Materials with anti-inflammatory and / or antimicrobial properties

Abstract

The present invention relates to a material comprising a thrombin-derived peptide and a poly (lactic acid-co-glycolic acid) polymer. The materials have antimicrobial (e.g., antibacterial) and / or anti-inflammatory properties. Also provided are medical products (e.g., sutures) comprising the materials, and methods of using the medical products to prevent and / or inhibit inflammation and / or infection or treat wounds in a subject.

Description

Technical Field

[0001] This invention relates to materials comprising thrombin-derived peptides and poly(lactic-co-glycolic acid) (PLGA) polymers. The materials possess antimicrobial (e.g., antibacterial) and / or anti-inflammatory properties and are useful when incorporated into medical products (e.g., sutures). Background Technology

[0002] Of the more than 300 million surgical procedures performed globally each year, nearly 10% result in surgical site infections (SSIs). SSIs impose a significant clinical and economic burden. While many factors contribute to SSIs, preoperative, intraoperative, and postoperative preventative measures can reduce their incidence. In addition to patient-specific endogenous factors, the causes of SSIs vary depending on anatomy, surgical procedure, and exogenous factors. Bacterial contamination is a controllable cause. In hospitals, despite routine disinfection and other preventative measures, infection can still occur even under aseptic surgical conditions due to the spread of bacteria from the patient's own microbiota. For example, up to 60% of bacteria recovered from infected surgical wounds have been reported to be antibiotic-resistant.

[0003] For medical implant materials (such as sutures), they provide ideal surfaces for bacterial adhesion and growth, and may be involved in the development of SSI pathogenesis. Furthermore, in the case of bacterial adhesion, the release of bacterial products (such as lipoteichoic acid (LTA) and LPS) can stimulate inflammation near the biomaterial. Therefore, efforts have been made to functionalize sutures to prevent bacterial colonization and subsequent infection. However, common anti-infective sutures containing antiseptic molecules such as triclosan have shown insufficient efficacy and may induce bacterial resistance. In addition, triclosan can cause side effects such as hormone interference. Another serious problem is the declining effectiveness of antibiotics and other antimicrobial agents due to the development of antimicrobial resistance (AMR). The development of resistance is particularly important in surgical procedures, where the combination of widespread antibiotic use, a high risk of systemic transmission, and bacterial sepsis leads to a high frequency of infections, necessitating the use of antibiotics as a "last resort."

[0004] Furthermore, during initial contact with the tissue, the given biological material itself may induce an immune response, which could lead to dysregulation of the inflammatory response, resulting in inefficient host defense and making the given biological material susceptible to infection.

[0005] Therefore, there is an urgent need for materials used in medical products to improve postoperative medical outcomes in reducing or preventing bacterial infections and related inflammation. Summary of the Invention

[0006] This disclosure provides materials comprising thrombin-derived peptides and poly(lactic-co-glycolic acid) (PLGA) polymers. These materials possess antimicrobial (e.g., antibacterial) and / or anti-inflammatory properties and are particularly useful when incorporated into medical products (e.g., sutures).

[0007] Interestingly, this invention discloses the binding of PLGA polymers to thrombin-derived peptides, which allows for the slow, continuous release of the peptides in vivo. This, in turn, allows the materials of this invention to exert their antibacterial and / or anti-inflammatory properties over an extended period of time. Furthermore, the release profile of the peptides from the polymer can be controlled by adjusting the ratio of lactic acid to glycolic acid in the PLGA polymer. Therefore, this allows for tailoring the release timing for specific applications.

[0008] Furthermore, the medical product of this invention is stable during storage and provides a continuous and sufficiently high release of peptides when applied to the relevant site of the body. This ensures the effective delivery of the polymer's antibacterial and anti-inflammatory effects to the treated tissue.

[0009] Furthermore, PLGA provides a microenvironment that promotes the activity of thrombin-derived peptides. Without being bound by theory, it is believed that PLGA provides an acidic microenvironment leading to the protonation of thrombin-derived peptides, which can promote their activity, for example, by enhancing antibacterial and / or anti-inflammatory activity.

[0010] In some aspects of this disclosure, a material is provided comprising a poly(lactic-co-glycolic acid) (PLGA) polymer and a peptide comprising or composed of the following amino acid sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16, where X3, X7, X10, X11, X13, X15, and X16 are any standard amino acids. X8 and X12 can be any amino acid. X1, X6, and X14 are G, A, V, L, I, P, F, M, Y, or W, and X2, X4, X5, and X9 are R, K, or H. The peptides described therein have a length of 10 to 30 amino acid residues.

[0011] In some aspects of this disclosure, medical products comprising materials as described herein are provided.

[0012] In some aspects of this disclosure, a method for producing a medical product as described herein is provided, the method comprising the steps of: submerging a starting material comprising or composed of PLGA in a coating solution comprising a soluble peptide as described herein, and subsequently drying the starting material.

[0013] In some aspects of this disclosure, a method is provided for preventing and / or inhibiting inflammation and / or infection in a subject in need, the method comprising exposing the subject to or implanting the subject with a medical product as described herein, and maintaining the exposure for a period of time.

[0014] In some aspects of this disclosure, a method for treating a wound in a subject in need is provided, the method comprising contacting the wound with or implanting a medical product as described herein into the wound.

[0015] In some aspects of this disclosure, medical products as described herein are provided for the prevention and / or inhibition of inflammation and / or infection in body parts of a subject.

[0016] In some aspects of this disclosure, medical products as described herein are provided for treating wounds in subjects in need. Attached Figure Description

[0017] Figure 1 Coating conditions, antimicrobial properties, and release curves of TCP-25 peptide-coated polyglactin sutures. Effects of coating conditions on peptide loading and antimicrobial activity of polyglactin sutures. Sutures were coated with TCP-25 under different coating concentrations (a), coating times (b), and coating temperatures (c). Peptides were eluted from the coated sutures, and protein concentrations were estimated (left panel). To investigate the effect on antimicrobial activity, the suture eluent was used for radial diffusion assays (RDA), as shown in the right panel. Diameter of the clear zone (excluding the 4 mm pore) represents the inhibitory effect of the released peptide (mean ± standard error [SEM] is shown for a, b, and c, n=3). (d) Cumulative release of TCP-25 from sutures in vitro. Conditions mimicking surgical wounds were obtained using transwell inserts, as shown, and TCP-25 release was estimated. (e) In vivo release of TCP-25 from sutures. Sutures coated with tetramethylrhodamine (TAMRA)-labeled TCP-25 were placed subcutaneously in SKH-1 mice. Peptide release was monitored long-term at 1, 6, 24, and 72 hours using IVIS imaging to quantify fluorescence. Superimposed thermograms obtained from the emitted light are shown. Bar graphs show the measured radiation emitted from the region of interest (showing averages, n=4). Dashed lines indicate the region of interest. (f) Scanning electron microscopy shows the surface of the pigliptin sutures before and after TCP-25 coating. P < 0.05; P < 0.001.

[0018] Figure 2QCM-D, coarse-grained simulation, and O-PTIR analyses show the peptide-polyglycine interaction. (a) QCM-D monitored the binding of TCP-25 to Vicryl fibers. Finely diced fibers were dissolved in ethanol and dropped onto SiO2 coated with poly-L-lysine to form a confluent fiber mat. The binding was monitored by frequency variation relative to pure MQ water. F (–100 ± 27 Hz) and dissipation variation ( D)(+42 ±11)1 The binding of the peptide to the fiber-functionalized sensor was confirmed. In contrast, a control experiment ruled out the possible adsorption of the peptide on the underlying poly-L-lysine surface, in which the interaction of the peptide with a fiber-free poly-L-lysine-functionalized SiO2 surface was monitored under the same experimental conditions, showing only a small frequency shift of -4 ± 1 Hz. Measurements were performed at room temperature (n=3). (b) Coarse-grained simulation of Vicryl assembly with TCP-25. (b) A 1 µs CG self-assembly simulation was performed on a 100-mer polygliptin 910 model with 50 copies to construct the Vicryl polymer model. Subsequently, 10 copies of TCP-25 were added to the system, and three independent 10 µs simulations were performed at 320 K. The figure shows the initial and final snapshots of one of the simulations. (c) (top) Minimum distance between the TCP-25 peptide and the Vicryl polymer surface in all three simulations. The thick line shows the mean of the 10 peptides, and the shaded area represents the standard deviation. (Bottom) Solvent-accessible surface area (SASA) of the Vicryl polymer, compared with simulations using TCP-25 (grey) and without TCP-25 (black). The thick line shows the average of three repeated simulations, and the shaded area represents the standard deviation. The probe radius used for the SASA calculation was 0.26 nm. (d) (Top) Average percentage of contact between each subunit of p-gliptin and the peptide at three time points during the simulation. COO, carboxyl terminus; G1 to G9, glycolide subunit; L6, lactide subunit; G10-OH, glycolide with a hydroxyl terminus. (Bottom) The same analysis was performed on each residue of the peptide. The distance cutoff value used for the contact measurements was 0.6 nm. (e) (Top) Magnified snapshot showing the entanglement of the TCP-25 peptide with the p-gliptin polymer at the end of one simulation. (Bottom) SASA of the peptide throughout the simulation, averaged for 10 peptides and 3 simulations. (f) With 2 c ¹ Normalized O-PTIR spectra of spectral data points, uncoated (control, bottom line in the figure), TCP-25 coated, and washed TCP-25 coated sutures obtained by 5 averages. Dashed lines show the TCP-25 coated sutures at 1656 c. The location of the characteristic band at ¹. (g) O-PTIR scale map is derived from 1656 c. The image obtained at ¹ (numerator of the ratio graph) and divided by 1760 c Image obtained at ¹ (denominator of ratio plot). Light gray represents the distribution of TCP-25 on the suture surface.

[0019] Figure 3 Structural analysis of TCP-25 eluted from coated sutures. (a) High-performance liquid chromatography (HPLC) analysis of fresh TCP-25 or TCP-25 eluted from coated sutures. (b) Circular dichroism (CD) spectra of TCP-25 from coated sutures alone or after incubation with LPS (left panel). The α-helix content of TCP-25 estimated based on the molar ellipticity at 222 nm is shown (right panel). Data are expressed as mean ± SEM (n=3). P-values ​​were determined using an unpaired t-test. The top line in the figure represents TCP-25, the lower line represents +2, and so on. (c) Intrinsic fluorescence spectrum of 10 M TCP-25 from coated sutures, showing the shift in peptide emission maxima after incubation with different concentrations of LPS (left panel). (d) Maximum emission wavelength of TCP-25 ( max The data were fitted to a function of different concentrations of LPS. Data are expressed as mean ± SEM (n=3). P < 0.05.

[0020] Figure 4 The in vitro and in vivo antibacterial effects of TCP-25 sutures. (a) Bioluminescent Staphylococcus aureus ( Staphylococcus aureus ) or Pseudomonas aeruginosa ( Pseudomonas aeruginosa (a) Bacteria were incubated with TCP-25 coated sutures or control sutures and imaged using IVIS. Representative images are shown (n=3). (b) Bioluminescence emitted by bacteria after treatment with TCP-25 sutures. Bioluminescent versions of Staphylococcus aureus ( S. aureus ) or Pseudomonas aeruginosa ( P. aeruginosa(c) Representative images showing bacterial viability assay results. Staining was performed using the LIVE / DEAD Baclight bacterial viability kit and imaging was performed using fluorescence microscopy. White indicates viable bacteria, and light gray indicates dead bacteria (n=3). (d) Scanning electron microscopy (SEM) images showing bacterial morphology after contact with TCP-25 sutures. (e) In vivo infection imaging showing the antibacterial properties of TCP-25 sutures in a suture-infected mouse model. Uncoated control sutures or TCP-25 sutures were subcutaneously implanted on the left or right side, respectively, and contaminated with bioluminescent Staphylococcus aureus (S. aureus). S. aureus (f) Line graphs show bacterial bioluminescence emission at 1, 6, 24, 48, and 72 hours post-infection. Data are expressed as mean SEM (n=5). P-values ​​were calculated using two-way ANOVA and Sidak tests. (g) Bar graphs show bacterial counts in tissue surrounding control or TCP-25 sutures. Sutures were implanted and infected with Staphylococcus aureus (S. aureus). S. aureus Following contamination, mice were sacrificed 72 hours later, and tissue adjacent to the sutures was collected for CFU counting using a viable bacterial count assay. Data are presented as mean ± SEM (n=5 mice per group). P-values ​​were calculated using an unpaired t-test. (h) In vivo IVIS imaging of infection and drug in mice. To combine in vivo drug localization with bioluminescent bacterial imaging, the sutures were coated with fluorescently labeled TCP-25. Representative images show bioluminescence (lum) and TCP-25 TAMRA fluorescence (flu) 6 hours after suture implantation. (n=5). P < 0.05; P < 0.01; P < 0.001.

[0021] Figure 5In vitro and in vivo effects of TCP-25 suture on endotoxin-induced inflammation. (a) NF-κB and AP-1 activation analysis by quantifying alkaline phosphatase secreted in THP1-Xblue™-CD14 cells (top bar). Cell viability was determined using an MTT assay (bottom bar). Lysed cells served as a positive control. Data are presented as mean ± SEM (n=3). P-values ​​were calculated using one-way ANOVA. (b) Long-term imaging of inflammation in NF-κB reporter mice. TCP-25 or control sutures were implanted on the left or right side, respectively, and contaminated with LPS. In vivo bioimaging of NF-κB reporter gene expression was performed using the IVIS Spectrum system. Representative thermal overlay photographs show bioluminescent signals at 3 and 24 hours post-implantation. Bar charts show the analysis of NF-κB reporter mouse emission. Data are presented as mean SEM (n=5). P-values ​​were determined by unpaired t-test. (c) Levels of TNF-α and IL-6 cytokines in fluid extracted from the implantation suture 24 hours post-implantation. Data are expressed as mean SEM (n=5 mice per group). P-values ​​were calculated using an unpaired t-test. P < 0.05, P < 0.01, P < 0.001. NS, no significance.

[0022] Figure 6 The anti-biofilm effect of TCP-25 coated sutures. (a) Representative fluorescence microscopy images after live / dead staining show Staphylococcus aureus (S. aureus) adhering to the suture surface. S. aureus ) or Pseudomonas aeruginosa ( P. aeruginosa Biofilm (white, live bacteria; light gray, dead bacteria). TCP-25 coated sutures or control sutures were added to the wells of a biofilm microtiter plate and allowed to grow for 48 hours. The sutures were stained using the LIVE / DEAD Baclight bacterial viability kit and imaged using a fluorescence microscope. Vicryl ® Plus was used as a baseline control. The bar chart shows the estimated total number of viable bacteria on the suture-adhered biofilm using viable count analysis. Data are expressed as mean ± SEM (n=3). P-values ​​were analyzed using one-way ANOVA. (b) Crystal violet staining shows the measurement of biofilm quality. Vicryl ® Plus was used as a baseline control. Data are expressed as mean ± SEM (n=3). P-values ​​were determined by one-way ANOVA. (c) Staphylococcus aureus growing on TCP-25 coated sutures or control sutures. S. aureus ) or Pseudomonas aeruginosa ( P. aeruginosa(d) Scanning electron microscopy of the biofilm. Representative SEM images show the suture surface. Arrows indicate bacterial biofilms formed on the suture surface. Bacterial viability analysis shows the anti-biofilm effect of the TCP-25 coated suture. Staphylococcus aureus was treated with TCP-25 coated suture or control suture. S. aureus ) and Pseudomonas aeruginosa ( P. aeruginosa Mature biofilms were stained using the LIVE / DEAD Baclight bacterial viability kit and then examined under a fluorescence microscope. White indicates live bacteria, and light gray indicates dead bacteria. Representative images (n=3) are shown. Vicryl ® Plus is used as a benchmark. P < 0.05, P < 0.01, P < 0.001. NS, no significance.

[0023] Figure 7 TCP-25 fragmentation induced by human neutrophil elastase in TCP-25 sutures. (a) Peptide fragmentation profile of TCP-25 after treatment with human neutrophil elastase. TCP-25 sutures were incubated with human neutrophil elastase and analyzed by nano-LC-MS / MS. The figure shows the sequences of the major peptides and the number (n=2) successfully identified by mass spectrometry at 0, 30, and 180 min. (b) Major peptides of TCP-25 (SEQ ID NO:1) obtained from coated sutures after digestion with human neutrophil elastase. These peptides have been reported to exhibit antibacterial effects. (c) Antibacterial activity against Escherichia coli was assessed by RDA, analyzing the antibacterial activity of peptide fragment products obtained after treatment of TCP-25 sutures with human neutrophil elastase. Bar charts show the quantification of clear areas. Data are presented as mean SEM (=3). (d) Anti-inflammatory activity of peptide fragments obtained after treatment of TCP-25 sutures with human neutrophil elastase. Activation of NF-κB and AP-1 was assessed in THP1-Xblue™-CD14 reporter cells. Data are presented as mean ± SEM (=3). P < 0.001. NS, no significance.

[0024] Figure 8Determination of tensile strength, hemolytic activity, and effects of long-term storage of TCP-25 sutures. (a) Effect of TCP-25 coating on suture tensile strength. The tensile strength of freshly coated, un-implanted (left panel) or tissue-implanted (right panel) sutures was measured. To investigate the effect of the TCP-25 coating on tensile strength in vivo, sutures were subcutaneously implanted in mice for 4 days. Data are presented as mean SEM (n=5). P-values ​​were calculated using the Mann-Whitney U test. (b) Hemolytic activity of control and TCP-25 coated sutures. Hemolysis was determined using human blood. The left bar chart shows the hemolytic activity of a 1 cm suture, and the right bar chart shows the hemolytic activity of a 10 cm suture (showing mean ± SEM, n=3). NS, no significant difference. (c) HPLC analysis of TCP-25 eluted from coated sutures (control, fresh TCP-25) or after long-term storage. TCP-25 sutures were stored at room temperature for 18 months, after which the eluted peptides were used for HPLC analysis. (d) Western blot analysis of TCP-25 eluted from coated sutures after 18 months of storage at room temperature (n=3). 1, 2, and 3 represent different sutures. (e) CD spectral results of TCP-25 from stored sutures with and without LPS are shown. Fresh TCP-25 was used as a control for comparison. (f) Analysis using Escherichia coli (E. coli) E. coli Radial diffusion assays were used to assess the effect of suture storage on the antimicrobial activity and release of TCP-25. Fresh TCP-25 was used as a control for comparison. Results show quantification of the clear area. Data are presented as mean SEM (n=3–4). NS, no significant difference.

[0025] Figure 9 TCP-25 sutures targeted inflammation induced by human wound fluid. (a) TCP-25 sutures reduced the pro-inflammatory capacity of human wound fluid in vitro. THP-1-XBlue™-CD14 cells were used in the reporter assay. Cells were stimulated with acute wound fluid (AWF) and chronic wound fluid (CWF) derived from infected wounds in human patients in the presence of TCP-25 sutures or control sutures. Activation of NF-κB and AP-1 was assessed by determining the amount of secreted alkaline phosphatase produced by the reporter cells. Data are presented as mean SEM (n=6). P-values ​​were calculated using paired t-tests. (b) TCP-25 sutures reduced the pro-inflammatory capacity of human wound fluid in vivo. TCP-25 coated sutures were contaminated with human chronic wound fluid and implanted into the backs of NF-κB reporter mice. Non-invasive IVIS imaging was performed to visualize NF-κB activation. Data are presented as mean SEM (n=5). P-values ​​were calculated using the Mann-Whitney U test. P < 0.05, P < 0.01.

