Process of obtaining bio-hybrid films for transdermal delivery of magnetic nanoparticles and bio-hybrid films

Description

[001] The present invention has application in the area of ​​pharmaceutical technology, chemical nanotechnology and biomedical area and describes the process of obtaining bio-hybrid magnetic films of sodium alginate with cobalt-gadolinium ferrite nanoparticles dispersed in the polymeric matrix, for transdermal delivery of magnetic nanoparticles. Fundamentals of the invention

[002] Nanocomposites are versatile and interesting alternatives for the biomedical field, as they optimize the structural (mechanical) and functional (electrical, optical, and magnetic) properties of their constituents. The use of natural polymers offers advantages such as abundance, biocompatibility, low cost, and versatility of use. The development of new materials with natural polymers is highly relevant, as it replaces the use of synthetic, petroleum-derived, and non-biodegradable polymers.

[003] Bio-hybrid systems based on biopolymers and inorganic nanoparticles are materials of great value to the medical and pharmaceutical fields, as their enhanced properties can be exploited for a wide variety of applications, such as wound dressings, drug delivery and tissue engineering.

[004] Transdermal delivery of molecules benefits from these systems and brings a number of advantages to the patient, such as reduced systemic side effects; reduced multiple dose administrations; improved adherence to treatment; avoidance of first-pass metabolism, in addition to being a non-invasive method.

[005] The incorporation of magnetic nanoparticles into the system imparts specific properties to the polymer matrices, allowing their use in Petition 870250057328, dated 07 / 07 / 2025, page 4 / 23 / 12 magnetic resonance imaging, targeted delivery of medications, wound care, and implants.

[006] An example of such uses is patent WO / 2020 / 081817A1 which describes the encapsulation of magnetic nanoparticles with a biologically active agent in polymeric matrices for the release of this agent after application of an alternating magnetic field. These biologically active agents can be antibiotics, antineoplastics, anti-inflammatories and other medications.

[007] Another example is US patent 8974826B2 which provides a therapeutic film delivery system for delivering active ingredients, such as peptides, directly into the circulatory system without exposure to the gastrointestinal tract.

[008] Another example is patent WO / 2007 / 003516 which presents a functionally coated medical device for therapeutic and / or diagnostic purposes, such as implants, stents and prostheses.

[009] Another example is patent WO / 2006 / 083796 which presents a nanocomposite containing nanomagnetic material arranged within a polymeric matrix with the aim of providing a therapeutic agent to the biological organism.

[010] US patent 20080213382A1 discloses a therapeutic composition in the form of liquid, gel, ointment, lotion, solid or semi-solid, composed of magnetic nanoparticles coated with polymers, a vehicle and an active agent such as chemotherapeutic, radiotherapy, vasopermeation enhancing, anti-inflammatory, anesthetic, analgesic, sedative, antibiotic and combinations thereof.

[011] Patent BR 10 2013 034036 7 describes the process for obtaining magnetic bio-hybrid films intended for the controlled release of active substances, as well as the respective bio-hybrid films based on magnetic graphite.

[012] As can be seen, the magnetic bio-hybrid systems presented use magnetic nanoparticles as adjuvants in Petition 870250057328, dated 07 / 07 / 2025, page 5 / 23 / 12, refers to the controlled release process of active substances, which are distinct from nanoparticles. Therefore, obtaining nanocomposites requires further steps for the effective incorporation of all components of the formula.

[013] In addition, many of the magnetic methods mentioned use toxic reagents in the synthesis process or in the preparation of the nanoparticle dispersion, such as organic solvents and surfactants, which are often undesirable for biomedical applications.

[014] Topical formulations are interesting treatment options due to their ability to deliver therapeutic agents to the systemic circulation and local region for extended periods, making them important for treating various diseases, including skin cancer. In relation to skin cancer, topical formulations reduce the systemic side effects associated with conventional chemotherapy and can potentially avoid invasive surgical procedures.

[015] Inorganic nanoparticles possess several properties that make them suitable candidates for skin cancer therapy, including small size, large surface area, bioactivity, biocompatibility, functionalization capability and intrinsic therapeutic property, which allows nanoparticles to act as anticancer agents.

[016] Furthermore, magnetic bio-hybrid systems can have their action enhanced through the application of an external magnetic field.

