TW202411241A - A fusion protein, a regulated wound dressing, and the use of the fusion protein in preparing a regulated wound dressing - Google Patents

Abstract

A fusion protein, a regulated wound dressing, and the use of the fusion protein for preparing a regulated wound dressing; the fusion protein comprises collagen and an antimicrobial peptide, wherein a first enzyme cleavage site is included between the collagen and the antimicrobial peptide, and the collagen and the antimicrobial peptide can be separated by being treated with a first enzyme to cleave the first enzyme cleavage site; the present invention uses a novel antimicrobial peptide to bridge collagen, and can regulate the release of antimicrobial peptides through enzymes to remove bacteria inside and outside cells and latent bacteria in autophagosomes, thereby providing multiple wound protection and preventing bacterial infection.

Description

A fusion protein, a regulated wound dressing, and use of the fusion protein in preparing a regulated wound dressing

The present invention relates to a fusion protein, a regulated wound dressing, and the use of the fusion protein in preparing the regulated wound dressing, in particular to the use of the regulated wound dressing comprising the fusion protein to provide multiple wound protection and prevent bacterial infection.

With the long-term use of antibiotics, wound infections in patients with low immunity or chronic diseases often present with high-risk drug-resistant bacteria, resulting in irreparable damage; such drug-resistant bacterial infections can be divided into general bacterial infections and latent (intracellular) bacterial infections; general bacterial infections cause wounds to become ulcerated, red and swollen and unable to heal, and increase the risk of latent bacterial infections; in latent bacterial infections, drug-resistant bacteria can hide in immune cells to evade drug treatment, ultimately causing bacteremia or infective endocarditis.

Antimicrobial peptides (AMPs) are polypeptide chains with a length of less than 50 amino acids. Through the positive amino acids at the end, AMPs can physically destroy bacterial cell walls, or enter bacteria through endocytosis to disrupt bacterial genome operations. Due to their physical properties, they are currently one of the best candidate drugs for overcoming drug-resistant bacterial infections and can effectively inhibit bacterial growth in the environment. Since AMPs are polypeptide chains that can be found in nature, in addition to being able to effectively inhibit bacterial growth, they are also relatively non-biotoxic. AMPs can be used to prevent bacterial infections in wounds, but long-term exposure to the wound environment can easily cause degradation of AMPs. In addition, traditional AMPs cannot treat bacteria lurking in the autophagosomes of cells.

In view of this, the inventor of the present case deeply understood the deficiencies and defects of the previous case and was eager to improve and innovate it. After years of research, he successfully developed a fusion protein, a regulated wound dressing, and the use of the fusion protein for preparing a regulated wound dressing in the present case.

To achieve the above object, the present invention provides a fusion protein comprising collagen and an antimicrobial peptide, wherein a first enzyme cleavage site is included between the collagen and the antimicrobial peptide, and the fusion protein is constructed in a manner that the collagen and the antimicrobial peptide can be separated by cleaving the first enzyme cleavage site through treatment with a first enzyme.

In one embodiment of the present invention, the first enzyme treatment is performed by thrombin.

In one embodiment of the present invention, the antimicrobial peptide is bridged to the penetrating peptide.

In one embodiment of the present invention, a second enzyme cleavage site is included between the antimicrobial peptide and the penetrating peptide, and the antimicrobial peptide and the penetrating peptide are constructed in a manner that the second enzyme cleavage site can be cleaved by a second enzyme to separate the antimicrobial peptide from the penetrating peptide.

In one embodiment of the present invention, the second enzyme treatment is performed by cathepsin.

The present invention also provides a controlled wound dressing, comprising a polyurethane layer, an absorption layer, and a collagen layer, wherein the absorption layer is located between the polyurethane layer and the collagen layer, and the collagen layer comprises the above-mentioned fusion protein.

In one embodiment of the present invention, the absorbent layer comprises alginate and chitosan.

In one embodiment of the present invention, the ratio of alginate to chitosan is 4:1-1:4.

In one embodiment of the present invention, the dosage of the fusion protein in the collagen layer is 5 μM to about 100 μM.

The present invention further provides a fusion protein for use in preparing a regulated wound dressing, wherein the fusion protein comprises the above-mentioned fusion protein. When the regulated wound dressing contacts the wound, the antimicrobial peptide is released inside and outside the cells through enzyme treatment in the wound to achieve an antimicrobial effect.

