Compounds for binding of microplastics and nanoplastics

Abstract

The present invention relates to a use of a chitosan for binding microplastics (MPs) and nanoplastics (NPs) in human being. Furthermore, the invention describes a food supplement and a pharmaceutical composition comprising chitosan and their therapeutic and non-therapeutic uses.

Description

[0001] P024280WG-01 Notarbartolo & Gervasi S.p.A.

[0002] “COMPOUNDS FOR BINDING OF MICROPLASTICS AND NANOPLASTICS”

[0003] *****

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to a use of at least one chitosan for binding microplastics (MPs) and / or nanoplastics (NPs). Furthermore, the invention describes a food supplement and a pharmaceutical composition comprising a chitosan and their therapeutic and non-therapeutic uses.

[0006] STATE OF THE ART

[0007] The massive and ever-increasing production of plastics (now exceeding 400 million tons per year) and the global surge in plastic consumption, coupled with inadequate waste management and recycling efforts, have resulted in an overwhelming accumulation of plastic debris.

[0008] This pervasive presence of plastic in the environment has led many to refer to the current period as the "plastic era", an era that follows the Stone, Copper, Bronze, and Iron Ages.

[0009] Unlike those materials, which were primarily localized to specific regions and purposes, plastics have become ubiquitous and are now a global environmental issue. The plastic era arguably began in the 19th century with the invention of synthetic polymers, which were rapidly adopted for their durability, versatility, and low cost. However, these same qualities have contributed to their persistence and accumulation in natural ecosystems, as plastics can take hundreds to thousands of years to degrade fully.

[0010] Projections indicate that by 2030, approximately 20 million tons of plastic waste will enter aquatic ecosystems annually. This staggering figure illustrates a growing environmental crisis, as current efforts to control and mitigate plastic pollution are far from sufficient to keep pace with the rising plastic production and disposal rates. Research by Borrelle et al. in Science (Borrelle SB et al. Predicted growth in plastic waste exceed efforts to mitigate plastic pollution. Science 2020; 80:1515-1518) highlights the alarming trend that the predicted growth in plastic waste will continue to outpace efforts to reduce pollution. This influx of plastic waste has far-reaching consequences, not only for marine life and biodiversity but also for human health, as P024280WG-01 Notarbartolo & Gervasi S.p.A. plastics break down into smaller particles that permeate both land and sea.

[0011] Plastics have now infiltrated the human food chain, as these materials are found in the air we breathe, the water we drink, and the food we consume.

[0012] This contamination occurs as plastic waste fragments into micro- and nanoplastics, which are small enough to enter the bodies of living organisms.

[0013] Microplastics (MPs), defined as particles between 100 and 5000 nanometers in size, and nanoplastics (NPs), which are smaller than 100 nanometers, are increasingly present in various food items.

[0014] Studies, such as those by Paul et al. in Nanoscale Advances (Paul MB et al. Micro- and nanoplastics-current state of knowledge with the focus on oral uptake and toxicity. Nanoscale Advances 2020; 2: 4350-4367), have detected these particles in seafood, honey, table salt, vegetables, fruits, milk, and even bottled mineral water. The ingestion of these particles raises concerns about their long-term health effects, as they may carry hazardous chemicals or serve as carriers for other environmental toxins that bind to plastic surfaces.

[0015] While the exact metabolic fate of micro- and nanoplastics in the human body is not fully understood, recent research indicates that these particles can cross biological barriers and enter the bloodstream. Leslie et al. (Leslie HA et al. Discovery and quantification of plastic particle pollution in human blood. Environ Inter. 2022; 107199) conducted a study measuring the concentration of microplastics in human blood, finding levels between 0.5 and 4.5 micrograms per milliliter.

[0016] These findings suggest that humans are continuously exposed to plastic particles, with unknown but potentially harmful effects.

[0017] Given their size, some particles may evade detection by the body's immune system, leading to persistent bioaccumulation in various organs and tissues. The presence of plastics in the bloodstream opens questions about the possible impact on cellular function, inflammatory responses, and overall health.

[0018] In addition to general systemic exposure, plastics have also been found in specific medical conditions. For example, recent research by Marfella et al. (Marfella R et al. Microplastics and nanoplastics in atheromas and cardiovascular events. New England P024280WO-01 Notarbartolo & Gervasi S.p.A.

[0019] Journal of Medicine, NEJM 2024: 390: 900-910) has identified microplastics and nanoplastics embedded in atheromas, fatty deposits within arteries. These plastic particles are suspected to contribute to the development of cardiovascular diseases, as they may provoke inflammatory responses and facilitate plaque buildup, ultimately leading to adverse cardiovascular events.

[0020] This discovery marks a critical connection between plastic pollution and serious health outcomes, underscoring the importance of addressing plastic contamination from both environmental and public health perspectives.

[0021] The pervasive presence of plastic contaminants in the environment and human body has spurred a growing field of research aimed at understanding the health implications of plastic exposure and finding ways to mitigate its impact.

[0022] The object of the present invention is to provide a composition for reducing the intake of microplastics (MPs) and nanoplastics (NPs) from water and food.

[0023] Specifically, this invention aims to effectively bind plastic contaminants, thereby limiting their absorption and adsorption within the intestinal tract.

