Improved wound care device

Abstract

To provide an antimicrobial amphiphilic hydrogel composition to be used for stopping bleeding from a wound.SOLUTION: An antimicrobial amphiphilic hydrogel composition contains a first crosslinkable amphiphilic component and an aqueous medium, the first crosslinkable amphiphilic component is a lyotropic liquid crystal in its chemically crosslinked state, and has an ordered nanostructure of a hydrophobic domain and a hydrophilic domain, the first crosslinkable amphiphilic component is a poloxamer triblock copolymer containing polyethylene oxide (PEO)-polypropylene oxide (PPO)-polyethylene oxide (PEO), the PEO forms a hydrophilic domain of amphiphilic hydrogel, the PPO forms a hydrophobic domain of amphiphilic hydrogel, the hydrogel contains an antimicrobial agent covalently bonded to the hydrophilic domain and / or the hydrophobic domain, the antimicrobial agent is an antimicrobial peptide, and the antimicrobial peptide is RRPRPRPRPWWWW-NH2.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to antimicrobial hydrogels. Specifically, the present disclosure relates to antimicrobial hydrogels for use in wound hemostasis. The present invention relates to an antibacterial amphiphilic hydrogel composition for the treatment of ulcers and ulcers. The antibacterial hydrogel is provided as a dispersion. That's fine. [Background technology]

[0002] Wound infections that involve the skin or tissues near the wound can interfere with the healing process and lead to systemic illness. Today, antibiotic therapy is the most common treatment for treating wound infections. For many years, such antibiotic therapy routines have caused systemic side effects in patients. Not only has it been shown to cause severe illness caused by antibiotic-resistant bacteria, It is causing a rapid increase in infections.

[0003] Hemostasis or hemostasis is the body's process for preventing and stopping bleeding. Involved in blood clotting and the formation of clots to stop bleeding. In wound care, microfibrils It is known to use microfibrillated collagen hemostatic agents to promote clot formation. Brillar collagen is available as sheets, powder, and sponges.

[0004] Mepilex® or Mepilex-Ag® (Molnlyc Commercially available wound dressings, such as those sold by Ke Health Care, contain silver as an antimicrobial agent. The silver is released into the wound, damaging the cell walls or or kill microorganisms by inhibiting their reproduction. Many other wound dressings incorporate antimicrobial molecules or traditional antibiotic drugs such as penicillin. It is used to prevent bacterial adhesion or infection at the wound site. The compound is characterized by its limited spectrum of activity, cytotoxicity to human cells, and short-term Its use is limited due to the potential for the development of antimicrobial resistance. The release of silver from these materials has harmful environmental effects.

[0005] Generally, the above-mentioned blood collecting devices may have antibacterial properties or may simply collect or draw up blood. Such a wound care device.

[0006] Patent Document 1 (AMFERIA AB) April 18, 2019, describes an antibacterial amphiphilic hydrogel. The hydrogel of Patent Document 1 is particularly useful in treating antibiotic-resistant infections. It is disclosed as a solid, monolithic material suitable for use as an antimicrobial wound care device. The document does not relate to hemostasis or bleeding wounds. may improve wound care alternatives for medical personnel and patients.

[0007] A device that combined antibacterial and hemostatic effects would be advantageous. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2019 / 074422A1 [Non-patent literature]

[0009] [Non-Patent Document 1] “Alternative drinking-water disinfectants: bromine, iodine and silver.” World Health Organization; 2018, “Based on the lowest median L(E)C50 value of the key environmental organisms both silver salt and silver nanoparticles would be classified as´´very toxic to aquatic organisms´´under EU Directive 93 / 67 / EEC (CEC, 1996)” Summary of the Invention

[0010] Therefore, the present invention preferably provides, alone or in any combination, The present invention seeks to reduce, alleviate, or eliminate one or more of the above-identified shortcomings and provides for wound hemostasis as follows: By providing an antimicrobial amphiphilic hydrogel composition for use in at least one of the above The present invention provides an antibacterial amphiphilic hydrogel composition for use in wound hemostasis. and a first cross-linkable amphiphilic component, the first amphiphilic component being capable of cross-linking the chemically cross-linked In this state, it is a lyotropic liquid crystal, and the order of the hydrophobic and hydrophilic domains is The hydrogel has a nanostructure and is covalently bonded to hydrophilic and / or hydrophobic domains. Antimicrobial amphiphilic hydrogel compositions containing antimicrobial agents. Surprisingly, the compositions exhibit hemostatic activity. It has been shown to have beneficial effects.

[0011] In contrast to other antibacterial hemostatic devices, amphiphilic hydrogels are non-biodegradable and antibacterial. The agent is covalently bound to the hydrogel, which allows the hydrogel and / or the antimicrobial agent to penetrate into the wound. This means that the collagen is stable over a long period of time and does not release any collagen. Rather than modifying the device, the present inventors have developed a compound that appears to combine antibacterial and hemostatic effects. Furthermore, by using an antibacterial amphiphilic material, this material is Absorbs both hydrophobic and hydrophilic substances, such as bacterial toxins released by bacteria that infect The small repeating domains also allow for a higher density of antibacterial agents to be attached to the hydrogen bonds. Compared with conventional antibacterial hemostatic devices, this amphiphilic The hydrogel is capable of swelling significantly from a wet state, thus preventing blood exudates and blood can be absorbed into the hydrogel.

[0012] Furthermore, the antibacterial agent may be an antibacterial peptide having 90-95% identity with RRPRPRPRP. Which, proline arginine-rich end leucine-rich repeat protein (proline-arginine-rich terminal leucine-rich repeat Antimicrobial peptide derived from PRELP for use in wound hemostasis Such proteins may have various properties, such as net charge and antimicrobial efficacy. In some respects, it has been shown to be similar to the human cathelicidin-derived LL-37. (Malmsten, M et al., “Highly Selective End-T  Antimicrobial Peptides Derived from PRELP”,PLoS ONE,2011,6(1):e16400.doi:10. 1371 / journal.pone.0016400). However, LL-37 It has previously been shown not to affect plasma coagulation (Harm, S et al., " Blood Compatibility-An Important but Often Forgotten Aspect of the Characterization f Antimicrobial Peptides for Clinical Appl. ication”, Int.J.Mol.Sci.,2019, 20,5426;doi :10.3390 / ijms20215426).

[0013] An antimicrobial hydrogel dispersion is also provided. The antimicrobial hydrogel dispersion comprises a first crosslinkable amphiphile. the first amphiphilic component in its chemically crosslinked state is a lyotropic component; It is a picric liquid crystal and has an ordered nanostructure of hydrophobic and hydrophilic domains. The rogels contain antimicrobial agents covalently bound to hydrophilic and / or hydrophobic domains. The component is present in the composition in the form of particles in a dispersion.

[0014] The particle dispersions can be used to improve wound coverage in irregularly shaped wounds and to bind antimicrobial agents. These include a larger surface area available for application, easier and faster application compared to traditional solid hydrogels, and The particles have further been shown to maintain their antimicrobial function in particulate form. The particles absorb both aqueous and non-aqueous solutions.

[0015] A method for preparing a dispersion comprising antimicrobial amphiphilic hydrogel particles is provided.

[0016] Also provided are devices having a coating of a dispersion of antimicrobial amphiphilic hydrogel particles. Due to the sprayable nature of the particle dispersion, coatings for use on the human body or body fluids are The design of the device is simplified.

[0017] The use of dispersions is also provided.

[0018] Additionally, a method of treating a wound is provided.

[0019] For wound hemostasis, RRP9W4, which is 90-95% identical to RRPRPRPRP, Proline arginine-rich end-chain peptides, such as antimicrobial peptides with The use of antimicrobial peptides derived from leucine-rich repeat proteins has been proposed. It is served.

