Method for producing a hydrogel-type medical device containing embedded copper nanoparticles

Abstract

Disclosed is a method for producing a hydrogel-type medical device, which comprises producing, in advance, copper nanoparticles coated with high-molecular-weight chitosan; and incorporating the particles during the final minutes of a step of crosslinking, which allows the coating to remain on the nanoparticles, preventing the dissolution thereof, and the nanoparticles to be uniformly incorporated into the latticed formation of the hydrogel. Also disclosed are the hydrogel and the use thereof in different types of wounds, burns and ulcerations.

Description

[0001] PROCESS FOR MANUFACTURING A HYDROGEL-TYPE MEDICAL DEVICE INTERLOADED WITH COPPER NANOPARTICLES

[0002] FIELD OF INVENTION

[0003] The present invention belongs to the field of biomedical product development, specifically to the process of making a medical device for the protection of skin wounds, which has a protective effect, induces healing, reduces discomfort associated with the inflammatory process, reduces manipulation of the damaged area, has antimicrobial activity to reduce infections, and self-elimination through bioabsorption of the components.

[0004] STATE OF THE ART

[0005] In the context of complex wound management, diabetes mellitus (DM) is a major contributing factor to slow-healing wounds, ulcers, and amputations in both hospital and outpatient settings (Mousley 2003). Among complicated diabetic wounds are diabetic foot ulcers (DFUs), the most common type of wound, where hyperglycemic conditions complicate and / or delay the physiological healing process (Bandyk 2018). Currently, patients with DFUs require advanced wound care, which involves not only assessing the wounds themselves but also considering the patient's physiological status and comorbidities.

[0006] Within this context, numerous polymeric materials, both natural and synthetic, have been studied for the development of new dressings. Examples of biopolymers (natural polymers) include alginate, dextran, hyaluronic acid (HA), chitosan, cellulose, gelatin, chitin, etc. (Sahana and Rekha 2018, Shah, Sohail et al. 2019, Maaz Arif, Khan et al. 2021). These polymers exhibit excellent biomedical properties such as good biocompatibility, non-immunogenicity, non-toxicity, hemostatic effects, and excellent biodegradability; some also possess antibacterial and wound-healing properties (Sezer and Cevher 2011). However, natural polymers often have poor mechanical properties. Therefore, to enhance the properties of each polymer and achieve better mechanical / biological performance, it is common to use two or more of these polymers combined in a single dressing.Among the biopolymers mentioned here, chitosan is presented as a promising material for use in dressings due to its biocompatible and non-toxic characteristics, flexibility and distinctive resistance (Bonini, Wiedenmann et al. 2006). In addition, it shows important antimicrobial properties, and it is postulated that this is due to its interaction with the negatively charged residues of the bacterial cell wall, which would lead to the penetration of the compound through the cell wall of the bacteria, for its subsequent binding to DNA and inhibition of the synthesis of RNA and proteins (Cava, Lam et al. 2011, Ivask, ElBadawy et al. 2014, Birsoy, Wang et al. 2015).Furthermore, chitosan has been reported to accelerate wound healing at all stages, stop bleeding by inducing thrombosis, improve the function of inflammatory cells, and activate fibroblasts to form a new collagen matrix in defective tissue regions (Beanes, Dang et al. 2003).

[0007] Hydrogels are three-dimensional polymeric structures that can hold up to 1000 times their weight in water without losing their structure. Hydrogels also possess a flexibility similar to natural tissue due to their high water content. While chitosan-based hydrogels exist, several studies have been conducted to improve the biological and mechanical properties of chitosan by combining it with other organic or inorganic materials. Starch is a biocompatible polymer that has been used to create hydrogels with a wide variety of applications (Ismali et al., 2013), but its use in tissue engineering has not yet been explored. In chitosan-based hydrogels, the addition of starch increases the size of the hydrogel's pores, leading to an increased swelling capacity (Ngoenkam et al., 2010).A hydrogel with a high swelling capacity can be used to retain bioactive compounds such as proteins, drugs, or nanoparticles that can subsequently act on a specific target (Catoira, Fusaro et al. 2019). Therefore, a hydrogel composed of chitosan and starch could be used to release antimicrobial compounds into an epithelial lesion, protecting the wound from pathogens.

[0008] The bioactive compound must be incorporated into the hydrogel during the synthesis process or subsequently through adsorption. In the first case, the active ingredient must be resistant to the chemicals and synthesis conditions, while in the second case, it must be smaller than the hydrogel's pores to penetrate them (Elahi et al., 2018). Nanoparticles are defined as particles ranging in size from 1 to 100 nm, which have distinct physicochemical and biological properties compared to larger materials composed of the same element. The chemical nature of nanoparticles can induce biological effects that can be combined with enhanced pharmacological activity to improve treatment efficacy. However, they generally do not provide structural support for cells on their own, nor do they constitute a porous, permeable, yet protective barrier, such as that required in the damaged area.That is why its application in these injuries is generally combined with three-dimensional dressings such as fibrous membranes or hydrogels (Mihai, Dima et al. 2019).

[0009] Hydrogels composed of chitosan and / or starch, both biocompatible and biodegradable polymers of natural origin, have been proposed for wound healing applications (Torres, Commeaux et al. 2013, Ahmed and Ikram 2016). They have been used in a triple polymer combination with PVA to form a hydrogel complemented with zinc oxide nanostructures with an antibacterial effect (Baghaie, Khorasani et al. 2017). This structure presented difficulties in terms of porosity, resulting in a material with poor fluid absorption. On the other hand, a mixture of starch and chitosan, chemically crosslinked with glutaraldehyde, has been proposed as a food coating, thanks to its antimicrobial effect and the production of a hydrogel with superior mechanical properties compared to the individual polymers.