[017] In the present invention, a biopolymer is used as a matrix and magnetic nanoparticles as a therapeutic agent, without the use of surfactants or organic solvents to obtain bio-hybrid films. The method for obtaining the films and the magnetic biohybrid films themselves will be described. The preparation method is characterized by being simple, with low-cost starting materials, without the need for complex machinery and few steps to complete the process. Petition 870250057328, dated 07 / 07 / 2025, page 6 / 23 / 12 List of Figures

[018] To obtain a complete visualization and better understanding of the object of this invention, some attached figures are presented, where: FIGURE 1: Digital photograph, for illustrative purposes, of the bio-hybrid films during the drying process (top) and the finished films (bottom). FIGURE 2: X-ray diffraction spectra of the synthesized films highlighting the peak of highest intensity. FIGURE 3: Infrared absorption spectra of the bio-hybrid films showing the main absorption bands. FIGURE 4: Micrographs of the surfaces of the bio-hybrid films, all at 50 μm magnification and sprayed with gold. FIGURE 5: EDS spectra with the elemental composition of the biohybrid films. FIGURE 6: Magnetization curves (MxH) of magnetic films obtained at room temperature. FIGURE 7: DMA data obtained for the bio-hybrid films. Logarithmic curves of the storage modulus and loss tangent (tan δ) curves as a function of temperature are presented. FIGURE 8: Graphs of cell viability by resazurin reduction for magnetic films, with and without the application of an external static magnetic field. Note: NIH3T3 are mouse fibroblasts and B16F10 are mouse skin cells with melanoma. FIGURE 9: In vivo nanotoxicity graph in a Drosophila melanogaster model. Note: Water and polymer-adjuvant are control samples and Alg 0 and Alg 0.01 are test samples (0 and 1% gadolinium, respectively). Description of the invention

[019] The present invention relates to the development of biohybrid magnetic films of sodium alginate with cobalt-gadolinium ferrite nanoparticles dispersed in the polymeric matrix. Petition 870250057328, dated 07 / 07 / 2025, page 7 / 23 / 12

[020] For the preparation of magnetic nanoparticles, the publicly available coprecipitation method was used, with a molar ratio between the salts in the proportion of 1:(2-x):x (Co:Fe:Gd) with x equal to 0, 1, 3, 5, 7, 9 and 11 %, in order to produce the CoFe2-xGdxO4 nanoparticles, called cobalt-gadolinium ferrite.

[021] Bio-hybrid films were produced by a sequence of steps, which began with the preparation of the aqueous sodium alginate solution. This solution, called alginate biopolymer, was prepared in the concentration range of 1 to 5% (w / v), preferably 2.5% (w / v), in 50% of the final volume of ultrapure water of the dispersion, with magnetic stirring for a time interval ranging from 8 to 12 hours, preferably 10 hours, until it became a viscous and homogeneous solution.

[022] Next, the magnetic nanoparticles were moistened with copaiba balsam, in the range of 0.1 to 1.0 μL / mL, preferably 0.6 μL / mL, followed by the addition of 50% of the final volume of the dispersion in absolute alcohol. This nanoparticle-balsam-alcohol mixture was sonicated, in a probe sonicator, for 5 minutes, with a 30-second pulse and an amplitude of 70%.

[023] The alginate biopolymer was poured onto the balsam-alcohol nanoparticle suspension and mixed with a glass rod, then sonicated with 30-second pulses and an amplitude of 80% for approximately 20 minutes, until a homogeneous dispersion was formed.

[024] This homogeneous dispersion was deposited in Petri dishes, preferably 30 mL per dish, for drying by solvent evaporation, at room temperature (20 to 25 °C, preferably 23 °C), for a period ranging from 72 to 120 hours.

[025] The films were removed carefully and stored at room temperature until use.

[026] Magnetic bio-hybrid films were characterized as systems composed of a sodium alginate base with dispersed cobalt-gadolinium ferrite nanoparticles, with a thickness of 0.05 to 0.08 μm, Petition 870250057328, dated 07 / 07 / 2025, page 8 / 23 / 12 preferably, 0.06 μm. These cobaltogadolinium ferrite nanoparticles are at a concentration in the range of 0.025 to 0.2 mg / mL, preferably 0.1 mg / mL. The concentration of gadolinium ions ranged from 1 to 11%, preferably 3%. Furthermore, these films were characterized for topical administration and for possessing biocompatibility and significant antineoplastic activity.

[027] Alginate films are important agents for the topical administration of medications because they deliver a fixed dose of the active ingredient to a defined area (when compared to creams, gels, lotions, and ointments) and have a long residence time at the application site. Furthermore, thin and flexible films can be produced, allowing the size to be adjusted to the area of ​​the lesion.

