Modified wound dressing

Abstract

Embodiments described herein relate to compounds for detecting wounds (e.g., chronic wounds or infected wounds), including compositions, substrates, kits, dressing materials and articles, and systems containing these compounds. Other embodiments relate to methods for using these compositions, kits and systems for diagnostic assays, and for diagnosing and / or detecting chronic or infected wounds based on enzymatic conversion of specific substrates contained in the compositions. Additional embodiments relate to methods for characterizing wounds based on the expression of multiple markers, and using this information to treat, manage and follow up patients with chronic or infected wounds.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 315,567, filed on March 30, 2016, the disclosure of which is incorporated herein by reference in its entirety and made a part of this application. Technical Field

[0003] Embodiments described herein relate generally to wound healing, and particularly to compositions and methods for detecting and treating wounds. Background Art

[0004] In mammals, skin damage triggers a series of organized complex cellular and biochemical events, leading to wound healing. Wound healing is a complex dynamic process that leads to the restoration of anatomical continuity and function: an ideally healed wound is one that is restored to normal anatomical structure, function and appearance. Typical wounds heal by a model consisting of the following four stages: an exudation stage, a proliferation stage, a repair stage and epithelial maturation (Hatz et al., Wound Healing and Wound Management, Springer-Verlag, Munich, 1994) or hemostasis, inflammation, proliferation and remodeling stages (Nwomeh et al., Clin. Plast. Surg. 1998, 25, 341).

[0005] Unfortunately, chronic and "infected" wounds are often difficult to heal. Chronic wounds include, for example, venous ulcers of the lower extremities, diabetic foot ulcers, and pressure sores (Krasner et al., Chronic Wound Care: A Clinical Source Book for Healthcare Professionals, HMP Communications, 2001). Patients with chronic wounds require extensive care, and wounds often result in a reduced quality of life; chronic wounds may become a problem that some patients must deal with for the rest of their lives. Patient comorbidities can also have a significant impact on the wound healing process, limiting or even stopping the process and becoming a factor in the reduced quality of life. Factors that can lead to difficult wound healing include pathophysiological problems, microbial infection, the presence of inactive tissue, poor tissue perfusion, chronic inflammatory conditions, and other underlying conditions such as diabetes (Bowler et al., Annals of Medicine 2002, 34, 419-427).

[0006] In many cases, chronic wounds are colonized by bacterial flora and / or pathogens such as fungi and viruses, in which case chronic wounds may become infected. Bacterial infection of wounds delays the healing process because bacteria produce enzymes and toxins and also compete for nutrients and oxygen with macrophages and fibroblasts, whose activity is essential for wound healing. Therefore, infection is a manifestation of a disturbed host / bacteria balance in favor of bacterial invasion. This causes a systemic septic response and also inhibits multiple processes involved in wound healing. The granulation stage of healing will only begin after the infection has resolved.

[0007] The inflammatory phase is particularly important to the wound healing process, where biochemical reactions at the wound site promote healing but also lead to tissue destruction due to the production of excess proteases. Although proteases play an important role in breaking down dead tissue, excess amounts can also have adverse effects on living tissue, causing additional inflammation.

[0008] The release of these proteolytic enzymes, such as matrix metalloproteinases (MMPs), elastases, and cathepsin G, is often associated with excessive neutrophil stimulation.

[0009] Elevated protease activity seems to be the reason for delaying wound repair, and may indicate wound infection. For example, the extracellular matrix (ECM) is a collection of extracellular molecules secreted by cells, which provides structure and biochemical support for surrounding cells, and due to excessive protease (e.g., MMP) activity and simultaneously due to reduced fibrinogen levels, the extracellular matrix is ​​often exhausted at the wound site. Increased protease activity also leads to growth factor degradation, thereby inhibiting the healing process. Therefore, infection and other problems increase the weight of chronic wounds and wounds are still difficult to treat (Yager et al., "Wound Repair Regeneration (Wound Repair Regen)" 1997,5,23-32; Widgerow et al., "Wound Repair Regeneration (Wound Repair Regen)" 2011,19,287-291).

[0010] Current methods of assessing wounds rely on the practitioner's training and experience. Wounds can be assessed visually, length and depth measurements can be taken, and digital photography can be used, where available, to track the appearance and size of the wound (Krasner et al., supra). In clinical practice, the diagnosis of infection is based on indirect parameters, such as the presence of local pain, fever, swelling, discharge, and redness. Many of these clinical indicators, such as inflammation and discharge, have low predictive value for wound infection. Swabbing the wound in a hospital laboratory followed by microbiological testing is an option to confirm bacterial colonization and identify the strain so that the correct course of antibiotics can be prescribed; however, the process is time-consuming and labor-intensive. Delays in the diagnosis of infection may delay the administration of antibiotics and may increase the risk of developing sepsis.

[0011] Additionally, there are few objective techniques for measuring wound healing. Although there are reports of MMP protease levels and activity in chronic wound fluid compared to healing wound fluid (Liu et al., Diabetes Care 2009, 32, 117-119; Bullen et al., J. Investig. Dermatol. 1995, 104, 236-240), these reports do not suggest a threshold at which a wound becomes chronic.

[0012] Therefore, there is an urgent but unmet need for sensitive and specific reagents, kits and assay techniques for characterizing chronic and infected wounds in human and animal subjects, including the use of such reagents and / or kits for scientific research as well as clinical applications, for example, for the effective and accurate diagnosis and treatment of diseases characterized by such wounds (e.g., venous ulcers, decubitus ulcers and diabetic ulcers). Summary of the invention

[0013] The technology disclosed herein provides compositions and methods for detecting infection and / or chronic wounds. The disclosed technology improves the exit assay by increasing the sensitivity, accuracy and specificity of detecting infected wounds; providing the ability of qualitative and quantitative measurements; and improving the speed of in-situ and real-time detection of infected wounds. The assay and method described herein are based in part on the use of specific reagents for detecting biomarkers and / or probes present in infected or chronic wounds. The detection process may involve the use of reagents that are specific to markers present in infected wounds rather than in uninfected or non-chronic wounds, and the detection step may involve qualitative or quantitative measurement of the signal generated when the marker acts on the probe. In embodiments where the detection method involves detecting an enzyme present in a wound, the probe preferably comprises a modified enzyme substrate that is specific to the enzyme, which produces a signal that can be optionally amplified. This greatly improves the efficiency and specificity of the detection. In addition, a variety of detection probes can be used, each of which is specific to one or more targets, such as an enzyme specific to the wound. This greatly helps to maximize the efficiency and accuracy of the diagnostic assay while minimizing the incidence of false positives (e.g., due to nonspecific interactions and / or target redundancy). In addition, the experimental results disclosed herein demonstrate that the novel probes and assays based thereon are capable of detecting and characterizing various types of wounds. Finally, the reagents of the disclosed technology can be used together with therapeutic molecules such as antibiotics, antifungals, etc. to monitor and evaluate the treatment and management of chronic wounds.

[0014] In one embodiment, provided herein is a wound dressing material comprising a compound of formula I, comprising the structure MLR, wherein M is a gel-forming polymer; R is a reporter molecule; L is a linker that is absent or present, and L connects M and R when present.

[0015] In another embodiment, provided herein is a compound comprising the structure MR, wherein M is a gel-forming polymer and R is a reporter molecule.

[0016] In another embodiment, provided herein is a compound comprising a structure MR, wherein M is a gel-forming polymer, R is a reporter molecule, and wherein M is covalently or non-covalently conjugated to R.

[0017] In another embodiment, provided herein is a wound dressing material comprising a compound of formula I, comprising the structure MLR, wherein M is a gel-forming polymer; R is a reporter molecule; and L is a linker covalently or non-covalently conjugated to M and R independently of each other.

[0018] In another embodiment, provided herein is a wound dressing material comprising a compound of formula I, comprising the structure MLR, wherein M is a gel-forming polymer; R is a reporter molecule; and L is a linker, wherein the reporter molecule R comprises an enzyme substrate.

[0019] In another embodiment, a wound dressing material is provided herein, comprising a compound of formula I, comprising the structure MLR, wherein M is a polymer that forms a gel; R is a reporter molecule; L is a linker, wherein the reporter molecule R comprises an enzyme substrate, which is a sugar, a polysaccharide, a nucleic acid, an amide, a peptide, a protein, a lipid, or a derivative thereof, or a combination thereof. In particular, under this embodiment, the substrate is a sugar, a polysaccharide, an amide, a peptide or a protein, or a derivative thereof. In particular, under this embodiment, the substrate is a peptide substrate (PEP), which comprises an amino acid or a peptide comprising multiple amino acids.

[0020] In another embodiment, there is provided herein a wound dressing material comprising a compound of formula I comprising the structure MLR, wherein M is a gel-forming polymer; R is a reporter molecule; L is a linker that is absent or present, and L, when present, connects M and R, wherein M is selected from cellulose, carboxymethyl cellulose, pectin, alginate, chitosan, hyaluronic acid, a polysaccharide or a gum-derived polymer, or a derivative thereof or any mixture or combination thereof. In particular, under this embodiment, the polymer is carboxymethyl cellulose (CMC) or a salt thereof.

[0021] In another embodiment, provided herein is a wound dressing material comprising a compound of formula I, comprising the structure MLR, wherein M is a gel-forming polymer; R is a reporter molecule; L is a linker that is absent or present, and L, when present, connects M and R, wherein M comprises from about 200 to about 4000 monomer units. In particular, under this embodiment, M comprises from about 500 to about 2000 monomer units.

[0022] In another embodiment, a wound dressing material is provided herein, comprising a compound of formula I, comprising the structure MLR, wherein M is a gel-forming polymer; R is a reporter molecule; L is a linker that is absent or present, and L connects M and R when present, wherein L comprises a monomer or a neutral polymer, which is an ethoxylated polyol, a polyvinyl pyrrolidone polymer, a polypropylene, a polyalkylene glycol, a polyamine, or an ether, amide or ester thereof. In particular, under this embodiment, L comprises 1 to 10 monomer units of an ethoxylated polyol, a polyvinyl pyrrolidone polymer, a polypropylene, a polyalkylene glycol, a polyamine, or an ether, amide or ester thereof. More specifically, under this embodiment, L comprises at least one polypropylene glycol subunit.

[0023] In another embodiment, provided herein is a wound dressing material comprising a compound of formula I comprising the structure MLR, wherein M is a gel-forming polymer; R is a reporter molecule; L is a linker that is absent or present, and L, when present, connects M and R, wherein R comprises a detectable label. In particular, under this embodiment, the detectable label is selected from the group consisting of: a luminescent molecule, a chemiluminescent molecule, a fluorescent dye, a fluorescent quencher, a lipid, a colored molecule, a radioisotope, a scintillator, biotin, avidin, streptavidin, protein A, protein G, an antibody or a fragment thereof, polyhistidine, Ni2+, a Flag tag, a myc tag, a heavy metal, and an enzyme.

[0024] In another embodiment, provided herein is a wound dressing material comprising a compound of formula I comprising the structure MLR, wherein M is a gel-forming polymer; R is a reporter molecule; L is a linker that is absent or present, and L, when present, connects M and R, wherein R comprises a fluorescent molecule selected from the group consisting of fluorescein, rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-3, Cy-5, Cy-7, Texas Red, Phar Red, allophycocyanin (APC), fluoresceinamine, eosin, dansyl, umbelliferone, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein ( JOE), 6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid, acridine, acridine isothiocyanate, r-amino-N-(3-vinylsulfonyl)phenylnaphthalimide-3,5, disulfonate (fluorescent yellow VS), N-(4-anilino-1-naphthyl)maleimide, o-aminobenzamide, brilliant yellow, coumarin, 7-amino 4-Methylcoumarin, 7-amino-4-trifluoromethylcoumarin (coumarin 151), tetrachlorotetrabromofluorescein, 4′,6-diamidino-2-phenylindole (DAPI), 5′,5″-diamidino-2-phenylindole (DAPI), 5′,5″-dibromopyrogallol-sulfonphthalein (bromopyrophenol red), 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin diethylenetriamine pentaacetate, 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC), eosin isothiocyanate , erythrosine B, erythrosine isothiocyanate, ethidium, 5-(4,6-dichlorotriazine-2-yl)aminofluorescein (DTAF), QFITC ​​(XRITC), fluorescamine, IR144, IR1446, malachite green isothiocyanate, 4-methylumbelliferone, o-cresolphthalein, nitrotyrosine, pararosaniline, phenol red, B-phycoerythrin, o-phthalaldehyde, pyrene, pyrene butyrate, 1-pyrene butyric acid succinimidyl ester, Reactive Red 4, lissamine rhodamine B sulfonyl chloride, rhodamine B, rhodamine 123, rhodamine X, sulfonyl rhodamine B, sulfonyl rhodamine 101, sulfonyl chloride derivatives of sulfonyl rhodamine 101, tetramethylrhodamine, riboflavin, rhodic acid and terbium chelate derivatives.

[0025] In another embodiment, provided herein is a wound dressing material comprising a compound of Formula I, which comprises the structure M-L-R, where M is a gel-forming polymer; R is a reporter molecule; L is a linker that is absent or present, and when present, L connects M and R, where R comprises a detectable label and a quencher molecule. In particular, under this embodiment, the reporter molecule comprising the quencher molecule is activated by an enzyme or its product.

[0026] In another embodiment, provided herein is a wound dressing material comprising a compound of Formula I, which comprises the structure M-L-R, where M is a gel-forming polymer; R is a reporter molecule; L is a linker that is absent or present, and when present, L connects M and R, where the reporter molecule or a portion thereof is released upon interaction with an enzyme. In particular, under this embodiment, the reporter molecule comprises a detectable label that is released upon interaction with an enzyme.

[0027] In another embodiment, provided herein is a wound dressing material comprising a compound of Formula I, which comprises the structure M-L-R, where M is a gel-forming polymer; R is a reporter molecule; L is a linker that is absent or present, and when present, L connects M and R, where the reporter molecule comprises a substrate specific for a wound-specific enzyme, which forms a product under the action of the enzyme. In particular, under this embodiment, the wound-specific enzyme is a protease. More particularly, in this embodiment, the reporter molecule comprises a substrate specific for a wound-specific enzyme selected from the group consisting of: MMP-1 (collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-8 (neutrophil collagenase), MMP-9 (gelatinase B), human neutrophil elastase (HNE), cathepsin G, urokinase-type plasminogen activator (uPA), and lysozyme. In particular, under this embodiment, the reporter molecule comprises a substrate specific for MMP-2 and MMP-9 or a combination thereof.

[0028] In another embodiment, provided herein is a composition comprising a carrier and a wound dressing material, the wound dressing material comprising a compound of Formula I, which comprises the structure M-L-R, where M is a gel-forming polymer; R is a reporter molecule; L is a linker that is absent or present, and when present, L connects M and R. In particular, under this embodiment, the composition is a pharmaceutical composition. More particularly, under this embodiment, the pharmaceutical composition comprises an antibiotic compound or a peptide that heals wounds. In particular, under this embodiment, the antibiotics are selected from the group consisting of: beta-lactams, fluoroquinolones, aminoglycosides, tetracyclines, glycylcyclines, and polymyxins, and / or the peptide that heals wounds is fibroblast growth factor (FGF) or platelet-derived growth factor (PDGF).

[0029] In another embodiment, provided herein is a composition comprising an article comprising a wound dressing material as described above.

[0030] In another embodiment, the present invention provides a method for diagnosing a wound state of a subject in need thereof, comprising contacting the wound with a wound dressing material as described above to allow the reporter molecule to be converted into a detectable signal and detecting the signal. In particular, under this embodiment, the conversion of the reporter molecule to a detectable signal is carried out by a wound-specific protease, for example, by a wound-specific protease, the wound-specific protease being selected from the group consisting of: MMP-1 (collagenase), MMP-2 (gelatinase A), MMP-3 (matrilysin 1), MMP-8 (neutrophil collagenase), MMP-9 (gelatinase B), human neutrophil elastase (HNE), cathepsin G, urokinase-type plasminogen activator (uPA) and lysozyme. In particular, in this embodiment, the method includes diagnosing chronic wounds or infected wounds.

[0031] In another embodiment, the present invention provides a method for diagnosing the wound state of a subject in need thereof, comprising contacting the wound with a wound dressing material as described above to allow the reporter molecule to be converted into a detectable signal and detecting the signal; evaluating the parameter of the activity or level of a wound-specific enzyme in the wound; comparing the parameter with a threshold level; and if the parameter level of the wound is above the threshold level, determining that the wound is chronic or infected. In this embodiment, the parameter is the amount or activity of a wound-specific protease, and the wound-specific protease is selected from the group consisting of: MMP-1 (collagenase), MMP-2 (gelatinase A), MMP-3 (matrilysin 1), MMP-8 (neutrophil collagenase), MMP-9 (gelatinase B), human neutrophil elastase (HNE), cathepsin G, urokinase-type plasminogen activator (uPA) and lysozyme. In particular, under this embodiment, the diagnostic method is performed in situ.

[0032] In another embodiment, provided herein is a method of treating a wound in a subject in need thereof, comprising contacting the wound with a wound dressing material as described above. In particular, under this embodiment, the dressing material is applied topically or dermally at the wound site.

[0033] In another embodiment, there is provided herein a method for preparing a compound according to the aforementioned formula I, wherein L is absent, comprising conjugating a gel-forming polymer M to a reporting region R, wherein M and R are each as described above. In particular, under this embodiment, the gel-forming polymer M is conjugated to the reporting region R via a covalent link, wherein the covalent link is selected from the group consisting of: a peptide link, a glycosidic link, an ester link, an oxyester link, an amide link, an amide link, an oxyamide link, an ether link, a sulfonyl link, a sulfinyl link, a sulfonamide link, an alkoxy link, an alkylthio link, an alkylamino link, or a combination thereof. In particular, under this embodiment, the gel-forming polymer M is conjugated to the reporting region R via a glycosidic link or a peptide link.

[0034] In another embodiment, provided herein is a method for preparing a compound according to the aforementioned formula I, wherein L is present, comprising conjugating a gel-forming polymer M with a linker L to produce a precursor molecule ML; conjugating the precursor molecule ML with a reporter region R, wherein M, L and R are each as described above. In particular, under this embodiment, the gel-forming polymer M is conjugated to the linker L via a covalent bond and / or the linker L is conjugated to the reporter region R via a covalent bond, wherein the covalent bond is selected from the group consisting of: an ester bond, an oxyester bond, an amide bond, an amide bond, an oxyamide bond, an ether bond, a sulfonyl bond, a sulfinyl bond, a sulfonamide bond, an alkoxy bond, an alkylthio bond, an alkylamino bond or a combination thereof.

[0035] In another embodiment, the present invention provides a method for preparing a compound according to the aforementioned formula I, wherein L is present, comprising conjugating a linker L to a reporter molecule R to produce a precursor molecule LR; conjugating the precursor molecule LR to a gel-forming polymer M, wherein M, L and R are each as described above. In particular, under this embodiment, the gel-forming polymer M is conjugated to the linker L via a covalent bond and / or the linker L is conjugated to the reporter region R via a covalent bond, wherein the covalent bond is selected from the group consisting of: an ester bond, an oxyester bond, an amide bond, an amide bond, an oxyamide bond, an ether bond, a sulfonyl bond, a sulfinyl bond, a sulfonamide bond, an alkoxy bond, an alkylthio bond, an alkylamino bond or a combination thereof.

[0036] In another embodiment, provided herein is a wound dressing material comprising a compound comprising the structure ML-PEP (Formula II), wherein M is a gel-forming polymer; PEP is a peptide and at least one amino acid; L is a linker that is absent or present, and when present, L connects M and PEP.

[0037] In another embodiment, provided herein is a wound dressing material selected from the group consisting of:

[0038]

[0039]

[0040]

[0041]

[0042]

[0043] Where n=200-4000.

[0044] It should be understood that other embodiments and configurations of the subject technology will be readily apparent to those skilled in the art from the following detailed description, in which various configurations of the subject technology are shown and described by way of example or illustration. As will be appreciated, the subject technology can have other and different configurations, and its several details can be modified in various other aspects, all without departing from the scope of the subject technology. Therefore, the drawings and detailed description are considered to be illustrative rather than restrictive in nature. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] For the purpose of understanding the present disclosure, reference will now be made to the accompanying drawings, which illustrate embodiments and examples of the present disclosure and, together with the following description, serve to explain the principles of the present disclosure, by way of example.

[0046] Figure 1 Quantification of the enzyme efficacy of polymer 12 using fluorescence-based studies is shown.

[0047] Figure 2 shows Figure 1 Images of 12 samples of the polymer used in the fluorescence-based studies.

[0048] Figure 2A Shown are samples viewed under ambient light.

[0049] Figure 2B The samples are shown observed under UV light.

[0050] Figure 2C Displays the labels for each sample.

[0051] Figure 3 Quantification of the enzyme efficacy of polymer 17 (pre-treated with PBS) using fluorescence-based studies (elution after a PBS soaking period) is shown.

[0052] Figure 4Quantification of the enzymatic efficacy of polymer 17 (pre-treated with PBS) using elution based on fluorescence studies (solids were resuspended in PBS after an initial PBS soak period) is shown.

[0053] Figure 5 Shown are mean closure times (error bars show SD) during scratch model testing for cell lines from patients A, F, and G. Red bars highlight samples in fiber form; blue bars represent samples in powder form.

[0054] Figure 6 shows confocal micrographs of the scratch assay of 12, CMC-PEG-NH2 powder, patient A, 0.066 mg / mL. The red dashed line represents the scratch area, and fibroblast proliferation can be observed between T=1 hour (no cells in the channel) and T=68 hours (cells fill the channel).

[0055] Fig. 6A Fibroblast proliferation observed at 1 hour is shown.

[0056] Figure 6B Fibroblast proliferation observed at 30 hours is shown.

[0057] Figure 6C Fibroblast proliferation observed at 50 hours is shown.

[0058] Fig.6D Shown is the fibroblast proliferation observed at 68 hours.

[0059] Figure 7 shows confocal micrographs of the scratch assay of 12, CMC-PEG-NH2 powder, 0.66 mg / mL of patient A. The red dashed line represents the scratch area, where fibroblast proliferation can be observed between T=1 hour (no cells present in the channel) and T=68 hours (cells filling the channel).

[0060] Fig. 7A Fibroblast proliferation observed at 1 hour is shown.

[0061] Figure 7B Fibroblast proliferation observed at 30 hours is shown.

[0062] Figure 7C Fibroblast proliferation observed at 50 hours is shown.

[0063] Fig.7D Shown is the fibroblast proliferation observed at 68 hours.

[0064] Figure 8 Showing results of a study using a collagen matrix model, graph shows lattice diameter over 7 days - Patient A.

[0065] Fig. 9 Showing results from a study using a collagen matrix model, graph shows lattice diameter over 7 days - Patient F.

[0066] Fig.10 Showing results from a study using a collagen matrix model, graph shows lattice diameter over 7 days - Patient G.

[0067] Fig.11 Shown are the results of a study using a collagen matrix model, with photographs indicating the difference in lattice diameter at day 3 and day 7 - Patient A.

[0068] Fig.12 Shown are the results of a study using a collagen matrix model, with photographs indicating the difference in lattice diameter at day 3 and day 7 - Patient F.

[0069] Fig.13 Shown are the results of a study using a collagen matrix model, with photographs indicating the difference in lattice diameter at day 3 and day 7 - Patient G.

[0070] Fig.14 Schematic diagram of the CMC and LC system showing potential peptide modifications for detecting proteases, where (a) shows the initial system setup showing homeotropic LC alignment (viewed under crossed polarized lenses, dark), (b) shows cleavage of the peptide releasing lipids, and (c) shows the cleaved lipids in contact with the LC, which will initiate planar LC rearrangement (viewed under crossed polarized lenses, colored).

[0071] Figure 15 shows a micrograph showing a TEM (transmission electron microscope) grid filled with liquid crystal 4'-n-n-pentyl-4-cyano-biphenyl (5CB) upon application of a CMC gel.

[0072] Fig.15A TEM grid filled with 5CB before application of CMC hydrogel - homeotropic LC arrangement is shown.

[0073] Fig. 15B Shown is a TEM grid filled with 5CB after application of CMC hydrogel at T=0; change to a planar LC arrangement.

[0074] Fig. 15C Shown are 5CB-filled TEM grids (planar LC alignment) after T=2 min application of CMC hydrogel (left) and 5CB-filled TEM grids (planar LC alignment) after T=5 min application of CMC hydrogel (right).

[0075] Fig.15DShown are 5CB-filled TEM grids after T=10 min application of CMC hydrogel (with planar LC alignment preserved) (left) and 5CB-filled TEM grids after T=90 min application of CMC hydrogel (with planar LC alignment preserved) (right). DETAILED DESCRIPTION

[0076] Various aspects will now be described more fully below. However, these aspects may be implemented in many different forms and should not be construed as limited to the embodiments set forth herein; on the contrary, these embodiments are provided to make this disclosure thorough and complete, and to fully convey its scope to those skilled in the art.

[0077] Throughout this disclosure, various patents, patent applications and publications are cited. The disclosures of these patents, patent applications and publications are incorporated into this disclosure in their entirety by reference, so as to more fully describe the prior art known to those skilled in the art before the disclosure date. In the event of any inconsistency between the cited patents, patent applications and publications and this disclosure, the disclosure shall prevail.

[0078] I. Definitions

[0079] Where a range of values ​​is provided, it is intended that each intervening value between the upper and lower limits of the range and any other stated or intervening value in the stated range is encompassed within the disclosure. For example, if a range of 1 μm to 8 μm is specified, it is intended that 2 μm, 3 μm, 4 μm, 5 μm, 6 μm and 7 μm are also specifically disclosed, as well as ranges of values ​​greater than or equal to 1 μm and ranges of values ​​less than or equal to 8 μm.

[0080] Unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include plural referents. Thus, for example, reference to "a polymer" includes a single polymer as well as two or more of the same or different polymers; reference to "an excipient" includes a single excipient as well as two or more of the same or different excipients, etc.

[0081] Unless otherwise indicated in the context of the present disclosure, or inconsistent with such an interpretation, the word "about" immediately preceding a numerical value means a range of plus or minus 10% of the value, e.g., "about 50" means 45 to 55, "about 25,000" means 22,500 to 27,500, etc. For example, in a numerical list such as "about 49, about 50, about 55," "about 50" means a range extending to less than half of the interval between the preceding value and the following value, e.g., greater than 49.5 to less than 52.5. In addition, the phrase "less than about" a value or "greater than about" a value should be understood according to the definition of the term "about" provided herein.

[0082] "Substantially" or "substantially" means almost completely or entirely, for example, 80% to 95% or more of some given amount, for example, at least 85%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or more%, by weight or volume or any other measured parameter. "Substantially free" means almost completely or completely absent from some given amount, for example, present at a level of less than about 1% to about 20% of some given amount, for example, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1% or less, by weight or volume or any other measured parameter. In some embodiments, "substantially free" refers to a level less than or equal to 1 to 5 weight % of the pharmaceutical composition.

[0083] II. Compositions and Systems for Wound Dressings

[0084] Provided herein are modified wound dressing materials for use in wound dressings for use in the treatment and diagnosis of wounds and wound management, wherein the wound dressing material, when used, demonstrates elevated levels of enzymes present in situ in the wound.

[0085] As used herein, "wound" refers to a physical disruption of the continuity or integrity of a tissue structure. "Wound healing" refers to the restoration of tissue integrity. It should be understood that this may refer to partial or complete restoration of tissue integrity. Thus, treatment of a wound refers to the promotion, improvement, progression, acceleration or other advancement of one or more stages or processes associated with the wound healing process.

[0086] Wounds can be acute or chronic. Chronic wounds, which include pressure ulcers, venous leg ulcers, and diabetic foot ulcers, can be simply described as wounds that fail to heal. Although the exact molecular pathogenesis of chronic wounds is not fully understood, it is thought to be multifactorial. These wounds are characterized by a prolonged inflammatory response, defective wound extracellular matrix (ECM) remodeling, and failure of re-epithelialization due to impairment of the normal responses of resident and migrating cells during acute injury.

