EP4007798A1

EP4007798A1 - Self-curing redox donor/acceptor adhesive composition

Info

Publication number: EP4007798A1
Application number: EP20851130.3A
Authority: EP
Inventors: Terry W. J. STEELE; Animesh Ghosh; Richard David Webster; Chew Shun Nigel TAN
Original assignee: Nanyang Technological University
Current assignee: Nanyang Technological University
Priority date: 2019-08-02
Filing date: 2020-07-30
Publication date: 2022-06-08
Also published as: KR102895746B1; EP4007798A4; WO2021025617A1; KR20220044536A

Abstract

An adhesive composition disclosed herein includes organic macromolecules each including nucleophilic functional groups, wherein each of the organic macromolecules is a linear macromolecule or a branched macromolecule, wherein each of the nucleophilic functional groups includes an amino group, a thiol group, a hydroxyl group, or a carboxyl group, and one or more electron donating moieties, wherein each of the nucleophilic functional groups grafted with the one or more electron donating moieties is converted to an electron accepting moiety, and each of the nucleophilic functional groups not grafted with the one or more electron donating moieties remains as the nucleophilic functional group, wherein the one or more electron donating moieties oxidizes into a diketone when the adhesive composition is subject to a stimulus, and wherein the diketone forms one or more cross-linkages with the nucleophilic functional groups. The present disclosure also provides its method of production and its uses. In a preferred embodiment, a self-curing and voltage-activated adhesive composition includes a dendritic polyamidomaine comprising amine functional groups which are grafted with 3,4-dihydroxybenzaldehyde via a Schiff base reaction.

Description

SELF-CURING REDOX DONOR/ACCEPTOR ADHESIVE COMPOSITION

Cross-Reference to Related Application

[0001] This application claims the benefit of priority of Singapore Patent Application No. 1Q201907184U, filed 2 August 2019, the content of it being hereby incorporated by reference in its entirety for all purposes.

Technical Field

[0002] The present disclosure relates to an adhesive composition, a method of producing the adhesive composition and its uses.

Background

[0003] Catechol may be a chemical cross-linking functional group present in chemical curing adhesives and tissue adhesives systems. However, catechol-based adhesives tend to require heat or a two-part mixing strategy to induce the cross-linking. For example, oxidizing agents such as periodate [ICC] , or metal chelators such as Fe³⁺ and Cu²⁺, may be utilized in order to activate the catechol to (i) quinone (e.g. oxidizing catechol by periodate) or (ii) chelate-mediated cross-linking macromolecules (e.g. using metal cations). The widespread applications of such approaches may be hindered due to cumbersome mixing equipment or mixing nozzles required. Such approaches are not viable for independent application (e.g. coating) and delayed cross-linking (adhesive curing) of the catechol-based adhesives, giving rise to unmet industrial and clinical needs. For instance, while the two-part curing may provide for rapid gelation, this still suffers from limited manipulation before application, local cytotoxicity, and narrow pH ranges. Thus, an unmet need exists for a one-pot catechol-mediated adhesive that allows self-curing (with a known lag-time) or through an external stimulus.

[0004] Apart from the above, there is a need for an adhesive, including a bioadhesive, to be stable. The adhesive should have shelf stability, that is, able to be stored for a significant period of time without degrading. The adhesive should also have self-curing capability, e.g. when applied to a hydrated substrate, with sufficient curing lag-time to allow manipulation of the liquid adhesive before cross-linking into a viscoelastic solid matrix, such that the cross-linking happens spontaneously after the lag-time or happens in response to an external stimulus not involving additional chemical reagents or mixing in any form. While self-curing chemical cross-linking techniques may have been developed, such as those involving cyanoacrylates (anionic polymerization), silicone (condensation), and acrylates (free radical polymerization), these conventional techniques tend not to allow adhesion (e.g. bioadhesion) to a hydrated or wet substrate or for underwater adhesion (e.g. underwater bioadhesion).

[0005] There is thus a need to provide for a solution that addresses one or more of the limitations mentioned above. The solution should at least provide for a self-curing adhesive composition that overcomes one or more of the limitations mentioned above, and a method of producing such self-curing adhesive composition.

Summary

[0006] In a first aspect, there is provided for an adhesive composition including: organic macromolecules each including nucleophilic functional groups, wherein each of the organic macromolecules is a linear macromolecule or a branched macromolecule, wherein each of the nucleophilic functional groups includes an amino group, a thiol group, a hydroxyl group, or a carboxyl group; and one or more electron donating moieties, wherein each of the nucleophilic functional groups grafted with the one or more electron donating moieties is converted to an electron accepting moiety, and each of the nucleophilic functional groups not grafted with the one or more electron donating moieties remains as the nucleophilic functional group, wherein the one or more electron donating moieties oxidizes into a diketone when the adhesive composition is subject to a stimulus, and wherein the diketone forms one or more cross-linkages with the nucleophilic functional groups.

[0007] In another aspect, there is provided for a method of producing an adhesive composition, wherein the adhesive composition includes organic macromolecules each including nucleophilic functional groups, wherein each of the organic macromolecules is a linear macromolecule or a branched macromolecule, wherein each of the nucleophilic functional groups includes an amino group, a thiol group, a hydroxyl group, or a carboxyl group; and one or more electron donating moieties, wherein each of the nucleophilic functional groups grafted with the one or more electron donating moieties is converted to an electron accepting moiety, and each of the nucleophilic functional groups not grafted with the one or more electron donating moieties remains as the nucleophilic functional group, wherein the one or more electron donating moieties oxidizes into a diketone when the adhesive composition is subject to a stimulus, and wherein the diketone forms one or more cross-linkages with the nucleophilic functional groups, wherein the method includes: providing the organic macromolecules including the nucleophilic functional groups; and mixing the organic macromolecules with a precursor of the one or more electron donating moieties.

[0008] In another aspect, there is provided for a method of adhering or sealing biological tissues, the method including: applying an adhesive composition to one or more biological tissues, wherein the adhesive composition includes organic macromolecules each including nucleophilic functional groups, wherein each of the organic macromolecules is a linear macromolecule or a branched macromolecule, wherein each of the nucleophilic functional groups includes an amino group, a thiol group, a hydroxyl group, or a carboxyl group; and one or more electron donating moieties, wherein each of the nucleophilic functional groups grafted with the one or more electron donating moieties is converted to an electron accepting moiety, and each of the nucleophilic functional groups not grafted with the one or more electron donating moieties remains as the nucleophilic functional group, wherein the one or more electron donating moieties oxidizes into a diketone when the adhesive composition is subject to a stimulus, and wherein the diketone forms one or more cross-linkages with the nucleophilic functional groups; subjecting the adhesive composition to the stimulus; and contacting the biological tissues together to adhere the biological tissues. [0009] In another aspect, there is provided for the use of an adhesive composition in the preparation of (i) an adhesive; or (ii) a kit including the adhesive composition for intestinal anastomosis, vascular anastomosis, tissue repair, and/or tissue implantation, wherein the adhesive composition includes: organic macromolecules each including nucleophilic functional groups, wherein each of the organic macromolecules is a linear macromolecule or a branched macromolecule, wherein each of the nucleophilic functional groups includes an amino group, a thiol group, a hydroxyl group, or a carboxyl group; and one or more electron donating moieties, wherein each of the nucleophilic functional groups grafted with the one or more electron donating moieties is converted to an electron accepting moiety, and each of the nucleophilic functional groups not grafted with the one or more electron donating moieties remains as the nucleophilic functional group, wherein the one or more electron donating moieties oxidizes into a diketone when the adhesive composition is subject to a stimulus, and wherein the diketone forms one or more cross-linkages with the nucleophilic functional groups.

Brief Description of the Drawings

[0010] The drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the present disclosure. In the following description, various embodiments of the present disclosure are described with reference to the following drawings, in which:

[0011] FIG. 1A depicts how a catechol moiety is conventionally used for cross-linking. Specifically, FIG. 1 A is a schematic diagram of catechol unit being chemically oxidized or electrochemically oxidized to an o-quinone unit, which undergoes irreversible covalent cross-linking with nucleophiles, such as primary and secondary amines. [0012] FIG. IB shows the synthesis and chemical structures of 3,4- dihydroxybenzaldehyde (3,4-DBA, which may be referred to simply as dihydroxybenzaldehyde or DBA), which is a catechol moiety, grafted onto G5- polyamidoamine (PAMAM). The G5-PAMAM grafted with DBA is herein referred to as a G5-DBAx conjugate. X denotes the molar percentage of the moiety grafted onto the G5-PAMAM, wherein an example of the moiety herein refers to DBA, and X may be 10 molar%, 20 molar%, 30 molar%, etc. X is calculated as moles of dihydroxybenzaldehyde divided by moles primary amines x 100%. The G5-DBAx adhesive composition is stable under anaerobic and non-aqueous conditions.

[0013] FIG. 1C shows the self-curing and electrochemical activation (electrocuring) through balancing of electrochemical donor (e.g. catechol moiety) and electrochemical acceptor (e.g. Schiff-base). The Schiff-base is formed from amine and aldehyde condensation. FIG. 1C also illustrates plausible cross-linking of G5-DBAx conjugates through electrocuring.

[0014] FIG. 2A demonstrates for size exclusion chromatography of G5-PAMAM (branched polymer used as control) and several G5-DBAx conjugates (X = 10 molar%, 20 molar%, and 30 molar%). Refractive index (RI) analysis as shown in the plot of solid lines detects polymer mass. UV absorbance (ABS@280 nm) detects for the electrochemical donor precursor grafted (e.g. catechol) as shown in the plot of broken lines, wherein overlapping elution time demonstrates successful coupling.

[0015] FIG. 2B demonstrates for cyclic voltammograms of G5-DBAio (black line) with no chemical activation and G5-BenzADHio (grey) with no catechol moiety, wherein G5-BenzADHio denotes G5-PAMAM grafted with 10 molar% of benzaldehyde. The catechol peak (electrochemical donor) gives a redox peak at E_pai = 0.3 V (Ag/AgCl reference electrode). The Schiff-base (electrochemical acceptor) gives a redox peak at E_Pa2 = 0.9 V (Ag/AgCl reference electrode). Scan rate is 100 mV s ¹. Electrolyte is phosphate buffered saline (pH 7.2).

[0016] FIG. 2C demonstrates for cyclic voltammograms of G5-DBAio conjugates (black line) that depicts the electrochemical activation of the electrochemical donor (catechol moiety, E_pai = 0.3 V), electrochemical acceptor (Schiff-base, redox acceptor internal additive, E_pa2 = 0.9 V) compared with free dihydroxybenzaldehyde (grey line, E_pao = 0.5 V), where no Schiff-base redox activation is observed. Branched polymers may facilitate electron transfer, but no redox behavior is observed with G5-PAMAM (light grey). Scan rate is 50 mV s ¹. Electrolyte is phosphate buffered saline (pH 7.2). [0017] FIG. 2D demonstrates for cyclic voltammograms of G5-DBAio at scan rates from 10 - 200 mV/s. E_pa2 at 0.9 V is dependent on scan rate, irreversible, and may be attributed to the electrochemical acceptor (e.g. Schiff-base). [0018] FIG. 3A demonstrates for the present self-curing and electrocurable adhesive compositions compared with a conventional two-part catechol curing adhesive composition. Real-time dynamic mechanical analysis of G5-DBA₂₀ conjugates in IX phosphate buffered saline (30% w/v) with three methods of adhesion activation are shown: (1) voltage activation applied at the two minute (i.e. min) timepoint (voltage -1 V vs. Ag/AgCl), (2) demonstration of self-curing with a 22 min lag-time after aqueous reconstitution/exposure to atmosphere, and (3) two-part curing with the same formulations activated by 0.1 mM sodium periodate solution. G’ denotes storage modulus (solid lines) and G” denotes loss modulus (dot dashed line).

[0019] FIG. 3B demonstrates for dynamic mechanical analysis of G5-DBA₂₀ at 30 wt% concentration in IX phosphate buffered saline electrolyte. No cross-linking or activation of the adhesive composition is observed from 0 to 2 min at a 0 V potential. Voltage activation begins at 2 min time point at both - I V and +1 V potentials (separate experiments). The -I V voltage activation demonstrates cross-linking in less than 10 seconds gelation (defined where G” = G’) occurring at 2.1 min after voltage activation. G’ denotes storage modulus (solid lines) and G” denotes loss modulus (dot dashed line). [0020] FIG. 3C shows various formulations of grafting, including G5-DBAio and G5- DBA20 at 30% w/v in phosphate buffered saline compared with non-adhesive control G5-PAMAM. G5-DBA₂₀ displays instantaneous activation by -1 V voltage, whereas the G5-DBAio does not. Thus, grafting percentage is a method to impart and control voltage activation. G’ denotes storage modulus (solid lines) and G” denotes loss modulus (dot dashed line).

[0021] FIG. 3D shows storage modulus (G’) values comparison of non-adhesive G5- PAMAM (branched polymer as control) with catechol-grafted conjugates compositions of G5-DBAio, G5-DBA₂₀, and G5-DBA30, respectively. White columns represent instantaneous activation by -1 V Ag/AgCl while grey columns represent two-part chemical curing by 0.1 M sodium periodate. The examples are based on formulations of 15% w/v and 30% w/v (per cent weight of polymer in gram divided by volume of saline in milliliters, %w/v). The saline solution is a good conductor of electricity. Ions in the saline solution increase conductivity of the adhesive composition and balance charges that arise from electron transfer reactions at the electrodes. The saline solution does not cause short-circuit, as the electrocuring process is carried out in an isolated setup (e.g. a zensor chip) and the current as well as voltage are very low. In addition, the rheometer probe used has a non-conductive ceramic end for contacting the electrocuring composite.

[0022] FIG. 4A demonstrates example of voltage activation of adhesive compositions using a commercial 3-electrode chip on a wet substrate of collagen film, evaluated by lap shear adhesion test.

[0023] FIG. 4B shows the representative stress/strain curves recorded for G5-DBA₂₀ samples that had undergone self-curing, voltage activation (applied - I V Ag/AgCl, also known as electrocuring), and two-part curing (chemical curing with 0.1 mM sodium periodate) for less than 30 min. Voltage activation exhibits significantly higher adhesion in 30 min or less.

[0024] FIG. 4C demonstrates the adhesion values for G5-DBA₂₀ samples under (i) self curing with 23 min lag-time, (ii) voltage activated electrocuring, and (iii) two-part curing after 60 min. No statistical difference is observed between voltage activation and two-part curing.

[0025] FIG. 5A demonstrates for the dynamic mechanical analysis of three present self curing adhesive compositions that allows tunable lag-times from 8 to 23 min before cross-linking after aqueous reconstitution. Lag-time is determined after reaching a complex viscosity of 2 Pa.s.

