KR102419318B1 - Biocompatible, ferrocene-containing polymer multilayer coatings having antifouling and ROS sensitive controlled release of therapeutic drug and the method for manufacturing the same - Google Patents

Abstract

The present invention relates to a multilayer coating material comprising a ferrocene polymer for biocompatibility, antifouling and controlled release of a ROS-sensitive drug and a method for preparing the same.

Description

Biocompatible, ferrocene-containing polymer multilayer coatings having antifouling and ROS sensitive controlled release of therapeutic drug and the method for manufacturing the same}

The present invention relates to a multilayer coating material comprising a ferrocene polymer for biocompatibility, antifouling and controlled release of a ROS-sensitive drug and a method for preparing the same.

Implantable medical devices such as catheters, stents, orthopedic implants and cardiovascular prostheses have become part of modern medicine to save lives or improve treatment outcomes. However, such a device is a risk factor for health care-associated infection (HAI), which is an infection related to medical practice in a medical institution including outpatient as well as inpatient treatment. The basic pathogenic mechanism for infection is bacterial colonization on the device surface, which can lead to the development of biofilms. Biofilms can create planktonic bacterial cells that damage surrounding tissues and spread infection, and can cause implant-defective negative foreign body response (FBR) and implant-related infections, resulting in an inflammatory cascade as a wound healing response. have. In addition, when a medical device is inserted into the human body, biomolecules such as blood cells and proteins rapidly interact with the surface due to the phenomenon of biofouling, and unnecessary substances such as biological materials, for example, proteins and cells, are removed from the medical device. It may adhere to the surface and cause functional deterioration. Therefore, it is very important to develop a material that can control and well integrate the surface properties of living tissue and prevent blood coagulation and bacterial adhesion, i.e., a material with "biocompatibility" and "anti-biofouling".

Drug delivery using implantable medical devices is an example of advances in biomedical science and offers a promising solution to achieve multiple therapeutic functions. Implant-based drug delivery should minimize side effects to vital organs compared to systemic administration of the drug, and should enable long-term drug release to the target site compared to standard topical application of the drug. Therefore, stimulation-responsive and sustainable drug delivery has received considerable attention due to its advantages of improving therapeutic action and reducing side effects. Among various stimuli such as pH, light, temperature, solvents, electricity and redox reactions, hydrogen peroxide (H ₂ O ₂ ), superoxide (O ₂ - ), reactive oxygen species (ROS) including hydroxyl radicals (·OH) ) is produced in abnormal metabolic environments such as inflammatory pathologies and tumors by redox stimulation. Thus, increased ROS levels in the pathological domain can control drug release at the target site using ROS-responsive medical devices.

On the other hand, layer-by-layer (LbL) is a simple and versatile deposition process that is widely applied to solve many problems such as biomolecule deposition, concentration, bioactivity, coating thickness and release rate. LbL films are created by sequentially depositing biomolecules in a solution containing functional groups that drive self-assembly. Most techniques rely on electrostatic interactions between oppositely charged polyelectrolytes during sequential deposition. Since the LbL film can be manufactured while controlling the thickness and surface properties, it is used in various fields such as drug delivery systems, anti-fouling membranes, and electrodes.

International Patent Publication WO-2010/065960

As a result of the present inventors' diligent efforts to develop a biocompatible polymer coating material for antifouling and ROS-sensitive drug controlled release, ferrocenylmethyl methacrylate (FMMA), poly (ethylene glycol) methacrylate (poly Poly (FMMA) as a tricopolymer using (ethylene glycol) methacrylate, PEGMA) and methacrylate (Methacrylic acid, MA) or 2-(dimethylamino)ethyl methacrylate (DMAEMA) -r-PEGMA-r-MA and poly(FMMA-r-PEGMA-r-DMAEMA) to prepare a multi-layer coating material by layer-by-layer (LbL), bacteria and blood cells After confirming that the biofilm such as adhesion is minimized and the controlled release of the therapeutic drug is possible, the present invention has been completed.

Biocompatible ferrocene containing polymers for controlled release of antifouling and ROS sensitive drugs according to the present invention, namely poly(FMMA-r-PEGMA-r-MA) (C series ferrocene polymer) and poly(FMMA-r-PEGMA-r -DMAEMA) (N series ferrocene polymer) synthesis method and a schematic diagram of a film on which a multilayer coating material including the same is deposited is shown in FIG. 1 .

The present invention is (A) ferrocenylmethyl methacrylate (Ferrocenylmethyl methacrylate, FMMA), poly (ethylene glycol) methacrylate (poly (ethylene glycol) methacrylate, PEGMA) and methacrylate (Methacrylic acid, MA) comprising poly(FMMA-r-PEGMA-r-MA) polymers; and (B) Ferrocenylmethyl methacrylate (FMMA), poly(ethylene glycol) methacrylate (PEGMA) and 2-(dimethylamino)ethyl methacrylate (2-( Dimethylamino)ethyl methacrylate, DMAEMA) to provide a biocompatible coating material having antibacterial, antifouling and antithrombotic effects comprising a bilayer composed of a poly(FMMA-r-PEGMA-r-DMAEMA) polymer aim to

Another object of the present invention is to provide a method for preparing a biocompatible coating material having the antibacterial, antifouling and antithrombotic effects.