[0087] The wound can be any internal wound, e.g., a bruise or internal ulcer, or an external wound, in particular a skin wound, while maintaining the external structural integrity of the skin, and the tissue can therefore be any internal or external body tissue. In one embodiment, the tissue is skin (e.g., human skin), i.e., the wound is a skin wound, e.g., a dermal or epidermal wound.

[0088] Human skin consists of two distinct layers, the epidermis and dermis, with the subcutaneous tissue beneath. The main functions of the skin are to protect internal organs and tissues from external trauma and infection by pathogens, and to sense and regulate body temperature.

[0089] The outermost layer of the skin, the epidermis, is about 0.04 mm thick, is avascular, consists of four cell types (keratinocytes, melanocytes, Langerhans cells, and Merkel cells), and is stratified into several epithelial cell layers. The innermost epithelial layer of the epidermis is the basement membrane, which is in direct contact with the dermis and fixes the epidermis to the dermis. All epithelial cell divisions that occur in the skin occur at the basement membrane. After cell division, the epithelial cells migrate to the outer surface of the epidermis. During this migration, the cells undergo a process called cornification, whereby the cell nucleus is lost and the cells are transformed into tough, flat, resistant, non-living cells. The migration is complete when the cells reach the outermost epidermal structure, the stratum corneum (a dry, waterproof squamous cell layer), which helps prevent the underlying tissue from drying out. This layer of dead epithelial cells is constantly shed and replaced by keratinocytes that move from the basement membrane to the surface. Because the epidermal epithelium is avascular, the nutrient supply of the basement membrane depends on the dermis.

[0090] The dermis is a highly vascularized tissue layer that provides nutrition to the epidermis. In addition, the dermis contains nerve endings, lymphatic vessels, collagen, and connective tissue. The dermis is approximately 0.5 mm thick and is primarily composed of fibroblasts and macrophages. These cell types are primarily responsible for the production and maintenance of collagen, a protein found in all animal connective tissues, including the skin. Collagen is primarily responsible for the resilient, elastic properties of the skin. The subcutaneous tissue found beneath the collagen-rich dermis provides skin mobility, insulation, calorie storage, and blood to the tissues above it.

[0091] Wounds can be divided into one of two major categories, namely partial-thickness wounds or full-thickness wounds. Partial-thickness wounds are confined to the epidermis and superficial dermis, with no damage to the dermal blood vessels. Full-thickness wounds involve destruction of the dermis and extend into deeper tissue layers, involving destruction of the dermal blood vessels. Healing of partial-thickness wounds occurs by simple regeneration of epithelial tissue. Wound healing of full-thickness wounds is more complex. The skin wounds considered in this article can be partial-thickness wounds or full-thickness wounds.

[0092] Wounds considered herein include cuts and lacerations, surgical incisions or wounds, punctures, abrasions, scratches, pressure wounds, abrasions, friction wounds (e.g., diaper rash, friction blisters), decubitus ulcers (e.g., pressure sores or ulcers); thermally affected wounds (burns from cold and heat sources (direct or by conduction, convection or radiation) and electrical sources), chemical wounds (e.g., acid or alkali burns) or pathogenic infections (e.g., viral, bacterial or fungal), including open or intact boils, rashes, scars and acne, ulcers, chronic wounds (including diabetic-related wounds such as leg and foot ulcers, venous leg ulcers and pressure sores), skin graft / transplant donor and recipient sites, immune response conditions such as psoriasis and eczema, gastric or intestinal ulcers, oral wounds (including oral ulcers), cartilage or bone damage, amputation wounds and corneal lesions.

[0093] Wound dressing materials and their preparation:

[0094] Embodiments described herein provide modified wound dressings that can be used to diagnose and / or treat chronic wounds. The dressing may comprise a gel-forming polymer, a non-gel-forming fiber, or a combination thereof. The wound dressing materials described herein are used in methods for detecting one or more enzyme levels in a mammalian wound. In some embodiments, the wound dressing materials described herein are used in methods for diagnosing chronic wounds in mammals. In some embodiments, the wound dressing materials described herein are used in methods for diagnosing infected wounds in mammals. In other embodiments, the wound dressing materials described herein are used in methods for treating wounds in mammals. In other embodiments, the wound dressing materials described herein are used in methods for treating chronic wounds in mammals.

[0095] In some embodiments, the wound dressing material has the structure of Formula I:

[0096] MLR

[0097] Formula I

[0098] wherein M is a gel-forming polymer; R is a region comprising a reporter molecule; and L is a linker connecting M and R. In one embodiment, the linker (L) is present. In another embodiment, the linker (L) is absent, in which case the wound dressing material comprises a compound of formula MR, wherein M and R are each as described above.

[0099] In some embodiments, the wound dressing material has the structure of Formula II:

[0100] ML-PEP

[0101] Formula II

[0102] wherein M is a gel-forming polymer; PEP is a peptide region comprising a reporter molecule and at least one amino acid; and L is a linker connecting M and PEP. In one embodiment, the linker (L) is present. In another embodiment, the linker (L) is absent, in which case the wound dressing material comprises a compound of formula MR, wherein M and R are each as described above.

[0103] In a specific embodiment, the wound dressing material of Formula I or Formula II does not contain a linker, wherein the reporter molecule (R) or peptide (PEP) is directly covalently or non-covalently linked to the polymer forming the gel. As understood in the art, covalent bonds involve sharing electrons between bonded atoms. In contrast, non-covalent bonds may include, for example, ionic interactions, electrostatic interactions, hydrogen bonding interactions, physicochemical interactions, van der Waals forces, Lewis acid / Lewis base interactions, or combinations thereof. In particular, in the absence of a linker, the peptide is linked or conjugated to the polymer forming the gel by covalent interactions.

[0104] The term "peptide" includes peptides and pharmaceutically acceptable salts of peptides. Typically, a peptide comprises a plurality of amino acid residues, such as 2, 3, 4, 5, 6, 8, 10 or more amino acid residues, which are bonded to each other by covalent bonds (e.g., peptide bonds). "Amino acid residue" refers to the individual amino acid units incorporated into the peptides of the present disclosure. As used herein, the term "amino acid" refers to naturally occurring or synthetic amino acids, as well as amino acid analogs, stereoisomers, and amino acid mimetics that are similar in function to naturally occurring amino acids. This definition includes naturally occurring amino acids, for example: 1. Histidine (His) 2. Isoleucine (Ile) 3. Leucine (Leu) 4. Lysine (Lys) 5. Methionine (Met) 6. Phenylalanine (Phe) 7. Threonine (Thr) 8. Tryptophan (Trp) 9. Valine (Val) 10. Arginine (Arg) 11. Cysteine ​​(Cys) 12. Glutamine (Gln) 13. Glycine (Gly) 14. Proline (Pro) 15. Serine (S er) 16. Tyrosine (Tyr) 17. Alanine (Ala) 18. Asparagine (Asn) 19. Aspartic acid (Asp) 20. Glutamic acid (Glu) 21. Selenocysteine ​​(Sec); unnatural amino acids: citrulline; cystine; γ-aminobutyric acid (GABA); ornithine; theanine and amino acid derivatives such as betaine; carnitine; carnosine creatine; hydroxytryptophan; hydroxyproline; N-acetylcysteine; S-adenosylmethionine (SAM-e); taurine; tyramine. In particular, amino acids containing reactive side chains such as cysteine; serine; threonine; lysine; arginine; aspartic acid / asparagine; glutamic acid / glutamine; glycine; alanine, etc. are used;

[0105] In certain embodiments, the peptide can be modified, for example, by adding, deleting, replacing one or more amino acids, by derivatizing one or more amino acids, or by cyclization and other modifications. In particular, the peptide is modified by adding, deleting or replacing one or more amino acids at the carboxyl terminal (C-terminus) or the amino terminal (N-terminus). In particular, the peptide is modified by adding at least one amino acid, especially an amino acid containing a reactive side chain, such as cysteine, serine, threonine, lysine, arginine, aspartic acid / asparagine, glutamic acid / glutamine, glycine, alanine, etc., at the C-terminus, wherein the reactive side chain can be used in combination with a label (such as a dye). In particular, under this embodiment, the peptide is modified to contain an additional cysteine ​​or serine residue at the C-terminus, wherein the sulfhydryl group of cysteine ​​or the hydroxyl group of serine is used for coupling with a fluorescent dye.

[0106] In a specific embodiment, peptides containing additional amino acids containing reactive side chains (e.g., SH groups of cysteine) can be coupled to dyes by click chemistry. Herein, the reaction between 1,2-aminothiol and 2-cyanobenzothiazole (CBT) can be used to prepare fluorescent fluorescein. Then, the fluorescence of fluorescein can be quantified by spectrometry after washing and used to determine the relative presence of molecules with 1,2-aminothiol. If it is necessary to quantify proteins with non-1,2-aminothiols, the target protein can be cleaved to produce fragments with N'Cys susceptible to 2-CBT. See Liang et al., J. Angew. Chem., Int. Ed., 48, 965, 2009.

[0107] Gel-forming polymer (M):

[0108] In some embodiments of the wound dressing material of Formula I or Formula II, the gel-forming polymer is a compound selected from the group consisting of cellulose, chemically modified cellulose, pectin, alginate, chitosan, modified chitosan, hyaluronic acid, a polysaccharide or a gum-derived polymer, or a derivative thereof or any mixture or combination thereof.

[0109] In some embodiments of the wound dressing material of Formula I or Formula II, the gel-forming polymer is selected from the group consisting of cellulose, carboxymethyl cellulose (CMC), oxidized cellulose (or its derivatives), cellulose ethyl sulfonate (CES), pectin, alginate, chitosan, modified chitosan, hyaluronic acid, polysaccharides or gum-derived polymers, or any combination or mixture thereof.

[0110] In some embodiments of the wound dressing material of Formula I or Formula II, the gel-forming polymer is cellulose or chemically modified cellulose, such as carboxymethyl cellulose, oxidized cellulose or derivatives thereof, cellulose ethyl sulfonate.

[0111] In one embodiment, the polymer forming the gel is a derivative of a polymeric compound, such as a cellulose derivative. The term "derivative" as used herein includes salts, amides, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs of the polymer forming the gel. For example, where the polymer is cellulose, the hydroxyl groups (-OH) of the cellulose can react partially or completely with various reagents to form derivatives with useful properties, such as cellulose esters and cellulose ethers (-OR). In one embodiment, the derivative of cellulose is selected from carboxymethyl cellulose, methyl cellulose, ethyl cellulose, methyl ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose and hydroxypropyl cellulose. Those skilled in the art can easily prepare these derivatives using known methods of this derivatization. In certain embodiments, the derivative can be administered to animals or humans without substantial toxic effects, and is pharmaceutically active or is a prodrug. Representative types of cellulose derivatives are described in U.S. Patents Nos. 7,544,640 and 9,561,188.

[0112] In another embodiment, the derivative is a salt of a polymeric compound, such as Li + 、Na + , K + , Rb + Mg 2+ , Ca 2+ , Sr 2+ Or Ba 2+ A salt, preferably Na + , K + Mg 2+ , Ca 2+ Salts of cellulose, cellulose esters and cellulose ethers, such as sodium or calcium salts, are known in the art.

[0113] In some embodiments, the polymeric compound forming the gel may contain a combination or mixture of one or more of the aforementioned compounds. The term "combination" includes compounds containing more than one component, which may be conjugated or non-conjugated to each other. In one embodiment, the polymeric compound forming the gel comprises a combination of one or more of the aforementioned compounds, which are conjugated to each other, for example, by covalent or non-covalent interactions. As a specific example, the polymer forming the gel may comprise a combination of pectin and carboxymethyl cellulose. See Ninan et al., Carbohydr Polym. 2013 Oct 15; 98(1): 877-85; PMID: 23987424.

[0114] In some embodiments, the compound includes a mixture of the aforementioned polymeric compounds. The term "mixture" refers to two or more substances mixed together without reacting to make them lose their respective properties. For example, a mixture of compound A and compound B can contain any weight ratio of compound A and compound B, so that the total weight of the mixture totals 100%, such as a weight ratio of compound A / compound B of 99:1 or a weight ratio of compound A / compound B of 1:99. A typical mixture may contain about 2, 3, 4, 5 or more of the aforementioned polymeric compounds.

[0115] In some embodiments of wound dressing materials of formula I or formula II, the polymer forming the gel is in the form of a powder or fiber, or a combination thereof. In some embodiments of wound dressing materials of formula I or formula II, the polymer forming the gel is in the form of a fiber. Gel-forming fibers are hygroscopic fibers that become moist, smooth or gel-like when absorbing wound exudate, thereby reducing the tendency of surrounding fibers to adhere to the wound. Gel-forming fibers can be types that maintain their structural integrity when absorbing exudate, or can be types that lose their fiber form and become a structureless gel. Gel-forming fibers preferably have an absorbency of at least 2 grams of 0.9% saline solution per gram of fiber (measured by the free swelling method).

[0116] In some embodiments, the wound dressing material may comprise non-gel forming fibers. In some embodiments, the non-gel forming fibers are selected from cellulose fibers (e.g., cotton or lyocell / TENCEL), polyester, nylon, viscose, aramid, acrylic, elastic fibers (LYCRA), polyolefins, polylactic acid, silk, and natural or synthetic wool. In some embodiments, the wound dressing material comprises a gel-forming polymer and non-gel forming fibers.

[0117] In some embodiments of wound dressing materials of Formula I or Formula II, the gel-forming polymer is in powder form. In certain embodiments of wound dressing materials of Formula I or Formula II, powdered gel-forming polymers are preferred over fibrous gel-forming fibers because the powdered gel-forming polymers have a higher degree of substitution (DoS). In some embodiments of wound dressing materials of Formula I or Formula II, fibrous gel-forming fibers are preferred over powdered gel-forming polymers.

[0118] In some embodiments of the wound dressing material of Formula I or Formula II, the gel-forming polymer has a DoS of at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 2.0, or more. The term DoS is understood in the art. For example, in the case of cellulose chemistry, each anhydroglucose (β-pyranose) unit has three reactive (hydroxyl) groups; thus, the DoS can range from zero (cellulose) to three (fully substituted cellulose).

[0119] Linker (L):

[0120] In some embodiments, where the wound dressing material comprises a linker, the linker can be covalently or non-covalently linked to the polymer forming the gel. As understood in the art, covalent bonds involve sharing electrons. In contrast, non-covalent bonds can include, for example, ionic interactions, electrostatic interactions, hydrogen bonding interactions, physiochemical interactions, van der Waals forces, Lewis acid / Lewis base interactions, or combinations thereof. In particular, the linker is linked or conjugated to the polymer forming the gel via a covalent interaction.

[0121] In one embodiment, the chemical linker is a carboxylic acid having 2 to 10 carbon atoms, particularly 4 to 8 carbon atoms, or particularly about 4 to 6 carbon atoms.

[0122] In another embodiment, the linker is a monomer or neutral polymer selected from the group consisting of ethoxylated polyols, polyvinyl pyrrolidone polymers, polypropylene, polyalkylene glycols, polyamines including ethers, amides and esters thereof.

[0123] In one embodiment, the neutral polymer is polypropylene, but its monomers containing propylene may also be used. Polypropylene (PP) is one of the most important and widely used polyolefins as a matrix material because it has low density, low production cost, design flexibility and recyclability. Because polypropylene is hydrophobic, it may be incompatible with polar surfaces, such as cellulose. This problem can be solved by incorporating functionalized polypropylene such as poly(propylene-grafted-maleic anhydride) (PP-g-MA) into a composite material, where the carboxylic anhydride groups can provide covalent bonding to cellulose. See Spoljaric et al., Composites: Part A 40, 791–799, 2009.

[0124] In one embodiment, the neutral polymer linker is a polyalkylene glycol, but monomers containing alkylene glycols may also be used. The term "polyalkylene glycol" refers to a linear or branched polyalkylene glycol polymer, such as polyethylene glycol, polypropylene glycol, and polybutylene glycol. A polyalkylene glycol subunit is a single polyalkylene glycol unit. For example, an example of a polyethylene glycol subunit is ethylene glycol -O-CH2-CH2-O-, or propylene glycol -O-CH2-CH2-CH2-O-, terminated with hydrogen at the chain ends. Other examples of poly(alkylene glycols) include, but are not limited to, PEG, PEG derivatives (e.g., methoxypoly(ethylene glycol) (mPEG)), poly(ethylene oxide), PPG, poly(tetramethylene glycol), poly(ethylene oxide-co-propylene oxide), or copolymers and combinations thereof.

[0125] In another embodiment, the neutral polymer is a polyamine, but amine-containing monomers thereof may also be used. The term "polyamine" refers to polymers having amine functionality in the monomer unit, incorporated into the backbone, as in polyalkyleneimines, or incorporated into pendant groups, as in polyvinylamines.

[0126] In particular, the linker is PEG or a PEG derivative, such as methoxypoly(ethylene glycol) (mPEG), poly(ethylene oxide), PPG, poly(tetramethylene glycol), poly(ethylene oxide-co-propylene oxide), or copolymers and combinations thereof.

[0127] In another embodiment, other hydrophilic or hydrophobic linkers can also be used as linkers, as long as they are flexible, such as, for example, not containing double bonds or cyclic structures or only containing a small amount of double bonds or cyclic structures. Representative examples include, for example, polyalkylene, polyhydroxyalkylene, polyalkylene succinate, polylactide, etc., and the chain length is about 2 to about 20 chain atoms. The chain length of polyalkylene glycol can vary between 3 units (MW about 150Da) at the edge, for example, about 100 units (MW about 5000). Polyalkylene glycol can vary between about 1 / 200 to about 1 / 1, especially about 1 / 50 to about 1 / 1.5 relative to the relative amount of polysaccharide, depending on the thickness required for the product and the required flexibility. Referring to U.S. Patent No. 9,089,614 and U.S. Pre-authorization Publication No. 2005-0079155.

[0128] In some embodiments of the wound dressing material of Formula I or Formula II, the linker L comprises 1 to about 20 monomer units, such as monomer units of natural polymers, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more monomer units. In specific embodiments, the linker comprises 1 to about 5 ethylene glycol units or derivatives thereof, 2 to about 5 ethylene glycol units or derivatives thereof, 2 to about 8 ethylene glycol units or derivatives thereof, 2 to about 10 ethylene glycol units or derivatives thereof, or 5 to about 10 ethylene glycol units or derivatives thereof.

[0129] In some embodiments, linker L comprises a chemical moiety that is a product of a nucleophilic reaction. Generally, the term "nucleophile" is considered in the art to represent a chemical group with a reactive electron pair that reacts with a compound (typically another nucleophile) by displacing a leaving group, such as typically occurring in aliphatic chemistry as a unimolecular (referred to as "SN1") or bimolecular ("SN2") reaction. Examples of nucleophiles include uncharged compounds, such as amines, thiols and alcohols, and charged groups, such as alkoxides, thiols, thiolates, carbon anions, and various organic and inorganic anions. Illustrative anionic nucleophiles particularly include simple anions, such as azide, cyanide, thiocyanate, acetate, formate or chloroformate, and bisulfite.

[0130] In some embodiments, the linker L comprises a maleimide-thiol adduct. Maleimide is particularly useful for conjugation with thiol-containing substances, for example, with thiol-containing amino acids such as cysteine. The thiol group reacts with maleimide to form a thioether via double bond addition. Maleimide is selective for cysteine ​​thiols relative to methionine, histidine or tyrosine. The reaction of maleimide with amines generally requires a higher pH than the reaction of maleimide with thiols. The hydrolysis of maleimide competes significantly with thiol modification, particularly above pH 8. See U.S. Pre-Grant Publication No. 2007-0087446.

[0131] In some embodiments, L comprises a haloacetamide-thiol conjugation product. Haloacetamides, such as iodoacetamide or bromoacetamide, can also be used to covalently bind to the thiol group of an amino acid (eg, cysteine).

[0132] In some embodiments, the linker L comprises a thiol or disulfide containing compound, which can be used similarly for maleimide. See Zalipsky et al., Bioconjug. Chem. 6, 150-165, 1995; Greenwald et al., Crit. Rev. Ther. Drug Carrier Syst. 17, 101-161, 2000; and Herman et al., Macromol. Chem. Phys. 195, 203-209, 1994. See also U.S. Pat. No. 7,432,330.

[0133] Reporter molecules and markers:

[0134] In some embodiments, the wound dressing material comprises a region containing a reporter molecule. In particular, the reporter molecule is a substrate for one or more wound-specific markers, such as an enzyme found in the wound environment. As used herein, a "wound-specific enzyme" is an enzyme differentially expressed in a wound. "Differential expression" means that the level or activity of an enzyme in the wound microenvironment is higher or lower than that in other sites (such as normal tissue or surrounding tissue). In particular, compared with normal or uninjured tissue, differential expression means a higher level of expression or activity of an enzyme in the wound microenvironment. The differential expression of an enzyme can be analyzed by conventional methods. For example, the enzyme level in a sample can be analyzed by ELISA determination or other immunoassays. The activity of an enzyme can be analyzed by measuring the loss rate of a substrate and / or the formation rate of a product, for example, using a mass spectrometer or HPLC. These techniques are known in the art and described in the Examples section.

[0135] In one embodiment, the marker is an enzyme selected from the group consisting of a hydrolase, a protease, an esterase, and a peroxidase.

[0136] In one embodiment, the marker is a hydrolase. As used herein, "hydrolase" or "hydrolytic enzyme" is an enzyme that catalyzes the hydrolysis of chemical bonds, such as esterases and nucleases (breaking ester bonds); glycolases (breaking glycosidic linkers); peptidases (breaking peptide bonds), etc.

[0137] In one embodiment, the marker is a protease. The protease can be sequence-specific or general protease. In particular, the term "sequence-specific protease" describes a protease (e.g., caspase) that recognizes the specific sequence of a peptide for its digestion, and is distinguished from general proteases (e.g., trypsin), which sequentially decompose peptides from one end or digest peptides in a sequence-nonspecific manner. For sequence specificity, the amino acid sequence of a peptide substrate can include four or more amino acid (aa) residues. Recognition site and digestion site can be close to each other.

[0138] As used herein, the term "substrate peptide of a protease" refers to a peptide comprising an amino acid sequence of a protein that is recognized by a protease as a substrate for its protease activity, e.g., a substrate that can be cleaved into one or more products. In some embodiments, the wound dressing material comprises a peptide region comprising a peptide sequence comprising a plurality of amino acids. The term "plurality" refers to two or more units, e.g., amino acids, although the individual units need not be structurally and / or functionally different.

[0139] In one embodiment, the peptide comprises natural amino acids. In other embodiments, synthetic peptides containing one or more unnatural amino acids may also be used.

[0140] In certain embodiments, multiple substrates may be used, each substrate being specific to a particular enzyme. In other embodiments, multiple substrates may also be used, each substrate being specific to multiple enzymes.

[0141] In one embodiment, the protease is an exopeptidase or an endopeptidase. Exopeptidases only degrade structures near the ends of peptide chains; endopeptidases are able to cleave internal bonds within peptides. These categories are also divided into subgroups: cysteine ​​proteases, serine proteases, threonine proteases, aspartic proteases, glutamic proteases, metalloproteases, etc. Each is able to digest specific protein connections by hydrolyzing peptide bonds.

[0142] In one embodiment, protease is specific to wound. As used herein, "wound-specific protease" is a protease differentially expressed in wounds. "Differential expression" means that the level or activity of protease in the wound microenvironment is higher or lower than that in other sites (e.g., normal tissue or surrounding tissue). In particular, differential expression means a higher level of expression or activity of protease in the wound microenvironment compared to uninjured tissue. The differential expression of protease can be analyzed by conventional methods. For example, the level of protease in the sample can be analyzed by ELISA or other immunoassays. The activity of the enzyme can be analyzed by measuring the loss rate of peptide substrates and / or the formation rate of the product, for example, using mass spectrometry or HPLC. These techniques are known in the art and described in the Examples section.

[0143] In one embodiment, the wound-specific enzyme is selected from the group consisting of: MMP-1 (collagenase), MMP-2 (gelatinase A), MMP-3 (matrilysin 1), MMP-8 (neutrophil collagenase), MMP-9 (gelatinase B), human neutrophil elastase (HNE), cathepsin G, urokinase-type plasminogen activator (uPA) and lysozyme.

[0144] In some embodiments, the substrate is a peptide sequence that is specific to collagenase. In some embodiments, the substrate is a peptide sequence that is specific to MMP-2. In some embodiments, the substrate is a peptide sequence that is specific to MMP-3. In some embodiments, the substrate is a peptide sequence that is specific to neutrophil collagenase. In some embodiments, the substrate is a peptide sequence that is specific to gelatinase. In some embodiments, the substrate is a peptide sequence that is specific to human neutrophil elastase. In some embodiments, the substrate is a peptide sequence that is specific to cathepsin G. In some embodiments, the substrate is a peptide sequence that is specific to urokinase-type plasminogen activator. In some embodiments, the substrate is a peptide sequence that is specific to lysozyme. In some embodiments, the substrate is a sugar that can be cleaved by lysozyme.

[0145] In a specific embodiment, the wound-specific protease is a matrix metalloproteinase (MMP) selected from the group consisting of MMP-1, MMP-2, MMP-8 and MMP-9 (collagenase) or a combination thereof. MMP-1 (UNIPROT Accession No. P03956 [human] and Q9EPL5 [mouse]) is also known as interstitial collagenase and fibroblast collagenase. MMP-2 (UNIPROT Accession No. P08253 [human] and P33434 [mouse]) is also known as gelatinase. MMP-8 (UNIPROT Accession No. P22894 [human] and O70138 [mouse]) is also known as PMNL collagenase (MNL-CL). MMP-9 (UNIPROT Accession No. P14780 [human] and P41245 [mouse]) is also known as gelatinase B (GELB).

[0146] In a specific embodiment, the MMP is MMP-2 or MMP-9, or a combination thereof.

[0147] In some embodiments, the matrix metalloproteinase (MMP) activity level is about 5U / mL to about 30U / mL, including all values ​​therebetween, such as about 6U / mL, about 7U / mL, about 8U / mL, about 9U / mL, about 10U / mL, about 11U / mL, about 12U / mL, about 13U / mL, about 14U / mL, about 15U / mL, about 16U / mL, about 17U / mL, about 18U / mL, about 19U / mL, about 20U / mL, about 21U / mL, about 22U / mL, about 23U / mL, about 24U / mL, about 25U / mL or more, indicating chronic wound infection. As understood in the art, activity units (U) are generally used to describe enzyme catalytic activity, wherein the unit (U) refers to the amount of enzyme that catalyzes the conversion of 1 micromole (μmole) of substrate per minute. Therefore, 1 enzyme unit (U) = 1 μmol / minute, wherein μmol refers to the amount of substrate converted.

[0148] In a specific embodiment, the MMP is MMP-2 or MMP-9, wherein an activity level of at least 10.5 U / mL for MMP-2 and MMP-9 is indicative of a chronic wound infection.

[0149] Any peptide that can be cleaved by a MMP can be used in accordance with the embodiments described herein. See, for example, Table 1 of U.S. Patent No. 7,148,194, which is incorporated herein by reference for that subject matter.

[0150] Table 1 shows various substrates and their specificity for different subtypes of human MMPs. The data are listed in Table 3 of Nagase et al. ("Substrate specificity of MMPs," in Matrix Metalloproteinase Inhibitors in Cancer Therapy," Clendeninn & Appelt, eds., Springer Science Media New York, 2001), which is incorporated herein by reference.

[0151] Table 1

[0152]

[0153]

[0154] *ND: Not determined.

[0155] In another embodiment, the wound-specific protease of the present invention is human neutrophil elastase (HNE) (UNIPROT Accession No. P08246 [human] and Q3UP87 [mouse]), which is a serine protease in the same family as chymotrypsin and has a broad substrate specificity. It is secreted by neutrophils and macrophages during inflammation, destroying bacteria and host tissues. In one embodiment, the substrate for detecting HNE has the core sequence Alanine-Alanine-Proline-Valine (AAPV). In another embodiment, the substrate of HNE is Ala-Pro-Glu-Glu-Ile / Met-Arg-Arg-Gln (APEEI / MRRQ) (Kasperkiewicz et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS USA), 111 (7): 2518-2523, 2014; Korkmaz et al., Methods Mol Biol., 844: 125-138, 2012).