[0026] FIG. 5B demonstrates for the complex viscosity of three present self-curing adhesive compositions that allows tunable lag-times from 8 to 23 mins before cross- linking after aqueous reconstitution. Lag-time is determined after reaching a complex viscosity of 2 Pa.s.

[0027] FIG. 6A demonstrates for the dynamic mechanical analysis of self-curing adhesive composition that allows 20 min of manipulation before cross-linking at 23 min after aqueous reconstitution.

[0028] FIG. 6B demonstrates that voltage activation allows instantaneous curing in less than 10 sec after exposure to a -1 V Ag/AgCl external stimulus using the present adhesive composition.

[0029] FIG. 7A demonstrates the dynamic mechanical analysis of G5-PAMAM non adhesive branched polymer that contains no electrochemical acceptor or catechol moiety. [0030] FIG. 7B demonstrates for the dynamic mechanical analysis of G5-PAMAM non-adhesive branched polymer that contains no electrochemical acceptor or catechol moiety with exposure to -1 V Ag/AgCl external stimulus. No instantaneous cross- linking is observed, which is defined as G” or G’ or both changes by 100X in less than 60 seconds.

[0031] FIG. 8A demonstrates for the dynamic mechanical analysis of the present self curing adhesive composition with the addition of ascorbic acid at three concentrations of 0.1, 10, and 1000 mM in 50 mM borate buffer at pH 8.0. It is observed that both gelation time and lag-time before cross-linking is decreased. This is surprising as ascorbic acid is typically used as a non-toxic anti-oxidant that retards catecholamine auto-oxidation. In other words, the addition of ascorbic acid was expected to have no effect of decreasing gelation time and lag-time based on such an understanding, but in this instance, both happened to decrease. Thus, the self-curing is not predictable based on the presence of oxygen. For example, at pH 8.0, the oxidation potential of ascorbic acid is + 0.33 V which is slightly higher than the oxidation potential of catechol (+ 0.3 V). To act as an effective anti-oxidant, the ascorbic acid should have an oxidation potential lower than that of catechol. As its redox potential is slightly higher than that of catechol, the ascorbic acid may not be effective as an anti-oxidant when mixed with catechol.

[0032] FIG. 8B demonstrates for the dynamic mechanical analysis of the present self curing adhesive composition compared across two buffer systems of phosphate buffered saline pH 7.4 (PBS) and 50 mM sodium borate buffer. Complexation of catechols to borate anions is typically used to retard spontaneous oxidation of catechols to quinones at neutral pH. However, in this instance, the electrochemical acceptor/donor system of Schiff-base/catechol (G5-DBA) is not retarded in the presence of borate buffer. Thus, the self-curing is not predictable based on the presence of oxygen. In an aqueous medium, the complexation of catechol by borate may be less effective due to hydrogen bonding stabilization of catechol and borate with the water molecules. The oxidation potential of catechol may decrease with the increase of pH. Thus, the ease of oxidation of, for example, a catechol moiety in the adhesive composition, may increase in a borate buffer (pH 8.0) compared to in a phosphate buffer (pH 7.2). This may render the decrease of the lag-time in borate buffer. [0033] FIG. 9A shows in the top row scheme extrapolated tautomers of aromatic aldehydes and ionic structures of Schiff-base catechol based on the known pK_a [CT] of substituted catechol (8.3-9) and pK_a[SB] of aromatic Schiff-base (11-12). The middle row scheme depicts a mechanism of oxygen induced quinone formation. The bottom row scheme depicts a mechanism of -1 V electrocuring with the protonated azomethine as electron donating moiety. The term “CT” denotes catechol.

[0034] FIG. 9B illustrates the density functional theory (DFT) calculations on the catechol and its tautomer intermediate have been performed to gauge if the reaction is thermodynamically favourable.

[0035] FIG. 9C shows stacked 1H NMR traces for starting materials (3,4-DBA and G5- PAMAM) and synthesized conjugates: lOmolar % DBA-co-PAMAM and 50 molar% DBA-co-PAMAM. Peak intensities and peak assignment with MestReNova software. [0036] FIG. 10A depicts heteronuclear two dimensional single (2D) quantum correlation (HSQC) NMR spectrum of 20% DBA-co-PAMAM, wherein the full range of spectrum is shown. The cross peak at 8.02 ppm x 162.33 ppm proves the proton at 8.02 ppm belongs to C-H group in azomethine (H-N=C-) linker.

[0037] FIG. 10B depicts heteronuclear two dimensional (2D) single quantum correlation (HSQC) NMR spectrum of 20% DBA-co-PAMAM, wherein a magnified portion of the spectrum is shown. The cross peak at 8.02 ppm x 162.33 ppm proves the proton at 8.02 ppm belongs to C-H group in azomethine (H-N=C-) linker.

[0038] FIG. IOC shows the 'H NMR traces for G5-DBA₂₀ with reductive amination. Peak intensities and peak assignment are analysed with Topspin software a' integration is set to 504. The sum of b’, c’, and d’ is 48 or an average grafting ratio of 16% as DBA:PAMAM.

[0039] FIG. 10D shows the ¹³C NMR traces for G5-DBA₂₀ with reductive amination. Peak intensities and peak assignment are analysed with Topspin software.

[0040] FIG. 10E shows the 2D heteronuclear single quantum correlation (HSQC) NMR spectrum of G5-DBA₂₀ with reductive animation for the full range of spectrum, where the cross peak at 8.02 ppm x 162.33 ppm shows no presence of H-N=C- linker.

[0041] FIG. 10F shows the 2D heteronuclear single quantum correlation (HSQC) NMR spectrum of G5-DBA₂₀ with reductive animation for a magnified portion of the spectrum, where the cross peak at 8.02 ppm x 162.33 ppm shows no presence of H- N=C- linker.

[0042] FIG. 11A is a plot of absorbance (ABS) against concentration of the 3,4-DBA used, demonstrating for ultraviolet (UV) extinction coefficient at 280 nm values determined by UV/Visible spectrum of 3,4-DBA.

[0043] FIG. 1 IB is a plot of absorbance (ABS) against wavelength demonstrating for ultraviolet (UV) extinction coefficient at 280 nm values determined by UV/Visible spectrum of 3,4-DBA.

[0044] FIG. llC is a kinetic plot of DBA grafting reactions in terms of ABS against wavelength.

[0045] FIG. 1 ID is a kinetic plot of DBA grafting reactions in terms of ABS (at l405 nm) against time. R² value for G5-DBAI_O,20,30 = 0.997, 0.996 and 0.996, respectively. Solid lines indicate the exponential decay fitting lines.

[0046] FIG. 11E depicts a representative stress/strain plot for 30 wt% G5-DBA₂₀ compositions: self-curing, electrocuring (-1 V), and two-part curing (with 0.1 mM NaKU) for measurement > 60 min. The solvent used in this instance is PBS buffer saline. [0047] FIG. 12A is a table depicting peak assignments of target conjugates.

[0048] FIG. 12B is a table depicting NMR and SEC-MALS-UV comparison of DBA grafting onto G5-PAMAM.

[0049] FIG. 13A shows the UV/Vis spectroscopy comparison between G5-DBA₂₀ (see line with arrow marked l = 405) and G5-DBA₂₀ reduced which has no presence of Schiff-base UV peak at 405 nm after reduction. The solvent used in this instance is PBS buffer saline.

[0050] FIG. 13B demonstrates for a real-time rheology test in terms of modulus against time for 30 wt% G5-DBA₂₀ and G5-DBA₂₀ reduced at self-curing. All compositions were in PBS.

[0051] FIG. 13C demonstrates for a real-time rheology test in terms of modulus against time for 30 wt% G5-DBA₂₀ and G5-DBA₂₀ reduced, (voltage off: 0-2min; voltage on: 2-30 min), gelation time (gt) at 3 min and 8.4 min for -1 V electrocuring, respectively. All compositions were in PBS.

[0052] FIG. 13D demonstrates for a real-time rheology test in terms of modulus against time for 30 wt% G5-DBA₂₀ and G5-DBA₂₀ reduced (voltage off: 0-2min; voltage on: 2-30 min), gelation time (gt) at 18 min and 18.1 min for +1 V electrocuring, respectively. All compositions were in PBS.

[0053] FIG. 14A shows cyclic voltammograms (CVs) of G5-PAMAM (as background), G5-Benzio (as reference), and G5-DBAio at 50 mV s l. Electrolyte: PBS (pH 7.2), working electrode: glassy carbon, reference electrode: Ag/AgCl, counter electrode: platinum (Pt). FIG. 14A shows real-time dynamic mechanical analysis of formulation of 30 wt% G5-DBA₂₀ conjugates in PBS electrolyte.

[0054] FIG. 14B shows a plot of modulus against time demonstrating for two-part curing (mixed with 0.1 mM NaI0₄ solution) and electrocuring (voltage off: 0-2 min; voltage on: 2-48 min, throughout) at -1 V, gt denotes gelation time.

[0055] FIG. 14C shows a plot of modulus against time demonstrating for self-curing (no voltage applied and no oxidant added) after rehydration. Storage modulus (G’, solid line) and loss modulus (G”, dot dashed line).

[0056] FIG. 15A shows CV of free 3,4-DBA, wherein the scan rate dependent test from 50 to 200 mV s ¹ is depicted. Electrolyte: PBS (pH 7.2), working electrode: glassy carbon, reference electrode: Ag/AgCl, counter electrode: Pt.

[0057] FIG. 15B demonstrates Ip increase depends on the square root of scan rate. Electrolyte: PBS (pH 7.2), working electrode: glassy carbon, reference electrode: Ag/AgCl, counter electrode: Pt. [0058] FIG. 15C shows repeated scanned results using glassy carbon (GC) working electrode. Electrolyte: PBS (pH 7.2), reference electrode: Ag/AgCl, counter electrode: Pt.

[0059] FIG. 15D shows repeated scanned results using Pt disk working electrode. Electrolyte: PBS (pH 7.2), reference electrode: Ag/AgCl, counter electrode: Pt. [0060] FIG. 15E shows CV of G5-DBAio, wherein the scan rate dependent test from

10 to 200 mV s ¹ is depicted. Electrolyte: PBS (pH 7.2), working electrode: glassy carbon, reference electrode: Ag/AgCl, counter electrode: Pt.

[0061] FIG. 15F shows CV of G5-Bemo conjugates, wherein the scan rate dependent test from 10 to 100 mV s ¹ is depicted. Electrolyte: PBS (pH 7.2), working electrode: glassy carbon, reference electrode: Ag/AgCl, counter electrode: Pt.

[0062] FIG. 16A shows real-time rheology test based on 30 wt% G5-DBA₂₀ at -1 V electrocuring utilizing PP05 aluminum disposable probe, sample loading takes 60s before starting measurement, voltage started 2 mins after the sample loading. All formulations were prepared in PBS.

[0063] FIG. 16B shows real-time rheology test based on 30 wt% and 45wt% G5-DBAio at -1 V electrocuring utilizing PP10 ceramic probe, sample loading takes 60s before starting measurement, voltage started 2 mins after the sample loading. All formulations were prepared in PBS.

[0064] FIG. 16C shows real-time rheology test based on 30 wt% and 45 wt% G5- DBAio at two-part curing with O.lmM Na utilizing PP10 ceramic probe, sample loading takes 60s before starting measurement, voltage started 2 mins after the sample loading. All formulations were prepared in PBS.

[0065] FIG. 16D shows real-time rheology test based on 30 wt% G5-Benz₂o at self curing utilizing PP10 ceramic probe, sample loading takes 60s before starting measurement, voltage started 2 mins after the sample loading. All formulations were prepared in PBS. [0066] FIG. 16E shows real-time rheology test based on 30 wt% G5-Benz₂o at -1 V electrocuring utilizing PP10 ceramic probe, sample loading takes 60s before starting measurement, voltage started 2 mins after the sample loading. All formulations were prepared in PBS.

[0067] FIG. 16F shows real-time rheology test based on 30 wt% G5-Benz₂o at +1 V electrocuring utilizing PP10 ceramic probe, sample loading takes 60s before starting measurement, voltage started 2 mins after the sample loading. All formulations were prepared in PBS.

[0068] FIG. 17A shows representative stress/strain plots of 30 wt% G5-DBA₂₀ compositions: self-curing, electrocuring (-1 V), and two-part curing (with 0.1 mM Na ). The solvent used in this instance is PBS buffer saline.

[0069] FIG. 17B demonstrates lap shear adhesion strength of 30 wt% G5-DBA₂₀ formulation in comparison of self-curing, electrocuring, and two-part curing in <30 min and >60 min curing period. The solvent used in this instance is PBS buffer saline. [0070] FIG. 18A depicts synthesis reaction of 20 molar% vanillin-PAMAM (G5- Van2o). The molar ratio of the synthesis required is 1 mole of PAM AM NFh surface group to 0.2 mole of Vanillin to achieve a 20% grafting onto the dendrimer. Vanillin (VAN) has the similar chemical structure as DBA (3,4-dihydroxybenzaldehyde) and vanillin has better stability in atmospheric environment than DBA.

[0071] FIG. 18B shows 'H NMR spectrum of 20 molar% vanillin-PAMAM.

[0072] FIG. 19A shows the steady state viscosity of 20 molar% vanillin-PAMAM at fixed shear rate of 10 s ¹ / 62.8 rad.s ¹.

[0073] FIG. 19B demonstrates for the electrorheology of 20 molar% van-PAMAM at 0 V for 50 min stability test and - I V for 50 min voltage activation.

[0074] FIG. 19C shows the amplitude sweep of 20 molar% vanillin-PAMAM at shear strain from 1 to 1000%. The amplitude sweep is done right after electrorheology test. The adhesive shear stress goes up till 200 Pa and pseudoplastic behavior is observed after shear strain of 70%.

[0075] FIG. 20A shows steady state viscosity of 20 molar% van-PAMAM at fixed shear rate of 10 s ¹ / 62.8 rad.s ¹ for 30 seconds.

[0076] FIG. 20B shows steady state viscosity of 20 molar% van-PAMAM at fixed shear rate of 10 s ¹ / 62.8 rad.s ¹ for 60 seconds.

[0077] FIG. 20C demonstrates for the electrorheology test on 20 molar% van-PAMAM for different voltage activation (-1V, -1.2V, -1.5V and -2V).

[0078] FIG. 20D shows the amplitude sweep of 20 molar% van-PAMAM at shear strain from 1 to 1000%. [0079] FIG. 21 A shows steady state viscosity of 20 molar% van-PAMAM at fixed shear rate of 10 s ¹ / 62.8 rad.s ¹ for 30 seconds.