[0020] Further advantageous embodiments are disclosed in the accompanying and dependent claims. It has been done. [Brief explanation of the drawings]

[0021] These and other aspects, features and advantages of the present invention will become apparent from and be better understood with reference to the accompanying drawings, in which: This will be apparent and elucidated from the following description of embodiments of the invention. [Figure 1] 1 is a synthesis scheme for diacrylate-modified Pluronic® triblock copolymers, where X and Y refer to the number of PEO and PPO groups. [Figure 2] 1 shows the reaction scheme for covalent attachment of antimicrobial peptides to diacrylate-modified Pluronic triblock copolymer F-127 via EDC / NHS activation. [Figure 3]Figure 3 shows zone inhibition tests of antimicrobial hydrogels versus control samples. Figure 3a shows a negative control amphiphilic hydrogel containing AMP; Figure 3b shows an amphiphilic hydrogel containing only physically absorbed AMP; and Figure 3c shows an amphiphilic antimicrobial hydrogel in which AMP is covalently bound to the amphiphilic hydrogel. The zone of inhibition is the darker area and can be seen to extend beyond the area of ​​the hydrogel (center circle), while Figure 3c shows the zone of inhibition directly beneath the hydrogel, indicating that the AMP has not leached out of the hydrogel. [Figure 4] Figure 1 shows a schematic of AMPs covalently attached to a chemically crosslinked amphiphilic hydrogel with repeatedly 3D printed, aligned regular hexagonal ordered nanostructures. [Figure 5] The results of a storage stability test in phosphate buffered saline (PBS) are shown. Figure 5a shows the percentage of dead cells (S. aureus) for the control amphiphilic hydrogel and the antibacterial amphiphilic hydrogel. Figure 5b shows the total surface coverage observed on the hydrogel. An asterisk (*) indicates a significant difference compared to the control sample at the 95% confidence level. [Figure 6] Results of a serum stability study in which hydrogels were exposed to 20% human serum are shown. Hydrogels were removed from the serum at the times indicated on the x-axis. The percentage of dead cells (S. aureus) on the surface of the hydrogel was determined by live / dead staining and is shown on the y-axis. At each time point except day 5, there was a significant difference at the 95% confidence level between the activated surface compared to the control. Each bar represents a collection of images taken of four samples. [Figure 7] Results of a blood clotting experiment (Experiment 1) are shown. The figure shows the platelet counts after 60 minutes of whole blood incubation at 37°C for two donors: initial blood, blood incubated without contact with hydrogel, blood contacted with amphiphilic hydrogel, and blood incubated with antimicrobial amphiphilic hydrogel. Data represent the mean ± standard error of the mean for n=4. [Figure 8] Control and AMP-activated particles are shown, plated on an agar plate streaked with S. aureus and incubated overnight (approximately 15 hours). Area A is the control area (hydrogel without AMP), and area B is the AMP-activated hydrogel particles. [Figure 9] 1 shows a schematic diagram of a spray device according to one embodiment. [Figure 10] Images of a blood clotting test (Experiment 1) using human blood on amphiphilic hydrogels with and without AMP are shown. From left to right: Donor 1's blood on the hydrogel without AMP, Donor 1's blood on the hydrogel with AMP, Donor 2's blood on the hydrogel without AMP, and Donor 2's blood on the hydrogel with AMP. Clotted blood is clearly visible on the two hydrogels containing the AMP sample. DETAILED DESCRIPTION OF THE INVENTION

[0022] The following description of the invention is directed to antimicrobial amphiphilic hydrogels, antimicrobial amphiphilic hydrogels in wound care, The use of antibacterial amphiphilic hydrogels and compositions containing such antibacterial amphiphilic hydrogels are described. The hydrogel comprises a first cross-linkable amphiphilic component. In its cross-linked state, the amphiphilic component: The result is a hydrogel containing an ordered structure of hydrophilic and hydrophobic domains. The gel contains antibacterial agents covalently bound to repeating hydrophilic and / or hydrophobic domains of the cross-linked hydrogel. Further details regarding the preparation of amphiphilic hydrogels and their advantages can be found in WO201 9 / 074422A1.

[0023] The repeat-ordered nanostructure of amphiphilic hydrogels consists of repeating and alternating hydrophobic-hydrophilic domains. The morphology and specific structure of the hydrophobic-hydrophilic domains are discussed below. The hydrogels are composed of ordered repeating molecules throughout the hydrogel, i.e., not just on the surface of the hydrogel. Cross-linked hydrogels are solids. Intermolecular cross-links irreversibly lock the ordered structure. The resulting hydrogels have high integrity and are mechanically elastic.

[0024] The antibacterial hydrogel is an ordered repetitive delivery system that delivers antibacterial agents onto the skin or wound surface in an orderly and repetitive manner. Due to the repeating nanostructure, it is particularly suitable for wound care applications. The fixation provides better long-term performance.

[0025] The hydrogel can be thought of as forming a matrix onto which the antimicrobial agent can be immobilized. In its crosslinked state, the gel is self-supporting (self-supporting) and three-dimensional. Therefore, the hydrogel is substantially non-degradable under physiological conditions. The polymer is non-biodegradable and is chemically or biodegradable, and is not susceptible to the chemical or physical properties encountered in relevant in vitro and in vivo conditions. For example, the hydrogel is not substantially degraded by enzymatic conditions. Furthermore, hydrogels have relatively low or no bioavailability. is virtually non-degradable, i.e., remains stable and solid, in high pH environments. do.

[0026] The hydrogel may form a first substantially uniform layer to which the antimicrobial agent is covalently attached. The agent may be covalently bonded to at least the repeating hydrophilic domains of the hydrogel throughout the layer. These can be applied not only to the surface of the hydrogel but also within the layer. This is a significant improvement compared to techniques for surface modification of hydrogels or other substrates that only result in immobilization. The small repeating domains also allow for a higher density of antibacterial agents to be present on the surface of the hydrogel. This makes it possible to do so.

[0027] The repeating ordered structure of hydrophilic and hydrophobic domains is at least nanoscale. They are ordered and, as discussed further below, may be arranged into larger, micro- or macro-structures depending on the production technique. The term ordered repetition refers to a defined periodicity. The present invention relates to hydrogels based on carbohydrates, polysaccharides, or other non-amphiphilic molecules. In contrast to gels, the nanostructure of the hydrogels described herein is an ordered repeating nanostructure. The hydrogel has a structure that is not randomly crosslinked. The hydrogel is amphiphilic. After crosslinking, the amphiphilic The hydrogel is a chemically crosslinked amphiphilic hydrogel.

[0028] The ordered repeating nanostructures provide antibacterial agents covalently bound to the hydrogel with defined orientation. If the antimicrobial agent itself is amphiphilic, the antimicrobial agent will be absorbed on the surface of the hydrogel and into the hydrogel. The amphiphilic nature of the hydrogel also allows it to be more effectively immobilized in aqueous and non-aqueous solutions. allows for the absorption of both

[0029] As mentioned above, in contrast to hydrogels that have surface treatments to define the surface chemistry, The hydrogels of the present disclosure have repeating ordered nanostructures both on the surface and within the bulk of the hydrogel. This allows the immobilization of antibacterial agents both on the surface and inside the hydrogel. It will be improved.

[0030] Antibacterial hydrogels are made up of cross-linkable copolymers, cross-linkable surfactants, cross-linkable proteins, and cross-linkable They can be formed by chemical cross-linking of organic amphiphilic materials, such as peptides and cross-linkable lipids As used herein, crosslinking refers to the bonding of molecules together using reactive chemical groups present on the molecules. Chemical cross-linking processes involve the use of light, such as ultraviolet light, heat, or other chemicals, such as enzymes. The covalent crosslinking of hydrogels is irreversible. Covalent crosslinks do not decompose or disintegrate at high temperatures. Covalent crosslinks also do not disintegrate at high temperatures. It is also stable against movement.

[0031] The first amphiphilic component of the hydrogel can be a crosslinkable amphiphilic polymer. A suitable amphiphilic material is a diacrylate-modified poloxamer as described in the experimental section below. mers, such as polyethylene oxide-polypropylene oxide-polyethylene oxide (DA-PEO x -PPO y -PEO x -DA, where x and y are each an integer of 1 to 4, and The number of EO groups and PPO groups is referred to as the number of EO groups and PPO groups. Specifically, the amphiphilic material is an amphiphilic tri Block copolymer, polyethylene oxide (100)-polypropylene oxide (70 )-Polyethylene oxide (100) (Pluronic® F127-BAS F Corporation), polyethylene oxide (30)-polypropylene oxide Polyethylene oxide (70)-polyethylene oxide (30) (Pluronic® P123- BASF Corporation).

