A system for the production of biomimetic magnetic nanocomposites for the immobilization of proteolytic enzymes and the targeted treatment of uterine infections in cattle.

Abstract

System (100) for the production of biomimetic magnetic nanocomposites for the immobilization of proteolytic enzymes and the targeted treatment of bovine uterine infections, comprising: a) a unit for producing a carrier matrix (102) configured for the production of magnetic nanoparticles; b) an enzyme preparation unit (104) configured to provide proteolytic enzymes selected from the group consisting of protease, trypsin, chymotrypsin, and papain; c) an immobilization unit (106) configured to immobilize the proteolytic enzymes on the modified renewable carrier matrix; and d) a characterization unit (108) configured to perform toxicological tests, comprising: - Equipment for testing antimicrobial activity, and - DPPH assay analysis system.

Description

FIELD OF THE INVENTION

[0001] The present disclosure relates to a system for producing biomimetic magnetic nanocomposites for the immobilization of proteolytic enzymes and the targeted treatment of uterine infections in cattle. BACKGROUND OF THE INVENTION

[0002] Uterine infections in cattle and buffalo pose a major reproductive challenge and manifest as puerperal metritis, clinical endometritis, and subclinical endometritis (SCE). These conditions, often exacerbated by dystocia and retained fetal membranes (RFM), result in reduced fertility and lower calf production. Current treatments rely heavily on antibiotics, contributing to increasing antimicrobial resistance.

[0003] Intrauterine infusion of proteolytic enzymes (trypsin, chymotrypsin, and papain in a 2:2:1 ratio) has proven to be a promising treatment alternative, as it effectively reduces polymorphonuclear cells (PMN) and inflammatory cytokines in the uterus. However, these enzymes, in their virgin form, have significant limitations: 1. Their activity is strongly dependent on pH and temperature conditions 2. They have low stability in the uterine environment 3. The production of raw formulations is time-consuming 4. The storage of these enzymes presents a major challenge.

[0004] These limitations necessitate the development of a more stable and effective delivery system for these proteolytic enzymes, particularly one that can maintain their therapeutic activity under variable uterine conditions while allowing for easier preparation and storage.

[0005] In view of the above, it is clear that there is a need for a system for the production of biomimetic magnetic nanocomposites for the immobilization of proteolytic enzymes and the targeted treatment of bovine uterine infections. SUMMARY OF THE INVENTION

[0006] The present disclosure relates to a system for producing biomimetic magnetic nanocomposites for immobilizing proteolytic enzymes and for the targeted treatment of uterine infections in cattle. The present invention provides a system for producing biomimetic magnetic nanocomposites for the effective treatment of uterine infections in cattle and buffalo. More specifically, the invention relates to the immobilization of proteolytic enzymes (trypsin, chymotrypsin, and papain) on modified renewable carrier matrices composed of magnetic nanoparticles. This novel immobilization approach not only protects the enzymes from denaturation during administration but also increases their catalytic activity and thermodynamic stability.The system transforms conventional novel enzymes into innovative forms by immobilizing them on biomimetic carriers, resulting in a more stable and effective therapeutic product. Initial toxicological studies and antimicrobial activity tests have demonstrated the safety and improved efficacy of these immobilized enzymes compared to their untreated counterparts. The invention provides a practical solution for commercial preparation and storage that overcomes the limitations of conventional crude enzyme formulations while maintaining therapeutic efficacy in the treatment of bovine uterine infections. This invention represents a significant advance in the field of enzyme-based therapeutics for veterinary medicine, combining the advantages of nanotechnology with enzymatic treatment to create a more stable and effective drug delivery system.

[0007] The present disclosure aims to provide a system for producing biomimetic magnetic nanocomposites for the immobilization of proteolytic enzymes and the targeted treatment of bovine uterine infections. The system comprises: a carrier matrix preparation unit (102) configured to produce magnetic nanoparticles; an enzyme preparation unit (104) configured to provide proteolytic enzymes selected from the group consisting of protease, trypsin, chymotrypsin, and papain; an immobilization unit (106) configured to immobilize the proteolytic enzymes on the modified renewable carrier matrix; and a characterization unit (108) configured to conduct toxicological tests, comprising: antimicrobial activity testing equipment and a DPPH assay analysis system.

[0008] An object of the present disclosure is to provide a system for producing biomimetic magnetic nanocomposites for the immobilization of proteolytic enzymes and the targeted treatment of bovine uterine infections.

[0009] Another object of the present disclosure is the development of a biomimetic magnetic nanocomposite system for immobilizing proteolytic enzymes (trypsin, chymotrypsin and papain), which increases their stability and catalytic activity under variable uterine conditions.

[0010] Another object of the present disclosure is to provide an improved drug delivery system using a modified renewable carrier matrix that enables safer and more effective intrauterine delivery of proteolytic enzymes for the treatment of uterine infections in cattle and buffaloes.