In one embodiment of the present invention, when the controlled wound dressing contacts the wound, the antimicrobial peptide is released into the autophagosome through enzyme processing in the autophagosome to eliminate latent bacteria.

In the present invention, in order to reduce the chance of degradation of antimicrobial peptides at wounds, collagen and antimicrobial peptides are bridged and an enzyme cleavage site, such as a thrombin action site, is inserted between them so that the antimicrobial peptides are released only when thrombin is secreted from the wound, thereby regulating the exposure time of the antimicrobial peptides and enhancing the stability of the antimicrobial peptides.

In addition, in order to solve the problem of latent bacteria mentioned above, antimicrobial peptides are bridged with cell penetrating peptides (CPP), and an enzyme cleavage site is inserted at the bridge between the antimicrobial peptide and the penetrating peptide, such as the insertion of the autophagosome-specific cathepsin cleavage site, to regulate the release of antimicrobial peptides in the autophagosome and eliminate latent bacteria.

[Definition of Terms] This specification widely uses many technical and scientific terms commonly used in the field of biotechnology. In the following description, in order to have a clear and consistent understanding of this specification and the scope of the patent application and the scope of these terms, the following definitions are provided. Other terms not specifically defined below have the meanings commonly understood by professionals in the relevant field.

Unless otherwise specified, the words "or", "and", and" used in this specification refer to "or/and". In addition, the words "include" and "include" are open conjunctions that are not restrictive. The above paragraphs are only systematic references and should not be interpreted as limiting the subject matter of the invention.

The "%" used in this manual refers to "weight percentage (wt%)" unless otherwise specified; a numerical range (such as 10%~11% of A) includes both upper and lower limits unless otherwise specified (i.e. 10%≦A≦11%); a numerical range without a defined lower limit (such as B less than 0.2%, or B below 0.2%) may refer to a lower limit of 0 (i.e. 0%≦B≦0.2%); the ratio of "weight percentage" of each component may also be replaced by the ratio of "parts by weight".

All numerical values disclosed in this specification may have a standard technical measurement error (standard deviation) of ± 10%. The term "about" is intended to mean ± 10%, ± 5%, ± 2.5%, or ± 1% relative to a given value, that is, "about" 20% means 20 ± 2%, 20 ± 1%, 20 ± 0.5%, or 20 ± 0.25%.

As used herein, "collagen" in some cases refers to a (e.g., monomeric) polypeptide that can bind to one or more collagen or collagen-like polypeptides to form a quaternary structure; non-limiting examples of collagen include human type 21 α1 collagen, human type 1 α2 collagen, and jellyfish (hydrozoan) collagen; in some cases, collagen can be treated with acid, alkali, or heat to prepare gelatin; the quaternary structure of natural collagen is a triple helix usually composed of three polypeptides; in some cases of three polypeptides forming natural collagen, two are usually the same and are designated as α chains; the third polypeptide is designated as β chain. In some cases, typical native collagen may be designated as AAB, where collagen is composed of two α ("A") chains and one β ("B") chain; in some cases, collagen may refer to α-chain polypeptides, β-chain polypeptides, or both α- and β-chain polypeptides.

The term "subject" as used herein refers to any mammal that needs or is considered to potentially need the controlled wound dressing of the present invention, including primates, rodents, pets, laboratory test animals, and captive wild animals. For example, this may include, but is not limited to: monkeys, humans, pigs, cattle, sheep, goats, equines, mice, rats, guinea pigs, hamsters, rabbits, cats (felines), and canines. Preferably, the subject is a mouse or a human.

The terms "anti", "inhibit" and similar terms used in this manual refer to preventing, delaying, improving, reducing or reversing the occurrence of symptoms.

The term "antibacterial" used in this manual includes bactericidal and bacteriostatic functions. Bactericidal refers to the process of achieving sterility by using chemical or physical methods to destroy microorganisms, including bacterial reproductive bodies, spores, and molds. Bacteriostatic refers to preventing or inhibiting the growth of bacteria.

The terms "treatment", "treating", and "therapy" used in this manual include alleviating, alleviating, or improving at least one symptom of a disease or physiological condition, preventing new symptoms, inhibiting a disease or physiological condition, stopping or slowing the progression of a disease, causing recovery from a disease or physiological condition, alleviating a physiological condition caused by a disease, or stopping a symptom or physiological condition of a disease in a therapeutic or preventive manner.

