DESCRIPTION

The present invention relates to a liposomal composition (solution or gel) with abilities to interact with atmospheric particulate, increasing the ability of the nasal mucosa, and of the various structures present in the nasal cavities, to slow down the speed of inhaled particles as much as possible, so as to reduce the absorption thereof.

A further object of the invention is the use of such a composition directly on face or endonasal masks or on individual nasal filters aimed at protecting the respiratory tract, to increase the filtration capacity and barrier effect thereof. FIELD OF THE INVENTION

Air pollution is reaching levels of attention which require the commitment of global and national institutions (which set limits for the concentration of pollutants in the air, which are often exceeded) but also greater awareness and prevention by individuals.

Air pollution is a proven cause of ailments, illnesses and premature deaths. New studies associate the recent estimates from the Global Burden of Disease (Lancet 2016) which place air pollution in fifth place worldwide among the causes of disease and mortality, just after diet, smoking, hypertension and diabetes: 4.2 million premature deaths a year. The harm and consequences are particularly evident in the pediatric population . The greatest danger is represented by fine and ultra-fine dust, mainly microparticles and nanoparticles (PM 10- PM 2.5 and less than 0.1 microns), the size of which is such as to penetrate up to the thoracic tract of the respiratory system (bronchi) and up to the pulmonary alveoli.

This fine and ultra-fine dust can be organic or inorganic in nature and appear in a solid or liquid state mixed with oily components.

The particles are capable of adsorbing various substances with toxic properties on the surface thereof, such as sulfates, nitrates, metals, in particular heavy metals, and volatile compounds, among which the presence of viruses was also hypothesized.

This possibility, so far without confirmation, has been of greater relevance since the spread of a pandemic such as the 2020 Coronavirus pandemic, which imposed the generalized use of individual protective masks.

The Italian .Society of Environmental Medicine (Sima) recently announced that traces of SARS-Cov-2 RNA have been found on particulate matter (PM), fine dust.

As illustrated by Leonardo Setti, coordinator of the scientific research group together with Gianluigi De Gennaro and Professor Alessandro Miani, president of Sima, "The first evidence related to the presence of the coronavirus on particles come from analyses carried out on 34 samples of PM10 in ambient air from industrial sites in the province of Bergamo, which were collected with two different air samplers for a continuous period of 3 weeks, from 21 February to March 13, 2020".

The results obtained do not demonstrate the presence of intact coronavirus in the particulate matter, but traces of virus RNA: this does not necessarily imply that the virus is active and has sufficient viral load to be contagious. However, it is significant that the presence of coronavirus viral RNA on atmospheric particulate matter has been demonstrated.

In any case, beyond this recent research, it is clear that the physio-pathological role of dust with micrometric and nanometric size is mainly expressed at the level of the respiratory system as the penetration capacity of the particles and the deposition thereof from the nose to the alveoli, responsible for the effect thereof, depends on the size thereof, the smaller the particle, the more toxic it is.

Particular attention is required for the elderly, cardiopathic and pulmonary patients, infants and children (especially with bronchial crises, cough and phlegm).

The harmfulness of fine and ultra-fine dust depends, as already mentioned above, on the size and ability thereof to reach the different parts of the respiratory system.

Epidemiological studies have ascertained a correlation between concentrations of fine dust in the air and the onset of chronic diseases of the respiratory tract: the micrometric and nanometric particles (PM10-PM2.5 and lower than 0.1 microns) act as a means of transport for substances characterized by high toxicity in living organs.

Many studies agree in associating an increase in particle pollution with an increase in daily mortality between 0.2 and 0.6% for every 10(μg/m3 of increase in PM10 concentration.(1)

From the bibliographic reference it is clear that exposure to particles can cause some diseases of the respiratory system, and the particles, depending on the size thereof, can influence some cellular parameters, and can have above all a pro-inflammatory effect which induces an aggravation of the respiratory diseases.