[0024] This approach offers a practical solution to mitigate the potential health risks associated with the ingestion of plastic particles.

[0025] SUMMARY OF THE INVENTION

[0026] Therefore, the solution proposed herein for the aforementioned aim is a use of at least one chitosan for binding and / or reducing the absorption of microplastics (MPs) and / or nanoplastics (NPs) in a human being, wherein said at least one chitosan has:

[0027] - an intrinsic viscosity in the range from 90 to 400 cPs (90 to 400 mPa s) as measured with an automated capillary viscometer in 0.25 M HAc / 0.25 M NaAc; and

[0028] - a degree of deacetylation of at least 70.

[0029] As reported in the experimental part, the inventor surprisingly found out that the binding was effectively done at the pH of the stomach of the human being, thus allowing the reduction of the absorption of the MPs and / or NPs in the tissues of the human being. The automated capillary viscometer used for evaluating the intrinsic viscosity was the Model AMV-200, Paar Physica USA Inc., Edison, N.J with a solution of acetic acid 0.25 M 10.25 M sodium acetate (0.25M HAc / 0.25M NaAc). P024280WO-01 Notarbartolo & Gervasi S.p.A.

[0030] Surprisingly the binding of the microplastics or of the nanoplastics occurred through hydrogen bonds.

[0031] Chitosan is a polymer derived from chitin that is composed of an alkaline deacetylated monomer of glucosamine and an acetylated monomer glucosamine and binding through (3-1 ,4 glycosidic and hydrogen bonds.

[0032] The degree of deacetylation of the at least one chitosan is at least 70, preferably in the range from 80 to 98, more preferably from 85 to 95.

[0033] In a further aspect, the invention relates to a food supplement comprising at least one chitosan and at least one physiologically acceptable ingredient, wherein said at least one chitosan has:

[0034] - an intrinsic viscosity in the range from 90 to 400 cPs (90 to 400 mPa s) as measured with an automated capillary viscometer in 0.25 M HAc / 0.25 M NaAc; and

[0035] - a degree of deacetylation of at least 70.

[0036] In another aspect, the present invention describes a pharmaceutical composition comprising at least one chitosan and at least a pharmaceutically acceptable carrier for use in the treatment and / or prevention of a disease derived from the MPs or NPs in a human being, wherein said at least one chitosan has:

[0037] - an intrinsic viscosity in the range from 90 to 400 cPs or mPa s as measured with an automated capillary viscometer in 0.25 M HAc / 0.25 M NaAc; and

[0038] - a degree of deacetylation of at least 70.

[0039] Advantageously and preferably the disease derived from the MPs or NPs in a human being is a gastrointestinal disease or a cardiovascular disease derived from the MPs or NPs in a human being.

[0040] In a still further aspect, it is provided a cosmetic use of the food supplement, herein described, for eliminating MPs or NPs from a human being. Said step of elimination derives from the binding of MPs or NPs carried out by the specific claimed chitosan in the human being and provides for the reduction of the absorption of said MPs and NPs in the tissues of the human being.

[0041] BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The characteristics and advantages of the present invention will be apparent from the P024280WO-01 Notarbartolo & Gervasi S.p.A. detailed description reported below, from the Examples given for illustrative and nonlimiting purposes, and from the annexed Figures 1 -3, wherein:

[0043] Figure 1 shows SEM images of chitosan HDPE MPs binding. (A) HPDE MPs without chitosans, (B) HDPE MPs after addition of Chito 1 at 1.0 mg / mL, (C) HDPE MPs after addition of Chito 2 at 1.0 mg / mL, (D) HDPE MPs after the addition of Chito 3 at 1.0 mg / mL, (E) HPDE after the addition of Chito 4 at 1 .0 mg / mL.

[0044] Figure 2 shows SEM images of chitosan PVC MPs binding. (A) PVC MPs without chitosans, (B) PVC MPs after addition of Chito 1 at 1.0 mg / mL, (C) PVC MPs after addition of Chito 2 at 1.0 mg / mL, (D) PVC MPs after the addition of Chito 3 at 1.0 mg / mL, (E) PVC after the addition of Chito 4 at 1 .0 mg / mL.

[0045] Figure 3 shows SEM images of chitosan PET MPs binding. (A) PET MPs without chitosans, (B) PET MPs after addition of Chito 1 at 1.0 mg / mL, (C) PET MPs after addition of Chito 2 at 1.0 mg / mL, (D) PET MPs after the addition of Chito 3 at 1.0 mg / mL, (E) PET after the addition of Chito 4 at 1 .0 mg / mL.

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] The inventor found out that he can use at least one specific chitosan for binding microplastics (MPs) and / or nanoplastics (NPs).

[0048] Therefore the present invention concerns the of at least one chitosan for binding and / or reducing the absorption of microplastics (MPs) and / or nanoplastics (NPs) in a human being, wherein said at least one chitosan has:

[0049] - an intrinsic viscosity in the range from 90 to 400 cPs (90 to 400 mPa s) as measured with an automated capillary viscometer in 0.25 M HAc / 0.25 M NaAc; and

[0050] - a degree of deacetylation of at least 70.

[0051] As it is clear from the experimental part the use for binding according to the invention is carried out in the stomach of said human being.