The "therapeutically effective amount", "therapeutic dose" and similar terms used in this specification are used herein to refer to the amount of a drug that is effective in treating, curing, preventing or improving a disease, disorder, or side effect, or reducing the rate of progression of a disease or disorder. The term also includes within its scope an amount that is effective in enhancing normal physiological functions.

The term "pharmaceutically acceptable" as used in this specification refers to a substance or composition that is compatible with other ingredients in its pharmaceutical formulation and does not aggravate the patient's symptoms.

The "pharmaceutically acceptable carrier" used in this specification includes one or more types of ingredients selected from the following: solvents, emulsifiers, suspending agents, disintegrating agents, binders, shaping agents, stabilizers, chelating agents, diluents, gelling agents, preservatives, lubricants, surfactants, and other similar or suitable oral carriers for the present invention.

The above-mentioned composition may also be appropriately added with one or more dissolution aids, buffers, colorants, flavoring agents, etc. commonly used in the pharmaceutical preparation field as needed.

The "pharmaceutically acceptable excipient" used in this specification includes, but is not limited to, at least one of a polymer, a resin, a plasticizer, a filler, a lubricant, a diluent, a binder, a disintegrant, a solvent, a co-solvent, a surfactant, a preservative, a sweetener, a flavoring agent, a pharmaceutical grade dye or pigment, and a viscosity agent.

As used herein, "cell culture" and similar terms are used herein to maintain cells in an artificial extracorporeal environment. However, it should be understood that the term "cell culture" is a general term and can be used to cover not only the cultivation of individual cells but also the cultivation of tissues or organs.

[Fusion protein] Please refer to Figure 1, which is a schematic diagram of a fusion protein of an embodiment of the present invention. As shown in the figure, the fusion protein of the present invention comprises collagen 1 and antimicrobial peptide 2, and a first enzyme cleavage site 3 is included between collagen 1 and antimicrobial peptide 2. The fusion protein is formed in a manner that the first enzyme 4 can be treated to cleave the first enzyme cleavage site 3 to separate collagen 1 and antimicrobial peptide 2.

In order to ensure that the antimicrobial peptide 2 can stably bind to the collagen 1 and be regulated by the first enzyme 4, the collagen binding domain (CBD) of the collagen 1 is bridged with the antimicrobial peptide 2, and the first enzyme cleavage site 3 is placed between the collagen binding domain and the antimicrobial peptide 2, so that the antimicrobial peptide 2 is regulated by the first enzyme 4; in one embodiment, the first enzyme cleavage site 3 can be a thrombin cleavage site, and the first enzyme 4 can be thrombin. When the fusion protein contacts the thrombin in the wound, the thrombin will recognize and cleave the thrombin cleavage site and release the antimicrobial peptide 2 to kill extracellular and intracellular bacteria; through the above-mentioned structure, the antimicrobial peptide 2 can be released by the thrombin on the wound, shortening the exposure time and delaying the decline.

The fusion protein of the present invention can be a controllable antimicrobial peptide, wherein such antimicrobial peptide can be cleaved by specific enzymes inside and outside the cell, thereby controlling the stability, release timing, and activity of the antimicrobial peptide; in one embodiment, by linking the collagen binding domain with the antimicrobial peptide, the antimicrobial peptide can be attached to the collagen to avoid direct exposure to the protein degradation environment, reduce the risk of the antimicrobial peptide being degraded by other proteases, regulate the activity of the antimicrobial peptide, and at the same time increase the adhesion of the antimicrobial peptide.

In one embodiment, the collagen binding domain is linked to the antimicrobial peptide via a thrombin recognition site, and when the wound bleeds and releases thrombin, the antimicrobial peptide can be released synchronously by the action of thrombin. In this embodiment, in order to release the antimicrobial peptide after the action of thrombin while still maintaining the function of the antimicrobial peptide, thrombin is preferably sequence-selected to recognize the cleavage site.

Please refer to Figures 1 and 2. Figure 2 is a schematic diagram of an antimicrobial penetrating peptide and a release mechanism of another embodiment of the present invention. As shown in the figure, the antimicrobial peptide 2 of the present invention can be bridged to the penetrating peptide 5. In one embodiment, a second enzyme cleavage site 6 is included between the antimicrobial peptide 2 and the penetrating peptide 5, and the antimicrobial peptide 2 and the penetrating peptide 5 are separated in a manner that the second enzyme 7 can be treated to cleave the second enzyme cleavage site 6.