[0010] The use of metallic nanoparticles as an antimicrobial agent to prevent infections in complex wounds has been extensively studied (Bui, Park et al. 2017, Wang, Hu et al. 2017, Khatami, Varma et al. 2018, Paladin and Pollini 2019). However, their use is still viewed with caution due to the potential harm they could cause to the environment and health; therefore, any proposal must always be accompanied by in-depth cytotoxicity and dosage analyses. In the specific case of copper nanoparticles (CuNps), toxicity studies have shown a dose-dependent effect, due to the accumulation of Cu(2+) ions in tissues, which stimulates the production of reactive oxygen species (Song, Vijver et al. 2015). However, when CuNps incorporated into chitosan bandages have been used, a low level of toxicity and a potent antibacterial effect have been observed, promoting wound healing (Gopal, Kant et al. 2014).The antimicrobial action of CuNps occurs through two main mechanisms: 1) substitution of potassium ions in membranes, which alters their redox potential; 2) induction of peptidoglycan hydrolysis in bacterial cell walls, changing the osmotic pressure of the pathogen (Mohandas, Deepthi et al. 2018).

[0011] Pressure ulcers are complex and heterogeneous, so no dressing currently available is ideal for all types of wounds. Dressings are needed that allow for appropriate gas and fluid exchange, have a microscopic structure conducive to cell support, migration, and proliferation, and protect the wound area from infection and contamination. Given the above, it is imperative to have improved medical devices that ensure the protection of the ulcerated area while the damaged tissue regenerates.

[0012] Although there are dressing-type devices available on the market, these lack multifunctionality, so it is still necessary to develop a dressing that allows combining in the same device the functions of protection, induction of healing, reduction of discomfort associated with the inflammatory process, reduction of manipulation of the damaged area, antimicrobial activity to reduce infections, self-elimination through bioabsorption of the components, and that does not require the use of other dressings or bandages.

[0013] References

[0014] Mousley, M. (2003). "Diabetes and its effect on wound healing and patient care." Nurs Times 99(42): 70, 73-74.

[0015] Sahana, T. G. and P. D. Rekha (2018). "Biopolymers: Applications in wound healing and skin tissue engineering." Mol Biol Rep 45(6): 2857-2867.

[0016] Shah, S. A., M. Sohail, S. Khan, M. U. Minhas, M. de Matas, V. Sikstone, Z. Hussain, M. Abbasi and M. Kousar (2019). "Biopolymer-based biomaterials for accelerated diabetic wound healing: A critical review." Int J Biol Macromol 139: 975-993.

[0017] Maaz Arif, M., S. M. Khan, N. Gull, T. A. Tabish, S. Zia, R. Ullah Khan, S. M. Awais and M. Arif Butt (2021). "Polymer-based biomaterials for chronic wound management: Promises and challenges." International Journal of Pharmaceutics 598: 120270.

[0018] Sezer, A. D. and E. Cevher (2011). Biopolymers as Wound Healing Materials: Challenges and New Strategies.

[0019] Bonini, M., A. Wiedenmann and P. Baglioni (2006). "Synthesis and characterization of magnetic nanoparticles coated with a uniform silica shell." Materials Science and Engineering: C 26(5): 745-750.

[0020] Cava, F., H. Lam, M. A. de Pedro and M. K. Waldor (2011). "Emerging knowledge of regulatory roles of D-amino acids in bacteria." Cell Mol Life Sci 68(5): 817-831.

[0021] Ivask, A., A. EIBadawy, C. Kaweeteerawat, D. Boren, H. Fischer, Z. Ji, C. H. Chang, R. Liu, T. Tolaymat, D. Telesca, J. I. Zink, Y. Cohen, P. A. Holden and H. A. Godwin (2014). "Toxicity Mechanisms in Escherichia coli Vary for Silver Nanoparticles and Differ from Ionic Silver." ACS Nano 8(1): 374-386 Birsoy, K., T. Wang, W. W. Chen, E. Freinkman, M. Abu-Remaileh and D. M. Sabatini (2015). "An Essential Role of the Mitochondrial Electron Transport Chain in Cell Proliferation Is to Enable Aspartate Synthesis." Cell 162(3): 540-551.

[0022] Beanes, S. R., C. Dang, C. Soo and K. Ting (2003). "Skin repair and scar formation: the central role of TGF-beta." Expert Rev Mol Med 5(8): 1-22.

[0023] Catoira, M. C., L. Fusaro, D. Di Francesco, M. Ramella and F. Boccafoschi (2019). "Overview of natural hydrogels for regenerative medicine applications." Journal of Materials Science: Materials in Medicine 30(10): 115.

[0024] Mihai, M. M., M. B. Dima, B. Dima and A. M. Holban (2019). "Nanomaterials for Wound Healing and Infection Control." Materials (Basel, Switzerland) 12(13): 2176.

[0025] Ahmed, E. M. (2015). "Hydrogel: Preparation, characterization, and applications: A review." Journal of Advanced Research 6(2): 105-121.

[0026] Ahmed, S. and S. Ikram (2016). "Chitosan Based Scaffolds and Their Applications in Wound Healing." Achievements in the Life Sciences 10(1): 27-37.

[0027] Baghaie, S., M. T. Khorasani, A. Zarrabi and J. Moshtaghian (2017). "Wound healing properties of PVA / starch / ch ¡tosan hydrogel membranes with nano Zinc oxide as antibacterial wound dressing material." J Biomater Sci Polym Ed 28(18): 2220-2241.

[0028] Bui, V. K. H., D. Park and Y.-C. Lee (2017). "Chitosan Combined with ZnO, TiO2 and Ag Nanoparticles for Antimicrobial Wound Healing Applications: A Mini Review of the Research Trends." Polymers 9(1): 21

[0029] Wang, L., C. Hu and L. Shao (2017). "The antimicrobial activity of nanoparticles: present situation and prospects for the future." International journal of nanomedicine 12: 1227-1249.

[0030] Khatami, M., R. S. Varma, N. Zafarnia, H. Yaghoobi, M. Sarani and V. G. Kumar (2018). "Applications of green synthesized Ag, ZnO and Ag / ZnO nanoparticles for making clinical antimicrobial wound-healing bandages." Sustainable Chemistry and Pharmacy.