[0080] FIG. 2 IB shows steady state viscosity of 20 molar% van-PAMAM at fixed shear rate of 10 s ¹ / 62.8 rad.s ¹ for 60 seconds.

[0081] FIG. 21C demonstrates for the electrorheology test on 20 molar% van-PAMAM for different voltage activation (-1V, -1.2V, -1.5V and -2V).

[0082] FIG. 2 ID shows the G” vs G’ on 20 molar% van-PAMAM for different voltage activation (-1V, -1.2V, -1.5V and -2V).

[0083] FIG. 2 IE shows the amplitude sweep of 20 molar% van-PAMAM at shear strain from 1 to 1000%. [0084] FIG. 2 IF shows the yield stress of 20 molar% van-PAMAM with 0.1 M ascorbic acid added. [0085] FIG. 21G is a comparison of yield stress for voltage activated adhesive with and without adding 0.1 M ascorbic acid.

[0086] FIG. 21H shows the LVR region of 20 molar% van-PAMAM at shear strain from 1 to 1000% to observe its modulus.

[0087] The following detailed description refers to the accompanying drawings that show, by way of illustration, specific details and embodiments in which the present disclosure may be practiced. [0088] Features that are described in the context of an embodiment may correspondingly be applicable to the same or similar features in the other embodiments. Features that are described in the context of an embodiment may correspondingly be applicable to the other embodiments, even if not explicitly described in these other embodiments. Furthermore, additions and/or combinations and/or alternatives as described for a feature in the context of an embodiment may correspondingly be applicable to the same or similar feature in the other embodiments.

[0089] The present disclosure relates to an adhesive composition, its method of production and uses. The adhesive composition can be used as an adhesive on various surfaces, including wet substrates. The term “adhesive composition” herein refers to the composition or formulation that is applied to the substrates for adhering the substrates. The term “adhesive” herein is distinguished from “adhesive composition”, wherein the adhesive is the resultant cured form of the present adhesive composition and the adhesive has the cross-linkages formed. The adhesive composition includes organic macromolecules each including nucleophilic functional groups, wherein each of the organic macromolecules is a linear macromolecule or a branched macromolecule, wherein each of the nucleophilic functional groups includes an amino group, a thiol group, a hydroxyl group, or a carboxyl group, and one or more electron donating moieties, wherein each of the nucleophilic functional groups grafted with the one or more electron donating moieties is converted to an electron accepting moiety, and each of the nucleophilic functional groups not grafted with the one or more electron donating moieties remains as the nucleophilic functional group, wherein the one or more electron donating moieties oxidizes into a diketone when the adhesive composition is subject to a stimulus, and wherein the diketone forms one or more cross-linkages with the nucleophilic functional groups.

[0090] In the present disclosure, the terms “redox donor”, “electrochemical donor”, “electron donor”, “redox donor internal additive” and “electron donating moiety”, are used interchangeably. The term “redox acceptor”, “electrochemical acceptor”, “electron acceptor”, “redox acceptor internal additive”, and “electron accepting moiety”, are used interchangeably.

[0091] The present adhesive composition may be referred to as a self-curing, redox donor/acceptor macromer or self-curing, redox donor/acceptor conjugate. The present adhesive composition may contain, for example, catechol moieties and Schiff-bases, formed via condensation of amine and aldehyde. The present adhesive composition can self-cure (i.e. cross-link) when reconstituted in an aqueous solution which may contain water and oxygen. There may be a lag-time before self-curing starts and the lag-time is advantageously tunable. The nature of the redox donor/acceptor moieties allows the additional capability of voltage-based activation of the self-curing adhesive composition. The present adhesive composition can be dried and stored under anaerobic and anhydrous conditions, wherein its adhesive capability can be “switched off’, and re-activated when reconstituted in an aqueous solution. The present adhesive composition can be stored for long periods at -20°C.

[0092] The present adhesive composition also provides other several advantages. For example, preparation of the present adhesive composition is economical and feasible for scaling up. Raw materials involved, e.g. unbranched polymers or branched dendrimers are in abundance and cheap. Formation of the electron donating moieties and electron accepting moieties may be achieved in a single step without using overly complicated processing conditions such as high temperature, high pressure, and/or specific catalysts and equipment.

[0093] Use of the present adhesive composition is advantageously straightfoward and convenient, rendering it feasible for structural, veterinary, and clinical use. The present adhesive composition exhibits desirable adhesion on wet substrates and allows many parameters for tuning the adhesion strength and curing speed by voltage, current, oxidants, and/or metal ions. The components of the present adhesive composition are non-toxic, and it may be stored at -20°C for several months as well. The present adhesive composition exhibits shelf stability even under ambient conditions, and activation may be specifically by voltage without oxygen.

[0094] Details of various embodiments of the present adhesive composition, its method of production and uses and advantages associated with the various embodiments are now described below. The advantages are demonstrated through the examples disclosed herein and shall not be iterated for brevity.

[0095] In various embodiments, the organic macromolecules may include a polymer or a dendrimer. The polymer may be a branched or unbranched polymer. Such organic macromolecules are advantageous as they provide for multiple nucleophilic functional groups on which other moieties may be grafted thereon. The organic macromolecules may include, without being limited to, polyamidoamine, polyethylenimine, polyester, polycaprolactone, chitosan, or a mixture thereof. A non-limiting example of the dendrimer may be a generation 5 polyamidoamine (G5-PAMAM). Other types of dendrimers that can have nucleophilic functional groups present thereon may be used. [0096] In various embodiments, the one or more electron donating moieties may include a 1,2-aryl-diol or a derivative thereof, an example is catechol. In certain embodiments, the one or more electron donating moieties may include a 1,4-aryl-diol or a derivative thereof.

[0097] In various embodiments, the one or more electron donating moieties may be derived from a compound having a catechol protecting group and an aldehyde group, wherein the aldehyde group may react with a nucleophilic functional group of the macromolecule to form the electron accepting moiety at one end of the compound that is attached to the macromolecule while the other end of the compound forms the electron donating moiety. Said differently, the compound may be used as a precursor for forming the one or more electron donating moieties. Non-limiting examples of such compound include 2-ethoxybenzo[d][l,3]dioxole-5-carbaldehyde, 2,2- dimethylbenzo [d] [ 1 ,3 ] dioxole-5-carbaldehyde, 2,2-diphenylbenzo [d] [ 1 ,3] dioxole-5- carbaldehyde, spiro [benzo [d] [ 1 ,3] dioxole-2, 1 '-cyclohexane] -5-carbaldehyde, vanillin, or iso-vanillin.

[0098] In various embodiments, the one or more electron donating moieties may be derived from a compound having a phenol protecting group and an aldehyde group, wherein the aldehyde group may react with a nucleophilic functional group of the macromolecule to form the electron accepting moiety at one end of the compound that is attached to the macromolecule while the other end of the compound forms the electron donating moiety. Said differently, the compound may be used as a precursor for forming the one or more electron donating moieties. Non-limiting examples of such compound include 4-hydroxy-3-((trimethylsilyl)oxy)benzaldehyde, 4-hydroxy-3- ((triethylsilyl)oxy)benzaldehyde, or 3-((tert-butyldimethylsilyl)oxy)-4- hydroxybenzaldehyde.

[0099] In various embodiments, the one or more electron donating moieties may be derived from catechol variants represented by the formula:

[00101] wherein Ri is fluorine, chlorine, bromine, -OCH3, -NH2, -NO2, or -CF3. The catechol variants have an aldehyde group which may react with a nucleophilic functional group of the macromolecule to form the electron accepting moiety at one end of the compound that is attached to the macromolecule while the other end of the compound forms the electron donating moiety. Said differently, the catechol variants may be used as a precursor for forming the one or more electron donating moieties. The one or more electron donating moieties may be derived using other precursors mentioned in various embodiments related to the present method of forming the adhesive composition, which is described further below. [00102] The electron donating moieties described above are advantageous as they are able to convert to a ketone for cross-linking with the nucleophilic functional groups when the present adhesive composition is subject to an external stimulus.

[00103] In various embodiments, when an electron donating moiety is grafted to a nucleophilic functional group on the macromolecule, an electron accepting moiety is formed. This electron accepting moiety advantageously gets reduced as the electron donating moiety gets oxidized for cross-linking and renders the present adhesive composition operable as an adhesive. The electron accepting moiety may include an imine, an alkene, an oxadiazole, a tetrazinealkyne, a fluoren-9-one, a thioazole, or a malononitrile. [00104] In various embodiments, 10 molar% to 50 molar%, 20 molar% to 50 molar%, 30 molar % to 50 molar%, 40 molar% to 50 molar%, 10 molar% to 40 molar' %, 10 molar% to 30 molar%, 10 molar% to 20 molar%, 20 molar% to 40 molar%, 20 molar% to 30 molar' %, etc. of the nucleophilic functional groups may be grafted with the one or more electron donating moieties, and 50 molar% to 90 molar%, 50 molar% to 80 molar%, 50 molar % to 70 molar%, 50 molar % to 60 molar%, 60 molar% to 90 molar' %, 70 molar% to 90 molar%, 80 molar% to 90 molar%, 60 molar% to 80 molar%, 60 molar% to 70 molar%, etc. of the nucleophilic functional groups remain as the nucleophilic functional group.

[00105] As described above, the electron donating moiety oxidizes into a monoketone or a diketone when the adhesive composition is subject to a stimulus. In various embodiments, the diketone may include quinone, para-quinone, 1,2-cyclo-diketone, or aliphatic-ketone. As one example, a catechol as the electron donating moiety may be oxidized into a quinone. As another example, vanillin as the electron donating moiety may be first oxidized into a monoketone, which in the presence of water may be further hydrolyzed and oxidized into quinone under electrochemical condition. The aliphatic- ketone may be an aliphatic-diketone. Aliphatic-ketone and aliphatic-diketone of the present disclosure refer to straight chain ketones having at least 4 or more carbon atoms, wherein 2 of the carbonyl carbons (i.e. ketone functional group) are adjacent carbon atoms. Advantageously, the diketone provides for cross-linking with nucleophilic functional groups of the macromolecules. The cross-linking may be intramolecular or intermolecular.

[00106] In various embodiments, the stimulus may be water containing oxygen, an oxidant, an electrical voltage, or a combination thereof. The oxidants may include periodates or peroxides.

[00107] The present adhesive composition may further include an additive including 1,2-aliphatic diol, catechol, tannic acid, 1,2-furan-diol, ascorbic acid, vicinal diol, polysaccharide-diol, or a mixture thereof.

[00108] As described above, the diketone forms one or more cross-linkages with the nucleophilic functional groups. Advantageously, the one or more cross-linkages may be formed after a lag-time of 300 seconds or more where the stimulus is water containing oxygen, or the one or more cross-linkages may be formed in 300 seconds or less where the stimulus is (i) electrical voltage or (ii) water containing oxygen and electrical voltage. In various embodiments, the electrical voltage may be direct current or alternating current. The electrical voltage may range from 0.1 V to 10 V, 1 V to 10 V, 5 V to 10 V, etc. Advantageously, for the present adhesive composition, the magnitude of voltage applied may be used to control the lag-time for the self-curing. [00109] In various embodiments, the adhesive composition may be for use as an adhesive to adhere biological tissues, or as a sealant in medical and/or veterinary applications including intestinal anastomosis, vascular anastomosis, tissue repair, and/or tissue implantation.

[00110] The present disclosure also provides for a method of producing an adhesive composition, wherein the adhesive composition includes organic macromolecules each including nucleophilic functional groups, wherein each of the organic macromolecules is a linear macromolecule or a branched macromolecule, wherein each of the nucleophilic functional groups includes an amino group, a thiol group, a hydroxyl group, or a carboxyl group, and one or more electron donating moieties, wherein each of the nucleophilic functional groups grafted with the one or more electron donating moieties is converted to an electron accepting moiety, and each of the nucleophilic functional groups not grafted with the one or more electron donating moieties remains as the nucleophilic functional group, wherein the one or more electron donating moieties oxidizes into a diketone when the adhesive composition is subject to a stimulus, and wherein the diketone forms one or more cross-linkages with the nucleophilic functional groups, wherein the method includes providing the organic macromolecules including the nucleophilic functional groups, and mixing the organic macromolecules with a precursor of the one or more electron donating moieties. Mixing the organic macromolecules with the precursor of the one or more electron donating moieties may be carried out in presence or absence of light. In other words, the present method is versatile and is not limited by the light conditions.

[00111] Embodiments and advantages described for the present adhesive composition of the first aspect can be analogously valid for the present method subsequently described herein, and vice versa. As the various embodiments and advantages have already been described above and examples demonstrated herein, they shall not be iterated for brevity. [00112] In various embodiments, providing the organic macromolecules including the nucleophilic functional groups may include providing a stock solution that includes the organic macromolecules in a first anhydrous organic solvent, and removing dissolved oxygen from the stock solution. In various embodiments, mixing the organic macromolecules with the precursor includes dissolving the precursor in a second anhydrous organic solvent to obtain a precursor solution and adding the stock solution having oxygen removed therefrom to the precursor solution under an inert environment. Examples of the first anhydrous organic solvent and the second anhydrous organic solvent mentioned above may include an anhydrous alcohol, such as methanol, ethanol, propanol or butanol that are anhydrous.

[00113] In the present method, mixing the organic macromolecules with the precursor may include mixing the precursor in an amount ranging from 10 molar % to 50 molar % of the nucleophilic functional groups of the organic macromolecules. Other embodiments of the amount are already described above and shall not be iterated for brevity. In various embodiments, the precursor may include 3,4- dihydroxybenzaldehyde (3,4-DBA) or a derivative thereof. Said differently, the precursor may include or may be a compound derived or modified from 3,4-DBA. The precursor may include a mixture of 3,4-DBA and one or more of its derivatives. Non limiting examples of the derivative are described as follows.

