Collagen crosslinking agent and its use

Abstract

A collagen crosslinker mixture containing the crosslinkers TRI-1, TRI-2 and TRI-3 ​​and / or two or more of TRI-1, TRI-2 and TRI-3 ​​is presented along with its synthesis. The collagen crosslinking effect of the mixture of TRI-1, TRI-2 and TRI-3 ​​is significantly enhanced over either molecule used individually. The mixture drastically improves collagen biostability, shows enhanced performance compared to commercially available collagen treatments, and is fast acting, requiring 60 seconds or less to show inhibition of collagenase. The mixture is also non-toxic and slightly beige in color, which is aesthetically ideal for treatments that may be visible to others. It has further been shown that the collagen crosslinker mixture is mixable with commercially available dental adhesives without compromising the desired properties of the crosslinker or adhesive.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from U.S. Provisional Patent Application No. 63 / 306,748, filed February 4, 2022.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant / Contract / Award 0053665 awarded by the National Institutes of Health (NIH). The Government has certain rights in this invention.

[0003] Technical Field The present teachings relate to novel biocompatible mixtures of organic crosslinkers (crosslinkers) that stabilize collagen and methods for making and using same. [Background technology]

[0004] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0005] Collagen is the major structural protein found in connective tissues throughout the human body, including skin, tendons, ligaments, tendons, bones, and teeth. Due to its wide range of uses in the body, collagen mixtures are diverse, with 28 types of collagen known to exist in the body. Despite their diversity, all types are known to contain at least one triple helix of three polypeptide chains. The most common type in the body, called type I collagen, is assembled into fibers that form the structural and mechanical scaffolding of bone, skin, tendons, cornea, blood vessel walls, and other connective tissues. Type I collagen is involved in the development, formation, and homeostasis of these tissues, providing structure and strength, and playing a role in tissue repair.

[0006] Collagen biosynthesis occurs naturally, but its rate slows with age. Collagen degradation also occurs naturally and is largely regulated by enzymes called collagenases. It is associated with undesirable outcomes such as sagging epidermal tissue and wrinkles. Therefore, the restoration and support of naturally degraded collagen has attracted attention in the so-called anti-aging industry. Treatments directed at collagen restoration and support, especially if they are topical, should ideally be colorless and non-toxic to maintain aesthetics and ensure that users are not harmed by the treatment.

[0007] Many treatments that attempt to restore and protect collagen work slowly over time. These include behavioral therapies such as avoiding tobacco, excess sugar, and excessive sun exposure, and supplemental treatments such as hyaluronic acid intake. However, collagen loss and degradation can also be induced by disease or trauma, and such acute collagen degradation can slow healing and prevent medical intervention. Some collagen treatments are applied directly to collagen in the body, and attempt to restore and protect collagen by strengthening collagen fibrils and increasing their intertwining with each other.

[0008] Such strengthening of the intertwining of collagen fibrils may be achieved by cross-linking. "Cross-linking" is a broad term in chemistry and biology that includes both a physical sense (wherein a cross-linking agent enhances weak attractive interactions between fibrils) and a chemical sense (wherein a cross-linking agent directly covalently bonds with multiple fibrils to create "bridges" that connect the fibrils). Cross-linking is a powerful means of strengthening polymers and proteins such as collagen, but caution is required as exogenous chemicals introduced into the body may result in toxicity and aesthetic problems. Furthermore, while many known cross-linking agents may exhibit some ability to improve the strength and / or intertwining of collagen fibrils, the cross-linking agents themselves are vulnerable to common chemical reactions that degrade them. Such cross-linking agents are considered to have low "bio-stability" because they are unlikely to remain stable when applied to tissues in the body. Thus, there is a need for a collagen cross-linking agent with high bio-stability that can be applied locally to sites of sudden and / or acute collagen degradation. Summary of the Invention

[0009] In various aspects, the present disclosure provides three novel collagen crosslinkers, referred to herein as TRI-1, TRI-2, and TRI-3. The present disclosure further provides mixtures containing two or more of the three crosslinkers. Although TRI-1, TRI-2, and TRI-3 ​​are novel and independently provide the advantages described herein, mixtures containing two or more, e.g., all three crosslinkers, provide higher crosslinking performance (e.g., collagenase inhibition) than any of the individual crosslinkers. In particular, the application of a mixture of two or more of the crosslinkers TRI-1, TRI-2, and TRI-3 ​​to collagen drastically improves collagen stability by improving collagen fibril crosslinking. The degree of collagen stabilization provided by the crosslinkers and / or mixtures thereof described herein is much higher compared to commercially available collagen treatments, as described in detail below. Collagenase inhibition can be directly inferred from the degree of collagen degradation when exposed to collagenase. Moreover, in various embodiments, the crosslinkers and / or mixtures thereof disclosed herein exhibit potent inhibition within 60 seconds of initial exposure to collagenase, thus protecting collagen much more rapidly than commercially available collagen treatments.

