PROCESS FOR OBTAINING AND USING HEALING FILMS

Description

1 / 27 PROCESS FOR OBTAINING HEALING FILMS AND THEIR USE FIELD OF APPLICATION

[0001] The present invention applies to the medical field with regard to the production of wound dressings, where the production is made from biopolymers and essential oil. BACKGROUND OF THE INVENTION

[0002] Tissue repair is a complex biological process involving multiple stages and requiring the involvement of different cellular subsets and signaling pathways, leading to the regeneration or healing of wounds. Although the cellular and molecular biochemical steps involved in tissue repair are remarkably conserved across different animal species, mammals, including humans, generally exhibit a limited response to tissue injury and tend to form scar tissue rather than fully regenerated healthy tissue. The impaired response is often attributed to factors such as infection, poor blood circulation, and coexisting chronic diseases like diabetes, which can interfere with the healing process and lead to complications.Therefore, there is a growing interest in developing innovative strategies and materials to improve wound healing, with the aim of facilitating and accelerating the tissue repair process, minimizing the risk of infections and adverse reactions.

[0003] The economic burden of chronic wounds, combined with the rise in antibiotic-resistant bacterial infections, has led to considerable research interest in the use of natural or herbal products for wound healing. These approaches have shown effectiveness. Petition 870250053375, dated 06 / 25 / 2025, page 4 / 30 2 / 27 in targeting various mechanisms involved in tissue repair, including immunomodulatory effects and anti-inflammatory properties, which are associated with fibroblast proliferation, extracellular matrix remodeling, and angiogenesis. In addition to well-known medicinal plant extracts, their essential oils have been traditionally used in folk medicine for wound treatment. Numerous studies have demonstrated that essential oils can accelerate wound closure, improve collagen deposition, and increase fibroblast proliferation, particularly when combined with biopolymers such as chitosan, alginate, gelatin, or collagen. These combinations have been processed into active dressings, which offer additional properties such as antioxidant, anti-inflammatory, or antimicrobial activities.

[0004] Sweet Fennel Oil (SFO), derived from the seeds of the fennel plant, has a long history of medicinal use due to its various beneficial properties, including antimicrobial, anti-inflammatory, and antioxidant effects. SFO shows significant antimicrobial activity against a broad spectrum of bacterial and fungal pathogens known to cause wound infections, having the ability to inhibit the growth of bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa, both commonly associated with wound infections.

[0005] Due to its antimicrobial, antioxidant, and anti-inflammatory effects, which can help reduce swelling, inflammation, oxidative stress, and pain, SFO shows promise as a natural and effective treatment for wound healing. However, the low Petition 870250053375, dated 06 / 25 / 2025, page 5 / 30 3 / 27 The solubility typically associated with essential oils hinders their potential application in aqueous pharmaceutical formulations. Furthermore, the high volatility of essential oils may limit their clinical efficacy due to the loss of pharmacologically active volatile compounds.

[0006] Recent studies have demonstrated that combining essential oils with nanotechnology-based materials can enhance their therapeutic efficacy and provide a versatile platform for wound healing applications. Nanotechnology-based materials have emerged as promising tools for innovative wound healing approaches due to their unique physical and chemical properties, such as a high surface area to volume ratio, enhanced biological interactions, adjustable surface charge, improved skin penetration of the drug, and the ability to provide sustained drug delivery.

[0007] Chitosan films are known for their excellent biocompatibility, as they are well tolerated by the body and do not provoke an immune response. Furthermore, chitosan films are known to accelerate the healing process and form a protective barrier over the wound, helping to prevent bacterial infections and reduce the risk of further injury. Polyvinyl alcohol (PVA) is a synthetic polymer derived from vinyl acetate commonly used in the production of wound healing films. PVA offers significant properties for dressings, including swelling capacity, water solubility, minimal toxicity, bioadhesive properties, biocompatibility, and elasticity. This makes it a material Petition 870250053375, dated 06 / 25 / 2025, page 6 / 30 4 / 27 is ideal for wound dressings, as it can be applied directly to the wound without causing further damage or irritation. Furthermore, PVA can be easily modified to incorporate other therapeutic agents, such as essential oils and nanoemulsions (NE). These properties allow both PVA and chitosan to create customized dressings that provide targeted treatment and enhance the healing process.

[0008] In view of the problems of the state of the art and the improvements that can be made, the present invention teaches the nanoencapsulation of SFO in nanoemulsions (NE), followed by its incorporation into chitosan and polyvinyl alcohol (PVA) dressings to increase the therapeutic efficacy of the oil and promote tissue regeneration instead of simply healing wounds. STATE OF THE ART

[0009] The final course work entitled “Poly(vinyl alcohol), chitosan and melaleuca oil hydrogels aiming at application as a cutaneous dressing” (D1) refers to the development of biomaterials, in the form of hydrogels containing chitosan, PVA and melaleuca essential oil, with the aim of being used as dressings to aid in healing.

[0010] However, although D1 reveals the presence of CS and PVA at a concentration of 1% and in a 1:1 ratio, it does not represent the same preparation, since it is observed that acidified water is used to solubilize the polymers and there is no preparation of nanoemulsions (NE) of fennel essential oil for subsequent incorporation into the polymeric matrix. Knowing that the active ingredients of essential oils are highly Petition 870250053375, dated 06 / 25 / 2025, p. 7 / 30 5 / 27 volatile compounds, their incorporation into certain solutions can alter the proportion of active ingredients in the essential oil. The system of the present invention presents the formulation of the nanoemulsified oil, showing results of optimization of the physicochemical properties of the oil and less loss of active ingredients, by evaporation, in relation to the increase in temperature according to the expected purpose of the system. Therefore, the product described in D1 constitutes a CS and PVA hydrogel film, with a preparation and obtaining method completely different from the present invention, which may lead to the compromise of the chemical groups of the elements involved in the production of the material, in order to obtain structural and functional characteristics different from those proposed here. Furthermore, document D1 does not present any description of action on skin lesions in a laboratory test model. Therefore, it is not known whether the product developed in D1 has therapeutic functionality.

