WO2023014293A2 - A ph-responsive composition for facilitating delivery of a cargo and a method of encapsulating cargo with a ph-responsive composition - Google Patents

Abstract

There is provided a pH-responsive composition for facilitating delivery of a cargo and a method of encapsulating cargo with a pH-responsive composition, the composition comprising, a copolymer comprising, a backbone moiety; and a plurality of side moieties grafted to the backbone moiety, the plurality of side moieties comprising at least one adenine-rich (A-rich) oligonucleotide and at least one cytosine-rich (C-rich) oligonucleotide; wherein the copolymer is configured to form a hydrogel when exposed to an environment with a pH falling in a first range of pH values, thereby facilitating encapsulation of the cargo within the hydrogel; and wherein the copolymer is configured to form a solution when exposed to an environment with a pH falling in a second range of pH values different from the first range of pH values, thereby facilitating release of the cargo.

Description

A PH-RESPONSIVE COMPOSITION FOR FACILITATING DELIVERY OF A CARGO AND A METHOD OF ENCAPSULATING CARGO WITH A PH- RESPONSIVE COMPOSITION

TECHNICAL FIELD

The present disclosure relates broadly to a pH-responsive composition for facilitating delivery of a cargo and a method of encapsulating cargo with a pH- responsive composition.

BACKGROUND

Diabetes refers to a condition of high blood glucose level, which increases the risk of dangerous complications, including stroke, heart disease and even death. According to the International Diabetes Federation (IDF), more than 400 million people are living with diabetes, and this will rise to 700 million by 2045, which is a heavy burden in global healthcare.

Currently, insulin, a hormone produced by the pancreas, is the most effective drug for diabetes treatment through stimulating the glucose transporter type 4 (GLUT-4) to transport glucose into cells, which eventually reduces the concentration of glucose in blood plasma. Due either to a lack of sufficient insulin or no insulin produced in the body, diabetes depends on the external supply of insulin, which could be achieved by subcutaneous injection (SC), oral delivery or inhalational methods.

SC has high bioavailability but needle insertion is associated with relatively low compliance and other side-effects, such as weight gain, skin infection, hypoglycemia and oedema. On the other hand, oral delivery presents relatively high patient compliance, but has low bioavailability (typically less than 1 %). For decades, oral insulin delivery has been the Holy Grail in protein drug development due to two challenging barriers in the gastrointestinal (Gl) tract. Firstly, the hostile acidic environment and digestive enzymes in the stomach cause the enzymatic hydrolysis of insulin. Secondly, the mucus layer and the underlying tight epithelial cell layers impede the transport of insulin into the blood stream for systemic circulation.

Efforts have been devoted to developing novel carriers to improve insulin loading contents, controlled release in a pre-designed manner, protection and transporting efficiency when crossing the Gl tract, and bioavailability. To realize these goals, micro/nano-carriers, metal-organic frameworks, ionic liquids, polymer hydrogels, nanoparticles, liposomes, microneedle injector, etc. were designed and loaded with insulin for oral delivery. However, these systems suffer from drawbacks, such as low compatibility, low loading capacity and incomplete insulin release. Synthetic beta cells mimicking natural beta cells to secrete insulin were designed, but the requirement for insulin replenishing impeded further application. Very recently, an intelligent self-orienting millimeter-scale applicator (SOMA) was fabricated for insulin delivery. In this design, the insulin tip was linked to a compressed spring, which was triggered by sucrose dissolution in the Gl tract, followed by insulin tip penetration. This concept combined the bioavailability of injection with a higher potential compliance; however, it also raised a concern of Gl perforation, which might induce long-term health problem.

Thus, there is a need for a pH-responsive composition for facilitating delivery of a cargo and a method of encapsulating cargo with a pH-responsive composition, which seek to address or at least ameliorate one of the above problems.

SUMMARY

In one aspect, there is provided a pH-responsive composition for facilitating delivery of a cargo, the composition comprising, a copolymer comprising, a backbone moiety; and a plurality of side moieties grafted to the backbone moiety, the plurality of side moieties comprising at least one adenine- rich (A-rich) oligonucleotide and at least one cytosine-rich (C-rich) oligonucleotide; wherein the copolymer is configured to form a hydrogel when exposed to an environment with a pH falling in a first range of pH values, thereby facilitating encapsulation of the cargo within the hydrogel; and wherein the copolymer is configured to form a solution when exposed to an environment with a pH falling in a second range of pH values different from the first range of pH values, thereby facilitating release of the cargo.

In one embodiment, the first range of pH values is from 1 .2 to 6.0; and the second range of pH values is 7.0 or more.

In one embodiment, the A-rich oligonucleotide is configured to form a A- motif duplex structure when exposed to an environment with a pH of less than 3.0, said A-motif duplex structure configured to act as a crosslinking unit to facilitate formation of the hydrogel.

In one embodiment, the A-rich oligonucleotide is configured to form a single-stranded structure when exposed to an environment with a pH of more than 4.0, said single-stranded structure configured to facilitate formation of the solution.

In one embodiment, the C-rich oligonucleotide is configured to form a i- motif quadruplex structure when exposed to an environment with a pH falling in the range of from 4.0 to 6.0; said i-motif quadruplex structure configured to act as a crosslinking unit to facilitate formation of the hydrogel.

In one embodiment, the C-rich oligonucleotide is configured to form a half i-motif strand structure when exposed to an environmental pH of 7.0 or more, said half i-motif strand structure configured to facilitate formation of the solution.

In one embodiment, the A-rich oligonucleotides and C-rich oligonucleotides are modified oligonucleotides having one or more functional groups configured to facilitate grafting to the backbone moiety, said one or more functional groups selected from the group consisting of acrydite, amine, dibenzoazacyclooctyne (DBCO)/alkyne, azide, thiol, and carboxyl group.

In one embodiment, the modified A-rich oligonucleotide comprises the general sequence: 5’-X- AAA AAA AAA (AAA)_n -3’, wherein n > 1 ; and the modified C-rich oligonucleotide comprises the general sequence: 5’-X- (W3 C4)2 -3’, wherein W = adenine (A) or thymine (T); and wherein X is a functional group selected from the group consisting of acrydite, amine, DBCO/alkyne, azide, thiol, carboxyl group, and combinations thereof. In one embodiment, the ratio of side moieties to ungrafted monomer units of the backbone moiety is from 1 :10 to 1 :100.

In one embodiment, the backbone moiety comprises a polymer having one or more functional groups configured to facilitate grafting of the plurality of side moieties.

In one embodiment, the backbone moiety comprises polyacrylamide.

In one embodiment, the composition further comprises a cargo, optionally wherein the cargo comprises a therapeutic agent.

In one embodiment, the cargo is insulin and the insulin encapsulated in the hydrogel is protected from hydrolysis by one or more enzymes.

In one embodiment, the composition further comprises a cell-penetrating peptide (CPP) configured to facilitate transportation of the cargo across a barrier comprising one or more layers of cells.

In one embodiment, the composition further comprises an enzyme inhibitor configured to suppress enzymatic hydrolysis of the cargo.

In one embodiment, the composition is configured to release the cargo at a controlled rate of release when in an in-vivo tissue environment.

In one embodiment, the polymer composition is in the form of an oral formulation for oral administration to a subject in need thereof.

In one aspect, there is provided a method of encapsulating cargo with a pH-responsive composition, the method comprising, providing a copolymer comprising a backbone moiety and a plurality of side moieties grafted to the backbone moiety, the plurality of side moieties comprising at least one adenine- rich (A-rich) oligonucleotide and at least one cytosine-rich (C-rich) oligonucleotide; providing the cargo; and forming a hydrogel by exposing the copolymer to a pH falling in a first range of pH values, thereby encapsulating the cargo within the hydrogel, wherein the hydrogel forms a solution when exposed to an environment with a pH falling in a second range of pH values different from the first range of pH values, thereby facilitating release of the cargo.

In one aspect, there is provided a method of forming a pH-responsive copolymer, the method comprising, providing a backbone moiety; and grafting a plurality of side moieties to the backbone moiety, the plurality of side moieties comprising at least one adenine-rich (A-rich) oligonucleotide and at least one cytosine-rich (C-rich) oligonucleotide; such that the copolymer is configured to form a hydrogel when exposed to an environment with a pH falling in a first range of pH values; and configured to form a solution when exposed to an environment with a pH falling in a second range of pH values different from the first range of pH values.

In one aspect, there is provided a pH-responsive composition as disclosed herein, for use as a medicament.

In one aspect, there is provided a pH-responsive composition as disclosed herein, for use in the treatment of diabetes, wherein the composition comprises insulin as the cargo.

In one aspect, there is provided a use of a pH-responsive composition as disclosed herein in the manufacture of a medicament for the treatment of diabetes, wherein the composition comprises insulin as the cargo.

In one aspect, there is provided a method of treating diabetes in a subject in need thereof, the method comprising, orally administering a therapeutically effective amount of a pH-responsive composition as disclosed herein, wherein the composition comprises insulin as the cargo.

DEFINITIONS

The term “biocompatible” as used herein is to be interpreted broadly to refer to the ability of a material to perform its intended function without inducing significant inflammatory response, immunogenicity, or cytotoxicity to native cells, tissues, or organs.

The term “biodegradable” as used herein is to be interpreted broadly to refer to substances that, when introduced into cells, are broken down by the cellular machinery or by hydrolysis into components that the cells can either reuse or dispose of without significant toxic effect on the cells. The components preferably do not induce inflammation or other adverse effects in vivo.

The term “encapsulate” as used herein is to be interpreted broadly to define accommodating a substance within a structure such that the substance is substantially isolated from an external surrounding environment as long as the structure is substantially intact.

The term “hydrogel” as used herein refers to a three-dimensional, hydrophilic or amphiphilic polymeric network capable of taking up a sufficient quantity of water. The polymeric network may be composed of copolymers, and may be substantially insoluble due to the presence of covalent chemical or physical (ionic, hydrophobic interactions, entanglements) crosslinks. The crosslinks provide the network structure and physical integrity. In the case of DNA copolymer-based hydrogel, the hybridization interaction (s) (e.g., Watson-Crick basepairing, non-Watson-Crick basepairing such as Hoogsteen binding, etc.) between DNA strands crosslinks copolymer chains to form three-dimensional networks, termed DNA hydrogel. The hybridization or dehybridization between DNA strands may be achieved by external triggers, such as ions, pH, light, enzymes, fuel strands, etc., resulting in the assembly or disassembly of the DNA hydrogel. The hybridization or dehybridization processes between DNA strands are reversible.

The term “nucleotide”, “nucleic acid”, and “nucleic acid molecule”, are used interchangeably, and can also include plurals of each respectively depending on the context in which the terms are utilized.

The term “polynucleotide” refers to a polymeric form of nucleotides, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.

The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. The term “therapeutic agent” as used herein is to be interpreted broadly to describe a substance which exhibits a therapeutic activity when administered to a subject.

The term “diagnostic agent” as used herein is to be interpreted broadly to describe a substance which can be used either alone or in combination with other agents and/or suitable equipment to practice a method, process, or procedure that provides diagnostic or prognostic information.

