System with probiotic biofilm and method thereof

Abstract

The present disclosure generally relates to a universal method for functionalizing diversified systems, including probiotic biofilms. A method of treating a system with a probiotic biofilm is provided. A medical device or implant comprising the probiotic biofilm is also provided.

Description

[0001] Related Applications

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 618,517, filed on January 8, 2024, which is hereby incorporated by reference in its entirety. 1. Field of the Technology

[0003] The present disclosure generally relates to a general method for functionalizing diverse systems, including probiotic biofilms. A method for treating a system with a probiotic biofilm is provided. A medical device or implant comprising a probiotic biofilm is also provided. 2. Background Art

[0004] Recent research reports that approximately 10% of implants fail. The main cause of implant failure is opportunistic pathogens, especially antibiotic-resistant bacterial infections. Traditional methods involve loading drugs, such as antibiotics, silver ions, and antimicrobial peptides, onto the implant surface. However, a fatal problem with this method is the limited time due to the non-replenishable reserve of reactive compounds. The drugs may have been depleted when antimicrobial release is most needed. Additionally, a second drawback is the problem of the continuous low-level drug release contributing to the development of drug resistance in the pathogens. Surface modification (e.g., plasma-related methods, atom transfer radical, and chemical vapor deposition) is another effective strategy to address the above problems. However, these methods have drawbacks in terms of residual catalyst contamination and are not suitable for different types of materials. Angew. Chem. Int. Ed. 2012, 51, 11293 - 11296 discusses a method of incorporating penicillin-producing fungi into active materials. However, their method requires pre-treatment of the substrate and may not be suitable for most materials. Additionally, the long-term release of antibiotics in the coating may lead to the generation of drug-resistant bacteria. Lactobacillus casei was cultured on alkali heat-treated titanium substrates (Sci. Adv. 2020, 6, eaba5723). This probiotic biofilm enhanced bone integration through immunomodulatory and anti-infective mechanisms. To avoid sepsis caused by live bacteria, they inactivated the probiotics. Therefore, a general method is needed for functionalizing implant surfaces to have long-term antibacterial and anti-apoptotic activities. 3. Summary of the Invention

[0005] The present disclosure provides a novel general strategy for functionalizing the surface of implants with probiotic biofilms. In one embodiment, it is demonstrated that dopamine chemistry allows probiotic attachment to the surface of any material. In this example, live probiotics are modified onto the surface of any material to achieve the activity of long-term drug release. Lactobacillus plantarum (L. Plantarum) is a widespread member of lactic acid bacteria, commonly present in fermented foods, and can kill methicillin-resistant Staphylococcus aureus (MRSA) by releasing substances such as antimicrobial peptides and organic acids. Through the research of the present disclosure, it is found that under alkaline conditions (pH = 8.6), L. Plantarum biofilm coatings can be modified onto the surface of any material using dopamine (1 mg / mL). The L. Plantarum biofilm coatings exhibit excellent antibacterial and anti-biofilm activities against MRSA. In addition, the biofilm is composed of exopolysaccharides (e.g., EPS in L. plantarum-12 and YL-11EPS), and these extracellular polysaccharides also show anti-apoptotic activities against human corneal epithelial cells.

[0006] The present disclosure relates to a method for coating a medical device suitable for implantation into an individual or application to the skin or mucosal tissue of an individual. The method includes applying a coating to at least a portion of the surface of the device, wherein the coating comprises Lactobacillus plantarum (L. Plantarum), to form a biofilm on at least a portion of the surface of the medical device coated with active L. Plantarum. In one embodiment, L. Plantarum is killed before implantation. According to the method of the present disclosure, a medical device coated with the coating can be obtained. The coated medical device according to the present disclosure is particularly suitable for use as the mucosal tissue or skin of an individual, including but not limited to, being used as an ocular implant, skin in the skin or oral cavity, or dental use. Finally, the present disclosure relates to the use of L. Plantarum to coat a medical device suitable for implantation into an individual or application to the skin or mucosal tissue of an individual.

[0007] The provided medical device comprises active biofilm-coated L. Plantarum co-cultured with dopamine under alkaline conditions.

[0008] In certain embodiments, L. Plantarum is Gram-positive and the concentration of co-culture with dopamine is 10 8 CFU / mL.

[0009] In certain embodiments, the concentration of dopamine is 0.5 - 1.0 mg / ml, 1.0 - 1.3 mg / ml, or 1.3 - 1.5 mg / ml.

[0010] In one embodiment, the concentration of dopamine is 1 mg / ml.

[0011] In one embodiment, the dopamine is dopamine nanoparticles.

[0012] In certain embodiments, the alkaline condition is pH 8.2 - 9.2.

[0013] In one embodiment, the alkaline condition is pH 8.6.

[0014] In certain embodiments, L. Plantarum is alive and provides a reservoir of reactive compounds that can be supplemented.

[0015] In certain embodiments, the biofilm coating includes polysaccharides.

[0016] In certain embodiments, the biofilm coating includes long-term antibacterial activity, low drug resistance activity, anti-apoptotic activity, or a combination thereof.

[0017] In certain embodiments, the biofilm coating includes anti-apoptotic activity.

[0018] In certain embodiments, the anti-apoptotic activity is directed against human corneal cells.