[028] In this invention, adjuvants were added to the alginate film, forming bio-hybrid systems. The addition of copaiba balsam to promote the dispersion of the nanoparticles did not interfere with the stability or homogeneity of the system, and has the advantage of excluding the use of surfactants and organic solvents of potential toxicity, which are employed in various magnetic systems. In addition, a biopolymer was used as a matrix and magnetic nanoparticles as a therapeutic agent and adjuvant in the controlled release process, without the need for additional steps for the incorporation of the formula components.

[029] Alginate films prepared without nanoparticles have a yellowish-brown color, and as ferrites are incorporated, the films acquire a gray color. All films were flexible, easily removed from Petri dishes, and homogeneous from a macroscopic point of view.

[030] The thickness of the films was around 0.05 to 0.08 μm, with a coefficient of variation below 10%, indicating the reproducibility of the preparation method, as shown in FIGURE1. Petition 870250057328, dated 07 / 07 / 2025, page 9 / 23 / 12 Characterization tests

[031] Next, analyses will be presented for the structural, morphological, magnetic, mechanical and toxicity characterization of these magnetic bio-hybrid systems. X-ray diffraction:

[032] The analyses were performed at room temperature. This assay allows for the structural characterization of the compounds and the identification of the cobalt-gadolinium ferrite phase in the films. In the X-ray diffraction analysis, FIGURE 2, as expected, the spectra were noisy due to the amorphous ordering of the samples, characteristic of sodium alginate. The peak at 2Θ = 35.5°, corresponding to the (311) plane of cobalt ferrite (COD 96-153-3164), was evidenced. This is also the peak of greatest intensity in the incorporation of gadolinium ions into the ferrite structure. No changes in phase formation were perceptible with the incorporation of nanoparticles into the alginate matrix, indicating that the cobalt-gadolinium ferrite structure is maintained. Infrared Spectroscopy:

[033] The spectra were recorded on an ATR platinum diamond-coupled spectrometer, in the region between 4000 and 400 cm-1. This technique allows the study of possible interactions between the biopolymers and solids used, providing information about the structure obtained. The spectra were similar and subtle shifts in the bands were observed with the incorporation of the nanoparticles, in addition to variations in intensity, related to a possible interaction between the alginate groups with the ions of the nanoparticles, FIGURE 3. Scanning Electron Microscopy and Energy Dispersive X-ray Spectroscopy:

[034] Surface morphology analysis of the films and semi-quantitative analysis of the chemical composition. The samples were sprayed with gold to improve image quality. FIGURE 4 shows the surface images of the Petition 870250057328, dated 07 / 07 / 2025, page 10 / 23 / 12 films that indicated similar, homogeneous surface morphologies without evidence of ferrite particles. EDS image mapping showed the elements that make up the alginate matrix and the main elements of the ferrites. The metals were distributed throughout the surface of the film, with the presence of some agglomerates. The EDS spectra, FIGURE 5, showed the presence of the metals Co, Fe, and Gd in the synthesized films. These analyses suggested a satisfactory distribution of cobalt-gadolinium ferrite nanoparticles in the polymeric matrix, indicating adequate interaction between the components of the bio-hybrid film and confirming the compatibility between the nanoparticles and the polymer. Magnetic Characterization by Vibrating Sample Magnetometer (VSM):

[035] Measurements were taken at a temperature of 300 K and a maximum magnetic field of 1.2 T. For a complete hysteresis cycle, 155 points were marked, with a 20-second interval. The hystereses, FIGURE 6, demonstrated ferromagnetic behavior, with low magnetic saturation values, indicating the dispersion of nanoparticles in the polymer matrix, promoting separation and reducing interactions between particles. In addition, the ferrite content is diluted in the membranes, reducing the magnetic signal. Increasing the concentration of gadolinium ions in the cobalt ferrite structure increased the coercivity values ​​due to better dipole-dipole interaction. For the formulations with nanoparticles dispersed at a concentration of 0.1 mg / mL, the Alg 0.03 sample showed the best magnetic characteristics.