The term “insulin” as used herein refers to human or non-human, recombinant, purified or synthetic insulin or insulin analogues, whether isolated from a natural source or made by genetically altered microorganisms, unless otherwise specified.

The term “subject” or “patient” as used herein refers to a mammal. Mammals may include but are not limited to rats, mice, cats, dogs, horses, sheep, cattle, cows, pigs, rabbits, non-human primates, and humans.

The term “treating” as used herein includes prophylaxis of a specific disease, disorder or condition, or alleviation of symptoms associated with a specific disease, disorder or condition and/or preventing or eliminating said symptoms. For example, as used herein the term “treating diabetes” may include altering glucose blood levels in the direction of normal levels and may include increasing or decreasing blood glucose levels depending on a given situation.

The term “therapeutically effective amount” or “effective amount” as used herein means the amount of a compound, agent, composition, construct that when administered to a subject for treatment is sufficient, in combination with another agent, or alone in one or more doses or administrations, to effect such treatment for the disease, disorder or condition. The “therapeutically effective amount” may vary depending on the compound, agent, composition, construct, the defect or disease, disorder or condition to be treated, and its severity and the age, weight, etc., of the subject to be treated.

The term “micro” as used herein is to be interpreted broadly to include dimensions from about 1 micron to about 1000 microns.

The term “nano” as used herein is to be interpreted broadly to include dimensions less than about 1000 nm. The term “particle” as used herein broadly refers to a discrete entity or a discrete body. The particle described herein can include an organic, an inorganic or a biological particle. The particle used described herein may also be a macroparticle that is formed by an aggregate of a plurality of sub-particles or a fragment of a small object. The particle of the present disclosure may be spherical, substantially spherical, or non-spherical, such as irregularly shaped particles or ellipsoidally shaped particles. The term “size” when used to refer to the particle broadly refers to the largest dimension of the particle. For example, when the particle is substantially spherical, the term “size” can refer to the diameter of the particle; or when the particle is substantially non-spherical, the term “size” can refer to the largest length of the particle.

The terms “coupled” or “connected” as used in this description are intended to cover both directly connected or connected through one or more intermediate means, unless otherwise stated.

The term “associated with”, used herein when referring to two elements refers to a broad relationship between the two elements. The relationship includes, but is not limited to a physical, a chemical or a biological relationship. For example, when element A is associated with element B, elements A and B may be directly or indirectly attached to each other or element A may contain element B or vice versa.

The term “adjacent” used herein when referring to two elements refers to one element being in close proximity to another element and may be but is not limited to the elements contacting each other or may further include the elements being separated by one or more further elements disposed therebetween.

The term “and/or”, e.g., “X and/or Y” is understood to mean either “X and Y” or “X or Y” and should be taken to provide explicit support for both meanings or for either meaning.

Further, in the description herein, the word “substantially” whenever used is understood to include, but not restricted to, “entirely” or “completely” and the like. In addition, terms such as “comprising”, “comprise”, and the like whenever used, are intended to be non-restricting descriptive language in that they broadly include elements/components recited after such terms, in addition to other components not explicitly recited. For example, when “comprising” is used, reference to a “one” feature is also intended to be a reference to “at least one” of that feature. Terms such as “consisting”, “consist”, and the like, may in the appropriate context, be considered as a subset of terms such as “comprising”, “comprise”, and the like. Therefore, in embodiments disclosed herein using the terms such as “comprising”, “comprise”, and the like, it will be appreciated that these embodiments provide teaching for corresponding embodiments using terms such as “consisting”, “consist”, and the like. Further, terms such as “about”, “approximately” and the like whenever used, typically means a reasonable variation, for example a variation of +/- 5% of the disclosed value, or a variance of 4% of the disclosed value, or a variance of 3% of the disclosed value, a variance of 2% of the disclosed value or a variance of 1% of the disclosed value.

Furthermore, in the description herein, certain values may be disclosed in a range. The values showing the end points of a range are intended to illustrate a preferred range. Whenever a range has been described, it is intended that the range covers and teaches all possible sub-ranges as well as individual numerical values within that range. That is, the end points of a range should not be interpreted as inflexible limitations. For example, a description of a range of 1% to 5% is intended to have specifically disclosed sub-ranges 1 % to 2%, 1 % to 3%, 1 % to 4%, 2% to 3% etc., as well as individually, values within that range such as 1 %, 2%, 3%, 4% and 5%. It is to be appreciated that the individual numerical values within the range also include integers, fractions and decimals. Furthermore, whenever a range has been described, it is also intended that the range covers and teaches values of up to 2 additional decimal places or significant figures (where appropriate) from the shown numerical end points. For example, a description of a range of 1 % to 5% is intended to have specifically disclosed the ranges 1.00% to 5.00% and also 1.0% to 5.0% and all their intermediate values (such as 1.01 %, 1.02% ... 4.98%, 4.99%, 5.00% and 1.1 %, 1 .2% ... 4.8%, 4.9%, 5.0% etc.,) spanning the ranges. The intention of the above specific disclosure is applicable to any depth/breadth of a range. When describing that a value falls in, within or on a given range of values, it will also be appreciated that the value may possibly also include the end points/values of the ranges i.e., the end points/values of the ranges are inclusive.

Additionally, when describing some embodiments, the disclosure may have disclosed a method and/or process as a particular sequence of steps. However, unless otherwise required, it will be appreciated that the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may be possible. The particular order of the steps disclosed herein should not be construed as undue limitations. Unless otherwise required, a method and/or process disclosed herein should not be limited to the steps being carried out in the order written. The sequence of steps may be varied and still remain within the scope of the disclosure.

Furthermore, it will be appreciated that while the present disclosure provides embodiments having one or more of the features/characteristics discussed herein, one or more of these features/characteristics may also be disclaimed in other alternative embodiments and the present disclosure provides support for such disclaimers and these associated alternative embodiments.

DESCRIPTION OF EMBODIMENTS

Exemplary, non-limiting embodiments of a pH-responsive composition for facilitating delivery of a cargo and a method of encapsulating cargo with a pH- responsive composition are disclosed hereinafter.

In various embodiments, there is provided a pH-responsive composition for facilitating delivery of a cargo, the composition comprising a copolymer, said copolymer comprising a backbone moiety and a plurality of side moieties grafted to the backbone moiety, the plurality of side moieties comprising at least one adenine-rich (A-rich) oligonucleotide and at least one cytosine-rich (C-rich) oligonucleotide; wherein the copolymer is configured to form a hydrogel when exposed to an environment with a pH falling in a first range of pH values, thereby facilitating encapsulation of the cargo within the hydrogel; and wherein the copolymer is configured to form a solution when exposed to an environment with a pH falling in a second range of pH values different from the first range of pH values, thereby facilitating release of the cargo. In various embodiments, there is provided a pH-responsive copolymer for facilitating delivery of a cargo. Advantageously, in various embodiments, the pH-responsive composition is capable of facilitating release of the cargo in a location of the gastrointestinal (Gl) tract. In one example, the pH-responsive composition is capable of facilitating release of the cargo in the small intestine.

In various embodiments, the first range of pH values is different from the second range of pH values. For example, there is substantially no overlap between the first and second ranges of pH values. The first and second ranges of pH values may be mutually exclusive and do not encompass each other. In other words, the second range of pH values may be completely outside of, and does not overlap with, the first range of pH values. In various embodiments, the second range of pH values are higher than the first range of pH values. In one example, the first range of pH values may be an acidic range of pH values and the second range of pH values may be a neutral to alkaline range of pH values. In another example, the first range of pH values may be a neutral to alkaline range of pH values and the second range of pH values may be an acidic range of pH values. The acidic range of pH values may range from about 0 to about 6.9. The neutral to alkaline range of pH values may range from about 7.0 to about 14.0. In various embodiments, the first range of pH values may be from about 1 .2 to about 6.0 and the second range of pH values may be about 7.0 or more, or between about 7.0 to about 7.4. In various embodiments, the copolymer is configured to form a hydrogel when exposed to an environment with an acidic pH falling in the range of from about 1 .2 to about 6.0; and is configured to form a solution when exposed to an environment with a physiological pH of about 7.4.

In various embodiments, the backbone moiety is a hydrogel precursor that is crosslinkable to form a hydrogel. In various embodiments, the backbone moiety is a polymer. Polymers suitable for forming a hydrogel may include naturally occurring materials, synthetic and modified materials. Non-limiting examples of naturally occurring materials may include albumin, alginate, collagen, chitosan, gelatin, hyaluronic acid, polysaccharides such as dextran, and cellulose. Nonlimiting examples of synthetic and modified materials include poly(l-glutamic acid), poly(lactic acid), polyethylene oxide), polyethylene glycol), poly(vinyl alcohol) (PVA), poly(dimethylaminoethylmethacrylate) (PDMAEMA), poly(N-vinyl pyrrolidone) (PNVP), poly(hydroxyalkyl methacrylate) such as poly(hydroxyethyl methacrylate) (PHEMA), polyethylene glycol) monomethyl ether (PEGME), poloxamers, poly(N-isopropylacrylamide), poly(acrylamide) (PAAm), zwitteronic polymers including polyampholytes and polybetaines. In various embodiments, the backbone moiety comprises materials that are biocompatible and/or biodegradable. In one embodiment, the backbone moiety comprises polyacrylamide.

In various embodiments, the backbone moiety, e.g., polymer, comprises one or more functional groups configured to facilitate grafting/attachment of the plurality of side moieties to the backbone moiety. The backbone moiety may be modified to introduce the one or more functional groups configured to facilitate grafting/attachment of the plurality of side moieties. The functional groups may include but are not limited to a hydroxyl group, a thiol group, an amino group, a carbonyl group, a carboxylic acid group, a A/-hydroxysuccinimide (NHS) group, an azide group and an alkyne group (such as di benzoazacyclooctyne (DBCO)), suitable for reacting and bonding to an oligonucleotide or a modified oligonucleotide.

In various embodiments, the plurality of side moieties comprises at least one A-rich oligonucleotide and at least one C-rich oligonucleotide. Advantageously, in various embodiments, oligonucleotides such as DNA oligonucleotides are biocompatible, biodegradable and designable, which renders them suitable for in vitro and in vivo applications such as gene therapy, immunotherapy and drug delivery etc. Even more advantageously, in various embodiments, oligonucleotides such as DNA oligonucleotides may be configured to switch nucleic acid structures, e.g., metal-ion-bridged duplexes, G- quadruplexes, i-motif, triplex structures, or programmed double-stranded hybrids of oligonucleotides.

In various embodiments, the plurality of side moieties comprises a plurality of A-rich oligonucleotides. In various embodiments, an A-rich oligonucleotide is characterised by the presence of one or more adenosine-rich stretches or stretches rich in adenosine derivatives. In various embodiments, the A-rich oligonucleotide may comprise a nucleic acid sequence having at least one stretch of two or more adenine-bearing nucleotides. The stretch of adenine-bearing nucleotides may comprise at least two, at least three, at least four, at least five, at least ten, at least fifteen, or at least twenty consecutive adenine-bearing nucleotides. The A-rich oligonucleotide may comprise non-adenine base(s) in between stretches of adenine-bearing nucleotides. In various embodiments, the A-rich oligonucleotide may comprise a nucleic acid sequence where at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 95%, or about 100% of the nucleotides are adenine-bearing nucleotides.

