![(a) Image to be processed (b) Reference image. Source: Own authorship. All pixels of the image are traversed making a comparison of the gray level (scale that goes from 0 to 255 denoting the light intensity of an image, with 0 representing the completely black color and 255 the white color, with intermediate values representing different levels of gray) of the corresponding pixels in the image to be processed, and the reference [29], [30]. If the corresponding pixels of the processed and reference images are considered similar, that is, they present differences in the gray scale values within a pre-determined range (in the present processing, the pixels that have a difference in their level of gray of up to 25), the pixel is understood as the "background" of the image (that is, a region where there are no microbubbles) and in the processed image this pixel is defined as white (value equal to 255). However, if the corresponding pixels are considered different (gray level difference greater than 25), the pixel is understood to be part of a droplet, and then in the processed image the corresponding pixel is defined as black (value equal to 0). Equation 1 shows the applied logic:](https://www.researchgate.net/publication/343227669/figure/fig2/AS:11431281102791264@1669543548954/a-Image-to-be-processed-b-Reference-image-Source-Own-authorship-All-pixels-of-the_Q320.jpg){kind=tracking}
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ISSN (Online): 2350-0530 International Journal of Research -GRANTHAALAYAH
ISSN (Print): 2394-3629 July 2020, Vol 8(07), 80 – 97
DOI: https://doi.org/10.29121/granthaalayah.v8.i7.2020.420
© 2020 The Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution,
and reproduction in any medium, provided the or iginal author and source are credited. 80
NEBULIZERS: AERODYNAMIC DROPLET DIAMETER
CHARACTERIZATION AND PHYSICOCHEMICAL PROPERTIES OF
DRUGS TO TREAT SEVERE ACUTE RESPIRATORY SYNDROME
CORONA VIRUS 2 (SARS-COV-2)
Walter Duarte de Araújo Filho *1 , Luciana Martins Pereira de Araújo 2, Anderson
Silva de Oliveira 1, 3, Vagner Cardoso da Silva 4, Aníbal de Freitas Santos Júnior 4
*1Department of Exact and Earth Sciences, State University of Bahia, Salvador, Bahia, Brazil
2 Jorge Amado University Center (UNIJORGE), Salvador, Bahia, Brazil
3 Coordination of Surveillance, Investigation and Monitoring, Directorate of Sanitary and
Environmental Surveillance of the State of Bahia, Salvador, Bahia, Brazil
4 Department of Life Sciences, State University of Bahia, Salvador, Bahia, Brazil
DOI: https://doi.org/10.29121/granthaalayah.v8.i7.2020.420
Article Type: Research Article
Article Citation: Walter Duarte de
Araújo Filho, Luciana Martins
Pereira de Araújo, Anderson Silva de
Oliveira, Vagner Cardoso da Silva,
and Aníbal de Freitas Santos Júnior.
(2020). NEBULIZERS:
AERODYNAMIC DROPLET
DIAMETER CHARACTERIZATION
AND PHYSICOCHEMICAL
PROPERTIES OF DRUGS TO TREAT
SEVERE ACUTE RESPIRATORY
SYNDROME CORONA VIRUS 2
(SARS-COV-2). International Journal
of Research -GRANTHAALAYAH,
8(7), 80-97.
https://doi.org/10.29121/granthaa
layah.v8.i7.2020.420
Received Date: 02 June 2020
Accepted Date: 26 July 2020
Keywords:
Drugs
SARS-Cov-2
Nebulizers
Direct Laminar Incidence
Physicochemical Properties
ABSTRACT
Currently, several drugs are being used systemically to treat Severe
Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). However, few
studies discuss the possibility of using the inhalation route for this
treatment. Pneumatic and ultrasonic nebulizers are increasingly used due
to the ease with which these media deliver drugs through an aerosol
suspension to deliver drugs in a localized manner in the respiratory tract,
providing greater efficiency of absorption. This study aims to characterize
the droplet diameters by bands of "breathable particles" generated by
nebulizers commercialized in Brazil (2 pneumatic and 1 ultrasonic), using
the direct laminar incidence (DLI) technique. In addition, to discuss the use
of drugs by inhalation based on the physicochemical and pharmacology
properties. In the nebulization procedure, the images of the dispersed aero
droplets were captured using the DLI technique. Droplet diameter
distribution histograms were elaborated, emphasizing the range of
droplets with diameters between 1.0 to 5.0 µm. The results attested that
each nebulizer has its own characteristic of delivering the aerodynamic
suspension in the nebulization process. In this study, DLI represents a
viable alternative for characterization of the aero dispersed droplets, of
drugs used worldwide to treat SARS-CoV-2 signs and symptoms.
Nebulizers: Aerodynamic Droplet Diameter Characterization and Physicochemical Properties of Drugs to Treat Severe
Acute Respiratory Syndrome Corona Virus 2 (Sars-Cov-2)
International Journal of Research -GRANTHAALAYAH 81
1. INTRODUCTION
The interaction with the environment exposes human beings to several microorganisms, among which stand
out viruses. There are more than 300,000 viruses hosted in mammals and about 200 of them are known to infecting
humans [1], [2]. With the expansion of this contact, the increase in the frequency of emerging viral infections is
notorious, such as Ebola [3], Zika [4], Severe Acute Respiratory Syndrome (SARS) [5] and, recently, in 2019, in China,
the Coronavirus 2019-nCoV [6]. Thus, the search for new drugs and treatments is essential to avoid spread,
especially in the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).
Currently, several drugs are being used systemically (oral or parenteral) to treat emerging viral infections,
especially for SARS-CoV-2. However, there are no studies that directly discuss the possibility of using the inhalation
route for this treatment. Physicochemical properties of drugs, such as water solubility, acid dissociation constant
(pKa), oil/water partition coefficient, molecular mass, granulometry, surface contact area, among others, are
essential for the development of pharmaceutical forms dispersible [7]. For inhalation application, the drugs must be
efficient aerosolization and rapid dissolution of the drug crystals. [8].