[0026] Figure 10 SDS-PAGE analysis of PLGA particles coated with TCP-25. The results are further described in Example 2.

[0027] Figure 11 Detection and localization of TCP-25 on PLGA particles. The coated particles were imaged using a fluorescence microscope to detect TCP-25-TAMRA (light gray). PLGA is shown as dark gray in the image. The results are further described in Example 2.

[0028] Figure 12 Bacterial growth assay on a medium containing dried TCP-25 coated PLGA particles. Viable cell counts (VCA) were performed on different bacteria incubated with TCP-25 coated PLGA particles (TCP-25PLGA) or uncoated PLGA particles (control), followed by inoculation onto TH agar plates. Results are further described in Example 2.

[0029] Figure 13 Antimicrobial effect of dried TCP-25 coated PLGA particles. Radial diffusion assay showed that TCP-25 coated PLGA particles were effective against Gram-negative Escherichia coli (E. coli). E. coli (left) and Gram-positive Staphylococcus aureus ( S. aureus (Right) shows antibacterial activity. The results are further described in Example 2. Invention Details

[0031] definition

[0032] In this specification, unless otherwise stated, "a" or "an" means "one or more species".

[0033] As used herein, the term “about” when referring to numerical values ​​means + / - 10%, preferably + / - 5%, and more preferably + / - 1%.

[0034] As used herein, the term "sequence identity" refers to the percentage of identical amino acids or nucleotides between the aligned candidate sequence and the reference sequence. Therefore, a candidate sequence with 80% amino acid identity to the reference sequence requires that, after alignment, 80% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence. The identity in this invention is determined with the aid of computer analysis, for example, but not limited to: the Clustal Omega computer alignment program for peptide sequence alignment (Sievers et al. (2011 October 11) Molecular Systems Biology 7:539, PMID: 21988835; Li et al. (2015 April 06) Nucleic Acids Research 43(W1):W580-4 PMID: 25845596; McWilliam et al., (2013 May 13) Nucleic Acids Research 41(Web Server issue):W597-600 PMID:23671338) and the suggested default parameters therein. The Clustal Omega software is available from EMBL-EBI (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ). Using this program and its default settings, align the mature (biologically active) portions of the query peptide and the reference peptide. Calculate the number of fully conserved residues and divide by the length of the reference peptide. The MUSCLE or MAFFT algorithm can be used for nucleotide sequence alignment. Sequence identity can be calculated in a similar manner to that shown for amino acid sequences. Therefore, the sequence identity provided in this paper is calculated over the entire length of the reference sequence.

[0035] The term "standard amino acid" refers to any one of the twenty genetically encoded amino acids commonly found in naturally occurring peptides. Standard amino acids are represented herein by their IUPAC single-letter and three-letter codes. The term "standard amino acid" is used both to refer to free standard amino acids and to standard amino acids incorporated into peptides. For the peptides shown, each encoded amino acid residue (where appropriate) is represented by a single letter.

[0036] As used herein, the term "any amino acid" refers to a compound chemically classified as an amino acid. Therefore, this term includes the twenty standard amino acids and their corresponding stereoisomers in their "D" form (compared to the natural "L" form), ω-amino acids, other naturally occurring amino acids, unconventional amino acids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, etc.), and chemically derived amino acids.

[0037] As used herein, the term "treatment" refers to any type of treatment or prevention of disease, including improvement of a subject's disease (e.g., one or more symptoms), delay of disease progression, delay of symptom onset, or slowing of symptom progression. Treatment can also be amenable or curative. Therefore, the term "treatment" also includes preventative treatment for an individual to prevent the onset of symptoms.

[0038] As used herein, the term "hydrogel" refers to a continuous phase of an aqueous solution and a hydrophilic polymer capable of swelling upon contact with water. A hydrogel comprises a nanostructure formed from said polymer and water, and typically contains more than 90% water. Hydrogels are generally transparent or translucent, regardless of their degree of hydration. Hydrogels are generally distinguishable from hydrocolloids, which typically comprise a hydrophobic matrix containing dispersed hydrophilic particles. The flow point of a hydrogel is typically at least 10 Pa, for example at least 15 Pa, for example between 10 and 80 Pa, for example between 40 and 60 Pa.

[0039] Material

[0040] This disclosure relates to materials comprising PLGA polymers as described elsewhere herein and peptides as described elsewhere herein, said materials having antibacterial and / or anti-inflammatory properties, and thus being particularly useful when included in medical products such as sutures, microparticles or nanoparticles.

[0041] In some aspects, a material is provided comprising a poly(lactic-co-glycolic acid) (PLGA) polymer and a peptide comprising or composed of the following amino acid sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16, where X3, X7, X10, X11, X13, X15, and X16 are any standard amino acids. X8 and X12 can be any amino acid. X1, X6, and X14 are G, A, V, L, I, P, F, M, Y, or W, and X2, X4, X5, and X9 are R, K, or H. The peptides described therein have a length of 10 to 30 amino acid residues.

[0042] In some embodiments, the material is anti-inflammatory. In some embodiments, the material is antibacterial. In some embodiments, the material is both anti-inflammatory and antibacterial.

[0043] In some embodiments, the material is capable of binding lipopolysaccharide (LPS). In some embodiments, the LPS is bacterial LPS.

[0044] In some embodiments, the material is biodegradable and / or bioabsorbable.

[0045] In some embodiments, the peptide is bound to the PLGA polymer. In some embodiments, the peptide is coated on the PLGA polymer. In some embodiments, the peptide is dissolved (e.g., completely or partially dissolved) in the PLGA polymer. In some embodiments, the peptide is dispersed between the fibers of the PLGA polymer.

[0046] In some embodiments, the material comprises the peptide at a concentration of at least 10 µg / cm², such as at least 25 µg / cm², such as at least 50 µg / cm², such as at least 75 µg / cm², such as at least 100 µg / cm², such as at least 150 µg / cm², such as at least 200 µg / cm², such as at least 250 µg / cm², such as at least 300 µg / cm², such as at least 350 µg / cm², such as at least 400 µg / cm², such as at least 450 µg / cm², such as at least 500 µg / cm².

[0047] In some embodiments, the material comprises the peptide at concentrations of 10 µg / cm² to 10000 µg / cm², such as 25 µg / cm² to 10000 µg / cm², such as 25 µg / cm² to 7500 µg / cm², such as 25 µg / cm² to 5000 µg / cm², such as 25 µg / cm² to 2500 µg / cm², such as 50 µg / cm² to 2000 µg / cm², such as 75 µg / cm² to 1500 µg / cm², such as 100 µg / cm² to 1000 µg / cm², such as 150 µg / cm² to 750 µg / cm², such as 200 µg / cm² to 500 µg / cm², such as 250 µg / cm² to 400 µg / cm².

[0048] In some embodiments, for every 1 mg PLGA in the material, the material contains at least 1 µg of the peptide described herein. In other words, each 1 mg PLGA may be bound to at least 1 µg of the peptide described herein. In some embodiments, for every 1 mg PLGA in the material, the material contains 1 to 200 µg of the peptide described herein. In some embodiments, for every 1 mg PLGA in the material, the material contains 5 to 200 µg of the peptide described herein. In some embodiments, for every 1 mg PLGA in the material, the material contains 1 to 100 µg of the peptide described herein. In some embodiments, for every 1 mg PLGA in the material, the material contains 5 to 50 µg of the peptide described herein.

[0049] In certain embodiments, the material is a suture. In some embodiments, for every 1 mg of PLGA in the material, the material contains at least 2 µg, for example, at least 3 µg, for example, at least 4 µg, for example, at least 5 µg, for example, at least 6 µg of the peptide described herein. In some embodiments, for every 1 mg of PLGA in the material, the material contains 1 to 10 µg, for example, 1 to 8 µg, for example, 1 to 6 µg, for example, 3 to 6 µg, for example, 5 to 6 µg of the peptide described herein.

[0050] In certain embodiments, the material is particulate, such as nanoparticles or microparticles. In some embodiments, for every 1 mg of PLGA in the material, the material contains at least 20 µg, for example, at least 30 µg, or at least 40 µg of the peptide described herein. In some embodiments, for every 1 mg of PLGA in the material, the material contains 20 to 60 µg, for example, 30 to 55 µg, or at least 40 to 50 µg of the peptide described herein.

[0051] In addition to the polymers and peptides disclosed herein, the materials disclosed herein may also contain additional therapeutic agents, such as antibiotics, anti-inflammatory or antiseptic agents, such as antibacterial agents, antifungal agents, antiviral agents and antiparasitic agents.

[0052] peptides

[0053] The peptides contained in the materials disclosed herein have useful antibacterial and / or anti-inflammatory properties.

[0054] The inventors have demonstrated, surprisingly, that the peptide must contain hydrophobic residues (preferably at specific positions) that allow for more efficient integration into the polymer phase of the PLGA polymer. This particularly relates to amino acids at positions X1, X6, and X14. Examples of hydrophobic amino acids include glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tyrosine (Y), and tryptophan (W).

[0055] It is also preferred that the peptide contains one or more protonable amino acids, particularly preferably X2, X4, X5, and X9 are R, K, or H.

[0056] In some respects, peptides contain or consist of the following amino acid sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16, where X3, X7, X10, X11, X13, X15, and X16 are any standard amino acids. X8 and X12 can be any amino acid. X1, X6, and X14 are G, A, V, L, I, P, F, M, Y, or W, and X2, X4, X5, and X9 are R, K, or H. The peptide is 10 to 40 amino acids long, for example, 10 to 30 amino acid residues.

[0057] As will be apparent to those skilled in the art, the peptide may be located at the N-terminus of X1. or The C-terminus of X16 contains additional amino acids.

[0058] In some embodiments, the peptide comprises or consists of the following amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16, wherein

[0059] X1, X6, and X14 are either F or W, and

[0060] X2, X4, X5, and X9 are either R or K.

[0061] In some embodiments, the peptide comprises or consists of the following amino acid sequence: FR-X3-KKW-X7-X8-K-X10-X11-X12-X13-F-X15-X16.

[0062] In some embodiments, the peptide comprises or consists of the following amino acid sequence: HVFR-X3-KKW-X7-X8-K-X10-X11-X12-X13-F-X15-X16.

[0063] Preferably, the peptide is capable of binding to both lipopolysaccharide and the LPS-binding hydrophobic pocket of CD14.

[0064] In some embodiments, the peptide is 10 amino acids long. In some embodiments, the peptide is 11 amino acids long. In some embodiments, the peptide is 12 amino acids long. In some embodiments, the peptide is 13 amino acids long. In some embodiments, the peptide is 14 amino acids long. In some embodiments, the peptide is 15 amino acids long. In some embodiments, the peptide is 16 amino acids long. In some embodiments, the peptide is 17 amino acids long. In some embodiments, the peptide is 18 amino acids long. In some embodiments, the peptide is 19 amino acids long. In some embodiments, the peptide is 20 amino acids long. In some embodiments, the peptide is 21 amino acids long. In some embodiments, the peptide is 22 amino acids long. In some embodiments, the peptide is 23 amino acids long. In some embodiments, the peptide is 24 amino acids long. In some embodiments, the peptide is 25 amino acids long. In some embodiments, the peptide is 26 amino acids long. In some embodiments, the peptide is 27 amino acids long. In some embodiments, the peptide is 28 amino acids long. In some embodiments, the peptide is 29 amino acids long. In some embodiments, the peptide is 30 amino acids long. In some embodiments, the peptide is 31 amino acids long. In some embodiments, the peptide is 32 amino acids long. In some embodiments, the peptide is 33 amino acids long. In some embodiments, the peptide is 34 amino acids long. In some embodiments, the peptide is 35 amino acids long. In some embodiments, the peptide is 36 amino acids long. In some embodiments, the peptide is 37 amino acids long. In some embodiments, the peptide is 38 amino acids long. In some embodiments, the peptide is 39 amino acids long. In some embodiments, the peptide is 40 amino acids long.

[0065] In some embodiments, the peptide is 10 to 40 amino acids long. In some embodiments, the peptide is 13 to 40 amino acids long. In some embodiments, the peptide is 16 to 30 amino acids long. In some embodiments, the peptide is 18 to 30 amino acids long. In some embodiments, the peptide is 18 to 25 amino acids long.

[0066] In a preferred embodiment, the peptide is a fragment of thrombin or has at least 90% sequence identity with a fragment of thrombin. In particular, the peptide may be the peptide shown in SEQ ID NO:1 or a fragment thereof, or a peptide having at least 90% sequence identity with SEQ ID NO:1 or a fragment thereof.

[0067] In some embodiments, the peptide comprises or consists of the following amino acid sequence: the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, or such as at least 99% sequence identity. In some embodiments, the peptide comprises or consists of a variant of the amino acid sequence shown in SEQ ID NO:1, wherein any amino acid has been substituted for another amino acid, provided that no more than five amino acids have been substituted, for example, 5, 4, 3, 2, or 1 amino acid have been substituted in this way in the amino acid sequence.

[0068] In some embodiments, the peptide comprises or consists of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity. In some embodiments, the peptide comprises or consists of a variant of the amino acid sequence shown in SEQ ID NO: 2, wherein any amino acid has been substituted for another amino acid, provided that no more than five amino acids have been substituted, for example, 5, 4, 3, 2, or 1 amino acid have been substituted in this way in the amino acid sequence.

[0069] In some embodiments, the peptide comprises or consists of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity. In some embodiments, the peptide comprises a variant of the amino acid sequence shown in SEQ ID NO: 3, wherein any amino acid has been substituted for another amino acid, provided that no more than five amino acids have been substituted, for example, five, four, three, two, or one amino acid has been substituted in this way in the amino acid sequence.

[0070] In some embodiments, the peptide comprises or consists of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity. In some embodiments, the peptide comprises or consists of a variant of the amino acid sequence shown in SEQ ID NO: 4, wherein any amino acid has been substituted for another amino acid, provided that no more than five amino acids have been substituted, for example, 5, 4, 3, 2, or 1 amino acid have been substituted in this way in the amino acid sequence.

[0071] In some embodiments, the peptide comprises or consists of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, or such as at least 99% sequence identity. In some embodiments, the peptide comprises or consists of a variant of the amino acid sequence shown in SEQ ID NO: 5, wherein any amino acid has been substituted for another amino acid, provided that no more than five amino acids have been substituted, for example, 5, 4, 3, 2, or 1 amino acid have been substituted in this way in the amino acid sequence.

[0072] In some embodiments, the peptide comprises or consists of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, or such as at least 99% sequence identity. In some embodiments, the peptide comprises or consists of variants of the amino acid sequence shown in SEQ ID NO: 6, wherein any amino acid has been replaced with another amino acid, provided that no more than five amino acids have been replaced, for example, 5, 4, 3, 2, or 1 amino acid have been so replaced in the amino acid sequence.

[0073] In some embodiments, the peptide comprises or consists of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity. In some embodiments, the peptide comprises or consists of a variant of the amino acid sequence shown in SEQ ID NO: 7, wherein any amino acid has been substituted for another amino acid, provided that no more than five amino acids have been substituted, for example, 5, 4, 3, 2, or 1 amino acid have been substituted in this way in the amino acid sequence.

[0074] In some embodiments, the peptide comprises or consists of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, or such as at least 99% sequence identity. In some embodiments, the peptide comprises or consists of a variant of the amino acid sequence shown in SEQ ID NO: 8, wherein any amino acid has been substituted for another amino acid, provided that no more than five amino acids have been substituted, for example, 5, 4, 3, 2, or 1 amino acid have been substituted in this way in the amino acid sequence.

[0075] In some embodiments, the peptide comprises or consists of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity. In some embodiments, the peptide comprises or consists of a variant of the amino acid sequence shown in SEQ ID NO: 9, wherein any amino acid has been substituted for another amino acid, provided that no more than five amino acids have been substituted, for example, 5, 4, 3, 2, or 1 amino acid have been substituted in this way in the amino acid sequence.

[0076] In some embodiments, the peptide is 18 to 30 amino acids long, preferably 18 to 25 amino acids, and comprises or consists of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity.

[0077] In some embodiments, the peptide is 18 to 30 amino acids long, preferably 18 to 25 amino acids, and comprises or consists of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity.

[0078] In some embodiments, the peptide is 18 to 30 amino acids long, preferably 18 to 25 amino acids, and comprises or consists of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity.

[0079] In some embodiments, the peptide is 18 to 30 amino acids long, preferably 18 to 25 amino acids, and comprises or consists of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity.

[0080] In some embodiments, the peptide is 18 to 30 amino acids long, preferably 18 to 25 amino acids, and comprises or consists of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity.

[0081] In some embodiments, the peptide is 18 to 30 amino acids long, preferably 18 to 25 amino acids, and comprises or consists of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity.

[0082] In some embodiments, the peptide is 18 to 30 amino acids long, preferably 18 to 25 amino acids, and comprises or consists of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity.

[0083] In some embodiments, the peptide is 18 to 30 amino acids long, preferably 18 to 25 amino acids, and comprises or consists of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity.

[0084] In some embodiments, the peptide is 18 to 30 amino acids long, preferably 18 to 25 amino acids, and comprises or consists of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity.

[0085] In one embodiment, the peptide is the peptide shown in SEQ ID NO:1. WO 2021 / 260164 discloses that SEQ ID NO:1 can be cleaved into a variety of peptides, including FYT21, GKY20, and HVF18, namely SEQ ID NO: 2, 3, and 4. The peptide also includes the amino acid sequence necessary for lipopolysaccharide (LPS) binding and CD14 binding. Therefore, in some embodiments, the peptide may comprise any one of these peptides (TCP-25FYT21, GKY20, and HVF18) or be composed of any one of these peptides (TCP-25FYT21, GKY20, and HVF18). Preferably, the peptide is 18-25 amino acids long, but the peptide length can be up to 30 amino acids as long as it is based on any of these peptides.

[0086] In some embodiments, the peptide contains an internal covalent bond between at least two amino acids. These peptides may exhibit enhanced stability compared to similar peptides with the same sequence but without said internal covalent bond.

[0087] Such peptides are described in WO 2023 / 067167, the contents of which are incorporated herein by reference in their entirety.

[0088] In some embodiments, the peptide is defined as any one of items 1 to 129 on pages 77-98 of WO 2023 / 067167. In some embodiments, the peptide comprises or is composed of the amino acid sequence described in the "Peptide Sequence" section on page 28 to page 35, line 16 of WO 2023 / 067167. In some embodiments, the peptide comprises a covalent bond as described in the "Internal Covalent Bonds" section on page 14 to page 27, line 25 of WO 2023 / 067167.

[0089] In some implementations, the peptide comprises or consists of the following sequences: VFRLKKWI-X1-KVI-X2-ZFG, X1 and X2 are amino acids linked by covalent bonds.

[0090] In some implementations, the covalent bond is a hydrocarbon staple.