In various embodiments, the A-rich oligonucleotide is configured to undergo a change in structure in response to a change in pH. The pH value at which the A-rich oligonucleotide undergoes a change in structure may be controlled by appropriately defining the length of the stretch of adenine-bearing nucleotides. The A-rich oligonucleotide may be configured to adopt a first structural conformation of an A-motif duplex structure when exposed to an environment with a pH falling in a third range of pH values. The A-rich oligonucleotide may be configured to adopt a second structural conformation of a single-stranded structure when exposed to an environment with a pH falling in a fourth range of pH values. The third range of pH values may be from about 1 .2 to about 3.0. The third range of pH may have start and end points selected from the following group of numbers: 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0. The fourth range of pH values may be from about 4.0 to about 7.4. The fourth range of pH may have start and end points selected from the following group of numbers: 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, and 7.4.

In various embodiments, the A-rich oligonucleotides are configured to form two parallel stranded duplexes in which the adenine-bearing nucleotides form a double helix (i.e., the A-motif duplex structure) when exposed to an environment with a pH of less than about 3.0. The A-motif duplex structure is configured to act as a crosslinking unit to facilitate formation of the hydrogel in various embodiments. That is, in various embodiments, the hybridization of the A-rich oligonucleotides to form the A-motif duplex structures crosslinks the copolymer chains, thereby facilitating formation of a three-dimensional network of hydrogel. In various embodiments, the A-rich oligonucleotide is configured to form a singlestranded structure when exposed to an environment with a pH of more than about 4.0. In various embodiments, the single-stranded structure is configured to facilitate formation of the solution. That is, in various embodiments, the dehybridization of the A-rich oligonucleotides to form the single-stranded structures reverses the crosslinking between the copolymer chains, thereby facilitating formation of the solution. In various embodiments, in a pH range of from about 3.0 to about 4.0, the A-motif duplex structure forms at a pH lower than about 3.5 (the p _a of adenine is 3.5) due to the protonation of adenines. In various embodiments, deprotonation happens at a pH above about 3.5, resulting in the formation of single stranded structures/sequences.

In various embodiments, the plurality of side moieties comprises a plurality of C-rich oligonucleotides. In various embodiments, a C-rich oligonucleotide is characterised by the presence of one or more cytosine-rich stretches or stretches rich in cytosine derivatives. In various embodiments, the C-rich oligonucleotide comprises a nucleic acid having at least one stretch of two or more cytosine- bearing nucleotides. The stretch of cytosine-bearing nucleotides may comprise at least two, at least three, at least four, at least five, at least ten, at least fifteen, or at least twenty consecutive cytosine-bearing nucleotides. The C-rich oligonucleotide may comprise non-cytosine base(s) in between stretches of cytosine-bearing nucleotides. In various embodiments, the C-rich oligonucleotide may comprise a nucleic acid sequence where at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 95%, or about 100% of the nucleotides are cytosine-bearing nucleotides.

In various embodiments, the C-rich oligonucleotide is configured to undergo a change in structure in response to a change in pH. The pH value at which the C-rich oligonucleotide undergoes a change in structure may be controlled by appropriately defining the length of the stretch of cytosine-bearing nucleotides. The C-rich oligonucleotide may be configured to adopt a first structural conformation of a quadruplex structure when exposed to an environment with a pH falling in a fifth range of pH values. The C-rich oligonucleotide may be configured to adopt a second structural conformation of a half i-motif strand structure when exposed to an environment with a pH falling in a sixth range of pH values. The fifth range of pH values may be from about 4.0 to about 6.0. The fifth range of pH may have start and end points selected from the following group of numbers: 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, and 6.0. The sixth range of pH values may be from about 7.0 to about 7.4. The sixth range of pH may have start and end points selected from the following group of numbers: 7.0, 7.1 , 7.2, 7.3, and 7.4.

In various embodiments, the C-rich oligonucleotides are configured to form a i-motif quadruplex structure when exposed to an environment with a pH of from about 4.0 to about 6.0. The i-motif corresponds to a “double duplex” in which two parallel duplexes are oriented in a head to tail orientation through the intercalation of hemi-protonated cytosine-cytosine (C-C+) base pairs. In various embodiments, the i-motif quadruplex structure is configured to act as a crosslinking unit to facilitate formation of the hydrogel. That is, in various embodiments, the hybridization of the C-rich oligonucleotides to form the i-motif quadruplex structures crosslinks the copolymer chains, thereby facilitating formation of a three-dimensional network of hydrogel. In various embodiments, the C-rich oligonucleotide is configured to form a half i-motif strand structure when exposed to an environmental pH of about 7.0 or more. In various embodiments, the half i- motif strand structure is configured to facilitate formation of the solution. That is, in various embodiments, the dehybridization of the C-rich oligonucleotides to form the half i-motif strand structures reverses the crosslinking between the copolymer chains, thereby facilitating formation of the solution. In various embodiments, in a pH range of from about 1 .2 to about 7.2, the formation of i-motif structures is minimal at pH of about 1 .2 and about 2.0, and the formation of i-motif structures by partial C-rich oligonucleotides is present at pH of about 3.0 and about 6.0. The p a value of cytosine is about 6.5, which means that deprotonation of cytosine happens at pH above about 6.5 resulting in i-motif disassembly into single stranded sequences.

In various embodiments, the side moieties e.g., A-rich and C-rich oligonucleotides may be modified to introduce one or more functional groups configured to facilitate grafting/attachment to the backbone moiety. The functional groups may include but are not limited to acrydite, amine, DBCO/alkyne, azide, thiol, and carboxyl group. In various embodiments, the A-rich oligonucleotide and C-rich oligonucleotide may be modified at its 5’-end and/or 3’-end.

In various embodiments, the modified A-rich oligonucleotides have the following general sequence:

SEQ ID NO. 1 : 5’-X- AAA AAA AAA (AAA)_n -3’, wherein n > 1 , and wherein X is a functional group selected from the group consisting of acrydite, amine, DBCO/alkyne, azide, thiol, carboxyl group, and combinations thereof. In various embodiments, there is no upper limit to the value of n in the general sequence of the A-rich oligonucleotide, however, it will be appreciated that the longer the sequence, the lower the yield of synthesis of the A-rich oligonucleotide.

In various embodiments, the modified A-rich oligonucleotides comprise one or more sequences selected from the group consisting of:

SEQ ID NO. 2: 5’-X- AAA AAA AAA AAA -3’;

SEQ ID NO. 3: 5’-X- AAA AAA AAA AAA AAA -3’;

SEQ ID NO. 4: 5’-X- AAA AAA AAA AAA AAA AAA -3’;

SEQ ID NO. 5: 5’-X- AAA AAA AAA AAA AAA AAA AAA -3’; and

SEQ ID NO. 6: 5’-X- AAA AAA AAA AAA AAA AAA AAA AAA -3’, wherein X is a functional group selected from the group consisting of acrydite, amine, DBCO/alkyne, azide, thiol, carboxyl group, and combinations thereof.

In various embodiments, the modified C-rich oligonucleotides have the following general sequence:

SEQ ID NO. 7: 5’-X- (W3 C4)2 -3’, wherein W = adenine (A) or thymine (T), and wherein X is a functional group selected from the group consisting of acrydite, amine, DBCO/alkyne, azide, thiol, carboxyl group, and combinations thereof. In various embodiments, the modified C-rich oligonucleotides comprise one or more sequences selected from the group consisting of:

SEQ ID NO. 8: 5’-X- TAA CCC CTA ACC CC -3’;

SEQ ID NO. 9: 5’-X- AAT CCC CAA TCC CC -3’;

SEQ ID NO. 10: 5’-X- ATA CCC CAT ACC CC -3’;

SEQ ID NO. 1 1 : 5’-X- TAT CCC CTA TCC CC -3’;

SEQ ID NO. 12: 5’-X- TAT CCC CAA TCC CC -3’;

SEQ ID NO. 13: 5’-X- ATA CCC CAA ACC CC -3’; and

SEQ ID NO. 14: 5’-X- AAA CCC CAA ACC CC -3’, wherein X is a functional group selected from the group consisting of acrydite, amine, DBCO/alkyne, azide, thiol, carboxyl group, and combinations thereof.

In various embodiments, the A-rich oligonucleotide and C-rich oligonucleotide are acrydite-modified oligonucleotides. In various embodiments, acrydite-modification is an attachment chemistry based on an acrylic phosphoramidite that can be added to oligonucleotides as a 5'-modification. In one example, the acrydite-modified A-rich oligonucleotide comprises SEQ ID NO. 5 (i.e., 5’-acry- AAA AAA AAA AAA AAA AAA AAA -3’) and the acrydite-modified C-rich oligonucleotide comprises SEQ ID NO. 14 (i.e., 5’-acry- AAA CCC CAA ACC CC - 3’).

In various embodiments, the crosslinking units of A-motif and i-motif structures may facilitate crosslinking between different strands of copolymers or between different locations within the same strand of copolymer. In various embodiments, A-motif and i-motif structures remain stable at body temperature, e.g., of about 37°C. In various embodiments, the change in state of the copolymer is reversible, i.e., from the hydrogel state to the solution state, and from the solution state to the hydrogel state. For example, the copolymer may change from solution state to hydrogel state when exposed to an environment with a pH falling in the first range of pH values and change back from hydrogel state to solution state when exposed to an environment with a pH falling in the second range of pH values.

In various embodiments, the ratio of side moieties/ DNA subunits to ungrafted monomer units of the backbone moiety may be from about 1 :10 to about 1 :100, from about 1 :15 to about 1 :95, from about 1 :20 to about 1 :90, from about 1 :25 to about 1 :85, from about 1 :30 to about 1 :80, from about 1 :35 to about 1 :75, from about 1 :40 to about 1 :70, from about 1 :45 to about 1 :65, from about 1 :50 to about 1 :60, or from about 1 :50 to about 1 :55. In various embodiments, the ratio of side moieties/ DNA subunits to ungrafted monomer units of the backbone moiety may be determined spectroscopically. In one embodiment, the ratio of DNA subunits to ungrafted monomer units e.g., unsubstituted acrylamide units is about 1 :53. It will be appreciated that the ratio of adenine and cytosine rich oligonucleotides in the copolymer may be adjusted to change the pH- responsive properties, e.g., the pH at which the structural conformation changes.

In various embodiments, the pH-responsive composition further comprises a cargo. The cargo may be a substance that exhibits a specific activity such as, for example, a physicochemical activity, surface-modification activity (e.g., solubility, hydrophobicity, hydrophilicity, lubrication, bioavailability and protection), therapeutic activity (by means of a therapeutic agent), targeting activity (by means of a targeting agent), labeling activity (by means of a labeling agent, e.g., for imaging and diagnostic purposes). In various embodiments, the cargo may comprise one or more substances that include but are not limited to organic compounds, inorganic compounds, small molecules, macromolecules, biomolecules, biomacromolecules, cells, and the like. In various embodiments, the cargo may comprise a therapeutic agent, a diagnostic agent, a labelling agent, a targeting agent, or a combination thereof.