[0031] Song, L., MG Vijver, WJGM Peijnenburg, TS Galloway and CR Tyler (2015). "A comparative analysis on the in vivo toxicity of copper nanoparticles in three species of freshwater fish." Chemosphere 139:181-189.

[0032] Gopal, A., V. Kant, A. Gopalakrishnan, S. K. Tandan and D. Kumar (2014). "Ch ¡tosan-based copper nanocomposite accelerates healing in excision wound model in rats." Eur J Pharmacol 731:8-19.

[0033] Mohandas, A., S. Deepthi, R. Biswas and R. Jayakumar (2018). "Chitosan based metallic nanocomposite scaffolds as antimicrobial wound dressings." Bioactive Materials 3(3): S1-J1. BRIEF DESCRIPTION OF THE FIGURES

[0034] Figure 1: Micrographs of copper-chitosan core-shell nanoparticles. (a) TEM images are shown and (b) AFM images are shown.

[0035] Figure 2: SEM micrographs of the lyophilized hydrogels.

[0036] Figure 3: Hydrogels obtained, from left to right: without nanoparticles; 100 pg / mL of nanoparticles; 150 pg / mL of nanoparticles; 200 pg / mL of nanoparticles.

[0037] Figure 4: SEM micrographs and EDS analysis of the modified hydrogels with concentrations of 50 to 300 mg / mL of the core-shell nanoparticles.

[0038] Figure 5: FTIR spectra of the hydrogels as the concentration of core-shell nanoparticles increased.

[0039] Figure 6: Representative photo of the pathogen inhibition assay by diffusion on agar.

[0040] Figure 7: Statistical analysis of the pathogen growth inhibition capacity induced by hydrogels.

[0041] Figure 8: Evaluation of cytotoxicity in human epithelial cells at different exposure times A) cytotoxicity measurement at 24 hours and B) at 48 hours. Statistical analysis: One-way ANOVA followed by Tukey's test for multiple comparisons. ****p<0.0001; ns: not significant, with respect to the control (no treatment) n=6.

[0042] Figure 9: Wound healing in healthy murine models. Graph of the diameter of mouse wounds in millimeters measured on days 0, 3, 6, 9, and 12. The graph shows the mean measurement per group and the standard deviation. Statistical analysis using a mixed-effects model, analyzed by Restricted Maximum Likelihood (REML), followed by multiple comparisons with Tukey's test, using GraphPad Prism software (v.8.0.2), α = 0.0001, comparing with the untreated control. Figure 10: Wound healing in healthy murine models. Representative images of the wounds of the animals in each group on day 3.

[0043] Figure 11: Wound healing in diabetic murine models with different treatments. A) Graph of the diameter of mouse wounds in millimeters measured on days 0, 3, 6, 9, and 12. The graph shows the mean measurement per group and the standard deviation. Statistical analysis using a mixed-effects model, analyzed by Restricted Maximum Likelihood (REML), followed by multiple comparisons with Dunnett's test, using GraphPad Prism software (v.8.0.2), p < 0.05, comparing with the untreated control. B) Representative images of the wounds of the animals in each group on day 3.

[0044] Figure 12: Histological evaluation of the wound. A) Graphical representation of the epithelial thickness of the wound tissue in animals with the different treatments on days 6 and 12. The graph shows the average measurement per group and the standard deviation. Statistical analysis was performed using GraphPad Prism software (v.8.0.2), two-way ANOVA, followed by Tukey's multiple comparisons test, * <0.05; ** <0.01; *** <0.001. B) Representative images of the histological sections of the wounds stained with H&E (20X) on days 6 and 12.

[0045] Figure 13: Representative images of histological sections of wounds stained with H&E on day 7. AB) Untreated control, reduced epidermal thickness and histological junctions. CD) Hydrogel-CuNp (200 pg / ml). Black lines: hair follicles; Red lines: sebaceous glands.

[0046] Figure 14: Rectal temperature of rabbits in each group over time. n=3.

[0047] Figure 15: Temperature distribution by group throughout the experiment during morning (AM) and afternoon (PM) hours. df: Group 1 Control cleaning with PBS, df: Group 2 Hydrogel only, df: Group 3 Hydrogel with copper nanoparticles (CuNp) at 150 pg / ml, df: Group 4 Commercial control. Statistical analysis by Kruskal-Wallis with the Bonferroni multiple comparison test, χ² <0.05.

[0048] Figure 16: Weight of the rabbits over time. A) Weight of each animal in each group at different time points, n=3. B) Average weight of each group at each time point. Statistical analysis by Two-way ANOVA, followed by Tukey's multiple comparison test, *** <0.0001.

[0049] Figure 17: Weight distribution by group throughout the experiment. df: Group 1 Control cleaning with PBS, df: Group 2 Hydrogel only, df: Group 3 Hydrogel with copper nanoparticles (CuNp) at 150 pg / ml, df: Group 4 Commercial control. Statistical analysis by One-way ANOVA, followed by Tukey's multiple comparison test, * <0.05.

[0050] DETAILED DESCRIPTION OF THE INVENTION

[0051] A manufacturing process and a medical device are presented, a type of polymeric hydrogel loaded with copper nanoparticles, which protects skin wounds, with an antimicrobial effect, promotes tissue recovery in a short time and with less discomfort, and the material is bioabsorbable, so that it does not need to be replaced.

[0052] This multipolymeric hydrogel is generated from high molecular weight chitosan polymers and starch, arranged in equivalent proportions and crosslinked with genipin. During the crosslinking process, the hydrogel volume was interloaded with copper nanoparticles under precise conditions and timing, ensuring an optimal structure for the device's function. The interload process of the nanoparticles stabilizes the structure of this biomaterial, contributing to the absorption of wound exudate, facilitating biosupport for healing that improves epithelialization, and reducing the possibility of infection by bacterial pathogens, whose growth is inhibited by the incorporation of copper nanoparticles.This device promotes tissue recovery with better epithelialization quality and fewer dressing changes associated with the progressive biodegradation of the material in the wound, since the composition of the hydrogel allows the degradation and final bio-absorption of the material, so that it is not necessary to replace or remove it continuously.