[0032] As mentioned above, the amphiphilic component is a diacrylate derivative of a triblock copolymer. The copolymer may then be chemically crosslinked. The process for the decoration is provided in the experimental section below. By reacting the polymer with acryloyl chloride to form a diacrylate derivative, Another method for forming crosslinkable amphiphilic polymers is to use methacrylate derivatives. Conductor formation or carboxyl-amine crosslinking may also be possible.

[0033] Crosslinkable amphiphilic polymers self-assemble in the presence of water to form lyotropic liquids. The ordered nanostructure called lyotropic liquid crystal (LLC) In its crosslinked form, i.e., after crosslinking, the hydrogel can be chemically crosslinked. The cross-linking of amphiphilic polymers can be considered as lyotropic liquid crystals (LLCs). Polymerized lyotropic liquid crystals with ll-defined structures It is thought that lyotropic liquid crystal (PLLC) is formed. obtain.

[0034] Non-solid cross-linked hydrogels are usually called micellar systems, where random cross-linking occurs throughout the hydrogel. The structure may be spherical micellar aggregates in the size range of 2 to 100 nm arranged in a matrix, Such a typical micelle hydrogel contains about 1% to about 19% (by weight) ) amphiphilic polymer, and about 99% to about 81% (by weight) water. The system does not form a cross-linked solid gel, but at amphiphilic polymer concentrations of 15-19% (wt%) In certain cases, such as in the range, this system exhibits very soft and flexible crosslinked mechanical properties. It can exist as a solid.

[0035] Hydrogels are known as ordinary micellar cubic systems, abbreviated as I1. , simple arrangement (P...) or body-centered (B...) arrangement of micellar structures in a cubic lattice or face-centered ( F...) arranged in a lyotropic liquid crystal, which is a cubic ordered array with an arrangement It may also have a structure of spherical micellar aggregates in the size range of 2 to 100 nm. Examples of typical micellar cubic structures with crystal symmetry include those containing about 20% to about 65% (by weight) of amphiphilic The polymer may contain about 80% to about 35% (by weight) of water. Another exemplary composition for obtaining a regular micellar cubic system with a simple arrangement of structures is 65% ( % water, 10% (wt%) butanol, and 25% (wt%) amphiphilic polymer - is.

[0036] The hydrogel is a bicontinuous cubic system known as a micellar cubic system with a Pn3m crystal structure. The lyotropic liquid crystal is an ordered array of 2-100 nm in size. Such bilayers having the Pn3m crystal structure may have the structure of spherical micellar aggregates in the range of 100-1500 nm. The micellar cubic system is composed of about 25% to about 65% (by weight) of an amphiphilic polymer and about It may contain 75% to about 35% (by weight) of water. Another example of obtaining such an LLC structure An exemplary composition is 33-38% (by weight) water, and the remainder is amphiphilic species or amphiphilic polymers. It is composed of mer.

[0037] The hydrogel is a bicontinuous cubic system known as a micellar cubic system with an Ia3d crystal structure. The lyotropic liquid crystal is an ordered array of 2-100 nm in size. The LLC may have a structure of spherical micellar aggregates in the range of 100 to 1500 nm. The preferred composition is 13-32% (by weight) water, and the remainder amphiphilic species or amphiphilic polymer. It consists of:

[0038] The hydrogels are made of ordered lyotropic materials, which are hexagonal geometries commonly called hexagonal systems. Cylindrical micelles arranged in a liquid crystal with diameters in the size range of 2 to 100 nm In such a normal hexagonal system, the amphiphilic polymer may have a small amount of The solvent may be present in an amount of about 30% to about 80% (by weight), with or without an organic solvent, and water may be present in an amount of about 60%. % to about 20% (by weight). Such conventional micellar hexagonal crystals may be present in amounts of about 35% to about 20% (by weight). About 40% (by weight) of an amphiphilic polymer, about 50% (by weight) of water, and about 10% to about 1 It may contain 5% (by weight) of an organic solvent.

[0039] Antibacterial hydrogels can also be produced using chemically crosslinked nanotubes with intermediate geometries and the following structures with zero curvature: They may have ordered nanostructures arranged as lyotropic liquid crystals, called lamellar systems. The distance between adjacent sheets is in the range of 2 to 100 nm. Such lamellar systems are composed of 20-80% (by weight) amphiphilic molecules, 15-6 0% (wt%) aqueous solution and a range of organic solvents such as butanol from 0 to 25% (wt%) An exemplary composition for obtaining a lamellar LLC is 20 % amphiphilic polymer, 55% (wt%) water, and 25% (wt%) butanol. It is an organic solvent.

[0040] The micellar and lyotropic liquid crystalline nanostructures of antibacterial hydrogels are composed of water molecules as continuous domains. The micellar aggregates may contain aqueous liquids such as ethanol, ethanolamine, and hydrophobic moieties trapped within the micellar aggregates. Micelle and lyotropic liquid crystalline nanostructures are characterized by the formation of aqueous liquids such as water trapped within micellar aggregates. The aqueous liquid may include water, saline, blood, These include, but are not limited to, sweat and other possible biological fluids. In its fully wet state, known as the "antibacterial hydrogel," the antibacterial hydrogel can absorb up to 3-4 times its own weight in aqueous The fully wet / swollen state is the state in which the hydrogel is able to absorb liquid. Depending on the type of bridge LLC structure, 20-90% aqueous solution and 10-80% amphiphilic organic component This refers to the original concentration (by weight) of the hydrogel. In a completely dry state, the hydrogel is % by weight of the aqueous solution, more usually less than 5% by weight of the aqueous solution, The hydrogel can absorb up to 8 to 10 times its own weight in aqueous solution. After absorbing the liquid, the antibacterial hydrogel The hydrogel swells and changes size; however, the shape and geometry of the hydrogel remain substantially constant. is maintained in a systematic manner.

[0041] Due to the amphiphilic nature of the antimicrobial hydrogel, it can also absorb hydrophobic liquids. In the presence of chloroform, the completely dried hydrogel can retain up to 20-30% of its own weight. It can absorb 30 times the amount of hydrophobic liquid such as chloroform. Dry state means less than 5% by weight of aqueous solution and more than 95% of hydrogel of amphiphilic organic molecules. The completely dry state refers to the concentration of the freeze-dried polymer that cannot be considered a hydrogel. It does not refer to the Ma Network.

[0042] The liquid absorption properties of the hydrogel can be tailored to absorb more or less water or hydrophobic liquids. This is possible by using amphiphilic molecules with different ratios of the chain length of the hydrophilic group to the hydrophobic group. This can be achieved by forming a hydrogel using, for example, an amphiphilic block copolymer. Wolfberry Copolymer DA-PEO x -PPO y -PEO x -DA (wherein x and y are PEO and The group (referring to the number of PEO and PPO groups) may contain more or fewer PEO or PPO groups. When the amount of PEO groups is higher than that of PPO groups, the hydrogel can be made up to 3-8 times as heavy as its initial weight. On the other hand, if the PPO group is more than the PEO group, the resulting hydrogel will have a high water absorption capacity. The resulting hydrogel absorbs approximately 0.5 to 1.5 times less water than its initial weight.

[0043] The antimicrobial agent is covalently bound to the repeating hydrophilic and / or hydrophobic domains. In the present invention, there are multiple antibacterial molecules, each of which has a repeating periodic hydrophilic and / or or is covalently bound to at least a portion of the hydrophobic domain.

[0044] More than 10%, for example more than 50%, or more than 90% of the antimicrobial agent present in the hydrogel is The antimicrobial agent may be covalently bonded to the gel, which allows for greater stability and increased hydrolysis of the antimicrobial agent. This results in reduced leaching from the filter.

