BIOINSPIRED SUPRAMOLECULAR MEDICAL ADHESIVES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of USSN 62/547,651, filed on

August 18, 2017, which is incorporated herein by reference in its entirety for all purposes. STATEMENT OF GOVERNMENTAL SUPPORT

[0002] This invention was made with government support under Grant No.

EB022031 awarded by the National Institutes of Health. The Government has certain rights in this invention.

BACKGROUND

[0003] Mussel adhesive proteins (MAPs) are remarkable underwater adhesive materials secreted by marine mussels that form tenacious bonds to the substrates upon which they reside. During the process of attachment to a substrate, MAPs are secreted as adhesive fluid precursors that undergo a crosslinking or hardening reaction which leads to the formation of a solid adhesive plaque. One of the unique features of MAPs is the presence of L-3,4-dihydroxyphenylalanine (DOPA), an unusual amino acid that is believed to be responsible for interfacial adhesion. Furthermore, several mechanisms of toughening and energy dissipation exist within the mussel tissue, arising from noncovalent interactions among subunits of the proteins. The unique combination of DOP A-inspired adhesion and noncovalent toughening represents a novel design principle for medical adhesives. [0004] Pregnancy and birth are universally the most wonderful events in people' s lives, however in some cases surgery of the unborn child may be necessary to permit a healthy future into our world. To perform surgery, pediatric surgeons have to puncture the fragile amniotic sac during operation. Unfortunately, an absence of viable adhesive materials to heal or seal the amniotic sac post-surgery often results in preterm birth due to leakage of essential amniotic fluids and complete fracture of the amniotic sac. Because fetal surgery is usually performed early in gestation, preterm birth results in a low survival rate and therefore directly limits significant health benefits. Surprisingly, two decades of active investigation into commercial and novel sealants has not yielded satisfying solutions.

[0005] In the medical arena, few adhesives exist that provide both robust adhesion in a wet environment and suitable mechanical properties to be used as a tissue adhesive or sealant. For example, fibrin-based tissue sealants (e.g. Tisseel VH, Baxter Healthcare) provide a good mechanical match for natural tissue, but possess poor tissue-adhesion characteristics. Conversely, cyanoacrylate adhesives (e.g. Dermabond, ETHICON, Inc.) produce strong adhesive bonds with surfaces, but tend to be stiff and brittle in regard to mechanical properties.

SUMMARY

[0006] Described herein are a set of polymers that show the different parameters that influence wet adhesion and provide a framework to design tough biomedical adhesives with high adhesive strength and the ability to detach and reattach to tissue. Importantly, in contrast to most existing medical adhesives, the tissue adhesive formulations described herein do not require the use of chemical cross-linkers, activators, initiators and other toxic compounds and do not require mixing of two solutions during use.

[0007] Accordingly, various embodiments contemplated herein may include, but need not be limited to, one or more of the following:

[0008] Embodiment 1 : A supramolecular adhesive polymeric material, wherein subunits (in certain embodiments repeating subunits) of said polymer comprise:

[0009] i) at least one monomer having a substituted 1,2 dihydroxy benzenediol or a substituted 1,2,3 trihydroxy benzenetriol;

[0010] ii) at least one monomer that can form directional non-covalent interactions with substantial interaction strength; and

[0011] iii) a combination substantially hydrophobic and hydrophilic monomers that permit controlled swelling of the material.

[0012] Embodiment 2: The adhesive material of embodiment 1, wherein the non- covalent interactions of the monomer that can form directional non-covalent interactions is based on hydrogen bonding. [0013] Embodiment 3 : The adhesive material of embodiment 2, wherein said hydrogen bonding is provided by a motif that provides at least 3 hydrogen bonds per motif.

[0014] Embodiment 4: The adhesive material according to any one of embodiments

2-3, wherein said hydrogen bonding is provided by a motif selected from the group consisting of a ureidopyrimidinone, a bis-urea (four H-bonds), a bis-urethane (four H- bonds), and a benzene tricarboxamide (H-bonds and aromatic stacking).

[0015] Embodiment 5 : The adhesive material of embodiment 2, wherein said non- covalent interactions are mediated by hydrogen bonding of a ureidopyrimidinone. [0016] Embodiment 6: The adhesive material of embodiment 5, wherein said non- covalent interactions are mediated by hydrogen bonding of a 2-ureido-4[lH]pyrimidinone.

[0017] Embodiment 7: The adhesive material according to any one of embodiments

1-6, wherein said monomer that can form directional non-covalent interactions comprises said ureidopyrimidinone coupled by a linker to a hydroxymethylmethacrylate.

[0018] Embodiment 8: The adhesive material of embodiment 7, wherein said linker comprises a hexamethylene diisocyanate linker.

[0019] Embodiment 9: The adhesive material according to any one of embodiments

1-8, wherein said substantially hydrophobic monomers comprise acrylates or methacrylates. [0020] Embodiment 10: The adhesive material of embodiment 9, wherein said substantially hydrophobic monomers comprise one or more monomers selected from the group consisting of 2-Ethylhexyl acrylate, 2-Ethylhexyl methacrylate, ethyl acrylate, ethyl methacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate, isooctyl acrylate, isooctyl methacrylate, methyl acrylate, methyl methacrylate, n-butyl acrylate, n-butyl methacrylate, t-butyl acrylate, t-butyl methacrylate, hexyl (meth)acrylate (meaning both methacrylate and acrylate of this monomer), benzyl (meth)acrylate, dodecyl (meth)acrylate, n-octadecyl (meth)acrylate, n-propyl (meth)acrylate, 2-ethyl hexyl (meth)acrylate, isodecyl

(meth)acrylate, and trimethylsilyl (meth)acrylate.