Legal limits vary and are often not uniform, but movement restrictions are generally related to concentrations above 50(μg/m ³ . In this regard, for air quality, the evaluation parameter of PM10 is the daily average. The daily limit value established by Italian law is 50(μg/mc of air, and cannot be exceeded more than 35 times in the calendar year (Italian Legislative Decreel55/2010), therefore it is important to count the days in which it has been exceeded since the beginning of the year.

To date, the main remedies against fine dust are only face or endonasal masks or nasal filters designed to protect the respiratory tract, but, as everyone's experiences teach, they are impractical for everyday and common use. See for example US patent document USD753821 related to a nasal dilator (2). TECHNICAL PROBLEM

From what has been said, the importance of providing a product in the form of a solution or gel intended for use in the nasal cavity is clear, which is capable of interacting directly with the nasal mucous membranes and the various structures present in the nasal cavities, to thereby reduce the absorption of atmospheric particulate by the user.

Similar importance is also given to a product in the form of a solution capable of being distributed directly on face or endonasal masks or on individual nasal filters aimed at protecting the respiratory tract, to increase the filtration capacity and barrier effect thereof.

SOLUTION TO THE PROBLEM CONNECTED TO THE INHALATION OF

PARTICLES

Therefore, the aim of the present invention is to provide a product capable of satisfying the aforementioned needs, including a preparation which could mimic, without interfering with the physiology and activity of the nasal cavities, the effectiveness and functionality of the capturing and filtering system of the nasal cavities, increasing the ability to slow down the speed of inhaled particles as much as possible, and ultimately, to capture them.

The present invention is based on the choice of using phosphatidylcholine, a phospholipid, as the basic material of such a product, taking into account the fact that the nasal membranes, like all membranes, also consist of phospholipids and that the same mucus has a phospholipid component.

According to a peculiar feature of the invention, the phosphatidylcholine was structured in the form of liposomes, since the liposomes have the ability not only to interact with membranes but also with organic and inorganic salts and with micrometric and nanometric particles, forming aggregates/clusters.

It should be noted that the solution of resorting to the use of phosphatidylcholine in the form of liposomes for a nasal spray contrasts the teachings provided by the prior art. Indeed, a dated article "Hydrolysis of phosphatidylcoline in aqueous liposome dispersions" in INTERNATIONAL JOURNAL OF PHARMACEUTICS,

ELSEVIER, NL, vol. 50, no. 1, of February 15, 1989, pages 1-6, emphasizes the possibility that the stability of a liposomal nasal spray comprising phosphatidylcholine and water was particularly brief due to the hydrolysis of the main component, phosphatidylcholine, which suggested that marketing a similar spray was practically impossible.

In spite of this, the Applicant, taking into account that liposome production technology has particularly evolved and spread in recent years also at an industrial level, with the extensive experience of its researchers, has taken on the burden of verifying experimentally, as illustrated in detail later, that such an assertion is incorrect, demonstrating that today it is possible to obtain liposomes containing phosphatidylcholine whose structure is highly stable at a temperature of 40 °C.

In light of such evidence, the inventors therefore decided not to resort to a simple aqueous solution of phosphatidylcholine, but to structure the phosphatidylcholine in the form of liposomes, considering that the liposomes advantageously have the ability not only to interact with membranes but also with organic and inorganic salts and with micrometric and nanometric particles, forming aggregates/clusters.

Liposomal nasal sprays are already known, but not of phosphatidylcholine. The article "Liposomial Nasal Spray versus Guideline-Recommended Steroid Nasal Spray in Patients with Chronic Rhinosinusitis: A comparison of Tolerability and Quality of Life" describes a "mucoadhesive compound for removal of toxins from mucosal membranes such as intranasal membranes wherein said compound comprises a mucoadhesive polymer - such as xanthan gum or hyaluronic acid- and a ligand group - such a liposome", However, the liposomes described always contain medicinal substances, such as vitamin A palmitate, dexpanthenol and tocopherol, all molecules recognized as having an anti-inflammatory effect as such and especially in association, but never mentions phosphatidylcholine .

