Catecholamine-based membranes, process for their preparation and uses thereof

Abstract

The present invention relates to a process for preparing free-standing catecholamine-based membranes, comprising the steps of: a) creating a catecholamine membrane at an air / liquid interface in the absence of any support in a liquid medium in which both catechol and amine are soluble at a pH between 6.5 and 10 under suitable stirring, by combining a catechol derivative with an amine selected from the group consisting of: known aliphatic amine hydrocarbons of formula (II); and aromatic amines of formula (IIbis); and b) isolating the membrane obtained from step (a) from the air / liquid interface. The resulting free-standing catecholamine-based membranes are robust, easy to handle and manipulate, highly flexible, adaptable to any kind of surface without breakage, and adhesive. Furthermore, the free-standing membranes of the present invention exhibit Janus characteristics and have unexpected nanopatterning on the water-contacting surface, which provides the membranes of the present invention with a rougher surface, which has some value in promoting cell adhesion. TIFF2024526426000026.tif27159

Description

[Technical field]

[0001] This application claims the benefit of European Patent Application No. 21382516.9, filed June 10, 2021. Technical Field

[0002] The present invention relates to the field of catecholamine-based membranes. In particular, the present invention provides a free-standing catecholamine membrane, a process for preparing the same, and uses thereof. [Background technology]

[0003] Catechol derivatives are widely distributed in natural, animal and plant systems and they are all characterized by sharing an aromatic core of formula (I): [ka]

[0004] One well-known example of a catechol derivative is 3,4-dihydroxyphenylalanine (DOPA). DOPA has a tendency to oxidize when exposed to air. The formed o-quinones can further react with various nucleophiles in different ways to form crosslinks. Well-known nucleophiles are amines that can react with o-quinones to form adducts by either Michael addition or Schiff base reaction.

[0005] The reaction of catechol derivatives with amines is of great importance in natural biological processes such as cross-linking of adhesive proteins by marine organisms, formation of the cytoskeleton by insects, and biosynthesis of melanin. This catechol-amine chemistry that occurs in natural organisms has attracted much attention in materials science due to the growing interest in discovering new materials.

[0006] Thus, several research groups have developed water-soluble polymers that form gels or water-resistant films upon reaction of catechol derivatives with amines. In many publications, the preparation of catecholamine-based coatings has been reported. For example, Iacomino M. et al. (Iacomino M. et al., 2017) reported the coating of substrates by dip-coating with a solution containing caffeic acid and a diamine crosslinker at pH 9. The coated substrates were efficient in promoting cell growth. Suarez-Garcia S. et al. (Suarez-Garcia S. et al., 2017) also reported the functionalization of substrate surfaces (glass, gold, silica, etc.) with thin films obtained by copolymerization of catechol with diamines.

[0007] However, the use of these coated substrates is limited by the stiffness imparted by the substrate itself, and the catecholamine coatings adhere strongly to the substrate and therefore cannot be peeled off without damaging the substrate, as the coatings are not robust enough to withstand the necessary peeling forces.

[0008] Due to the limitations presented by these coatings, free-standing polymer films with well-defined structural and adaptive functions have attracted increasing attention in recent years. Various methods, such as layer-by-layer (LbL), Langmuir-Blodgett deposition, spin-coating and casting, have been developed to prepare free-standing polymer films.

[0009] Therefore, research on polymer films is still completely limited due to the lack of a simple and efficient synthesis method at present. For example, self-supporting polymer films can be prepared from self-assembled monolayers crosslinked by surface-initiated polymerization reactions, and have very thin properties as well as excellent chemical stability and sensitivity. However, this method requires the use of electron beams for crosslinking, and the harsh conditions may affect the wide application of this method. To alleviate this condition, methods of constructing or asymmetrically modifying the air-liquid interface to form polydopamine films at the air-water interface have also been considered. However, this film material was found to be prone to generating cracks during the preparation process and the stability of the film was poor. Other research groups have studied the synthesis using polyethyleneimine (PEI) as a support material. For example, Ponzio F et al. (Ponzio F.et al., 2016) disclosed the use of PEI to impart robustness to dopamine-based films. The reaction conditions require a basic pH, and the presence of PEI provides toxic by-products in biological environments, limiting the use of these films.

[0010] In view of the above, there remains a need to provide catecholamine-based membranes that are flexible, robust and safe, as well as processes for their preparation. Summary of the Invention [Problem to be solved by the invention]

[0011] We have developed a new process to obtain free-standing catecholamine-based membranes that have highly advantageous mechanical characteristics but also degradability, adhesion and cell proliferation profiles that make them particularly useful in therapeutic and diagnostic applications.

[0012] As shown below, the process of the present invention is based on the specific selection of amines as crosslinkers, either aliphatic or aromatic amines, which are hydrocarbon chains with terminal amines at each end of the chain, and stringent reaction conditions of pH and stirring. Notably, the pH reaction conditions are very mild, allowing in situ functionalization with biological molecules, which is a slight advantage when compared to prior art processes that work under basic conditions.

[0013] Thus, in a first aspect, the present invention provides a process for preparing a catecholamine-based membrane, the process comprising the steps of: a) reacting a catechol derivative with an amine selected from the group consisting of: a.1) Known aliphatic amines of formula (II) AB-A'(II) (In the formula, A and A' are the same or different and represent -NR1R'1; B represents the following: (C1-C 20 ) alkylene; -OH, halogen, -NO2, cyano, -O-(C1-C 10 (C1-C) substituted with one or more substituents selected from the group consisting of alkyl, -C(O)OR2, and -NR3R'3 20 ) alkylene; (C2-C 20 ) Alkenylene; -OH, halogen, -NO2, cyano, O-(C1-C 10 (C2-C) substituted with one or more substituents selected from the group consisting of alkyl, -C(O)OR4, and -NR5R'5 20 )Alkenylene;(C2-C 20 ) Alkynylene; -OH, halogen, -NO2, cyano, O-(C1-C 10 ) alkyl, -C(O)OR6, and -NR7R'7, substituted with one or more substituents selected from the group consisting of (C2-C 20 ) alkynylene; the known 3- to 20-membered heteroalkylenes; and -OH, halogen, -NO2, cyano, -O-(C1-C 10)alkyl, -C(O)OR8, and -NR9R'9; the known 3- to 20-membered heteroalkylenes substituted with one or more substituents selected from the group consisting of: -OH, halogen, -NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 10 , and -NR 11 R' 11 a known 3- to 20-membered heteroalkenylene substituted with one or more substituents selected from the group consisting of: A is attached to the first atomic ring member that forms part of the B biradical hydrocarbon backbone; A' is attached to the last atomic ring member that forms part of the B biradical hydrocarbon backbone; The "first" and "last" atomic ring members are identified by reading the B biradical hydrocarbon backbone from left to right or vice versa; R1, R'1, R3, R'3, R5, R'5, R7, R'7, R9, R'9, R 11 , and R' 11 are the same or different, -H; (C1-C 10 )Alkyl;(C1-C 10 )Haloalkyl;(C2-C 10 ) alkenyl; (C2-C 10 )Alkynyl; -OH, -NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 12 , and -NR 13 R' 13 (C1 to C 10 ) alkyl; R2, R4, R6, R8, R 10 and R 12 -H;(C1-C 10 )Alkyl;(C1-C 10 )Haloalkyl;(C2-C 10 ) alkenyl; (C2-C 10 )Alkynyl; -OH, -NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 14 , and -NR 15 R'15 (C1 to C 10 ) alkyl; R 13 , R' 13 , R 14 , R 15 and R' 15 are the same or different, -H; (C1-C 10 )Alkyl;(C1-C 10 )Haloalkyl;(C2-C 10 ) alkenyl; and (C 10 ) alkynyl; The "known 3- to 20-membered heteroalkylene" is C(R x )2, CR x , -N-, -NR' x It means a known saturated chain consisting of 3 to 20 members selected from the group consisting of -, -S-, and -O-, provided that (a) at least one of the ring members is -N-, -NR x -, -S-, or -O-; (b) the first and last ring members forming the heteroalkylene backbone are carbon atoms; The "known 3- to 20-membered heteroalkenylene" is C(R x )2, -CR x -, -N-, -NR' x It means a known unsaturated chain consisting of 3 to 20 ring members selected from the group consisting of -, -S-, and -O-, provided that (a) at least one of the ring members is -N-, -NR x (b) the first and last ring members forming the heteroalkylene backbone are carbon atoms; (c) it contains one or more double bonds; R x -H; -OH; (C1-C 10 )Alkyl;(C2-C 10 ) alkenyl; (C2-C 10 ) alkynyl; (C1-C 10 )Haloalkyl;O-(C1-C 10 ) alkyl; -O-(C2-C 10 )Alkenyl;-O-(C2-C 10 ) Alkynyl; Nitro, -NRx1 R x2 ;-NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 16 , and -NR 17 R' 17 (C1 to C 10 ) alkyl; and halogen; and R X1 , Rx2, R 16 , R 17 , R' 17 , and R' x is -H, (C1~C 10 ) Alkyl, (C2-C 10 ) alkenyl, (C2-C 10 ) alkynyl, and (C1-C 10 ) haloalkyl); and a.2) Aromatic amines of formula (IIbis): [ka] (Wherein, W is -NR t R' t or * represents SSL; * ) indicates that the S atom of the W radical is bonded to a carbon atom that forms part of an aromatic ring; and L is -OH, -NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 28 , and -NR 28 R' 28 (C1 to C 10 ) alkyl (provided that at least one of the substituents is -NR 28 R' 28 -OH, -NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 28 , and -NR 28 R' 28(C1-C10)haloalkyl substituted with one or more substituents selected from the group consisting of, provided that at least one of the substituents is -NR 28 R' 28 -OH, -NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 28 , and -NR 28 R' 28 (C2 to C 10 ) alkenyl (wherein at least one of the substituents is -NR 28 R' 28 -OH, -NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 28 , and -NR 28 R' 28 (C2 to C 10 ) alkynyl (wherein at least one of the substituents is -NR 28 R' 28 and the known 5- or 6-membered aromatic rings (each one of the ring members is -CR v -, -N-, -O-, -NR' v -S-; R m , R' m , R t and R' t are the same or different, H, (C1 to C 10 )Alkyl;(C1-C 10 )Haloalkyl;(C2-C 10 ) alkenyl; (C2-C 10 )Alkynyl; -OH, -NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 29 , and -NR 30 R' 30 (C1 to C 10 ) alkyl; and each one of the ring members is -CR z -, -N-, -NR' z-O-, -S-, ... R v At least one of the following is -NR 31 R' 31 and other R v (single possible) is H, -NR 31 R' 31 , (C1~C 10 )Alkyl;(C1-C 10 )Haloalkyl;(C2-C 10 ) alkenyl; (C2-C 10 ) alkynyl; and -OH, -NO2, cyano, -O-(C1-C 10 ) alkyl, C(O)OR 32 , and -NR 33 R' 33 (C1 to C 10 ) alkyl; R z and R' z H, (C1~C 10 )Alkyl;(C1-C 10 )Haloalkyl;(C2-C 10 ) alkenyl; (C2-C 10 )Alkynyl; -OH, -NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 34 , and -NR 35 R' 35 (C1 to C 10 ) alkyl; R 28 , R' 28 , R 29 , R 30 , R' 30 , R 31 , R' 31 , R 32 , R 33 , R' 33 , R 34 , R 35 , and R' 35 -H;(C1-C 10 )Alkyl;(C1-C10 )Haloalkyl;(C2-C 10 ) alkenyl; and (C 10 ) alkynyl; to create a catecholamine film at the air / liquid interface in the absence of any support, the crosslinking reaction being carried out in a liquid medium in which both the catechol and the amine are soluble at a pH of 6.5-10, particularly 6.5-8, and under stirring, particularly with adequate stirring; b) isolating the membrane resulting from step (a) from the air / liquid interface.

[0014] It is worth mentioning that, through the process of the present invention, membrane formation occurs at the liquid-air interface, avoiding the use of substrates (e.g., glass, metal) or polymers that provide support in situ (e.g., polyethyleneimine, PEI).

[0015] Stirring contributes to a continuous influx of O in the reaction medium, which also determines the oxidation of the catechol derivatives, and also allows the diffusion of the oxidized catechol moieties that crosslink with amino-based ligands at the interface.

[0016] Comparative testing of aliphatic amines having terminal amines at each end of the hydrocarbon chain of formula (II) with aliphatic monoamines having a terminal amine at only one end of the hydrocarbon chain (such as hexylamine) is shown below to act under the specific reaction conditions of the present invention to provide fragile films that are unable to isolate from the air / water interface.

[0017] A further advantage of the process of the present invention is that it is cheap and environmentally friendly, requiring only two starting materials in aqueous media, and avoiding the use of hazardous solvents.

[0018] Advantageously, the inventors have also found that aromatic amines of formula (IIbis) can provide free-standing membranes when reacted with catechol derivatives.

[0019] The process of the present invention allows for an exquisite control of the characteristics obtained. One of the characteristics that can be controlled is the final thickness of the membrane. This control can be easily achieved via concentration and reaction time. With increasing concentration of the starting reagents and longer reaction times, thicker membranes are formed. With this control, it is possible to produce membranes with thicknesses ranging from 50 nm to 3 μm, as measured by scanning electron microscopy (SEM) and atomic force microscopy (AFM), as shown below. It is also worth mentioning that, compared to thin membranes, thick membranes are easier to handle. Nevertheless, in all cases it was possible to isolate floating membranes (via functionalization) for their characterization and use.

[0020] The particular features of the process of the present invention make it possible to obtain catecholamine-based membranes at the air / liquid interface.Thus, the membranes that can be obtained by the process of the present invention are free-standing membranes (also called self-supporting membranes or self-supporting membranes), which means that they do not require a support for polymerization.

[0021] Moreover, the membrane obtained from the process of the present invention exhibits several advantageous properties. As shown below, the membrane obtained from the process of the present invention is robust, easy to handle and manipulate, highly flexible, and adaptable to any kind of surface without breakage. In fact, the membrane can be easily cut into various shapes (e.g., square, rectangular) using a sharp tool (e.g., scalpel or scissors, etc.) or produced in a pre-made mold with the desired shape. In contrast, the membrane obtained from aliphatic monoamines with terminal amines at only one end of the hydrocarbon chain is very fragile without support, and despite the efforts of the inventors, no regular membrane was obtained.

[0022] Moreover, the free-standing membranes of the present invention exhibit Janus properties. They exhibited different roughness depending on the side, as well as asymmetric chemical nature. In particular, the inventors discovered that the side in contact with the liquid medium (water in the examples) exhibited higher roughness due to the nanopatterning created based on catecholamine nanoparticles (produced during the process of the present invention), while the air-contacting side was smooth. Surprisingly, the inventors discovered that when the membranes of the present invention were formed and washed using ultrapure water, the nanopatterning of the liquid-contacting side of the membrane remained unchanged, i.e., the nanoparticles remained embedded in the surface. This unexpected nanopatterning on the liquid-contacting side results in the membranes of the present invention having a rougher surface. It is worth mentioning that this nanopatterning was not observed in the synthesized comparative membranes and substrates of PEI, both sides had similar topographies as observed by SEM. In these comparative examples, both sides exhibited similar surfaces without any different features between them.

[0023] Such morphological asymmetric properties add value to the membranes of the invention, since the liquid contacting (in the examples water contacting) surface promotes better cell adhesion compared to the smoother surfaces observed in comparable synthetic membranes with PEI or using substrates, where cells adhere much less and grow less densely. In the case of substrates, the membranes show similar characteristics on both sides, with a roughness of about 3.5 nm on both sides, which avoids the formation of nanoparticles that cannot then be embedded in the surface to form nanopatterning.

[0024] Furthermore, the self-supporting membranes of the prior art obtained using PEI cannot be used in contact with animals because they produce toxic by-products. Advantageously, the membranes of the present invention are safe and suitable for use in contact with animals or humans. As shown below, cells can adhere and grow on the membranes of the present invention even when they degrade, in contrast to when the membrane contains PEI (see examples below).

[0025] In summary, the membranes of the present invention exhibit improved characteristics with respect to catecholamine-based membranes reported in the prior art.

[0026] In view of the above, in a second aspect, the present invention provides a free-standing catecholamine-based membrane obtainable by the process of the first aspect of the invention.

[0027] As shown below, the particular properties of the membranes of the invention make them particularly efficient in supporting cell attachment and differentiation, which is indicative of their inherent regenerative capacity.

[0028] This is due in large part to the roughness and Janus properties exhibited by the membranes of the present invention, which further endow them with the ability to be readily functionalized with molecules of interest having either a therapeutic profile or visualization means.