[0021] Embodiment 11 : The adhesive material according to any one of

embodiments 9-10, wherein said substantially hydrophobic monomers comprise butyl methacrylate.

[0022] Embodiment 12: The adhesive material according to any one of

embodiments 1-9, wherein said hydrophilic monomers comprise one or more monomers selected from the group consisting of PEG substituted methacrylate, vinyl acetate, vinyl alcohol, N-vinyl-2-pyrrolidone, and (meth)acrylic acid.

[0023] Embodiment 13 : The adhesive material of embodiment 12, wherein said hydrophilic monomers comprise PEG substituted methacrylate.

[0024] Embodiment 14: The adhesive material according to any one of

embodiments 1-13, wherein said monomer having a substituted 1,2 dihydroxy benzenediol or a substituted 1,2,3 trihydroxy benzenetriol is selected from the group consisting of dopamine 4-(2-methacrylamide-ethyl)-l,2-benzenediol, 3 -(2-methacrylami de-ethyl)- 1,2- benzenediol, 4-(2-methacrylamide-ethyl)-2 -benzenediol, 4-(2-methacrylamide-ethyl)-l,2,3 benzenetriol, and 5-(2-methacrylamide-ethyl)-l,2,3 benzenetriol.

[0025] Embodiment 15: The adhesive material of embodiment 14, wherein the monomer is dopamine 4-(2-methacrylamide-ethyl)-l,2-benzenediol. [0026] Embodiment 16: The adhesive material according to any one of embodiments 1-15, wherein said adhesive material comprises one or more subunits according to the formula:

wherein: n ranges from about 5 to about 25 mol%; m ranges from about 5 to about 15 mol%; o ranges from about 25 wt% to about 75 wt%; p is adjusted to provide about 50 weight percent of hydrophilic monomer; a ranges from about 3 up to about 100 repeating units; and q ranges from 1 up to about 12.

[0027] Embodiment 17: The adhesive material of embodiment 16, wherein n is about 10 mol%. [0028] Embodiment 18: The adhesive material according to any one of embodiments 16-17, wherein m is about 10 mol%.

[0029] Embodiment 19: The adhesive material according to any one of embodiments 16-18, wherein o is about 50 wt%. [0030] Embodiment 20: The adhesive material according to any one of

embodiments 16-19, wherein p in mol% is about 100 - n (mol%) - m (mol%) - o (mol%).

[0031] Embodiment 21 : The adhesive material according to any one of

embodiments 16-20, wherein a is about 20 in repeating units. [0032] Embodiment 22: The adhesive material according to any one of

embodiments 16-21, wherein q is about 6.

[0033] Embodiment 23 : The adhesive material according to any one of

embodiments 16-22, wherein said adhesive material comprises a plurality of said subunit(s).

[0034] Embodiment 24: The adhesive material of embodiment 23, wherein said subunits comprising said plurality are contiguous.

[0035] Embodiment 25: The adhesive material of embodiment 23, wherein said adhesive material consists of said plurality of subunits.

[0036] Embodiment 26: The adhesive material of embodiment 23, wherein said subunits comprising said plurality are not contiguous. [0037] Embodiment 27: The adhesive material according to any one of

embodiments 1-26, wherein said adhesive material has a molar mass of at least 1000 g/mol.

[0038] Embodiment 28: The adhesive material of embodiment 27, wherein said adhesive material has a molar mass of at least 5000 g/mol.

[0039] Embodiment 29: The adhesive material of embodiment 27, wherein said adhesive material has a molar mass of at least 10000 g/mol.

[0040] Embodiment 30: The adhesive material according to any one of

embodiments 1-29, wherein the adhesive material is characterized by an adhesive tissue shear strength of at least about 25 kPa.

[0041] Embodiment 31 : The adhesive material of embodiment 30, wherein the adhesive material is characterized by an adhesive tissue shear strength of at least about 40 kPa.

[0042] Embodiment 32: The adhesive material of embodiment 31, wherein the adhesive material is characterized by an adhesive tissue shear strength of about 110 kPa.

[0043] Embodiment 33 : The adhesive material according to any one of

embodiments 1-32, wherein the material can regain a substantial portion of its original adhesive strength after catastrophic rupture within a period of time upon re-adhesion. [0044] Embodiment 34: The adhesive material of embodiment 33, wherein the contact time after catastrophic rupture is at least about 10 minutes, or at least about 15 minutes, or at least about 30 minutes, or at least about 45 minutes, or at least about 1 hour or longer. [0045] Embodiment 35: The adhesive material according to any one of

embodiments 1-34, wherein said adhesive material is a solid material.

[0046] Embodiment 36: The adhesive material of embodiment 35, wherein said material comprises a solid polymer film.

[0047] Embodiment 37: The adhesive material according to any one of

embodiments 1-32, wherein said adhesive material comprises a liquid material.

[0048] Embodiment 38: The adhesive material of embodiment 37, wherein said adhesive material comprises an injectable liquid material.

[0049] Embodiment 39: The adhesive material according to any one of

embodiments 37-38, wherein said material is provided as a liquid by dissolution in a water- soluble hydrogen bond disrupting biocompatible solvent.

[0050] Embodiment 40: The adhesive material of embodiment 39, wherein said solvent comprises a solvent selected from the group consisting of PEG having a molecular weight of 1000 g/mol or lower, DMSO and ethyl lactate.

[0051] Embodiment 41 : The adhesive material of embodiment 40, wherein said PEG comprises a PEG having molecular weight of 900 g/mol or lower, or 800 g/mol or lower, or 700 g/mol or lower, or 600 g/mol or lower, or 500 g/mol or lower, or 400 g/mol or lower, or 300 g/mol or lower, or 200 g/mol or lower.