[0052] The first plastic contaminants are Microplastic (MPs), which following common hydrolytic processes (mechanical, physical or biological) become Nanoplastics (NPs). Surprisingly according to the invention, the use of the at least one chitosan in the human gastrointestinal tract allowed the binding / inactivation of MPs thus reducing the NPs formation and availability, specifically for the absorption in the other tissues of the P024280WO-01 Notarbartolo & Gervasi S.p.A. human being.

[0053] Surprisingly the binding of the microplastics or of the nanoplastics occurred through hydrogen bonds.

[0054] In gravimetric terms, after the use of the at least one chitosan of the invention the average daily food intake corresponded to about 2.5 Kg (approximately 35% as water) that in turn corresponded to 0.24 pg / Kg (or Liter) of MPs / NPs. This value was 3 orders of magnitude lower than the minimal concentrations of MPs tested as will be evident in the experimental part.

[0055] In an aspect, therefore the present invention concerns a use of at least one chitosan for binding and / or reducing the absorption of microplastics (MPs) and / or nanoplastics (NPs) in a human being, wherein said at least one chitosan has:

[0056] - an intrinsic viscosity in the range from 90 to 400 cPs (90 to 400 mPa s) as measured with an automated capillary viscometer; and

[0057] - a degree of deacetylation of at least 70.

[0058] In the context of the present application, the expression “chitosan” indicates a linear polysaccharide composed of randomly distributed |3-(1 — >4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). Chitosan is, in general, produced by deacetylation of chitin, which is the structural element in the exoskeleton of crustaceans (such as crabs and shrimp) and cell walls of fungi.

[0059] In the context of the present application, the expression “MW’ indicates the molecular weight as “viscosity average molecular weight”. The measure of molecular weight was derived from the intrinsic viscosity “q” according to Mark-Houwink-Sakurada equation: q = KMva

[0060] Where q is the intrinsic viscosity; Mvis viscosity average molecular weight; K and a are constant for given solute-solvent system and temperature. In the present invention hence the “viscosity average molecular mass” or “MW of chitosan is determined by measuring the viscosity in terms of cPs (centipoise) (mPa s) in 0.25 M HAc / 0.25 M NaAc using an automated capillary viscometer (preferably Model AMV-200, Paar Physica USA Inc., Edison, N.J.) according to Mark-Houwink-Sakurada equation.

[0061] According to the invention the “intrinsic viscosity” is a property of the chitosan and can P024280WO-01 Notarbartolo & Gervasi S.p.A. be measured from a dilute solution of macromolecules. The intrinsic viscosity contains information on the macromolecular shape, flexibility, and (for nonspherical particles) molar mass of macromolecules.

[0062] According to the invention the “intrinsic viscosity” is expressed with cP / cPs (centipoise / centipoises), that correspond to mPas according to the following formula 1cP=1 mPa s.

[0063] The definition “degree of acetylation (DA)” of chitosan represents the proportion of N- acetyl-d-glucosamine units with respect to the total number of units. It allows to define the two terms chitin and chitosan.

[0064] The degree of deacetylation of the at least one chitosan is at least 70, preferably in the range from 80 to 98, more preferably from 85 to 95.

[0065] In the present invention when “MPs” are indicated, it is meant microplastics defined as particles having a size in the range from 100 and 5000 nanometers as measured with SEM and “NPs” nanoplastics defined as particles having a size lower than 100 nanometers as measured with SEM.

[0066] Within the framework of the present description and in the subsequent claims, except where otherwise indicated, all the numerical entities expressing amounts, parameters, percentages, and so forth, are to be understood as being preceded in all instances by the term "about". Also, all ranges of numerical entities include all the possible combinations of the maximum and minimum values and include all the possible intermediate ranges, in addition to those specifically indicated herein below.

[0067] The present invention may present in one or more of the aspects of the present invention one or more of the characteristics disclosed hereinafter, which may be combined as desired according to the application requirements.

[0068] From the experimental part below, it will be clear that the inventor was able to reveal the binding of MPs / Chitosan. In fact the SEM documented the binding of MPs / Chitosan, while the stereomicroscopic allowed the measurement of the percentage of entrapment of the different MPs made by the claimed chitosan.

[0069] In particular, the stereomicroscopic analysis allowed to confirm the binding of chitosan with MPs. P024280WO-01 Notarbartolo & Gervasi S.p.A.

[0070] Surprisingly, chitosan polymers having an intrinsic viscosity (cPs (mPa s)) in the range from 90 to 400 cPs (mPa s) were found effective to bind plastics. These chitosan polymers were effective with respect to chitosan polymers characterized by much lower viscosity, approximately between 10 to 70 cPs (mPa s). These latter polymers resulted to be practically ineffective.

[0071] Without being bound to any theory, the inventor deems that the capacity of the chitosan to bind plastic particles belongs to the amount of hydrogen bonds in the polymer chitosan. The hydrogen bonds, being present in a given amount, were responsible for entrapping MPs and NPs. According to the inventor hence the number of these bonds have a cut-off so that under this value they were not in a sufficient amount to maintain a stable binding capacity. Therefore, the inventor concluded that chitosan showing high intrinsic viscosity were needed to bind efficiently MPs and NPs.