In order to ensure that the antimicrobial peptide 2 (also called antimicrobial penetrating peptide) bridged with the penetrating peptide 5 can stably bind to collagen 1 and be regulated by the first enzyme 4, the collagen binding domain (CBD) of collagen 1 is bridged with the antimicrobial penetrating peptide, and the first enzyme cleavage site 3 is placed between the collagen binding domain and the antimicrobial penetrating peptide, so that the antimicrobial penetrating peptide is regulated by the first enzyme 4.

In order to ensure that the antimicrobial peptide 2 can be released by the penetrating peptide 5 to perform its function after the antimicrobial penetrating peptide enters the cell, in this embodiment, in order to release the antimicrobial peptide 2 in the autophagosome, the second enzyme cleavage site 6 can be an enzyme cleavage site exclusive to the autophagosome, for example, it can be a tissue protease cleavage site, and the second enzyme 7 can be a tissue protease; please refer to the release mechanism of Figure 2. When the antimicrobial penetrating peptide enters the cell, the tissue protease in the cell will recognize and cleave the tissue protease cleavage site to release the antimicrobial peptide 2 to kill the bacteria in the cell (in the autophagosome); through the above-mentioned structure, the antimicrobial penetrating peptide can release the antimicrobial peptide 2 through the tissue protease in the cell after entering the cell, and the release of the antimicrobial peptide 2 in the autophagosome is regulated to eliminate latent bacteria.

The fusion protein of the present invention can be a controllable antimicrobial penetrating peptide, wherein such antimicrobial penetrating peptide can be cleaved by specific enzymes inside and outside the cell, thereby controlling the stability, release timing, and activity of the antimicrobial penetrating peptide; the above-mentioned technical features and advantages of antimicrobial peptides are also applicable to antimicrobial penetrating peptides, which will not be elaborated here. The following describes other technical features and advantages of antimicrobial penetrating peptides.

In one embodiment, the antimicrobial penetrating peptide is linked to a thrombin recognition site, and when the wound bleeds and releases thrombin, the antimicrobial penetrating peptide can be released synchronously by the action of thrombin. The antimicrobial penetrating peptide can enter the cell through the macrophage effect caused by the penetrating peptide. The antimicrobial penetrating peptide entering the cell will merge with the phagosome or autophagosome, and the penetrating peptide and the antimicrobial peptide connection site will be cleaved by a dedicated enzyme in the phagosome or autophagosome, releasing and activating the antimicrobial peptide to kill bacteria; and, in order to ensure that the dedicated enzyme in the phagosome or autophagosome can cleave the penetrating peptide and the antimicrobial peptide connection site and can maintain the activity of the antimicrobial peptide, the site is specifically simulated.

In addition, the specific amino acid sequences of the collagen, antimicrobial peptide, penetrating peptide, enzyme cleavage site, etc. used in the present invention are not limited. Unless otherwise specified, the collagen, antimicrobial peptide, penetrating peptide, enzyme cleavage site used in the present invention can be any known biological material, and the amino acid sequence information thereof can be easily obtained by those having common knowledge in the field to which the present invention belongs.

In one embodiment, the antimicrobial peptide may have a positively charged region in its molecular structure, such as one or more of AA230, DPK-060 or LL-37, with DPK-060 being preferred. By using the above antimicrobial peptides, the antimicrobial peptides have better antimicrobial efficacy after being released inside or outside the cells.

In one embodiment, the types of penetrating peptides include, for example, polyarginine, TAT, HIV-Tat, R9-TAT, Pep-1, Pep-7, penetratin, transporter, Antp, Rev, FHV envelope protein, buforin II, MAP, K-FGF, Ku70, SynBl, HN-1, TP10, pVEC, BGSC, and BGTC, among which TAT is preferred. By using the above penetrating peptides, when the penetrating peptides are linked to antimicrobial peptides, it helps to effectively transfer the antimicrobial peptides across the cell membrane.

[Controlled Wound Dressing]

Please refer to FIG. 3 , which is a schematic diagram of a controllable wound dressing according to an embodiment of the present invention. As shown in the figure, the controllable wound dressing of the present invention comprises a polyurethane (PU) layer 10, an absorption layer 9, and a collagen layer 8. The absorption layer 9 is located between the polyurethane layer 10 and the collagen layer 8. The collagen layer 8 comprises the above-mentioned fusion protein. With the above-mentioned structure, the fusion protein can be stably fixed to the collagen layer in contact with the wound surface, and the antimicrobial peptide is used to resist bacterial infection of the wound, thereby achieving multiple wound protection and preventing bacterial infection.