[0052] Embodiment 42: The adhesive material of embodiment 40, wherein said

PEG comprises a PEG having molecular weight of about 400 g/mol. [0053] Embodiment 43 : The adhesive material according to any one of

embodiments 39-42, wherein said solvent comprises ethyl lactate.

[0054] Embodiment 44: The adhesive material according to any one of

embodiments 1-43, wherein said adhesive material is sterile.

[0055] Embodiment 45: The adhesive material according to any one of

embodiments 1-44, wherein said adhesive material is provided as a coating on biomedical material or device to render said biomedical material or device adhesive to tissue. [0056] Embodiment 46: A method for attaching two substrates to one another, said method comprising contacting each substrate with a supramolecular adhesive polymeric material according to any one of embodiments 1-44 for a time sufficient that the substrates are attached to one another. [0057] Embodiment 47: The method of embodiment 46, wherein at least one of the two substrates comprises a biological tissue surface.

[0058] Embodiment 48: The method of embodiment 47, wherein each of the two substrates comprises a biological tissue surface.

[0059] Embodiment 49: The method according to any one of embodiments 46-48, wherein one or both surfaces comprises a dermal or mucosal tissue surface.

[0060] Embodiment 50: The method according to any one of embodiments 46-48, wherein one or both surfaces comprises an internal membrane tissue surface.

[0061] Embodiment 51 : The method according to any one of embodiments 46-50, wherein at least one of the two substrates comprises a mammalian tissue surface. [0062] Embodiment 52: The method of embodiments 46-47, and 49-51, wherein at least one of the two substrates comprises a non-biological surface.

[0063] Embodiment 53 : The method according to any one of embodiments 46-52, wherein said adhesive is applied in solid form.

[0064] Embodiment 54: The method of embodiment 53, wherein said adhesive comprises a solid polymer film.

[0065] Embodiment 55: The method according to any one of embodiments 46-54, wherein said adhesive material is used to seal a rupture, a hole, a tear, or an incision in a tissue.

[0066] Embodiment 56: The method according to any one of embodiments 46-51, and 53-55, wherein said adhesive material is used to mechanically adhere two tissues together.

[0067] Embodiment 57: The method according to any one of embodiments 46-56, wherein said adhesive material is applied as a liquid onto wet tissue, whereupon it transforms into a solid adhesive. [0068] Embodiment 58: The method according to any one of embodiments 46-47, and 49-56, wherein said adhesive material is applied as a coating to a biomedical material or device to render the latter adhesive to tissue.

[0069] Embodiment 59: The method according to any one of embodiments 46-47, and 49-56, wherein said adhesive material is used to adhere a biomedical material or device to a tissue surface.

[0070] Embodiment 60: The method according to any one of embodiments 46-56, wherein said method promotes healing of a wound.

[0071] Embodiment 61 : The method according to any one of embodiments 46-56, wherein said adhesive material protects and seals anastomosis and/or suture lines in internal body cavities.

[0072] Embodiment 62: The method according to any one of embodiments 46-56, wherein said adhesive material prevents dehiscence of an anastomosis or a surgical wound in a patient. [0073] Embodiment 63 : The method according to any one of embodiments 46-56, wherein said method comprises occluding a fistula within a patient.

[0074] Embodiment 64: A method of sealing an amniotic sack after fetal surgery, said method comprising applying to puncture or incision in said amniotic sack a

supramolecular adhesive polymeric material according to any one of embodiments 1-44 where said adhesive seals a puncture or incision in said amniotic sack.

[0075] Embodiment 65: The method of embodiment 64, wherein said applying comprises injecting a liquid formulation of said adhesive material.

[0076] Embodiment 66: The method of embodiment 65, where said liquid formulation transforms into a solid adhesive on contact with a tissue. [0077] Embodiment 67: A kit, said kit comprising a container containing supramolecular adhesive polymeric material according to any one of embodiments 1-44.

[0078] Embodiment 68: The kit of embodiment 67, wherein said container comprise a sterile package.

[0079] Embodiment 69: The kit according to any one of embodiments 67-68, wherein said kit comprises a container containing a diluent that can be combined with said adhesive material to form a liquid adhesive material. [0080] Embodiment 70: The kit of embodiment 69, wherein said diluent is sterile.

[0081] Embodiment 71 : The kit according to any one of embodiments 69-70, wherein said diluent comprises a water-soluble hydrogen bond disrupting biocompatible solvent. [0082] Embodiment 72: The kit of embodiment 71, wherein said diluent comprises a solvent selected from the consisting of PEG having a molecular weight of 1000 g/mol or lower, DMSO and ethyl lactate.

[0083] Embodiment 73 : The kit of embodiment 72, wherein said PEG comprises a

PEG having molecular weight of 900 g/mol or lower, or 800 g/mol or lower, or 700 g/mol or lower, or 600 g/mol or lower, or 500 g/mol or lower, or 400 g/mol or lower, or 300 g/mol or lower, or 200 g/mol or lower.

[0084] Embodiment 74: The kit of embodiment 72, wherein said PEG comprises a

PEG having molecular weight of about 400 g/mol.

[0085] Embodiment 75: The kit of embodiment 71, wherein said diluent comprises ethyl lactate.

[0086] Embodiment 76: The kit according to any one of embodiments 67-75, wherein said kit includes an applicator device for transfer of said adhesive from said container onto a biological tissue.

[0087] Embodiment 77: The kit according to any one of embodiments 67-76, wherein said kit comprises instructional materials teaching the use of said adhesive on a biological tissue.

