Methods of obtaining uniaxial and coaxial nanofibers electrofired with silver nanoparticles, uniaxial and coaxial nanofibers electrofired with silver nanoparticles and their use

Description

METHODS FOR OBTAINING UNIAXIAL AND COAXIAL NANOFIBERS ELECTROSPOUND WITH SILVER NANOPARTICLES, UNIAXIAL AND COAXIAL NANOFIBERS ELECTROSPOUND WITH NANOPARTICLES OF SILVER AND ITS USE FIELD OF THE INVENTION

[001] The present invention relates to a method for obtaining uniaxial and coaxial electrospun nanofibers incorporated with silver nanoparticles (AgNPs), with potential antimicrobial activity, and their use for the production of materials with applications in the biomedical / hospital field. The invention is related to the fields of nanotechnology and tissue engineering.

[002] The application area of ​​the present invention is nanomaterials, particularly in the development of uniaxial and coaxial nanofibers for the production of smart fabrics for the manufacture of personal protective equipment (PPE). FUNDAMENTALS OF THE INVENTION

[003] In general, one of the major challenges faced in combating microbial infections relates to controlling the spread of microorganisms. In this scenario, personal protective equipment, such as gloves, masks, gowns, and lab coats, are the main protective items. However, the materials available on the market only provide protection, but are not capable of inhibiting the growth or killing microorganisms. Thus, even if PPE is sufficient for protection during its use, contamination can occur during its removal or disposal. It is worth noting that this situation has worsened considerably in recent years, having in Petition 870260056447, dated 10 / 06 / 2026, page 5 / 45 2 / 38 In view of the pandemic associated with the coronavirus, the scientific community has been working hard to develop PPE that is capable not only of protecting but also of inhibiting microorganisms.

[004] In view of this, electrospun nanofibers incorporating silver nanoparticles are promising alternatives given their low production cost, ease of acquisition, and the possibility of being applied not only in the manufacture of PPE, but also in the coating of hospital fabrics, such as sheets and curtains, controlling the spread of microorganisms, especially those resistant to multiple drugs. Thus, the present invention brings the development of nanoscale fibers, obtained from the electrospinning technique, with great potential for the production of fabrics with antimicrobial properties.

[005] Structurally, nanofibers are polymeric filaments with diameters less than 1 µm. These ultrathin materials exhibit unique characteristics, such as high surface area per unit volume, high porosity, and high flexibility. Thus, they are nanomaterials with potential applications in the biomedical field, tissue engineering, and controlled drug delivery. Furthermore, they can be used in industrial processes, high-efficiency filter media, protective clothing, and other technological applications.

[006] The nanofibers obtained in the present invention are incorporated with silver nanoparticles (AgNPs), whose antimicrobial action has been reported in the literature. In fact, the AgNP-incorporated nanofibers of the present invention Petition 870260056447, dated 10 / 06 / 2026, page 6 / 45 3 / 38 showed excellent potential in killing the Sarscov2 virus. Furthermore, the process for obtaining them, developed by the present invention, allows the nanofibers to be obtained and the AgNPs to be formed before electrospinning.

[007] In general, the state of the art presents a series of polymer combinations (polymer blends) capable of forming nanofibers with suitable diameters and mechanical properties. However, when seeking the incorporation of silver nanoparticles, even the most modern methods still employ post-loading. In these methods, the nanoparticles are prepared separately from the nanofibers and then adsorbed onto the fabric. In such traditional methods, nanofibers incorporating silver nanoparticles are obtained using toxic reducing agents, such as tannic acid and sodium borohydride. Another widely used methodology for obtaining AgNPs in fabrics is the method of post-treatment of nanofibers, such as, for example, using heat treatment at high temperatures, microwave radiation, or ultraviolet radiation.Therefore, these traditional methods / processes for obtaining these nanofibers are more laborious and time-consuming, as they involve a greater number of steps, reagents, and instrumentation, and are thus contrary to the principles of green chemistry.

[008] Furthermore, although some more modern state-of-the-art works cite the term in situ in the description of the process for obtaining silver-embedded nanofibers, in general, such works employ the aforementioned Petition 870260056447, dated 10 / 06 / 2026, page 7 / 45 4 / 38 post-loading, in which the nanoparticles are prepared separately from the nanofibers and then adsorbed onto the fabric. That is, in these production processes, at least one reducing agent or post-treatment is used, unlike the present invention. Among the limitations of such work, one can also highlight the reduced capacity for incorporating nanoparticles, as well as the self-aggregation of silver nanoparticles, which leads to the inhibition of the antimicrobial activity of the material obtained.

[009] The state of the art reports some advances in the field of nanomaterials, especially on the electrospinning technique, as presented in the review article of MINGHUAN LIU et al., 2017 (Electrospun Nanofibers for Wound (Healing), which cites several related works in the field. However, in all the cited works, at least reducing agents (often toxic, such as tannic acid, for example) are added, or some type of post-treatment (such as microwave radiation) is performed. In general, although the works mention the term in situ, in practice, all employed one of the following options: additional reducing agents, heat treatment after obtaining the nanofibers, or ultraviolet irradiation. In contrast, the present invention enables the production of reproducible nanofibers containing AgNPs without the need for the addition of toxic reducing agents or post-treatment. Thus, the present method involves a smaller number of steps, reagents, and instrumentation. It is worth highlighting that in the present invention, the polymer constituting the nanofibers themselves, polyvinyl alcohol (PVA), will act in the reduction and stabilization of silver. Petition 870260056447, dated 10 / 06 / 2026, p. 8 / 45 5 / 38

[010] The work of HUSSEIN, MAM; et al., 2021 (“Dual-Drug Delivery of Ag-Chitosan Nanoparticles and Phenytoin Via Core-Shell Pva / Pcl Electrospun Nanofibers”) describes the application of electrospun nanofibers, with a coaxial core-shell structure, in the production of membranes with antimicrobial activity and cell proliferation properties. The nanofibers obtained are composed of polycaprolactone (PCL) and polyvinyl alcohol (PVA) polymers, and are incorporated with silver-chitosan nanoparticles (AgNPs-CHT) in the fiber core. However, the methodology is presented incompletely, lacking information about the reducing agent, as well as the contact time with the reducing agent for silver reduction. Another difference is that the authors use only PVA / PCL in the production of coaxial nanofibers, while chitosan (CHT) is only used to obtain AgNPs.Conversely, in the present invention, the final product is obtained with a reduced number of steps, without the need to add additional reducing agents. Furthermore, the AgNPs obtained by the authors have larger diameters than those obtained in the present invention, a fact that limits their antimicrobial activity.

[011] WO 2021 / 055939 describes core-envelope coaxial fibers with electrical conductivity, which may include silver nanoparticles in the fiber envelope, obtained by electrospinning. It also describes the use of PCL as candidate polymers in the composition of nanofibers, as well as their use in the production of fabrics. The document provides a general and simplistic approach to obtaining electrospun fibers. Petition 870260056447, dated 10 / 06 / 2026, page 9 / 45 6 / 38 coaxial fibers consisting of an electrically conductive inner material (“core”) and a second outer polymer. Furthermore, polymer blends are used in the construction of the coaxial nanofibers, where the use of PVA has not been described. Distinctly, the differentiating factor of the present invention is the way in which the silver nanoparticles are obtained and the polymer blends used, a fact not observed in WO 2021 / 055939.