In various embodiments, the cargo may comprise a therapeutic agent. The therapeutic agent may include but is not limited to a drug, a chemotherapeutic agent, an amino acid, a peptide, a polypeptide, a protein, an antigen, an antibody, a nucleic acid, a nucleic acid construct, a gene, a cardiovascular agent, a cofactor, a cytokine, a growth factor, a heparin, a hormone, a ligand, a lipid, a metabolite, a phospholipid, a prostaglandin, a receptor agonist, a receptor antagonist, a toxin, a vitamin, an agonist, an analgesic, an antagonist, an antibiotic, an antidepressant, an anti-diabetic agent, an anti-histamine, an antihypertensive agent, an anti-inflammatory drug, an anti-metabolic agent, an antimicrobial agent, an antioxidant, an anti-platelet agent, an antiproliferative agent, an anti-psychotic agent, an antisense, an anti-thrombogenic agent, an enzyme, an epitope, an immunoglobulin, an inhibitor, an oligonucleotide and any combination thereof. In various embodiments, the cargo encapsulated in the hydrogel is protected from hydrolysis by one or more enzymes, e.g., digestive enzymes in the Gl tract of a subject.

In various embodiments, the cargo comprises insulin. The insulin may be human insulin, recombinant human insulin, insulin from a non-human animal source (e.g., bovine, porcine) or any other insulin, including insulin analogs. Insulin formulations may include mixtures of different insulins to the extent they are compatible with a subject to be administered with said insulin formulations. In various embodiments, the insulin encapsulated in the hydrogel is protected from hydrolysis by one or more enzymes, e.g., digestive enzymes in the Gl tract of a subject.

In various embodiments, the cargo may comprise a labeling agent. The labelling agent may be a substance which produces a detectable signal which can be traced to a specific location of the substance in a subject's body. The labelling agent may include but is not limited to a fluorescent agent, a radioactive agent, a magnetic agent, a chromophore, a phosphorescent agent and a heavy metal cluster. In various embodiments, the cargo may comprise a targeting agent. The targeting agent may be a substance which has a specific affinity to a desired bodily site (e.g., particular organ, cells and/or tissues). The targeting agents may include but is not limited to porphyrins, hormones, antibodies and fragments thereof, and receptor ligands which bind to receptors that are expressed at specific bodily sites.

In various embodiments, the cargo is not chemically attached to the copolymer regardless of its state (i.e., hydrogel or solution state). In various embodiments, the cargo may be physically encapsulated within the three- dimensional polymeric network formed by the copolymer in the hydrogel state. When the pH-responsive composition is exposed to a pH falling within the second range of pH values (e.g., physiological pH), the cargo is released in a controlled manner as the three-dimensional polymeric network of the hydrogel changes to the solution state. In other embodiments, the cargo may be temporarily attached/bound to the hydrogel formed by the copolymer via one or more functional groups and released when the hydrogel changes to a solution.

In various embodiments, the pH-responsive composition may further comprise one or more penetration enhancing agents e.g., cell-penetrating peptide (CPP). In various embodiments, the one or more penetration enhancing agents is encapsulated in the hydrogel together with the cargo. The penetration enhancing agent may be configured to facilitate transportation of the cargo across a barrier comprising one or more layers of cells by promoting the transfer of a molecule from the extracellular space to the intracellular space. Non-limiting examples of the cell-penetrating peptide include peptides composed of six (R(6)), eight (R(8)) and ten (R(10)) residues of arginine. In one example, the cellpenetrating peptide is D-R8. Other CPPs including human immunodeficiency virus (HIV)1 Tat-(48-60), an amphipathic penetratin derived from Antennapedia Homeoprotein, R9F2, decalysine, transportan, etc. may also facilitate penetration.

In various embodiments, the pH-responsive composition may further comprise one or more enzyme inhibitors configured to suppress enzymatic hydrolysis of the cargo. In various embodiments, the one or more enzyme inhibitors may be inhibitors of digestive enzymes produced by the gastrointestinal tract of a subject. In various embodiments, the one or more enzyme inhibitor is encapsulated in the hydrogel together with the cargo. Non-limiting examples of the enzyme inhibitor include protease inhibitors such as trypsin inhibitor, which may be used to suppress the enzymatic hydrolysis of insulin in the small intestine. Other examples include Bowman-Birk inhibitors and Kunitz-type inhibitors, etc.

In various embodiments, there is provided a pharmaceutical composition comprising a pH-responsive composition for facilitating delivery of one or more therapeutic agents, e.g., insulin. The pH-responsive composition comprises a copolymer, said copolymer comprising a backbone moiety and a plurality of side moieties grafted to the backbone moiety, the plurality of side moieties comprising at least one adenine-rich (A-rich) oligonucleotide and at least one cytosine-rich (C-rich) oligonucleotide; wherein the copolymer is configured to form a hydrogel when exposed to an environment with a first range of pH values, thereby facilitating encapsulation of the one or more therapeutic agents within the hydrogel; and wherein the copolymer is configured to form a solution when exposed to an environment with a second range of pH values different from the first range of pH values, thereby facilitating release of the one or more therapeutic agents.

In various embodiments, the first range of pH values may correspond to the pH condition of an intermediate location, e.g., stomach with an acidic range of pH condition, that the pharmaceutical composition has to pass through in order to arrive at a target location where the therapeutic agent is intended to be released. In various embodiments, the second range of pH values may correspond to the pH condition of the target location, e.g., small intestine with a substantially neutral range of pH condition, where the therapeutic agent is intended to be released. When passing through the intermediate location, the pH condition of the intermediate location corresponding to the first range of pH values causes the copolymer to form a hydrogel, thereby encapsulating and protecting the therapeutic agent from degradation, e.g., hydrolysis by one or more enzymes in the Gl tract. When passing through the target location, the pH condition of the target location corresponding to the second range of pH values causes the copolymer to form a solution, thereby releasing the therapeutic agent at the target location.

In various embodiments, the pH-responsive composition is in the form of an oral formulation for oral administration to a subject in need thereof. In various embodiments, the oral formulation may be in the form of a tablet, capsule, solution, suspension, syrup, or emulsion. In various embodiments, the oral formulation may be in the form of a liquid or hydrogel contained within a capsule that is to be swallowed by the subject. In various embodiments, the oral formulation may be in the form of a liquid, e.g., solution, suspension, syrup, or emulsion, to be drunk by the subject. In various embodiments, the oral formulation may be in the form of a dry powder that can be reconstituted with water or another suitable liquid before use.

In various embodiments, the oral formulation may be a pharamaceutical composition. Advantageously, oral administration may improve patient compliance as compared to other routes of administration. For example, in the case of diabetes, subcutaneous insulin injection currently serves as the most effective strategy for diabetes treatment. While subcutaneous insulin injection may provide high bioavailability of insulin, however, only about 20% of the insulin reaches the hepatic portal circulation. Further, low patient compliance and sideeffects, e.g., pain and skin infection, hypoglycemia, weight gain, edema, lipodystrophy remain as acute drawbacks associated with subcutaneous insulin injection. Various embodiments of the pH-responsive composition disclosed herein may be used to deliver insulin to a patient via the oral route to improve patient compliance, avoid pain and skin infection, improve hepatic portal levels of insulin and curtail peripheral hyperinsulinemia, minimise immune response and weight gain, which are some of the drawbacks associated with subcutaneous injection.

In various embodiments, the pH-responsive composition may overcome the challenges of oral administration due to the hostile environment in gastrointestinal gut (e.g., susceptibility to gastrointestinal enzymes and low pH etc.) and the underlying tight junctions of epithelial cells impeding the effectiveness of oral administration (e.g., low bioavailability of drugs, difficulty in delivering drugs with high molecular weight (about 6 kDa) low diffusion across mucin barrier in the Gl tract etc.). In various embodiments, the pH-responsive composition may be used as a DNA hydrogel delivery system. The pH-responsive DNA copolymer may be configured to form a hydrogel at acidic pH (1 .2 to 6.0) and transit to solution state at physiological pH (7.2) to deliver cargo orally into the gastrointestinal gut of the subject. In various embodiments, the DNA hydrogel delivery system is based on the pH-triggered formation of A-motif (pH 1 .2 to 3.0) and i-motif (pH 4.0 to 6.0) structures and separation into A (adenine)-rich and C (cytosine)-rich sequences at physiological pH. For example, the cargo may be a protein drug such as insulin. Insulin may be encapsulated in the DNA hydrogel and protected from hydrolysis by digestive enzymes in the stomach with acidic pH. The physiological pH in the small intestine may then trigger the release of insulin from the DNA hydrogel. In various embodiments, the pH-responsive composition is configured to release the cargo at a controlled rate of release in an environment with a pH falling within the second range of pH values. In various embodiments, the pH- responsive composition is configured to release the cargo, e.g., one or more therapeutic agents, at a controlled rate of release when in an in-vivo tissue environment. The one or more therapeutically active agents may be brought into contact with the copolymer at a pH falling in the second range of the pH values (e.g., a substantially neutral pH value) where the copolymer is in the solution state and then incorporated into the hydrogel by lowering the pH to within the first range of pH values (e.g., an acidic pH value) to initiate formation of the hydrogel. The pH-responsive composition may be administered to a subject and configured to release the one or more therapeutically active agents when exposed to an environment with a pH falling within the second range of pH values (e.g., a physiological pH value of 7.4).

In various embodiments, the pH-responsive composition is configured to release the cargo over a period of up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, up to 14 hours, up to 15 hours, up to 16 hours, up to 17 hours, up to 18 hours, up to 19 hours, up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours, or up to 24 hours. The release of cargo may be at a controlled rate of release.

In various embodiments, there is provided a pH-responsive composition for use as a medicament. In various embodiments, there is provided a pH- responsive composition for use in the treatment of diabetes, wherein the composition comprises insulin as the cargo. In various embodiments, there is provided a use of a pH-responsive composition in the manufacture of a medicament for the treatment of diabetes, wherein the composition comprises insulin as the cargo. In various embodiments, there is provided a method for treating or alleviating one or more symptoms of a disease, disorder or condition, the method comprising orally administering a pH-responsive composition to a subject in need thereof. In various embodiments, there is provided a method of treating diabetes in a subject in need thereof, the method comprising, orally administering a therapeutically effective amount of a pH-responsive composition, wherein the composition comprises insulin as the cargo.

In various embodiments, the medicament I pharmaceutical composition comprising the pH-responsive composition may further comprise one or more pharmaceutically acceptable excipients. Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, diluent, disintegrating agent, binding agent, mucomembranous adhesion agent, filler, extender, antitack agent, antioxidant, buffer agent, complexing agent, carrier, coloring agent, flavoring agent, coating materials, plasticizer, organic solvent, stabilizing agent, antiseptic, lubricant, solubilizing agent, fluidizer, chelating agent, and combination thereof.