[0045] The antimicrobial agent may be an amphipathic antimicrobial agent, i.e., the antimicrobial molecule has a hydrophilic region and a hydrophobic region. The antimicrobial agent may be selected to rupture bacterial cell walls by electrostatic forces. The antimicrobial agent may be a polymeric biocide or an antimicrobial peptide. The antimicrobial polymer molecule may be an antimicrobial polymer molecule such as an antimicrobial peptide (AMP). Destroys or inhibits the growth and proliferation of microorganisms by damaging the cell membranes of the organisms. AMPs are generally short peptides, i.e., 1-50 amino acids in length. AMP consists of linear AMP, branched AMP, and These generally have a net positive charge and have hydrophilic and phosphatase regions. The positively charged amphiphilic structure of AMPs allows the peptides to have both a negatively charged It is known that the damaged cell wall allows bacteria to penetrate the damaged bacterial membrane. The amphiphilic nature of AMPs combined with the ordered and repeating nanostructure of the hydrogel This results in orientation and higher fixation of the AMP. That is, the AMP adheres to the underlying hydrogel. This makes the antimicrobial hydrogel non-leachable for the antimicrobial agent. AMPs act as a matrix, forming both the hydrophilic and hydrophobic domains of amphiphilic hydrogels. The AMP may be covalently bound to adjacent hydrophilic and hydrophobic domains. The N-terminus of the AMP may be covalently bound to the hydrophobic domain of the hydrogel. The C-terminus of the AMP may be covalently attached to the hydrophilic domain of the hydrogel. .

[0046] AMPs may be covalently bound to the amphiphilic hydrogel or physically absorbed into the hydrogel. As shown in the rightmost image of Figure 3B in WO2019 / 074422A1, Thus, even after washing in 50% ethanol for 3 weeks, the amphiphilic hydrogels remained physically Not all of the absorbed fluorescently tagged AMPs are released. The philicity and the interaction of AMP with the hydrophilic and hydrophobic domains of the hydrogel This results in improved antibacterial performance and long-term stability during use.

[0047] The antimicrobial agent may be silver (Ag). For example, the antimicrobial agent may be a repeating ordered molecule of the hydrogel. Silver nanoparticles immobilized within or on hydrophilic and / or hydrophobic domains Silver has the drawback of being highly toxic to mammalian cells, and is have adverse environmental effects when released into the system, however, they are generally less severe than AMPs. It is also known to be a relatively low-cost antimicrobial agent.

[0048] It will be apparent to those skilled in the art that carboxyl groups will be present in amphiphilic hydrogels. Therefore, AMPs can be added to amphiphilic hydrogels without further modification of the hydrogel. There is no reason to attempt to covalently bond

[0049] Immobilization generally occurs via covalent bonds between multiple carboxyl groups on the hydrophilic domains of the hydrogel. When the antimicrobial agent is an antimicrobial peptide, the AMP and the hydrogel are repeatedly bonded. The AMP forms a strong amide bond between the hydrophilic domain and the hydrogel. 1-ethyl-3-(3-dimethylaminopropyl) carboxyl groups present in the functional domain ) via carbodiimide (EDC)-N-hydroxysuccinimide (NHS) activation Such covalent attachment of AMPs via EDC / NHS activation can be achieved by covalently attaching them to the hydrogel. The reaction scheme for the conjugation can be seen in Figure 2. AMPs or other antimicrobial agents are It may further be physically absorbed into the hydrogel, however, in such a case, the hydrogel There is no covalent bonding of the antimicrobial agent to the hydrophilic or hydrophobic regions of the rogel. The bound antimicrobial agent is subject to relatively rapid degradation and leaching / release from the hydrogel. In the zone inhibition test, physically absorbed AMPs tended to leach / release from the hydrogel. On the other hand, covalently bonded or covalently bonded It can be seen that AMP (valently attached) does not exude.

[0050] AMPs or other amphiphilic antimicrobial agents interact with the ordered repeating hydrophobic domains of the hydrogel. This results in improved orientation and immobilization of the antimicrobial agent. Therefore, due to the hydrogel having ordered repeating hydrophilic and hydrophobic domains, , reducing or eliminating the release of AMP into the surrounding environment while also providing protection against AMP stability and degradation. Such architectures are likely to improve the stability and activity of AMPs. It is widely asserted that

[0051] As shown in the experimental section below, the antibacterial hydrogel kills up to 99.99% of bacteria. Without being bound by theory, a further advantage is that the hydrophilicity of the antibacterial hydrogel The functional domain attracts negatively charged bacteria, effectively killing them. In wound care applications, this also facilitates the removal of wound dressings containing antimicrobial hydrogels. This can result in the removal of dead and / or attached bacteria. The results of experiments on Gram-negative bacteria and methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant (mult) bacteria showed that the antibacterial hydrogel also inhibited MRSA and multidrug-resistant (mult) bacteria. Drugs for bacteria such as MDR (medicinal drug resistant) E. coli This suggests that it can also kill drug-resistant strains.

[0052] As shown in the experimental section, the antimicrobial peptide can be one or more of the following: RRP RPRPRPWWWW-NH2(RRP9W4N, Red Glead Discover y AB, Lund, Sweden), RRPRPRPRP-NH2 (RRP9N, Re d Glead Discovery AB, Lund, Sweden), RRPRPRP WWWWRP-NH2(RRP7W4RPN, Red Glead Discovery AB, Lund, Sweden), RRPRPWWRPWWRP-NH2(RRP5W2 RPW2RPN, Red Glead Discovery AB, Lund, Sweden The sequences of RRP9W4N and RRP9N are provided in WO2012 / 033450A1. The sequences of RRP7W4RPN and RRP5W2RPW2RPN are disclosed in WO2019 The antimicrobial peptide has the amino acid sequence RRPRPRPR P (the sequence provided in WO2012 / 033450A1), e.g., at least 90% identical to an amino acid sequence having at least 95% identity, and optionally a C-terminal or N-terminal At least three consecutive tryptophan groups added to either or between Antibacterial agents containing fewer than 20 amino acids, including a stretch of phenylalanine or phenylalanine residues The antimicrobial peptide may be a peptide. The antimicrobial peptide may include N-terminal amidation. The antimicrobial peptide may be At least one, e.g., at least three, hydrophobic amino acids forming a hydrophobic region, e.g., Antimicrobial peptides, including stretches of phenylalanine or tryptophan residues. The hydrophobic regions allow interaction with the hydrophobic regions of the hydrogel. However, other antimicrobial peptides may be suitable for use as antimicrobial agents.

[0053] Antimicrobial agents may be synthetically derived AMPs, as in the previous paragraph, or biologically derived AMPs. Biologically derived AMPs include kininogen proteins, proline and arginine rich end leucine rich repeat p protein (PRELP), growth factor proteins, coagulation system proteins, complement factor C3a , von Willebrand factor, vitronectin, superoxide dismutase, purine On-proteins, protein C inhibitor, fibronectin, laminin, chemokines, Some examples of biologically derived AMPs are histidine- and histidine-rich glycoproteins. These peptides are the tocathelicidin-derived LL-37 peptide and omiganan pentahydrochloride. All of these can potentially be incorporated into the hydrogel either covalently bound or physically absorbed. The antimicrobial agent, alone or in combination with other peptides, is a peptide with fewer than 37 amino acids, i.e., wild-type L The antibacterial agent may be an LL-37 derivative having a length shorter than that of L-37. is proline arginine-rich end leucine-rich r Preferably, the antibacterial agent is a peptide derived from the human rheumatoid arthritis protein (PRELP). , which has the same charge and antibacterial efficacy as LL-37. As described in the experimental section, Preferably, the PRELP-derived peptide is comparable to LL-37 in terms of antimicrobial potency and net charge. It is a petite, RRP9W4.

[0054] Antimicrobial agents can be attached to hydrogels through various processes. As shown, the antimicrobial agent is attached by immersing the hydrogel in a solution containing the antimicrobial agent. The antimicrobial agent can be applied to the hydrogel via a surface application process, as opposed to immersion. Alternatively, a solution containing an antimicrobial agent may be applied dropwise to the surface of the hydrogel. A solution containing an antimicrobial agent may be sprayed onto the hydrogel. In addition, the amount of antimicrobial agent required for surface antimicrobial activation is generally higher for dripping and spraying compared to immersion. This is because most of the hydrogel is not activated by the antimicrobial agent. This is the case.