[0011] Another object of the present disclosure is a stable commercial preparation of immobilized enzymes that overcomes the storage and preparation problems associated with crude enzyme formulations while maintaining therapeutic efficacy.

[0012] To further clarify the advantages and features of the present disclosure, a more particular description of the invention will be given by reference to specific embodiments illustrated in the accompanying drawings. These drawings are understood to represent only typical embodiments of the invention and are therefore not to be considered limiting of the scope of the invention. The invention will be described and explained with additional specificity and detail with the accompanying drawings. BRIEF DESCRIPTION OF THE ILLUSTRATIONS

[0013] These and other features, aspects, and advantages of the present disclosure will be better understood when the following detailed description is read with reference to the accompanying drawings, in which like characters represent like parts throughout the drawings, wherein: Fig. 1 shows a block diagram of a system for producing biomimetic magnetic nanocomposites for the immobilization of proteolytic enzymes and the targeted treatment of bovine uterine infections according to an embodiment of the present disclosure; and Fig. Figure 2 shows a block diagram illustrating the preparation of nanoparticle-immobilized enzymes for the treatment of uterine infections in dairy animals according to an embodiment of the present disclosure.

[0014] Those skilled in the art will understand that the elements in the drawings are shown for convenience and are not necessarily drawn to scale. For example, the flowcharts illustrate the method by key steps to enhance understanding of aspects of the present disclosure. Furthermore, one or more components of the device may be represented in the drawings by conventional symbols, and the drawings may show only the specific details relevant to understanding embodiments of the present disclosure in order not to clutter the drawings with details that would be readily apparent to those skilled in the art familiar with the present description. DETAILED DESCRIPTION:

[0015] To facilitate an understanding of the invention, reference will now be made to the embodiment illustrated in the drawings and described in specific terms. It should be understood, however, that this is not intended to limit the scope of the invention, and such changes and further modifications to the illustrated system, and such further applications of the principles of the invention embodied therein, are contemplated as would normally occur to one skilled in the art to which the invention pertains.

[0016] It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not to be considered limiting.

[0017] When this specification refers to "one aspect," "another aspect," or the like, it means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Therefore, the terms "in one embodiment," "in another embodiment," and similar expressions throughout this specification may or may not all refer to the same embodiment.

[0018] The terms "comprises," "including," or other variations thereof are intended to cover non-exclusive inclusion, such that a process or method comprising a list of steps not only comprises those steps, but may also include other steps not expressly listed or included in such process or method. Likewise, one or more devices or subsystems or elements or structures or components introduced with "comprises...a" do not preclude, without further limitation, the existence of other devices or other subsystems or other elements or other structures or other components or additional devices or additional subsystems or additional elements or additional structures or additional components.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are for illustrative purposes only and are not intended to be limiting.

[0020] Embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings.

[0021] Fig. 1 shows a block diagram of a system (100) for producing biomimetic magnetic nanocomposites for the immobilization of proteolytic enzymes and the targeted treatment of bovine uterine infections according to an embodiment of the present disclosure.

[0022] Referring to Fig. 1, the system (100) comprises a carrier matrix preparation unit (102) configured to produce magnetic nanoparticles; an enzyme preparation unit (104) configured to obtain proteolytic enzymes selected from the group consisting of protease, trypsin, chymotrypsin, and papain; an immobilization unit (106) configured to immobilize the proteolytic enzymes on the modified renewable carrier matrix; and a characterization unit (108) configured to perform toxicological tests, comprising equipment for testing antimicrobial activity and a DPPH assay analysis system.

[0023] In one embodiment, the immobilization unit (106) is configured to maintain the viability of the enzyme and improve catalytic activity and thermodynamic stability.

[0024] In one embodiment, the carrier matrix preparation unit (102) is configured to produce nanoparticles suitable for in utero drug delivery.

[0025] In one embodiment, the system is configured to enable the administration of refurbished enzymes to infected dairy cows and buffaloes.

[0026] In one embodiment, the system (100) further comprises a testing unit (110) configured to facilitate in vitro testing of the produced immobilized enzymes, the testing unit (110) comprising equipment for comparing the activities of immobilized enzymes with those of untreated enzymes.

[0027] In one embodiment, the system (100) further comprises a validation unit (112) configured to test the immobilized enzymes on artificial substrates.

[0028] The present invention relates to a system configured to renovate the virgin proteolytic enzyme by immobilizing it on a biomimetic support that stabilizes the enzyme under variable conditions. This invention develops biomimetic magnetic nanocomposites for the immobilization of proteolytic enzymes and the targeted treatment of bovine uterine infections.

[0029] Fig. Figure 2 shows a block diagram illustrating the preparation of nanoparticle-immobilized enzymes for the treatment of uterine infections in dairy animals according to an embodiment of the present disclosure.