Advantageously, the use of phosphatidylcholine in liposomal form, according to another peculiar feature of the present invention, allows precisely exploiting the tendency liposomes have to aggregate and form clusters with salts and micro and nanometric particles, together with the tendency of liposomal formulations to precipitate or settle once charged, transforming them into a positive factor, which can be used to reduce the speed of inhaled particles, and possibly capture them definitively, avoiding the transfer thereof to deeper areas of the respiratory system.

In a preferred embodiment, the above-mentioned liposomal composition of phosphatidylcholine was made viscous, by means of xanthan gum, in order to reproduce, as far as possible, the mucus present in the nasal cavities.

A viscous liposomal product with xanthan gum was thus developed which experimentally proved capable of incorporating in 0.2 ml of product, corresponding to one spray of a spray dispenser for nasal use, 46.6 μg of particulate, without no trace of sedimentation within 24 hours.

A viscous liposomal product with xanthan gum was thus developed which experimentally proved capable of incorporating in 0.2 ml of product, corresponding to one spray of a spray dispenser for nasal use, 46.6 ug of particulate, 'without no trace of sedimentation within 24 hours.

As an alternative to the use of xanthan gum, the opportunity to use hyaluronic acid was then verified in the light of the following considerations: hyaluronic acid has the ability to coordinate water, like xanthan gum, but in an even more accentuated manner; hyaluronic acid has a negative charge like xanthan gum, whereby the features of the formulation consisting of liposomes and hyaluronic acid, in particular the overall charge thereof, remain unchanged with respect to the formulation containing liposomes and xanthan gum .

Therefore, two different formulations were examined: a formulation in which hyaluronic acid completely replaces the xanthan gum, whereby the concentration of the same hyaluronic acid is in the same range 0.1-0.4%; and a formulation in which xanthan gum is not totally replaced by hyaluronic acid but is present in the same concentration, for example 0.15%, so as to have a final concentration of 0.3%.

Thereby, the presence of xanthan gum is maintained as a replacement for mucus, an important component of the nasal cavity for the capture of atmospheric particles, and the moisturizing and bioadhesive capacities are enhanced.

The description of the invention will be better followed with reference to the accompanying drawings which show some anatomical details of the respiratory system and, merely by way of non-limiting example, some embodiments of the invention.

LIST OF FIGURES

In the drawings: fig. 1 is a diagrammatic view of the deposition site of particulate in the human body according to the particle size from 0.1 to 10 microns and the ability thereof to reach the different parts of the respiratory system; fig. 2 shows the respiratory system which controls the gaseous exchanges between blood and atmospheric air; fig. 3 is a figure which graphically summarizes all the information and features of the nasal cavities; fig. 4 shows a sample A of 2% liposomal solution obtained by means of the high pressure technique, 600- 800 atm, 5 times, at time 0; fig. 5 shows the previous liposomal solution to which 3.5 mg of charcoal has been added, after 30 minutes (sample C); fig. 6 compares samples A and C, where the difference is highlighted due to the presence of a gray supernatant consisting of non-settled charcoal; fig. 7 shows a sample B, viscous liposome product with xanthan gurn; fig. 8 shows a sample D, viscous liposome product with xanthan gum after addition of charcoal, after 30 minutes; fig. 9 compares samples B and D after 30 minutes: fig. 10 compares samples B and D after 24 hours. DETAILED DESCRIPTION OF THE INVENTION

Below we report some information on the respiratory system and in particular on the structure and function of the nasal cavities, from which the inventors took inspiration to reach the solution object of the present invention.

The respiratory system, described in figure 1, controls the gaseous exchanges between blood and atmospheric air: it therefore has a dual function: to allow oxygen to enter and to eliminate carbon dioxide. Breathing consists of two phases: inhalation, through which we inhale air and therefore the oxygen present therein; and exhalation, with which we expel air rich in carbon dioxide.

The respiratory tract comprises: the nasal cavities; the pharynx; the larynx; the trachea and bronchi. The surface of the nasal cavity is provided with vibratile cilia. These have the task, also through the production of mucus, to purify and moisten inhaled air.