In the last decades, aerosol therapy has achieved significant progress, which has determined that its use makes
it a faster, more effective and safer modality for the treatment of various respiratory diseases [9], [10]. This form of
administration allows the drug to arrive directly at the site of action, avoiding the need to use the digestive tract or
parenteral route. The administration of drugs by inhalation offers the following advantages: possibility to use
significantly less amount of drug; higher blood levels, with lesser side effects; and, faster action [11], [12].
In the respiratory tree, the greatest resistance occurs in the upper airways (lining of the mouth, tongue and
oropharynx) due to the tendency of this region to retain large particles. This becomes an obstacle when it is desired
that the particles carrying active drug penetrate the lower airways [13], [14]. The movement of particles is governed
by two types of factors: the intrinsic of the aerosol and, those inherent to the patient.
One of the intrinsic factors of aerosol is the aerodynamic behaviour of particles in the respiratory system,
governed mainly by size: The diameter of the particles is the most important factor in determining whether they can
enter the lower airway and reach the site of action [15], [16]. Very small particles (less than 1.0 µm) have low drug
transport capacity and a high probability of being exhaled into the environment without reaching the respiratory
epithelium. Very large particles (> 5.0 µm) have a high tendency to agglomerate and quickly impact the upper airway;
therefore, they are associated with the possibility of generating greater systemic effects. Particles of intermediate
size, 1.0 to 5.0 µm, are "ideal" because, through deposition, sedimentation and diffusion mechanisms, they can reach
the walls of the lower airways [17]. Particles with this characteristic constitute the "breathable mass" of an aerosol.
Therapeutic aerosols are "polydispersed", which means that the aerosol cloud is made up of particles of very
different sizes [18], [19]. As the dispersion is asymmetric (non-Gaussian), an aerosol can be characterized in terms
of a central measure of trend, the mass means aerodynamic diameter (MMAD) and a dispersion measure, the
geometric standard deviation (GSD). For the administration of particles in the peripheral pathways, the system that
provides an aerosol with the smallest MMAD and the narrowest GSD will be the most suitable.
In addition, other intrinsic aerosol factors are relevant, such as, input speed: the higher the speed, the greater
the likelihood of deposition in the upper airways; electrostatic charge: the influences of attraction and repulsion by
the electric charge can be between the particles, or between the particles and the airway walls; and, Hygrophilia: It
is the property of particles to increase in size when they are in an environment saturated with water, such as the
airway. It can be seen that due to the size, some types of drugs are "inefficient", because a large part is wasted on the
route not reaching the action site for the procedure to be effective [20]. It is noted that part of the dose inhaled by
the patient is divided into droplets that can be larger or smaller than 5.0 µm; for those larger than 5.0 µm that are
deposited in the upper airways, there is digestive absorption that culminates in systemic effects and renal excretion.
For droplets smaller than 5.0 µm there is a deposition in the lower airways leading to the expected therapeutic
effect. The proportion of particle distribution in the respiratory system depends on the type of generation method
and on the countless variables that participate in its performance characteristics [21]. Based on what was previously
treated, the size of the droplets generated in the nebulization process has a crucial role in the effective ness of the
drug's action during treatment [22]. The design of each nebulizer has specific characteristics that make them behave
differently in the nebulization process [23].
Reference techniques are used for characterizing droplet size, such as, Laser diffraction (LD), Cascade impactor
and Phase doppler anemometry (PDA). In Laser Diffraction (LD) technique, the particles present in the aerodynamic
Walter Duarte de Araújo Filho, Luciana Martins Pereira de Araújo, Anderson Silva de Oliveira, Vagner Cardoso da Silva,
and Aníbal de Freitas Santos Júnior
International Journal of Research -GRANTHAALAYAH 82
suspension absorb or disperse light incident according to its size, shape and refractive index. The scattered light is
subsequently collimated by the lenses of a Fourier transformer, and focused on a detector on the center axis
positioned at a distance from the lens equivalent to the focal length. The non-diffracted light is focused on the central
detector. In this way, a diffraction pattern of all contributing particles is recorded as a function of the dispersion
angle [24], [25].
The technique based on inertial behaviour, also known as Cascade Impactor (CI), also represents one of the
ways to measure the diameter of the droplets in suspension aerodynamics. This method is based on particle inertia
and is used to characterize aerosols through a device where the particles are separated by impact at various levels.
The current of air that entrains the particles is accelerated through a nozzle against a flat plate. The gas current lines
change abruptly, which does not happen with some particles, especially the larger ones, which, due to their high
inertia, precipitate in the dish. The passage of the particles through successive stages causes their deposition in
fractions with successively smaller diameters. In each floor mass fractions of particles are obtained with the same
inertia, that is, the same aerodynamic diameter, which, after mathematical manipulation, generated the size
distribution of the initial sample [26], [27].
PDA is one of the most accurate for the characterization of particle size in aerodynamic dispersion. In this
technique, several detectors positioned at different scattering angles are used to sample slightly different spatial
portions of the scattered light signal per particle. A system of two-phase shift detectors transmits information about
the particle diameter, shrinkage index and geometric shape characteristics. PDA is capable of characterizing particles
of wide dynamic size, from 0.3 to 8.0 µm, with an accuracy of 5% [28].
Thus, there is a problem to be answered: How to quantify the efficiency of nebulizers in delivering dispersed
aero droplets with diameters in the range between 1.0 and 5.0 µm (breathable droplets)? In search of an answer to
this question, DLI technique was used to characterize the population of droplets generated in the nebulization
process, emphasizing the region between 1.0 and 5.0 µm, which corresponds to the ideal range absorption of the
drug in the lower respiratory tract, which allows a better response to therapeutic treatment.
In the current pandemic scenario of the SARS-CoV-2, this paper presents a method to measure the diameter of
droplets generated by nebulizers in misting procedures by DLI technique. This paper also discusses the possibility
of using drugs by nebulizers, based on the physicochemical properties of drugs already used in clinical practice.