[0010] In various embodiments, the crosslinker and / or mixture thereof is also colorless, which is aesthetically ideal for treatments that may be visible to the human eye, and non-toxic. In various embodiments, the collagen crosslinker and / or mixture thereof of the present disclosure actually exhibits high biocompatibility and even improves dentin cell proliferation. It is further shown that the collagen crosslinker and / or mixture thereof of the present disclosure can be mixed with commercially available dental adhesives without compromising its desired colorless and fast-acting, non-toxic collagen stabilization properties or polymerization properties of the dental adhesive.

[0011] This specification also discloses a method for making the crosslinker mixture of the present disclosure. [Brief description of the drawings]

[0012] [Figure 1A-1B]1A and 1B show simplified synthetic pathways for each of the three components of a biocompatible collagen crosslinker mixture according to various embodiments of the present disclosure. Figure 1A shows a generalized synthetic pathway in which the reaction of catecholamines and tricarbonylbenzene produces molecules of generalized form TRI-1a, TRI-2a, and TRI-3a, depending on the nature of the R substituents, which are independently selected. Figure 1B shows a more specific example of the generalized synthesis modeled in Figure 1A, in which the reaction of trimesoyl chloride and dopamine produces TRI-1, TRI-2, and TRI-3. [Figure 2A-2B] Figures 2-9 show the experimental protocol provided in the "Examples" below. Figures 2A and 2B are representative bar graphs showing the in vivo stability of dentin collagen to collagenase with the crosslinkers shown in the figures. Figure 2A shows the percentage of weight loss of dentin collagen with crosslinkers; Figure 2B shows the amount of hydroxyproline released from dentin collagen with crosslinkers. [Diagram 3] FIG. 3 is a bar graph illustrating the extent of collagenase activity on dentin collagen comparing continuous exposure to known crosslinkers for the times shown in the figure with exposure to the crosslinker mixture of the present disclosure. [Figure 4A-4B] Figures 4A and 4B compare the modulation of matrix metalloproteinase activity of dentin collagen by the crosslinker mixture disclosed herein and various known crosslinkers. Figure 4A shows fluorescent microscopy images of dentin samples exposed to the crosslinker mixture disclosed herein or known crosslinkers for 60 seconds (top image) or 1 hour (bottom image); Figure 4B is a bar graph showing matrix metalloproteinase activity of the dentin collagen sample of Figure 4A after exposure to the crosslinker mixture disclosed herein or various crosslinkers for the times shown in the figure. [Diagram 5] FIG. 5 is a bar graph showing relative intensity by absorbance spectroscopy of L929 cells treated with the crosslinkers shown in FIGS. 4A and 4B, comparing the ability of the crosslinker mixture disclosed herein and various known crosslinkers to stabilize dentin collagen and improve cell proliferation for the concentrations and exposure periods shown in the figure. [Figure 6A-6B]6A and 6B show bar graphs depicting the number of live and dead L929 cells after the cells were exposed to dentin collagen treated with the crosslinker mixture disclosed herein or various known crosslinkers for an extended period of 1 or 3 days, respectively. [Figure 7A-7B] Figures 7A and 7B show fluorescent microscopy images of live (top) and dead (bottom) L929 cells after 1 and 3 days of exposure to materials released from dentin collagen treated with the crosslinker mixture disclosed herein and various known crosslinkers. [Figure 8A-8B] Figures 8A and 8B are comparative photographs of dentin films before (Figure 8A) and after (Figure 8B) collagenase digestion, which were treated with only a commercial dental adhesive, only a crosslinker mixture disclosed herein, or dental adhesive and various weight percentages of a crosslinker mixture disclosed herein. [Figure 9] FIG. 9 is a bar graph comparing the degree of conversion of a commercial dental adhesive mixed with a fixed concentration of either the crosslinker mixture disclosed herein or the known crosslinker, GSE. Detailed Description

[0013] Corresponding reference numerals are used in the several figures.

[0014] The following description illustrates the claimed invention by way of example and not by way of limitation. The description describes several aspects, adaptations, variations, alternatives and uses of the claimed invention, including what we currently contemplate as the best mode of carrying out the claimed invention, clearly enabling one of ordinary skill in the art to make and use the claimed invention. It is further to be understood that the claimed invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The claimed invention is capable of other embodiments and can be practiced or carried out in various ways. It is also to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0015] As used herein, the term "crosslinker" refers to a substance that strengthens the attractive interactions between polymer or protein chains by promoting covalent bond formation, ionic bond formation, hydrogen bond formation, hydrophobic interactions or physical entanglements.

[0016] As used herein, the term "biocompatible" refers to materials and / or therapies that are effective as intended and do not have toxic, harmful, or other known significant detrimental effects on biological systems.

[0017] As used herein, the term "small molecule" refers to an organic compound having a molecular weight of 1,000 daltons or less, and as used herein, the term "polymer" refers to an organic compound having a molecular weight of more than 1,000 daltons.

[0018] As used herein, the term "biomolecule" refers to biological materials such as proteins, peptides, carbohydrates, and polymers that are naturally formed from biologically derived monomers.

[0019] As used herein, the term "collagen" refers to the known types of collagen found in bones, teeth, skin and other tissues of animal species, without affecting the collagen mixture and morphology of the surrounding chemical or biological matrix.

[0020] As used herein, the term "dentin" refers to the primary component of teeth found just beneath the enamel, an organic mixture that is 90% type I collagen.