- For this reason, it is better to breathe through the nose rather than the mouth, especially if the air is cold.

Therefore, it can be understood how the operation of the nasal cavity is important as a first barrier to the intrusion of particles.

The proposed solution refers to the nose, especially to the two nasal cavities, to a part of the constituents thereof, in particular the vibratile cilia, mucus and hairs.

Although it may be mildly disgusting to many, the nasal mucus produced by the respiratory tract is a defense mechanism of the respiratory system.

The composition of the nasal mucus is relatively simple: it is a viscous gel consisting of 95% water, 1% inorganic ions, 0.3-0.5% phospholipids, 3% glycoproteins (mucins) and 0.1-0.5% antimicrobial proteins such as lactoferrin, lysozyme, β-defensin, IgA, IgG.

Mucus is secreted by special glands, called muciparous glands, positioned along the nasal cavities and the trachea. The primary function thereof is twofold: firstly, it coats and keeps sensitive epithelial membranes of the respiratory tract moist and lubricated secondly, the viscous nature of the mucus allows it to block and capture foreign bodies inhaled with air, preventing them from entering the lungs and expelling them via ciliary mucus clearance, coughing and sneezing.

The glycoproteins contained in the mucus are complex proteins comprising carbohydrates within the structure thereof; the composition of the glycoproteins allows the formation of very strong molecular bonds capable of trapping particles: this explains the particular viscosity of the liquid.

- In addition to lubricating and keeping the mucous membrane surfaces moist, mucus has the function of capturing particles, microorganisms, exfoliated mucosal cells and leukocytes present in the event of inflammatory reactions, facilitating the elimination thereof.

Mucus consists of water, mucins and inorganic substances; although the composition thereof differs in relation to the production site, the structure and production process of the main component thereof, mucins, are similar in the various mucous membranes. Mucins are high molecular weight glycoproteins with a percentage by weight of carbohydrates which reaches 50% of the total.

In particular, mucin is a glycoprotein present in the mucous secretions of the respiratory and gastrointestinal tract. The glycosylation of mucins increases the frictional friction between them and the external solvent, with a consequent increase in the viscosity of the solution. Thereby mucin, which is very viscous, becomes protective element which forms a kind of protective layer on the respiratory epithelium.

The viscosity increases because the carbohydrate part of the glycoprotein, being highly hydrophilic, actracts a lot of water, increasing interactions therewith. In the two nasal cavitliesof the internal nose experts recognize three anatomical reference regions, which are: the vestibule, the olfactory region and the respiratory region.

The inventors were mainly interested in the vestibule, which is the very first part of the nasal cavities. It is an enlarged area, provided with a characteristic mucous lining and especially in adults, it is also the region of the internal nose from which nasal hairs can originate.

The purpose of the latter is to filter out potentially harmful particles from the air we breathe and even humidify the air in cold and dry conditions.

The cilia present in the trachea, called vibratile cilia, move in the caudo-cranial direction, together 'with the mucus they filter the substances introduced through breathing; the mucus traps atmospheric dust (dust, pollen, bacteria, etc.) so that the respiratory tract is kept clean.

Fig. 3 graphically summarizes all the information and features of the nasal cavities.

To define a daily particulate absorption, it is important to consider that the nose heats and humidifies over 12,000 liters of air per day, equal to 12 m ³ of air.

From the foregoing, a solution was hypothesized which could mimic, without interfering with the physiology and activity of the nasal cavities, the effectiveness and functionality of the capturing and filtering system of the nasal cavities.

The different steps which allowed achieving the prefixed result are described below. FIRSTSTEP The first activity was to develop a product/device which had the ability to interact with the nasal mucosa and the various structures present in the nasal cavities, increasing the ability to slow down the speed of inhaled particles as much as possible, and ultimately, to capture them.

From the consideration that nasal membranes, like all membranes, also consist of phospholipids and that the same mucus has a phospholipid component, the inventors hypothesized a product based on phosphatidylcholine, which is a phospholipid, and that the same phosphatidylcholine was then appropriately structured in the form of liposomes.