[0053] Hydrogel can be effective on various types of wounds, including traumatic and post-operative wounds, chronic and diabetic wounds, ulcers, and burns. Its high porosity, fluid absorption, antimicrobial effect, and biodegradable composition make it suitable for use in both humans and various animal species.

[0054] First, the copper nanoparticles are prepared and coated with high molecular weight chitosan. The preparation process comprises: i. A chitosan mixture is prepared with a range between 1.3 and up to 1.6% weight / vol, which is dissolved in 1.5% acetic acid, heated to 60°C until completely dissolved;

[0055] i. In parallel, a solution of CuSCUShbO (between 23 and 100 g) is prepared in 500 ml of distilled water, while stirring; iii. 100 ml of the previous chitosan-acetic acid mixture is added dropwise to the CuSCUShbO solution, and stirring is constant until fully incorporated; iv. The temperature is increased in a water bath to between 80° and 90°C for 1 h, and 100 ml of NaOH (625 mM) is added dropwise until pH 9.0 is reached; v. The mixture is stirred until a gray-brown color is observed, and 100 ml of ascorbic acid (between 410 and 450 mM) is added dropwise until a reddish color is reached, stirring is maintained at between 80° and 90°C for 30 min in the water bath; and vi. The final mixture is centrifuged at 12000 g and the precipitate is dried at 80° C for 24 h.

[0056] The process for preparing the hydrogel includes at least the following steps: a) First, a chitosan solution of 1.0 to 2% w / v in 1.5% acetic acid with a pH of 4.5 is prepared and stirred at 500-800 rpm for 3 hours at 25°C; b) A starch solution of 1.0 to 3% w / v is prepared by continuous stirring at 500-800 rpm for 1 hour at 100°C; c) 35-65% of the chitosan solution and 35-65% of the starch solution are mixed and stirred at 500-800 rpm for 1.5 to 3 hours until complete homogenization is achieved; d) Genipin is added in a range of 0.01 to 0.05% of the mixture volume and stirring is continued for 4 days at 25°C to ensure the initial crosslinking phase of the hydrogel;e) The coated nanoparticles are incorporated between 20 and 40 minutes before the end of the crosslinking process with genipin. The previously prepared nanoparticles are weighed for dispersion, maintaining ranges of 50 to 500 milligrams of nanoparticles per liter of hydrogel. The nanoparticles must be carefully dispersed in the stirring mixture; f) Three freezing cycles are performed at -20°C for 12 hours, followed by thawing at 25°C, as part of the physical / chemical crosslinking phase of the biomaterial;g) Excess water is removed from the samples using filter paper, and the fully cross-linked biomaterial is dispensed into molds of inert solid material, in the required dimensions, and frozen at -80°C for 4 to 12 hours. Subsequently, the units are dried by freeze-drying for 16 to 24 hours, depending on the desired material thickness. A lightweight, spongy, and dry biomaterial with a high liquid absorption capacity is obtained.

[0057] This process achieves a homogeneous dispersion of the nanoparticles in the biomaterial, maintaining its porosity and promoting its structural stability.

[0058] The processes described, taken together, ensure that a hydrogel containing copper nanoparticles interconnected and homogeneously arranged within the device is obtained. For this procedure to be effective, it is essential that the coated nanoparticles be incorporated in the final minutes of the crosslinking stage. This ensures that the coating remains on the nanoparticles and is not dissolved by the other components, allowing the particles to become uniformly integrated into the hydrogel's network structure.

[0059] The second protective element is the hydrogel obtained through the process. Its components allow for its degradation and bio-absorption, so that continuous replacement or removal is not necessary in wounds, burns, or ulcerations.

[0060] The hydrogel device comprises at least: a. a freeze-dried chitosan-starch hydrogel matrix; b. 0.005 to 0.05 wt / v of coated copper nanoparticles; and c. 0.01 to 0.05% of a crosslinking agent.

[0061] The chitosan-starch matrix crosslinked with copper nanoparticles is bioadherent, facilitating the exchange of exudates and gases in tissue damaged by wounds, burns, ulcers, bedsores, or other epithelial injuries. It exhibits inflammation-regulating, healing, and epithelialization-enhancing properties in damaged tissue. Furthermore, the copper nanoparticles possess antimicrobial activity that reduces the growth of pathogenic bacteria characteristic of skin infections.

[0062] This hydrogel allows combining in a single device the functions of protection, induction of healing, reduction of discomfort associated with the inflammatory process, reduction of manipulation of the damaged area, antimicrobial activity for the reduction of infections, self-elimination through bioabsorption of the components, and does not require the use of other dressings or bandages.

[0063] This hydrogel combines the three elements that make this multifunctional dressing more effective: high absorption and swelling capacity, antimicrobial activity, and promotion of the physiological healing process.

[0064] The hydrogel matrix is ​​composed of equal parts of the naturally occurring polymers chitosan and starch, chemically cross-linked, resulting in a homogeneous structure with excellent fluid absorption properties. The copper nanoparticles, which must be prepared beforehand for in situ incorporation into the hydrogel, exhibit antimicrobial activity and a lack of toxicity in in vitro and in vivo human epithelial cell models.

[0065] The final material is freeze-dried, which enhances permeability, ensuring the absorption of exudate and gas exchange at the wound site. This promotes proper moisture and aeration balance in exposed tissue, facilitating the recovery process and reducing associated discomfort.

[0066] The resulting hydrogels are of varying sizes, depending on the mold in which they were lyophilized, and have a light blue hue that depends on the concentration of copper nanoparticles. These hydrogels possess a network structure with uniform pores, which strengthens as the copper nanoparticles cross-link, reaching a maximum nanoparticle incorporation of 0.05 wt / v. However, cytotoxicity analyses in human fibroblast models (HDF cell line) showed an optimal cell viability range following treatment with hydrogels loaded with copper nanoparticles between 0.005 and 0.02 wt / v.