[0029] As shown below, cell adhesion is favored on rough surfaces, which enhances cell proliferation and growth. This property is of particular interest for tissue regeneration applications, as the membranes can be used as a platform for growing new tissue in damaged areas.

[0030] Thus, in a third aspect, the present invention provides a catecholamine-based membrane as defined in the second aspect of the invention further comprising one or more therapeutic molecules for use in therapy, or alternatively a catecholamine-based membrane as defined in the second aspect of the invention further comprising one or more detection labels for use in diagnosis.

[0031] A fourth aspect of the invention relates to a catecholamine-based membrane as defined in the second aspect of the invention, further comprising one or more molecules of interest selected from the group consisting of cells, growth factors, and combinations thereof, for use in regenerating tissue.

[0032] In a fifth aspect, the present invention provides the use of a catecholamine-based membrane as defined in the second aspect of the invention as an adhesive (eg in the form of a patch).

[0033] In a sixth aspect, the present invention provides the use of a catecholamine-based membrane as defined in the second aspect of the invention as a vehicle for a molecule of interest.

[0034] In a final aspect, the present invention provides articles such as medical devices and / or electronic components (eg electrodes or sensors) coated partially or entirely with the free-standing film of the second aspect of the invention. [Brief description of the drawings]

[0035] [Figure 1] 3 corresponds to SEM images of different membranes. In the images it is possible to observe different topographies between the water contact surface (e and h) and the air contact surface (d and g). Furthermore, the flexibility of the membranes is confirmed by the absence of cracks or tears. a) Rolled membrane with high flexibility (1-7), b) Rolled membrane with high flexibility (3-7), c) Rolled membrane with air contact surface (2-7), d) Water contact surface of the membrane (2-7), e) Water contact surface of the membrane (3-7), f) Flexibility shown by membrane (5-7) with the air contact surface exposed, g) Air contact surface of the membrane (1-11), and h) Water contact surface of the membrane (1-11). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] All terms used herein in this application are to be understood in their ordinary meaning as known in the art unless otherwise specified. Other, more specific definitions of certain terms used in this application are set forth below and are intended to be applied uniformly throughout the specification and claims, unless a definition expressly set forth otherwise provides a broader definition.

[0037] For purposes of the present invention, any range given includes both the lower and upper endpoints of the range.

[0038] In a first aspect, the present invention provides a process for preparing a catecholamine-based membrane.

[0039] In the context of the present invention, the terms "catecholamine-based membrane" and "catecholamine-based film" have the same meaning and can be used interchangeably, referring to a material made of catechol derivatives crosslinked with an amine compound. In the context of the present invention, the catechol derivative is any compound containing a moiety of formula (I), and the amine is an aliphatic amine of formula (II) or an aromatic amine of formula (IIbis). Crosslinking can be confirmed by any suitable routine technique, such as infrared (FT-IR) or UV-visible spectroscopy. As shown below, the inventors confirmed successful crosslinking of the amine of formula (II) or (IIbis) with the catechol derivative by detecting the formation of a covalent bond between the catechol derivative and the amine-based ligand. This covalent interaction relies on the functional group of the catechol reacting with the terminal amino group. Typically, the polymerization of the catechol derivative with an amino-based molecule can be confirmed using FT-IR spectroscopy. For example, the FT-IR spectroscopy can be performed using a FT-IR spectroscopic technique with a wavelength of 3600-3000 cm. -1 The decrease in intensity bands associated with hydroxyl groups, appearing in the range of 1800–1650 cm, is a signal of copolymerization. Furthermore, the incorporation of amino-based ligands on the catechol rings forming the bridges is indicated by the decrease in intensity bands in the range of 1800–1650 cm. -1 Finally, the incorporation of aliphatic carbons present in amino-based ligands can be confirmed through the appearance of new bands in the range of 3000–2500 cm -1 The signal can be tracked by detecting a specific band in the range of

[0040] According to the present invention, the catechol derivative can be any that contains the moiety of formula (I). That is, a catechol derivative in the context of the present invention is any compound that contains or consists of a six-membered aromatic ring (o-benzenediol) that exhibits two adjacent hydroxyl groups. This catechol skeleton of formula (I) is present in various natural products, such as drugs that mimic them (such as MDMA), hormones / neurotransmitters, and catechins found in tea. Many pyrocatechin derivatives have been suggested for therapeutic applications. In addition, there are many catechol derivatives available commercially from several companies (such as Sigma-Aldrich, Alfa Aesar, Fisher Scientific, among others). Illustrative, non-limiting examples include pyrocatechol (CAS RN 120-80-9), pyrogallol (CAS RN 87-66-1), 4-methylcatechol (CAS RN 452-86-8), caffeic acid (CAS RN 331-39-5, 501-16-6, 71693-97-5), dopamine (CAS RN 51-61-6, 62-31-7), quercetin (CAS RN 117-39-5, 6151-25-3, 849061-97-8), hexahydroxytriphenylene (CAS RN 4877-80-9), catechin (CAS RN The following compounds are considered to be essential oils: catechin (CAS RN 7295-85-4, 154-23-4, 18829-70-4, 225937-10-0), gallic acid (CAS RN 149-91-7, 5995-86-8), tannic acid (CAS RN 1401-55-4), and epigallocatechin gallate (CAS RN 989-51-5).

[0041] In the context of this invention, the term "alkyl" (or "alkylene" when referred to as a "B" biradical) refers to a saturated straight or branched hydrocarbon chain containing the number of carbon atoms indicated in the claims and detailed description. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonanyl, decanyl, and the like.

[0042] In the context of this invention, the term "alkenyl" (or "alkenylene" when referred to as a "B" biradical) refers to a saturated straight or branched hydrocarbon chain containing one or more double bonds, with the number of carbon atoms indicated in the claims and detailed description. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.

[0043] In the context of this invention, the term "alkynyl" (or "alkynylene" when referred to as a "B" biradical) refers to a saturated straight or branched hydrocarbon chain containing the number of carbon atoms and one or more triple bonds as indicated in the claims and detailed description. Examples of alkyl groups include, but are not limited to, ethynyl, 1-propynyl, 2-butynyl, 1,3-butadinyl, 4-pentynyl, and 1-hexynyl.

[0044] The terms "known heteroalkylene" and "known heteroalkenylene" refer to heteroalkylenes and heteroalkenylenes that are known in the art and are intended to exclude those heterosystems that are not chemically possible.

[0045] In the context of the present invention, the term "known 3- to 20-membered heteroalkylene" refers to any known saturated chain containing 3 to 20 atoms selected from carbon and heteroatoms (i.e., atoms other than carbon atoms, such as N, O, or S), with the proviso that the first and last atoms forming the unsaturated chain are carbon atoms (to which A and A' are attached). For example, the ring atoms are -C(R x )2-, -CR x -, -N-, -NR' x -, -S-, and -O-, with the proviso that at least one of the ring members is -N-, -NR x -, -S-, or -O-. The heteroalkylene chain may be linear or branched. R x -H; -OH; (C1-C 10 )Alkyl;(C2-C 10 ) alkenyl; (C2-C 10) alkynyl; (C1-C 10 )Haloalkyl;O-(C1-C 10 ) alkyl; -O-(C2-C 10 )Alkenyl;-O-(C2-C 10 ) Alkynyl; Nitro, -NR x1 R x2 ;-OH, -NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 16 , and -NR 17 R' 17 (C1 to C 10 ) alkyl; and halogen. 16 , R 17 , R' 17 , and R' x is -H, (C1~C 10 ) Alkyl, (C2-C 10 ) alkenyl, (C2-C 10 ) alkynyl, and (C1-C 10 ) haloalkyl.

[0046] In the context of the present invention, the term "heteroalkenylene" of the known 3- to 20-membered type refers to any of the known unsaturated chains containing one or more double bonds and consisting of 3 to 20 ring atoms selected from carbon and heteroatoms (i.e., atoms other than carbon atoms, such as N, O or S), with the proviso that at least the first and last atoms forming the chain are carbon atoms (to which A and A' are attached). For example, the ring atoms are -C(R x )2-, -CR x -, -N-, -NR' x -, -S-, and -O-, with the proviso that at least one of the ring members is -N-, -NR' x -, -S-, or -O-. The heteroalkylene chain may be linear or branched. R x -H; -OH; (C1-C 10 )Alkyl;(C2-C 10 ) alkenyl; (C2-C 10) alkynyl; (C1-C 10 )Haloalkyl;O-(C1-C 10 ) alkyl; -O-(C2-C 10 )Alkenyl;-O-(C2-C 10 ) Alkynyl; Nitro, -NR x1 R x2 ;-OH, -NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 16 , and -NR 17 R' 17 (C1 to C 10 ) alkyl; and halogen. 16 , R 17 , R' 17 , and R' x is -H, (C1~C 10 ) Alkyl, (C2-C 10 ) alkenyl, (C2-C 10 ) alkynyl, and (C1-C 10 ) haloalkyl.

[0047] In the context of this invention, the term "haloalkyl" refers to a saturated straight or branched hydrocarbon chain containing the number of carbon atoms indicated in the claims and the detailed description, where at least one of the carbon atoms is substituted with at least one halogen.

[0048] The carbon number of alkyl, alkenyl, alkynyl, alkylene, alkenylene, and alkynylene is represented by "C" (the symbol for carbon atom) followed by a subindex number indicating the number of carbon atoms. Thus, "C1" means that the alkyl has one carbon atom; for example, "(C1-C 20 When referring to a range in the form "alkyl," it means that the hydrocarbon has from 1 to 20 carbon atoms.

[0049] The term "known ring system" refers to ring systems that are known in the art and is therefore intended to exclude ring systems that are chemically impossible.

[0050] According to the present invention, a ring system formed by an "isolated" ring means that the ring system is formed by two, three or four rings, said rings being connected via a bond from an atom of one ring to an atom of the other ring. The term "isolated" also encompasses embodiments in which the ring system has only one ring. Non-limiting examples of known ring systems consisting of one ring are those derived from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, phenyl, biphenylyl and cycloheptenyl.

[0051] According to the present invention, the expression "fused ring" includes fully fused, partially fused, or spiro-fused rings.

[0052] According to the present invention, when a ring system is "fully fused", it is meant that the ring system is formed by two, three or four rings in which two or more atoms are common to two adjacent rings. Illustrative non-limiting examples are 1,2,3,4-tetrahydronaphthyl, 1-naphthyl, 2-naphthyl, anthryl, or phenanthryl.

[0053] According to the present invention, when a ring system is "partially fused", it is meant that the ring system is formed by three or four rings, at least two of which are fully fused (i.e., two or more atoms are common to two adjacent rings), and the remaining ring(s) are connected via a bond from an atom of one of the rings to an atom of one of the fused rings.

[0054] According to the present invention, when a ring system is "spiro-fused", it means that the ring system contains at least two rings that share a common atom. The simplest spiro compounds are bicyclic (having only two rings) or have a bicyclic moiety as part of a larger ring system, in either case the two rings are connected through one common atom as defined. Spiro compounds can be fully carbocyclic (all carbon) or heterocyclic (having one or more non-carbon atoms that form part of the backbone of the ring).

[0055] In the context of this invention, the terms "halo" and "halogen" are used interchangeably and refer to a halogen radical selected from the group consisting of chloro, fluoro, bromo and iodo.

[0056] In one embodiment, the catechol derivative has formula (III): [ka] (In the formula, R 18 , R 19 , R' 18 and R' 19 are the same or different: -H; -OH; -NR 20 R' 20 ; halogen (C1~C 10 ) alkyl; (C2~C 10 ) alkenyl; -OH, halogen, nitro, cyano, (C1-C 10 ) Alkyl, (C1-C 10 ) haloalkyl, -NR 21 R' 21 , -C(O)OR 22 , and -O-(C1-C 10 ) alkyl substituted with one or more substituents selected from the group consisting of (C1-C 10 ) alkyl; -OH, halogen, nitro, cyano, (C1-C 10) Alkyl, (C1-C 10 ) haloalkyl, -NR 23 R' 23 , -C(O)OR 24 , and -O-(C1-C 10 ) alkyl substituted with one or more substituents selected from the group consisting of (C2-C 10 ) alkenyl; Each one of the rings is (a)-C(R y ) 2,- , -CR y -, -N-, -NR' y (b) is saturated, partially unsaturated, or aromatic; (c) is a known ring system consisting of one or two rings, which may be isolated, partially isolated, or fused; is selected from the group consisting of R y Each of the is -H, -OH, (C1-C 10 ) Alkyl, (C2-C 10 ) alkenyl, (C2-C 10 ) alkynyl, (C1-C 10 ) haloalkyl, -O-(C1-C 10 ) Alkyl, Nitro, -NR 25 R 25 and halogen; R'y is -H, (C1-C 10 ) Alkyl, (C2-C 10 ) alkenyl, (C2-C 10 ) alkynyl, and (C1-C 10 ) haloalkyl; R 20 , R' 20 , R 21 , R' 21 , R 23 , R' 23 , R 25 , and R' 25 are the same or different, H; (C1-C 10 )Alkyl;(C1-C 10 )Haloalkyl;(C2-C 10 ) alkenyl; (C2-C10 ) Alkynyl; -OH, halogen, nitro, cyano, (C1-C 10 ) Alkyl, (C1-C 10 ) haloalkyl, -NR 26 R' 26 , -C(O)OR 27 , and -O-(C1-C 10 ) alkyl substituted with one or more substituents selected from the group consisting of (C1-C 10 ) alkyl; R 22 and R 24 H, (C1~C 10 ) Alkyl, (C1-C 10 )Haloalkyl, (C2-C 10 ) alkenyl; and (C 10 ) alkynyl; R 26 , R' 26 and R 27 are the same or different, -H; (C1-C 10 )Alkyl;(C1-C 10 )Haloalkyl;(C2-C 10 ) alkenyl; and (C 10 ) alkynyl) It is of the following.

[0057] In another embodiment, optionally in combination with any of the embodiments provided above or below, the catechol derivative of formula (III) is R 18 and R 19 are the same or different, -H; -OH; (C1-C 10 ) alkyl; (C1-C 10 ) alkyl; and (C2-C 10 In an alternative embodiment, optionally in combination with any of the embodiments provided above or below, the catechol derivative of formula (III) is selected from the group consisting of R 18 and R 19 One of the rings is (a)-C(Ry)2,- , -CR y -, -N-, -NR' y (b) a 5-6 member ring selected from the group consisting of -, -S-, and -O-; (c) a known ring system consisting of two rings that are isolated, partially isolated, or fused, wherein R y and R' y is as defined above. In an alternative embodiment, optionally in combination with any of the embodiments provided above or below, the catechol derivative of formula (III) is 18 and R 19 One of the rings is (a)-C(R y ) 2,- , -CR y -, -N-, -NR' y (b) a known ring system consisting of two rings that are saturated, partially unsaturated, or aromatic; and (c) a fused ring system, wherein R y and R' y is as defined above. In an alternative embodiment, optionally in combination with any of the embodiments provided above or below, the catechol derivative of formula (III) is 18 and R 19 One of the rings is (a)-C(R y ) 2,- , -CR y -, -N-, -NR' y -, -S-, and -O-, provided that at least one of the rings is not a heteroatom (-N-, -NR' y (b) a known ring system consisting of two rings that are saturated, partially unsaturated, or aromatic; and (c) fused, wherein R y and R' y is as defined above. In an alternative embodiment, optionally in combination with any of the embodiments provided above or below, the catechol derivative of formula (III) is 18 and R19 One of the rings is (a)-C(R y ) 2,- , -CR y -, -N-, -NR' y (b) a known ring system consisting of two rings that are saturated, partially unsaturated, or aromatic, and (c) fused, each of which is a 6-member ring selected from the group consisting of -, -S-, and -O-, with the proviso that at least one of the rings contains an -O-heteroatom, wherein R y and R' y is as defined above. In an alternative embodiment, optionally in combination with any of the embodiments provided above or below, the catechol derivative of formula (III) is 18 and R 19 One of the rings is (a)-C(R y ) 2,- , -CR y -, -O-, with the proviso that at least one of the rings contains an -O- heteroatom; (b) is saturated, partially unsaturated, or aromatic; and (c) is a known ring system consisting of two rings that are fused, wherein R y and R' y is as defined above. In an alternative embodiment, optionally in combination with any of the embodiments provided above or below, the catechol derivative of formula (III) is 18 and R 19 Each of the rings is (a)-C(R y ) 2,- , -CR y -, -O-, with the proviso that only one of the rings contains an -O- heteroatom; (b) is saturated, partially unsaturated, or aromatic; and (c) is a known ring system consisting of two rings that are fused, wherein R y and R' y is as defined above.

[0058] In one embodiment, optionally in combination with any of the embodiments provided above or below, the catechol of formula (III) is R 18 or R 19 one of which is -H and the other is as defined in any of the above embodiments.