[0011] The scientific article entitled “Chitosan nanoparticle encapsulation of antibacterial essential oils (D2)” reveals a summary of carbohydrate-based biopolymers available as anti-biofilm materials, focusing on chitosan. Specifically, the article focuses on chitosan nanoparticle-based encapsulation with nanoemulsions of various essential oils, citing its use in biomedical materials. Additionally, the article focuses on the encapsulation of basil essential oil (Ocimum basilicum), mandarin essential oil (Citrus reticulata), Carum copticum (“Ajwain”) essential oil, dill seed essential oil (Anethum graveolens), peppermint oil (Mentha piperita), green tea oil (Camellia sinensis), oil Petition 870250053375, dated 06 / 25 / 2025, p. 8 / 30 6 / 27 essential oil of cardamom, clove essential oil (Eugenia caryophyllata), cumin seed essential oil (Cuminum cyminum), lemongrass essential oil (Cymbopogon commutatus), summer oregano essential oil (Satureja hortensis), thyme essential oil, cinnamon essential oil (Cinnamomum zeylanicum) and nettle essential oil (Urtica dioica) with chitosan nanoparticles. Additionally, chitosan nanoparticles are used for the encapsulation of the main essential components carvacrol and cinnamaldehyde, the encapsulation of a nanoemulsion of eucalyptus oil (Eucalyptus globulus), the encapsulation of a nanoemulsion of mandarin essential oil, and the nanofiber obtained by electrospinning of collagen-chitosan hydrolysate with essential oils of lemon balm (Melissa officinalis) and dill (Anethum graveolens).

[0012] However, article D2 does not develop a nanoemulsion system with sweet fennel essential oil (Foeniculum vulgare Mill, Family Apiaceae), nor does it describe functional activity on skin lesions, as described in the present invention.

[0013] Frequently, studies are published on the development of dressings of various kinds. Classic polymers such as CS and PVA are widely used because they are safe and sustainable platforms for application to skin lesions. However, depending on the nature of the active ingredients used, the resulting system is completely different. The present invention is efficient in tissue repair and healing of skin lesions, increasing vascularization in the treated area and significantly increasing collagen deposition, suggestive of a completed inflammatory phase. Petition 870250053375, dated 06 / 25 / 2025, page 9 / 30 7 / 27 proliferative in the final stage and remodeling phase underway, both in healthy animals and diabetic animals. Therefore, the material described in D2, although it uses similar production technology and physicochemical tests, does not constitute or suggest a system similar to that of the present invention and does not demonstrate the same functionality.

[0014] Document BR1020170027368 (D3) discloses a polymeric film with healing and antimicrobial action. The present invention relates to a polymeric film with healing and antimicrobial action intended for the treatment of skin lesions, composed of chitosan, poly(vinyl alcohol), ε-polylysine and oleic acid. It is presented as a semi-occlusive film, with the desired morphological, spectroscopic and mechanical properties, capable of inhibiting the growth of Gram-negative and Gram-positive bacteria, and may be indicated for the treatment of various types of skin lesions. It is noted that the product of document D1 is composed of a natural polymer, chitosan, a synthetic polymer, poly(vinyl alcohol), oleic acid as a plasticizer and healing agent, and ε-polylysine as an antimicrobial agent.

[0015] The aforementioned document BR1020170027368 (D3) presents its innovation as being: “The innovation of this product lies in the choice of the antimicrobial agent and the plasticizer and healing agent”. This characteristic of the described material is far from the present invention. D3 describes that the developed product has healing and antimicrobial action, due to the activity of the materials used in its formulation, such as CS, oleic acid and ε-polylysine. However, only in vitro data are presented. Petition 870250053375, dated 06 / 25 / 2025, page 10 / 30 8 / 27 of the CS cytotoxicity analysis. Such data do not reflect the absolute reality of wound healing activity when compared to in vivo laboratory tests.

[0016] The present invention, on the other hand, exhibits significant activity on tissue repair after skin injury, as observed after in vivo analysis, reducing healing time, optimizing the repair pattern, promoting collagen deposition, and restoring the vascular pattern in less time in healthy and diabetic animal models. Therefore, based on the analysis of the D1 data, in comparison with the present invention, it can be stated that there is no overlap or similarity between the inventions.

[0017] The doctoral thesis entitled “Development of breadfruit and chitosan coating associated with essential oil nanoemulsion and its application in the post-harvest conservation of golden papaya” (D4) reveals the development and evaluation of the physical, physicochemical properties and antifungal potential in the form of bilayers based on breadfruit starch, chitosan, sodium alginate and sweet fennel essential oil and its influence on the post-harvest conservation of Golden papaya cultivar.