[00114] In various embodiments, the precursor, such as a derivative of 3,4-DBA, may include, without being limited to, vanillin, isovanillin, piperonal, l,3-benzodioxole-5- carboxaldehyde, 2-bromo- 1 ,3-benzodioxole-5-carboxaldehyde, 6-methyl- 1,3- benzodioxole-5-carboxaldehyde, l,3-benzodioxole-5-carboxaldehyde, 2,2-dichloro- l,3-benzodioxole-5-carboxaldehyde, 7 -methyl- l,3-benzodioxole-5-carboxaldehyde, 2- nitro-l,3-benzodioxole-5-carboxaldehyde, 4-hydroxy- l,3-benzodioxole-5- carboxaldehyde, 7-(methylthio)-l,3-benzodioxole-5-carboxaldehyde, 1,3- benzodioxole-4,6-dicarboxaldehyde, 6-chloro-l,3-benzodioxole-5-carboxaldehyde, 5- formyl-methyl ester- l,3-benzodioxole-2-carboxylic acid, 6-formyl- 1,3-benzodioxole- 5-sulfonic acid, 7-chloro-l,3-benzodioxole-5-carboxaldehyde, 4-methoxy-2-oxo-l,3- benzodioxole-5-carboxaldehyde, 6-nitro-l,3-benzodioxole-5-carboxaldehyde, naphtho[ 1 ,2-d]- 1 ,3-dioxole-5-carboxaldehyde, 7-(trifluoromethyl)- 1 ,3-benzodioxole- 5-carboxaldehyde, 6-(2-thienyl)-l,3-benzodioxole-5-carboxaldehyde, 2-alkoxy-l,3- benzodioxole-5-carboxaldehyde, 2,2-dialkyl- l,3-benzodioxole-5-carboxaldehyde, spiro[l,3-benzodioxole-2,l-cycloalkane]-5-carboxaldehyde, or a mixture thereof. Other examples of the precursor have already been described above, such as the compound having a catechol protecting group and an aldehyde group, the compound having a phenol protecting group and an aldehyde group, and the catechol variants. The term “alkoxy” in such embodiments refer to an alkyl group singularly bonded to oxygen. Examples include methoxy, ethoxy, n-propoxy, and isopropoxy. The term “dialkyl” in such embodiments refer to the presence of two alkyl groups.

[00115] In various embodiments, the precursor may include 2,3- dihydroxybenzaldehyde (2,3-DBA) or a derivative thereof. Said differently, the precursor may include or may be a compound derived or modified from 2,3-DBA. The precursor may include a mixture of 2,3-DBA and one or more of its derivatives. [00116] In various embodiments, the precursor may include 2,5- dihydroxybenzaldehyde (2,5-DBA) or a derivative thereof. Said differently, the precursor may include or may be a compound derived or modified from 2,5-DBA. The precursor may include a mixture of 2,5-DBA and one or more of its derivatives. [00117] In various embodiments, the precursor may include any combination of 3,4- DBA, 2,3-DBA, 2,5-DBA, and their respective derivatives.

[00118] The present method may further include adding an anhydrous ether after mixing the organic macromolecules with the precursor to precipitate the adhesive composition, washing the adhesive composition which precipitated with the anhydrous ether, and drying the adhesive composition under vacuum. The vacuum drying helps remove any residual solvent.

[00119] The present disclosure further provides for a method of adhering or sealing biological tissues, the method includes applying an adhesive composition to one or more biological tissues, wherein the adhesive composition includes organic macromolecules each including nucleophilic functional groups, wherein each of the organic macromolecules is a linear macromolecule or a branched macromolecule, wherein each of the nucleophilic functional groups includes an amino group, a thiol group, a hydroxyl group, or a carboxyl group, and one or more electron donating moieties, wherein each of the nucleophilic functional groups grafted with the one or more electron donating moieties is converted to an electron accepting moiety, and each of the nucleophilic functional groups not grafted with the one or more electron donating moieties remains as the nucleophilic functional group, wherein the one or more electron donating moieties oxidizes into a diketone when the adhesive composition is subject to a stimulus, and wherein the diketone forms one or more cross-linkages with the nucleophilic functional groups, subjecting the adhesive composition to the stimulus, and contacting the biological tissues together to adhere the biological tissues.

[00120] Embodiments and advantages described for the present adhesive composition of the first aspect and the method described above can be analogously valid for the present method of adhering or sealing biological tissues subsequently described herein, and vice versa. As the various embodiments and advantages have already been described above and examples demonstrated herein, they shall not be iterated for brevity.

[00121] In the present method, subjecting the adhesive composition to the stimulus may include exposing the adhesive composition to water containing oxygen or an oxidant, applying an electrical voltage to the adhesive composition, or a combination thereof. Subjecting the adhesive composition to the stimulus may include exposing the adhesive composition to moisture to have the one or more cross-linkages formed after a lag-time of 300 seconds or more, or applying an electrical voltage to the adhesive composition to have the one or more cross-linkages formed in 300 seconds or less, or exposing the adhesive composition to moisture and applying an electrical voltage to the adhesive composition to have the one or more cross-linkages formed in 300 seconds or less.

[00122] The present method may further include applying pressure to the biological tissues.

[00123] The present disclosure also provides for the use of an adhesive composition in the preparation of (i) an adhesive; or (ii) a kit including the adhesive composition for intestinal anastomosis, vascular anastomosis, tissue repair, and/or tissue implantation, wherein the adhesive composition includes organic macromolecules each including nucleophilic functional groups, wherein each of the organic macromolecules is a linear macromolecule or a branched macromolecule, wherein each of the nucleophilic functional groups includes an amino group, a thiol group, a hydroxyl group, or a carboxyl group, and one or more electron donating moieties, wherein each of the nucleophilic functional groups grafted with the one or more electron donating moieties is converted to an electron accepting moiety, and each of the nucleophilic functional groups not grafted with the one or more electron donating moieties remains as the nucleophilic functional group, wherein the one or more electron donating moieties oxidizes into a diketone when the adhesive composition is subject to a stimulus, and wherein the diketone forms one or more cross-linkages with the nucleophilic functional groups. The kit may include an isotonic aqueous solution which serves as the stimulus. The isotonic aqueous solution may contain water with oxygen.

[00124] Embodiments and advantages described for the present adhesive composition of the first aspect and the methods described above can be analogously valid for the present use described herein, and vice versa. As the various embodiments and advantages have already been described above and examples demonstrated herein, they shall not be iterated for brevity.

[00125] The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the present disclosure. [00126] In the context of various embodiments, the articles “a”, “an” and “the” as used with regard to a feature or element include a reference to one or more of the features or elements.

[00127] In the context of various embodiments, the term “about” or “approximately” as applied to a numeric value encompasses the exact value and a reasonable variance. [00128] As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.

[00129] Unless specified otherwise, the terms "comprising" and "comprise", and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, unrecited elements.

Examples

[00130] The present disclosure relates to a self-curing, redox donor/acceptor adhesive composition for use in, for example, bonding to wet substrates. The adhesive composition may include, for example, an unbranched or branched polymer containing nucleophilic functional groups, a redox acceptor attached to a redox donor, wherein the redox donor forms the cross-linking group and the redox donor is electrochemically balanced by the redox acceptor. The redox donor can spontaneously form a cross- linking group that covalently bonds to a nucleophilic group. The terms “redox donor”, “electrochemical donor”, “electron donor”, “redox donor internal additive”, and “electron donating moiety”, are used interchangeably herein. The term “redox acceptor”, “electrochemical acceptor”, “electron acceptor”, “redox acceptor internal additive”, and “electron accepting moiety”, are used interchangeably herein.

[00131] The composition is inert and inactive in a water-free, anhydrous state, but upon aqueous reconstitution, cross-linking may be activated after a lag-time of more than 300 seconds without any energy inputs, wherein after the lag-time, self-curing is activated. [00132] The lag-time can be controlled through voltage or current activation, wherein electrochemical redox curing may be activated in less than 10 seconds after voltage or current input.

[00133] The present disclosure also describes a fabrication method and workings of the present self-curing adhesive composition.

[00134] The present adhesive composition, method of fabricating the adhesive composition, and uses thereof, are described in further details, by way of non-limiting examples, as set forth below.

[00135] Example 1: General Description of Present Adhesive Composition [00136] The present adhesive composition is a self-curing, redox donor/acceptor adhesive composition that is safe and non-toxic, rapidly cross-links upon application of external stimulus, able to self-cure with a controllable lag-time after aqueous reconsitution, and allows for manipulation before and during curing (e.g. the adhesive composition may be shaped or molded before and during curing).

[00137] In various embodiments disclosed herein, the present adhesive composition contains (i) one or more macromolecules grafted with (ii) one or more electron donating moieties (e.g. at least ten catechol moieties), (iii) one or more electron accepting moieties (e.g. at least ten of such moieties), and (iv) one or more nucleophilic functional groups on the macromolecules (e.g. at least ten nucleophilic functional groups). The nucleophilic functional group includes, without being limited to, amines, thiols, and carboxylic acids. [00138] The present adhesive composition may be rapidly cross-linked using voltage activation in addition to aqueous reconstitution. The present adhesive composition may also covalently cross-link after a lag-time by self-curing mechanism upon exposure to oxygen and/or aqueous reconstitution.

[00139] The present adhesive composition may be stored under anhydrous, anaerobic conditions in a liquid or solid form. For example, the present adhesive composition present as a solid can be activated by dissolving in saline or phosphate buffered saline solution. The buffered saline solution may have a concentration of 10% to 70% (w/v or g/mL). The activated mixture solution may then be smeared on one substrate and adhered to another substrate with application of a gentle pressure on the substrates for a duration more than the lag-time. In another example, to adhere the substrates, a 1 V differential may be applied to the present adhesive composition (i.e. the mixture solution) smeared thereon. In another example, the present adhesive composition, as a solid, can be applied directly to a wet substrate, such that the wet substrate donates water through an osmotic gradient to the adhesive composition which is anhydrous. Then, another substrate may be adhered by applying a gentle pressure for a duration more than the lag-time. In another example, the present solid adhesive composition may be applied directly to the wet substrate, such that the wet substrate donates water through an osmotic gradient to the anhydrous adhesive composition and another substrate may then be adhered after applying a 1 V differential to the adhesive composition applied thereon. Application of the voltage differential may be through any electrochemical techniques, including but not limited to interdigitated electrodes, semiconductive substrates, bipolar virtual electrodes, or combination thereof. Hence, the present adhesive composition is advantageous in that it can be an adhesive compatibly usable with wet surfaces.

[00140] Advantageously, the macromolecules act as the backbone and may be a natural or synthetic bio-macromolecule that is absent of any toxic components, in particular aldehyde, acrylates, urethanes, or cyanoacrylate groups. The present adhesive composition is designed and fabricated with the consideration of integrating both self curing and voltage activation capabilities for long term effectiveness by covalent cross- linking.

[00141] Example 2A: Materials [00142] G5-PAMAM dendrimer (5th generation, 28 kDa, 128 primary amines on dendrimer) was supplied by Dendritech, USA. 3,4-dihydroxybenzaldehyde (97%, denoted as DBA) and sodium borohydride (NaBtU) was supplied by Sigma, Singapore. Disposable TE100 3-electrode chip was purchased from Zensor® R&D Company, Taipei, China.

[00143] Example 2B: Synthesis of the Present Adhesive Composition Using 3,4- Dihydroxybenzaldevde (DBA)

[00144] The present adhesive composition is synthesized by spontaneous coupling of, 3,4-dihydroxybenzaldehyde as one example of the electron donating moiety, with amine-functionalized dendrimers as one example of the macromolecule having the nucleophilic functional groups.

[00145] Briefly, generation 5 polyamidoamine (G5-PAMAM) was degassed (with nitrogen) in a methanol stock solution to remove dissolved oxygen. 3,4- dihydroxybenzaldeyde (DBA) was dissolved in anaerobic, anhydrous methanol and subsequently added dropwise to G5-PAMAM stock solution (e.g. over a period of 10 min) that was rapidly stirred to obtain 10 to 50 molar% amine grafted G5-PAMAM (moles DBA/moles amine per dendrimer x 100%). The reaction solution was stirred at room temperature under dark conditions in an inert gas atmosphere overnight. The present adhesive composition was obtained by precipitating in anhydrous diethyl ether, followed by washing the precipitation with diethyl ether for two times and then drying under vacuum to remove any residual solvent. The present adhesive composition can be stored under anaerobic, anhydrous conditions at, for example, 20°C. The resultant adhesive composition was characterized by 'H NMR and size exclusion chromatography. The amount of catechol groups and Schiff-base group were determined by 'H NMR.

[00146] Example 2C: Synthesis of Present Adhesive Composition (G5-DBA20 Conjugates) with Reductive Amination

[00147] Schiff-bases on G5-DBA₂₀ are chemically reduced by reductive amination with 2x equivalent of sodium borohydride (NaBlU) in anhydrous methanol. NaBlU solution (1 M) is added dropwise over 5 min and stirred for 1 hr. DI water (0.5 mL) is subsequently added and stirred for 1 hr to quench excess reducing agent. The reduced products are precipitated and purified as above in anhydrous diethyl ether, redissolved into anhydrous methanol and precipitated again in anhydrous diethyl ether. This process is repeated three more times before drying under vacuum.

[00148] Example 2D: Synthesis of the Present Adhesive Composition Using Vanillin

[00149] In this example, the present adhesive composition is synthesized by spontaneous coupling of vanillin as one example of the electron donating moiety with amine-functionalized dendrimers as one example of the macromolecule with nucleophilic functional groups. The present adhesive composition is stable under ambient conditions (e.g. 20 to 28°C).

[00150] Briefly, generation 5 polyamidoamine (G5-PAMAM) was degassed (with nitrogen) in a methanol stock solution to remove dissolved oxygen. Vanillin (4- Hydroxy-3-methoxybenzaldehyde) was dissolved in anaerobic, anhydrous methanol and subsequently added dropwise to a G5-PAMAM stock solution (e.g. over a period of 10 min) that was rapidly stirred to obtain 20 molar% amine grafted thereon (moles vanillin/moles amine per dendrimer x 100%). The reaction solution was stirred at room temperature under dark conditions in an inert gas atmosphere overnight. The present ambient stable adhesive composition was obtained by precipitating in anhydrous diethyl ether, followed by washing the precipitation with diethyl ether for two times then drying under vacuum to remove any residual solvent. The present adhesive composition can be stored under anhydrous conditions at 25°C. FIG. 18A illustrates the chemical structure of reactants and products of the synthesis reaction. The resultant adhesive composition was characterized by 'H NMR and size exclusion chromatography. The amount of vanillin groups and Schiff-base groups were determined by 'H NMR. The resultant adhesive composition does not self-cure when dissolved in aqueous formulations. In other words, the present adhesive composition formed using vanillin has an advantage for applications that specifically require electrochemical activation (e.g. application of a voltage) to induce formation of cross- linkages and curing, as the present adhesive composition formed using vanillin is sufficiently stable (does not self-cure) even when dissolved in an aqueous solution. [00151] Further details and discussion on characterization, properties and performance of present adhesive composition formed using vanillin are provided in example 9 below. Purpose of using vanillin is to graft vanillin onto PAMAM and determine the stability in atmospheric environment and electrocuring of this adhesive composition, which is one of the adhesive compositions provided for by the present disclosure.