[0067] The evaluation of resistance to pathogens, such as Staphylococcus aureus, shows that hydrogels loaded with copper nanoparticles in a range between 0.01 to 0.03% w / v inhibit the growth of these pathogenic bacteria, in both antibiotic-sensitive and antibiotic-resistant strains.

[0068] When analyzing the healing and anti-inflammatory capacity in vivo in murine models, we observed that treatment with hydrogels significantly reduced wound diameter within the first three days of treatment, as they absorbed exudate and reduced inflammation. These findings were demonstrated in both healthy mice and diabetic mouse models. Furthermore, histopathological studies showed improved epithelialization during healing in both the first and second weeks post-treatment in animals treated with hydrogels interleaved with nanoparticles, compared to those that received hydrogels without nanoparticles, commercial dressings, or untreated animal groups.

[0069] Repeated application of hydrogels (acute treatment) was shown not to cause fever (pyrogens), loss of appetite, or weight loss in acute tolerance studies in rabbits.

[0070] The advantages of this hydrogel over others available on the market are the following: It combines two natural polymers with hydrophilic characteristics that have already demonstrated high absorption of aqueous material and healing capacity, which is not currently being used for a starch-chitosan hydrogel; It contains copper nanoparticles as an antimicrobial agent.This differentiates it from other similar proposals that primarily use silver; its dry presentation ensures spontaneous hydration with wound exudates and a moisture balance that does not require the application of other dressings; its spontaneous degradation and long duration for days significantly reduce wound manipulation; its antimicrobial and healing-inducing effect, thanks to the combined properties of chitosan and copper nanoparticles, as well as its high swelling capacity, provided by starch, make this hydrogel a unique product for wound healing; materials are used that allow obtaining a functional hydrogel at low cost, and its synthesis process guarantees its scalable production.

[0071] APPLICATION EXAMPLES

[0072] Example 1: Manufacturing of nanoparticles

[0073] The process of preparing the coated nanoparticles comprises the following steps: a) A mixture of chitosan in 1.5% w / v acetic acid is prepared and heated to 60°C until completely dissolved; b) In parallel, a solution of 25g CuSC ShbO₄ in 500 ml of distilled water is prepared and stirred; c) 100 ml of the previous chitosan-acetic acid mixture is added dropwise to the CuSCUShbO₄ solution and stirred constantly until fully integrated; d) The temperature is increased in a water bath to 85°C for 1 h, and 100 ml of NaOH (625 mM) is added dropwise until pH 9.0 is reached; e) The mixture is stirred until a gray-brown color is observed, and 100 ml of ascorbic acid (430 mM) is added dropwise until a reddish color is reached, stirring is maintained at a range of 85°C for 30 min in the water bath; and f) The final mixture is centrifuged at 12000 g and the precipitate is dried at 80°C for 24 h.

[0074] The core-shell nanoparticles obtained are semi-spherical in shape and approximately 50 nanometers in size, as shown in the TEM micrographs (Figure 1A). It can be observed that the copper is covered by a layer of organic matter that is more translucent when the electron beam passes through the sample. This information was corroborated by AFM analysis (Figure 1B), which shows copper nanostructures with an organic layer. These nanostructures are agglomerated by the organic layer, resulting in sizes greater than 100 nm.

[0075] Example 2: Manufacturing of hydrogels and incorporation of nanoparticles.

[0076] The hydrogel manufacturing process comprises the following stages:

[0077] 1. A chitosan solution of 2.5% weight / vol in 1.5% acetic acid with a pH of 4.5 is prepared, which is stirred at 600 rpm for 3 hours at 25°C;

[0078] 2. A starch solution of 2% w / v is prepared by continuous stirring at 600 rpm for 1 hour at 100°C;

[0079] 3. Mix 50% of the chitosan solution and 50% of the starch solution, stirring at 600 rpm for 2 hours until complete homogenization is achieved;

[0080] 4. Genipin is added in a range between 0.03% of the volume of the mixture and stirring was continued for 4 days at 25°C to ensure the initial crosslinking phase of the hydrogel;

[0081] 5. The coated nanoparticles are incorporated between 20 and 40 minutes before the end of the crosslinking process with genipin. The previously prepared nanoparticles are weighed for dispersion, maintaining ranges of 50 to 500 milligrams of nanoparticles per liter of hydrogel. The nanoparticles must be carefully dispersed in the stirring mixture;

[0082] 6. Three freezing cycles are performed at -20°C for 12 hours, followed by thawing at 25°C, as part of the physical / chemical crosslinking phase of the biomaterial.

[0083] 7. Excess water is removed from the samples using filter paper, and the fully cross-linked biomaterial is dispensed into inert solid molds of the required dimensions and frozen at -80°C for 4 to 12 hours. The units are then freeze-dried for 16 to 24 hours, depending on the desired thickness. This results in a lightweight, spongy, and dry biomaterial with high liquid absorption capacity.

[0084] The resulting hydrogels have a porous structure with a particle size distribution ranging from 10 to 250 nm, as shown in Figure 2. EDS analysis was also performed to determine the elements present before incorporating the core-shell particles. The results show the highest concentration of oxygen, which belongs to the chitosan matrix (from the functional groups -OH and -COOH).

[0085] Subsequently, different concentrations of nanoparticles, from 50 to 300 mg / mL, were added to the hydrogels. Figure 3 shows different hydrogels with increasing concentrations of nanoparticles; the gels retain their color due to the coating provided by the nanoparticles, which prevents their rapid oxidation.

[0086] These core-shell nanoparticle modified hydrogels were characterized by SEM and FTIR as shown in Figures 4 and 5.

[0087] SEM analysis of the hydrogels revealed core-shell particles, with nanoparticles (approximately 50 nm) observed on the hydrogel surface forming agglomerates. These structures are due to the copper being coated with a chitosan layer, which protects the copper from oxidation and preserves its antimicrobial properties.