[0018] It is understood that D4 constitutes a treatment system for controlling fungi in agricultural cultivation, specifically for Golden papaya. The use of chitosan in structuring one of the carrier layers of the developed and evaluated active ingredients shows how this polymer has been widely used as a platform for releasing active ingredients. However, fennel essential oil does not provide similarity to the present invention. The nanoemulsification process of essential oils has been increasingly used due to its Petition 870250053375, dated 06 / 25 / 2025, page 11 / 30 9 / 27 The ability to optimize the activity of the active ingredients in the oil, the preservation against evaporation of these active ingredients, the increased action due to the increased contact surface, the thermal stability achieved, the increased bench time, and the low production cost offer so many benefits. However, each system produced results in a material of varying quality. In the doctoral thesis (D4), there is no mention of the optimal parameters for obtaining the nanoemulsion used, but the concentrations of fennel oil used did not exceed 2% of said oil. This shows, once again, that the system in D4 is not the same as that of the present invention, since different essential oils, non-ionic surfactant, and energy rates in the specific ultrasonicator are used, which are well defined.

[0019] Therefore, it can be concluded that the present invention differs from the prior art documents presented here, since none of them refers to the use of sweet fennel essential oil nanoemulsion, carried in a chitosan and polyvinyl alcohol biofilm polymer matrix, evidencing its entire obtaining and functional characterization as a healing biocompound through the treatment of acute and diabetic wounds, in experimental models. SUMMARY OF THE INVENTION

[0020] The present invention applies to the medical field with regard to the production of dressings for skin wounds, where the production is made from biopolymers and nanoemulsified essential oil.

[0021] The present invention discloses dressings in the form of natural biofilms with nanoencapsulated sweet fennel essential oil with efficacy evaluated in vivo, from Petition 870250053375, dated 06 / 25 / 2025, p. 12 / 30 10 / 27 Skin lesion in a healthy and diabetic murine model. The nanoemulsion film (NE) of sweet fennel oil was produced by the ultrasonication method and incorporated into a chitosan and polyvinyl alcohol polymeric system. The use of the nanoemulsion polymeric film in dorsal lesions in the models proved to be quite efficient with regard to lesion retraction, re-epithelialization of the affected area, obtaining an organized and keratinized epidermis, an organized dermis with signs of completion of the proliferative phase, moving to the remodeling phase, with significant collagen deposition and the presence of elongated cells in the region. BRIEF DESCRIPTION OF THE FIGURES

[0022] The present invention may be better understood through the brief description of the following figures: Figure 1 illustrates that treatment of skin lesions with NE accelerates healing kinetics, with a comparison between a Lesion Kinetics graph (1A) and images of Murines (1B) on days zero, 3, 5 and 7 after the skin lesion was made; it is observed that on day 7 animals treated with NE presented a lesion area of ​​2% (purple line) of the initial lesion, while animals treated with PBS1x presented a lesion area of ​​18.13% (pink line) of the initial lesion; at this point in the treatment the difference observed between the healing areas was significantly different (asterisk).

[0023] Figure 2 illustrates the histology of healthy animals from the control group, treated with 1x PBS, showing an ongoing picture of acute inflammation; (A) illustrates the skin containing an area of ​​the cutaneous lesion performed on healthy animals, treated for 7 days with 1x PBS and stained with Petition 870250053375, dated 06 / 25 / 2025, page 13 / 30 11 / 27 HE; recently completed re-epithelialization is observed (A,1 and B,1); intense hyperplasia of the epidermis (B,2 - 0.085 cm), dermis with a disorganized appearance, with much cellular debris (B,3 and C,3), intense inflammatory infiltrate (B,4 and C,4), rare neoangiogenesis under the epidermis (B,5 and C,5) and discreet in the dermis (B,6 and C,6); expanded adipose tissue is also observed, invading the area of ​​the epicenter of the lesion (A,7); N=6.

[0024] Figure 3 illustrates the histology of healthy control group animals treated with 1x PBS, with absence of collagen in the epicenter area of ​​the lesion, on the 7th day of treatment with saline solution; a rare presence of collagen is observed (B,1), apparently beginning to be distributed in a region of disorganized dermis (B, 2); Adipose tissue invading the lesion area (B, 3); N=6.

[0025] Figure 4 illustrates the histology of healthy animals from the group treated with NE, showing an optimized healing pattern, consistent with the inflammatory phase in the process of completion; area of ​​the lesion on the 7th day of treatment with NE; 10x magnification (A) and highlight I (B); it is possible to observe epidermal hyperplasia (B,1 - 0.020 cm); dermis with a relatively organized appearance (B,2); discrete neoangiogenesis (B,3); discrete inflammatory infiltrate (C,4); rare elongated cells (B,5); rare appendages (B,6); connective tissue suggestive of an inflammatory stage in the process of completion and expanded adipose tissue in the epicenter of the lesion (C,7), invading the area of ​​the epicenter of the lesion; N=6.

[0026] Figure 5 illustrates the histology of healthy animals from the group treated with NE with collagen deposition in the extracellular matrix of the connective tissue in the healing process; area of ​​the lesion on the 7th day of treatment with NE; Petition 870250053375, dated 06 / 25 / 2025, page 14 / 30 12 / 27 10x magnification (A) and highlight I (B), respectively; it is possible to note a significant collagen deposit in the extracellular matrix under the epicenter of the lesion (A,1 and B,1); at the epicenter of the lesion, the connective tissue of the dermis area shows initiated collagen deposition (B,2); a hyperplastic, re-epithelialized and keratinized epidermis is noted (B,3); N=6.

[0027] Figure 6 illustrates the analysis of the kinetics of skin lesion healing among the experimental groups: white (CS:PVA), nanoemulsion (NE), and nanoemulsion film (CS:PVA:NE), with a comparison between a Lesion Kinetics graph (6A) and images of the animals (6B) on days zero, 3, 5, and 7 after the skin lesion was created; it can be observed that on day 7, animals treated with CS:PVA presented a lesion area of ​​5.0% (pink line), animals treated with NE presented a lesion area of ​​6.9% (red line), while animals treated with CS:PVA:NE presented a lesion area of ​​25.6% (purple line). At this point in the treatment, the difference observed between the healing areas with the NE and CS:PVA treatments was significantly different (asterisk) from the CS:PVA:NE treatment.