[0019] In certain embodiments, the biofilm coating inhibits methicillin-resistant Staphylococcus aureus (“MRSA”), Streptococcus mutans ATCC 35668, Porphyromonas gingivalis ATCC 33277, or Rothia dentocariosa ATCC9126.

[0020] In certain embodiments, the medical device is further coated with an effective amount of bioactive materials including antibiotics, silver ions, antimicrobial peptides, etc.

[0021] In certain embodiments, the medical device is an implant.

[0022] In certain embodiments, the medical device is an implant for the external environment of the human body.

[0023] In certain embodiments, the medical device is a contact lens or a bandage.

[0024] A method of coating a medical device is provided, comprising the steps of: (i) providing a medical device having a surface; (ii) immobilizing live L. Plantarum on the surface by polymerization of dopamine to provide a biofilm coating; (iii) formatting the biofilm coating.

[0025] In certain embodiments, the surface includes metal, polymer, organic compound, inorganic compound, or a combination thereof.

[0026] In certain embodiments, L. Plantarum is genetically modified.

[0027] In certain embodiments, the medical device is irradiated with ultraviolet light or pasteurized.

[0028] In certain embodiments, L. Plantarum is a Gram-positive bacterium and is co-cultured with dopamine at a concentration of 10 8 CFU / mL.

[0029] In certain embodiments, the concentration of dopamine is 0.5 - 1.0 mg / mL, 1.0 - 1.3 mg / mL, or 1.3 - 1.5 mg / mL.

[0030] In certain embodiments, the concentration of dopamine is 1 mg / mL.

[0031] In certain embodiments, the alkaline condition is pH 8.2 - 9.2.

[0032] In certain embodiments, the alkaline condition is pH 8.6.

[0033] A method for preventing infection of a subject with a medical device is provided, including implanting the medical device. 4. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The patent or patent application document contains at least one color drawing. Upon request and payment of the necessary fees, the patent office will provide a copy of the patent or patent application publication containing the color drawing.

[0035] Figure 1 Schematic diagram of the development of an active L. Plantarum biofilm coating.

[0036] Figures 2A - 2F. (a) Schematic diagram of dopamine nanoparticles aggregating bacteria in solution. (b) Transmission electron microscopy (TEM) examination of L. plantarum with and without dopamine solution. (c) Self-aggregation activity of Gram-positive bacteria (L. GG, L. plantarum, S. mutants, and MRSA), Gram-negative bacteria (E. coli, F. nucleatum, P. gingivalis, and P. aeruginosa), and fungi (C. albicans and S. cerevisiae) with and without dopamine. (d) Schematic diagram of dopamine nanoparticles aggregating bacteria on the surface. (e) Scanning electron microscopy (SEM) images of L. plantarum with and without dopamine. (f) Biofilm production of Gram-positive bacteria (L. GG, L. plantarum, S. mutants, and MRSA), Gram-negative bacteria (E. coli, F. nucleatum, P. gingivalis, and P. aeruginosa), and fungi (C. albicans and S. cerevisiae) with and without dopamine.

[0037] Figures 3A - 3C. (a) Schematic diagram of implantable biomaterials on the human body. (b) Production of L. plantarum biofilms with and without dopamine on different substrates. (c) SEM images of L. plantarum with and without dopamine on different substrates.

[0038] Figures 4A - 4I. In vitro antibacterial activity of the active coating surface against MRSABAA40. (a) Schematic comparison of the active coating and the classical coating. (b) Antibacterial activities of the control group, PDA, LP, and PDA@LP groups. (c) SEM images of MRSA on the surfaces of the control group, PDA, LP, and PDA@LP. (d) Quantitative measurement of the long - term antibacterial activity of bacterial growth in Mueller - Hinton broth on different surfaces. (e) Development of resistance of MRSA (USA300) to LP coating, PDA@LP coating, and the antibiotic levofloxacin. (f) Quantitative measurement of the long - term low - drug - resistance activity of the killing kinetics of different groups against persister strains generated by treatment with 10×MIC levofloxacin. (g) Inhibitory activity of the control group, PDA, LP, and PDA@LP groups against MRSA biofilm production. (h) Formation of MRSA biofilms on the surfaces of the control group, PDA, LP, and PDA@LP was observed by confocal microscopy. (i) Morphology of MRSA on the surfaces of the control group, PDA, LP, and PDA@LP was observed by SEM images. The biofilm was stained with live / dead BacLight TM kit staining.

[0039] Figures 5A - 5H. In vitro anti - apoptotic activity of the active coating surface against human corneal cells. (a) Schematic diagram of the experimental process. (b) Antioxidant properties of the control group, PDA, LP, and PDA@LP coatings. (c) Quantitative analysis of the fluorescence intensity of intracellular reactive oxygen species. (d) Quantitative measurement of anti - apoptotic activity. Quantitative analysis of the fluorescence intensity of TUNEL. (e) Quantitative measurement of mitochondrial membrane potential. (f) Confocal microscopy images of intracellular reactive oxygen species in human corneal cells. (g) TUNEL staining of human corneal cells in different groups to measure the apoptosis level. (h) Measurement of mitochondrial membrane potential of human corneal cells on different surfaces with JC1 dye.