Another object of the present invention is to provide a multilayer film comprising a substrate and a biocompatible coating material having an antibacterial, antifouling and antithrombotic effect deposited on the substrate.

Another object of the present invention is to provide a medical device comprising the biocompatible coating material having the antibacterial, antifouling and antithrombotic effect deposited on the substrate.

As used herein, the term “anti-fouling” or “anti-biofouling” refers to adhesion of proteins, plasma, cells, tissues and/or microorganisms, including blood proteins, to a substrate with respect to the amount of adhesion to a reference polymer. means that the composition reduces or prevents the amount of Preferably, the device surface will be substantially anti-biofouling in the presence of human blood. Preferably the amount of coalescence is 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90% relative to the reference polymer. %, 95%, 99%, 99.5%, or 99.9%.

As used herein, the term “anti-biofilm” refers to a collection of single or multiple species encapsulated in self-produced extracellular polymeric substances (EPS), in particular reducing or preventing bacterial infection. it means.

As used herein, the term “coating” refers to any temporary, semi-permanent or permanent layer or layers that treat or cover a surface. The coating may be a chemical modification of the underlying substrate or may include the addition of a new material to the surface of the substrate. Coating includes any increase in thickness to the substrate or change in the chemical composition of the surface of the substrate. The coating may be a gas, vapor, liquid, paste, semi-solid or solid. In addition, the coating can be applied as a liquid and solidified into a solid coating.

The biocompatible coating material according to the present invention has excellent antibacterial, antithrombotic, antifouling and ROS-sensitive drug controlled release, and thus can be used to manufacture a highly versatile medical material.

1(A) shows ferrocene containing polymers according to the invention, for example poly(FMMA-r-PEGMA-r-MA) (C series ferrocene polymer) and poly(FMMA-r-PEGMA-r-DMAEMA) (N series ferrocene polymers), and FIG. 1(B) shows a schematic diagram of a multilayer film comprising said ferrocene-containing polymer for biocompatible, antifouling and ROS sensitive drug controlled release.
2 shows the thickness of a (Poly Ns/Cs) ₁₀ LBL film.
3 shows the surface properties of (Poly Ns/Cs) _n LBL films. (A) Changes in surface zeta potential, (B) thickness growth, (C) (Poly N2/C2) _n LBL film FTIR spectra. (D) SEM image of (Poly N2/C2) _n LBL film.
Figure 4 (A) shows the adhesion properties of the (Poly N2/C2) _n LBL film to S. aureus and E. coli, and Figure 4 (B) is the (Poly N2 / C2) _n LBL film biofilm formation inhibition. SEM images showing the ability, Figure 4 (C) shows LIVE / DEAD BacLight bacterial viability analysis, Figure 4 (D) shows live and Figure 4 (F) shows fluorescence microscopy images of dead bacteria.
Figure 5 (A) shows a drug loading model on (Poly N2 / C2) _n LBL film according to the present invention, Figure 5 (B) is the release profile of the loaded drug and Figure 5 (B) (Poly N2 / C2) _n It shows the decomposition rate according to the change of the LBL film thickness.
6(A) shows the release profile of PTX from (Poly N2/C2) _n LBL films, and FIG. 6(B) shows toxicity of PTX, PTX loaded and unloaded (Poly N2/C2) _n LBL films. and anticancer effect.
Figure 7(A) shows the hemolysis rate of uncoated and (Poly N2/C2) _n LBL film surfaces, Figure 7 (B) is bare and Figure 7 (C) is for (Poly N2/C2) ₁₀ LBL films. FE-SEM images of platelet adhesion are shown.

Aha, a biocompatible ferrocene-containing polymer for controlled release of antifouling and ROS-sensitive drugs and uses thereof according to specific embodiments of the present invention will be described in detail. However, this is presented as an example of the invention, thereby not limiting the scope of the invention, it is apparent to those skilled in the art that various modifications to the embodiment are possible within the scope of the invention.

Throughout this specification, unless otherwise specified, "including" or "containing" refers to including any component (or component) without particular limitation, and excludes the addition of other components (or components). cannot be construed as

According to the first embodiment,

The present invention is (A) ferrocenylmethyl methacrylate (Ferrocenylmethyl methacrylate, FMMA), poly (ethylene glycol) methacrylate (poly (ethylene glycol) methacrylate, PEGMA) and methacrylate (Methacrylic acid, MA) comprising poly(FMMA-r-PEGMA-r-MA) polymers; and

(B) Ferrocenylmethyl methacrylate (FMMA), poly(ethylene glycol) methacrylate (PEGMA) and 2-(dimethylamino)ethyl methacrylate (2-(Dimethylamino) To provide a biocompatible coating material having antibacterial, antifouling and antithrombotic effects including a bilayer composed of a poly(FMMA-r-PEGMA-r-DMAEMA) polymer containing )ethyl methacrylate, DMAEMA) .