In various embodiments, there is provided a DNA copolymer comprising A-rich and C-rich oligonucleotides acting as DNA tethers grafted to a backbone polymer. The DNA copolymer may form hydrogel at a pH falling within the first range of pH values (e.g., from about 1.2 to about 6.0), crosslinked by either A- motif or i-motif structures, and change to solution state at a pH falling within the second range of pH values (e.g., physiological pH of 7.4). In various embodiments, the DNA hydrogel may be used as a carrier for cargo, e.g., therapeutic agent, such that the cargo is encapsulated within the DNA hydrogel and administered via oral delivery. In various embodiments, the DNA sequences in the DNA copolymer are biocompatible and biodegradable, thereby rendering them safe for administration into a subject’s body. It will be appreciated that the pH-responsive properties of the DNA hydrogel could be finely tuned by using different oligo structures or compositions, such as A-motif, i-motif and triplex structures. Advantageously, the DNA copolymer may be used for drug delivery to a desired region of the body such as a particular location of the Gl tract (e.g., stomach, duodenum, or small intestine) by rational design of the pH-responsive DNA hydrogels.

In various embodiments, the DNA copolymer may be used to deliver insulin to a target location in the small intestine via oral administration. The encapsulated insulin may be protected by the DNA hydrogel in a hostile environment, e.g., in a simulated environment mimicking the stomach by using artificial gastric fluid (AGF). In various embodiments, the DNA copolymer remain as a hydrogel at the pH value of 5.0, e.g., in a simulated environment mimicking the duodenum by using articifial duodenal fluid (ADF). In various embodiments, in a simulated environment mimicking the small intestine by using artificial intestinal fluid (AIF), the DNA hydrogel dissociated into solution state due to the separation of A-motif and i-motif structures into A-rich and C-rich random coils at physiological pH, accompanied by the full release of insulin. Advantageously, the DNA copolymer may be superior to other oral insulin delivery systems based on the “shrinking-swollen” transitions of polymer hydrogel.

In various embodiments, there is provided a method of encapsulating cargo with a pH-responsive composition, the method comprising, providing a copolymer comprising a backbone moiety and a plurality of side moieties grafted to the backbone moiety, the plurality of side moieties comprising at least one adenine-rich (A-rich) oligonucleotide and at least one cytosine-rich (C-rich) oligonucleotide; providing the cargo; and forming a hydrogel by exposing the copolymer to a pH falling in a first range of pH values, thereby encapsulating the cargo within the hydrogel, wherein the hydrogel forms a solution when exposed to an environment with a pH falling in a second range of pH values different from the first range of pH values, thereby facilitating release of the cargo.

In various embodiments, there is provided a method of forming a pH- responsive copolymer, the method comprising, providing a backbone moiety; and grafting a plurality of side moieties to the backbone moiety, the plurality of side moieties comprising at least one adenine-rich (A-rich) oligonucleotide and at least one cytosine-rich (C-rich) oligonucleotide; such that the copolymer is configured to form a hydrogel when exposed to an environment with a pH falling in a first range of pH values; and configured to form a solution when exposed to an environment with a pH falling in a second range of pH values different from the first range of pH values.

In various embodiments, the A-rich and C-rich oligonucleotides may be copolymerized with monomers of the backbone moiety using suitable processes depending on the chemical properties of the backbone moiety. For example, where the backbone moiety is polyacrylamide, the copolymer may be formed by free radical polymerization in the presence of an initiator and accelerator. It will be appreciated that other different methods may be employed to prepare DNA hydrogels with adenine and cytosine rich oligonucleotides. In one example, amine modified A-rich and C-rich oligonucleotides may be grafted to carboxymethylcellulose (CMC) backbones via amide bonds. In another example, azide modified A-rich and C-rich oligonucleotides may be conjugated to alkyne modified polymer backbones via click reaction.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a schematic illustration showing preparation and pH-triggered insulin release of a pH-responsive DNA hydrogel. At acidic pH (1.2 to 6.0), pH- responsive adenine (A)-rich and cytosine (C)-rich sequences form A-motif and i- motif crosslinked DNA copolymer hydrogel, while at physiological pH (7.2), dissociation of the two DNA configurations generates a solution state. As a demonstration of its suitability as an oral drug delivery system, insulin is encapsulated by the DNA hydrogel in artificial gastric fluid and released in artificial intestinal fluid.

FIG. 2A is a schematic illustration of an adenine to adenine (A:A) base pair and electrostatic interaction in the base pair. In this configuration, A-motif is stabilized by the hydrogen bonds (reverse Hoogsteen interaction) between protonated adenines and electrostatic attraction between the positively charged protons at the N(1 ) position of adenines and the negatively charged phosphate groups.

FIG. 2B is a schematic illustration of a hemi-protonated cytosine to cytosine C:C⁺ base pair. At slightly acidic pH (4.0-6.0), sequences rich in cytosine (pKa=6.5), form an i-motif structure consisting of two parallel-stranded C:C+ hemi-protonated base-paired duplexes that are intercalated in an antiparallel manner.

FIG. 3A is a graph showing circular dichroism (CD) spectra of A-rich sequences under various pH values. FIG. 3B is a graph showing CD spectra of C-rich sequences under various pH values.

FIG. 3C is a graph showing CD spectra of DNA copolymers comprising A- rich and C-rich sequences under pH values of 1 .2, 5.0 and 7.2.

FIG. 4A is a graph showing CD spectra of A-rich sequences at room temperature and 37°C.

FIG. 4B is a graph showing CD spectra of C-rich sequences at room temperature and 37°C

FIG. 5A is a graph showing absorption spectra of different concentrations of polyacrylamide in the presence of a constant concentration of acrydite modified nucleic acids, corresponding to 0.5 x 10’⁶ M: (a) 0, (b) 0.5 x 10’⁵ M, (c) 1 x 10’⁵ M, (d) 2.5 x 10’⁵ M, (e) 5 x 10’⁵ M, (f) 7.5 x 10’⁵ M, (g) 1.0 x 10’⁴ M, (h) 1 .25 x 10’ ⁴ M, (i) 1.5 x 10’⁴ M, (j) 1 .75 x 10’⁴ M, and (k) 2.0 x 10’⁴ M.

FIG. 5B is a graph showing a calibration curve corresponding to an absorbance ratio AA2oonm/AA26Onm as a function of acrylamide/DNA ratio for determination of loading of acrylamide monomer by nucleic acids tethers of the polymer chains.

FIG. 6A is a photograph (top) and scanning electron microscope (SEM) micrograph (bottom) of a DNA hydrogel at pH 1 .2. (Scale bar: 20 pm).

FIG. 6B is a photograph (top) and SEM image (bottom) of a DNA hydrogel at pH 5.0. (Scale bar: 20 pm).

FIG. 6C is a collage of two photographs showing pH-triggered dissociation of a DNA hydrogel (top) into solution (bottom).

FIG. 7 is a graph showing rheology studies of a DNA hydrogel under pH 1.2, 5.0, and 7.2.

FIG. 8A is a graph showing absorption spectra of a DNA hydrogel at pH

7.2 and 37°C.

FIG. 8B is a graph showing dissociation profiles of DNA hydrogels at pH

7.2 and 37°C.

FIG. 9A is a graph showing absorption spectra of a DNA hydrogel at pH

1.2 and 37°C. FIG. 9B is a graph showing absorption spectra of the DNA hydrogel at pH

7.2 and 37°C.

FIG. 9C is a graph showing dissociation profiles of DNA hydrogels at 37°C at pH 1 .2, followed by pH 7.2.

FIG. 10A is a graph showing absorption spectra of a DNA hydrogel at pH

1.2 and 37°C.

FIG. 10B is a graph showing absorption spectra of the DNA hydrogel at pH 5.0 and 37°C.

FIG. 10C is a graph showing absorption spectra of the DNA hydrogel at pH 7.2 and 37°C.

FIG. 10D is a graph showing dissociation profiles of DNA hydrogels at 37°C at pH 1.2, subsequently pH 5.0, and then pH 7.2.

FIG. 1 1 A is a graph showing absorption spectra of FITC-insulin under pH

1.2.

FIG. 1 1 B is a graph showing a concentration calibration curve of FITC- insulin under pH 1 .2.

FIG. 1 1 C is a graph showing absorption spectra of FITC-insulin under pH 5.0.

FIG. 1 1 D is a graph showing a concentration calibration curve of FITC- insulin under pH 5.0.

FIG. 1 1 E is a graph showing absorption spectra of FITC-insulin under pH

7.2.

FIG. 11 F is a graph showing a concentration calibration curve of FITC- insulin under pH 7.2.

FIG. 12A is a graph showing a release profile of FITC-insulin from DNA hydrogel in artificial intestinal fluid (AIF) (pH 7.2, 37°C).

FIG. 12B is a graph showing a release profile of FITC-insulin from DNA hydrogel in artificial gastric fluid (AGF) (pH 1.2, 37°C), followed by AIF (pH 7.2, 37°C).

FIG. 12C is a graph showing a release profile of FITC-insulin from DNA hydrogel in AGF (pH 1.2, 37°C), subsequently in artificial duodenal fluid (ADF) (pH 5.0, 37°C), and then in AIF (pH 7.2, 37°C). FIG. 12D is a graph showing results of an ELISA assay of insulin released from DNA hydrogel under various pH values. Standard insulin solution was used as a positive control.

FIG. 13A is a graph showing release profiles of FITC-insulin from DNA hydrogels under a pH value of 7.2.

FIG. 13B is a graph showing release profiles of FITC-insulin from DNA hydrogels as pH changes from 1 .2 to 7.2 over time.

FIG. 13C is a graph showing release profiles of FITC-insulin from DNA hydrogels as pH changes from 1 .2 to 5.0 and then to 7.2 over time.

FIG. 14 is a graph showing CD spectra of insulin under various pH values, and the corresponding calculated <|)208/<|>223 ratio.

FIG. 15 is a graph showing cell viability at different concentrations of DNA copolymers.

FIG. 16 is a graph showing hemolysis activity at different concentrations of DNA copolymers.

FIG. 17A is a graph showing profiles of blood glucose levels versus time in diabetic rats following the administration of (■) free insulin solution (30 lll/kg), (•) SC injection (3 lU/kg), and (A) insulin@DNA hydrogel (30 lU/kg). Data are presented as the average ± standard deviation (n = 5). *P < 0.05 as compared to the group treated with oral administration of free insulin solution.

FIG. 17B is a graph showing changes of serum insulin levels versus time in diabetic rats after administration of (■) free insulin solution (30 lll/kg), (•) SC injection (3 lll/kg), and (A) insulin@DNA hydrogel (30 lll/kg).

FIG. 17C is a collage of micrographs of Hematoxylin and Eosin (H&E) stained stomach, intestine, liver and kidney sections of diabetic rats treated with insulin@DNA hydrogel. Rats without any treatment served as the control group. (Scale bar: 100 pm).