[0021] The term "collagenase," as used herein, refers to an enzyme that degrades collagen. Collagenase, as used herein, most commonly refers to collagenase type I, a protease that cleaves glycine-amino acid bonds frequently found in collagen.

[0022] As used herein, the term "MMP" refers to matrix metalloproteases, a group of enzymes capable of degrading several extracellular matrix proteins, bioactive molecules, and, in particular, collagen.

[0023] As used herein, the term "HYP" refers to hydroxyproline, which is the main component of collagen in the human body.

[0024] As used herein, the term "MTS" refers to (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, a reagent commonly used in colorimetric assays of cell cultures.

[0025] As used herein, the term "EDTA" refers to ethylenediaminetetraacetic acid, a well-known chelating agent and selective inhibitor of a variety of enzymes.

[0026] The term "acetonitrile" as used herein refers to the compound also known as methyl cyanide, of the formula CH3CN.

[0027] As used herein, the term "EtOH" refers to ethanol.

[0028] As used herein, the term "THF" refers to tetrahydrofuran.

[0029] As used herein, the term "TEA" refers to triethylamine, which should not be confused with triethanolamine.

[0030] As used herein, the term "EGCG" refers to epigallocatechin gallate, a known cross-linking agent.

[0031] As used herein, the term "QC" refers to quercetin, a known cross-linking agent.

[0032] The term "GEN," as used herein, refers to genipin, a known cross-linking agent.

[0033] As used herein, the term "EDC / NHS" refers to the known cross-linking agent 3-dimethylaminopropylcarbodiimide hydrochloride / N-hydroxysuccinimide.

[0034] As used herein, the term "GSE" refers to grape seed extract, which is known to have a protective effect on collagen.

[0035] As used herein, the terms "CT" and "CONTROL" refer to a control study of a particular experiment or study.

[0036] As used herein, the term "GA" refers to glutaraldehyde.

[0037] As used herein, the term "NCTC" refers to the National Collection of Type Cultures, an organization that collects, stores, and distributes reference materials for biological cultures.

[0038] As used herein, the term "ATCC" refers to the American Type Culture Collection, an organization that collects, stores, and distributes biological culture reference materials.

[0039] As used herein, the term "FBS" refers to fetal bovine serum, which is a growth supplement for cell culture media.

[0040] As used herein, the term "MALDI" refers to matrix-assisted laser desorption / ionization, a technique of mass spectrometry that uses pulsed laser irradiation to ionize a sample, and is typically applied to macromolecules and biomolecules.

[0041] As used herein, the term "ESI" refers to electrospray ionization, a technique in mass spectrometry in which a sample is ionized by applying a high electric potential to the sample.

[0042] As used herein, the term "TOF" refers to "time of flight," a type of mass spectrometry in which the mass / charge ratio of ions in a sample is determined by the time it takes for a given ion in a fixed electric field to reach a detector at a known distance from the point of ionization.

[0043] As used herein, the term "CLSM" refers to confocal laser scanning microscopy, a microscopy technique that provides precise control over optical resolution and depth of field.

[0044] As used herein, the term "ANOVA" refers to "analysis of variance," a standard family of statistical models often used to determine whether two or more population means are statistically equivalent.

[0045] The term "catecholamines" as used herein refers to a class of compounds defined by a benzene ring characterized by two adjacent alcohol groups and an alkylamine. These include intact catecholamines or catecholamines characterized by further substitution. Form:

[0046] [ka] (wherein R1-R4 are each independently selected from hydrogen, halogen, and alkyl groups, including linear alkyl groups of various chain lengths).

[0047] Specifically included are compounds of the formula: R5 is hydrogen or an alkyl group. x can represent one or two hydrogens, and the amine is a primary, secondary, tertiary, or quaternary amine. In the quaternary amine form, a suitable anion can counter the charge of the amine. Thus, as used herein, the term catecholamine is not limited to the three well-known neurotransmitters, dopamine, epinephrine, and norepinephrine, although these substances are included within the scope of the term.

[0048] As used herein, the term "dopamine" is a contraction of the molecule 3,4-dihydroxyphenethylamine. The term "dopamine hydrochloride" refers to the hydrochloride salt of dopamine, which has the chemical formula CH 12 NO2Cl.

[0049] As used herein, the term "tricarbonylbenzene" refers to a molecule characterized by a benzene containing three carbonyl substituents.

[0050] [ka] (wherein R1 to R3 are each independently selected from hydrogen, halogen, and an alkyl group, and X1 to X3 are each independently selected from hydrogen, oxygen, nitrogen, halogen, alkyl oxide, and alkyl amine).

[0051] Specifically included are molecules of the formula: The tricarbonylbenzenes of the present disclosure are preferentially selected such that the primary site of reactivity for nucleophiles is the carbonyl center.

[0052] As used herein, the term "trimesoyl chloride" refers to a molecule also known as 1,3,5-benzenetricarbonyl trichloride.

