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WO2019164450A1 - Complexe, hydrogel et méthode - Google Patents

Complexe, hydrogel et méthode Download PDF

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Publication number
WO2019164450A1
WO2019164450A1 PCT/SG2019/050096 SG2019050096W WO2019164450A1 WO 2019164450 A1 WO2019164450 A1 WO 2019164450A1 SG 2019050096 W SG2019050096 W SG 2019050096W WO 2019164450 A1 WO2019164450 A1 WO 2019164450A1
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WIPO (PCT)
Prior art keywords
hydrogel
load
nucleic acid
linker
complex
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Ceased
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PCT/SG2019/050096
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English (en)
Inventor
Fangwei Shao
Jingyuan WU
Xiao Hu
Xing ZHU
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Nanyang Technological University
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Nanyang Technological University
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Publication of WO2019164450A1 publication Critical patent/WO2019164450A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention relates to a complex, a hydrogel, a 3D cell culture, use of the hydrogel for the proliferation of a cell, a method of forming the complex, a method of forming the hydrogel, a pharmaceutical composition comprising the hydrogel, the pharmaceutical composition for use in therapy, use of the hydrogel or the pharmaceutical composition in the manufacture of a medicament for treating cancer or for gene therapy, and a method of delivering a load to a patient.
  • Hydrogels lock water into a jelly-like texture usually with their three-dimensional (3D) crosslinking networks.
  • the high-water content environment in a hydrogel is similar to that of an extracellular matrix (ECM) and natural tissue.
  • ECM extracellular matrix
  • hydrogels have great potential in the fields of biosensing, drug delivery, 3D cell culture, regenerative medicine and tissue engineering.
  • synthetic hydrogels are either based on: 1 ) synthetic polymers hydrogels; or 2) small molecules, which suffer from low biocompatibility and low biodegradability.
  • these hydrogels usually require complicated steps to bioconjugate a natural molecule component, such as a peptide or a sugar, onto the hydrogel so that the resultant hydrogel can be suitable for 3D cell culture and tissue engineering.
  • Nanoscale carriers are regarded as a promising solution for administrating cancer theranostics, which combines specific targeted therapy based on specific targeted diagnostic tests, due to their innate selectivity against healthy cells from the size dependent enhanced permeability and retention effect (EPR) and their ability to deliver a high quantity of therapeutics per targeting event.
  • EPR enhanced permeability and retention effect
  • Currently available nano-vesicles, such as liposomes and inorganic/organic nanoparticles, have been found to significantly enhance the potency of chemodrugs and demonstrate high specificity in targeting tumors.
  • the multi-modulus synergistic delivery of diverse therapeutics remains a key challenge.
  • liposomes are capable of encapsulating a load and may have a high payload, there is a compromise on anti-cancer activity due to the lack of specific targeting.
  • metal- and organic polymer-based nanoparticles are capable of covalent and/or non-covalent surface binding of small molecule drugs and antisense oligonucleotides (ODN), their biosafety and inherent toxicity limit their widespread use.
  • a complex comprising a central moiety and a plurality of branches extending from the central moiety, wherein the plurality of branches comprises at least one loading branch and at least three crosslinking branches; wherein the loading branch comprises a nucleic acid sequence that is capable of connecting to a first load; and wherein each crosslinking branch comprises a nucleic acid sequence that is capable of connecting to a linker.
  • the complex has a tetrahedral structure.
  • each crosslinking branch is capable of connecting to a second load.
  • the nucleic acid sequence of the loading branch is complementary to an oligonucleotide sequence of the first load.
  • the oligonucleotide sequence of the first load comprises a messenger RNA (mRNA) or an aptamer.
  • the linker comprises a first segment of nucleic acid sequence that is complementary to the crosslinking branch and a second segment of nucleic acid sequence which is capable of connecting to a nucleic acid sequence of a second segment of nucleic acid sequence of a linker of an adjacent complex.
  • the second segment of nucleic acid sequence of the linker is capable of connecting to a third load.
  • the second segment of nucleic acid sequence of the linker comprises a palindromic sequence.
  • the second segment of nucleic acid sequence of the linker and the second segment of nucleic acid sequence of the linker of the adjacent complex are non-palindromic.
  • the first load, the second load and/or the third load comprises a small molecule, a macromolecule, a targeting motif, an exogenous factor, a therapeutic drug or a combination thereof.
  • the central moiety comprises at least one functional group that is capable of connecting to the plurality of branches.
  • the at least one functional group is a hydroxyl group, a thiol group, an amine group a carboxylic acid group or an amide group.
  • a hydrogel comprising: a linker connected to a complex; wherein the complex comprises a central moiety and a plurality of branches extending from the central moiety, wherein the plurality of branches comprises a loading branch and at least three crosslinking branches; wherein the loading branch comprises a nucleic acid sequence that is capable of connecting to a first load; wherein each crosslinking branch comprises a nucleic acid sequence that is complementary to a first segment of the linker; wherein the first segment of the linker comprises a nucleic acid sequence that is complementary to the crosslinking branch and the linker further comprises a second segment of nucleic acid sequence which is capable of connecting to a nucleic acid sequence of a second segment of nucleic acid sequence of a linker of an adjacent complex.
  • the complex has a tetrahedral structure.
  • the hydrogel further comprises at least one load, wherein the at least one load comprises a small molecule, a macromolecule, a targeting motif, an exogenous factor, a therapeutic drug or a combination thereof.
  • the at least one load comprises a second load, wherein the second load is connected to each crosslinking branch.
  • the at least one load comprises a third load, wherein the third load is connected to the second segment of nucleic acid sequence of the linker.
  • the at least one load comprises a fourth load which is encapsulated at least partially within the hydrogel.
  • the at least one load comprises the first load, wherein the first load is connected to the loading branch.
  • the nucleic acid sequence of the loading branch is complementary to an oligonucleotide sequence of the first load.
  • the oligonucleotide sequence of the first load comprises a messenger RNA (mRNA) or an aptamer.
  • mRNA messenger RNA
  • aptamer an aptamer
  • the second segment of nucleic acid sequence of the linker comprises a palindromic sequence.
  • the hydrogel comprises a particle diameter of about
  • the hydrogel is substantially spherical in shape.
  • the second segment of nucleic acid sequence of the linker and the second segment of nucleic acid sequence of the linker of the adjacent complex are non-palindromic.
  • the hydrogel comprises a particle diameter of about 500 nm to about 800 nm.