[0055] In addition to the antimicrobial agent, the hydrogel may include at least one therapeutic agent. Due to the alternating hydrophilic and hydrophobic domains, the therapeutic agent can be hydrophobic, hydrophilic, or can be amphiphilic, polar or non-polar. Antibacterial hydrogels contain hydrophobic domains. The hydrophilic domains can host (accept) the hydrophilic therapeutic agent. The therapeutic agent may be a peptide with anti-inflammatory, antibiotic, or anti-cancer properties. The molecule may be, but is not limited to, a drug molecule or a small biomolecule such as a peptide or protein. This property of selective release of therapeutic agents from antimicrobial hydrogels, in addition to their antimicrobial properties, may contribute to wound care. Can be used in medical devices such as smears and wound healing, or other antimicrobial / drug-releasing applications At least one therapeutic agent may be present in the hydrophobic and / or hydrophilic domains of the antimicrobial hydrogel. Multiple therapeutic agents can be provided in the hydrogel, either covalently bound to the hydrogel or physically absorbed. In such cases, a first therapeutic agent may be covalently attached to the hydrogel, and a second The third, etc., therapeutic agent may be physically absorbed. In contrast to the antimicrobial agent, at least one The therapeutic agent does not need to be immobilized on or within the hydrogel and is substantially free from the surface. May be freely leached.

[0056] The antimicrobial hydrogel does not adhere or stick to biological surfaces such as skin or wound beds. This improves performance in a variety of applications. Wound care products, such as wound dressings, seal infections and A soft, excess wound tissue is used to contain and prevent the wound environment from harboring microorganisms. The antibacterial hydrogel must be able to absorb exudate released from broken skin. It can be used as a wound dressing to absorb uncontrollable exudate that is produced. Wound exudate can contain pus, blood, water, and sweat. High and versatile absorption of antibacterial hydrogels These properties, combined with their antibacterial properties, make them particularly suitable as wound care articles. The hydrogel is a hydrogel containing an amphiphilic component but without an antimicrobial agent covalently bonded to the amphiphilic component. The antibacterial hydrogel absorbs substantially the same amount of water as the gel. The antibacterial hydrogel has sufficient wound exudate absorption capacity even when it contains an antibacterial agent. Hydrophobic and hydrophilic substances, such as bacterial toxins released by dying bacteria, It is further possible for the material to absorb both

[0057] The antimicrobial amphiphilic hydrogel may be prepared as a dispersion of particles in a continuous medium, The particles may be present as a suspension of particles in a solution. The amphiphilic hydrogel particles can be processed by grinding or the like to obtain an aggregate of amphiphilic hydrogel particles. The particles may have a diameter of about 0.01 mm to about 0.5 mm. The particles may be milled, for example, by a milling process. The particles do not need to be regular spherical particles, but may be irregular. Particles smaller than the ranges specified above may also be obtained by other manufacturing methods. For example, particles with a diameter in the range of 10 nm to 1 μm can be dispersed by ultrasonic treatment. It may be obtainable by dispersion methods.

[0058] An alternative process for obtaining particles is to first disperse the non-crosslinked polymer in solution before crosslinking. For example, a non-crosslinked amphiphilic polymer may form a solution, e.g., an LLC. The LLC hydrogel formed in water may then be applied to, for example, a blade. It can be rapidly dispersed in solution by mixing and subsequent UV crosslinking. The particles range in size from 10nm to 0.5mm depending on the process used to form the particles. The size range can be:

[0059] The particles may be present in dry form or may be swollen, for example, with an aqueous solution. The hydrogel particles may then be functionalized with an antimicrobial agent such as an AMP. The antimicrobial agent may be covalently bonded to at least the hydrophilic regions of the hydrogel. The antimicrobial agent may be an amphiphilic agent bound to both the hydrophilic and hydrophobic regions of the gel. The amphiphilic hydrogel particles functionalized with antimicrobial agents have a larger surface area and are flat and solid. Such a solution can expose the bacterial membrane to a larger portion of the antimicrobial agent than a hydrogel. The particles are claimed to be more effective, for example, antibacterial agents.

[0060] Particles of the amphiphilic hydrogel may be dispersed in a solution, such as an aqueous solution. Particles of the hydrophilic hydrogel may be dispersed in saline. As can be seen in Figure 5, The hydrogel remained antibacterial after 10 weeks in PBS. The amphiphilic hydrogel particles are biocompatible, i.e., non-toxic to cells. In contrast to many hydrogels, the amphiphilic nature of the hydrogel allows it to be dispersed in a suitable buffer solution. Therefore, hydrogel particles can be easily dissolved in non-polar solvents such as essential oil-based systems. It can be combined with an alcohol and dispersed in an aqueous solution.

[0061] The particles in the dispersion are swollen, i.e., they absorb the solution. In contrast to hydrogel dispersions, these are truly suspensions of individual hydrogel particles in solution. Some hydrogels are swollen and contain hydrogels. Since the hydrogel contains discrete particles that form the hydrogel, the hydrogel itself can be dissolved in a dispersion or suspension. In this case, the compositions are separated from one another and are separated from a continuous medium, e.g. For example, it comprises a plurality of crosslinked hydrogel particles separated from an aqueous solution.

[0062] The solution and particles dispersed therein can be sprayed. The spraying device can be, for example, a manual sprayer. A spray mechanism such as a pump and a solution containing a plurality of amphiphilic hydrogel particles as described above are combined. wherein the antimicrobial agent, such as an antimicrobial peptide, is present in the hydrophilic and / or hydrophobic regions of the particle. It is covalently bound to the hydroxyl group.

[0063] The spray device is shown schematically in Figure 9. The device sprays a solution 100, such as saline solution. The device includes a membrane 2 separating the solution from the plurality of antimicrobial hydrogel particles 300. 00. Prior to first use, the device is agitated 500, such as by shaking. , causing the membrane 200 to collapse, and the hydrogel particles to spread into the solution 100, forming hydrogel particles 30 Such a device forms a suspension of hydrogel particles 300. They are kept separate until the device is ready to use, ensuring storage stability. Improve.

[0064] Spraying amphiphilic hydrogels has advantages over placing solid pieces of hydrogel on the wound. Spray particles can cover irregular surfaces better than solid materials, resulting in improved coverage. Furthermore, the spray can be applied to the wound area without being held in place by additional dressings or the like. It can be used to quickly cover the wound. Therefore, spraying is a useful treatment for acute wounds. It may be ideal for use.

[0065] The sprayable compositions of antimicrobial amphiphilic hydrogel particles are also suitable for use in stents, catheters, skin Implants, contact lenses, personal hygiene products, diapers, wound dressings, ostomy dressings ( Ostomy dressing, ostomy base plate plate, incision film, surgical drapes, patches , bandages, band-aids, casts, adhesive, adhesive tape, adhesive plasters aster, sticking-plaster, and court -plaster), or any combination thereof, for surgical instruments or medical devices It is an ideal coating medium for the manufacturing or processing process of any of the devices listed above. The process may include spraying antimicrobial amphiphilic hydrogel particles onto the surface of the device; This may include post-treatment such as drying or scrubbing or wiping. The surface may be a surface intended to come into contact with the human body or bodily fluids.

[0066] Surprisingly, the inventors have found that the antimicrobial amphiphilic hydrogel induces and / or inhibits hemostasis in wounds. Therefore, the antibacterial hydrogel can be used to improve the The present invention may be used to increase and / or promote blood clotting and / or clot formation in the setting of atherosclerosis. As described in the experimental section and shown in Figure 10, the antibacterial hydrogels showed significantly improved antibacterial activity compared to the control samples. , forming a clearly visible blood clot. The promotion of hemostasis may be in mammals. For example, hemostasis of human blood can be promoted as shown in Experiment 1.

[0067] As described in the Background Art section, antibacterial amphiphilic hydrogels are disclosed in WO2019 / 0 However, this document does not include any indications beyond the antimicrobial treatment of wounds. Further uses of amphiphilic hydrogels are not described. Furthermore, there is no suggestion that antimicrobial hydrogels can be used to treat all wounds. Wounds such as burns and pressure sores do not bleed, but do bleed. susceptible to bacterial and other microbial infections. This difference is not relevant to bleeding wounds, see This does not seem to be taken into account in the literature. Their known therapeutic uses, i.e., prevention or treatment of infection, differ only from the enhancement or promotion of hemostasis. rather than a specific target population of bleeding wounds, and Related.