[0072] In a preferred embodiment, the MPs / NPs, which are bound, preferably through hydrogen bonds, and entrapped by chitosan, are selected from the group consisting of HDPE (High Density Polyethylene), PVC (Polyvinyl chloride), and PET (Polyethylene terephthalate), Polyethylene (PE), Polypropylene (PP), Polymethyl methacrylate (PMMA), Nylon (PA), Polyurethane, and Acrylates Copolymer, preferably HDPE (High Density Polyethylene), PVC (Polyvinyl chloride), and PET (Polyethylene terephthalate).

[0073] In a further aspect, the invention relates to a food supplement comprising at least one chitosan and at least one physiologically acceptable ingredient, wherein said at least one chitosan has:

[0074] - an intrinsic viscosity in the range from 90 to 400 cPs (mPa s) as measured with an automated capillary viscometer in 0.25 M HAc / 0.25 M NaAc; and

[0075] - a degree of deacetylation of at least 70.

[0076] In a preferred embodiment, the physiologically acceptable ingredient in the food supplement is selected from the group consisting of an auxiliary agent, a filler, an amino acid, a protein, a fatty acid, a carbohydrate, a vitamin, a mineral, a botanical extract obtained from plants or cell cultures, an enzyme, a binding agent, a colouring agent, a preservative, an acidifier, a flavour and an aroma. P024280WO-01 Notarbartolo & Gervasi S.p.A.

[0077] In a still further aspect, the present invention relates to a pharmaceutical composition comprising at least one chitosan and at least a pharmaceutically acceptable carrier for use in the treatment and / or prevention of a disease derived from the MPs or NPs in a human being, wherein said at least one chitosan has:

[0078] - an intrinsic viscosity in the range from 90 to 400 cPs (mPa s) as measured with an automated capillary viscometer in 0.25 M HAc / 0.25 M NaAc; and

[0079] - a degree of deacetylation of at least 70.

[0080] In the present invention when the definition “a disease derived from the MPs or NPs in a human being” is used, it is intended to refer to a disease that can derive from the absorption of the MPs and / or NPs in the tissues of the human being due to circulation of the blood after the consumption of food and / or drinks containing such MPs and / or NPs.

[0081] Among the diseases derived from the MPs or NPs in a human being the following can be cited: cardiovascular diseases, hematological diseases, intestinal disease, cerebral diseases, andrological diseases, gynecological diseases, oncological diseases, connective tissue diseases, liver diseases, urinary diseases, sensorial diseases, thyroid diseases, endocrine diseases, respiratory diseases, gastroenteritis (viral or bacterial), appendicitis, diverticulitis, ischemic colitis, pancreatitis, cholecystitis, hepatitis (viral or toxic), COVID-19 (acute phase), seasonal flu, pneumonia, (bacterial or viral), sepsis, bacterial endocarditis, meningitis, encephalitis, pyelonephritis, DVT (Deep Vein Thrombosis), allergic reaction (severe), food poisoning and cystitis.

[0082] Preferably and advantageously such a disease derived from MPs or NPs in a human being is a gastrointestinal disease or a cardiovascular diseases derived from the MPs or NPs in a human being.

[0083] In a preferred embodiment, the use according to the invention or the food supplement or the pharmaceutical composition for use provides for at least one chitosan having the intrinsic viscosity in the range from 110 to 400 cPs (mPa s), more preferably from 250 to 400 cPs (mPa s) as measured with an automated capillary viscometer in 0.25 M HAc / 0.25 M NaAc.

[0084] In a further preferred embodiment, the use according to the invention or the food P024280WO-01 Notarbartolo & Gervasi S.p.A. supplement or the pharmaceutical composition for use provides for at least one chitosan having a Molecular Weight (MW) in the range from 100 to 450 KDa, preferably from 140 to 450 KDa or from 100 to 140 KDa according to Mark-Houwink-Sakurada equation.

[0085] In a more preferred embodiment, the at least one chitosan has a length in the range from 310 to 1400 nm, preferably from 400 to 1400 nm as measured with atomic force microscopy (Zhang H et al. Directly determining the molecular weight of chitosan with atomic force microscopy. Front Nanosci Nanotech 2016; 2(3):123-127) and a number of monomers in the range from 590 to 2700, preferably from 900 to 2700 as measured with atomic force microscopy in the food supplement or in the pharmaceutical composition according to the invention.

[0086] As it can be appreciated by referring the examples and the Figures 1 , 2 and 3, chitosan (for example Chito 1 and Chito 2) with an intrinsic viscosity lower than 70 cPs, a MW under 95 KDa, a length less than 295nm and a number of monomer below 570, at the concentration of 0.5 mg / mL and 1.0 mg / mL, was found ineffective, while chitosan according to the invention (for example Chito 3 and Chito 4) with an intrinsic viscosity in the range from 90 to 400 cPs, preferably having a MW in the range from 100 to 450 KDa, more preferably a length in the range from 310 to 1400 nm and still more preferably a number of monomer in the range from 590 to 2700 was able to entrap MPs, preferably HDPE, PVC and PET.

[0087] The entrapment of such MPs occurred preferably when the MPs were in the range from 100 to 500 pm of size as above defined. In this case the entrapment was surprisingly for at least 62% of the initial amount of MPs and when their concentrations in the acidic stomach environment were in the range from 0.5 to 1 .0 pg / mL.

