evaluations to be done. The formulations are based on the available experimental data. Yet, according
to our analysis, the published experiments were all done on sub-optimal NAgC. Hence, following the
presented framework, future tests with our recommended more optimized NAgC should lead to
improved formulations. We analyse three core aspects: (a) material composition options, (b) associated
effective treatment dosages, and (c) the delivery method. Building towards potential clinical evaluation,
we discuss the evidence for the safety of the proposed treatment based on published tests and guidelines
in the EU and USA for inhalation of silver nanoparticles. From the clinical therapeutic point of view,
we argue that our treatment formulations may be most effectively applied as a first-line intervention at
an early stage of respiratory infections, i.e., when mostly affecting the upper respiratory system and
bronchial tree. The proposed treatment is a measure with the potential to suppress proliferation of the
viral infection across the respiratory system, thereby giving more time for proper immune system
response and lowering the risk for aggravation and spread of the infection. For example, the proposed
formulations could be used to control local outbreaks of COVID-19 via early stage home treatment.
In the context of bacterial pneumonia, we note that similar NAgC dosages should also provide anti-
bacterial effectiveness. We propose that for hospital ventilator associated pneumonia (VAP), inhalation
delivery of NAgC can be implemented prophylactically to lower VAP risk. For completeness, we
discuss manufacturing and commercial availability for near term wide public usage.
If it was some other new drug substance – having the same overwhelmingly positive in-vitro and animal
trials data, combined with similarly established strong and diverse safety testing (including EU and
USA national safety bodies reviews) – it would have been heralded as one of the most promising anti-
microbial potential drug in history, which would have been rushed to be developed by big pharma
companies. That this did not happen with nano-silver colloids until now, is only due to a market failure
– the inability to patent any of it. Unfortunately, the gap between promising academic research and
marketable regulatory approved products has been left to be filled by “alternative medicine” charlatans
– giving bad publicity to the whole field. In order to overcome this market failure situation, the present
paper also serves as a proclamation of our open-source drug development program to realise it.
Method: We integrate several components of analysis: (a) We deduce from the published literature the
most effective composition of the nano-silver colloid, showing high sensitivity to both particle size and
manufacturing stabilizing factors. Therefore, specifying only colloidal mass concentration (μg/ml) is
not enough and may even be misleading. (b) We deduce the required inhibitory concentration IC50 in
the target respiratory system locations. (c) We argue that the proper approach for respiratory infections
is to think of the delivery as “topical”, i.e., deposition from the air on the respiratory system surface,
and that the relevant concentration, where the IC50 is to be computed for, is the concentration in the
liquid mucus covering the surface of the respiratory system (with different thickness in different
portions of it). (d) For computing the require delivery dosage, we take into account deposition fraction

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losses and inhalation time fraction of the breathing cycle. We evaluate independent targeting of: (i) the
trachea-bronchial tree (mucus volume of ~1cc), and (ii) the alveoli (total mucus volume of ~10cc).
Results: The dosage is highly sensitive to the silver nanoparticle size, with 3nm - 7nm being the optimal
size. In manufacturing, PVP stabilization should be avoided, as it lowers the NAgC anti-viral
effectiveness significantly (but is still fine for anti-bacterial applications). Effective anti-viral inhibitory
concentration (IC) of 10μg/ml is estimated as a reasonable target concentration to achieve in the mucus
fluid of the respiratory system. With colloidal silver of 5nm particles, delivering inhalation of standard
5μ diameter droplets aerosol (e.g., using off-the-shelf ultrasonic mesh nebulizers), we assert that IC can
be achieved with depositing a total of just 0.33cc of a 30μg/ml NAgC concentration in the bronchial
tree. Yet, after accounting for effective deposition fraction (~30%) and due to the fact that active
inhalation time is just about 1/3 of the breathing cycle (if common continuous nebulizers are used, rather
than breath-activated), a dosage of 3.3ml need to be continuously nebulized for inhalation by the user.
We recommend that exhalation through the nose can be implemented as a method to deliver the exhaled
fraction of NAgC also to the nasal cavity when the nebulized inhalation is done orally.
Conclusions: We conclude that effective IC is achievable, both in the bronchial tree and in the alveoli
(though the specific aerosol prescription may differ). The optimal size of silver nanoparticles for anti-
viral effectiveness is in the range of 3-7nm, which is significantly smaller the about 10nm size of all the
best experiments published in the literature. Hence, there is a potential for even better efficacy and/or
lower dosage if the optimal NAgC is used. Since respiratory infections commonly start in the upper
airways, it would be best to use the presented method early on as a first line treatment to suppress the
progression of the infection. The required formulations are presently not available on the market but
are easy to mass produce OTC in principle. Using off-the-shelf ultrasonic nebulizers and providable
OTC colloidal silver formulations, we posit that our suggested method can be used precautionarily at
home by anyone feeling the early signs of a potential infection. In addition, due to the anti-bacterial
properties of colloidal silver, our method can serve in hospital intensive care units (ICU) as a new
standard of care prophylactic treatment for ventilator acquired pneumonia (VAP).