[0053] The term "TRI" is used herein as a contraction of the term "tri-amide" and refers broadly to the collagen crosslinker mixture of the present disclosure. The specific terms "TRI-1," "TRI-2," and "TRI-3" are used herein to refer to molecular components of the collagen crosslinker mixture of the present disclosure and can be distinguished by the fact that they are characterized by three amide groups, two amide groups and a carboxylic acid, and two amide groups and an ester, respectively.

[0054] 1A-1B, in various embodiments, the present disclosure provides three newly discovered generalized forms of low molecular weight collagen crosslinkers (TRI-1a, TRI-2a, and TRI-3a), three specific variants of these crosslinkers (TRI-1, TRI-2, and TRI-3), and mixtures containing two or more of TRI-1, TRI-2, and TRI-3. When applied to collagen, these three crosslinkers and / or mixtures of two or more stabilize collagen fibrils and prevent collagen degradation by collagenases and the like. While TRI-1, TRI-2, and TRI-3 ​​are each novel, mixtures of two or more, such as all three, also have a synergistic effect, providing greater collagen protection than the individual components used alone. The structures of each of TRI-1, TRI-2, and TRI-3 ​​contain catechol-like end groups that are believed to aid in their adsorption to collagen. Additionally, these structures are largely free of reactive ester groups found in the prior art. This relatively low reactivity is believed to contribute to the in vivo stability afforded by the TRI-1, TRI-2 and TRI-3 ​​crosslinkers, individually and / or in combination, and, as disclosed below, in various embodiments, it is possible to obtain all three of these molecules in a single synthetic procedure in the desired stable and highly synergistic combinations.

[0055] As shown in Figure 1A, in various embodiments, a collagen crosslinker mixture including the generalized molecules TRI-1a, TRI-2a, and TRI-3a can be synthesized by a simple reaction of tricarbonylbenzene compounds, which are inexpensive, commercially available compounds, with catecholamines. Figure 1A provides a highly generalized scheme for the synthesis of the crosslinker mixtures of this disclosure, with the substituents "R" and "X" representing sites where one of ordinary skill in the art can envision routine minor modifications without significantly altering the properties of the mixture and without departing from the scope of this disclosure. Further modifications of the disclosed synthesis and reagents can also be envisioned by one of ordinary skill in the art without significantly altering the properties of the mixture and are considered to be within the scope of this disclosure.

[0056] Figure 1B shows specific synthetic schemes carried out in various manners. A representative total synthesis is shown in the section "Synthesis A" below. In particular, Figure 1B shows that trimethoxysuccinyl chloride and dopamine hydrochloride undergo an addition-elimination reaction in step 1 to obtain a fully substituted compound TRI-1 containing three amide groups and a partially substituted TRI-2 characterized by two amide groups and one carboxylic acid group. This mixture containing TRI-1 and TRI-2 is recovered and washed, and then through reaction with a reducing agent and alcohol, a mixture of TRI-1, TRI-2, and a molecule TRI-3 characterized by two amide groups and one ethyl ester is obtained. Accordingly, the present disclosure provides a synergistic mixture composed of novel collagen crosslinking agents TRI-1, TRI-2, and TRI-3 that can be manufactured in two steps. By carefully controlling the synthesis method, the ratios of TRI-1, TRI-2, and TRI-3 in the mixture can be varied, thereby affecting the effectiveness of the mixture for stabilizing natural collagen such as dentin, skin, and bone.

[0057] In various embodiments, TRI-1 (C 33 H 33 N3O9), TRI-2 (C 25 H 24 N2O8) and TRI-3 (C 27 H 28 N2O8) are mixed in various ratios to provide various advantages described in the specification, mainly such as the in vivo stability of collagen. The range of weight percentages is approximately zero to 100 respectively, and more precisely a:b:c = x:y:(100 - x - y) (where 0 ≦ x < 90 and 10 < y ≦ 100). In various embodiments, the percentage ratio a:b:c is about 50:25:25.

[0058] In various embodiments, the reactions of steps 1 and 2 of FIG. 1B can be better controlled by placing the reaction vessel over a NaCl / ice bath under nitrogen flow. In various embodiments, the solvent used in step 1 of FIG. 1B is a polar aprotic solvent such as acetonitrile, and the solvent used in step 2 of FIG. 1B is THF. In various embodiments, the reaction of step 1 can be accelerated by adding triethylamine. In various embodiments, the reaction of step 2 is reduced with LiAlH4 and followed up with hydrochloric acid and ethanol. While representative reagents for the general reactions shown in the specification are listed, one of skill in the art will recognize permissible substitutions and modifications, all of which are within the scope of the present disclosure.

[0059] For example, in various exemplary embodiments, polar aprotic solvents other than acetonitrile can be used, including, but not limited to, dichloromethane, tetrahydrofuran, ethyl acetate, dimethylformamide, dimethylsulfoxide, and acetone. Similarly, polar aprotic solvents other than THF can be used during addition of the reducing agent.

[0060] In various embodiments, alkaline compounds other than triethylamine can be used, including, but not limited to, pyridine, N,N-diisopropylethylamine, and potassium carbonate.

[0061] The NaCl / ice bath is prepared in various embodiments with a ratio of 99 g NaCl to 300 g ice. The temperature of the NaCl / ice bath is −21.3° C. in various embodiments, but may be any temperature between −21.3° C. and 0° C. The NaCl / ice bath may be any mixture that provides a low temperature environment instead of a mixture of NaCl and ice.