[0040] 4.1 Definitions

[0041] As used herein, the term "subject" refers to an individual (e.g., human, animal, or other organism) who is treated by the methods or compositions of the present disclosure. Subjects include, but are not limited to, mammals (e.g., rodents, primates, horses, cows, pigs, dogs, cats, etc.), including humans. The term "subject" generally refers to an individual who has received or has been treated with a medical device of the present disclosure (e.g., coated with probiotic microorganisms, and optionally one or more other agents) to treat a disease characterized by the presence of pathogenic bacteria. A "subject" can be a "non-human animal", including but not limited to vertebrates such as rodents, non-human primates, sheep, cows, ruminants, lagomorphs, pigs, goats, horses, dogs, cats, rats, etc.

[0042] As used herein, the term "effective amount" refers to the amount of a composition (e.g., probiotic microorganisms) that is sufficient to produce a beneficial or desired result. The effective amount can be administered in one or more administrations, applications, or doses, either once or multiple times, and is not limited to a particular formulation or route of administration. A skilled artisan can relatively easily determine the effective amount of probiotic microorganisms or other therapeutic compositions.

[0043] As used herein, the term "treated surface" refers to the act of exposing a surface, e.g., a catheter, to one or more compositions of the present invention. Methods of treating a surface include, but are not limited to, spraying, atomizing, immersing, and coating.

[0044] As used herein, the term "medical device" includes any material or device that is used in, on, or through the body of a subject or patient, e.g., during a medical treatment (e.g., for a disease or injury). Medical devices include, but are not limited to, medical implants, wound care devices, drug delivery devices, and body cavity and personal protection devices. Medical implants include, but are not limited to, urinary catheters, intravascular catheters, dialysis shunts, wound drainage tubes, skin sutures, vascular grafts, implantable meshes, intraocular devices, heart valves, etc. Wound care devices include, but are not limited to, general wound dressings, biological graft materials, tape closures and dressings, and surgical incision masks. Drug delivery devices include, but are not limited to, needles, drug delivery skin patches, drug delivery mucosal patches, and medical sponges. Body cavity and personal protection devices include, but are not limited to, tampons, sponges, surgical and examination gloves, and toothbrushes. Contraceptive devices include, but are not limited to, intrauterine devices (IUDs), diaphragms, and condoms.

[0045] As used herein, the term "coating" refers to a layer of material that covers a medical device or a portion thereof. The coating can be applied to a surface or incorporated into an implant material. 5. Detailed Description

[0046] The present disclosure relates to methods and materials for treating surfaces with non-pathogenic organisms. In certain embodiments, these methods and materials are used to establish cultures on a subject and / or on the surface of a medical device. In certain embodiments, the culture comprises a biofilm.

[0047] A biofilm is defined as an aggregate of a population of microbial cells that are adhered together by an extracellular matrix (EPS) and attached to a surface. Microbial cells growing in a biofilm are physiologically different from the planktonic cells of the same organism, which are single cells that may float or swim in a liquid medium. Under physiological conditions, in a nutrient-rich medium, a biofilm continuously disperses planktonic cells of the same organism into the medium, and these cells can colonize new locations and compete with other organisms for growth in the environment. Thus, probiotic biofilms are effective in mediating bacterial interference, thereby preventing pathogenic bacteria colonization on the probiotic biofilm-coated surface. However, there are many challenges in forming a stable and highly covered probiotic biofilm. Specifically, forming a probiotic biofilm on an in vivo medical device that can withstand physiological conditions for a long time has been a challenge. The present disclosure has successfully established a highly covered and stable probiotic biofilm on a medical device.

[0048] Live L. Plantarum was immobilized on the surface by dopamine and ultimately formed a biofilm covering the surface. This active biofilm coating can adhere to different surfaces (such as metals, polymers, and inorganics) because dopamine can adhere to almost all solid surfaces through self-polymerization. At the same time, the active biofilm coating has long-term drug release and exhibits excellent antibacterial, low drug resistance, and anti-biofilm activities against multi-drug resistant bacteria by releasing substances such as antimicrobial peptides and organic acids. Finally, the active biofilm coating is rich in polysaccharides, making the active biofilm surface have anti-apoptotic activity. Therefore, this active L. Plantarum biofilm coating is a general method with strong long-term antibacterial and low drug resistance activities and anti-apoptotic activity against human corneal epithelial cells.

[0049] To avoid the potential toxicity and immunogenicity of probiotics when used in the human body and to enhance the stability and bioactivity of the L. Plantarum biofilm coating, the following strategies were implemented. First, the toxic components of probiotics can be eliminated through genetic engineering. Second, the probiotic biofilm coating can be inactivated by ultraviolet light or pasteurization before implantation into the human body. Third, for implants used in the external environment of the human body, such as contact lenses and bandages, the first lines of defense, such as the skin, cornea, and mucous membranes, will resist the entry of bacteria such as probiotics into the human body, thereby reducing the risk of toxicity or inflammation. In addition, the live L. Plantarum biofilm coating with dopamine showed more stability compared to those without dopamine. Dopamine can bind to the membrane of L. Plantarum through Schiff base and Michael reactions, making it a mild and environmentally friendly reaction that will maintain the bioactivity of the live L. Plantarum biofilm coating.

[0050] In certain embodiments, L. Plantarum produces polysaccharides (Psl or Pel) when co-cultured with dopamine. In addition, reactive compounds with antibacterial activity were identified by high performance liquid chromatography (HPLC). This disclosure demonstrates that the L. Plantarum biofilm coating has strong antibacterial, biocompatible, and anti-apoptotic activities in vitro. A small animal corneal infection model was further provided to test its antibacterial, biocompatible, and anti-apoptotic activities in vivo.