Introduction
When there are a core scientific and biologically grounded effects, the initial association with fringe
unsubstantiated “alternative medicine” should not deter scientists from investigating the potential for
success that may come when applying rigorous calculations and analytical reasoning. The antimicrobial
properties of silver nanoparticles are well established [4,5,25,26,28]. Applications of nano-silver are
medically FDA approved in the field of wound care. A senseless uncontrolled “alternative medicine”
practice of ingesting colloidal silver led to widespread disregard by the pharmacological and academic
establishment of potential new applications for the antimicrobial properties of silver nanoparticles
(beyond wound treatment). In particular, there is no rigorous analysis in the literature concerning the
potential of inhalation use for the prevention and/or treatment of respiratory infections.
The goal of this article is to analytically substantiate potential antimicrobial colloidal silver
formulations, delivered by inhalation, to minimise the aggravation of respiratory system infections. In
the context of the present discussion, we shall distinguish between two scenarios: (A) viral respiratory
infections (including the recent COVID-19 / SARS-CoV-2); and (B) bacterial infections risk associated
with hospital ventilator associated pneumonia (VAP) in intensive care units (ICU) patients on
ventilation breathing support.
A common pathogenesis aspect of respiratory infections is that they are commonly initiated mildly in
the nasopharynx and/or upper bronchial tree portions of the respiratory system [1,2]. Aggravation of
the condition occurs once the pathogens and associated inflammation migrates to lower portions of the
respiratory system. Moreover, as the infection spreads, the increased immune response is exacerbated
and may cause a greater damage [3]. Therefore, we contend that a desirable effective treatment would
be to suppress the proliferation of the pathogens at early stages of the infection when it is still mostly

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confined to the upper respiratory system. e.g., (i) when most patients are still at home with mild
symptoms, and (ii) from day-one of patient’s arrival to the hospital ICU before the signs of any VAP
infection. In this article, we will propose potential formulations both for upper respiratory system
treatment and for lower respiratory system (alveoli) treatment.

Figure-1: Pathogenesis, (A) Sites of influenza entry in the respiratory tract. Influenza first
infects the upper airway and the ciliated cells in the bronchus and bronchioles [1]. (B)
Pathogenesis of hospital bacterial ventilator associated pneumonia (VAP).

Formulations Calculation
It is well established in the scientific literature that silver has both antibacterial and antiviral properties
[4,8], including explicitly to influenza viruses. Clinical usage and testing of these properties has thus
far focused mostly on wound care. There are is no published theoretical or clinical research on medicinal
inhalation of silver particles that we are aware of. In this article we intend to guide and lay the grounds
for such clinical evaluations to be done, including (a) determination of optimal material properties of
nanoparticles for most effective anti-viral composition, (b) the required effective inhibitory
concentration (IC) that is required to be obtained at the target respiratory system location, and (c) the
required dosage for practical implementation by inhalation delivery. To calculate the required delivery
dosage, we go through a stepped procedure of analysis, evaluating the effect of each stage between the
aerosol production by the aerosolizing device to the final target tissue deposition.

Figure 2: Outline of the dosage calculation logical structure

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As elaborated below, we reach the following conclusions:
• Colloid: For antiviral applications, nanoparticle size should be in the range of 3nm – 7nm. In
manufacturing, there is great significance to the stabilizing ("capping”) agent used. It appears
that polymers (PVP, PVE) should be avoided. For antibacterial applications there is less
sensitivity to nanoparticle size and stabilizing method, since the eluted silver ions are the
predominant source of antibacterial effect and not the originating nanoparticles.
• Target inhibitory concentration (IC): For antiviral applications, effective target IC is about
10µg/ml. For antibacterial applications similar dosages are needed.
• Tissue Deposition Fraction: Under oral breathing of 5µm aerosol droplets, Pharynx 30%,
Bronchial tree 30%, Alveoli 25%.
• Breathing cycle losses: Inhalation is about 1/3 time of the breathing cycle. Hence, using a
continuous aerosol source (most common home medicinal nebulizers) only about 30% of the
nebulized substance is assumed to be actually inhaled. Exhalation through the nose can be
implemented as a method to deliver the NAgC also to the nasal cavity when the nebulized
inhalation is done orally.

In order to achieve a desired IC in the target tissue, assuming the usage of colloidal silver in water, the
summary equation for calculating the require aerosol for delivery is the following:

(Aerosol dosage) = (Target IC [µg/ml]) * (Mucus Volume [ml])

/ (Tissue deposition fraction) / (Inhalation time losses)
/ (colloid concentration [µg/ml])

For antiviral applications via oral inhalation of colloidal silver 5nm particles, examples of possible
formulations according to our analysis are summarized in table-1. We see that with continuous
nebulizer, a continuous nebulized dosage of 3.3ml is needed in order to effectively deposit. Nebulizing
a quantity of 3ml of drug solution for inhalation delivery (with 10% - 20% effective lung deposition) is
a common practice in hospital intensive care [36]. Hence, we similarly expect our ~3ml nebulized
dosage to be a well-tolerated volume. We note that, because of the factor 10X difference in mucus
volume, targeting alveoli IC50 requires significantly higher concentration of colloidal silver source.
Since smaller droplets are more preferentially deposited in the alveoli, it may be recommended to use
a 3nm droplets aerosol when targeting the alveoli.
Table 1: Dosage calculation when using a 5µ droplets aerosol for bronchial tree target

Tissue IC Mucus deposition Colloid Inhaled Continuous

target in target Volume fraction concentration dosage nebulized dosage
[µg/ml] [ml] [µg/ml]) [ml] [ml]
Bronchial tree 10 1 0.3 30 1.1 3.3

Table 2: Dosage calculation when using a 3µ droplets aerosol for alveoli target

Tissue IC Mucus deposition Colloid Inhaled Continuous

target in target Volume fraction concentration dosage nebulized dosage
[µg/ml] [ml] [µg/ml]) [ml] [ml]
Bronchial tree 33 1 0.1 150 2.2 6.6
Alveoli 10 10 0.3 150 2.2 6.6

For anti-bacterial applications, such as for prophylactic treatment of tracheal tube or tracheostomy
ventilated patients in hospital ICU, the data in the literature suggests that similar dosages are effective.