[0088] In certain embodiments with the teaching provided herein, it is possible to provide polymers with different polymer backbones such as polyester, polyanhydride, and the like to render these biodegradable. Illustrative, but non-limiting areas of application of the adhesive materials described herein include, but are not limited to, general tissue adhesive patches, injectable formulations, surgical sealants, Hernia Mesh, hemostatic patch, pneumostatic patch, fetal membrane sealant, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 shows the structure of bioinspired supramolecular polymer adhesive. Panel a: Chemical structure of the polymeric material. The influence of the co- monomers is depicted within the circled areas. Wet tissue adhesion is mediated by dopamine methacryl amide (DMA, in red). Hydrogen bonding crosslinks are established via ureidopyrimidinone-hydroxymethylmethacrylate (UPy, in orange) monomers. The equilibrium swelling of the polymeric material is controlled through a combination of hydrophilic PEG-methacrylate (of different chain lengths) and hydrophobic butyl methacrylate (combined in green). Panel b: Wet adhesion mechanism of dopamine. First dopamine is auto oxidized into the quinone form via dissolved oxygen, than the latter can react with amino acid residues such as lysine and cysteine that are present at the surface to form covalent bonds between the polymeric adhesive and the tissue surface. Panel c:

Reversible formation of crosslinks via dimerization through four-fold H-bonding of the UPy motif. Panel d:. Image of a flexible polymeric film (polymer 3) cut into a 'Cal' logo shape.

[0090] Figure 2 shows a schematic of the free radical polymerization of bioinspired supramolecular polymer adhesive. Polymers were synthesized from monomers that were either directly purchased (PEG-methacrylate) or synthesized as described in literature (dopaminemethacrylamide and UPy-HEMA) via free radical polymerization in DMF at 80 0C for 5 hours under oxygen free conditions. Three times precipitation from a DCM/MeOH mixture (4: 1) into cold diethyl ether resulted in the pure polymer.

[0091] Figure 3 shows a table with the polymer characterization and swelling data.

All data presented in the table are measured by ¹ H- MR (monomer molecular percentages), GPC (molecular weights and PDI) and via equilibrium swelling experiments of polymeric films (-200 μπι thickness) in phosphate buffered saline at room temperature (n = 3).

[0092] Figure 4 shows the methods that were followed for the measurement of wet tissue adhesion according to ASTM-2255-05. Panel A: For solid patch adhesives bovine pericardium tissue was adhered to one side of a polycarbonate rectangular bar (left part of image) and the adhesive patch material as square film (-200 μπι thickness) on another polycarbonate bar (right part of image). A lap joint was formed by overlapping the two lap bars and held together with a small binder clip. Solid patch adhesion shear experiments were performed with 10 lap joints. Panel B: For liquid adhesives bovine pericardium tissue was adhered to two polycarbonate lap bars left image) and the adhesive liquid formulation (-40 μΙ_, of 33 wt% polymer solution in ethyl lactate, right image) was added via a syringe onto one of the two tissues. A lap joint was formed by overlapping the two lap bars

(overlap area = 1 cm x 1 cm) and held together with a small binder clip. Liquid formulation adhesion shear experiments were performed with 15 lap joints. Panel C: After preparation lap joints were submerged into PBS solution at 37°C for 1 hour in a term stated bath (left image). Than the lap joints were loaded in shear at 5 mm/min until catastrophic rupture occurred (right image). The shear strength that is reported was calculated by the maximum load divided by the adhesive area. Furthermore, the shear strain at the maximum load was also recorded. Panel D: After the tensile test the two lap bars were studied to observe whether adhesive failure and/or cohesive failure led to rupture. Panel E: Most samples did not optically reveal what the mode of failure was. Therefore the lap bars were subjected to Arnow's stain (Yoo et al. (2016) Nat. Comm., 7: 11923.) in which a red coloration indicates the presence of Dopa. The observation of Dopa on the tissue surface indicates cohesive failure (failure of the adhesive polymer matrix) and the absence of Dopa at the tissue surface indicates adhesive failure (failure at the interface between adhesive and tissue).

[0093] Figure 5 shows results of adhesion shear tests to bovine pericardium (a tissue that is high in collagen). The tests were performed according to ASTM-2255-05 with a sample size of 10. Higher adhesion strength was found when increasing the hydrophobic content (compare polymer 1 to 3) while Dopa and UPy contents were constant due to higher interaction strength of H-bonding in a more hydrophobic environment. Lower adhesion strength was found for CI and C3 (that both did not contain Dopa) indicating that Dopa is responsible for tissue - adhesive material interactions. Similar adhesion strength was found for CI and C3 indicating that adhesion in these examples is due to non-specific interactions (such as hydrophobic interactions). Higher adhesion strength for C2 compared to 3 was contributed to the higher Dopa content of C2. The cohesive strength is higher for 3 compared to 1 and C2 since excessive swelling resulting in loss of most of the patch was observed for 1 and C2.

[0094] Figure 6 shows the effect of PEG side chains on tissue adhesion. Polymers

2,3 and 4 all contain similar Dopa and UPy contents and have similar PEG weight percentages. However, the PEG side chain length differs (3 repeating units for polymer 2; 9 repeating units for polymer 3 and 20 repeating units for polymer 4). Different adhesive strength were observed for adhesive patches of 2 ,3 and 4, with a high adhesion strength for 4. Low equilibrium swelling in PBS buffer was observed for 2 and similar swelling was observed for 3 and 4. The dry state of 4 was rubbery, while the swollen state was found to be brittle, probably due to nano phase separation. Staining of the tissue for catechol motifs (Arnow's stain, see for example Yoo et al. (2016) Nat. Comm., 7: 11923.) indicated that after the tensile test catechol was present on the tissue for all different patches. This result indicates that in all cases the adhesive failed due to cohesive forces rather than adhesive forces. Additionally, the stained (for a similar length of time) patch side after the tensile test showed a much darker patch for 3 and 4 compared to 2 indicating lower availability of Dopa for 2.