Therefore, the stability of a nasal drop product with liposomes S80 (CAS 8030-76-0) was first monitored by carrying out a series of aging tests at a temperature of 40° C, as reported in the following table 0 TABLE 0 - Note: Produced after 6 months, it does not separate at room temperature, nor at 40°C.

The choice to develop a product based on phosphatidylcholine liposomes, as previously mentioned, arose from the fact that liposomes have the ability not only to interact with membranes, but also with organic and inorganic salts and with micrometric and nanometric particles, forming aggregates/clusters.

Normally this interaction with salts, and with micrometric and nanometric particles, leads to an inclusion thereof and in the most intense cases to a precipitation of the charged liposomes. The formation of clusters, the subsequent precipitation or rather the sedimentation of the liposomal formulations is considered from a purely technological/pharmaceutical viewpoint as a negative event to be avoided, even if this negative phenomenon takes a long time, depending on other factors such as temperature, exposure to light, etc.

The inventors have transformed this tendency to aggregate liposomes, to interact with particles, into a positive factor, which can be exploited to reduce the speed of the inhaled particles, possibly capturing them definitively by the respiratory system. See (3) "Liposomes in Contact and Interacting with Silica Nanoparticles: From Decorated Vesicles to Internalized Particles." by Raphael Michel, published by Cuvillier Verlag, and (4) "Stability Aspects of Liposomes" by Yadav A.V., Murthy M.S. Shete A. S and Sfurti Sakhare, in Indian Journal of Pharmaceutical Education and Research, October-December 2C11/Vol45/Issue. According to an advantageous feature of the invention, such a product/device can also be sprayed on protective masks, increasing the capacity thereof as a further filter/barrier .

Finally, the product may have the ability to capture pollen and mites. OPERATING METHOD

Keeping in mind the above information and the latest considerations on liposome instability, the liposomal formulation was prepared and is shown in TABLE 1 :

- TABLE 1 BASIC FORMULATION : 2% LIPOSOMES

With the aid of light scattering analysis it was possible to determine the average size of the liposomes, from which considering the concentration of lipids as well as the molecular weight of phosphatidylcholine it was possible to obtain the number of liposomes per ml of preparation using the dictates of the article "A model based in the radius of vesicles to predict the number of unilamellar lipodomes" by Montanari et al, published in International Journal of Research in Pharmacy and Chemistry, IJRPC 2014, 4(2), 484-489.

Subsequently, the size analysis was further examined by defining more in particular the composition of the particle distribution which turned out to consist of two families of particles which we will indicate as peak 1 and peak 2.

The liposomes were prepared by means of the high pressure technique, 600-800 atm, 5 times.

The ability of the liposomal solution to interact with particulate was evaluated as follows: 3.5mg of charcoal was added (concentration 233.33 μg/ml) to 15 ml of this liposomal solution, defined Sample A; the solution with additive, which became Sample C, after 30 minutes remained gray in the upper part, in the bottom instead it had the settling of the largest charcoal particles with a size greater than 10 microns

Figures 4 and 5 visually explain what occurred:

Figure 4 shows a sample A consisting of 2% liposomes at time 0: the particularly white color can be easily noted.

Fig. 5 shows the sample C obtained by adding charcoal to sample A. It should be noted that sample C refers to the supernatant of the liposomes to which the charcoal was added, taken 24 hours after the addition, therefore without the particles which settled.

To better characterize the phenomenon indicated above, samples A and C were subjected to light scattering analysis with a DLS apparatus.

TABLE 2 shown below indicates the size analyses carried out in two modes:

INTENSITY mode with which the largest particles are highlighted; and VOLUME mode with which the smaller and often more numerous particles than the larger ones are highlighted.

Both samples at the time of light scattering analysis were diluted from 1 ml to 30 ml with distilled water. TABLE 2 A

CHARACTERIZATION with light scattering of the 2% liposomes of SAMPLE A

SIZE DISTRIBUTION 2% Liposomes

* Sample A refers to phosphatidylcholine liposomes as such.