[0088] It was observed that as the amount of nanoparticles increased, the pores of the hydrogels became clogged with them. Since the hydrogel matrix and the core-shell have the same components, the particles were able to diffuse within the hydrogel pores without losing their protective coating. EDS analysis indicated a gradual increase in copper content in the hydrogels, from 0.4% copper for the 100 mg / mL sample to 6.6% copper for the 300 mg / mL sample. These results were corroborated by FTIR (Figure 5), which showed variations in the functional groups present in the hydrogels. The hydrogel with the highest amount of nanoparticles exhibited a characteristic broad band at 3451 cm⁻¹ (stretch vibrations of the -OH group), indicating a higher proportion of these groups in the hydrogels.In all hydrogels, the organic components chitosan and genipin exhibit the characteristic signals of 2921 cm⁻¹ (symmetric and asymmetric C-H stretching vibrations), 1669 cm⁻¹ (C=O carbonyl stretching vibration), and 1559 and 1441 cm⁻¹ (symmetric and asymmetric bending vibrations of the -OCH₃ and -CH₃ groups). Peaks at 1156 and 1015 cm⁻¹ (symmetric and asymmetric CO₂ stretching vibrations) are also present; all these signals correspond to the hydrogel matrix. Additionally, a signal at 640 cm⁻¹ corresponds to the Cu-O bond vibration, indicating the interaction between the copper and chitosan of the matrix and the nanostructure layer. As a greater quantity of core-shell nanoparticles is added, an increase in these signals is observed, indicating a rise in the concentration of these nanostructures and a modification of the hydrogel.

[0089] Example 3: Antimicrobial activity; inhibition of pathogens.

[0090] The pathogen growth inhibition capacity of hydrogels was evaluated using the agar diffusion technique. Pathogenic bacterial strains of Staphylococcus aureus 43300, resistant to the antibiotic oxacycline, were studied. The bacterial culture was homogeneously dispersed on Mueller-Hinton agar plates at McFarland concentrations equivalent to 0.5 in a volume of 100 lp. Samples of 6 mm in diameter and 3 mm in height were taken from each of the manufactured hydrogels and applied to the agar plate previously inoculated with the pathogenic bacteria. After 24 h, the diameter of the inhibition halos (translucent circumference) around each applied sample was measured (Figure 6). Three replicates were performed per experiment. No significant differences were observed between the samples of the unloaded hydrogel, the oxacycline antibiotic control, and the commercial control.However, statistically significant differences were observed between the antibiotic control and the unloaded hydrogel compared to all hydrogels loaded with copper nanoparticles, with the effect being highly significant in the hydrogels loaded with 100 to 300 pg / mL (p<0.0001). Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparisons test (Fig. 7).

[0091] The trials evaluated corresponded to:

[0092] 1-CuNp-free hydrogel.

[0093] 2- Hydrogel-CuNp 50 pg / ml

[0094] 3- Hydrogel-CuNp 100 pg / ml

[0095] 4- Hydrogel-CuNp 150 pg / ml

[0096] 5- Hydrogel-CuNp 200 pg / ml

[0097] 6- Hydrogel-CuNp 300 pg / ml CC- Commercial control. ox- Oxacycline antibiotic sensidisco control.

[0098] Example 4: Evaluation of cytotoxicity in human epithelial cells at 24 and 48 h.

[0099] The cytotoxicity of the generated material was determined by evaluating the viability of human dermal fibroblasts (HDF cell line) treated with different hydrogels loaded with copper nanoparticles. The cells were treated with the corresponding hydrogel for 24 and 48 hours, and culture viability was measured at both time points using the MTT method. Hydrogels obtained by chemical / physical crosslinking of chitosan-starch that were not loaded with copper nanoparticles or showed significant differences in cell viability compared to untreated cells.

[0100] Measurements at both time points show that hydrogels loaded with copper nanoparticles at concentrations of 50, 100, and 150 pg / ml exhibited a significant increase in viability compared to untreated cells (p < 0.0001). However, hydrogels loaded with copper nanoparticles at concentrations of 200 and 300 pg / ml showed a significant decrease in viability compared to untreated cells (p < 0.0001). Figure 8A shows cytotoxicity measurements at 24 hours, and Figure 8B shows measurements at 48 hours.

[0101] Example 5: Evaluation of healing capacity in a murine animal model.

[0102] Healthy murine model:

[0103] To evaluate the effect of hydrogels on tissue regeneration in a healthy murine model, CF-1 mice were used. The 24 mice with an average weight of 42 g were randomly distributed into 4 groups of 6 animals each as follows: o Group 1: No treatment o Group 2: Hydrogel alone o Group 3: Hydrogel with copper nanoparticles (CuNp) at 150 pg / ml o Group 4: positive control, Tegaderm Hydrogel 3M (commercial hydrogel).

[0104] All mice were anesthetized using a 120 mg / kg dose of ketamine / xylazine administered intraperitoneally, and a 6 cm diameter incision was made on their back using a biopsy punch of the same diameter. After imaging, different dressing treatments were applied to study skin regeneration according to the previously described groups. The dressings were applied for the treatments described above to study skin regeneration. Mice in each group were marked and numbered 1 to 4 to allow for personalized monitoring of their healing process. The diameter of the wounds was measured every three days (days 3, 6, 9, and 12), and two animals per group were sacrificed on day 6 to obtain tissue samples from the wound for subsequent histological analysis. The remaining animals were sacrificed at the end of the experiment, on day 12, and the same samples were taken.

[0105] The initial and final weights in grams (g) of the healthy mice were recorded for the experiment; the averages per group are shown in Table 1.

[0106] Table 1: Average weight of individuals in the different groups.

[0107] Treatment of wounds in healthy mice with the hydrogel containing copper nanoparticles at 150 showed a significant decrease in wound size by day 3, similar to the commercial control. However, this significant decrease was also observed by day 6 post-treatment, a day on which the commercial control showed no difference from the untreated control (Figure 9). Furthermore, it was also observed that by day 3, the wounds of mice treated with the CuNps-loaded hydrogel had a cleaner and healthier appearance, similar to the commercial control, which was not observed in the untreated control groups or the hydrogel-only group (Figure 10).