[012] Document IN 2021 / 41030688 describes core-shell nanofibers obtained by coaxial electrospinning, which include antimicrobial agents in the shell, such as plant extracts and antibiotics, and a process for obtaining them. Medical uses are also described, such as in the production of wound dressings. The document provides a general overview of coaxial electrospinning, mainly in constitutional terms, but falls short in operational terms, as it only mentions process protection. In contrast, the focus of the present invention is the final product obtained. The electrospinning technique itself is already widely established in the state of the art, and was employed in the present context only as a means to obtain the products.

[013] The work of KHODKAR, F. & EBRAHIMI, NG; 2017 (“Preparation and Properties of Antibacterial, Biocompatible Core-shell Fibers Produced by Coaxial Electrospinning”) describes core-shell fibers with antimicrobial properties through the incorporation of silver nanoparticles. The document also describes the use of dimethylformamide (DMF) as a reducing agent in obtaining AgNPs of the Ag-PCL type and the use of PVA / PCL blends. Petition 870260056447, dated 10 / 06 / 2026, page 10 / 45 7 / 38 In contrast, the present invention utilizes PVA / CHT polymer blends, and no additional reducing agents or heat treatment are used after obtaining the nanofibers. Furthermore, the fibers obtained by KHODKAR, F. & EBRAHIMI have larger diameters than those obtained in the present invention, which, in some cases, cannot even be considered nanofibers.

[014] Document CN 2013 / 10664371 describes core-shell metal / polymer nanofibers with silver or copper nanoparticles in the fiber envelope, obtained by electrospinning. The document also describes the methods for obtaining them and their possible uses in medical and biological engineering. In contrast, in the present invention, the metallic nanoparticles are coated with polymers, wherein these polymers act as the reducing and stabilizing agent that will support the metallic nanoparticles in their nanofibrous matrix. Furthermore, document CN 2013 / 10664371 does not use electrospinning, or any other method for obtaining fibrous matrices, so the material obtained could not be used in the production of dressings or in the manufacture of personal protective equipment.On the other hand, in the present invention, the use of electrospinning allows the materials obtained to be applied in the production of personal protective equipment, without the need for sequential processing.

[015] Document WO 2020 / 159946 describes coaxial electrospun nanofibers with antimicrobial properties, with a core-shell structure. The document also describes the use of silver nanoparticles as an antimicrobial agent. Petition 870260056447, dated 10 / 06 / 2026, page 11 / 45 8 / 38 present in the fiber core. WO 2020 / 159946 also claims nanofibrous structures comprising an amphiphilic block copolymer, a hydrophobic polymer, and an antimicrobial agent. The document discloses the production of nanofibers, whose application is limited to obtaining dressings for the treatment of infections. In the present invention, block copolymers are not used, but rather biopolymers, and the antimicrobial agent is obtained during the process. Furthermore, in the present invention, the nanofibers obtained can be used both in obtaining dressings (such as in the treatment of wounds, for example) and in obtaining personal protective equipment. In addition, biopolymers and processing conditions consistent with the principles of green chemistry are used.

[016] US patent 11,124,897 uses coaxial electrospinning for the controlled carrying and delivery of antibiotics and / or other bioactive agents loaded into the core and / or bark layer of the fibrous platform, released in a controlled manner. However, US patent 11,124,897 is limited to obtaining dressings for the treatment of infections. In contrast, the present invention uses biopolymers and processing conditions consistent with the principles of green chemistry.

[017] US patent 2017 / 0042822 refers to a nanofibrous membrane and more particularly to a production process from PCL and hyaluronic acid (HA), the matrix being capable of inhibiting the growth of microorganisms. Ultraviolet radiation is used to induce the formation of AgNPs. However, in the present invention, the AgNPs are obtained without the need for any post-treatment of the nanofibers. Petition 870260056447, dated 10 / 06 / 2026, page 12 / 45 9 / 38 or solutions, thus having the advantage of being a simpler and more accessible procedure.

[018] Document CN105332163 relates to the preparation of electrospun nanofibers containing adsorbed silver nanoparticles. To achieve this objective, the process is proposed to be carried out in four steps, namely: (1) preparation of a mixed solution of polyethylene oxide (PEO) and carboxymethylcellulose (CMC); (2) preparation of PEO / CMC composite nanofibers by electrospinning; (3) adsorption of ions from a silver nitrate solution; (4) obtaining silver nanoparticles by reduction using ultraviolet light. The main disadvantage to be highlighted is the number of steps required to obtain the final sample, increasing the chances of contamination, reducing reproducibility, and raising energy and production costs. Furthermore, silver nanoparticles are obtained by photoinduction, making it necessary to use UV light sources. In this scenario, the dependence on the distribution of silver ions by the matrix can also be highlighted, something that is difficult to control and / or reproduce. In contrast, the present invention uses a technique that involves only two steps, one being the preparation of the working solutions and the other, the single-step electrospinning. In this way, the working time, as well as the production costs, are reduced, and the method presents high reproducibility.

[019] Document EP 3 165 511 discloses the formation of drying films, in which the formation of silver nanoparticles is achieved by employing organic reducing agents. Petition 870260056447, dated 10 / 06 / 2026, page 13 / 45 10 / 38 However, the method employed in obtaining the films does not impart to the material the nanofibrous character obtained by electrospinning. Furthermore, nanoparticles depend on the use of reducing agents which, in most cases, are toxic to the human body. In contrast, the present invention uses a technique that enables the production of a nanofibrous material whose main characteristics include high surface area, porosity, and uniformity, essential for the biomedical field of application. Moreover, the silver nanoparticles are obtained without the use of any toxic reducing agents.

[020] US patent 7,410,650 B2 uses a methodology involving at least four steps: preparation of the solution in an organic solvent; then, a silver salt and a reducing agent are added to the dispersing solution; the dispersing solution is then stirred to form silver nanoparticles; the silver salt reacts with the reducing agent to distribute the silver nanoparticles uniformly in the organic solution; subsequently, a polymer resin for spinning is added to the organic solution. Finally, a wet spinning process is used to obtain the nano-silver fibers. Among the main disadvantages of the patent are the several steps involved in the process of obtaining the final material and the formation of silver nanoparticles with diameters of approximately 100 nm. However, for the inhibition of microorganisms, several studies have shown that nanoparticles with diameters smaller than 30 nm are ideal.In this respect, the present invention discloses a reproducible method in which the silver nanoparticles obtained have sizes smaller than 30 nm, making them ideal for... Petition 870260056447, dated 10 / 06 / 2026, page 14 / 45 11 / 38 Microorganism control.

[021] US patent 9,505,027 B2 focuses on the immobilization of silver nanoparticles on solid substrates with different surface properties. The method involves two steps, the first being the covalent modification of the polymer used. Furthermore, the nanoparticles are immobilized within the structure. Among the main disadvantages of the patent, the first point to highlight is the covalent modification of the polymer, which increases production costs and hinders the achievement of the final product, since the success of the initial modification becomes a limiting step in obtaining the final product. In addition, the nanoparticles are immobilized on a solid substrate, which limits their area of ​​action. In contrast, the present invention does not involve any type of polymer modification. Furthermore, the nanoparticles are formed in a nanofibrous matrix that does not impede their movement / release from the material, increasing their area of ​​action.