[0091] In some embodiments, X1 and X2 are alkenylated amino acids. In some embodiments, X1 and X2 are two C-alkenylated amino acids. In some embodiments, X1 and X2 are two α-substituted alkenyl amino acids. In some embodiments, X1 and X2 are α,α-disubstituted alkenyl amino acids. In some embodiments, the covalent bond is an olefin tether formed between the alkenyl residues.

[0092] In some implementations, the internal hydrocarbon staple bond is formed by linking two (S)-2-(4'-pentenyl)-alanines.

[0093] In some implementations, one or more of the standard amino acids contained in the peptide are modified or derivatized.

[0094] In some embodiments, one or more of the standard amino acids contained in the peptide are PEGylated. In some embodiments, one or more of the standard amino acids contained in the peptide are amidated. In some embodiments, one or more of the standard amino acids contained in the peptide are acylated. In some embodiments, one or more of the standard amino acids contained in the peptide are acetylated. In some embodiments, one or more of the standard amino acids contained in the peptide are alkenylated. In some embodiments, one or more of the standard amino acids contained in the peptide are alkylated.

[0095] In some embodiments, the C-terminal amino acid contained in the peptide is PEGylated. In some embodiments, the C-terminal amino acid contained in the peptide is amidated. In some embodiments, the C-terminal amino acid contained in the peptide is acylated. In some embodiments, the C-terminal amino acid contained in the peptide is acetylated. In some embodiments, the C-terminal amino acid contained in the peptide is alkenylated. In some embodiments, the C-terminal amino acid contained in the peptide is alkylated.

[0096] The peptides disclosed herein may also be pharmaceutically acceptable acid or base addition salts of peptides as disclosed above. The acids used to prepare pharmaceutically acceptable acid addition salts of peptides are those that form non-toxic acid addition salts, namely salts containing pharmaceutically acceptable anions, such as hydrochlorides, hydrobromides, hydroiodides, nitrates, sulfates, hydrogen sulfates, acids, acetates, lactates, citrates, acid citrates, tartrates, hydrogen tartrates, succinates, maleates, fumarates, gluconates, glycosides, benzoates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and bis(hydroxynaphthyl)ate [i.e., 1,1'-methylene-bis(2-hydroxy-3-naphthylcarbamate)].

[0097] Without being bound by theory, it may be advantageous for the peptide to be cationic (i.e., having a net positive charge) at the central pH for binding with polymers. Therefore, in some embodiments, the peptide is cationic at pH 7.0.

[0098] polymer

[0099] The polymers contained in this disclosure comprise or consist of poly(lactic acid-co-glycolic acid) (PLGA) (a copolymer). PLGA is a synthetic copolymer of lactic acid (α-hydroxypropionic acid) and glycolic acid (glycolic acid).

[0100] Those skilled in the art know how to synthesize PLGA polymers to obtain specific, desired properties. For example, a wide range of performance characteristics can be controlled by manipulating three key properties of the copolymer, such as solubility, crystallinity, thermal stability, strength, toughness, elasticity, and degradation rate: composition (glycolic acid to lactic acid ratio), lactic acid stereoisomer composition (L- or DL-lactide), and the molecular weight of the peptide. Examples of these performance characteristics and their corresponding numerical values ​​can be found in Avgoustakis, 2005, “Polylactic-Co-Glycolic Acid (PLGA)”, Encyclopedia of Biomaterials and Biomedical Engineering, doi:10.1081 / E-EBBE-120013950.

[0101] PLGA polymers can be synthesized into random copolymers or block copolymers.

[0102] In some embodiments, the PLGA polymer is synthesized as a block copolymer. In some embodiments, the monomers of the PLGA polymer follow formula I: (I), Where x is the number of lactic acid units and y is the number of glycolic acid units.

[0103] As described above, the values ​​of x and y can be selected by a technician to impart specific desired properties to the polymer. In some embodiments, x is 1 and y is 1. In some embodiments, x is 1 and y is 2. In some embodiments, x is 2 and y is 1. In some embodiments, x is 2 and y is 2.

[0104] Preferably, PLGA polymers can be synthesized as random copolymers by mixing monomers glycolic acid and lactic acid in a specific molar ratio and polymerizing them.

[0105] By selecting a specific ratio between lactic acid and glycolic acid in the PLGA polymer, the release profile of the bound peptide, as described herein, from the polymer can be controlled. This allows for tailoring the release timing for specific applications.

[0106] In particular, a higher glycolic acid to lactic acid ratio promotes faster polymer degradation, thus enabling a more rapid release of the conjugated peptide described herein to the site of application. This can be very useful at sites of injury requiring rapid delivery of the conjugated peptide, such as acute wounds. Conversely, a higher lactic acid to glycolic acid ratio leads to slower polymer degradation, resulting in a prolonged release of the conjugated peptide over time. Furthermore, the higher lactic acid content in PLGA polymers can protect the conjugated peptide from exogenous proteases due to the slower polymer degradation. This can be particularly useful in bodily environments characterized by high protease activity, such as wounds.

[0107] In some embodiments, the PLGA polymer comprises glycolic acid and lactic acid in a ratio of 5:95 to 95:5, or is composed of glycolic acid and lactic acid in a ratio of 5:95 to 95:5 (the ratio represents the ratio of glycolic acid to lactic acid). A 5:95 ratio means that for every 5 units of glycolic acid in the PLGA polymer, the polymer contains 95 units of lactic acid.

[0108] In some embodiments, the PLGA polymer comprises glycolic acid and lactic acid in a ratio of 80:20 to 95:5 or is composed of glycolic acid and lactic acid in a ratio of 80:20 to 95:5 (the ratio represents the ratio of glycolic acid to lactic acid).

[0109] In some embodiments, the PLGA polymer comprises glycolic acid and lactic acid in a ratio of 90:10 to 15:85 or is composed of glycolic acid and lactic acid in a ratio of 90:10 to 15:85 (the ratio represents the ratio of glycolic acid to lactic acid).

[0110] In some embodiments, the PLGA polymer comprises glycolic acid and lactic acid in a ratio of 95:10 or is composed of glycolic acid and lactic acid in a ratio of 95:10 (the ratio represents the proportion of glycolic acid to lactic acid). In some embodiments, the PLGA polymer comprises glycolic acid and lactic acid in a ratio of 90:10 to 5:95 or is composed of glycolic acid and lactic acid in a ratio of 90:10 to 5:95 (the ratio represents the proportion of glycolic acid to lactic acid). In some embodiments, the PLGA polymer comprises glycolic acid and lactic acid in a ratio of 90:10 or is composed of glycolic acid and lactic acid in a ratio of 90:10 (the ratio represents the proportion of glycolic acid to lactic acid).

[0111] In some embodiments, the PLGA polymer comprises or consists of glycolic acid and lactic acid in a 50:50 ratio (the ratio represents the proportion of glycolic acid to lactic acid). This specific ratio can lead to relatively rapid degradation and release of the bound peptide and can be used in applications requiring peptide release over days to weeks.

[0112] In some embodiments, the PLGA polymer comprises glycolic acid and lactic acid in a ratio of 30:70 or is composed of glycolic acid and lactic acid in a ratio of 30:70 (the ratio represents the ratio of glycolic acid to lactic acid).

[0113] In some embodiments, the PLGA polymer comprises or consists of glycolic acid and lactic acid in a 25:75 ratio (the ratio represents the ratio of glycolic acid to lactic acid). This specific ratio can result in slower degradation and release of the bound peptide and can be used in applications requiring peptide release over several weeks to months.

[0114] In some embodiments, the PLGA polymer comprises glycolic acid and lactic acid in a ratio of 15:85 or is composed of glycolic acid and lactic acid in a ratio of 15:85 (the ratio represents the proportion of glycolic acid to lactic acid). This specific ratio can result in slow degradation and prolonged release of the bound peptide and can be used in applications requiring long-term delivery, such as implants and microspheres for chronic conditions.

[0115] Without being bound by theory, the peptides described herein primarily bind glycolic acid compared to lactic acid in the polymer. In some embodiments, a 50:50 glycolic acid to lactic acid ratio is sufficient to achieve maximum binding of the peptide to the polymer, meaning that increasing the amount of glycolic acid in the polymer compared to lactic acid will result in a minimal increase in peptide binding.

[0116] In some embodiments, the PLGA polymer comprises glycolic acid and lactic acid in a ratio of 50:50 to 5:95 or is composed of glycolic acid and lactic acid in a ratio of 50:50 to 5:95 (the ratio represents the ratio of glycolic acid to lactic acid).

[0117] In some embodiments, the PLGA polymer is polygliptin 910, also known as Vicryl. Polygliptin 910 consists of glycolic acid and lactic acid in a ratio of 90:10 (the ratio represents the ratio of glycolic acid to lactic acid).

[0118] In some embodiments, the PLGA polymer comprises glycolic acid and lactic acid in a ratio of 93:7 or is composed of glycolic acid and lactic acid in a ratio of 93:7 (the ratio represents the proportion of glycolic acid to lactic acid).

[0119] The degradation rate of the polymer may also be affected by the molecular weight of PLGA.

[0120] In some embodiments, the PLGA polymer has a molecular weight of 5 to 250 kDa. In some embodiments, the PLGA polymer has a molecular weight of 5 to 200 kDa. In some embodiments, the PLGA polymer has a molecular weight of 10 to 150 kDa. In some embodiments, the PLGA polymer has a molecular weight of 20 to 100 kDa. In some embodiments, the PLGA polymer has a molecular weight of 30 to 60 kDa.

[0121] In some embodiments, the PLGA polymer has a molecular weight of approximately 5 kDa. In some embodiments, the PLGA polymer has a molecular weight of approximately 10 kDa. In some embodiments, the PLGA polymer has a molecular weight of approximately 15 kDa. In some embodiments, the PLGA polymer has a molecular weight of approximately 30 kDa. In some embodiments, the PLGA polymer has a molecular weight of approximately 40 kDa. In some embodiments, the PLGA polymer has a molecular weight of approximately 60 kDa. In some embodiments, the PLGA polymer has a molecular weight of approximately 100 kDa.

[0122] In a preferred embodiment, the PLGA polymer is carboxyl-terminated. Therefore, in a preferred embodiment, the PLGA polymer follows formula A: (Formula A) In some embodiments, the PLGA polymer is ester-terminated. Therefore, in some embodiments, the PLGA polymer follows formula B: (Formula B) Without being bound by theoretical constraints, ester terminalization increases the hydrophobic properties of polymers, making them less soluble in water and thus slowing down water absorption. This slow water absorption may affect the rate of polymer degradation in biological environments. Therefore, ester-terminated PLGAs may exhibit a longer degradation half-life.

[0123] Without being bound by theory, when PLGA hydrolyzes into lactic acid and glycolic acid during degradation (e.g., at a wound site), this may lower the pH of the microenvironment at that site. In some embodiments, the peptides of the present invention can have increased affinity for bacteria and LPS in such a low-pH environment due to protonation, thereby providing better therapeutic efficacy when delivered via PLGA-containing materials as described herein (e.g., when coated on PLGA-containing sutures or PLGA-containing particles). Furthermore, the stability of the peptides during application to a therapeutic area (e.g., a wound) may also be affected by peptide protonation due to the decrease in microenvironment pH caused by PLGA.

[0124] Combining the peptide with PLGA-containing materials (such as PLGA-containing sutures or PLGA-containing particles) can also increase the stability of the peptide during long-term storage.

[0125] Medical products

[0126] In some aspects of this disclosure, medical products comprising the materials described elsewhere herein are provided.

[0127] In some embodiments, the medical product is selected from sutures, strips, films, stents, grafts, hydrogels, nanoparticles, microparticles, and dressings such as mesh, patches, or bandages.

[0128] In some embodiments, the medical product is a suture. In some embodiments, the medical product is a strip. In some embodiments, the medical product is a film. In some embodiments, the medical product is a scaffold. In some embodiments, the medical product is a graft. In some embodiments, the medical product is a hydrogel. In some embodiments, the medical product is particles, such as microparticles or nanoparticles.

[0129] In some embodiments, the material comprising the PLGA polymer and the TCP25 peptide of the present invention is particulate, such as microparticles or nanoparticles. In some embodiments, the average diameter of the particles is from 100 nm to 100 µm. In some embodiments, the average diameter of the particles is from 100 nm to 50 µm. In some embodiments, the average diameter of the particles is about 100 nm. In some embodiments, the average diameter of the particles is about 500 nm. In some embodiments, the average diameter of the particles is about 1 µm. In some embodiments, the average diameter of the particles is about 5 µm. In some embodiments, the average diameter of the particles is about 10 µm. In some embodiments, the average diameter of the particles is about 20 µm. In some embodiments, the average diameter of the particles is about 30 µm. In some embodiments, the average diameter of the particles is about 40 µm. In some embodiments, the average diameter of the particles is about 50 µm.

[0130] Particle size can be selected based on the desired release characteristic, allowing for further control over the release profile. For example, smaller particles have a larger surface area to volume ratio, exhibiting faster hydrolysis and greater surface binding, resulting in more rapid modulation of the activity of bound proteins. Larger particles degrade more slowly, thus allowing for sustained activity of bound proteins.

[0131] The PLGA polymer can be as described elsewhere herein. In some embodiments where the material is particulate, the particulate PLGA polymer can comprise or consist of glycolic acid and lactic acid in a ratio of 5:95 to 95:5, for example 5:95 to 95:10, for example 30:70, for example 50:50 (the ratio represents the ratio of glycolic acid to lactic acid).

[0132] The medical product can be any medical product containing such microparticles or nanoparticles. In some embodiments, the medical product is a strip, film, scaffold, graft, spray, powder, fibrin glue, hydrogel, or dressing containing or coated with microparticles or nanoparticles. For example, the medical product can be a hydrogel containing said particles, or a spray or powder containing said particles or composed of said particles. Preferably, the medical product is compatible with wound application, such as at a surgical site, or with implants containing the medical product inserted into the body.

[0133] In some embodiments, the medical product is a dressing. In some embodiments, the medical product is a mesh. In some embodiments, the medical product is a patch. In some embodiments, the medical product is a bandage.

[0134] Medical products are preferably pharmaceutically acceptable, i.e., non-toxic. They can withstand routine pharmaceutical processes, such as sterilization, and / or may contain common adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, fillers, etc.

[0135] The medical products disclosed herein can be used for the combined treatment or prevention of inflammation and infection, such as for treating infection-related inflammation in individuals of need. The infection may be caused by a microorganism. The microorganism may be selected from bacteria, fungi, viruses, and protozoa.

[0136] In some implementations, the medical product is antimicrobial. In some implementations, the medical product inhibits bacterial growth. In some implementations, the medical product prevents bacterial growth.

[0137] The bacteria can be any infectious bacteria. For example, the bacteria can be Gram-negative or Gram-positive. Therefore, the bacteria can belong to, for example, genera selected from the following: Staphylococcus genus ( Staphylococcus ), Enterococcus spp. Enterococcus Streptococcus spp. Streptococcus Corynebacterium spp. Corynebacterium Escherichia coli spp. Escherichia Klebsiella spp. Klebsiella ), Oligotrophomonas spp. Stenotrophomonas ), Shigella spp. Shigella Moraxella spp. Moraxella Acinetobacter spp. Acinetobacter Haemophilus spp. Haemophilus ), Pseudomonas spp. Pseudomonas ) and Citrobacter spp. Citrobacter In some embodiments, the bacteria are selected from Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and Escherichia coli ( Escherichia coli The group consists of [a group of bacteria]. In another embodiment, the bacteria are Gram-negative bacteria.

[0138] The bacteria can even be multidrug-resistant bacteria. The medical product disclosed herein can provide antibacterial effects against several multidrug-resistant bacteria (i.e., bacteria resistant to several known antibiotics).

[0139] In some embodiments, the medical product is antibacterial and / or anti-inflammatory. In some embodiments, the medical product is antibacterial. In some embodiments, the medical product is anti-inflammatory. In some embodiments, the medical product is both antibacterial and anti-inflammatory.

[0140] In some embodiments, the medical product inhibits or prevents the growth or formation of biofilms. In some embodiments, the biofilm is a bacterial biofilm.

[0141] In some embodiments, the medical product reduces endotoxin-induced NF-κB and AP-1 activation and / or TNF-α induction. In some embodiments, the medical product reduces endotoxin-induced NF-κB and AP-1 activation. In some embodiments, the medical product reduces TNF-α induction. In some embodiments, the medical product reduces endotoxin-induced NF-κB and AP-1 activation as well as TNF-α induction.

[0142] The medical product disclosed herein is capable of continuously releasing a useful concentration of peptides to the relevant body site to exert its antimicrobial (e.g., antibacterial) and / or anti-inflammatory effects.

[0143] In some embodiments, when the medical product comes into contact with a body part, the medical product is capable of continuously releasing the peptide to that body part for at least 12 hours. In some embodiments, when the medical product comes into contact with a body part, the medical product is capable of continuously releasing the peptide to that body part for at least 24 hours. In some embodiments, when the medical product comes into contact with a body part, the medical product is capable of continuously releasing the peptide to that body part for at least 36 hours. In some embodiments, when the medical product comes into contact with a body part, the medical product is capable of continuously releasing the peptide to that body part for at least 48 hours. In some embodiments, when the medical product comes into contact with a body part, the medical product is capable of continuously releasing the peptide to that body part for at least 60 hours. In some embodiments, when the medical product comes into contact with a body part, the medical product is capable of continuously releasing the peptide to that body part for at least 72 hours.

[0144] The rate at which the peptide is released to the body site is preferably approximately constant over the time period.

[0145] In some implementations, when the medical product comes into contact with a body part, the medical product can continuously release the peptide to that part for 12 hours to 100 days.

[0146] In some implementations, the body part is a wound.

[0147] In some embodiments, the medical product is capable of continuously releasing the peptide to a cumulative concentration of at least 75 µg / mL over a period of approximately 24 hours. In some embodiments, the medical product is capable of continuously releasing the peptide to a cumulative concentration of at least 100 µg / mL over a period of approximately 24 hours. In some embodiments, the medical product is capable of continuously releasing the peptide to a cumulative concentration of up to 200 µg / mL over a period of approximately 24 hours.

[0148] In some embodiments, the medical product is capable of continuously releasing the peptide to its theoretical maximum concentration (c) over a period of approximately 24 hours. max The cumulative percentage is 5% to 60%, for example, 10% to 50%.

[0149] In some embodiments, the medical product is capable of continuously releasing the peptide to a cumulative concentration of at least 100 µg / mL over a period of approximately 48 hours. In some embodiments, the medical product is capable of continuously releasing the peptide to a cumulative concentration of at least 125 µg / mL over a period of approximately 48 hours. In some embodiments, the medical product is capable of continuously releasing the peptide to a cumulative concentration of up to 250 µg / mL over a period of approximately 48 hours.

[0150] In some embodiments, the medical product is capable of continuously releasing the peptide to its theoretical maximum concentration (c) over a period of approximately 48 hours. max The cumulative percentage is 10% to 70%, for example, 15% to 60%.

[0151] In some embodiments, the medical product is capable of continuously releasing the peptide to a cumulative concentration of at least 125 µg / mL over a period of approximately 72 hours. In some embodiments, the medical product is capable of continuously releasing the peptide to a cumulative concentration of at least 150 µg / mL over a period of approximately 72 hours. In some embodiments, the medical product is capable of continuously releasing the peptide to a cumulative concentration of up to 300 µg / mL over a period of approximately 72 hours.