In the biocompatible coating material having antibacterial, antifouling and antithrombotic effects according to the present invention, the ferrocenylmethyl methacrylate, poly(ethylene glycol) methacrylate, and methacrylate or 2-(dimethylamino) It is characterized in that the molar ratio of ethyl methacrylate is 1:5-10:5-20, preferably 1:7-8:17-18.

In the biocompatible coating material having antibacterial, antifouling and antithrombotic effects according to the present invention, the coating material comprises the poly(FMMA-r-PEGMA-r-MA) polymer and poly(FMMA-r-PEGMA-r). -DMAEMA) polymer is sequentially laminated by layer-by-layer (LbL), characterized in that it is composed of 2 to 10, preferably 2.5 to 6 double layers. In the layered self-assembly method, the poly (FMMA-r-PEGMA-r-MA) polymer and the poly (FMMA-r-PEGMA-r-DMAEMA) polymer are applied by immersion, spray or spin coating. ) method can be used. According to the lamination order, the poly(FMMA-r-PEGMA-r-MA) polymer is first laminated according to the lamination target, and then the poly(FMMA-r-PEGMA-r-DMAEMA) polymer is laminated by a layer-by-layer method. , LbL) can be stacked, and vice versa.

In the biocompatible coating material having antibacterial, antifouling and antithrombotic effects according to the present invention, the antifouling is Pseudomonas aeruginosa, Salmonella spp, Shigella spp, Vibrio enteritis. parahaemolyticus), Vibrio choreae, Escherichia coli O157, Campylobacter jejuni, Clostridium difficile, Clostridium perfringens, Elsinia enterocoYersinia enterocoYersinia Helicobacter pylori, Entemoeba histolytica, Bacillusu cereus, Clostridium botulinum, Haemophilus influenzae, Streptococcus pneumoniae, Chlamydia pneumoniae, Chlamidia pneumoniae Legionella pneumoniae, Branhamella catarrhalis, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Storeptcoccus pyogenes, Corynebacterium diphtheriae, Bordetella pertussis diphtheriae , Chramidia psittaci, methicillin resistant Staphylococcus aureus, MRSA, Escherichia coli, Klebsiella pneumoniae, Enterobacter spp, Proteus spp Acinetobacter (Acinetobacter) er spp), Enterococcus faecalis, Staphylococcus saprophyticus, and B type (Storeptcoccus agalactiae) characterized in that it is formed by one or more bacteria selected from the group consisting of.

In the biocompatible coating material having an antibacterial, antifouling and antithrombotic effect according to the present invention, the coating material is a ship or hull coating, a fuel tank, an oil pipeline, an industrial pipe, a pharmaceutical facility, a drug delivery device, e.g. For example inhalers, contact lenses, dental implants, coatings for in vivo sensors, textiles such as hospital drapes, gowns or bedding, ventilation ducts, door handles, separation devices such as microbial suspensions, biomolecular separation, proteins It is characterized for use in membranes for fractionation, cell separation, wastewater treatment, water purification, bioreactors, or food processing.

According to the second embodiment,

The present invention also intends to provide a method for preparing a method for preparing a biocompatible coating material having the antibacterial, antifouling and antithrombotic effect.

In the method for preparing a coating material having antibacterial, antifouling and antithrombotic effects according to the present invention, the method comprises:

(a) Ferrocenylmethyl methacrylate (FMMA), poly(ethylene glycol) methacrylate (PEGMA) and methacrylate (Methacrylic acid, MA) 1: 5-10 : preparing a poly(FMMA-r-PEGMA-r-MA) polymer solution by dissolving in a solvent in a molar ratio of 5-20;

(b) Ferrocenylmethyl methacrylate (FMMA), poly(ethylene glycol) methacrylate (PEGMA) and 2-(dimethylamino)ethyl methacrylate (2-(Dimethylamino) ) ethyl methacrylate, DMAEMA) is dissolved in a solvent in a molar ratio of 1: 5-10: 5-20 to prepare a poly (FMMA-r-PEGMA-r-DMAEMA) polymer solution; and

(c) the poly(FMMA-r-PEGMA-r-MA) polymer solution and the poly(FMMA-r-PEGMA-r-DMAEMA) polymer solution were sequentially prepared by layer-by-layer (LbL) It characterized in that it comprises the step of manufacturing a coating material composed of a double layer by laminating. In the step (c), the stacking sequence is a poly(FMMA-r-PEGMA-r-MA) polymer solution first stacked according to the stacking target, and then a poly(FMMA-r-PEGMA-r-DMAEMA) polymer solution stacked. or vice versa may be used.

In the method for producing a coating material having antibacterial, antifouling and antithrombotic effects according to the present invention, the poly(FMMA-r-PEGMA-r-MA) polymer solution and poly(FMMA-r-PEGMA-r-DMAEMA) ) The polymer solution is characterized in that it is sequentially laminated by layer-by-layer (LbL) and consists of 2 to 10, or preferably 2.5 to 6, double layers.