[022] US patent 2009 / 0130186 provides wound dressing sets, systems and methods that utilize nanomaterials, such as silver nanoparticles embedded in a hydrophilic polymer sponge material, such as chitosan. Among the main disadvantages of said patent are the use of a series of steps, each step being performed by different routes, and the use of additional reducing agents. In contrast, the present invention proposes a method for obtaining the final material in a simple manner and without the addition of additional toxic reducing agents. Petition 870260056447, dated 10 / 06 / 2026, page 15 / 45 12 / 38

[023] Moreover, others from the state of the art, such as WEI, X., et al., 2021, “Controlled Release of Monodisperse Silver Nanoparticles via in Situ Cross-Linked Polyvinyl Alcohol as Benign and Antibacterial Electrospun Nanofibers” (Colloids Surfaces B Biointerfaces); WILEY BLOSI, M., et al, 2021, “Polyvinyl Alcohol / Silver Electrospun Nanofibers: Biocidal Filter Media Capturing Virus-Size Particles” (J. Appl. Polym. Sci); YANG, Y., et al. 2020, “A Facile Method for the Fabrication of Silver Nanoparticles Surface Decorated Polyvinyl Alcohol Electrospun Nanofibers and Controllable Antibacterial Activities” (Polymers); KOWSALYA, E.; et al., 2019, “Electrospun Nanofibers for Potential The articles "Antimicrobial Food Packaging Applications" (Food Packaging Shelf Life) and AUGUSTINE, R.; et al., 2018, "Incorporated with Green Synthesized Silver Nanoparticles for Wound Dressing Applications" (J. Mater. Sci. Mater. Med) also describe obtaining nanofibers incorporated with nanoparticles, but they do not present the advantage of the present invention of obtaining them by reduction in the polymer component of the nanofibers themselves.

[024] Considering the state of the art, the present invention brings as an innovation the obtaining of uniaxial and coaxial nanofibers capable of incorporating silver nanoparticles “in situ”, that is, the AgNPs participate in the nanofiber formation step without employing any additional reducing agent (which are generally toxic), post-heat treatment or UV irradiation, something unprecedented in the literature until then, essential in the development of PPEs that act in the protection and control of microorganisms, that is, in the production of “smart PPEs”. In the present Petition 870260056447, dated 10 / 06 / 2026, page 16 / 45 13 / 38 invention, the polymer itself is used as a reducing and stabilizing agent that supports the metallic nanoparticles in its nanofibrous matrix. In this way, a simplified, inexpensive and efficient method is provided for obtaining uniform electrospun nanofibers with silver nanoparticles, in which a reduced number of steps is employed, and which presents processing conditions consistent with the principles of green chemistry.

[025] In summary, the present invention presents a method for obtaining nanofibers, formed by nanoparticles pre-nucleated in the polymer solution, and the AgNPs are formed before electrospinning. This method does not employ post-loading, traditionally used in the state of the art, and therefore does not employ treatments and / or compounds considered toxic. Furthermore, the silver nanoparticles are incorporated into the core of the coaxial nanofibers, and not into their envelope, which provides low polydispersity and excellent distribution in the produced fabric, in addition to being presented in colloidal form and used in polymeric combinations. In this way, a nanoscale material is obtained with high reproducibility, high surface area, high porosity and high flexibility, compared to materials obtained by widely known methods in the state of the art. BRIEF DESCRIPTION OF THE INVENTION

[026] In summary, the present invention relates to a method for obtaining uniaxial and coaxial electrospun nanofibers incorporating silver nanoparticles (AgNPs) with antimicrobial activity, to said nanofibers obtained Petition 870260056447, dated 10 / 06 / 2026, page 17 / 45 14 / 38 by the same and its uses intended for the production of materials with application in the biomedical / hospital field. The invention is related to the fields of nanotechnology and tissue engineering.

[027] The method for obtaining the present invention comprises the steps of (a) producing a combination of polymers (PVA and CHT) to form the polymer blend of the fiber core, in which pre-nucleated silver nanoparticles are formed in the polymer blend; and (b) obtaining by electrospinning uniaxial or coaxial nanofibers, with an average diameter on the order of 300-350 nm, consisting of the core blends (PVA / CHT / AgNPs) and the envelope (PCL), in which the silver nanoparticles (AgNPs) are formed before electrospinning.

[028] Additionally, the present invention relates to uniaxial and coaxial electrospun nanofibers incorporating silver nanoparticles (AgNPs) obtained by the aforementioned method, with antimicrobial activity, with an average diameter in the order of 300 to 350 nm, consisting of core blends (PVA / CHT / AgNPs) and envelope blends (PCL).

[029] Finally, the present invention relates to the use of said nanofibers for the production of materials with application in the biomedical / hospital field. BRIEF DESCRIPTION OF THE FIGURES

[030] Figure 1 shows in (AB) 10% (m / V) PCL nanofibers; (C) Diameter distribution of the nanofibers PCL.

[031] Figure 2 shows in (AB) 10% (w / V) PCL nanofibers and AgNPs by the post-loading method; (C) Distribution of PCL nanofiber diameters; (D) Petition 870260056447, dated 10 / 06 / 2026, page 18 / 45 15 / 38 X-ray energy dispersive spectroscopy (EDS) of nanofibers.

[032] Figure 3 shows in (AB) 12% (w / V) PVA nanofibers and AgNPs in situ; (C) Diameter distribution of PCL nanofibers; (D) EDS of nanofibers.

[033] Figure 4 shows the electronic absorption spectra of AgNPs released from PVA-AgNP nanofibers obtained in situ; (B) Release Kinetics.

[034] Figure 5 shows the diameter size distribution by number of AgNPs released from PVA-AgNP nanofibers in situ.

[035] Figure 6 in (AB) shows the scanning electron microscopy of the PVA / CHT nanofibers (70 / 30); (C) Distribution of nanofiber diameters.

[036] Figure 7 in (AB) shows the scanning electron microscopy of the PVA / CHT / AgNPs nanofibers in situ (70 / 30); (C) Distribution of nanofiber diameters.

[037] Figure 8 shows the infrared spectroscopy (ATR) spectra of PVA, PVA / CHT and PVA / CHT / AgNPs nanofibers (in situ).

[038] Figure 9 shows (A) scanning electron microscopy of coaxial nanofibers embedded with AgNPs in situ and (B) diameter distribution of coaxial nanofibers embedded with AgNPs in situ.

[039] Figure 10 in (AB) shows the X-ray diffractograms in (A) constituent polymers of nanofibers and (B) uniaxial and coaxial nanofibers.

[040] Figure 11 in (A and B) shows the thermogravimetric analysis (TGA) curves for the precursor polymers and nanofibers; (B and D) the curves of Petition 870260056447, dated 10 / 06 / 2026, page 19 / 45 16 / 38 derivative thermogravimetry (DTG) for precursor polymers and nanofibers.

[041] Figure 12 shows the antibacterial activity of PVA and PVA / CHT nanofibers incorporated with AgNPs in controlling gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria. DETAILED DESCRIPTION OF THE INVENTION

[042] The present invention relates to a method for obtaining uniaxial and coaxial electrospun nanofibers incorporated with silver nanoparticles (AgNPs) of antimicrobial activity, to said nanofibers obtained therefrom and to their uses for the production of materials with application in the biomedical / hospital field. The invention is related to the fields of nanotechnology and tissue engineering.

[043] The aforementioned method of obtaining the present invention comprises the following steps: (a) to produce a combination of polymers (PVA and CHT) to form the core polymer blend of the fiber, in which pre-nucleated silver nanoparticles are formed in the polymer blend; and (b) to obtain uniaxial or coaxial nanofibers by electrospinning, with an average diameter on the order of 300-350 nm, consisting of core blends (PVA / CHT / AgNPs) and envelope blends (PCL), in which silver nanoparticles (AgNPs) are formed before electrospinning.

[044] To demonstrate the potential of the aforementioned method for obtaining nanofibers, the aforementioned steps will be detailed further from the perspective of the procedures / implementations. Petition 870260056447, dated 10 / 06 / 2026, page 20 / 45 17 / 38 performed, as well as the results obtained. It should be noted that the following description is only intended to elucidate the understanding of the proposed invention and reveal, in more detail, the implementation of the invention without limiting it to the same. Thus, variables similar to the example are also within the scope of the invention.