[0152] In some embodiments, the medical product is capable of continuously releasing the peptide to its theoretical maximum concentration (c) over a period of approximately 72 hours. max The cumulative percentage is 20% to 80%, for example, 25% to 70%.

[0153] The cumulative concentration can be measured by a method including the following steps: 1. Place the medical product into the transwell insert in the top chamber of the transwell; 2. Add a certain volume of elution buffer to the outer chamber of the transwell substrate to bring the porous filter membrane of the transwell and the medical product into contact with the elution buffer; 3. Seal the surface of the transwell to prevent the elution buffer from evaporating; 4. While shaking, heat the transwell to a physiologically relevant temperature; 5. Measure the concentration of peptides in the elution buffer in the outer chamber of the substrate at a set time point after performing step 4; 6. Optionally, step 5 may be repeated once or more at subsequent time points; 7. Calculate the cumulative concentration of the released peptide based on the concentrations measured in step 5 and optionally step 6.

[0154] In some implementations, the elution buffer is 10 mM Tris at pH 7.4.

[0155] In some implementations, the heating and shaking steps are performed at 37°C and shaking at 60 rpm.

[0156] In some implementations, the set time point after performing step 4 is 12 hours, for example, 24 hours, for example, 36 hours, for example, 48 hours, for example, 60 hours, for example, 72 hours or longer.

[0157] In some embodiments, the step of measuring the peptide concentration in the elution buffer in step 5 is performed by measuring the absorbance of the elution buffer. In some embodiments, the absorbance is at 280 nm (Å). 280 ) was measured at the location.

[0158] The medical product disclosed herein can maintain the stability of the peptides it contains during storage. The storage can be wet storage or dry storage.

[0159] In some embodiments, the peptide is stable for at least 12 months during storage at room temperature (e.g., wet or dry storage). In some embodiments, the peptide is stable for at least 18 months during storage at room temperature (e.g., wet or dry storage). In some embodiments, the peptide is stable for at least 24 months during storage at room temperature (e.g., wet or dry storage).

[0160] In some embodiments, the peptide is classified as stable when it degrades by no more than 15% after storage (e.g., after wet or dry storage). In some embodiments, the peptide is classified as stable when it degrades by no more than 10% after storage (e.g., after wet or dry storage). In some embodiments, the peptide is classified as stable when it degrades by no more than 5% after storage (e.g., after wet or dry storage). In some embodiments, the peptide is classified as stable when it degrades by no more than 2% after storage (e.g., after wet or dry storage). In some embodiments, the peptide is classified as stable when it degrades by no more than 1% after storage (e.g., after wet or dry storage).

[0161] In some embodiments, the peptide is stable when stored in an aqueous solution (such as an aqueous buffer, e.g., 10 mM Tris buffer, pH 5 or 7). In some embodiments, the peptide is stable when stored in a gel (such as a hydrogel).

[0162] In some embodiments, a peptide is classified as stable when, after storage (e.g., after dry storage), it exhibits at least 85%, for example at least 90%, for example at least 95%, or approximately the same antimicrobial activity as an unstored peptide (e.g., the same fresh peptide, such as a newly synthesized peptide). This antimicrobial activity can be assessed by using suitable microorganisms (e.g., ... E. coli The evaluation was conducted using the radial diffusion method.

[0163] In addition to the polymer and peptide materials described herein, the medical products disclosed herein may also contain additional therapeutic agents, such as antibiotics, anti-inflammatory or antiseptic agents, such as antibacterial agents, antifungal agents, antiviral agents and antiparasitic agents.

[0164] Treatment

[0165] The medical products disclosed herein can be used for treatment methods. In particular, the medical products of the present invention can be used for the combined treatment or prevention of inflammation and infection, for example, for treating infection-related inflammation in individuals in need.

[0166] Therefore, in some aspects of this disclosure, methods for preventing and / or inhibiting inflammation and / or infection in a subject in need are provided, as disclosed herein, the methods comprising contacting the subject with a medical product as described elsewhere herein or implanting the medical product into the subject and maintaining the contact for a period of time.

[0167] In some embodiments, the contact is maintained for at least one week. In some embodiments, the contact is maintained for at least two weeks. In some embodiments, the contact is maintained for at least three weeks. In some embodiments, the contact is maintained for at least one month. In some embodiments, the contact is maintained for at least two months. In some embodiments, the contact is maintained for at least three months. In some embodiments, the contact is maintained until the medical product dissolves and / or is absorbed.

[0168] In some embodiments, the method is used to prevent inflammation and / or infection in a subject in need. In some embodiments, the method is used to suppress inflammation and / or infection in a subject in need. In some embodiments, the method is used to suppress inflammation and / or prevent infection in a subject in need. In some embodiments, the method is used to prevent inflammation and / or suppress infection in a subject in need.

[0169] During or after surgery, medical products (such as sutures or mesh) are necessary for closing, ligating, or closure of internal wounds (such as internal surgical wounds, i.e., deep closures) or for fixing or supporting weakened or damaged internal tissues (such as hernias). The term "internal" wound or damaged tissue refers to damage inside the body, i.e., not external damage, such as wounds on the skin surface. These medical products must generally be able to be dissolved or absorbed by the body of the subject to which they are implanted, as removal from the outside of the body is not possible. The medical products disclosed herein are preferably biodegradable and / or bioabsorbable and exhibit a variety of beneficial properties in tissue healing and / or support, such as antibacterial and / or anti-inflammatory properties, and are therefore useful for these applications.

[0170] In some embodiments, the medical product comes into contact with an internal body part of the subject. In some embodiments, the internal body part of the subject is a hernia. In some embodiments, the internal part is intradermal. In some embodiments, the internal part is subcutaneous. In some embodiments, the internal part is intravenous.

[0171] In some aspects of this disclosure, medical products, as described elsewhere herein, are provided for the prevention and / or inhibition of inflammation and / or infection in body parts of a subject.

[0172] In some embodiments, the body part is a hernia. In some embodiments, the body part is intradermal. In some embodiments, the body part is subcutaneous. In some embodiments, the body part is intravenous.

[0173] In some embodiments, the medical product is used to prevent inflammation and / or infection of the body part of the subject. In some embodiments, the medical product is used to suppress inflammation and / or infection of the body part of the subject. In some embodiments, the medical product is used to prevent inflammation and / or suppress infection of the body part of the subject. In some embodiments, the medical product is used to suppress inflammation and / or prevent infection of the body part of the subject.

[0174] In some aspects of this disclosure, a method for treating a wound in a subject in need is also provided, the method comprising contacting the wound with a medical product as described elsewhere herein or implanting the medical product into the wound.

[0175] In some aspects of this disclosure, medical products, as described elsewhere herein, are provided for treating wounds in subjects in need.

[0176] In some implementations, the wound is a surgical wound. Surgical wounds are described in more detail in the "Surgical Wounds" section below.

[0177] In some embodiments, the wound is a burn wound. In some embodiments, the wound is a non-healing ulcer. In some embodiments, the wound is a chronic wound. In some embodiments, the wound is selected from radiation-induced wounds, laser-induced wounds, and cryotherapy-induced wounds.

[0178] In some implementations, the wound is caused by a disease selected from diabetes, cancer, and vasculitis.

[0179] In some implementations, the subjects are mammals. In other implementations, the subjects are humans.

[0180] The treatment methods described herein involve humans and other mammals such as horses, dogs, cats, cattle, pigs, and camels. Therefore, the medical products disclosed herein are intended for human treatment and veterinary applications.

[0181] surgical wound

[0182] The medical products of this invention are particularly suitable for the prevention or treatment of inflammation and / or infection in surgical wounds.

[0183] In some implementations, the medical product is a suture.

[0184] Surgical wounds can be caused by cuts or incisions made to the skin, such as by a scalpel, during surgery (e.g., laparoscopic or open surgery). Surgical wounds can also result from drainage tubes placed during surgery.

[0185] Surgical wounds can be classified into four categories. These categories depend on the degree of contamination or cleanliness of the wound, the risk of infection, and the location of the wound on the body.

[0186] Class I wounds are classified as clean wounds. These wounds are not infected, do not show any signs of inflammation, and are usually closed. If drainage is required, closed drainage is recommended. It is important to note that Class I wounds do not involve the respiratory, digestive, reproductive, or urinary tracts. Examples of clean wounds include inguinal hernia repair or thyroidectomy.

[0187] Class II wounds are classified as clean-contaminated wounds, meaning they have a low level of contamination. These wounds involve entry into the respiratory, digestive, reproductive, or urinary tracts, but are only performed under controlled conditions.

[0188] Class III wounds are classified as contaminated wounds, typically caused by breaches of aseptic technique or gastrointestinal leakage. Incisions resulting from acute or non-suppurative inflammation are also considered Class III wounds.

[0189] Class IV wounds are considered dirty or infected. These injuries typically occur when traumatic wounds are poorly treated, severely suppurating, and show obvious signs of infection. Class IV wounds can also result from tissue loss of vitality. This is usually caused by microorganisms found during surgery or in perforated organs.

[0190] In some implementations, the surgical wound is a Class I wound. In some implementations, the surgical wound is a Class II wound. In some implementations, the surgical wound is a Class III wound. In some implementations, the surgical wound is a Class IV wound.

[0191] Production methods

[0192] The production methods for peptides are well-known in the industry.

[0193] Peptides can be produced using recombinant methods well-known in the industry (see, for example, Sambrook & Russell, 2000, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor, New York).

[0194] Alternatively, peptides can be chemically synthesized, for example, by linking multiple amino acids together via amide bonds. Typically, peptides are chemically synthesized through a condensation reaction between the carboxyl group of one amino acid and the amino group of another. Protecting group strategies can be used to prevent unwanted side reactions with various amino acid side chains.

[0195] Well-known liquid-phase or solid-phase peptide synthesis techniques are known to those skilled in the art (such as standard f-Boc or Fmoc solid-phase peptide synthesis).

[0196] The covalent connection of the side chains of two non-adjacent internal amino acids can be introduced by any method known to those skilled in the art, such as any method described by Li et al., 2020.

[0197] The peptides of this invention can also be ordered from companies that specialize in producing custom peptides, such as AmbioPharm Inc. (US).

[0198] Similarly, methods for producing materials containing peptides and PLGA polymers are also known in the art.

[0199] In some aspects, this disclosure provides a method for producing a medical product as described elsewhere herein, the method comprising immersing a starting material comprising or consisting of PLGA in a coating solution comprising a dissolving peptide as defined elsewhere herein, and subsequently drying the starting material.

[0200] In some embodiments, the starting material is selected from sutures, strips, films, scaffolds, grafts, hydrogels, nanoparticles, and dressings such as meshes, patches, or bandages.

[0201] In some embodiments, the concentration of the peptide in the coating solution is at least 0.5%. In some embodiments, the concentration of the peptide in the coating solution is at least 1%. In some embodiments, the concentration of the peptide in the coating solution is at least 2%. In some embodiments, the concentration of the peptide in the coating solution is at least 3%. In some embodiments, the concentration of the peptide in the coating solution is at least 4%.

[0202] In some embodiments, the concentration of the peptide in the coating solution is 0.5%-4%. In some embodiments, the concentration of the peptide in the coating solution is 1%-4%. In some embodiments, the concentration of the peptide in the coating solution is 2%-4%. In some embodiments, the concentration of the peptide in the coating solution is 2%-3%.

[0203] In a preferred embodiment, the concentration of the peptide in the coating solution is approximately 2%, such as 2%.

[0204] In some embodiments, the starting material is immersed in the coating solution for at least 10 minutes. In some embodiments, the starting material is immersed in the coating solution for at least 20 minutes. In some embodiments, the starting material is immersed in the coating solution for at least 30 minutes. In some embodiments, the starting material is immersed in the coating solution for at least 1 hour. In some embodiments, the starting material is immersed in the coating solution for about 2 hours, such as 2 hours.

[0205] In some embodiments, the starting material is immersed in the coating solution for 10 minutes to 4 hours. In some embodiments, the starting material is immersed in the coating solution for 20 minutes to 3 hours. In some embodiments, the starting material is immersed in the coating solution for 30 minutes to 2 hours. In some embodiments, the starting material is immersed in the coating solution for 1 hour to 2 hours.

[0206] In some embodiments, the starting material is immersed in the coating solution at a temperature of 10-50°C. In some embodiments, the starting material is immersed in the coating solution at a temperature of 15-40°C. In some embodiments, the starting material is immersed in the coating solution at a temperature of 20-37°C. In some embodiments, the starting material is immersed in the coating solution at a temperature of 20-30°C. In some embodiments, the starting material is immersed in the coating solution at a temperature of 20-25°C.

[0207] In a preferred embodiment, the starting material is immersed in the coating solution at a temperature of about 21°C (e.g., 21°C).

[0208] In a particular implementation scheme, The concentration of the peptide in the coating solution is approximately 2%; The starting material is immersed in the coating solution for approximately 2 hours; and The starting material is immersed in the coating solution at a temperature of about 21°C.

[0209] In a preferred embodiment, the starting material is suture thread.

[0210] Therefore, in some embodiments, the peptide is coated onto the starting material. As used herein, the term "coat, coated, or coating" refers to applying the peptide to the surface of the starting material. Thus, the peptide can be painted or sprayed using a solution containing the composition. Alternatively, the material can be immersed in a reservoir containing the peptide.

[0211] In some embodiments, the starting material is impregnated with a composition containing peptides. "Impregnation" refers to the absorption or adsorption of the composition by the starting material.

[0212] project

[0213] 1. A material comprising a poly(lactic-co-glycolic acid) (PLGA) polymer and a peptide comprising or composed of the following amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16, where X3, X7, X10, X11, X13, X15, and X16 are any standard amino acids. X8 and X12 can be any amino acid. X1, X6, and X14 are G, A, V, L, I, P, F, M, Y, or W, and X2, X4, X5, and X9 are R, K, or H. The peptides described therein have a length of 10 to 30 amino acid residues.

[0214] 2. The material according to Project 1, wherein the peptide comprises or is composed of the following amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16, where X1, X6, and X14 are either F or W, and X2, X4, X5, and X9 are either R or K.

[0215] 3. The material according to any one of the preceding items, wherein the peptide comprises or is composed of the amino acid sequence FR-X3-KKW-X7-X8-K-X10-X11-X12-X13-F-X15-X16.

[0216] 4. The material according to any one of the preceding items, wherein the peptide comprises or is composed of the amino acid sequence HVFR-X3-KKW-X7-X8-K-X10-X11-X12-X13-F-X15-X16.

[0217] 5. The material according to any one of the preceding items, wherein the peptide is 18 to 30 amino acids in length, preferably 18 to 25 amino acids, and comprises or consists of any amino acid sequence selected from or composed of any amino acid sequence selected from the following sequences: SEQ ID NO:1 or an amino acid sequence having at least 90% sequence identity therewith, SEQ ID NO:2 or an amino acid sequence having at least 90% sequence identity therewith, SEQ ID NO:3 or an amino acid sequence having at least 90% sequence identity therewith, SEQ ID NO:4 or an amino acid sequence having at least 90% sequence identity therewith, SEQ ID NO:5 or an amino acid sequence having at least 90% sequence identity therewith, SEQ ID NO:6 or an amino acid sequence having at least 90% sequence identity therewith, SEQ ID NO:7 or an amino acid sequence having at least 90% sequence identity therewith, SEQ ID NO:8 or an amino acid sequence having at least 90% sequence identity therewith, and SEQ ID NO:9 or an amino acid sequence having at least 90% sequence identity therewith.

[0218] 6. The material according to any one of the preceding items, wherein the peptide is 18 to 30 amino acids in length, preferably 18 to 25 amino acids, and comprises or consists of SEQ ID NO:1.

[0219] 7. The material according to any one of the foregoing items, wherein the peptide comprises or is composed of the following sequences: VFRLKKWI-X1-KVI-X2-ZFG in X1 and X2 are amino acids linked by covalent bonds.

[0220] 8. The material according to Item 7, wherein the covalent bond is a hydrocarbon staple bond.

[0221] 9. The material according to any one of items 7 to 8, wherein X1 and X2 are alkenylated amino acids, such as two C-alkenylated amino acids, such as two α-substituted alkenyl amino acids and / or α,α-disubstituted alkenyl amino acids, and the covalent bond is an olefinic tethered chain formed between the alkenyl residues.

[0222] 10. The material according to any one of items 7 to 9, wherein the internal hydrocarbon staple bond is formed by linking two (S)-2-(4'-pentenyl)-alanines.

[0223] 11. The material according to any one of the preceding items, wherein one or more of the standard amino acids contained in the peptide are modified or derivatized.

[0224] 12. The material according to any one of the preceding items, wherein one or more of the standard amino acids contained in the peptide are PEGylated, amidated, acylated, acetylated, alkenylated and / or alkylated.

[0225] 13. The material according to any one of the preceding items, wherein the peptide is cationic at pH 7.0.

[0226] 14. The material according to any one of the foregoing items, wherein the monomers of said PLGA polymer follow formula I:

[0227] Where x is the number of lactic acid units and y is the number of glycolic acid units.

[0228] 15. The material as described in Item 14, where x is 1 and y is 1.

[0229] 16. The material as described in Item 14, where x is 1 and y is 2.

[0230] 17. The material described in Item 14, where x is 2 and y is 1.

[0231] 18. The material according to any one of the preceding items, wherein the PLGA polymer comprises or consists of glycolic acid and lactic acid in a ratio of 5:95 to 95:5 (glycolic acid: lactic acid).

[0232] 19. The material according to any one of the preceding items, wherein the PLGA polymer comprises or consists of glycolic acid and lactic acid in a ratio of 5:95 to 90:10 (glycolic acid: lactic acid).

[0233] 20. The material according to any one of items 1 to 18, wherein the PLGA polymer comprises or consists of glycolic acid and lactic acid in a ratio of 80:20 to 95:5 (glycolic acid: lactic acid).

[0234] 21. The material according to any one of items 1 to 19, wherein the PLGA polymer comprises or consists of glycolic acid and lactic acid in a ratio of 15:85 to 90:10 (glycolic acid: lactic acid).

[0235] 22. The material according to any one of the preceding items, wherein the PLGA polymer comprises or consists of glycolic acid and lactic acid in a ratio of 90:10 (glycolic acid: lactic acid).

[0236] 23. The material according to any one of the preceding items, wherein the PLGA polymer is polyglucagon 910.

[0237] 24. The material according to any one of items 1 to 19, wherein the PLGA polymer comprises or consists of glycolic acid and lactic acid in a ratio of 93:7 (glycolic acid: lactic acid).

[0238] 25. The material according to any one of items 1 to 19, wherein the PLGA polymer comprises or consists of glycolic acid and lactic acid in a ratio of 25:75 or 30:70 (glycolic acid: lactic acid).

[0239] 26. The material according to any one of items 1 to 19, wherein the PLGA polymer comprises or consists of glycolic acid and lactic acid in a ratio of 50:50 (glycolic acid: lactic acid).

[0240] 27. The material according to any one of the preceding items, wherein the PLGA polymer is carboxyl-terminated.

