Process of obtaining a lyophilized composition of chitosan-based nanoparticles containing bromelin, lyophilized composition of chitosan-based nanoparticles containing bromelin and use in the treatment of wounds

Abstract

The present invention relates to a process for obtaining a lyophilized composition of chitosan-based nanoparticles containing bromelain. Said composition has proven stable with respect to enzymatic activity using small molecules, such as sugars or amino acids, as lyophilized protectants. Additionally, the present invention reveals its use in the preparation of a wound care medication.

Description

PROCESS FOR OBTAINING A LYOPHILIZED CHITOSAN-BASED NANOPARTICLE COMPOSITION CONTAINING BROMELAIN, LYOPHILIZED CHITOSAN-BASED NANOPARTICLE COMPOSITION CONTAINING BROMELAIN AND ITS USE IN WOUND TREATMENT Field of invention:

[001] The present invention relates to a process for obtaining a freeze-dried composition of chitosan-based nanoparticles containing bromelain.

[002] This composition proved to be stable with respect to enzymatic activity using sugars as lipoprotectors.

[003] The composition referred to does not contain organic solvents or chemical additives, such as preservatives, and is obtained by a green process and based on natural and biocompatible excipients.

[004] Additionally, the present invention discloses its use in the preparation of a medicament for treating wounds. Fundamentals of the invention:

[005] Bromelain, a complex of substances extracted mainly from pineapple (Ananas comosus L.), is recognized for its anti-inflammatory, antithrombotic, fibrinolytic, antitumor activity, and immunomodulatory effects. Following Seligman's 1962 study demonstrating its anti-inflammatory action, several studies support the use of bromelain extracts in various conditions (Seligman, 1962; Taussig and Batkin, 1988; Salas et al., 2008; Chobotova et al., 2010; Amid et al., 2011; Ferreira et al., 2011). In particular, some studies Petition 870260066355, dated 06 / 07 / 2026, page 6 / 44 2 / 33 have demonstrated the potential use of bromelain in wound healing processes. Maurer (2001) demonstrated that this enzyme has benefits for wound healing, specifically reducing edema, hematomas, and pain. In burns, bromelain acts by hydrolyzing devitalized tissue, both in vivo and in vitro, which increases the healing capacity.

[006] Wounds are formed from the disruption of the integrity of the skin, mucous surfaces, or organ tissues (Young and McNaught, 2011), and although skin lesions vary, they share a common mechanism for repair and healing. Wound healing is a regular biological process in the human body, as human skin has a natural capacity to promote self-regeneration after damage (Guo and DiPietro, 2010; Pereira and Bártolo, 2016), and when triggered by an injury, this mechanism comprises an elaborate cascade of physiological events designed to ultimately heal the skin (Strondtbeck, 2001).

[007] The first stage, hemostasis, occurs immediately upon injury and usually lasts a few hours. The second stage, inflammation, begins soon after hemostasis and is usually completed within the first 24 to 72 hours after injury; however, it can last up to 5-7 days after injury (Haas, 1995). Proliferation and repair, the third phase, typically occurs 1 to 3 weeks after injury. The fourth and final stage, remodeling, begins approximately 3 weeks after injury and can take months to several years to reach physiological completion. Petition 870260066355, dated 06 / 07 / 2026, page 7 / 44 3 / 33

[008] In 1962, Winter concluded that hydrated wounds in piglets epithelialize the skin twice as fast as wounds exposed to air (Winter, 1962). Since then, much has been learned about wound healing mechanisms and the factors that affect them (Cooper, 1990; Cuzzell and Stotts, 1990; Salas Campos et al., 2005; Winter and Scales, 1963). Currently, it is known that healing is a complex process that can be accelerated and improved by the use of techniques, products, and dressings (Cooper, 1990; Kumar et al., 2007).

[009] Modern dressings have been developed to facilitate the healing process rather than simply covering the wound (Dhivya et al., 2015), and their essential characteristic is to retain and create a moist environment around the wound to facilitate its healing (Boateng et al., 2008). Most modern treatments are interactive dressings, as they interact with the wound to provide an optimal environment at the dressing interface (Sarabahi, 2012). These interactive dressings include semi-permeable films and foams, hydrofibers, hydrogels, hydrocolloids, and alginates (Dhivya et al., 2015; Sarabahi, 2012).

[010] Bioactive or biological dressings are also modern dressings and are produced from biomaterials, which play an important role in the healing process (Boateng et al., 2008; Dhivya et al., 2015). These dressings are known for their biocompatibility, biodegradability and non-toxic nature (Dhivya et al., 2015), and are generally derived from natural tissues or Petition 870260066355, dated 06 / 07 / 2026, page 8 / 44 4 / 33 artificial (Bartlett, 1981; Boateng et al., 2008; Dhivya et al., 2015). In some cases, bioactive dressings can be incorporated with active compounds, such as antimicrobials and growth factors, improving the healing process (Boateng et al., 2008; Dhivya et al., 2015).

[011] Due to the characteristics described above, bromelain has potential applications in the cosmetic, pharmaceutical, and food industries. However, the use of proteins as an active ingredient in pharmaceutical products is a challenge due to their physical and chemical instability.

[012] The processes to which proteins are subjected generate stress conditions that can lead to a reduction or loss of biological activity, or even alter their immunogenic potential (Sanchez-Ruiz and Makhatadze, 2001). Pereira et al. (2014) demonstrated that compositions with bromelain, when stored at 37 °C, lost almost all of their enzymatic activity. They attributed this fact to the self-degradation (autolysis or autodigestion) of bromelain, since 37 °C has been considered the optimal temperature for its proteolytic activity (Pereira et al., 2014).

[013] In recent years, the development of biodegradable nanoparticulate systems for drug delivery has grown significantly. These colloidal carriers offer several advantages, such as the possibility of protecting the incorporated active ingredient against in vivo degradation, relative stability in biological fluids, and the ability to modulate drug release, and therefore are Petition 870260066355, dated 06 / 07 / 2026, page 9 / 44 5 / 33 considered quite promising (Lemarchand et al., 2004; Le Droumaguet et al., 2012).