[0051] The inventors recognized that forming a biofilm on at least a portion of the surface of a medical device suitable for implantation, particularly an implant pre-coated with L. Plantarum, reduces the formation of pathogenic biofilms on the implant in addition to preventing the attachment of pathogenic bacteria. In addition, L. Plantarum coated as a biofilm on a medical device can modulate the immune system towards an anti-inflammatory response.

[0052] The method according to the present disclosure provides a stable coating for a medical device, particularly the surface of a medical device, including an implant. In addition, the method represents a simple process using L. Plantarum.

[0053] As used herein, the term "medical device" includes all medical devices suitable for implantation into an individual. The material of the medical device can be selected from any material suitable for implantation into an individual or for application to the skin or mucous membrane tissue of an individual. Generally, the material of the medical device is an inert material, including metals and polymers.

[0054] A medical device can be a device that resides temporarily or permanently within an individual or on the skin or mucosal tissue of an individual, and is made of, for example, titanium and its alloys, zirconia, or stainless steel. The material can be a metal-based degradable material, such as a magnesium-based degradable material. Alternatively, or in combination, a degradable biopolymer can be used, such as a composite material based on polylactic acid.

[0055] In one embodiment, the material of the medical device is a metal or an alloy. For example, the metal or alloy consists of or contains titanium. In another embodiment, the medical device contains a magnesium-based degradable material.

[0056] In one embodiment, the medical device is an implant for dental use. Implants for dental use include abutments, subcutaneous implants, transosseous implants, endosseous implants, and jaw frame implants, as well as other maxillofacial implants for bone reconstruction. In one case, the medical device is made of metal for dental use in the oral cavity, such as titanium. Additionally, if the medical device comes into contact with the mucosal tissue or skin of an individual, the medical device can be a urinary catheter, various intravenous catheters, vaginal and intestinal devices (such as gastric tubes, sigmoidoscopes, colonoscopes, gastroscopes), and external prostheses.

[0057] The coating taught by the present disclosure is non-toxic to an individual receiving an implant or a medical device. In one embodiment, the medical device is placed on a heating device and heated before microorganisms are coated on its surface.

[0058] In one embodiment, the steps of coating and drying the coating material coated with L. Plantarum are repeated at least once, such as at least twice, three times, four times, five times, six times, seven times, eight times, nine times, twenty times, or more. Repeating the coating of the coating material on the device at least once can improve the formed coating. In particular, when the coating is coated at least twice or more, the stability of the coating can be improved.

[0059] In one embodiment, the medical device to be coated is heated, such as on a heating device as described above. The temperature of the heating device is at least 50°C to about 90°C. For example, the temperature of the device is between 60°C and 90°C, between 65°C and 85°C, or between 70°C and 80°C.

[0060] The suspension for coating is typically a suspension of L. Plantarum in water or other suitable solvents. When adding the material to the medical device, the material is dried, for example, by heating. A person skilled in the art is well aware of the suitable means to achieve drying so as to obtain a biofilm coating on the surface of the medical device. As previously mentioned, the step of coating the suspension on the surface of the medical device can be repeated one or more times. Thus, a stable coating is obtained. The coating is non-toxic to the individual. In addition, the coating will be biocompatible with the host tissue and coexist symbiotically with the host immune cells in homeostasis. That is to say, the coating according to the present disclosure also has benefits in terms of immune response and immune response alteration. In addition, L. Plantarum plays an important role in supporting the host immune system. This technology will guide and enhance the function of the host immune system.

[0061] In addition, L. Plantarum interferes with pathogenic bacteria attempting to attach to the medical device, especially pathogenic bacteria attempting to form a biofilm on the medical device, thereby keeping the pathogenic bacteria in a free state rather than a biofilm state, allowing the host immune cells to easily kill them. Different from the mechanism of action of antibiotics, factors that usually inhibit adhesion do not kill infectious bacteria. Therefore, through this technology, the evolutionary opportunity of antibacterial resistance in bacteria will be significantly reduced.

[0062] L. Plantarum in the coating step can be alive. Alternatively, the coated L. Plantarum is killed, for example, by preheating. In one embodiment, the coating is a suspension of live L. Plantarum. Alternatively, a suspension of killed L. Plantarum is coated.

[0063] In one embodiment, the method is a method of making the coating a complete coating on the outer surface of the medical device, especially for medical devices for skin or eye use.

[0064] On the other hand, the present disclosure relates to a coated medical device obtained by the method according to the present disclosure. As mentioned above, for the coated medical device according to the present disclosure, the coating determined by the present disclosure shows better performance than other coatings, including improved stability and being non-toxic to the individual receiving the medical device.

[0065] In one aspect, the present disclosure relates to a coated medical device for implantation into an individual. In one embodiment, the coated medical device according to the present disclosure is for application to the skin or mucosal tissue of an individual.

[0066] In one embodiment, medical devices suitable for application to an individual's skin or mucosal tissue are, for example, urinary catheters, vaginal and intestinal devices (gastric tubes, sigmoidoscopes, colonoscopes, gastroscopes), and external prostheses. The coated medical device is superior to other coatings in avoiding infection with pathogenic microorganisms in applications as implants or in applications in contact with an individual's mucosal tissue or skin.