[0156] In some embodiments, human neutrophil elastase activity level is about 5U / mL to about 30U / mL, including all values ​​therebetween, such as about 6U / mL, about 7U / mL, about 8U / mL, about 9U / mL, about 10U / mL, about 11U / mL, about 12U / mL, about 13U / mL, about 14U / mL, about 15U / mL, about 16U / mL, about 17U / mL, about 18U / mL, about 19U / mL, about 20U / mL, about 21U / mL, about 22U / mL, about 23U / mL, about 24U / mL, about 25U / mL or more, indicating chronic wound infection. In some embodiments, at least 9.6 human neutrophil elastase activity levels indicate chronic wound infection. In some embodiments, at least 22.9U / mL human neutrophil elastase activity levels indicate chronic wound infection.

[0157] The MMP and HNE subgroups have different mechanisms when interacting with proteins in the wound, so as one might expect, each has a different way of inhibiting wound healing.

[0158] In another embodiment, the wound-specific enzyme is lysozyme. Lysozyme (UNIPROT registration number P61626 [people] and P08905 [mouse]) is a glycoside hydrolase, and its main function is to destroy the cell wall of bacteria. It hydrolyzes the (1 → 4)-β-connections between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan, and the (1 → 4)-β-connections between N-acetyl-D-glucosamine residues in chitodextrin. The natural substrate of lysozyme is the peptidoglycan layer of the bacterial cell wall. However, various low molecular weight substrates including cell wall degradation products and synthetic compounds have been used for various photometric, isotopic and immunological lysozyme assays. et al., (EXS), 75:105-10, 1996. The following low molecular weight lysozyme substrates can be purchased from Sigma Aldrich, St. Louis, MO: 4-methylumbelliferyl β-DN,N',N"-triacetyl-chitotriosidoside (Sigma catalog number M5639) and 4-nitrophenyl β-DN,N',N"-triacetyl-chitotriosidoside (Sigma catalog number N8638).

[0159] In some embodiments, the lysozyme activity level is from about 1000 U / mL to about 10000 U / mL L, including all values ​​therebetween, for example, about 1100 U / mL, about 1200 U / mL, about 1300 U / mL, about 1400 U / mL, about 1500 U / mL, about 1600 U / mL, about 1700 U / mL, about 1800 U / mL, about 1900 U / mL, about 2000 U / mL, about 2100 U / mL, about 2200 U / mL, about 2300 U / mL, about 2400 U / mL, about 2500 U / mL, about 2600 U / mL In some embodiments, the lysozyme activity level of at least 4800U / mL indicates chronic wound infection.

[0160] In another embodiment, the wound-specific enzyme is a peroxidase, more specifically myeloperoxidase (MPO). MPO (UNIPROT accession number P05164 [human] and P11247 [mouse]) is a peroxidase found in neutrophils. In the presence of hydrogen peroxide (H2O2) and halides (most commonly chlorides), it produces antibacterial substances hypochlorite, singlet oxygen (1O2), chlorine (Cl2) and hydroxyl radicals (OH·). MPO can be detected using tetramethylbenzidine or 4-benzamido-2,5-dimethoxyaniline. See Andrews et al., Anal Biochem, 127 (2): 346-50, 1982; Klebanoff et al., J. Leukocyte Biol., 77, 598-625, 2005.

[0161] In another embodiment, the wound-specific enzyme is cathepsin G (UNIPROT Accession Nos. P08311 [human] and P28293 [mouse]), which is one of three serine proteases of the chymotrypsin family that are stored in azurophilic granules. The cathepsin G-specific substrate has the sequence Ala-Ala-Pro-Phe or Ala-Ala-Pro-Met (SigmaAldrich Catalog Nos. S7388 and M7771).

[0162] In some embodiments, the cathepsin G activity level is about 10U / mL to about 100U / mL, including all values ​​therebetween, such as about 15U / mL, about 20U / mL, about 25U / mL, about 30U / mL, about 35U / mL, about 40U / mL, about 45U / mL, about 50U / mL, about 55U / mL, about 60U / mL, about 65U / mL, about 70U / mL, about 75U / mL, about 80U / mL, about 85U / mL, about 90U / mL, about 95U / mL, about 100U / mL, about 110U / mL, about 120U / mL, or more, indicating chronic wound infection. In some embodiments, the cathepsin G activity level is at least 50U / mL, at least 40U / mL, at least 30U / mL, at least 20U / mL, at least 15U / mL or at least 10U / mL, indicating chronic wound infection.

[0163] In some embodiments, the wound-specific enzyme is urokinase-type plasminogen activator (UNIPROT Accession Nos. P00749 [human] and P06869 [mouse]), which is a serine protease involved in extracellular matrix degradation and possibly tumor cell migration and proliferation. Urokinase-specific substrates have a basic motif Arg-Val or Lys-Val. See Rijken et al., Biochem Biophys Res Commun., 174(2):432-8, 1991.

[0164] In some embodiments, the one or more enzymes are esterases. Esterases are hydrolases that decompose esters into acids and alcohols in a chemical reaction with water. In a specific embodiment, the substrate of the esterase is fluorescein diacetate-5-maleimide.

[0165] In some embodiments, the composition comprises substrates capable of detecting multiple enzymes, e.g., at least 2, at least 3, at least 4 or more of the aforementioned enzymes. Such compositions may include, for example, multiple substrates conjugated to the same gel polymer or different gel polymers.

[0166] In certain embodiments, labeled substrate. As used herein, the term "label" refers to any substance attached to an epitope binding agent or other substrate materials, wherein the substrate can be detected by a detection method. Non-limiting examples of suitable labels include luminescent molecules, chemiluminescent molecules, fluorescent dyes, fluorescence quenchers, colored molecules, radioisotopes, scintillators, biotin, avidin, streptavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni2+, Flag tags, myc tags, heavy metals and enzymes (including alkaline phosphatase, peroxidase and luciferase). These methods are well known in the art.

[0167] In certain embodiments, the substrate is labeled with a label, which is a detectable label. A detectable label is a moiety whose presence can be determined directly or indirectly. Typically, detection of a label involves the generation of a detectable signal, such as energy emission. The label can be chemical, peptide or nucleic acid in nature, but is not limited thereto. The nature of the label used depends on a variety of factors, including the nature of the analysis performed, the type of energy and detector used, and the type of polymer, analyte, probe, and first and second analyte specific binding partners. The label should be spatially and chemically compatible with the component to which it is bound.

[0168] The label can be directly detected, for example, by its ability to emit and / or absorb electromagnetic radiation of a specific wavelength. The label can be indirectly detected, for example, by its ability to bind, recruit, and in some cases cleave another part, which can itself emit or absorb light of a specific wavelength (e.g., epitope tags such as FLAG epitopes, enzyme tags such as horseradish peroxidase, etc.). Generally, the detectable label can be selected from the group consisting of: a directly detectable label, such as a fluorescent molecule (e.g., fluorescein, rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-3, Cy-5, Cy-7, Texas Red, Phar Red, allophycocyanin (APC), fluoresceinamine, eosin, dansyl, umbelliferone, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrho TAMRA, 6-carboxy-X-rhodamine (ROX), 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, acridine, acridine isothiocyanate, r-amino-N-(3-vinylsulfonyl)phenylnaphthalimide-3,5, disulfonate (fluorescent yellow VS), N-(4-anilino-1-naphthyl)maleimide, o-aminobenzamide, brilliant yellow, coumarin , 7-amino-4-methylcoumarin, 7-amino-4-trifluoromethylcoumarin (coumarin 151), tetrachlorotetrabromofluorescein, 4′,6-diamidino-2-phenylindole (DAPI), 5′,5″-diamidino-2-phenylindole (DAPI), 5′,5″-dibromopyrogallol-sulfonphthalein (bromopyrophenol red), 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin diethylenetriamine pentaacetate, 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid, 4,4'-diisothiocyanatostilbene-2,2' -disulfonic acid, 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC), eosin isothiocyanate, erythrosine B, erythrosine isothiocyanate, ethidium bromide, 5-(4,6-dichlorotriazine-2-yl)aminofluorescein (DTAF), QFITC ​​(XRITC), fluorescamine, IR144, IR1446, malachite green isothiocyanate, 4-methylumbelliferone, o-cresolphthalein, nitrotyrosine, pararosaniline, phenol red, B-phycoerythrin, o-phthalaldehyde, pyrene, pyrene butyrate, 1-pyrene butyrate succinimidyl ester, Reactive Red 4 ( Brilliant Red 3B-A), lissamine rhodamine B sulfonyl chloride, rhodamine B, rhodamine 123, rhodamine X, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivatives of sulforhodamine 101, tetramethylrhodamine, riboflavin, rhodic acid and terbium chelate derivatives), chemiluminescent molecules, bioluminescent molecules, chromogenic molecules, radioisotopes (e.g., P32 or H3, 14C, 125I and 131I), electron spin resonance molecules (e.g., nitroxyl radicals), optical or electron density molecules, charge transduction or transfer molecules, electromagnetic molecules (e.g., magnetic or paramagnetic beads or particles), semiconductor nanocrystals or nanoparticles (e.g., quantum dots, such as described in U.S. Pat. No. 6,207,392 and commercially available from Quantum Dot Corporation and Evident Technologies), colloidal metals, colloidal gold nanocrystals, nuclear magnetic resonance molecules, etc.

[0169] The detectable label can also be selected from the group consisting of: an indirectly detectable label, such as an enzyme (e.g., alkaline phosphatase, horseradish peroxidase, β-galactosidase, glucoamylase, lysozyme, luciferase such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456); a sugar oxidase, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase; a heterocyclic oxidase (e.g., uricase and xanthine oxidase, which is compatible with the use of hydrogen peroxide to detect the presence of a luciferase); Enzymes that oxidize dye precursors (such as HRP, lactoperoxidase or microperoxidase) coupled), enzyme substrates, affinity molecules, ligands, receptors, biotin molecules, avidin molecules, streptavidin molecules, antigens (e.g., epitope tags such as FLAG or HA epitopes), haptens (e.g., biotin, pyridoxal, digoxigenin and dinitrophenol), antibodies, antibody fragments, microbeads, etc. Antibody fragments include Fab, F(ab)2, Fd and antibody fragments containing CDR3 regions.

[0170] In some embodiments, substrate is conjugated with donor and acceptor fluorophores respectively to form FRET pairs. FRET can be used, for example, in array form to determine whether a specific second antibody is combined regardless of the identity of the analyte to which it is combined. Alternatively, the second binding partner can be detectably labeled without labeling the first binding partner. The labeling of the second binding partner can also be used to establish the direction of the nucleic acid connected thereto. Individual FRET usually requires only one excitation source (therefore one wavelength) and usually requires only one detector. The detector can be set to the emission spectrum of the donor or acceptor fluorophore. If FRET is detected by quenching the donor fluorescence, it is set to the donor fluorophore emission spectrum. Alternatively, if FRET is detected by the emission of the acceptor fluorophore, it is set to the acceptor fluorophore emission spectrum. In some embodiments, the FRET emission of the donor and acceptor fluorophores can be detected. In other embodiments, the donor is excited with polarized light, and the polarization of the two emission spectra is detected.

[0171] In one embodiment, the detectable label is compatible with a FRET-based assay. FRET requires the use of a FRET fluorophore pair. A FRET fluorophore pair is two fluorophores that, when close to each other, can undergo FRET to produce or eliminate a detectable signal. Examples of donors include Alexa 488, Alexa 546, BODIPY 493, Oyster 556, Fluor (FAM), Cy3, and TMR (Tamra). Examples of acceptors include Cy5, Alexa 594, Alexa 647, and Oyster 656. For example, Cy5 works with Cy3, TMR, or Alexa 546 as donors. FRET should be possible for any fluorophore pair with a fluorescence maximum that is 50 to 100 nanometers apart from each other.

[0172] In addition to the barcode labels discussed herein, the substrate can be labeled in a sequence-nonspecific manner. For example, if the polymer is a nucleic acid such as DNA, its backbone can be stained with a backbone label. Examples of backbone stains for labeling nucleic acids in a sequence-nonspecific manner include intercalating dyes such as phenanthridines and acridines (e.g., ethidium bromide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimers-1 and -2, monoazide ethidium and ACMA); minor groove binders such as indoles and imidazoles (e.g., Hoechst 33258, Hoechst 33342, Hoechst 34580 and DAPI); and various nucleic acid dyes such as acridine orange (also intercalated), 7-AAD, actinomycin D, LDS751 and hydroxystilbene. All of the aforementioned nucleic acid dyes are commercially available from suppliers such as Molecular Probes.

[0173] Other examples of nucleic acid stains include the following from Molecular Probes: cyanine dyes, such as SYTOX BLUE, SYTOX GREEN, SYTOX ORANGE, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PICOGREEN, OLIGREEN, RIBOGREEN, SYBR GOLD, SYBR GREEN I, SYBR GREEN II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24, -21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82, -83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, -63 (red).

[0174] In some embodiments, the reporter molecule comprises a chromophore or a fluorophore. In other embodiments, the chromophore is an azo moiety, a nitro moiety, a triarylmethane moiety, a methine, anthraquinone, a polyene moiety, or a phthalocyanine. In some embodiments, the reporter molecule is a dye. Contemplated dyes may be, but are not limited to, rhodamine, coumarins, cyanines, xanthenes, polymethines, pyrenes, dipyrromethene boron difluoride, naphthylimide, phycobiliproteins, polydinoflagellates chlorophyll proteins, conjugates thereof, and combinations thereof. Non-limiting examples of dyes include fluorescein, 6-FAM, rhodamine, Texas Red, California Red, iFluor594, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6F, carboxyparamethylaminophenol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarins, Cy-Chrome, DyLight 350, DyLight 405, DyLight 488, DyLight 549, DyLight 594, DyLight 633, DyLight 649, DyLight 680, DyLight 750, DyLight 800, Phycoerythrin, PerCP (Peridinyl Chlorophyll-a Protein), PerCP-Cy5.5, JOE (6-Carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), NED, ROX (5-(and-6-)-Carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa 350、Alex 430. Alexa 488. Alexa 532. Alexa 546. Alexa 568. Alexa 594. Alexa 633. Alexa 647、Alexa 660、Alexa 680, 7-amino-4-methylcoumarin-3-acetic acid, FL, FL-Br2, FL, FL-Br2, 530 / 550, 558 / 568, 630 / 650, 650 / 665, R6G, TMR, TR, its conjugates and combinations thereof. In some embodiments, the reporter molecule is dimethylaminoazobenzenesulfonic acid (dabsyl) or a dabsyl derivative. In some embodiments, the reporter molecule is fluorescein, a fluorescein derivative or a fluorescein-containing compound.

[0175] In some embodiments, the reporter molecule is a lipid. In some embodiments, the lipid is a synthetic phospholipid derivative. In some embodiments, the synthetic phospholipid derivative is DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC or DEPC. In some embodiments, the synthetic phospholipid derivative is DLPC, DMPC or DPPC. In some embodiments, the synthetic phospholipid derivative is DLPC. In some embodiments, the synthetic phospholipid derivative is DMPC. In some embodiments, the synthetic phospholipid derivative is DPPC.

[0176] In some embodiments, the reporter molecule is contained in a detectable fragment that is cleaved from the wound dressing material upon contact with the enzyme. In some embodiments, the reporter molecule is not contained in a fragment that is cleaved from the wound dressing material upon contact with the enzyme. In some embodiments, the reporter molecule is observed by the naked eye. In some embodiments, the reporter molecule is observed under UV light. In some embodiments, the reporter molecule is observed using fluorescent light.

[0177] In some embodiments of the wound dressing material of Formula I, R optionally comprises a quencher fragment. In some embodiments, the quencher fragment prevents the reporter molecule from fluorescing. In some embodiments, the quencher fragment is a protecting group. In some embodiments, the quencher fragment is an acetate group.

[0178] In certain embodiments, disclosed herein are modified wound dressing materials comprising target sequences for one or more enzymes. In some embodiments, the enzyme-catalyzed cleavage releases a detectable fragment. The detectable fragment may comprise a reporter molecule. Qualitative or quantitative measurement of the level of the detectable fragment enables determination of the presence or absence of infection in a wound.

[0179] In some embodiments, the enzyme-catalyzed cleavage releases an undetectable fragment. In some embodiments, the interaction of the enzyme with the wound dressing material cleaves the quencher fragment and allows the reporter molecule bound to the wound dressing material to fluoresce. Qualitative or quantitative measurement of the fluorescence enables determination of the presence or absence of infection in the wound.

[0180] In certain embodiments, disclosed herein are peptide-modified wound dressing materials containing target sequences for one or more proteases. Protease-catalyzed cleavage releases detectable peptide fragments. The detectable peptide fragments contain reporter molecules. Qualitative or quantitative measurement of the levels of detectable peptide fragments enables determination of the presence or absence of elevated proteases in a wound.

[0181] In some embodiments, the enzyme-catalyzed cleavage releases an undetectable fragment. In some embodiments, the interaction of the enzyme with the wound dressing material cleaves the quencher fragment and allows the reporter molecule bound to the wound dressing material to fluoresce. Qualitative or quantitative measurement of the fluorescence enables determination of the presence or absence of infection in the wound.

[0182] In some embodiments of the wound dressing material of Formula I, R comprises a quencher fragment. In some embodiments, the quencher fragment prevents the reporter molecule from fluorescing. In some embodiments, the quencher fragment is a protecting group. In some embodiments, the quencher fragment is an acetate group. In some embodiments of the wound dressing material of Formula I, R comprises a chemical moiety that is a product of a nucleophilic reaction. In some embodiments, R comprises a maleimide-thiol adduct. In some embodiments, R comprises a haloacetamide-thiol conjugate product. In some embodiments, R comprises a haloacetamide conjugate product.

[0183] In some embodiments, the wound dressing material has the structure of Formula Ia:

[0184]

[0185] wherein R is a region comprising a reporter molecule; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more; and n is an integer selected from 200 to 4000, such as 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 2500, 3000, 12000, 2500, 3000, 13000, 15000, 25000, 25000 200, 201, 202, 203, etc. In other embodiments, n is an integer selected from 300 to 3500. In yet other embodiments, n is an integer selected from 400 to 3200. In some embodiments, R is a peptide region comprising a reporter molecule and at least one amino acid.

[0186] In some embodiments, the wound dressing material has the structure of Formula Ib:

[0187]

[0188] wherein R is a region comprising a reporter molecule; and n is an integer selected from 200 to 4000, such as 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800 In some embodiments, R is a peptide region comprising a reporter molecule and at least one amino acid. In some embodiments, R is a peptide region comprising a reporter molecule and one amino acid.

[0189] In some embodiments, the wound dressing material has the structure of Formula IIa:

[0190] ML-PEP

[0191] Formula IIa

[0192] Wherein M is a gel-forming polymer selected from cellulose, chemically modified cellulose, pectin, alginate, chitosan, hyaluronic acid, a polysaccharide or a gum-derived polymer or any combination thereof; PEP is a peptide region comprising a reporter molecule and at least one amino acid; and L is a linker connecting M and PEP, wherein L comprises one or more polyethylene glycol subunits or polypropylene subunits.

[0193] In some embodiments, the wound dressing material has the structure of Formula IIb:

[0194]

[0195] Wherein PEP is a peptide region comprising a reporter molecule and at least one amino acid; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more; n is an integer selected from 200 to 4000. In other embodiments, n is an integer selected from 300 to 3500. In yet other embodiments, n is an integer selected from 400 to 3200.

[0196] In some embodiments, the wound dressing material has the structure of Formula IIc:

[0197]

[0198] wherein PEP is a peptide region comprising a reporter molecule and at least one amino acid; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more; n is an integer selected from 200 to 4000, such as 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 110 203, etc. In other embodiments, n is an integer selected from 300 to 3500. In yet other embodiments, n is an integer selected from 400 to 3200.

[0199] Composition:

[0200] Embodiments described herein also relate to compositions containing compounds of Formula I or Formula II. These compositions can be prepared using conventional methods.

[0201] After formulation, the obtained raw material composition of the compound of formula I or formula II can be further modified into a desired form, such as gel, balm, lotion, cream, paste, ointment, etc. using conventional methods, such as using carriers, gelling agents, lubricants, surfactants, moisturizers, viscosity enhancers, emulsifiers, etc. See, for example, WO2011 / 126384 and WO2013 / 004953, which are incorporated by reference.

[0202] The carrier for the composition may include, but is not limited to, water, glycerol, diglycerol, glycerol derivatives, glycols, glycol derivatives, sugars, ethoxylated and / or propoxylated esters and ethers, urea, sodium PCA, alcohols, ethanol, isopropanol, and combinations thereof. In one embodiment, the carrier is propylene glycol. Typically, the composition contains the carrier in an amount of from about 1% by weight of the composition to about 99.9% by weight of the composition, more typically from about 2% by weight of the composition to about 95% by weight of the composition, and more typically from about 5% by weight of the composition to about 90% by weight of the composition.

[0203] Thermoreversible gelling agents are defined as ingredients that are soluble, partially soluble or miscible in a hydrophilic carrier at elevated temperatures (e.g., 50° C.), wherein the agent has the ability to thicken the carrier when cooled to 25° C., but when it is desired to be applied to a substrate, the viscosity decreases at 50° C. Suitable hydrophilic carriers include water, glycols, such as propylene glycol. Thermoreversible gelling agents for compositions may include fatty acid salts, such as sodium stearate, sodium palmitate, potassium stearate. These salts may be added to the composition, or may be generated in situ by adding fatty acids and neutralizing with an appropriate base. An example of an in situ formation composition is to provide stearic acid and sodium hydroxide to produce sodium stearate. Other common thermoreversible gelling agents can include, for example, polyethylene glycol and derivatives such as PEG-20, PEG-150 distearate, PEG-150 pentaerythritol tetrastearate, disteareth-75 IPDI, disteareth-100 IPDI, fatty alcohols such as cetyl alcohol, fatty acids such as stearic acid, hydroxystearic acid and derivatives thereof, and combinations thereof.

[0204] In addition to the carrier and the thermoreversible gelling agent, the composition may contain various other ingredients and components. Examples of other ingredients that may be included in the composition are lubricants, sterols or sterol derivatives, natural and synthetic fats or oils, viscosity enhancers, rheology modifiers, polyols, surfactants, alcohols, esters, silicones, clays, starches, celluloses, particles, wetting agents, film formers, slip modifiers, surface modifiers, skin protectants, moisturizers, sunscreens, etc.

[0205] Pharmaceutical compositions and / or preparations:

[0206] Embodiments described herein also relate to pharmaceutical compositions and / or preparations comprising one or more aforementioned Formula I or Formula II compounds and carriers. The term "pharmaceutically acceptable" is used herein to refer to those compounds, salts, compositions, dosage forms, etc., which are suitable for contact with human and / or other mammalian tissues within the scope of reasonable medical judgment without excessive toxicity, stimulation, allergic reaction or other problems or complications, commensurate with reasonable benefit / risk ratio. In some respects, "pharmaceutically acceptable" means approved by a federal or state government regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for mammals (e.g., animals), more particularly for humans.

[0207] The pharmaceutical composition can be prepared by any suitable method known in the art. Examples of such compositions include those suitable for the following: (a) topical application, such as preparations (e.g., gauze, pads, swabs, dressings), creams, ointments, gels, lotions, etc.; (b) parenteral administration, such as subcutaneous, intramuscular or intravenous injection as a sterile solution or suspension; (c) oral administration, external application (e.g., immersion, including aqueous and non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pellets mixed with feed, pastes for tongue application, etc.

[0208] In certain embodiments, the pharmaceutical composition may include one or more antibiotics. As used herein, the term "antibiotic" or "antimicrobial agent" refers to a substance that inhibits the growth of or destroys microorganisms. Preferably, the antibiotic can be used to inhibit the toxicity of infectious agents and / or treat infectious diseases. Antibiotics also refer to semisynthetic substances in which the natural form produced by microorganisms such as yeast or fungi is structurally modified.

[0209] Preferably, the antibiotic is selected from the group consisting of: β-lactams (including β-lactamase inhibitors and cephalosporins), fluoroquinolones, aminoglycosides, tetracyclines and / or glycylcyclines and / or polymyxins. Any combination of antimicrobial agents may also be used, for example, at least one β-lactam and at least one fluoroquinolone; at least one aminoglycoside and one cephalosporin; at least one β-lactam and one β-lactamase inhibitor, optionally in combination with an aminoglycoside, etc.

[0210] As used herein, the term "β-lactam" inhibitors include natural and semisynthetic penicillins and penicillin derivatives, such as benzathine penicillin, benzylpenicillin (penicillin G), phenoxymethylpenicillin (penicillin V), procaine penicillin and oxacillin; methicillin, dicloxacillin and flucloxacillin; temocillin; amoxicillin and ampicillin; azlocillin, carbenicillin, ticarcillin, mezlocillin and piperacillin; biapenem, doripenem, ertapenem, imipenem, meropenem, panitumumib, phenoxyethylpenicillin ... Penem and PZ-601; cephalexin, cephalothin, cefazolin, cefaclor, cefuroxime, cefoperazone, cefotetan, cefoxitin, cefotaxime and cefpodoxime; cefepime and cefpirome; cefdroxil, cefixime, cefprozil, cephalexin, cephalothin, cefuroxime, cefoperazone, cefepime and cefpirome; cefoxitin, cefotetan, cefmetazole and fluoxetine; tigemonam, nocardin A and pyrotoxin; clavulanic acid, latamoxef and fluoxetine. Fluoroquinolones include ciprofloxacin, garenoxacin, gatifloxacin, gemifloxacin, levofloxacin and moxifloxacin. Aminoglycosides include, for example, kanamycin, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin B, neomycin C, neomycin E (paromomycin) and streptomycin, including synthetic derivatives clarithromycin and azithromycin. Tetracyclines include naturally occurring compounds (e.g., tetracycline, chlortetracycline, oxytetracycline, demeclocycline) or semi-synthetic agents (e.g., lymecycline, meclocycline, methacycline, minocycline, hydropyracycline). Glycylcyclines (e.g., minocycline / tigecycline) are derived from tetracycline. Polymyxins include, for example, polymyxin B and polymyxin E (colistin).

[0211] In certain embodiments, the composition can contain a concentration of 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL In some embodiments, the present invention provides an antibiotic of at least 20 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL, 400 mg / mL, 500 mg / mL or more. For example, imipenem and ertapenem can be used in a concentration of 50, 30, 20, 15, 10, 5 and 1 mg / mL.

[0212] Wound Dressing:

[0213] In certain embodiments, disclosed herein are wound dressings comprising wound dressing materials as described herein, such as compounds of Formula I or Formula II. In some embodiments, the wound dressing consists essentially of wound dressing materials as described herein, such as compounds of Formula I or Formula II.

[0214] In one embodiment, the wound dressings disclosed herein are biocompatible, biodegradable, non-immunogenic, and readily commercially available.

[0215] In one embodiment, the compound of formula I or formula II is provided in the form of particles, such as fiber particles or powder particles, which optionally contain a drug. In particular, the material preferably contains CMC fibers.

[0216] Composition can preferably comprise the homogeneous mixture of dressing material and other compounds.For example, in one embodiment, the homogeneous mixture comprises the mixed solution or dispersion of dressing material and suitable vehicle (such as solvent), or the solid composition prepared by removing solvent from such solution or dispersion.Under this embodiment, dressing material accounts for at least 5 weight % of material, more preferably at least 10 weight %, 20 weight %, 30 weight %, 50 weight %, 75 weight %, 90% or higher %.In certain preferred embodiments, the material is substantially composed of dressing material.