2. MATERIALS AND METHODS
NEBULIZERS AND DROPLETS IN AERODYNAMIC SUSPENSION
In the development of the work, three household nebulizers were randomly chosen, commercialized in the
Brazilian market, two pneumatic and one ultrasonic. They will be designated by the letters A, B and C. To quantify
the droplet size, a Stage Micrometer (Edmund Optics. 30-101, USA) was used, attached to the eyepiece of the Axiovert
A1 tri-eyed microscope (Carl Zeiss, Jena, Germany). The images were obtained using a high-speed digital camera
(HiSpec 47; Fastec Imaging) with a resolution of 1696 x 1710 pixels, at an acquisition rate of 200 fps.
A 0.9% saline solution was used to constitute the aqueous vehicle of the nebulizers, with an estimated viscosity
of 0.90 mPa. The images were obtained by directly focusing the aerosol generated on a glass slide coupled to the
microscope at a fixed distance of 0.1, at a temperature of 22o C, atmospheric pressure of 102.1 kPa, and with relative
humidity of the air around 68%.
The images of the droplets close to the slide were captured and stored in a memory unit. They were acquired
at a rate of 200 (fps) with a total acquisition time of 2 s, totalling 400 images for each researched nebulizer by LDI
technique. Figure 1 presents an image obtained from a population of droplets flush with the slide, excluding the
droplets condemned on its surface. The image was acquired with a 200X increase, an acquisition rate of 200 fps, a
temperature of 22o C, an atmospheric pressure of 102.1 kPa and a relative humidity of around 68%.
Nebulizers: Aerodynamic Droplet Diameter Characterization and Physicochemical Properties of Drugs to Treat Severe
Acute Respiratory Syndrome Corona Virus 2 (Sars-Cov-2)
International Journal of Research -GRANTHAALAYAH 83
Figure 1: Image of droplets in aerodynamic suspension acquired by the experimental device. Source: Own
authorship.
To quantify the size of the droplets, the images were processed using a protocol established in MATLAB®
version 2015b.
IMAGE PROCESSING USED IN THE ACQUISITION OF EXPERIMENTAL DATA
The first step in processing was to segment the images. The objective was to try to differentiate all the regions
that represent the droplets from the rest of the image. This segmentation is done by comparing the image to be
processed with a reference image (image from the same region filmed during the experiment, but without the
presence of droplets). An example of a real image (a) next to a reference image (b) can be seen in Figure 2.
Figure 2: (a) Image to be processed (b) Reference image. Source: Own authorship.
All pixels of the image are traversed making a comparison of the gray level (scale that goes from 0 to 255
denoting the light intensity of an image, with 0 representing the completely black color and 255 the white color, with
intermediate values representing different levels of gray) of the corresponding pixels in the image to be processed,
and the reference [29], [30]. If the corresponding pixels of the processed and reference images are considered
similar, that is, they present differences in the gray scale values within a pre-determined range (in the present
processing, the pixels that have a difference in their level of gray of up to 25), the pixel is understood as the
“background” of the image (that is, a region where there are no microbubbles) and in the processed image this pixel
is defined as white (value equal to 255). However, if the corresponding pixels are considered different (gray level
difference greater than 25), the pixel is understood to be part of a droplet, and then in the processed image the
corresponding pixel is defined as black (value equal to 0). Equation 1 shows the applied logic:
()=�255(−)<25
0 (−)≥25 (Equation 1)
Walter Duarte de Araújo Filho, Luciana Martins Pereira de Araújo, Anderson Silva de Oliveira, Vagner Cardoso da Silva,
and Aníbal de Freitas Santos Júnior
International Journal of Research -GRANTHAALAYAH 84
Figure 3 presents an example of the original image (a) together with the image obtained after applying the
described technique (b). This initial processing provides an image in which the background is white and the droplets
are black (binary image, only with values 255 or 0). However, until this stage, normally only the droplet contour
regions were identified, leaving a small region in the center still identified as background. In this step, all regions
that are surrounded by black (regions that are theoretically in the middle of the interfaces found in a droplet, and
therefore are also part of the droplet) they are also filled with black. This procedure is performed using region filling
using the “in-fill” function, a function of the MATLAB® image processing library, which uses an algorithm based on
morphological reconstruction [30]. With this, an image is obtained in which the droplets are all represented in black
and the background of the image all in white. In Figure 3 (c), the image processed in the previous step can be seen,
together with the image after applying the “infill” function [31]. In the next step, each of the black regions in the
image is identified separately (with values 0), which represent each of the droplets. The feature extraction step,
involving the calculation of the area, in pixels, of each of these regions, is performed with the aid of the “regionprops”
function in MATLAB®. The different regions considered as individual droplets are shown in Figure 3 (d), with each
droplet represented by a different color. Each droplet identified has its diameter in pixels (DP) determined, based
on the area in pixels (AP) it occupies (Equation 2).
Figure 3: (a) Original image; (b) Image obtained after applying the segmentation technique used; (c) Image
obtained after the application of the “infill” function and (d) Different regions of different colours are considered
individual droplets. Source: Own authorship.
(Equation 2)
Areas considered to be very small are disregarded as they represent possible noise from image processing. An
example with images before and after eliminating possible noise is shown in Figure 4.
Figure 4: (a) Image before eliminating possible noise (b) Image after eliminating possible noise. Source: Own
authorship.
The diameters found so far are still in pixels as a unit, so it becomes necessary to convert them to a unit of
suitable length for analysis (micrometres, for example). This is done through a reference measurement of the image
Nebulizers: Aerodynamic Droplet Diameter Characterization and Physicochemical Properties of Drugs to Treat Severe
Acute Respiratory Syndrome Corona Virus 2 (Sars-Cov-2)
International Journal of Research -GRANTHAALAYAH 85
using the Stage Micrometer. A real length taken as standard in the image is then measured in pixels, which allows
the transformation to be made for all calculated diameters [31]. At this point in the processing, all droplet diameters
identified in the image are distributed. What is done next is to create histograms (with the “hist” function) to
represent the distribution of the diameters found in the image in the desired way, as shown in Figure 5. The process
is then repeated for all images, creating a histogram of diameters for each image. Finally, an additional histogram is
created considering now all the droplets found in all the images.
Figure 5: Histogram representing the relative frequency as a function of the droplet diameter range. Source:
Own authorship.