[00152] Example 3A: UV/Visible Characterization

[00153] Free DBA and G5-DBAx was dissolved in methanol and filtered to obtain clear solution for UV-Vis spectroscopy (UV-Vis-NIR Lambda 950). Reaction kinetics were evaluated with diluted solutions of PAMAM and free DBA in degassed methanol mixed with the designated mol ratio in a 1 cm³ spectroscopy cell to obtain the desired conjugation. Once mixed, the reaction mixture was sealed by a cap and placed in spectrophotometer. UV spectrum was obtained at certain intervals of time to investigate the reaction kinetics. UV-Vis spectroscopy (NanoDrop 2000 Spectrophotometer, Thermo Scientific™) for reduced G5-DBA₂₀ is characterized in saturated methanol. [00154] Example 3B: SEC-MALLS-UV Quantification of Catechol [00155] Size exclusion chromatography (SEC) consists of an Agilent 1100 solvent pump with in-line detectors of 1) UV/Vis detector (280 nm), 2) multi-angle laser light scattering (MALLS), and refractive index (RI) detector for evaluation of catechols, molar mass, and mass detection, respectively. A PLGel aqueous MIXH column thermostat set at 60°C with 1% w/v formic acid eluent at a flow rate of 1 mL/min. Conjugates samples are dissolved in eluent at 2-10 mg/mL, followed by 0.2 pm syringe filtration before injection (50 pL). Dn/dc of 0.185 is applied for PAMAM dendrimers mass detection. Dn/dc denotes specific refractive index increment. The UV extinction coefficient of catechol units are reported in FIG. 11A.

[00156] Example 3C: NMR Spectroscopy Analysis

[00157] Bruker Advance NMR at 400 MHz evaluated the G5-DBAx and reduced G5- DBAx in DMSO-d6 and methanol-d4, respectively. MestReNova and Topspin software aids the peak assignment and peak integration of 1H NMR and 13C NMR. Conjugates percentage calculation is listed in FIG. 12B. 2D NMR is employed to confirm the Schiff-base formation for G5-DBA₂₀ and reduction of G5-DBA₂₀ conjugate.

[00158] Example 3D: Cyclic Voltammetry (CV)

[00159] AUTOLAB with Nova 2.1 software records the cyclic voltammograms. The electrochemical cell equipped with a 3 mm glassy carbon (GC) (or 3 mm Pt disk) working electrode a platinum counter electrode, and a Ag/AgCl reference electrode (filled with 3.0 M KC1 solution) are placed in a Faraday cage. GC is pretreated with 1.5, 0.3, and 0.05 mih AI2O3 and 1.0 mhi diamond polishing powder, followed by rinse through with DI water. The cyclic voltammograms data are recorded in PBS solution at the scan rate of 50 mV/s. Ah cyclic voltammograms are scanned from negative potential to positive potential. CV scanning is performed over three cycles, and the second cycle data was collected for analysis.

[00160] Example 3E: Real-Time Electrorheology and Rheology [00161] Dynamic rheometer (MCR102, Anton Paar, Singapore) coupled with a portable potentiostat (Vertex, Ivium Technologies, The Netherlands) activates the voltage potential across the G5-DBAX samples with the aid of a disposable 3-electrode polypropylene-based Zensor® chip, embedded with a 3-mm diameter GC as working electrode (WE), an outer annular crescent GC as counter electrode (CE), and a Ag/AgCl pellet as reference electrode (RE). A ceramic rheometer probe with 10-mm diameter parallel-plate geometry PP10 probe serves as the measuring probe and directly interfaces to the 3-mm diameter WE in contact with 20 pL of dissolved sample (G5- DBAx conjugates in PBS solution). The PP10 probe has an optimized 0.30 mm gap size, as gap sizes greater than 0.5 mm were previously found to have excessive gelation times due to diminishing electric field strength. The loss modulus and the storage modulus of the formulations are recorded at 1 Hz strain rate and 1% strain under oscillatory dynamic analysis. The potentiostat was applied to maintain the activation voltage at 0, -1, and +1 V, respectively. Measurement period was controlled within 60 min to avoid the Zensor® chip’s insulator coating being dissolved/damaged. Similar parameters are employed for the chemical curing samples with 2 pL NaI0₄ solution (dissolved in PBS in 0.1 mM) added to the 20 pL G5-DBAx formulations, 5 seconds of pipet mixing, followed by real-time rheology analysis. For self-curing samples, real time rheology is initiated after dissolving in PBS buffer. Ah samples are evaluated in triplicate and minimum torque measurements are 20 nN.m.

[00162] Example 3F: Characterization via Lap Shear Adhesion of Adhesives Against Collagen Films

[00163] Collagen film sections (2.5 x 3 cm²) and Zensor® chip are mounted on microscope slides with double sided tape. G5-DB Ax composition (20 pL dissolved in PBS) is sandwiched between 3-electrode Zensor® chip and collagen films. A tape of thickness 100 pm serves as a thickness spacer. For the electrocuring samples, -1 V is applied. For the chemical curing samples, 2 pL NaKL solution (dissolved in PBS in 0.1 mM) is mixed for 5 seconds within the G5-DBAx formulation, followed by covering with collagen films. For the control samples, there were no voltage applied, and no NaI0₄ solution added. Lap shear failure is evaluated by a tensile tester fitted with a 50 N force cell (Chatillon Force Measurement Products, USA), with a linear elongation of 3 mm/min.

[00164] Example 4: Electrochemical Activation of the Present Adhesive Composition

[00165] Cyclic voltammetry (CV) was determined by linear voltage sweeps using an AUTOLAB with Nova 2.1 software, as already described above.

[00166] Overview of the present adhesive composition can be understood from FIG. 1 A to 1C, which is not an adhesive in its liquid or solid form under anaerobic, anhydrous conditions. Varying amounts of electron accepting moiety and/or electron donating moiety can be present or grafted on the macromolecule to tune the properties of the adhesive composition as shown in FIG. 2A. After grafting on G5-PAMAM, the electrochemical activation of the catechol (donor) and Schiff-base (acceptor) is seen at £pai= 0.29 V (catechol) and E^a = 0.91 V (Schiff-base), as shown in FIG. 2B to 2D. Grafting of a non-catechol control, benzaldehyde, to G5-PAMAM (G5-BenzADH) demonstrates only activation at E_pa2 = 0.91 V (Schiff-base), as seen in FIG. 2B. Activation of 3,4-dihydroxybenzaldehyde with no grafting of G5-PAMAM, shows only presence of catechol at anodic peaks (£p_ao = 0.52 V) and cathodic peaks (£p_Co = -0.02 V) (vs. Ag/AgCl), as seen in FIG. 2C. Thus, the Schiff-base (acceptor) is only formed upon grafting 3,4-dihydroxybenzaldehyde on to G5-PAMAM in a one- step non- aqueous reaction.

[00167] Example 5: Properties of Present Adhesive Composition [00168] The viscoelastic properties of the present adhesive composition, which can be voltage activated, were determined by using a rheometer (Anton-Paar, MCR101) coupled with a ceramic non-conductive probe, and a portable potentiostat (Ivium, CompactStat) was applied to activate electrocuring of dendrimer samples. A polypropylene-based 3 -electrode chip (Zensor, Taiwan), embedded with a 3 -mm diameter glassy carbon working electrode (WE), an outer annular crescent glassy carbon counter electrode (CE), and a Ag/AgCl pellet as reference electrode (RE), was selected as the electrode platform.

[00169] The loss modulus and the storage modulus of the compositions were recorded at 1 Hz strain rate for the amplitude sweep and 10% amplitude under oscillatory dynamic analysis. The potentiostat was applied to maintain the activation voltage at -1 V. Measurement period was controlled within 60 min. Before reconstitution of the present adhesive composition, the composition is not an adhesive in its dry form. After reconstitution in phosphate buffered saline (or other aqueous and/or aqueous/solvent liquid) and exposed to oxygen, a 20 microliter sample is applied to the 3-electrode chip, where the sample is in contact with the working electrode, counter electrode, and reference electrode and subsequently activated with an external stimulus (-1 V or +1 V). The conductive attributes of the present adhesive composition and electrochemical interactions of the electron accepting moiety and electron donating moiety allow for voltage activation, wherein a catechol-mediated cross-linking can be observed via a real-time rheology evaluation as shown in FIG. 3A to 3C. To confirm catechol- mediated cross-linking, a two-part curing is initiated with dilute sodium periodate to activate the electron donating moiety and electron accepting moiety, wherein catechol- mediated cross-linking is seen instantaneously. Electrocuring of the present adhesive composition is statistically non-inferior to the two-part curing by periodate oxidation at DBA grafting ratios of 20 molar% and 30 molar% after 30 min of +1 V voltage activation as seen in FIG. 3D.

[00170] Example 6: Stability of Present Adhesive Composition and Self-Curing Ability After Fan-Time

[00171] The present adhesive composition demonstrates self-curing with a tunable lag time as seen in FIG. 5A and 5B. Lag-time is assessed after aqueous reconstitution, rapidly mixing, and pipetting on the substrate (start time point) and the interval of time passage until one of the two criteria is met, whichever occurs first (stop time point): (1) an order of magnitude increase in G’ (storage modulus) or (2) when the complex viscosity exceeds 2 Pa.s at room temperature (25 ± 2°C). Formulation A, which is stored under anhydrous, anaerobic conditions at -20°C showed a 23 min lag-time after reconstitution in phosphate buffered saline (pH 7.2). Formulation B, which is stored under anhydrous, non-anaerobic conditions at -20°C for two months, showed a 12 min lag-time after reconstitution in phosphate buffered saline. Formulation C, which is stored under anhydrous, non-anaerobic conditions at -20°C for three months, showed an 8 min lag-time after reconstitution in phosphate buffered saline. These demonstrate that the lag-time can be tuned through adjustment of aerobic, atmosphere exposure. The lag-time can be further tuned with the addition of redox mediators and redox catalysts. FIG. 8A demonstrates that the self-curing lag-time can be decreased to less than 1 min with an external additive of ascorbic acid, which is a non-toxic anti-oxidant. FIG. 8B demonstrates that complexing agents such as boron do not retard the self-curing mechanism in this instance, which is in contrast to what is observed for cross-linking mechanism that depends on oxygen mediated auto-oxidation processes. At pH 8.0, the oxidation potential of ascorbic acid is + 0.33 V. Due to the low oxidation potential, ascorbic acid may be oxidized by oxygen present in air. The decrease in lag-time and gelation time with the increased concentration of ascorbic acid may be due to ease of reduction of Schiff base by the increased abundance of electrons and protons generated by the air oxidation of ascorbic acid. In addition, in an aqueous medium, complexation of catechol by borate may be less effective due to hydrogen bonding stabilization of catechol and borate with the water molecules. This may be why the typical retarding effect of self-curing by complexation with borate is not observed in the present case. [00172] Example 7: Adhesion to Wet Substrates

[00173] The present adhesive composition demonstrates lap-shear adhesion to the wet substrate, e.g. a wetted collagen film. Voltage activation of the present adhesive composition is exemplified with a disposable 3-electrode chip (glassy carbon as working and counter electrodes, and a Ag/AgCl reference electrode) as seen in FIG. 4A. Electrocuring of the present adhesive composition can be rapidly activated by a voltage of -1 V (referenced to Ag/AgCl). The combination of the electron donating moiety and electron accepting moiety allows electrical conductivity and a redox balance for generation of quinone species, which cross-links available nucleophiles. Without the combination, no voltage-initiated curing may be possible. Cross-linking occurs by oxidation of the electron donating moiety, e.g. catechol, which can be demonstrated by a two-part curing method with dilute periodate. Tack (defined as load greater than 1 N/cm²) is observed for both two-part curing and electrocuring methods, but not self curing before 30 min as seen in FIG. 4B. Adhesion strength by -1 V voltage activation is statistically non-inferior to oxidation by dilute periodate (two-part curing method), as seen in FIG. 4C.

[00174] In connection with the above, a kit for adhering wet substrates may be developed using a dried adhesive composition of the present disclosure and the kit may include a reconstitution solution (e.g. saline). Such kit may be straightforward and convenient to use. For example, a user may first add the reconstitution solution to the dry adhesive composition and the mixture may be applied on a wet substrate. The liquid adhesive composition can be manipulated easily during the lag-time. Manipulation can be by means of an aerosol spray, brush-based coatings, syringe-delivery, knife casting, or combination thereof. The kit for adhering wet substrates may include the present adhesive composition dissolved in an anhydrous solvent within an anaerobic container. Such a kit may be very easy to use. For example, a user may apply pressure to the container and release the mixture on a water permeated substrate exposed to an aerobic atmosphere. Adhesion of a second substrate to the first substrate may be carried out by contacting the first substrate with the second substrate where the present adhesive composition was applied and exerting a gentle pressure on one or both substrates. [00175] Example 8A: Further Discussion of Voltage Activation [00176] Low voltage activation has been demonstrated on conventional adhesives. However, activation of diazirine was required, wherein voltage activation occured in the range of water electrolysis (-1.6 V vs. -1.23 V, respectively), which led to significant foaming in the matrix from ¾ and O2 evolution. The electron donating moiety of the present disclosure, e.g. catechols, in comparison, are electrochemically activated well below the voltage requirements for water electrolysis. Herein, a dendrimer as the macromolecule may be conjugated/grafted with catechols, forming a reducible electron accepting moiety that can be voltage activated for quinone-mediated cross-linking and adhesion. This configuration elevates catechols to a one-pot adhesive composition with material properties that can be controlled with an electric external stimuli. The electron donating and accepting moieties of the present disclosure can mediate electron transduction, wherein catechol can be oxidized to reactive intermediates, e.g. o-quinone, which are then responsible for intermolecular cross-linking. Several catechol small molecules are available with handles for dendrimer grafting. A strategy to simultaneously graft catechol (donor) and an acceptor group is to reduce the synthesis to a one-pot reaction. Protocatechuic aldehyde or 3,4-dihydroxybenzaldehyde (DBA) is a naturally derived catechol that spontaneously forms Schiff-bases (azomethines) in the presence of amines. Schiff-bases of the present disclosure can be rapidly reduced under aqueous conditions with mild reducing agents (e.g. sodium borohydride). Simple mixing of DBA with G5-PAMAM (FIG. IB) grafts the catechol thereon, forming the acceptor (Schiff-base), in a 1:1 molar ratio to form zwitterionic tautomers that are thermodynamically stable (FIG. 9A and 9B). G5-PAMAM is utilized as the polymer macromolecule, as it offers numerous design advantages. The surface primary amines on the spherical dendrimer limit intramolecular cross-linking while preventing linear entanglements. It is also soluble in both aqueous and organic solvents, unlike chitosan macromers. PAMAM branched polymer can be a model system for exploring structure activity relationships while maintaining an excess of amines for quinone-mediated cross-linking, and further modifications can be incorporated for bioadhesive or underwater applications. Grafting ratios of 10 to 30 molar%, and up to 44 molar% catechol conjugates are explored (G5-DBAx wherein X = 10 molar%, 20 molar%, 30 molar' %, etc.).