[045] In the present invention, the following polymers are employed: 85% deacetylated chitosan with a viscometric molar mass (Mv) of 87 kDa acquired from GoldenShell Biochemical (China), poly-ε-caprolactone (PCL - Sigma Aldrich) with a molar mass of 80,000 gmol-1 and polyvinyl alcohol (PVA - Neon) with a molar mass of 104,500 gmol-1. In addition, silver nitrate (AgNO3), dimethylformamide, dichloromethane and acetic acid are employed, all obtained from Sigma Aldrich. The water used in all stages is ultrapure.

[046] Electrospun nanofibers were also tested for crosslinking polyvinyl alcohol (PVA) and chitosan. In general, different carboxylic acids have been reported as crosslinking agents for PVA. In the present invention, the most favorable tricarboxylic acid crosslinking agent was citric acid. However, other crosslinking agents can be selected from carboxylic acids of different types, such as mono-, di-, tri- and polycarboxylic acids, and also from their derivatives such as acid anhydrides and acid chlorides, among which the following can be highlighted: formic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, malic acid, fumaric acid, tartaric acid, suberic acid, terephthalic acid, 4 Petition 870260056447, dated 10 / 06 / 2026, page 21 / 45 18 / 38 sulfophthalic acid, sulfosuccinic acid, furanodicarboxylic acid, butane-1,2,3,4-tetracarboxylic acid, maleic anhydride, ethylenediaminetetraacetic dianhydride, 3,3',4,4'-benzophenone tetracarboxylic dianhydride, 4,4-oxyphthalic anhydride, and pyromellitic dianhydride. Crosslinking involving citric acid was performed using 20% ​​by mass of citric acid relative to the total mass of the polymers (PVA and CHT) and a temperature of 110°C. It is worth noting that the process involves the addition of citric acid (20% by mass relative to the other polymers) during the preparation of the solutions for electrospinning. After obtaining the nanofibrous matrix, the crosslinking process is carried out. For this, the nanofibers are left in an oven for a period of 16 hours.

[047] Nanofibers made up of PVA and CHT polymer blends (PVA / CHT and PVA / CHT / AgNPs; tables 2, 3 and 4) are obtained by uniaxial electrospinning. In addition, nanofibers with coaxial injection [PCL(PVA / CHT)] and [PCL(PVA / CHT / AgNPs)] with a core-shell structure are also obtained, the latter from the experimental arrangement as shown in Table 1. Table 1: Preparation of solutions for electrospinning. Material %(m / V) Solvent Conditions PCL 10.0 DMF / DCM (1:1) 1h at 25°C CHT 10.0 H2O + 2% acetic acid 3h at 60°C PVA 12.0 H2O 3h at 80°C * PVA-AgNO3 12.0; 0.25 H2O 3h at 80°C * * Reflux Initial screening: obtaining uniaxial nanofibers.

[048] PCL, CHT and PVA nanofibers are primarily Petition 870260056447, dated 10 / 06 / 2026, page 22 / 45 19 / 38 evaluated in isolation. All experimental conditions employed are presented in Table 1.

[049] After obtaining the polymer solutions, as per Table 1, each is subjected to the electrospinning process separately to obtain nanofibers of “isolated polymers”. For this, each solution is carefully transferred to a conventional 10 mL syringe (internal diameter equal to 14 mm). In addition, the syringes are coupled to a metallic capillary (needle) whose tip is previously cut (internal diameter between 0.10 and 2.00 mm). The mixture is kept in the syringe under constant pressure (flow varying between 0.25 mL h-1 and 2.00 mL h-1). To obtain the fibers, an electrical potential difference (voltage) (varying between 10 and 25 kV) is applied between the tip of the needle and the flat spherical metallic trimmer (diameter = 25 cm) separated by a distance (varying between 5 and 30 cm).

[050] AgNO3 is used as a precursor to obtain silver nanoparticles (AgNPs), the main antimicrobial agent in the formulation.

[051] After obtaining nanofibers containing “single polymers” (initial screening), the polymer combination step (PVA and CHT) is carried out, that is, the formation of polymer blends, in order to obtain improved properties compared to the isolated species. Information regarding each polymer blend is presented in Table 2. Table 2: Incorporation of drugs and obtaining polymer blends for electrospinning. Sample Material %(m / V) Solvent Conditions % Blend Petition 870260056447, dated 10 / 06 / 2026, page 23 / 45 20 / 38 PVA / CHT PVA 12.0 H2O 3h at 80°C * 70 CHT 10.0 H2O / 2% CH3COOH 3h at 60°C 30 PVA / CHT / AgNPs PVA / AgNO3 12.0 / 0.25 H2O 3h at 80°C * 70 CHT 10.0 H2O / 2%CH3COOH 3h at 60°C 30 *Reflux

[052] To obtain the blends, the solutions (70% PVA and 30% CHT (v / v) are mixed, always adding the chitosan solution to the PVA solution. The sample is kept under agitation for fifteen minutes to ensure the homogeneity of the polymer blend.

[053] After initial screenings and definition of electrospinning parameters for all samples, nanofibers are obtained. In general, the solutions are transferred to a conventional 10 mL syringe with a 0.7 mm diameter metal capillary (needle). The mixture is kept in the syringe under constant pressure with a characteristic flow rate of 0.50 mL h-1. To obtain the fibers, an electrical potential difference (ddp) of approximately 23 kV is applied between the needle tip and the flat spherical metal trimmer (diameter = 25 cm) separated by a distance of cm. The same electrospinning parameters are used to obtain nanofibers made up of the blends presented in Table 2. Obtaining Coaxial Nanofibers

[054] Next, coaxial nanofibers are obtained, that is, those with a core-envelope structure. For this, the same parameters described above are followed. In all cases, the core was made up of Petition 870260056447, dated 10 / 06 / 2026, page 24 / 45 21 / 38 polymer blends are presented in Table 2 (PVA / CHT and PVA / CHT / AgNPs), and other solutions / blends used are presented in Table 3. The envelope was composed of PCL solution (see Table 3). For coaxial electrospinning, two syringes are necessary, one for the inner solution (core) and the other for the outer solution (envelope). Thus, a coaxial needle is used to combine the solutions. Table 3: Composition of coaxial nanofibers. Sample Shell composition Core composition Final sample 5 PCL PVA / CHT PCL[PVA / CHT] 6 PCL PVA / CHT / AgNPs PCL[PVA / CHT-AgNPs]

[055] In general, the internal (core) and external (shell) solutions are transferred to two conventional 10 mL syringes with the coaxial needle attached, with a metal capillary (needle) diameter of 0.7 mm. The mixture is kept in the syringes under constant pressure with a characteristic flow rate of 0.50 mL h-1. To obtain the fibers, an electrical potential difference (ddp) of approximately 23 kV is applied between the needle tip and the flat spherical metal trimmer (diameter = 25 cm) separated by a distance of cm.