[0067] In one embodiment, the coated medical device is made of or contains a metal, particularly titanium. In particular, the coated medical device according to the present disclosure does not require specific pretreatment of the surface of the metal or alloy, particularly a metal or alloy containing titanium.

[0068] The coated medical device according to the present disclosure is made of a suitable material. As described above, suitable materials include metals and polymers, such as biocompatible and biodegradable metals or alloys and polymers. The coated medical device can be used temporarily or permanently in the individual.

[0069] In one embodiment, the present disclosure teaches enhancing the formation of a high-density and stable L. Plantarum biofilm to treat and prevent pathogen colonization that causes device-related infections. In certain embodiments, the present disclosure also relates to methods and compositions for storing and using medical devices coated with non-pathogenic biofilms. In certain embodiments, the present disclosure also relates to methods and compositions for modifying implantable medical devices with bacterially resistant polymers and / or antibacterial agents.

[0070] In one embodiment, the present disclosure teaches co-administration of bioactive agents or therapies in combination with the products or methods of the present disclosure. In certain embodiments, the co-administered agents or therapies are simultaneous. In other embodiments, the first agent / therapy is administered first, and then the second agent / therapy is administered. Those skilled in the art can understand that the formulations and / or administration routes of various agents or therapies may vary. The appropriate dosage for co-administration can be readily determined by those skilled in the art. In certain embodiments, when the agents or therapies are co-administered, the respective agents or therapies are administered at a lower dosage than the appropriate dosage when administered alone. Thus, co-administration is particularly desirable in embodiments where the co-administered agents or therapies reduce the required dosage of potentially harmful (e.g., toxic) agents.

[0071] In one embodiment, live probiotics are maintained in cell culture. In certain embodiments, the live probiotics are continuous cell lines, primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), bacterial cultures in solid or liquid media, and any other cell population maintained in vitro.

[0072] In one embodiment, live probiotics are maintained in liquid culture. In one embodiment, the liquid medium can be any liquid composition suitable for providing nutrients to the organisms growing therein. A solidifying agent, such as agar, can be added to produce a "solid medium", such as a petri dish or a slant, also known as an agar plate and an agar slant.

[0073] In one embodiment, an antibacterial agent is used in combination with the products or methods of the present disclosure.

[0074] In one embodiment, a preservative is used in combination with the products or methods of the present disclosure. In certain embodiments, the antibacterial substances inhibit the action of microorganisms, including but not limited to α-terpineol, methylisothiazolinone, cetylpyridinium chloride, chlorophenol, adipic acid, chlorhexidine and other cationic biguanides, methane chloride, iodine and iodides, triclosan, taurine amide, nitrofurantoin, methacrylic acid, silver, benzoyl peroxide, alcohol and carboxylic acids and their salts. Those skilled in the art know that these preservatives can be used in combinations of two or more to obtain a synergistic effect.

[0075] In certain embodiments, the combinations of preservatives include chlorhexidine, chlorhexidine and chlorophenol, chlorhexidine and methylisothiazolinone, chlorhexidine and α-terpineol, methylisothiazolinone and α-terpineol; thymol and chlorophenol; chlorhexidine and cetylpyridinium chloride; or chlorhexidine, methylisothiazolinone and thymol. These combinations provide broad-spectrum activity against various organisms.

[0076] In one embodiment, a protecting agent is used in combination with the products or methods of the present disclosure. In certain embodiments, the protecting agent is a composition or compound that protects the activity or integrity of an active agent (such as an enzyme, probiotic microorganism) when exposed to certain conditions (such as drying, freezing). In certain embodiments, the protecting agent protects organisms (such as probiotic microorganisms) during the freezing process, i.e., it is a "cryoprotectant". Examples of protecting agents include but are not limited to non-fat milk powder, trehalose, glycerol, betaine, sucrose, glucose, lactose, dextran, polyethylene glycol, sorbitol, mannitol, polyethylene propylene, potassium glutamate, monosodium glutamate, Tween 20 detergent and Tween 80 detergent, and amino acid hydrochlorides.

[0077] In one embodiment, a gelling agent is used in combination with the products or methods of the present disclosure. In certain embodiments, the gelling agent is a composition that forms a colloidal semi-solid (such as a lubricating gel) when dissolved, suspended or dispersed in a fluid (such as an aqueous fluid such as water or a buffer solution). Examples of gelling agents include but are not limited to hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl guar gum, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, carbomer, alginate, gelatin and poloxamer.

[0078] In one embodiment, excipients are used in combination with the products or methods of the present disclosure. In certain embodiments, excipients are inactive ingredients (i.e., having no pharmacological activity) added to an active ingredient formulation. The above-mentioned gelling agents and protective agents are generally referred to as "excipients".

[0079] In one embodiment, a kit is provided, which is a delivery system for delivering materials. In the context of reaction materials such as probiotic microorganisms, such a delivery system includes, but is not limited to, systems that allow the storage, transportation, or delivery of appropriate reagents (e.g., cells, buffers, media, selection reagents, etc., in appropriate containers) and / or devices (e.g., catheters, syringes, reaction tubes or plates, culture tubes or plates) and / or support materials (e.g., media, written instructions for using the materials) from one location to another. For example, the kit includes one or more containers containing relevant reaction reagents and / or support materials. In one embodiment, the provided kit is a dispersion kit, which is a delivery system that includes two or more separate containers, each containing a part of the total kit components. The containers can be delivered together or separately to the intended recipient. For example, one container can contain a dry composition of microorganisms with a specific use and a gelling agent, while another container contains a sterile fluid, such as water or buffer, for dissolving or resuspending the dry composition.