  • the central moiety comprises at least one functional group that is capable of connecting to the plurality of branches.
  • the at least one functional group is a hydroxyl group, a thiol group, an amine group a carboxylic acid group or an amide group.
  • the hydrogel further comprises a shielding strand of nucleic acid hybridized to the loading branch.
  • the hydrogel is modified to be pH responsive.
  • the hydrogel further comprises a cell at least partially encapsulated within the hydrogel.
  • a 3D cell culture comprising a hydrogel as described above and a cell.
  • a hydrogel as described above for the proliferation of a cell.
  • a method of forming a complex comprising connecting at least one loading branch to a central moiety followed by connecting at least three crosslinking branches to the central moiety; wherein the loading branch comprises a nucleic acid sequence that is capable of connecting to a first load; and wherein each crosslinking branch comprises a nucleic acid sequence that is capable of connecting to a linker.
  • a method of forming a hydrogel comprising: mixing a linker with a complex in a liquid; wherein the complex comprises a central moiety and a plurality of branches extending from the central moiety, wherein the plurality of branches comprises at least one loading branch and at least three crosslinking branches; wherein the loading branch comprises a nucleic acid sequence that is capable of connecting to a first load; wherein each crosslinking branch comprises a nucleic acid sequence that is complementary to a first segment of the linker; wherein the first segment of the linker comprises a nucleic acid sequence that is complementary to the crosslinking branch and the linker further comprises a second segment of nucleic acid sequence which is capable of connecting to a nucleic acid sequence of a second segment of nucleic acid sequence of a linker of an adjacent complex.
  • the method further comprises the step of varying the concentration of the complex.
  • the second segment of nucleic acid sequence of the linker comprises a palindromic sequence.
  • the second segment of nucleic acid sequence of the linker and the second segment of nucleic acid sequence of the linker of the adjacent complex are non-palindromic.
  • the ratio of the complex to the linker is 1 :1.
  • the ratio of the complex to the linker is 1 :3.
  • the ratio of the complex: the linker of the complex: the linker of the adjacent complex is 1 : 0.5: 0.5.
  • the mixing takes place at room temperature.
  • the concentration of the complex is a nanomolar concentration, preferably 100 nM.
  • the method further comprises loading at least one load to the hydrogel, wherein the at least one load comprises a small molecule, a macromolecule, a targeting motif, an exogenous factor, a therapeutic drug or a combination thereof.
  • the at least one load comprises the first load and wherein the nucleic acid sequence of the loading branch is complementary to an oligonucleotide sequence of the first load.
  • the oligonucleotide sequence of the first load comprises a messenger RNA (mRNA) or an aptamer.
  • the crosslinking branch, the second segment of nucleic acid sequence of the linker or both comprises a nucleic acid sequence that is complementary to an oligonucleotide sequence of the at least one load.
  • loading the at least one load to the hydrogel comprises encapsulating the at least one load at least partially within the hydrogel.
  • loading the at least one load to the hydrogel comprises connecting the at least one load to the loading branch.
  • the method further comprises modifying the hydrogel so that the hydrogel is pH responsive.
  • a pharmaceutical composition comprising a hydrogel as described above and at least one load.
  • a pharmaceutical composition as described above for use in therapy there is provided a pharmaceutical composition as described above for use in therapy.
  • a hydrogel as described above or a pharmaceutical composition as described above in the manufacture of a medicament for treating cancer or for gene therapy there is provided.
  • a method of delivering a load to a patient comprising administering to a patient, a therapeutically effective amount of a hydrogel as described above.
  • the hydrogel further comprises at least one load.
  • Figure 1 illustrates the characterization of D used in Examples 1 to 4: (A) HPLC trace of D; (B) MALDI-TOF mass spectrum of D (peak at 17866.0992 m/z);
  • FIG. 2 is a schematic illustration of a self-assembling method in accordance with embodiments of the present invention, wherein D and L self- assemble into a hydrogel (DNG);
  • DNG hydrogel
  • Figure 3 illustrates the sequences of the oligonucleotides or DNA sequences used for DNG formation in Examples 1 to 4;
  • Figure 4 illustrates optimization of the physical crosslinking between D and L molecules by adjusting the [D]/[L] ratio and the effect on the mechanical strength, as indicated by the G”/G’ value;
  • Figure 5 illustrates SEM images of lyophilized DNA hydrogels with different [D] (labelled as [DDNA]) and [D] vs [L] ratio:
  • Y- shaped DNA hydrogel (labelled as Y-tile) at 200 mM;
  • FIG. 7 illustrates (A) Rheological modulus of DDH.
  • G’ elastic modulus
  • G” viscous modulus.
  • Inserts Photos of DDH assembled with indicated [D] in an inverted glass vial. When [D] is 200 mM, the resultant DDH was adequately robust to hold its shape. As such, the resultant DDH was taken out of the inverted glass vial and the photo shows DDH placed on a surface.
  • G Elastic modulus
  • G viscous modulus
  • B Elastic modulus and viscous modulus of hydrogel under rheological strain sweep from 0.01 % to 10000% at 25 °C with a fixed frequency of 1 Hz over 960s;
  • C Rheological data of DDH prepared in DMEM with or without S modification;
  • Figure 9 illustrates 1 mM DDH (A-E) and 1 mM Y-DNA hydrogel (F and G) incubated with different amounts of DNase I: (A) 0, (B) 0.25, (C) 0.5U, (D) 1 .2, (E) 2.5, (F) 0, (G) 1 .2 U per pg DNA;
  • Figure 1 1 illustrates a 3D cell culture comprising FIEK 293 in C- DDH1 :
  • A 3D Fluorescence images of FIEK 293 cells cultured in C-DDH1 (top) or FIEK 293 cells cultured in 2D cell medium or solution (bottom) for 48 hours. Cells were stained by LysoTracker® Deep Red;
  • B Microscopy images of FIEK 293 cells cultured in C-DDH1 (top) or FIEK 293 cells cultured in solution (bottom) for 48 hours.
  • C-DDH1 Cell colony was circled in microscopy image of FIEK 293 cells cultured in C-DDH1 ;
  • C Cell viability of FIEK 293 cells in solution (square annotation) and C-DDH1 (circle annotation).
  • Figure 13 illustrates a photo image of C-DDH2 in an inverted glass vial.
  • Figure 14 illustrates a microscopy image of A549 cells cultured in C-DDH2.