[0241] 28. The material according to any one of the preceding items, wherein the peptide is coated on the fibers of the PLGA polymer, dissolved in the fibers of the PLGA polymer, and / or dispersed between the fibers of the PLGA polymer.

[0242] 29. The material according to any one of the preceding items, wherein the material is in particulate form, such as microparticles or nanoparticles.

[0243] 30. The material according to item 29, wherein the average diameter of said particles is 100 nm to 100 µm, for example 100 nm to 50 µm.

[0244] 31. The material according to any one of the preceding items, wherein the material comprises the peptide at a concentration of at least 25 µg / cm², such as at least 50 µg / cm², such as at least 75 µg / cm², such as at least 100 µg / cm², such as at least 150 µg / cm², such as at least 200 µg / cm², such as at least 250 µg / cm², such as at least 300 µg / cm², such as at least 350 µg / cm².

[0245] 32. The material according to any one of the preceding items, wherein for every 1 mg of the PLGA polymer, the material comprises at least 1 µg of the peptide.

[0246] 33. The material according to any one of the preceding items, wherein for every 1 mg of the PLGA polymer, the material comprises 1 µg to 200 µg, for example 5 µg to 200 µg of the peptide.

[0247] 34. The material according to any one of the preceding items, wherein for every 1 mg of the PLGA polymer, the material comprises 1 to 10 µg, for example 1 to 8 µg, for example 1 to 6 µg, for example 3 to 6 µg, for example 5 to 6 µg of the peptide.

[0248] 35. The material according to any one of the preceding items, wherein for every 1 mg of the PLGA polymer, the material comprises 20 to 60 µg, for example 30 to 55 µg, for example 40 to 50 µg of the peptide.

[0249] 36. The material according to any one of the preceding items, wherein the material is anti-inflammatory and / or antimicrobial, such as antibacterial.

[0250] 37. The material according to any one of the preceding items, wherein the material is capable of binding lipopolysaccharide (LPS), such as bacterial LPS.

[0251] 38. The material according to any one of the preceding items, wherein the material is biodegradable and / or bioabsorbable.

[0252] 39. A medical product comprising the material described in any one of the preceding items.

[0253] 40. The medical product according to item 39, wherein the medical product is selected from sutures, strips, films, scaffolds, grafts, hydrogels, nanoparticles and dressings such as meshes, patches or bandages.

[0254] 41. The medical product according to any one of items 39 to 40, wherein the medical product is a suture.

[0255] 42. The medical product according to item 39 is a strip, film, scaffold, graft, hydrogel, or dressing comprising particles described in any one of items 29 to 30.

[0256] 43. The medical product according to any one of items 39 to 41, wherein the medical product is antimicrobial (e.g., antibacterial) and / or anti-inflammatory.

[0257] 44. The medical product according to any one of items 39 to 43, wherein the medical product is antimicrobial (e.g., antibacterial) and anti-inflammatory.

[0258] 45. The medical product according to any one of items 39 to 44, wherein the medical product inhibits the growth of bacteria.

[0259] 46. ​​The medical product according to item 45, wherein the bacteria is selected from Staphylococcus aureus (… Staphylococcus aureus Pseudomonas aeruginosa (Pseudomonas aeruginosa) and Escherichia coli ( Escherichia coli ).

[0260] 47. The medical product according to any one of items 39 to 46, wherein the medical product inhibits or prevents the growth or formation of biofilms (such as bacterial biofilms).

[0261] 48. The medical product according to any one of items 39 to 47, wherein the medical product reduces endotoxin-induced NF-κB and AP-1 activation and / or TNF-α induction.

[0262] 49. The medical product according to any one of items 39 to 48, wherein when the medical product comes into contact with a body part, the medical product is capable of continuously releasing the peptide to the body part for at least 24 hours, for example at least 48 hours, for example at least 72 hours.

[0263] 50. The medical product according to item 49, wherein the body part is a wound.

[0264] 51. The medical product according to any one of items 39 to 50, wherein the medical product is capable of continuously releasing the peptide to a cumulative concentration of at least 75 µg / mL, for example, at least 100 µg / mL, over a period of about 24 hours.

[0265] 52. The medical product according to any one of items 39 to 51, wherein the medical product is capable of continuously releasing the peptide to a cumulative concentration of at least 100 µg / mL, for example, at least 125 µg / mL, over a period of about 48 hours.

[0266] 53. The medical product according to any one of items 39 to 52, wherein the medical product is capable of continuously releasing the peptide to a cumulative concentration of at least 125 µg / mL, for example, at least 150 µg / mL, over a period of about 72 hours.

[0267] 54. The medical product according to any one of items 39 to 53, wherein the peptide is stable for at least 12 months, for example at least 18 months, or for example at least 24 months, during dry storage at room temperature.

[0268] 55. The medical product according to item 54, wherein the peptide is stable when it degrades by no more than 10%, for example, no more than 5%, after dry storage.

[0269] 56. A method for producing a medical product according to any one of items 39 to 55, the method comprising the steps of immersing a starting material comprising PLGA or composed of PLGA in a coating solution comprising a soluble peptide according to any one of items 1 to 13, and subsequently drying the starting material.

[0270] 57. The method according to item 56, wherein the starting material is selected from sutures, strips, films, scaffolds, grafts, hydrogels, particles such as microparticles or nanoparticles, and dressings such as meshes, patches, or bandages.

[0271] 58. The method according to any one of items 56 to 57, wherein the starting material is a suture.

[0272] 59. The method according to any one of items 56 to 57, wherein the starting material is PLGA-containing particles.

[0273] 60. The method according to any one of items 56 to 59, wherein the concentration of the peptide in the coating solution is at least 0.5%, for example 0.5-4%, for example at least 1%, for example at least 2%, for example 2-4%, preferably 1% or 2%.

[0274] 61. The method according to any one of items 56 to 60, wherein the starting material is immersed in the coating solution for at least 10 minutes, for example at least 20 minutes, for example at least 30 minutes, for example at least 1 hour, or for example at least 2 hours.

[0275] 62. A method for preventing and / or inhibiting inflammation and / or infection in a subject in need, the method comprising exposing or implanting the subject with any of the medical products described in any one of items 39 to 55, and maintaining the exposure for a period of time.

[0276] 63. The method according to item 62, wherein the medical product comes into contact with an internal part of the subject's body, such as with the subject's hernia.

[0277] 64. A method for treating a wound in a subject in need, the method comprising contacting the wound with or implanting a medical product, as described in any one of items 39 to 55, into the wound.

[0278] 65. The method described in item 64, wherein the wound is a surgical wound.

[0279] 66. The method according to any one of items 62 to 65, wherein the subject is a mammal, such as a human.

[0280] 67. The method according to any one of items 62 to 66, wherein the contact is maintained for at least one month, for example at least two months, or until the medical product is dissolved and / or absorbed.

[0281] 68. The medical product according to any one of items 39 to 55, for the prevention and / or inhibition of inflammation and / or infection in a body part of a subject.

[0282] 69. The medical product used according to item 68, wherein the body part is a hernia.

[0283] 70. The medical product used according to item 68, wherein the body part is intradermal.

[0284] 71. The medical product used according to item 68, wherein the body part is subcutaneous.

[0285] 72. The medical product according to any one of items 39 to 55, for treating wounds of a subject in need.

[0286] 73. The medical product for use according to item 72, wherein the wound is a human wound.

[0287] 74. The medical product for use according to any one of items 72 to 73, wherein the wound is a surgical wound.

[0288] Example 1 – TCP-25 Coated Suture

[0289] Materials and Methods

[0290] Material The TCP-25 (SEQ ID NO:1) peptide was synthesized by Ambiopharm (Spain). Tetramethylrhodamine (TAMRA)-labeled TCP-25 was synthesized by Biopeptide Co. (San Diego, CA, USA). The purity of the peptide was confirmed to be 95% by mass spectrometry analysis (MALDI-ToF Voyager).

[0291] microorganism The strains used include Escherichia coli (E. coli) Escherichia coli ) ( ATCC 25922), Pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1 and Staphylococcus aureus (Staphylococcus aureus)( ATCC29213). Bioluminescent Pseudomonas aeruginosa ( P. aeruginosaXen41 (PerkinElmer, Akron, OH) and Staphylococcus aureus ( S. aureus ) SAP229 is used in experiments requiring infection imaging. In some experiments, Staphylococcus aureus (S. aureus) is also used. S. aureus (2404, 2278, 2405, 2528, 1779), Pseudomonas aeruginosa ( P. aeruginosa ) ( 27.1, 23.1, 13.2, 10.5, 62.1) S. epidermidis ( 2282) and E. faecalis (2374) clinical isolates. These isolates were obtained from skin or wound infection samples from the Department of Bacteriology, Lund University Hospital, Sweden.

[0292] Preparation of TCP-25 coated sutures Vicryl sutures (3-0, ETHICON, Johnson & Johnson International, Belgium) were cut into 10 cm fragments. A 2% TCP-25 solution was prepared by dissolving the peptide in sterile water. The suture fragments were coated in the TCP-25 solution (5 mL) on a shaker at room temperature for 1 hour. After coating, the sutures were placed in a Class II biosafety cabinet at room temperature (20°C). Air dry at 1°C for 1 hour. Use sterile water coating only for control sutures.

[0293] To examine the effect of TCP-25 coating concentration on antibacterial properties, sutures were coated with 0.1%, 0.5%, 2.0%, or 4.0% TCP-25 solutions for 2 hours. To examine the effect of coating time on antibacterial properties, sutures were coated with 2% TCP-25 solution at room temperature for 1, 2, 4, 8, and 24 hours. To examine the effect of coating temperature on antibacterial properties, sutures were coated with 2% TCP-25 solution at 21°C, 37°C, or 50°C for 2 hours. Coated sutures were stored at –80°C under dry conditions until further use. To elute the peptide, coated sutures were added to Tris (10 mM, pH 7.4) and shaken on a shaker at room temperature for 30 minutes. The eluent was stored at –80°C. The TCP-25 loading of the sutures was determined as previously described (Champeau et al., 2015). Briefly, the sutures were weighed before and after TCP-25 coating. The drug loading was calculated using the following equation: Drug loading (%) = ((Weight after coating – Weight before coating) / Weight before coating) 100.

[0294] Protein estimation:To estimate protein concentration, the Nanodrop method (ND1000, Thermo Scientific) was used at 280 nm, with the TCP-25 extinction factor (8480) employed. ¹c ¹) and molecular weight (3088, 62 Da).

[0295] Radial diffusion measurement (RDA) Use 10 mL of tryptone soy broth (TSB) to remove E. coli ( E. coli The bacteria were cultured to mid-log phase. Afterward, they were washed with 10 mM Tris (pH 7.4). The bacteria (4 × 1) were then... CFU was added to 15 mL of a bottom agarose gel containing 0.03% (w / v) TSB, 1% (w / v) low-electroosmotic (EEO) agarose (Sigma, St Louis MO, USA), and 0.02% (v / v) Tween 20 (Sigma). The bottom gel was then placed into a 144 mm diameter petri dish. Once the gel solidified, 4 mm diameter wells were cut into the bottom gel using a biopsy punch. A sample containing 6 μL of test sample (eluted from sutures) was added to each well. The plate was then incubated at 37°C for 3 hours, and 15 mL of top gel (a distilled aqueous solution of 6% TSB and 1% low-EEO agarose) was added to cover the bottom gel. The plate was incubated at 37°C for 18 hours. Antimicrobial activity was determined as the ratio of the clear area to the well diameter (excluding the 4 mm wells).

[0296] SDS-PAGE: To investigate the effect of TCP-25-coated sutures on storage, eluted peptides were studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, samples (5 µL) were loaded onto 10%–20% Tris-Tricine gels and electrophoresed at 100 V for 90 min. 2 µg of TCP-25 dissolved in 10 mM Tris (pH 7.4) was used as a control. The gels were stained with Coomassie Brilliant Blue (Invitrogen, Rockford, IL, USA).

[0297] Protein blot Immediately after electrophoresis, perform Western blotting as described previously (Saravanan et al., 2018).

[0298] Peptide release assay: For the in vitro peptide release assay, some modifications were made to simulate a wound scenario. The Transwell insertion system (VWR) was used. ®The tissue culture plate insert (6-well, 0.4µm pore size, VWR International) was used in conjunction with a 6-well plate. Twenty 3 cm long TCP-25 coated suture fragments were placed on the porous filter membrane in the head chamber. 2 mL of elution buffer (10 mM Tris, pH 7.4) was added to the basal outer chamber. The elution buffer contacted the porous filter membrane, thereby contacting the sutures (e.g., ...). Figure 1 (As shown in d). Cover the plate and seal it with sealing film to prevent solution evaporation. Place the plate on a shaker at 37°C and shake at 60 rpm. At the specified time, take 20 µL of sample from the outer portion of the substrate and immediately replace it with 20 µL of fresh Tris buffer. Measure the absorbance at 280 nm using a spectrophotometer (Nanodrop, Thermofisher). 280 The results represent the cumulative TCP-25 release (mg / mL).

[0299] Scanning electron microscope (SEM): In short, bacteria were incubated with coated and uncoated sutures at 37°C for 15 minutes, after which they were transferred to new tubes. The sutures were washed twice in 0.1 M Sorensen phosphate buffer (pH 7.4). The samples were further processed for SEM as described previously (Stromdahl et al., 2021). For SEM of sutures with biofilm, after incubation in a microtiter plate for 48 hours and removal of the sutures (as described in the biofilm section), the sutures were washed in Sorensen buffer and processed as described above for SEM.

[0300] Quartz crystal microbalance for dissipation monitoring (QCM-D) QCM-D measurements were performed using a Biolin Scientific (Gothenburg, Sweden) QSense E4 system equipped with four standard flow modules (QSense, Biolin Scientific), each featuring a silica surface (QSense, Biolin Scientific, QSX 303 SiO2, 4.95 ± 0.05 MHz, 14 mm diameter, 0.3 mm thickness, mass sensitivity 17.7 ng c). ²). Before use, the flow chamber, tubing, and O-rings were thoroughly cleaned with 2% Hellmanex solution, followed by multiple rinses with Milli-Q (MQ) water using an ultrasonic bath, then rinsed in pure ethanol and subsequently dried with a stream of N2. The SiO2 surface was sequentially cleaned with 2% Hellmanex, MQ water, and ethanol, then dried with N2 and subjected to plasma cleaning in residual air (Model PDC-32G, HarrickPlasma, USA) for 5 minutes. Immediately afterwards, the SiO2 surface was incubated at room temperature in 0.1% (w / w) poly-L-lysine (MW 150–300 kDa, Sigma-Aldrich, Merck, New Jersey, USA) solution in MQ water (pH 9.5) for 30 minutes. Afterward, the SiO2 surface was thoroughly rinsed with MQ water and dried with nitrogen. Subsequently, 100 mg of finely chopped fibers (50–100 μm in length) placed in 1 mL of ethanol were deposited on the poly-L-lysine-functionalized SiO2 surface and incubated overnight at room temperature. This causes solvent evaporation, followed by fiber fixation onto the positively charged poly-L-lysine-functionalized surface. Finally, the fiber-functionalized surface is thoroughly rinsed in MQ water and sealed in the QCM-D measurement chamber. In 0.1 mL mi ¹ The MQ water flow (controlled by a peristaltic pump) reaches a stable frequency ( F) and dissipation (F) D) After offsetting the baseline, add 0.001% (w / w) peptide in MQ aqueous solution to 0.1 mL mi ¹ The flow rate of the injection into the measurement chamber. Adsorption of peptides on the fiber-functionalized sensor is measured by frequency variations relative to pure MQ water (¹). F) 100 ± 27 Hz and dissipation variation ( D)(+42 ±11)1 To confirm this, the sample was then rinsed with MQ water in the absence of peptides. In a control experiment, the possible adsorption of the peptides on the underlying poly-L-lysine surface was ruled out, where the interaction between the peptides and the fiber-free poly-L-lysine-functionalized SiO2 surface was monitored under the same experimental conditions. Measurements were performed at room temperature.

[0301] Computer Modeling and Simulation of Vicryl with TCP-25 (Coarse-grained Parameterization of Vicryl): A coarse-grained (CG) model of the Vicryl polymer was developed using the Martini 2.2 force field (Marrink et al., 2007). Vicryl, or polygliptin 910, is a copolymer composed of 90% glycolide and 10% lactide. Its molecular weight is approximately 80 kDa, corresponding to approximately 600-mer (Chandrasekhar 2017). To simplify the system, we first constructed an atomic model of 10-mer polygliptin containing 9 glycolide subunits and 1 lactide subunit using the CHARMM-GUI Polymer Builder (Choi et al., 2021) and the CHARMM36m all-atom force field (Huang and MacKerell, 2013). The polymer was dissolved in a TIP3P water molecule box and neutralized with 0.15 M NaCl salt. We then performed energy minimization and equilibrium according to the CHARMM-GUI standard scheme (Jo et al., 2008). A 1 µs production simulation was conducted at 310 K and 1 atm, with temperature coupling via a Nosé-Hoover thermostat (Nosé, 1984) and isotropic pressure coupling via a Parrinello-Rahman barometer (Parrinello and Rahman, 1981). Electrostatic interactions were calculated using the particle mesh Ewald (PME) method (Essmann et al., 1995), with van der Waals interactions truncated at 1.2 nm and a force-switching smoothing function applied between 1.0 and 1.2 nm. The simulation time step used was 2 fs.

[0302] To generate an equivalent model of polygliptin in the CG Martini force field, we initially followed a mapping scheme derived from previous studies (Pannuzzo et al., 2022). Each glycolide and lactide subunit was mapped as a bead, represented by a Na-type bead. Unlike previous studies, our polygliptin model has a negatively charged carboxyl group at one end of the polymer and a hydroxyl group at the other. The former was mapped as two small beads of the SQa and SNa types, while the latter was mapped as a single P3-type bead. The generated polygliptin CG model was then dissolved in Martini water molecules and neutralized with 0.15 M NaCl salt. Energy minimization was performed using the steepest descent method, and short equilibrium simulations of 100 ps were conducted. Production simulations of 1 µs were then performed using a 10-fs time step. Electrostatic interactions were calculated using the reaction field method with a cutoff of 1.1 nm, van der Waals interactions with a cutoff of 1.1 nm, and a potential shift Verlet scheme. Temperature and pressure were maintained at 310 K and 1 atm, respectively, using a velocity recalibrating thermostat (Bussi et al., 2007) and isotropic coupling with a Parrinello-Rahman barometer. The bond, bond angle, and dihedral angle distributions from the CG simulation were then compared with those from the all-atom simulation. Iterative modifications were made to the bond parameters to match the distributions from the all-atom simulation.

[0303] We multiplied the final model to generate 50-mer and 100-mer polygliptin polymers with the same 9:1 glycolide:lactide ratio. These longer polymers underwent similar energy minimization, equilibration, and 1 µs production run schemes as described above. The polymer structures at the end of these simulations were then used for self-assembly simulations to build large Vicryl polymer aggregates. Therefore, we dissolved 50 copies of the polygliptin polymer in Martini water and neutralized the system with 0.15 M NaCl salt. The system underwent the same energy minimization and equilibration schemes. We then performed a 1 µs simulation at 320 K using the parameters described above to allow for polymer self-assembly. We then extracted the structures of the polymer aggregates from these simulations for subsequent TCP-25 simulations.