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 Target IC Determination for Antiviral Applications
In the context of inhalations, silver particles suspensions or solution in water is the relevant
configuration. The key starting point for calculating any effective dosage is to establish the required
target inhibitory concentration (IC) of the effective agent. For example, in microbiology, IC50 is the
concentration of a chemical, usually a drug, that is required for 50% inhibition in vitro.

As elaborated on below, optimal anti-viral effectiveness is obtained with nanoparticles with size <12nm.
In addition, the nanoparticles size manufacturing method stabilization (capping) is affecting the anti-
viral potency. In particular, popular PVP capping is significantly inhibiting the antiviral effect.
Therefore, for analysing the published data we have limited the consideration only to experiments with
nanoparticles size <15nm and to non-PVP stabilized NAgC. The evidence summarized in Table-3
below seems to indicate that an IC of 10µg/ml would be an effective concentration to be desired at the
target respiratory system location. In particular we note that one of the referenced experiments was
conducted on a Corona virus. Hence, we speculate that similar concentrations of 10 µg/ml might be
effective also for SARS. Yet, our analysis below suggests that the optimal size of silver
nanoparticles for anti-viral effectiveness is in the range of 3-7nm, not the 10nm of all the
experiments published in the literature. Hence, there is a potential for even better efficacy and/or
lower dosage if the optimal NAgC is used. In the published experiments (Table-3), likely all the anti-
viral effectiveness comes from the tails of the particle size distribution within the noted range <10nm.

Table 3: Required inhibitory concentration (IC) of silver nanoparticles with size <12nm,
with non-PVP stabilized NAgC. The published literature seems to imply that ~10µg/ml is
a good candidate target IC.

Virus Nanoparticle size IC Reference

(peak of distribution) [µg/ml]
[nm]
Influenza H3N2 9.5 <12.5 [27]
Monkeypox (MPV) 10 <12.5 [32]
Corona TGEV 10 ~10 [24]
Tacaribe (TCRV) 10 <10 [37]

Moreover, it appears that nanoparticles of size less than 10nm have much higher anti-viral effectiveness
than larger nanoparticles (e.g., 25nm) [6,9]. The effect is much more significant than what is observed
for anti-bacterial properties. We speculate that this may have to do with the virus size themselves being
on the order of 100nm (HIV size is about 120nm, and SARS virus is also about 100nm). i.e., in order
to be effective in interacting with the virus, the silver nanoparticles need to be significantly smaller than
the virus, such as less than 10nm. This speculation is given support by direct imaging of nanoparticles
binding to viruses [5]. Interestingly, the observed sizes of nanoparticles bound to the virus (see Fig.5)
were exclusively within the range of 1–10 nm, with peak virus attachment effectiveness for
nanoparticles size in the range of 3nm – 7nm. The fact that no nanoparticles greater than 10 nm in
diameter were observed to interact with the virus is significant, since the size of ~40% of the overall
population in the sample was beyond this range [5]. Consequently, experiments done with larger
particles NAgC are expected to have skewed too high IC50, since their whole effectiveness come from
the tail of the distribution of particles with sizes <10nm. Hence, for the remainder of this article, we
shall focus on analysing data only for ~10nm peak concentration NAgC experiments.

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 (a) (b)

Figure 3: (a) image of an HIV-1 virus interaction with silver. (b) image of HIV-1 viruses
without silver nanoparticle treatment. (c) Composite size distribution of silver nanoparticles
bound to the HIV-1 virus, derived from all tested preparations, seems to peak at nanoparticle
size of about 4nm.

Therefore, we proclaim that for significant antiviral effectiveness the size of silver nanoparticles
needs to be less than 10nm, preferably in the range 3nm-7nm size. As seen in Fig.3, multiple silver
nanoparticles get attached to a virus. It is thought that a virus function becomes disturbed only when
sufficiently covered with nanoparticles [10].
As alluded to above, a major problem in deriving clear numerical conclusions from the published
literature is that the IC – given in weight fraction units (µg/ml) – is very sensitive to the nanoparticle
size. For the same weight fraction, smaller nanoparticles have higher number density than larger
nanoparticles (nanoparticle number density is roughly proportional to 1/R 3, where R is the particle
diameter). Since higher density of nanoparticles have higher probability of interacting with pathogens,
we would expect smaller nanoparticles to have higher effectiveness than larger nanoparticles.
Therefore, required IC of smaller nanoparticles will be smaller than IC of larger nanoparticles, which
experimentally is indeed the case. Consequently, since every published research article used different
size nanoparticles in its experiments, the resulting IC values indeed may vary significantly between one
publication to another – an artifact of the different nanoparticles size used in different experiments
and not an inconsistency of the NAgC potential effect.

Assuming a roughly fixed size of nanoparticles in the colloid, the antiviral potency is expected to
increase with concentration (i.e., with mass concentration µg/ml of the silver colloids) which appears
to be supported by experimental results [6], as illustrated in Fig.4.

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 Figure 4: Concentration dependence of HIV-1 infection inhibition by silver nanoparticles
[6]. Note that the concentration units in this graph are mg/ml. i.e., 5mg/ml = 500µg/ml. We
comment that this experiment was conducted with nanoparticles of size ~40nm, which may
explain the very high concentration need to achieve IC50 with this colloidal silver.