PHYSICOCHEMICAL AND PHARMACOLOGICAL PROPERTIES OF DRUGS
Physicochemical properties of drugs currently used to treat SARS-CoV-2 signs and symptoms, such as water
solubility, acid dissociation constant (pKa), oil/water partition coefficient, molecular mass, granulometry, surface
contact area, among others, were compiled into databases PubChem [32] and Drug Bank [33]. Data on physical and
chemical constants and data on pharmacokinetics and pharmacodynamics were extracted. In addition, the Brazilian
Pharmacopoeia [34] and Brazilian laws were consulted which provide for in vitro performance tests and tests to
prove therapeutic equivalence for oral inhaled drugs and nasal sprays and aerosols [35], [36].
3. RESULTS AND DISCUSSIONS
The three nebulizers surveyed use the value of the Average Aerodynamic Mass Diameter (MMAD) present in
the manuals as a standard for characterizing the droplets. This variable represents the average value of the
aerodynamic size of the droplets generated by each of the researched nebulizers, since it takes into account the size
of the mass diameter of each drop.
In the development of this work, Median Aerodynamic Diameter Count (CMAD) was used to characterize the
size of the aerodynamic dispersion droplets produced by the nebulizers studied here. This parameter represents the
median of the droplets, i.e., it characterizes the amount of 50% of the droplets that are above the average and the
50% of the droplets that are below the average. For each of the surveyed nebulizers, a statistical analysis was made
of the number of droplets generated as a function of the diameter, and a characteristic histogram related to the
droplet diameter was elaborated, emphasizing the range between 1.0 to 5.0 µm which corresponds to the ideal range.
Table 1 presents the information about the diameter of the droplets conveyed in the factory manuals, percentage of
the droplets located in the ideal range, and characteristic average diameter of the droplets in suspension for each
nebulizer (sample) using the parameter (CMAD) using the technique DLI.
Walter Duarte de Araújo Filho, Luciana Martins Pereira de Araújo, Anderson Silva de Oliveira, Vagner Cardoso da Silva,
and Aníbal de Freitas Santos Júnior
International Journal of Research -GRANTHAALAYAH 86
Table 1: Information on the diameter of the droplets provided by the manuals accompanying the equipment;
Percentage of droplets in the ideal range; Characteristic average diameter of the droplets in suspension for each
nebulizer (sample) using the parameter (CMAD) using the DLI technique. Source: Own authorship.
Nebulizer
Instruction
manual
(µm)
Percentage of particles in the ideal range
(%)
CMAD parameter using the DLI technique
(µm)
A
4.0
43.95
5.5
B
<5.0
34.48
5.9
C
0.5-10
58.46
4.5
It can be seen in Table 1 that the information conveyed in the manufacturer’s manuals of the researched
nebulizers, brings only the values of the MMAD parameter as a reference, without any information on the
methodology used in the determination of that value. In contrast, the table shows the droplet categorization by
diameter ranges using the CMAD parameter showing quantitatively the population of droplets located in the ideal
range 1.0 to 5.0 µm.
HISTOGRAMS OF NEBULIZERS
Figures 6 to 8 show the histograms of the distribution of the droplets produced by nebulizers A, B and C, from
the recording of images captured in the nebulization process in 2.0 s. In each of the histograms, the corresponding
range of breathable particles (green bands) is emphasized, that is, droplets with diameters between 1.0 to 5.0 µm.
Figure 6: Histogram expressing a range of breathable particles from nebulizer A.
Figure 7: Histogram expressing a range of breathable particles from nebulizer B.
Nebulizers: Aerodynamic Droplet Diameter Characterization and Physicochemical Properties of Drugs to Treat Severe
Acute Respiratory Syndrome Corona Virus 2 (Sars-Cov-2)
International Journal of Research -GRANTHAALAYAH 87
Figure 8: Histogram expressing a range of breathable particles from nebulizer C.
Analysing the histograms, it is possible to perceive the polydisperse character of the droplets generated by the
nebulizers taken as a sample, and that each equipment has its own characteristic of droplet production in the ideal
range (1.0 to 5.0 µm). This represents an important indication for the choice of equipment, since as it has already
been treated it is in this range of diameters that a better response to treatment occurs due to a greater effective
absorption of the drug by the pulmonary alveoli. The analysis of pure and simple histograms can represent an initial
way of characterizing nebulizers based on the ability of each one to deliver droplets in the ideal range.
FREQUENCY DISTRIBUTIONS OF THE DIAMETER OF THE DROPS GENERATED BY THE NEBULIZERS
The histogram with the frequency densities is useful in cases where you want to visualize the fit of the
distribution density curves. Each frequency density value (on the vertical axis) is equal to the absolute frequency of
the values in the corresponding class (on the horizontal axis), divided by the length of this class. Normal Distribution
is widely used in numerous applications with experiments and physical phenomena having symmetry properties
around the mean. Although its theoretical model allows positive and negative values of the diameter of droplets, this
distribution can be used in practice because low values of standard deviation result in almost zero probabilities of
obtaining negative values. The Log Normal, Gamma and Normal Inverse distributions only allow positive values, so
there is an interest in using these distributions in experiments that result in only positive values (such as droplet
diameters). Another parameter is the Akaike Information Criterion (AIC) criterion of goodness of fit of a set of values
to a specific probability distribution: the lower the value of AIC, the better the fit of the model. This criterion verifies
the goodness of the adjustment taking into account the number of parameters in the model.
Figures 9 to 11 show the histograms with the frequency densities, associated with the frequency distribution
curves: Normal, Log-Normal, Gamma and Inverse Normal for each of the researched nebulizers. These data are of
great importance for the characterization of the behaviour of each nebulizer, in addition to the calculation of the
important AIC parameter that quantitatively characterizes the correlation between the data.
Figure 9: Histogram with the density curves adjusted for nebulizer A.