[014] Due to its adhesiveness, biocompatibility, biodegradability, and low toxicity, chitosan is an attractive material for various uses, mainly in the pharmaceutical field. This polysaccharide is used in modified drug delivery systems for various therapeutic classes, such as antibiotics, anti-inflammatories, antihypertensives, as well as peptides and proteins (Bernkop-Schnürch, 2000; Florea et al., 2006; Boonyo et al., 2007; Sandri et al., 2010). The ease of adhesion of chitosan, as well as its antifungal, bacteriostatic character and its oxygen permeability, are very attractive properties for topical use (Argüelles, 2004; Jayakumar et al., 2011).

[015] It is known that the use of chitosan for the treatment of wounds and burns has been studied, based on its hemostatic capacity and its effect of accelerating wound repair with a better final aesthetic result.

[016] Based on the above, previous studies have shown the instability of bromelain when applied directly as topical compositions, even when stored at low temperatures (Lourenço et al., 2016; Pereira et al., 2014; Spir et al., 2015). Therefore, it is understood that this enzyme could benefit from chitosan-based nanostructuring for such purposes.

[017] For economic and manufacturing reasons, a new Petition 870260066355, dated 06 / 07 / 2026, page 10 / 44 6 / 33 The product in a liquid composition would be the simplest alternative, however, possible chemical, physical and biological processes may occur in a way that could impair its stability and consequent performance.

[018] The freeze-drying process provides solid compositions without heating above room temperature, which promotes the stability of the proteins present. The low-moisture solid state avoids or reduces possible chemical and biological reactions and mechanical stress, resulting in stable compositions for months to years without loss of desired performance.

[019] The present invention therefore aimed to encapsulate bromelain in chitosan nanoparticles and lyophilize the composition in order to increase enzymatic stability, with the aim of producing a medicine to treat wounds.

[020] Unexpectedly, it was revealed that the process involving the use of a lyophilized chitosan-bromelain nanoparticle-based composition containing lyophilisates maintained stable particle parameters, resulting in a composition with a short reconstitution time in water and a higher rate of bromelain incorporation when compared with the liquid form. State of the art:

[021] Some prior art documents refer to natural active ingredients for wound treatment. These active ingredients known in the literature are propolis, Centella asiatica, Punica granatum L, Rosmarinus officinalis L and Calendula. Petition 870260066355, dated 06 / 07 / 2026, page 11 / 44 7 / 33 officinalis, among others. Bromelain, whose properties also make it ideal for wound treatment, is extensively reviewed, Ataide et al. Natural actives for wound healing: a review. Phytotherapy Research, 2018.

[022] Ekambaram et al. (2017) describes a multiphasic combined delivery system produced by coaxial electrospinning of different biocompatible polymers, with optimization of the ratio and specificity of the polymers for the specific function, using bromelain as a debriding agent and salviolic acid B as an angiogenesis and re-epithelialization stimulator. The in vitro release profile illustrated the sustained release of debriding protease and bioactive component in a timely manner. The manufactured product showed angiogenic potential through in vitro endothelial cell migration and increased new capillaries from existing blood vessels in response to an in ovo chicken chorioallantoic membrane assay. With the engineered fiber, accelerated wound healing was also achieved in vivo in a full-thickness rat skin wound model.

[023] Other documents refer to the use of bromelain associated with chitosan in nanoparticles.

[024] The article “BROMELAIN-IMMOBILIZED AND LACTOBIONIC ACID-MODIFIED CHITOSAN NANOPARTICLES FOR ENHANCED DRUG PENETRATION IN TUMOR TISSUES” (2018) discusses the use of chitosan modified with lactobionic acid, which modifies its properties. The modified chitosan was then used to produce a dispersion of nanoparticles containing Petition 870260066355, dated 06 / 07 / 2026, page 12 / 44 8 / 33 doxorubicin, which were subsequently coated with bromelain to increase their ability to penetrate tumors after degradation of the tumor extracellular matrix. The stability of the composition was not studied in that work. The present invention addresses the problem of bromelain stability by proposing a composition in which chitosan-bromelain nanoparticles are freeze-dried in the presence of a lyoprotectant. In addition, the present invention does not involve chemically modified chitosan, which reduces excipient costs and maintains the natural appeal of the composition.

[025] Document CN 107475226 discloses a bromelain complex with a polyanionic polysaccharide (alginate, pectin, CMC, gum arabic or xanthan gum), which may use a single polysaccharide or a mixture. According to the patent abstract, the inventors complex bromelain with an anionic polysaccharide, thus increasing its stability. However, the drying process of the complex, its average size and characteristics are not detailed. Unlike the above, the present invention uses a polycationic polysaccharide (chitosan has a positive charge), in addition to forming and characterizing nanoparticles with bromelain.

[026] Some prior art documents also describe the production of lyophilized bromelain powders.

[027] Document CN 1834239 proposes a method for obtaining lyophilized bromelain powder. The enzyme is extracted from pineapple in steps involving juice filtration. Petition 870260066355, dated 06 / 07 / 2026, page 13 / 44 9 / 33 of the fruit, adsorption with zinc oxide, ultrafiltration, and lyophilization. The innovation relates to the purification method achieved by using zinc oxide, and is intended for application in possible oral formulations. However, data on stability, storage, and lyophilization conditions are not disclosed, although these impact the enzymatic activity of the product.

[028] In the present invention, there is no need for purification of the extract because bromelain is commercially acquired in its already purified form. Furthermore, the product consists of a pharmaceutical composition of bromelain encapsulated in chitosan, presented as a dry powder (lyophilized), stable at room temperature for more than 90 days in relation to particle size and enzymatic activity.