• Charcoal was then added to the sample of liposomes A at the final concentration of 233 μg/ml.

• The analyzed part is the supernatant.

TABLE 2B

CHARACTERIZATION with light scattering of 2% liposomes after addition of charcoal (table 2) SAMPLE C

To TABLE 2a, 2b above we add the graph of the particle size distributions of samples A and C. Graphic ref. sample A, sample C.

Conclusion

A) The first observation which can be made is that in Sample C there is an increase in particles smaller than 100 nm, but these particles have a size greater than that of the starting liposomes, an evident indication of an interaction between the liposomes and fine and hyper-fine particles (50-10nm) of the charcoal.

B) A percentage decrease in particles with size around 200 nm can be noted as well, both in "Intensity" and "Volume" modes, again indicating an interaction between liposomes and fine (less than 200 nm) and hyper-fine particles (50-10 nm) of charcoal.

A more in-depth analysis highlights the following: in intensity mode an increase in the size of the smallest particles is observed: in fact, it passes from 46 nm to 75 nm, moreover the percentage number of the same also increases from 2.2% to 12.2%; there is a % decrease in the largest particles, from 97.8% to 87.8%; in volume mode an increase in the size of the smallest particles is observed: it passes from 44.6 nm to 72.6 nm; moreover, the percentage number of the same also increases from 59.7% to 73.9%; there is a % decrease of the largest particles, passing from 40.3% to 26.1%.

SECOND STEP

From the experiment indicated above, we obtained the indication of a possible interaction between the fine particles of the charcoal, but wanting to enhance this primary, partly unexpected effect, we improved the formulation taking into account some peculiarities indicated above, in particular: we developed a product containing xanthan gum to reproduce, as much as possible, the mucus present in the nasal cavities.

In fact, xanthan gum has some peculiar features:

1.It interacts with many cations: it has a negative charge resulting from the presence of a pyruvic acid anionic group in the structure. See "The primary structure of the molecule (Noble and Urlacher,1 999) and "Microbial

Polysaccharides in Food Industry",(Namita Jindal, Singh Khattar), in Biopolymers for Food Design 2018.

2.It coordinates many water molecules, 1 g coordinates about 300 g of water, for this reason it is highly moisturizing, a feature in common with mucus. See "Microbial Polysaccharide in Food Industry" by Jindal and Khattar, in Biopolymers for Food Desing, 2018, par.8.1, and the article by Karaman, Kesler, Goksel, Dogan & Kayacier (2014) "Rheological and some Physicochemical Properties of Selected Hydrocolloids and their Interactions with Guar Gum: Characterization using

Principal Component Analysis and Viscous Synergism Index, " in International Journal of Food Properties 17:8, 1655-1667, DOI:

10.1080/10942912.2012.675612.

3.it interacts with membranes, and is highly mucus adhesive.

These features make it a good substitute for mucus, in particular for MUCIN, present in the mucus, which lines the nasal epithelium.

To confirm what has been said, there are products on the market used for the treatment of lack of saliva, (XEROSTOMIA), where xanthan gum is used to replace MUCIN.

OPERATING STEP

Considering the above, we prepared a viscous liposomal formulation with xanthan gum shown below in TABLE 3:

TABLE 3

2% VISCOUS LIPOSOME FORMULATION WITH 0.3% XANTHAN GUM Sample B

With the aid of light scattering analysis, it was possible to determine the average size of the liposomes, from which considering the concentration of lipids as well as the molecular weight of phosphatidylcholine, it was possible to obtain the number of liposomes per ml of preparation, always using the method of Monanari et Al. already mentioned.

Subsequently, the size analysis was further examined by defining more in particular the composition of the AVERAGE particle distribution which turned out to consist of two families of particles indicated below as peak. .1 and peak 2.

The liposomes were prepared by the high pressure extrusion technique, 600-800 atm, 5 times; once prepared they were mixed with xanthan gum. Fig. 7 shows sample B at time 0.