[0304] Coarse-grained simulations of Vicryl and TCP-25:The TCP-25 peptide was modeled using the NMR structure of HVF18 (PBD: 5Z5X) (Saravanan et al., 2018) as a template. The missing N-terminal residue (GKYGFYT) was constructed using Modeller version 9.21 (Saliand Blundell, 1993), and the model with the lowest discrete optimized protein energy was selected. The TCP-25 model was converted to a CG representation using a Martini 2.2 force field combined with a standard ElNeDYn elastic network model to preserve secondary structure (Periole et al., 2009). Ten copies of the TCP-25 peptide were added to a box containing a Vicryl polymer aggregate composed of 100-mer pioglitazone generated in the previous step. Figure 2 a) The TCP-25 peptide was placed at least 2 nm away from the polymer surface and from each other. The system was then dissolved in Martini water and neutralized with 0.15 M NaCl. Energy minimization was performed using the steepest descent method. Equilibrium simulations were performed at 100 ns, with a force constant of 500 kJ / mol. ¹ n ² Positional constraints are applied to each bead of the polymer, resulting in 1000 kJ mo. ¹ n The positional constraints were applied to each backbone bead of the peptide. Three independent 10 µs production simulations were then performed at different initial velocities. The temperature was maintained at 320 K using a velocity-recalibrated thermostat (Bussi et al., 2007), and the pressure was maintained at 1 atm using isotropic pressure coupling with a Parrinello-Rahman barometer (Parrinello et al., 1981). Electrostatic interactions were calculated using a reaction field scheme with a distance cutoff of 1.1 nm, while van der Waals interactions were cut off at 1.1 nm using a potential shift Verlet scheme. An integration time step of 10-fs was used. Similar simulations were performed using the same scheme for systems with higher TCP-25 peptide concentrations (20 copies), shorter pioglitazone chains (50-mer), and lower temperatures (310 and 298 K). All simulations were performed using GROMACS 2022 (Abraham et al., 2015) and visualized using VMD (Humphrey et al., 1996).

[0305] Optical photothermal infrared microspectroscopy:Optical photothermal infrared microspectroscopy (O-PTIR) is an analytical technique based on the photothermal effect induced by scanning infrared laser and measured by scattering probe light (Klementieva et al., 2020). O-PTIR was performed on the SMIS beamline of the SOLEIL synchrotron (France). Sutures were deposited directly on a glass slide and used for measurement. The photothermal effect was detected by modulating the intensity of a CW 532 nm laser induced by infrared laser. The infrared source was a pulsed tunable quantum cascade laser with an intensity set to 22%, and the scan range was 1800 to 1300 nm. ¹, with a repetition frequency of 80 kHz. More details about the technical basis and instrumentation can be found in previous work (Paulus et al., 2022).

[0306] Frozen slices: To facilitate TCP-25 fluorescence imaging, Vicryl sutures (3-0, ETHICON, Johnson & Johnson International, Belgium) were coated as previously described (Section 4.3), except that TCP-25 (doped with 5% TCP-25-Cy3) was used for coating. The coated sutures were then mounted in an OCT complex for cryosectioning. Suture cryosections (8 µm thick) were prepared using a cryostat (Leica Biosystems). Slides were washed in PBS (5 min at room temperature), dried, and mounted with an anti-quenching mounting medium (PermaFluor, ThermoFisherScientific). The sections were then imaged using a fluorescence microscope (AxioScope.A1, Carl Zeiss, Germany).

[0307] High performance liquid chromatography: Following the report by Petruk et al., 2020, 1 µg or 2 µg of TCP-25 eluted from sutures was analyzed by reversed-phase high-performance liquid chromatography (HPLC). 2 µg of freshly dissolved TCP-25 in 10 mM Tris (pH 7.4) was used as a control. Samples from three different elutions were analyzed.

[0308] Intrinsic fluorescence analysis: Following a previous description (Stromdahl et al., 2018), the binding of LPS to TCP-25 was analyzed by measuring the intrinsic fluorescence of the peptide. Increased LPS concentrations (2–100 μg mcg) were used. ¹) Titrate 10 μM TCP-25 eluted from the suture. Assuming a single binding site, calculate Kd using GraphPad Prism v9.

[0309] Circular dichroism (CD) spectrum: The secondary structure of TCP-25 eluted from sutures was evaluated using a Jasco J-810 spectropolarimeter (Jasco, USA). The spectropolarimeter was equipped with a Jasco CDF-426S Peltier and set to 25°C. The sample cell optical path length was 0.2 cm. Spectra were acquired between 190 and 260 nm (scanning speed 20 nm / min). ¹). Experiments used either standalone TCP-25 (10 µM, 200 µL) or with the addition of 100 μg m ¹ TCP-25 of LPS. The acquired spectra were corrected for the contribution of buffers with or without LPS and converted to the mean residue ellipticity θ (mdeg cm² dmo). ¹). The content of α-helical structures was calculated according to a previous report (Morrisett et al., 1973). The experiment was performed three times, each time using freshly eluted TCP-25.

[0310] viable cell count assay: Viable bacterial counts were performed according to a previous report (Saravanan et al., 2018). Briefly, bacteria were incubated with sutures coated in TCP-25 or control sutures under various conditions at 37°C for 5 minutes, and 1 and 2 hours. The buffers used in this assay were 10 mM Tris (pH 7.4) containing 5 mM glucose and 10 mM Tris (pH 7.4) containing 1.3% glycerol supplemented with 20% human plasma or 20% acute wound fluid. Serially diluted samples were inoculated onto TH broth agar and incubated overnight at 37°C.

[0311] Antibacterial effect of TCP-25 sutures on bioluminescent bacteria: Bioluminescent bacteria were cultured in TH medium to an OD of 0.4. The bacteria were then washed with 10 mM Tris (pH 7.4) and resuspended in either 10 mM Tris (pH 7.4) containing 5 mM glucose or 10 mM Tris (pH 7.4) containing 1.3% glycerol. The bacterial suspension (200 µL containing 1.3% glycerol) was then cultured. CFU and 1 cm long TCP-25 coated sutures or control sutures were placed in white polystyrene 96-well plates. The plates were placed in an incubator at 37°C. Bioluminescent signals were imaged over a long period using IVIS (PerkinElmer, USA) and quantified by photometry.

[0312] Live-dead assay: Perform live-dead bacterial staining as described previously (Puthia et al., 2020). Briefly, add 200 µL of the bacteria *Pseudomonas aeruginosa* to a 1 cm length of TCP-25 coated suture or control suture. P. aeruginosa (PAO1) or Staphylococcus aureus ( S. aureus The suture fragments were incubated in a (ATCC 29213) suspension at 37°C for 30 minutes. 50 µL of a mixture of components A and B was added to the sample, and the mixture was then incubated at room temperature in the dark for 15 minutes. The suture fragments were removed from the stained suspension, placed on a glass slide, and analyzed using a fluorescence microscope.

[0313] NF-κB / AP-1 assay: The activation of nuclear factor kappa B / activating protein (NF-κB / AP-1) was studied using THP1-Xblue™-CD14 reporter cells (InvivoGen, San Diego, USA). Assays were performed as previously reported (Saravanan et al., 2018). Briefly, 180 µL of 1 × 10⁻¹ medium was used. Cells were seeded into 96-well plates and treated with 10 μM TCP-25 eluted from sutures, LPS (from *E. coli* O111:B4, Sigma-Aldrich), or 5 µL of human wound fluid. After incubation at 37°C and 5% CO2 for 20 hours, 20 μL of culture medium was transferred from each well to a new 96-well plate containing 180 μL of QUANTI-Blue reagent (InvivoGen). The plates were then incubated at 37°C for 1 to 2 hours. The amount of secreted embryonic alkaline phosphatase (SEAP) was measured at OD600 nm.

[0314] Cell viability assay: Assess the viability of THP-1 cells in the above assays as described previously (Saravanan et al., 2018).

[0315] Cytokine assay: Mice in the suture-induced inflammation model were sacrificed 24 hours after suture implantation. The implanted sutures were recovered from the mice and added to pre-chilled Eppendorf tubes. 50 μL of Tris buffer was added to the sutures and vortexed for 10 minutes for elution. Finally, the tubes were centrifuged (2000 × g, 4 °C, 5 min), and the supernatant was collected for cytokine analysis. Tumor necrosis factor-α and interleukin-6 (TNF-α and IL-6, respectively) were assessed using a mouse inflammation kit (Becton Dickinson AB, Franklin Lakes, NJ) according to the manufacturer's description.

[0316] Hemolytic activity: Fresh venous blood was collected from a healthy donor in a lepirudin tube (50 µg m ¹) Sutures coated with TCP-25 (1 cm or 10 cm) were placed into tubes containing 0.5 or 1 mL of 25% human blood diluted with phenol red-free RPMI-1640-GlutaMAX-I (Gibco). The hemolytic activity of the sutures alone was analyzed by placing only the buffer-coated 1 or 10 cm sutures. Blood (25%) in RPMI was used as a negative control. A positive control was obtained by mixing 75 μL of blood solution with 225 µL of phenol red-free RPMI-1640-GlutaMAX-I containing 5% Tween-20. Samples were incubated at 37 °C (5% CO2) for 1 h and centrifuged at 800 × g. 100 μL of each sample was transferred to a flat-bottomed 96-well plate, and absorbance was measured at 450 nm. The percentage of hemolysis was calculated according to a previous report (Stromdahl et al., 2021).

[0317] Peptide release in mice: In vivo release of TCP-25 was studied using 10–12 week old hairless male SKH-1 mice. Mice were anesthetized with 4% isoflurane (Baxter). All procedures were performed under sterile conditions. TAMRA-labeled TCP-25-coated sutures were used for fluorescence bioimaging. A 2 cm fragment was subcutaneously implanted into the back of the hairless SKH-1 mouse using a needle. Fluorescence intensity was acquired using an IVIS imaging system (Perkin Elmer) to observe TCP-25 release over a long period. Data were analyzed using Living Image 4.0 software (Perkin Elmer).

[0318] A mouse model of suture suture infection: SKH-1 hairless mice (8–10 weeks old, female) were used as an experimental model for suture infection. Anesthesia was performed using a mixture of isoflurane (Baxter), 4% for induction and 2% for maintenance. All procedures were performed under sterile conditions. The mouse backs were wiped with alcohol swabs and then with sterile gauze. A 5 mm incision was made in the dorsal skin, and small pockets were created using scissor tips. Using a needle, a 2 cm length of TCP-25 suture or control suture was placed in the pockets on either side of the back. Using a pipette, the sutures were inoculated with bioluminescent Staphylococcus aureus (S. aureus) in the pockets. S. aureus (SAP229) or Escherichia coli ( Escherichia coli ) ( ATCC 25922)(20 µL Tris buffer contains 1 CFU contamination. Close the incision with tissue glue.

[0319] In some experiments, in addition to bacterial visualization, TAMRA-labeled TCP-25-coated sutures were used to monitor the tissue distribution of TCP-25, followed by IVIS imaging in bioluminescent (for bacteria) and fluorescent (for TCP-25) modes.

[0320] NF-κB reporter mouse model of suture-induced inflammation: The anti-inflammatory effects of TCP-25 coated sutures were evaluated using BALB / c tg (NF-RE-Luc)-Xen reporter mice (Taconic Biosciences, Albany, NY, USA). Male mice (8–10 weeks old) were used in this study. As described above for the suture infection model, a 5 mm incision was created on the skin of the mouse's back under aseptic conditions, and a small pocket was made. Using a needle, a 2 cm length of TCP-25 suture or control suture was placed in the pocket on each side of the back. Using a pipette, the suture was contaminated with 2 µg LPS (in 20 µL Tris buffer) in the pocket. The incision was closed with tissue glue. For inflammation analysis, long-term in vivo imaging with IVIS was used to determine NF-κB activation. 15 minutes before IVIS imaging, 100 μL of D-fluorescein (PerkinElmer, 150 mgkb) was injected intraperitoneally. ¹body weight). Bioluminescence was quantified using Living Image 4.0 software (PerkinElmer). In experiments involving contamination of TCP-25 coated sutures with human chronic wound fluid, the above procedure was followed, except that 10 μL of wound fluid was used instead of LPS to contaminate the sutures in the subcutaneous pocket.

[0321] Nanoscale LC–MS / MS analysis:For peptide digestion, a 1 cm long TCP-25 coated suture fragment was incubated with HNE (0.1 µg, in 20 µL 10 mM Tris, pH 7.4) at 37 °C for 30 min and 3 h. After loading the sample into an Evosep pipette tip, the HNE-digested TCP-25 peptide was separated by nanoflow reversed-phase chromatography using the Evosep One liquid chromatography (LC) system (Evosep). Separation was performed using a 60 SPD method (gradient length 21 min) on an Evosep column (8 cm x 150 µm) packed with ReproSil-Pur C18-AQ particles (1.5 μm). The Evosep One system was connected to a capacitor source mounted on a timsTOF Pro mass spectrometer (Bruker Daltonics). The instrument was operated in DDA PASEF mode. Using PEAKS Pro version to search for raw files against the human Uniprot database (released 2021-03-09), the following settings were used: MS tolerance 30 ppm, MSMS 0.02 Da, enzyme-free, methionine oxidation (variable), and a maximum of one post-translational modification per peptide.

[0322] Biomembrane research: To investigate the anti-biofilm activity of TCP-25 coated sutures, Staphylococcus aureus (Staphylococcus aureus) was used. S. aureus (ATCC 29213) and Pseudomonas aeruginosa ( P. aeruginosa PAO1 biofilm. To investigate the direct effect of TCP-25 sutures on mature biofilms, Staphylococcus aureus was cultured on 96-well round-bottom vinyl flexible plates (Corning, Kennebunk, USA). S. aureus Biofilm formation. Add 100 µL of culture medium, 0.5% tryptone soybean broth, and TBS to each well, supplemented with 0.2% glucose. Add 5 µL of 1x1... CFU m ¹Bacteria. Similarly, *Pseudomonas aeruginosa* was cultured in M63 medium supplemented with 0.5% casein amino acids, 0.2% glucose, and 1 mM MgSO4 on a flat-bottomed 96-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany). P. aeruginosa For both strains, add 5 µL of 1 x 1 solution to the culture medium. CFU m ¹Bacteria. After adding bacteria, the plates were sealed with microplate sealing film and placed in a humidified container to prevent evaporation. The container was then incubated at 37°C for 48 hours to obtain mature biofilm. The mature biofilm in the wells was washed twice (100 µL Tris) to remove planktonic cells, and then another 100 µL of Tris was added to the wells. For biofilm treatment, a 1 cm long fragment of TCP-25 suture or control suture was added to the well. The plates were sealed, placed in a humidified container, and incubated at 37°C for another 2 hours. After treatment, to count viable bacteria, the biofilm was disrupted by scraping with a pipette tip. Then, 10 µL aliquots were removed from each well, serially diluted, and inoculated for CFU assay. To visualize the effect of the treatment on biofilm-associated bacteria, the biofilm was removed from the wells by scraping and stained with a Live / Dead Bacterial Viability Kit (ThermoFischer Scientific). A mixture of components A and B (0.3 L each) was added to the biofilm sample and mixed. The sample was incubated in the dark (15 minutes, room temperature), and 10 μL was placed on a glass slide and observed under a fluorescence microscope.

[0323] To assess the effect of TCP-25 sutures on biofilm growth in microtiter plate wells and on the sutures themselves, a 1 cm long TCP-25 suture fragment was added to the bottom of the well 30 minutes after the addition of bacterial culture medium for biofilm formation. Incubation was then performed as described above (37°C, 48 h). After incubation, the suture fragment was removed, and the wells were washed twice (100 µL Tris buffer) to remove planktonic cells. The extracted suture fragments were further processed for staining with the Live / Dead Bacterial Viability Kit, viable cell counting for CFU assays, or SEM. CFU counting of suture-associated biofilms was performed by placing the suture in a tube containing 100 µL Tris buffer. The sample was then sonicated (1 min x 3 times) to disrupt the biofilm. Samples were then taken and processed according to the aforementioned procedure for viable cell counting. For Live / Dead analysis, the above protocol was used, with the modification of placing the suture in a premixed staining agent to avoid biofilm disruption due to vortexing.

[0324] To assess biomass in each well, the plate was further treated by washing each well twice with distilled water, followed by the addition of 150 µL of 1% crystal violet. The plate was incubated for 15 minutes, after which the wells were rinsed again with distilled water. Then, 200 µL of 96% ethanol was added to the wells, and the plate was incubated for another 15 minutes. 120 µL was then transferred from each well to a new microtiter plate, and the solution was measured at OD. 600 The following analysis was performed to determine the absorbance in the well.

[0325] Tensile strength test: Using Instron ®8511.20 (Instron Corp) analyzed the tensile strength of the suture. A 10 cm suture segment was mounted on the sample holder by wrapping the suture three times around a cylindrical bolt and then tying three single knots to secure the suture. The same process was repeated at both ends of the test fixture, and the instrument was moved using a hydraulic controller to ensure no slack occurred in the suture, while taking care not to apply a force >1 N to the suture. The distance between the centers of the cylindrical bolts was measured using vernier calipers and recorded as L0 or the original length. The sample was then pulled axially at a predefined slope speed of 0.25 mm / sec, and the failure force and displacement (L1) were digitally recorded using a 250 N load cell. The maximum breaking force was obtained from the force-displacement data.

[0326] In addition, the % elongation was calculated using the following formula: % elongation = (L1 - L0) / L0 x 100, where L1 = displacement at failure, and L0 = the original length of the line before the test (measured as the distance between the centers of the clamping bolts). Data acquisition was performed using an MTS FlexTest 40 controller and MTS TestSuite Multipurpose Elite software.

[0327] Patient's wound fluid: This study used wound fluid from chronic venous lower extremity ulcers and acute wounds, the collection method of which has been previously described (Lundqvist et al., 2004). The wound fluid used in the experiments was collected from *Pseudomonas aeruginosa* (…). P. aeruginosa ) and Staphylococcus aureus ( S. aureus Patients who test positive for the virus. In short, centrifuge the wound fluid at 10,000 rpm using a benchtop centrifuge. Prepare aliquots and store at -20°C until further use.

[0328] Data Analysis: For normally distributed data, the Student's t-test was used to determine the difference in means between the two groups; otherwise, the Mann-Whitney test was used. For more than two groups, one-way ANOVA and post-hoc tests (Tukey) were used to compare means for normally distributed data; otherwise, the Kruskal-Wallis test was used. Data are expressed as mean ± SEM. A single legend includes details of the statistical analysis used in the experiment. Data analysis was performed using GraphPad Prism software v8. A p-value < 0.05 was considered statistically significant.

[0329] Ethical Statement: The experiments included in this study were conducted in accordance with the Swedish Animal Welfare Act SFS1988:534. Approval was obtained from the Malmö / Lund Animal Ethics Committee (License Nos. M252-11, M131-16, M88-91 / 14, M5934-19, 8871-19, M5935-19, 8643-20). The use of human wound fluid materials was approved by the Lund University Ethics Committee (LU 708-01 and LU 509-01). Informed consent was obtained from all donors. The use of human blood was approved by the Lund University Ethics Committee (License No. 657-2008).