[029] Document RU 2677232 refers to a pharmaceutical composition of bromelain encapsulated in chitosan gel of low and / or high molecular weight, presented in the form of macroparticles. The bromelain is immobilized in a 50 mM Tris-glycine buffer solution at pH 8.5-9.0, incubated, and the resulting precipitate is washed with a 50 mM Tris-HCl buffer at pH 7.5. The invention is intended for the treatment of wounds, providing macroscopic particles of varying sizes. However, it does not present data on the physicochemical characterization of the particles or even on the stability of the composition or the lyophilization conditions. Additionally, the composition has a final pH between 8.5 and 9.0. Petition 870260066355, dated 06 / 07 / 2026, page 14 / 44 10 / 33 unsuitable for use on skin, pH 5.5.

[030] The present invention is distinguished by being a reproducible composition of lyophilized low molecular weight chitosan nanoparticles. Data on the stability of the composition, as well as the characterization of the nanoparticles, are provided and it has a pH compatible with that of the skin.

[031] It is also known that the addition of lyophilisates, including sugars, is addressed in the state of the art. In particular, the article “FREEZE-DRYING OF NANOPARTICLES: FORMULATION, PROCESS AND STORAGE CONSIDERATIONS” (2006) compiles information on the lyophilization of nanoparticles. Maltose is mentioned for stabilizing caprolactone-encapsulated vectors, but no reference is made to its role in protein delivery.

[032] Therefore, no document describes the lyophilization of bromelain nanoparticles on a chitosan base with the addition of maltose as a lyophilisate, nor does it offer extensive and detailed descriptions of the process capable of making it reproducible and increasing enzymatic stability. Brief description of the invention:

[033] The present invention relates to a process for obtaining a freeze-dried composition of chitosan-based nanoparticles containing bromelain.

[034] This composition proved to be stable with respect to enzymatic activity using sugars as lipoprotectors.

[035] Additionally, the present invention discloses its Petition 870260066355, dated 06 / 07 / 2026, page 15 / 44 11 / 33 use in the preparation of a medicine to treat wounds.

[036] The composition obtained in the form of lyophilized nanostructured bromelain powder can be used as a final pharmaceutical form or as an active pharmaceutical ingredient incorporated into another product. Brief description of the figures:

[037] To obtain a complete visualization of the object of this invention, the figures to which reference is made are presented, as follows: Figure 1. Scanning electron microscopy (A) and transmission electron microscopy (B) of chitosan-bromelain nanoparticles. Figure 2. Fourier Transform Infrared (FTIR) spectra of bromelain (black), chitosan nanoparticles (red), and chitosan-bromelain nanoparticles (green). Figure 3. Average size of nanoparticles (A), polydispersity index (B) and zeta potential (C) during accelerated stability study, where Chi = chitosan nanoparticles, Chi-brom = chitosan-bromelain nanoparticles. Error bars represent standard deviation of three measurements. Figure 4. Protein concentration (A) and enzymatic activity (B) in the chitosan-bromelain nanoparticle suspension during an accelerated stability study, where Chi-brom = chitosan-bromelain nanoparticles. Error bars represent the standard deviation of three measurements. Figure 5. Bromelain encapsulation efficiency in nanoparticles according to protein concentration (A) Petition 870260066355, dated 06 / 07 / 2026, page 16 / 44 12 / 33 and enzymatic activity (B), where Gly = glycine, Malt = maltose. The error bars represent the standard deviation of the three measurements. Figure 6. Pareto chart of the factorial design relating the studied variables (inputs) to the responses of average particle size (A), polydispersity index (B), D10 (C), D50 (D), D90 (E), zeta potential (F) and encapsulation efficiency in terms of proteins (G) and enzymatic activity (H). Figure 7. Graphs of the main effect of the factorial design relating the studied variables (inputs) in relation to the responses of average particle size (A), polydispersity index (B), D10 (C), D50 (D), D90 (D), zeta potential (F) and encapsulation efficiency in terms of proteins (G) and enzymatic activity (H). Figure 8. Scanning electron microscopy of lyophilized powder with glycine (A), maltose (B) and after reconstitution with glycine (C) and with maltose (D). Figure 9. Transmission electron microscopy of lyophilized samples after reconstitution containing glycine (A) and maltose (B). Figure 10. Average size of nanoparticles (A), polydispersity index (B) and zeta potential (C) after reconstitution of the lyophilized chitosan-bromelain nanoparticles during the stability study, where Gly = glycine, Malt = maltose. Error bars represent standard deviation of three measurements. Petition 870260066355, dated 06 / 07 / 2026, page 17 / 44 13 / 33 Figure 11. Protein concentration (A) and enzymatic activity (B) after reconstitution of the lyophilized chitosan-bromelain nanoparticles during the stability study, where Gly = glycine, Malt = maltose. Error bars represent standard deviation of three measurements. Figure 12. Graph of the drying ramp used during the freeze-drying process of the formulation. Detailed description of the invention:

[038] The present invention relates to a process for obtaining a lyophilized composition based on chitosan nanoparticles containing bromelain.

[039] This composition proved to be stable with respect to enzymatic activity using sugars as lipoprotectors.

[040] Additionally, the present invention discloses its use in the preparation of a medicament for treating wounds.

[041] The composition obtained in the form of lyophilized nanostructured bromelain powder can be used as a final pharmaceutical form or as an active pharmaceutical ingredient incorporated into another product.

[042] More specifically, the aforementioned process comprises steps which are described as follows: i. Production of chitosan-bromelain nanoparticles ii. 2) Lyophilization of chitosan-bromelain nanoparticles.

[043] Each of the steps is best described and defined below: i. Production of chitosan-bromelain nanoparticles Petition 870260066355, dated 06 / 07 / 2026, page 18 / 44 14 / 33 a) Add a TPP solution at a concentration of 0.5 mg / mL dropwise to a chitosan solution at a concentration of 2.5 mg / mL while stirring until completely homogenized; b) Add a bromelain solution at a concentration of 10 mg / mL to the solution obtained in step (a) while stirring until completely homogenized; c) Obtain chitosan and bromelain nanoparticles; ii. Lyophilization of chitosan-bromelain nanoparticles d) Dissolve the lyoprotectant directly in the nanoparticle solution obtained in step (c); (e) The mixture of nanoparticles with lyoprotectant obtained in step (d) stored in an ultra-freezer at a temperature of -80 °C; f) The nanoparticles frozen at -80°C, obtained in step (e), were inserted into the freeze dryer with the shelf set to -45°C and kept there until they reached the same temperature as the shelf; g) The nanoparticles from step (f) follow a drying ramp, increasing the temperature by 5°C from -45°C to +10°C; h) Obtaining a freeze-dried composition of chitosan-based nanoparticles containing bromelain.