  • Figure 15 illustrates a series of confocal fluorescence microscopy images of a A549 cell spheroid cultured in C-DDH2 at the height of 16 pm to 80 pm from the lowest focus plane.
  • the cells were stained by CellMaskTM Deep Red Plasma Membrane Stain. The bar is 40 pm.
  • A The entire cell spheroid was above the confocal focus. Thus, no fluorescent signal could be observed on the image due to the defocus;
  • B The bottom of the cell spheroid was on the focus site.
  • Figure 16 illustrates merged fluorescent image (left) and the 3D construct (right) of (A) the 2D cell culture; (B) wide view area of the DNA hydrogel; (C) spheroids. Scale bar: (A) 20pm, (B) 50pm, (C)10pm;
  • Figure 17 illustrates merged fluorescent and bright field image of the spheroids at different heights in the live/dead cell assay. Scale bar: 10pm;
  • Figure 18 illustrates microscope images of re-cultured A549 cells in DNA hydrogel medium at different timings: (A) 24 hours; (B) 96 hours; (C) 192 hours; (D) fluorescent image of live/dead assay;
  • Figure 19 illustrates the characterization of the dendritic DNA (D1 and D2) used in Examples 5 to 9: (A) FIPLC trace of D1 and D2; (B) MALDI- TOF mass spectrum of D1 (peak at 17873.5 m/z); (C) MALDI-TOF mass spectrum of D2 (peak at 20701 .4 m/z);
  • Figure 20 is a schematic illustration of the assembly of a DNA nanohydrogel by dendritic DNA and the therapeutics loaded by encapsulation of small molecule drugs, by hybridization of cell-targeting aptamers and by encoding antisense oligonucleotides, and finally delivery of multiple modulus therapeutics to disease cells;
  • Figure 21 illustrates the DNA sequences used for nanohydrogel formation and functionalization in Examples 5 to 9;
  • Figure 24 illustrates the stability of DNG: (A) DLS spectra of the hydrodynamic size of DNG before and after re-swelling; (B) DLS spectra of DNG in 1 X PBS at 0, 1 , 7 and 30 days;
  • Figure 25 illustrates the selective cell uptake of D-DNG: (A) Schematic illustration of Dox-loaded on DNG; (B) Flow cytometry data of DNG or D-DNG incubated with A549 cells (1 -4) and FIEK 293 cells (5-8); (C) Co localization of Dox and FAM labelled DNG in A549 cells;
  • Figure 26 illustrates the characterization of DOX-loaded DNA nanogel (D-DNG):
  • A DOX molecules per base pair (bp) loaded to D-DNG is linear to the concentrations of incubation stock solution of DOX;
  • B Flydrodynamic diameters of D-DNG upon loading via various Dox incubation solution ([DOX]) remain constant;
  • C Release experiment of free DOX, DOX loaded-DNG (D-DNG) with DNase I treatment or without DNase I in 1 X PBS buffer. DOX percentage inside the dialysis membrane is plotted against dialysis time;
  • Figure 27 illustrates confocal fluorescent images of A549, MCF-7 and HEK 293 cells incubated with FAM-labelled DNG;
  • Figure 28 illustrates the functionalization of DNG by the aptamer:
  • A Schematic illustration of A-DNG synthesis;
  • B Flow cytometry analysis of DNG and A-DNG on A549, MCF-7 and HEK 293 cell lines, respectively;
  • C ICso of DOX, DNG, D-DNG, and D-A-DNG of A549 cell line, MCF-7 cell line or HEK 293 cell.
  • * means at 50 mM cannot get (or determine) ICso;
  • Figure 29 illustrates the cytotoxicity of DNG on A549 cells (left) and HEK 293 cells (right);
  • Figure 30 illustrates DLS analysis of A-DNG (A) and G-DNG (B);
  • Figure 31 illustrates a confocal image of A549 cells after incubation with A-DNG
  • Figure 32 illustrates a nanohydrogel for gene therapy (G-DNG):
  • A Scheme illustrates the assembly and therapeutic strategy of G-DNG;
  • B Confocal fluorescent images of wild type (WT-DNG) and scramble DNG (SC- DNG) in cancerous A549 and somatic HEK293 cell lines;
  • C The inhibition of WT- (labelled as WT-DNG), SC- (labelled as SC-DNG) or EP-DNG (labelled as EP-ODT) on survivin mRNA levels in A549 cells. The relative abundance of survivin mRNA was determined by RT-PCR and normalized to expression levels in untreated cells;
  • D Cell viability of WT-DNG and SC-DNG on A549 cells or HEK 293 cells;
  • Figure 33 illustrates in vitro detection of SV gene by FRET fluorescence: (A) Fluorescent spectra of WT-DNG titrated by DNA target, SV;
  • Figure 34 illustrates pH responsive DNG (pH-DNG) for DOX delivery:
  • A Scheme of i-motif encoded DNG for DOX encapsulation and pH stimulus drug release;
  • B Amount of DOX released (or DOX release percentage) from pH-DNG at pH 7.4 (square annotation) and 6.2 (circle annotation) over 2 days;
  • C pH induced disassociation of pH-DNG upon time at pH 6.2 PBS buffer;
  • D Cell cytotoxicity of D-pH-DNG towards A549 cell lines;
  • Figure 35 illustrates the characterization of pH-DNG: (A) AFM image of pH-DNG; (B) DLS spectrum of pH-DNG; (C) DOX loading into pH- DNG via incubation in DOX stock solution at various concentrations ([DOX]).
  • the term“about” typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically +/- 2% of the stated value, even more typically +/- 1 % of the stated value, and even more typically +/- 0.5% of the stated value.
  • the term“complex” refers to a building block for a hydrogel.
  • exogenous factor refers to a stimulus that is not present at least partially within the hydrogel, but is present due to external addition and/or presence in an environment that is external to the hydrogel, such as an exo- or sub-cellular environment.
  • the term“medical condition” refers to a condition, disease, disorder, dysfunction, abnormality or deficit.
  • the term “patient” refers to a living human or non human organism that is receiving medical care or that should receive medical care due to a disease, or is suspected of having a disease.
  • the term“therapeutically active agent” refers to a molecule, compound, complex, adduct or composite, which exerts one or more pharmaceutical activities, and is used to prevent, ameliorate or treat a medical condition.