[0062] In various embodiments, the reaction can be carried out under any inert environment, such as Ar instead of N2.

[0063] In various embodiments, reducing agents other than LiAlH4 can be used, for example weaker reducing agents such as sodium borohydride.

[0064] Experiments have shown that the application of collagen crosslinker mixtures containing any one of crosslinkers TRI-1, TRI-2, and TRI-3, or two or more of TRI-1, TRI-2, and TRI-3, such as mixture 10, which contains all three crosslinkers TRI-1, TRI-2, and TRI-3, to mammalian collagen improves the in vivo stability of the collagen. Numerous studies evaluating the individual crosslinkers TRI-1, TRI-2, and TRI-3, as well as crosslinker mixtures, such as crosslinker mixture 10, have demonstrated positive effects, particularly on dentin collagen. The following examples provide detailed information about the synthesis protocol of collagen crosslinker mixtures that have been found to be beneficial for dentin collagen, and the specific studies that have been conducted on the interaction between mixture 10 and dentin collagen. EXAMPLES

[0065] Synthesis A: In one exemplary embodiment, acetonitrile and TEA were dried over freshly activated 3 Å and 4 Å molecular sieves, respectively, for 24 h prior to use. Dopamine hydrochloride (3.3 eq; 2.0381 g, 10.75 mmol) was dissolved in 40 mL of dry CH3CN under N2. To the stirred solution was added dry TEA (12 eq; 3.8857 g, 5.35 mL, 38.4 mmol). The reaction vessel was transferred to an ice / NaCl bath (a mixture of 99 g NaCl and 300 g ice, −21.3° C.) and stirring was continued for an additional 15 min, after which a solution of trimesoyl chloride (1 eq; 0.8535 g, 3.22 mmol) in 20 mL of dry CH3CN was added dropwise over approximately 30 min. The reaction mixture was continued to stir under N2 in a gradually melting ice / NaCl bath for 44 h. The reaction mixture was suction filtered and the filtrate was rotary evaporated under vacuum to remove the acetonitrile solvent. The solids from the vacuum filtration and the solids recovered from the evaporation were combined and washed with 50 mL of 2 M aqueous HCl (pH<2) with the aid of sonication, stirring and physical trituration. After vacuum filtration, the solids were washed thoroughly with deionized (DI) water. The solids (1.2656 g) were dried under vacuum at 30° C. for 48 h. The sample was analyzed using a MALDI TOF mass spectrometer calibrated with CsI3. The sample (4.96 mg) was dissolved in 200 μL of CHCl3 and the solution was mixed (1:1) with a commercially available MALDI matrix 2,5-dihydroxybenzoic acid solution and spotted onto a MALDI plate. TRI-1 (C 33 H 33 N3O9, monoisotopic mass: 615.2217, theoretical value [M+H] + :616.2290) [M+H] + The main peak at m / z 616.2272 corresponds to TRI-2 (C 25 H 24 N2O8, monoisotopic mass: 480.1533, theoretical value [M+H] + :481.1605) [M+H] + A minor peak was observed at m / z 481.1679, corresponding to m / z 481.1605. The molar ratio of TRI-1:TRI-2 is calculated to be 88.15:11.85.

[0066] The above product mixture (307.8 mg) was dispersed in THF (15 mL) and the mixture was stirred under nitrogen atmosphere in an ice / NaCl bath (mixture of 99 g NaCl and 300 g ice, −21.3° C.) for about 15 min. To the mixture was added 3 mL of 2.4 M LiAlH4 in THF dropwise. The ice bath was removed and the reaction mixture was heated to reflux and kept at reflux for 2 h. After cooling to room temperature, the ice bath was replaced and the reaction mixture was stirred for about 15 min. The reaction products and excess hydride were decomposed by dropwise addition of water (3 mL) and the orange reaction mixture became a thick gel. After dropwise addition of dilute aqueous HCl (15 mL; diluted from 4 ml concentrated HCl), the solution became clear and the color changed to pale yellow. After stirring the mixture under N2 for several days, an oily droplet-like phase was observed in the water / THF mixture. After adding a few drops of concentrated HCl, the solvent was removed and the remaining sticky oily / fatty droplets were collected using ethanol. The ethanol solution was analyzed by ESI mass spectrometry. 33 H 33 N3O9, monoisotopic mass: 615.2217, theoretical value [M+H] + :616.2290) [M+H] + The main peak at m / z 616.22914 corresponds to (m / z 616.2290) and TRI-2 (C 25 H 24 N2O8, monoisotopic mass: 480.1533, theoretical value [M+H] + :481.1605) [M+H] + (m / z 481.1605) and TRI-3 ​​(C 27 H 28 N2O8, monoisotopic mass: 508.1846, theoretical value [M+H] + :509.1918) [M+H] + Two moderate peaks were observed at m / z 481.16046 and 509.19188, corresponding to m / z 509.1918. The molar ratio of TRI-1:TRI-2:TRI-3 ​​is calculated to be 51.4:22.7:25.9.