[044] The ratio of chitosan, TPP, bromelain is 4:6:1 (v / v). Agitation in steps (a) and (b) is at 350 rpm, and the duration of step (b) is 40 min.

[045] In the aforementioned process, the lipoprotectant is selected from small molecules, such as sugars or Petition 870260066355, dated 06 / 07 / 2026, page 19 / 44 15 / 33 amino acids, maltose, glycine, preferably maltose.

[046] In step (g) the temperature is changed when the composition temperature reaches the shelf temperature. only advancing to the next step when the formulation temperature reaches the shelf temperature.

[047] The nanoparticles obtained in step (c) of chitosan-bromelain have an average diameter of 84.5 to 100.9 nm, a polydispersity index of 0.18 to 0.23, a zeta potential of 21.9 to 27.1 mV and a particle concentration on the order of 1012 particles / mL. Also in step c, bromelain is encapsulated in chitosan nanoparticles and shows an encapsulation effectiveness of 83.4 to 97.8% of the protein concentration, which corresponds to an encapsulation efficiency of 75.5 to 81.6% of the enzymatic activity.

[048] In one embodiment of the present invention in step (d) the lyoprotectant is at a concentration of 3% (w / v) relative to the volume of the chitosan-bromelain nanoparticle solution.

[049] In the process of the present invention, the composition has a collapse temperature ranging from -56°C to 28°C, preferably above -40°C, with the addition of maltose and glycine, respectively.

[050] Also the subject of the present invention is a freeze-dried composition of chitosan-based nanoparticles containing freeze-dried bromelain obtained by the process as defined above and comprising bromelain, chitosan, lyophilized protective agent, TPP and residual moisture between 3.5 and Petition 870260066355, dated 06 / 07 / 2026, page 20 / 44 16 / 33 4.4 (m / m).

[051] The said composition comprises a lipoprotectant selected from small molecules, which may be sugars or amino acids such as maltose or glycine, preferably maltose.

[052] Additionally, the composition that is the subject of the present invention comprises nanoparticles with an average diameter of 88.5 and 127.8 nm, a polydispersity index of 0.29 to 0.34, and a zeta potential of 20.0 to 23.2 mV.

[053] The composition obtained by the process is in the form of lyophilized powder, exhibiting an encapsulation efficiency with maltose of 96.3% to 98.8%, which is equivalent to an enzymatic activity of 73.3 to 99.5%.

[054] The use of the composition obtained as defined in the process described in the preparation of a medicament for treating wounds is still an object of the present invention. Example of implementation: Step 1: Production of chitosan-bromelain nanoparticles Materials

[055] Bromelain from pineapple stem, azocasein, Bradford reagent, and low molecular weight chitosan, preferably 50,000-190,000 Da (based on viscosity) being 75-85% deacetylated (catalog number 448869) were purchased from Sigma-Aldrich® (St. Louis, USA). All other reagents were purchased in analytical grade. Azocasein is the reagent used in the enzymatic activity test and Bradford reagent is the reagent used in the Petition 870260066355, dated 06 / 07 / 2026, page 21 / 44 17 / 33 protein concentration test.

[056] The bromelain solution was prepared by dissolving bromelain in distilled water (10 mg / mL) and filtering it with 0.22 pm membranes.

[057] The chitosan solution was prepared in the following proportion: 2.5 mg / mL of chitosan in 1% acetic acid, pH 5.0, and subsequently filtered through 0.45µm membranes.

[058] The TPP solution was prepared in the following proportion: 0.5 mg / mL in ultrapure water, subsequently filtered through a 0.22µm membrane. The TPP solution is at 30% (w / w) relative to the total amount of chitosan.

[059] The nanoparticles were produced by the ionic crosslinking technique (Shu and Zhu, 2000), using sodium tripolyphosphate (TPP) as a crosslinking agent. A ratio of 30% (w / w) of TPP relative to the total amount of chitosan was used, with stirring at 350 rpm. Bromelain solution (10 mg / mL in water) was added under stirring at 350 rpm for 40 minutes, after dropwise addition of the TPP solution to the chitosan solution. Immediately after the addition of TPP, 1 ml of bromelain solution was added to produce bromelain-chitosan nanoparticles or 1 ml of distilled water to produce chitosan nanoparticles.

[060] The proportions of the solutions used in carrying out the invention, without, however, being limited to this embodiment, were 4 mL of chitosan solution + 6 mL of TPP solution + 1 mL of bromelain solution. Petition 870260066355, dated 06 / 07 / 2026, page 22 / 44 18 / 33

[061] Nanoparticles with and without bromelain produced with different types of chitosan were physically characterized by DLS, zeta potential and NTA. A. Size distribution, polydispersity index, and zeta potential

[062] The average diameter and polydispersity index (PDI) of the nanoparticles were evaluated using the Zetasizer Nano ZS equipment (Malvern, UK). The average size and polydispersity index were determined by dynamic light scattering (DLS), while the zeta potential was measured by laser Doppler microelectrophoresis (Zetasizer Nano ZS, Malvern, UK).

[063] All analyses were performed on triplicate readings.