[0080] 6. Examples

[0081] 6.1 Materials and Methods

[0082] 6.1.1 Materials

[0083] Lactobacillus plantarum (ATCC 8014) was obtained from ATCC. Dopamine and test kits (live / dead detection, Tunel detection) can be purchased directly from chemical reagent companies such as Sigma, Fisher, TCI, J&K, Aladdin, etc.

[0084] 6.1.2 Methods

[0085] Preparation of PDA@LP coating. A new general strategy was developed to functionalize the implant surface with active probiotic biofilms. Specifically, L. Plantarum (Gram-positive bacteria) was selected and co-cultured with dopamine (1 mg / mL) under alkaline conditions (pH = 8.6) in a shaker incubator for 1 hour at a rotation speed of 100 rpm, and then washed three times with PBS ( Figure 1 ).

[0086] Characterization. To observe the morphology of probiotics after co - culture with dopamine, transmission electron microscopy (TEM) and field emission scanning electron microscopy (FE - SEM) were used. The probiotics were washed three times with PBS after co - culture with dopamine. To fix the bacteria, 10% glutaraldehyde was used. After standing for 1 hour, it was washed with PBS. Finally, dehydration was carried out successively with gradient ethanol (20%, 30%, 50%, 70%, 90%, and 100%) for 15 minutes each.

[0087] Dopamine aggregation experiment. Bacteria were cultured overnight in the medium and then adjusted to an OD 600 of 1.0 (about 10 8 CFU / mL) in a new 1 - mL tube, and then the initial CFU was measured as A0. Subsequently, the suspension was allowed to stand for 1 hour to aggregate flocs, and the CFU in the supernatant was measured as At. The bacterial aggregation index was calculated by the following formula: Aggregation index %=(1 - A t / A0)×100.

[0088] Crystal violet biofilm experiment. Bacteria with and without dopamine treatment were cultured on 96 - well microplates and other material surfaces (ceramics, silica gel, PHEMA, PLGA, steel, titanium). Then the wells were washed three times with PBS and stained with 1% crystal violet solution (Sigma - Aldrich, St. Louis, MO, USA) at room temperature for 30 minutes. Next, it was washed at least three times to remove the dye not bound to the biofilm. The biofilm - bound dye was dissolved in ethanol - acetone (70:30, v / v) for 10 minutes, and then the OD 595 .

[0089] In vitro antibacterial coating experiment. 1 mL of a bacterial (MRSA) suspension in the mid - logarithmic growth phase was collected, centrifuged, and washed with PBS. Then, the bacteria were resuspended and diluted to 1.0×10 8 CFU / mL in PBS. Then ten microliters of the bacterial solution was pipetted onto the coating. After that, the coating was cultured in an incubator for 4 hours with the humidity maintained above 90%. After culture, the recovered bacteria were serially diluted 10 - fold to a series of bacterial suspension concentrations, and then agar plate culture was carried out, and CFU counting was performed after overnight incubation. The log reduction of the pathogen was calculated by the following formula: Log reduction = log (control group cell count) – log (cell count on the coating sample). The morphology of bacteria on the coating was examined under FE - SEM. The samples were fixed with 4% phenol for 1 hour and then dehydrated with a gradient ethanol solution. After the samples were freeze - dried, they were gold - plated before SEM observation. 50 microliters of the MRSA solution was mixed with the mid - logarithmic growth phase (1.0×10 8(CFU / mL) was added to 5 mL of MHB with different coatings. The solution was cultured at 37 °C, and the bacterial survival was detected using an ultraviolet spectrophotometer at a wavelength of 600 nm. The O.D. (optical density) of the mixture was checked from 0 to 10 hours.

[0090] Drug resistance stimulation study. Drug resistance was induced by repeatedly treating bacteria with antibacterial agents. Here, MRSA (ATCC BAA40) was cultured to test the drug resistance of the cell-free supernatant of the PDA@LP coating. The MIC of the cell-free supernatant of the PDA@LP coating against bacteria was tested until the 16th passage of bacterial growth. Before each MIC measurement, the bacteria were co-cultured at a sub-MIC concentration (1 / 8 of the MIC in the previous passage for the same antibacterial agent) and grown to the logarithmic growth phase. By observing the change in the ratio of MIC to the first passage, the drug resistance behavior of the bacteria could be shown. Compared with traditional antibiotics, an antibiotic, levofloxacin, was selected and the drug resistance of the bacteria was tested.

[0091] Killing kinetics of antibiotic-generated persister strains. 1 mL of an MRSABAA40 suspension in the mid-logarithmic growth phase was challenged with 10×MIC of levofloxacin in MHB for 18 hours. Half of the bacterial solution was collected by centrifugation and washed three times with PBS, and then challenged with the cell-free supernatant. The other half continued to be exposed to the antibiotic as a control. The recovered bacteria were serially diluted 10-fold to a series of bacterial suspension concentrations and then agar plate cultured, and CFU counting was performed after overnight incubation.