Walter Duarte de Araújo Filho, Luciana Martins Pereira de Araújo, Anderson Silva de Oliveira, Vagner Cardoso da Silva,
and Aníbal de Freitas Santos Júnior
International Journal of Research -GRANTHAALAYAH 88
Analysing the graph represented by Figure 9, it can be concluded that the predominance of frequency density
in the region between 2.0 and 5.0 µm, is relevant for nebulizer A. In this range there is a large delivery of breathable
droplets i.e., (1.0 to 5.0 µm). It turns out that the Inverse Nor-mal density and Log Normal density curves are almost
identical they better capture the modal class of the microbubble diameters (from 3 to 4 µm).
Figure 10: Histogram with the density curves adjusted for nebulizer B.
Analysing the graph represented by Figure 10, it is observed that there is a greater concentration of the
frequency density distribution in the region between 3.0 and 9.0 µm, with a peak between 4.0 to 5.0 µm for the
nebulizer B. It is verified that the Inverse Normal and Log Normal density curves are almost identical, they better
capture the modal class of the micro-bubble diameters from 4.0 to 5.0 µm. The graph represented by Figure 45 shows
the behaviour of the F nebulizer according to the tested distributions.
Figure 11: Histogram with the density curves adjusted for nebulizer C.
Analysing the graph represented by Figure 11, a peak be-tween 3.0 and 4.0 µm is observed for the F nebulizer.
It can be seen that the Inverse Normal and Log Normal density curves are almost identical - they better capture the
class modal of the microbubble diameters (from 3.0 to 4.0 µm), in addition to adjusting better to values close to 1.0
µm (to the left of the graph). From the aspects presented of the adjusted distributions, in above figures, we can verify
a summary of the statistical parameters for each nebulizer studied. This analysis shows a predominance of the Log
Normal distribution, according to the literature on the distribution that best fits the micro droplet size data. Table 2
shows the Mean, Median, Standard Deviation, and Adjusted Distribution of the data.
Nebulizers: Aerodynamic Droplet Diameter Characterization and Physicochemical Properties of Drugs to Treat Severe
Acute Respiratory Syndrome Corona Virus 2 (Sars-Cov-2)
International Journal of Research -GRANTHAALAYAH 89
Table 2: Descriptive statistics with adjusted distributions and Akaike criteria (AIC), in addition to the values
relative to Mean, Median, and Standard Deviation.
Nebulizer
Mean
Median
Standard deviation (σ)
Adjusted Distribution
A
6.43
5.51
3.17
Log Normal
B
6.94
5.95
3.09
Log Normal
C
4.88
4.51
2.15
Log Normal
THE DIRECT LAMINAR INCIDENCE (DLI) TECHNIQUE IN THE CHARACTERIZATION OF AERO
DISPERSED DROPS
Microscopy is considered an absolute method of analysing particle size, since it is the method in which
individual particles are observed and measured [19]. The other analysis techniques such as Laser diffraction (LD),
Cascade Impactor (CI), and Phase Doppler Anemometry (PDA) as previously described are calibrated using optical
microscopes and high intensity lasers, which characterize indirect measurements [26]. This finding suggests that
optical microscopy is more suitable when working with dispersed aero particles with diameters between 0.8 to
150.0 µm depending on the wavelength of the light source, although a practical lower limit of 3.0 µm it is frequently
cited [19].
Thus, digital processing of aerosol microscopy images produced by nebulizers can provide reliable results.
Based on this information, the microscopic measurement technique was applied as a direct form of measurement
associated with image processing, which translates to the DLI technique. Analysing the experimental results, it was
found that:
• The histograms presented together with the third column of Table 1, show that each nebulizer has a
different behaviour in relation to the delivery of droplets in the ideal region, besides presenting different
values of the parameter CMAD. This may be associated with the equipment design or technical
anomalies liable to be circumvented by the manufacturers.
• The DLI technique represents a viable alternative for the characterization of aero dispersed droplets,
since its operation does not require sophisticated and expensive equipment, which makes it quite
competitive in relation to other measurement techniques.
• The difference in behaviour of each nebulizer in delivering droplets in the ideal range, signals that the
choice of a certain equipment may present an advantage over the other with regard to the therapeutic
response of the treatment, since this response depends on the proportion of droplets in the breathable
range, i.e., between 1.0 to 5.0 µm (where the greatest pulmonary deposition).
• The frequency density distribution of the droplet diameter associated with each of their searched
nebulizers, shows the high degree of dispersion of the aerodynamic droplet population. This fact is
confirmed by the high values of the standard deviations (σ) associated with each distribution. The
proposed methodology with the characterization of the droplets generated by size ranges, introduces a
way to evaluate the behaviour of the nebulizers with reference to the ideal region, i.e., 1.0 to 5.0 µm.
• Table 2 shows the behaviour of nebulizers in relation to the statistical data Average, Median, Mode,
Standard Deviation, and Adjusted Distribution. It can be seen that there is a predominance of the Log
Normal distribution as described in the literature that involves the study of dispersed aero droplets.
PHYSICOCHEMICAL AND PHARMACOLOGICAL PROPERTIES OF DRUGS, WITH POTENTIALS USE BY
NEBULIZATION, TO TREAT SARS-CoV-2 SIGNS AND SYMPTOMS
Inhaled drug administration requires attention on application as well as on administration. The
physicochemical properties of the formulation and the interactions with the nebulizer were relevant factors, as they
can directly affect the properties of the aerosol and, consequently, the bioavailability of the drug and the
effectiveness after nebulization [37]. The administration of drug for inhalation is typically associated with high
pulmonary efficacy and minimal systemic side effects and, pharmacokinetic processes, such as, drug particle/droplet
deposition; pulmonary drug dissolution; absorption to lung tissue; pulmonary tissue retention and tissue
metabolism; and, absorptive drug clearance to the systemic perfusion are clinically relevant [38].
Walter Duarte de Araújo Filho, Luciana Martins Pereira de Araújo, Anderson Silva de Oliveira, Vagner Cardoso da Silva,
and Aníbal de Freitas Santos Júnior
International Journal of Research -GRANTHAALAYAH 90
One of the proposals of this article is to analysis of the potential of drugs, based on physical-chemical and
pharmacological properties, for inhaled administration, as an aid to the multidisciplinary health team, in the
treatment of respiratory tract diseases. Tables 3 and 4 show, respectively, the main physical-chemical and
pharmacological properties of drugs used in the treatment of respiratory tract diseases, which are currently being
investigated to treat SARS-CoV-2 signs and symptoms, worldwide.