[056] In summary, the nanofibers thus obtained exhibit high surface area, high porosity, and high flexibility. Additionally, the material obtained from nanofibers electrospun with silver nanoparticles shows great potential for use in the manufacture of “smart fabrics” with potential applications in the field Petition 870260056447, dated 10 / 06 / 2026, page 25 / 45 22 / 38 biomedical / hospital. In the tests performed, the tissues showed excellent potential for inhibiting the growth of gram-positive and gram-negative bacteria, in addition to showing excellent potential in killing Sars-cov2. Characterization of Nanofibers

[057] All nanofibers obtained (uniaxial and coaxial) were evaluated by optical microscopy, scanning electron microscopy (SEM) and infrared spectroscopy (ATR). In addition, nanofibers containing AgNPs were also evaluated by energy-dispersive X-ray spectroscopy (EDS), thermogravimetric analysis (TGA), X-ray diffraction (XRD) and mechanical properties (texturometer). In vitro release study of AgNPs from nanofibers

[058] For conducting in vitro drug release studies, samples of nanofibers with dimensions 5x5 cm² panels were placed in contact with 10 ml of the release medium (phosphate-buffered saline; pH 7.4). The temperature and agitation of the system were fixed at 37°C and 50 rpm, respectively. An aliquot of the sample was withdrawn at specific time intervals. Each aliquot was analyzed by electron absorption, measuring the absorbance at 430 nm from the previously constructed calibration curve. Antibacterial activity of nanofibers against S. aureus and E. coli. Growth Method

[059] The growth method is carried out using three to five well-isolated colonies of the same morphological type selected from the agar plate. The surface of Petition 870260056447, dated 10 / 06 / 2026, page 26 / 45 23 / 38 Each colony is touched with a loop, and the microorganisms are transferred to a tube containing 4 to 5 mL of a suitable culture medium, such as tryptic soy broth. The culture is then incubated in broth at 35°C until it reaches or exceeds the turbidity of a 0.5 McFarland standard solution (generally two to six hours). Subsequently, the turbidity of the growing culture is adjusted with sterile saline or broth to obtain an optical turbidity comparable to that of the 0.5 McFarland standard solution. This results in a suspension containing approximately 1 to 2 x 10⁸ CFU / mL of E. coli ATCC® 25922. A spectrophotometer is used to perform this step. - Inoculation of Test Plates

[060] Under ideal conditions, a sterile cotton swab is dipped into the adjusted suspension, up to 15 minutes after adjusting the turbidity of the inoculum suspension. The swab should be rotated several times and pressed firmly against the inner wall of the tube, above the liquid level. This will help remove any excess inoculum from the swab. The dry surface of the Mueller-Hinton agar plate is inoculated by rubbing the swab across the entire sterile surface of the agar. The procedure is repeated by rubbing two more times, rotating the plate approximately 60° each time, in order to ensure uniform distribution of the inoculum. As a final step, a swab is passed along the edge of the agar plate.

[061] The lid can be left ajar for three to five minutes, though never more than 15 minutes, in order to allow any excess moisture to be absorbed before the drug-impregnated discs are applied. Reading the License Plates and Interpreting the Results Petition 870260056447, dated 10 / 06 / 2026, page 27 / 45 24 / 38

[062] After 16 to 18 hours of incubation, each plate is examined. If the plate was satisfactorily seeded, and the inoculum is correct, the resulting inhibition halos will be uniformly circular and there will be a confluent mat of growth. If individual colonies are apparent, the inoculum was too light and the test should be repeated. The diameters of the total inhibition halos (judged with the naked eye) are measured, including the diameter of the disc. The halos were measured in millimeters using a caliper that is placed against the back of the inverted petri dish. The petri dish was positioned a few inches above a non-reflective background and illuminated with reflected light. Virucidal Assay Table 4: Cell and Virus tested: Coronavirus strain MHV, genus Betacoronavirus (same genus as SARS-CoV-1, SARS-CoV-2, MERS and other species). Virus Cell Lines Coronavirus MHV Cell: L929-NCTC clone 929 [L cell, L-929, derivative of strain] (ATCC® CCL-1TM)

[063] The assays were performed in an NB-2 (Biosafety Level 2) laboratory following the ANVISA Recommendations Art. 1 and Art. 3 of IN 04 / 13 and IN 12 / 16 and methodologies described in the standards (BS ISO 21702:2019 “Measurement of antiviral activity on plastics and other non-porous surfaces”, BS EN 14476:2013+A2:2019 and the Robert Koch Institute - RKI) and complying with Good Laboratory Practices (GLP). The tests were performed in quadruplicate (four repetitions) biological: Culture medium for viruses and cell line was Dulbecco minimum essential medium (DMEM) containing 10% Petition 870260056447, dated 10 / 06 / 2026, page 28 / 45 25 / 38 of fetal bovine serum.

[064] Coronavirus (MHV-3 strain) titration was performed according to the DICT50 (Tissue Culture Infectious Doses 50%) method, where sequential dilutions of the virus on a base of 10 were performed in quadruplicate in sterile 96-well microplates. Subsequently, L929 cells were added at a concentration of 2 x 10⁵ cells / well. After 48 hours, the cytopathic effect (CPE) of viral infection was verified, in comparison with cellular and viral controls.

[065] The first stage of the tests was to perform the “Determination of the Maximum Non-Toxic Concentration (MTC)” in the different cells tested, to determine the concentration that does not cause toxicity to the cells, since the test substance must be active only against the virus and not against the cells.

[066] Test samples “PVA / AgNPs (uniaxial), PVA / CHT AgNPs (uniaxial), PCL[PVA / CHT] and PCL[PVA-CHT-AgNPs] (coaxial) were treated individually. The materials were cut into five equal pieces and added to Coronavirus, in DMEM Culture medium, previously titrated and subjected to different contact times (30 minutes, 1, 8, 24 and 64 hours), under gentle agitation.

[067] The supernatants of each material and after each reaction time were added to 96-well microplates with 100 pL of each test sample, 100 pL of virus that were titrated (101 to 1010). The entire system was added to L929 cells (2x105 cells / mL) previously cultured in 96-well plates and incubated at 37°C in an incubator with 5% CO2 for 48 hours.

[068] After 48 hours of incubation the plates were read Petition 870260056447, dated 10 / 06 / 2026, page 29 / 45 26 / 38 through an inverted microscope in search of the characteristic cytopathic effect of the virus, and the titers were calculated based on the method of REED and MUENCH, 1938. The results are expressed as a percentage of viral inactivation (Table 5) compared to the untreated viral control (virus titer).

[069] The controls used were: • Negative: cell control (2 x 10⁵ cells / mL) in DMEM medium, virus-free and without test sample; • Virus control: Virus titration (101 to 1012) and cell culture in DMEM medium; • Positive test: presence of virus, each sample tested and cell line in DMEM medium. Table 5: Results are expressed as a percentage of viral inactivation compared to the untreated viral control. Log Reduction Factor Percentage of Inactivation / Reduction 1 10 90% 2 100 99% 3 1000 99.9% 4 10,000 99.99% VIRUCIDAL 5 100,000 99.99% 6 1,000,000 99.99% https: / / microchemlab.com / information / log-and-percent-reductionsmi crobiology-and-antimicrobial-testing RESULTS Optimization and characterization of uniaxial and coaxial nanofibers

[070] Internal and external nanofiber solutions Petition 870260056447, dated 10 / 06 / 2026, page 30 / 45 27 / 38 coaxial fibers were initially evaluated independently. Scanning electron microscopy of the PCL fibers is shown in Figure 1.

[071] In general, PCL fibers in the absence of CUR were obtained with a high degree of uniformity (Figures 1A and 1B) and an average diameter of 800 nanometers.

[072] The interaction of PCL was also evaluated after post-loading with AgNPs prepared by ultrasound methods. The results obtained are presented in Figure 2.

[073] Figures 2A and 2B clearly show that despite the formation of fibers, they exhibited high heterogeneity regarding their thicknesses. Furthermore, the presence of silver clusters in the fiber structure is noted (Figure 2B), with diameters much larger than expected, thus representing an unfavorable factor. In addition, Figure Figure 2C shows an average diameter of approximately 1000 nanometers. Finally, Figure 2D shows the peak at 2.98 eV corresponding to metallic silver incorporated into the nanofibers. Despite the presence of silver, the heterogeneity of the fibers, associated with the diameter of the obtained AgNPs, makes it clear that the post-loading incorporation method, traditionally used in the state of the art, is not useful. Furthermore, it is observed that the thickness of the PCL fibers varies significantly with the incorporation of AgNPs.