[0028] Figure 7 illustrates the histology of the treatment of healthy animals with CS:PVA film, showing a healing pattern similar to that observed in the control group treatment with PBS 1x; lesion area on the 7th day of treatment. 10x magnification (A) and highlight I (B); moderate to intense hyperplasia of the epidermis is observed (B,1 - 0.150 cm); dermis, almost non-existent, with a disorganized appearance (B,2); discreet to moderate neoangiogenesis under the epidermis and dermis (B,3); Petition 870250053375, dated 06 / 25 / 2025, page 15 / 30 13 / 27 moderate to intense inflammatory infiltrate (B,4); adipose tissue invading the area of ​​the lesion epicenter (B,5); N=9.

[0029] Figure 8 illustrates the histology of healthy animals treated with CS:PVA film, showing no collagen at the epicenter of the lesion; lesion area on the 7th day of treatment. 10x magnification (A) and highlight I (B), rare presence of collagen is observed (1); disorganized area (2) and adipose tissue invading the epicenter of the lesion (3); N=9.

[0030] Figure 9 illustrates the histology of healthy animals treated with CS:PVA:NE film, showing an efficient and optimized healing pattern, indicating the completion of the inflammatory phase; lesion area on the 7th day of treatment; 10x magnification (A) and highlight I (B), complete re-epithelialization is observed, with discrete hyperplasia of the epidermis (B,1 - 0.020 cm); dermis with a relatively organized appearance (B,2); discrete neoangiogenesis (B,3); rare inflammatory infiltrate (B,4); elongated cells (B,5) and adipose tissue under the dermis, originating from the hypodermis (B,6). N=9.

[0031] Figure 10 illustrates the histology of healthy animals treated with CS:PVA:NE films; lesion area on the 7th day of treatment; 10x magnification (A) and highlight I (B); moderate presence of collagen is observed (1); dense dermis area (2); disorganized appearance (3); N=9.

[0032] Figure 11 illustrates a comparison between treatments performed on healthy animals with CS:PVA (A), NE (B) and CS:PVA:NE (C), with the epicenter of the lesion in the group treated with CS:PVA:NE film showing the best healing pattern; N=9.

[0033] Figure 12 illustrates the analysis of the kinetics of skin lesion healing among the experimental groups. Petition 870250053375, dated 06 / 25 / 2025, page 16 / 30 14 / 27 control (PBS 1x), treated with NE (NE); blank (CS:PVA), and nanoemulsion film (CS:PVA:NE) with a comparison between a Lesion Kinetics graph (12A) and images of the animals (12B) on days zero, 3, 5 and 7 after the cutaneous lesion was created, it is observed that on day 7, animals treated with PBS 1x presented a lesion area of ​​16.0% (black line), of the initial lesion, animals treated with NE presented a lesion area of ​​16.91% (bright pink line), animals treated with CS:PVA presented a lesion area of ​​17.6% (burgundy line), of the initial lesion, while animals treated with CS:PVA:NE presented a lesion area of ​​20.0% (baby pink line), of the initial lesion; at this point in the treatment there was no significant difference between the healing areas with the treatments with PBS 1x, CS:PVA and CS:PVA:NE.

[0034] Figure 13 illustrates the histology of diabetic animals, from the control group, treated with saline solution, presenting lesions with a chronic appearance, characteristic of diabetic wounds; area of ​​the lesion on the 7th day of treatment, at 4x magnification (A) and highlight I (B); hyperplastic epidermis (0.100 cm), recently re-epithelialized (B,1) is observed; the dermis region, almost non-existent, completely disorganized (B,2); invaded by adipose tissue (B,3); intense inflammatory infiltrate, with an appearance of late pro-inflammatory response (B,4); neoangiogenesis in the epidermis (B,5); under the epidermis (B,6) and also in the dermis area (B,7).

[0035] Figure 14 illustrates the histology of diabetic animals with 7 days of treatment with PBS 1x showing no collagen deposits in the dermal area; lesion area at 4x magnification (A) and highlight I (B); dermal area almost non-existent (B,1); disorganized (A,2 and B,2); without the presence Petition 870250053375, dated 06 / 25 / 2025, page 17 / 30 15 / 27 significant collagen (A,3 and B,3); invaded by adipose tissue (A,4 and B,4).

[0036] Figure 15 illustrates the histology of diabetic animals treated with NE, showing lesions with a more advanced inflammatory response than the control condition, with fewer characteristics of diabetic wounds; the tissue was stained with HE (A) and TG (B); Area of ​​the lesion on the 7th day of treatment, at 10x magnification (A and B); less hyperplastic epidermis (0.075 cm), re-epithelialized, but still disorganized is observed (A,1); the dermis region, with dense connective tissue (A,2); adipose tissue pushed downwards (A,3); moderate inflammatory infiltrate, with an aspect of late pro-inflammatory response (A,4); neoangiogenesis in the dermis area (A,5) and moderate collagen deposition (B, 6).

[0037] Figure 16 illustrates the histology of diabetic animals after 7 days of treatment with CS:PVA film exhibiting a relatively better healing pattern than the control condition; 10x magnification (A) and highlight I (B), observe a fibrinoleukocytic crust (A,1); slightly hyperplastic re-epithelialized epidermis (A,2 - 0.104cm); relatively disorganized dermis (B,3); moderate to intense inflammatory infiltrate (B,4); rare neoangiogenesis under the epidermis (B,5).