  • the therapeutically active agent may be an oligonucleotide, a nucleic acid construct, an antisense, a plasmid, a polynucleotide, an amino acid, a peptide, a polypeptide, a hormone, a steroid, an antibody, an antigen, a radioisotope, a chemotherapeutic agent, a toxin, an anti-inflammatory agent, a growth factor or a combination thereof.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a disease such as cancer.
  • Those in need of such treatment include those already with the disease as well as those prone to getting it or those in whom a disease is to be prevented.
  • range format is merely for convenience and brevity and should not be construed as a limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 3, 4, 5, and 6. Ranges are not limited to integers, and can include decimal measurements. This applies regardless of the breadth of the range.
  • a complex comprising a central moiety and a plurality of branches extending from the central moiety, wherein the plurality of branches comprises at least one loading branch and at least two crosslinking branches; wherein the loading branch comprises a nucleic acid sequence that is capable of connecting to a load; and wherein each crosslinking branch comprises a nucleic acid sequence that is capable of connecting to a linker.
  • the central moiety comprises an organic moiety such as an organic molecule.
  • the central moiety comprises multiple functional groups that are capable of connecting to the plurality of branches. Each functional group may be independently a hydroxyl group, a thiol group, an amine group, a carboxylic acid group or an amide group. In various embodiments, there may be at least three functional groups, such as four functional groups or five functional groups. Each functional group may be capable of connecting to the loading branch or the crosslinking branch.
  • the central moiety comprises an organic molecule, an inorganic metal complex, or a polymer.
  • the organic molecule may comprise at least one alkyl group, at least one alkene group and/or at least one aromatic group.
  • each alkyl group or alkene group may be optionally substituted with a heteroatom, such as chlorine, fluorine, bromine, nitrogen, sulphur or oxygen.
  • the organic molecule comprises tetra alkyl-ethylene glycol, wherein the alkyl may be a C1 to C20 alkyl.
  • the aromatic group may be an aromatic or heteroaromatic ring system such as benzene, naphthalene, anthracene, benzanthracene, phenanthrene, benzophenanthrene, pyrene, chrysene, perylene, fluoranthene, benzofluoranthene, naphthacene, pentacene, benzopyrene, biphenyl, biphenylene, terphenyl, fluorene, spiro-bifluorene, dihydrophenanthrene, dihydropyrene, tetrahydropyrene, cis- or trans indenofluorene cis- or trans-monobenzoindenofluoren, cis- or trans- dibenzoindenofluoren, truxene, isotruxene, spirotruxene, spiroiso- truxene, furan
  • the inorganic metal complex may comprise at least one metal such as an alkali metal, an alkaline earth metal, a transition metal or a lanthanide.
  • the metal may be lithium, sodium, potassium, magnesium, calcium, barium, aluminium, iron, nickel and copper.
  • the inorganic metal complex may comprise at least one organic molecule as described above.
  • the polymer may be a biopolymer.
  • the biopolymer may be an amino acid, a nucleic acid, a polypeptide, a carbohydrate, or an analogue thereof.
  • the biopolymer may be poly-lysine.
  • the term“plurality of branches” refers to at least three branches, wherein the at least three branches comprises at least one loading branch and at least two crosslinking branches.
  • the plurality of branches comprises three branches, four branches, five branches, six branches or seven branches.
  • the complex may simultaneously realize two functions (bi-entity), particularly, (i) the ability to connect to a load (i.e. loading) because of the presence of the at least one loading branch; and (ii) the ability to crosslink (or gelate) because of the presence of the at least two crosslinking branches (or gelation branches).
  • each crosslinking branch can crosslink to form a three-dimensional (3D) network for gelation
  • the loading branch can be separately programmed for encoding and loading at least one exogenous subject (or load) and/or at least one functionality to the hydrogel.
  • the at least one functionality of the loading branch may be capable of reacting with one or more functional groups on a load, cell and/or biological tissue.
  • prior art branched DNA such as X- and Y-shaped DNA are unable to differentiate these two functions and when used to form a hydrogel, often crosslink with each other simultaneously to form a closed network. Consequently, when prior art branched DNA are made into a hydrogel, complicated sequence design of the X- and/or Y- tiles may be required to introduce exogenous subjects and functionalization to these hydrogels.
  • the inventors of the present invention believe that the loading and/or encoding of at least one exogenous subject and/or at least one functionality via hybridization onto a hydrogel framework has not been achieved by prior art DNA hydrogels.
  • the complex may advantageously have a defined composition.
  • prior art polymer-based dendritic molecules do not have a defined composition because they may have a range of composition in terms of size, molecular weight, formula and/or number of building blocks. Consequently, the complex may be a desirable building block for hydrogels.
  • the present invention significantly improves the programmable functionalization of hydrogels, particularly compared to currently available DNA hydrogels.
  • the complex i.e. dendritic DNA
  • the complex i.e. dendritic DNA
  • programmable loading of at least one load with spatial, entity and stoichiometric control to the hydrogel may be achieved.
  • the at least one load (such as the first load, the second load, the third load and/or the fourth load) may be the same or different. Consequently, the hydrogel of the present invention may possess desirable characteristics or properties conferred by the at least one load. For instance, better manipulation of cell growth, migration, proliferation and/or differentiation for various applications may be achieved.
  • connection refers to the ability of a moiety (loading branch, crosslinking branch, linker, or load) or a part thereof to bind, attach, crosslink, conjugate or hybridize to another moiety or a part thereof.
  • each of these moieties may be modified (or selectively designed) because the nucleic acid may be modified.
  • the nucleic acid may be modified via the phosphate group on the 5’-end and/or the hydroxyl group on the 3’ -end.
  • nucleotide sequence may be modified by changing the length and/or the bases making up the nucleotide sequence.
  • the loading branch, the crosslinking branch and the linker may be modified to encode a nucleotide sequence that is complementary to a load
  • the loading branch, the crosslinking branch and/or the linker may be modified to encode an antisense sequence.
  • hybridization may occur when the nucleotides of the target are
  • the loading branch and the crosslinking branch may be independently modified so that each functional group of the focal core may connect to the loading branch or the crosslinking branch.
  • the hydroxyl group may form a phosphodiester bond to connect an oligonucleotide (such as the loading branch and/or the crosslinking branch) via solid phase phosphoramidite chemistry (such as solid phase DNA/RNA synthesis).
  • the at least one functional group of the focal core comprises a thiol group
  • the thiol group may form a linkage bond with a maleimide group.
  • the loading branch and/or the crosslinking branch may be modified to comprise the maleimide group.
  • the carboxylic acid group or the amine group may form an amide bond in the presence of at least one activating agent.