[074] In view of this information, the interaction of PVA nanofibers with AgNPs was evaluated. The results obtained for the tests involving the in situ synthesis of AgNPs are presented in Figure 3.

[075] PVA nanofibers exhibited a high degree Petition 870260056447, dated 10 / 06 / 2026, page 31 / 45 28 / 38 uniformity with respect to thickness, absence of beads, and an average thickness of 400 nanometers. Furthermore, the EDS spectrum clearly demonstrates the presence of AgNPs in the fiber (signal at 2.98 eV). Additionally, the yellowish coloration of the obtained fibers indicates successful in obtaining AgNPs in situ. In this case, the fibers were obtained with greater homogeneity and less thickness.

[076] Under these conditions, the release kinetics of AgNPs from PVA nanofibers was evaluated, as shown in Figure 4.

[077] In general, the AgNPs were rapidly released from the PVA nanofibers. Figure 4A shows the emergence of an absorption peak with a maximum at 430 nanometers. Furthermore, a continuous increase in absorption intensity is observed up to 500 seconds. The kinetic data, Figure 4B, show a practically linear increase in the absorbance of the AgNPs up to 500 seconds. Below Figure 4, the stages of release over time are represented. The appearance of a yellowish coloration, characteristic of spherical AgNPs, is noted.

[078] The liberation solution was subsequently evaluated by dynamic light scattering to determine the hydrodynamic diameter (DH), polydispersity index, and Zeta potential of the liberated AgNPs. The results obtained are presented in Figure 5. Furthermore, the Zeta potential analyses (in triplicate) can be observed in Table 6. Table 6: Zeta potential analyses INPUT DATA File name: AgNPs.dts Petition 870260056447, dated 10 / 06 / 2026, page 32 / 45 29 / 38 Dispersant Name: Water Dispersant RI 1.330 Viscosity (cP) 0.8872 Dielectric Constant of Dispersant 78.5 SYSTEM Temperature (°C) 25.0 Count rate (kcps) 20.2 Cell definition disposable transparent zeta cell Zeta run 35 Measurement position (mm) 2.00 Attenuator 5 RESULTS (triplicate) Zeta Potential (mV): Zeta Potential (mV): Zeta Potential (mV): -4.45 -5.94 -4.97 Zeta Deviation (mV): Zeta Deviation (mV): Zeta Deviation (mV): 0.00 0.00 0.00 Conductivity Conductivity Conductivity (mS / cm): (mS / cm): 17.5 (mS / cm): 20.3 19.0 Quality of the Quality of the Quality of the result: good result: good good Mean Zeta Potential (mV) ± Standard Deviation: -5.12 ± 0.62

[079] The AgNPs released from the PVA-AgNP nanofibers in situ presented average diameters of approximately 20 nanometers and a polydispersity index of 0.5. According to the literature, AgNPs with the characteristics obtained are ideal for obtaining antimicrobial effects. Furthermore, the surface potential obtained was approximately -5.12 ± 0.62 mV, being close to Petition 870260056447, dated 10 / 06 / 2026, pages 33 / 45 30 / 38 neutrality, as expected (Table 6).

[080] Despite the excellent characteristics presented by the AgNPs obtained by the in situ methodology, the release kinetics occurred in a very short time interval. Therefore, to improve this release profile, PVA / CHT (70 / 30) nanofibers were produced. CHT is a natural and biocompatible polymer, extensively studied in the reconstruction of the extracellular matrix, in addition to presenting intrinsic antimicrobial effects. In this sense, its hydrophobic character will tend to decrease the release rate of the incorporated drugs. The results obtained are presented in Figure 6.

[081] The PVA / CHT nanofibers shown in Figure 6 were obtained with a degree of uniformity in relation to thickness, despite the presence of beads. In addition, it is noted that the fibers obtained presented very thin diameters, on the order of 150 nanometers, being ideal for application in wound treatment, ECM reconstruction, incorporation and release of drugs.

[082] In view of obtaining ultrafine fibers from the PVA / CHT blend (70:30), nanofibers of the same type were subsequently obtained, but in the presence of AgNO3 to obtain AgNPs in situ, in the same way as the nanofibers shown in Figure 6 were prepared. The results obtained are shown in Figure 7.

[083] The microscopy images shown in Figure 7 demonstrate that the presence of the silver salt did not affect the production of nanofibers, resulting in homogeneous fibers with fewer defects than those obtained in the absence of AgNO3 (Figure 6). Despite this, the diameter of the fibers remained practically the same. Petition 870260056447, dated 10 / 06 / 2026, pages 34 / 45 31 / 38 doubled due to the presence of salt and the consequent formation of AgNPs. It is worth noting that the formation of AgNPs can be attributed to the yellow coloration presented by the obtained fiber. The infrared spectra of the PVA, PVA / CHT, and PVA / CHT / AgNP nanofibers are shown in Figure 8.

[084] Figure 8 shows the infrared spectroscopy (ATR) spectra of PVA, PVA / CHT, and PVA / CHT / AgNPs nanofibers. The signals related to PVA are highlighted in Figure 8, while the signals related to CHT (at lower intensity) appear at 895 and 1150 cm⁻¹ and are associated with the vibration of the -COC- group of the saccharides. The peaks at 1030 and 1080 cm⁻¹ were attributed to the -CO stretching vibration, while those at 1652, 1587, and 1318 cm⁻¹ were attributed to the -NH bending of the amine groups I, II, and III, respectively. The symmetrical deformation mode of the -CH₃ group appears at 1374 cm⁻¹ and the stretching mode for the -CH bond at 2922 cm⁻¹. Finally, the broadband at approximately 3430 cm-1 corresponds to the -OH stretching vibration of the CHT.

[085] Considering the set of results obtained, it is verified that both PCL nanofibers and PVA / CHT nanofibers were successfully obtained by the coaxial electrospinning method. Furthermore, based on the thickness of the fibers obtained, it is concluded that PCL should be used in the envelope, i.e., in the shell of the coaxial nanofibers. On the other hand, the ultrafine PVA / CHT / AgNPs nanofibers in situ are consistent with the characteristics necessary for the composition of the core of the coaxial nanofibers.

[086] Based on the results obtained in the previous item (Optimization of uniaxial nanofibers), core-shell type nanofibers containing a core were obtained. Petition 870260056447, dated 10 / 06 / 2026, pages 35 / 45 32 / 38 consisting of PVA / CHT / AgNPs (70:30 w / w) and an envelope consisting of PCL, named PCL[PVA / CHT / AgNPs]. The results obtained are presented in Figure 9.

[087] The PCL[PVA / CHT / AgNPs] nanofibers (Figure 9) showed a degree of uniformity in relation to thickness (average diameter equal to 300 nm), despite the presence of beads. Furthermore, it is noted that the fibers obtained presented intermediate diameters between the uniaxial nanofibers referring to the core and the sheath, being suitable for application in wound treatment, extracellular matrix reconstruction, incorporation and release of drugs, or even in the production of personal protective equipment. The X-ray diffractograms obtained are presented in Figure 10.