[064] Through DLS measurements, chitosan-bromelain nanoparticles showed a smaller size than chitosan nanoparticles and a reduction in zeta potential (Table 1). Table 1. Size distribution of chitosan bromelain nanoparticles. Results are presented as mean ± standard deviation of three measurements. DLS = dynamic light scattering; NTA = nanoparticle tracking analysis; Z-ave = mean size; Dio = 10th percentile; D50 = 50th percentile; D90 = 90th percentile. DLS NTA Size Z-ave 100.9 ± 0.5 177.2 ± 4.8 Petition 870260066355, dated 06 / 07 / 2026, page 23 / 44 19 / 33 (nm) Dio 56.1 ± 0.5 101.1 ± 1.3 D50 112.0 ± 2.5 143.0 ± 5.5 D90 235.0 ± 2.5 273.5 ± 19.7 Polydispersity Index 0.222 ± 0.012 - Zeta Potential (mV) +21.9 ± 0.5 — Concentration (particles / mL) - (1.25 ± 0.03) x 1012 B. Encapsulation efficiency

[065] To determine encapsulation efficiency, the nanoparticles were ultracentrifuged (Centrifuge 5810R, Eppendorf, Germany) at 4,000 g for 10 minutes using 0.5 mL ultrafiltration devices with a 100 kDa membrane (Arnicon® Ultra 100k, Millipore, Germany), thus separating the bromelain that remained free from the encapsulated part. The total protein concentration and enzymatic activity were determined in the initial bromelain solution and in the filtrate (resulting from the described filtration), and the efficiency was calculated according to equation 1: EE = χ 100 Equation 1

[066] The initial bromelain solution showed 2.0 ± 0.3 mg / mL of total protein and 23.3 ± 1.6 U / mL. Protein concentration and enzymatic activity were also determined in the resulting filtered solution (0.25 ± 0.01 mg / mL and 4.5 ± 0.6 U / mL, respectively) for calculating efficiency. Petition 870260066355, dated 06 / 07 / 2026, page 24 / 44 20 / 33 encapsulation, which was 87.0 ± 5.1% of the total protein, corresponding to 80.7 ± 1.1% of the enzymatic activity of bromelain (Table 2). Table 2. Total protein concentration, enzymatic activity, and encapsulation efficiency of bromelain. Total Proteins Enzymatic Activity Initial Bromelain Solution 2.0 ± 0.3 mg / mL 23.3 ± 1.6 U / mL Resulting Filtered Solution 0.25 ± 0.01 mg / mL 4.5 ± 0.6 U / mL Encapsulation Efficiency 87.0 ± 5.1% 80.7 ± 1.1% C. Scanning and transmission electron microscopy

[067] The morphological characteristics of chitosan-bromelain nanoparticles were observed using a Leo 440i scanning electron microscope with a 6070 energy-dispersive X-ray detector (LEO Electron Microscopy, England). Scanning electron microscopy images were obtained using an accelerating voltage of 15 kV. Before analysis, the samples were dried at room temperature under vacuum for 24 hours and coated with gold (92 Å) using a Polaron SC7620 Sputter Coater (VG Microtech, England). The particles were also observed using a transmission electron microscope (TEM JEOL-1400 Plus, 120kV), with the gratings prepared by negative contrast. Petition 870260066355, dated 06 / 07 / 2026, page 25 / 44 21 / 33

[068] In the scanning electron microscopy image (Figure 1A), the chitosan-bromelain nanoparticles presented a spherical shape, a smooth surface, and a diameter of approximately 1 µm, which was larger than the size obtained by DLS and NTA measurements. This shows that even a gentle drying process at room temperature can lead to the aggregation of nanoparticles, resulting in the formation of microparticles (Rampino et al., 2013). In the transmission electron microscopy image (Figure 1), the particles continue to present a spherical shape with a diameter smaller than 50 nm, but it is possible to observe the presence of agglomerates with several particles joined together at approximately 100 nm, which is consistent with the results obtained by DLS and NTA. D. Fourier Transform Infrared Spectroscopy

[069] Infrared spectra of chitosan and chitosan-bromelain nanoparticles, and free bromelain (Figure 2), were obtained using a Fourier transform infrared spectrophotometer (Shimadzu Scientific Instruments, Model 8300, Japan), operating from 4000 to 650 cm-1, with a resolution of 4 cm-1. All measurements were performed in triplicate.

[070] In the bromelain spectrum (black), a peak can be observed at 3280 cm-1, corresponding to enzymatic peptide bonding. A peak can also be observed at 1634 cm-1, indicative of a C=O group, and at 1515 cm-1, indicative of Petition 870260066355, dated 06 / 07 / 2026, page 26 / 44 22 / 33 NH group, which confirms the presence of amino acids. The peak at 1236 cm-1 can be attributed to the CN linkage, in aliphatic amine (Devakate et al., 2009; Soares et al., 2012; Ataide et al., 2017a; Ataide et al., 2017b).

[071] The spectra of the nanoparticles show an absorption band around 3400 cm-1, equivalent to hydrogen interaction and OH vibration (Wu et al., 2005), with greater intensity in the chitosan-bromelain nanoparticle when compared to the chitosan nanoparticle. In both spectra, a peak around 795 cm-1 is also observed, which can be attributed to the presence of vibrations related to the bond between phosphorus and oxygen (PO and POP) (Knaul et al., 1999; Antoniou et al., 2015). In the chitosan nanoparticle (red), it is possible to observe the shift of the C=O vibration peaks of amide II and NH of amine I, which indicates the interaction between the amino groups of chitosan with the phosphate groups of TPP (Knaul et al., 1999; Antoniou et al., 2015).Comparing the same region of the chitosan nanoparticle and the chitosan-bromelain nanoparticle (green), an increase in the intensity of both peaks is observed, which can be attributed to the presence of bromelain, since bromelain also absorbs at the same frequency (Devakate et al., 2009; Soares et al., 2012). Furthermore, once again, a shift in the peaks corresponding to the NH group is observed, indicating interaction with the amino group. E. Stability studies

[072] For the study of nanoparticle stability Petition 870260066355, dated 06 / 07 / 2026, p. 27 / 44 23 / 33 were prepared with a 10 mg / mL bromelain solution, and solutions of chitosan and chitosan-bromelain nanoparticles. The 3 solutions were packaged in glass vials and stored under 4 conditions: bright environment (27±2°C, exposed to light), dark environment (27±2°C, protected from light), refrigerator (5±2°C) and incubator (45±2°C). For the preliminary stability test, the samples were evaluated for 15 consecutive days, starting from day 0 (when they were prepared and packaged). The accelerated stability test was performed sequentially, under the same conditions, evaluating the samples on days 30, 45, 60, 90, 120, 150 and 180 (Anvisa, 2005).