[0088] Higher MPs concentration such as 2.0 pg / mL were still bound by Chito 3 and Chito 4 of the below examples although in lower entity and for at least the 42% of the total amount in the acidic stomach environment.

[0089] The chitosan binding increased when MPs concentration in the acidic stomach environment was in the lowest range, and the inventor deems that almost all the entire plastic polymers are bound when their concentration is below or equal to 0.5 pg / mL. P024280WO-01 Notarbartolo & Gervasi S.p.A.

[0090] All the preferred aspects for the use of the invention above indicated are the same of the food supplement.

[0091] In a further aspect, it is provided a cosmetic use of the food supplement, herein described, for eliminating MPs or NPs from a human being.

[0092] In a further aspect of the present invention, a pharmaceutical composition for use in the treatment and / or prevention of gastrointestinal diseases or cardiovascular diseases derived from the MPs in a human being is described.

[0093] In a preferred embodiment, in the use according to the invention and in the cosmetic use of the food supplement according to the invention or in the pharmaceutical composition for use, as herein described, the at least one chitosan is administered, preferably orally administered, in the amount from 0.5 to 1 .0 g / day.

[0094] In the stomach, at pH around 3, any chitosan administered in the amount in the range from 0.5 to 1 .0 g / day can reach a concentration between 0.5 and 1 .0 mg / mL.

[0095] These levels are sufficient for the binding of many MPs / NPs contained in foods, which are consistent with MPs having size equal to or higher than 100 nm up to 5 mm or NPs that are nanoplastics having size below 100 nm.

[0096] Since the binding of MPs / NPs belongs to the capability of chitosan to entrap these particles, the inventor deems that the binding is obtained regardless of their dimension and could be complete in the case of lower dimensions (NPs).

[0097] In a preferred embodiment the food supplement or the pharmaceutical composition of the invention are used for entrapping MPs or NPs in a human being.

[0098] It is worthy to underline that the entrapment of NPs can avoid the formation of a “protein corona” which can make them available to the enterocytes (Wang H et al. Formation of a monolayer protein corona around polystyrene nanoparticles and implication for nanoparticles. Nano-Micro SMALL 2019 doi.org / 10.1002 / small.201900974).

[0099] The invention advantageously allowed the entrapment of plastic contaminants as MPs or NPs in the stomach and so avoided or at least reduced their bioavailability (for absorption / adsorption) due to the volume increase. Most importantly the food supplement or the pharmaceutical composition of the invention in view of the presence of the claimed chitosan allows in the cephalic gut that the combination chitosan / MPs P024280WO-01 Notarbartolo & Gervasi S.p.A.

[0100] (or NPs) becomes a gel due to the pH increase, thus forming the bulk of chyme containing the chitosan polymers, that will be transferred into the colon and then secreted with feces.

[0101] Solid formulations of the food supplement or the pharmaceutical composition for oral administration are food bars, tablets, pills, powders, granules and capsules. These solid formulations are prepared by mixing at least one chitosan as described in the present invention with one or more suitable excipients such as starch, calcium carbonate, sucrose, lactose and / or gelatin. Except for the simple excipients, lubricants, for example magnesium stearate, talc can be used.

[0102] Liquid formulations of the food supplement or the pharmaceutical composition for oral administration are suspensions, solutions, emulsions and syrups, and the above- mentioned formulations can contain various excipients such as wetting agents, sweeteners, aromatics and preservatives in addition to generally used simple diluents such as water.

[0103] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0104] EXPERIMENTAL PART

[0105] EXAMPLES

[0106] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention.

[0107] Example 1. Chitosan Viscosity as indicative of the molecular weight.

[0108] The MW (molecular weight) of chitosans can be determined using different methods and one of the most frequent is the viscosity in terms of cPs (centipoise).

[0109] The viscosities in 0.25 M HAc / 0.25 M NaAc were measured using an automated capillary viscometer (Model AMV-200, Paar Physica USAInc., Edison, N.J.). The capillary diameter was 0.9 mm, and the inclination angle was 15°. These conditions, along with the use of solution concentrations lower than 1 % (w / V), were selected so that corrections for kinetic energy and shear were negligible.

[0110] The intrinsic viscosity was determined by both Huggins and Kraemer plots (Pavlov GM, P024280WG-01 Notarbartolo & Gervasi S.p.A.

[0111] Gosteva AA. Current analysis of Huggins and Kraemer plots for determining the intrinsic viscosity of macromolecules and corresponding dimensionless parameters. Polymer Science, Series A 2023; 64: 586-590).

[0112] Four chitosans (Chito 1 , Chito 2, Chito 3, and Chito 4), having the identical DDA (degree of deacetylation), were analyzed.

[0113] Four measures were carried out for each chitosan.

[0114] The viscosity ranges are reported in Table 1 below.

[0115] Table 1. Intrinsic viscosity range of chitosans used for MPs binding

[0116] Four chitosans were used at concentrations of 0.5 mg / mL and 1.0 mg / mL for the binding with MPs at concentrations between 0.5 to 2.0 pg / mL.

[0117] They were dissolved in distilled water at 35 °C and at pH 3 (simulating the stomach environment of a human being) of following the addition of acetic acid 0.1 N.

[0118] The measure of molecular weight was derived from the intrinsic viscosity “q” according to Mark-Houwink-Sakurada equation: q = KMva

[0119] Where q is the intrinsic viscosity; Mvis viscosity average molecular weight; K and a are constant for given solute-solvent system and temperature.