[0059] In one embodiment, optionally in combination with any of the embodiments provided above or below, the catechol derivative of formula (III) is R' 18 and R' 19 is -H and R 18 and R 19 is as defined in any of the above embodiments.

[0060] In one embodiment, optionally in combination with any of the embodiments provided above or below, the catechol derivative is selected from the group consisting of pyrocatechol, dopamine, pyrogallol, caffeic acid, 4-methylcatechol, and catechin.

[0061] In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II):

[0062] In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where A and A' are the same. In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where A and A' are the same or different and represent NHR'1. In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where A and A' are the same and represent NHR'1. In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where at least one of A and A' represents -NH2. In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where A and A' are the same and represent -NH2.

[0063] In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B is (C1-C 20 ) alkylene; (C1-C 20 ) alkylene; or the known 3- to 20-membered heteroalkylene.

[0064] In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B is (C1-C 20 ) alkylene; (C1-C 20 ) alkylene; or the known 3- to 20-membered heteroalkylene, where A and A' are the same (such as -NH2).

[0065] In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B is (C1-C 15 ) alkylene; (C1-C 15 ) alkylene; or the known 3- to 15-membered heteroalkylene.

[0066] In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B is (C1-C 15 ) alkylene; (C1-C 15 ) alkylene; or the known 3- to 15-membered heteroalkylene, where A and A' are the same (such as -NH2).

[0067] In one embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B is (C1-C 15 ) alkylene, (C2-C 15 ) Alkylene, (C4-C 15 ) alkylene or (C5-C 15 ) alkylene.

[0068] In one embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B is (C1-C 15 ) alkylene, (C2-C 15 ) Alkylene, (C4-C 15 ) alkylene or (C5-C 15 ) alkylene, where A and A' are the same (such as -NH2).

[0069] In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B represents a known 3- to 15-membered heteroalkylene, particularly a known 3- to 10-membered heteroalkylene, or a known 4- to 8-membered heteroalkylene. In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B represents a known 3- to 15-membered heteroalkylene, particularly a known 3- to 10-membered heteroalkylene; A and A' are the same (such as -NH2). In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B represents a known 3- to 15-membered heteroalkylene, particularly a known 3- to 10-membered heteroalkylene, and the ring members are selected from carbon, S, and N atoms as defined above. In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B represents a known 3- to 15-membered heteroalkylene, in particular a 3- to 10-membered heteroalkylene, each one of the ring members being C(R x )2, CR x , -NR' x -, and -S-, wherein R x and R' x is as defined above.

[0070] In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B represents a known 3- to 15-membered heteroalkylene, in particular a known 3- to 10-membered heteroalkylene, the ring members being selected from carbon, S, and N atoms as defined above; and A and A' are the same, e.g., -NH2.

[0071] In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B represents a known 3- to 15-membered heteroalkylene, in particular a known 3- to 10-membered heteroalkylene, two of the ring members are S atoms as defined above, and the others are carbon atoms. In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B represents a known 3- to 15-membered heteroalkylene, in particular a known 3- to 10-membered heteroalkylene, two of the ring members are S atoms as defined above, and the others are carbon atoms; and A and A' are the same, for example -NH2. In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II) where B represents a known 4- to 15-membered heteroalkylene, particularly a known 4- to 10-membered heteroalkylene, and the ring members are carbon and S atoms, as defined above. In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II) where B represents a known 4- to 15-membered heteroalkylene, particularly a known 4- to 10-membered heteroalkylene, and the ring members are carbon and -NR', as defined above. x -atom. In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B represents a known 4- to 15-membered heteroalkylene, in particular a known 4- to 10-membered heteroalkylene, the ring members being carbon and S atoms as defined above; and A and A' are the same, for example -NH2. In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B represents a known 4- to 15-membered heteroalkylene, in particular a known 4- to 10-membered heteroalkylene, the ring members being carbon and -NR', as defined above. xatom; and A and A' are the same, for example -NH2. In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B represents a known 4- to 15-membered heteroalkylene, in particular a known 4- to 10-membered heteroalkylene, two of the ring members being S atoms as defined above, and the remaining ring members being carbon atoms. In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B represents a known 4- to 15-membered heteroalkylene, in particular a known 4- to 10-membered heteroalkylene, one of the ring members being -NR', as defined above. x A is an atom, and the remaining ring members are carbon atoms. In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B represents a known 4- to 15-membered heteroalkylene, in particular a known 4- to 10-membered heteroalkylene, two of the ring members are S atoms as defined above, and the remaining ring members are carbon atoms; and A and A' are the same, for example -NH2. In these embodiments where the heteroalkylene is a heteroalkylene containing two S atoms, the heteroalkylene may also be referred to as an "alkylene disulfide". Illustrative non-limiting examples are cystamine, 4-aminophenyl disulfide, and bisaminopolyethylene glycol disulfide, among others.

[0072] In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B represents a known 4- to 15-membered heteroalkylene, in particular a known 4- to 10-membered heteroalkylene, one of the ring members being -NR' as defined above. x - and the remaining ring members are carbon atoms; and A and A' are the same, for example -NH2.

[0073] In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II) where B represents a known 4- to 15-membered heteroalkylene, in particular a known 4- to 10-membered heteroalkylene, and each one of the ring members is selected from the group consisting of C(R x )2, CR x , -NR' x -, and -S-, wherein R x and R' x is as defined above.

[0074] In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine is a known aliphatic amine of formula (II), where B represents a known 3- to 15-membered heteroalkylene, in particular a known 3- to 10-membered heteroalkylene; (a) One or two of the ring members are -S- atoms and the other ring members are C(R x )2 and CR x or alternatively, (b) one or two of the ring members is -NR' x - and the other ring members are C(R x )2 and CR x wherein Rx is as defined above; (c) One of the ring members is a -S- atom and the other ring member is a C(R x )2 and CR x or alternatively, (d) one of the ring members is -NR' x - and the other ring members are C(R x )2 and CR x is a carbon atom selected from the group consisting of: (c) Two of the ring members are -S- atoms and the other ring member is C(R x )2 and CR x or alternatively, (f) Two of the ring members are -NR' x- and the other ring members are C(R x )2 and CR x is a carbon atom selected from the group consisting of: In the formula, R x and R' x is as defined above.

[0075] In one embodiment, optionally in combination with any of the embodiments provided above or below, the known aliphatic amines of formula (II) are selected from the group consisting of amines of formula (II1), amines of formula (II2), and amines of formula (II3): [ka] In the formula, a is an integer from 1 to 20; b, d, and e are independently integers from 1 to 10, more specifically, b, d, and e are the same; f and g are independently integers from 1 to 10, more specifically, f and g are the same.

[0076] In one embodiment, optionally in combination with any of the embodiments provided above or below, the known aliphatic amine of formula (II) is selected from the group consisting of hexamethylenediamine, octamethylenediamine, dodecamethylenediamine, cystamine, tris-(3-aminopropyl)amine, and tris-(2-aminopropyl)amine, more specifically selected from hexamethylenediamine, dodecamethylenediamine, octamethylenediamine and cystamine.

[0077] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis): In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis1): [ka] In the formula, R m , R' mand W is as defined above.

[0078] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1) comprising at least two amino groups.

[0079] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), and W is -NR t R' t Represents.

[0080] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), and W is -NR t R' t R m , R' m , R t and R' t In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), and W is -NR t R' t R m , R' m , R t and R' t are the same and represent -H.

[0081] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), and W is -NR t R' t R m and R' mare the same and represent -H, and Rt and R't are the same and are as defined above.

[0082] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), and W is -NR t R' t R t and R' t are the same and are other than hydrogen.

[0083] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), and W is -NR t R' t R m and R' m is hydrogen; R t and R' t are the same and are other than hydrogen.

[0084] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), and W is -NR t R' t R m and R' m are the same and represent -H and R t and R' represents an aromatic ring as defined above in the first aspect of the invention. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), W is -NR t R' t R m and R' m are the same and represent -H and R t and R' tEach one of represents an aromatic ring having 6 members as defined above in the first aspect of the invention.

[0085] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), and W is -NR t R' t R t and R' t represents an aromatic ring as defined above.

[0086] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), and W is -NR t R' t R t and R' t Each one of represents an aromatic ring having 6 members as defined above.

[0087] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), and W is -NR t R' t R t and R' t each one of represents an aromatic ring having six members as defined above, all ring members being -CR z -, wherein R z is as defined above.

[0088] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), and W is -NR t R' t R t and R' trepresent aromatic rings that are the same and have six members, all of the ring members being -CR z -ring member, where R z is as defined above.

[0089] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), and W is -NR t R' t R m and R' m are the same and represent -H and R t and R' t each one of represents an aromatic ring having six members, all of the ring members being -CR z -, wherein R z is as defined above. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), W is -NR t R' t R m and R' m are the same and represent -H and R t and R' t are the same and represent an aromatic ring having six members, all of the ring members being -CR z -ring member, where R z is as defined above.

[0090] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is one of the formulae (IIbis) or (IIbis1), where W=NR t R' t and R m , R' m , R t and R' t One or more of the following has the formula (IV): [ka] where n is 1 or 2, especially 1, and the other(s) are -H.

[0091] Alternatively, in another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), and W is * In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), W represents * R stands for SSL m and R' m In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), and W is * R stands for SSL m and R' m are the same and represent -H. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), W is * R stands for SSL m and R' m are the same and L represents an aromatic ring as defined above. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), W is * R stands for SSL m and R' m are the same and represent -H, and L represents an aromatic ring as defined above. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), W is * R stands for SSLm and R' m are the same and L represents an aromatic ring having 6 members as defined above. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), W is * R stands for SSL m and R' m are the same and represent -H, and L represents an aromatic ring having 6 members as defined above. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), W is * R stands for SSL m and R' m are the same, L represents an aromatic ring having 6 members as defined above and may be the same or different, -CR v In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), and W is represented by: * R stands for SSL m and R' m are the same and represent -H, L represents an aromatic ring having 6 members as defined above and are the same or different, in particular are the same and represent -CR v -Represented by:

[0092] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), wherein L represents an aromatic ring as defined above.

[0093] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), wherein L represents an aromatic ring having 6 members, as defined above.

[0094] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), and W is * R stands for SSL m and R' m are the same, and L represents an aromatic ring having 6 members as defined above, which may be the same or different and is represented by -CRv-.

[0095] In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), and L has the following formula (IV): [ka] wherein n is 1 or 2, and one of the carbon atoms forming the aromatic ring is bonded to the -S- atom. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the amine of formula (IIbis) is one of formulae (V), (VI) or (VII): [ka]

[0096] In one embodiment, optionally in combination with any of the embodiments provided above or below, the amine is selected from the group consisting of hexamethylenediamine, octamethylenediamine, dodecamethylenediamine, cystamine, tris-(3-aminopropyl)amine, tris-(2-aminopropyl)amine, and 4,4',4''-triaminotriphenylamine.

[0097] The catechol derivative and amine solutions can be prepared separately and then mixed together, or one of the catechol derivative and amine can be prepared in solution form and the other can be obtained directly from a supplier and added.

[0098] The appropriate solvent system for preparing the solution(s) depends on the polar nature of the catechol and amine, which is part of the general knowledge of the person skilled in the art. Both the catechol and amine must be dissolved in the same solvent system to carry out the crosslinking reaction. In one embodiment, the catechol and amine are prepared in the form of an aqueous solution. The term "aqueous solution" encompasses not only solutions consisting of water alone, but also those in combination with water and other polar solvents, such as water + alcohol or aqueous buffers. The aqueous solution is obtained by simply mixing the compound with water. In any case, when mixing the catechol and amine, this step is carried out under stirring (to properly promote the formation of a film at the air / liquid interface).

[0099] In one embodiment, optionally in combination with any of the embodiments provided above or below, the pH of the solution is 6.5-8, or 7-7.5. In another embodiment, optionally in combination with any of the embodiments provided above or below, the pH is 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8., 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10. There are well-known commercially available buffers that can be added to the reaction medium to provide the intended pH value. Illustrative non-limiting examples are phosphate buffered saline (PBS), carbonate-bicarbonate and citrate, among others. The buffer may be added to the reaction medium in any order with respect to the addition of the catechol derivative and the amine.

[0100] In one embodiment, optionally in combination with any of the embodiments provided above or below, the liquid medium comprises an aqueous-based buffer, more specifically an aqueous-based buffer in which no nitrogen atoms or amino groups are present, even more specifically an aqueous-based buffer selected from the group consisting of phosphate buffers, carbonate buffers, and citrate buffers.

[0101] In another embodiment, optionally in combination with any of the embodiments provided above or below, the liquid medium is water.

[0102] After adjusting the pH, the stirring is also adjusted. Stirring may be performed during the entire crosslinking step. In this embodiment, as the skilled person will understand, the stirring speed must not create turbulence that would prevent the proper formation of the membrane. The skilled person can easily check whether the speed is appropriate: if no turbulence is observed in the reaction medium, this indicates that the speed is appropriate. If the skilled person observes the onset of turbulence in the liquid medium during the adjustment, this indicates that the speed is not appropriate and that it is necessary to reduce the speed until the turbulence disappears. The suitable stirring means used during the entire crosslinking step are magnetic (or mechanical) means, using the lowest possible speed to avoid turbulence at the air / liquid interface, homogeneous during the entire procedure, and avoiding irregular movements of magnets or mechanical parts involved in the stirring procedure. The skilled person can routinely determine the appropriate stirring speed depending on the volume of the reactor, the liquid medium, and the stirring means. For example, for the examples provided below, where the volume is about 20 mL, the inventors established a suitable stirring of less than 500 rpm using a magnetic stirrer. In one embodiment, optionally in combination with any of the embodiments provided above or below, the stirring speed is less than or equal to 450 rpm, less than or equal to 400 rpm, or less than or equal to 350 rpm.

[0103] Alternatively, stirring can be performed during part of the crosslinking reaction. In this embodiment, the stirring rate can be any rate at which, once stirring is stopped, the reaction medium is left for a period of time, e.g., the oxidized catechol moieties that crosslink with the amino-based ligand diffuse to the interface, align, and form a film. In this alternative embodiment, stirring can be stopped, e.g., when a visual color change is detected due to oxidation of the catechol moieties. The copolymerization reaction with the amino-based ligand is then left to complete, terminating in the formation of a floating film at the air / liquid interface.

[0104] In one embodiment, optionally in combination with any of the embodiments provided above or below, agitation is performed during the entire crosslinking step or during part of the crosslinking reaction.

[0105] In one embodiment, optionally in combination with any of the embodiments provided above or below, stirring is performed in a turbulent manner during the entire crosslinking step, in particular the stirring speed is equal to or less than 450 rpm, equal to or less than 400 rpm, or equal to or less than 350 rpm.

[0106] In another embodiment, optionally in combination with any of the embodiments provided above or below, the stirring is performed using the lowest speed possible to avoid turbulence at the air / liquid interface, to be homogeneous throughout the entire procedure, and to avoid irregular movement of magnets or mechanical parts involved in the stirring procedure, more specifically, the stirring speed is equal to or less than 450 rpm, equal to or less than 400 rpm, or equal to or less than 350 rpm.

[0107] In another embodiment, optionally in combination with any of the embodiments provided above or below, stirring is performed only during a portion of the crosslinking reaction, in particular stirring is performed in a turbulent manner until a color change is observed and then stopped, more particularly stirring is performed for 10 minutes to 2 hours and then stopped.In another embodiment, optionally in combination with any of the embodiments provided above or below, the amine of formula (II) or (IIbis) is in excess relative to the catechol derivative in a molar ratio, in particular the molar ratio of the amine compound of formula (II) to the catechol derivative is comprised between 1.1:1 and 3:1, in particular between 1.2:1 and 2:1.

[0108] In another embodiment, optionally in combination with any of the embodiments provided above or below, the crosslinking step is carried out at a temperature between 10 and 60° C., in particular at a temperature between 10 and 50° C., between 12 and 40° C., or between 15 and 38° C.

[0109] In another embodiment, optionally in combination with any of the embodiments provided above or below, the crosslinking step is carried out for at least 24 hours. In one embodiment, optionally in combination with any of the embodiments provided above or below, step (a) is carried out at a pH value of 7 to 7.5 and stirring equal to or less than 300 rpm. In another embodiment, optionally in combination with any of the embodiments provided above or below, step (a) is carried out at a pH value of 7 to 7.5, stirring equal to or less than 300 rpm, and the molar ratio of the amine compound of formula (II) to the catechol derivative is comprised between 1.1:1 and 3:1, in particular between 1.2:1 and 2:1.

[0110] One skilled in the art can adjust routine parameters such as reaction time or concentration of reagents, which helps to adjust the final thickness of the film.