In the method for manufacturing a coating material having antibacterial, antifouling and antithrombotic effects according to the present invention, the layered self-assembly method comprises an immersion, spray or spin coating method. do it with

In the method for preparing a coating material having antibacterial, antifouling and antithrombotic effects according to the present invention, the solvent is THF, xylene, toluene, methylene chloride, CH ₃ OH, CH ₃ CH ₂ OH, CH ₃ CH ₂ CH ₂ OH, hexane, ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, butylene glycol, diethylene glycol monomethyl ether, diethylene glycol monobutyl ether, propylene glycol monomethyl ether or DMSO is characterized.

According to the third embodiment,

An object of the present invention is to provide a multilayer film comprising a substrate and a biocompatible coating material having antibacterial, antifouling and antithrombotic effects deposited on the substrate.

In the multilayer film comprising a biocompatible coating material having antibacterial, antifouling and antithrombotic effects according to the present invention, the substrate is silicone, polyurethane (PU), Teflon (PTFE, Teflon), Polyvinyl chloride (PVC), polystyrene (polystyrene), nylon (Nylon), polyethylene terephthalate (PET), polyacrylate (polyacrylate), polypropylene (PP) and polyethylene (PE), poly Thermosetting selected from the group consisting of a thermoplastic polymer selected from the group consisting of carbonate (PC), polyether ether ketone (PEEK), and polysulfone (PS), and phenol resin (Phenol) and epoxy resin polymers, non-degradable synthetic polymers and natural rubbers; Polyglycolide (PGA), polylactide (PLLA), polycaprolactone (PCL), PLGA, PLCL, polydioxanone (PDO), polytrimethylene carbonate (PTMC), polyanhydride, polyorthoester, a degradable polymer selected from the group consisting of polyphospha and copolymers thereof; Natural polymers selected from the group consisting of hyaluronic acid, alginic acid, chitosan, collagen, gelatin and polyamino acids; stainless steel (SS), titanium (Ti), zirconium (Zr), cobalt-chromium (Co-Cr), platinum-chromium, tantalum, titanium, nitinol, gold, platinum, silver, magnesium, iron and alloys thereof. a metal selected from the group; Hydroxypapatite (HA) or beta-tricalcium phosphate (b-TCP) is characterized in that it is selected from the group consisting of ceramics and complexes thereof.

According to the fourth embodiment,

An object of the present invention is to provide a medical device including the biocompatible coating material having the antibacterial, antifouling and antithrombotic effects.

The medical device may be a textile, a surgical device, a medical or dental device, a blood oxygenator, a ventilator, a pump, a drug delivery device, tubing, wiring, an electrode, a contraceptive device, a feminine hygiene product, an endoscope. , grafts, stents, stent grafts, pacemakers, implantable cardioverter/defibrillators, devices for cardiac resynchronization therapy, cardiovascular device leads, ventricular assist devices and drivelines, heart valves, vena cava Filters, endovascular coils, catheters, catheter connectors and valves, intravenous delivery lines and manifolds, shunts, wound drains, dialysis membranes, infusion ports, cochlear implants, endotracheal tubes , tracheostomy tubes, ventilator breathing tubes and circuits, guide cores, fluid collection bags, drug delivery bags and tubing, implantable sensors, ophthalmic devices, orthopedic devices, dental implants, periodontal Implants, breast implants, penile implants, maxillofacial implants, cosmetic implants, valves, appliances, supports, suture materials, needles, hernia treatment meshes, tension-free vaginal tapes and vaginal slings, neural prosthetic devices, tissue regeneration or a cell culture device.

In the medical device according to the present invention, the medical device is characterized in that it comprises a lumen, a cavity, a porous structure, or a combination thereof.

In the medical device according to the present invention, the medical device is a peripherally inserted central venous catheter (PICC), a central venous catheter (CVC), or a vascular insertion catheter selected from the group consisting of a hemodialysis catheter. characterized by being.

<Example>

Example 1. Synthesis of functional ferrocene polymer

Two functional ferrocene polymers (C series ferrocene polymer: poly(FMMA-r-PEGMA-r-MA) and N series ferrocene polymer: poly(FMMA-r-PEGMA-r-DMAEMA)) were synthesized using radical polymerization method. did. First, PEGMA, MA and DMAEMA were passed through an inhibitor-removal column. FMMA (0.4 mmol, 0.114 g), PEGMA (3 mmol, 1.425 g) and MA (3 mmol, 0.258 g) for C series ferrocene polymers or DMAEMA (3 mmol, 0.472 g) for N series ferrocene polymers are listed in Table 1 below. It was dissolved in THF (9 mL) according to the indicated molar ratios. AIBN (0.12 mmol, 0.02 g) was added as a radical initiator, and the resulting mixture was bubbled with a vapor of Ar gas and then sealed with Teflon tape to degas for 5 minutes. The polymerization reaction was carried out at 70° C. with stirring for 24 hours, and the resulting polymer solution (concentration; 10%) was cooled to 25° C. and stored at 4° C. before use.