[073] During the preliminary stability study, general parameters such as pH value and macroscopic characteristics were evaluated. The chitosan and chitosan-bromelain nanoparticle suspension did not show macroscopic or pH changes. Thus, the samples continued in the accelerated stability study. After 7 days of study (Figure 3), the chitosan-bromelain nanoparticles showed a significant increase (p <0.05, Student's t-test) in average diameter under all conditions studied, accompanied also by a significant increase in the polydispersity index and a decrease in zeta potential. The increase in particle size is regularly attributed to particle agglomeration, which can be induced by the adsorption of active molecules onto the nanoparticles (Magenheim and Benita, 1991; Abdelwahed et al., 2006).

[074] Protein concentration and enzymatic activity Petition 870260066355, dated 06 / 07 / 2026, page 28 / 44 24 / 33 were also determined during the accelerated stability study. Chitosan nanoparticles did not show significant protein concentration or enzymatic activity at any time or condition studied. Chitosan-bromelain nanoparticles showed a decrease in protein concentration and enzymatic activity (Figure 4) over time, where enzymatic activity decreased significantly (p <0.05, Student's t-test) after 7 days. This decrease was slightly less pronounced when samples were stored at low temperatures (Figure 4), consistent with results reported by other studies using bromelain (Pereira et al., 2014; Spir et al., 2015; Lourenço et al., 2016).Given these results, chitosan-bromelain nanoparticles are unstable when stored in liquid form, and therefore, lyophilization is an important process to increase the preservation of particle size for long-term storage (Fonte et al., 2016; Almalik et al., 2017) and the enzymatic activity of bromelain (Arakawa et al., 2001). Step 2: Lyophilization of chitosan bromelain nanoparticles A. Evaluation of the addition of lipoprotectants and collapse temperature

[075] Trehalose, maltose and glycine were chosen as possible lipoprotectants, due to their collapse temperatures when alone in solution, and that Petition 870260066355, dated 06 / 07 / 2026, page 29 / 44 The collapse temperature of 25 / 33 particles was -24.3°, -25.1°, and -12.7°, respectively. Subsequently, they were dissolved in a 3% (w / v) chitosan-bromelain nanoparticle suspension to evaluate its effect on the collapse temperature of the chitosan-bromelain nanoparticle suspensions. The collapse temperature of chitosan-bromelain nanoparticles with and without lyoprotectants was determined using a microscope coupled to a lyophilization module, Lyostat 2, model FDCS 196 (Linkam Instruments, Surrey, UK), equipped with a liquid nitrogen freezing system (LNP94 / 2) and a programmable temperature controller (TMS94, Linkam). Chitosan bromelain nanoparticles exhibited a collapse temperature of -56°C, which decreased to -49.3°C, -34.8°C, and -28.0°C with the addition of trehalose, maltose, and glycine, respectively.

[076] Although the two sugars (maltose and trehalose) have close collapse temperatures when alone (Table 3), it was unexpected that they resulted in solutions with such different collapse temperatures when combined with the dispersion of nanoparticles. Table 3. Collapse temperature of potential lyoprotectants in isolated solutions and in nanoparticles. Isolated Solution Nanoparticle Solution Without Lyoprotectant - -56 °C Trehalose -24.3 °C -49.3 °C Maltose -25.1 °C -34.8 °C Petition 870260066355, dated 06 / 07 / 2026, page 30 / 44 26 / 33 Glycine -15.2 °C -28.0 °C

[077] In general, the freeze-drying process below 40°C is not advisable (Carpenter et al., 1997; Tattini Jr et al., 2006) and, therefore, maltose and glycine were evaluated in the factorial design as lyophiliscs, since they allow freeze-drying to begin at -40°C. B. Experimental planning of the lyophilization process

[078] To optimize the lyophilized composition of the experimental points, a 2³ factorial design with chitosan-bromelain duplicates was performed using only nanoparticles. The variables (inputs) evaluated were the type of lyophilized agent (glycine or maltose), the concentration of lyophilized agent (3 or 5%, w / v), and the lyophilization process itself (before and after the process). The samples were subjected to flash freezing with liquid nitrogen and lyophilized in the Lyostar 3 Freeze-Drier (SP Scientific, USA). Primary drying was performed by increasing the temperature from -45°C to 10°C by 5°C (Figure 12), waiting for the vial temperature to reach the shelf temperature. After drying, the samples were reconstituted with distilled water to the same initial volume and were physico-chemically characterized by dynamic light scattering and encapsulation efficiency, which were used as responses in the factorial design.The freeze-dried products were subjected to thermogravimetric analysis (TGA-50M, Shimadzu, Japan) in order to determine the moisture content. Petition 870260066355, dated 06 / 07 / 2026, page 31 / 44 27 / 33 residual. The dried nanoparticle samples were precisely weighed (approximately 10 mg) and then heated at 10 °C / min from 25 °C to 300 °C under a nitrogen atmosphere with a flow rate of 50 mL / min. Residual moisture was determined by steady-state weight loss (%) at a temperature of approximately 100 °C (Sylvester et al., 2018).