[0095] Figure 7. Tissue shear adhesion tests of the adhesives as patches. Panel a:

Displayed are the shear strength results for polymers 1 - 4 and control polymers CI - C3. The average values are displayed above the bars and the vertical thin bar shows the standard deviation. Panel b: Images of the lap bar with the adhesive patch after the tensile test. The corresponding polymer is displayed in the lower part of the image. Note that in the case of 1, C2 and C3 there was almost not polymer film observed after the test, probably due to swelling and solution. In the case of C3 part of the swollen polymer was observed on the backside of the tensile bar. Panel c: Arnow's staining of the tissue side after the tensile test. Dopa is observed evenly on the tissue side where the patch was attached for 2 - 4 and C2 indicating that all these lap joints failed cohesively. The tissue surface of 1 shows partly larger concentrations of red indicating and uneven failure, possibly at the glued surface due to excessive swelling during incubation prior to the tensile test. The edges where patch of C2 was attached shows pronounced redness indicating larger content of dopa due to swelling and accumulation around the edges of the tensile bar. Note that the neat tissue showed yellowing due to random nitration of the tissue, however redness was not observed indicating the absence of diols and triols in the tissue.

[0096] Figure 8 illustrates the creation of liquid adhesives with water insoluble polymers and uses thereof. The polymers that were synthesized as swellable in aqueous conditions but not soluble. When these polymers would be dissolved in a water soluble solvent, the solvent would dissipate in water and an adhesive polymer would be precipitated at the same time. Preparation of a stable solution of polymer was facilitated by disrupting the hydrogen bonds to decrease the cross linking density. Therefore hydrogen bond accepting solvents are an ideal choice. Furthermore, for biomedical applications it is desirable that the solvent is biocompatible. Three different biocompatible hydrogen bonding accepting solvents were tested: PEG (average molecular weight = 400 g/mol), DMSO and ethyl lactate. Without being bound to a particular theory or solvent, it appears that ethyl lactate is the most safe solvent in this list and polymer solutions were prepared at 33 weight percent polymer in ethyl lactate. A similar trend in the adhesion strengths was observed compared to the patches, however all adhesion strengths were lower. This is believed to be due to residual H-bonding disrupting solvent that is present in the adhesive that acts as plasticizer. This analysis is further strengthened by the observation of Dopa on both sides of the tissue after staining (with Arnow's stain) indicating cohesive failure that is directly related to the cross linking density.

[0097] Figure 9. Tissue shear adhesion tests of injectable formulations in ethyl lactate. Panel a: Displayed are the shear strength results for polymers 1 - 5 and control polymers CI - C3. The average values are displayed above the bars and the vertical thin bar shows the standard deviation. Panel b: Arnow's staining of the tissue side after the tensile test. Dopa is observed evenly on the tissue side where the patch was attached for 1, 3 and C2 indicating that all these lap joints failed cohesively. Redness outside the area where the lap bar were overlapped indicates that excess liquid adhesive that flowed out during lap joint formation adhered and attached to the surrounding tissue. See Figure 7, panel c, for an example of a stained neat tissue.

[0098] Figure 10. Tissue shear adhesion healing results for adhesive patches upon repeated shear tests and re-adhesion. Healing experiments were performed by first performing a shear adhesion test. Immediately after the test the samples were overlapped and clamped together again and left in PBS buffered solution for 1 hour at 37 °C before performing another shear adhesion test.

[0099] Figure 11 shows the analysis of a 1H- MR spectrum of bioinspired supramolecular polymer adhesive. Shown is the MR spectrum of polymer 3.

DETAILED DESCRIPTION

[0100] Inspired by the adhesive strength of Mytilus californianus in wet

environments we developed a new (injectable) polymeric material based on orthogonal chemistries that results in tissue-like elastic properties, high toughness and excellent wet tissue adhesion (see image). Our work demonstrates that orthogonal supramolecular polymers are an attractive approach to tissue adhesives. Initial results showed that this novel polymeric sealant has high adhesive strength to bovine pericardium tissue compared to (commercial) medical sealants {see, e.g., comparison of Tables 1, and 2, with Table 5).

[0101] The supramolecular adhesive polymeric materials described herein are biocompatible and effectively adhere to wet tissue surfaces. Accordingly it is believed these materials make extremely effective bioadhesives. [0102] Inspired by the adhesive strength of Mytilus californianus in wet

environments we developed a new (injectable) polymeric sealant based on orthogonal chemistries that results in tissue-like elastic properties, high toughness and high wet tissue adhesion strength.

[0103] The adhesive materials described herein offer numerous advantages over previously published adhesives and commercial biomedical adhesives. Such advantages include, inter alia, an exceedingly high wet tissue adhesive shear strength (up to 110 kPa), a one-step application approach (while most adhesives require a two-step approach) and the absence of chemical activators, photoinitiators, cross-linkers, oxidants, toxic chemicals or alkaline conditions during application.

Adhesive polymers.

[0104] In various embodiments the adhesive polymers comprise a polymethacrylate random copolymer design comprising consists of 4 different monomers that control wet adhesion {e.g., dopamine methacrylamide), supramolecular crosslinks for cohesive forces (via dissipative H-bonding, e.g., ureidopyrimidinone-hydroxymethylmethacrylate, UPy- HEMA), and controlled swelling (a mixture between butyl methacrylate and PEG- methacrylate of various side-chain lengths) {see, e.g., Figure 1).