division FMMA PEGMA MA EMAEMA Poly C series Poly C1 0.4 3 3 - Poly C2 0.4 3 5 - Poly C3 0.4 3 7 - Poly N
series Poly N1 0.4 3 - 3 Poly N2 0.4 3 - 5 Poly N3 0.4 3 - 7

Example 2. Preparation of multilayer film by LBL assembly using ferrocene polymer

Using the functional ferrocene polymer prepared in Example 1, a multilayer film was prepared on glass by the LBL assembly method. First, the surface of the glass substrate is treated with oxygen plasma for 5 minutes so that it becomes negatively charged, and the glass substrate is subjected to 1.0 mg/mL of each ferrocene polymer solution prepared in Example 1, for example, a Poly N series solution at 25°C. After repeating the LBL method of immersion for 10 minutes at a temperature of 10 minutes, then rinsing with water 3 times and immersing in Poly C series solution at 25° C. for 10 minutes, 1 to 10 times, and then drying in air to form 1 to 10 layers of LBL coating expressed as follows. did. At this time, the pH of the ferrocene polymer immersion solution was fixed to 7.4.

(Poly Ns/Cs)n (where n represents 1 to 10 immersion cycles)

Example 3. Confirmation of surface properties of multilayer LbL film using ferrocene polymer

Among the multilayer films prepared under a series of ferrocene polymers according to Example 1, the thickness of (Poly Ns/Cs) ₁₀ was measured with a contact surface profiler (alpha step, USA), and a zeta potentiometer (ELS-Z2, Japan) was used. was used to obtain the zeta potential value. Fourier transform infrared transmission spectrum was analyzed by ATR-FTIR (Cary 630, USA).

As a result, in the case of the (Poly N1/C1) ₁₀ film, it was formed with the thinnest thickness of 15±2 nm, and the bonding of the film is not strong due to the weak electrostatic attraction, so it can be decomposed during the washing process, and (Poly N3/C3) ₁₀ In the case of the film, it was confirmed that there is a possibility that the growth of the film may be hindered due to the electrostatic repulsion effect during film formation due to the strong ionic strength. On the other hand, the thickness of the (Poly N2/C2) ₁₀ film was about 130 ± 17 nm, which was formed as the thickest film, confirming that (Poly N2/C2) _n was optimized for the multilayer film (FIG. 2).

In addition, as a result of measuring the zeta potential after alternately depositing the Poly N series and Poly C series, it was confirmed that the LbL film was successfully formed by the layer-by-layer assembly method by changing the charge in each step (Fig. 3A), (Poly As a result of measuring the coating thickness as a function of N2/C2) _n its double layer, the thickness of the multilayer film increased as the number of depositions increased, confirming that the Poly N series and Poly C series were successfully deposited (Fig. 3B-C). ). In addition, as a result of cross-sectional FE-SEM image analysis, (Poly N2/C2) ₁₀ films were deposited to about 132 nm, and these results were consistent with the thickness results of the contact surface profiler ( FIG. 3D ).

<Experimental example>

Experimental Example 1. Anti-biofilm effect of multilayer LbL film using ferrocene polymer against pathogenic bacteria

1-1. antibacterial effect

To evaluate the antifouling properties of the (Poly N2/C2) _n LBL film prepared in Example 2, first, pathogenic bacterial strains Staphylococcus aureus and Escherichia coli were cultured in LB medium at 37° C. for 24 hours with shaking. . Dilute exponentially growing bacterial cultures (1:100) in fresh LB medium containing 0.5% glucose and aliquots (1 mL) of the diluted bacterial cultures containing LBL film pieces ( ^0.5x0.5 cm2). It was added to the wells of a 24-well plate and subjected to static incubation at 37°C for 24 hours. The medium was removed and the adherent bacterial cells were washed 3 times with sterile PBS, and then the remaining adherent cells were further incubated at 60 °C for 10 min and stained with 0.1% crystal violet (CV) for 15 min at room temperature. Excess CV staining was removed by washing the cells with sterile PBS. For CV staining, ethanol:acetone (95:5, v/v; 0.5 mL) was added to each well to dissolve CV staining and absorbance was measured at 562 nm. The antibacterial ability of the LBL film against pathogenic bacteria was observed using the Live/Dead BacLight Bacterial viability kit.

As a result, in the case of the (Poly N2/C2) _n LBL film according to the present invention, the number of bacterial cells was significantly reduced, and in particular, it was confirmed that the number of bacterial cells was significantly reduced when it was prepared with 6 or more double-layer structures. (Fig. 4A). In addition, in the case of the bare glass substrate, bacterial aggregates or biofilms were observed, whereas in the case of the LBL film coated with the functional ferrocene polymer according to the present invention, it was confirmed that bacterial colonization was hardly observed (FIG. 4B,D,F).

1-2. Anti-fouling effect

The live and dead bacteria of the biofilm were stained with a mixture (1:1) of fluorescent Syto9 (green, live stain) and propidium iodide nucleic acid (red, dead stain) dyes (1:1) for 15 min, and using a fluorescence microscope (Poly N2 /C2) We observed the antibacterial effect of _n LBL films. Samples for scanning electron microscopy (SEM) observation were prepared by air drying and platinum coating.