Corona Virus Evidence

Transmissible gastroenteritis virus (TGEV), a porcine coronavirus, causes very high mortality in piglets
and thus has tremendous impact on pig industry. Vaccination has been extensively applied to prevent
pigs from TGEV infection. However, it appears the immune efficacy is not ideal and the potential
dissemination as well as prevalence of infectious agent in piglets remain [24]. Fig.5 presents the results
of testing the effect of NAgC on corona virus TGEV [24].

Figure 5: (adapted from [24]) Corona virus TGEV (MOI 0.5) were incubated with indicated
doses of Ag nano-materials at 37 °C for 1 h in DMEM. Then the pretreated complexes were
used to infect cells at 37 °C for 1 h in 96-well plates. Cell viability was measured by MTT
at 48 hpi. The indicated AgNP are nanoparticles of size <20nm.
The potential anti-viral efficacy of NAgC on human corona virus (SARS) seems promising from tests
done on silver impregnated nonwoven material by Swiss manufacturer HeiQ, as noted in Fig.6.

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 Figure 6: (adapted from [31]) Antiviral efficacy test (ISO 18184): Nonwoven material for
face masks treated with silver impregnated HeiQ Viroblock NPJ03, tested according to ISO
18184 standard.

Distinguishing Ionic vs Colloidal Silver Solutions

It is important to distinguish between ionic silver solution and colloidal silver. Ionic silver is the case
were atomic silver ions are dissolved in water. Colloidal silver is the case where we have nano-size
silver chunks of silver matter, commonly in sizes between 1nm – 100nm diameter. When it comes to
antibacterial properties, both ionic silver and colloidal silver IC50 levels are well documented, and it
is thought that the silver ions are the most effective agent [23]. In contrast, it has been argued that the
antiviral properties of the colloidal particles are about 10x more potent than ionic silver [6]. This is very
important to realise, since any colloidal silver also contains a significant amount of ionic silver
component (as further discuss below). A key implication is that for evaluating the antiviral potency
of a colloidal silver, it would be a good approximation to focus analysis on the particle
concentration alone and disregard the ionic component.

Storage time may have an effect on colloidal silver water suspensions, since silver ions are eluted over
time (e.g., during storage) as illustrated in the Fig.7 [7]. It appears that within about 24h of storage in
water about 10% of the colloidal particle mass is lost to ions, and within 10 days of storage saturation
is reached with about 50% of the colloid mass lost to ions. Yet, we speculate that, intuitively, the colloid
particle number density remains the same, only that each colloid silver particle mass is reduce by about
50%. for an approximation of spherical nano-particle, 50% mass reduction translates to the diameter
smaller only by a factor ~0.8 which is not significant at the level of cluster size resolution for our
application.

Figure 7: Ageing saturation of colloidal silver in water (adapted from [7])

The analysis in this article relies phenomenologically on experimental data. While it is not essential for
our line of arguments, for the sake of completeness we note that there is growing research on the

Page 8 of 19
potential mechanisms of action of the antiviral properties of metallic nanoparticles [5]. In particular, it
appears that the mechanism involves attachment of the nanoparticles themselves to the virus, and not
the effect of eluted Ag+ ions.

Figure 8: Supposed antiviral mechanisms of action of colloidal silver (adapted from [5])

Significance of Selected Stabilization Coating of the Nanoparticles

A much-overlooked aspect in the NAgC field discussions is that different manufacturing methods
involve substances other than silver [28], primarily used for the particle size stabilization (or
“capping”), which are part of the particle surface composition and do influence the anti-viral
effectiveness of the NAgC. i.e., the “silver nanoparticle” is actually a composite object (see Figs.9,10).
As discussed below, polymer capping with PVP (and likely also PVA) seem to lower significantly the
anti-viral potential of the silver nanoparticles, while not disturbing the anti-bacterial effect. This can be
understood by the different anti-microbial mechanism of action. The capping molecules of all kinds do
not prevent the effective anti-bacterial silver ions (Ag+) eluting, yet they do modify binding interaction
of the full nano-particle with viruses.

Figure 9: (left) A silver nanocrystal with the average diameter of 3.9 nm, packed in the
fcc quasi-spherical structure. (middle) The molecular structure and G45a4 atom type
labeling of PVA polymer. (right) A MD snapshot of equilibrated PVA1540–AgNP in
aqueous solution. (Silver core atoms are shown as mauve balls, the PVA backbone in green,
and buffering ions Na+ and Cl as blue and orange balls.) (adapted from [30]).

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 Figure 10: Silver nanoparticles prepared by green synthesis method using GSH as
reducing agent and stabilizer [29]. Smaller capping layer might be less disturbing the anti-
viral potential.

There are a few indications in the literature that not all capping agents have the same consequence –
when it comes to anti-viral effectiveness. As seem in Fig.11, it appears that PVP capped NAgC had
practically no anti-viral effect on the Corona TGEV, while a non-PVP NAgC had strong anti-viral
effectiveness. A similar result is illustrated in Fig.12, where a factor 10X concentration is needed for
PVP coated NAgC sample to reach the same effect as a non-PVP NAgC.

(PVP)

Figure 11: (adapted from [24]) Corona virus TGEV was first incubated with various Ag
nanomaterials (NMs) in vitro for 1 h. Next, ST cells were infected by TGEV combined
with the indicated doses of Ag NMs for 1 h, and then washed three times with PBS and
cultured in DMEM for another 48h. The PVP capped 10nm silver colloid shows no effect,
while the AgNP size <20nm show best anti-viral effectiveness.

Figure 12: (adapted from [5]) Inhibition of HIV-1 and toxicity data. A) Assessment of
HIV-1 mediated syncytium formation in MT-2 cells. B) Percentage of HIV-1
transmission in cMAGI cells.