[00177] Reaction efficiency and final grafting ratios are assessed by 'H NMR (FIG. 9C and 10A to 10F, and FIG. 12A and 12B) and size exclusion chromatography (FIG. 2A and FIG. 12B). Generation of the Schiff-base is completed after 8 hrs as assessed by UV/vis at 405 nm (FIG. 11D). This peak is extinguished after borohydride reduction, which removes the Schiff-base accepting moiety as a control (FIG. 13A). Molar mass of G5-DBAx positively correlates with the grafting ratio with a reduction of the UV/RI peak elution volume (FIG. 2A). Redox properties of G5-DBAx conjugates are examined by cyclic voltammetry (CV) in isotonic PBS electrolyte (FIG. 14A and FIG. 15A to 15F). A catechol-free benzaldehyde is also grafted on G5-PAMAM to serve as a catechol-free control (G5-Benz₂o, FIG. 16D to 16F), and borohydride reduced G5- DBA20 serves as Schiff-base free control (reduced G5-DBA₂₀, FIG. 13A to 13D). Free DBA (FIG. 15A) exhibits an irreversible and diffusion-controlled redox behavior, supported by the following observations: (1) the peak current ratio (I^E _pao/I^Ep_co) is less than 1 at the scan rate of 50 mV s ¹, (2) both the anodic peaks (E_pao = 0.52 V) and the cathodic peaks (E_pco = -0.02 V) (vs. Ag/AgCl) increase progressively with the increasing scan rates (v), (3) the plot of peak current as a function of v^1/2 is linear for both peak E_pao and E_pco (FIG. 15B). The successive CV cycles (either using glassy carbon or Pt as working electrode) exhibited non-repeatable current signal (FIG. 15C and 15D), suggesting that the electrode surface is fouled by electropolymerized DBA film, which in turn inactivates the electrode surface sensitivity. At the scan rate of 50 mV s ¹, the cyclic voltammograms of G5-DBAio retains the irreversible redox behavior of DBA. G5-DBAio displays two anodic peaks (E_pai = 0.29 V and E_pa2 = 0.91 V) and a min cathodic peak (FIG. 14A). Scan rate dependent CV confirms the existence of the E_pa2 peak (FIG. 15E). The origin of E_pa2 peak is attributed to the aldehyde/Schiff-base, as it is observed in free DBA, G5-Bemo (E_pa3 in FIG. 15F), G5-DBAio, but not in G5- PAMAM (FIG. 14A). All scans of G5-Bemo are plotted the first cycle (FIG. 15E and 15F) since the peak current decreases after the second cycle. E_pa2 results from the known redox activation of Schiff-bases under similar conditions

[00178] Real-time rheology is applied to evaluate the mechanical properties (storage modulus-G’ and loss modulus-G”) before and after curing stimuli. The customized rheology platform incorporates a ceramic probe to avoid catechol-metal chelates, as aluminum probes/surfaces are found to instantly initiate curing (FIG. 16A). Voltages of +1 V and -I V effects on G5-DBAx composition’s viscoelastic properties, gelation (when G’ = G”), and lag-time (period before gelation) are compared to a typical two- part curing method (FIG. 14B). Two-part curing with periodate on G5-DBA₂₀ achieves gelation within 3 min and exhibits a kPa shear modulus after 25 min. Negative voltage (cathode as WE) displays instantaneous initiation (FIG. 14B), which is removed after reduction of Schiff-base (FIG. 13C). Voltage initiation rapidly increases G’, with gelation seen at 2.9 min after voltage activation. At 0 V (control), the G5-DBA₂₀ observes self-curing with a lag-time of 25 ± 5 min following aqueous reconstitution (FIG. 14C). Self-curing is found to be tunable with exposure to aerobic environments in a dry state, where oxygen exposure can increase ratios of catechol/quinone (FIG. 9A). Rapid initiation of cross-linking (fast increase of G’) is observed at -1 V for 15 and 30 wt%formulations (FIG. 3D). High solute (45 wt%) reconstituted compositions are solid aqueous gels but dissolve in methanol (data not shown). When +1 V is applied, a lag time of 2-3 min is noted before cross-linking with a retarded gelation time of 18 min. The voltage polarity simply swaps the working electrode into a cathode (-1 V, acceptor reduction) or anode (+1 V, donor oxidation), where the working/counter electrodes have a surface area ratio of 4/1, respectively. In the catechol-free G5-Benz₂o formulation, no cross-linking is observed at either polarity, therefore, cross-linking is solely mediated by the oxidation of catechol (FIG. 16A to 16F). However, the Schiff- base-free reduced G5-DBA₂₀ loses the ability to be instantly reduced and also has a delayed gelation time of 8.4 min vs. 3 min. Taken together, these observations demonstrate that reduction of the Schiff-base is the rate limiting reaction under the -1 V conditions, whereby a cathode working electrode facilitates rapid initiation by providing a larger area. Oxygen mediated pathways are responsible for the self-curing behavior, and the +1 V is likely a mixture of both mechanisms. However, minimum grafting of catechol is required before voltage mediated cross-linking is observed, as G5-DBAio in FIG. 16A to 16F display no gelation within 30 min but can be chemically cured. FIG. 3D compares the G’ values of all electrocuring formulations to two-part curing with periodate. G5-DBA₂₀ and G5-DBA₃₀ activation by electrocuring or periodate displayed no significant differences after 60 min.

[00179] Lap shear adhesion is evaluated with wet collagen films, which serve as a mimic of wet tissue substrates. G5-DBA₂₀ at 30 wt% is applied to the collagen and activated with disposable 3-electrode chips (FIG. 4A, FIG. 17A and 17B). G5-DBA₂₀ composition is cured by two methods: electrocuring (-1 V) vs. two-part curing (periodate). Electrocuring at +1 V is not evaluated due to the inferior material properties and gelation time, as shown in the electrorheology analyses. Lap shear adhesion strength at failure evaluates the cross-linked matrix since cohesive failure is seen throughout.

[00180] Tack evaluation under 30 min demonstrates that electrocuring G5-DBA₂₀ formulation has a significant increase over two-part curing, and no adhesion is present for the self-curing formulation. The self-curing control sample (atmosphere exposure with neither voltage nor periodate applied) displays only viscous liquid material properties (FIG. 17A). The lap shear adhesion strength comparisons after 60 min are indicated in FIG. 17B. The self-curing formulation has an increase of adhesion strength to ~4 N cm ² (40 kPa), which is about 4 times higher than 30 min case. Electrocuring continuously for 60 min is non-inferior to two-part curing, which both present ~5 N cm ² (50 kPa) adhesion strength. In summary, an azomethine/catechol adhesive is synthesized under ambient conditions to yield an acceptor/donor pair. The spontaneous reaction creates a redox-responsive adhesive that can be applied and activated by a number of external stimuli. The inclusion of both electron donating and accepting moieties creates an adhesive that can self-cure after a designed lag-time. Activation can be triggered under both oxidative and reducing environments. For example, gelation time decreased in the following oxidative conditions: ambient atmosphere, +1 V, or periodate. A reducing environment triggered cross-linking instantaneously, -1 V or aluminum substrates (A1 Al³⁺ + 3e ).

[00181] A considerable observation was the short-term stability in aqueous solvents, but this allows development of self-curing adhesion that is semi-stable in a dry state. A bioadhesive that allows liquid manipulation and then self-cures with a known lag-time is a current unmet need for tissue repair. Activation of the lag-time ‘clock’ is as simple as aqueous reconstitution. The lag-time of the dried and precipitated formulation drifted over a period of 3 months when exposed to aerobic environments. This suggests that the lag-time may be optimized in oxygen-free or limited exposure conditions (FIG. 14C).

[00182] For the first time, a catechol-adhesive allows activation by electrocuring, instead of traditional two-part curing method. This allows activation while avoiding side-effects from oxidation agents or highly concentrated metal chelators. Electrocuring adhesive strength was comparable to the periodate two-part curingmethods. Two-part curing is a standard method of activation of mussel biomimetic adhesives, with modest adhesion strength 40 kPa. Polydopamine-co-acrylate cured with periodate achieved up to 70 kPa after one day of curing. Electrocuring allows a simpler approach with no additive mixing and more precise control over initiation and gelation time. Electrocuring was better observed at 20-30% grafting ratios. Too little (10 molar%) grafting prevented voltage-activation while too much (greater than 40 molar%) may render the present adhesive composition less aqueous soluble that may stem from spontaneous cross-linking under storage. Nevertheless, such configuration (10 molar% or less and 40 molar% or more) may be used for applications that require them. The electrocuring formulations herein have advantages over conventional formulations. The present adhesive composition could be activated at -1 V (vs. Ag/AgCl), but conventional formulations tend to need a higher voltage of -1.6 V that evolves gases which may weaken the adhesive matrix and limit strength. [00183] Unexpectedly, voltage-activated cross-linking was accelerated when the working electrode was set as the cathode, effectively providing a larger surface area for reductive reactions. This supports the acceptor/donor mechanism illustrated in FIG. IB and 1C. G5-PAMAM contains both primary and tertiary amines, providing a local alkaline environment (pH 9-10). If zwitterionic, the Schiff-base catechol may attract electrons to the protonated azomethine (FIG. 9A). At this high pH, protons are limited to the zwitterion or other tautomers known to exist under aqueous conditions (see examples 8B to 8H below). A -1 V electrochemical gradient at the working electrode attracts protons from atmosphere 0₂-mediated oxidation of catechol, which is thermodynamically favorable (but the internal electronic reduction/oxidation is not, see FIG. 9B). With Schiff-bases reduced at this -I V potential, catechol to quinone oxidations are uninhibited. As quinones are responsible for cross-linking, their formation decreases the time to gelation. Supporting this is the empirical result that the aryl-Schiff-bases are reduced with sodium borohydride (FIG. 13 A to 13D), where borohydride anion is estimated to have a formal potential of E⁰’ = -0.7 to -0.43 V. [00184] Many formulations of chitosan-catechol conjugates have been synthesized, as chitosan is a cheap and scalable branched polymer that has been incorporated in many biomaterials. However, chitosan has a solubility limit in acidic mediums that renders the requirement of additional grafting. Even with catechol grafting, the functionalized chitosan unfortunately achieve a 6 wt% solution (polymer in aqueous solvent), which is too dilute for voltage-activation (G5-DBA₂₀ at 10 wt% displays no electrocuring). Grafting 19 to 80 molar% of the total amines with catechol on chitosan displays similar material properties as the reported G5-DBA₂₀ formulations. It is possible to utilize electrochemical synthesis of chitosan-catechol, where reductive amination grafts chitosan to the surface followed by oxidative activation for catechol-chitosan grafting towards anti-oxidant surfaces. Fortunately, the technology herein may allow a more straightforward approach for similar purposes. G4-PAMAM was grafted with PEG- catechol to form high modulus hydrogels up to 80 kPa, which is workable with two- part curing with periodate and cure duration of 48 hrs.

[00185] Example 8B: Results Discussion of Voltage Activation - pKa and

Tautomers of Aryl- Aldehyde Schiff-Bases [00186] The Schiff-base catechol (phenolic tautomer, FIG. 9A) predominates in non- aqueous solvents like methanol employed in the synthesis, which displays similar UV/Vis peaks to the aryl-aldehyde Schiff-base base formed from salicylaldehyde and alkyl-amines. The semiquinone tautomer (FIG. 9A scheme in top row, extrapolated from aryl-aldehyde Schiff-base) primarily exists in aqueous solutions (tautomer ratio of 0.088) and is similar in structure to quinone methides that can self-polymerize. Extrapolated pKa values from substituted catechols (e.g. ethyl catechol, pKai = 8.32) and aryl-aldehyde Schiff-bases (pKa ranging from 11-12) advocates that the Schiff- base catechol is in a zwitterionic state at the pH’s of 8.5-11. Atmospheric oxygen can induce free radicals on catechols and spontaneously from quinones, as shown in FIG. 9A scheme in middle row. The electrocuring method at -1 V is illustrated through FIG. 9A scheme in bottom row.

[00187] Example 8C: Results Discussion of Voltage Activation - Thermodynamic Modelling of Schiff-Base Catechol to Reduction Intermediate [00188] Density functional theory (DFT) calculations on the catechol molecule and its potential reaction intermediate are calculated in FIG. 9B. All calculations of geometry optimization, vibrational frequency analysis, wavefunction stability analysis, and single point energy were conducted via a B3LYP method with 6-311G(d,p) basis set and D3 dispersion correction under Becke-Jonson damping. The Gibbs free energy, which is the difference between the ground energy of the intermediate and the ground energy of catechol is calculated to be 1.4018 kJ/mol. This small energy barrier suggests an unspontaneous reaction between these two states thermodynamically [00189] Example 8D: Results Discussion of Voltage Activation - Quantitation of DBA Grafting by ¹H NMR Before and After Reductive Amination [00190] DBA 'H and ¹³C NMR peaks are indicated in FIG. 12A. As for the conjugates (i.e., G5-DBAx), the expected characteristic signals come from: (1) azomethine (H- N=C-) group, as seen at 8.02 ppm overlaps with exchangeable -H atoms (-NH) from PAMAM, preventing integration, (2) -OH groups in DBA is obscured in the conjugate at 7.85-7.98 ppm, (3) aromatic protons at 7.21-7.08 ppm, 6.98-6.85 ppm, and 6.76-6.63 ppm, respectively, which can be the representative of catechol group. As for the internal standard, -CH2-CO- in PAMAM structure provides proton signal at 2.30-1.90 ppm, which is calibrated as 504 -H atoms. Hence, the DBA/P AMAM molar ratio (i.e., grafting ratio) is calculated by the following method. Number of type ‘g’ protons in G5- PAMAM = 504. Number of type ‘b’ or ‘c’ or ‘d’ protons in DBA = 1. Z — intensity of peak ‘g’, Z_b — intensity of peak ‘b’, Z_c — intensity of peak ‘c’, Z_d — intensity of peak ‘d’. Conjugation percentage of DBA (%) = (Z_b+c+d 3)// /504 x 100%. For example, G5- DBA50 (FIG. 9C), Z_g is assigned 504 protons/P AMAM and integration of Z_b, Z_c, and Z_d yields 53.4, 53.9, and 54.1 protons/P AMAM, respectively. After averaging, conjugation percentage of 53.8% is calculated. 2D NMR, heteronuclear single quantum coherence (HSQC) spectrum is applied to confirm the presence of the Schiff-base before (FIG. 10A and 10B) and after (FIG. 10E and 10F) reduction amination. G5- DBA20 conjugate contains a cross-peak of signal at 8.02 ppm x 162.33 ppm, which is attributed to the imine coupling. The zoom-in spectrum displays both the imine (blue circle at left bottom comer) and catechol protons (green circle at right top corner). After reductive amination, no ¹³C peak is observed near 162 ppm (FIG. IOC and 10D) and further analysis with 2D NMR HSQC (FIG. 10E and 10F) observes no peak in the general region, suggesting little to no Schiff-bases remain after treatment by the NaBH4 reducing agent.