[0120] The corresponding retrieved MW are reported in Table 2. P024280WO-01 Notarbartolo & Gervasi S.p.A.

[0121] Table 2. Chitosan viscosity and relative MW in KDa following Mark-Houwink-Sakurada algorithm.

[0122] No overlapping of the viscosities or MW was found among the 4 chitosans, which allowed to correlate the binding / entrapping of MPs regardless of if viscosity or MW are used.

[0123] In relation to the binding capacity, it was important to calculate the length in nm of the different chitosans.

[0124] The polymers’ length depends on the number of monomers.

[0125] The chitosan monomer has an average MW of 167.5 Daltons and a linear length of 0.52 nm (Zhang H et al. Directly determining the molecular weight of chitosan with atomic force microscopy. Front Nanosci Nanotech 2016; 2(3): 123-127).

[0126] Therefore the lengths of the four chitosans were calculated and the results are reported in Table 3.

[0127] Table 3 below reports the range of monomers number and length of the different chitosans.

[0128] Table 3. MW, number of monomers and length of Chito 1 , 2, 3 and 4

[0129] The values indicated that chitosan due to the dynamic bonds among monomers could bind and entrap some MPs (>100 nm) and also NPs (< 100 nm).

[0130] The following Example 2 and Example 3 were aimed to show the capability of chitosan to bind MPs, which are responsible for the formation of NPs due to mechanical, physical and metabolic processes (bacterial enzymes).

[0131] It was evident that Chito 3 and Chito 4 were characterized by more consistent length than Chito 1 and Chito 2. P024280WG-01 Notarbartolo & Gervasi S.p.A.

[0132] Example 2. SEM (Electronic microscopy) analysis of the binding between Chitosans with HDPE (High Density Polyethylene), PVC (Polyvinyl chloride), and PET (Polyethylene terephthalate).

[0133] The MPs of three different plastic materials were used.

[0134] HDPE, PVC and PET are among the most common plastic contaminants of foods both in the form of NPs and MPs. The dimension of MPs considered was between 100 pm and 500 pm which are those more freguently found in foods with different forms (fibers, fragments or films).

[0135] The tested concentrations of MPs were 0.5, 1 .0 and 2.0 pg / mL.

[0136] These were consistent with high levels of daily intake since it was calculated that it corresponded to 4.1 pg / week which meant about 0.6 pg / day [Pletz M. Ingested microplastics: do humans eat one credit card per week? J Hazardous Materials letter 3 (2022) 100071.]

[0137] The MPs of HDPE, PVC and PET were obtained using 20 g of plastic material (respectively plastic caps, packaging films, and food containers).

[0138] The materials were finely ground using a Cgoldenwald High Speed Grinder (2400 W at 28,000 rpm) for a total of 5 times, with 3-minute intervals between each grinding session to avoid overheating the material.

[0139] Once the powders were obtained, a part (half of the total amount) was added with chitosans (from Chito 1 to Chito 4) solutions.

[0140] Chitosans were dissolved in distilled water at 35 °C and at pH 3 following the addition of acetic acid 0.1 N, thus simulating the stomach environment of a human being.

[0141] Once the combinations Chitosans / MPs were dried (at 35 °C for 24 hours) the particles were isolated using a set of stainless-steel sieves with mesh sizes of 1000, 500, 250, 100 pm, allowing for the separation of particles by size.

[0142] The powders (half of the total) without any addition of chitosans were kept as a control and followed the same filtration process to isolate MPs.

[0143] For SEM study (Scanning Electron Microscopy) was used a JEOL-6610LV with microanalysis (Microanalysis INCA Energy 350-Xmax 50).

[0144] The mesh sizes of 100 pm and 250 pm were analyzed which allow to obtain images P024280WO-01 Notarbartolo & Gervasi S.p.A. up to 500 pm.

[0145] SEM instrument characteristics: Scanning Electron Microscope with tungsten filament electron gun, with the possibility of working from 0.5 to 30 kV and a maximum resolution of 3.0 nm. From x5 to x50,000 magnifications were considered. The instrument can work in high vacuum modes for maximum resolution and low vacuum for samples with humidity or non-conductive surfaces.

[0146] Equipped with secondary electron detectors and backscattered electron detectors (composition, topography and shading). 5-axis asynchronous mechanical eucentric stage with eucentric rotation and tilt, which can hold samples up to 20 cm in diameter. Fully computerized, it is managed through a PC, with automatic image archiving in BMP, TIFF or JPG formats.

[0147] SEM microanalysis characteristics: A microanalysis apparatus built into the SEM microscope JEOL-6610LV. Using an active area of 50 mm2and an SDD detector type Xmax 50 with an energy resolution below 125 eV at 5.9 keV and 20,000 cps. including a tiny window that enables the investigation and detection of ultralights (C, N, O, etc.) to choose specific spots or regions on the microscopic image under study.

[0148] Sample preparation for scanning microscopy: Two requirements were met by samples meant for SEM: they were conductive and dry. It was important to dry the sample in a way that maintained as much of its original structure as possible.

[0149] One of the microscopes already had the cryofixation technology, which was a modern physical fixation technique, coupled to it. The sample needed to be coated in a substance that rendered it conductive and enabled microscopic observation in both situations.