  • activating agents are commonly used in bioconjugate chemistry and are known in the art.
  • the at least one activating agent may be a carbodiimide (such as N,N'- dicyclohexylcarbodiimide (DCC), N,N'-dicyclopentylcarbodiimide, N,N'- diisopropylcarbodiimide (DIC), l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC)), an anhydride (such as a symmetric, mixed, or cyclic anhydride), an activated ester (such as phenyl activated ester derivatives, p-hydroxamic activated ester, hexafluoroacetone (HFA)), an acylazole (such as acylimidazoles using CDI, acylbenzotriazoles), an acyl azide, an acid halide, a phosphonium salt (such as HOBt, PyBOP, HO At), an aminium/uronium salt (DCC), N
  • the nucleic acid may be deoxyribonucleic acid (DNA), modified DNA, ribonucleic acid (RNA), modified RNA, locked nucleic acid (LNA), peptide nucleic acids (PNA), threose nucleic acid (TNA), hexitol nucleic acid (HNA), bridge nucleic acid, cyclohexenyl nucleic acid, glycerol nucleic acid, morpholino, phosphomorpholino, aptamer and catalytic nucleic acid versions thereof.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • LNA locked nucleic acid
  • PNA peptide nucleic acids
  • TAA threose nucleic acid
  • HNA hexitol nucleic acid
  • bridge nucleic acid cyclohexenyl nucleic acid
  • morpholino morpholino
  • aptamer and catalytic nucleic acid versions thereof
  • the nucleic acid may comprise a nitrogenous base or a modified nitrogenous base such as, 2'- o-methyl DNA, 2'-o-methyl RNA, 2'-fluoroDNA, 2'-fluoro-RNA, 2'-methoxy- purine, 2'-fluoro-pyrimidine, 2'-methoxymethyl-DNA, 2'-methoxymethyl-RNA, 2'-acrylamido-DNA, 2'-acrylamido-RNA, 2'-ethanol-DNA, 2'- ethanol-RNA, 2'- methanol-DNA, 2'-methanol-RNA, and a combination thereof.
  • a nitrogenous base such as, 2'- o-methyl DNA, 2'-o-methyl RNA, 2'-fluoroDNA, 2'-fluoro-RNA, 2'-methoxy- purine, 2'-fluoro-pyrimidine, 2'-methoxymethyl-DNA, 2'-methoxymethyl-RNA, 2'-acrylamido-DNA, 2'
  • the nucleic acid may comprise a phosphate backbone or a modified phosphate backbone, such as a phosphorothioate backbone, phosphoroborate backbone, methyl phosphonate backbone, phosphoroselenoate backbone, or phosphoroamidate backbone.
  • a phosphate backbone or a modified phosphate backbone such as a phosphorothioate backbone, phosphoroborate backbone, methyl phosphonate backbone, phosphoroselenoate backbone, or phosphoroamidate backbone.
  • the nucleic acid may be single-stranded, partially single-stranded or partially double-stranded, e.g., due to secondary structures, such as a hairpin.
  • the nucleic acid may be oligonucleotides comprising less than 50, or less than 40, or less than 30 or less than 20 bases in length. In various embodiments, the nucleic acid may comprise 6 to 40 bases.
  • the nucleic acid comprises at least 6 nucleotides so that there may be adequate stability for hybridization and at most 40 nucleotides to maintain synthetic strength (or structural integrity).
  • the number of nucleic acid may facilitate the formation of a desirable structure, such as a compact structure.
  • the nucleic acid may comprise 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 nucleotides.
  • the nucleic acid molecule comprises 20 nucleotides.
  • DNA may be used as the nucleic acid to form the complex (i.e.
  • the linker and/or a hydrogel thereof i.e. a DNA-based hydrogel or a dendritic DNA hydrogel. Consequently, the DNA-based complex, the linker and/or the DNA-based hydrogel may advantageously have unique programmability, innate biocompatibility and biodegradability properties, thereby making them suitable for various applications, which will be elaborated later on.
  • the complex, linker and the hydrogel consists essentially of DNA.
  • DNA is a genetic material and also an inherently polymeric material made from repeating units called nucleotides.
  • the rigidity of the DNA may be controlled by changing the number of base pairs in the DNA.
  • DNA is water soluble, various reactions can occur in an aqueous environment, which is therefore more environmentally friendly due to the minimization or elimination of organic solvents.
  • the complex has a tetrahedral structure.
  • the complex has an inherent 3D conformation which allows more efficient cross-linking with another complex. Consequently, the concentration of the complex required for gelation into a hydrogel may be significantly lower than that of prior art hydrogels.
  • prior art hydrogels made from X- and Y-shaped DNA may have adequate mechanical stiffness for 3D cell suspension, but a relatively high gelation concentration is required, which may therefore limit cell proliferation due to the densely-packed DNA framework in the hydrogel.
  • X-,and Y-shaped DNA are not as well oriented as the complex.
  • the tetrahedral structure is made up of the loading branch and three crosslinking branches. Consequently, each of the three crosslinking branches may form a 3D framework of hydrogel via self- assembly with the linker, while the loading branch may be programmed separately for encoding or loading a load.
  • the tetrahedral structure may be made up of two loading branches and two crosslinking branches or three loading branches and one crosslinking branch.
  • the complex may comprise at least three crosslinking branches.
  • each of the crosslinking branches may be the same or different.
  • the at least three crosslinking branches are identical to each other.
  • the nucleotide sequence of the loading branch may be changed independently of the nucleotide sequence of the crosslinking branch.
  • different variants of the complex may be synthesized by changing the length of the loading branch, the length of the crosslinking branch, the specific sequence of the loading branch, the specific sequence of the crosslinking branch and a combination thereof. Consequently, the different variants of the complex may lead to different hydrogels. As such, the properties of the hydrogel may be tuned by using a different complex.
  • the crosslinking branch is complementary to a load (or target).
  • the crosslinking branch may comprise an extension (or overhang) that is complementary to the load and not complementary to the linker so that the extension may be connected to the load.
  • the extension may encode an antisense sequence.
  • the antisense sequence may connect to the load.
  • the extension is at least a 6 nt sequence.
  • the extension is a 40 nt sequence or less.
  • the extension comprises 6 to 40 nt.
  • the extension comprises 10 to 30 nt.
  • the other parts of the crosslinking branch is not complementary to the load.