[079] All lyophilized products had a good appearance and showed no shrinkage. The nanoparticles with glycine and maltose were immediately reconstituted after the addition of water, and the resulting solutions were clear. Without the lyophilized agent, however, resuspension took more than 20 seconds, and the solutions were slightly cloudy, even after vortexing. The lyophilized powders were analyzed by thermogravimetric analysis to determine residual moisture. The compositions with glycine at concentrations of 3% and 5% showed 4.1% and 3.2% mass loss, respectively, while the compositions with maltose showed 5.6% and 3.8%. The lyophilized products were reconstituted in distilled water to analyze the physical characteristics of the particles by dynamic light scattering (Table 4) and the encapsulation efficiency (Figure 5), which were considered outputs for the factorial design.After lyophilization, glycine at a concentration of 3% (w / v) managed to maintain the polydispersity index within the desired values, however the zeta potential decreased. On the other hand, maltose showed the opposite behavior, increasing the polydispersity index and demonstrating... Petition 870260066355, dated 06 / 07 / 2026, pages 32 / 44 28 / 33 has little effect on the zeta potential. Table 4. Nanoparticle size, PDI, and zeta potential before and after the lyophilization process (experimental design). Results are presented as mean ± standard deviation of two compositions, measured three times each. Z-ave = average particle size, PDI = polydispersity index, control = nanoparticle without lyophilization agent. Nanoparticles Diameter (nm) PDI Zeta (mV) Control Before 84.5±6.1 0.231±0.030 27.1 ± 5.1 After 2386±320.5 0.485±0.043 20.2 ± 0.3 Glycine 3% Before 93.7±12.5 0.257±0.020 28.6 ± 3.5 After 161.7±24.7 0.214±0.025 15.7 ± 3.8 Glycine 5% Before 94.2±10.7 0.238±0.006 30.0 ± 2.1 After 223.6±29.1 0.466±0.004 18.6 ± 0.1 Maltose 3% Before 74.5±3.7 0.385±0.039 19.7 ± 4.5 After 90.2±2.4 0.324±0.019 23.0 ± 0.3 Maltose 5% Before 70.6±0.3 0.503±0.006 20.5 ± 0.9 After 76.8±1.1 0.483±0.019 21.6± 0.2

[080] The encapsulation efficiency before the lyophilization process increased in terms of protein concentration after the addition of glycine and maltose, while the encapsulation efficiency in terms of enzymatic activity decreased after the addition of glycine and increased after the addition of maltose (Figure 5). After lyophilization, a small increase in protein encapsulation efficiency was observed for all samples. The encapsulation efficiency in terms of enzymatic activity also Petition 870260066355, dated 06 / 07 / 2026, pages 33 / 44 29 / 33 increased in all compositions.

[081] The responses (outputs) (average particle size, PDI, D10, D50, D90, zeta potential, and encapsulation efficiency) were statistically analyzed using Pareto diagrams (Figure 6), and the main effects of the variables on the responses were also analyzed (Figure 7). Lyoprotectants significantly interfered with almost all responses, excluding zeta potential and encapsulation efficiency in terms of enzymatic activity. Glycine increased the average particle size, increasing the D10, D50, and D90 distribution percentages; however, it also decreased the polydispersity index, indicating a narrower size distribution. On the other hand, maltose decreased the average nanoparticle size and increased the polydispersity index.The concentration of lipoprotective agents was an important variable observed for PDI, D10, and protein encapsulation, with low concentrations (3%, w / v) decreasing PDI and increasing D10 and protein encapsulation.

[082] Excluding protein encapsulation, lyophilization significantly affects all responses. The process increased the average particle size by increasing the distribution percentile, also increasing polydispersity and encapsulation of enzymatic activity. The zeta potential decreased with the lyophilization process, which could cause some problems in the stability of the nanoparticles, which should be studied. Considering all the Petition 870260066355, dated 06 / 07 / 2026, pages 34 / 44 30 / 33 effects, compositions with glycine or maltose at a concentration of 3% (w / v) appear to have the potential to better stabilize chitosan-bromelain nanoparticles. C. Production and characterization of lyophilized nanoparticles

[083] Based on factorial design, it was found that compositions containing 3% (w / v) glycine and maltose as lyophilisates were the most promising for stabilizing chitosan-bromelain nanoparticles. Therefore, a new batch of samples with these excipients was produced and lyophilized following the parameters previously determined. Immediately after the lyophilization process was complete, the samples were characterized and subjected to a stability study. For this study, the dried samples were kept in lyophilization vials sealed with plastic film and stored at room temperature (25° ± 2°) and in a refrigerator (5° ± 2°) protected from light.

[084] The samples were characterized in terms of average size, polydispersity index, and zeta potential, before and immediately after lyophilization (Table 5). The addition of glycine increased the average size of the nanoparticles and the zeta potential and decreased the polydispersity index when compared to nanoparticles without the addition of lyophilizing agents. On the other hand, the addition of maltose has the opposite effect, decreasing the average size and the zeta potential and increasing the polydispersity index. As observed in the experimental design, after the process of Petition 870260066355, dated 06 / 07 / 2026, pages 35 / 44 31 / 33 freeze-drying: the average size of the nanoparticles and the PDI increased, while the zeta potential decreased. Encapsulation efficiency increased in all cases after the freeze-drying process. Table 5. Nanoparticle size, PDI, and zeta potential before and after the lyophilization process. Results presented as mean ± standard deviation of two compositions, measured three times each. Z-ave = average particle size, PDI = polydispersity index, EE Ptn = total protein encapsulation efficiency, EE Ativ = enzymatic activity encapsulation efficiency. Nanoparticles Diameter (nm) PDI Zeta (mV) E Protein EE Activity None 96.7± 0.8 0.179±0.005 25.8±2.5 97.8% 81.6% Glycine 3% Before 101.6±0.6 0.165±0.007 27.3±1.6 96.8% 52.7% After 271.9±4.5 0.223±0.005 11.0±1.1 101.3% 87.4% Maltose 3% Before 91.3± 1.0 0.286±0.006 21.3±2.1 97.3% 82.4% After 127.8±1.7 0.288 ± 0.009 20 ± 0.8 98.8% 93.3%

[085] After the lyophilization process was complete, the morphology of the samples was evaluated by scanning electron microscopy (Figure 8) in dry form (Figure 8A and *B) and after reconstitution (Figure 8C and 8D). Due to equipment limitations, the samples after reconstitution show small spots, making it impossible to adequately determine the nanoparticle boundaries.