As a result, it was confirmed that live bacteria were observed in the case of the bare glass substrate, whereas almost no bacteria were observed in the (Poly N2/C2) _n LBL film according to the present invention. On the other hand, compared to the (Poly N2/C2) ₂ LBL film layer, the (Poly N2/C2) _2.5 LBL film layer showed more inhibition of bacterial adsorption, and the (Poly N2/C2) LBL film layer exceeding ₆ In the case of (Poly N2/C2), it was confirmed that there was no significant effect change according to the increase of the layer (FIG. 4C).

Example 5. Preparation of multilayer LbL film using drug-loaded ferrocene polymer and analysis of drug release

NR as a drug model and PTX as a therapeutic drug for cancer treatment were used. First, in order to incorporate the drug into the multilayer film, each drug (500 μg/mL in THF) was mixed with C and N series ferrocene polymer solutions (1 mg/mL in THF) and then reacted with each other under rotational stirring for 1 hour at room temperature. . The THF of the drug-interacting polymer solution was evaporated and then dissolved in DIW according to a specific concentration. Additional drugs were removed through a dialysis tube, and a film was prepared in the same manner as in the existing LBL multilayer thin film manufacturing method. (Fig. 5A). The drug-loaded multilayer film was immersed in 2 mL of 1xPBS under various conditions and placed in an incubator at 37°C. At the determined time point, 1 mL of each drug containing PBS was collected and 1 mL of fresh PBS was added. To confirm the loading of NRs on the multilayer film, a fluorescence microscope (Eclipse NI-Uo, Japan) equipped with a 40x objective lens and a general filter optimized for red fluorescent dye was used. The release of NR as determined by fluorescence was evaluated by immersing (Poly N2/C2) ₁₀ films in PBS buffer under different pH and H ₂ O ₂ presence conditions to confirm the pH and ROS-responsive release profiles of LBL films. The amounts of NR and PTX emitted from each multilayer film were measured using a fluorescent plate reader (VICTOR X5, USA) and high performance liquid chromatography (HPLC, USA). The drug release profile was calculated by dividing the amount of drug released at the determined time points different by the amount of drug release over 72 hours.

As a result, for 72 hours (Poly N2/C2) in 0.1% H ₂ O ₂ at pH7.4, pH7.4, and 0.1% H ₂ O ₂ at pH2 (Poly N2/C2) for ₁₀ LBL films, The amounts were identified as 4.2±0.4, 11.0±1.1, 20.5±0.9 and 26.1±1.7 ng/mL, respectively ( FIG. 5B ). In addition, the thickness of the LBL film was decreased by 5-8%, 16-23%, and 22-24% in DI water, pH 7.4 PBS, and 0.1% H ₂ O ₂ in PBS pH 7.4, whereas 0.1 at pH 2 and pH 2 In the case of % H ₂ O ₂ , the thickness of the LBL was decreased by 81 to 83% and 85 to 88%, respectively. Therefore, it was confirmed that the (Poly N2/C2) ₁₀ LBL film according to the present invention was well prepared for stimulatory drug release while maintaining pH 7.4 and ROS environment.

On the other hand, PTX-loaded (Poly N2/C2) ₁₀ film release occurred faster in PBS with pH 2 or pH 5.5 compared to pH 7.4, and the presence of 0.1% H ₂ O ₂ , regardless of the pH value, slowed the drug release rate. improved dramatically. Therefore, it was confirmed that PTX-loaded Poly N2/C2) ₁₀ had a desirable pH/ROS dual reactive drug release ability ( FIG. 6A ).

Example 6. Evaluation of toxicity and anti-tumor effect of multilayer LbL film using ferrocene polymer

Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Human breast cancer cells MCF-7 were used as a cancer model. The breast cancer cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin, and maintained in an incubator at 37° C. in a 5% CO ₂ atmosphere. Cells were seeded in 96-well plates (1×10 ⁴ cells/well) and incubated overnight. Then, PTX, PTX-loaded (Poly N2/C2) _n LBL film or blank (Poly N2/C2) _n LBL film was added to the cells and incubated for 24 hours. The medium was replaced with RPMI containing MTT (1 mg/mL) and the cells were incubated for an additional 2 hours. Finally, the medium was replaced with DMSO to dissolve the formazan crystals. Absorbance was detected at 570 nm using a microplate reader (Bio Tek, USA). Cell viability was expressed as a percentage using the following equation:

A570 in drug-treated cells/A570 in untreated cells × 100

All experiments were performed in triplicate, and data are presented as mean±standard deviation.

As a result, it was confirmed that the film according to the present invention was not cytotoxic as cell viability did not decrease in all multilayer films that were not loaded with PTX. On the other hand, in the case of PTX loading, the more the PTX-loaded polymer multilayer thin film was piled up, the lower the cell viability was, and the anticancer effect of the PTX-loaded multilayer thin film was consistent with the amount released in the environment of 0.1% H2O2 at pH 5.5. appeared (Fig. 6B). Therefore, it was demonstrated that the LBL film according to the present invention is a good drug delivery system for PTX and can realize fine-tuned therapeutic efficacy as the drug loading and release kinetics can be easily controlled.