  • different variants of the linker may be synthesized by changing the length of the first segment, changing the length of the second segment, changing the specific sequence of the first segment, changing the specific sequence of the second segment or a combination thereof. Consequently, the properties of the hydrogel may be varied by changes to the linker, therefore reducing the synthetic demands associated with synthesizing different variants of the complex.
  • the nucleic acid sequence of the loading branch may be complementary to an oligonucleotide sequence of the load.
  • the nucleic acid sequence of the loading branch may be an antisense oligonucleotide.
  • the nucleic acid sequence of the loading branch is complementary to a target messenger RNA (mRNA) or an aptamer.
  • the loading branch may be modified to encode a therapeutic oligonucleotide.
  • the oligonucleotide sequence of the load may comprise a mRNA or an aptamer.
  • the mRNA is survivin RNA. It would be understood by a person skilled in the art that the oligonucleotide sequence of the load may be selected (or designed) based on the application (or use), such as treatment of a medical condition.
  • the linker comprises a first segment of nucleic acid sequence (i.e. first segment) that is complementary to the crosslinking branch and a second segment of nucleic acid sequence (i.e. second segment) which has a different nucleic acid sequence from the first segment.
  • the second segment of the linker is capable of connecting to a second segment of a linker of an adjacent complex.
  • the first segment of the linker comprises a 15 nucleotide (nt) sequence.
  • the number of nucleotides in the first segment of the linker is between 10 and 40.
  • the second segment of the linker is complementary to a load (or target).
  • the second segment of the linker may comprise an extension (or overhang) that is complementary to the load and not complementary to the crosslinking branch so that the extension may be connected to the load.
  • the extension may encode an antisense sequence.
  • the antisense sequence may connect to the load.
  • the extension is at least a 6 nt sequence.
  • the extension is a 40 nt sequence or less.
  • the extension comprises 6 to 40 nt.
  • the extension comprises 10 to 30 nt.
  • when the second segment comprises the extension the other parts of the second segment is not complementary to the load.
  • the second segment of the linker may comprise a non-palindromic sequence or a palindromic sequence.
  • the second segment of the linker of a complex (or a first complex) and the second segment of a linker of an adjacent complex (or a second complex) may be non-palindromic.
  • the second segment of the linker of the complex may be complementary to the second segment of the linker of the adjacent complex.
  • the second segment of the linker of the complex may connect to the second segment of the linker of the adjacent complex to form the hydrogel.
  • the second segment of the linker of the complex may comprise a nucleotide sequence as set forth in SEQ ID NO. 9 and the second segment of the linker of the adjacent complex may comprise a nucleotide sequence as set forth in SEQ ID NO. 10.
  • the second segment of the linker comprises a palindromic sequence.
  • the second segment of the linker of a complex may connect to the second segment of a linker of an adjacent complex (or a second complex) to form the hydrogel, wherein the second segment of the linker of the complex and the second segment of the linker of the adjacent complex are palindromic.
  • the second segment of the linker of the complex and the second segment of the linker of the adjacent complex may be identical and palindromic.
  • the second segment of the linker of the complex and the second segment of the linker of the adjacent complex may comprise a nucleotide sequence as set forth in SEQ ID NO. 7.
  • the use of the palindromic sequence may lead to a hydrogel having a particle size of about 250 nm or less. This may be because when the linker possesses a palindromic sequence, it can self-anneal to efficiently connect to the complex, as well as offer an end-closing effect by forming a hairpin structure. Consequently, further crosslinking to form a larger hydrogel may be prevented or limited.
  • the palindromic sequence comprises about 6 nt to about 15 nt. In various embodiments, the palindromic sequence comprises 12 nt.
  • the second segment of the linker has a melting temperature at least 10°C above the working temperature. Consequently, the linker may be thermally stable.
  • the term “melting temperature” refers to the temperature at which the second segment of the linker may denature. In various embodiments, the term “melting temperature” refers to the temperature at which nucleic acids, such as DNA strands are half denatured, meaning that half of the double-stranded DNA duplexes dissociate into single-stranded DNA.
  • the term “working temperature” refers to the temperature at which a user would perform an assay, a protocol or method described herein. In various embodiments, the working temperature may be room temperature or physiological temperature. For instance, when the second segment of the linker has a guanine-cytosine (G-C) content of about 40%, the second segment of the linker has a melting temperature about 10°C above the working temperature.
  • G-C guanine-cytosine
  • the complex as described above may act as a building block for a hydrogel.
  • the hydrogel may be formed by self-assembling of a plurality of complexes.
  • hydrogels made of the complex as a building block may have at least the following advantages compared to hydrogels made of prior art branched DNA such as X- and Y-shaped DNA.
  • X- and Y-shaped DNA have a near planar structure (Urn, S. H.; Lee, J. B.; Park, N.; Kwon, S. Y.; Umbach, C. C.; Luo, D., Nat. Mater.
  • the complex possesses an inherent 3D conformation.
  • Both the tetrahedral geometry of the focal core carbon atom and the electrostatic repulsion of the four strands of DNA (i.e. DNA strands) would naturally orientate the four DNA strands away from each other in 3D space. Consequently, the inherent 3D conformation may allow a significantly more efficient cross-linking among the dendritic DNA (i.e. complex), which can significantly lower the gelation concentration and enhance the mechanical strength of the resultant hydrogel.
  • a hydrogel comprising: a linker connected to a complex; wherein the complex comprises a central moiety and a plurality of branches extending from the central moiety, wherein the plurality of branches comprises a loading branch and a crosslinking branch; wherein the loading branch comprises a nucleic acid sequence that is capable of connecting to a load; wherein the crosslinking branch comprises a nucleic acid sequence that is complementary to a first segment of the linker; wherein the first segment of the linker comprises a nucleic acid sequence that is complementary to the crosslinking branch and the linker further comprises a second segment of nucleic acid sequence which is capable of connecting to a nucleic acid sequence of a second segment of nucleic acid sequence of a linker of an adjacent complex.
  • the complex has a tetrahedral structure as described above.
  • the tetrahedral structure is made up of the loading branch and three crosslinking branches.
  • the central moiety is as described above.
  • the central moiety comprises at least one functional group that is capable of connecting to the plurality of branches.
  • the at least one functional group is a hydroxyl group, a thiol group, an amine group a carboxylic acid group or an amide group.
  • nucleic acid of the complex and/or the linker may be modified, a versatile spectrum of gelation networks via physical hybridization (such as physical DNA hybridization) and specific/selective loading of one or more (multiple) loads with quantitative and spatial control may be achieved.