[0067] Protocol 1: Dentin collagen harvesting Collagen exists in various locations in the body. Therefore, it is best to select one type of collagen to demonstrate the beneficial effect of the collagen crosslinker and / or crosslinker mixture disclosed herein. Because dentin stabilization is a very frequent problem in dental medicine, dentin collagen is selected to demonstrate the collagen-in vivo stabilization property of the crosslinker mixture disclosed herein.

[0068] Dentin collagen films were harvested and prepared according to the following protocol: Thirty-six non-carious human third molars were harvested and the enamel layer was removed with a low-speed diamond saw under water cooling, after which 12 teeth were cut into dentin blocks (6 × 6 × 4 mm). These 12 dentin blocks were then processed into 876 dentin films (6 μm thick) using a tungsten carbide knife attached to a microtome. These films were thoroughly demineralized in 10% phosphoric acid and rinsed with deionized water for 30 min. The demineralized dentin collagen films were then randomly divided into 12 experimental groups (N = 73 per group).

[0069] Protocol 2: Exposure of dentin thin film to crosslinking agent The crosslinking ability and biostability promoted by the collagen crosslinker mixture disclosed herein was compared with other known crosslinkers: EGCG, QC, GEN and EDC / NHS. All crosslinkers were freshly prepared using 0.6 wt% in EtOH. The group treated with EtOH only was the control. The crosslinkers were applied for two treatment times: 60 seconds and 1 hour. After treatment, all dentin collagen films were thoroughly rinsed with EtOH for 30 minutes (3 x 10 minutes) to remove unreacted compounds.

[0070] Example 1 Degradation of dentin collagen by exogenous collagenase Following treatment and rinsing, the dentin collagen films were dried (48 h in vacuum) and exposed to 0.1% collagenase (type 1, from Clostridium histolyticum, ≥125 U / mg) for 1 h at 37 °C (6 × 10 films per group). The remaining films were similarly dried and analyzed for weight loss (WL) by comparing the weight before and after collagenase digestion. The digestion solution was collected and processed to quantify the released HYP. This process included exposure to 5% Ehrlich's reagent, which binds and stains HYP, the intensity of which is proportional to the concentration of HYP. Colorimetric analysis was performed by measuring absorbance at 555 nm in a microplate reader, and the μg of HYP per mg of collagen in the digestion was calculated. Data were analyzed by two-way ANOVA and Games-Howell post-hoc test (α = 0.05). All data for weight loss for each sample are shown separately for 60 s and 1 h after digestion.

[0071] The results are shown in Figures 2A and 2B. Figure 2A shows the percent weight change measured before and after collagenase digestion. The CT sample, which uses only ethanol to protect the dentin collagen, clearly showed a very large percent weight change, while the QC and GEN samples did not fare much better. The samples exposed to the TRI crosslinker mixture showed by far the smallest percent weight loss due to degradation. Similarly, Figure 2B shows that the samples exposed to TRI released the least HYP during degradation, while the competing crosslinkers were significantly inferior in this regard, with the control sample performing worst as expected.

[0072] Example 2 Direct inactivation of exogenous collagenase by TRI Direct inactivation of collagenase by TRI was also measured using a collagenase assay kit. The assay was performed in 96-well microplates containing 0.2 U / mL of Clostridium histolyticum collagenase and incubated with the treatment solution for up to 24 h according to the manufacturer's instructions. EDTA 5 mM, a known collagenase inhibitor, was used as a positive control. A no treatment group was included as a negative control. Fluorescence kinetics of inactivation was measured in triplicate at 490 nm / 520 nm at 1, 2, 4, 16 and 24 h of incubation at 37°C. Fluorescence at each time point was determined by subtracting background fluorescence. Collagenase activity was expressed in arbitrary fluorescence units. Activity was then calculated as a percentage of inhibition according to the untreated negative control after 4 h of incubation (assuming 100% collagenase activity). Statistical analysis was performed with one-way ANOVA and Games-Howell post-hoc test (α=0.05).

[0073] The results are shown in Figure 3. All crosslinkers, including EDTA, inhibited collagenase to some degree after 1 hour, with the TRI sample inhibiting collagenase the most. Samples exposed to the other crosslinkers did not show comparable collagenase inhibition even after 24 hours, indicating that TRI was not only very effective for this purpose, but also inhibited collagenase very rapidly.

[0074] Example 3 Endogenous MMP activity by in situ zymography Three dentin collagen films from each group were analyzed for endogenous MMPs in dentin collagen. Fluorescein-conjugated gelatin from the assay kit was prepared immediately before use according to the manufacturer's protocol. Immediately after treatment, the collagen films were spread on microscope slides, covered with a small drop of gelatin (3 μL) and incubated for 24 h in a humidified chamber at 37 °C protected from light. Slides containing the films were covered with a cover slip and visualized by confocal laser scanning microscopy in fluorescence mode (40x objective, 0.95 NA) with excitation at 488 nm and emission at 530 nm. For each collagen film, three images from the same z-layer were acquired randomly. All images (9 images per group) were analyzed and quantified for the relative intensity of green fluorescence, which indicates the activity of endogenous MMPs. Data were analyzed by two-way ANOVA and Games-Howell post-hoc test (α = 0.05).