In one embodiment, the controllable wound dressing design includes a three-layer structure. The outermost polyurethane layer 10 has waterproof and breathable properties, which can prevent secondary infection; the middle absorption layer 9 is composed of alginate and chitosan, which can absorb excess exudate on the wound and provide a micro-moist environment that is most suitable for wound healing; the collagen layer 8 can provide the raw materials required for the regeneration of wound tissue and use antimicrobial peptides to resist bacterial infection of the wound.

In one embodiment, the collagen layer 8 is used to bridge the collagen and antimicrobial peptides, and a thrombin action site is inserted between them, so that the antimicrobial peptides are released only when thrombin is secreted from the wound, thereby regulating the exposure time of the antimicrobial peptides to the wound.

In one embodiment, the collagen layer 8 is used to bridge the collagen and the antimicrobial penetrating peptide, and a thrombin action site is inserted therebetween, so that the antimicrobial penetrating peptide is released only when the wound secretes thrombin, thereby regulating the timing of the antimicrobial penetrating peptide being exposed to the wound, and utilizing the penetrating peptide to allow the antimicrobial peptide to penetrate the cell, and inserting a tissue protease specific to the autophagosome therein, so that the antimicrobial peptide is controlled to be released in the autophagosome to eliminate latent bacteria. This can solve the problem in the prior art that the antimicrobial peptide cannot be regulated to directly penetrate the cell at a specific time, and has the technical effect of providing multiple wound protection and preventing bacterial infection.

The absorption layer 9 is used to absorb excess exudate to optimize the wound environment. The content ratio of alginate to chitosan is not limited. In one embodiment, the ratio of alginate to chitosan in the absorption layer 9 is 4:1 to 1:4, preferably 1:3. With the above ratio, the absorption layer 9 can achieve the best absorption capacity. For specific experimental data, please refer to the following embodiments.

In addition, although the embodiment of the present invention is a dressing, the present invention is not limited thereto, and the fusion protein of the present invention can be applied to any suitable product, such as a patch, a patch, a gel, a hydrogel, etc.

[Use of fusion protein for preparing controlled wound dressing]

The present invention provides a fusion protein for preparing a regulated wound dressing. When the regulated wound dressing contacts a wound, the antimicrobial peptide is released inside and outside the cell through enzyme treatment in the wound to achieve antibacterial effect, and the antimicrobial peptide is released into the autophagosome through enzyme treatment in the autophagosome to eliminate latent bacteria. In particular, the dressing containing collagen and antimicrobial peptide can resist bacterial infection on the wound of a patient at multiple levels and assist wound recovery.

In one embodiment, the enzyme in the wound may be thrombin, and the enzyme in the autophagosome may be tissue protease, but the present invention is not limited thereto.

[Method for protecting and treating wounds] The present invention discloses a method for protecting and treating wounds, which involves administering a therapeutically effective amount of a regulated wound dressing to an individual in need thereof to protect and treat the wound; in this embodiment, the fusion protein is used as a regulated wound dressing, which is used to protect or treat the wound, and achieves antibacterial effects and removes latent bacteria.

The present invention relates to administering a controlled wound dressing to an individual (e.g., a human patient) to treat a wound. The treatment method according to the present invention can also treat, cure, alleviate, relieve, alter, remedy, improve, enhance or affect the disease, the symptoms or conditions of the disease, the disability caused by the disease, or the progression of the disease.

In the treatment method of the present invention, an effective amount of the controlled wound dressing is administered to an individual in need thereof. Specifically, the amount varies depending on the purpose of administration, the health and physical condition of the individual to be treated, age, the classification group of the individual to be treated (e.g., human, non-human primate, primate, etc.), the evaluation of the medical situation by the treating clinician, and other relevant factors. It is expected that the amount will be within a relatively wide range, which can be determined by routine experiments.

The controlled wound dressing of the present invention can be used via an external application route and can be administered with or without the addition of a modifier.