Page 10 of 19
The significance of both the particle size and the stabilization coating is exemplified in the
experimental results on Tacaribe virus (TCRV) illustrated in Fig.13.

Figure 13: (adapted from [37]) TCRV replication following exposure to NAgCs. TCRV
was treated with uncoated (Ag) and polysaccharide-coated (PS-Ag) 10nm and 25nm
NAgC for 1h.

Target IC Determination for Antibacterial Applications

For bacterial infections, the consensus in the literature seems to be that the antibacterial effect is
primarily due to the silver ions [15] and not the silver nano particles. Yet, it should be remembered, as
we highlighted above, that any silver nanoparticle colloid actually elutes silver ions into the solution.
Hence, even when testing a nanoparticle colloidal silver source, one cannot attribute the antibacterial
effects to the nanoparticles direct interactions. Therefore, in the context of antibacterial properties, it is
better to regard the nano-silver content as a general statement of silver material concentration.
Considering colloidal silver anti-bacterial properties (see Fig.14), it appears that typical IC50 (~7
µg/ml) for small nanoparticles (~7nm) is similar to that of the typical antiviral IC50 according to our
above analysis.

Figure 14: Antibacterial IC of colloidal silver [19].

Target Tissue Fluids Volume

For calculating the intake dosage which needs to be deposited in order to achieve the target IC, we need
to know the volume of mucus liquid (also called epithelial lining fluid – ELF) into which the delivered
droplets of colloidal silver are mixed after deposition. Nasal mucus is about 10–15 µm thick [38] and
has two layers: the lower, 6 µm thick liquid layer (also called: periciliary liquid), is covered by the more
viscous gel phase.
The total surface area of the bronchial tree is about 1m2 (10,000 cm2). But for aerosol droplets of about
5 µm diameter, most of the deposition occurs within the top 15 generations of the bronchial tree. Using
the data of the surface areas and mucus thickness from the table in Fig.15, we estimate the combined

Page 11 of 19
mucus volume in this top half of the bronchial tree to be about 1cc. The alveoli total surface area is
about 100m2 (i.e., 1,000,000 cm2) according to some estimations (or 140m2 according to others) with
mucus thickness of about 0.07 µm, resulting in total mucus volume of between 7cc to 10cc.

Figure 15: Surface area and mucus thickness of various parts of the bronchial tree [21].

Figure 16: (adapted from [22]) Visualization and quantification of nebulized aerosol
deposition in mouth-lung casts under healthy and abnormal breathing conditions.

Deposition Fraction in Target Tissue

The deposition fraction factor depends on
i. Target tissue location: extrathoracic / trachea-bronchial (TB) tree / pulmonary (alveoli).
ii. Mode of inhalation (nasal/oral). In early stages, the infection is mainly in the bronchial tree. In
particular, the infection and destruction of cilia cells in the trachea-bronchial tree by viral
infection is a key aggravation factor in viral pneumonia infections, such as COVID-19 (SARS-
CoV-2).
iii. Size of aerosol droplets.
As illustrated in the Fig.17 [14], there is a significant difference in bronchial tree deposition between
nasal and oral breathing. Peak nasal breathing TB deposition is only about 10%, while mouth breathing
TB deposition is about 30%. Hence, we recommend oral breathing inhalation.

Page 12 of 19
The aerosol droplet size dependence of the deposition fraction is also different in each tissue region. In
order to achieve optimal utilization, or to facilitate more practical inhalation conditions, it would be
preferred to use aerosol of a size corresponding to peak probability of deposition in the target tissue. As
illustrated in the Fig.17, (a) peak bronchial tree deposition fraction (of about 30%+) is obtained with
aerosol droplets size of about 6 microns, while (b) peak alveoli deposition (of about 30%+) is obtained
with aerosol droplets size of about 3 microns.
Standard mass-produced devices presently on the market have droplets of size 5 micron on average,
which would work reasonably fine. For 5 µm droplets, bronchial tree deposition fraction is about
30% and alveoli deposition fraction is about 25%.

Figure 17: Primary structures of the respiratory system and associated nanoparticle
deposition fractions at rest breathing [14].

Inhalation Timing Losses

Inhalation duration is only about 1/3 of the full breathing cycle (Fig.18). Therefore, if one is using an
aerosol source which is continuously active, only about 1/3 of the aerosolized dosage is really counted
for effective tissue delivery. This would be the common situation for home users when using standard
commercial medicament aerosol which are available for purchase in pharmacies. Of course, having a
breath actuated nebulizer (also available commercially) would correspondingly save on wasted silver
colloid solution.

Page 13 of 19
 Figure 18

The lungs comprise innate mechanisms to remove deposited particles. Hence, we need to examine
whether the nanoparticles resize long enough in the respiratory system surface tissue to exercise their
anti-viral potential. For indicative reference, we take as reference the behaviour of inhaled antibiotics
(tested in intensive care ventilated patients), as exemplified in Fig.19. We note that the inhalation
deposited antibiotics reside for an effective peak duration of about 2h in the lungs. The NAgC
experimental data that we analysed were commonly for 1h treatments (e.g., for the corona virus
presented in Fig.5). Hence, the experience with inhaled antibiotics give good confidence that there will
be sufficient time to our proposed NAgC inhalation to exercise its antiviral potency.