  • physical hybridization such as physical DNA hybridization
  • specific/selective loading of one or more (multiple) loads with quantitative and spatial control may be achieved.
  • the hydrogel further comprises a load, such as a biologically active moiety, a therapeutically active agent, a labelling moiety or a combination thereof.
  • the load comprises a biologically active moiety.
  • the load comprises a therapeutically active agent.
  • the load comprises a labelling moiety.
  • the load is selected from the group consisting of a biologically active moiety, a therapeutically active agent, a labelling moiety and a combination thereof.
  • the load may be functionalized so that it is capable of connecting with the loading branch, the linker and/or the crosslinking branch.
  • the load may be functionalized with at least one oligonucleotide.
  • the at least one oligonucleotide of the load is capable of connecting with the loading branch, the linker and/or the crosslinking branch.
  • the load may comprise a small molecule such as a small molecule drug, a macromolecule such as a macromolecule drug (such as a biomacromolecule drug), a targeting motif, an exogenous factor, a therapeutic drug, or a combination thereof.
  • the load may be characterized in more than one category selected from the group consisting of a small molecule drug, a macromolecule drug, a targeting motif, an exogenous factor, and a therapeutic drug.
  • doxorubicin for instance, doxorubicin
  • Dox may be a small molecule drug and a therapeutic drug.
  • the small molecule drug may be Dox.
  • the small molecule drug may have an aqueous solubility at or above micromolar range so that it can be encapsulated at least partially within the hydrogel.
  • the macromolecule drug may be folate, a protein or peptide.
  • the protein may be a small protein and the peptide may be a short peptide or a glycopeptide.
  • the macromolecule drug may have an aqueous solubility at or above micromolar range and a thermal stability above 60°C.
  • the targeting motif may be an aptamer (or targeting aptamer).
  • the aptamer may be an oligonucleotide (e.g., DNA, RNA, or an analogue or derivative thereof) that binds to a target, such as a polypeptide, carbohydrate, cancer cell (or tumor cell) or other target.
  • the targeting function of the aptamer may be based on the 3D structure of the aptamer, and/or its oligonucleotide sequence.
  • the exogenous factor (or exogenous molecule or exogenous agent) may be a hybridizing agent.
  • the hybridizing agent may be a regulatory agent (or a regulatory subject).
  • the exogenous factor and/or hybridizing agent and/or regulatory agent may be an aptamer, pH, a carbohydrate, a divalent metal ion such as Ca 2+ , Mg 2+ , a metabolite such as adenosine triphosphate (ATP) or an enzyme such as protease and tumor-relevant enzymes.
  • the exogenous factor may be a small molecule such as ATP or a sugar such as glucose.
  • the regulatory agent may be attached or connected to the loading branch of the complex by hybridization.
  • the regulatory agent is an aptamer
  • the presence of the aptamer may allow the hydrogel to specifically bind to diseased cells, such as but not limited to cancerous cells, and consequently induce cell death, thereby regulating the amount of cancerous cells and functioning as a regulatory agent.
  • the regulatory agent may be able to retain its biological activity and regulate cell growth even after attachment to the loading branch of the complex.
  • the therapeutic drug may be an anti cancer drug such as Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE- PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Adrucil (Fluorouracil), Afatinib Dimaleate, Afinitor (Everolimus), Aldara (Imiquimod), Aldesleukin, Alemtuzumab, Alimta (Pemetrexed Disodium), Aloxi (Palonosetron Hydrochloride), Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Aminolevulinic Acid, Anastrozole, Apre
  • an anti cancer drug such as
  • the therapeutic drug may be capable of promoting wound healing or vascularization.
  • the therapeutic drug may be a drug that reduces ischemia, e.g., due to peripheral artery disease (PAD) or damaged myocardial tissues due to myocardial infarction.
  • PAD peripheral artery disease
  • the drug may comprise a protein or fragment thereof, e.g., a growth factor or angiogenic factor, such as vascular endothelial growth factor (VEGF), e.g., VEGFA, VEGFB, VEGFC, or VEGFD, and/or IGF, e.g., IGF-1 , fibroblast growth factor (FGF), angiopoietin (ANG) (e.g., Angl or Ang2), matrix metalloproteinase (MMP) or delta-like ligand 4 (DLL4).
  • VEGF vascular endothelial growth factor
  • VEGFA vascular endothelial growth factor
  • VEGFB vascular endothelial growth factor
  • VEGFC vascular endothelial growth factor
  • IGF e.g., IGF-1 , fibroblast growth factor (FGF), angiopoietin (ANG) (e.g., Angl or Ang2), matrix metalloproteinas
  • the therapeutic drug may be an anti proliferative drug such as mycophenolate mofetil (MMF), azathioprine, sirolimus, tacrolimus, paclitaxel, biolimus A9, novolimus, myolimus, zotarolimus, everolimus, or tranilast.
  • MMF mycophenolate mofetil
  • azathioprine sirolimus
  • tacrolimus tacrolimus
  • paclitaxel biolimus A9
  • biolimus A9 novolimus
  • myolimus myolimus
  • zotarolimus everolimus
  • tranilast tranilast
  • the therapeutic drug may be an anti inflammatory drug such as a corticosteroid anti-inflammatory drug (e.g., beclomethasone, beclometasone, budesonide, flunisolide, fluticasone propionate, triamcinolone, methylprednisolone, prednisolone, and prednisone); or a non-steroidal anti-inflammatory drug (NSAID) (e.g., acetyls alicylic acid, diflunisal, salsalate, choline magnesium trisalicylate, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, fluribiprofen, oxaprozin, loxoprofen, indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac,
  • NSAID non-steroidal
  • the therapeutic drug may be capable of preventing or reducing transplant rejection, such as an immunosuppressant.
  • the immunosuppressant may be a calcineurin inhibitor (e.g., cyclosporine, Tacrolimus (FK506)); a mammalian target of rapamycin (mTOR) inhibitor (e.g., rapamycin, also known as Sirolimus); an antiproliferative agent (e.g., azathioprine, mycophenolate mofetil, mycophenolate sodium); an antibody (e.g., basiliximab, daclizumab, muromonab); or a corticosteroid (e.g., prednisone).