[0068] The antibacterial amphiphilic hydrogel has been shown to be effective in treating blood coagulation disorders, i.e., disorders in which the ability of blood to clot is impaired. These compounds may be particularly useful in inducing or promoting hemostasis in patients with bleeding disorders.

[0069] As shown in Figure 7, the hemostatic effect of antibacterial hydrogels is actually due to their biochemical interactions with the body. Hydrogel alone did not significantly or substantially affect platelet counts in the samples measured. No significant decrease was caused.

[0070] The antimicrobial amphiphilic hydrogel simultaneously induces hemostasis and promotes healing at and / or in the wound. It may be used to inhibit bacterial growth. It has experimentally confirmed serum stability properties and In combination with the improved hemostatic effect, the antibacterial amphiphilic hydrogel induces hemostasis in the wound. This makes it an ideal wound dressing for use in treating

[0071] A method for treating a bleeding wound may include: applying a disposing the antibacterial amphiphilic hydrogel according to the specification; thereby Forming a clot at the interface between the gel and the blood; thereby preventing bacterial growth in the wound. Substantially restrict or inhibit

[0072] A sprayable dispersion of antimicrobial amphiphilic hydrogel particles would ideally combine antimicrobial and hemostatic properties. The sprayable dispersion may be used for combined antibacterial and hemostatic wound treatment. The hydrogel particles are ideal for use in combination with wound care compositions. The amphiphilic hydro ... It has been shown to have improved antibacterial and hemostatic effects compared to a substantially flat sheet of Rogel. Furthermore, the hydrogel is non-biodegradable, and the antibacterial agent remains in the solution for several days, as shown in Figure 6. These effects can be maintained for a long time. This is because it is used to stop bleeding wounds. This is particularly relevant to devices for treating bleeding wounds, where the device provides a greater amount of are essentially subject to contact with bodily fluids.

[0073] The results presented herein demonstrate that proline arginine-rich end Antibacterial peptide derived from leucine-rich repeat protein (PRELP) These results suggest that the acetaldehyde-containing acetaminophen may be surprisingly effective in preventing hemostasis in wounds. Thus, RRP9W4 has been shown to have hemostatic effects. The adhesive may be applied to a substrate.

[0074] Although the present invention has been described above with reference to specific embodiments, the present invention is not limited to the specific embodiments described herein. Rather, the present invention is defined by the appended claims. Only limited to.

[0075] In the claims, "comprises / comprising" The term "component" does not exclude the presence of other elements or steps. Although the invention may be included in different claims, they may possibly be advantageously combined and may be included in different claims. Inclusion in a claim does not imply that a combination of features is not feasible and / or advantageous. In addition, reference to the singular does not exclude a plurality. Terms such as "first" and "second" exclude plurals Reference signs in the claims are provided merely as a clarifying example and are not to be construed as limiting the scope of the invention. and should not be construed as limiting the scope of the claims. [Example]

[0076] Experimental section The following examples are illustrative only and should not be construed as limiting the scope of the invention in any way. Rather, the present invention is limited only by the appended claims.

[0077] Experiment 1 Fabrication of amphiphilic hydrogels Crosslinked amphiphilic hydrogels were prepared using WO20 for Pluronic® F127. It was prepared according to the method of Experiment 1 of JP 19 / 074422A1. The reaction scheme is shown in Figure 1. Briefly, a mixture of Pluronic F-127 (30 wt%) and water (70 wt%) A micellar cubic liquid crystal phase was formed using Irgacure 2959 as a photoinitiator. This was added to a mixture of Pluronic F-127 (2 wt%). A thick, homogeneous gel was formed. Mixing was performed manually with a spatula in a 20 mL glass vial until the gel was uniform. The pellet was spread onto a glass slide and kept in a sealed container overnight to set in the correlation phase. The gel was then UV polymerized (90 W, λ = 252 nm) for 10 minutes to form a 4-5 mm thick gel. Flexible polymer hydrogels were formed. The gels were cut into the desired shape and then immersed in Mili-Q water. Washing for 48 hours removed unwanted by-products and their subsequent depletion prior to further analysis and AMP binding. It was allowed to fully swell.

[0078] Pluronic F127(EO 100 PO 70 EO 100 ) in WO2019 / 0 Chemically prepared with a polymerizable diacrylate head group as shown in Experiment 1 of 74422A1 The functionalized and modified polymer was used to fabricate crosslinked F127 hydrogels for AMP modification. Ta.

[0079] AMP immobilization on amphiphilic hydrogels AMP was prepared according to the immersion method described in Experiment 1 of WO2019 / 074422A1. The control amphiphilic hydrogel was prepared without AMP. The reaction scheme is shown in Figure 2. Briefly, the antimicrobial peptide (AMP) RRPRPRPRPWW WW-NH2(RRP9W4N, Red Glead Discovery AB, Lun A solution of 200 μM of hydroxybenzoates (H2O, Sweden) was prepared in sterile water. For the covalent attachment of AMP to the hydrogel, clean hydrogels were treated with MES buffer prior to AMP modification. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide mixed in buffer (pH 6) Final concentrations of EDC and N-hydroxysuccinimide (NHS) in the solution The mixture was immersed in 2 mg / ml of water and allowed to react for 30 minutes at room temperature while being gently shaken. Wash the gel three times in PBS (pH 7.4) and add 1 ml of a 200 μM AMP solution in sterile water. The surface was washed three times with sterile water to remove unreacted peptide. and was used for all tests performed in this study.

[0080] Blood coagulation tests Eppendorf tubes, pipette tips, and loop tubes for collecting blood Heparinization was performed to avoid unnecessary blood activation. The polymeric amine and heparin conjugates were then incubated alternately to obtain a denatured layer. By layer-by-layer assembly method The Corline method (Corline Biomedical AB, Upps Fresh blood from two healthy volunteers was collected at 1 IU / ml heparin solution (Leo Pharma A / S, Ballerup, Denmark) Fresh blood was used after collection. 1 ml of blood was collected in an Eppendorf tube with EDTA at the baseline (named initial). It was used as.

[0081] The samples were conditioned by adding 1 ml of PBS and incubated at 600 rpm for 30 minutes before the experiment. The hydrogels (control and AMP-modified) were placed in Eppendorf tubes. To soak the tubes, 100 μL of PBS was added. Then, 1 ml of fresh blood was added to each tube. The tube was then rotated vertically in an incubator at 37°C for 60 minutes. As a blank control, 1 ml of blood was injected into an Eppendorf tube without any hydrogel. After the experiment, the blood was carefully taken from the tube and diluted with EDTA. The platelet count was measured immediately after the experiment using a Sysmex XP-30 The blood samples were determined using a blood analyzer (Kobe, Japan). It was carried out in succession.

[0082] Results and Discussion One of the most important aspects of proper wound management is controlling bleeding. In the presence of a wound, which can trap platelets and release inflammatory chemokines, This is accompanied by the formation of a fibrin clot, which activates the inflammatory response and subsequent wound healing. Therefore, we investigated the effect of AMPs in the hydrogel. We investigated how the presence of two different drivers could affect blood clotting. A whole blood test was performed using fresh blood from a donor to measure platelet counts before and after 1 hour of exposure to blood. The number of AMP-hydrogels was quantified compared to the AMP-free hydrogels. Highly visible clot formation was observed. As expected from the observation, platelet counts showed no significant differences. The results (shown in Figure 7) showed significantly lower platelet counts in the presence of AMP. The remarkable result was that when AMP-hydrogel was used as a wound patch, it It has been suggested that it can promote blood clotting and clot formation, which may contribute to its excellent antibacterial effect. Apart from this, we believe that this is a desirable additional property introduced by the AMP-hydrogel. This can be done.