Figure 19: Comparison of lung concentration (measured by HPLC) and bacterial burden of
colistin between aerosolized and intravenous administration. Samples taken 1 hour after the
third aerosol in the aerosol group and the fourth infusion in the intravenous group and 49
hours after the bacterial inoculation. Diagram derived from data of Lu et al. [34,35]

Page 14 of 19
Clinical Safety
We are normally and daily exposed to silver intake via food and water. The human body has inherent
normal mechanisms for disposal of silver. Most foods contain traces of silver in the 10–100 μg/kg range.
The median daily intake of silver in a research from 84 self-selected diets, including drinking-water,
was 7.1 μg. Higher figures have been reported ranging from 20 to 80 μg of silver per day [16]. Therefore,
our recommended single treatment dosage of 1.1 μg deposition in the lungs is within the range of normal
dietary daily intake. The biological half-life in humans (liver) ranges from several days up to 50 days.
Most of absorbed silver is being excreted with the bile in the faeces [16]. Published scientific
experiments on colloidal silver antiviral and antibacterial properties, which also contain examination of
cell culture toxicity, consistently show non-toxicity at the relevant IC50 silver concentrations.
When addressing substance safety and/or toxicity, the professional literature distinguishes between
acute exposure of less than 14 days and prolong exposure of more than 14 days. In our context, it is
only the acute (<14 days) analysis which matters.
There are several occupational guidelines and exposure limits in the United States for airborne silver
dust. All are defined on a mass basis. OSHA71 has adopted the threshold limit value on a time-weighted
average (TLV-TWA) for a 40-hr/week exposure from the American Conference of Governmental
Industrial Hygienists (ACGIH) of 100 μg /m3 for metallic silver dust. Under normal breathing (500L/h
air breathing), this amounts to work environment (8 hours per day) inhalation of about 400μg daily of
silver nanoparticles. This is about 400 times our single dosage amount of 1.1μg nanosilver inhalation.
A 28-day inhalation safety trial with rats did not show any significant changes in body weight and no
significant changes in the hematology and blood biochemical values in either the male or female rats
under exposure to silver nanoparticles at a concentration near the ACGIH silver dust limit.
Most importantly, in the context of inhalation delivery, it has been shown that even after 90 days of
continuous exposure to high dosage of silver nanoparticles inhalation (total of 1,143μg silver per day),
cumulated tissue levels return to normal within about 12 weeks of recovery (See Fig.20). It is a very
strong indication of short-term inhalation exposure safety, in the sense of having no permanent
cumulation in body tissue.

Figure 20: Tissue silver concentrations in female rats exposed to the high AgNP dose
(381 μg/m3, silver nanoparticles ~15nm diameter, for 6 h/day, amounts to an inhalation
dosage 1,143 μg silver per day) in a 90 day inhalation study, followed by a 12 week
recovery period [17,18].

Page 15 of 19
Commercial Availability
There are many manufacturing methods of NAgC [28]. Some of the manufacturing methods require
little more than elementary lab equipment. From commercially available sources, and the simplicity of
the known techniques, we conclude that the price of a single treatment of 3.3ml dosage at 30 µg/ml
concentration should cost not more than $1. Mass production capabilities are essentially already
available. What is missing at the manufacturing level is primarily standardization of dosages. What is
missing at the commercial level is primarily the completion of clinical trials and regulation. We believe
this is a small challenge to conduct in comparison for the potential benefits to humanity.

Discussion
Though marred by charlatan claims of unprofessional “alternative medicine” commercial products,
there is well-established scientific research on the antibacterial and antiviral properties of colloidal
silver. Yet, its potential application for the treatment of respiratory infections was never properly
explored, to the best of our knowledge. The surveyed literature indicates that colloidal silver of particle
sizes between 3nm – 7nm can be highly effective to suppress viral mechanisms of infection. We further
conclude that IC50 concentrations of such colloids is about 10 µg/ml. Yet, these values we obtained by
making somewhat indirect inferences and therefore more focused research is called for. We estimate
that these formulations can be effective for prevention and treatment of respiratory viral infections at
early stages, including COVID-19 / SARS-CoV-2.
In order to set the scale of potential benefits, we flash out the remarkable results of an Influenza H3N2
in-vivo experiment done on mice by Xiang et al. (one of the very few in vivo animal experiments in the
NAgC literature), comparing the effectiveness of intranasal administration of NAgC to that of Tamiflu
(Oseltamivir) [27]. Roche, the makers of Tamiflu, claim that Tamiflu also reduces the number of
patients who have serious complications from the flu, such as pneumonia (by 44%) or hospitalization
(by 63%). A NAgC treatment, if as effective as in the mice model, could cost less than 1/10 fraction of
Tamiflu, can be available essentially OTC (as the market availability of NAgC already is), and easily
manufactured locally, and patent-free, in any country in the world. As shown in Fig.21 below (adapted
from [27]), it appears that potentially NAgC can be as effective as Tamiflu. But no investment in clinical
human trials was ever done to investigate the possibility. We can only speculate that this is due to the
lack of financial incentives for any conventional pharma company to make the required investment in
development and regulatory procedures. One of the main goals of this paper is to motivate and guide
the future clinical and regulatory development of NAgC treatment formulations.

Figure-21: In-vivo intranasal AgNP administration protected the mice from infection with
H3N2 IFV. (A) survival rate changes (%). (B) Changes in body weight (%) Adapted from
[27].

For bacterial infections, particularly in the context of ICU prevention of hospital ventilator associated
pneumonia (VAP), the same formulations can be applicable. An additional risk reduction benefit of
colloidal silver inhalation treatment for ventilated patients is the possibility of suppression of biofilm
formation inside the endotracheal or tracheostomy tube.