[0038] Figure 17 illustrates the histology of diabetic animals after 7 days of treatment with CS:PVA film showing discrete collagen deposit in the dermis area; 10x magnification (A) and highlight I (B), the dermis area with discrete collagen deposit is observed (B,1); relatively disorganized (B,2); adipose tissue discreetly invading the epicenter of the lesion (B,3). Petition 870250053375, dated 06 / 25 / 2025, page 18 / 30 16 / 27

[0039] Figure 18 illustrates the histology of diabetic animals with 7 days of treatment with CS:PVA:NE film, showing the optimized and effective healing pattern, 10x magnification (A); highlight I (B) and highlight II (C), fibrinoleukocytic crust (A,1), hyperplastic, organized and keratinized epidermis (A,2; B,2 and C,2), clean dermis (B,3 and C,3); in the process of organization (C,4), inflammatory infiltrate (C,5); elongated cells (C,6); severe neoangiogenesis under the epidermis (C,7) and in some points of the dermis (C,8) adipose tissue under the dermis area, at the epicenter of the lesion (B,9).

[0040] Figure 19 illustrates the histology of diabetic animals after 7 days of treatment with CS:PVA:NE film showing collagen deposition in the dermis area; 10x magnification (A) and highlight I (B); expanded dermis is observed (B,1); moderate collagen deposition (B,2); disorganized dermis (B,3) and adipose tissue under the dermis area, at the epicenter of the lesion (B,4).

[0041] Figure 20 illustrates a comparison between treatments performed on diabetic animals with PBS 1x (A), NE (B) and CS:PVA:NE (C), with the epicenter of the lesion in the group treated with CS:PVA:NE film showing the best healing pattern. DETAILED DESCRIPTION OF THE INVENTION

[0042] The invention can be better understood through the following detailed description, in accordance with the attached figures.

[0043] The present invention describes a nanoencapsulation process for sweet fennel essential oil (Foeniculum vulgare Mill, Apiaceae) for the production of Petition 870250053375, dated 06 / 25 / 2025, page 19 / 30 17 / 27 Skin dressings. According to the present invention, polymers, essential oils, surfactants, solvents, plasticizers, preservatives and water are used.

[0044] The polymers are selected from a group comprising chitosan, poly(vinyl alcohol) (PVA), comprising a mixture thereof.

[0045] Essential oils are selected from sweet fennel oil, clove oil, copaiba oil, cypress oil, palmarosa oil, marjoram oil, tea tree oil or a mixture thereof, with sweet fennel oil being used preferentially.

[0046] Furthermore, the non-ionic surfactants are selected from Hydrogenated Castor Oil, Ethoxylated Castor Oil, Macrogolglycerol Hydroxystearate, Kolliphor 40, PEG-40 and equivalents. A plasticizing agent is preferably 70% sorbitol, an acidifying agent is preferably acetic acid, and a preservative is preferably benzalkonium chloride.

[0047] The present invention is developed from the following steps: (a) Prepare essential oil nanoemulsions; a1. Prepare the organic phase; a2. Prepare the aqueous phase; and a3. Add the organic phase to the aqueous phase. (b) Prepare healing films; b1. First solubilization; b2. Second solution treatment; b3. Elimination of bubbles; b4. Film molding. Petition 870250053375, dated 06 / 25 / 2025, page 20 / 30 18 / 27

[0048] In step (a), the oil is used in quantities ranging from 5 to 9% w / w, preferably 7%. Initially, a pure essential oil, preferably sweet fennel, is used. Then (a2), the aqueous phase is prepared by dissolving the surfactant in water in a proportion ranging from 8 to 12% w / w, preferably 10%. Finally (a3), the aqueous phase is added to the organic phase for processing in the ultrasonic cleaner (US).

[0049] In step (b), for the preparation of the healing films, 0.4g to 0.8g of polymers are solubilized in a mixture of 15 to 25mL of water acidified with 1 to 2% acetic acid, under constant stirring (between 800 and 1200 rpm, preferably 1000 rpm) at room temperature (18 to 22°C), for 50 to 70 minutes. Next (b2), after the polymers have been solubilized, 5 to 10 mL of the NE produced in step (a) are added with 2.0 to 5.0 g of the plasticizer sorbitol and 0.03 to 0.08% of the preservative benzalkonium chloride. The mixture is stirred again (1000 rpm) at room temperature (18 to 22 °C) for 50 to 70 minutes, and then placed in a vacuum chamber (b3) to eliminate bubbles for 50 to 70 minutes. Finally (b4), the mixture is transferred to plates with removable bottoms and placed in a dry oven at a temperature ranging from 40 to 50 °C for 20 to 30 hours.

[0050] The invention will be illustrated by the following examples, without being limited to or by them. TESTS AND EXAMPLES

[0051] The formulation was subjected to ultrasonication to obtain nanometric droplets. To evaluate its Petition 870250053375, dated 06 / 25 / 2025, page 21 / 30 19 / 27 characteristics, both formulations were subjected to dynamic light scattering (DLS) analysis, measuring 38nm according to the average droplet size analysis and PDI 0.152, the polydispersity index (PdI). EXAMPLE 1: Ultrasonication

[0052] Ultrasound was performed to obtain nanometric droplets.