[0108] Diabetic murine model:

[0109] A diabetic mouse model with an average blood glucose level of 313.3 mg / dL was established, and the healing effect of hydrogels was analyzed in healthy mice. The 40 mice, with an average weight of 42 g, were randomly assigned to 5 groups of 8 animals each, as follows: Group 1: No treatment; Group 2: Hydrogel alone; Group 3: Hydrogel with copper nanoparticles (CuNp) at 100 pg / ml; Group 4: Hydrogel with copper nanoparticles at 200 pg / ml; Group 5: Positive control, Tegaderm Hydrogel 3M (commercial hydrogel).

[0110] The wound healing trial lasted 12 days. All mice were anesthetized using a 120 mg / kg dose of ketamine / xylazine administered intraperitoneally. Two 6-cm diameter incisions were then made on the back using a biopsy punch of that size, and different dressing treatments were applied to study skin regeneration according to the groups described previously. Wound size (diameter) was measured on day 3 by taking images with a graduated magnifying glass and recording the wound diameter in millimeters. On day 6, half of the animals in each group (4 animals) were sacrificed, and tissue samples were taken for histological analysis. The remaining animals were monitored until day 12, when they were sacrificed, and final tissue samples were taken.

[0111] The initial and final weights in grams (g) and blood glucose levels in mg / dL of the diabetic mice were recorded; the averages per group are shown in Table 2.

[0112] Table 2: Record of initial and final weights and blood glucose levels by group. Treatment of wounds in diabetic mice with the hydrogel containing copper nanoparticles at 100 and 200 pg / ml showed a significant decrease in wound size on day 3 post-treatment (Figure HA), which was not observed in the other treatments, whether with hydrogel alone, the commercial control, or no treatment. Furthermore, on day 3, the wounds were also smaller and had more defined borders in the groups treated with the CuNp-loaded hydrogel compared to the other groups (Figure 11B).

[0113] Tissue samples from wounds taken on days 6 and 12 were fixed in 10% formaldehyde solution for 24 hours at room temperature, dehydrated in a graduated series of alcohols, and embedded in paraffin blocks. Subsequently, they were cut into 4 µm sections, deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E) to observe re-epithelialization and granulation tissue formation.

[0114] The tissues were observed under a light microscope, and two images were taken for each wound, calibrated to 200 micrometers with 20X magnification. The thickness of the epithelium was measured from the basement membrane to the stratum corneum, taking three data points per wound, i.e., obtaining six data points per mouse. The data were plotted, representing the average epithelial thickness in micrometers (µm) ± the standard deviation (SD) (Figure 12).

[0115] A statistically significant greater epidermal thickness was identified in the wound tissue of animals treated with the hydrogel containing CuNp at 200 pg / ml on days 6 and 12 of treatment compared to the untreated control and the hydrogel without nanoparticles (Figure 12A), which can be observed in the representative histology images in Figure 12B).

[0116] In addition to quantifying the thickness of the epithelium, the presence of appendages such as hair follicles and sebaceous glands was observed, identifying a greater presence of them in the tissue treated with the CuNp Hydrogel 200 pg / ml (Figure 13), consistent with the results obtained previously where the control shows reduced epidermal thickness and histological junctions (Figure 13 AB), while the tissue treated with the CuNp Hydrogel 200 pg / ml shows greater epidermal thickness and a greater presence of appendages (Figure 13 CD).

[0117] Example 6: Safety model in rabbits, pyrogen assessment.

[0118] The study was conducted according to the recommendations of ISO 10993-1 Food and Drug Administration (FDA), 2016). Rectal temperature was measured in rabbits after dermal application of the hydrogels under study.

[0119] The rabbits were randomly distributed into 4 experimental groups with 3 animals per experimental group:

[0120] Group 1: Control of cleaning with saline solution (PBS)

[0121] Group 2: Hydrogel only

[0122] Group 3: Hydrogel with copper nanoparticles (CuNp) at 150 pg / ml

[0123] Group 4: Commercial control (Tegaderm Hydrogel 3M).

[0124] The pyrogen assessment trial lasted 15 days. Animals underwent 8 mm diameter incisions on their backs, made with a biopsy punch of that size, and were treated with different dressings according to the previously described groups. Statistical analysis was performed using descriptive statistics with measures of central tendency, dispersion, line graphs, and boxplots for the variables temperature and weight. The normality and sphericity of the repeated measures data were analyzed. Based on these results, ANOVA was performed using the ezANOVa package in R, along with Kruskal-Wallis tests and Bonferroni or Dunn multiple comparisons as appropriate.

[0125] The rectal temperature of the animals was measured every 12 hours for 15 days after the start of the trial and the data were graphed according to the time of the measurements corresponding to the morning (AM) and afternoon (PM).

[0126] According to the analyses performed, the distribution of animal temperatures during the 15 days of the experiment remained within normal ranges (considering the normal range for rabbits to be between 38.5°C and 40°C), and no differences were observed between groups at any time point (Figure 14). Although morning temperatures were higher than afternoon temperatures, they still remained within normal ranges. By grouping the temperatures for each day by animal, group, and time of day, and analyzing their distribution (Figure 15), a difference was identified between the temperatures of the group treated only with hydrogel (group 2) compared to the group treated with hydrogel loaded with copper nanoparticles (group 3), the latter being significantly lower.However, it is worth noting that, as previously mentioned, despite these differences, the temperatures of both groups are within normal ranges, with the temperature of the animals treated with the hydrogel loaded with CuNps being far from the upper limit.

[0127] In addition, the animals' weight was analyzed during the treatment. The animals were weighed on days 0, 2, 5, 10, and 15, and their weight was recorded in grams (g) (Figure 16A). According to the data obtained and the analyses performed, it was identified that all the weights of each group on day 15 were significantly higher than on day 0, indicating that the animals gained weight during the course of this experiment (Figure 16B).

[0128] By analyzing the distribution of weights by group throughout the experiment, we can see that group 2 has a significantly higher weight than group 3; however, this is because group 2 started the trial with a higher average weight than group 3 and not due to an effect of the hydrogel, the copper nanoparticles, or the treatment with them (Figure 17).