Page 16 of 19
References
1. Georg Behrens and Matthias Stoll, “Pathogenesis and Immunology”, in Influenza Report,
http://www.influenzareport.com/ir/pathogen.htm

2. Jeffery S. Garland, “Ventilator-Associated Pneumonia in Neonates: An Update”, NeoReviews

June 2014, 15 (6) e225-e235; DOI: https://doi.org/10.1542/neo.15-6-e225 .

3. Ma. Eugenia Manjarrez-Zavala, Dora Patricia Rosete-Olvera, Luis Horacio Gutiérrez-

González, Rodolfo Ocadiz-Delgado and Carlos Cabello-Gutiérrez (February 6th 2013).
“Pathogenesis of Viral Respiratory Infection, Respiratory Disease and Infection – A” New
Insight, Bassam H. Mahboub, IntechOpen, DOI: 10.5772/54287.

4. Aderibigbe BA. “Metal-Based Nanoparticles for the Treatment of Infectious Diseases”.

Molecules. 2017;22(8):1370. Published 2017 Aug 18.
https://doi.org/10.3390/molecules22081370 .

5. Galdiero, S.; Falanga, A.; Vitiello, M.; Cantisani, M.; Marra, V.; Galdiero, M. “Silver
Nanoparticles as Potential Antiviral Agents”. Molecules 2011, 16, 8894-8918.
https://doi.org/10.3390/molecules16108894.

6. Lara, Humberto H et al. “Mode of antiviral action of silver nanoparticles against HIV-1”
Journal of nanobiotechnology vol. 8 1. 20 Jan. 2010. https://doi.org/10.1186/1477-3155-8-1 .

7. K. Loza a, J. Diendorf a, C. Sengstock b, L. Ruiz-Gonzalez c, J. M. Gonzalez-Calbet c, M.

Vallet-Regi c, M. Köller b and M. Epple, “The dissolution and biological effects of silver
nanoparticles in biological media”, J. Mater. Chem. B, 2014, 2, 1634-1643. DOI:
10.1039/C3TB21569E.

8. Haggag EG, Elshamy AM, Rabeh MA, Gabr NM, Salem M, Youssif KA, Samir A, Bin
Muhsinah A, Alsayari A, Abdelmohsen UR. “Antiviral potential of green synthesized silver
nanoparticles of Lampranthus coccineus and Malephora lutea”. Int J Nanomedicine.
2019;14:6217-6229. https://doi.org/10.2147/IJN.S214171.

9. Elechiguerra, J.L., Burt, J.L., Morones, J.R. et al. Interaction of silver nanoparticles with HIV-
1. J Nanobiotechnol 3, 6 (2005). https://doi.org/10.1186/1477-3155-3-6.

10. Morris, Dorothea et al. “Antiviral and Immunomodulatory Activity of Silver Nanoparticles in
Experimental RSV Infection”. Viruses vol. 11,8 732. 8 Aug. 2019, doi:10.3390/v11080732.

11. Tareq Hussein, Shatha Suleiman Ali Saleh, Vanessa N. dos Santos, Brandon E. Boor, Antti J.
Koivisto, and Jakob Löndahl, “Regional Inhaled Deposited Dose of Urban Aerosols in an
Eastern Mediterranean City”, Atmosphere 2019, 10(9), 530;
https://doi.org/10.3390/atmos10090530

12. Garcia-Diaz, Maria & Birch, Ditlev & Wan, Feng & Nielsen, Hanne. (2017). “The role of mucus
as an invisible cloak to transepithelial drug delivery by nanoparticles”. Advanced Drug
Delivery Reviews. 124.10.1016/j.addr.2017.11.002.

13. Beule A. G. (2010). “Physiology and pathophysiology of respiratory mucosa of the nose and
the paranasal sinuses”. GMS current topics in otorhinolaryngology, head and neck surgery, 9,
Doc07. https://doi.org/10.3205/cto000071.

Page 17 of 19
14. Cheng Y. S. (2014). “Mechanisms of pharmaceutical aerosol deposition in the respiratory
tract”. AAPS PharmSciTech, 15(3), 630–640. https://doi.org/10.1208/s12249-014-0092-0.

15. Jung, W. K., Koo, H. C., Kim, K. W., Shin, S., Kim, S. H., & Park, Y. H. (2008). “Antibacterial
activity and mechanism of action of the silver ion in Staphylococcus aureus and Escherichia
coli”. Applied and environmental IC50robiology, 74(7), 2171–2178.
https://doi.org/10.1128/AEM.02001-07.

16. “Silver in Drinking-water, Background document for development of WHO Guidelines for
Drinking-water Quality”;
https://www.who.int/water_sanitation_health/dwq/cheIC50als/silver.pdf.

17. Fewtrell L. Silver: water disinfection and toxicity. Geneva: Centre for Research into
Environment and Health, World Health Organization; 2014.
http://www.who.int/water_sanitation_health/dwq/cheIC50als/Silver_water_disinfection_toxic
ity_2014V2.pdf

18. Song KS, Sung JH, Ji JH, Lee JH, Lee JS, Ryu HR, Lee JK, Chung YH, Park HM, Shin BS,
Chang HK, Kelman B, Yu IJ (2013), “Recovery from silver-nanoparticle-exposure-induced
lung inflammation and lung function changes in Sprague-Dawley rats”. Nanotoxicology 7,
169-189.

19. Martínez-Castañón, G.A., Niño-Martínez, N., Martínez-Gutierrez, F. et al. Synthesis and

antibacterial activity of silver nanoparticles with different sizes. J Nanopart Res 10, 1343–1348
(2008). https://doi.org/10.1007/s11051-008-9428-6.