[0330] Results and Discussion

[0331] Peptide release characterization of TCP-25 coated polyglucan sutures

[0332] To preserve the antimicrobial and anti-inflammatory effects of TCP-25, we decided to employ a simplified approach in this proof-of-concept work, avoiding the addition of formulation components. Therefore, a simple method involving immersing sutures in a TCP-25 peptide solution followed by drying was used to coat pigliptin sutures with TCP-25. To determine the optimal coating conditions, sutures were placed in 0.1%, 0.5%, 2.0%, and 4.0% TCP-25 solutions for 2 h, followed by air drying. The peptides were then eluted from the sutures, and the peptide concentration was determined. Sutures coated with a 2% TCP-25 solution showed significantly higher peptide recovery (…). Figure 1 a, left figure). Sutures coated with 4% TCP-25 solution did not show better TCP-25 recovery than sutures coated with 2% solution, which may be due to the oligomerization of TCP-25 molecules at high concentrations, as previously mentioned (Petruk et al., 2020).

[0333] Radial diffusion assay (RDA) was used to analyze the release and antimicrobial activity of the eluted peptides. Results showed that sutures coated with 2% TCP-25 exhibited the highest antimicrobial activity. Figure 1 a, right figure). Therefore, a 2% TCP-25 coating concentration was found to be optimal. Next, the effects of coating time and temperature on suture peptide content and antibacterial activity were investigated. Coating times of 1, 2, 4, 8, and 24 h all produced similar TCP-25 recovery rates in the sutures. Figure 1 b, left figure). Accordingly, using RDA, the inhibition zones for coating times of 1, 2, 4, and 8 hours were determined. Although the differences between time points were small, the inhibition zone for a 2-hour coating time was relatively higher than that for other coating times (b). Figure 1 b, right figure). The sutures of the coating at 21°C showed a relatively higher TCP-25 recovery rate than those of the coatings at 37°C and 50°C. Figure 1 c, left figure).

[0334] All coating temperatures showed no significant effect on antibacterial activity, as similar inhibition zones were observed in the RDA. Figure 1 (c, right figure) SDS-PAGE analysis of the peptides eluted from the sutures showed no degradation of TCP-25 at any of the coating times and temperatures. Furthermore, the TCP-25 loading of the sutures was investigated at coating concentrations of 0.5%, 2.0%, and 4.0%. A coating concentration of 0.5% TCP-25 resulted in low loading efficiency, while a 2% coating concentration showed significantly higher TCP-25 loading efficiency. Interestingly, a 4% coating concentration exhibited similar TCP-25 loading efficiency to the 2% concentration. Based on these results, a 2% coating concentration and coating at 21°C for 2 hours were selected as the final coating conditions for this study.

[0335] Next, to assess peptide release from the suture, we first used an in vitro model (Del Amo et al., 2019). A transwell filter insertion system was used, and the TCP-25 suture was held on a porous filter membrane in the head chamber. Elution buffer was added to fill the basal outer chamber. The elution buffer was then contacted with the filter and the TCP-25 suture (e.g., ...). Figure 1 (As shown in d). Following a rapid release in the initial 24 hours, sustained release of TCP-25 was observed over 72 hours. Figure 1 d). Further investigation of TCP-25 release from sutures was conducted using hairless SKH-1 mice. TAMRA-labeled TCP-25 was applied to the sutures and implanted subcutaneously. Long-term fluorescence bioimaging was then performed using the IVIS spectrum in vivo imaging system. Release of the peptide was observed immediately in the vicinity of the implant; most of the peptide appeared to be locally retained within and around the suture. Figure 1 e). TCP-25TAMRA fluorescence intensity was observed after 1 h, which gradually decreased over time, but a significant signal was still obtained even 72 h after implantation. Figure 1 e, bar chart). Finally, the surface morphology of the uncoated control and TCP-25 coated sutures was studied by scanning electron microscopy (SEM). Typical polyglucan suture weave structures were observed in both the control and coated sutures. Figure 1 f). No significant differences were observed in the surface of the TCP-25 coated suture compared to the control suture surface.

[0336] The peptide-peglitin interaction was characterized using QCM-D, computer modeling and simulation, and O-PTIR analysis.

[0337] To investigate the interaction between TCP-25 and Vicryl suture fibers, a dissipative monitoring quartz crystal microbalance (QCM-D) was employed. Finely cut suture fibers (50–100 μm) were dispersed in ethanol and then dropped onto a SiO2-based substrate for QCM-D measurements. To allow for accurate monitoring of peptide binding to the suture fibers, the underlying SiO2 surface was modified with poly-L-lysine at low ionic strength, resulting in a thin, net positively charged surface (Ringstad et al., 2006), which had previously been shown to exhibit low adsorption for a range of host defense peptides (Malekkhaiat Haffner et al., 2019).

[0338] In fact, cationic poly-L-lysine surface coatings are also effective in inhibiting TCP-25 adsorption, although the peptide concentration corresponds to the plateau value of other surface binding isotherms (Singh et al., 2013). Figure 2 a). In stark contrast, the fiber-coated surface exhibited very significant peptide binding, manifested as a strongly reduced frequency change ( F). Therefore, a frequency shift of –100 ± 27 Hz was observed on the fiber coating surface, while only –4 ± 1 Hz was observed on the underlying poly-L-lysine surface. Although these results cannot be quantitatively interpreted as to the amount of peptides bound to the fibers due to uncertainties in fiber surface area and solvent amount during crystal oscillations during measurement, it is clear that the peptides bind to and have a high affinity for the suture fibers, much higher than that of host defense peptides typically found to bind to bare SiO2 surfaces (Lozeau et al., 2018).

[0339] Finally, we note that the peptide binding kinetics are relatively slow (consistent with the release results discussed above), and that initiating flushing after peptide loading does not lead to immediate peptide release; both of these effects suggest that peptide binding occurs not only on the outer surface of the fiber, but also in pore and defect structures.

[0340] To understand the molecular mechanism of the interaction between TCP-25 and the polygliptin suture, we constructed a coarse-grained (CG) model of the polygliptin 910 copolymer comprising 90% glycolide and 10% lactide subunits; parameters were formed within a Martini force field framework based on atomic resolution sampling. We simulated the spontaneous self-aggregation of the polymer consisting of 100-mer chains, followed by the addition of 10 copies of the TCP-25 peptide.

[0341] The peptides rapidly adsorbed onto the surface of the polygliptin polymer. Figure 2 b). Within a time frame of less than 200 ns, all peptides in the system are interacting with the polymer ( Figure 2c, above figure), leading to the gradual burial of the polymer's solvent-accessible surface area (SASA). Figure 2 c, Figure below). Contact analysis shows that the negatively charged carboxyl termini of the polygliptin polymer chain interact most strongly with the cationic peptide. Figure 2 d, above figure). Interestingly, we observed that once peptides adsorb onto the outer surface of the polymer, they become entangled with the polymer chains and gradually integrate into the polymer phase. Figure 2 e), which is consistent with the QCM-D results above. This is evidenced by the significant decrease in SASA of the TCP-25 peptide during simulation. Polar (Y3, Y6, H8) and hydrophobic (F5, F10, W15, F23) residues play important roles in peptide integration into the polymer phase. Figure 2 d, as shown in the image below.

[0342] At higher peptide concentrations, we observed slower overall peptide adsorption, likely due to peptide aggregation in the solution prior to binding, consistent with the results obtained using different coating concentrations. At shorter chain lengths (50-mer), we found no difference in the ability of the peptide to integrate into the polymer phase. At physiological temperatures (310 K) and room temperature (298 K), we observed slightly slower peptide adsorption rates; however, by the end of the simulation, the peptides reached similar SASA values, indicating that lower temperatures do not significantly affect the ability of the peptide to integrate into the polymer. We also found that the binding of the peptide to the polygliptin polymer replaced Na+ ions on the polymer surface, further highlighting the interaction between the peptide and the polymer's carboxyl groups. Although we observed peptide aggregation in solution and on the polymer surface, depolymerization occurred once the peptide was integrated into the polymer phase.

[0343] In summary, our results are consistent with previous experimental studies on the interaction between polylactic acid-co-glycolic acid (PLGA) and cationic peptides, namely that peptides can not only bind to the surface but also be integrated and distributed within the polymer phase (Giles et al., 2013). This could explain why TCP-25 sutures retain their bioactivity after long-term storage.

[0344] To further verify the interaction between TCP-25 and Vicryl sutures, optical photothermal infrared (O-PTIR) spectral measurements were performed on uncoated and TCP-25 coated sutures. Spectral analysis showed that the coated sutures exhibited a wavelength of 1665 cm⁻¹. -1 A new band appeared, indicating the presence of TCP-25 on the fiber filament. Figure 2(f and g). Importantly, washing the suture in water for 30 min did not significantly affect the newly formed band, further supporting the interaction between TCP-25 and the suture. Finally, fluorescence imaging of cryosections of the TCP-25-Cy3-coated sutures revealed the distribution and localization of TCP-25 on the suture fibers. TCP-25 appeared to be densely distributed on and between the Vicryl fibers.

[0345] Peptide structure and function analysis

[0346] Interactions between peptides and other materials can have detrimental effects on their structure and function. To investigate whether TCP-25 coating of pigliptin sutures affects the structure and activity of peptides, we analyzed TCP-25 eluted from the coated sutures using high-performance liquid chromatography (HPLC). No differences were observed in the elution profiles of the major peptides, indicating that the coating procedure had no significant effect on peptide stability. Figure 3 a). The ability of TCP-25 to bind to and neutralize LPS is crucial to its anti-inflammatory activity. We further investigated whether TCP-25 retained its ability to interact with and bind to LPS after coating. The peptide was eluted from the coating suture, and its structural changes in the presence of LPS were investigated using circular dichroism (CD) analysis. The results showed that TCP-25 eluted from the coating suture exhibited similar α-helix-induced changes as observed in the control peptide, a finding indicating compatibility with the interaction between TCP-25 and LPS. Figure 3 b). After excitation at 280 nm, the change in the intrinsic fluorescence of the peptide was determined by LPS-induced structural changes. LPS-peptide binding was observed and shown as the emission maxima ( max blue shift () Figure 3 c) This is achieved by fitting TCP-25. max Determined as a function of different LPS concentrations. Kd was observed to be 11.15 ± 1.76 μg / mL, consistent with previous results. Figure 3 d)(Stromdahl et al., 2021).

[0347] Antibacterial properties and efficacy of TCP-25 coated sutures in vitro and in mouse models of suture infection

[0348] We used several in vitro assays to determine the antimicrobial efficacy of TCP-25 coated sutures. Bioluminescent Staphylococcus aureus (Staphylococcus aureus) was used. S. aureus ) and Pseudomonas aeruginosa ( P. aeruginosaThis was used to demonstrate the antibacterial efficacy. Compared to control sutures, bacteria treated with TCP-25-coated sutures showed a significant reduction in bioluminescence, visualized by IVIS bioimaging. This effect was observable 5 minutes after TCP-25 suture application, demonstrating the release and rapid antibacterial action of TCP-25. Figure 4 a).

[0349] In another experiment using bioluminescent bacteria, luminescence was measured using a photometer after the addition of sutures. Upon addition of TCP-25 coated sutures, a rapid and sustained decrease in bacterial luminescence was observed. Figure 4 b). Next, a live-dead assay was used to investigate membrane integrity. Analysis showed that only TCP-25 coated sutures resulted in Staphylococcus aureus (S. aureus) infection. S. aureus ) and Pseudomonas aeruginosa ( P. aeruginosa Significant permeability of bacteria Figure 4 c). SEM was used to further investigate the effect of TCP-25 coated sutures on bacterial morphology. Bacteria adhering to TCP-25 coated sutures showed significant morphological changes, such as bacterial lysis and aggregation, and the presence of cell debris was observed. Figure 4 d). In contrast, smooth and normal cell wall surfaces were observed on bacteria in the control suture group. Overall, these results indicate that TCP-25 maintains its rapid antibacterial activity even after being coated on piggliflozin sutures.

[0350] Next, to answer whether TCP-25 coated sutures maintain their antibacterial efficacy in vivo, we used a mouse model of suture infection simulating clinical SSIs. Staphylococcus aureus (S. aureus) was selected. S. aureus It was used in in vivo experiments because it was significantly correlated with SSIs (Saleh and Schmidtchen, 2015). Control or TCP-25 coated sutures were subcutaneously implanted into the left or right side of BALB / c mice and contaminated with bioluminescent Staphylococcus aureus (SSIs). S. aureus IVIS spectroscopy is used for non-invasive, long-term in vivo bioimaging of infections. A significant decrease in bioluminescence intensity was observed on the TCP-25 coated suture side compared to the control side. Figure 4 e). Bioluminescence measurements showed that bacterial infection persisted at the control suture site, while the TCP-25 coated suture maintained a significantly low level of infection throughout the experiment, even up to the final 72-hour observation point. Figure 4 f). At the 72-hour endpoint, analysis of the tissue surrounding the suture site showed that the TCP-25 coated sutures resulted in a significant reduction in bacterial counts. Figure 4 g).

[0351] To visualize the distribution of TCP-25 and further confirm that the antibacterial effect was indeed caused by the TCP-25-coated sutures, fluorescently labeled TCP-25 (TCP-25-TAMRA) was applied, followed by subcutaneous implantation and treatment with Staphylococcus aureus (Staphylococcus aureus). S. aureus Contamination. We observed that TCP-25-coated sutures inhibited bacterial growth, and TCP-25 fluorescence co-localized with the suture site. Figure 4 h).

[0352] To further investigate whether TCP-25 coated sutures exhibit resistance to Escherichia coli (E. coli) Escherichia coli The in vivo antibacterial efficacy of ) and the use of CLSI-controlled strains of Escherichia coli in sutures ( E. coli) (ATCC 25922) contamination was detected, and the tissue around the suture site was analyzed at 72 h. CFU assay showed that TCP-25 coated sutures resulted in a significant reduction in bacterial count.

[0353] In vitro and in vivo effects of TCP-25 coated sutures on endotoxin responses

[0354] We further investigated the anti-inflammatory efficacy of TCP-25-coated sutures using in vitro and in vivo models. Reporter gene assays were performed using THP1-XBlue™-CD14 cells. Reporter cells were stimulated with LPS containing or without TCP-25 from the coated sutures. In the presence of TCP-25 eluted from the sutures, a significant reduction in endotoxin-induced NF-κB and AP-1 activation was observed. Figure 5 a, the above figure). The results of the MTT assay showed that these concentrations of TCP-25 were not toxic to cells. Figure 5 (a, see the image below).

[0355] Next, it was important to understand whether the TCP-25 coated sutures exhibited similar anti-inflammatory efficacy in vivo. To investigate this effect, we used an NF-κB reporter mouse model in which the sutures were subcutaneously implanted and contaminated with LPS, followed by long-term in vivo inflammatory bioimaging using IVIS spectroscopy. High levels of local NF-κB activation were observed on the side implanted with the uncoated control suture. In contrast, the TCP-25 coated sutures resulted in a significant reduction in this NF-κB-driven inflammation at 3 and 24 h post-implantation. Figure 5 (b) At the end of the experiment, we recovered the sutures from the mice and eluted the absorbed proteins for cytokine analysis. Compared with the control sutures, significantly reduced levels of TNF-α and IL-6 cytokines were observed in the fluid extracted from the implanted TCP-25 coated sutures. Figure 5 c).

[0356] The effect of TCP-25 coated sutures on bacterial biofilm

[0357] To investigate whether TCP-25 coated sutures have an anti-biofilm effect, we used two different experimental methods.

[0358] In the first method, our aim was to evaluate the effect of the TCP-25 coating on sutures on biofilm growth in microtiter plate wells and on the sutures themselves. To achieve this, control or TCP-25 coated sutures were added to the wells of a microtiter plate 30 minutes after bacterial inoculation, during biofilm growth. Biofilm growth was allowed at 37°C. After 48 h, live / dead staining, bacterial counts of the suture-attached biofilm, and crystal violet staining of the microtiter plate biofilm were performed. Under a fluorescence microscope, Staphylococcus aureus (S. aureus) was visible on the sutures after live / dead staining. S. aureus ) and Pseudomonas aeruginosa ( P. aeruginosa Bacterial biofilm formation (white staining) Figure 6 a). Less biofilm formation and light gray staining were visible on TCP-25 coated sutures, indicating the presence of dead bacteria. A viable bacterial count was used to assess the total number of viable bacteria on the biofilm adhering to the suture. TCP-25 coated sutures showed a significantly lower bacterial count ( Figure 6 a. Bar chart).

[0359] Similarly, crystal violet staining of the plates showed a significantly lower biofilm formation in the pores containing TCP-25-coated sutures. Importantly, compared to the baseline antimicrobial sutures containing triclosan (Vicryl... ® The comparison of Plus showed the effect on Staphylococcus aureus (Plus) S. aureus It has similar anti-biofilm activity, but it is less effective against Pseudomonas aeruginosa. P. aeruginosa Biofilm formation activity was not significant. Figure 6 a and b). Finally, SEM analysis of the sutures showed that the amount of biofilm adhering to the TCP-25 coated sutures was significantly lower ( Figure 6 c).

[0360] In the second method, the aim is to investigate the effect of TCP-25 coated sutures on mature biofilms. Staphylococcus aureus was cultured on plates... S. aureus ) or Pseudomonas aeruginosa ( P. aeruginosa Biofilms were collected and exposed to TCP-25-coated sutures for 2 h. Live / dead staining showed a significant increase in dead bacterial cells (red staining) in samples exposed to TCP-25-coated sutures. Figure 6 (d and e). As expected, viable counts of biofilms exposed to TCP-25-coated sutures showed a significant reduction in bacterial counts. Figure 6 d and e, bar charts). The baseline triclosan-containing suture shows resistance to Staphylococcus aureus (…). S. aureus It exhibits considerable anti-biofilm activity, while being effective against Pseudomonas aeruginosa (…). P. aeruginosa The activity of the biofilm was not significant.

[0361] Effects of neutrophil elastase on TCP-25 coated sutures

[0362] Human neutrophil elastase (HNE) is an important enzyme produced during wound healing, infection, and inflammation. We have previously demonstrated that HNE digests TCPs in vitro and generates multiple fragments (Puthia et al., 2020). We have also shown that HNE can generate active TCP-25 fragments in TCP-25 hydrogels (Puthia et al., 2020). We wanted to investigate whether HNE could generate bioactive TCP-25 fragments from TCP-25-coated sutures. TCP-25-coated sutures were directly treated with HNE, followed by elution, and fragmentation profiles were analyzed by nano-LC-MS / MS. HNE digestion of TCP-25 on the sutures produced numerous peptides at different time points. Figure 7 a).

[0363] In addition to fragments, intact TCP-25 (SEQ ID NO:1) was observed throughout all study time periods. Many of these fragments are known to be biologically active and exert antimicrobial and anti-inflammatory activities (Puthia et al., 2020). Interestingly, the CD14 binding region was preserved in many fragments. Figure 7 b). As assessed by RDA, the peptide fragment "cocktail" produced by HNE digestion retained its antibacterial activity over a digestion period of up to 6 hours. Figure 7 c). Furthermore, the generated peptide fragments retained their anti-inflammatory activity, as observed in the THP-1 cell model system. Figure 7 d).