[0129] Treatment with the hydrogels, whether alone or loaded with CuNp, had no effect on the animals' temperature or weight during this trial. We can conclude that the 150 pg / ml CuNp hydrogel is non-pyrogenic, demonstrating its safety with respect to this parameter for wound treatment.

Claims

CLAIMS 1. A process for manufacturing a hydrogel-type medical device CHARACTERIZED in that it comprises: I. Preparation of copper nanoparticles coated with high molecular weight chitosan; and II. Preparation of the hydrogel that incorporates the copper nanoparticles.

2. A process for manufacturing a hydrogel-type medical device, according to claim 1, CHARACTERIZED in that the manufacturing of the copper nanoparticles comprises the following steps: i. A chitosan mixture is prepared in a range between 1.3 and up to 1.6% w / v, which is dissolved in 1.5% acetic acid, heating to 60°C until completely dissolved; i. In parallel, a solution of CuSO45H2O (between 23 and 1 g) is prepared in 500 ml of distilled water, maintaining constant stirring; iii. 100 ml of the previous chitosan-acetic acid mixture is added dropwise to the CuSO45H2O solution, maintaining constant stirring until complete integration; iv. The temperature is increased in a water bath to a range of 80° to 90° C for 1 h, and 100 ml of NaOH (625 mM) is added dropwise until pH 9.0 is reached; v. The mixture is stirred until a gray-brown color is observed, and 100 ml of ascorbic acid (in a range of 410 to 450 mM) is added dropwise until a reddish color is reached; stirring is maintained at a range of 80° to 90° C for 30 min in the water bath; and vi. The final mixture is centrifuged at 12000 g and the precipitate is dried at 80° C for 24 h.

3. A process for manufacturing a hydrogel-type medical device, according to claim 1, CHARACTERIZED in that the manufacturing of the hydrogel comprises at least the following steps: a) preparing a chitosan solution of between 1.0 and 2% w / v in 1.5% acetic acid with a pH of 4.5, which is stirred at 500-800 rpm for 3 hours at 25°C; b) preparing a starch solution of between 1.0 and 3% w / v, by continuous stirring at 500-800 rpm for 1 hour at 100°C; c) mixing 35-65% of the chitosan solution and 35-65% of the starch solution, stirring at 500-800 rpm for 1.5 to 3 hours until complete homogenization is achieved; d) Genipin is added in a range of 0.01 to 0.05% of the mixture volume and stirring is continued for 4 days at 25°C to ensure the initial crosslinking phase of the hydrogel;e) The previously prepared coated nanoparticles are incorporated between 20 and 40 minutes before the end of the crosslinking process with genipin. They are weighed for dispersion, maintaining ranges of 50 to 500 milligrams of nanoparticles per liter of hydrogel. The nanoparticles must be carefully dispersed in the stirring mixture; f) Three freezing cycles are performed at -20°C for 12 hours, followed by thawing at 25°C, as part of the physical / chemical crosslinking phase of the biomaterial; and g) Excess water is removed from the samples using filter paper, and the fully crosslinked biomaterial is dispensed into molds of inert solid material, in the required dimensions, and frozen at -80°C for a range of 4 to 12 hours, and then... dried by freeze-drying in a range of 16 to 24 hours, depending on the desired thickness of the material.

4. Process for manufacturing a hydrogel-type medical device, according to claim 1, CHARACTERIZED in that the incorporation of the coated nanoparticles in the last minutes of the crosslinking stage allows the coating to remain on them and not be dissolved in the other components, in addition to allowing uniform incorporation into the crosslinking formation of the hydrogel.

5. Use of the process for manufacturing a hydrogel-type medical device, according to claim 1, CHARACTERIZED in that it serves to achieve a homogeneous dispersion of the nanoparticles in the biomaterial, maintaining its porosity and promoting its structural stability.

6. Use of the process for manufacturing a hydrogel-type medical device, according to claim 1, CHARACTERIZED in that it serves to obtain a homogeneous structure with high absorption and swelling capacity, antimicrobial activity, and promotes the physiological healing process.

7. A hydrogel-type medical device CHARACTERIZED in that it comprises at least: a) a lyophilized chitosan-starch hydrogel matrix; b) 0.005 to 0.05 wt / v of coated copper nanoparticles; and c) 0.01 to 0.05% of a crosslinking agent.

8. A hydrogel-type medical device, according to claim 7, CHARACTERIZED in that the crosslinking agent is genipin.

9. A hydrogel-type medical device, according to claim 7, CHARACTERIZED in that it comprises equal parts of the naturally occurring polymers chitosan and starch, chemically cross-linked with Genipin, possess a network structure with a uniformity of pores, which is strengthened as the copper nanoparticles cross-link, reaching a maximum incorporation of nanoparticles at 0.05% weight / vol.

10. A hydrogel-type medical device, according to claim 7, CHARACTERIZED in that it comes in various sizes and has a blue-light blue hue dependent on the concentration of copper nanoparticles.

11. A hydrogel-type medical device, according to claim 7, CHARACTERIZED in that it has antimicrobial activity and absence of toxicity in in vitro human epithelium cell models and in vivo animal models.

12. A hydrogel-type medical device, according to claim 7, CHARACTERIZED in that it allows the degradation and final bio-absorption of the material, making its replacement or continuous removal unnecessary in wounds, burns or ulcerations.

13. Use of the hydrogel-type medical device, according to claim 7, CHARACTERIZED in that it serves for different types of wounds, including traumatic and post-operative wounds, chronic and diabetic wounds, ulcers and burns.

14. Use of the hydrogel-type medical device, according to claim 13, CHARACTERIZED in that it serves for the absorption of exudates and gas exchange at the wound site, facilitating biosupport for healing, improving epithelialization, and reducing the possibility of infection with bacterial pathogens.

15. Use of the hydrogel-type medical device, according to claim 13, CHARACTERIZED in that its repeated application does not generate fever (pyrogens), loss of appetite or weight loss in acute tolerance studies.

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