[086] The reconstituted samples were also analyzed using a transmission electron microscope (Figure 9). In these images, it is possible to see that both particles Petition 870260066355, dated 06 / 07 / 2026, pages 36 / 44 32 / 33 have a spherical shape, with the lyophilized particles containing glycine exhibiting a larger average diameter and a more homogeneous size distribution when compared to the composition containing maltose. However, the composition with maltose preserved more of the characteristics of the initial chitosan-bromelain nanoparticle suspension, i.e., the presence of clusters of approximately 100 nm composed of several smaller particles.

[087] The samples showed residual moisture of 3.5% and 4.4% for the glycine and maltose samples respectively, which were lower than the values ​​found during the planning, but still higher than the recommended limit of 2% (Abdelwahed et al., 2006; Sylvester et al., 2018). After 90 days of stability study, the residual moisture of the glycine samples increased to 6.2% and 7.6% when stored at room temperature and in a refrigerator, respectively. The maltose samples showed a reduction in residual moisture, which was 2.4% and 1.8% when stored at room temperature and in a refrigerator, respectively.

[088] On days 0, 30, 60, and 90, samples stored at room temperature and in a refrigerator were reconstituted with the same initial volume (3 mL of MilliQ water) and were monitored for average size, polydispersity index, and zeta potential (Figure 10), and protein concentration and enzymatic activity (Figure 11). Despite showing the highest polydispersity index at the beginning of the Petition 870260066355, dated 06 / 07 / 2026, pages 37 / 44 33 / 33 stability study, the chitosan-bromelain nanoparticle composition containing maltose maintained stable particle parameters throughout the study period (Figure 10), with the composition stored in a refrigerator (5° ± 2°) showing the most stable results. In contrast, the compositions with glycine were not stable, reaching a PDI of 1.0 when stored at room temperature (25° ± 2°).

[089] Regarding the parameters related to bromelain stability (enzymatic activity and total proteins), it was possible to note that the protein concentration and enzymatic activity did not show significant changes at the beginning and end of the stability study (days 1 and 90), Figure 11.

Claims

1. Process for obtaining a lyophilized composition of chitosan-based nanoparticles containing bromelain, characterized in that it comprises the following steps: i. Production of chitosan-bromelain nanoparticles a) Add by drop an aqueous solution of TPP at a concentration of 0.5 mg / mL to a chitosan solution at a concentration of 2.5 mg / mL in 1% acetic acid under stirring until complete homogenization; b) Add an aqueous solution of bromelain at a concentration of 10 mg / mL under stirring to the solution obtained in step (a) until complete homogenization; c) Obtain the chitosan and bromelain nanoparticles; ii.Freeze-drying of chitosan-bromelain nanoparticles d) Dissolve the lyoprotectant directly in the nanoparticle solution obtained in step (c); e) Store the mixture of nanoparticles with lyoprotectant obtained in step (d) in an ultra-freezer at a temperature of -80 °C; f) The nanoparticles frozen at -80°C, obtained in step (e), were inserted into the freeze-dryer with the shelf set to -45°C and maintained until reaching the same temperature as the shelf; g) The nanoparticles from step (f) follow a drying ramp, increasing the temperature by 5°C from -45 °C to +10 °C; h) Obtaining the freeze-dried composition of chitosan-based nanoparticles containing bromelain.

2. Process according to claim 1 characterized by the ratio of chitosan, TPP, bromelain being of Petition 870260066355, dated 06 / 07 / 2026, page 39 / 44 2 / 3 4:6:1(v / v).

3. Process according to claim 1 characterized in that agitation in steps (a) and (b) is at 350 rpm.

4. Process according to claims 1 and 3 characterized by step (b) having a duration of 40 min.

5. Process according to claim 1, characterized in that the lipoprotective agent is selected from small molecules, which may be sugars or amino acids such as maltose or glycine.

6. Process according to claim 1, characterized in that in step (g) the temperature is changed when the temperature of the composition reaches the shelf temperature.

7. Process according to claim 1, characterized in that in step (c) the chitosan bromelain nanoparticles have an average diameter of 84.5 to 100.9 nm, a polydispersity index of 0.18 to 0.23, a zeta potential of 21.9 to 27.1 mV and a particle concentration on the order of 1012 particles / mL.

8. Process, according to claim 1, characterized in that additionally in step c, bromelain is encapsulated in chitosan nanoparticles and exhibits an encapsulation effectiveness of 83.4 to 97.8% of the protein concentration, which corresponds to an encapsulation efficiency of 75.5 to 81.6% of the enzymatic activity.

9. Process according to claim 1, characterized in that in step (d) the lyoprotectant is at a concentration of 3% (w / v) relative to the volume of the chitosan-bromelain nanoparticle solution.

10. Process, according to claim 9, characterized in that it has a collapse temperature that varies from -56°C to -28°C, preferably above -40°C, with the addition of maltose and glycine, respectively.

11. Freeze-dried composition of chitosan-based nanoparticles containing freeze-dried bromelain obtained by the process defined in claims 1 to 10, characterized by comprising bromelain, chitosan, lyophilized protective agent, TPP and residual moisture between 3.5 and 4.4 (m / m).

12. Composition according to claim 11 characterized in that the lipoprotective agent is selected from small molecules, which may be sugars or amino acids such as maltose or glycine.

13. Composition according to claim 11 characterized in that it comprises nanoparticles with an average diameter of 88.5 and 127.8 nm, a polydispersity index of 0.29 to 0.34, and a zeta potential of 20.0 to 23.2 mV.

14. Composition according to claim 11 characterized in that it is in the form of lyophilized powder.

15. Composition according to claim 11, characterized in that it has an encapsulation efficiency with maltose of 96.3% to 98.8%, which is equivalent to an enzymatic activity of 73.3% to 99.5%.

16. Use of the composition obtained as defined in claims 1 to 10, characterized by being in the preparation of a medicament for treating wounds.

17. Use of the composition obtained as defined in claims 11 to 15, characterized by being in the preparation of a medicament for treating wounds.

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