[0075] The results are shown in Figures 4A and 4B. MMP activity was highest in the CT samples as expected, and all samples exposed to crosslinkers inhibited MMP activity to some extent. Samples treated with TRI inhibited MMPs the most.

[0076] Protocol 3: Preparation of cell cultures Cell cultures were prepared and the cytotoxicity of TRI was determined by either directly exposing cells to TRI or indirectly exposing cells to substances released from dentin lamina treated with TRI. NCTC clone 929 fibroblasts were cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% FBS and a 1% solution of 1:1 penicillin:streptomycin. Cells were cultured at 2 × 10 4 cells / cm 2 and incubated at 37° C., 5% CO 2 and 95% relative humidity until a monolayer of cells spread across the bottom of the well.

[0077] Example 4 Effect of direct treatment on cell proliferation Direct treatments were performed in 96-well plates by replacing the original medium with medium containing treatment solutions diluted at 1:1000, 1:2000 and 1:4000 (v / v) ratios, while the control group contained only the original medium. Cell proliferation was measured using the MTS assay CellTiter 96 AQ ueous The One Solution Cell Proliferation Assay was used to assess the proliferation of cells according to the protocol guidelines specified by the manufacturer. Two hours after addition of the MTS reagent, the absorbance at 460 / 30 nm was measured using a multimode microplate reader. Statistical analysis was performed using two-way ANOVA for each concentration used, followed by Tukey's post-hoc test (α=0.05).

[0078] The results are shown in Figure 5. Even at the highest concentration, cell cultures treated with TRI show superior cell growth characteristics compared to control samples, whereas GA (glutaraldehyde) shows high cytotoxicity as expected.

[0079] Example 5 Effect of released substances on cell proliferation Twenty-four teeth were sectioned to obtain 24 dentin discs (approximately 6 mm in diameter). The dentin discs were surface etched using 32% phosphoric acid gel for 15 seconds on each side, rinsed thoroughly, and placed in 100 μL of treatment solution. After 1 minute of treatment, the dentin discs were rinsed with sterile distilled water for 30 seconds. The treated dentin discs were immediately placed on a permeable polycarbonate membrane insert (6.5 mm diameter, 3.0 μm pore size) on top of the cultured cells and covered with 100 μL of medium. After incubation, cell viability assays stained live cells with calcein-AM and dead cells with ethidium homodimer. To distinguish overlapping cells during cell counting, Hoechst 33342 was used as a cell-permeant nuclear counterstain, following the guidelines specified by the manufacturer. Cells were imaged at 20x magnification using a fluorescent high-content microscope. Cell counts were performed using ImageJ (NIH Image J 1.8.0). Cell proliferation and the percentage of dead cells were statistically compared to day 1 controls using one-way ANOVA and Dunnett's test (α = 0.05).

[0080] The results of this analysis are shown in Figures 6A-B and 7A-B. Compared to the control, treatment with TRI resulted in a high number of live L929 cells and minimal dead L929 cells. In contrast, GA is cytotoxic.

[0081] Example 6 Addition of TRI to dental adhesives and its effect on collagen degradation and degree of conversion of the adhesive The inventors recognize that the use of TRI crosslinkers in various applications may require mixing with other materials. For example, the use of TRI crosslinkers in stabilizing dentin collagen is likely to involve the use of dental adhesives as part of medical treatment. To date, there are no collagen crosslinkers available that provide strong dentin collagen stabilization, long shelf life due to inherent chemical stability, colorless for aesthetic appeal, non-toxic and do not interfere with polymerization of dental adhesives.

[0082] Therefore, in a preliminary study, we tested the addition of a 0.6% TRI solution mixed directly with a commercial dental adhesive at 0, 5, 10, and 15% (v / v), which correspond to final TRI concentrations of 0, 0.03, 0.06, and 0.09% TRI when mixed with the dental adhesive. Dentin collagen was treated with each of these mixtures for 60 seconds, followed by thorough rinsing with EtOH for 30 minutes.

[0083] These samples were tested for exposure to collagenase. Visual results are shown in Figures 8A and 8B, which show the microcentrifuge tubes of each sample before (Figure 8A) and after (Figure 8B) 1 hour of collagenase digestion. The two samples on the far right in each figure are CT solutions, which contained no TRI or adhesive. Visual assessment confirms that in the absence of TRI (0%), dentin collagen was completely digested after 1 hour, while each sample containing TRI showed protection of collagen. Thus, there does not appear to be any detrimental effect of TRI on collagenase inhibition by mixing with dental adhesive.

[0084] The reverse was also tested, i.e., whether TRI has a detrimental effect on the polymerization ability of dental adhesive monomers. Fourier transform infrared spectroscopy (FTIR) with a universal attenuated total reflectance (ATR) accessory was used for the photopolymerization test. An LED light curing unit was used to irradiate the specimens. Five adhesive samples were tested: Prime&Bond Elect (PB) without crosslinker, PB with 5% and 10% (v / v) TRI, and PB with 5% and 10% (v / v) GSE. The adhesive samples were placed on the diamond crystal top plate of the ATR attachment at a thickness of 0.3 mm, covered with a plastic cover slip, and light cured for 10 seconds. The conversion at 10 minutes after curing was 811 / 1716 cm -1 Calculations were performed using band ratios (n=5).