[00191] Example 8E: Results Discussion of Voltage Activation - Quantitation of DBA Grafting and Molar Mass Analysis

[00192] Catechol quantitation (FIG. 11 A to 11C), Schiff-base reaction kinetics in (FIG. llC and 11D), SEC (FIG. 2A), and extended stress-strain plots (FIG. 11E) are displayed. Absorbance (ABS) at l280 _pi is attributed to -OH auxochrome on DBA units which is the characteristic ABS peak of catechol species (FIG. 11B) gives 3 peaks at 230, 280, and 310 nm. A new peak at 405 nm is attributed to Schiff-base (-N=CH-^) coupling. UV extinction coefficient value (FIG. 11A, 8280_nm = 14556 g^.mL.cm ¹) of catechol was obtained via Beer-Lambert Law. The absorption spectrum displays similar absorption peaks (280, 316, 405 nm) to an investigated system of salicylaldehyde and alkylamines (285, 320, 415 nm), which has a predominate phenolic tautomer in methanol. Higher grafting ratio of G5-DBAx took longer time to reach equilibrium, where the equilibrium log K_f of 2-4 drives the reaction to completion, albeit slowly. To achieve more than 75% conversion of Schiff-base formation, grafting ratios took 71 min, 153 min and 160 min, for G5-DBAio, G5-DBA20 and G5-DBA30, respectively. Reaction efficiency and final grafting ratios were assessed by SEC and results displayed in FIG. 2A. With the known eixo_nm. the grafting ratio is quantified and molar mass determination is listed in FIG. 12B. As is seen in FIG. 2A, the molar mass of G5-DBAx positively correlates with the grafting ratio, leading to the shift of UV peaks at lico_phi and RI peaks to smaller elution volumes.

[00193] Example 8F: Results Discussion of Voltage Activation - Assessment of G5-DBA20 Before and After Reductive Amination

[00194] The G5-DBA₂₀ before and after reductive amination is compared by UV/Vis wavelength scan. After reduction of the Schiff-base to the secondary amine, the peak at 405 nm is extinguished (FIG. 13A). The reduced G5-DBA₂₀ is compared via self-curing (FIG. 13B), -1 V electrocuring (FIG. 13C), or +1 V electrocuring (FIG. 13D). No changes are observed for self-curing or for +1 V electrocuring. However, instantaneous activation via reductive electrocuring (-1 V) is no longer present, and the gelation time is retarded from 3 to 8 min as compared with DBA20 conjugate. However, the gelation time is still faster than +1 V, and this is speculated to be voltage activated electrocuring via the tautomerized quinone-methide.

[00195] Example 8G: Results Discussion of Voltage Activation - Cyclic Voltammetry Analyses of DBA, G5-DBAio, and G5-Benzio

[00196] DBA is subjected to a number of scan rates and two kinds of working electrodes (FIG. 15A to 15D), displaying irreversible redox behavior. The behavior is retained when DBA is grafted to G5-PAMAM via a Schiff-base coupling (FIG. 15E). Synthesis of the non-catechol Schiff-base coupling of G5-Benzio observes no oxidation peak from 0.3-0.4 (FIG. 15F), suggesting the Schiff-base is redox active even in the absence of catechol, and it responsible for T_pa2 in FIG. 15E.

[00197] Example 8H: Results Discussion of Voltage Activation - Redox Curing of G5-DBAio, G5-DBA20 and G5-Benz20

[00198] G5-DBA₂₀ was observed to instantly activate upon contact to aluminium surfaces (FIG. 16A), which precluded employment of metal probes on the rheometer. Catechols can form chelate complexes with Al(III) and metal oxides that are present on aluminium surfaces such as the disposable aluminium probes. This necessitated non- conductive, 10 mm ceramic probes, which covered the working and counter electrodes of the 3-electrode disposable chip (see FIG. 4A). Despite high solute concentrations, low grafting ratios of G5-DBAio did not undergo electrocuring (FIG. 16B), but are capable of chemical curing with periodate (FIG. 16C), suggesting that electrocuring is dependent on intramolecular dendrimer spacing. If no catechol is present, gelation is not observed under self-curing, -1 V, or 1 V electrocuring (FIG. 16D to 16F) despite high solute concentrations of G5-Benz₂o.

[00199] Example 9A: Performance of Using Vanillin for Present Adhesive Composition

[00200] 'H NMR was used to analyse the grafting ratio of vanillin onto PAMAM surface group synthesis after work up experiment (FIG. 18B). DMSO-d₆ solvent is used for this. The percentage of vanillin grafted onto PAMAM may be calculated as follows:

Vanti!in grafting onto

[00201] The sample obtained is also readily used for electrorheology test and stability test. 0 V and - I V were used and duration tested was for 50 min.

[00202] Example 9B: Electrorheology Test on 20% Vanillin-PAMAM using 0 V and -1 V for 50 min

[00203] First, rheology test is conducted on 20% vanillin-PAMAM by electrorheology in 1 x PBS with 30% w/w samples using Anton Paar Physica Rheometer MCR102 with parallel plate (PP10_C) geometry. The parameters for rheology test are strain is kept constant at 10%, frequency at 1 Hz, and measurement gap is maintained at 0.30 mm. Approximately 20uL sample is applied for rheology test. In-situ electrocuring was conducted with potentiostat, it was given 50 min to ensure its stability against atmospheric environment, then -1 V of voltage was applied at 50 min and activated for another 50 min. Total experiment time of 100 mins.

[00204] All results are repeated to ensure it is repeatable. Due to DBA has prone for self-curing vanillin is introduced as it is stable in atmospheric environment. It is used to replace the DBA and check for stability test as well as electrorheology test. -IV is applied to observe whether it can be activated. The sample is fresh and steady state rotational viscosity test is done in FIG. 19A before the electrorheology. Viscosity ranged from 48 mPa.s to 104 mPa.s. Average viscosity for Test 1 = 64.69 mPa.s, for Test 2 = 77.16 mPa.s, for Test 3 = 50.88 mPa.s.

[00205] Subsequently, there is no increase of storage modulus within 50 min stability test which shows that the vanillin is stable in atmospheric environment and no self curing observed. After which, -I V voltage activation is applied at 50 min. G’ started to increase at the 53min, 65min and 67min in 3 separate sample replicates, respectively FIG. 19B. The conjugation of vanillin is 25% as observed from NMR. It shows that the vanillin is shelf-stable, but can be induced to start cross-linking. FIG. 19C displays linear stress/strain behavior, which is evidence of a cross-linked viscoelastic gel. [00206] Example 9C: Electrorheology Test on Reduced 20% Vanillin-PAMAM Using 0 V for 50 min Stability Test, Followed by Different Voltage Activation [00207] Electrorheology test was carried out on 20 molar% van-PAMAM at higher voltages. The shelf- stability is maintained for 20 molar% van-PAMAM. No water electrolysis or gas evolution, hence rendering an advantage over diazirine-based electrocuring.

[00208] Vanillin grafted PAMAM (Vanillin-PAMAM or van-PAMAM) is utilized to replace DBA grafted PAMAM (DBA-PAMAM) for investigation of self curing problem. Vanillin may be used as adhesive that is shelf-stable and does not self-cure. However, cross-linking is slower than DBA-PAMAM under voltage activation of -1 V. Methods are explored to increase the cross-linking kinetics of vanillin-PAMAM upon voltage activation. The first plan is to apply +1 V to oxidise the adhesive composition first and then apply -1 V to observe its mechanical behavior. The second plan is to increase the voltage activation (-1.2V, -1.5V and -2V) accordingly.

[00209] First, rheology test is conducted on 20 molar% vanillin-PAMAM by electrorheology in 1 x PBS with 30% w/w samples using Anton Paar Physica Rheometer MCR102 with parallel plate (PP10_C) geometry. The parameters for rheology test are strain is kept constant at 10%, frequency at 1 Hz, and measurement gap is maintained at 0.30 mm. Approximately 20 uL sample is applied for rheology test. In-situ electrocuring was conducted with potentiostat], it was given 50 min to ensure it’s stability against atmospheric environment, then +1 V of voltage was applied at 15 min and - I V activated for another 15 min. Total experiment time of 90 min. Besides the time dependent rheology data, viscosity was also measured before voltage activation (steady state rotational viscosity test), break down OH bonds created during 50 min stability test as well as amplitude sweep after electrocuring test.

[00210] All results are repeated in triplicate to ensure it is repeatable. Due to DBA has prone for self-curing vanillin is introduced as it is stable in atmospheric environment. It is used to replace the DBA and check for stability test as well as electrorheology test. -1 V is applied to observe whether it can be activated. One month shelf-stability tests are explored by fridge incubation in the presence of atmosphere. Refrigeration prevent microorganism growth. Steady state rotational viscosity test is done and presented in FIG. 20A. Viscosity ranged from 10 mPa.s to 100 mPa.s, suggesting no spontaneous cross-linking. Subsequently, there is no increase of storage modulus within 50 min stability test which shows that the vanillin is stable in atmospheric environment and no self curing observed.

[00211] After 50 min of stability test, the reversible OH bonds may build up within the adhesive. Thus, steady state rotational viscosity test is introduced again to break down the bonds before electrocuring test. As observed from FIG. 20B, the viscosity dropped from 100,000 mPa.s to 100 mPa.s (which is the original viscosity measured from the first steady state rotational viscosity test).

[00212] After steady state rotational viscosity test, electrocuring test is done with different voltage activation (-1V, -1.2V, -1.5V and -2V). Before applying negative voltage, the adhesive is activated using +1 V for 15 min in order to see whether by applying +1 V can oxidised the adhesive before voltage activation. As observed from FIG. 20C, +1 V activates cross-linking, although a latent period exists. Van-PAMAN demonstrates cross-linking at all voltages tested of -IV, -1.2V, -1.5V and -2V.

[00213] Amplitude sweep is done right after electrorheology test. The adhesive yield stress correlates with voltage with a yield stress of 200 Pa at -2V. As observed in FIG. 20D (modulus as a function of shear strain), a linear stress/strain graph shows that the linear viscoelastic region undergoes pseudoplastic behavior at 70% shear strain. [00214] Example 9D: Electrorheology Test on 20% Vanillin-PAMAM with 0.1 M Ascorbic Acid as Proton Donor and Reaction Accelerator

[00215] First, rheology test is conducted on 20% van-PAMAM by electrorheology in 1 x PBS with 30% w/w samples using Anton Paar Physica Rheometer MCR102 with parallel plate (PP10_C) geometry. The parameters for rheology test are strain is kept constant at 10%, frequency at 1 Hz, and measurement gap is maintained at 0.30 mm. Approximately 20 uL sample is applied for rheology test. In-situ electrocuring was conducted with potentiostat, it was given 50 min to ensure it’s stability against atmospheric environment, then +1 V of voltage was applied at 15 min and -I V activated for another 15 min. Total experiment time of 90 min. Besides the time dependent rheology data, viscosity was also measured its before voltage activation (steady state rotational viscosity test), break down OH bonds created during 50 min stability test as well as amplitude sweep after electrocuring test.

[00216] Van-PAMAM is utilized to replace DBA grafted PAMAM (DBA-PAMAM) for investigation of self curing problem. Addition of a proton donor such as ascorbic acid (vitamin c) is studied to increase cross-linking kinetics and improve the mechanical behavior upon voltage activation. Molar concentration of ascorbic acid is fixed at 0.1 M while to sample to ascorbic acid vol/vol ratio required is 20 uL:2 uL. Vanillin is introduced as it is stable in atmospheric environment. It is used to replace the DBA and check for stability test as well as electrorheology test. With a fixed concentration of ascorbic acid, -1 V, -1.5 V and -2 V is applied to observe whether the van-PAMAM adhesive sample can be activated. Viscosity ranged from 10 mPa.s to 100 mPa.s before the 50 min stability test. The sample is kept in fridge for about 1 month and steady state rotational viscosity test is done and presented in FIG. 21 A. After 50 min of stability test, the OH bonds may build up within the adhesive. Thus, steady state rotational viscosity test is introduced again to break down the bonds before electrocuring test. As observed from FIG. 2 IB, the viscosity dropped from 300,000 mPa.s to 400 mPa.s (which is the original viscosity measured from the first steady state rotational viscosity test).

[00217] After steady state rotational viscosity test, electrocuring test is done with different voltage activation (-1 V, -1.5 V and -2 V). -1.2 V is omitted as it does not show any difference with -1.5 V. As observed from FIG. 21C, the time to reach gelation (where G’ = G”) correlates with the voltage applied. The adhesive transitions from a liquid-like to solid-like viscoelastic material. The adhesive has a lag-time with -I V activation. However, when applying voltage activation of -1.5 V and -2 V, it shows that the van-PAMAM obtained gelation time within min. Viscoelastic vector graph display the crossing of the gelation point in FIG. 2 ID.

[00218] Amplitude sweep is done right after electrorheology test, the adhesive reach gelation and it is still solid-like sticky material. The adhesive yield stress goes up till 712-826 Pa (observed in FIG. 2 IE) and breakdown of adhesive at shear strain of 100% for -2 V activated adhesive sample. As observed in FIG. 2 IF, yield strain correlates to voltage applied. All voltage activated adhesives display linear viscoelastic ranges typical of cross-linked matrices. FIG. 21G displays acomparison of Van-PAMAM with and without 0.1 M ascorbic acid as a function of voltage.

[00219] FIG. 21H displays the shear modulus as a function of shear strain, where all formulations display pseudoelastic behavior. In conclusion, by adding 0.1 M Ascorbic acid to 20 molar% van-PAMAM, shelf- stability is maintained and cross-linking kinetics can be vastly improved. The mechanical properties of 20 molar% van-PAMAM obtained approach that observed for 20 molar% DBA-PAMAM. No water electrolysis or gas evolution was observed, giving it an advantage over diazirine based electrocuring.

[00220] Example 10: Summary, Commercial and Potential Applications [00221] In various examples, the present adhesive composition may include (i) a macromolecule (e.g. a branched or unbranched polymer), wherein the macromolecule is grafted with a electron donating moiety (e.g. at least 10 such moieties) which forms a cross-linking group, that upon 1 or 2-electron oxidation per precursor, turns into a cross-linking functional group. The macromolecule may be further grafted with electron accepting moieties (e.g. at least 10 such moieties), that allow a 1 or 2-electron reduction per functional group. The macromolecule may contain nucleophilic groups (e.g. amino groups on the branched or unbranched polymer) that spontaneously react with the cross-linking functional groups, and optionally (ii) additives such as 1,2- aliphatic diols, catechol, tannic acid, 1,2-furan-diols, ascorbic acid, vicinal diols, and poly saccharide-diols .