[0111] Once the crosslinking step is complete and the catecholamine film is created at the air / water or air / liquid interface in the absence of any support, it is isolated from the medium. This can be done manually (at laboratory scale), for example using tweezers, or mechanically (at industrial scale), for example using a ring-shaped tool.

[0112] In a second aspect, the present invention provides a catecholamine-based membrane obtainable by the process of the invention as defined above in either its first aspect or in any of the specific embodiments described above.

[0113] Thus, all embodiments provided above in relation to the process are also embodiments of this second aspect of the invention.

[0114] The terms "obtainable" and "obtained" have the same meaning and are used interchangeably. In each case, the term "obtainable" encompasses the term "obtained."

[0115] The chemical asymmetry exhibited by the membranes of the present invention, as already explained above, offers different functionalization options and greater versatility as a platform. By having different chemical groups on each side of the membrane, specific molecules can be immobilized depending on their activity. For example, the rougher, water-contacting side can be functionalized with growth factors, while the air-contacting side (with lower roughness) can be functionalized with antibacterial moieties to avoid infection during the regeneration process. For this reason, the versatility of these membranes allows functionalization with (bio)molecules of different chemical nature depending on the side, since the chemical groups exposed on each side of the membrane are different.

[0116] Thus, in one embodiment, the catecholamine-based films of the present invention include one or more molecules of interest, such as a therapeutic molecule (e.g., a peptide, protein, or antibody, such as, inter alia, an antimicrobial moiety (e.g., cetrimide or silver nanoparticles), or a growth factor (e.g., FGF-2 or TGFβ3)) or, inter alia, a detection label (e.g., a nanoparticle such as calcein, a metal nanoparticle, or an antibody).

[0117] In one embodiment, optionally in combination with any of the embodiments provided above or below, the catecholamine-based membrane further comprises one or more molecules of interest selected from the group consisting of therapeutic molecules, cells, growth factors, detection labels (fluorescently active moieties, nanoparticles or antibodies), and combinations thereof.

[0118] The present invention also relates to a process for preparing a catecholamine-based membrane of the present invention comprising one or more molecules of interest, comprising: (i.1) carrying out step (a) of the process defined in the first aspect of the invention or any embodiment of this aspect, (i.2) adding a molecule of interest to the reaction medium; and (i.3) carrying out step (b) of the process of the first aspect of the invention; or or, (ii.1) incubating the catecholamine-based membrane of the second aspect of the invention with a molecule of interest. The process is also provided.

[0119] Step (i.2) is carried out by adding the molecule of interest either directly to the reaction medium obtained from the supplier or in the form of an aqueous suspension (following the manufacturer's instructions). The mild conditions used in the process of the invention advantageously allow the functionalization of the membrane in the unique reaction medium in which it was formed, advantageously simplifying the process and reducing time and costs. Since the membrane is formed at the air / water or air / liquid interface, the addition step can be carried out by pouring the solution into the reaction medium or, if the membrane covers the entire surface of the reaction medium, by injecting a solution of the molecule through the membrane into the reaction medium. The latter does not have a negative effect on the integrity of the membrane, which is a further indication of the robustness and improved mechanical properties of the membranes of the invention.

[0120] In the case of step (ii.1), the membrane is already isolated from the interface. The membrane may therefore be incorporated into a medium containing a solution containing the molecule of interest (and the surface in contact with the medium is functionalized), or alternatively, the membrane is incorporated into the medium and a solution containing this molecule is injected into the solution. The functionalized surface can be preselected and a membrane is deposited with the desired surface in contact with the liquid phase (aqueous phase) containing the molecule of interest. The membrane has exposed functional groups on its surface that can be used as anchoring points.

[0121] Functionalization of the membrane surface with the molecule of interest can occur by covalent attachment, adsorption, or electrostatic interactions. In the case of covalent attachment, functionalization can occur via nucleophilic or electrophilic attack, depending on the chemical nature of the molecule of interest. For example, the presence of an acyl chloride (e.g., stearoyl chloride) can act as an electrophile in the presence of an amino terminal group. On the other hand, an amino group (e.g., hexadecaamine) or a thiol group (e.g., 1,8-octanedithiol) can act as a nucleophilic molecule covalently attached to the ring of a catechol derivative. Furthermore, the presence of a carboxylic acid in the backbone of the molecule of interest can be used to form an amide bond (peptide-like bond) with the terminal amino group exposed on the surface of the membrane. Finally, physical adsorption of the molecule of interest can be induced by entrapment of the molecule in the backbone of the membrane. In one embodiment of the present invention, the molecule of interest is covalently attached to the membrane surface of the second aspect of the present invention.

[0122] In one embodiment, optionally in combination with any of the embodiments provided above or below, the catecholamine-based membrane of the present invention further comprises one or more molecules of interest selected from the group consisting of therapeutic molecules and detection labels (fluorescently active moieties, nanoparticles or antibodies). More specifically, the molecules of interest are covalently bound to the membrane, in particular by amide bonds.

[0123] Covalent attachment can be achieved by any routine protocol, such as generating a conjugated amide between the free amino groups of the membrane of the invention and the free carboxyl groups of the molecule of interest. In this case, the coupling reaction can be carried out using (1-ethyl-3-(3-dimethylamino)propylcarbodiimide, hydrochloride (EDC) and N-hydroxysuccinimide (NHS). As a general procedure, the formed membrane can be introduced into a larger diameter Petri dish containing a buffer solution within the ranges mentioned above. The EDC is then injected into the aqueous phase. After a suitable time, such as 30 minutes, an active (bio)molecule (e.g., antimicrobial moiety, fluorescent dye, growth factor, etc.) is injected into the aqueous phase along with the NHS agent. The reaction is stirred slowly at less than 500 rpm for a sufficient time, e.g., 60 minutes, to ensure the formation of the covalent bond. The functionalized membrane can then be extracted from the Petri dish and washed. The functionalization can be independently controlled and induced on both sides of the membrane, thereby enhancing the chemical Janus behavior of the membrane. Other catechol derivatives with different functional groups, such as terminal thiols, amines, or acyl chlorides, can also be functionalized through nucleophilic or electrophilic attacks on exposed functional groups on the membrane or ring of the catechol derivative. The procedure is similar to that described above. Some parameters, such as pH or solvent, can be routinely adjusted or selected to activate the reaction between the molecule of interest and the membrane.

[0124] As also explained above and discussed in detail below, the particular Janus characteristics of the membranes of the present invention allow efficient functionalization from both sides, a further unique property with respect to membranes already reported in the prior art.

[0125] The membrane of the invention itself exhibits improved cell adhesion when compared to prior art membranes, supporting its utility as an adhesive.

[0126] Moreover, due to the specific Janus distribution, the membranes of the present invention can also be functionalized with different types of molecules, which supports their usefulness as vehicles for therapeutic or diagnostic molecules.

[0127] In addition to the above, and due to the specific nature of the material, the membrane of the present invention can also be used as an adhesive or to coat any article (e.g., medical devices, electronic supports, sensors) that requires a catecholamine membrane due to such adhesive properties. The membrane of the present invention can be used as part of other substrates or devices, and can impart improved properties to the final product. For example, prostheses can be entirely or partially covered with a membrane to enhance biocompatibility and reduce side effects or biorejection. Furthermore, through the use of the membrane of the present invention, a sensor or electronic support can be attached to a biological tissue or device. The sensor or electronic support can be part of the membrane in one platform, or can form a hybrid complex, in which case the sensor or electronic support is attached to the desired area with the help of the membrane applied as an adhesive patch.

[0128] Throughout the description and claims, the word "comprise" and variations of that word are not intended to exclude other technical features, additives, components, or steps. Furthermore, the term "comprise" encompasses "consisting of." Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of the detailed description or may be learned by practice of the present invention. The following examples are provided by way of illustration and are not intended to limit the present invention. Moreover, the present invention includes all possible combinations of the specific preferred embodiments described herein.

[0129] Working Example 1.Material Catechol compounds (pyrocatechol (1), caffeic acid (2), dopamine (3), 4-methylcatechol (4), pyrogallol (5), and catechin (6)) and amine compounds of formula (II) (hexamethylenediamine (7), octamethylenediamine (8), dodecamethylenediamine (9), cystamine (10), tris-(3-aminopropyl)amine (11), tris-(2-aminopropyl)amine (12), and 4,4',4''-triaminotriphenylamine (13)), as well as the comparative hexylamine and polyethyleneimine (PEI), were purchased from Sigma-Aldrich (Merck, Madrid, Spain) and used without further purification. Type 1 ultrapure water from a Milli-Q filtration system (Millipore, Burlington, MA, USA) was used in all experiments unless otherwise specified. In some experiments, different water-based buffers were used: phosphate-buffered saline (PBS, pH 7.4, Sigma-Aldrich) and aqueous carbonate buffer (pH 9.1) were prepared by dissolving 1.89 g NaHCO3 and 265 mg Na2CO3 in 250 mL Milli-Q water.

[0130] 2. Method 2.1 Physicochemical characterization Fourier transform infrared (FT-IR) spectroscopy. FT-IR spectra were recorded using a room temperature detector and a mid-infrared source (4000–400 cm -1 ) were recorded using a Tensor27 FTIR spectrometer (Bruker Optik, GmbH, Berlin, Germany).

[0131] A) Characterization of reagents by depositing solid samples onto a diamond attenuated total reflectance (ATR, model MKIIGoldenGate, Specac) window reflectance. This method was used for IR characterization of reagents.

[0132] B) Film characterization. A Hyperion 2000 FTIR microscope (BrukerOptik, GmbH, Ettlingen, Germany) equipped with a nitrogen-cooled mercury cadmium telluride (MCT) detector (InfraRedAssociates, Inc., Stuart, FL, USA) was used in reflection mode with a 15x reflection objective, a gold mirror as reference, and a resolution of 4 cm. -1 The sample was deposited on a gold surface for analysis.

[0133] All data obtained were processed using Opus version 7.2.139.1294 (Bruker) software. Gold-coated glass substrates were provided for FT-IR analysis.

[0134] X-ray photoelectron spectroscopy (XPS). Measurements were performed using a Phoibos 150 analyzer (SPECS EAS10P GmbH, Berlin, Germany) under ultra-high vacuum conditions (reference pressure 10 -10 mbar, residual pressure approx. 10 -7 The measurements were carried out at 1000 nm (1000 nm mbar). Monochromatic Al Kα radiation was used as the X-ray source (1486.6 eV and 300 W). The electron energy analyzer was operated with a pass energy of 50 eV. The hemispherical analyzer was positioned perpendicular to the sample surface. Data were collected eV per eV with a dwell time of 0.5 s. An electron flood gun with an energy of less than 20 eV was used to compensate for the charge. Samples were deposited on silicon substrates. All data were processed with CasaXPS version 2.3.17PR1.1 (Casa Software LTD, Teignmouth, UK) and OriginPro version 8.0988 (OriginLab Corporation, Northampton, MA, USA) software.

[0135] Scanning electron microscopy (SEM). Images were taken using a scanning electron microscope (SEM) (FEI Quanta650FEG, Thermo Fisher Scientific, Eindhoven, The Netherlands) in secondary electron mode at a beam voltage of 20 kV and a chamber pressure of 10 -5 The images were obtained at 100 nm and 1000 nm at 1000 nm resolution. The working distance was set at 10 mm and different magnifications were tested for the final images. Samples were prepared by depositing a free-standing film on an aluminum stub. Before the analysis was performed, the samples were metallized by depositing a thin platinum coating (5 nm) on the surface using a sputter coater (Leica EMACE600). An aluminum tapped support (pin stub) was provided.

[0136] Atomic force microscope (AFM). Surface topography imaging of various samples was performed using a beam-shaped silicon cantilever (Nanosensors, nominal force constant: 5 Nm -1 The measurements were performed in air on an Agilent 5500 AFM / SPM microscope (Keysight Technologies, Santa Clara, CA, USA) in tapping mode using a 300 nm tip (tip radius: approx. 7 nm) in combination with PicoScan5 version 1.20 (Keysight Technologies) software. An external XY positioning system (closed loop, 12NPXY100E, (nPoint, USA)) was used. Image processing was performed using the open source software: WSxM version 3.1 (Nanotec Electronica, Madrid, Spain) and Gwyddion version 2.46 (CMI, Brno, Czech Republic).

[0137] Contact angle (CA). Static contact angle measurements were performed with a 15 μL water droplet using an EasyDrop contact angle meter (KRUSS GmbH, Hamburg, Germany). Each film was measured at three different points to obtain an average over the surface. Measurements were taken approximately 1 min after deposition of the droplet.

[0138] Optical and fluorescent microscopy (OM). Images were obtained in transmission mode using a Zeiss Axio Observer Z1m (Carl Zeiss AG, Jena, Germany). Membranes were attached onto glass substrates. Optical images were taken in bright field and fluorescent images were obtained by switching between the different fluorescent probes DAPI and Alexa488.

[0139] Ultraviolet-visible spectroscopy. Ultraviolet-visible spectroscopy (UV-vis) was performed using a Cary 4000 UV-vis spectrometer (Agilent Technologies, Santa Clara, CA, USA) with a wavelength range of 200–800 nm and a quartz cuvette with a path length of 1 cm (QS10 mm). The baseline was corrected using blank samples of pure solvent. All measurements were performed under ambient conditions.

[0140] 2.2 Biological characterization a) Cell culture. For membrane testing, various cell lines were used: human cervical cancer cells HeLa (ATCC® CCL-2), fibroblast cell line NIH / 3T3 (ATCC® CRL-1658), adipose-derived mesenchymal ASC cells (ATCC® PCS-500-011) were purchased from LGC Standards SLU (Barcelona, ​​Spain). All basal media (DMEM, Glutamax, and glucose) were supplemented with 10% fetal bovine serum, 100 μL / mL penicillin, and 100 μg / mL streptomycin (LifeTechnologies, ThermoFisher Scientific, Waltham, MA, USA). All cell cultures were maintained in a humidified incubator at 37 °C under a 5-10% CO2 atmosphere.

[0141] b) Membrane degradation. The stability of the membranes in mouse plasma was measured at various time intervals over a period of 3 months. For this purpose, the membranes were incubated with mouse plasma (BioIVT, Hicksville, NY, USA) at pH 7.4 at 37°C. The samples were then placed in Sectra / Por® dialysis tubing (Spectrum, New Brunswick, NJ, USA) and dialyzed against 20 mL of 1xPBS with a molecular weight cut-off (MWCO) of 1-3 kDa, pH 7.4. Three aliquots were taken at predefined time points (5 min, 10 min, every 10 min remaining until 1 h, every 30 min until 10 h, 12 h, 16 h, 20 h, 1 day, 2 days, 3 days, 5 days, 7 days, and weekly after completion of 3 months) and measured by UV-Vis spectroscopy. The measurements focused on the detection of a band in the range of 250-450 nm, which corresponds to the signal associated with the catechol moiety. The appearance of the band in the aforementioned area as well as its growth may be related to the progressive degradation of the membrane. The liquid extract obtained from the outside of the dialysis bag was tested in vitro using different cell lines (HeLa and NIH / 3T3) to determine the cytotoxicity of potential by-products resulting from the degradation of the membrane.

[0142] c) In vitro cell viability test. Cells were added to 48-well plates at a density of 15000 cells / well and allowed to adhere for 24 hours. Different types of membranes cut into 0.8 mm disks were then added to the wells in quadruplicate and the plates were returned to the incubator. MTT and PrestoBlue™ assays for cellular respiration were performed after 24 and 72 hours of incubation.

[0143] (i) For the MTT assay, 20 μL of 5 mg / mL thiazoyl blue tetrazolium bromide (MTT, Sigma-Aldrich) solution was added to each well and incubated for 2 h. The supernatant was aspirated without disturbing the cells, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan salt from the metabolism of MTT in metabolically active cells. After complete dissolution, the absorbance was read at 540 nm using a BioTek MX plate reader (BioTek Instruments Inc., Winooski, VT, USA). The percentage of cell viability was calculated by dividing the absorbance of each well by the corresponding value of the control wells treated with vehicle (1xPBS) only. At least four independent experiments were performed on different days.

[0144] (ii) For PrestoBlue™ assay, 10 μL of resazurin was added to the medium to a final concentration of 15 μM, and fluorescence was read at an excitation wavelength of 531 nm and an emission wavelength of 572 nm using a Victor3 plate reader (PerkinElmer, Waltham, MA, USA). As with the MTT assay, at least four independent experiments were performed, and cell viability was calculated relative to vehicle-treated cells.