[0083] RRP9W4N is proline arginine-rich end leuci It is an antimicrobial peptide derived from ne-rich repeat protein (PRELP), The blood clotting effect is particularly surprising, as it is similar in many ways to LL-37, while It has been shown that LL-37 does not affect plasma coagulation (Harm, et al. al.,Blood Compatibility-An Important but O ften Forgotten Aspect of the Characterizat ion of Antimicrobial Peptides for Clinical Application,Int.J.Mol.Sci.,2019,20,5426; doi:10.3390 / ijms20215426).

[0084] Experiment 2 Sprayable dispersions of amphiphilic hydrogels A sprayable formulation of particulate hydrogel was prepared according to the following procedure.

[0085] DA-F127 is a Pluronic® F127 patent application WO2019 / 0 DA-F127 was synthesized according to experiment 1 of JP 74422A1, and DA-F127 was prepared using 30 wt. % Pluron ic® and water having a composition of 70% by weight of water. After mixing, the initiator Ir gacure® 2959 (1-[4-(2-hydroxyethoxy)-phenyl ]-2-hydroxy-2-methyl-1-propan-1-one) was added to the formed gel. The gel was cooled and then cooled. After refrigeration, the gel was cross-linked at 365 nm for 6 minutes. The crosslinked hydrogel was washed in water for at least 2 days.

[0086] After washing, the hydrogel was ground to a coarse paste with a pestle and mortar. water and disperse it using an Ultra-Turrax® disperser to obtain a finer particle size and The resulting solution was then stable for conveniently performed experiments.

[0087] A known weight of particles (swollen form) is filtered by suction, and the granules are then added to 15 ml of Falcon Chloride. This was usually about 2 grams. To prepare the solution, 10 ml of freshly prepared EDC / NHS (2 mg / ml) in MES buffer was added to the flask. The tube was sonicated for a few minutes and then placed on a shaking plate. After 30 minutes, the solution was filtered by suction and washed with water to separate the particles. The C / NHS was washed away. The granules were then weighed to record any loss, followed by 400 μM 10 ml of AMP (dissolved in PBS) was added to the granules, which were also quickly sonicated. and then placed on a shaker plate for approximately 2 hours.

[0088] The solution was again filtered by suction and washed with 30 ml of water, but this time the remaining The washings were collected to measure the amount of peptide, which activates the granules and allows them to react with the rest of the peptide. It can be weighed into solution to give a known concentration for the experiment.

[0089] Antibacterial effect against Staphylococcus aureus (S. aureus) on agar plates 200 mg each of control granules (non-activated) and AMP-activated granules were placed in separate eppendorf tubes. The tubes were placed in four tubes each, and 180 μl of PBS solution was added to each tube. Then, the tubes are briefly centrifuged to bring all particles into solution, followed by ultrasonication in a water bath. Treated to redisperse the particles.

[0090] On the agar plate, 10 8 Staphylococcus aureus, which is the concentration of CFU (colony forming units) / ml The plate was streaked with a culture of S. aureus and allowed to dry for a few minutes. The solution of the AMP-activated antibody was plated on each agar plate, along with one control and one AMP-activated version. The solution was gently spread onto the agar plate to compare the solutions each time. The agar plate was then spread evenly and evenly until no liquid was observed around the granules. Leave it open to air dry for a few minutes until the solution reaches the top of the agar after inversion. The agar plate was then inverted and left overnight (approximately 15 hours). Incubated.

[0091] The next day, place the center of each "spread" into an Eppendorf tube containing 1 ml of PBS. CFU experiments were performed by taking biopsy punches. The tubes were vortexed and Shake for at least 10 minutes. Then, 10 serial dilutions were made and the appropriate 10 μL drops were added. The diluted solution was dropped onto an agar plate and incubated for approximately 15 hours (until colonies became easy to count). Incubated.

[0092] Results and Discussion Figure 8 shows an example of what the plate looks like after overnight incubation. The difference in the number of bacteria present in / under the AMP-activated granules may be due to the presence of far fewer bacteria. The color difference is visible in the pre-conversion archive. The published black and white drawings have different textures. In addition, the areas where the coverage rate of the AMP activated particles was poor, i.e., the sprayable particles In areas where the bacteria did not spread onto the plate, it was easy to see that bacteria were growing.

[0093] TIFF2025128123000001.tif32170

[0094] The CFU results are shown in Table 1. Here, CFU was calculated by counting the number of cells in 1 ml of PBS used to wash the biopsy. The results showed that when AMP-activated granules were applied, the number of bacteria increased by approximately 9% compared to the control granules. A significant reduction of 9.993% is clearly shown.

[0095] After washing the granules, UV-vis measurement of the AMP residue showed that 1.7 grams of particles had a concentration of approximately 5.7 The amphiphilic hydrogel formed by the PEG-10 ... This is compared to 0.07 mg of peptide per 0.07 g of solid sheet. The peptide was absorbed approximately 3.35 times more than in the solid hydrogel in the form of particles / granules.

[0096] To demonstrate proof of concept, particles dissolved in PBS were placed in a simple mechanical spray bottle and the The particles were sprayable and had a diameter of approximately 25 cm. 2 of surface area quickly and relatively uniformly The thickness uniformity was visually inspected.

[0097] Experiment 3 Blood clotting in hydrogel samples with different peptides The hydrogels were functionalized with four different types of antimicrobial peptides (AMPs). P was PGLa, LL-37, temporin B, and RRP9W4. Hydrogels without any AMPs attached were used.

[0098] Fresh citrated horse blood was purchased from Hatunalab AB, Sweden. , was used to demonstrate whether the presence of AMP can induce clotting. 1 ml of blood was added to the sputum along with 0.1 ml of 250 mM calcium chloride (citrate anticoagulant). (to reconstitute the binder) and mixed.

[0099] The samples were placed in a 24-well plate and 1 ml of PBS was added to the hydrogel. After 15 minutes, the sample was transferred to an Eppendorf tube containing blood. The tubes were placed on a shaker and incubated at 37°C for 60 minutes.

[0100] As a blank control, 1 ml of blood was placed in an Eppendorf tube without any hydrogel. After the experiment, the blood was carefully taken from the tube and collected with tweezers. Carefully remove the hydrogel from the tube using a (observed on the side of the tube wall), the hydrogel was washed three times with flushes of PBS, Unattached clots and other blood cells were removed before imaging. Samples were run in duplicate.

[0101] Results and Discussion This study examines how the presence of AMPs on hydrogels affects blood clotting Here, any observable clots were identified as cytoplasmic blood clots with different peptides covalently attached. To determine whether the RRP could be formed on a hydrogel, a horse whole blood test was performed. Large, visible clots were observed in the hydrogels with 9W4, whereas other AMPs Hydrogels containing PGLa, LL-37, and temporin B and any AMPs No clot formation was observed in the control hydrogel without any

[0102] In this test, only blood clots that were firmly attached to the hydrogel surface were identified as hydrogel-blood contacts. Other clots formed during this study were primarily in vitro. They either stuck to the wall or peeled easily from the surface. See the table below, where: a * indicates the formation of a clot immobilized in the hydrogel.

[0103] TIFF2025128123000002.tif55170

[0104] Due to the simple and qualitative nature of Experiment 3, the results above cannot be considered conclusive. However, PGLa, LL-37, and temporin B all have linear structures. It is similar to RRP9W4 in terms of structure, net charge, and hydrophobicity, but has a strong adhesion to the hydrogel surface. This confirms the surprising nature of the RRP9W4 results, as they did not form any new clots. In particular, temporin B and RRP9W4 have the same length (13 amino acids) and similar hydrophobicity. (4.22 kcal / mol and 9.15 kcal / mol, respectively), It should be noted that the RRP9W4 hydrogel showed different coagulation results. The net charges of LL-37 and LL-37 (+6 and +6, respectively) are similar, but according to previous results LL-37 did not show any coagulation effect, but RRP9W4 showed a surprising coagulation effect. He continued.

Claims

1. 1. An antimicrobial amphiphilic hydrogel composition for use in wound hemostasis, comprising: a first cross-linkable amphiphilic component, said first amphiphilic component being capable of cross-linking said chemically cross-linked amphiphilic component; In this state, the liquid crystal is a lyotropic liquid crystal and has a hydrophobic domain and a hydrophilic domain. The hydrogel has an ordered nanostructure, and the hydrogel has the hydrophilic domains and / or the hydrophobic domains. containing an antimicrobial agent covalently bound to the Antimicrobial amphiphilic hydrogel compositions.