[0030] In the present invention, the native proteolytic enzymes are renovated by immobilizing them on a biomimetic support that ensures the stability of the enzymes under different conditions, as described in Fig.2. This immobilization not only protects the enzymes from denaturation during administration but also has the potential to significantly increase their activity. A conceptual application with biocatalytic modules consisting of multiple enzymes is being tested by modifying or hydrolyzing various hemicellulosic polysaccharides. Model enzymes for validating these bio-nanocomposites include protease, trypsin, chymotrypsin, and papain.

[0031] The main focus of this invention is the development of a carrier matrix for immobilizing proteolytic enzymes on a modified renewable carrier matrix to maintain the enzyme's activity while improving its catalytic efficiency and thermodynamic stability. Furthermore, a nanoparticle-based matrix is ​​developed that serves as an effective drug delivery carrier and can be used for the development of drug vials for safer intrauterine administration. Furthermore, a combination of immobilized enzymes, specifically chymotrypsin, trypsin, and papain, is developed that can provide even more effective results. The primary material objective of this invention is to improve and refine existing implementations.

[0032] The present invention utilizes renewable resources to support the immobilization of proteolytic enzymes for dairy animal health. The base material, immobilized with a model enzyme as a mutated surface, was prepared. Preliminary toxicological evaluations, including antimicrobial activity and DPPH tests, were conducted prior to specific in vitro testing.

[0033] The developed immobilized enzymes are intended to be administered to infected dairy cows and buffaloes as an alternative to untreated enzymes. Tests with artificial substrates have demonstrated the superior performance of the immobilized enzymes compared to their native counterparts, and similar results are expected in clinical trials. The toxicological studies conducted confirmed the safety of the immobilized product for animals during the trials. Clinical trials have shown initial success with the replacement of trypsin with the immobilized product, which exhibits higher activity than the original enzyme.

[0034] This bio-nanointerface formulation has the potential to significantly improve animal welfare and benefit the livestock industry by providing effective therapeutics for uterine infections in dairy animals. This, in turn, will help farmers reduce economic losses and increase their income. The effective dissemination and utilization of these findings is critical to maximizing their impact. The present invention will benefit not only dairy cattle, but also other animal species and industries that rely on animal health.

[0035] The drawings and the foregoing description provide examples of embodiments. Those skilled in the art will understand that one or more of the described elements may well be combined to form a single functional element. Alternatively, certain elements may be separated into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, the order of the processes described herein may be changed and is not limited to the manner described herein. Furthermore, the acts of a flowchart need not be performed in the order shown; nor do all acts necessarily need to be performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of the embodiments is in no way limited by these specific examples.Numerous variations are possible, whether explicitly stated in the description or not, such as differences in structure, dimensions, and use of materials. The scope of the embodiments is at least as broad as indicated in the following claims.

[0036] Advantages, other benefits, and solutions to problems have been described above with respect to specific embodiments. However, the advantages, benefits, solutions to problems, and components that may cause an advantage, benefit, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature or component of any or all of the claims. REFERENCES: 102 Carrier matrix preparation unit 104 Enzyme preparation unit 106 Immobilization Unit 108 Unit for characterization 110 test units 112 Validation Unit 202 Fe 3 O 4 @SiO 2 @CPTS@TSC nanocomposite 204 Immobilization of trypsin 206 Immobilization of chymotrypsin 208 Immobilization of papain 210 Nanoparticle-immobilized enzymes for the treatment of uterine infections in dairy animals

Claims

[1] System (100) for the production of biomimetic magnetic nanocomposites for the immobilization of proteolytic enzymes and the targeted treatment of bovine uterine infections, comprising: a) a unit for producing a carrier matrix (102) configured to produce magnetic nanoparticles; b) an enzyme preparation unit (104) configured to provide proteolytic enzymes selected from the group consisting of protease, trypsin, chymotrypsin and papain; c) an immobilization unit (106) configured to immobilize the proteolytic enzymes on the modified renewable support matrix; and d) a characterization unit (108) configured to perform toxicological tests, comprising: - Equipment for testing antimicrobial activity, and - DPPH assay analysis system. [2] The system (100) of claim 1, wherein the immobilization unit (106) is configured to maintain the viability of the enzyme and increase catalytic activity and thermodynamic stability. [3] The system (100) of claim 1, wherein the carrier matrix preparation unit (102) is configured to produce nanoparticles suitable for in utero drug delivery. [4] The system (100) of claim 1, wherein the system is configured to enable the administration of renovated enzymes to infected dairy cows and buffaloes. [5] The system (100) of claim 1 further comprises a testing unit (110) configured to facilitate in vitro testing of the prepared immobilized enzymes, the testing unit (110) comprising equipment for comparing the activities of immobilized enzymes with those of untreated enzymes. [6] The system (100) of claim 1 further comprises a validation unit (112) configured to test the immobilized enzymes on artificial substrates.

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