[0145] d) Adhesion, proliferation and cell differentiation. The adhesion of cells on the surface membranes was tested for all the aforementioned cell lines. Using a sterile biopsy punch, the membranes were cut into 0.8 mm disks and placed in a 48-well plate containing PBS. Then, the PBS was removed so that the membranes covered the bottom of the wells. The membranes were seeded with cells (25000 cells / membrane) in 50 μl of medium. The membranes were then incubated for 30 minutes and after this time the wells were filled with 350 μl of medium. The membranes containing the cells were incubated for different times: 1 day, 3 days, 7 days, 14 days and 30 days.

[0146] After each time point, the membranes were washed at least five times in PBS to remove non-adherent cells. Different criteria were established to evaluate adhesion, proliferation and cell differentiation for each cell type and membrane. Regarding adhesion, the membranes were washed at least five times in PBS to remove non-adherent cells. In addition, the cells on the surface were counted using image analysis software (ImageJ), and the cells adhered at different time points on the two different surfaces were counted and the density of the cells was determined. Proliferation was evaluated by analyzing the formation of cell networks and preferential growth in specific directions, which occurs in typical in vitro cell culture, with light and / or electron microscopy. Finally, cell differentiation of ASC cells was evaluated, and the formation of various cell types was identified through the observation of different morphologies and cell structures using light and / or electron microscopy. In experiments of cell differentiation with ASC cell lines, the medium was enriched with growth factors (TGFβ3 and BMP6). For observation with fluorescence and electron microscopy, the cells were treated with formaldehyde for 1 h and then washed three times with PBS. The membranes containing the cells were stored in a refrigerator with PBS. For fluorescence imaging, cells were pre-stained with Live / Dead kit test (Thermo Fisher Scientific) according to the manufacturer's specifications. Specifically, for SEM analysis, PBS was removed and then washed with increasing concentrations of EtOH solutions (30%, 50%, 70%, 80%, 96% and 100%). Next, cells were washed three times with hexamethyldisilazane and dried overnight at room temperature. Finally, the membrane containing cells was mounted on an SEM stub for image acquisition.

[0147] e) In vivo assay Animal experiments were approved by the local ethical committee in accordance with local and state laws. For this assay, Sprague Dawley rats (12 males, 12 females) were purchased (Envigo, Indianapolis, IN, USA). First, the in vivo adhesion of the membrane was tested. To do this, the membrane was placed in the knee joint and excess blood and other body fluids were dried with gauze. The membrane of the present invention was left to adhere. The wound was then sutured and the implanted membrane was examined 3 and 7 days after the rat was euthanized. In another experiment, joint injury was induced in rats to remove the cartilage and the membrane was directly adhered to the bone to confirm the in vivo adhesion. These experiments were performed with functionalized and non-functionalized membranes, and with and without cells (ASC cell line) on the membrane (n=5). Meanwhile, a tolerability test was performed by implanting the membrane subcutaneously and in the muscle to observe possible inflammation, hematoma, or erythema. These tests were carried out for 4 and 8 days, checking the condition of the skin with the naked eye (observation of irritation or redness) and, after euthanasia, observing any damaging effect on the surrounding tissue. All tests were carried out on different formulations of the membrane of the present invention and the PEI-based membrane for comparison purposes.

[0148] 3. Synthesis of the Membrane of the Invention The corresponding catechol and amine derivatives were placed as solids in the reaction vessel, with the amine derivative in excess of the amine in molar ratio (1:1.5). The addition of PBS was then performed to adjust the pH of the solution to 7.4. The reaction vessel was covered with Parafilm with a hole of 2 mm diameter to allow the introduction of oxygen. The formation of free-standing floating membranes was carried out at room temperature under magnetic stirring at 300 rpm. The polymerization reaction took place at the liquid-air interface through the oxidation of the catechol derivative and its reaction with the amine-based ligand, without the need for the formation of a substrate in situ by physical substrates or other secondary molecules. After 24 hours, the free-standing floating membranes were ready to be isolated. The membranes used in the following in vitro and in vivo tests were synthesized in a biological safety cabinet (Biosafety Class II Cabinet Telstar BioVanguard) to ensure sterility (avoiding airborne contaminants).

[0149] After the synthesis of the membrane, it was treated with absolute ethanol overnight.

[0150] Table 1 summarizes the reagents and IR properties.

[0151] [Table 1] TIFF2024526426000011.tif168159

[0152] 4. Synthesis of Membranes for Comparative Purposes For comparative purposes, membranes were also synthesized according to the protocol above, but with one of the reagents and / or reaction conditions changed as shown in Table 2 below.

[0153] [Table 2]

[0154] 5. Membrane Functionalization The (bio)molecules were immobilized via a coupling reaction using (1-ethyl-3-(3-dimethylamino)propylcarbodiimide, hydrochloride (EDC) and N-hydroxysuccinimide (NHS). This coupling reaction results in the formation of amide bonds between the amino groups exposed on the membrane and the carboxyl groups of the functionalized (bio)molecule. In a typical procedure, the formed membrane was placed in a Petri dish containing PBS with a larger diameter compared to the membrane under magnetic stirring (500 rpm). Then, an aqueous solution of EDC (5 mM) was injected into the aqueous phase. After 30 min, the active (bio)molecule was injected into the aqueous phase together with the NHS agent. Antimicrobial moieties (vanillic acid) or fluorescent dyes (calcein) were dissolved in water (10 mM) and then mixed with NHS (10 mM). The reaction was stirred slowly for 60 min. The functionalized membranes were then isolated from the Petri dish, washed at least five times with a flux of distilled water, and dried under vacuum. This approach allowed for a variety of functionalizations with high yields (approximately 55–60%, depending on the functionalizing molecule) due to the membrane composition rich in surface-exposed amino groups. The functionalization could be controlled and induced independently on both sides of the membrane, thereby enhancing the chemical Janus behavior of the membrane. The membranes were first paced.

[0155] A variety of functionalizations can be achieved by this approach, and two specific cases are presented below as representative examples.

[0156] 6. Results and Discussion 6.1.Mechanical properties of the membrane The membrane of the present invention can be easily isolated by using common tweezers, and can be properly handled and transferred to another container for storage.The membrane obtained exhibits high flexibility, without breaking, even when one end of the membrane is bent 180 degrees and touched to the other end of the membrane.This is performed on both wet and dry membranes, and no cracks or tears are observed by SEM.This bending operation is performed manually for at least 50 cycles to confirm the flexibility of the membrane.Furthermore, a twisting operation is performed to confirm the limit of the flexibility of the membrane, which shows excellent robustness.

[0157] First, through several measurements using FT-IR and XPS, we confirmed that the membrane was a catecholamine-based membrane. Depending on the chemical nature of the catechol derivative and the amine-based ligand, FT-IR spectra in different frequency ranges were evaluated. First, catechol (3500–3000 cm -1 ) and amines (1700-1500cm -1 The presence of catechol- or amine-based groups was the first indication of the formation of crosslinked polymers. Later, specific bands were identified within specific ranges depending on the type of catechol or amine-based molecule. For example, the band corresponding to the amide bond is located at 1650–1500 cm -1 range and 1290~1200cm -1 , especially 1-7 (1500-1260 cm -1 ), 1-8 (1499~1268cm -1 ), 1-9 (1519~1261cm -1 ), 1-10 (1508~1260cm -1 ), 1-11(1547~1281cm -1 ), 1-12 (1511~1263cm -1 ), 2-7 (1546~1270cm -1 ), 2-8 (1515~1260cm -1 ), 2-9 (1635~1210cm -1 ), 2-10 (1595~1261cm -1 ), 2-11(1529cm -1 ), 2-12 (1538~1204cm -1 ), 3-7 (1511~1231cm -1 ), 3-8(1231cm -1 ), 3-9(1252cm -1 ), 3-10 (1564~1251cm -1 ), 3-11(1209cm -1 ), 3-12 (1509~1236cm -1 ), 4-7 (1567~1251cm -1 ), 4-8(1246cm -1 ), 4-9 (1597~1250cm -1 ), 4-10 (1583~1247cm -1 ), 4-11(1267cm-1 ), 4-12 (1527~1223cm -1 ), 5-7(1219cm -1 ), 5-8 (1530~1276cm -1 ), 5-9 (1230cm -1 ), 5-10 (1583~1201cm -1 ), 5-11(1261cm -1 ), 5-12 (1509~1233cm -1 ), 6-7 (1576~1240cm -1 ), 6-8 (1521~1277cm -1 ), 6-9(1284cm -1 ), 6-10 (1654~1255cm -1 ), 6-11(1209cm -1 ) and 6-12 (1587-1210 cm -1 ) and imines appear between 1700 and 1600 cm -1 In particular, the range of 1-7 (1700 cm -1 ), 1-9(1623cm -1 ), 1-11(1611cm -1 ), 2-7(1701cm -1 ), 2-8(1628cm -1 ), 2-9(1635cm -1 ), 3-7(1638cm -1 ), 3-9(1678cm -1 ), 3-11(1661cm -1 ), 3-12(1611cm -1 ), 4-7(1652cm -1 ), 4-8(1624cm -1 ), 4-12(1627cm -1 ), 5-7(1654cm -1 ), 5-9 (1672cm -1 ), 6-7 (1703cm -1 ), 6-8(1705cm -1 ), 6-9 (1653cm -1 ) and 6-12 (1643 cm -1 ) and the incorporation of aliphatic carbons occurs at 3000–2500 cm -1 In particular, in the range of 1-7 (2850 cm -1 ), 1-8(2810cm-1 ), 1-9(2842cm -1 ), 1-11(2632cm -1 ), 1-12(2853cm -1 ), 2-7(2761cm -1 ), 2-8(2747cm -1 ), 2-9(2623cm -1 ), 2-11(2617cm -1 ), 2-12(2821cm -1 ), 3-7(2777cm -1 ), 3-8(2781cm -1 ), 3-9(2671cm -1 ), 3-11(2861cm -1 ), 3-12(2817cm -1 ), 4-7(2891cm -1 ), 4-8(2761cm -1 ), 4-9(2817cm -1 ), 4-11(2732cm -1 ), 4-12(2801cm -1 ), 5-7(2791cm -1 ), 5-8(2745cm -1 ), 5-9(2891cm -1 ), 5-11(2893cm -1 ), 5-12(2781cm -1 ), 6-7(2893cm -1 ), 6-8(2871cm -1 ), 6-9(2872cm -1 ), 6-11(2878cm -1 ) and 6-12 (2873 cm -1 For membranes synthesized using amino-based ligands with aromatic moieties (e.g., 1-13), the incorporation of the ring into the copolymer structure was observed at 2765 cm -1 In addition, the bond between the catechol-ligand and the amino group exposed from the aromatic ligand was detected through the appearance of specific bands at 1500 and 1327 cm. -1 Finally, the appearance of two new bands at 1175 and 1120 cm -1The shift in the bands of 3500 and 3000 cm was due to the formation of new bonds with the aromatic terminal amino groups. The incorporation of catechol and amino moieties into the resulting bridged structures shifted the bands from 3500 to 3000 cm, respectively. -1 and 1700-1500 cm -1 It is worth mentioning that this was confirmed by bands ranging from

[0158] XPS then confirmed that the chemical environment of the molecules had been modified, confirming the formation of specific bonds by copolymerization between catechol and amino derivative molecules. For this reason, high-resolution measurements were performed on the C, N, O signals to determine the percentage of CNOH, thus indicating the final composition of the films. It is worth mentioning that in all the films of the invention, the amount of coexisting quinone / hydroxyl groups was higher on the water-contacting surface compared to the amino end groups that prevailed on the air-contacting surface. This difference in the exposure of functional groups depending on the contacting surface is interesting during functionalization, which allows the selection of the respective surface for the immobilization of specific molecules.

[0159] In contrast to the membranes of the present invention: -PEI-based membranes required a high pH (about 11); they were very inflexible due to the formation of cracks when manipulated and bent, and exhibited a plastic-like appearance due to the smooth and bright appearance of the resulting membranes. Furthermore, topographic AFM measurements highlighted low roughness on both sides, indicating low conformability of the membrane to the surface; - Membrane coated substrates: synthesis on substrates was successful, but the high adhesion to the substrate and the fragile nature of the formed membrane made it very difficult to detach it from the substrate. Only small fragments could be released from the surface, thus preventing further biological applications. For this reason, the manipulation and handling of the membranes carried out on substrates is more difficult and less efficient compared to the membranes of the present invention; When aliphatic monoamines, having a terminal amine at only one end of the hydrocarbon chain, were used, no membranes were obtained, despite attempts being made.

[0160] 6.2. Morphological characterization (optical microscopy, SEM, AFM) The free-standing membranes of the present invention showed morphologically Janus characteristics. When their topography was examined by SEM, it was found that both sides of the film were different. The surface in contact with water had embedded nanoparticles of the same catecholamine material. These nanoparticles were formed as a by-product and were found embedded in the water contact surface as precipitates. The precipitated NPs were centrifuged and isolated for characterization by FT-IR, confirming their catechol-amine composition. Furthermore, the formation of the membrane was followed over time, and aliquots were taken from the interface at various time points and observed by SEM. The SEM images showed the process of NPs being embedded. Conversely, the surface exposed to air did not show embedded NPs and remained smooth and intact. Analysis of the images obtained by SEM (Figure 1) highlighted the formation of membranes with different surface patterning. As will be explained in the next section, this patterning affects not only the membrane's biological tissue but also its eventual adhesion to cells.

[0161] It was also noteworthy that once the membranes of the present invention were formed and washed using ultrapure water, the nanopatterning on the water-contacting side of the membrane remained unchanged, i.e., the nanoparticles remained embedded in the surface. This unexpected nanopatterning on the water-contacting side provided the membrane with a rougher surface, which is favorable for cell adhesion (as shown further below).

[0162] It is worth mentioning that this nanopatterning was not observed in the synthesized PEI comparison membranes and substrates, both of which had similar topographies as observed by SEM. In these comparative examples, both sides presented similar surfaces without any different features between them.

[0163] In particular, the PEI-based membranes exhibited chemical Janus properties, as the PEI was completely separated within the PEI-formed structure, forming two different domains and different porosity depending on the side. However, no nanopatterning was detected. Due to the plastic-like morphology (smooth, rigid side) exhibited by the PEI membrane, the roughness of the PEI-based membranes was very low on both sides, showing similar properties when SEM images were analyzed. This low roughness and lack of nanopatterning makes cell adhesion to the membrane difficult, as discussed in the next section.

[0164] For the membranes obtained from the substrate, in this comparative membrane, the two sides of the membrane were very similar and therefore did not show Janus properties, since the use of the substrate avoided the formation of a floating membrane at the interface (the membrane of the present invention) and because of its polarity, molecular ordering in the water and air phases could not occur.

[0165] Further testing was carried out by SEM and AFM to investigate the surface of the free-standing membranes in detail. First, SEM was used to analyze the thickness of the membranes of the present invention (Table 3). It is worth mentioning that the thickness of the membranes increased with the extension of the reaction time. One explanation for this effect is that the amount of polymerized material increased with time. Next, AFM was carried out to determine the roughness of some membranes by measuring the topographical characteristics of both the air-contacting and water-contacting surfaces (Table 4).

[0166] Additionally, the AFM topography profiles were evaluated to confirm the thickness measurements obtained by SEM.

[0167] As a qualitative observation, the SEM images showed different roughness depending on the contact surface of the membrane. In particular, the water contact surface showed an unexpectedly high roughness. For this reason, AFM was performed on the water contact surface. The membrane of the present invention showed the highest surface roughness, with embedded nanoparticles. Several areas were investigated with regular profiles of various regions with a height of about 275 nm. In terms of morphology, the nanoparticles embedded in the membrane correlated with the SEM observations. In the case of the air contact surface, the surface roughness was found to be less than 10 nm, resulting in a smoother surface. Finally, the height of the profile ranged from 200 to 600 nm depending on the membrane, which coincides within the range of thickness estimated by SEM.

[0168] Finally, the roughness of both sides of the different films was measured, as shown in Table 4 below.

[0169] [Table 3] Thickness values ​​of the membrane of the present invention at different reaction times at an amine-based ligand concentration of 10 mM.

[0170] [Table 4]

[0171] Interestingly, the surface in contact with water exhibited a greater roughness due to the embedded nanoparticles, whereas the surface in contact with air is smoother. This property confers an added value to the membrane, as the water-contacting surface promotes better adhesion of cells compared to smooth surfaces (where cells adhere much less and become less dense due to cell proliferation), as observed in the comparative membranes synthesized with PEI (see below). Moreover, the membranes synthesized using the substrate showed similar characteristics on both sides, with a roughness of about 3.5 nm on both sides. This is due to the fact that the membranes are synthesized in contact with the substrate, which avoids the formation of nanoparticles that cannot be embedded in the surface, forming a nanopattern.