In one embodiment, the dosage of the fusion protein in the regulated wound dressing may be about 5 μM or more, preferably about 7.752 μM or more, more preferably about 9.304 μM or more, and even more preferably 10 μM or more; in one embodiment, the dosage of the fusion protein in the regulated wound dressing may be about 100 μM or less, preferably about 80 μM or less, even more preferably about 65 μM or less, even more preferably about 63.8 μM or less, and even more preferably 60 μM or less; in one embodiment, the dosage of the fusion protein in the regulated wound dressing may be about 5 μM to about 100 μM, preferably about 7.752 μM to about 80 μM, even more preferably about 9.304 μM to about 63.8 μM, and even more preferably about 10 μM to about 60 μM; by administering the above effective dose, the regulated wound dressing can remove bacteria inside and outside the cells and latent bacteria in the autophagosome, provide multiple wound protection and prevent bacterial infection. For specific experimental data, please refer to the following examples.

Several embodiments and comparative examples are given below to illustrate the controllable wound dressing of the present invention, but they are not intended to limit the present invention. Anyone skilled in the art can make various modifications and improvements without departing from the spirit and scope of the present invention. In addition, the materials used below are all commercially available and easily available.

Experiment 1: Verification of the thrombin release mechanism

Please refer to Figure 4. In order to verify whether the designed release mechanism is effective and to understand the antibacterial duration, the present thrombin cleavage experiment was conducted to observe the release amount of the antibacterial agent over time. In order to simulate the condition of the wound, the present experiment used the thrombin concentration in the wound and combined it with the enzyme kinetic model to predict the antibacterial agent release curve at different cut positions. It can be inferred that the release amount of the antibacterial agent reaches the maximum value after about 12 hours.

FIG4 is a simulation diagram of the antibacterial release curve of an embodiment of the present invention. As shown in FIG4 , the blue line S is the full-length peptide (full-length peptide of fusion protein), and its concentration decreases with time, indicating that the fusion protein is cleaved by thrombin, resulting in a decrease in the concentration of the full-length peptide; the purple line A is the collagen binding domain, and the green line B is the linking peptide (including the first enzyme cleavage site), and its concentration increases with time, indicating that the fusion protein is cleaved by thrombin and releases the collagen binding domain. The red line C represents the antimicrobial peptide, whose concentration increases with time, indicating that the antimicrobial peptide is released after the fusion protein is cleaved by thrombin, and the concentration of the antimicrobial peptide increases and reaches the maximum value after about 12 hours; the pink line AB represents the collagen binding domain-linking peptide, and the yellow line BC represents the linking peptide-antimicrobial peptide, whose concentration increases first and then decreases, indicating the process of thrombin cleavage.

Experiment 2: Verification of the antimicrobial ability of antimicrobial peptides

In order to detect the antibacterial ability of the antimicrobial peptide DPK-060 and the minimum effective concentration in the regulated wound dressing, this experiment conducted a minimum inhibitory concentration (MIC) test, the results of which are shown in Table 1 below:

[Table 1] Antimicrobial peptides MIC ₉₀ (μM) E. coli S. aureus DPK-060 7.752 9.304 In Table 1, MIC ₉₀ represents the lowest concentration of antimicrobial peptide that can inhibit 90% microbial growth. As can be seen from the table above, the MIC ₉₀ of antimicrobial peptide DPK-060 against E. coli and S. aureus are 7.752 μM and 9.304 μM, respectively.

Experiment 3: Verify that antimicrobial peptides are not cytotoxic

According to the safety standard of ISO-10993, hemolysis should be less than 2% and the reduction of cell viability should not exceed 30%.

Please refer to FIG. 5 , which is a hemolytic test result of the antimicrobial peptide DPK-060 according to an embodiment of the present invention. As shown in the figure, the present invention uses mouse blood cells in the hemolytic test and finds that the antimicrobial peptide DPK-060 is not hemolytic even when the concentration is more than five times higher than MIC ₉₀ (63.8 μM).

Please refer to FIG. 6 , which is a cytotoxicity test result of the antimicrobial peptide DPK-060 according to one embodiment of the present invention. As shown in the figure, in the cytotoxicity test, the present invention uses a macrophage cell line (U937) and an MTT test to find that the antimicrobial peptide DPK-060 does not harm macrophages even if the MIC is more than five times higher than MIC ₉₀ (63.8 μM).

Experiment 4: Preparation of TAT-eGFP and eGFP proteins

In order to prepare TAT (penetrating peptide)-eGFP and eGFP proteins for cell penetration test, the target gene was cloned into pET15b plasmid and then transformed into E. coli BL21 strain. The expression of the gene was induced by IPTG. Finally, FPLC was performed to purify eGFP and TAT-eGFP proteins. SDS-PAGE and Coomassie blue staining were used to confirm whether the target protein was successfully expressed.