2. The antibacterial agent is a proline arginine- having a length of less than 37 amino acids. rich end leucine-rich repeat protein (PRELP 2. The antimicrobial amphiphilic hydrogel composition for use according to claim 1, wherein the antimicrobial peptide is derived from thing.

3. The antimicrobial peptide is an antimicrobial peptide containing less than 20 amino acids, and the amino acid sequence RRPRPRPRPRP, Item 3. An antibacterial amphiphilic hydrogel composition for use according to item 2.

4. 4. The method according to claim 2, wherein the antimicrobial peptide is RRP9W4N. Antimicrobial amphiphilic hydrogel composition for use.

5. 5. Any of claims 1 to 4, wherein the composition is a suspension of amphiphilic hydrogel particles in a solution.

10. An antibacterial amphiphilic hydrogel composition for use according to any one of claims 1 to 9.

6. A composition according to any one of claims 1 to 5 for use in wound hemostasis in patients suffering from coagulation disorders. An antimicrobial amphiphilic hydrogel composition for use according to any one of claims 1 to 4.

7. 1. An antimicrobial hydrogel dispersion comprising: a first cross-linkable amphiphilic component, said first amphiphilic component being capable of cross-linking said chemically cross-linked amphiphilic component; In this state, the liquid crystal is a lyotropic liquid crystal and has a hydrophobic domain and a hydrophilic domain. The hydrogel has an ordered nanostructure, and the hydrogel has the hydrophilic domains and / or the hydrophobic domains. an antimicrobial agent covalently attached to the the first component is present in the composition in the form of particles in a dispersion; Antibacterial hydrogel dispersion.

8. 8. The composition of claim 7, wherein the composition is a suspension of the amphiphilic hydrogel particles in a solution. Antibacterial hydrogel dispersion of.

9. The antimicrobial agent is encapsulated in the hydrophilic and optionally hydrophobic domains of the first amphiphilic component.

9. The method of claim 7 or 8, wherein the antimicrobial agent is a substantially amphiphilic antimicrobial agent covalently immobilized on the surface of the immobilized antimicrobial agent. The antibacterial hydrogel.

10. 10. The antimicrobial hydrogel dispersion of claim 9, wherein the antimicrobial agent is an antimicrobial peptide.

11. The antibacterial agent is a proline arginine- having a length of less than 37 amino acids. rich end leucine-rich repeat protein (PRELP 11. The antimicrobial hydrogel dispersion of claim 10, wherein the antimicrobial peptide is derived from Bacillus subtilis.

12. The antimicrobial agent forms hydrophobic regions for interacting with the hydrophobic regions of the hydrogel. and an antibody comprising at least one, e.g., at least three, stretches of hydrophobic amino acids that form 12. The antibacterial hydrogel dispersion of claim 10 or 11, which is a bacterial peptide.

13. The antimicrobial peptide is an antimicrobial peptide containing less than 20 amino acids, and the amino acid sequence at least 90%, e.g., 95%, identity to RRPRPRPRPRP.

13. The antimicrobial hydrogel dispersion of any one of claims 10 to 12.

14. The antimicrobial peptide according to any one of claims 10 to 13, wherein the antimicrobial peptide is RRP9W4N. The antibacterial hydrogel dispersion.

15. An antibacterial hydrogel according to any one of claims 7 to 14 for use in hemostasis of a wound. dispersion.

16. 15. The method of claim 7, wherein the hydrogel is substantially non-degradable under physiological conditions.

16. An antimicrobial hydrogel dispersion according to any one of claims 1 to 15, or for use according to claim 15. 。

17. 16. A method according to claim 15 for use in hemostasis of wounds in patients suffering from a coagulation disorder.

10. An antibacterial hydrogel dispersion for use in

18. A method for producing the antibacterial amphiphilic hydrogel dispersion according to any one of claims 7 to 14. There is: providing a first cross-linkable amphiphilic component; The crosslinkable amphiphilic component is converted into a lyotropic liquid crystal. forming an amphiphilic hydrogel (Crystal: LLC); treating the amphiphilic hydrogel to form granular hydrogel particles; Crosslinking either the (LLC) amphiphilic hydrogel or the granular hydrogel particles. That and; In the hydrophilic and / or hydrophobic regions of the amphiphilic hydrogel particles, covalently attaching a fungicide to the hydrogel; providing said dispersion of particles in a continuous medium; A method comprising:

19. 19. The method of claim 18, wherein the antimicrobial agent is an antimicrobial peptide.

20. The antibacterial agent is a proline arginine- having a length of less than 37 amino acids. rich end leucine-rich repeat protein (PRELP 20. The method of claim 19, wherein the antimicrobial peptide is derived from Bacillus subtilis.

21. The antimicrobial peptide is an antimicrobial peptide containing less than 20 amino acids, and the amino acid sequence at least 90%, e.g., 95%, identity to RRPRPRPRPRP.

21. The method according to claim 19 or 20.

22. 22. The method according to any one of claims 19 to 21, wherein the antimicrobial peptide is RRP9W4N. How to post.

23. Surgical instruments, stents, catheters, skin grafts, contact lenses, personal hygiene products, Diapers, wound dressings, ostomy dressings, ostomy - Base plate (ostomy baseplate), incision film (incision film) on film), surgical drapes, patches, bandages, band-aids, casts, adhesives, glue adhesive tape, adhesive plaster, sticking- plaster, and court-plaster, or any combination thereof 15. The anti-corrosion coating of claim 7 for use as a composite coating. Bacterial amphiphilic hydrogel dispersions.

24. A device comprising at least one surface that comes into contact with the human body and / or body fluids, said surface At least a portion of the surface is coated with the antibacterial amphiphilic hydrogel according to any one of claims 7 to 14. A device comprising a coating of the cellulose dispersion.

25. 1. A method of treating a wound comprising: providing to the wound an antimicrobial amphiphilic hydrogel composition, said composition comprising: a first cross-linkable amphiphilic component, said first amphiphilic component being capable of cross-linking said chemically cross-linked amphiphilic component; In the state where the polymer is dissolved, it is a lyotropic liquid crystal and has hydrophobic and hydrophilic domains. and the hydrogel has an ordered nanostructure of the hydrophilic domains and / or the hydrophobic domains. Contains a covalently bound antimicrobial agent in the main The method, wherein the composition causes blood to clot in the wound.

26. The antibacterial agent is a proline arginine- having a length of less than 37 amino acids. rich end leucine-rich repeat protein (PRELP 26. The method of claim 25, wherein the antimicrobial peptide is derived from Bacillus subtilis.

27. The antimicrobial peptide is an antimicrobial peptide containing less than 20 amino acids, and the amino acid sequence at least 90%, e.g., 95%, identity to RRPRPRPRPRP.

26. The method according to claim 26.

28. 28. The method of claim 26 or 27, wherein the antimicrobial peptide is RRP9W4N.

29. 1. A method of treating a wound comprising:

15. Providing the wound with an antimicrobial hydrogel dispersion according to any one of claims 7 to 14. wherein the dispersion causes blood to clot in the wound.

30. Proline and ATP having a length of less than 37 amino acids for use in wound hemostasis. ginine-rich end leucine-rich repeat protei n(PRELP)-derived antimicrobial peptide.

31. The antimicrobial peptide is an antimicrobial peptide containing less than 20 amino acids, and the amino acid sequence at least 90%, e.g., 95%, identity to RRPRPRPRPRP.

30. An antimicrobial peptide for use according to claim 30.

32. 32. The use of claim 30 or 31, wherein the antimicrobial peptide is RRP9W4N. Antimicrobial peptides.

33. The peptides are useful in wound dressings, ostomy dressings, , ostomy baseplate, incision film incision film), surgical drapes, patches, bandages, band-aids, casts, Adhesive, adhesive tape, adhesive plaster, sticking plaster king-plaster, and court-plaster, or coated, painted, sprayed, or otherwise applied to a substrate such as any combination of 33. An antimicrobial peptide for use according to any one of claims 30 to 32.