[088] First, the precursor polymers of the nanofibers were evaluated for XRD (Figure 10A). The diffractogram of CHT, Figure 10A, reveals the presence of a peak at 11.8° and another at 20.2°, representing the intrinsic symmetrical regions of CHT. The XRD of PVA was also evaluated, and a signal around 20° is observed, corresponding to the semicrystalline nature of pure PVA. Finally, the PCL homopolymer crystallizes easily, and the diffraction pattern reveals the presence of significant crystallinity with peaks at 21.9° and 24.2° corresponding to the (110) and (200) planes of the orthorhombic crystal structure.

[089] The uniaxial PVA / CHT, PVA / AgNPs, and PVA / CHT / AgNPs nanofibers (Figure 10B) showed an amplified signal between 15° and 30°. Furthermore, the signals from the CHT crystalline regions appear discreetly. Thus, the interactions between the polymer chains disrupt the crystalline arrangements present. Petition 870260056447, dated 10 / 06 / 2026, pages 36 / 45 33 / 38 in the polymers. Furthermore, it is observed that the presence / formation of AgNPs further reduces the intensity of the broadened band, indicating their interaction with the polymer chains. On the other hand, the XRD of the coaxial nanofibers (PCLPVA / CHT / AgNPs) shows, in addition to the broadened band between 15° and 30°, referring to the constituent polymers of the core, the main signals at 21.9° and 24.2° which are attributed to the intrinsic crystallinity of PCL. In this way, the presence of PCL crystallinity indicates the efficient formation of the coaxial nanofiber, denoting the absence of interactions between the core and envelope chains, a desired fact.

[090] The thermal stability of the nanofibers and their precursor polymers was evaluated by TGA, Figure 11A and C, and their respective DTG curves in Figure 11B and D. The first weight loss stage in the range of 30 to 120°C in all TGA profiles was attributed to the loss of crystalline water. Events related to polymer degradation were observed in the range of 150 - 500°C. The thermogravimetric analysis (TGA) curve for pure PVA shows three mass loss regions. As mentioned above, the first region between 50 and 200°C can be attributed to the loss of absorbed water molecules, while the second between 200 and 340°C is related to the loss of water bound to the polymer matrix. The third region between 340 and 450°C is associated with the decomposition and carbonization of the polymer. For CHT degradation occurs above 250°C with a maximum Tmax of 280°C. For PCL, a degradation step can be observed in the range of 350 to 450°C, which is consistent with the literature. The results obtained for the uniaxial nanofibers (Figure 11A and B) confirm the presence of both polymers in the structure. Petition 870260056447, dated 10 / 06 / 2026, pages 37 / 45 34 / 38 nanofibers. In the case of coaxial nanofibers, peaks corresponding to all constituent polymers are also observed, according to the proportions used. The results confirm the composition of the nanofibers. The mechanical properties of the nanofibers obtained are presented in Table 7. Table 7: Mechanical properties of nanofibers: modulus of Young's tensile strength and elongation at break. Samples Young's Modulus (MPa) Tensile Strength (10-3 MPa) Elongation at Break (%) Free PVA 0.164 ± 0.008 10.8 ± 1.0 21.9 ± 6.9 PVA AgNPs 0.084 ± 0.001 9.3 ± 0.10 113.6 ± 15.6 PVA / CHT 0.020 ± 0.004 1.25 ± 0.10 6.2 ± 2.0 PVA / CHT / AgNPs 0.030 ± 0.003 9.50 ± 0.05 15.5 ± 2.4 PCL[PVA / CHT] 0.013 ± 0.005 1.45 ± 0.05 12.1 ± 0.3 PCL[PVA / CHT / AgNPs] 0.049 ± 0.002 3.70 ± 0.70 16.1 ± 1.5

[091] In general, it is observed that the inclusion of chitosan in PVA nanofibers leads to a decrease in their mechanical performance. However, the incorporation of AgNPs increases the Young's modulus and elongation at break in PVA and PVA / CHT nanofibers. Similar results are observed for coaxial nanofibers, indicating that the incorporation of AgNPs improves the interaction between the polymeric materials of the blend, possibly due to their interaction, reduction, allocation and "link" with both. Antibacterial activity of nanofibers

[092] PVA and PVA / CHT nanofibers in the absence and presence of AgNPs were evaluated for their antibacterial activity. The results obtained are presented in Petition 870260056447, dated 10 / 06 / 2026, pages 38 / 45 35 / 38 Figure 12.

[093] In general, it is observed that the white nanofibers, i.e., those that do not contain AgNPs, did not show antibacterial activity, given the absence of an inhibition halo. Thus, the amount of CHT (30% w / w), known for its antimicrobial activity, was not sufficient to exert inhibitory activity on the growth of microorganisms. On the other hand, all samples containing AgNPs showed considerable antibacterial activity.

[094] In general, dynamic light scattering analysis of AgNPs obtained from nanofibers in in vitro release studies indicates the existence of nanoparticles with diameters in the range of 20 to 25 nanometers. In this scenario, previous studies have shown that AgNPs with a high surface area have easier access to bacterial cells, leading to greater interaction. Furthermore, electron absorption analysis of AgNPs showed a maximum absorption length of 435 nanometers and a distinctly yellow coloration, characteristic of spherical AgNPs. Thus, the shape of the nanoparticles has a significant effect on their antimicrobial efficacy. Previous studies have shown that spherical AgNPs require a total of 12.5 μg of silver to reach the bacterial inhibition zone. On the other hand, rod-shaped nanoparticles need 50 to 100 μg of silver to achieve the same result.Thus, obtaining spherical AgNPs with diameters on the order of 20 to 25 nanometers using a methodology of direct formation / deposition of AgNPs onto nanofibers reveals a promising process for... Petition 870260056447, dated 10 / 06 / 2026, pages 39 / 45 36 / 38 Obtaining materials for wound treatment.

[095] A useful dressing must possess some essential characteristic components such as an adequate water vapor transmission rate to create a moist environment in wounds, preventing the risk of dehydration and exudate accumulation. Furthermore, the material requires gas permeability, as the repair process requires O2. Other characteristics involve the ability to adsorb fluid for the removal of excess bacterial exudates and nutrients from wounds, a barrier to infection and suppression of bacterial growth, and a scarcity of any cytotoxic effects on the patient. Therefore, electrospun nanofibers have unique properties that meet all the necessary requirements for obtaining a “smart dressing” for the treatment of cell regeneration in acute or chronic wounds.Previous studies have reported the incorporation of AgNPs into PCL nanofibers, but have not shown in situ incorporation of these nanofibers, nor in the blends used in the present invention. Furthermore, in vivo studies have revealed that electrospinning accelerated the rate of wound healing.

[096] Finally, the bacterial activity against both Gram-positive and Gram-negative strains is highlighted, with inhibition of Gram-negative bacteria generally being more difficult or involving higher doses of the active drugs. However, the results obtained in Figure 12 make it clear that the nanofibers incorporated with AgNPs are active in both strains and at similar Ag0 concentrations, showing promise in the treatment and control of bacteria. Virucidal activity of nanofibers Petition 870260056447, dated 10 / 06 / 2026, pages 40 / 45 37 / 38