We evaluated the ability of the viscous liposomal solution (Sample B) to interact with the particulate as follows:

1) 3.5mg of charcoal (concentration 233.33 μg/ml) was added to 15 ml of Sample B, obtaining a compact mixture of black color called Sample D;

2) As can be seen in fig.8, after 30 minutes, Sample D remained black, without any sedimentation in the bottom even after 24 hours;

3) To better characterize the phenomenon indicated above, samples B and D were subjected to light scattering analysis with a DLS apparatus.

4) Tables 4a, 4b shown below indicate the size analyses carried out in two modes: a) INTENSITY mode with which the largest particles are highlighted; b) VOLUME mode with which the smaller and often more numerous particles than the larger ones are highlighted. c) Both samples at the time of light scattering analysis were diluted from 1 ml to 30 ml with distilled water.

TABLE 4a

SIZE DISTRIBUTION: SAMPLE B

CHARACTERIZATION with light scattering of 2% viscous liposomes with 0.3% xanthan gum

Charcoal was added to the sample of viscous liposomes at the final concentration of 233 μg/ml.

It is important to consider that no sediment formation was noticed even after 24 hours from the addition.

TABLE 4b

SIZE DISTRIBUTION: SAMPLE D

CHARACTERIZATION with light scattering of 2% viscous liposomes after addition of charcoal As regards Sample D, it should be noted that:

1 peak: in intensity mode there is an increase in the size of the smallest particles from 71 to 123 ran, furthermore also the percentage number of the same increases, passing from 7.9% to 25.5%;

2 peak: in intensity mode there is a consistent increase in size from 265 to 479.4 ran, with a consequent decrease in %, passing from 92% to 74.5%;

Still Sample D:

1 peak in volume mode shows an increase in the size of the smallest particles from 67.1 to 114.9 ran; furthermore, the percentage number of the same shows a slight decrease from 59.7 to 52.0%.

2 peak: in volume mode shows an increase in the size of the largest particles from 262 to 486.6 ran and the number thereof increases, passing from 40.3 to 48.0%.

To the table above we add the graph of the particle size distributions of samples B and D Graphical ref. sample B, sample D.

Conclusion

The information reported in the Intensity and Volume modes indicates a consistent capture/interaction ability of the viscous liposomal preparation with xanthan gum with the charcoal particles.

Furthermore, Sample D does not show any sedimentation of carbon particles after 30 minutes and even after 24 hours, which means that 15 ml of 2% viscous liposomes with 0.3% xanthan gum stably interact with the charcoal particles. Figure 8 shows Sample D 30 minutes after preparation; and figure 10 shows Sample D 24 hours after preparation.

The results obtained from this last experiment allow us to make a calculation to define how much charcoal was incorporated by the mixture of viscous liposomes with xanthan gum.

In fact, we know that:

3.5 mg of charcoal (concentration 233.33 μg/ml) was added to 15 ml of Sample B; therefore, given that the sample did not show any sedimentation of charcoal particles, even after 10 days, we can conclude that we have had a complete clusterization. This means that 0.2 ml of product, comparable to one spray from a spray dispenser for nasal use, could interact, i.e., retain 46.6 μg of particulate.

This result must be taken into consideration and must be compared with the limit values related to particulate air pollution where the daily limit value established by Italian law is 50 μg/m ³ which cannot be exceeded more than 35 times in the calendar year (Italian Legislative Decree.155/2010). We know that the nose heats and humidifies at least 12,000 liters (12 2 ³ ) of air per day, therefore considering pollution of 50 μ/m ³ , in one day we could inhale at least 600 μg of particulate.

Considering as a first approximation an administration of 200 pl per spray, to sequester 600μg of particulate we should use about 2.5 ml of product. This conclusion is made simply to have an idea, however rough, of the volumes to be administered with a spray vial, also imagining that the natural capture system is completely inactive.

THIRD STEP

From the experiment reported previously with solution D, we obtained the indication of a stable interaction between the fine particles of the charcoal and the 2% viscous liposomal solution with 0.3% xanthan gum: in fact, sample D containing a concentration of 223 μg/ml of charcoal did not present any charcoal sedimentation even 10 days after preparation.