Example 7. Evaluation of biocompatibility and blood component adsorption for multilayer LbL film using ferrocene polymer

Uncoated and coated (Poly N2/C2) _n LBL film samples (0.5x0.5 cm ² in diluted blood containing 5% fresh anticoagulated blood and physiological saline) were incubated at 37 °C for 3 hours to form a multilayer film. Hemolysis rates were assessed. The analysis was performed according to standard procedures related to the evaluation of hemolytic properties of substances (ASTM F756-17, 2017). Normal saline and distilled water were used as negative and positive controls, respectively. After centrifugation at 1000xg for 10 minutes, the absorbance of the supernatant was recorded at 540 nm. Hemolysis rate (HR) was calculated according to the following equation.

(Wherein, A ₁ , A ₂ and A ₃ represent the absorbance of the sample, the positive control and the negative control, respectively)

As a result, when the (Poly N2/C2) _n LBL film according to the present invention was coated, it exhibited low hemolytic activity (hemolysis rate <2%) and was confirmed to have excellent blood compatibility ( FIG. 7A ).

Fresh blood was collected from healthy rabbits according to the guidelines of the Ethics Committee of the Korean Academy of Sciences, and the blood was centrifuged at 1000 rpm for 15 minutes to obtain platelet-rich plasma (PRP). After immersing the unmodified (Poly N2/C2) ₁₀ LBL film substrate in PBS solution (pH 7.4) for 24 hours, 60 uL of fresh PRP was dropped into the center of the sample and incubated at 37° C. for 1 hour. Rinse the substrate with PBS and immediately immerse in formaldehyde solution (2%) for 24 h to fix adherent platelets and use a series of ethanol solutions (50%, 60%, 70%, 80%, 90%, and 100%). was dehydrated. Dehydrated samples were air dried and platinum coated for 40 s prior to SEM imaging.

As a result, in the case of bare glass, a thrombus mass was formed on the surface (Fig. 7B), whereas when coated with the (Poly N2/C2) ₁₀ LBL film according to the present invention, the amount of platelets was greatly reduced and it was confirmed that it was hardly visible ( 7C).

The examples were looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (10)

(A) poly(FMMA) containing ferrocenylmethyl methacrylate (FMMA), poly(ethylene glycol) methacrylate (PEGMA) and methacrylic acid (MA) -r-PEGMA-r-MA) polymer; and
(B) Ferrocenylmethyl methacrylate (FMMA), poly(ethylene glycol) methacrylate (PEGMA) and 2-(dimethylamino)ethyl methacrylate (2-(Dimethylamino) ) containing a bilayer composed of a poly (FMMA-r-PEGMA-r-DMAEMA) polymer containing ethyl methacrylate, DMAEMA)
A biocompatible coating material with antibacterial, antifouling and antithrombotic effects.
The method of claim 1,
The molar ratio of ferrocenylmethyl methacrylate, poly(ethylene glycol) methacrylate, and methacrylate or 2-(dimethylamino)ethyl methacrylate is 1: 5-10: 5-20 sign,
A biocompatible coating material with antibacterial, antifouling and antithrombotic effects.