[0217] The other components of the material may include 0% to 25% by weight, for example, about 1% to about 20% by weight of one or more other biocompatible polysaccharides, such as alginates, such as sodium alginate or calcium alginate, starch derivatives such as sodium starch glycolate, cellulose derivatives such as methylcellulose or carboxymethylcellulose, or mucopolysaccharides such as hyaluronic acid or its salts, chondroitin sulfate or heparan sulfate. The material may also include up to about 25% by weight, for example, about 1% to about 20% by weight of one or more structural proteins, the structural proteins being selected from the group consisting of fibronectin, fibrin, laminin, elastin, collagen and mixtures thereof. Preferably, the protein comprises collagen, and more preferably, the protein is substantially composed of collagen. The material may also include up to about 20% by weight, preferably about 2% to about 10% by weight of water. The material may also contain 0% to 40% by weight, for example, about 5% to about 25% by weight of a plasticizer, preferably a polyol such as glycerol or sorbitol.

[0218] In certain embodiments, the material may also include up to about 10% by weight, such as about 0.01% by weight to about 5% by weight, typically about 0.1% by weight to about 2% by weight of one or more therapeutic wound healing agents, such as nonsteroidal anti-inflammatory drugs (e.g., acetaminophen), steroids, local anesthetics, antimicrobials, or growth factors (e.g., fibroblast growth factor or platelet-derived growth factor). Antimicrobials may, for example, include preservatives, antibiotics, or mixtures thereof. Preferred antibiotics include tetracycline, penicillin, oxytetracycline, erythromycin, bacitracin, neomycin, polymyxin B, mupirocin, clindamycin, and mixtures thereof. Preferred preservatives include silver (including colloidal silver), silver salts (including salts of one or more anionic polymers constituting the material), silver sulfadiazine, chlorhexidine, povidone iodine, triclosan, sucralfate, quaternary ammonium salts, and mixtures thereof. When the wound dressing material decomposes in use, these medicated wound dressing materials according to the present invention provide sustained release of therapeutic agents.

[0219] All the above percentages are based on dry weight. Preferably, the weight ratio of the wound dressing material to other adjuvants and materials is about 1:99 to about 99:1. More preferably, the weight ratio is in the range of about 1:9 to about 9:1, more preferably in the range of about 4:1 to about 1:4, and even more preferably in the range of about 2:1 to about 1:2.

[0220] The material may be in any convenient form, such as a powder, microspheres, flakes, mats or films.

[0221] In certain embodiments, the material is in the form of a semisolid or gel ointment for topical administration.

[0222] In certain embodiments, the material is in the form of a freeze-dried or solvent-dried bioabsorbable sponge for application to chronic wounds. Preferably, the average pore size of the sponge is in the region of 10 to 500 μm, more preferably about 100 to 300 μm. A suitable sponge is prepared by freeze-drying or solvent-drying an aqueous dispersion comprising a compound of Formula I or Formula II and a suitable therapeutic agent.

[0223] In other embodiments, the material is in the form of a flexible film, which may be continuous or interrupted (eg, perforated). The flexible film preferably comprises a plasticizer to make it flexible, such as glycerol.

[0224] The ready availability of two gel-forming polymers (e.g. cellulose derivatives) with a range of controllable properties means that the properties of the compositions of the invention can be controlled to a particular extent. In particular, the bioresorption rate, porosity and density of the material can be controlled.

[0225] In one embodiment, provided herein is a sheet-like wound dressing material comprising an active layer of a composition containing a compound of Formula I or Formula II. The active layer is typically the wound contact layer in use, but in some embodiments, it may be separated from the wound by a liquid-permeable top sheet. In one embodiment, the area of ​​the active layer is about 1 cm 2 To about 400cm 2 , in particular about 4 cm 2 To about 100cm 2 .

[0226] In another embodiment, the wound dressing material further comprises a backing sheet extending above the active layer, facing the active layer opposite the wound. Preferably, the backing sheet is larger than the active layer so that an edge region with a width of 1 mm to 50 mm, preferably 5 mm to 20 mm, extends around the active layer to form a so-called island dressing. In this case, the backing sheet is preferably coated with a pressure-sensitive medical grade adhesive at least in its edge region.

[0227] In embodiments where the dressing material comprises a backsheet, the backsheet is substantially liquid impermeable. In another embodiment, the backsheet is semipermeable, for example, the backsheet is preferably permeable to water vapor but impermeable to liquid water or wound exudate. Preferably, the backsheet is also impermeable to microorganisms. A suitable continuous backsheet preferably has a moisture vapor transmission rate (MVTR) of the backsheet alone of 300 to 5000 g / m at 37.5°C and a relative humidity difference of 100% to 10%. 2 / 24 hours, preferably 500 to 2000 g / m 2 The thickness of the backsheet is preferably in the range of 10 to 1000 microns, more preferably in the range of 100 to 500 microns.

[0228] The MVTR of the entire dressing is lower than the MVTR of the backsheet alone because the perforated sheet partially blocks the transfer of water through the dressing.

[0229] Suitable polymers for forming the backsheet include polyurethanes and polyalkoxyalkyl acrylates and methacrylates. Preferably, the backsheet comprises a continuous layer of a predominantly closed cell, high density, closed polyurethane foam. A suitable backsheet material is a polyurethane film.

[0230] In wound dressings comprising a backsheet comprising an adhesive, the adhesive layer should be moisture vapor permeable and / or patterned to allow water vapor to pass through. The adhesive layer is preferably a continuous moisture vapor permeable pressure sensitive adhesive layer conventionally used for island wound dressings, such as pressure sensitive adhesives based on acrylate copolymers, polyvinyl ethyl ethers and polyurethanes. Polyurethane-based pressure sensitive adhesives may optionally be used.

[0231] In another embodiment, the dressing may include other layers of a multi-layer absorbent article, which may be constructed between the active layer and the protective sheet. For example, these layers may include a perforated plastic film to provide support for the active layer in use, in which case the holes in the film are preferably aligned with the holes in the hydrogel layer.

[0232] Further, in other embodiments, the dressing may include an absorbent layer between the active layer and the protective sheet, especially if the dressing is used for exuding wounds. The optional absorbent layer may be any layer conventionally used in the field of wound healing to absorb wound fluid, serum or blood, including gauze, nonwoven fabrics, superabsorbents, hydrogels and mixtures thereof. Preferably, the absorbent layer comprises a layer of absorbent foam, such as an open-cell hydrophilic polyurethane foam. In other embodiments, the absorbent layer may be a nonwoven fiber web, such as a carded web of viscose staple fibers.

[0233] In certain embodiments, the wound dressing may be protected by a removable cover sheet. The cover sheet is typically formed of a flexible thermoplastic material. Suitable materials include polyesters and polyolefins. Preferably, the surface of the cover sheet facing the adhesive is a release surface. That is, the surface that is only weakly adhered to the active layer and the adhesive on the back sheet facilitates the release of the hydrogel layer from the cover sheet. For example, the cover sheet may be formed of a non-adhesive plastic such as a fluoropolymer, or may be provided with a release coating, such as a silicone or fluoropolymer release coating.

[0234] In one embodiment, the wound dressing is sterile and packaged in a microorganism-impermeable container.

[0235] Reagent test kit:

[0236] In certain embodiments, the disclosed technology provides a kit comprising a compound of Formula I or Formula II in one or separate compartments, optionally with excipients, carriers or oils. The kit may also include other ingredients in one or more compartments, such as gelling agents, lubricants, surfactants, moisturizers, viscosity enhancers, emulsifiers, etc. The kit may optionally include instructions for preparing a product for diagnosing, detecting or treating wounds (e.g., chronic or infected wounds). The kit may also include instructions for using the components alone or together in wound treatment.

[0237] In a related embodiment, the disclosed technology provides a kit comprising a package and at least one absorbent article (as described above) comprising the above composition. Alternatively, the kit may comprise each component separately, optionally together with auxiliary information, which may be used in or with the package.

[0238] Other embodiments disclosed herein relate to the use of the composition for preparing a dressing for treating a wound.Preferably, the wound is a chronic wound, such as a wound selected from the group consisting of a venous ulcer, a decubitus ulcer and a diabetic ulcer.

[0239] surface:

[0240] Embodiments of the disclosed technology also provide surfaces comprising the aforementioned compounds of Formula I or Formula II, wherein the reporter or peptide is directed to allow binding to a partner such as an enzyme. Preferably, the surface is the surface of a solid support. Many different solid supports are known to those skilled in the art. Useful solid supports include natural polymeric carbohydrates and synthetically modified, cross-linked or substituted derivatives thereof, such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar gum, cellulose esters, especially cellulose esters containing nitric acid and carboxylic acids, mixed cellulose esters and cellulose ethers; nitrogen-containing natural polymers, such as proteins and derivatives, including cross-linked or modified gelatin; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers, which can be prepared with a suitable porous structure, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinyl acetate and partially hydrolyzed derivatives thereof, polyacrylamide, Polymethacrylates, copolymers and terpolymers of the above polycondensates, such as polyesters, polyamides, and other polymers, such as polyurethanes or polyepoxides; porous inorganic materials, such as sulfates or carbonates of alkaline earth metals and magnesium, including barium sulfate, calcium sulfate, calcium carbonate, alkali metals and alkaline earth metals, aluminum and magnesium silicates; and oxides or hydrates of aluminum or silicon, such as clay, alumina, talc, kaolin, zeolites, silica gel or glass (these materials can be used as filters for the above polymeric materials); and mixtures or copolymers of the above types, such as graft copolymers obtained by initiating the polymerization of synthetic polymers on pre-existing natural polymers.

[0241] In one embodiment, the support is a well of an array plate, such as a microarray. Methods for constructing such arrays are known in the art, such as Cao et al., Appl Environ Microbiol., 77(23):8219-8225, 2011. Each compound of pattern I or formula II (or a separate reporter molecule) can be placed in triplicate to eliminate irregular data caused by physical defects of the array.

[0242] system:

[0243] Embodiments of the disclosed technology also provide diagnostic systems comprising the aforementioned compositions and / or kits.

[0244] The various components of the diagnostic system can be provided in various forms. For example, a compound of Formula I or Formula II (e.g., a compound containing a peptide reporter molecule) can be provided as a lyophilized reagent. These lyophilized reagents can be pre-mixed before lyophilization so that when reconstituted, they form a complete mixture for determination, wherein the proportion of each component is appropriate. In addition, the diagnostic system of the present invention may contain a reconstitution reagent for reconstitution of the lyophilized reagent of the kit.

[0245] Embodiments described herein also relate to LC detection systems. The LC detection system utilizes monitoring of changes in the arrangement of 5CB liquid crystals (LC) as a detection method. In order to adapt the LC detection system to the CMC structure, lipids are added to the portion of the peptide sequence that will be cleaved. Once released, the lipids will cause changes in the arrangement of 5CB. The arrangement of 5CB can be detected by cross-polarized lenses and displayed from dark to light; the system will include accommodating the LC in the correct arrangement until the point of use, and may also include detection and / or visualization using cross-polarized lenses and a microscope.

[0246] In some embodiments, the wound dressing comprises a wound dressing material having the structure of Formula Ia:

[0247]

[0248] wherein R is a region comprising a reporter molecule; m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and n is an integer selected from 200 to 4000. In other embodiments, n is an integer selected from 300 to 3500. In still other embodiments, n is an integer selected from 400 to 3200. In some embodiments, R is a peptide region comprising a reporter molecule and at least one amino acid.

[0249] In some embodiments, the wound dressing comprises a wound dressing material having the structure of Formula Ib:

[0250]

[0251] Wherein R is a region comprising a reporter molecule; n is an integer selected from 200 to 4000. In other embodiments, n is an integer selected from 300 to 3500. In still other embodiments, n is an integer selected from 400 to 3200. In some embodiments, R is a peptide region comprising a reporter molecule and at least one amino acid. In some embodiments, R is a peptide region comprising a reporter molecule and one amino acid.

[0252] In another aspect, provided herein is a wound dressing comprising a wound dressing material having the structure of Formula II:

[0253] ML-PEP

[0254] Formula II

[0255] Wherein M is a gel-forming polymer; PEP is a peptide region containing a reporter molecule and at least one amino acid; and L is a linker connecting M and PEP.

[0256] In some embodiments, the wound dressing comprises a wound dressing material having the structure of Formula IIa:

[0257] ML-PEP

[0258] Formula IIa

[0259] Wherein M is a gel-forming polymer selected from cellulose, chemically modified cellulose, pectin, alginate, chitosan, modified chitosan, hyaluronic acid, a polysaccharide or a gum-derived polymer, CES, oxidized cellulose (or its derivatives) or any combination thereof; PEP is a peptide region comprising a reporter molecule and at least one amino acid; L is a linker connecting M and PEP, wherein L comprises one or more polyethylene glycol subunits or polypropylene subunits.

[0260] In some embodiments, the wound dressing comprises a wound dressing material having the structure of Formula IIb:

[0261]

[0262] Wherein PEP is a peptide region comprising a reporter molecule and at least one amino acid; m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n is an integer selected from 200 to 4000. In other embodiments, n is an integer selected from 300 to 3500. In still other embodiments, n is an integer selected from 400 to 3200.

[0263] In some embodiments, the wound dressing comprises a wound dressing material having the structure of Formula IIc:

[0264]

[0265] Wherein PEP is a peptide region comprising a reporter molecule and at least one amino acid; m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n is an integer selected from 200 to 4000. In other embodiments, n is an integer selected from 300 to 3500. In still other embodiments, n is an integer selected from 400 to 3200.

[0266] Method for preparing compounds of formula I or formula II:

[0267] Embodiments provided herein also relate to methods of preparing compounds of Formula I or Formula II, including precursors thereof. The term "precursor" includes any compound used as a reactant to produce an intermediate or final product.

[0268] In one embodiment, provided herein is a method for preparing a compound of formula I comprising structure MR, wherein M is a gel-forming polymer comprising a plurality of monomers selected from the group consisting of cellulose, carboxymethylcellulose (CMC), oxidized cellulose (or a derivative thereof), cellulose ethylsulfonate (CES), pectin, alginate, chitosan, modified chitosan, hyaluronic acid, a polysaccharide or a gel-derived polymer, or any combination or mixture thereof, and R is a reporter region, the method comprising conjugating the gel-forming polymer to a reporter molecule, for example, by a covalent bond. In one embodiment, the reporter molecule R is a substrate for a wound-specific marker, such as a wound-specific enzyme, such as a hydrolase, and more specifically a protease, as described above. Under this embodiment, the substrate for the wound-specific marker includes, for example, a hydrolyzable substrate, such as an amino acid, a sugar, a peptide, a polysaccharide, a nucleic acid, a lipid, or a combination thereof.

[0269] In one embodiment, the polymer forming the gel is conjugated to the reporter molecule through a peptide, glycoside, amide, ester, ether, anhydride or similar connection. As used herein, a "peptide bond" is formed by a condensation reaction between two amino acids, wherein the acid portion of one reacts with the amino portion of the other to produce a peptide bond (-CO-NH-) between the two amino acids. As used herein, a "glycosidic bond" is formed between a hemiacetal or hemiketal group of a sugar (or a molecule derived from a sugar) and a hydroxyl group of some compounds (e.g., alcohol). Substances containing glycosidic bonds are glycosides. The term "glycoside" is now expanded to also include compounds having bonds formed between several chemical groups other than the hemiacetal (or hemiketal) group of a sugar and a hydroxyl group, such as -SR (thioglycoside), -SeR (selenoglycoside), -NR1R2 (N-glycoside), or even -CR1R2R3 (C-glycoside). The term "amide" as used herein refers to --N(R 1 )--C(═O)--or--C(═O)--N(R 1 )--, where R 1 It is defined herein to include hydrogen as well as other groups. The term "substituted amide" refers to the case where R1 is not hydrogen, while the term "unsubstituted amide" refers to the case where R1 is hydrogen. The term "ester" refers to a compound derived from an acid (organic or inorganic) in which at least one hydroxyl group is replaced by an alkoxy group. Esters have the general formula -C(=O)-OR 1 or R 1 -C(=O)-O–, where R 1It is defined herein to include hydrogen as well as other groups. Representative types of "esters" include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl and heterocyclic esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids and boric acids. The term "sulfonyl" represents a group of formula -SO2-alkyl or -SO2-aryl, wherein "alkyl" includes a saturated monovalent hydrocarbon group having a straight chain, branched or cyclic portion or a combination thereof, and contains 1 to 20 carbon atoms, preferably 1 to 5 carbon atoms, and "aryl" includes an organic group derived from an aromatic hydrocarbon by removing one hydrogen, such as phenyl or naphthyl, which is optionally substituted with 1 to 5 substituents, the substituents being independently selected from the following groups: halogen, hydroxyl, thiol, amino, nitro, cyano, acyl, acyloxy, sulfonyl, sulfinyl, alkylamino, carboxyl, ester, ether, amide, sulfonic acid, sulfonamide, alkylthio, oxyester. The term "sulfinyl" refers to a group of formula -SO-alkyl or -SO-aryl, wherein "alkyl" and "aryl" are as defined above. The term "sulfonamide" refers to a group of formula -SO2NH2. The term "oxyester" refers to a group of formula -O-COO-alkyl or -O-COO-aryl, wherein "alkyl" and "aryl" are as defined above. The term "ether" refers to a group of formula alkyl-O-alkyl or alkyl-O-aryl or aryl-O-aryl, wherein "alkyl" and "aryl" are as defined above. The term "amide" refers to a group of formula -CONRR', wherein R and R' are independently selected from hydrogen, "alkyl" or "aryl". The term "oxyamide" refers to a group of formula -O-CONRR', wherein R and R' are independently selected from hydrogen, "alkyl" or "aryl". The term "alkoxy" as used herein includes -O-alkyl, wherein "alkyl" is as defined above. As used herein, the term "alkylthio" includes alkyl groups, wherein "alkyl" is as defined above. As used herein, the term "alkylamino" includes -NHalkyl or -N(alkyl)2 groups, wherein "alkyl" is as defined above.

[0270] Methods of conjugating reactive moieties to produce glycosides, peptides, esters, oxyesters, amides, amidos, oxyamidos, ethers, sulfonyls, sulfinyls, sulfonamides or other linkages such as alkoxy, alkylthio, alkylamino, etc. are known in the art and are further described in the Examples.

[0271] In another embodiment, provided herein is a method for preparing a compound of Formula I comprising the structure MLR, wherein M and R are each as described above, and L is a linker which is a monomer or polymer of a neutral polymer, such as a polymer selected from the group consisting of ethoxylated polyols, polyvinyl pyrrolidone polymers, polypropylene, polyalkylene glycols, polyamines, including ethers, amides and esters thereof.

[0272] In one embodiment, M is conjugated to L via a first ester, oxidized ester, amide, amido, oxyamido, ether, sulfonyl, sulfinyl, sulfonamide, alkoxy, alkylthio, alkylamino, or similar linkage. Likewise, under this embodiment, the linker L is conjugated to the reporter region R via a second ester, oxidized ester, amide, amido, oxyamido, ether, sulfonyl, sulfinyl, sulfonamide, alkoxy, alkylthio, alkylamino, or similar linkage. The two linkages may be the same or different, for example, M may be conjugated to L via an ester linkage, while L may be conjugated to R via a peptide linkage.

[0273] In one embodiment, a compound of Formula I having structure MLR is synthesized by first conjugating a gel-forming polymer M with a linker L to produce a precursor ML, and then conjugating the precursor ML with a reporter region R to produce a compound of Formula I.

[0274] Alternatively, a compound of Formula I having the structure MLR is synthesized by first conjugating a linker L to a reporter region R to produce a precursor LR, which is then conjugated to a gel-forming polymer M to produce a compound of Formula I.

[0275] Furthermore, compounds of Formula I having structure MLR can be synthesized in a single reaction chamber or in multiple reaction chambers.

[0276] A representative retrosynthetic overview of potential reaction schemes for synthesizing compounds of Formula I is as follows:

[0277] Retrosynthetic Scheme I

[0278]

[0279] Diagnosis and treatment methods:

[0280] In one embodiment, the compositions, dressing materials, articles, kits and systems described herein can be used to diagnose or treat wounds, particularly chronic or infected wounds. Although any type of wound can be diagnosed and / or treated, these embodiments are particularly suitable for diagnosing and treating wounds with wound fluid exudation. For example, the wound can be a chronic or acute wound. Representative examples of chronic wounds include, for example, venous ulcers, pressure sores, decubitus ulcers, diabetic ulcers, and chronic ulcers of unknown etiology. Representative examples of acute wounds include, for example, acute traumatic lacerations that may be caused by intentional surgical incisions.

[0281] As used herein, the term "wound fluid" refers to any wound exudate or other fluid (suitably substantially excluding blood) present on the wound surface or removed from the wound surface by suction, absorption or washing. The determination, measurement or quantification is suitably performed on wound fluid that has been removed from the patient's body, but may also be performed in situ on the wound fluid. The term "wound fluid" does not generally refer to blood or tissue plasma away from the wound site. The wound fluid is mammalian wound fluid, suitably human wound fluid.

[0282] In one embodiment, the diagnostic method comprises contacting the wound with at least one composition comprising a compound of Formula I or Formula II, a dressing material comprising such a compound, an article comprising such a material or compound, a kit comprising such a material or compound, or a system comprising these materials or compounds as described herein; and measuring a parameter associated with the wound. In a specific embodiment, the parameter measured is the level or activity of a wound-specific hydrolase. In particular, the parameter measured is the activity of a hydrolase.

[0283] In the aforementioned embodiments, measurement can be carried out in situ or non-in situ. As used herein, the term "in situ" refers to a process, event, object or component that exists or occurs in the background of a system or device, including the surrounding environment, for example, a biomaterial in contact with a composition, product, system or device. As an example, an in situ reaction can refer to the reaction of various components (for example, a compound of Formula I or Formula II) present in a device, including components provided by human skin tissue (for example, wound exudate containing an enzyme). The term is contrasted with non-in situ, and non-in situ refers to outside the environment.

[0284] In a second embodiment, the measurement is performed ex situ, for example fluid is removed from the wound for analysis in a device or apparatus of the invention.

[0285] Suitably, the measurements are made in situ.

[0286] In one diagnostic embodiment, the method includes determining the level of a reporter molecule, such as a product of a substrate acted upon by a wound-specific enzyme. More specifically, the method includes determining the level of a hydrolase product. As used herein, the term "determining" includes measuring the activity or level of the hydrolase; determining whether the activity or level is above or below a predetermined range; and / or comparing the activity or level to a control standard. The control standard may include determining the level or activity of the hydrolase in biopsy material obtained from an uninjured site or a healthy subject.

[0287] In a specific embodiment, the term "determining" includes measuring a parameter (e.g., activity or level) of at least one wound-specific protease selected from the group consisting of: MMP-1 (collagenase), MMP-2 (gelatinase A), MMP-3 (matrilysin 1), MMP-8 (neutrophil collagenase), MMP-9 (gelatinase B), human neutrophil elastase (HNE), cathepsin G, urokinase-type plasminogen activator (uPA) and lysozyme or a combination thereof; determining whether the parameter exceeds a first predetermined threshold; and / or comparing the value of the parameter to a control standard. The control standard may include determining the protease parameter in biopsy material obtained from an uninjured site or from a healthy subject. In a related embodiment, the term "determining" includes determining whether the weighted average (weighted sum) of the parameters associated with a plurality of the aforementioned proteases exceeds a predetermined threshold of the weighted average.

[0288] In a particular embodiment, the parameter is the activity level of an analyte (eg a protease) in the wound fluid. Typically, the activity of a single analyte is expressed in terms of units / ml.

[0289] In another embodiment, the parameter is the level of an analyte (eg, a protease) in the wound fluid.In general, the term amount also refers to the activity of the particular analyte.

[0290] As used herein, the term "combined amount" or "combined activity" refers to a single numerical value resulting from the application of a mathematical function to a plurality of values, such as those obtained for a number of individual analytes. For example, the term "combined amount" or "combined activity" may refer to the sum or product of a set of individual values. Typically, the term "combined amount" or "combined activity" refers to the sum of a set of individual values. For example, in suitable embodiments, the amount of elastase refers to elastase-like activity (e.g., U / mL), and the amount of metalloproteinase (MMP) refers to the total concentration of the corresponding analyte (e.g., in ng / mL).

[0291] As used herein, the term "quantification" refers to measuring the absolute amount of a particular analyte or substrate in a sample within the tolerance of experimental error.

[0292] The term "marker" or "analyte" refers to any chemical entity identified or determined using an instrument, device, kit or method as defined herein. The marker or analyte determined or identified by the instrument, device, kit or method of the present invention is a cleavage product of the aforementioned enzyme.

[0293] As used herein, the term "predetermined range" refers to a data range or profile that is understood by a skilled artisan to be indicative of a particular subclass of patients. For example, a predetermined range may be a typical data range or profile for wounds that respond well to a particular wound treatment (e.g., antibiotic treatment). Alternatively, a predetermined range may appropriately refer to a typical data range for wounds that do not respond well to a particular wound treatment (e.g., antibiotic treatment).

[0294] When used in this article, the term "predetermined threshold value" refers to the minimum level of indicating non-healing wounds determined by the technician based on statistical analysis of the levels determined for known healing and non-healing wounds, such as further explained above. In order to make the test clinically useful, the threshold value should be set at an appropriate level so as to correctly identify non-healing wounds with high protease activity. Increasing the threshold value will increase the chance that only non-healing wounds exceed the threshold value. However, if the threshold value is too high, wounds that cannot heal due to high levels of protease will not be identified, and clinically this will mean that they will not receive the required protease regulation treatment.

[0295] As used herein, the term "control standard" or "control" refers to a data set or profile that can be used as a reference or comparison to define or standardize another data point or data set. For example, the term "control" or "control standard" can be a data set or profile that indicates a particular subclass of patients. Suitably, the control standard can be a data set or profile that indicates the state of a healing or non-healing wound.

[0296] Suitably, in other aspects or embodiments of the invention, a "control" or "control standard" can be a data set or profile that can be used as a comparison tool, which allows a technician to determine whether a wound is likely to respond or not to a wound treatment (such as an antibiotic treatment). In one embodiment, the control standard is a data set or profile indicating a patient who responds poorly to wound treatment. Typically, the control standard is a data set or profile indicating a patient who responds well to wound treatment. Compared to patients who tend to respond poorly to treatment, patients who tend to respond well to wound treatment as disclosed herein show a lower combined amount or activity of hydrolases. For example, patients who tend to respond well to wound treatment as disclosed herein show a lower combined amount of at least one wound-specific hydrolase.

[0297] In one embodiment, the threshold matrix metalloproteinase (MMP) activity is about 5 U / mL to about 30 U / mL, including all values ​​therebetween, such as about 6 U / mL, about 7 U / mL, about 8 U / mL, about 9 U / mL, about 10 U / mL, about 11 U / mL, about 12 U / mL, about 13 U / mL, about 14 U / mL, about 15 U / mL, about 16 U / mL, about 17 U / mL, about 18 U / mL, about 19 U / mL, about 20 U / mL, about 21 U / mL, about 22 U / mL, about 23 U / mL, about 24 U / mL, about 25 U / mL or more, indicating chronic wound infection. As understood in the art, activity units (U) are generally used to describe enzyme catalytic activity, wherein the unit (U) refers to the amount of enzyme that catalyzes the conversion of 1 micromole (μmole) of substrate per minute. Therefore, 1 enzyme unit (U) = 1 μmol / minute, wherein μmol refers to the amount of substrate converted.

[0298] In one embodiment, a threshold human neutrophil elastase activity of about 5 U / mL to about 30 U / mL, including all values ​​therebetween, for example, about 6 U / mL, about 7 U / mL, about 8 U / mL, about 9 U / mL, about 10 U / mL, about 11 U / mL, about 12 U / mL, about 13 U / mL, about 14 U / mL, about 15 U / mL, about 16 U / mL, about 17 U / mL, about 18 U / mL, about 19 U / mL, about 20 U / mL, about 21 U / mL, about 22 U / mL, about 23 U / mL, about 24 U / mL, about 25 U / mL or more is indicative of a chronic wound infection.

[0299] In a specific embodiment, a threshold human neutrophil elastase activity level of at least 9.6 is indicative of a chronic wound infection. In some embodiments, a human neutrophil elastase activity level of at least 22.9 U / mL is indicative of a chronic wound infection.