[0092] Antibiofilm experiment. 150 μL of MRSABAA40 bacteria in tryptic soy broth (TSB) containing 1% glucose (initial inoculum of 10^8 CFU per well) was added to each well of a 24-well plate. After culturing for 24 hours, different coatings were co-cultured with the above biofilms across the wells overnight. Then, biofilm production was tested by crystal violet. For FESEM imaging of the biofilm, after treatment with the control group, PDA, LP, and PDA@LP coatings, it was fixed with 4% phenol overnight and then dehydrated with a gradient ethanol solution. For confocal imaging, the biofilm was stained with the BacLight TM Live / Dead kit and observed under a confocal microscope.

[0093] Antioxidant efficiency of the coatings. Control group, PDA, LP, and PDA@LP samples were dispersed in 100 μM DPPH solution. Then the samples were incubated in the dark for half an hour. Next, the wavelength of DPPH was scanned with an ultraviolet-visible spectrophotometer. The degradation of DPPH was calculated by the following formula:

[0094]

[0095] Among them, A B is the absorption of the blank (DPPH + ethanol), and A H is the absorption of the coating (DPPH + ethanol + coating).

[0096] ROS scavenging in vitro. Human corneal cells were seeded in six-well plates, then stimulated with H2O2 (200 μmol / L) and co-cultured with PDA, LP, and PDA@LP coatings. Then, 10 μM DCFH-DA was added to the cells and incubated in the dark at 37 °C for 30 minutes. After that, the cells were washed three times with serum-free medium to remove the unloaded DCFH-DA probe, and then imaged using a laser confocal microscope.

[0097] TUNEL test experiment. Human corneal cells were seeded in confocal dishes, cultured with Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) containing 10% fetal bovine serum, and stimulated with H2O2. Then, green fluorescent apoptotic cells were imaged using dUTP-FITC in a TUNEL kit (Beyotime, C1088).

[0098] JC-1 dye was used for mitochondrial membrane potential. Human corneal cells were induced by H2O2 (200 μmol / L) to develop apoptosis. The mitochondrial membrane potential was tested using a kit from Beyotime. It was prepared by adding 2 μL Mito-Tracker Red CMXRos and 5 μL AnnexinV-FITC, incubated at 25 °C for 30 minutes, and then placed in an ice bath. Finally, the sample smear was observed under a Leica DMi8 fluorescence microscope.

[0099] Example products, systems, and methods are as follows:

[0100] 1. A medical device comprising L. Plantarum with an active biofilm coating co-cultured with dopamine under alkaline conditions.

[0101] 2. The medical device of item 1, wherein L. Plantarum is a Gram-positive bacterium and the concentration of co-culture with dopamine is 10 8 CFU / mL.

[0102] 3. The medical device of any one of the foregoing, wherein the concentration of dopamine is 0.5 - 1.0 mg / mL, 1.0 - 1.3 mg / mL, or 1.3 - 1.5 mg / mL.

[0103] 4. The medical device of any one of the foregoing, wherein the concentration of dopamine is 1 mg / mL.

[0104] 5. The medical device of any one of the foregoing, wherein dopamine is dopamine nanoparticles.

[0105] 6. The medical device of any preceding item, wherein the alkaline condition is pH 8.2-9.2.

[0106] 7. The medical device of any preceding item, wherein the alkaline condition is pH 8.6.

[0107] 8. The medical device of any preceding claim, wherein the L. Plantarum is alive and provides a replenishable reserve of reactive compounds.

[0108] 9. The medical device of any preceding claim, wherein the biofilm coating comprises a polysaccharide.

[0109] 10. The medical device of any of the preceding items, wherein the biofilm coating comprises long-term antimicrobial activity, low drug resistance activity, anti-apoptotic activity, or a combination thereof.

[0110] 11. The medical device of any preceding claim, wherein the biofilm coating comprises anti-apoptotic activity.

[0111] 12. The medical device of any preceding claim, wherein the anti-apoptotic activity is directed against human corneal cells.

[0112] 13. The medical device of any of the preceding items, wherein the biofilm coating inhibits methicillin-resistant Staphylococcus aureus ("MRSA"), Streptococcus ATCC 35668, Porphyromonas gingivalis ATCC 33277, or Ribonucleobacterium ATCC9126.

[0113] 14. The medical device of any of the preceding items, further coated with an effective amount of a biologically active material including antibiotics, silver ions, and antimicrobial peptides.

[0114] 15. The medical device of any of the preceding items, which is an implant.

[0115] 16. The medical device of any of the preceding items, which is an implant for use in an environment external to the human body.

[0116] 17. Any of the above medical devices, which is a contact lens or a wound dressing.

[0117] 18. A method for coating a medical device, comprising the steps of: (i) providing a medical device having a surface; (ii) providing a biofilm coating on the surface by immobilizing active L. Plantarum via dopamine polymerization; (iii) further coating an effective amount of a bioactive material; and (iv) formatting the biofilm coating.

[0118] 19. The method of claim 18, wherein the surface comprises a metal, a polymer, an organic compound, an inorganic compound, or a combination thereof.

[0119] 20. The method of any one of the foregoing, wherein L. Plantarum is genetically modified.

[0120] 21. The method of any one of the foregoing, wherein the medical device is irradiated with ultraviolet light or pasteurized.

[0121] 22. The method of any one of the foregoing, wherein L. Plantarum is a Gram-positive bacterium and the concentration of co-culture with dopamine is 10 8 CFU / mL.