[0172] In summary, the morphological results clearly demonstrated that the inventive films have Janus characteristics from a morphological point of view. These surface techniques (SEM and AFM) not only showed different topographies depending on the surface, but also evaluated the roughness. Also, for each film type, the film thickness was measured by SEM and AFM. In all cases, similar results were obtained, demonstrating the reproducibility and generality of the inventive approach to the formation of catecholamine-based films.

[0173] Finally, the films were characterized using optical microscopy, which observed the interaction of light with the film's surface, which manifested as different colored reflections depending on the surface thickness, confirming both the film thickness difference and the nanopatterning on the surface.

[0174] The above was confirmed by measuring FT-IR, XPS, and contact angle in each case. This characterization was performed for all of the inventive and comparative membranes. Similar results were obtained in all cases.

[0175] FT-IR spectroscopy results provided information on the species present in the film and were read on the whole material to confirm the chemical composition.

[0176] The spectra were analyzed and shared the following characteristics: 3500-3000 cm -1 The broad band in the range of 1700–1500 cm corresponds to the stretching vibration of hydroxyl (–OH) groups, and the amine (–NH) groups correspond to the stretching vibration of 1700–1500 cm -1 and their peaks can be recognized in the spectra of catechol derivatives and amine-based compounds, respectively. -1 The peaks in the range of 1700 cm are associated with the asymmetric and symmetric stretching vibrations of aliphatic carbons (C-H). -1 The peak at 1500-1400 cm corresponds to the presence of quinone (C=O). -1 Signals in the range of 1200-1450 cm can be assigned to C=CH and C=C vibrations from the catechol / quinone ring of the catechol derivative.-1 Peaks in the range belong to secondary amines attached to alkyl or aromatic rings.

[0177] The chemical composition of the film therefore coincides with the functional groups of the reagents. In all combinations performed, the FT-IR spectra showed similar results, with the presence of specific bands for amino and catechol groups. Furthermore, for each specific catechol derivative, other representative signals could be assigned according to the functional groups of the molecule. These results also confirmed the successful copolymerization between catechol-based and amino-based molecules according to the established methodology.

[0178] Finally, the wettability of the membrane was tested using a contact angle device. Before measurements were taken, the membrane was placed in a 170 °C oven for 2 hours to dry it completely and stored under vacuum to remove all water molecules. After this drying process, the membrane was weighed until it reached a minimum weight indicating that all water had been lost. These measurements determined the hydrophobic / hydrophilic characteristics of the membrane. As observed with other characterization techniques, the wettability differed depending on the surface being measured. The water contact surface has a more hydrophilic behavior with a contact angle of about 25 degrees, while the air contact surface is higher with a contact angle of about 40 degrees, indicating a high hydrophobicity. Nevertheless, the membrane as a whole shows an intermediate hydrophilic / hydrophobic behavior. This surface property can be changed by using the exposed functional groups present on the membrane to anchor hydrophobic or hydrophilic moieties. The wettability can be used to tune the adhesion of the membrane depending on the final tissue to which it will be applied. For example, a high hydrophilicity of the membrane at the water-contacting surface (where more cells can grow) is favorable for direct adhesion of the membrane to the damaged tissue, while a higher hydrophobicity at the air-contacting surface avoids undesirable adhesion to other tissues in the surrounding area.

[0179] 6.3.Functionalization 6.3.1. Functionalization with antibacterial molecules One of the main problems when biomaterials have to be applied in medicine is the risk of infection, which is becoming particularly relevant in regeneration processes. For this reason, incorporating antibacterial moieties into the membrane could improve the regeneration process of damaged tissues without side effects.

[0180] Therefore, we functionalized membranes with vanillic acid, which has well-known antibacterial activity, through an EDC / NHS coupling reaction following the procedure described above. Tests were performed on six different membranes (1-7, 1-8, 2-7, 2-8, 3-9, 3-10) as proof-of-concept.

[0181] To confirm the covalent adhesion, the films were characterized by various means. FT-IR spectroscopy revealed a peak at 1665 cm, which is assigned to the formation of an amide bond through the free amino group and the carboxylic acid of vanillic acid. -1 In addition, a specific peak derived from vanillic acid was incorporated in the spectrum corresponding to the functionalized membrane.

[0182] Additional XPS measurements showed significant changes in the spectra corresponding to C1 and O1 when comparing non-functionalized and functionalized membranes. These results confirmed that antimicrobial moieties were incorporated into the membrane. It is worth mentioning that the functionalized membranes were vigorously washed to remove any unreacted material.

[0183] Finally, contact angle measurements were performed on the surfaces of non-functionalized and functionalized films (Table 5). These results highlight that the contact angle values ​​increase when the film is functionalized. These measurements indirectly indicate that the surface film is modified.

[0184] [Table 5]

[0185] 6.3.2. Functionalization with Fluorescent Moieties Another interesting functionalization that adds value to the membranes is the functionalization with fluorescent molecules that are useful for monitoring and visualization by the bioimaging techniques used. Thus, the membranes (1-7, 1-8, 2-7, 2-8, 3-9, 3-10) were functionalized with a fluorescent dye (calcein). Fixation of the dye to the membranes was achieved by an EDC / NHS coupling reaction, following the procedure described above. After functionalization synthesis, the membranes were characterized using a fluorescence microscope.

[0186] The membranes were found to be successfully functionalized with fluorescent dyes. The fixation throughout the coupling reaction was stable and remained unchanged after several weeks in both dried and soaked membranes, since no loss of the fluorescent signal was observed by fluorescence microscopy. These tests were performed by keeping the functionalized membranes in water and measuring the fluorescence at various time points for at least 4 weeks. In addition, the fluorescence of membranes completely dried and stored under vacuum was also checked within the same period. The robust functionalization and retention of fluorescent activity was confirmed by a) measuring the fluorescence by fluorescence microscopy and b) measuring the fluorescence after dye delivery by dialysis experiments. In this last case, the membranes were placed in a dialysis tubing bag (Spectrum® with pore size 4-6 kDa) and degradation tests were performed at room temperature under magnetic stirring (500 rpm). Different aliquots were taken from the outside of the dialysis bag at different time points and measured in a fluorometer. The fluorescence spectra were determined by measuring the fluorescence bands associated with the dyes (calcein, λ emission = 521 nm), there was no significant dye release after 3 months, indicating that the functionalized film was stable in water.

[0187] It is worth mentioning that the as-functionalized membrane of the present invention exhibited very high fluorescence at an exposure time of about 450 ms, which was sufficient to acquire images without saturating the signal. Moreover, as in the case of the antibacterial moiety, FT-IR spectroscopy confirmed the functionalization of the membrane with the fluorescent dye, revealing typical bands corresponding to the calcein reagent (1750, 1630, 1445, and 1280 cm). -1Furthermore, contact angle measurements showed that after functionalization of the film, the water contact angle increased by about 30°, indicating successful surface modification.

[0188] 6.4. Biological characterization 6.4.1.Decomposition The degradability of all the membranes of the present invention was tested in physiological medium (PBS, pH 7.4, 37° C.) using dialysis experiments. At various time points, aliquots were taken from the outside of the dialysis bag and measured by UV-vis to detect the appearance of bands corresponding to catechol groups (in the range of 250-450 nm, depending on the catechol derivative).

[0189] This monitoring was carried out for 4 months, and surprisingly, no signal from catechol was measured. This means that the membranes of the present invention are robust and do not degrade significantly. This stability is essential for their application in tissue regeneration, in order to have the membrane for all regeneration processes. The membrane retains its structure for a sufficient time to promote tissue regeneration.

[0190] Aliquots of the extracts obtained from the dialysis experiments were also tested in vitro on different cell lines. These results demonstrated that no cytotoxic by-products were released from the membranes during the degradation process.

[0191] In contrast to the membranes of the present invention, the PEI-based membranes of the prior art degraded significantly faster when compared to the membranes of the present invention, with traces of PEI detected after 2 weeks in dialysis experiments. This delivery of PEI affected the robustness of the membranes. Not only this, PEI has also been reported to be toxic in biological environments and is highly cytotoxic in different cell lines. This toxicity has hindered the application of PEI-based membranes in tissue regeneration and other biological applications.

[0192] 6.4.2. Cell viability All membranes of the present invention, both functionalized and non-functionalized, were tested in vitro in all cell lines mentioned in the experimental section above. The results showed excellent biocompatibility without toxicity, even over long periods of time, exceeding 14 days. Cells were able to grow, proliferate and differentiate (the latter was used in the case of ASC cells). Extracts obtained from the 4-month degradability assay, as mentioned above, were also tested in various cell lines. Tests were performed for 72 hours, showing that no toxic products were delivered.

[0193] It is worth noting that the cells grow and proliferate until the membrane is completely covered, without any signs of membrane-induced cell death.

[0194] Further testing of PEI-based membranes was performed. The results showed higher cytotoxicity compared to the membranes of the present invention. After 24 hours, all membranes of the present invention showed cell viability of more than 90% in all cell lines tested. However, PEI-based membranes showed increased toxicity, with cell viability of about 65%. This high toxicity is associated with the rapid degradation of PEI-based membranes, which induces the release of PEI, which has a well-known cytotoxic effect. This is one of the main reasons why PEI-based membranes cannot be used for biological applications.

[0195] 6.4.3. Cell attachment and proliferation Different cell lines were seeded on the membranes placed on the bottom of cell culture plates and incubated with cell culture medium for 21 days at different times. The membranes were extracted and washed at various time points for their characterization.

[0196] In the case of the membrane of the present invention, cells were found to fully adhere to the membrane surface (both sides) and grow preferentially on the side in contact with water, which is the nanopatterned side. This preferential growth was observed by a higher cell density on the water-contacting side compared to the air-contacting side (45% increase). Various images were taken by optical and electron microscopes. The cells adhered to the membrane through the same mechanism observed on common cell culture plates, and showed their native cell morphology and growth in a preferred orientation. The cells were labeled with fluorescent moieties and observed using a fluorescent microscope and fixed for observation by SEM. The membrane was washed to remove non-adherent cells.

[0197] Finally, PEI-based membranes showed very low cell adhesion and very low cell density. Compared to the membranes of the present invention, the cell density on the PEI-based membranes was reduced by about 70%. This, together with the toxic nature of PEI, may be related to the surface characteristics of the membranes made of this polyimine, which may hinder the interaction between the material and the cells.

[0198] 6.4.4.Ex vivo adhesion The adhesion of the membrane was also tested on ex vivo tissues (porcine cartilage and skin), taken by the Veterinary Faculty of the Autonomous University of Barcelona directly from euthanized animals for use in other purposes. The aim was to establish the adhesion of the membrane in different types of living tissue and in wet conditions. For this, different membranes of the invention (1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 2-7, 2-8, 2-9, 2-10, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 3-7, 3-8, 3-9, 3-10, 4-7, 4-8) were cut to different sizes and attached to the tissue. Tests were carried out with non-functionalized and functionalized membranes, as well as with and without cells. Using gauze, the attached membrane remained in the area without movement and the area could be washed without affecting the membrane. To test adhesion under extreme conditions, biological tissue with the film adhered to it was immersed in water for several months and it was observed that the film still adhered to the tissue without peeling off from the tissue.

[0199] In vivo In vivo assays were performed using female and male Dawley Sprague rats. Seven membranes of different compositions (1-7, 1-12, 2-7, 3-7, 3-11, 5-1 and 5-11) were tested in the presence and absence of ASC cells (see above).

[0200] The membranes were placed on cartilage, bone, and muscle and showed excellent adhesion to the tissues without any rejection signals, and no inflammation, erythema, or edema was observed. These preliminary results demonstrated excellent in vivo tolerability and biocompatibility.

[0201] For comparison purposes, a PEI-based film was tested and showed very low adhesion to biological tissue due to very low roughness and lack of interaction between the film and the tissue. Furthermore, it lacked flexibility, which made it difficult to apply the film accurately and resulted in the film not being able to adequately cover the biological tissue. Due to the low adhesion to the tissue and the stiffness, it was not possible to continue the test and it had to be stopped.

[0202] Citation List Non-patent literature: Iacomino M. et al., “Multifunctional Thin Films and Coatings from Caffeic Acid and a Cross-Linking Diamine”, 2017, Langmuir, 33, 9, 2096-2102 (doi:10.1021 / acs.langmuir.6b04079); Ponzio F. et al., “Polydopamine deposition at fluid interfaces”, 2016, Society of Chemical Industry, pages 1-7 (doi 10.1002 / pi.5124);and Suarez-Garcia S. et al., “Copolymerization of a Catechol and a Diamine as a Versatile Polydopamine-Like Platform for Surface Functionalization:The Case of a Hydrophobic Coating”, 2017, Biomimetics, 2(4), 22 (doi:10.3390 / biomimetics2040022);

[0203] Terms For reasons of completeness, the various aspects of the invention are set out in the following numbered clauses: Clause 1. A process for preparing a catecholamine-based membrane, comprising the steps of: a) reacting a catechol derivative with an amine selected from the group consisting of: a.1) The known aliphatic amine hydrocarbons of formula (II) AB-A'(II) (In the formula, A and A' are the same or different and represent -NR1R'1; B is (C1~C 20 ) alkylene; -OH, halogen, -NO2, cyano, -O-(C1-C 10 (C1-C) substituted with one or more substituents selected from the group consisting of alkyl, -C(O)OR2, and -NR3R'3 20 ) alkylene; (C2-C 20 ) Alkenylene; -OH, halogen, -NO2, cyano, O-(C1-C 10 (C2-C) substituted with one or more substituents selected from the group consisting of alkyl, -C(O)OR4, and -NR5R'5 20 )Alkenylene;(C2-C 20 ) Alkynylene; -OH, halogen, -NO2, cyano, O-(C1-C 10 ) alkyl, -C(O)OR6, and -NR7R'7, substituted with one or more substituents selected from the group consisting of (C2-C 20 ) alkynylene; the known 3- to 20-membered heteroalkylenes; and -OH, halogen, -NO2, cyano, -O-(C1-C 10 )alkyl, -C(O)OR8, and -NR9R'9; the known 3- to 20-membered heteroalkylenes substituted with one or more substituents selected from the group consisting of: -OH, halogen, -NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 10 , and -NR 11 R' 11 represents a known 3- to 20-membered heteroalkenylene substituted with one or more substituents selected from the group consisting of: A is attached to the first atomic ring member that forms part of the B biradical hydrocarbon backbone; A' is attached to the last atomic ring member that forms part of the B biradical hydrocarbon backbone; The "first" and "last" atom ring members are identified by reading the B biradical hydrocarbon backbone from left to right or vice versa; R1, R'1, R3, R'3, R5, R'5, R7, R'7, R9, R'9, R 11 , and R' 11 are the same or different, -H; (C1-C 10 )Alkyl;(C1-C 10 )Haloalkyl;(C2-C 10 ) alkenyl; (C2-C 10 ) alkynyl; and -OH, -NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 12 , and -NR 13 R' 13 (C1 to C 10 ) alkyl; R2, R4, R6, R8, R 10 and R 12 -H;(C1-C 10 )Alkyl;(C1-C 10 )Haloalkyl;(C2-C 10 ) alkenyl; (C2-C 10 ) alkynyl; and -OH, -NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 14 , and -NR 15 R' 15 (C1 to C 10 ) alkyl; R 13 , R' 13 , R 14 , R 15 and R' 15 are the same or different, -H; (C1-C 10 )Alkyl;(C1-C 10 )Haloalkyl;(C2-C 10 ) alkenyl; and (C 10) alkynyl; The "known 3- to 20-membered heteroalkylene" is C(R x )2, CR x , -N-, -NR' x means a known saturated chain consisting of 3 to 20 members selected from the group consisting of -, -S-, and -O-, provided that (a) at least one of the ring members is -N-, -NR' x (b) the first and last ring members forming the heteroalkylene backbone are carbon atoms; The "known 3- to 20-membered heteroalkenylene" is C(R x )2, -CR x -, -N-, -NR' x means a known unsaturated chain having 3 to 20 ring members selected from the group consisting of -, -S-, and -O-, provided that (a) at least one of the ring members is -N-, -NR' x (b) the first and last ring members forming the heteroalkylene backbone are carbon atoms; (c) it contains one or more double bonds; R x -H; -OH; (C1-C 10 )Alkyl;(C2-C 10 ) alkenyl; (C2-C 10 ) alkynyl; (C1-C 10 )Haloalkyl;O-(C1-C 10 ) alkyl; -O-(C2-C 10 )Alkenyl;-O-(C2-C 10 ) Alkynyl; Nitro, -NR x1 R x2 ;-NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 16 , and -NR 17 R' 17 (C1 to C 10 ) alkyl; and halogen; R X1 , Rx2, R 16 , R 17 , R' 17 , and R' xis -H, (C1~C 10 ) Alkyl, (C2-C 10 ) alkenyl, (C2-C 10 ) alkynyl, and (C1-C 10 ) haloalkyl); and a.2) Aromatic amines of formula (IIbis): [ka] (Wherein, W is -NR t R' t or * represents SSL; * ) indicates that the S atom of the W radical is bonded to a carbon atom that forms part of an aromatic ring; L is -OH, -NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 28 , and -NR 28 R' 28 (C1 to C 10 ) alkyl (provided that at least one of the substituents is -NR 28 R' 28 -OH, -NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 28 , and -NR 28 R' 28 (C1-C10)haloalkyl substituted with one or more substituents selected from the group consisting of, provided that at least one of the substituents is -NR 28 R' 28 -OH, -NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 28 , and -NR 28 R' 28 (C2 to C 10 ) alkenyl (wherein at least one of the substituents is -NR 28 R' 28 -OH, -NO2, cyano, -O-(C1-C 10) alkyl, -C(O)OR 28 , and -NR 28 R' 28 (C2 to C 10 ) alkynyl (wherein at least one of the substituents is -NR 28 R' 28 -CR v -, -N-, -O-, -NR' v -S-, ... R m , R' m , R t and R' t are the same or different, H, (C1 to C 10 )Alkyl;(C1-C 10 )Haloalkyl;(C2-C 10 ) alkenyl; (C2-C 10 )Alkynyl; -OH, -NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 29 , and -NR 30 R' 30 (C1 to C 10 ) alkyl; and -CR z -, -N-, -NR' z -O-, -S-, ... R v At least one of the following is -NR 31 R' 31 and other R v (single possible) is H, -NR 31 R' 31 , (C1~C 10 )Alkyl;(C1-C 10 )Haloalkyl;(C2-C 10 ) alkenyl; (C2-C 10 ) alkynyl; and -OH, -NO2, cyano, -O-(C1-C 10 ) alkyl, C(O)OR 32 , and -NR33 R' 33 (C1 to C 10 ) alkyl; R z and R' z H, (C1~C 10 )Alkyl;(C1-C 10 )Haloalkyl;(C2-C 10 ) alkenyl; (C2-C 10 )Alkynyl; -OH, -NO2, cyano, -O-(C1-C 10 ) alkyl, -C(O)OR 34 , and -NR 35 R' 35 (C1 to C 10 ) alkyl; R 28 , R' 28 , R 29 , R 30 , R' 30 , R 31 , R' 31 , R 32 , R 33 , R' 33 , R 34 , R 35 , and R' 35 -H;(C1-C 10 )Alkyl;(C1-C 10 )Haloalkyl;(C2-C 10 ) alkenyl; and (C 10 ) alkynyl) to create a catecholamine film at the air / liquid interface in the absence of any support, the crosslinking reaction being carried out in a liquid medium in which both the catechol and the amine are soluble under stirring, particularly under adequate stirring, and the pH is between 6.5 and 10, particularly between 6.5 and 8; and b) isolating the membrane obtained from step (a) from the air / liquid interface. A process including.