[0150] Sample coating in low vacuum: This process applied two different coatings: carbon wire coating in case X-ray microanalysis was needed, and gold sputtering to achieve optimal imaging circumstances.

[0151] Mesh size: The combination of mesh 100 pm and 250 pm was used for SEM images selection.

[0152] Once the sample was prepared, the SEM divides the images into quadrants.

[0153] The instrument, through the microscope, was capable of analyzing each quadrant. P024280WO-01 Notarbartolo & Gervasi S.p.A.

[0154] Using visual analysis under a microscope, it was possible to see whether there were particles in each quadrant that stood out due to their particular shape.

[0155] As a result, the region was magnified, and its form and morphology were used to determine whether the particle was MPs or chitosan or the combination MPs / chitosan. Images were analyzed for 45 different quadrants: 15 for each type MPs as follows 3 quadrants were relative to the controls (0.5, 1.0 and 2.0 pg / mL) without any chitosan and 12 quadrants were corresponding to the chitosans (Chito 1 , Chito 2, Chito 3 and Chito 4) as represented in the Table 4.

[0156] Table 4. Number of quadrants analyzed visually in the SEM. Scoring: the entity of binding was simply reported as = 0 when no binding was visually P024280WO-01 Notarbartolo & Gervasi S.p.A. evident or as = 1 in case of some polymers entrapment was found between 10 to 20 images / quadrant and = 2 when > 20 images were presenting some binding.

[0157] The scores of the binding determined visually by SEM are reported in Table 5.

[0158] Table 5. Score of bindings between MPs of HDPE, PVC, PET and chitosans. The entity of binding was evident only for Chito 3 and Chito 4, while Chito 1 and Chito 2 never showed the capacity to bind any of the MPs analyzed.

[0159] The presence of chitosans modified the images of MPs particles which appear to be not bound, partially or totally bound.

[0160] Some examples of the SEM images of chitosan binding are reported respectively as follows: HPDE in Figures 1 , PVC in Figures 2 and PET in Figures 3.

[0161] The Figures are relative to the MPs as such (no chitosan addition) and following the P024280WO-01 Notarbartolo & Gervasi S.p.A. highest concentrations of chitosan (1 mg / mL) only since this concentration was considered more suitable to make evident the bindings.

[0162] At the opposite, the concentration used for MPs was the lowest (0.5 pg / mL).

[0163] In other terms, in the Figures the highest concentration of chitosans were reported compared to the lowest concentration of MPs. This was considered the best condition to make evident the possible bindings between polymers.

[0164] In all the figures the MPs size is reported (on the right in the bottom).

[0165] The binding with Chito 3 and Chito 4 was evident for MPs of HDPE, PVC and PET, while for Chito 1 and Chito 2 little to no binding was detectable.

[0166] Surprisingly high viscosity chitosan polymers (> 80 cPs) were needed for an efficient binding.

[0167] Example 3. Stereomiscroscopic analysis of the binding between MPs of HDPE, PET and PVC and Chitosans.

[0168] For stereomicroscopic examination, a semi-automatic instrument (Leica M205FA) with a high-resolution color digital camera (Leica DFC310FX; 1.4 Mpixel, CCD) was employed.

[0169] To quantify the number of MPs in each sample, the Imaged software (Confocal UniOvi Imaged) was used, which allows for estimating the size of fibers and amorphous fragments.

[0170] Different concentrations of MPs were evaluated, respectively 0.5, 1 , 2 pg / mL.

[0171] The stereomicroscopic analysis allowed to identify MPs >50 pm since lower dimensions did not consent a clear identification.

[0172] The mesh sizes combination of 100 pm and 250 pm (containing approximately particles between 100 and 500 pm) were used for the analysis. Depending upon the way that particles crossed the filter it was possible to detect also particles with one dimension higher than 500 pm.

[0173] The MPs concentrations for the study were 0.5, 1.0 and 2.0 pg / mL which were consistent with the quantities contained in foods.

[0174] Chitosans were added to the MPs under analysis only at the concentration of 0.5 mg / mL and 1 mg / mL. They were dissolved in distilled water at 35 °C for 24 hours and P024280WO-01 Notarbartolo & Gervasi S.p.A. at pH 3 (simulating the pH of the stomach of a human being) following the addition of acetic acid 0.1 N.

[0175] The MPs of the different products were characterized by different colors: light yellow- orange for chitosan, violet for HDPE, gray for PVC and black for PET. The Confocal UniOvi Imaged software allows to calculate the entity of binding between particles. Three determinations were carried out for each sample.

[0176] The statistical analysis was carried out according to ANOVA and Tukey test.

[0177] The entrapment of MPs was evident only for Chito 3 and Chito 4 since for all the other chitosans (Chito 1 and Chito 2) the binding was almost inconsistent. Tables 6 and 7 below summarize the results respectively at the concentrations of 0.5 and 1 .0 mg / ml.

[0178] Table 6. Binding of different MPs with chitosans (Chitol to Chito 4) at 0.5 mg / mL; values as % of binding: mean of 3 determinations (corrections with 5 % cut-off)a(a => 2.6 % was consider as 5 %;< 2.6 % was considered 0 %) P024280WO-01 Notarbartolo & Gervasi S.p.A.