[00222] The present adhesive composition possesses shelf-stability under non-aqueous, anaerobic conditions, and upon aqueous reconstitution, it is able to spontaneously cross link after a lag-time of 30 to 300 seconds or spontaneously cross-links in less than 30 seconds after exposure to v oltage or current.

[00223] In various examples, the macromolecule may not contain an aldehyde, acrylate, diazo, diazirine, genipin, and/or a cyan group. The macromolecule may be selected from the group consisting of an amino group functionalized synthetic polymer, branched polysaccharide, a polyamino acid, and a combination thereof. In various examples, the macromolecule may be selected from the group consisting of polyamidoamine, polyethylenimine, Boltom polyesters, polycaprolactone, chitosan, and combinations thereof. For example, the macromolecule may be or may consist of a polyamidoamine. The macromolecule may be or may contain a synthetic macromer and a conductive macromer. The term “macromer” herein refers to a macromolecule formed when one or more terminal groups of dendrimers or polymers are linked with each other. An example of a macromer may be a cluster of polymers covalently linked with one another through such terminal groups.

[00224] In various examples, the electron donating moiety may include a 1,2-aryl-diol (e.g. catechol). In various examples, the amount of catechol grafted on the macromolecule is in the range of about 1% (w/w) to about 50% (w/w). In various examples, the amount of catechol grafted on the macromolecule is in the range of about 10 molar% to about 50 molar% of the nucleophilic functional groups on the macromolecule.

[00225] In various examples, the cross-linking functional group derived from the electron donating moiety may be quinone, 1,2-cyclo-diketone, aliphatic-ketone, or a combination thereof.

[00226] In various examples, the electron donating moiety may be conjugated to the macromolecule via an electron accepting moiety that is derived from the nucleophilic functional groups present on the macromolecule. The electron accepting moiety may include or may consist of Schiff-bases, imines (e.g. a,b-unsaturated imines), oxime, ferrocenes, and/or sacrificial anodes. The sacrificial anode refers to any other moiety that may act as an electron donor. The nucleophilic functional groups may be selected from the group consisting of an amino group, a thiol group, hydroxyl group, and combinations thereof. The nucleophilic functional groups may include or may consist of an amino group.

[00227] The present adhesive composition may have a surface electrical resistance ranging from 0.1 to 1 xlO⁸ ohm/mm.

[00228] In various examples, a method of producing the present adhesive composition is provided. The method may include providing an adhesive composition as described above, and providing a rehydration mixture that includes 30-90 w/v% water, mixing the adhesive composition and the rehydration mixture, and activating a lag-time of the adhesive composition, wherein after the lag-time, self-curing of the adhesive composition gets activated, and/or providing a semiconductor substrate and/or conductive electrode and activating the adhesive composition after application of voltage or current gradient.

[00229] In various examples, the step of providing the adhesive composition may include dissolving the adhesive composition in an aqueous solution. The step of providing the adhesive composition may include adding an aqueous solution to an organic solution including the adhesive composition. The concentration of the adhesive composition in the aqueous solution may be in the range of about 10% w/v to about 50% w/v. The step of providing the adhesive composition may include applying the adhesive composition on a wet substrate to form a coating.

[00230] In various examples, a kit for adhering biological tissues may have been described. The kit may include an adhesive composition as described above, and an isotonic aqueous reconstitution solution.

[00231] In various examples, uses of the present bioadhesive composition are described. For example, disclosed is a method of adhering biological tissues, the method includes applying the present adhesive composition and covalently cross- linking the cross-linking functional groups on a first biological tissue to form a coating, contacting a second biological tissue with the resultant coating, and applying pressure to one or both of the first biological tissue and the second biological tissue to adhere the first biological tissue to the second biological tissue. Use of the present adhesive composition may include application as a wet substrate adhesive and/or sealant for medical and veterinary applications, supplementing or replacing sutures or staples in internal surgical procedures, intestinal anastomosis, vascular anastomosis, tissue repair, and/or tissue implantation.

[00232] While the present disclosure has been particularly shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as defined by the appended claims. The scope of the present disclosure is thus indicated by the appended claims and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced.

Claims

1. An adhesive composition comprising: organic macromolecules each comprising nucleophilic functional groups, wherein each of the organic macromolecules is a linear macromolecule or a branched macromolecule, wherein each of the nucleophilic functional groups comprises an amino group, a thiol group, a hydroxyl group, or a carboxyl group; and one or more electron donating moieties, wherein each of the nucleophilic functional groups grafted with the one or more electron donating moieties is converted to an electron accepting moiety, and each of the nucleophilic functional groups not grafted with the one or more electron donating moieties remains as the nucleophilic functional group, wherein the one or more electron donating moieties oxidizes into a diketone when the adhesive composition is subject to a stimulus, and wherein the diketone forms one or more cross-linkages with the nucleophilic functional groups.

2. The adhesive composition of claim 1, wherein the organic macromolecules comprise a polymer or a dendrimer.

3. The adhesive composition of claim 1 or 2, wherein the organic macromolecules comprise polyamidoamine, polyethylenimine, polyester, polycaprolactone, chitosan, or a mixture thereof.

4. The adhesive composition of any one of claims 1 to 3, wherein the one or more electron donating moieties comprise a 1,2-aryl-diol, a 1,4-aryl-diol, or a derivative thereof.

5. The adhesive composition of any one of claims 1 to 4, wherein the electron accepting moiety comprises an imine, an alkene, an oxadiazole, a tetrazinealkyne, a fluoren-9-one, a thioazole, or a malononitrile.

6. The adhesive composition of any one of claims 1 to 5, wherein 10 molar% to 50 molar% of the nucleophilic functional groups are grafted with the one or more electron donating moieties, and 50 molar % to 90 molar% of the nucleophilic functional groups remain as the nucleophilic functional group.

7. The adhesive composition of any one of claims 1 to 6, wherein the diketone comprises quinone, para-quinone, 1,2-cyclo-diketone, or aliphatic-ketone.

8. The adhesive composition of any one of claims 1 to 7, wherein the stimulus comprises water containing oxygen, an oxidant, an electrical voltage, or a combination thereof.

9. The adhesive composition of any one of claims 1 to 8, further comprising an additive comprising 1,2-aliphatic diol, catechol, tannic acid, 1,2-furan-diol, ascorbic acid, vicinal diol, polysaccharide-diol, or a mixture thereof.

10. The adhesive composition of any one of claims 1 to 9, wherein: the one or more cross-linkages are formed after a lag-time of 300 seconds or more where the stimulus is water containing oxygen; or the one or more cross-linkages are formed in 300 seconds or less where the stimulus is (i) electrical voltage or (ii) water containing oxygen and electrical voltage.

11. The adhesive composition of claim 10, wherein the electrical voltage ranges from 1 to 10 V.

12. The adhesive composition of any one of claims 1 to 11 for use as an adhesive to adhere biological tissues, or as a sealant in medical and/or veterinary applications comprising intestinal anastomosis, vascular anastomosis, tissue repair, and/or tissue implantation.

13. A method of producing an adhesive composition, wherein the adhesive composition comprises organic macromolecules each comprising nucleophilic functional groups, wherein each of the organic macromolecules is a linear macromolecule or a branched macromolecule, wherein each of the nucleophilic functional groups comprises an amino group, a thiol group, a hydroxyl group, or a carboxyl group; and one or more electron donating moieties, wherein each of the nucleophilic functional groups grafted with the one or more electron donating moieties is converted to an electron accepting moiety, and each of the nucleophilic functional groups not grafted with the one or more electron donating moieties remains as the nucleophilic functional group, wherein the one or more electron donating moieties oxidizes into a diketone when the adhesive composition is subject to a stimulus, and wherein the diketone forms one or more cross-linkages with the nucleophilic functional groups, wherein the method comprises: providing the organic macromolecules comprising the nucleophilic functional groups; and mixing the organic macromolecules with a precursor of the one or more electron donating moieties.

14. The method of claim 13, wherein providing the organic macromolecules comprising the nucleophilic functional groups comprises providing a stock solution comprising the organic macromolecules in a first anhydrous organic solvent, and removing dissolved oxygen from the stock solution.

15. The method of claim 14, wherein mixing the organic macromolecules with the precursor comprises dissolving the precursor in a second anhydrous organic solvent to obtain a precursor solution and adding the stock solution having oxygen removed therefrom to the precursor solution under an inert environment.

16. The method of claim 14 or 15, wherein the first anhydrous organic solvent and the second anhydrous organic solvent comprise an anhydrous alcohol.

17. The method of any one of claims 13 to 16, wherein mixing the organic macromolecules with the precursor comprises mixing the precursor in an amount ranging from 10 molar % to 50 molar % of the nucleophilic functional groups.

18. The method of any one of claims 13 to 17, wherein the precursor comprises 3,4- dihydroxybenzaldehyde, 2,3-dihydroxybenzaldehyde, 2,5-dihydroxybenzaldehyde, or a derivative thereof.

19. The method of any one of claims 13 to 18, wherein the precursor comprises 2- ethoxybenzo [d] [ 1 ,3] dioxole-5-carbaldehyde, 2,2-dimethylbenzo [d] [ 1 ,3 ] dioxole-5- carbaldehyde, 2,2-diphenylbenzo[d][l,3]dioxole-5-carbaldehyde, spiro [benzo[d] [1,3] dioxole-2, 1 '-cyclohexane] -5-carbaldehyde, vanillin, isovanillin, piperonal, 1 ,3-benzodioxole-5-carboxaldehyde, 2-bromo- 1 ,3-benzodioxole-5- carboxaldehyde, 6-methyl- 1 ,3-benzodioxole-5-carboxaldehyde, 1 ,3-benzodioxole-5- carboxaldehyde, 2,2-dichloro- 1 ,3-benzodioxole-5-carboxaldehyde, 7-methyl- 1,3- benzodioxole-5-carboxaldehyde, 2-nitro-l,3-benzodioxole-5-carboxaldehyde, 4- hydroxy-l,3-benzodioxole-5-carboxaldehyde, 7-(methylthio)-l,3-benzodioxole-5- carboxaldehyde, l,3-benzodioxole-4,6-dicarboxaldehyde, 6-chloro-l,3-benzodioxole- 5-carboxaldehyde, 5-formyl-methyl ester-l,3-benzodioxole-2-carboxylic acid, 6- formyl- 1 , 3 -benzodioxole-5 - sulfonic acid, 7 -chloro- 1 , 3 -benzodioxole- 5 - carboxaldehyde, 4-methoxy-2-oxo-l,3-benzodioxole-5-carboxaldehyde, 6-nitro-l,3- benzodioxole-5-carboxaldehyde, naphtho [ 1 ,2-d] - 1 ,3 -dioxole-5-carboxaldehyde, 7 - (trifluoromethyl)-l,3-benzodioxole-5-carboxaldehyde, 6-(2-thienyl)-l,3- benzodioxole-5-carboxaldehyde, 2-alkoxy-l,3-benzodioxole-5-carboxaldehyde, 2,2- dialkyl-l,3-benzodioxole-5-carboxaldehyde, spiro [1,3 -benzodioxole-2,1 - cycloalkane] -5-carboxaldehyde, 4-hydroxy-3-((trimethylsilyl)oxy)benzaldehyde, 4- hydroxy-3-((triethylsilyl)oxy)benzaldehyde, 3-((tert-butyldimethylsilyl)oxy)-4- hydroxybenzaldehyde, a catechol variant represented by the formula: wherein Ri is fluorine, chlorine, bromine, -OCH3, -NH2, -NO2, or -CF3_, or a mixture thereof.

20. The method of any one of claims 13 to 19, further comprising adding an anhydrous ether after mixing the organic macromolecules with the precursor to precipitate the adhesive composition; washing the adhesive composition which precipitated with the anhydrous ether; and drying the adhesive composition under vacuum.

21. A method of adhering or sealing biological tissues, the method comprising: applying an adhesive composition to one or more biological tissues, wherein the adhesive composition comprises organic macromolecules each comprising nucleophilic functional groups, wherein each of the organic macromolecules is a linear macromolecule or a branched macromolecule, wherein each of the nucleophilic functional groups comprises an amino group, a thiol group, a hydroxyl group, or a carboxyl group; and one or more electron donating moieties, wherein each of the nucleophilic functional groups grafted with the one or more electron donating moieties is converted to an electron accepting moiety, and each of the nucleophilic functional groups not grafted with the one or more electron donating moieties remains as the nucleophilic functional group, wherein the one or more electron donating moieties oxidizes into a diketone when the adhesive composition is subject to a stimulus, and wherein the diketone forms one or more cross-linkages with the nucleophilic functional groups; subjecting the adhesive composition to the stimulus; and contacting the biological tissues together to adhere the biological tissues.

22. The method of claim 21, wherein subjecting the adhesive composition to the stimulus comprises exposing the adhesive composition to water containing oxygen or an oxidant, applying an electrical voltage to the adhesive composition, or a combination thereof.

23. The method of claim 21 or 22, wherein subjecting the adhesive composition to the stimulus comprises exposing the adhesive composition to water containing oxygen to have the one or more cross-linkages formed after a lag-time of 300 seconds or more; or applying an electrical voltage to the adhesive composition to have the one or more cross-linkages formed in 300 seconds or less; or exposing the adhesive composition to water containing oxygen and applying an electrical voltage to the adhesive composition to have the one or more cross-linkages formed in 300 seconds or less.

24. The method of any one of claims 21 to 23, further comprising applying pressure to the biological tissues.

25. The use of an adhesive composition in the preparation of (i) an adhesive; or (ii) a kit comprising the adhesive composition for intestinal anastomosis, vascular anastomosis, tissue repair, and/or tissue implantation, wherein the adhesive composition comprises: organic macromolecules each comprising nucleophilic functional groups, wherein each of the organic macromolecules is a linear macromolecule or a branched macromolecule, wherein each of the nucleophilic functional groups comprises an amino group, a thiol group, a hydroxyl group, or a carboxyl group; and one or more electron donating moieties, wherein each of the nucleophilic functional groups grafted with the one or more electron donating moieties is converted to an electron accepting moiety, and each of the nucleophilic functional groups not grafted with the one or more electron donating moieties remains as the nucleophilic functional group, wherein the one or more electron donating moieties oxidizes into a diketone when the adhesive composition is subject to a stimulus, and wherein the diketone forms one or more cross-linkages with the nucleophilic functional groups.

26. The use of claim 25, wherein the kit comprises an isotonic aqueous solution which serves as the stimulus.