[0204] Clause 2. The process of clause 1, wherein the amine is an amine of formula (II).

[0205] Clause 3. The process of any of the preceding clauses, wherein A and A' are the same or different and represent NHR'1.

[0206] Clause 4. The process of any one of the preceding clauses, wherein the known aliphatic amine of formula (II) is one in which A and A' are the same.

[0207] Clause 5. The process of any of the preceding clauses, wherein A and A' are the same and represent NHR'1.

[0208] Clause 6. The process of any of the preceding clauses, wherein at least one of A and A' represents -NH2.

[0209] Clause 7. The process according to any of the preceding clauses, wherein A and A' are the same and represent NHR'1, in particular -NH2.

[0210] Article 8. B: (C1~C 20 ) alkylene; (C1-C 20 ) alkylene, or the known 3- to 20-membered heteroalkylene defined in clause 1.

[0211] Article 9. B: (C1~C 15 ) alkylene; (C1-C 15 ) alkylene or the known 3- to 15-membered heteroalkylenes defined in clause 1.

[0212] Article 10. B: (C1~C 15 ) alkylene, (C2-C 15 ) Alkylene, (C4-C 15 ) alkylene or (C5-C 15 ) alkylene.

[0213] Clause 11. The process of any of the preceding clauses, wherein B represents a known 3- to 15-membered heteroalkylene, in particular a known 3- to 10-membered heteroalkylene or a known 4- to 8-membered heteroalkylene.

[0214] Clause 12. B represents a known 3- to 15-membered heteroalkylene, particularly a 3- to 10-membered heteroalkylene, each one of the ring members being C(R x )2, CR x , -NR' x -, and -S-, wherein R x and R' x (a) a process as described in any of the preceding clauses, wherein

[0215] Clause 13. B represents a known 4- to 15-membered heteroalkylene, in particular a known 4- to 10-membered heteroalkylene, each one of the ring members being C(R x )2, CR x , -NR' x -, and -S-, wherein R x and R' x 2. A process as described in any of the preceding clauses, wherein said process is as defined in clause 1.

[0216] Clause 14. B represents a known 3- to 15-membered heteroalkylene, in particular a known 3- to 10-membered heteroalkylene; (a) One or two of the ring members are -S- atoms and the other ring members are C(R x )2 and CR x or alternatively, (b) one or two of the ring members is -NR' x - and the other ring members are C(R x )2 and CR x wherein Rx is as defined in clause 1; (c) One of the ring members is a -S- atom and the other ring member is a C(R x)2 and CR x or alternatively, (d) one of the ring members is -NR' x - and the other ring members are C(R x )2 and CR x is a carbon atom selected from the group consisting of: (c) Two of the ring members are -S- atoms and the other ring members are C(R x )2 and CR x or alternatively, (f) Two of the ring members are -NR' x - and the other ring members are C(R x )2 and CR x is a carbon atom selected from the group consisting of: In the formula, R x and R' x is as defined in clause 1, A process as described in any of the preceding clauses.

[0217] Clause 15. The known aliphatic amines of formula (II) are selected from the group consisting of amines of formula (II1), amines of formula (II2), and amines of formula (II3): [ka] wherein a is an integer from 1 to 20; b, d, and e are independently integers from 1 to 10, more specifically, b, d, and e are the same; f and g are independently integers from 1 to 10, more specifically, f and g are the same; and more specifically, selected from the group consisting of hexamethylenediamine, octamethylenediamine, dodecamethylenediamine, cystamine, tris-(3-aminopropyl)amine, and tris-(2-aminopropyl)amine. The process of any one of the preceding clauses, wherein the compound is selected from the group consisting of:

[0218] Clause 16. The process of clause 1, wherein the amine is an aromatic amine of formula (IIbis).

[0219] Clause 17. The process according to the preceding clause, wherein the amine is an aromatic amine of formula (IIbis1): [ka] (wherein Rm, R'm, and W are as defined in clause 1).

[0220] Clause 18. The amine is an aromatic amine of formula (IIbis) or formula (IIbis1), and W is -NR t R' t The process according to any one of clauses 1, 16-17,

[0221] Clause 19. The amine is an aromatic amine of formula (IIbis) or formula (IIbis1), W is -NR t R' t and Rm, R'm, Rt and R't are the same.

[0222] Article 20. Rm, R'm, R t and R' t are the same and represent -H.

[0223] Article 21. Rt and R' t are the same and other than hydrogen.

[0224] Article 22. R m and R' m is hydrogen and R t and R' t are the same and other than hydrogen.

[0225] Article 23. R t and R' t represents an aromatic ring as defined in clause 1.

[0226] Article 24. R t and R't represents an aromatic ring having 6 members as defined in clause 1.

[0227] Article 25. R t and R' t each of which represents an aromatic ring having six members, all of which are -CR z - and R z (a) a process as described in any of the preceding clauses, wherein

[0228] Article 26. R t and R' t are the same and represent a 6-membered aromatic ring, all of the ring members being -CR z -Ring member, R z (a) a process as described in any of the preceding clauses, wherein

[0229] Clause 27. The amine is of formula (IIbis) or (IIbis1), W=NR t R' t and R m , R' m , R t and R' t The process of any of the preceding clauses, wherein one or more of has formula (IV): [ka] where n is 1 or 2, especially 1, and the other(s) are -H.

[0230] Clause 28. The process according to the preceding clause, wherein the amine of formula (IIbis) is one of formulas (V) or (VI): [ka] Clause 29. The amine is an aromatic amine of formula (IIbis) or formula (IIbis1), W being * The process described in any of the preceding clauses represents SSL.

[0231] Clause 30. The amine is an aromatic amine of formula (IIbis) or formula (IIbis1), W being * R stands for SSL m and R' m is the same as the process described in any of the preceding clauses.

[0232] Clause 31. The amine is an aromatic amine of formula (IIbis) or formula (IIbis1), W being * R stands for SSL m and R' m represents -H, the process described in any of the preceding clauses.

[0233] Clause 32. The process according to any of the preceding clauses, wherein the amine is an aromatic amine of formula (IIbis) or formula (IIbis1), and L represents an aromatic ring as defined in clause 1.

[0234] Clause 33. The process according to any of the preceding clauses, wherein L represents an aromatic ring having 6 members as defined in clause 1.

[0235] Clause 34. L represents an aromatic ring having 6 members as defined in clause 1, and may be the same or different, -CR v - a process as described in any of the preceding clauses, as represented by

[0236] Clause 35. The process of any of the preceding clauses, wherein L has formula (IV): [ka] (wherein n is 1 or 2, and one of the carbon atoms forming the aromatic ring is bonded to the -S- atom).

[0237] Clause 36. The process according to any of the preceding clauses, wherein the amine of formula (IIbis) corresponds to the amine of formula (VII): [ka]

[0238] Clause 37. The process of any of the preceding clauses, wherein the amine is selected from the group consisting of hexamethylenediamine, octamethylenediamine, dodecamethylenediamine, cystamine, tris-(3-aminopropyl)amine, tris-(2-aminopropyl)amine, and 4,4',4''-triaminotriphenylamine.

[0239] Clause 38. The process of any one of the preceding clauses, wherein the catechol derivative is a derivative of formula (III): [ka] (In the formula, R 18 , R 19 , R' 18 and R' 19 are the same or different, -H; -OH; -NR 20 R' 20 ; halogen (C1~C 10 ) alkyl; (C2~C 10 ) alkenyl; -OH, halogen, nitro, cyano, (C1-C 10 ) Alkyl, (C1-C 10 ) haloalkyl, -NR 21 R' 21 , -C(O)OR 22 , and -O-(C1-C 10 ) alkyl substituted with one or more substituents selected from the group consisting of (C1-C 10 ) alkyl; -OH, halogen, nitro, cyano, (C1-C 10 ) Alkyl, (C1-C 10 ) haloalkyl, -NR 23 R' 23 , -C(O)OR 24 , and -O-(C1-C 10) alkyl substituted with one or more substituents selected from the group consisting of (C2-C 10 ) alkenyl; Each one of the rings is:(a)-C(R y ) 2,- , -CR y -, -N-, -NR' y (b) is saturated, partially unsaturated, or aromatic; and (c) is a known ring system consisting of one or two rings, which may be isolated, partially isolated, or fused. selected from the group consisting of; R y Each of the is -H, -OH, (C1-C 10 ) Alkyl, (C2-C 10 ) alkenyl, (C2-C 10 ) alkynyl, (C1-C 10 ) haloalkyl, -O-(C1-C 10 ) Alkyl, Nitro, -NR 25 R 25 and halogen; R'y is -H, (C1-C 10 ) Alkyl, (C2-C 10 ) alkenyl, (C2-C 10 ) alkynyl, and (C1-C 10 ) haloalkyl; R 20 , R' 20 , R 21 , R' 21 , R 23 , R' 23 , R 25 , and R' 25 are the same or different, H; (C1-C 10 )Alkyl;(C1-C 10 )Haloalkyl;(C2-C 10 ) alkenyl; (C2-C 10 ) Alkynyl; -OH, halogen, nitro, cyano, (C1-C 10 ) Alkyl, (C1-C 10 ) haloalkyl, -NR 26 R' 26, -C(O)OR 27 , and -O-(C1-C 10 ) alkyl substituted with one or more substituents selected from the group consisting of (C1-C 10 ) alkyl; R 22 and R 24 H, (C1~C 10 ) Alkyl, (C1-C 10 )Haloalkyl, (C2-C 10 ) alkenyl; and (C 10 ) alkynyl; R 26 , R' 26 and R 27 are the same or different, -H; (C1-C 10 )Alkyl;(C1-C 10 )Haloalkyl;(C2-C 10 ) alkenyl; and (C 10 ) alkynyl.

[0240] Clause 39. The catechol derivative is a derivative of formula (III): -R 18 and R 19 are the same or different, -H; -OH; (C1-C 10 ) alkyl; (C1-C 10 ) alkyl; and (C2-C 10 ) alkenyl, or alternatively, -R 18 and R 19 is a known ring system consisting of two rings, each one of which is (a)-C(R y ) 2,- , -CR y -, -N-, -NR' y (b) is saturated, partially unsaturated, or aromatic; (c) is isolated, partially isolated, or fused; wherein Ry and R' y is as defined in clause 38; or alternatively, -R 18 and R 19 is a known ring system consisting of two rings, each one of which is (a)-C(R y ) 2,- , -CR y -, -N-, -NR' y (b) is saturated, partially unsaturated, or aromatic; (c) is fused; y and R' y is as defined in clause 38; or alternatively, -R 18 and R 19 is a known ring system consisting of two rings, each one of which is (a)-C(R y ) 2,- , -CR y -, -N-, -NR' y -, -S-, and -O-, provided that at least one of the rings is selected from the group consisting of a heteroatom (-N-, -NR' y (b) is saturated, partially unsaturated, or aromatic; (c) is fused; y and R' y is as defined in clause 38; or alternatively, -R 18 and R 19 is a known ring system consisting of two rings, each one of which is (a)-C(R y ) 2,- , -CR y -, -N-, -NR' y (b) is saturated, partially unsaturated, or aromatic; (c) is fused; wherein R y and R' y is as defined in clause 38; or alternatively, -R 18 and R19 One of the known ring systems consists of two rings, each of which is (a)-C(Ry) 2,- , -CR y -, -O-, with the proviso that at least one of the rings contains an -O- heteroatom; (b) is saturated, partially unsaturated, or aromatic; (c) is fused; y and R' y is as defined in clause 38; or alternatively, -R 18 and R 19 One of the known ring systems consists of two rings, each of which is (a)-C(Ry) 2,- , -CR y -, -O-, with the proviso that only one of the rings contains an -O- heteroatom; (b) is saturated, partially unsaturated, or aromatic; (c) is fused; R y and R' y is as defined in Article 38; 2. A process as described in any one of the preceding clauses.

[0241] Clause 40. The catechol derivative is of formula (III), wherein R 18 or R 19 A process according to any of the preceding clauses, wherein one of said is -H and the other is as defined in any of the above clauses, in particular clause 38 or 39.

[0242] Clause 41. The catechol derivative of formula (III) is R' 18 and R' 19 is -H, and R 18 and R 19 is as defined in any of clauses 38, 39 or 40.

[0243] Clause 42. The process of any of the preceding clauses, wherein the catechol derivative is selected from the group consisting of pyrocatechol, caffeic acid, dopamine, 4-methylcatechol, pyrogallol, and catechin.

[0244] Clause 43. The process of any one of the preceding clauses, wherein the pH is 6.5-8 or 7-7.5, or the pH is selected from the group consisting of 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.

[0245] Article 44. a) stirring is performed during the entire crosslinking step or during part of the crosslinking reaction, or alternatively b) the stirring is carried out in a manner that does not cause turbulence during all steps of crosslinking, in particular the stirring speed is less than or equal to 450 rpm, less than or equal to 400 rpm, or less than or equal to 350 rpm, or alternatively c) the stirring is performed using the lowest possible speed to avoid turbulence at the air / liquid interface, to be homogeneous throughout the entire procedure, and to avoid irregular movements of magnets or mechanical parts involved in the stirring procedure, more specifically, the stirring speed is equal to or less than 450 rpm, equal to or less than 400 rpm, or equal to or less than 350 rpm, or alternatively d) stirring is performed only during part of the crosslinking reaction, in particular stirring is performed so as not to provide turbulence until a color change is observed and then stopped, even more particularly stirring is performed for a period of 10 minutes to 2 hours and then stopped, or alternatively e) the stirring speed is equal to or less than 400 rpm, or equal to or less than 350 rpm; 2. A process as described in any one of the preceding clauses.

[0246] Clause 45. The process of any one of the preceding clauses, wherein the amine of formula (II) or (IIbis) is in molar excess relative to the catechol derivative.