[0179] Chito 3 and Chito 4 were found significantly more active than Chito 1 and Chito 2 (ANOVA p< 0.05). The analysis was considering the data of all the MPs (HDPE, PVC, and PET) together and not for each polymer. The entire picture of the analysis was as follows: Chito 1 = Chito 2< Chito 3 = Chito 4. Chito 3 and Chito 4 binding was at least the 60 % (from 60 % to 73 %) when MPs concentrations were 0.5 and 1 pg / mL, instead it was at least 42 % (from 42 % to 78 %) in case of MPs concentration of 2 pg / mL.

[0180] Table 7. Binding of different MPs with chitosans (Chito 1 to Chito 4) at 1 mg / mL; values as % of binding: mean of 3 determinations (corrections with 5 % cut-off)a(a = > 2,6 % was consider as 5 %;<2.6 % was considered 0 %)

[0181] Chito 3 and Chito 4 were found significantly more active than Chito 1 and Chito 2 (ANOVA p < 0.05). The analysis was considering the data of all the MPs (HDPE, PVC, and PET) together and not for each polymer. The results of the analysis were as P024280WO-01 Notarbartolo & Gervasi S.p.A. follows: Chito 1 = Chito 2 < Chito 3 = Chito 4.

[0182] When the concentration of MPs was between 0.5 to 2.0 pg / mL Chito 3 and Chito 4 were binding at least 63 % (from 63 % to 87 %).

[0183] The binding of these chitosans was dose dependent since the values obtained with 1 .0 mg / mL were significantly higher than those related to 0.5 mg / mL (ANOVA p< 0.05).

[0184] From the above description and the above-noted examples, the advantage attained by the product described and obtained according to the present invention are apparent.

Claims

1. P024280WO-01 Notarbartolo & Gervasi S.p.A.CLAIMS1. A use of at least one chitosan for binding and / or reducing the absorption of microplastics (MPs) and / or nanoplastics (NPs) in a human being, wherein said at least one chitosan has:- an intrinsic viscosity in the range from 90 to 400 cPs (90 to 400 mPa s) as measured with an automated capillary viscometer; and- a degree of deacetylation of at least 70.

2. The use according to claim 1 , wherein said use for binding is carried out in the stomach of said human being.

3. The use according to claim 1 or claim 2, wherein said MPs are selected from the group consisting of HDPE, PET and PVC.

4. A food supplement comprising at least one chitosan and at least one physiologically acceptable ingredient, wherein said at least one chitosan has:- an intrinsic viscosity in the range from 90 to 400 mPa s as measured with an automated capillary viscometer; and- a degree of deacetylation of at least 70.

5. The food supplement according to claim 4, wherein said at least one physiologically acceptable ingredient is selected from the group consisting of an auxiliary agent, a filler, an amino acid, a protein, a fatty acid, a carbohydrate, a vitamin, a mineral, a botanical extract obtained from plants or cell cultures, an enzyme, a binding agent, a colouring agent, a preservative, an acidif ier, a flavour and an aroma.

6. A cosmetic use of the food supplement according to claim 4 or 5 for eliminating MPs or NPs from a human being.

7. A pharmaceutical composition comprising at least one chitosan and at least a pharmaceutically acceptable carrier for use in the treatment and / or prevention of a disease derived from the MPs or NPs in a human being, preferably a gastrointestinal disease or a cardiovascular disease, wherein said at least one chitosan has:- an intrinsic viscosity in the range from 90 to 400 mPa s as measured with an automated capillary viscometer; and- a degree of deacetylation of at least 70.23P024280WO-01 Notarbartolo & Gervasi S.p.A.

8. The use according to anyone of claims 1 -3 or the food supplement according to anyone of claims 4.-5 or the pharmaceutical composition for use according to claim 7, wherein the intrinsic viscosity of said at least one chitosan is in the range from 110 to 400 mPa s, preferably from 250 to 400 mPa s as measured with an automated capillary viscometer.

9. The use according to anyone of claims 1 -3, 8 or the food supplement according to anyone of claims 4,5, 8 or the pharmaceutical composition for use according to anyone of claims 7-8, wherein said at least one chitosan has a Molecular Weight in the range from 100 to 450 KDa, preferably from 140 to 450 KDa or from 100 to 140 KDa as obtained according to Mark-Houwink-Sakurada equation.

10. The use according to anyone of claims 1 -3,8,9 or the food supplement according to anyone of claims 4, 5, 8, 9 or the pharmaceutical composition for use according to anyone of claims 7-9, wherein said at least one chitosan has a length in the range from 310 to 1400 nm, preferably from 400 to 1400 nm as measured with atomic force microscopy and a number of monomers in the range from 590 to 2700, preferably from 900 to 2700 as measured with atomic force microscopy.11 . The use according to anyone of claims 1 -3,8,9, 10 or the food supplement according to anyone of claims 4,5,8,9,10 or the pharmaceutical composition for use according to anyone of claims 7-10, wherein the degree of deacetylation of the at least one chitosan is in the range from 80 to 98, more preferably from 85 to 95.

12. The cosmetic use according to claim 6 or the pharmaceutical composition for use according to any one of claims 7-11 , wherein said at least one chitosan is administered, preferably orally administered, in the amount from 0.5 to 1 .0 g / day.

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