So far, there are no vaccines and drugs for the treatment of Coronavirus, however, several countries have been
making efforts to create clinical protocols that minimize the damage caused in physiological systems, in human
organisms, especially cardiovascular and respiratory damage [39]. Various therapeutic alternatives have been used
to treat SARS-CoV-2, among which antimicrobials (Azithromycin and Ciprofloxacin), antimalarials (Chloroquine and
Hydroxychloroquine), Antiparasitic (Ivermectin and Nitazoxanide), antirheumatics (Hydroxychloroquine and
Tocilizumab), anticoagulants (Enoxaparin and unfractionated Heparin), Non-steroidal anti-inflammatory - NSAID
(ibuprofen), Steroidal anti-inflammatory (Prednisolone and Methylprednisolone), antiretrovirals (Lopinavir and
Ritonavir), antivirals (Nitazoxanide, Oseltamivir, Remdesivir and Ribavirin). However, often, such drugs are only
available in solid (powder, tablet and capsule), semi-solid (suspension) and, some of them, liquid (injectable and
ophthalmic) solutions. In the current scenario of the pandemic caused by the Coronavirus, the off-label use of these
drugs can be a viable alternative for administration and therapeutic success [40], [41]. In addition, the
pharmacotechnicals development of new pharmaceutical formulations is a relevant tool, as it encompasses the study
of aspects related to chemical substance (drug), pharmaceutical form and industrial technology [42].
The main properties and parameters for pharmaceutical formulations useful for administration via nebulization
to be considered when designing an effective inhaler are: aerosol properties (mass median aerodynamic diameter;
geometric standard deviation; fine particle fraction and, air/particle velocity); particle properties (volume diameter;
bulk density; tap density; shape and, charge); physicochemical properties (solubility and hygroscopicity); and, lung
properties (geometry of respiratory tree - airway structure and diameter of airways; influence of disease state on
airway structure; breathing pattern – mouth or nasal breathing) [8]. The data obtained showing that the drugs
enoxaparin, oseltamivir, ribavirin is the most easily soluble in water. Azithromycin, ibuprofen, prednisolone and
methylprednisolone, also show partial solubility in water. Such information about solubility can facilitate its use by
inhalation. However, studies of technological and biopharmaceutical feasibility are necessary to obtain stable and
effective formulations.
Regarding the molecular mass (MM), a range of 307.28 to 748.98 g/mol (Daltons) is observed for the evaluated
drugs. Enoxaparin has a molecular mass between 3,800 to 5,000 Daltons. In this context, the lower the molecular
mass, the transport of the molecule through the respiratory tract can be accelerated, reaching the bronchial tree with
greater speed and, therefore, being more available for absorption. Ibuprofen, ribavirin and nitozoxamide have MM
lower than 300 g/moL, which strengthen the possibility of using this route. However, several other physicochemical
parameters must be analysed for potential pharmaceutical formulations, suitable for this purpose.
Oil/water partition coefficient (OAPC) is used in drug design as a measure of a solute’s hydrophobicity and a
proxy for its membrane permeability [43]. In pharmaceutical industry, to estimate how a drug may transfer between
different biological environments and are regularly used to predict a molecule’s hydrophobicity [44]. Lower OAPC,
less the solubility, in fats, of chemical substances. Drugs such as ciprofloxacin, enoxaparin, prednisolone,
methylprednisolone, nitazoxamide, oseltamivir, remdesevir and ribavirin have a partition strength of less than 3
and, this can strengthen its use by inhalation, carried in 0.9% saline solution. A pharmacotechnical alternative for
the formulation of drugs for use by inhalation would be the incorporation into liposomes. However, some drugs,
when diluted in a larger volume, either in a nebulization chamber or even in the lung, such as corticosteroids in
liposomes, tend to lose their content in a biphasic pattern determined by their partition coefficient [45].
All the drugs studied had a surface area of less than 250 angstrom, that is, between 0.003 and 0.02 µ. In this way,
drugs used worldwide to treat SARS-CoV-2 signs and symptoms could be transported by nebulization. In this study,
DLI technique represents a viable alternative for the characterization of aero dispersed droplets, for administration
of these drugs. However, according to data in table 4, some drugs have bioavailability below 40%, with lopinavir
(25%) and azithromycin (37%) standing out. In addition, aspects of safety in the use of drugs should be evaluated.
Among the main adverse effects are Cardiotoxicity (azithromycin, ciprofloxacin, chloroquine and
hydroxychloroquine) and toxic to the respiratory tract (ibuprofen, lopinavir, remdesevir, ritonavir and toclizumab).
The drugs nitazoxamide and oseltamivir are the least toxic and, therefore, may be potential drugs to be
administered by nebulization. In the literature, some studies indicate the inhaled use of Azithromycin [7],
Nebulizers: Aerodynamic Droplet Diameter Characterization and Physicochemical Properties of Drugs to Treat Severe
Acute Respiratory Syndrome Corona Virus 2 (Sars-Cov-2)
International Journal of Research -GRANTHAALAYAH 91
Ciprofloxacin in the treatment of non-cystic fibrosis bronchiectasis [46], Enoxaparin [47], Prednisolone [48],
Ribavirin in Chronic Obstructive Pulmonary Disease [49] and Oseltamivir as influenza prophylaxis [50], [51].
Nitazoxanide is related, in literature, as a new drug candidate for the treatment of Middle East respiratory syndrome
coronavirus [52]. For treatment of SARS-CoV-2 signs and symptoms, the literature indicated the
Hydroxychloroquine as an aerosol might markedly reduce and even prevent severe clinical symptoms infection [53].
In addition, benefit of the Remdesivir for treatment of Coronavirus symptoms, by combination of pulmonary and
intravenous administration were studied [54].
In this context, the possibility of innovative treatments for SARS-CoV-2 signs and symptoms is a real necessity
and the inhalation route is a potential alternative. Therefore, it is relevant to know the physicochemical properties
of drugs already used in clinical practice, which may have the possibility of administration by nebulization.