[0122] 23. The method of any one of the foregoing, wherein the concentration of dopamine is 0.5 - 1.0 mg / mL, 1.0 - 1.3 mg / mL or 1.3 - 1.5 mg / mL.

[0123] 24. The method of any one of the foregoing, wherein the concentration of dopamine is 1 mg / mL.

[0124] 25. The method of any one of the foregoing, wherein the alkaline condition is pH 8.2 - 9.2.

[0125] 26. The method of any one of the foregoing, wherein the alkaline condition is pH 8.6.

[0126] 27. A method for preventing infection of a subject with a medical device, comprising implanting the medical device of item 1.

[0127] The description of the foregoing specific embodiments will fully disclose the general nature of the present disclosure, such that others can easily modify and / or adapt these specific embodiments by applying the knowledge of the relevant technical fields (including the content of the documents cited and incorporated herein) to suit various applications without undue experimentation and without departing from the general concept of the present disclosure. Therefore, these adjustments and modifications are intended to fall within the meaning and equivalent scope of the embodiments of the present disclosure, based on the teachings and guidance presented herein. It should be understood that the language or terms herein are for the purpose of description and not limitation, that is, the terms or language in this specification should be interpreted by those skilled in the art in combination with the teachings and guidance presented herein and the knowledge of the relevant technical fields.

[0128] Although the various embodiments of the present disclosure have been described above, it should be understood that they are presented by way of example and not limitation. Various changes in form and detail may be made thereto by those skilled in the art without departing from the spirit and scope of the present disclosure. Therefore, the present disclosure should not be limited by any of the exemplary embodiments described above, but should be defined only by the following claims and their equivalents.

[0129] All references cited herein are incorporated herein by reference in their entirety to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference for all purposes.

[0130] References

[0131] 1.Angew.Chem.Int.Ed.2012,51,11293-11296.

[0132] 2.Sci.Adv.2020,6,eaba5723.

Claims

1. A medical device comprising L. Plantarum with an active biofilm coating co-cultured with dopamine under alkaline conditions.

2. The medical device according to claim 1, wherein L.Plantarum is a Gram-positive bacterium and the concentration of co-culture with dopamine is 10 8 CFU / mL.

3. The medical device according to claim 1, wherein the concentration of dopamine is 0.5 - 1.0 mg / mL, 1.0 - 1.3 mg / mL, or 1.3 - 1.5 mg / mL.

4. The medical device according to claim 1, wherein the concentration of dopamine is 1 mg / mL.

5. The medical device according to claim 1, wherein dopamine is dopamine nanoparticles.

6. The medical device according to claim 1, wherein the alkaline condition is pH 8.2 - 9.

2.

7. The medical device according to claim 6, wherein the alkaline condition is pH 8.

6.

8. The medical device according to claim 1, wherein L. Plantarum is viable and can provide a replenishable reserve of reactive compounds.

9. The medical device according to claim 1, wherein the biofilm coating comprises polysaccharides.

10. The medical device according to claim 1, wherein the biofilm coating comprises long-term antibacterial activity, low drug resistance activity, anti-apoptotic activity, or a combination thereof.

11. The medical device according to claim 1, wherein the biofilm coating comprises anti-apoptotic activity.

12. The medical device according to claim 1, wherein the anti-apoptotic activity is directed against human corneal cells.

13. The medical device according to claim 1, wherein the biofilm coating inhibits methicillin-resistant Staphylococcus aureus ("MRSA"), Streptococcus ATCC 35668, Porphyromonas gingivalis ATCC 33277, or Actinobacillus actinomycetemcomitans ATCC 9126.

14. The medical device according to claim 1, wherein the medical device is further coated with an effective amount of a bioactive material comprising an antibiotic, silver ions, an antimicrobial peptide, or the like.

15. The medical device according to claim 1, wherein the medical device is an implant.

16. The medical device according to claim 1, wherein the medical device is an implant for the external environment of the human body.

17. The medical device according to claim 1, wherein the medical device is a contact lens or a wound dressing.

18. A method of coating a medical device, comprising the steps of: (i) providing a medical device having a surface; (ii) providing a biofilm coating by fixing live L. Plantarum on the surface through dopamine polymerization; (iii) further coating an effective amount of a bioactive agent; (iv) formatting the biofilm coating.

19. The method according to claim 18, wherein the surface comprises a metal, a polymer, an organic compound, an inorganic compound, or a combination thereof.

20. The method according to claim 18, wherein L. Plantarum is genetically modified.

21. The medical device according to claim 1, wherein the medical device is irradiated with ultraviolet light or pasteurized.

22. The method according to claim 18, wherein L.Plantarum is a Gram-positive bacterium and the concentration of co-culture with dopamine is 10 8 CFU / mL.

23. The method according to claim 18, wherein the concentration of dopamine is 0.5 - 1.0 mg / mL, 1.0 - 1.3 mg / mL, or 1.3 - 1.5 mg / mL.

24. The method according to claim 18, wherein the concentration of dopamine is 1 mg / mL.

25. The method according to claim 18, wherein the alkaline condition is pH 8.2 - 9.

2.

26. The method according to claim 18, wherein the alkaline condition is pH 8.

6.

27. A method for preventing infection in a subject having a medical device, comprising implanting the medical device according to claim 1.

Images