[0105] Accordingly, in certain embodiments a supramolecular adhesive polymeric material is provided wherein subunits of the polymer comprise:

i) at least one monomer having a substituted 1,2 dihydroxy benzenediol or a substituted 1,2,3 trihydroxy benzenetriol {e.g., a monomer that permits wet tissue adhesion via covalent bond formation with the tissue surface);

ii) at least one monomer that can form directional non-covalent interactions with substantial interaction strength; and

iii) a combination substantially hydrophobic and hydrophilic monomers that permit controlled swelling of the material. [0106] In certain embodiments the non-covalent interactions of the monomer that can form directional non-covalent interactions is based on hydrogen bonding, and in certain embodiments is provided by a motif that provides at least 3 hydrogen bonds per motif. In certain embodiments the hydrogen bonding is provided by a motif selected from the group consisting of a ureidopyrimidinone, a bis-urea (four H-bonds), a bis-urethane (four H- bonds), and a benzene tricarboxamide (H-bonds and aromatic stacking). In certain embodiments the non-covalent interactions are mediated by hydrogen bonding of a ureidopyrimidinone (UPy) {e.g., a a 2-ureido-4[lH]pyrimidinone). In certain embodiments the monomer that can form directional non-covalent interactions comprises a ureidopyrimidinone coupled by a linker (e.g., a hexamethylene diisocyanate linker) to a hydroxymethylmethacrylate.

[0107] As an H-bonding motif the UPy is by far the most used for supramolecular polymers because of the easy of synthesis and the high interaction strength. However, besides H-bonding there are other categories of non-covalent interactions that can be used as supramolecular "cross linker" in aqueous conditions. Such interactions include, but are not limited to hydrophobic interactions (we also utilize these interactions), metal ligand interactions, host guest interactions and engineered recombinant proteins (see, e.g., Webber, et al. (2016) Nat. Mater. 15(1): 13-26; Krieg et al. (2016) Chem. Rev., 116(4):2414-2477; and the like). In particular, Krieg et al, supra., describes in detail all possible motifs for supramolecular cross linking. Certain suitable motifs include, but are not limited to bis-urea (four H-bonds), bis-urethane (four H-bonds), benzene tricarboxamide (H-bonds and aromatic stacking contribute to the strength), and the like. As general design principle it is preferable to have at least 3 hydrogen bonds per motif because below the interaction strength is can be too low to give sufficient cohesive strength and can result in excessive swelling. Unfortunately we cannot refer to a specific preferable binding constant because binding constants are typically reported in chloroform and it is known that aqueous conditions result in much lower constants.

[0108] In certain embodiments the substantially hydrophobic monomers comprise acrylates or methacrylates. Illustrative substantially hydrophobic monomers include, but are not limited to one or more monomers selected from the group consisting of 2-Ethylhexyl acrylate, 2-Ethylhexyl methacrylate, ethyl acrylate, ethyl methacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate, isooctyl acrylate, isooctyl methacrylate, methyl acrylate, methyl methacrylate, n-butyl acrylate, n-butyl methacrylate, t-butyl acrylate, t- butyl methacrylate, hexyl (meth)acrylate (meaning both methacrylate and acrylate of this monomer), benzyl (meth)acrylate, dodecyl (meth)acrylate, n-octadecyl (meth)acrylate, n- propyl (meth)acrylate, 2-ethyl hexyl (meth)acrylate, isodecyl (meth)acrylate, and trimethylsilyl (meth)acrylate. In certain embodiments the substantially hydrophobic monomers comprise butyl methacrylate. [0109] For a good adhesive patch material it is desirable to have materials properties that approximately match the mechanical properties of the tissue. The mechanical properties are a result (but not limited to) of cross linking density, swelling and glass transition temperature. Without being bound to a particular theory, it is believed the choice of hydrophobic monomer will mostly effect the glass transition temperature. It is noted, however, that also the choice of the length of the PEG side chain can change the glass transition temperature. A glass transition temperature (roughly) below room temperature results in a rubbery material properties that can easily be deformed (important for application in the body). A glass transition temperature above room temperature results in glassy properties upon application with low yield strain (poor mechanical properties), low availability of Dopa motifs (thus poor adhesion) and low compatibility with tissue.

[0110] Accordingly, in certain embodiments substituted methacrylate monomers with carbon linkers shorter than C4 will result in too high glass transition temperatures and the higher linkers (such as hexyl and octyl) may give rise to higher adhesion strengths than presently observed. In certain embodiments for the shorter substituted methacrylate monomers (such as methyl methacrylate and ethyl methacrylate) it is desirable to choose a long chain PEG monomer to achieve a glass transition temperature below room

temperature. In certain embodiments the linker can be outside the preferable alpha range described herein {see below).

[0111] In certain embodiments the hydrophilic monomers comprising the adhesive material comprise one or more monomers selected from the group consisting of PEG substituted methacrylate, vinyl acetate, vinyl alcohol, N-vinyl-2-pyrrolidone, and

(meth)acrylic acid. In certain embodiments the hydrophilic monomers comprise PEG substituted methacrylate. In certain embodiments the hydrophilic monomers do not include methacylic acid because this may disrupt the H-bonding (cross linking density, cohesive strength) too much.

[0112] In certain embodiments the monomer having a substituted 1,2 dihydroxy benzenediol or a substituted 1,2,3 trihydroxy benzenetriol is selected from the group consisting of dopamine 4-(2-methacrylamide-ethyl)-l,2-benzenediol, 3-(2-methacrylamide- ethyl)-l,2-benzenediol, 4-(2-methacrylamide-ethyl)- ,2-benzenediol, 4-(2-methacrylamide- ethyl)-l,2,3 benzenetriol, and 5-(2-methacrylamide-ethyl)-l,2,3 benzenetriol. In certain embodiments the monomer comprises dopamine 4-(2-methacrylamide-ethyl)-l,2- benzenediol. [0113] In certain embodiments the adhesive material comprises one or more subunits according to the formula:

[0114] In certain embodiments where n ranges from about 5 to about 25 mol%. In certain embodiments n is about 10 mol%. It is noted that at, or below, the lower boundary in certain embodiments the low availability of Dopa may result in poor adhesive strength. At, or below, the higher boundary there may be no additional advantage for the adhesive strength and negative potential for covalent cross-linking of the material during long term exposure to aqueous environments (as is the case in the body).