[0300] In one embodiment, the threshold lysozyme activity level is from about 1000 U / mL to about 10000 U / mL, including all values ​​therebetween, such as about 1100 U / mL, about 1200 U / mL, about 1300 U / mL, about 1400 U / mL, about 1500 U / mL, about 1600 U / mL, about 1700 U / mL, about 1800 U / mL, about 1900 U / mL, about 2000 U / mL, about 2100 U / mL, about 2200 U / mL, about 2300 U / mL, about 2400 U / mL, About 2500U / mL, about 2600U / mL, about 2700U / mL, about 2800U / mL, about 2900U / mL, about 3000U / mL, about 3250U / mL, about 3500U / mL, about 3750U / mL, about 4000U / mL, about 4250U / mL, about 4500U / mL, about 4750U / mL, about 5000U / mL, about 5250U / mL, about 5500U / mL, about 5750U / mL, about 6000U / mL or more indicates a chronic wound infection. In a specific embodiment, a lysozyme activity level of at least 4800U / mL indicates a chronic wound infection.

[0301] In one embodiment, the threshold cathepsin G activity level is about 10U / mL to about 100U / mL, including all values ​​therebetween, such as about 15U / mL, about 20U / mL, about 25U / mL, about 30U / mL, about 35U / mL, about 40U / mL, about 45U / mL, about 50U / mL, about 55U / mL, about 60U / mL, about 65U / mL, about 70U / mL, about 75U / mL, about 80U / mL, about 85U / mL, about 90U / mL, about 95U / mL, about 100U / mL, about 110U / mL, about 120U / mL or more, indicating chronic wound infection. In some embodiments, the cathepsin G activity level is at least 50U / mL, at least 40U / mL, at least 30U / mL, at least 20U / mL, at least 15U / mL or at least 10U / mL, indicating chronic wound infection.

[0302] Embodiments disclosed herein also relate to treating chronic or infected wounds using the compositions, materials, articles, dressings, kits and / or systems described herein. Treatment embodiments include contacting a composition, material, article, dressing, kit, system or device of the present invention with a subject in need thereof. Optionally, the method may include determining whether the subject is responsive to treatment.

[0303] A skilled person will be able to readily identify whether a wound is "responsive to treatment". In particular, a skilled person will be able to readily determine the levels of proteases identified in the claims of the present invention that predict or indicate a good response or a poor response to wound treatment, particularly treatment with a wound dressing comprising oxidized cellulose. The terms "responsive" and "responder" as used herein refer to a wound that is considered to respond well to wound treatment, particularly treatment with a drug such as an antibiotic. Similarly, "unresponsive" and "non-responder" refer to a wound that is not considered to respond well to wound treatment, particularly treatment with a drug such as an antibiotic. For example, a patient who exhibits better than 50% wound closure after 4 weeks of wound treatment is considered to respond to the treatment.

[0304] In certain embodiments, a patient may be diagnosed and treated simultaneously using a composition, article, system or device described herein. As used herein, the term "simultaneously" means performing the objectives, such as diagnosis and treatment, together.

[0305] In certain embodiments, the patient can be diagnosed and treated sequentially with the compositions, articles, systems or devices described herein. As used herein, the term "sequentially" means that the objectives, e.g., diagnosis and treatment, are separated in time or space, e.g., diagnosis before treatment or diagnosis after treatment or a combination thereof, e.g., first diagnosis ==> treatment ==> second diagnosis.

[0306] The embodiments described herein also enable a caregiver or patient to quickly and reliably determine whether a wound is likely to be non-healing, and select appropriate treatment based on the determination. For example, a non-healing wound may require application of a special wound dressing, such as a wound dressing containing a specific therapeutic agent, to promote healing. Therefore, the embodiments described herein also provide a method for treating a wound (e.g., a chronic or infected wound), comprising determining whether a wound is healing or non-healing, and then, if it is non-healing, applying a wound dressing containing a therapeutic agent to the wound.

[0307] The embodiments described herein provide methods and assays for diagnosing or detecting an infected wound. The methods are applicable to detecting bacterial infectious agents. In one embodiment, the wound is infected with Gram-negative bacteria. Exemplary Gram-negative bacteria include Proteobacteria such as Escherichia coli, Salmonella, Pseudomonas, and Helicobacter, as well as cyanobacteria. When classified by drug, they include Pseudomonas aeruginosa and Hemophilus influenzae, which cause respiratory disorders, Escherichia coli and Proteus mirabilis, which cause urinary tract disorders, and Helicobacter pylori and Bacillus Gaertner, which cause digestive system disorders, and Micrococcus such as Neisseria meningitidis, Moraxella catarrhalis, and Neisseria gonorrhea.

[0308] In another embodiment, the wound is infected with Gram-positive bacteria. "Gram-positive bacteria" refers to bacteria that contain teichoic acids (e.g., lipoteichoic acid and / or wall teichoic acid) or functionally equivalent sugar polymers (e.g., rhamnopolysaccharide, teichuronic acid, arabinogalactan, lipomannan, and lipoarabinomannan) in their cell walls. Non-limiting examples of functionally equivalent sugar polymers are described in Weidenmaier et al., Nature, 6 : 276-287, 2008.

[0309] Bacteria include pathogens that infect mammalian hosts (e.g., cattle, mice, horses, primates, felines, dogs, and human hosts). Examples of such pathogens include, for example, members of bacterial species such as Bacteroides, Clostridium, Streptococcus, Staphylococcus, Pseudomonas, Haemophilus, Legionella, Mycobacterium, Escherichia, Salmonella, Shigella, Vibrio, or Listeria. Some clinically relevant examples of pathogens that cause disease in a human host include, but are not limited to, Bacillus anthracis, Bacillus cereus, Bordetella pertussis, Borrelia burgdorferi, Brucella aborus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, and Clostridium spp. tetani), Corynebacterium diphtheriae, Enterococcus faecalis, vancomycin-resistant Enterococcus faecalis, Enterococcus faecium, Escherichia coli, enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia colicoli O157:H7, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Proteus, Pseudomonas aeruginosa aeruginosa), Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermis, Staphylococcus saprophyticus, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VSA), Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae cholerae and Yersinia pestis.

[0310] In another embodiment, the infecting bacteria is selected from the group consisting of Clostridium difficile, Carbapenem-Resistant Enterobacteriaceae (CR-Klebsiella spp; CR-E. coli) and Neisseria gonorrhoeae. In another embodiment, the infecting bacteria is selected from the group consisting of multidrug-resistant Acinetobacter, drug-resistant Campylobacter, extended spectrum β-lactamase (ESBL)-producing enterobacteriaceae, vancomycin-resistant enterococcus, multidrug-resistant Pseudomonas aeruginosa. In another embodiment, the infectious bacteria is selected from the group consisting of vancomycin-resistant Staphylococcus aureus, erythromycin-resistant Group A Streptococcus, and clindamycin-resistant Group B Streptococcus.

[0311] In certain embodiments, chronic or infected wounds are found in host subjects. Preferably, the host is a mammal, such as a rodent, a human, a livestock animal, a companion animal, or a non-domesticated or wild animal. In one embodiment, the subject can be a rodent, such as a mouse, a rat, a guinea pig, etc. In another embodiment, the subject can be a livestock animal. Non-limiting examples of suitable livestock animals can include pigs, cattle, horses, goats, sheep, llamas, and alpacas. In yet another embodiment, the subject can be a companion animal. Non-limiting examples of companion animals can include pets, such as dogs, cats, rabbits, and birds. In another embodiment, the subject can be a zoo animal. As used herein, "zoo animals" refers to animals that can be found in zoos. These animals can include non-human primates, large cats, wolves, and bears. In an exemplary embodiment, the subject is a human.

[0312] In one aspect, provided herein is a method for detecting the level of one or more enzymes in a mammalian wound, the method comprising the steps of: (a) contacting a wound dressing material described herein with a mammalian wound; (b) visually comparing the wound dressing in contact with the mammalian wound to one or more reference samples; and (c) obtaining a qualitative determination of the concentration of a reporter molecule in the wound dressing material in contact with the mammalian wound.

[0313] In some embodiments, a method for detecting the level of one or more enzymes in a mammalian wound consists essentially of the following steps: (a) contacting a wound dressing material described herein with a mammalian wound; (b) visually comparing the wound dressing in contact with the mammalian wound to one or more reference samples; and (c) obtaining a qualitative determination of the concentration of a reporter molecule in the wound dressing material in contact with the mammalian wound.

[0314] In another aspect, provided herein is a method for detecting the level of one or more enzymes in a mammalian wound, the method comprising the steps of: (a) contacting a wound dressing material described herein with a mammalian wound; (b) obtaining a quantitative measurement of the concentration of a reporter molecule in the wound dressing material in contact with the mammalian wound; and (c) comparing the quantitative measurement to one or more reference samples.

[0315] In some embodiments, the method for detecting the level of one or more enzymes in a mammalian wound consists essentially of the following steps: (a) contacting a wound dressing material described herein with a mammalian wound; (b) obtaining a quantitative measurement of the concentration of a reporter molecule in the wound dressing material in contact with the mammalian wound; and (c) comparing the quantitative measurement to one or more reference samples.

[0316] In one aspect, provided herein is a method for detecting the level of one or more proteases in a mammalian wound, the method comprising the steps of: (a) contacting a wound dressing material described herein with a mammalian wound; (b) visually comparing the wound dressing material in contact with the mammalian wound to one or more reference samples; and (c) obtaining a qualitative determination of the concentration of a reporter molecule in the wound dressing material in contact with the mammalian wound.

[0317] In some embodiments, the method for detecting the level of one or more proteases in a mammalian wound consists essentially of the following steps: (a) contacting a wound dressing material described herein with a mammalian wound; (b) visually comparing the wound dressing material in contact with the mammalian wound to one or more reference samples; and (c) obtaining a qualitative determination of the concentration of a reporter molecule in the wound dressing material in contact with the mammalian wound.

[0318] In another aspect, provided herein is a method for detecting the level of one or more proteases in a mammalian wound, the method comprising the steps of: (a) contacting a wound dressing material described herein with a mammalian wound; (b) obtaining a quantitative measurement of the concentration of a reporter molecule in the wound dressing material contacted with the mammalian wound; and (c) comparing the quantitative measurement to one or more reference samples.

[0319] In some embodiments, the method for detecting the level of one or more proteases in a mammalian wound consists essentially of the following steps: (a) contacting a wound dressing material described herein with a mammalian wound; (b) obtaining a quantitative measurement of the concentration of a reporter molecule in the wound dressing material in contact with the mammalian wound; and (c) comparing the quantitative measurement to one or more reference samples.

[0320] In another aspect, provided herein is a method for diagnosing a chronic wound in a mammal, the method comprising the steps of: (a) contacting a wound dressing material described herein with a mammalian wound; (b) visually comparing the wound dressing material in contact with the mammalian wound with one or more reference samples; and (c) obtaining a qualitative determination of the concentration of a reporter molecule in the wound dressing material in contact with the mammalian wound.

[0321] In some embodiments, the method for diagnosing a chronic wound in a mammal consists essentially of the following steps: (a) contacting a wound dressing material described herein with a mammalian wound; (b) visually comparing the wound dressing material in contact with the mammalian wound to one or more reference samples; and (c) obtaining a qualitative measurement of the concentration of a reporter molecule in the wound dressing material in contact with the mammalian wound.

[0322] In another aspect, provided herein is a method for diagnosing a chronic wound in a mammal, the method comprising the steps of: (a) contacting a wound dressing material described herein with a mammalian wound; (b) obtaining a quantitative measurement of the concentration of a reporter molecule in the wound dressing material in contact with the mammalian wound; and (c) comparing the quantitative measurement with one or more reference samples.

[0323] In some embodiments, the method for diagnosing a chronic wound in a mammal consists essentially of the following steps: (a) contacting a wound dressing material described herein with a mammalian wound; (b) obtaining a quantitative measurement of the concentration of a reporter molecule in the wound dressing material in contact with the mammalian wound; and (c) comparing the quantitative measurement to one or more reference samples.

[0324] In another aspect, provided herein is a method for treating a mammalian wound, the method comprising the steps of: (a) contacting a wound dressing material described herein with a mammalian wound; (b) visually comparing the wound dressing material in contact with the mammalian wound with one or more reference samples; (c) obtaining a qualitative measurement of the concentration of a reporter molecule in the wound dressing material in contact with the mammalian wound; and (d) administering a drug therapy to the mammal; wherein the drug therapy includes antibiotic therapy only if the concentration of the reporter molecule indicates that the mammalian wound is a chronic wound.

[0325] In some embodiments, the method for treating a mammalian wound consists essentially of the following steps: (a) contacting a wound dressing material described herein with a mammalian wound; (b) visually comparing the wound dressing material in contact with the mammalian wound to one or more reference samples; (c) obtaining a qualitative measurement of the concentration of a reporter molecule in the wound dressing material in contact with the mammalian wound; and (d) administering a drug therapy to the mammal; wherein the drug therapy includes antibiotic therapy only if the concentration of the reporter molecule indicates that the mammalian wound is a chronic wound.

[0326] In another aspect, provided herein is a method for treating a mammalian wound, the method comprising the steps of: (a) contacting a wound dressing material described herein with a mammalian wound; (b) obtaining a quantitative measurement of the concentration of a reporter molecule in the wound dressing material in contact with the mammalian wound; (c) comparing the wound dressing material in contact with the mammalian wound to one or more reference samples; and (d) administering a drug therapy to the mammal; wherein the drug therapy includes antibiotic therapy only if the concentration of the reporter molecule indicates that the mammalian wound is a chronic wound.

[0327] In some embodiments, the method for treating a mammalian wound consists essentially of the following steps: (a) contacting a wound dressing material described herein with a mammalian wound; (b) obtaining a quantitative measurement of the concentration of a reporter molecule in the wound dressing material in contact with the mammalian wound; (c) comparing the wound dressing material in contact with the mammalian wound to one or more reference samples; and (d) administering a drug therapy to the mammal; wherein the drug therapy includes antibiotic therapy only if the concentration of the reporter molecule indicates that the mammalian wound is a chronic wound.

[0328] Preferably, diagnosis and treatment are performed in situ. Therefore, the embodiments described herein allow for easy, non-invasive diagnosis and treatment of wounds. For example, diagnosis can be performed in real time, and treatment can be applied to infected wounds or patients (systemically), and the progress of wound treatment can be monitored in real time, for example, the dissipation of the reporter molecule generation signal caused by wound healing.

[0329] Example

[0330] The structures, materials, compositions and methods described herein are intended to be representative embodiments of the present invention, and it should be understood that the scope of the present invention is not limited by the scope of the embodiments. Those skilled in the art will recognize that the disclosed structures, materials, compositions and methods may be varied to implement the present invention, and these variations are considered to be within the scope of the present invention.

[0331] List of abbreviations:

[0332] As stated above, and throughout the specification of the present invention, unless otherwise indicated, the following abbreviations shall be understood to have the following meanings:

[0333] ACN or MeCN Acetonitrile

[0334] Bn Benzyl

[0335] BOC or Boc tert-butyl carbamate

[0336] t-Bu tert-butyl

[0337] CMC Carboxymethyl Cellulose

[0338] DCE Dichloroethane (ClCH 2 CH 2 Cl)

[0339] DCM Dichloromethane (CH 2 Cl 2 )

[0340] DIPEA or DIEA diisopropylethylamine

[0341] DMAP 4-(N,N-dimethylamino)pyridine

[0342] DMF Dimethylformamide

[0343] DMSO Dimethyl sulfoxide

[0344] equiv equivalent

[0345] Et Ethyl

[0346] EtOH

[0347] EtOAc Ethyl acetate

[0348] Fmoc Fluorenylmethoxycarbonylamide

[0349] HBTU N,N,N',N'-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate

[0350] HPLC High Performance Liquid Chromatography

[0351] Me Methyl

[0352] MeOH Methanol

[0353] MS

[0354] NMR Nuclear Magnetic Resonance

[0355] PBS Phosphate buffered saline

[0356] RP-HPLC Reversed Phase High Pressure Liquid Chromatography

[0357] TFA Trifluoroacetic acid

[0358] THF Tetrahydrofuran

[0359] For the polymers shown in the following examples, n is an integer selected from 400 to 3200.

[0360] Example 1: Preparation of polymer 1 (using CMC fibers)

[0361]

[0362] Sodium carboxymethylcellulose (NaCMC) fiber (443mg, 1.08mmol) is dissolved in deionized water (44ml) to obtain a 1% solution. Dowex 650C Monosphere ion exchange resin is added to provide acidified CMC (CMC-H). Monosphere is removed by filtration, and then tetrabutylammonium hydroxide (TBAH) 40% (aqueous solution) is added until pH is 8 to 9. The resulting solution is stirred for 30 minutes, and then lyophilized overnight. The lyophilized material (0.38g) is dissolved in 40ml dry DMF under nitrogen and stirring, and gently heated in a time period of about 1 hour. The resulting solution is turbid and off-white. The solution is cooled to about 4°C. 2-chloro-N-methylpyridinium iodide (CMP-I) (0.33g, 1.3mmol) is added to the stirred solution. Soon thereafter, compound 1-b (0.5g, 2mmol) and 3 drops of dry DCM and a few drops of dry triethylamine are also added. The reaction mixture was kept at 4 ° C and stirred for 3 hours, after which it became darker, almost brown. 95% acetone (aqueous solution) (80 ml) was slowly added and stirred at 4 ° C for 20 minutes, then kept at 4 ° C overnight. The resulting white precipitate was filtered out from the brown solution. The material was then washed with acetone five times by the following method: 40 ml 99.5% acetone was added, mixed and ultrasonicated for about 1 minute, acetone was filtered out and the solid product was collected. This produced a solid off-white spongy material. The remaining acetone was removed under reduced pressure. The solid was washed in ethanol (20 ml × 1) and then in acetone (20 ml × 3). During each washing, the washing solution was ultrasonicated for 1 minute, and then filtered to collect the solid. The solid (compound 1-c) was placed on a high vacuum rotary evaporator for 45 minutes. Weight = 0.288g. FTIR: 3360, 3320 (NH / OH), 2875 (CH), 1650, with shoulder peak (amide C=O of BOC), 1590 (C=O of CMC).

[0363] To evaluate the solubility of compound 1-c, the following method was followed:

[0364] Compound 1-c was weighed into a mass spectrometry vial (approximately 0.0013 g per vial). Approximately 1 ml of the desired solvent was added to the vial. The solubility of the sample was visually assessed at the following time points: initially, after gentle heating at 40°C for 1 minute, after sonication for 10 seconds, and after standing overnight at room temperature. The data are shown below: x represents insoluble material.

[0365] Solvents initial After heating After ultrasonic treatment overnight DCM x x x x EtOH x x x x MeOH x x x x THF x x x x Deionized water x Solid dispersion Well dispersed, turbid solution Well dispersed, turbid solution DMF x x x x

[0366] Compound 1-c (0.05 g, 0.11089 mmol) was weighed and cut into pieces as small as possible. A 10% TFA solution in DCM (10 ml) was added and the mixture was stirred at room temperature for 1 hour. Volatile solvents and reagents were removed using a high vacuum rotary evaporator. The remaining solid was subjected to three azeotropes to remove any remaining TFA by lowering its boiling point: toluene (10 ml×3) was added to the solid product and stirred, and then removed using a high vacuum rotary evaporator to provide polymer 1, which was an off-white solid. Weight = 0.0524 g. FTIR: 3326 (NH / OH), 2891 (CH), 1668 (C=O carbamate stretching). Elemental analysis (TFA salt): Carbon: expected to be about 43%; actual = 38.90%. Hydrogen: expected to be about 5.8%; actual = 5.43%. Nitrogen: expected to be about 4.4%; actual = 0.28%. The degree of substitution is 0.28 / 4.38=0.06.

[0367] The solubility of polymer 1 was determined in a similar manner as compound 1-c. The data are shown below: x represents insoluble material.

[0368] Solvents initial After heating After ultrasonic treatment overnight DCM x x x x EtOH x x x x MeOH x x x x THF x x x x Deionized water x x x x DMF x x x x

[0369] A qualitative assay for the amination of polymer 1 was evaluated using the Kaiser test based on ninhydrin. Three reagents were prepared: (1) 500 mg (0.5 g) ninhydrin in 10 ml EtOH; (2) 80 g phenol in 20 ml EtOH; (3) 2 ml 0.001 M KCN ​​diluted to 100 mL with pyridine. A sample of polymer 1 (10 to 20 mg) was placed in a round bottom flask. Three to five drops of each reagent were added to the flask. The flask was heated to 100 °C using an oil bath and reflux condenser and stirred for 5 minutes. Any color change was observed visually. A dark blue / purple color was detected, indicating the presence of amine.

[0370] Example 2: Preparation of polymer 2

[0371]

[0372] A solution of Fmoc-Phe-OH (0.0267 g, 0.069 mmol), HBTU (0.0262 g, 0.069 mmol), DIPEA (0.015 ml, 0.090 mmol) and dry DMF (0.82 ml) was prepared. A 15 ml plastic separation column was installed with a 10 μm polyethylene glass frit and a Luer Tip was connected. Polymer 1 (0.034 g, 0.097 mmol) was added to the column, followed by the amino acid solution. The lid was placed on the column and further sealed with parafilm. The column was placed on a blood rotor at room temperature overnight. The solution was drained under reduced pressure and the solid product was washed with EtOH to provide polymer 2 as a white solid. Weight = 0.0266 g. Kaiser test was used to confirm the absence of terminal amines. FTIR: 3324 (NH / OH), 3281 (OH), 2898 (CH), 1720 (aromatic C=O), 1666 (C=O urethane stretching), 1660 (amide stretching of C=O Fmoc).

[0373] The solubility of polymer 2 was determined in a similar manner as compound 1-c. The data are shown below: x represents insoluble material.

[0374] Solvents initial After heating After ultrasonic treatment overnight DCM x not applicable not applicable not applicable EtOH x not applicable not applicable not applicable DMF x not applicable not applicable not applicable

[0375] Example 3: Preparation of polymer 3

[0376] Polymer 2 is placed in a solution of piperidine:DMF (1:4v / v, 6ml) for about 2 hours. The deprotected product obtained is then washed with EtOH to confirm the presence of free amine before the Kaiser test. The deprotected product (0.080g, 0.15mmol) is added to a 15ml plastic separation column, which is equipped with 10μm polyethylene glass frit and Luer Tip. A solution of Fmoc-Phe-OH (0.063g, 0.16mmol), HBTU (0.062g, 0.16mmol), DIPEA (0.23ml, 1.8mmol) and dry DMF (6ml) is prepared and added to the column. The lid is placed on the column and further sealed with paraffin film.

[0377]

[0378] The column was placed on a blood rotor at room temperature overnight. The solution was drained under reduced pressure and the solid product was washed with EtOH (10 ml x 3) to provide the coupled product as a red solid. Weight = 0.0691 g. Kaiser test was used to confirm the absence of terminal amines. The coupled product was washed with DCM. FTIR: 3315 (NH / OH), 2866 (CH), 1730 (aromatic C=O), 1651+ shoulder (C=O, amide of F) and / or (amide stretching of C=O F-Fmoc), 1587 (C=O carbamate stretching).

[0379] To remove the Fmoc group, the coupled product was placed in a solution of piperidine:DMF (1:4 v / v, 6 ml) for about 2 hours. The resulting deprotected product was then washed with EtOH (10 ml×3) and DCM (10 ml) to provide polymer 3 as a brittle red solid. Weight = 0.0638 g, yield = 62%. Kaiser test was used to confirm the presence of terminal amines. FTIR: 3315 (NH / OH), 2866 (CH), 1730 very small peaks (aromatic C=O), 1651 no shoulders (C=O, F amide), 1587 (C=O carbamate stretching). [Note: FTIR shows that there is still a small amount of Fmoc in the final product].

[0380] Example 4: Preparation of polymer 4

[0381] Synthesis of intermediate 4-c

[0382]

[0383] Fmoc-Lys-OH·HCl (0.0405 g, 0.100 mmol) was placed in a round-bottom flask, and a 5:2 mixture of 1,4-dioxane:10% K 2 CO 3 The mixture was stirred, dansyl chloride (0.033 g, 0.10 mmol) was added, and then stirred at room temperature overnight, and the atmosphere was passed through. The solution was diluted with 150 ml of water. The layers were separated, and the aqueous layer was extracted with ether (10 ml × 3). The combined organic layers were dried and concentrated. The crude material was purified using flash column chromatography (10:90 MeOH / DCM) to provide compound 4-c. 1 H NMR (400 MHz, with a few drops of CD 3 OD CDCl 3)8.55,7.79,7.65,7.52,7.26,7.17,6.69,6.59,5.19,5.06,4.78,4.28,4.17,4.08,3.97,3.57,3.55,3.35,3.05,2.87,2.14,1.99,1.63,1.41,1.21,0.83,0.03.

[0384] Synthesis of polymer 4

[0385]

[0386] Prepare a solution of compound 4-c (0.0160g, 0.024mmol), HBTU (0.00925g, 0.0244mmol), DIPEA (0.23ml, 1.8mmol) and dry DMF (6ml). Install a 15ml plastic separation column with 10μm polyethylene glass frit and Luer Tip. Add polymer 3 (0.0230g, 0.084mmol) to the column, and then add the amino acid solution. Place the lid on the column and further seal it with paraffin film. The column is placed on a blood rotor at room temperature overnight. The solution is discharged under reduced pressure, and the solid product is washed with EtOH (10ml×3) to provide a coupled product (red solid), which is washed with DCM. The Kaiser test is used to confirm the absence of terminal amines. FTIR: 3346 (NH / OH), 2912 (CH), 1730 (C=C dansyl aromatic), 1652 (C=O, amino acid amide), 1591 (C=O carbamate).

[0387] The coupled product was placed in a solution of piperidine:DMF (1:4 v / v) (6 ml) for about 2 hours. The resulting deprotected product was then washed with EtOH (10 ml×3) and then with DCM (10 ml) to provide polymer 4 as a red / brown brittle solid. Weight = 0.0368 g, yield = 56%. Kaiser test was used to confirm the presence of terminal amines. FTIR: 3335 (NH / OH), 2918 (CH), 1651 (C=O, amide of amino acid or Fmoc), 1589 (C=O carbamate).

[0388] Example 5: Preparation of polymer 5 (using powdered CMC)

[0389]

[0390] Sodium carboxymethylcellulose (NaCMC) (2.0 g, 8.33 mmol) of DoS 0.7 was dissolved in deionized water (180 mL) to obtain a 1% solution. Dowex 650C Monosphere ion exchange resin was added under stirring for 10 minutes. The Monosphere was removed by filtration, and tetrabutylammonium hydroxide (TBAH) 40% (aqueous solution) was then added in 0.1 mL aliquots until the pH was 8 to 9 (3.5 mL, 36.14 mmol). The resulting solution was stirred for 30 minutes and then lyophilized within 7 days.

[0391] The lyophilized material was dissolved in dry DMF (240 mL) under nitrogen atmosphere and stirring, and gentle heating was required over a period of about 2 hours. The solution was cooled to about 4 ° C, and 2-chloro-N-methylpyridinium iodide (CMP-I) (1.5 g, 5.8 mmol) was then added under vigorous stirring. 2,2'-(ethylenedioxy)bis(ethylamine) (1.3567 g, 9.17 mmol) was added to the reaction along with dry triethylamine (5 mL). The reaction was maintained at 4 ° C and stirred for a minimum of 3 hours, then 95% ethanol (aqueous solution) (80 mL) was added and stirred at 4 ° C for 10 minutes. 99% acetone (200 mL) was slowly added under stirring to precipitate the product, which was filtered and washed with acetone (3×200 mL). The product was concentrated in vacuo to give an off-white solid. Weight = 1.908 g, yield = 68%. FTIR: (ν max / cm- 1 )3267 (NH / OH), 2916 / 2873 (CH), 1739 (acetone), 1650 (C=O, amide of coupling product), 1588 (C=O of CMC), 1401 / 1314 / 1257 / 1037. 13C CP MAS NMR: CMC 0.7DoS (starting material): δC (13,000 Hz, CP MAS) 61.7 (C6), 74.5 (C7, C2, C5), 82.5 (shoulder, C3), 97.0 (C4), 103.3 (C1), 177 (C=O); CMC-PEG diamine: δC (13,000 Hz, CP MAS) 13.4 (C14), 20.1 (unassigned), 23.4 (unassigned), 30.6 (C9), 61.7 (C6), 70 (shoulder, C7), 74.5 (C2, C5), 82.5 (C3), 95 (C4), 103.3 (C1), 113 (unassigned), 142 (unassigned), 152 (unassigned), 169.7 (C=O of linker), 177.1 (C=O of CMC). Elemental analysis: If the DoS of the starting material is 0.7, then the expected product: mass 306 g / mol: C 45%, H 7%, N 6%; (d) actual: C 43%, H 7%, N 4%. Therefore, for all monomers, about 47% have been substituted and now contain linker groups. Qualitative Kaiser test: positive, indicating the presence of free amines. According to the calibration curve based on valine as standard reference, the UV data obtained at 570 nm was equivalent to 4.6 μmol of amine.