[036] Studies of magnetic properties are presented in TABLE 1, where we have: Saturation magnetization (Ms), magnetic remanence (Mr), coercivity (Hc) and Mr / Ms ratio as a function of Gd3+(x) concentration recorded at room temperature. Petition 870250057328, dated 07 / 07 / 2025, page 11 / 23 9 / 12 TABLE 1 Sample Concentration Gd (mg) Saturation Magnetization MS (emu / g) Magnetic Remanence Ml (emu / g) Coercivity HfJkOe) Mr / Ms Alg 0 0 0.23 0.11 0.88 0.478 Alg 0.01 0.074 0.19 0.09 1.13 0.473 Alg 0.03 0.253 0.26 0.14 1.41 0.538 Alg 0.05 0.423 0.23 0.12 1.22 0.522 Alg 0.07 0.626 0.23 0.13 1.26 0.565 Alg 0.09 0.806 0.21 0.10 1.11 0.476 Alg 0.11 0.879 0.21 0.11 1.26 0.523 Dynamic Mechanical Thermal Analysis (DMTA):

[037] The mechanical properties of the films as a function of temperature were evaluated in rectangular films (20 χ 5 χ 0.06 mm) in tensile mode with an oscillation frequency of 1 Hz, a static force of 10 mN, an oscillation amplitude of 15.0 pm, and an automatic tension adjustment of 125%. Measurements were performed with a heating rate of 3 °C / min⁻¹ in a range of -10 to 200 °C. This analysis allows us to understand the elastic and viscous characteristics of polymeric materials as a function of temperature. These properties are related to the sample composition, morphology, and nature of phases. In the synthesized bio-hybrid films, it can be concluded that the gadolinium concentration influenced the stiffness of the membranes, FIGURE 7. Cell viability through resazurin reduction:

[038] Assay performed with B16F10 (mouse skin cells with melanoma) and NIH3T3 (mouse fibroblasts) cell lines cultured in RPMI 1640 medium, pH 7.4, supplemented with 10% (v / v) fetal bovine serum, to verify the cytotoxic potential of the prepared films. All cultures were maintained in a humidified incubator with a controlled atmosphere of Petition 870250057328, dated 07 / 07 / 2025, page 12 / 23 / 12% CO2 (v / v) at 37 °C and the experiments were performed in quadruplicate. Cell groups were seeded in 2 sets of 96-well microplates with 2x104 cells per well. After 24 hours of incubation for adhesion, the cells were treated with an average of 0.73 mg of film.

[039] A set of neoplastic and healthy cells received the films and, after 1 hour of contact, the resazurin reagent was added and kept in incubation for another 4 hours. The second set of neoplastic and healthy cells received the films and was subjected to a static magnetic field formed by the use of two parallel ferrite magnets with dimensions 150x100x12 mm for 1 hour. Then, the resazurin reagent was added and the cultures were kept in an incubator for another 4 hours.

[040] After the 5 hours of treatment were completed, the plates were subjected to absorbance readings at wavelengths of 570 nm and 600 nm.

[041] Cell viability was calculated using Equation (1): % cell viability = [(02 xA1( - (01 xA2)] x 100 (1) [(02 pPI) - (01 %P2)]

[042] Where: A1 is the absorbance of the treated cells at 570 nm; A2, the absorbance of the treated cells at 600 nm; O1 is the molar extinction coefficient (E) of resazurin oxidized at 570 nm (pre-determined at 117216); O2 is the value of the molar extinction coefficient (E) of resazurin oxidized at 600 nm (pre-determined at 80586); P1 is the average of the absorbance values ​​of the negative control at 570 nm and P2 is the average of the absorbance values ​​of the negative control at 600 nm.

[043] Statistically significant differences in cell viability were assessed by TwoWay-ANOVA statistical analysis, with Bonferroni post-test, using specific software.

[044] The cell viability test allowed us to assess the degree of cell death induction caused by the substances. In this application example, a set Petition 870250057328, dated 07 / 07 / 2025, page 13 / 23 / 12 of cells received the application of an external static magnetic field in order to magnetize the nanoparticles present in the films and generate an additional magnetic force.

[045] Static magnetic fields are safe, convenient, and inexpensive, and have proven effects on biological systems, such as bone fracture healing, osteogenic differentiation, and cell regulation.

[046] In this application example, in mouse cells, a similar behavior was observed for cytotoxic activity, independent of the action of the static magnetic field. Therefore, in this study, the application of the external magnetic field did not contribute to the cytotoxic activity of the nanoparticles, FIGURE 8.