[0085] The results are shown in Figure 9. According to the results, PB+5% TRI adhesive showed the same degree of conversion (DC) value (66.23±1.22%) as PB adhesive (66.41±0.85%), PB+10% TRI adhesive showed a slightly lower DC value (64.28±1.39%) than PB adhesive, while PB adhesive with 5% or 10% GSE showed a DC value of zero. The results suggest that TRI crosslinker has minimal interference with the photopolymerization of PB adhesive at concentrations below 10%, whereas GSE at concentrations of 5-10% completely stops the photopolymerization of PB adhesive.

[0086] From the above, it can be seen that the several objects and advantages of the present invention have been achieved and other advantageous results obtained. The collagen crosslinker mixture of the present disclosure is non-cytotoxic and actually promotes cell proliferation. It further exhibits strong inhibition of collagenases and MMPs, stabilizing collagen from degradation even at low concentrations and when mixed with commercial dental adhesives. It is colorless and fast acting, stabilizing collagen against collagenases after only 60 seconds, making it ideal for use in repairing and protecting collagen-containing tissues in the body, including those visible to the human eye.

[0087] Various modifications may be made in the above configurations without departing from the scope of the invention, and it is intended that all matter contained in the above description or shown in the drawings shall be interpreted as illustrative and not in a limiting sense.

Claims

1. structure: 【Chemistry 1】 (In the formula, R １ ~R １５ are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amine, linear alkyl, branched alkyl, cyclic alkyl, heteroalkyl, alkenyl, and alkynyl; R １６ ~R １８ are each independently an alkyl group. Collagen crosslinker TRI-1 having the formula: structure: 【Chemistry 2】 (In the formula, R １ ~R １１ are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amine, linear alkyl, branched alkyl, cyclic alkyl, heteroalkyl, alkenyl, and alkynyl; R １２ and R １３ are independently an alkyl group. A collagen crosslinker TRI-2 having the formula: structure: 【Chemistry 3】 (In the formula, R １ ~R １２ are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amine, linear alkyl, branched alkyl, cyclic alkyl, heteroalkyl, alkenyl, and alkynyl; R １３ and R １４ are independently an alkyl group. Collagen crosslinking agent TRI-3 ​​having A collagen crosslinker mixture comprising at least one of:

2. 2. The mixture of claim 1, wherein all "--R" substituents of TRI-1, TRI-2 and TRI-3 ​​are hydrogen.

3. 2. The mixture of claim 1, wherein the ratio of TRI-1:TRI-2:TRI-3 ​​is about 2:1:

1.

4. 1. A method for making a collagen crosslinker mixture, comprising: mixing a tricarbonylbenzene compound with a catecholamine to produce a mixture containing molecules of TRI-1 and TRI-2, wherein TRI-1 has the structure: 【Chemistry 4】 (In the formula, R １ ~R １５ are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amine, linear alkyl, branched alkyl, cyclic alkyl, heteroalkyl, alkenyl, and alkynyl; R １６ ~R １８ are independently an alkyl group. and TRI-2 has the structure: 【Chemistry 5】 (In the formula, R １ ~R １１ are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amine, linear alkyl, branched alkyl, cyclic alkyl, heteroalkyl, alkenyl, and alkynyl; R １２ and R １３ are independently an alkyl group. having reacting the mixture with a reducing agent; adding an alcohol to the mixture to obtain a mixture comprising TRI-1, TRI-2 and TRI-3, wherein TRI-3 ​​has the structure: 【Chemistry 6】 (In the formula, R １ ~R １２ are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amine, linear alkyl, branched alkyl, cyclic alkyl, heteroalkyl, alkenyl, and alkynyl; R １３ and R １４ are independently an alkyl group. having A method comprising:

5. 5. The method of claim 4, wherein the tricarbonylbenzene is trimesoyl chloride.

6. 5. The method of claim 4, wherein the catecholamine is dopamine.

7. The reducing agent is LiAlH ４ The method according to claim 4, wherein

8. The method of claim 4 , wherein the alcohol is ethanol.

9. Dental adhesives; and structure: 【Chemistry 7】 (In the formula, R １ ~R １５ are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amine, linear alkyl, branched alkyl, cyclic alkyl, heteroalkyl, alkenyl, and alkynyl; R １６ ~R １８ are independently an alkyl group. Collagen crosslinker TRI-1 having the structure 【Chemistry 8】 (In the formula, R １ ~R １１ are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amine, linear alkyl, branched alkyl, cyclic alkyl, heteroalkyl, alkenyl, and alkynyl; R １２ and R １３ are independently an alkyl group. and the collagen crosslinker TRI-2 having the structure: 【Chemistry 9】 (In the formula, R １ ~R １２ are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amine, linear alkyl, branched alkyl, cyclic alkyl, heteroalkyl, alkenyl, and alkynyl; R １３ and R １４ are independently an alkyl group. a collagen crosslinker mixture comprising at least one of the collagen crosslinkers TRI-3 ​​having the formula:

1. A dentin treatment mixture comprising:

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