[0115] In certain embodiments m ranges from about 5 to about 15 mol%. In certain embodiments m is about 10 mol%. In certain embodiments at, or below, the lower boundary the cohesive forces may not be adequate to prevent excessive swelling resulting in poor cohesion. At, or above, the higher boundary in certain embodiments, the material may have a glass transition temperature that is too high leading to brittle mechanical properties (difficult to apply as patch) and low swelling upon tissue contact probably resulting in low Dopa availability (thus poor or very slow onset of adhesion). [0116] In certain embodiments o ranges from about 25 wt% to about 75 wt%. In certain embodiments o is about 50 wt%. In certain embodiments at, or below, the lower end of the range, the material may have great adhesion and mechanical properties, however at some low weight percentage of hydrophilic monomer the material can be too brittle and won't swell upon contact with tissue resulting in poor adhesion. In certain embodiments at or above the higher limit the material may have poor mechanical properties and high swelling. [0117] In certain embodiments p is adjusted to provide about 50 weight percent of hydrophilic monomer. In certain embodiments p (in mol%) is about equal to 100 - n (mol%) - m (mol%) - o (mol%). In certain embodiments the molar amounts of Dopa and hydrogen bonding monomer are also abstracted since these are substantially hydrophobic. Besides adjusting for the hydrophilic content the choice of this monomer can influence the glass transition temperature and the H-bonding equilibrium (and therefore H-bonding interaction strength).

[0118] In certain embodiments a ranges from about 3 up to about 100 repeating units. In certain embodiments a is about 20 repeating units. In certain embodiments at, or below, the lower boundary the material may show low swellability resulting in low Dopa availability and brittle properties. In certain embodiments, at, or above, the higher boundary there can be a high potential for nano-phase separation resulting in low Dopa availability since most Dopa is located in the hydrophobic phase and not available for tissue adhesion (thus poor adhesion) as is the case for polymer 5. [0119] Formula I, shown above, provides a diamine linker. As noted, the length of the linker can be significantly varied. The linker has a neglectable influence on the mechanical properties (cohesive forces) since the UPy motif itself is responsible for the interaction strength. However, in certain embodiments the additional urethane can lead to lateral stacking of dimerized UPy motifs that can further give rise to nano phase separation resulting in increased mechanical strength. For efficient stacking of the dimerized UPy motifs it is desirable that the length of the diamine linker is constant. There is a body of work on bisurea and bisuerethane linkers and how they influence stacking. In case of diamine linkers the length probably does not matter for the mechanical properties. The type of diamine (linear versus branched) does matter to stacking, however it is believed there is little influence on the adhesion in the swollen state for our materials so both would be optional, although, in certain embodiments, linear is preferable.

[0120] Another illustrative, but non-limiting option (potentially cost and time saving), is to react 2-isocyanatoethyl methacrylate directly with 6-methylisocytosine to give a UPy methacrylate monomer with a two carbon spacer between the methacrylate and the UPy and no additional urethane. Similar adhesion and mechanical properties can be expected for materials containing this monomer.

[0121] In certain embodiments the adhesive material comprises a plurality of said subunit(s). In certain embodiments the subunits comprising said plurality are contiguous. In certain embodiments the adhesive material consists of the plurality of subunits. In certain embodiments the subunits comprising the plurality are not contiguous. In certain embodiments the adhesive material has a molar mass of at least about 1000 g/mol, or at least about 5000 g/mol, or at least about 10000 g/mol. In certain embodiments the the adhesive material is characterized by an adhesive tissue shear strength of at least about 25 kPa, or at least about 40 kPa, or an adhesive tissue shear strength of about 1 10 kPa.

[0122] In certain embodiments the adhesive material can regain a substantial portion of its original adhesive strength after catastrophic rupture within a period of time upon re- adhesion. In certain embodiments the contact time after catastrophic rupture is at least about 10 minutes, or at least about 15 minutes, or at least about 30 minutes, or at least about 45 minutes, or at least about 1 hour or longer.

[0123] In certain embodiments the adhesive material is a solid material (e.g., a solid polymer film). In certain embodiments the adhesive material comprises a liquid material (e.g., an injectable liquid material). In certain embodiments the adhesive material is provided as a liquid by dissolution in a water-soluble hydrogen bond disrupting

biocompatible solvent (e.g., PEG having a molecular weight of 1000 g/mol or lower, DMSO, ethyl lactate, and the like). In certain embodiments the PEG comprises a PEG having molecular weight of 900 g/mol or lower, or 800 g/mol or lower, or 700 g/mol or lower, or 600 g/mol or lower, or 500 g/mol or lower, or 400 g/mol or lower, or 300 g/mol or lower, or 200 g/mol or lower. In certain embodiments the PEG has a molecular weight ranging from about 200 g/mol, or from about 300 g/mol, or from about 400 g/mol up to about 800 g/mol, or up to about 600 g/mol, or up to about 500 g/mol. In certain

embodiments the PEG comprises a PEG having molecular weight of about 400 g/mol. In certain embodiments said solvent comprises ethyl lactate. [0124] In certain embodiments any of the adhesive materials or formulations thereof is sterile.

[0125] In certain embodiments the adhesive material is provided as a coating on biomedical material or device to render said biomedical material or device adhesive to tissue. [0126] Figure 2 shows one illustrative, but non-limiting synthesis scheme for the adhesive materials described herein. Figure 1 1 shows a representative analysis of a 1H- MR spectrum of one bioinspired supramolecular polymer adhesive (polymer 3). Using the teachings provided herein, numerous variants on the adhesives and synthesis schemes will be available to one of skill in the art.