Please refer to FIG. 7 and FIG. 8 . FIG. 7 shows the expression of TAT-eGFP according to one embodiment of the present invention, and FIG. 8 shows the expression of eGFP according to one embodiment of the present invention. As shown in the figures, TAT-eGFP and eGFP proteins were successfully prepared in this experiment.

Experiment 5: Verification of penetrating peptide penetration ability

In another embodiment of the present invention, the antimicrobial peptide in the fusion protein is bridged to the penetrating peptide to prepare the antimicrobial penetrating peptide. To verify the penetrating ability of the penetrating peptide, the penetrating peptide is bridged to green fluorescent protein (GFP) in this experiment, and the penetrating peptide is co-cultured with a macrophage cell line (U937) and the penetrating peptide bridged with GFP to facilitate observation of the penetration of the penetrating peptide.

Please refer to FIG. 9 , which is a result of testing the cell penetration ability of the penetrating peptide according to one embodiment of the present invention. As shown in the figure, the experimental results clearly observed that GFP entered the macrophage cell line U937, confirming the penetration ability of the penetrating peptide.

Experiment 6: Verification of intracellular separation mechanism

Since bacteria can lurk in the lysosome of the cell and further lurk in the phagosome or autophagosome through an inhibitory mechanism, the antimicrobial penetrating peptide of the present invention can enter the cell through endocytosis and reach the lysosome (or phagosome, autophagosome) to fight against the latent bacteria. In this experiment, the antimicrobial peptide and the penetrating peptide contain a cleavage site of cathepsin S, which is unique to lysosomes, and are constructed in a manner that can be treated with cathepsin S to cleave the cathepsin S cleavage site to separate the antimicrobial peptide and the penetrating peptide, and release the antimicrobial peptide to kill bacteria.

In order to verify that the cathepsin S cleavage site designed in this experiment is effective, molecular docking was used to study the preference of cathepsin S. Please refer to Table 2 below. Since its binding affinity is mainly affected by the amino acid at the P2 position of the cleavage site; this experiment systematically changed the P2 amino acid in the P2-P1-P1' tripeptide and used AutoDock to evaluate its affinity for the cathepsin S active pocket.

[Table 2] Cathepsin S cleavage site DPK-060 (antimicrobial peptide) P4 P3 P2 P1 P1' P2' P3' P4' A A L G G K H K In Table 2, cathepsin S cleavage sites were designed according to their cleavage preferences, and the substrate amino acids were numbered using the Schechter and Berger nomenclature; the cleavage site of cathepsin S is between P1-P1'.

Please refer to Figure 10, which is the result of the docking of cathepsin S and the tripeptide according to one embodiment of the present invention. As shown in the figure, blue is the tripeptide LGG and pink is the tripeptide VGG. Cathepsin S is colored according to the surface hydrophobicity (red indicates the most hydrophobic and blue indicates the most hydrophilic). From the above results, it can be seen that when the amino acid at the P2 position is hydrophobic, its conformation is very similar and is located at the target binding site, indicating that the cleavage site designed in this experiment is reasonable.

Experiment 7: Testing the optimal ratio of the absorption layer

One embodiment of the present invention provides a controlled wound dressing, comprising a polyurethane layer, an absorbent layer, and a collagen layer; wherein the absorbent layer may be a composite material formed by ion crosslinking of alginate (ALG) and chitosan (CHI). In order to understand the optimal ratio of the absorbent layer combination, this experiment used 1% alginate solution and 1% chitosan solution, and prepared controlled wound dressings with different ratios and tested their swelling rates. The results are shown in Table 3 below.

[Table 3] Absorption layer swelling rate test results ALG:CHI (1%:1%) 4:1 3:1 2:1 1:1 1:2 1:3 1:4 Average swelling rate (%) (n=3) 353.5 668.0 703.1 1403.6 1637.8 1926.8 1762.3 Standard deviation of swelling rate (n=3) 54.7 53.1 112.6 215.4 69.9 69.7 22.1 In Table 3, swelling rate (%) = [(W _s -W _d )/W _d ] x 100% (W _d : weight of dry dressing; Ws : weight of dressing after absorption.)

From the table above, we can see that when the ratio of alginate to chitosan is 1:3, the best absorption capacity can be achieved, and it has an absorption capacity of 19 times its own weight.