[097] The results obtained in the virucidal assays are presented in Table 8. The reduction factors are described in Table 6. Table 8: Assays with Coronavirus at different contact times with three tested products. Coronavirus (MHV): 10Λ8,25 DICT50 / mL and the reduction in viral infectivity was > log 5 (virucidal). Products TIMES (h) Percentage of inhibition Coronavirus MHV Log reduction ± SD PVA control (uniaxial) 0.5 90% 0.75 ± 0.50 1 90% 1.00 ± 0.00 8 90% 1.00 ± 0.43 24 90% 0.75 ± 0.43 48 90% 1.00 ± 0.00 64 90% 1.00 ± 0.00 PVA-CHT control (uniaxial) 0.5 99% 1.50 ± 0.50 1 99% 1.75 ± 0.43 8 99% 2.00 ± 0.70 24 99% 2.00 ± 0.00 48 99% 2.00 ± 0.00 64 99% 2.00 ± 0.00 PCL / PVA-CHT control (coaxial) 0.5 99% 1.50 ± 0.50 1 99% 1.50 ± 0.50 8 99% 1.75 ± 0.43 24 99% 2.00 ± 0.00 48 99% 2.00 ± 0.00 64 99% 2.00 ± 0.00 PVA + AgNPs 0.5 99.9995% 5.50 ± 0.50 Petition 870260056447, dated 10 / 06 / 2026, pages 41 / 45 38 / 38 (uniaxial) 1 99.9995% 5.50 ± 0.50 8 99.9992% 5.25 ± 0.43 24 99.9995% 5.50 ± 0.50 48 99.9995% 5.50 ± 0.50 64 99.9995% 5.50 ± 0.50 PVA-CHT + AgNPs (uniaxial) 0.5 99.9992% 5.25 ± 0.43 1 99.9992% 5.25 ± 0.43 8 99.9990% 5.50 ± 0.50 24 99.9995% 5.50 ± 0.50 48 99.9995% 5.50 ± 0.50 64 99.9995% 5.50 ± 0.50 PCL / PVA-CHT + AgNPs (coaxial) 0.5 99.997% 5.00 ± 0.70 1 99.9992% 5.25 ± 0.43 8 99.9995% 5.50 ± 0.50 24 99.9995% 5.50 ± 0.50 48 99.9995% 5.50 ± 0.50 64 99.9995% 5.50 ± 0.48

[098] The products “PVA / AgNPs (uniaxial), PVA / CHT-AgNPs (uniaxial) and PCL[PVA / CHT-AgNPs] (coaxial)” inhibited up to 99.999% (Viral infectivity reduction of > 5 log) of viral contamination, and therefore can be used as a proven virucidal agent for Coronavirus group viruses at all times tested.

[099] In fact, the results presented in Table 8 confirm the potential of colloidal silver in controlling the virus, but also the potential of nanofibers made up of uniaxial PVA / CHT and PCL[PVA / CHT-AgNPs] in obtaining personal protective equipment and other materials for the virucidal control of Sars-cov-2.

Claims

1. Method for obtaining uniaxial electrospun nanofibers incorporated with silver nanoparticles (AgNPs) characterized by comprising the following steps: (a) producing a combination of solutions of poly(vinyl alcohol) (PVA) polymers with a concentration of 12.0% (w / v) and chitosan (CHT) with a concentration of 10.0% (w / v) in the proportion of 70:30 (v / v) (PVA:CHT) for the formation of the polymer blend of the fiber with incorporated AgNPs, wherein AgNPs are formed by adding AgNO3 salt to the PVA solution subjected to reflux at 80 °C for 3 hours to reduce the Ag+ ion to Ag0, followed by adding the CHT solution to the PVA / AgNPs solution; and (b) obtain uniaxial nanofibers by electrospinning, consisting of the blend (PVA / CHT / AgNPs), in which the AgNPs are formed before electrospinning.

2. Method according to claim 1, characterized in that in step (a) the AgNPs prenucleated in 0.25% (w / v) AgNOa and combined with 12% (w / v) PVA are in colloidal form.

3. Method according to claim 1, characterized in that in step (a) the CHT optionally has a viscometric molar mass (Mv) of 87 kDa, and is prepared with 2% acetic acid in water at 60°C for 3h; and the PVA optionally has a molar mass of 104,500 g mol1.

4. Method according to claim 1, characterized in that in step (a) the preparation of the PVA / CHT / AgNPs blend is carried out at 25°C for 3h.

5. Method according to claim 1, Petition 870250116628, dated 12 / 17 / 2025, page 44 / 63 2 / 4 characterized in that in step (b) the nanofibers are obtained using a metal capillary diameter needle of 0.7 mm, under constant pressure with a flow rate of 0.50 mL h-1, applying an electrical potential difference (ddp) of the order of 23 kV between the tip of the needle and the flat spherical metal trimmer.

6. Method for obtaining coaxial electrospun nanofibers incorporated with silver nanoparticles (AgNPs) characterized by comprising the following steps: (a) producing a combination of solutions of poly(vinyl alcohol) (PVA) polymers with a concentration of 12.0% (w / v) and chitosan (CHT) with a concentration of 10.0% (w / v) in a 70:30 (v / v) ratio (PVA:CHT) to form the polymer blend of the fiber core with incorporated AgNPs, wherein AgNPs are formed by adding AgNO3 salt to the PVA solution subjected to reflux at 80 °C for 3 hours to reduce the Ag+ ion to Ag0, followed by adding the CHT solution to the PVA / AgNPs solution; (b) obtaining the 10.0% w / v PCL solution for the fiber sheath; and (c) obtain coaxial core-shell structure nanofibers by electrospinning of PCL[PVA / CHT / AgNPs] composition; wherein silver nanoparticles are formed prior to electrospinning.

7. Method according to claim 6, characterized in that in step (a) the AgNPs prenucleated in 0.25% (w / v) AgNOa and combined with 12% (w / v) PVA are in colloidal form.

8. Method according to claim 6, Petition 870250116628, dated 12 / 17 / 2025, page 45 / 63 3 / 4 characterized in that in step (a) the CHT optionally has a viscometric molar mass (Mv) of 87 kDa, and is prepared with 2% acetic acid in water at 60°C for 3h; the PVA optionally has a molar mass of 104,500 gmol-1; and the PCL optionally has a molar mass of 80,000 gmol-1.

9. Method according to claim 6, characterized in that in step (a) the preparation of the PVA / CHT / AgNPs blend is carried out at 25°C for 3h.

10. Method according to claim 6, characterized in that in step (b) the PCL solution is prepared in DMF / DCM solvent (1:1) at 25°C for 1h.

11. Method according to claim 6, characterized in that in step (c) the nanofibers are obtained using two syringes, one for the internal (core) PVA / CHT / AgNPs solution of step (a) and the other for the external (shell) PCL solution of step (b) and using a coaxial needle with a metallic capillary diameter of 0.7 mm, under constant pressure with a flow rate of 0.50 mL h-1, applying an electrical potential difference (ddp) of 23 kV between the needle tip and the flat spherical metallic trimmer.

12. Uniaxial electrospun nanofibers incorporating silver nanoparticles (AgNPs) obtained according to the method defined in any one of claims 1 to 6, characterized in that the AgNPs are formed by adding AgNO3 salt to a PVA solution subjected to reflux at 80 °C for 3 hours to reduce the Ag+ ion to Ag0, followed by adding CHT solution to the PVA / AgNPs solution, and obtained by uniaxial electrospinning after the formation of the AgNPs, and have an average diameter of 350 nm, with uniform thickness.

13. Electrospun coaxial nanofibers incorporating silver nanoparticles (AgNPs) obtained according to the method defined in any of claims 6 to 12, characterized in that the AgNPs are formed by adding AgNO3 salt to PVA solution subjected to reflux at 80 °C for 3 hours to reduce the Ag+ ion to Ag0 followed by the addition of CHT solution to the PVA / AgNPs solution, have a PCL envelope, with structure (PCL[PVA / CHT AgNPs]) and have an average diameter of 300 nm, with uniform thickness.

14. Use of uniaxial or coaxial electrospun nanofibers incorporated with silver nanoparticles (AgNPs), as defined in claims 12 or 13, characterized by being for the manufacture of antimicrobial / antiviral fabrics for application in the biomedical / hospital field.

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