EXAMPLES

Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. It should be appreciated that other modifications related to structural and chemical changes may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments. The example embodiments should not be construed as limiting the scope of the disclosure.

MATERIALS AND METHODS

Materials used for experiments

Human recombinant insulin, FITC-labeled insulin, trypsin inhibitor, magnesium chloride, ammonium persulfate (APS), N,N,N',N'- tetramethylethylenediamine (TEMED), acrylamide solution (40%) and other chemicals were purchased from Sigma-Aldrich, unless otherwise noted. Insulin ELISA kit was purchased from R&D Systems, Inc. Cell-penetrating peptide, D- R8, was purchased from GL Biochem (Shanghai) Ltd. Desalted 5' end acrydite modified nucleic acid strands were purchased from Integrated DNA Technologies Inc. (Coralville, IA). Ultrapure water purified by a Milli-Q system was used to prepare all of the solutions. The sequences used in the study are:

(1 ) 5’-acry- AAA AAA AAA AAA AAA AAA AAA -3’

(2) 5'-acry- AAA CCC CAA ACC CC -3’

Circular Dichroism (CD) Spectra of A-motif and i-motif

CD spectra of A-motif and i-motif under different pH values (1.2, 2.0, 3.0, 4.0, 5.0, 6.0, 7.2) and temperatures (room temperature, 37°C) were recorded in phosphate-buffered saline (PBS) buffer by using JASCO J -815 CD Spectrometer.

Synthesis of Acrylamide/Acrydite-Nucleic Acid Copolymers

A 400-pL buffer solution (PBS, 10 mM, MgCl2, 10 mM, pH 7.2) that included 2% acrylamide and the acrydite-modified DNA strand (1 ) (0.8 mM) and (2) (0.8 mM) was prepared. Nitrogen was bubbled through the solution. Twentyeight microliters of a 0.2-mL aqueous solution that included APS (20 mg) and TEMED (10 pL) were added to the mixture. The resulting solution was allowed to polymerize at room temperature for 5 min before the solution was further polymerized at 4°C for 12 h. The resulting copolymer was purified from unreacted monomer units, salts and initiators, using an Amicon (Millipore) spin filter unit (MWCO 10 kDa). The purified copolymer was removed from the filter and dried under a gentle nitrogen flow. The dried copolymer was redispersed in PBS buffer for further experiments, unless otherwise noted. The concentrations of the copolymer and the ratio of the acrylamide/acrydite-nucleic acid units were determined spectroscopically.

In Vitro Cytotoxicity Evaluation and Hemolysis Assay

Cytotoxicity of DNA copolymers on cells were measured using CellTiter- Glo® 2D Cell Viability Assay (Promega). Primary human dermal fibroblasts (HDF, ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum (Life Technologies) and 1 % penicillin-streptomycin (Life Technologies) at 37°C in a 5% CO2 incubator. HDFs were allowed to expand to 80-90% confluence before passaging with 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA, Life Technologies). Cells were seeded in 96-well plates at a density of ~ 10⁴ cells/well and cultured for 24 h. The culture medium was replaced with 100 pL of the prepared DNA copolymer solutions, and the plates were returned to the incubator and maintained in 5% CO2 at 37°C for 48 h. 100 pL CellTiter-Glo® Reagent was added to each well. The plates were incubated at room temperature for 10 min to stabilize luminescent signal, and luminescence was measured with a Cytation™ 5 plate reader (BioTek Instruments). Cell viability was expressed as the ratio of the number of viable cells with treatment to that without treatment. Experiments were conducted in triplicates, and consistent results were obtained.

Fresh mouse red blood cells (RBCs) were diluted with PBS buffer to give an RBC stock suspension (4 vol% blood cells). 100-pL aliquots of RBC suspension were mixed with 100 pL of DNA copolymer solutions. After 1 h of incubation at 37°C, the mixtures were centrifuged at 2000 rpm for 5 min. 100 pL of the supernatant were transferred to a 96-well plate. Hemolytic activity was determined as a function of hemoglobin release by measuring the absorbance of the supernatant at 576 nm using the microplate reader. Absorbance of red blood cells lysed with 0.5% Triton-X was taken as 100% hemolysis. A control solution that contained only PBS was used as a reference for 0% hemolysis. The hemolysis activity was calculated by the following equation: 100

Encapsulation of Insulin in the DNA Hydrogel

Human recombinant insulin was dispersed in 0.01 M HCI to a concentration of 25 mg/ml. A certain amount of recombinant insulin solution together with D-R8 (50 mg/ml) and trypsin inhibitor (7 mg/ml) were added to DNA copolymers to form insulin@DNA hydrogel composite. Loading capacity (LC) of insulin was calculated with the following equation. encapsulated insulin LC% = — . ; _£ - - - x 100% weight of copolymers

In Vitro Dissociation Profiles of DNA Hydrogels and Release Profiles of FITC-lnsulin from DNA Hydrogels

A 10-pL DNA copolymer solution (10 mM PBS, pH 7.2, containing 10 mM MgCh) was changed to a hydrogel of pH 1.2 or 5.0 by adding HCI (1.5% v/v, 1 pL) or acetic acid (1 % v/v, 1 pL), respectively. The DNA hydrogel was transferred to PBS buffer (pH 7.2, 300 pL) and incubated at 37°C with shaking (500 rpm). The dissociated DNA copolymer in solution was measured every 10 min using Agilent Cary 3500 UV-Vis spectrophotometer. The same procedure was adopted for the measurement of dissociation profiles of DNA hydrogel in PBS buffer with pH 1.2 or pH 5.0. For the sequential changing of the pH from 1.2 to 5.0 to 7.2, the pH 1 .2 PBS buffer (300 pL) was first carefully removed, leaving behind the DNA hydrogel, and replaced by pH 5.0 PBS buffer (300 pL), which was subsequently replaced by pH 7.2 PBS buffer (300 pL) after UV-Vis measurements.

A 2-pL FITC-insulin solution (5 mg/mL) was added to the DNA copolymer solution (10 pL). The mixture was changed to the hydrogel state of pH 1.2 by adding HCI (1 .5% v/v, 1 pL). The mixture was transferred to PBS buffer (pH 7.2, 300 pL), and incubated at 37°C with shaking (500 rpm). The FITC-insulin released in solution was measured every 10 min using Agilent Cary 3500 UV-Vis spectrophotometer. The same procedure was adopted for the measurement of FITC-insulin release profiles in PBS buffer with pH 1.2 or pH 5.0. For the sequential changing of the pH from 1 .2 to 5.0 to 7.2, the pH 1 .2 PBS buffer (300 pL) was first carefully removed, leaving behind the DNA hydrogel, and replaced by pH 5.0 PBS buffer (300 pL), which was subsequently replaced by pH 7.2 PBS buffer (300 pL) after UV-Vis measurements.

In Vivo Studies

Male Sprague-Dawley (SD) rats, aged 8 weeks (220-260 g), were obtained from Invivos Pte Ltd (Singapore). The rats were housed in individual cages with water ad libitum. The experiments on animals were conducted according to the Guidelines on the Care and Use of Animals for Scientific Purposes by National Advisory Committee for Laboratory Animal Research (Singapore, 2004). All animal procedures were approved by Institutional Animal Care and Use Committee, Biological Resource Center, Singapore (Approved IACUC Protocol No.: 181330).

Rats with diabetes were induced by a single intraperitoneal injection of freshly dissolved streptozotocin (STZ, Merck) (65 mg/kg, citrate buffer, 10 mM, pH 4.5) after 12 h of fasting. Diabetes was identified by polydipsia, polyuria and by measuring serum glucose concentration after 3 weeks of STZ injection. Rats with a blood glucose level above 17 mM were considered to be diabetic and were used in this study.

Oral administration of insulin@DNA hydrogel was performed on diabetic rats after 12 h of fasting. Water was allowed ad libitum. The diabetic rats were divided into 5 per group. The rats were intragastrically administered with free insulin solution (control, 30 lU/kg) or insulin@DNA hydrogel (30 lU/kg) by using an oral gavage needle, or subcutaneously injected with insulin (3 lU/kg). Blood samples were collected from the tail veins of the rats at different time internals after drug administration. Blood glucose level was monitored with Accu-Chek Performa test strips. Serum insulin levels were tested by using an insulin ELISA kit. Relative bioavailability (BA) of the insulin@DNA hydrogel after oral administration was calculated with the following equation,

where [AL/C]_Orai and [AL/C]sc are the area under the serum insulin concentration versus time curves for oral administration and subcutaneous injection of insulin, respectively, and dosesc and oseorai are the insulin doses administered by subcutaneous injection (3 lU/kg) and oral administration (30 lU/kg), respectively.

RESULTS AND DISCUSSION pH-Responsive DNA Hydrogel System

As the genetic information carrier and the translational code for protein synthesis, DNA oligo nucleotides are biocompatible, designable and biodegradable, which render them with merits for various in vivo and in vitro studies, such as gene therapy, immunotherapy, cancer drug delivery, etc. External stimuli, for example, metal ions, pH, light, temperature and fuel/anti-fuel strands, provide triggers to switch nucleic acid structures, e.g., metal-ion-bridged duplexes, G-quadruplexes, i-motifs, triplex structures, or programmed doublestranded hybrids of oligonucleotides, to the desired functions.

In this study, a pH-responsive DNA hydrogel system was used to encapsulate insulin and deliver it orally to treat diabetes. The DNA hydrogel was designed to possess high stability in hostile acidic stomach environment for insulin protection, and liberate insulin in small intestine by using pH as the trigger.

Double helices crosslinked by Watson-Crick interactions are relatively unstable under extremely acidic pH values, such as pH 1.0-3.0. The pH- responsive DNA hydrogel system contains A-rich sequences, which form doublestranded, parallel A-motif structures via reverse Hoogsteen base pairing under extremely acidic pH values (< 3.0). Separation of A-motif structures into singlestranded A-rich sequences takes place at pH value above 4.0. Furthermore, half i-motif strands (C-rich), which crosslink into i-motif quadruplex structures held together by hemi-protonated and intercalated cytosine base pairs (C:C⁺) through three hydrogen bonds under slightly acidic pH (4.0- 6.0), are also introduced to the pH-responsive DNA hydrogel system.

At neutral pH, the crosslinked i-motif units dissociate into C-rich random coils. Therefore, under acidic pH (1.2-6.0), the A-rich strands and half i-motif tethers grafted polyacrylamide copolymers form a hydrogel through the generation of A-motif (pH< 3.0) or i-motif (pH 4.0 to 6.0) bridging units, and the protein drug, insulin, is encapsulated in the hydrogel and protected from digestive enzymes in stomach.

At physiological pH, the DNA hydrogel turns to solution state due to dissociation of A-motif and i-motif crosslinking bridges into single-stranded A-rich and C-rich sequences, respectively, accompanied by the complete release of insulin in an environment with a substantially neutral pH of 7.2, e.g., small intestine (see FIG. 1 ), which is more advantageous than other insulin delivery systems based on shrinking-swollen transition of polymer hydrogels.