[0053] For the production of the nanoemulsion, an ultrasonicator (US) was used, as it generates high shear stress (rotor-stator). Differential pressure (ultrasound or high-pressure homogenizer - HAP) can also be used, deforming and fragmenting the emulsified droplets into smaller sizes. The high-pressure homogenizer has the advantage of greater control in reducing the size of the droplets. The high-pressure homogenizer (HAP) basically consists of a pump, which injects the liquid to be homogenized under high pressure into a homogenizing valve. In the HAP, the sample is forced to pass through small channels under a pressure that can vary from 500 - 15000 psi. The dispersion flows through microchannels resulting in a very fine emulsion, which causes fragmentation of the dispersed droplets. Therefore, the radius of the generated droplets gradually decreases according to the increase in the shear rate. EXAMPLE 2: Development of chitosan + PVA (CS:PVA) films

[0054] For the development of chitosan-based films, 0.3 g of chitosan and 0.3 g of PVA were weighed. 30 mL of water + 1.5% acetic acid were added to the polymers. The preparation was carried out in a 100 mL Scott bottle. Petition 870250053375, dated 06 / 25 / 2025, p. 22 / 30 20 / 27 The compounds were kept under constant stirring at room temperature until complete solubilization, approximately 60 minutes. After this step, 3.0 g of sorbitol + 0.05% benzalkonium chloride were added to the system. The system was stirred at room temperature for solubilization. After complete solubilization, the solution was placed in a vacuum chamber to eliminate bubbles for 60 minutes. Then, the solution was divided and transferred to previously cleaned and dried plastic Petri dishes. The films were placed in a dry oven at 45 °C for 24 h. EXAMPLE 3: Development of chitosan + PVA + NE films (CS:PVA:NE)

[0055] For the development of chitosan, PVA, and NE-based films, 0.3 g of chitosan and 0.3 g of PVA were weighed. 20 mL of water + 1.5% acetic acid (% of the final system volume) were added to the polymers. The preparation was carried out in a 100 mL Scott bottle. The compounds were kept under constant stirring at room temperature until complete solubilization, approximately 60 minutes. After this step, 10 mL of the desired NE was added, followed by the addition of 3.0 g of sorbitol, which acts as a plasticizer, and 0.05% benzalkonium chloride, which acts as a preservative. The system was subjected to stirring at room temperature for solubilization. After complete solubilization, the solution was placed in a vacuum chamber to eliminate bubbles for 60 minutes. Then, the solution was divided and transferred to previously cleaned and dried metal plates with removable bottoms. The films were placed in a dry oven at 40°C for 24 hours. Petition 870250053375, dated 06 / 25 / 2025, page 23 / 30 21 / 27 TEST 1: Dynamic light scattering (DLS)

[0038] The formulations were subjected to dynamic light scattering (DLS) analysis to evaluate their characteristics, measuring the average droplet size and the polydispersity index (PdI); the average size ranged between 30 and 38 nm and PDI was approximately 0.152. TEST 2: In vivo evaluation of the films produced

[0039] The efficacy of the present invention was evaluated in vivo, from skin lesions in a healthy and diabetic murine model.

[0040] Figure 1 illustrates that treatment of skin lesions with NE accelerates healing kinetics, with a comparison between a Lesion Kinetics graph (1A) and images of Murine animals (1B) on days zero, 3, 5 and 7 after the skin lesion was created; it can be observed that on day 7 animals treated with NE presented a lesion area of ​​2% (purple line) of the initial lesion, while animals treated with PBS1x presented a lesion area of ​​18.13% (pink line) of the initial lesion; at this point in the treatment the difference observed between the healing areas was significantly different (asterisk).

[0041] Figure 2 illustrates the histology of healthy animals from the control group, treated with 1x PBS, presenting an ongoing picture of acute inflammation; (A) illustrates the skin containing an area of ​​the cutaneous lesion performed in healthy animals, treated for 7 days with 1x PBS and stained with HE; recently completed re-epithelialization is observed (A,1 and B,1); intense hyperplasia of the epidermis (B,2 - 0.085 cm), dermis with a disorganized appearance, with much cellular debris (B,3 and C,3), intense inflammatory infiltrate (B,4 and C,4), rare Petition 870250053375, dated 06 / 25 / 2025, page 24 / 30 22 / 27 neoangiogenesis under the epidermis (B,5 and C,5) and discreet in the dermis (B,6 and C,6); expanded adipose tissue is also observed, invading the area of ​​the lesion epicenter (A,7); N=6.

[0042] Figure 3 illustrates the histology of healthy control group animals treated with 1x PBS, with absence of collagen in the epicenter area of ​​the lesion, on the 7th day of treatment with saline solution; a rare presence of collagen is observed (B,1), apparently beginning to be distributed in a region of disorganized dermis (B, 2); Adipose tissue invading the lesion area (B, 3); N=6.

[0043] Figure 4 illustrates the histology of healthy animals from the group treated with NE, showing an optimized healing pattern, consistent with the inflammatory phase nearing completion; lesion area on the 7th day of treatment with NE; 10x magnification (A) and highlight I (B), Leica microscope. DM500; epidermal hyperplasia can be observed (B,10,020 cm); dermis with a relatively organized appearance (B,2); discreet neoangiogenesis (B,3); discreet inflammatory infiltrate (C,4); rare elongated cells (B,5); rare appendages (B,6); connective tissue suggestive of an inflammatory stage in the final stages and expanded adipose tissue at the epicenter of the lesion (C,7), invading the area of ​​the epicenter of the lesion; N=6.