[0247] Clause 46. The process of any one of the preceding clauses, wherein the crosslinking step is carried out at a temperature of 10 to 60° C.

[0248] Clause 47. The process of any one of the preceding clauses, wherein the crosslinking step is carried out for at least 24 hours.

[0249] Clause 48. The process of any one of the preceding clauses, wherein the liquid medium comprises an aqueous based buffer, more particularly an aqueous based buffer in which no nitrogen atoms or amino groups are present, even more particularly an aqueous based buffer selected from the group consisting of phosphate buffer, carbonate buffer, and citrate buffer.

[0250] Clause 49. The process of any one of the preceding clauses, wherein the liquid medium is water.

[0251] Clause 50. A free-standing catecholamine-based membrane obtainable by the process defined in any one of the preceding clauses.

[0252] Clause 51. The catecholamine-based membrane of the preceding clause, further comprising one or more molecules of interest selected from the group consisting of therapeutic molecules, cells, growth factors, detection labels (fluorescently active moieties, nanoparticles or antibodies), and combinations thereof.

[0253] Clause 52. A catecholamine-based membrane according to clause 51, wherein the molecule of interest is covalently bound to the membrane, particularly by an amide bond.

[0254] Article 53. (i.1) carrying out step (a) of the process defined in clause 1; (i.2) adding a molecule of interest to the reaction medium; and (i.3) Implementing step (b) of the process defined in clause 1 Contains or or alternatively (ii.1) incubating a catecholamine-based membrane as defined in clause 50 with a molecule of interest; 5. A process for preparing a catecholamine-based membrane as defined in clause 51 or 52.

[0255] Clause 54. Use of a catecholamine-based membrane according to clause 50 as an adhesive.

[0256] Clause 55. Use of the catecholamine-based membrane according to clause 50 as a vehicle for a molecule of interest, such as a therapeutic or labelled molecule.

[0257] Clause 56. A catecholamine-based membrane as defined in either clause 51 or 52, wherein the molecule of interest is a therapeutic molecule for use in therapy, or alternatively a free-standing catecholamine-based membrane as defined in either clause 47 or 48, wherein the molecule of interest is a detection label for use in diagnosis.

[0258] Clause 57. A catecholamine-based membrane as defined in either clause 51 or 52, wherein the molecule of interest is a cell, a growth factor, or a combination thereof, for use in tissue regeneration.

[0259] Clause 58. Articles partially or completely coated with a film as defined in any one of clauses 50 to 52.

Claims

1. A process for preparing a self-supporting catecholamine-based film, comprising: a) reacting a catechol derivative with an amine selected from the group consisting of: a.1) a known aliphatic amine hydrocarbon of formula (II) A-B-A' (II) wherein: A and A' are the same or different and represent -NR １ R' １ and B is (C １ ~C ２０ ) alkylene; -OH, halogen, -NO ２ , cyano, -O-(C １ ~C １０ ) alkyl, —C(O)OR ２ and -NR ３ R' ３ (C １ ~C ２０ ) alkylene; (C ２ ~C ２０ ) alkenylene; -OH, halogen, -NO ２ , cyano, O-(C １ ~C １０ ) alkyl, —C(O)OR ４ and -NR ５ R' ５ (C ２ ~C ２０ ) alkenylene; ２ ~C ２０ ) Alkynylene; -OH, halogen, -NO ２ , cyano, O-(C １ ~C １０ ) alkyl, —C(O)OR ６ and -NR ７ R' ７ (C ２ ~C ２０ ) alkynylene; the known 3- to 20-membered heteroalkylene; and -OH, halogen, -NO ２ , cyano, -O-(C １ ~C １０ ) alkyl, —C(O)OR ８ and -NR ９ R' ９ the known 3- to 20-membered heteroalkylenes substituted with one or more substituents selected from the group consisting of: the known 3- to 20-membered heteroalkenylenes; and -OH, halogen, -NO ２ , cyano, -O-(C １ ~C １０ ) alkyl, —C(O)OR １０ and -NR １１ R' １１ represents a known 3- to 20-membered heteroalkenylene substituted with one or more substituents selected from the group consisting of; A is bonded to a first atomic ring member forming part of the B biradical hydrocarbon skeleton; A' is bonded to the last atomic ring member forming part of the B biradical hydrocarbon skeleton; wherein the "first" and "last" atomic ring members are identified by reading the B biradical hydrocarbon skeleton from left to right or vice versa; R １ 、R' １ 、R ３ 、R' ３ 、R ５ 、R' ５ 、R ７ 、R' ７ 、R ９ 、R' ９ 、R １１ 、and R' １１ are the same or different and are selected from the group consisting of -H; (C １ ~C １０ ) alkyl; (C １ ~C １０ ) haloalkyl; (C ２ ~C １０ ) alkenyl; (C ２ ~C １０ ) alkynyl; and one or more substituents selected from the group consisting of -OH, -NO ２ , cyano, -O-(C １ ~C １０ ) alkyl, -C(O)OR １２ , and -NR １３ R' １３ and are selected from the group consisting of (C １ ~C １０ ) alkyl substituted with one or more substituents selected from the group; R ２ 、 R ４ 、 R ６ 、 R ８ 、 R １０ and R １２ are each independently selected from the group consisting of -H; (C １ ~C １０ )alkyl; (C １ ~C １０ )haloalkyl; (C ２ ~C １０ )alkenyl; (C ２ ~C １０ )alkynyl; and one or more substituents selected from the group consisting of -OH, -NO ２ , cyano, -O-(C １ ~C １０ )alkyl, -C(O)OR １４ , and -NR １５ R’ １５ ; and are each independently selected from the group consisting of (C １ ~C １０ )alkyl substituted with one or more substituents selected from the group; R １３ , R' １３ , R １４ , R １５ and R' １５ are the same or different, -H; (C １ ~C １０ ) alkyl; (C １ ~C １０ ) haloalkyl; ２ ~C １０ ) alkenyl; and (C ２ ~C １０ ) alkynyl; The "known 3- to 20-membered heteroalkylene" is C(R ｘ ) ２ , C.R. ｘ , -N-, -NR' ｘ means a known saturated chain consisting of 3 to 20 ring members selected from the group consisting of -, S-, and -O-, provided that (a) at least one of the ring members is -N-, -NR ｘ (b) the first and last ring members forming the heteroalkylene backbone are carbon atoms; The "known 3- to 20-membered heteroalkenylene" is C(R ｘ ) ２ , -CR ｘ -, -N-, -NR' ｘ means a known unsaturated chain consisting of 3 to 20 ring members selected from the group consisting of -, S-, and -O-, provided that (a) at least one of the ring members is -N-, -NR ｘ (b) the first and last ring members forming the heteroalkylene backbone are carbon atoms; (c) the unsaturated chain contains one or more double bonds; R ｘ is -H; -OH; (C １ ~C １０ )-alkyl; (C ２ ~C １０ )-alkenyl; (C ２ ~C １０ )-alkynyl; (C １ ~C １０ )-haloalkyl; O-(C １ ~C １０ )-alkyl; -O-(C ２ ~C １０ )-alkenyl; -O-(C ２ ~C １０ )-alkynyl; nitro, -NR ｘ１ R ｘ２ ; -NO ２ , cyano, -O-(C １ ~C １０ )-alkyl, -C(O)OR １６ , and -NR １７ R’ １７ substituted by one or more substituents selected from the group consisting of; (C １ ~C １０ )-alkyl; and independently selected from the group consisting of halogen; R Ｘ１ , Rx ２ , R １６ , R １７ , R' １７ , and R' ｘ are independently selected from the group consisting of -H, (C １ ~C １０ ) alkyl, (C ２ ~C １０ ) alkenyl, (C ２ ~C １０ ) alkynyl, and (C １ ~C １０ ) haloalkyl); a.2) an aromatic amine of formula (IIbis): 【Chemical 1】 wherein W is -NR ｔ R' ｔ or ＊ represents S-S-L; wherein, ( ＊ ) indicates that the S atom of the W radical is bonded to a carbon atom forming part of the aromatic ring; L is substituted with one or more substituents selected from the group consisting of -OH, -NO ２ , cyano, -O-(C １ ~C １０ ), alkyl, -C(O)OR ２８ , and -NR ２８ R’ ２８ ), (wherein at least one of the substituents is -NR １ ~C １０ ), alkyl (however, at least one of the substituents is -NR ２８ R’ ２８ ); -OH, -NO ２ , cyano, -O-(C １ ~C １０ ), alkyl, -C(O)OR ２８ , and -NR ２８ R’ ２８ ), (wherein at least one of the substituents is -NR ２８ R’ ２８ ); -OH, -NO ２ , cyano, -O-(C １ ~C １０ ), alkyl, -C(O)OR ２８ , and -NR ２８ R’ ２８ ), (wherein at least one of the substituents is -NR ２ ~C １０ ), alkenyl (however, at least one of the substituents is -NR ２８ R’ ２８ ); -OH, -NO ２ , cyano, -O-(C １ ~C １０ ), alkyl, -C(O)OR ２８ , and -NR ２８ R’ ２８ ), (wherein at least one of the substituents is -NR ２ ~C １０ ), alkynyl (however, at least one of the substituents is -NR ２８ R’ ２８ ); and -CR ｖ -, -N-, -O-, -NR’ ｖ selected from the group consisting of known aromatic rings having 5 or 6 members selected from the group consisting of -S-; R ｍ 、 R', ｍ 、 R ｔ and R' ｔ are the same or different and are selected from the group consisting of H, (C １ ~C １０ ), alkyl; (C １ ~C １０ ), haloalkyl; (C ２ ~C １０ ), alkenyl; (C ２ ~C １０ ), alkynyl; -OH, -NO ２ , cyano, -O-(C １ ~C １０ ), alkyl, -C(O)OR ２９ , and -NR ３０ R' ３０ substituted with one or more substituents selected from the group consisting of (C １ ~C １０ ), alkyl; and -CR ｚ -, -N-, -NR' ｚ , -O-, and -S-, and are selected from the group consisting of known aromatic rings having 5 or 6 members; R ｖ At least one of them is -NR ３１ R' ３１ and the other R ｖ (if plural) is H; -NR ３１ R' ３１ ; (C １ ~C １０ ) alkyl; (C １ ~C １０ ) haloalkyl; (C ２ ~C １０ ) alkenyl; (C ２ ~C １０ ) alkynyl; and one or more substituents selected from the group consisting of -OH, -NO ２ , cyano, -O-(C １ ~C １０ ) alkyl, C(O)OR ３２ , and -NR ３３ R' ３３ substituted (C １ ~C １０ ) alkyl selected from the group consisting of; R ｚ and R' ｚ are each independently selected from the group consisting of H; (C １ ~C １０ alkyl); (C １ ~C １０ haloalkyl); (C ２ ~C １０ alkenyl); (C ２ ~C １０ alkynyl); and one or more substituents selected from the group consisting of -OH, -NO ２ , cyano, -O-(C １ ~C １０ alkyl), -C(O)OR ３４ , and -NR ３５ R' ３５ ; and are each independently selected from the group consisting of (C １ ~C １０ alkyl) substituted with one or more of the above substituents; R ２８ , R' ２８ , R ２９ , R ３０ , R' ３０ , R ３１ , R' ３１ , R ３２ , R ３３ , R' ３３ , R ３４ , R ３５ , and R' ３５ is independently selected from the group consisting of -H; (C １ ~C １０ ) alkyl; (C １ ~C １０ ) haloalkyl; (C ２ ~C １０ ) alkenyl; and (C ２ ~C １０ ) alkynyl); crosslinked to create a catecholamine film at the air / liquid interface in the absence of any support, and the crosslinking reaction is carried out in a liquid medium in which both catechol and the amine are soluble at a pH of 6.5 to 10, with stirring, particularly suitable stirring; and b) isolating the film obtained from step (a) from the air / liquid interface A process comprising the steps of.

2. The process according to claim 1, wherein the amine is an amine of formula (II).

3. A and A' are the same and are -NHR', １ in particular -H, ２ The process according to claim 1, wherein it represents.

4. B is (C １ ~C １５ ) alkylene; (C １ ~C １５ ) alkylene substituted as defined in claim 1, or known 3- to 15-membered heteroalkylene as defined in claim 1, the process according to claim 1.

5. The process according to claim 1, wherein the amine is selected from the group consisting of hexamethylenediamine, octamethylenediamine, dodecamethylenediamine, cystamine, tris-(3-aminopropyl)amine, tris-(2-aminopropyl)amine, and 4,4',4''-triaminotriphenylamine.

6. The process according to claim 1, wherein the catechol derivative is of formula (III): 【Chemical Formula 2】 wherein R １８ , R １９ , R' １８ and R' １９ are the same or different, -H; -OH; -NR ２０ R' ２０ ; halogen (C １ ~C １０ ) alkyl; (C ２ ~C １０ ) alkenyl; -OH, halogen, nitro, cyano, (C １ ~C １０ ), alkyl, (C １ ~C １０ ), haloalkyl, -NR ２１ R’ ２１ , -C(O)OR ２２ , and -O-(C １ ~C １０ ), alkyl, substituted with one or more substituents selected from the group consisting of: (C １ ~C １０ ); -OH, halogen, nitro, cyano, (C １ ~C １０ ), alkyl, (C １ ~C １０ ), haloalkyl, -NR ２３ R’ ２３ , -C(O)OR ２４ , and -O-(C １ ~C １０ ), alkyl, substituted with one or more substituents selected from the group consisting of (C ２ ~C １０ ), alkenyl; and Each one of the rings is: (a) -C(R ｙ ) ２，－ , -CR ｙ -, -N-, -NR' ｙ (b) is saturated, partially unsaturated, or aromatic; and (c) is a known ring system consisting of one or two rings that are isolated, partially isolated, or fused. selected from the group consisting of; R ｙ One of each of them is selected independently from the group consisting of -H, -OH, (C １ ~C １０ ), alkyl, (C ２ ~C １０ ), alkenyl, (C ２ ~C １０ ), alkynyl, (C １ ~C １０ ), haloalkyl, -O-(C １ ~C １０ ), alkyl, nitro, -NR ２５ R ２５ , and halogen; R'y is selected from the group consisting of -H, (C １ ~C １０ ), alkyl, (C ２ ~C １０ ), alkenyl, (C ２ ~C １０ ), alkynyl, and (C １ ~C １０ ), haloalkyl, R ２０ 、R' ２０ 、R ２１ 、R' ２１ 、R ２３ 、R' ２３ 、R ２５ 、およびR' ２５ are the same or different and are H; (C １ ~C １０ ) alkyl; (C １ ~C １０ ) haloalkyl; (C ２ ~C １０ ) alkenyl; (C ２ ~C １０ ) alkynyl; and -OH, halogen, nitro, cyano, (C １ ~C １０ ) alkyl, (C １ ~C １０ ) haloalkyl, -NR ２６ R' ２６ , -C(O)OR ２７ , and -O-(C １ ~C １０ ) alkyl, and are selected from the group consisting of one or more substituents substituted on (C １ ~C １０ ) alkyl; R ２２ and R ２４ are independently selected from the group consisting of H, (C １ ~C １０ )alkyl, (C １ ~C １０ )haloalkyl, (C ２ ~C １０ )alkenyl; and (C ２ ~C １０ )alkynyl; R ２６ , R' ２６ and R ２７ are the same or different, -H; (C １ ~C １０ ) alkyl; (C １ ~C １０ ) haloalkyl; (C ２ ~C １０ ) alkenyl; and (C ２ ~C １０ ) alkynyl selected from the group consisting of) is a compound of.

7. The process according to claim 1, wherein the catechol derivative is selected from the group consisting of pyrocatechol, dopamine, pyrogallol, caffeic acid, 4-methylcatechol, and catechin.

8. The process according to claim 1, wherein the pH is from 6.5 to 8, or from 7 to 7.

5.

9. The process according to claim 1, wherein the known aliphatic amine hydrocarbon of formula (II) is in a molar ratio in excess with respect to the catechol derivative.

10. A self-supporting catecholamine-based film obtainable by the process according to any one of claims 1 to 9.

11. The self - supporting catecholamine - based membrane according to claim 10, further comprising one or more target molecules selected from therapeutic molecules and detection labels. **Claim 12** Use of the catecholamine - based membrane according to claim 10 as an adhesive. **Claim 13** Use of the catecholamine - based membrane according to claim 10 as a vehicle for target molecules such as therapeutic labels or detection labels. **Claim 14** The self - supporting catecholamine - based membrane according to claim 11, wherein the target molecule is a therapeutic molecule for use in therapy, or alternatively, the self - supporting catecholamine - based membrane according to claim 11, wherein the target molecule is a detection label for use in diagnosis. **Claim 15** An article partially or completely coated with the membrane defined in claim 10.

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