Furthermore, to facilitate the transport of insulin through epithelium layers for systematic circulation, a cell-penetrating peptide (CPP), D-R8, has also been co-encapsulated in the hydrogel along with trypsin inhibitor, which suppresses the enzymatic hydrolysis of insulin in the small intestine.

A-rich sequence is a tail component of mRNA in all eukaryotic cells and it plays a key role in the stability of mRNA and translation initiation. At neutral or alkaline pH, A-rich sequences exhibit single-stranded, right-handed structure stabilized by TT-TT stacking of adenines, while at acidic pH values (<3.0), the protonation of adenines (pK_a=3.5) changes the structure to parallel duplex A- motif. In this configuration, A-motif is stabilized by the hydrogen bonds (reverse Hoogsteen interaction) between protonated adenines and electrostatic attraction between the positively charged protons at the N(1 ) position of adenines and the negatively charged phosphate groups (see FIG. 2A). At slightly acidic pH (4.0- 6.0), the sequences rich in C (pK_a=6.5), form i-motif structure consisting of two parallel-stranded C:C⁺ hemi-protonated, base-paired duplexes that are intercalated in an antiparallel manner (see FIG. 2B). The i-motif structure is related to the oncogene and is recently detected in the nuclei of human cells. Besides the key regulatory roles in the human genome, i-motif may be used to construct pH-responsive DNA nanomachines, hydrogels, nanoparticles assembly, etc.

By rationally combining the pH-triggered generation/dissociation of A-motif and i-motif structures, a DNA hydrogel that is configured to be in a gel state at pH 1 .2-6.0 and to dissociate into solution state at pH 7.2 may be provided. Advantageously, the pH-responsive properties of the DNA hydrogel fulfill the requirements for oral insulin delivery, including the resistance to severe pH environment and enzyme degradation in stomach, fast release and permeability through epithelium layer in small intestine.

Characterization of A-motif and i-motif structures

In order to verify the configuration of A-motif and i-motif under various acidic pH values, the circular dichroism (CD) spectra of both structures were recorded.

At pH above 4.0, A-rich sequences showed strong positive maximum at 217 nm with a shoulder at 230 nm, a weak positive band at 270 nm and negative bands centered at 250 nm and 205 nm, which are characteristics of singlestranded A-rich sequences. At a pH range of 1 .2-3.0, an intense, positive band maximum at 264 nm with a shoulder at 272 nm and a negative band centered at 243 nm were observed, which indicated the parallel A-motif duplex structures (see FIG. 3A).

As for i-motif, at pH 4.0 and 5.0, a strong positive maximum at 286 nm and a negative band at 255 nm represent the characteristics of i-motif structure. At pH 3.0, both bands decreased in intensities, and they became negligible at pH 1 .2 and 2.0, indicating the destruction of i-motif structures. The maximum positive and negative bands blue shifted to 284 nm and 251 nm at pH 6.0, and 275 nm and 249 nm at pH 7.2, accompanied by the intense decrease in the magnitude (see FIG. 3B).

From the CD spectra of FIGs. 3A and 3B, it could be concluded that duplex A-motif forms at pH 1.2, 2.0, and 3.0, while i-motif bridging units function at pH 4.0, 5.0 and 6.0. Both structures dissociate at pH 7.2. These unique pH- responsive properties of A-motif and i-motif configurations serve as the basis for the design of DNA hydrogels for oral insulin delivery. After polymerization, the CD spectra of DNA copolymers were attained at pH 1 .2, 5.0, and 7.2 (see FIG. 3C). The characteristic peaks of the A-motif duplex and i-motif quadruplex in DNA copolymers were consistent with that measured independently (see FIG. 3A, 3B). The A-motif and i-motif structures are stable at 37°C (see FIG. 4A and 4B), which is practical for in vivo studies.

Synthesis of Acrylamide/Acrydite-Nucleic Acid Copolymers

After verifying the pH-responsive properties of A-motif and i-motif structures, a polyacrylamide copolymer, bearing A-rich and C-rich sequences as pH-triggered crosslinking/dissociation domains, was designed and prepared. Accordingly, acrydite-modified A-rich and C-rich sequences were copolymerized with acrylamide monomers by free radical polymerization, in the presence of an initiator and accelerator, to form the copolymer. The ratio of DNA subunits to unsubstituted acrylamide units was determined spectroscopically to be 1 :53 (for the determination of the loading, see FIGs. 5A and 5B).

The copolymer existed in a solution state at pH 7.2 due to no crosslinking between DNA tethers. Changing pH of the copolymer solution to 1.2 led to the formation of a hydrogel (see FIG. 6A, top), bridged by the parallel A-motif duplexes. Similarly, adjusting the pH of copolymer solution to 5.0 resulted in a hydrogel (see FIG. 6B, top), crosslinked by the i-motif tetrads. Scanning electron microscopy (SEM) images of both hydrogels showed porous crosslinked networks (see FIGs. 6A and 6B, bottom), consistent with the morphology of a hydrogel matrix. Subjecting both hydrogels to pH 7.2 buffer at 37°C led to dissociation of the hydrogels (see FIG. 6C), which was due to the separation of A-motif duplexes and i-motif structures to single stranded A-rich sequences and half i-motif tethers, respectively.

To gain physical insight into the DNA hydrogel and the solution state, rheological studies were conducted (see FIG. 7). At pH 1 .2, the DNA hydrogel showed a storage modulus G’ of 360 ± 5 Pa and a loss modulus G” of 2 Pa, indicating the formation of a hydrogel (G7G” = 180). At pH 5.0, the storage modulus G’ of the DNA hydrogel decreased to 96 ± 10 Pa and the loss modulus G” was 3 Pa, giving G7G” = 32 (consistent with a hydrogel matrix). The higher G’ value at pH 1 .2 versus pH 5.0 was due to the more rigid, longer A-motif duplex (formation at pH 1.2) as compared to the i-motif structure (formation at pH 5.0). This implied that the stiff antiparallel A-motif increased the DNA hydrogel’s stiffness, and the crosslinking units of the hydrogel switch to A-motif or i-motif at different pH values, i.e., at pH 1.2, A-motif was formed, and i-motif unwound, whereas at pH 5.0, i-motif was formed and A-motif dehybridized. At pH 7.2, the DNA hydrogel transitioned to a liquid phase (G’ ~ G” ~ 1 -2 Pa) due to the dissociation of cross-linking units into single-stranded A-rich and C-rich sequences.

In Vitro Dissociation Profiles of DNA Hydrogels and Release Profiles of FITC-lnsulin from DNA Hydrogels

Detailed dissociation profiles of DNA hydrogels in buffers of pH 1 .2, 5.0 and 7.2 at 37°C were recorded by measuring the absorbance of released DNA- tethered copolymers at A = 260 nm (see FIGs. 8A, 8B, 9A to 9C, 10A to 10D). Negligible dissociation was observed when subjecting the hydrogel to pH 1 .2 or pH 5.0 buffer (including a successive step from pH 1 .2 to pH 5.0), while fast and complete disaggregation was noticed when subjecting the hydrogel to pH 7.2 buffer. The copolymer solution was transportable by using syringe, and immediate hydrogel formation was observed when the solution was injected into buffer of pH 1 .2 or pH 5.0 (the copolymer solution was stained red for better illustration). This very fast hydrogel gelation may advantageously contribute to its utility in oral drug delivery, which could protect drugs, especially proteins, from denaturation and digestion by enzymes in the acidic gastric tract.

Gelation and dissociation profiles of DNA hydrogel under various pH values suggested that proteins, such as insulin, could be encapsulated in the hydrogel at acidic pH and released at physiological pH. To investigate the pH- triggered release characteristics, insulin was encapsulated in DNA hydrogel. The insulin@DNA hydrogel sample was transferred to solutions simulating the Gl tract environment, including artificial gastric fluid (AGF) (pH 1.2), artificial duodenal fluid (ADF) (pH 5.0), or artificial intestinal fluid (AIF) (pH 7.2), which an orally delivered protein drug will encounter in vivo. Insulin release experiments were conducted at 37 °C, unless otherwise stated. Since the absorbance between insulin and DNA tethers overlaps at A = 280 nm, the absorbance of FITC-labeled insulin at ~ 450 nm was recorded by ultraviolet-visible (UV-Vis) spectroscopy after exposure to the aforementioned conditions (see FIGs. 1 1 A to 1 1 F) to characterize insulin release from DNA hydrogel.

When insulin@DNA hydrogel was transferred to AIF, immediate insulin release was observed and was complete within 60 min (see FIG. 12A). Only 6% of FITC-insulin was released from DNA hydrogel in AGF in 60 min, while total insulin release was achieved in AIF after 60 min (see FIG. 12B). Before reaching the small intestine from the stomach, orally delivered insulin would pass through the duodenum, where pH value is in the range of 4.0-6.0.

Besides the effective protection of insulin from harsh environment, it would also be important to ensure that insulin is only released at the target site. To test the selective release of insulin in AIF, insulin@DNA hydrogel sample was introduced to AGF, followed by ADF, and lastly to AIF, mimicking the transport from the stomach to the duodenum and then the small intestine of the Gl tract. FIG. 12C shows that only 3% and 4% of insulin release from DNA hydrogel was detected in AGF and ADF in 60 min, respectively, while 100% insulin release was observed in AIF in 60 min. This demonstrated the tunability of the DNA hydrogel system for pH-responsive insulin delivery. The corresponding UV-Vis spectra of released FITC-insulin from DNA hydrogel are presented in FIGs. 13A to 13C. The enzyme-linked immunosorbent assay (ELISA), which is commonly used to accurately detect proteins in a liquid sample, was employed to measure the released insulin from DNA hydrogel under different pH values (see FIG. 12D). By using a standard insulin solution as a positive control, about 80% of insulin released from DNA hydrogel in AIF was detected (the difference might be due to the non-specific absorption of insulin in vials), while only 4% of insulin was detected in AGF or ADF, confirming the high encapsulation efficacy of the DNA hydrogel against hostile acidic environment, and the precisely tuned release in AIF.

To determine conformational changes of insulin under various pH values, the CD spectra of insulin under various pH values were recorded (see FIG. 14). The band at 208 nm primarily arises from a-helix structure, and the 223 nm band represents the [3-structure. The ratio between two bands (0208/0223) gives a qualitative measure of overall conformation structure of insulin. As shown in FIG. 14, the 208/ 223 values obtained at various pHs were calculated, indicating that insulin retained its overall tertiary structure.

Encapsulation of Insulin in the DNA Hydrogel

Loading capacity of insulin in DNA hydrogel was calculated to be 44 wt% from the encapsulated insulin weight divided by the weight of DNA copolymers. Advantageously, the relatively high insulin loading was superior to that achieved by most of the previously reported materials.

In Vitro Cytotoxicity Evaluation and Hemolysis Assay

The DNA copolymers were determined to be negligible in in vitro cytotoxicity, as cells exhibited high viability of above 80% across different concentrations of the DNA copolymer (see FIG. 15). In addition, the hemolytic effect of DNA copolymers was found to be less than 0.5% (see FIG. 16).