According to claim 1,
The coating material comprises the poly(FMMA-r-PEGMA-r-MA) polymer and the poly(FMMA-r-PEGMA-r-DMAEMA) polymer, or the poly(FMMA-r-PEGMA-r-DMAEMA) polymer and poly( FMMA-r-PEGMA-r-MA) polymer is sequentially laminated by layer-by-layer (LbL), characterized in that it is composed of 2 to 10 double layers,
A biocompatible coating material with antibacterial, antifouling and antithrombotic effects.
4. The method of claim 3,
said poly(FMMA-r-PEGMA-r-MA) polymer and poly(FMMA-r-PEGMA-r-DMAEMA) polymer, or poly(FMMA-r-PEGMA-r-DMAEMA) polymer and poly(FMMA-r- PEGMA-r-MA) polymer is sequentially laminated by layer-by-layer (LbL), characterized in that it consists of 2.5 to 6 bilayers,
A biocompatible coating material with antibacterial, antifouling and antithrombotic effects.
According to claim 1,
The antifouling is Pseudomonas aeruginosa, Salmonella spp, Shigella spp, Vibrio parahaemolyticus, Vibrio choreae, E. coli O-157 (Escherichia coli O157) Campylobacter jejuni, Clostridium difficile, Clostridium perfringens, Yersinia enterocolitica, Helicobacter pylori, Entemoeba histolytica, Bacillusu cereus Clostridium botulinum, Haemophilus influenzae, Streptococcus pneumoniae, Chlamidia pneumoniae, Legionella pneumoniae, Branhamella catarrhalis, Mycobacterium tuberculosis Mycoplasma pneumoniae, Storeptcoccus pyogenes, Corynebacterium diphtheriae, Bordetella pertussis, Chramidia psittaci, methicillin resistant Staphylococcus a , Escherichia coli, Klebsiella pneumoniae, Enterobacter spp, Proteus spp, Acinetobacter spp, Enterococcus faecalis, Staphy lococcus saprophyticus), characterized in that it is formed by one or more types of bacteria selected from the group consisting of B type (Storeptcoccus agalactiae),
A biocompatible coating material with antibacterial, antifouling and antithrombotic effects.
A method for preparing a biocompatible coating material having an antibacterial, antifouling and antithrombotic effect according to any one of claims 1 to 5, the method comprising:
(a) Ferrocenylmethyl methacrylate (FMMA), poly(ethylene glycol) methacrylate (PEGMA) and methacrylate (Methacrylic acid, MA) 1: 5-10 : preparing a poly(FMMA-r-PEGMA-r-MA) polymer solution by dissolving in a solvent in a molar ratio of 5-20;
(b) Ferrocenylmethyl methacrylate (FMMA), poly(ethylene glycol) methacrylate (PEGMA) and 2-(dimethylamino)ethyl methacrylate (2-(Dimethylamino) ) ethyl methacrylate, DMAEMA) is dissolved in a solvent in a molar ratio of 1: 5-10: 5-20 to prepare a poly (FMMA-r-PEGMA-r-DMAEMA) polymer solution; and
(c) said poly(FMMA-r-PEGMA-r-MA) polymer solution and poly(FMMA-r-PEGMA-r-DMAEMA) polymer solution, or poly(FMMA-r-PEGMA-r-DMAEMA) polymer solution and A method comprising the step of sequentially stacking a poly (FMMA-r-PEGMA-r-MA) polymer solution by layer-by-layer (LbL) to prepare a coating material composed of a double layer.
7. The method of claim 6,
The coating material comprises the poly(FMMA-r-PEGMA-r-MA) polymer and the poly(FMMA-r-PEGMA-r-DMAEMA) polymer, or the poly(FMMA-r-PEGMA-r-DMAEMA) polymer and poly( A method, characterized in that the FMMA-r-PEGMA-r-MA) polymer is sequentially laminated by layer-by-layer (LbL) and is composed of 2 to 10 double layers.
7. The method of claim 6,
The layered self-assembly method, characterized in that it comprises a immersion (immersion), spray (spray) or spin coating (spin coating) method, the method.
Board;
A multilayer film comprising a biocompatible coating material having an antibacterial, antifouling and antithrombotic effect according to any one of claims 1 to 5 deposited on the substrate,
The substrate is silicon (silicone), polyurethane (polyurethane, PU), Teflon (PTFE, Teflon), polyvinyl chloride (polyvinyl chloride, PVC), polystyrene (polystyrene), nylon (Nylon), polyethylene terephthalate (PET), Select from the group consisting of polyacrylate, polypropylene (PP) and polyethylene (PE), polycarbonate (PC), polyether ether ketone (PEEK), and polysulfone (PS) Non-degradable synthetic polymers and natural rubbers of thermosetting polymers selected from the group consisting of thermoplastic polymers and phenolic resins and epoxy resins; Polyglycolide (PGA), polylactide (PLLA), polycaprolactone (PCL), PLGA, PLCL, polydioxanone (PDO), polytrimethylene carbonate (PTMC), polyanhydride, polyorthoester, a degradable polymer selected from the group consisting of polyphospha and copolymers thereof; Natural polymers selected from the group consisting of hyaluronic acid, alginic acid, chitosan, collagen, gelatin and polyamino acids; Stainless steel (SS), titanium (Ti), zirconium (Zr), cobalt-chromium (Co-Cr), platinum-chromium, tantalum, titanium, nitinol, gold, platinum, silver, magnesium, iron and alloys thereof a metal selected from the group; A multilayer film, characterized in that it is selected from the group consisting of hydroxyapatite (HA) or beta-tricalcium phosphate (b-TCP) ceramics and composites thereof.
A medical device comprising a biocompatible coating material having an antibacterial, antifouling and antithrombotic effect according to any one of claims 1 to 5,
The medical device may be a textile, a surgical device, a medical or dental device, a blood oxygenator, a ventilator, a pump, a drug delivery device, tubing, wiring, an electrode, a contraceptive device, a feminine hygiene product, an endoscope. , grafts, stents, stent grafts, pacemakers, implantable cardioverter/defibrillators, devices for cardiac resynchronization therapy, cardiovascular device leads, ventricular assist devices and drivelines, heart valves, vena cava Filters, endovascular coils, catheters, catheter connectors and valves, intravenous delivery lines and manifolds, shunts, wound drains, dialysis membranes, infusion ports, cochlear implants, endotracheal tubes , tracheostomy tubes, ventilator breathing tubes and circuits, guide cores, fluid collection bags, drug delivery bags and tubing, implantable sensors, ophthalmic devices, orthopedic devices, dental implants, periodontal Implants, breast implants, penile implants, maxillofacial implants, cosmetic implants, valves, appliances, supports, suture materials, needles, hernia treatment meshes, tension-free vaginal tapes and vaginal slings, neural prosthetic devices, tissue regeneration or a cell culture device.

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