[0364] Overall, the results indicate that HNE digestion of coated sutures yields multiple TCP fragments from TCP-25, many of which are biologically active.

[0365] Tensile strength and hemolytic effect of TCP-25 coated sutures

[0366] We then wanted to investigate whether the TCP-25 coating on the polyglucan sutures had any adverse effect on their tensile strength. Mechanical testing of the sutures was performed using the Instron tensile strength testing system. No change in the tensile strength of the sutures was observed after the TCP-25 coating was applied. Figure 8 a, left figure). Host tissue response leads to the degradation and dissolution of pigliptin sutures (Reul, 1977).

[0367] We also wanted to examine whether the TCP-25 coating adversely affected suture degradation in tissue. TCP-25 coated sutures or control sutures were subcutaneously implanted into mice and removed four days later. Mechanical testing of the recovered TCP-25 coated sutures or control sutures from mouse tissue showed no difference in tensile strength, indicating that the TCP-25 coating did not adversely affect suture degradation in tissue. Figure 8 (b, right figure). After subcutaneous implantation, similar loss of tensile strength was observed in both the coated sutures and the control sutures compared to the unimplanted sutures. Furthermore, we investigated the hemolytic activity of the TCP-25 sutures. Results using sutures of 1 or 10 cm length showed a hemolysis rate of less than 10% in human blood, indicating blood compatibility. Figure 8 b).

[0368] Effects of storage on TCP-25 coated sutures

[0369] Furthermore, we wanted to investigate whether peptides would degrade after long-term storage of TCP-25 coated sutures. TCP-25 sutures were stored at room temperature for 18 months, after which the eluted peptides were analyzed. HPLC analysis showed no significant storage-related effects, and the peptides eluted from the stored TCP-25 sutures exhibited peaks similar to those of the fresh TCP-25 control peptides. Figure 8 c). A non-significant storage-related effect was noted, as several additional small peaks were observed in the chromatogram of TCP-25 eluted from the storage suture compared to the freshly prepared control peptide.

[0370] To confirm that the additional peaks in the chromatogram of the eluted TCP-25 did not correspond to significant peptide degradation, we applied Western blot analysis. Western blot analysis of the eluted peptide revealed multiple high-molecular-weight bands, confirming peptide oligomerization. Figure 8 d), which has been reported previously (Petruk et al., 2020). Next, we analyzed whether long-term storage of TCP-25-coated sutures had any negative impact on the LPS-binding ability of TCP-25. Circular dichroism analysis showed that the peptide retained its LPS-binding ability even after long-term storage. Figure 8 e). Importantly, RDA analysis of the eluted peptides showed no loss of antimicrobial activity after long-term storage of TCP-25 sutures. Figure 8 f). The oligomerization or aggregation of TCP-25 at the suture line during storage may explain its retained biological activity.

[0371] TCP-25 from coated sutures targets clinical bacterial isolates from human wounds and those derived from human wound fluid. Induced inflammation

[0372] After demonstrating antimicrobial and anti-inflammatory efficacy in vitro and in animal models, we finally investigated whether TCP-25-coated sutures could also target clinical bacterial isolates from wounds and inflammation associated with human wounds. A significant inhibitory zone was observed around the TCP-25-coated sutures when incubated with human wound isolates from various clinical sources. Furthermore, the use of wound-derived Staphylococcus aureus (S. aureus)... S. aureus ) and Pseudomonas aeruginosa ( P. aeruginosa Acute wound fluid (AWF) and chronic wound fluid (CWF) from patients colonized with bacteria such as AWF activate inflammation in THP-1 reporter cells. TCP-25 eluted from coated sutures leads to a reduction in human wound fluid-induced inflammation in THP-1 cells. Figure 9 a, the above figure). The results of the MTT assay showed that these concentrations of TCP-25 were not toxic to cells. Figure 9 (a, see the image below).

[0373] Finally, in vivo, TCP-25-coated sutures were contaminated with human chronic wound fluid and implanted into the backs of NF-κB reporter mice. Non-invasive IVIS imaging was performed to visualize NF-κB activation. The control sutures contaminated with human chronic wound fluid induced significantly more inflammation than the TCP-25-coated sutures. Figure 9 b).

[0374] In summary, these results demonstrate that TCP-25 coated sutures have the potential to reduce infection and inflammation in complex human wound conditions.

[0375] in conclusion

[0376] Combining computational molecular modeling studies, in vitro antimicrobial assays, biochemical and biophysical assays, and in vivo models, we demonstrate a proof-of-concept that it is possible to functionalize pigglitin suture materials with a dual-action host defense peptide that can simultaneously target bacteria and excessive inflammatory responses. This broad capability has biological and clinical significance. It is understood that, upon initial contact with tissue, a given biomaterial itself induces an immune response, which can lead to dysregulation of the inflammatory response, resulting in inefficient host defense and making the given biomaterial susceptible to infection (Busscher et al., 2012).

[0377] Furthermore, in the presence of bacterial adhesion, the release of bacterial products such as lipoteichoic acid (LTA) and LPS can stimulate inflammation near the biomaterial. The possible mechanisms of suture-related infections are only partially understood, with research primarily focusing on bacterial adhesion and biofilm formation. However, applying these understandings to sutures to create a local suture environment that both combats bacteria and controls the immune response may be a novel and attractive strategy for optimizing and enhancing infection control. Another serious problem is the declining effectiveness of antibiotics and other antimicrobial agents due to the development of antimicrobial resistance (AMR). The development of resistance is particularly significant in surgical procedures, where the combination of extensive intraoperative antibiotic use, high risk of systemic transmission, and bacterial sepsis leads to a high frequency of infections, necessitating the use of antibiotics as a "last resort." Considering these issues, there is an unmet need for novel bio-guided strategies based on the multi-pronged approach of TCP-25, targeting bacteria in novel ways to minimize resistance problems and control excessive inflammatory processes.

[0378] Currently, several antimicrobial agents are in use or being developed for sutures (Chua et al., 2022). Based on their widespread use, we decided to use triclosan as the baseline antimicrobial agent in this study (Schweizer 2001). As mentioned in the introduction, there is growing concern about the use of this preservative. Therefore, in the case of Staphylococcus aureus (… S. aureus Triclosan resistance is frequently reported (Suller and Russell, 2000). Furthermore, Nadafpour et al., 2021, investigated bacterial colonization on various suture materials used in dental implants. Compared to vicryl alone, vicryl... ® Plus sutures showed E. coli ( E. coli ) and Staphylococcus aureus ( S. aureus The highest buildup of ) and the Vicryl triclosan coating ®Plus sutures did not show any advantage over commonly used silk sutures in reducing bacterial counts. A meta-analysis by Elsolh et al. (Elsolh et al., 2017) determined whether antibiotic-impregnated sutures (including triclosan) could prevent surgical site infections and complications after abdominal surgery. They found no evidence to support the routine use of these sutures. More recently, a systematic review and meta-analysis of randomized controlled trials (S. National Institute of Health Research Unit on Global, Lancet Infect Dis 2022, 22, 1242) showed no benefit from the use of triclosan-coated sutures. The authors noted that recommendations in global and national guidelines regarding the routine use of triclosan-coated sutures should be reassessed. In this context, it should be mentioned that in 2016, the U.S. Food and Drug Administration (FDA) banned the addition of triclosan to household products and subsequently banned its use in over-the-counter antiseptic products without premarket review. The European Commission did not approve triclosan as an active ingredient in Group 1 biocidal products in 2016. Given the questionable risk-benefit profile of triclosan, several other antimicrobial sutures are in use or under development (Chua et al., 2022). These sutures incorporate classic antiseptics such as chlorhexidine, polyhexamethylene biguanide (PHMB), octenidine, and povidone-iodine. Other substances used are derived from natural products such as chitosan, aloe vera, silver nanoparticles, or various antibiotics. Notably, octenidine and chlorhexidine have been applied to Vicryl via impregnation coating, as used in this study.

[0379] Unlike the aforementioned antimicrobial drugs, this work endows Vicryl sutures with previously unexplored pharmacological functions based on TCP-25's ability not only to kill bacteria but also to clear various bacterial products and simultaneously inhibit downstream CD14 / TLR-mediated inflammatory responses (Saravanan et al., 2018). Notably, our results highlight the specific interaction between TCP-25 and Vicryl suture fibers. Using QCM-D, we demonstrated the binding and affinity of the peptide to the suture fibers, further confirmed by molecular modeling and simulation using a coarse-grained model of the TCP-25 and p-ligliptin 910 copolymer. Furthermore, O-PTIR spectroscopy confirmed the presence of TCP-25 on the surface of the coated suture fibers, and fluorescence imaging demonstrated a dense distribution of TCP-25 on and between the Vicryl fiber filaments. Taken together, these findings provide a comprehensive characterization of the TCP-25-p-ligliptin interaction, supporting both in vitro and in vivo data.

[0380] It should be mentioned that the dual-action concept based on TCP-25 has shown promise in the context of wound healing and infection (Puthia et al., 2020; Stromdahl et al., 2021), and the TCP-25 hydrogel is currently in clinical development, undergoing Phase I human safety studies. By utilizing clinically approved absorbable sutures and peptides with established safety profiles, our research paves the way for the rapid clinical translation of TCP-25-coated Vicryl sutures, addressing the urgent need for effective coating biomaterials to combat infection and inflammation in the surgical setting. Looking ahead, the subsequent development of the final suture product should include further evaluation and standardization of the coating procedure, peptide stability assays, optimal peptide release kinetics analysis, biocompatibility assessments, and comprehensive preclinical and clinical studies.

[0381] Example 2 – TCP-25 Coated Particles

[0382] PLGA particles coated with TCP-25

[0383] PLGA particles (1 mg, average diameter 50 μm, lactate / glycolic acid ratio 50 / 50 (Sigma, No. 805122)) were suspended in 400 μL of 1% TCP-25 solution. The mixture was incubated at room temperature with continuous shaking at 1000 rpm for 2 hours. After incubation, the particles were centrifuged at 15,000 rpm for 20 minutes at 20 °C to precipitate the coated particles. The supernatant was carefully removed, and the concentration of TCP-25 in the supernatant was measured to assess the coating efficiency. The precipitated particles were then resuspended in 500 μL of 10 mM Tris buffer, vortexed for 5–10 seconds, and centrifuged again at 15,000 rpm for 15 minutes at 20 °C. The washing step was repeated at least twice to remove unbound peptides. After the final wash, the PLGA particles were dried at 30 °C for 1 hour using SpeedVac.

[0384] SDS-PAGE analysis of TCP-25 coated PLGA particles

[0385] For SDS-PAGE analysis, the dried PLGA precipitate was resuspended in 500 μL of 10% SDS solution and incubated at 99 °C for 5 min. The mixture was then centrifuged for 2 min, and 10 μL of the resulting supernatant was collected for analysis. Additionally, 8 μL of supernatant from the initial coating step and 10 μL of supernatant from each of the three washing steps were loaded onto a 10–20% Tricine gel. Electrophoresis was run at 100 V for 1 hour and 40 min to assess the presence of TCP-25 on the particles.

[0386] like Figure 10 The results show that, after the washing step, TCP-25 binds to the particles and is released through the SDS solution.

[0387] Detection and localization of TCP-25 on PLGA particles

[0388] To detect TCP-25 on PLGA particles, the particles were coated with TCP-25 doped with TAMRA-TCP25 (1% of total TCP-25). The coating conditions were similar to those described above. The coated particles were imaged using a fluorescence microscope to detect TCP-25-TAMRA, as shown below. Figure 11 As shown (TCP-25-TAMRA is light gray in the figure).

[0389] Fluorescence microscopy results showed that TCP-25 was bound to PLGA particles.

[0390] It is expected that binding with PLGA particles will reduce the degradation of TCP-25, thereby preserving the LPS binding capacity of TCP-25 even after long-term storage, as observed with suture binding in Example 1 above.

[0391] Viable cell count (VCA) of dried TCP-25 coated PLGA particles.

[0392] To evaluate the antibacterial activity of TCP-25 coated PLGA particles, a viable count (VCA) assay was performed. *Pseudomonas aeruginosa* (…) was included in the assay. Pseudomonas aeruginosa PA01 (50,000 CFU / tube) or E. coli ( E. coli ATCC25922 was added to dry TCP-25 coated or uncoated PLGA particles. The bacteria and particles were incubated in 10 mM Tris buffer (pH 7.4) at +37°C and 5% CO2 for 2 hours. After incubation, serial dilutions of 10X, 100X, 1,000X, 100,000X, and 1,000,000X were prepared in Tris buffer. The diluted samples were then plated on TH agar plates and incubated overnight at +37°C and 5% CO2. Bacterial viability was determined by counting the colonies formed.

[0393] The results showed that the TCP-25 coating particles reduced bacterial growth by more than 99%. Figure 12 This shows a typical example of bacterial growth on TH agar, where no bacterial colonies were detected after TCP-25-PLGA treatment.

[0394] Antimicrobial effect of dried TCP-25 coated PLGA particles

[0395] For radial diffusion assay (RDA), dried TCP-25 coated PLGA particles are scraped from the bottom of the tube and spread onto a substrate inoculated with E. coli. Escherichia coli or Staphylococcus aureus (Staphylococcus aureus) The particles were uniformly distributed on the gel surface and were measured to assess the antimicrobial activity of the coated particles through the inhibition zone formed around the particles.

[0396] The TCP-25 coating on PLGA particles produces an antibacterial effect, which is due to... Figure 13 The PLGA particles shown are surrounded by Gram-negative Escherichia coli (E. coli). E. coli ) and Gram-positive Staphylococcus aureus ( S. aureus It can be seen from the absence of bacterial growth in all samples.

[0397] Example 3 – Amount of PLGA sutures and particulate-absorbable TCP-25

[0398] Measurements showed that 1 mg PLGA suture absorbs approximately 5-6 μg of TCP-25, while 1 mg PLGA particles absorb approximately 40-50 μg of TCP-25.

[0399] Sequence Overview

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Claims

1. A material comprising a poly(lactic-co-glycolic acid) (PLGA) polymer and a peptide, said peptide comprising or consisting of the following amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16, where X3, X7, X10, X11, X13, X15, and X16 are any standard amino acids. X8 and X12 can be any amino acid. X1, X6, and X14 are G, A, V, L, I, P, F, M, Y, or W, and X2, X4, X5, and X9 are R, K, or H. The peptide is 10 to 40 amino acid residues long, for example, 10 to 30 amino acid residues.

2. The material according to claim 1, wherein the peptide comprises or is composed of the following amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16, where X1, X6, and X14 are either F or W, and X2, X4, X5, and X9 are either R or K.

3. The material according to any one of the preceding claims, wherein the peptide is 18 to 30 amino acids in length, preferably 18 to 25 amino acids, and comprises or consists of any amino acid sequence selected from the group consisting of: SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity therewith, SEQ ID NO: 2 or an amino acid sequence having at least 90% sequence identity therewith, SEQ ID NO: 3 or an amino acid sequence having at least 90% sequence identity therewith, SEQ ID NO: 4 or an amino acid sequence having at least 90% sequence identity therewith, SEQ ID NO: 5 or an amino acid sequence having at least 90% sequence identity therewith, SEQ ID NO: 6 or an amino acid sequence having at least 90% sequence identity therewith, SEQ ID NO: 7 or an amino acid sequence having at least 90% sequence identity therewith, SEQ ID NO: 8 or an amino acid sequence having at least 90% sequence identity therewith, and SEQ ID NO: 9 or an amino acid sequence having at least 90% sequence identity therewith.

4. The material according to any one of the preceding claims, wherein the peptide comprises or is composed of the following sequence: VFRLKKWI-X1-KVI-X2-ZFG in X1 and X2 are amino acids linked by covalent bonds.

5. The material according to any one of the preceding claims, wherein the PLGA polymer comprises glycolic acid and lactic acid in a ratio of 5:95 to 95:5 (glycolic acid: lactic acid) or is composed of glycolic acid and lactic acid in a ratio of 5:95 to 95:5 (glycolic acid: lactic acid).

6. The material according to any one of the preceding claims, wherein the PLGA polymer comprises glycolic acid and lactic acid in a ratio of 5:95 to 90:10 (glycolic acid: lactic acid) or is composed of glycolic acid and lactic acid in a ratio of 5:95 to 90:10 (glycolic acid: lactic acid).

7. The material according to any one of claims 1 to 6, wherein the PLGA polymer comprises glycolic acid and lactic acid in a ratio of 25:75 (glycolic acid: lactic acid), such as 30:70 (glycolic acid: lactic acid), such as 50:50 (glycolic acid: lactic acid), or is composed of glycolic acid and lactic acid in a ratio of 25:75 (glycolic acid: lactic acid), such as 30:70 (glycolic acid: lactic acid), such as 50:50 (glycolic acid: lactic acid).

8. The material according to any one of the preceding claims, wherein the PLGA polymer comprises glycolic acid and lactic acid in a ratio of 90:10 (glycolic acid: lactic acid) or is composed of glycolic acid and lactic acid in a ratio of 90:10 (glycolic acid: lactic acid), preferably wherein the PLGA polymer is polygliptin 910.

9. The material according to any one of the preceding claims, wherein the peptide is coated on the fibers of the PLGA polymer, dissolved in the fibers of the PLGA polymer, and / or dispersed between the fibers of the PLGA polymer.

10. The material according to any one of the preceding claims, wherein the material is in particulate form, such as microparticles or nanoparticles.

11. The material of claim 10, wherein the average diameter of the particles is from 100 nm to 100 μm, such as from 100 nm to 50 μm.

12. The material according to any one of the preceding claims, wherein the material is anti-inflammatory and / or antimicrobial, such as antibacterial.

13. A medical product comprising the material described in any one of the preceding claims.

14. The medical product of claim 13, wherein the medical product is selected from the group consisting of sutures, strips, films, scaffolds, grafts, hydrogels, particles such as microparticles or nanoparticles, and dressings such as meshes, patches or bandages, preferably wherein the medical product is a suture.

15. The medical product of claim 14, wherein the medical product is a strip, film, scaffold, graft, hydrogel, or dressing comprising particles of any one of claims 10 to 11.

16. The medical product according to any one of claims 13 to 15, wherein the medical product is antimicrobial, such as antibacterial, and / or anti-inflammatory.

17. The medical product according to any one of claims 13 to 16, wherein when the medical product comes into contact with a body part, the medical product is capable of continuously releasing the peptide to the body part for at least 24 hours, such as at least 48 hours, such as at least 72 hours.

18. The medical product according to any one of claims 13 to 17, wherein the peptide is stable for at least 12 months, such as at least 18 months, such as at least 24 months, during dry storage of the medical product at room temperature.

19. A method for producing a medical product according to any one of claims 13 to 18, the method comprising the steps of: immersing a starting material comprising or composed of PLGA in a coating solution comprising a dissolved peptide according to any one of claims 1 to 12, and subsequently drying the starting material.

20. The method of claim 19, wherein the starting material is PLGA-containing particles.

21. The method according to any one of claims 19 to 20, wherein the medical product is a suture.

22. The medical product according to any one of claims 13 to 18, for preventing and / or inhibiting inflammation and / or infection in a body part of a subject, preferably wherein the body part of the subject is a wound, such as a surgical wound.

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