[0127] As shown in the Figures, different polymers were synthesized to determine the influence of various parameters on tissue adhesion, on swelling, and the effects of PEG chain length. Thus, for example, Figure 3 characterizes swelling, Figures 4 and 5 characterize tissue adhesion, Figure 6 characterizes the effects of PEG chain length, Figure 7 characterizes tissue shear adhesion of an adhesive patch (see, also Table 1), while Figure 8 characterizes tissue shear adhesion of an adhesive liquid (see also Tables 2 and 4). Figure 10, characterizes repeated adhesion (see also Table 5). In addition, control polymers were synthesized to elucidate the effect of Dopa and H-bonding on the adhesive properties.

[0128] Our work demonstrates that H-bonding and Dopa motifs are orthogonal to each other and give an attractive new approach to tissue adhesives. Furthermore, by investigating various parameters that influence adhesion it was possible to deduce general design criteria for further improvement of this class of adhesives. [0129] To that end, the general design that is disclosed can be easily extended to other polymer backbones to impart additional function such as controlled biodegredation, release of (bioactive) agents and tuning of the mechanical properties.

Table 1. Shear adhesion results for adhesive patches. All measurements were performed in

10 fold.

Table 2. Shear adhesion results for injectable 33 wt% polymer formulations in ethyl lactate. All measurements were performed in 15 fold.

Control 1 15.4 8.4 7 6.2

Control 2 23.4 7.8 12.2 5.6

Control 3 18 7.4 10.9 4

Table 3. Shear adhesion healing results for adhesive patches upon repeated shear tests and re-adhesion. All measurements were performed in 10 fold.

Table 4. Shear adhesion healing results for injectable 33 wt% polymer formulations in ethyl lactate.

Table 5. Comparison between biomedical adhesives. Shear strength of various adhesives.

Illustrative uses.

[0130] The adhesive polymers described herein find numerous uses. In certain embodiments the adhesive materials (adhesive polymers) are effective bioadhesives.

[0131] Accordingly in certain embodiments, methods for attaching two substrates to one another are provided where the methods comprise contacting each substrate with a supramolecular adhesive polymeric material as described herein for a time sufficient that the substrates are attached to one another. In certain embodiments one or both of the two substrates comprises a biological tissue surface. Thus, for example, in certain

embodiments, one or both surfaces comprises a dermal or mucosal tissue surface., or one or both surfaces comprises an internal membrane tissue surface. In certain embodiments the one or both substrates comprises a mammalian tissue surface (e.g., a tissue surface of a human or of a non-human mammal). In certain embodiments the methods provide for adhesion of a tissue surface to a non-biological surface (e.g., a surface of a prosthesis such as a Hernia Mesh, a hemostatic patch, a pneumostatic patch, and the like). In certain embodiments the adhesive is applied in solid form (e.g., as a solid polymer film). In certain embodiments the adhesive material is used to seal a rupture, a hole, a tear, or an incision in a tissue and/or to mechanically adhere two tissues together (e.g., to promote healing of a wound or surgical site). In certain embodiments the adhesive material is applied as a liquid onto wet tissue, whereupon it transforms into a solid adhesive. In certain embodiments the adhesive material is applied as a coating to a biomedical material or device to render the latter adhesive to tissue. In certain embodiments the adhesive material protects and seals anastomosis and/or suture lines in internal body cavities. In certain embodiments the adhesive material prevents dehiscence of an anastomosis or a surgical wound in a patient. In certain embodiments the method comprises occluding a fistula within a patient. [0132] In certain embodiments methods of sealing an amniotic sack after fetal surgery are provided where the methods involve applying to puncture or incision in the amniotic sack a supramolecular adhesive polymeric material described herein where the adhesive seals a puncture or incision in said amniotic sack. In certain embodiments such application comprises injecting a liquid formulation of said adhesive material. In certain embodiments the liquid formulation transforms into a solid adhesive on contact with a tissue (see, e.g. Figure 8). Kits

[0133] In certain embodiments kits containing the supramolecular adhesive polymeric material described herein are provided. In certain embodiments the kits comprise a container containing a supramolecular adhesive polymeric material as described herein. In certain embodiments the container comprises a sterile package. In certain embodiments the kit comprises a container containing a diluent (e.g., a sterile diluent) that can be combined with the adhesive material to form a liquid adhesive material. In certain embodiments the diluent comprises a water-soluble hydrogen bond disrupting

biocompatible solvent. In certain embodiments the diluent comprises a solvent selected from the consisting of PEG having a molecular weight of 1000 g/mol or lower, DMSO and ethyl lactate. In certain embodiments the PEG comprises a PEG having molecular weight of 900 g/mol or lower, or 800 g/mol or lower, or 700 g/mol or lower, or 600 g/mol or lower, or 500 g/mol or lower, or 400 g/mol or lower, or 300 g/mol or lower, or 200 g/mol or lower. In certain embodiments the PEG has a molecular weight ranging from about 200 g/mol, or from about 300 g/mol, or from about 400 g/mol up to about 800 g/mol, or up to about 600 g/mol, or up to about 500 g/mol. In certain embodiments the PEG comprises a PEG having molecular weight of about 400 g/mol. In certain embodiments the diluent comprises ethyl lactate.

[0134] In certain embodiments the kit includes an applicator device for transfer of said adhesive from said container onto a biological tissue.

0135] In addition, the kits can optionally include labeling and/or instructional materials providing directions (i.e., protocols) for the use of the adhesive materials described herein. The instructional materials may also, optionally, teach preferred amounts, application methods, indications and counter indications, etc. [0136] While the instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

[0137] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.