In Vivo Studies

In vivo pharmacokinetics and hypoglycemic effect of the insulin@DNA hydrogel was evaluated by oral administration to streptozotocin (STZ) induced diabetic rats. The blood glucose level of diabetic rats after administration of free insulin solution, SC injection or oral delivery by insulin@DNA hydrogel was examined as a function of time (see FIG. 17A). No reduction in blood glucose level was observed after oral administration of free insulin solution (30 lU/Kg), indicating that there is almost no oral pharmacological availability of insulin in the hostile acidic environment with digestive enzymes. With the SC injection of insulin solution, the blood glucose level of diabetic rats dropped severely to about 20% after 2 h, but eventually returned to the original level after 10 h due to the short circulating half-life of insulin in blood. Reduction in blood glucose level was observed with oral administration of insulin@DNA hydrogel, but at a rate that was lower than that in the case of SC injection of insulin solution. This outcome with oral administration of insulin@DNA hydrogel is desirable. The severe reduction in blood glucose level in the case of SC injection should be avoided to prevent hypoglycemic episode. It is also noteworthy that decrease in blood glucose level induced by insulin@DNA hydrogel was maintained for over 12 h.

Intestinal absorption of insulin delivered by DNA hydrogel was further evaluated by measuring the serum insulin level in diabetic rats (see FIG. 17B). A rapid spike in insulin level was observed with SC injection of insulin solution in 1 h; subsequently, the insulin level dropped to the original level in 4 h. With the oral administration of insulin@DNA hydrogel, the insulin level increased more slowly but steadily, peaking at 6 h post-administration. Taking the bioavailability of insulin administrated by SC injection as 100%, the oral bioavailability of insulin@DNA hydrogel was determined to be 5.3%.

For in vivo toxicity studies, stomach, small intestine, liver and kidney sections of diabetic rats treated with insulin@DNA hydrogel was isolated after 14 days. The sections stained with H&E are shown in FIG. 17C. In general, there was no toxicity after oral administration of insulin@DNA hydrogel as compared to the control ones. In addition, a biochemical assay of insulin@DNA hydrogel to diabetic rats was conducted, and the parameters were comparable to these in control group (see Table 1 ). These results demonstrated the excellent biocompatibility of DNA hydrogel in vivo and the potential in drug delivery.

Table 1. Blood chemistry parameters of diabetic rats treated with insulin@DNA hydrogel (30 lU/kg) after 14 days. Rats without any treatment served as the control group.

CREA: creatinine, ALT: alanine aminotransferase, AST: aspartate transaminase, TBIL: total bilirubin. *p > 0.05 as compared to the control group.

Therefore, in vivo tests revealed that the pH-responsive DNA hydrogel acted as an effective oral insulin delivery system to diabetic rats.

In summary, the insulin@DNA hydrogel demonstrated attractive performance in several aspects. First, the specially designed pH-responsive DNA oligos served as crosslinking tethers in hydrogel, making the hydrogel resistant to the hostile acidic environment and effectively protecting the encapsulated insulin from enzymatic hydrolysis. Second, over a wide pH range from 1 .2 to 6.0, which corresponded to the pH of stomach and duodenum of Gl tract, the hydrogel minimized insulin leakage. pH-triggered DNA hydrogel dissociation at 7.2 led to the full release of encapsulated insulin in the jejunum part of the small intestine. This approach allowed for better control in insulin release than hydrogel contraction/expansion mechanism. Third, the biocompatibility and degradability of DNA oligos offer safe and practical in vivo drug delivery applications.

APPLICATIONS

In the described example embodiments, a DNA copolymer comprising A- rich and C-rich DNA tethers is provided, said DNA copolymer configured to form hydrogel in an acidic environment (e.g., in the pH range of from about 1 .2 to about 6.0) crosslinked by either A-motif or i-motif structures, and to change to solution state in an environment with substantially neutral pH (e.g., at physiological pH of 7.4).

Advantageously, the pH-responsive DNA hydrogel may be used as a carrier for encapsulation and oral delivery of a cargo, e.g., a therapeutically active agent such as insulin. In a hostile environment mimicking the stomach (AGF), in vitro studies demonstrated that the encapsulated insulin may be protected by the DNA hydrogel from digestive enzymes. The composite remained as a hydrogel at the pH value of 5.0, e.g., mimicking the environment in duodenum (ADF). In an environment mimicking the small intestine (AIF), the DNA hydrogel dissociated into solution state due to the separation of A-motif and i-motif structures into A- rich and C-rich random coils at physiological pH, accompanied by the full release of insulin, which made it superior to other oral insulin delivery systems based on the “shrinking-swollen” transitions of polymer hydrogel.

Even more advantageously, in vivo studies demonstrated the potential application of DNA hydrogel in drug delivery. In addition, DNA sequences are biocompatible and biodegradable, and the pH-responsive properties of DNA hydrogel could be finely tuned by using different oligo structures or compositions, such as A-motif, i-motif and triplex structures. Besides the small intestine, drug delivery to other sections (e.g., stomach or duodenum) could also be realized by rational design of the pH-responsive DNA hydrogels.

Accordingly, the pH-responsive DNA hydrogel may be used as an oral drug delivery product, especially drugs based on peptides and proteins. The pH- responsive DNA hydrogels may also be used in other applications such as chemical or biochemical sensors, stimuli-responsive mechanical sensors, valves or actuators, soft robotics, shape memory/modulation, and cell culture.

It will be appreciated by a person skilled in the art that other variations and/or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.

Claims

44 CLAIMS

1 . A pH-responsive composition for facilitating delivery of a cargo, the composition comprising, a copolymer comprising, a backbone moiety; and a plurality of side moieties grafted to the backbone moiety, the plurality of side moieties comprising at least one adenine-rich (A-rich) oligonucleotide and at least one cytosine-rich (C-rich) oligonucleotide; wherein the copolymer is configured to form a hydrogel when exposed to an environment with a pH falling in a first range of pH values, thereby facilitating encapsulation of the cargo within the hydrogel; and wherein the copolymer is configured to form a solution when exposed to an environment with a pH falling in a second range of pH values different from the first range of pH values, thereby facilitating release of the cargo.

2. The composition according to claim 1 , wherein the first range of pH values is from 1.2 to 6.0; and wherein the second range of pH values is 7.0 or more.

3. The composition according to claim 1 or 2, wherein the A-rich oligonucleotide is configured to form a A-motif duplex structure when exposed to an environment with a pH of less than 3.0, said A-motif duplex structure configured to act as a crosslinking unit to facilitate formation of the hydrogel.

4. The composition according to any one of claims 1 to 3, wherein the A-rich oligonucleotide is configured to form a single-stranded structure when exposed to an environment with a pH of more than 4.0, said single-stranded structure configured to facilitate formation of the solution.

5. The composition according to any one of claims 1 to 4, wherein the C-rich oligonucleotide is configured to form a i-motif quadruplex structure when 45 exposed to an environment with a pH falling in the range of from 4.0 to 6.0; said i-motif quadruplex structure configured to act as a crosslinking unit to facilitate formation of the hydrogel.

6. The composition according to any one of claims 1 to 5, wherein the C-rich oligonucleotide is configured to form a half i-motif strand structure when exposed to an environmental pH of 7.0 or more, said half i-motif strand structure configured to facilitate formation of the solution.

7. The composition according to any one of claims 1 to 6, wherein the A-rich oligonucleotides and C-rich oligonucleotides are modified oligonucleotides having one or more functional groups configured to facilitate grafting to the backbone moiety, said one or more functional groups selected from the group consisting of acrydite, amine, DBCO/alkyne, azide, thiol, and carboxyl group.

8. The composition according to claim 7, wherein the modified A-rich oligonucleotide comprises the general sequence: 5’-X- AAA AAA AAA (AAA)_n - 3’, wherein n > 1 ; and the modified C-rich oligonucleotide comprises the general sequence: 5’-X- (W3 C4)2 -3’, wherein W = adenine (A) or thymine (T); and wherein X is a functional group selected from the group consisting of acrydite, amine, DBCO/alkyne, azide, thiol, carboxyl group, and combinations thereof.

9. The composition according to any one of claims 1 to 8, wherein the ratio of side moieties to ungrafted monomer units of the backbone moiety is from 1 :1 O to 1 :100.

10. The composition according to any one of claims 1 to 9, wherein the backbone moiety comprises a polymer having one or more functional groups configured to facilitate grafting of the plurality of side moieties.

1 1. The composition according to claim 10, wherein the backbone moiety comprises polyacrylamide. 46

12. The composition according to any one of claims 1 to 1 1 , further comprising a cargo, optionally wherein the cargo comprises a therapeutic agent.

13. The composition according to claim 12, wherein the cargo is insulin and wherein the insulin encapsulated in the hydrogel is protected from hydrolysis by one or more enzymes.

14. The composition according to any one of claims 1 to 13, further comprising a cell-penetrating peptide (CPP) configured to facilitate transportation of the cargo across a barrier comprising one or more layers of cells.

15. The composition according to any one of claims 1 to 14, further comprising an enzyme inhibitor configured to suppress enzymatic hydrolysis of the cargo.

16. The composition according to any one of claims 1 to 15, wherein the composition is configured to release the cargo at a controlled rate of release when in an in-vivo tissue environment.

17. The composition according to any one of claims 1 to 16, wherein the polymer composition is in the form of an oral formulation for oral administration to a subject in need thereof.

18. A method of encapsulating cargo with a pH-responsive composition, the method comprising, providing a copolymer comprising a backbone moiety and a plurality of side moieties grafted to the backbone moiety, the plurality of side moieties comprising at least one adenine-rich (A-rich) oligonucleotide and at least one cytosine-rich (C-rich) oligonucleotide; providing the cargo; and forming a hydrogel by exposing the copolymer to a pH falling in a first range of pH values, thereby encapsulating the cargo within the hydrogel, wherein the hydrogel forms a solution when exposed to an environment with a pH falling in a second range of pH values different from the first range of pH values, thereby facilitating release of the cargo.

19. A method of forming a pH-responsive copolymer, the method comprising, providing a backbone moiety; and grafting a plurality of side moieties to the backbone moiety, the plurality of side moieties comprising at least one adenine-rich (A-rich) oligonucleotide and at least one cytosine-rich (C-rich) oligonucleotide; such that the copolymer is configured to form a hydrogel when exposed to an environment with a pH falling in a first range of pH values; and configured to form a solution when exposed to an environment with a pH falling in a second range of pH values different from the first range of pH values.

20. The composition according to any one of claims 1 to 17, for use as a medicament.

21 . The composition according to any one of claims 1 to 17, for use in the treatment of diabetes, wherein the composition comprises insulin as the cargo.

22. Use of the composition according to any one of claims 1 to 17 in the manufacture of a medicament for the treatment of diabetes, wherein the composition comprises insulin as the cargo.

23. A method of treating diabetes in a subject in need thereof, the method comprising, orally administering a therapeutically effective amount of a pH-responsive composition according to any one of claims 1 to 17, wherein the composition comprises insulin as the cargo.