[0044] Figure 5 illustrates the histology of healthy animals from the group treated with NE, showing collagen deposition in the extracellular matrix of the connective tissue undergoing healing; lesion area on the 7th day of treatment with NE; 10x magnification (A) and highlight I (B), respectively, Leica DM500 microscope; it is possible to note a significant collagen deposit in the extracellular matrix under the region of Petition 870250053375, dated 06 / 25 / 2025, page 25 / 30 23 / 27 epicenter of the lesion (A,1 and B,1); at the epicenter of the lesion, the connective tissue of the dermis area shows initiated collagen deposition (B,2); a hyperplastic, re-epithelialized and keratinized epidermis is noted (B,3); N=6.

[0045] Figure 6 illustrates the analysis of the kinetics of skin lesion healing among the experimental groups: white (CS:PVA), nanoemulsion (NE), and nanoemulsion film (CS:PVA:NE), with a comparison between a Lesion Kinetics graph (6A) and images of the animals (6B) on days zero, 3, 5, and 7 after the skin lesion was created; it can be observed that on day 7, animals treated with CS:PVA presented a lesion area of ​​5.0% (pink line), animals treated with NE presented a lesion area of ​​6.9% (red line), while animals treated with CS:PVA:NE presented a lesion area of ​​25.6% (purple line). At this point in the treatment, the difference observed between the healing areas with the NE and CS:PVA treatments was significantly different (asterisk) from the CS:PVA:NE treatment.

[0046] Figure 7 illustrates the histology of the treatment of healthy animals with CS:PVA film showing a healing pattern similar to that observed in the control group treatment with PBS 1x; lesion area on the 7th day of treatment. 10x magnification (A) and highlight I (B); moderate to intense hyperplasia of the epidermis is observed (B,1 - 0.150 cm); dermis, almost non-existent, with a disorganized appearance (B,2); slight to moderate neoangiogenesis under the epidermis and dermis (B,3); moderate to intense inflammatory infiltrate (B,4); adipose tissue invading the area of ​​the epicenter of the lesion (B,5); N=9.

[0047] Figure 8 illustrates the histology of healthy animals treated with CS:PVA film showing no signs of the disease. Petition 870250053375, dated 06 / 25 / 2025, page 26 / 30 24 / 27 collagen at the epicenter of the lesion; lesion area on the 7th day of treatment. 10x magnification (A) and highlight I (B), rare presence of collagen is observed (1); disorganized area (2) and adipose tissue invading the epicenter of the lesion (3); N=9.

[0048] Figure 9 illustrates the histology of healthy animals treated with CS:PVA:NE film, showing an efficient and optimized healing pattern, indicating the completion of the inflammatory phase; lesion area on the 7th day of treatment; 10x magnification (A) and highlight I (B), complete re-epithelialization is observed, with discrete hyperplasia of the epidermis (B,1 - 0.020 cm); dermis with a relatively organized appearance (B,2); discrete neoangiogenesis (B,3); rare inflammatory infiltrate (B,4); elongated cells (B,5) and adipose tissue under the dermis, originating from the hypodermis (B,6). N=9.

[0049] Figure 10 illustrates the histology of healthy animals treated with CS:PVA:NE films; lesion area on the 7th day of treatment; 10x magnification (A) and highlight I (B); moderate presence of collagen is observed (1); dense dermis area (2); disorganized appearance (3); N=9.

[0050] Figure 11 illustrates a comparison between treatments performed on healthy animals with CS:PVA (A), NE (B) and CS:PVA:NE (C), with the epicenter of the lesion in the group treated with CS:PVA:NE film showing the best healing pattern; N=9.

[0051] Figure 12 illustrates the analysis of the kinetics of skin lesion healing among the experimental groups: control (PBS 1x), treated with NE (NE); blank (CS:PVA), and nanoemulsion film (CS:PVA:NE) with a comparison between a Lesion Kinetics graph (12A) and images of the animals (12B) on days zero, 3, 5, and 7 after the lesion was created. Petition 870250053375, dated 06 / 25 / 2025, page 27 / 30 25 / 27 cutaneous, it is observed that on day 7, animals treated with PBS 1x presented a lesion area of ​​16.0% (black line) of the initial lesion, animals treated with NE presented a lesion area of ​​16.91% (shocking pink line), animals treated with CS:PVA presented a lesion area of ​​17.6% (burgundy line) of the initial lesion, while animals treated with CS:PVA:NE presented a lesion area of ​​20.0% (baby pink line) of the initial lesion; at this point in the treatment there was no significant difference between the healing areas with the treatments with PBS 1x, CS:PVA and CS:PVA:NE.

[0052] Figure 13 illustrates the histology of diabetic animals, from the control group, treated with saline solution, presenting lesions with a chronic appearance, characteristic of diabetic wounds; area of ​​the lesion on the 7th day of treatment, at 4x magnification (A) and highlight I (B); hyperplastic epidermis (0.100 cm), recently re-epithelialized (B,1) is observed; the dermis region, almost non-existent, completely disorganized (B,2); invaded by adipose tissue (B,3); intense inflammatory infiltrate, with an appearance of late pro-inflammatory response (B,4); neoangiogenesis in the epidermis (B,5); under the epidermis (B,6) and also in the dermis area (B,7).

[0053] Figure 14 illustrates the histology of diabetic animals with 7 days of treatment with PBS 1x showing no collagen deposits in the dermis area; lesion area at 4x magnification (A) and highlight I (B); dermis area almost non-existent (B,1); disorganized (A,2 and B,2); without significant collagen presence (A,3 and B,3); invaded by adipose tissue (A,4 and B,4).