Wanting to stress this detail, we prepared a new liposomal formulation, which we will indicate as SAMPLE E, increasing the concentration of liposomes to 3%, to possibly enhance the capture by the latter of ultrafine particles, which are particularly dangerous because they can reach the pulmonary alveoli due to the nanometric size thereof.

TABLE 5

FORMULATION 3% viscous liposomes: SAMPLE E 4.5 mg of charcoal (concentration 300μg/ml) was added to 15 ml of Sample E, giving rise to a product which we will call Sample F.

Sample F showed no sedimentation after 30 minutes and after 24 hours.

Taking into account the foregoing, 0.2 ml of product, comparable to one spray from a spray dispenser for nasal use, could interact with, i.e., retain, 60 μg of particulate matter.

As can be seen in the figure, Sample F remained black after 30 minutes, without any sedimentation in the bottom even after 24 hours.

5) To better characterize the phenomenon indicated above, samples E and F were subjected to light scattering analysis with a DLS apparatus.

6) Tables 5a, 5b below indicate the size analyses carried out in two modes: d) INTENSITY mode with which the largest particles are highlighted; e) VOLUME mode with which the smaller and often more numerous particles than the larger ones are highlighted. f) Both samples at the time of the light scattering analysis were diluted 1 ml to 40 ml with distilled water. TABLE 5a

SIZE DISTRIBUTION analysis

TABLE 5b CHARACTERIZATION with light SCATTERING after the addition of charcoal

In intensity mode, an increase in the size of the smallest particles is noted, passing from 75 nm to 135 nm, moreover the percentage number of the same increases as well, passing from 10% to 33.5%.

There is an increase in the size of the largest particles, passing from 270 nm to 490 nm, with a consequent decrease in %, passing from 90% to 66.5%. In volume mode, an increase in the size of the smallest particles is noted, passing from 72.1 nm to 135.9 nm. Furthermore: the percentage number thereof does not vary much: in fact, it passes from 60% to 65.11%.

The larger particles further increase the size thereof, passing from 273.2 nm to 495 nm, the number thereof decreases slightly, passing from 40.0 to 34.9.

We know that the nose heats and humidifies at least 12,000 liters (12 m ³ ) of air per day, therefore considering a pollution of 50 p/m ³ , in one day we could inhale at least 600 μg of particulate.

Considering as a first approximation an administration of 200 μl per spray, to sequester 600μg of particulate we should use about 2 ml of product for 24 hours, assuming that the natural system for removing and blocking the particles is completely inactive.

FOURTH STEP

Preparation of the formulation with preservative

To allow the preservation of the formulation of 3% viscous liposomes with 0.3% xanthan gum, the necessary preservatives have been added: Sodium benzoate and potassium sorbate with citrate buffer.

Table 6 follows.

TABLE 6 Complete product formula with sodium benzoate and potassium sorbate

4.5 mg of charcoal (concentration 300μg/ml) was added to 15 ml of the preparation. The product showed no settling after 30 minutes and after 24 hours.

TABLE 7

Complete product formula with PARABENS with citrate buffer

4.5mg of charcoal (concentration 300 μg/ml) was added to 15 ml of the preparation.

The product showed no settling after 30 minutes and after 24 hours.

FIFTH STEP

The initial product formulated as a mucoadhesive humectant nasal spray was modified into a nasal gel: in this regard, the concentration of xanthan gum was increased to obtain the following formulas:

TABLE 8

Complete GEL product formula with sodium benzoate and potassium sorbate TABLE 9

Complete gel product formula with PARABENS USE OF HYALURONIC ACID IN ADDITION TO OR AS REPLACEMENT OF XANTHAN GUM AS VISCOSIFYING AGENT

As previously mentioned, in the context of the experimental tests carried out, the opportunity arose to use hyaluronic acid as a total or partial replacement for xanthan gum as a viscosifying agent .

Therefore, the two different formulations were developed, which follow:

TABLE 10

Total replacement of xanthan gum TABLE 11