Table 3: Physic-chemical properties of drugs used in diseases of the respiratory tract and, currently being
investigated to treat SARS-CoV-2 signs and symptoms [32], [33], [34].
Drug
Pharmaceutical
forms available
in Brazil
Solubility
Water
Solubilit
y
(mg/L, at
25 °C)
Molecula
r mass
(Daltons)
Oil /
water
partition
coefficien
t
Area
superficia
l (Å)
pKa
Azithromycin
tablets, oral
suspension and
solution for
injection
soluble in ethanol
and Dimethyl
sulfoxide
(DSMO)/minimall
y soluble in water
2.37
748.98
3.03
180.08
8.5
Ciprofloxacin
tablets,
solution for
injection and
ophthalmic
solution
Practically
insoluble in water.
Soluble in dilute
acetic acid, very
poorly soluble in
ethyl alcohol.
< 1 x 10
-3
331.34
0.28
72.88
6.09
(carboxyli
c acid
group);
8.74
(nitrogen,
in the
piperaziny
l ring)
Chloroquine
tablets
(diphosphate)
and solution
for injection
(hydrochloride
)
Very little soluble
in water. Soluble
in diluted acids.
0.14
319.87
4.63
28.16
10.1
Hydroxychloroquin
e
tablets
NR
2.61 x
10
-4
434.0
3.6
48.4
9.67
Enoxaparin
solution for
injection
(subcutaneous)
soluble in water,
alcohol, ether,
acetone,
chloroform and
benzene.
Hygroscopic
powder.
> 0.2
3800 –
5000
-13.2
NR
NR
Ibuprofen
tablets and oral
suspension
Practically
insoluble in water,
easily soluble in
ethyl alcohol and
alcohol methyl.
Soluble in aqueous
solutions of
21
206.29
3.97
37.3
5.3
Walter Duarte de Araújo Filho, Luciana Martins Pereira de Araújo, Anderson Silva de Oliveira, Vagner Cardoso da Silva,
and Aníbal de Freitas Santos Júnior
International Journal of Research -GRANTHAALAYAH 92
alkaline
hydroxides.
Ivermectin
tablets
insoluble in water
insoluble
875.1
4.1
170.06
12.47
Lopinavir
tablets
Practically
insoluble in water,
soluble in
methanol and
ethanol; soluble in
isopropanol
7.7 x 10
-3
628.8
5.94
120
13.39
Prednisolone
solution for
injection and
oral suspension
Moderately
soluble in dioxane,
methanol; slightly
soluble in
methanol, acetone,
chloroform
223
360.4
1.6
94.8
NR
Methylprednisolone
solution for
injection
Moderately
soluble in dioxane,
methanol; slightly
soluble in acetone,
chloroform; very
slightly soluble in
ether.
120
374.5
1,525
94.83
12.59
Nitazoxanide
tablets and oral
suspension
NR
0.00018
9
307.28
1.63
142
8.3
Oseltamivir
capsules and
powder for oral
suspension
soluble in water
6.86 x
10-4
312.4
1.1
90.65
7.7
Remdesivir
solution for
injection
NR
3,39 x
10
-4
602.6
2.2
203.55
10.23
Ritonavir
tablets and oral
solution
Freely soluble in
methanol and
ethanol; soluble in
isopropanol.
1.26 x
10-4
720.9
3.9
145.78
13.68
Ribavirin
tablets and
suspension for
injection
(subcutaneous)
soluble in water
and slightly
soluble in alcohol
≥ 0.1
244.2
-1.9
143.72
11.88
Table 4: Pharmacological properties of drugs used in diseases of the respiratory tract and, currently being
investigated to treat SARS-CoV-2 signs and symptoms [32], [33], [34].
Drug
Therapeutic class
Pharmacokinetic processes
Main adverse
effects
Absorption /
Bioavailability
Distribution
Metabolism
Elimination
Azithromycin
Antibiotic
(macrolide)
Not affected
by food;
OB: 37%
Widely
distributed
in tissues;
VD: 31.1
L/Kg; T1/2:
approx. 68
hours; BPP:
7-51 %
Hepatic
(CYP 3A4)
Bile (main
route) -
drug
unaltered)
/ Urine
(6%)
Thrombocytopenia,
arrhythmias
(ventricular
tachycardia)
Ciprofloxacin
Antibiotic
(fluoroquinolone)
OB: 70-80 %
VD: 2.00-
3.04 L/Kg;
T1/2: 4
hours; BPP:
20-40%
Hepatic
(CYP 1A2)
Urine
(45%) and
feces (62%)
Tachycardia,
dyspnoea,
thrombocytopenia
Nebulizers: Aerodynamic Droplet Diameter Characterization and Physicochemical Properties of Drugs to Treat Severe
Acute Respiratory Syndrome Corona Virus 2 (Sars-Cov-2)
International Journal of Research -GRANTHAALAYAH 93
Chloroquine
Antimalarial
OB: 67-114 %
VD: 200-
800 L/Kg;
T1/2: 20-60
days; BPP:
46-74%
Hepatic (CYP
2C8, CYP 3A4,
CYP 3A5, CYP
2D6 and CYP
1A1)
Urine (50%
unaltered)
Cardiotoxicity
(atrioventricular
block,
cardiomyopathies)
Hydroxychloroquine
Antimalarial and
Antirheumatic
OB: 67-74%
VD: 5,522
L/Kg
(blood);
44,257 L/Kg
(plasma);
T1/2: 537
hours
(blood);
2,963 hours
(plasma);
BPP: 50%
Hepatic
(CYP 3A4)
Renal (40-
50%);
urine (16-
21%) / skin
(5%) and
feces (24-
25%)
Cardiomyopathy,
prolongation of QT
interval, emotional
lability
Enoxaparin
Anticoagulant
100 %
VD: 4.3
L/Kg); T1/2:
4.5 hours;
BPP: 80 %
Hepatic
(desulfation
and
polymerization)
Urine (40%
of the dose)
Haemorrhages,
thrombocytopenia,
haematuria
Ibuprofen
Non-steroidal
anti-
inflammatory
OB: 80-90 %
VD: 0.1
L/Kg; T1/2:
1.2 - 2
hours; BPP:
99 %
Hepatic
(CYP 2C9)
Urine
(90%)
Bronchospasm,
CHF,
gastrointestinal
disorders,
hypertension,
anaphylaxis
Ivermectin
Antiparasitic
absorption
moderate,
especially
with a
diet rich in
fats
VD: 3.5
L/Kg; T1/2:
16 hours;
BPP: 93 %
Hepatic
Feces
Diarrhea, nausea,
urticaria,
onchocerciasis,
hypotension
Lopinavir
Antiretroviral
OB: 25%
VD: 16.9
L/Kg; T1/2:
6.9 ± 2.2
hours; BPP:
> 98 %
Hepatic
(CYP 3A4)
Feces
SRT infections,
diarrhea, anemia,
angioedema
Prednisolone
Steroidal anti-
inflammatory
70 %
VD: 29.3
L/Kg; T1/2:
2.1-3.5
hours); BPP:
70-90 %
Hepatic
Urine (98
%)
CHF, arrhythmias,
leukocytosis,
cushing's
syndrome,
hypopituitarism
Methylprednisolone
(MP)
Steroidal anti-
inflammatory
OB: MP
acetate
(89.9%) and
rectal
(14.2%)
VD: 1.38 L /
Kg; T1/2: 2.3
hours; BPP:
76.8 %
Hepatic
Urine
Nitazoxanide
Antihelmintic and
Antiviral
70 %
(suspension)
VD: not
found; T1/2:
7.3 hours;
BPP: > 99)
Hepatic
Urine, bile
and feces.