[0054] Figure 15 illustrates the histology of diabetic animals treated with NE, showing response lesions. Petition 870250053375, dated 06 / 25 / 2025, pages 28 / 30 26 / 27 more advanced inflammatory conditions than the control condition, with fewer characteristics of diabetic wounds; the tissue was stained with HE (A) and TG (B); Lesion area on the 7th day of treatment, at 10x magnification (A and B); less hyperplastic epidermis (0.075 cm), re-epithelialized, but still disorganized (A,1); the dermis region, with dense connective tissue (A,2); adipose tissue pushed downwards (A,3); moderate inflammatory infiltrate, with an aspect of late pro-inflammatory response (A,4); neoangiogenesis in the dermis area (A,5) and moderate collagen deposition (B, 6).

[0055] Figure 16 illustrates the histology of diabetic animals after 7 days of treatment with CS:PVA film exhibiting a relatively better healing pattern than the control condition; 10x magnification (A) and highlight I (B), a fibrinoleukocytic crust is observed (A,1); discreetly hyperplastic re-epithelialized epidermis (A,2 - 0.104cm); relatively disorganized dermis (B,3); moderate to intense inflammatory infiltrate (B,4); rare neoangiogenesis under the epidermis (B,5).

[0056] Figure 17 illustrates the histology of diabetic animals after 7 days of treatment with CS:PVA film showing discrete collagen deposit in the dermis area; 10x magnification (A) and highlight I (B), the dermis area with discrete collagen deposit is observed (B,1); relatively disorganized (B,2); adipose tissue discreetly invading the epicenter of the lesion (B,3).

[0057] Figure 18 illustrates the histology of diabetic animals after 7 days of treatment with CS:PVA:NE film, showing an optimized and effective healing pattern, 10x magnification (A); highlight I (B) and highlight II (C), note Petition 870250053375, dated 06 / 25 / 2025, pages 29 / 30 27 / 27 if fibrinoleukocyte crust (A,1), hyperplastic, organized and keratinized epidermis (A,2; B,2 and C,2), clean dermis (B,3 and C,3); in the process of organization (C,4), inflammatory infiltrate (C,5); elongated cells (C,6); severe neoangiogenesis under the epidermis (C,7) and in some points of the dermis (C,8) adipose tissue under the dermal area, at the epicenter of the lesion (B,9).

[0058] Figure 19 illustrates the histology of diabetic animals after 7 days of treatment with CS:PVA:NE film showing collagen deposition in the dermis area; 10x magnification (A) and highlight I (B); expanded dermis is observed (B,1); moderate collagen deposition (B,2); disorganized dermis (B,3) and adipose tissue under the dermis area, at the epicenter of the lesion (B,4).

[0059] Figure 20 illustrates a comparison between treatments performed on diabetic animals with PBS 1x (A), NE (B) and CS:PVA:NE (C), with the epicenter of the lesion in the group treated with CS:PVA:NE film showing the best healing pattern.

[0060] The present invention has been disclosed in this descriptive report in terms of its preferred embodiment. However, other modifications and variations are possible from the present description, and are still within the scope of the invention disclosed herein. Petition 870250053375, dated 06 / 25 / 2025, page 30 / 30

Claims

1 / 2 CLAIMS 1. Process for obtaining healing films CHARACTERIZED by comprising the steps: (a) Preparing essential oil nanoemulsions; a1. Preparing the organic phase; a2. Preparing the aqueous phase; a3. Addition of the organic phase to the aqueous phase; (b) Preparing healing films; b1. First solubilization; b2. Second solubilization; b3. Elimination of bubbles; b4. Molding of the films.

2. Process according to claim 1, CHARACTERIZED in that in step (a) the oil is used in amounts ranging from 5 to 9% w / w, in its pure form; and in that the aqueous phase is prepared from the solubilization of the surfactant in water in a proportion ranging from 8 to 12% w / w; and in that the aqueous phase is added to the organic phase for processing in the ultrasonic cleaner (US).

3. Process according to claims 1 and 2, CHARACTERIZED in that in step (b), 0.4 g to 0.8 g of polymers are solubilized in a mixture of 15 to 25 mL of water acidified with 1% to 2% acetic acid, under constant stirring between 800 and 1200 rpm at a temperature of 18 to 22 °C, for 50 to 70 minutes; whereby, after the polymers have been solubilized, 5 to 10 mL of nanoemulsion containing 2.0 to 5.0 g of plasticizer and 0.03 to 0.08% preservative are added, the mixture is stirred between 800 and 1200 rpm at a temperature of 18 to 22°C for 50 to 70 minutes, and placed in a vacuum chamber to eliminate bubbles, Petition 870250000004, dated 01 / 01 / 2025, p. 35 / 63 2 / 2 for 50 to 70 minutes; and the mixture is transferred to plates with removable bottoms and placed in a dry oven at a temperature ranging from 40 to 50 °C for 20 to 30 h.

4. Process, according to any one of claims 1 to 3, CHARACTERIZED in that the polymers are selected from a group comprising chitosan, poly(vinyl alcohol) (PVA) being a mixture thereof; the essential oils are selected from sweet fennel oil, clove oil, copaiba oil, cypress oil, palmarosa oil, marjoram oil, melaleuca oil or a mixture thereof, sweet fennel oil being preferably; the surfactant is a non-ionic surfactant selected from the group comprising ethoxylated castor oil, hydrogenated castor oil, macrogolglycerol hydroxystearate, PEG-40, Kolliphor 40; the preservative is preferably benzalkonium chloride; the plasticizer is preferably 70% sorbitol; and the solvent is preferably acidified water.

5. Use of the healing films obtained as defined in claims 1 to 4, CHARACTERIZED by being for treating skin wounds. Petition 870250000004, dated 01 / 01 / 2025, pp. 36 / 63

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