[047] The Alg 0.03 sample showed great cytotoxic activity on B16F10 and NIH3T3 cells. The bio-hybrid film of alginate and cobalt ferrite-gadolinium nanoparticles (0.74% Gd3+) may have its action related to the different prominent characteristics presented by the system, such as single phase cobalt ferrite with incorporation of gadolinium ions in the network, small size (52 nm), higher saturation magnetization (0.26 emu / g), higher coercivity (1.41 kOe) and higher matrix fluidity (Ta = 110°C). Nanotoxicity test in a Drosophila melanogaster model:

[048] Canton S strain flies aged 0-2 days post-hatching were used. The flies were separated into groups of 20 individuals with a 1:1 ratio between males and females and housed in an incubator at 25 °C in a 12 / 12h light / dark cycle, in triplicate. For treatment administration, 1 g of enriched mashed potato (75% instant mashed potato, 15% yeast extract, 9.3% glucose and 0.07% methylparaben) was hydrated with 5 mL of bio-hybrid film solution. The polymer-adjuvant (0.8 mg / mL), Alg 0 (0.7 mg / mL) and Alg 0.03 (0.154 mg / mL) films were tested, along with a control group whose hydration was done with type I water.

[049] The treatment methods were changed every 2 days and the number of dead individuals was counted at each change. The total exposure time Petition 870250057328, dated 07 / 07 / 2025, page 14 / 23 / 12 of the flies to feeding with the treatments was 15 days.

[050] Significant statistical differences were assessed by Longrank analysis (Mantel-Cox), using specific software.

[051] In order to investigate the biocompatibility of the films in more complex models such as animals, the assay was carried out which revealed that there was no statistically significant difference between the control groups (water and polymer adjuvant) and the test groups (Alg 0 and Alg 0.03), indicating that at these concentrations, the formulations are not toxic to animals, FIGURE 9, although they are cytotoxic in neoplastic cells.

[052] Structural analyses confirmed the incorporation of magnetic nanoparticles into the polymer matrix, with distribution throughout the surface, suggesting adequate interaction between the components of the bio-hybrid film and reinforcing the potential for topical use of the films in the treatment of skin cancer.

Claims

1. PROCESS FOR OBTAINING BIO-HYBRID FILMS for transdermal delivery of magnetic nanoparticles characterized by comprising the following steps: a) Preparation of an aqueous solution of sodium alginate in the concentration range of 1 to 5% (w / v), preferably 2.5% (w / v), in 50% of the final volume of ultrapure water of the dispersion, with magnetic stirring for a time interval ranging from 8 to 12 hours, preferably 10 hours, until it becomes a viscous and homogeneous solution; b) Wetting of the previously synthesized cobalt-gadolinium ferrite magnetic nanoparticles with copaiba balsam, in the range of 0.1 to 1.0 μL / mL, preferably 0.6 μL / mL, followed by the addition of 50% of the final volume of the dispersion in absolute alcohol; c) Sonication of the nanoparticle-balsam-alcohol mixture for 5 minutes, with a 30-second pulse and 70% amplitude;d) Mixing the aqueous sodium alginate solution with the magnetic nanoparticles, pouring the biopolymer solution over the nanoparticle-balsam-alcohol suspension, mixing, and sonicating with 30-second pulses at an amplitude of 80% for 20 minutes, until a homogeneous dispersion is formed; e) Depositing the dispersion onto Petri dishes, preferably 30 mL per dish, and drying until the solvent evaporates, at a temperature between 20 and 25 °C, preferably 23 °C, for a period between 72 and 120 hours; f) Removing the prepared films and storing them at room temperature until use.

2. PROCESS FOR OBTAINING BIO-HYBRID FILMS, according to claim 1, characterized by the presence of cobalt-gadolinium ferrite magnetic nanoparticles in a concentration in the range of 0.025 to 0.2 mg / mL, preferably 0.1 mg / mL. Petition 870250008514, dated 01 / 31 / 2025, page 39 / 46 2 / 2 3. PROCESS FOR OBTAINING BIO-HYBRID FILMS, according to claim 2, characterized in that the concentration of gadolinium ions in the cobalt-gadolinium ferrite nanoparticles varies from 1 to 11%, preferably 3%.

4. BIO-HYBRID FILMS produced by any of the preceding claims, characterized by having a thickness of 0.05 to 0.08 μm, ferromagnetic behavior, low magnetic saturation and increased coercivity with increasing gadolinium ion concentration, greater rigidity with increasing gadolinium ion concentration, cytotoxic activity with and without the application of an external magnetic field, and biocompatibility in Drosophila melanogaster models.

5. USE of MAGNETIC BIO-HYBRID FILMS produced according to the preceding claims, characterized by being administered topically in the treatment of skin cancer.

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