[0392] The solubility of polymer 5 was determined in a similar manner as compound 1-c. The data are shown below: x represents insoluble material.

[0393] Solvents initial After heating After ultrasonic treatment overnight DCM x x x x EtOH x x x x MeOH x x x x Deionized water x x Become softer dispersion pH 9 buffer x x x Hydrogel formation pH 4 buffer x x x A certain degree of hydrogel formation

[0394] Example 6: Preparation of polymer 6

[0395]

[0396] Polymer 5 was soaked in dry DMF (6 mL) in a filter tube for 20 minutes. Compound 4-c (0.0416 g, 0.063 mmol) and HBTU (0.2893 g, 0.8571 mmol) and DIPEA (0.2 mL, 0.8571 mmol) were added. The tube was fixed and rotated on a laboratory mixer at room temperature overnight. The supernatant was filtered and the remaining solid was washed with DMF (3×3 mL) and methanol (3×5 mL) and then dried in vacuo. Weight = 0.1285 g (yield 45%). Kaiser test was used to confirm the presence of terminal amine. FTIR: 3313 (NH, OH), 2862 (OH), 1747 (C=O of amide), 1587 (C=O of CMC), 1404 / 1315 / 1023. Elemental analysis: Carbon: expected 51%; found = 40%. Hydrogen: expected 5%; found = 6%. Nitrogen: Expected 5%; Actual = 4%.

[0397] The solubility of polymer 6 was examined in the following manner. A small amount of polymer 6 was placed in two separate glass bottom petri dishes. The solubility of polymer 6 was examined in the following manner. A pH 4 universal laboratory buffer or a pH 9K 2 HPO 4 / MgCl 2 Buffer (20 μL each) was used to saturate the material. Each was observed under a Zeiss Axioimager light microscope (lens at x10 magnification and eyepiece at x10 magnification).

[0398] Both samples formed hydrogels when in contact with the buffer. Under the microscope, the powder aggregates appeared to gel and contained a red color from the dansyl groups. Some areas of color were more concentrated than others, but a distribution of color was seen throughout the sample. The experiment was repeated for the starting material, compound 4-c, and polymer 5. No color was visible in the polymer 5 sample; a hydrogel powder structure was observed. The sample of compound 4-c was consistently red and did not form a hydrogel.

[0399] Example 7: Preparation of polymer 7

[0400]

[0401] Sodium carboxymethylcellulose (NaCMC) powder (2.0 mg, 8.3 mmol, DoS = 0.7) was dissolved in deionized water (200 mL) with stirring, sonicated for 60 seconds, and gently heated over the course of 30 minutes. 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) (1.92 g, 10.01 mmol) was added to the solution with stirring. The pH was found to be 8 and adjusted to pH = 6 by adding 1.0 M HCl (3 drops). L-cysteine ​​(0.50 g, 4.1 mmol) was added to the reaction with stirring. The pH was then found to be 8 and adjusted to pH 6 (about 1 mL) using HCl again. The reaction was stirred overnight and dialyzed (12-14 KDa Medicell dialysis membrane) against 1 M HCl (700 mL), 1 M HCl plus 1% NaCl (700 mL), 0.5 M HCl (700 mL), each for 60 min at 10° C. in the dark. The resulting solution was lyophilized to provide polymer 7. FTIR: 3296 (NH,OH), 2972 / 2930 (CH), 2733 (small peak), 2522 (SH of C), 2089 (very small peak), 1730 (C═O acid of Cys), 1681 (amide), 1633 (C═O of CMC), 1469 (shoulder), 1382 / 1346 / 1221 / 1107, 1051. 13C CP MAS NMR: 14 (C11), 18 (unassigned), 24 (unassigned), 43 (C9), 58 (unassigned), 62 (C6), 74 (C2, 3, 5, 7), 82 (C4), 103 (C1), 173 (carbonyl of C8 amide). Elemental analysis: If the DoS of the starting material is 0.7, then the expected product: mass 299 g / mol: C 40%, H 6%, N 3%, S 6%; actual: C 34%, H 8%, N 8%, S 1.4%. Therefore, for all monomers, about 16% contain linker groups.

[0402] The solubility of polymer 7 was determined in a similar manner as compound 1-c. Polymer 7 was found to be insoluble in water, DMF, acetone, MeOH, EtOH, and DCM.

[0403] Example 8: Preparation of polymer 8

[0404]

[0405] Polymer 5 (1.00 g, 2.86 mmol) was ground into a fine powder and mixed with DMF (25 ml) for 20 minutes. At room temperature, Fmoc-Cys (StBu) -OH (3.7 g, 8.6 mmol), HBTU (2.87 g, 8.57 mmol) and DIPEA (1.10 ml, 8.57 mmol) were added under stirring, and air was passed (centrifuge tubes and laboratory rotors were used to promote mixing overnight). The mixture was ultrasonically treated for 30 seconds and then stirred overnight. The solid was filtered and washed with DCM (20 mL × 5) and MeOH (20 mL × 5). The resulting product was vacuum dried to provide compound 8-b, which was an off-white solid. 13 C CP MAS NMR: 20 (unassigned), 30 (C9), 32 (C19), 39 (C10, 11, 12, 13), 47 (C18, 22), 55 (C16), 61 (C6), 70-74 (broad peak, C2, 3, 5, 7), 82 (shoulder, C4), 92 (C17), 96 (unassigned), 103 (C1), 120 (C24), 127 (C25, 26, 27), 141 (C28), 145 (shoulder, C23), 157 (C20, C=O carbamate), 171 (carbonyl of C8 amide), 177 (carbonyl of C8 CMC), 191 (unassigned), 221 / 227 (possibly a contaminant).

[0406] Compound 8-b (0.5 g) was placed in a piperidine:DMF (20:80 v / v) (10 ml) solution on a laboratory rotor for about 2 hours. The resulting deprotected product was then washed with EtOH (50 mL×3) and DCM (50 mL×3) to provide compound 8-c as an off-white solid. The process was repeated to completely remove the Fmoc group.

[0407] Compound 8-c (0.30 g, 0.54 mmol) was added to a round-bottomed flask and purged with nitrogen. Tris (2-carboxyethyl) phosphine (TCEP) (0.307 g, 1.07 mmol) was dissolved in deionized water (1.3 ml) and added to the reaction with MeOH (2.6 ml) under stirring. The system was kept under stirring for 1 hour at room temperature and nitrogen, then filtered and washed with the following solution: 2: 1 MeOH / water (90 ml), 1: 2 MeOH / water (90 ml), 100% water (90 ml), 100% MeOH (90 ml). The resulting solid was vacuum dried to provide polymer 8, which was an off-white solid. The process was repeated to completely reduce the disulfide. 13 C CP MAS NMR: 30 (C9, 19), 39 (C10, 11, 12, 13), 47 (C18, 22), 54 (C16), 60 (C6), 70-74 (broad peak, C2, 3, 5, 7), 82 (shoulder, C4), 92 (C17), 97 (unassigned), 102 (C1), 120 (C24), 127 (C25, 26, 27), 141 (C28), 156 (C20, C=O carbamate), 171 (carbonyl of C8 amide), 177 (carbonyl of C8 CMC), 191 (unassigned), 221 (unassigned). Elemental analysis: If the DoS of the starting material is 0.7, then the expected product: mass 390 g / mol: C 43%, H 6%, N 6%, S 5%; actual: C 46%, H 7%, N 4%, S 3.5%. Therefore, for all monomers, about 50% contain linker groups.

[0408] Example 9: Preparation of polymer 9

[0409]

[0410] Polymer 5 (1.0 g, 2.714 mmol) was dispersed in PBS (25 mL) with gentle heating, stirring and sonication. The reaction vessel was purged with nitrogen. EDTA (0.044 g, 0.15 mmol) was added to a solution of Traut's reagent (0.600 g, 4.304 mmol) in PBS (50 mL). Once completely dissolved, the solution was added to the reaction mixture, which was stirred for 1 hour at room temperature under a nitrogen atmosphere. The resulting product was isolated by filtration, washed with PBS (3×10 mL) and methanol (3×10 mL). The white / off-white powdery solid product was then vacuum dried and stored under nitrogen. Weight = 1.2413 g (91%). The yellow color from the Ellman test indicated that some free thiol was still present, which may be present in the unreacted remaining starting material. In the quantitative test, there was about 0.06 mmol / g of SH groups. FTIR: 3315 (NH / OH), 2957 / 2934 / 2870 (CH), 1644 (C=O, amide of coupling product), 1593 (C=O of CMC), 1412 / 1322 / 1022. At 2059 cm- 1 , the SH peak is very small. δC (13,000 Hz, CP MAS): 176.7 (C8, C=O of CMC), 172.2 (C8, C=O of amide), 156.0 (C15, small peak) 121.0, 103.0 (C1), 81.3 (C7, shoulder), 74.4 (C2, C3, C4, C5), 61.7 (C6), 38.9 + shoulder (C9, C10, C11, C12, C13, C14). Elemental analysis: If the DoS of the raw material is 0.7, the expected product: mass 414 g mol- 1 : C43%, H 7%, N 6%, S 4%. Actual: C40%, H 7%, N 4%. Therefore, for all monomers, about 47% contain a linker group.

[0411] Example 10: Preparation of polymer 10

[0412]

[0413] Polymer 9 (0.150 g, 0.316 mmol) was weighed into a round bottom flask and purged with nitrogen. Ellman's reagent (0.375 g, 0.947 mmol) was dissolved in PBS (20 mL) and added to polymer 9. The reaction was stirred at room temperature for 2 hours. The intermediate disulfide product 10-a was filtered and washed with PBS (3×20 mL) and methanol (1×20 mL), then vacuum dried to produce intermediate 10-a as a light white / yellow powdery solid. Weight = 0.133 g (yield 60%). The yellow color from the Ellman's test indicates that some free thiol is still present, which may be present in the unreacted remaining starting material. In the quantitative test, there are about 0.33 mmol / g of SH groups. FTIR: 3310 (NH / OH), 2911 / 2875 (CH), 1727 (C=O carboxylic acid), 1648 (C=O, amide of coupling product), 1592 (C=O of CMC). 1 δC (10,000 Hz, CP MAS): 222.37 (spinning sideband), shoulder at 172.5 (C8, C=O of CMC), 172.5 (C8, C=O of amide, possibly also some acid groups of Ellman's reagent), 156.0 (C15, small peak), 123.7 (spinning sideband), 144.5 (aromatic region of Ellman's reagent), 103.0 (C1), 96.79, 82.25 (C7, shoulder), 74.5 (C2, C3, C4, C5), 61.4 (C6), 39.3 plus shoulder (C9, C10, C11, C12, C13, C14), 32.2 sharp peak (possibly a contaminant). Elemental analysis: If the DoS of the feedstock is 0.7, the expected product: mass 553 g mol- 1 : C 43%, H 6%, N 6%, S 6%. Actual: C 39%, H 6%, N4%, S 1.45%. Therefore, for all monomers, about 17% contain linker groups.

[0414] The intermediate disulfide product 10-a (0.1 g, 0.1422 mmol), mercaptobenzoic acid (0.11 g, 0.7112 mmol) and methanol:water (4:1, 5 mL) were combined and stirred at room temperature under nitrogen for 2 hours. The resulting product was filtered, washed with methanol (3×10 mL), DCM (3×10 mL), and then washed with methanol (3×10 mL), and then vacuum dried to produce polymer 10 as a yellow solid powder. Weight = 0.0821 g (yield 87%). The strong yellow color from the Ellman test indicated that some free thiol was still present, which may be present in the unreacted remaining starting material. In the quantitative test, there were about 2.97 mmol / g of SH groups. FTIR: 3293 (NH / OH), 2910 / 2881 / 2849 (CH), 1718 (C=O carboxylic acid), 1638 (C=O, amide of coupling product), 1586 (C=O of CMC), 1553. δC (10,000 Hz, CP MAS): 177.4 (C8, C=O of CMC), 171.9 (C8, C=O of amide, possibly also some acid groups of benzoic acid), 130 / 143 small broad peaks, 102.7 (C1), 96.79, 81.6 (C7, shoulder), 74.1 (C2, C3, C4, C5), 62.5 (C6), 39.0 plus shoulder (C9, C10, C11, C12, C13, C14), 32.1 sharp peak (possibly a contaminant). Elemental analysis: If the DoS of the raw material is 0.7, the expected product: mass 520 g mol- 1 : C 44%, H 6%, N 6%, S 6%. Actual: C 37%, H 6%, N 3%.

[0415] A further washing series in water was performed to ensure that any byproducts were washed away from the structure in the swollen (hydrogel-like) state. The solid was washed sequentially with pH 9 phosphate buffer (100 mL), deionized water (20 mL), pH 9 phosphate buffer (5 mL), methanol (20 mL) and then dried in vacuo to provide polymer 10 as a white / cream solid powder. Weight = 0.0482 g (yield 51%). The light yellow color from the Ellman test indicated that some free thiol was still present, probably in the remaining unreacted starting material. In the quantitative test, about 0.31 mmol / g of SH groups were present. FTIR: 3267 (NH / OH), 2904 / 2866 (CH), 2119 minor peaks (SH), 1638 (C=O, amide of the coupled product), 1587 (C=O of CMC), 1547. Elemental analysis: If the DoS of the starting material is 0.7, then expected product: mass 520 g mol- 1: C 44%, H 6%, N 6%, S 6%. Actual: C 38%, H 6%, N 3%, S 0.65%.

[0416] The solubility of polymers 9 and 10 was determined in a similar manner as compound 1-c. The data are shown below: x represents insoluble material

[0417] Compound Solvents initial After heating After ultrasonic treatment overnight 9 pH 9 buffer X Not tested Not tested After mixing, hydrogel is formed 10 pH 9 buffer x Not tested Not tested Because the particles are so small, it is difficult to distinguish

[0418] Example 11: Preparation of polymer 11

[0419]

[0420] Aminophenylfluorescein (0.005 g, 0.0180 mmol), HBTU (0.0136 g, 0.036 mmol) and polymer 5 (0.013 g, 0.036 mmol) were added to 25 mL RBF with a stirring bar and stirred with N 2 (g) Purge. Dry DMF (5 mL) was added and the reaction was stirred overnight at room temperature, covered with foil to avoid light. The reaction solution was filtered through a filter tube and washed several times with DMF. The supernatant remained orange; it was stored with the washes and dried under vacuum. The solid product was recovered and dried under vacuum. In order to maximize the coupling, and given that the supernatant and washes had some orange color, the reaction was repeated using the solids recovered from the supernatant and the washes as reactants. Weight = 0.012 g (yield 43%). FTIR: 3367 (NH / OH), 2932 (CH), 1702, 1655 (C=O, amide), 1555 shoulder (C=O of CMC), 1494 (aromatic CC of APF), 1437 / 1413 / 1387 / 1308, 1194 (CC), 1106 (CO of APF), 838 (aromatic out-of-plane CH bend).

[0421] Example 12: Preparation of polymer 12

[0422]

[0423] First, polymer 8 was treated with TCEP to decouple any disulfide bonds that had formed. TCEP (0.0243 g, 0.0848 mmol, 4 equiv) was dissolved in 1 mL of water and added to polymer 8 (0.01 g, 0.0212 mmol) and stirred at room temperature for 30 minutes. The solid was filtered and washed with water (3 x 10 mL) and freeze-dried to give a solid off-white / cream powder, which was then washed with DMF (3 x 10 mL).

[0424] Compound 12-a (0.0108 g, 0.0212 mmol, 1 equivalent) was dissolved in 3 mL of dry DMF and added with N 2 (g) in the purged polymer 8. The reaction was stirred at room temperature overnight and covered with foil to avoid light. After stirring overnight, the reaction mixture became red and only a very small amount of white solid material (0.0022 g) was recovered during filtration. The solution phase was concentrated in vacuo to provide a crude polymer 12 as a red waxy solid.

[0425] The product and starting material may be present in the crude polymer 12, so washing is required to further purify the crude material. The mixture is insoluble in hexane, ethanol and chloroform. However, when immersed in deionized water and after gentle heating and ultrasonic treatment, the supernatant turns orange, which may be the starting material compound 12-a. The solution was subjected to the following steps 5 times: (1) adding deionized water (5 mL), (2) gentle heating with stirring, (3) 1 minute ultrasonic treatment repeated 3 times, and (4) filtering through a filter column and a glass filter. Another 50 mL of deionized water was passed through the solid sample for washing. It was then filtered and freeze-dried overnight to remove water. Product weight: 0.01701 g (yield 85%). FTIR: 3347 (NH / OH), 2915 / 2851 (CH), 1756 (C=O of ester / C=O of fluorescein), 1716 (C=O of ester / C=O of fluorescein), 1643 (C=O, amide of coupling product), 1608 (C=O of CMC / aromatic C=C stretching), 1492 (aromatic C=C stretching), 1369 (CO stretching of ester), 1246 (CO stretching), 1152 (t-OH), 1110 (CO / -OH of ester), 842 (aromatic out-of-plane CH bending).

[0426] Enzyme efficacy of polymer 12

[0427] Enzyme efficacy was tested with esterase (approximately 50 units / mL achieved by mixing neat esterase (0.01 mL) in PBS (0.99 mL).

[0428] Fluorescence microscopy showed that a clear difference in fluorescence was observed between the polymer 12 sample and the control sample (polymer 12 in PBS without enzyme), demonstrating the activity of the enzyme on the polymer to release the ester group from the attached fluorescein.

[0429] The use of confocal microscopy resulted in visualization of the fluorescence differences between polymer 12 and control samples. Microscope settings: Smart Gain = 716v, Smart Offset = -2.1%, Magnification = x20, Pinhole Size = 105.05 μm.

[0430] In order to quantify and accurately determine the enzyme efficacy, further experiments were set up using 96-well plates and a disk analyzer (Fluostar Optima BMGLabtech, excitation set to 485nm, emission set to 590nm, and gain set to 1500). Polymer 12 was dispersed in 320 μL PBS and vortexed to mix. 40 μL suspension was distributed to 8 wells and the measured value was used as a baseline reading. The following solutions were prepared: 58 units / mL of esterase in PBS (weak enzyme solution), 116 units / mL of esterase in PBS (strong enzyme solution), 1M NaOH (aqueous solution).

[0431] N = 2 wells had 40 μL of PBS only added to the test dispersion (control).

[0432] N=2 wells had 40 μL of weak enzyme solution pipetted into the test dispersion.

[0433] N = 2 wells had 40 μL of strong enzyme solution pipetted into the test dispersion.

[0434] N=2 wells had 40 μL of 1 M NaOH solution pipetted into the test dispersion.

[0435] Another fluorescence reading was recorded immediately after the final well was added and then after 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 minutes. The plate was then covered and left overnight so that a reading could be obtained after 24 hours. However, the fluorescence detector had reached its limit and no meaningful results were achieved at this time point. Table 2 and Figure 1 Fluorescence readings minus the baseline readings (particle suspension only) are shown for each time point.

[0436] Table 2

[0437]

[0438] The negative control showed a slight increase in fluorescence over 1 hour.

[0439] As expected, the fluorescence of the weak and strong esterase solutions increased over time. The strong esterase solution appeared to have reached maximum fluorescence intensity, measurable by the instrument at approximately 30 minutes (machine saturation point was 65,000). Figure 1 Show the graph for strong esterase up to 30 minutes and plot its trend line and that for weak esterase over the entire 60 minutes.

[0440] After this experiment, each solution (only n=1) was removed from its well and transferred into a glass bottom microscope well for visual inspection. It was also placed in a UV chamber set to the long wavelength setting and images were captured (Figure 2a to c).

[0441] Overall, this evidence suggests that the synthetic approach was successful and that the enzymatic cleavage step produced a positive response that could be quantified by fluorescence spectroscopy. Even without the addition of esterase, the sample of polymer 12 was fluorescent, suggesting that there may have been some excess conjugation.

[0442] Example 13: Preparation of polymer 13 (using CMC fibers)

[0443]

[0444] NaCMC fibers (2x AQUACEL dressings 10x10 cm) (1.97 g, 8.33 mmol) with a DoS of about 0.2 to 0.3 were broken down into small open fibers and dispersed in a solution of ethanol / deionized water (80:20 v / v) (180 mL). Dowex 650C Monosphere ion exchange resin was added to provide acidified CMC (CMC-H) and mixed for about 30 minutes. The Monosphere was carefully removed by filtration and tetrabutylammonium hydroxide (TBAH) 40% (aqueous solution) was added until the pH was 8 to 9. The resulting solution was stirred for 30 minutes and then concentrated in vacuo. The dried material was dissolved in dry DMF (150 mL) under nitrogen. Stirring overnight was required and the resulting solution was very viscous and had a clear / yellow color. Further dry DMF (100 mL) was added to reduce the viscosity, the mixture was cooled to about 4°C and stirred, and then 2-chloro-N-methylpyridinium iodide (CMP-I) (1.4875 g, 5.8 mmol) was added. Shortly thereafter, compound 5-b (1.3567 g, 9.17 mmol) was added along with dry triethylamine (5 mL). The reaction was maintained at 4°C and stirred overnight. The solid was filtered and then washed with acetone (3×100 mL) followed by DMF (3×100 mL), with ultrasonic treatment during the washing steps to promote dispersion of the fibers in the washing solution. The solid was further dried in vacuo to obtain polymer 13 as off-white fluffy / powdery fibers. Weight = 1.8365 g (yield 60%). Kaiser test was used to confirm the presence of terminal amines (reading at 570 nm equal to 3.08 μmol amine). SS NMR: δ (10,000 Hz, CP MAS) 176.9 (C=O) 153.4, 142.4, 104.1 (C1), 96.8 (C4), 83.6 (C3), 74.2 (C7, C2, C5), 69.4 (C6), 61.9 (C6, shoulder). FTIR: 3325 (NH / OH), 2872 (CH), 1648 (C=O, amide of coupling product), 1589 (C=O of CMC), 1543, 1408 / 1367 / 1265 / 1022. Elemental analysis: If the DoS of the raw material is 0.7, then the expected product: mass 318 g mol-1: C 45%, H 7%, N 6%. Actual: C 42.5%, H 7%, N 3%. Thus, of all monomers, approximately 35% contained a linker group.

[0445] The solubility of polymer 13 was determined in a similar manner as compound 1-c. The data are shown below: x represents insoluble material.

[0446] Solvents initial After heating After ultrasonic treatment overnight pH 9 buffer x Not tested Not tested After mixing, hydrogel is formed

[0447] Example 14: Preparation of polymer 14

[0448]

[0449] Under nitrogen atmosphere, polymer 13 (0.250 g, 1.396 mmol) was placed in a RB flask. Under nitrogen atmosphere, compound 14-a (90 mg, 0.679 mmol) was dissolved in dry DMF (5 mL) and added to polymer 13 with stirring. The reaction mixture was stirred at room temperature under nitrogen atmosphere for 90 minutes, then filtered and washed with DMF (5×5 mL) and methanol (5×5 mL). The product was concentrated in vacuo to give polymer 14 as an off-white powdery solid. Weight = 0.1852 g (yield 52%). Kaiser test was used to confirm the presence of terminal amine (reading at 570 nm equal to 1.86 μmol amine). FTIR: 3251 (NH / OH), 2915 / 2874 (CH), 1649 (C=O, amide of coupled product), 1583 (C=O of CMC), 1405 / 1316 / 1262 / 1020. Elemental analysis: If the DoS of the raw material is 0.7, the expected product: mass 519 g mol- 1 : C 48%, H 5.8%, N 5.6%. Actual: C 42.8%, H 6.95%, N 4.25%. Therefore, for all monomers, about 53% contain a linker group.

[0450] The solubility of polymer 14 was determined in a similar manner as compound 1-c. The data are shown below: x represents insoluble material.

[0451]

[0452] Example 15: Preparation of polymer 15

[0453]

[0454] Under nitrogen, polymer 5-a (prepared using 2.0g NaCMC as described in Example 5) was dissolved in dry DMF (150mL). In addition to multiple ultrasonic treatment cycles for 1 minute, it is necessary to stir and heat to about 50°C over a period of about 2 hours to provide a clear solution. The solution is cooled to about 4°C and stirred, and then 2-chloro-N-methylpyridinium iodide (CMP-I) (1.4875g, 5.8mmol) is added. The solution becomes a sticky "jelly" and mixes vigorously, and 20mL of dry DMF is added to decompose and dilute the gel. Shortly thereafter, 4,7,10-trioxa-1,13-tridecanediamine (12.02g, 2.02mL, 9.17mmol) and dry triethylamine (5mL) are added. The reaction is maintained at 4°C and stirred overnight. Acetone (100mL) is cooled to 4°C and the reaction mixture is slowly added dropwise under stirring. The mixture is filtered with 20mL aliquots to obtain a clear white gel-like solid. The solid was washed in acetone (3 x 100 mL), ethanol (3 x 100 mL), water (1 x 100 mL), hexane (1 x 100 mL), and then washed again with water (1 x 100 mL), followed by concentration for about 15 minutes. The resulting product was lyophilized over a 3 day period to provide polymer 5-a as a light colored fluffy solid. Weight = 1.794 g (yield 49%). Kaiser test was used to confirm the presence of terminal amine (reading at 570 nm equal to 1.86 μmol amine). 13 C NMR (101 MHz, none) δ 176.51 (C8, C=O of CMC), 171.09 (C8, C=O of amide), 163.79 (new peak), 153.15, 143.12, 103.40 (C1), 82.39 (C7), 75.81 / 73.99 / 70.07 (C2, C3, C4, C5), 60.81 (C6), 42.54 / 36.84 (C11, C12, C13, C14, C15, C16), 31.45 (C9, C14), 28.25. FTIR: Peaks at 3239 (NH / OH), 2865 (CH), 1650 (C=O, amide of coupled product), 1586 (C=O of CMC), 1404 / 1388 / 1315 / 1256 / 1053. Elemental analysis: If the DoS of the starting material is 0.7, the expected product: mass 369 g mol-1: C 47.5%, H 7%, N 4%. Actual: C 45.5%, H 7.4%, N 5.1%. Therefore, of all monomers, about 87.5% contain linker groups.