In summary of the above results, the fusion protein of the present invention comprises collagen, antimicrobial peptide and a first enzyme cleavage site, and may further comprise a penetrating peptide and a second enzyme cleavage site. By linking the collagen binding domain with the antimicrobial peptide (or antimicrobial penetrating peptide), the antimicrobial peptide and antimicrobial penetrating peptide can be attached to the collagen, thereby avoiding direct exposure to the protein degradation environment, reducing the risk of being degraded by other proteases, regulating the activity of the antimicrobial peptide and antimicrobial penetrating peptide, and at the same time increasing the adhesion of the antimicrobial peptide and antimicrobial penetrating peptide, thereby clearing bacteria inside and outside the cell and latent bacteria in the autophagosome, providing multiple wound protection and preventing bacterial infection.

The embodiments described above are only for illustrating the technical ideas and features of the present invention, and their purpose is to enable persons skilled in the art to understand the contents of the present invention and implement them accordingly. They cannot be used to limit the patent scope of the present invention. That is, any equivalent changes or modifications made based on the spirit disclosed by the present invention should still be included in the patent scope of the present invention.

1: Collagen 2: Antimicrobial peptide 3: First enzyme cleavage site 4: First enzyme 5: Penetrating peptide 6: Second enzyme cleavage site 7: Second enzyme 8: Collagen layer 9: Absorption layer 10: Polyurethane layer

FIG1 is a schematic diagram of a fusion protein according to an embodiment of the present invention.

FIG. 2 is a schematic diagram and release mechanism of an antimicrobial penetrating peptide according to another embodiment of the present invention.

FIG. 3 is a schematic diagram of a controllable wound dressing according to an embodiment of the present invention.

FIG. 4 is a simulation diagram of the antibacterial release curve of an embodiment of the present invention.

FIG5 is a hemolytic test result of the antimicrobial peptide DPK-060 according to an embodiment of the present invention.

FIG6 is a cytotoxicity test result of the antimicrobial peptide DPK-060 according to an embodiment of the present invention.

FIG. 7 shows the expression of TAT-eGFP according to one embodiment of the present invention.

FIG8 shows the expression of eGFP according to an embodiment of the present invention.

FIG. 9 is a result of testing the cell penetration ability of penetrating peptides according to an embodiment of the present invention.

FIG. 10 shows the result of docking tissue proteinase S with a tripeptide according to an embodiment of the present invention.

1: Collagen

2: Antimicrobial peptides

3: First enzyme cleavage site

4: First enzyme

Claims (11)

A fusion protein comprises collagen and an antimicrobial peptide, wherein a first enzyme cleavage site is contained between the collagen and the antimicrobial peptide, and the fusion protein is constructed in such a way that the collagen and the antimicrobial peptide can be separated by cleaving the first enzyme cleavage site through treatment with a first enzyme. The fusion protein as described in claim 1, wherein the first enzyme treatment is performed by thrombin. The fusion protein as described in claim 1, wherein the antimicrobial peptide is bridged to the penetrating peptide. The fusion protein as described in claim 3, wherein a second enzyme cleavage site is included between the antimicrobial peptide and the penetrating peptide, and is constructed in a manner that the antimicrobial peptide and the penetrating peptide can be separated by being treated with a second enzyme to cleave the second enzyme cleavage site. The fusion protein as described in claim 4, wherein the second enzyme treatment is performed by cathepsin. A controlled wound dressing comprises a polyurethane layer, an absorption layer, and a collagen layer, wherein the absorption layer is located between the polyurethane layer and the collagen layer, and the collagen layer comprises the fusion protein as described in any one of claims 1 to 5. A controlled wound dressing as described in claim 6, wherein the absorbent layer comprises alginate and chitosan. The controlled wound dressing as described in claim 7, wherein the ratio of the alginate to the chitosan is 4:1 to 1:4. A regulated wound dressing as described in claim 6, wherein the dosage of the fusion protein in the collagen layer is 5 μM to 100 μM. A fusion protein is used for preparing a regulated wound dressing, wherein the fusion protein comprises a fusion protein as described in any one of claims 1 to 5. When the regulated wound dressing contacts a wound, the antimicrobial peptide is released inside and outside the cells through enzyme treatment in the wound to achieve an antimicrobial effect. The use as described in claim 10, wherein when the regulated wound dressing contacts the wound, the antimicrobial peptide is released into the autophagosome through enzyme processing in the autophagosome to eliminate latent bacteria.