abdominal colic,
diarrhea, headache
Oseltamivir
Antiviral
OB: 75 %
VD: 23-26
L/Kg; T1/2:
1-3 hours);
BPP: 42 %
Hepatic
Renal (90
%).
Nausea, vomiting,
headache and body
pains
Walter Duarte de Araújo Filho, Luciana Martins Pereira de Araújo, Anderson Silva de Oliveira, Vagner Cardoso da Silva,
and Aníbal de Freitas Santos Júnior
International Journal of Research -GRANTHAALAYAH 94
Remdesivir
Antiviral
NR
VD: NR;
T1/2: 20
hours
(metabolite)
; BPP: NR
NR
Urine (74
%) and
feces (18
%)
RF, hypotension
Ritonavir
Antiretroviral
NR
VD: 0.41 ±
0.25 L/Kg;
T1/2: 1-3
hours; BPP:
> 98 %
Hepatic
(CYP3A4 and
CYP2D6)
Feces (86
%).
IRT, cellulite,
folliculitis and
furunculosis
Ribavirin
Antiviral
OB: 45-64 %
VD: NR;
T1/2: 120-
170 hours;
BPP: NR
Renal
Urine (61
%) and
feces (12
%)
Anemia, anorexia,
depression
Tocilizumab
Antirheumatic
80-96 %
VD: 6.4 L /
Kg; T1/2: 11
– 13 days;
BPP: NR
Hepatic
Renal
(linear
depuration)
SRT infections,
leukopenia
OB: Oral bioavailability; VD: apparent volume of distribution; BPP: Binding to plasmatic proteins; T1/2: Time
oh half-life; NR (not reported); CHF: Congestive Heart Failure; Superior Respiratory Tract (SRT); Inferior Respiratory
Tract (IRT); MP: Methylprednisolone; RF: Respiratory failure.
6. CONCLUSIONS AND RECOMMENDATIONS
The quantification of the diameter of the droplets produced by the nebulizers taken as a sample using DLI
technique led to the elaboration of the characteristic histograms of each nebulizer researched, relating the frequency
of events as a function of the droplet diameter. In the histograms presented, the ideal range corresponding to the
breathable droplets was emphasized, that is, those with diameters between 1.0 to 5.0 µm. Allied to that, graphs of
the histograms associated with the Normal, Log distribution curves Normal, Gamma and Inverse Normal to assess
the most appropriate type of distribution according to with the database. It was concluded that the distribution that
comes closest is Log Normal according to the literature dealing with the topic under study. The polydispersed
character of the population of the droplets generated by the nebulizers was also proven, attested by the high
Deviation Standard (σ) of the diameters related to the mean.
The choice of CDMA over MMAD arose from the need to use a parameter alternative to quantify the population
of breathable droplets, CDMA made this possible because it works with the representation of the median of the
droplets, and not a value absolute mean mass diameter of the droplet population represented by MMAD.
DLI technique proved to be efficient for the characterization of dispersed aero droplets, combining optical
procedures aimed at image acquisition, computational processing techniques. In addition, it proved to be
operationally viable for its simple and accessible character by using low cost materials and equipment compared to
the other techniques already mentioned.
The question raised at the beginning of the work regarding the qualification of efficiency nebulizers to deliver
droplets with diameters between 1.0 to 5.0 µm, which corresponds to the range of breathable droplets, was
answered according to the results experimental results achieved. This parameter is of fundamental importance, as
it provides a differentiation of nebulizers according to their ability to deliver droplets in the range above, which
provides a better therapeutic response during inhalation treatment.
In this scenario, there is still much to speculate in the search for an effective, accessible and safe therapeutics
for use to treat SARS-CoV-2 signs and symptoms. Knowledge of physicochemical and pharmacological properties is
essential for the development of new innovative formulations for administration by inhalation. DLI technique
represents a viable alternative for the characterization of aero dispersed droplets, for administration of these drugs.
Nebulizers: Aerodynamic Droplet Diameter Characterization and Physicochemical Properties of Drugs to Treat Severe
Acute Respiratory Syndrome Corona Virus 2 (Sars-Cov-2)
International Journal of Research -GRANTHAALAYAH 95
SOURCES OF FUNDING
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit
sectors.
CONFLICT OF INTEREST
The author have declared that no competing interests exist.
ACKNOWLEDGMENT
The authors are grateful for State University of Bahia (UNEB), “Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior – Brasil (CAPES)” and Research Group: Biopharmaceutics and Drugs.
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