[0455] Example 16: Preparation of polymer 16

[0456]

[0457] Polymer 15 (1.000 g, 2.273 mmol) was ground into a fine powder and mixed with DMF (15 mL) for 20 minutes. At room temperature, compound 8-a (1.14 g, 2.639 mmol), HBTU (2.59 g, 6.82 mmol) and DIPEA (1.19 mL, 6.82 mmol) were added under stirring and air was passed through (centrifuge tubes and laboratory rotors were used to promote mixing overnight). The mixture was sonicated for 30 seconds and then allowed to stir overnight. The solid was filtered and washed with DCM (5×20 mL) and methanol (5×20 mL). The resulting coupling intermediate was dried under vacuum. The intermediate (850 mg) was added to a solution of piperidine:DMF (20:80 v / v) (20 mL) and nitrogen was bubbled to stir the mixture for 1 hour, then the solid was filtered and washed with ethanol (3×50 mL) and DCM (3×50 mL). The process was then repeated and the amine intermediate was dried under vacuum. The amine intermediate was added to a RB flask and purged with nitrogen. TCEP (428 mg) was dissolved in deionized water (2.6 mL) and added to the amine intermediate under stirring, followed by methanol (5.2 mL). The system was kept under stirring for 1 hour at room temperature under nitrogen, then filtered and washed with the following solution: 2:1 methanol: water (90 mL), 1:2 methanol: water (90 mL), 100% water (90 mL), 100% methanol (90 mL). The resulting solid was vacuum dried to provide polymer 16, which was an off-white powdery solid. Weight = 0.3537 g (yield 30%). The yellow color of the Ellman test indicates the presence of free thiol. FTIR: 3349 (NH / OH), 2917 / 2872 (CH), 1716 (acetone), 1650 (C=O, amide of coupling product), 1593 (C=O of CMC), 1539, 1438 / 1313 / 1255 / 1028. δC (10,000 Hz, CP MAS): 171.8 (C8, C19), 156.5 (residue C=O of Fmoc), 142.3 (residue CHAr of Fmoc), 127.9 (residue CHAr of Fmoc), 120.08, 103.3 (C1), 82.6 (C7), 74.5 (C2, C3, C4, C5), 69.9 (x), 61.5 (C6), 49.5 (C20), 36.37 (C9, C18, C21), 29.52 (C10-18). Elemental analysis: If the DoS of the raw material is 0.7, the expected product: mass 440.7 gmol- 1 : C 45%, H 7%, N 5.4%, S 4.1%. Actual: C 47.1%, H 6.74%, N 4.11%, S 0.80%. Therefore, for all monomers, about 20% contain linker groups.

[0458] Solubility evaluation showed that polymer 16 was 2 HPO 4 / MgCl 2 ) to form a hydrogel.

[0459] Example 17: Preparation of polymer 17

[0460]

[0461] Initially, polymer 16 was treated with TCEP to decouple any disulfide bonds that had formed. TCEP (0.1685 g, 0.196 mmol, 4 eq) was dissolved in 1 mL of water and added to polymer 16 (0.0754 g, 0.147 mmol) and stirred at room temperature for 30 minutes. The solid was filtered, washed with water (3 x 10 mL), and freeze-dried to give a solid off-white / cream powder, which was then washed with DMF (3 x 10 mL).

[0462] Compound 12-a (0.0250 g, 0.049 mmol, 1 equivalent) was dissolved in 2 mL of dry DMF and added with N 2(g) purged polymer 16. The reaction was stirred overnight at room temperature, covered with foil to avoid light. After stirring overnight, the reaction did not change color. The off-white solid powder was filtered and washed as follows, each step was repeated 5 times: (1) adding deionized water (5 mL), (2) gently heating under stirring, (3) 1 minute ultrasonic treatment was repeated 3 times, (4) filtering through a filter column and a glass filter, and (5) further rinsing with deionized water (20 mL). The resulting solid was filtered and freeze-dried for 3 hours to provide polymer 17, which is an off-white product. Weight = 0.063 g (yield 44%). FTIR: 3315 (NH / OH), 2911 / 2869 (CH), 1922, 1756 (C=O of ester / fluorescent C=O), 1720 (C=O of ester / fluorescent C=O), 1647 (C=O, amide of coupling product), 1591 (C=O of CMC / aromatic C=C stretching), 1420 (aromatic C=C stretching), 1366 (CO stretching of ester), 1248 (CO stretching), 1050 (CO / -OH of ester), 894 (aromatic out-of-plane CH bending). δC (10,000 Hz, CP MAS): 171.0 (C8, C19, C40), 153.1 (C22, C37), 141.7 (C-CAr), 127.0 (C-CAr), 102.4 (C1), 81.9 (C7, C33), 74.5 (C2, C3, C4, C5), 68.6 (C23, C34), 61.5 (C6), 46.7 (C20), 36.5 (C9, C18), 32.1 (C21), 29.5 (C10-17). Elemental analysis: If the DoS of the raw material is 0.7, then the expected product: mass 797.7 g mol- 1 : C 52%, H 5%, N 3.5%, S 2.1%. Actual: C 42.8%, H 6.95%, N 4.02%, S 0.52%. Therefore, for all monomers, about 25% contain linker groups.

[0463] Enzyme efficacy of polymer 17

[0464] Enzyme efficacy was tested with esterase (approximately 58 units / mL achieved by mixing neat esterase (0.01 mL) in PBS (0.99 mL).

[0465] In order to quantify and accurately determine the enzyme efficacy, the experiment was set up using a 96-well plate and a disk analyzer (Fluostar Optima BMGLabtech, excitation set to 485nm, emission set to 590nm, and gain set to 1500). Preparation of samples: 0.4mg of polymer 17 was soaked in 640μL PBS for 24 hours. After soaking, the PBS solution did not change and remained clear. The mixture was centrifuged to remove most of the solids, at which time the sample of polymer 17 appeared to have become a hydrogel. The eluate was then filtered and analyzed; the solid hydrogel was set aside for additional experiments. Before each aliquot, 40μL of the eluate was dispensed into the well and mixed thoroughly. The measured value was taken as a baseline reading. The following solutions were prepared: 58 units / mL of esterase PBS solution (weak enzyme solution), 116 units / mL of esterase PBS solution (strong enzyme solution).

[0466] N = 4 wells had 40 μL of PBS only added to the test dispersion (control).

[0467] N = 4 wells had 40 μL of weak enzyme solution pipetted into the test dispersion.

[0468] N = 4 wells had 40 μL of strong enzyme solution pipetted into the test dispersion.

[0469] Another fluorescence reading was recorded immediately after the final well addition and then every 60 seconds for 160 minutes. Figure 3 The fluorescence readings for each time point are shown minus the baseline readings (particle suspension only). The instrument did not reach a saturation point during this experiment as it had previously. As expected, the fluorescence of the weak and strong esterase solutions increased over time.

[0470] The fluorescence of the negative control sample (sample containing only PBS and no enzyme) increased slightly over time.

[0471] Further, for the above experiments, additional control experiments were performed using the solid filtrate. The solid was added to 640 μL of PBS, vortexed and sonicated until a good dispersion was observed. The solution was analyzed using the same kinetic method as above (results are shown in Figure 4 ). Overall, this evidence indicates that the synthetic method was successful and that the enzymatic cleavage step produced a positive response that could be quantified by fluorescence spectroscopy. In the absence of esterase, polymer 17 did not fluoresce as well as polymer 12, indicating that less fluorescent label was attached to the polymer.

[0472] Example 18: Preparation of polymer 18

[0473]

[0474] First, polymer 8 was treated with TCEP to decouple any disulfide bonds that had formed. TCEP (0.0243 g, 0.0848 mmol, 4 eq) was dissolved in 1 mL of water and added to polymer 8 (0.01 g, 0.0212 mmol) and stirred at room temperature for 30 minutes. The solid was filtered, washed with water (3 x 10 mL), and freeze-dried to give a solid off-white / cream powder, which was then washed with DMF (3 x 10 mL).

[0475] Methoxypolyethylene glycol maleimide (Compound 18-a) (0.01 g, 0.0543 mmol, 2.5 equivalents) was dissolved in 2 mL of dry DMF and added with N 2 (g) purged polymer 8. The reaction was stirred overnight at room temperature, covered with foil to avoid light. After stirring overnight, the reaction mixture turned red and no solid phase was found when filtered. The filter was rinsed with DMF (5 mL), the solutions were combined and concentrated in vacuo to give a clear / yellow oil (weight = 0.0106 g). Chilled deionized water (2 mL) was slowly dripped into the oil at 4°C and then freeze-dried to give polymer 18 as an off-white / pink waxy solid. Weight = 6.42 mg. Non-quantitative Kaiser test indicated the absence of free amine. FTIR: about 3350 (small peak, NH / OH), 2881 (CH), 2740 (CH), 1964, 1710 (5-membered cyclic ketone), 1665 (C=O, amide), 1359 (CO ester stretching), 1240 (CO stretching), 1145 / 1100 (CO / -OH of ester), 842 (aromatic out-of-plane CH bending).

[0476] Solubility evaluation showed easy solubility in chloroform, PBS and DMF.

[0477] Example 19: Preparation of polymer 19

[0478]

[0479] Polymer 5 (300 mg, 0.81 mmol) was ground into a fine powder and mixed with DMF (20 mL) for 20 minutes. Fmoc-Ala-OH (1.00 g, 2.31 mmol), HBTU (1.84 g, 4.86 mmol), EDC (100 mg, 0.52 mmol) and DIPEA (6.2 mL, 4.86 mmol) were added to a large centrifuge tube and purged with nitrogen. A laboratory rotor was used to facilitate mixing overnight. The solid was filtered and washed with DCM (5×20 mL) and methanol (5×20 mL). The resulting product was dried to provide polymer 19-a as an off-white powder. Weight = 0.2932 g. A positive Kaiser test indicated incomplete coupling. FTIR: 3300 (NH / OH), 2850 (CH), 1748 (amide), 1648 (C=O, amide of coupling product), 1589 (C=O of CMC), 1403 / 1022,896.

[0480] Polymer 19-a (207 mg) was placed in a solution of piperidine:DMF (20:80 v / v) (20 mL) and purged with nitrogen and stirred for 1 hour, after which the solid was filtered and washed with ethanol (3×50 mL) and DCM (3×50 mL). The process was then repeated, and the product was dried to provide polymer 19 as an off-white powder. Weight = 0.345 g (yield 60%). A positive Kaiser test result confirmed the presence of free amine. FTIR: 3300 (NH / OH), 2850 (CH), 1748 (amide), 1648 (C=O, amide of coupled product), 1589 (C=O of CMC), 1403 / 1022, 896.

[0481] Example 20: Preparation of polymer 20

[0482]

[0483] Polymer 19 (200 mg, 0.470 mmol) was ground into a fine powder and mixed with DMF (20 mL) for 20 minutes. Fmoc-Ala-OH (0.439 g, 1.41 mmol), HBTU (0.539 g, 1.41 mmol), EDC (270 mg, 1.41 mmol) and DIPEA (0.018 mL, 1.41 mmol) were added to a large centrifuge tube and purged with nitrogen. A laboratory rotor was used to facilitate mixing overnight. The solid was filtered and washed with DCM (5×20 mL) and methanol (5×20 mL). The resulting product was dried to provide polymer 20-a as an off-white powder. Weight = 0.1576 g. A positive Kaiser test indicated incomplete coupling. FTIR: peaks at 3300 (NH / OH), 2900 / 2850 (CH), 1748 (amide), 1648 (C=O, amide of coupling product), 1584 (C=O of CMC), 1542(), 1445 / 1403 / 1022, 899.

[0484] Polymer 20-a (158 mg) was placed in a solution of piperidine:DMF (20:80 v / v) (20 mL) and purged with nitrogen and stirred for 1 hour, after which the solid was filtered and washed with ethanol (3×50 mL) and DCM (3×50 mL). The process was then repeated and the product was dried to provide polymer 20 as an off-white powder. Weight = 0.1391 g (yield 58%). A positive Kaiser test result confirmed the presence of free amine. FTIR: peaks at 3300 (NH / OH), 2900 / 2850 (CH), 1748 (amide), 1648 (C═O, amide of coupled product), 1584 (C═O of CMC), 1403 / 1025, 900.

[0485] Example 21: Preparation of polymer 21

[0486]

[0487] Polymer 20 (150 mg, 0.2785 mmol) was ground into a fine powder and mixed with DMF (20 mL) for 20 minutes. Fmoc-Pro-OH (0.282 g, 0.836 mmol), HBTU (0.317 g, 0.8356 mmol), EDC (160 mg, 0.836 mmol) and DIPEA (0.20 mL, 0.836 mmol) were added to a filter column and sealed. A laboratory rotor was used to facilitate mixing overnight. The solid was filtered and washed with DCM (5×20 mL) and methanol (5×20 mL). The resulting product was dried to provide polymer 21-a, which was an off-white powder. Weight = 0.10967 g. A positive Kaiser test result indicated the presence of unreacted amine. FTIR: peaks at 3300 (NH / OH), 2850 (CH), 1748 (amide), 1648 (C=O, amide of coupling product), 1584 (C=O of CMC), 1403 / 1025.

[0488] Polymer 21-a (100 mg) was placed in a solution of piperidine:DMF (20:80 v / v) (20 mL) and stirred for 1 hour, then the solid was filtered and washed with ethanol (3×50 mL) and DCM (3×50 mL). The process was then repeated, and the product was dried to provide polymer 21 as an off-white powder. Weight = 0.08020 g (yield 47%). Positive Kaiser test results indicated the presence of free amine. FTIR: peaks at 3300 (NH / OH), 2850 (CH), 1748 (amide), 1648 (C=O, amide of coupled product), 1584 (C=O of CMC), 1453 / 1403 / 1024, 962 / 906 / 841.

[0489] Example 22: Preparation of polymer 22

[0490]

[0491] Polymer 21 (100 mg, 0.165 mmol) was ground into a fine powder and mixed with DMF (10 mL) for 20 minutes. Fmoc-Val-OH (0.167 g, 0.494 mmol), HBTU (0.187 g, 0.494 mmol), EDC (95 mg, 0.494 mmol) and DIPEA (0.064 mL, 0.494 mmol) were added to a filter column and sealed. A laboratory rotor was used to facilitate mixing overnight. The solid was filtered and washed with DCM (5×20 mL) and methanol (5×20 mL). The resulting product was dried to provide polymer 22-a, which was an off-white powder. Weight = 0.0603 g. A positive Kaiser test result indicated the presence of unreacted amine.

[0492] Polymer 22-a (60 mg) was placed in a solution of piperidine:DMF (20:80 v / v) (20 mL) and stirred for 1 hour, then the solid was filtered and washed with ethanol (3×50 mL) and DCM (3×50 mL). The process was then repeated and the product was dried to provide polymer 22 as an off-white powder. Weight = 0.0553 g (yield 48%). A positive Kaiser test result indicated the presence of free amine. FTIR: peaks at 3300 (NH / OH), 2850 (CH), 1748 (amide), 1648 (C═O, amide of coupled product), 1585 (C═O of CMC), 1453 / 1403 / 1024 / 1095 / 1058, 961, 841.

[0493] Example 23: Preparation of polymer 23

[0494]

[0495] Polymer 22 (50 mg, 0.707 mmol) was ground into a fine powder and mixed with DMF (10 mL) for 20 minutes. Fmoc-Cys(StBu)-OH (716 mg, 2.122 mmol), HBTU (805 mg, 2.122 mmol), EDC (407 mg, 2.122 mmol) and DIPEA (0.274 mL, 2.122 mmol) were added to a filter column and sealed. A laboratory rotor was used to facilitate mixing overnight. The solid was filtered and washed with DCM (5×20 mL) and methanol (5×20 mL). The resulting product was dried to provide polymer 23-a as an off-white powder. Weight = 0.0253 g. A positive Kaiser test result indicated the presence of unreacted amine. FTIR: 3300 (NH / OH), 2850 (CH), 1748 (amide), 1648 (C=O, amide of coupling product), 1585 (C=O of CMC), 1453 / 1403 / 1024 / 1095 / 1058, 961, 841.

[0496] Polymer 23-a (25 mg) was placed in a solution of TCEP in water (5 mL) and stirred at room temperature for 2 hours, then the solid was filtered and washed with ethanol (3×50 mL) and DCM (3×50 mL). The process was then repeated and the product was dried to provide polymer 23 as an off-white powder. A positive Kaiser test result indicated the presence of free amine. The material was used for Example 24 without further characterization.

[0497] Example 24: Preparation of polymer 24

[0498]

[0499] TCEP (0.0357 g, 0.125 mmol, 4 eq) was dissolved in 1 mL of water and added to polymer 23 (25.3 mg, 0.0312 mmol), and the mixture was stirred at room temperature for 30 minutes. The solid was filtered, washed with water (10 mL x 3) and freeze-dried to give a solid off-white / cream powder, which was then washed with DMF (10 mL x 3).

[0500] Fluorescein diacetate-5-maleimide (Compound 12-a) (0.016 g, 0.0312 mmol, 1 equivalent) was dissolved in 3 mL of dry DMF and added to the mixture that had been treated with N 2 (g) Purged pretreated polymer 23. The reaction was stirred at room temperature overnight, covered with foil to protect from light. A laboratory rotor was used to facilitate mixing overnight. The solid was filtered and washed with DCM (5×20 mL), water (5×20 mL), and methanol (5×20 mL). The resulting product was dried to provide polymer 24 as an off-white powder. Weight = 0.0156 g (yield 32%). FTIR: (ν max / cm -1 )3286 (NH / OH), 2872 (CH), 1722 (acetone), 1647 (C=O, amide of coupling product), 1589 (C=O of CMC), 1545 (CC aromatic fluorescein), 1409 / 1317 / 1250 / 1024 / 894. 13 C CP MAS NMR: CMC 0.7DoS (starting material): δC (13,000 Hz, CP CPMAS) 61.7 (C6), 74.5 (C7, C2, C5), 82.5 (shoulder, C3), 97.0 (C4), 103.3 (C1), 177 (C=O); CMC-PEG diamine: δC (13,000 Hz, CPMAS) 13.4 (C14), 20.1 (unassigned), 23.4 (unassigned), 30.6 (C9), 61.7 (C6), 70 (shoulder, C7), 74.5 (C2, C5), 82.5 (C3), 95 (C4), 103.3 (C1), 113 (unassigned), 142 (unassigned), 152 (unassigned), 169.7 (C=O of linker), 177.1 (C=O of CMC).

[0501] Example 25: Preparation of polymer 25 (fiber)

[0502]

[0503] Polymer 13 was mixed with DMF (30 mL) for 20 minutes. Under stirring at room temperature, Fmoc-C(StBu)-OH (1.5 g, 3.5 mmol), HBTU (3.09 g, 8.14 mmol) and DIPEA (1.05 mL, 8.14 mmol) were added, and air was introduced (a centrifuge tube and a laboratory rotor were used to facilitate mixing overnight). After rotating overnight, the solid was filtered and washed with DCM (5 × 50 mL), methanol (5 × 50 mL), and again with DCM (5 × 50 mL). The resulting product was dried to afford Polymer 25, which was used directly in the next example (Example 26).

[0504] Example 26: Preparation of Polymer 26

[0505]

[0506] Polymer 26 was prepared using a similar procedure as described in Example 12. Appearance: off-white, powder / solid. Weight = 1.258 g (yield 59%). FTIR: 3339 (N-H / O-H), 2872 (C-H), 1736 (acetone), 1648 (C=O, amide of the coupling product), 1549 (C=O of CMC), 1419, 1363, 1313, 1201. δC (10,000 Hz, CP MAS): 30 (C9, 19), 39 (C10, 11, 12, 13), 47 (C18, 22), 54 (C16), 60 (C6), 70 - 74 (broad peak, C2, 3, 5, 7), 83 (shoulder peak, C4), 92 (C17), 97, 104 (C1), 120 (C24), 128 (C25, 26, 27), 142 (C28), 156 (small peak, C20, C=O carbamate), 171 (carbonyl of C8 amide). Elemental analysis: If the DoS of the starting material is 1 and is completely converted to the linker group, the expected product: mass 781 g mol- 1 : C 55%, H 7%, N 5%, S 8%. Actual: C 46%, H 7%, N 2%, S (not determined). Based on S and calculating the %N present / % possible N% (2 / 3.5) = 57% of the linker group coupling. Based on S and calculating the %N present / % possible N% (2 / 3.5) = 57% of the linker group coupling. Thus, for all monomers, approximately 40% contain the linker group.

[0507] Example 27: Preparation of Polymer 27

[0508]

[0509] Prepare a solution of aminophenylfluorescein (5 mg, 0.018 mmol) and HBTU (14 mg, 0.036 mmol) in dry DMF (2.0 mL). 2 (5-a) (0.13 mg, 0.036 mmol) was added to the reaction mixture and the mixture was stirred at room temperature for about 24 hours with a foil cover to protect from light. The product was filtered, washed with ethanol (10 mL×3) and DMF (5×10 mL), and then concentrated in vacuo to give a solid red / orange product (27) (12.0 mg, 43%). FTIR: (νmax / cm-1) 3367 (NH / OH), 2932 (CH), 1702 (amide of APF), 1655 (CONH, amide of coupling product), 1555 (HNCO, CMC), 1494, 1437, 1413, 1387 (aromatic C=C bending), 1106 (OCH R , alkoxy APF), 838, 759, 722, 557 (aromatic CH bending).

[0510] Example 28: Preparation of polymer 28

[0511]

[0512] CMC-Cys (7) (10 mg, 0.029 mmol, 1 eq) was stirred with TCEP (16.8 mg, 0.058 mmol, 4 eq) in deionized water (1 mL) at room temperature for 1 hour to decouple any unwanted disulfide bonds. The solid was filtered, washed with water (10 mL×3), freeze-dried, and then washed with DMF (10 mL×3). Diacetate fluorescein-5-maleimide (12-a) (11 mg, 0.029 mmol, 1 eq) was dissolved in dry DMF (3 mL) and added to CMC-CYS under a nitrogen atmosphere. The reaction was stirred at room temperature overnight and protected from light. The product was washed with water (3×100 mL) and DMF (1×100 mL) to give an opaque waxy solid (28) (0.017 g, 85%). FTIR: (νmax / cm- 1 )3365 (OH), 2971 / 2910 (NH, CH), 2883 (SH), 1728 (COOH, acid), 1677 (CONH, amide), 1640 (HNCO, peptide), 1598 (HNCOO, CMC), 1409, 1371 (COO), 1307 (aromatic C=C bend), 1216, 1019 (CN, tertiary amine); Solubility: insoluble in water, pH 9 phosphate buffer and common laboratory solvents (acetone, methanol, ethanol, THF, DCM and DMF).

[0513] Example 29: Preparation of polymer 29

[0514]

[0515] CMC-PEG-NH 2 (15) (1.000 g, 2.27 mmol) was ground into a fine powder and mixed with DMF (15 mL) for 20 minutes. Fmoc-C(StBu)-OH (1.14 g, 2.639 mmol, 1.2 equiv), HBTU (2.59 g, 6.8181 mmol) and DIPEA (1.19 mL, 6.8181 mmol) were added under stirring at room temperature overnight. The product was filtered, washed with DCM (5×20 mL) and methanol (5×20 mL), and then concentrated in vacuo. The protected product was then subjected to StBu deprotection, and each deprotection step was repeated 3 times, followed by vacuum concentration after each repetition to obtain a white solid powder (29-a) (0.236 g, 5%). Solid 13 C NMR (10,000 MHz, CP MAS) 176.52 (C-8 of CMC part), 171.84 (C-8, C-19), 156.45, 142.32, 127.85-120.08 (C-Ar, Fmoc), 113.93, 103.26 (C-1), 82.60 (C-4), 74.50 (C-2, C-3, C-5), 69.92 (C-7) , 61.45(C-6), 54.93(C-20), 49.52, 47.30, 41.58, 38.72-23.08(C-9, C-10, C-11, C-12, C-13, C-14, C-15, C-16, C-17, C-18, C-20); FTIR (νmax / cm-1) 3310 (OH), 2915 (NH, CH), 2868 (SH), 1731 (COOH, acid), 1650 (CONH, amide of coupling product), 1592 (HNCOO, CMC), 1538 (HNCOO, peptide), 1441, 1417, 1361 (COO), 1310 (aromatic C=C bending), 1252 (amide CO stretching), 1031 (CN, tertiary amine), 841; expected values ​​of elemental analysis of deprotected product: C: 45.3%, H: 7.5%, N: 4.7%, S: 7.1%; found values ​​C: 47.1%, H: 7.8%, N: 6.7%, S: 10.1%, so the coupling yield is 11%; solubility: insoluble in water and common laboratory solvents (acetone, methanol, ethanol, THF, DCM and DMF), soluble in pH 9 phosphate buffer over time; Ellmans test is positive.

[0516] The intermediate (29-a) (75.4 mg, 0.147 mmol, 3 eq) was stirred with TCEP (168.5 mg, 0.196 mmol, 4 eq) in deionized water (1 mL) at room temperature for 1 hour to decouple any unwanted disulfide bonds. The solid was filtered, washed with water (10 mL×3), freeze-dried, and then washed with DMF (10 mL×3). Diacetate fluorescein-5-maleimide (12-a) (25.0 mg, 0.049 mmol, 1 eq) was dissolved in dry DMF (2 mL) and added to CMC-PEG-NH-CYS (29-a) under a nitrogen atmosphere. The reaction was stirred at room temperature overnight and protected from light. The reaction mixture was concentrated in vacuo, the product was washed five times with water (approximately 75 mL) and freeze-dried to give an off-white powder (29) (63.0 mg, 44%). Solid 13 C NMR (10,000 MHz, CP MAS) 176.38 (C-8 of CMC part), 171.00 (C-8, C-19, C-38), 153.12 (C-22), 141.67, 135.49-119.80 (C-Ar, Fmoc, fluorescein), 102.43 (C-1), 81.92 (C-4), 74.47 (C-2, C-3, C-5), 70.30 (C-7), 68.61, 61.45 (C-6), 46.73 (C-42), 36.47-29.48 (C-9, C-10, C-11, C-12, C-13, C-14, C-15, C-16, C-17, C-18, C-20); FTIR (νmax / cm- 1 )3315 (OH), 2911 (NH, CH), 2869 (SH), 1922, 1720 (COOR, conjugated ester, fluorescein), 1647 (CONH, amide of coupling product), 1591 (HNCOO, CMC), 1542 (CONH, peptide), 1420, 1366 / 1309 (aromatic C=C bending), 1248 (CO stretching), 1213, 1050 (CN, tertiary amine), 842, 581 (aromatic CH bending); expected values ​​of elemental analysis: C: 51.1%, H: 5.8%, N: 3.7%, S: 2.1%; measured values: C: 42.8%, H: 7.0%, N: 4.0%, S: 0.5%, so the coupling yield is 24%.

[0517] Example 30: Preparation of polymer 30 (fiber)

[0518]

[0519] NaCMC fibers (2.0 g, 8.33 mmol) of DoS 0.3 were dispersed in a solution of ethanol: deionized water (80:20 v / v) (180 mL) to obtain a 1% suspension. Dowex 650C Monosphere ion exchange resin was added under stirring for 30 minutes. The Monosphere was removed by filtration, and then tetrabutylammonium hydroxide (TBAH) 40% (aqueous solution) was added in 0.1 mL aliquots until the pH was 8 to 9 (3.0 mL, 1.16 mmol). The resulting solution was stirred for 30 minutes and then concentrated in vacuo. The film-like material was dissolved in dry DMF (100 mL) under nitrogen atmosphere and stirring, and gentle heating was required overnight to obtain a viscous gel-like solution with a uniform texture. The solution was cooled to about 4°C and CMP-1 (1.48 g, 5.8 mmol) was added under vigorous stirring. 2,2'-(Ethylenedioxy)bis(ethylamine) (1.36 g, 9.17 mmol) was added to the reaction along with dry triethylamine (3 mL). The reaction was maintained at 4 °C and stirred for at least 3 hours, then the solid was filtered and washed with 99% acetone (3×100 mL) and DCM (3×100 mL), with sonication for 1 minute during each wash cycle to help loosen the fiber clumps and release any contaminants. The product was concentrated in vacuo to give a white fibrous solid (30-a) (1.8 g, 60%). Solid 13 C NMR (10,000 MHz, CP MAS) 176.93 (C-8 of CMC part), 171.96 (C-8), 153.41, 142.40, 104.11 (C-1), 96.80, 83.55 (C-4), 74.18 (C-2, C-3, C-5), 69.39 (C-7), 61.89 (C-6), 40.48 (broad peak, C-10, C-11, C-12, C-13, C-14); FTIR (νmax / cm- 1 )3325 (OH), 2872 (NH, CH), 1648 (CONH, amide of coupling product), 1589 (HNCOO, CMC), 1543, 1408, 1367 (CHO), 1314, 1265 (amide CO stretching), 1022 (CN, tertiary amine), 896; expected value of elemental analysis: C: 41.7%, H: 7.0%, N: 2.3%; found value: C: 42.6%, H: 6.3%, N: 2.9%, so the coupling yield is 127%; solubility: insoluble in water and common laboratory solvents (acetone, methanol, ethanol, THF, DCM and DMF), soluble in pH 9 phosphate buffer and PBS over time; Kaiser test positive, 3.08 μmol amine.