20. Fröhlich, E., Mercuri, A., Wu, S., & Salar-Behzadi, S. (2016). Measurements of Deposition,
Lung Surface Area and Lung Fluid for Simulation of Inhaled Compounds. Frontiers in
pharmacology, 7, 181. https://doi.org/10.3389/fphar.2016.00181.

21. Hasan, MD Anwarul & Lange, Carlos. (2007). Estimating In Vivo Airway Surface Liquid
Concentration in Trials of Inhaled Antibiotics. J Aerosol Med. 20. 282-293.
10.1089/jam.2007.0603.

22. Vazquez-Muñoz, R., Arellano-Jimenez, M.J., Lopez, F.D. et al. “Protocol optimization for a
fast, simple and economical chemical reduction synthesis of antimicrobial silver nanoparticles
in non-specialized facilities”. BMC Res Notes 12, 773 (2019). https://doi.org/10.1186/s13104-
019-4813-z

23. Tien, Der & Tseng, Kuo-Hsiung & Liao, Chih-Yu & Tsung, Tsing-Tshih. (2009).
“Identification and quantification of ionic silver from colloidal silver prepared by electric spark
discharge system and its antiMicrobial potency study”. Journal of Alloys and Compounds. 473.
298-302. 10.1016/j.jallcom.2008.05.063.

24. Xiaonan Lv, Peng Wang, Ru Bai ,Yingying Conga ,Siqingaowa Suo, Xiaofeng Ren, Chunying
Chen, “Inhibitory effect of silver nanomaterials on transmissible virus-induced host cell
infections”, https://doi.org/10.1016/j.biomaterials.2014.01.054

25. Nakamura, S., Sato, M., Sato, Y., Ando, N., Takayama, T., Fujita, M., & Ishihara, M. (2019).
“Synthesis and Application of Silver Nanoparticles (Ag NPs) for the Prevention of Infection in
Healthcare Workers”, International journal of molecular sciences, 20(15), 3620.
https://doi.org/10.3390/ijms20153620 .

Page 18 of 19
 26. Seyyed Amir Siadati; Mohsen Afzali; Mehdi Sayyadi. "Could silver nano-particles control the
2019-nCoV virus?; An urgent glance to the past". Chemical Review and Letters, 3, 1, 2020, 9-
11. doi: 10.22034/crl.2020.224649.1044 .

27. Xiang D, Zheng Y, Duan W, Li X, Yin J, Shigdar S, O’Conner ML, Marappan M, Zhao X,
Miao Y, Xiang B, Zheng C., “Inhibition of A/Human/Hubei/3/2005 (H3N2) influenza virus
infection by silver nanoparticles in vitro and in vivo”. Int J Nanomedicine. 2013;8(1):4103-
4114, https://doi.org/10.2147/IJN.S53622 .

28. Quang Huy Tran et al, "Silver nanoparticles: synthesis, properties, toxicology, applications and
perspectives”, 2013 Adv. Nat. Sci: Nanosci. Nanotechnol. 4 033001.
https://doi.org/10.1088/2043-6262/4/3/033001 .

29. Balavandy, S.K., Shameli, K., Biak, D.R.B.A. et al. “Stirring time effect of silver nanoparticles
prepared in glutathione mediated by green method”. Chemistry Central Journal 8, 11 (2014).
https://doi.org/10.1186/1752-153X-8-11 .

30. Kyrychenko, Alexander et al. “Poly(vinyl alcohol) as a water protecting agent for silver
nanoparticles: the role of polymer size and structure”. Physical chemistry chemical physics:
PCCP 19 13 (2017): 8742-8756 . DOI:10.1039/c6cp05562a .

31. Antiviral efficacy test (ISO 18184): Nonwoven material for disposable face masks treated with
HeiQ Viroblock NPJ03 has been tested according to ISO 18184 standard.
https://heiq.com/technologies/heiq-viroblock/ .

32. Rogers, J.V., Parkinson, C.V., Choi, Y.W. et al. “A Preliminary Assessment of Silver
Nanoparticle Inhibition of Monkeypox Virus Plaque Formation”. Nanoscale Res Lett 3, 129
(2008). https://doi.org/10.1007/s11671-008-9128-2 .

33. Ji JH, et al., “Twenty-eight-day inhalation toxicity study of silver nanoparticles in Sprague-
Dawley rats”, Inhal Toxicol. 2007 Aug;19(10):857-71.
https://doi.org/10.1080/08958370701432108 .

34. Dhanani, J., Fraser, J.F., Chan, H. et al. “Fundamentals of aerosol therapy in critical care”.
Crit Care 20, 269 (2016). https://doi.org/10.1186/s13054-016-1448-5 .

35. Lu, Q., Girardi, C., Zhang, M. et al. Nebulized and intravenous colistin in experimental
pneumonia caused by Pseudomonas aeruginosa . Intensive Care Med 36, 1147–1155 (2010).
https://doi.org/10.1007/s00134-010-1879-4 .

36. Dugernier, J., Reychler, G., Wittebole, X. et al. “Aerosol delivery with two ventilation modes
during mechanical ventilation: a randomized study”. Ann. Intensive Care 6, 73 (2016).
https://doi.org/10.1186/s13613-016-0169-x .

37. Speshock, J.L., Murdock, R.C., Braydich-Stolle, L.K. et al. Interaction of silver nanoparticles
with Tacaribe virus. J Nanobiotechnol 8, 19 (2010). https://doi.org/10.1186/1477-3155-8-19 .

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