[0520] CMC-PEG-NH 2 (30-a) (1.0 g, 2.86 mmol) was mixed with DMF (30 mL) for 20 min. Fmoc-C(StBu)-OH (1.5 g, 3.5 mmol), HBTU (3.09 g, 8.14 mmol) and DIPEA (1.10 mL, 8.57 mmol) were added under stirring at room temperature overnight. The product was filtered and washed with DCM (5×50 mL), methanol (5×50 mL), washed again with DCM (5×50 mL), and then concentrated in vacuo to give a white solid powder (30-b) (1.23 g, 59%). Note: The protected product was not subjected to Fmoc or StBu deprotection at this stage. Solid 13 C NMR (10,000 MHz, CP MAS) 176.38 (small peak, C-8 of CMC part), 171.93 (C-8, C-15), 156.28 (C-20), 141.73, 127.61-120.01 (C-Ar, Fmoc), 104.17 (C-1), 96.81, 87.27, 82.86 (C-4), 74.29 (C-2, C-3, C-5), 69.17 (C-7), 62.17 (C-6), 60.00 (C-21), 54.22 (C-16), 46.87 (C-22), 46.87, 39.36-29.49 (C-10, C-11, C-12, C-13, C-14, C-17, C-19); FTIR (νmax / cm- 1 )3339 (OH), 2872 (NH, CH), 1736 (COOH, acid), 1648 (CONH, amide of coupling product), 1549 (HNCOO, CMC), 1419, 1363 (COO), 1201, 1031 (CN, tertiary amine), 841; expected values ​​of elemental analysis: C: 44.6%, H: 6.7%, N: 1.6%, S: 2.5%; found values: C: 46.0%, H: 6.5%, N: 2.2%, S: not enough material; solubility: insoluble in water and common laboratory solvents (acetone, methanol, ethanol, THF, DCM and DMF), soluble in pH 9 phosphate buffer over time; Ellmans test was positive.

[0521] CMC-PEG-Cys(Fmoc)StBu (30-b) (0.5965 g, 1.265 mmol) in fiber form was stirred with TCEP (1.45 g, 5.060 mmol, 4 eq) in deionized water (3 mL) for 30 minutes at room temperature to decouple any unwanted disulfide bonds. The solid was filtered, washed with water (10 mL×3), freeze-dried, and then washed with DMF (10 mL×3). Fluorescein diacetate-5-maleimide (12-a) (0.125 g, 0.240 mmol, 1 eq) was dissolved in dry DMF (15 mL) and added to CMC-PEG-NH-CYS under a nitrogen atmosphere. The reaction was stirred overnight at room temperature and protected from light. The reaction mixture was concentrated in vacuo, and the product was washed with DCM (3×50 mL), methanol (3×50 mL) and concentrated in vacuo. The off-white solid fiber was then washed with water (5 x 75 mL) with gentle heating, stirring and sonication, then filtered and lyophilized to give off-white solid fiber (30) (1.10 g, 72%). 13 C NMR (10,000 MHz, CP MAS) 174.88 (C-8 of CMC part), 172.30 (C-8, C-15, C-34), 141.36, 129.34-126.65 (C-Ar, Fmoc, fluorescein), 104.58 (C-1), 86.94, 82.52 (C-4), 78.56-72.96 (C-2, C-3, C-5, C-7), 69.74, 61.45, 60.35 (C-6), 38.69-21.02 (C-9, C-10, C-11, C-12, C-13, C-14); FTIR (νmax / cm- 1 )3315 (OH), 2868 (NH, CH), 1728 (COOR, conjugated ester, fluorescein), 1647 (CONH, amide of coupled product), 1542 (CONH, amide of peptide coupling, NHCOO CMC), 1419, 1364 (aromatic C=C bending), 1264 (CO stretching), 1152 (OCH R ), 1018 (CN tertiary amine), 879; expected values ​​of elemental analysis: C: 50.9%, H: 6.9%, N: 5.5%, S: 1.0%; measured values: C: 30.8%, H: 8.6%, N: 1.3%, S: <0.3%, so the coupling yield is 24%.

[0522] Example 31: Preparation of polymer 31

[0523]

[0524] CMC-PEG-NH-AAPVC(Fmoc)StBu(23-a) (525 mg, 439 mmol) in powder form was stirred with TCEP (503.4 mg, 0.1756 mmol, 4 eq) in deionized water (23 mL) at room temperature for 30 minutes to decouple any unwanted disulfide bonds. The solid was filtered and washed with water (50 mL×3), methanol (50 mL×3), ethanol (50 mL×3), and then washed with water (50 mL×3), then freeze-dried and washed with DMF (50 mL×3). Fluorescein-5-maleimide (25 mg, 59 mmol, 0.1 eq) was dissolved in dry DMF (70 mL) and added to CMC-PEG-NH-AAPVC(Fmoc) under a nitrogen atmosphere. The reaction was stirred at room temperature for 3 hours and protected from light. The reaction mixture was concentrated in vacuo, washed with DCM (3×50 mL), methanol (3×50 mL), deionized water (3×50 mL), washed again with DCM (3×50 mL), and washed again with methanol (3×50 mL) and methanol (3×50 mL) and concentrated in vacuo to give an off-white solid (31) (436 mg, 68%). 13 C NMR (10,000 MHz, CP MAS) 176.70 (C-8, C-39, C-41 of CMC part), 171.39 (C-8, C-15, C-18, C-21, C-26, C-30, C-45), 153.37 (C-33, C-51), 142.19 (C-36, C-37, C-Ar), 127.57-120.48 (C-Ar), 103.85 (C-1), 102.63 (C-Ar), 97.55, 82.31 (C-4), 74.34 (C-2, C-3, C-5, C-7), 69.85 (C-23), 60.80 (C-6, C-27), 52.23-48.37 (C-16, C-19, C-25, C-35), 42.10 (C-9, C- 14, C-38), 38.70-25.05 (C-9, C-10, C-11, C-12, C-13, C-14), 18.45 (C-17, C-20, C-29), 14.11; FTIR (νmax / cm- 1)3340 / 3294 (OH), 2914 / 2862 (NH, CH), 1737 (COOH, acid), 1645 (CONH, amide of coupling product / COOR, conjugated ester, fluorescein), 1593 (HNCOO, CMC), 1543 (CONH, peptide), 1414, 1375 (COO), 1343 (aromatic C=C bending), 1262 (CO stretching), 1230, 1056 (CN, tertiary amine / OCHR), 897, 841, 764, 556 (aromatic CH bending); expected values ​​of elemental analysis: C: 53.4%, H: 6.0%, N: 5.4%, S: 1.5%; measured values: C: 47.1%, H: 6.7%, N: 4.1%, S: 0.8%, so the coupling conversion rate is 52%.

[0525] Example 32: Preparation of polymer 32

[0526]

[0527] CMC-PEG-NH-AAPVC(Fmoc)StBu(23-a) (175 mg, 146 mmol) in powder form was stirred with TCEP (168 mg, 585 mmol, 4 eq) in deionized water (23 mL) for 30 minutes at room temperature to decouple any unwanted disulfide bonds. The solid was filtered, washed with water (50 mL×3), methanol (50 mL×3), ethanol (50 mL×3), and then washed with water (50 mL×3), then freeze-dried, and then washed with DMF (50 mL×3). N-Bromophenylmaleimide (37 mg, 0146 mmol, 1 eq) was dissolved in dry DMF (20 mL) and added to CMC-PEG-NH-AAPVC(Fmoc) under a nitrogen atmosphere. The reaction was stirred at room temperature for 3 hours and protected from light. The reaction mixture was concentrated in vacuo, washed with DCM (3×50 mL), methanol (3×50 mL), deionized water (3×50 mL), washed again with DCM (3×50 mL), and washed again with methanol (3×50 mL) and methanol (3×50 mL) and concentrated in vacuo to give an off-white solid (32) (129 mg, 69%). 13C NMR (10,000 MHz, CP MAS) 171.16 (C-8, C-15, C-18, C-21, C-26, C-30, C-38, C-39), 153.33 (C-32), 142.03, 127.45-113.03 (C-Ar, Fmoc, fluorescein), 102.74 (C-1), 97.09, 82.15 (C-4), 74.39 (C-2, C-3 , C-5, C-7), 69.84, 61.21, 57.42 (C-6, C-31), 47.91 (C-16, C-19, C-27), 38.76-22.56 (C-9, C-10, C-11, C-12, C-13, C-14) 18.33-14.16 (C-17, C-20, C-29); FTIR (νmax / cm- 1 )3322 (OH), 2901 / 2872 (NH, CH), 2103, 1788 (COOH, acid), 1644 (CONH, amide of coupling product / COOR, conjugated ester, fluorescein), 1594 (HNCOO, CMC), 1544 / 1514 (CONH, peptide), 1349 / 1304 (aromatic C=C bending), 1251 (CO stretching), 1230, 1025 (CN, tertiary amine / OCHR), 898, 843, 738, 556 (aromatic CH bending); expected values ​​of elemental analysis: C: 50.0%, H: 6.1%, N: 6.1%, S: 1.8%, Br: 4.3%; found values: C: 46.3%, H: 6.7%, N: 5.2%, S: 0.33%, Br: not detected, so the coupling conversion rate is 17%.

[0528] Embodiment 33

[0529] Enzymatic cleavage of proteases / peptides

[0530] The activity of wound-specific proteases, including other proteases such as trypsin, chymotrypsin, and thermolysin, was determined. Trypsin cleaves P1-P1', where P1 is Lys or Arg, and P1' is non-specific (except when followed by proline). Chymotrypsin cleaves P1-P1', where P1 is any aromatic amino residue, Trp, Tyr, or Phe, and P1' is non-specific. Thermolysin is a metalloendopeptidase that cleaves P2-P1-P1'-P2', where P1 is non-specific, P1' is Leu, Phe, Ile, Val, Met, Ala, and P2' is not Pro.

[0531] Esterases hydrolyze the ester linkage of carboxylic acid esters, so their performance is different from proteases, which specifically hydrolyze amino acid sequences. The substrate used for the enzyme assay is CMC-PEG-NH-Phe-Phe-Lys (dansyl), and the enzyme activity is determined using a UV-visible method. The Phe-Phe-Lys (dansyl) sequence should be hydrolyzed by chymotrypsin. Trypsin is used as a negative control because it should not cleave the sequence.

[0532] The activity of the enzyme on the substrate CMC-PEG-NH-Cys-maleimide diacetate fluorescein was analyzed using confocal fluorescence microscopy. The solid particles were first observed separately, and then observed after adding an esterase solution (about 50 units / mL). The microscope image shows the fluorescence increase observed when the enzyme solution is added to the sample. The advantage of using this method is that the particles can be visualized and the difference in fluorescence intensity when the enzyme solution is added can be observed, which confirms the coupling of diacetate fluorescein to the CMC polymer. Alternatively, a multi-disc analyzer can be used to evaluate the cleavage of the substrate and the results are quantitatively measured with Michaelis-Menton kinetics.

[0533] In an experiment, the effect of esterase on CMC-PEG-NH-Cys-maleimide-diacetate fluorescein powder was measured. First, CMC-PEG-NH-Cys-maleimide-diacetate fluorescein powder (0.2 mg) was dispersed in PBS (320 μL). Ultrasonic treatment, vortex mixing and gentle heating were required to disperse the compound. 40 μL of substrate suspension was added to the wells of a 96-well plate, followed by the addition of weak esterase solution (58 U / mL), strong esterase solution (116 U / mL) or PBS (negative control) (40 μL per well). A cycle of 5 minutes within 2 hours was selected for fluorescence measurement.

[0534] It is of interest to use two esterase solutions of different strengths so that any differences in reaction rates can be analyzed. Changes in fluorescence intensity are calculated by subtracting the zero time point reading (baseline) from each subsequent time point.

[0535] Increased fluorescence was observed in samples incubated with weak (58 U / mL) and strong (116 U / mL) esterase solutions, which increased over time. The strong esterase solution showed a 3.5-fold increase in rate over the weak esterase solution before reaching a saturation point (intensity of 45,000 units) at approximately 30 minutes.

[0536] Effect of spacer length

[0537] To investigate the effect of longer PEG chain lengths on the esterase assay, fluorescence measurements of 77 (CMC-'longer' PEG-NH-Cys(Fmoc)-maleimidodiacetate fluorescein) were recorded. In this assay, 4 replicates were performed for each sample and 1 minute cycles were recorded over a 2 hour period. As with the shorter substrates, the longer substrate (77) was incubated with the esterase to detect fluorescence. The 116U / mL (strong) esterase sample peaked after 40 minutes; the 58U / mL (weak) esterase sample peaked after 80 minutes. The 116U / mL esterase reacted at twice the rate of the 58U / mL sample, so doubling the enzyme concentration resulted in doubling the rate. The reaction rate was significantly lower than that of the shorter PEG linker equivalent. This reduction was expected due to less peptide loading onto the CMC.

[0538] Incubation of CMC-PEG-NH-Cys(Fmoc)-maleimidodiacetate fluorescein (82) in fiber form with esterase also produced similar results, with both esterase solutions (58 and 116 U / mL) leading to an increase in fluorescence over time and a higher rate of fluorescent product production at higher enzyme concentrations.

[0539] Cleavage of CMC-PEG-NH-AAPVC-maleimidylfluorescein by elastase (powder, alginate fiber and hydrocolloid forms): The AAPV peptide sequence is a substrate for elastase with predicted cleavage sites at P1 of Ala and Val. For compounds containing this AAPV sequence, an elastase assay was performed in a similar manner to the esterase assay. Three forms of the substrate were tested: (a) powder form; (b) wet-spun alginate containing the compound in fiber form, and (c) hydrocolloid gel containing 0.8% or 8% compound.

[0540] Incubation of the compound with elastase alone showed a significant increase in fluorescence over time. The same experiment was also performed to evaluate the effect of adding an elastase solution to a hydrocolloid gel having 0.8% and 8% loaded CMC-PEG-NH-AAPVC-maleimide fluorescein in powder form. The results showed that the fluorescence of the modified CMC-loaded hydrocolloid gel increased over time compared to the control (hydrocolloid gel alone). The fluorescence of the higher loading level (8%) was much stronger than that of the lower loaded (0.8%) sample. Two different enzyme strengths were tested; in this case, the fluorescence of the sample increased only slightly when a strong elastase solution (0.5 mg / mL) was used compared to a weak elastase solution (0.1 mg / mL). This shows that even at 0.1 mg / mL, elastase is excessive and the use of modified CMC has a more significant effect on the fluorescence response.

[0541] LC-MS analysis of cleavage fragments

[0542] An experiment was set up to attempt to characterize the fragments produced when elastase was added to CMC-PEG-NH-AAPVC-maleimide fluorescein in powder form. Elastase (0.5 mg / mL in PBS) was added to 78 and incubated at 37°C; aliquots were collected at several time points (1 minute to 3 hours) and stored by immediate freezing in liquid nitrogen. Each thawed aliquot was subjected to HPLC to separate the fragments, followed by mass spectrometry to analyze their masses. This analysis confirmed the presence of the expected fragments that were cleaved during incubation with elastase. As predicted, the cleavage sites were located at P1 of Ala and Val. There was also evidence of some Pro fragments, suggesting an additional cleavage site at the P1 position of Pro. This result is consistent with the increase in fluorescence recorded during the fluorometer assay and further establishes evidence that the system is able to detect specific enzymes and give a detectable signal.

[0543] Embodiment 34

[0544] Cell Research - Biocompatibility of Medical Devices

[0545] An important aspect to consider when designing a medical device is its biosafety and other factors such as cytotoxicity, sensitization, blood compatibility, pyrogenicity, transplantation, genotoxicity, carcinogenicity, reproductive and developmental toxicity, biodegradation, etc. Studies on the interaction between new materials and cells grown in vitro can be a good indicator of the toxicity of these substances, so a range of these methods are used (Eisenbrand et al., Food Chem. Toxicol. 2002, 40, 193-236). Appropriate cell types must be selected so that the test is suitable for the body area and the function related to the final use of the device. The methods can be quantitative or purely visual, and modern technology allows complex views of cell interactions, such as time-lapse video imaging of cells using microscopy.

[0546] Cells associated with wound healing: Fibroblasts are important in the wound healing process. About 24 hours after injury in the late inflammatory phase, they begin to move to the wound surface. They change the wound environment throughout the proliferation and epithelial regeneration phase by producing mediators including proteases (such as MMPs). Finally, once the new wound extracellular matrix reaches sufficient strength, fibroblast levels drop back to normal levels during the remodeling phase (Bainbridge et al., Journal of Wound Care (J.WoundCare) 2013, 22, 407-408). Therefore, fibroblasts are suitable cell lines used in simulating the wound healing process.

[0547] In vitro Methods Used: Two in vitro cellular methods were used to screen the peptide-modified CMC materials for a preliminary idea of ​​their biocompatibility.

[0548] Source and culture of human dermal fibroblasts: Human dermal fibroblasts have been previously isolated and stored under appropriate conditions.

[0549] Cultures of normal fibroblasts were obtained with informed consent from the patients. Patients with diabetes, systemic immunosuppression, or signs of local infection were excluded from the study. Three patient cell lines were included in this study: patients A, F, and G. A 6-mm biopsy was taken from the patient's thigh. Cultures were established by single-cell suspension techniques after enzymatic degradation of the samples. Briefly, the tissues were incubated overnight with neutral protease (2 mg / mL; Boehringer Mannheim, Lewes, UK) to separate epidermal tissue from dermal tissue. The dermal tissue samples were then disaggregated overnight using the bacterial Clostridium histolyticum A collagenase (1 mg / mL; Boehringer Mannheim). Fibroblast cultures were maintained in fibroblast-serum-containing medium (F-SCM) containing Dulbecco's modified Eagle's medium (DMEM) supplemented with L-glutamine (2 mM), non-essential amino acids (1x), antibiotics (100 U / mL penicillin G; 100 mg / mL streptomycin sulfate; 0.25 mg / mL amphotericin B) and 1% (v / v) fetal calf serum (FCS). The cultures were maintained at 37° C. in a humidified atmosphere of 5% CO2. At confluence, fibroblasts were trypsinized and replated (1.5×105 cells per T75 flask).

[0550] Fibroblast scratch assay method:

[0551] The scratch assay is a technique in which a confluent monolayer of fibroblasts is grown on a flat surface, which is then "scratched" or "wounded" to create a channel separating two fibroblast regions. The sample solution is added to the top of the cells, and the channel is monitored over time using a confocal microscope to observe how the cells respond (Liang et al., "Natural Protocols (Nat. Protocols)" 2007, 2, 329-333). If the surrounding area containing the sample is suitable for cells, the cells will proliferate and migrate to fill the channel within the time period; if the sample is not suitable, the cells will not migrate and die.

[0552] Human dermal fibroblasts were seeded into 24-well tissue culture plates (2×104 cells per well), cultured to 80 to 90% confluence, and the monolayer was wounded by scraping along the surface of the tissue culture plastic with a 200 μL pipette tip. The monolayer was washed with PBS, and the compounds were added to DMEM at 0.66 mg / mL or 0.066 mg / mL. The cells were then fed again with F-SCM and incubated under standard culture conditions on a motorized, heated, and gasified stage of a confocal microscope with a Cell-IQ system.

[0553] Images were collected every 20 minutes and movies were created using Cell-IQ software. Assays were done in triplicate for each cell line; patients A, F, and G.

[0554] Collagen Matrix Model Methods:

[0555] The collagen matrix model is an in vitro tool that represents the dermis during the reorganization phase of healing. Here, a series of fibroblast-filled collagen lattices (FPCLs) are used to compare peptide-modified cellulose and controls with respect to the effects on collagen matrix reorganization. Under normal conditions, it is expected that the diameter of the FPCL will be reduced due to fibroblast reorganization, indicating that the cell process is proceeding normally and "healing" can occur (Carlson et al., Wound Repair Regen. 2004, 12, 134-147).

[0556] Fibroblasts derived from culture by trypsin digestion were used to construct fibroblast-filled collagen lattices (FPCLs). Type I rat tail collagen was purchased from First Link. 1.5×105 fibroblasts (in 750 μL F-SCM) were added to a 2×DMEM (40 parts 10×DMEM, 10 parts NaHCO 3 100 μ L), 100 μ L of 100 μ L of 0.1 M NaOH (750 μ L), 2.25 μ L of type I collagen (1.7 mg / mL) and test compound (0.66 mg / mL). The plates were incubated at 37° C. for 60 minutes to allow collagen polymerization. They were then separated from the edge of the plates and 2 mL of F-SCM was added. The FPCL was maintained at 37° C. in a 5% CO2 humidified atmosphere.

[0557] The circular shape was retained during FPCL reconstitution, allowing the diameter of the FPCL to be measured at days 3 and 7. For each sample, experiments were performed on cells from three different patient samples (n=3).

[0558] Seven modified CMC materials were tested as well as CMC powder and CMC fiber as a baseline (Table 3).

[0559] Table 3: Compounds analyzed during in vitro cell testing

[0560] Compound Description and Number form <![CDATA[CMC-PEG-NH 2 ,12]]> powder <![CDATA[CMC-longer PEG-NH 2 ,83]]> powder <![CDATA[CMC-PEG-NH 2 ,81]]> fiber CMC-PEG-NH-Cys-Mal-fluorescein diacetate, 74 fiber CMC-PEG-NH-AAPVC-Mal-fluorescein, 78 powder CMC-PEG-NH-AAPVC-Mal-bromophenol, 80 powder Unmodified CMC powder

[0561] Fibroblast scratch assay results:

[0562] In general, fibroblasts remained viable and proliferated to fill the scratch channel during the test period. Figure 5 ) shows the time it takes for fibroblasts to completely close the scratch channel. Figure 5 As shown, all samples closed the channel within 70 hours. The CMC powder sample was used as a reference as this is known to be safe for wound contact applications. 2 The fiber was the only sample where the scratch closed in a shorter period of time than the control. 2 The powder scratch closure time was roughly comparable to that of other modified CMC samples (approximately 40 to 60 h), suggesting that the cells may have some preference for the physical structure of the samples in fiber form.

[0563] The scratches of patient A containing 0.066mg / mL 12, CMC-PEG-NH2 powder (Figure 6) and patient A containing 0.66mg / mL 12, CMC-PEG-NH2 powder (Figure 7) all closed over time because fibroblasts replicated and migrated into the channel. These images show that fibroblasts are not affected by sample insolubility or higher concentration samples, and are still able to thrive in and around modified CMC. Figures 6 and 7 represent the observations of almost all tests conducted on all samples and cell lines from patients A, F and G. Although CMC-longer PEG-NH2 powder (#83) produced abnormal results in one study, fibroblasts did proliferate and migrate when the test was repeated with samples obtained from three different patients.

[0564] Example 35: Collagen Matrix Model

[0565] Fibroblasts remained viable in all samples during the collagen matrix model study. Only slightly reduced reorganization was observed for the modified CMC samples compared to the control, with the exception of 83, CMC-longer PEG-NH2 powder, which showed only limited fibroblast reorganization compared to the other samples tested ( Figure 8 , Fig. 9 and Fig.10A significant difference was observed between compound 83 (CMC-longer PEG-NH2 powder; lattice diameter of about 40 mm to 60 mm at day 7) and compound 12 (CMC-PEG-NH2 powder; lattice diameter of about 15 mm to 35 mm at day 7), with the results ranging from Figures 8 to 10 It is obvious from Figures 11 to 13 The raw data evident from the photographs in the Figures demonstrate that each of Compounds 12, 83, 81, 74, 78, 80, CMC powder, and CMC fibers has an effect on the survival of patients A ( Fig.11 )、B( Fig.12 ) and C( Fig.13 ) The effect of lattice diameter on the cell samples obtained at day 3 and day 7.

[0566] in conclusion

[0567] This study demonstrated that the modified CMC materials were nontoxic to fibroblasts. Furthermore, there were no differences between fiber and powder forms, longer or shorter PEG spacer linkers, or the addition of peptides and detectable fragments. Fibroblasts survived in all cases of the collagen matrix model, with the exception of CMC-longer PEG-NH2 powder 83 which showed a diminished effect compared to the other compounds, and for most samples reconstitution was only slightly slower than the control.

[0568] Example 36: Liquid Crystal (LC) Studies

[0569] Liquid crystal experiment set up

[0570] A study was conducted in which a CMC gel was placed on a 5CB liquid crystal and the anchoring was monitored over time using polarized light microscopy. In order to set up the LC study, a chamber needed to be created to allow the LC to be held within a set area and to allow for visualization using polarized light microscopy. TEM grid confinement of 5CB was performed based on published studies (Nazarenko et al., Physical Review E 1999, 60, R3495-R3497; Brake et al., Langmuir 2003, 19, 6436-6442).

[0571] To fabricate the 5CB-TEM grid experimental system, the glass slide used as the substrate is first preferably free of impurities such as grease. It is cleaned using piranha solution, a strong oxidizing agent that removes all organic matter from the glass. Due to the strong oxidizing properties of piranha solution, appropriate safety precautions are taken to avoid contact with the skin and to avoid explosions.

[0572] Concentrated sulfuric acid (about 30 mL, 98% grade) was added to the container containing the glass to be cleaned, and then hydrogen peroxide (about 10 mL, 30%) was slowly added and left at room temperature for 1 hour. Pour out the piranha solution and wash the glass thoroughly with water and ethanol. Finally, carefully neutralize the discarded piranha solution. Next, the glass was coated with octadecyltrichlorosilane (OTS). OTS is a long-chain self-assembling amphiphilic molecule that will coat the surface of the slide and make the slide surface hydrophobic. Therefore, 5CB is aligned with the vertical alignment anchor along the bottom of the chamber. A TEM grid was placed on the OTS-coated glass to keep the grid of the LC solution in it. 5CB was carefully added to the TEM grid using a capillary to ensure that each grid was fully filled, but not overfilled, forming a solution dome on top of the grid. When adding 5CB to the TEM grid, the 5CB alignment was checked using the cross-polarized lens of an optical microscope. 5CB is vertically aligned at this stage, which is due to the alignment of the LC with the OTS-coated slide.

[0573] The principle of enzyme detection relies on changes in LC orientation upon release of lipids (e.g. DLPC). A potential system using peptide-modified CMCs and LCs in this manner is shown in Fig.14 To this end, Figure 15 shows a micrograph showing a TEM grid filled with 5CB when a CMC gel was applied.

[0574] Other Implementations

[0575] The preceding examples may be repeated by substituting the generically or specifically described reactants and / or operating conditions of the disclosed techniques for those used in the preceding examples.

[0576] From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of the disclosed technology, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosed technology to adapt it to various usages and conditions.

[0577] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used to practice or test disclosed technology, suitable methods and materials are described in the preceding paragraphs. In addition, materials, methods and embodiments are only illustrative and are not intended to be restrictive. If a conflict occurs, this specification (including definitions) shall prevail.

[0578] All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All disclosed foreign patents and patent applications cited herein are incorporated by reference. All disclosed references, documents, manuscripts, scientific literature cited herein are incorporated by reference. All identifiers and registration numbers relevant to NCBI, GENBANK, EBI, PUBMED databases cited herein are incorporated by reference.

[0579] Although preferred embodiments of the disclosed technology have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided as examples only. Now, many variations, changes and substitutions will occur to those skilled in the art. It should be understood that various alternatives of the embodiments of the disclosed technology described herein may be adopted when practicing the disclosed technology. The appended claims are intended to define the scope of the disclosed technology, and are intended to cover methods and structures within the scope of these claims and their equivalents.

Claims

1. A wound dressing material selected from the group consisting of: Where n=200-4000.

2. A wound dressing material according to claim 1, for use in a method for treating or diagnosing a wound.

Images