  • a calcineurin inhibitor e.g., cyclosporine, Tacrolimus (FK506)
  • mTOR mammalian target of rapamycin
  • an antiproliferative agent e.g., azathioprine, mycophenolate mofetil
  • the therapeutic drug may be an anti thrombotic drug, e.g., an anti-platelet drug, an anticoagulant drug, or a thrombolytic drug.
  • the anti-platelet drug may be an irreversible cyclooxygenase inhibitor (e.g., aspirin or triflusal); an adenosine diphosphate (ADP) receptor inhibitor (e.g., ticlopidine, clopidogrel, prasugrel, or tricagrelor); a phosphodiesterase inhibitor (e.g., cilostazol); a glycoprotein IIB/IIIA inhibitor (e.g., abciximab, eptifibatide, or tirofiban); an adenosine reuptake inhibitor (e.g., dipyridamole); or a thromboxane inhibitor (e.g., thromboxane synthase inhibitor, a thromboxane receptor inhibitor,
  • ADP a
  • the anticoagulant drug may be a coumarin (e.g., warfarin, acenocoumarol, phenprocoumon, atromentin, brodifacoum, or phenindione); heparin and derivatives thereof (e.g., heparin, low molecular weight heparin, fondaparinux, or idraparinux); factor Xa inhibitors (e.g., rivaroxaban, apixaban, edoxaban, betrixaban, darexaban, letaxaban, or eribaxaban); a thrombin inhibitor (e.g., hirudin, lepirudin, bivalirudin, argatroban, or dabigatran); an antithrombin protein; batroxobin; hementin; or thrombomodulin.
  • a coumarin e.g., warfarin, acenocoumarol, phenprocoumon, atromentin, bro
  • the therapeutic drug may be capable of treating or preventing an infection, e.g., an antibiotic.
  • the antibiotic may be a beta-lactam antibiotic (e.g., penicillins, cephalosporins, carbapenems), polymyxin, rifamycin, lipiarmycin, quinolone, sulfonamide, macrolide, lincosamide, tetracycline, aminoglycoside, cyclic lipopeptide (e.g., daptomycin), glycylcycline (e.g., tigecycline), oxazonidinone (e.g., linezolid), or lipiarmycine (e.g., fidazomicin).
  • a beta-lactam antibiotic e.g., penicillins, cephalosporins, carbapenems
  • polymyxin e.g., rifamycin, lipiarmycin, quinolone, sulfonamide, macro
  • the therapeutic drug may be suitable for gene therapy.
  • the therapeutic drug may comprise siRNA molecules to combat cancer.
  • the labelling moiety may be a fluorescent moiety, a radio-labelled moiety, a phosphorescent moiety, a heavy metal cluster moiety or a combination thereof.
  • the at least one load may comprise two or more loads, such as two or more therapeutic drugs from a single class, or one or more therapeutic drugs from different classes.
  • one or more different loads (such as a first load, a second load, a third load, a fourth load) may be loaded to the hydrogel.
  • diverse loads may be loaded to the hydrogel simultaneously (and/or synergistically) via multiple modules (or modes) to achieve high potency and selective targeting for applications such as, treatment of at least one medical condition, such as but not limited to cancer.
  • multiple and synergistic loading modules may be achieved by the hydrogel. Consequently, the hydrogel may be capable of delivering simultaneous (and/or synergistic) treatments of diverse therapeutics.
  • the at least one load may be loaded to the hydrogel by encapsulation (a first mode); and/or loaded to the loading branch (a second mode), and/or loaded to the crosslinking branch (a third mode) and/or loaded to the linker (a fourth mode).
  • the loading branch, the crosslinking branch and/or the linker may be modified to encode a nucleotide sequence that is complementary to a load (or target).
  • the at least one load may be functionalized so that it is capable of connecting with the loading branch, the linker and/or the crosslinking branch.
  • the crosslinking branch may be encoded with a cytosine (C)-rich sequence, thereby making the sequence responsive to the presence of stimuli, such as pH.
  • the load may be functionalized with at least one oligonucleotide.
  • the hydrogel may comprise a first load (such as a small molecule drug) encapsulated at least partially within the hydrogel, a second load (such as a macromolecule drug or targeting motif) can be connected to the loading branch, and a third load (such as an exogenous factor) can be connected to the linker.
  • a synergistic effect may be achieved when two or more loads are loaded to the hydrogel.
  • the same load may be loaded to the hydrogel simultaneously via multiple modes. For instance, a load can be connected to the loading branch, the crosslinking branch and/or the linker. As such, a greater amount of the load may be loaded to the hydrogel.
  • the hydrogel may serve as a matrix material for controlled release of the one or more loads.
  • the load may be essentially any drug suitable for local, regional, or systemic administration.
  • the load may be administered orally, buccally, sublingually, rectally, intravenously, intra-arterially, intraosseously, intramuscularly, intracerebrally, intracerebroventricularly, intrathecally, subcutaneously, intraperitoneally, intraocularly, intranasally, transdermally, epidurally, intracranially, percutaneously, intravaginaly, intrauterineally, intravitreally or transmucosally.
  • the hydrogel may be a biodegradable and biocompatible hydrogel that permits release of the load.
  • the load may be released as the hydrogel degrades due to a stimuli such as pH, metal ions, exogenous factors (such as an enzyme or small molecules), light, heat, cooling, ion strength, magnetism.
  • the enzyme DNase I may be present in an individual (or a host, a subject or a patient) when the hydrogel is administered.
  • the ion strength may be affected by the number, quantity and/or type of metal ion.
  • the hydrogel may allow for the controlled release of the load.
  • the hydrogel may be used on any surface or area.
  • the hydrogels comprising one or more load may be used on or in any internal or external biological tissues, lumens, orifices, or cavities.
  • the biological tissues, lumens, orifices, or cavities may be human or other mammalian tissues, lumens, orifices, or cavities.
  • the biological tissues may be natural or artificially generated. Therefore, the biological tissues may be in vivo or in vitro.
  • the biological tissues may be skin, bone, ocular, muscular, vascular, or an internal organ, such as but not limited to lung, intestine, heart, liver, or cancerous tissue, including tumors, associated with any biological tissue, including the foregoing.
  • the load may be substantially evenly distributed in the hydrogel, such as by encapsulation.
  • the distribution of the load encapsulated in the hydrogel may be tailored to obtain a desired release of the load from the hydrogel after deployment.
  • desired release of the load may be tailored by adding an amount of an enzyme, such as but not limited to DNase I.
  • the load may be delivered to a desired or target location or specific location, such as organs, tissues, cells, extracellular matrix components, and/ or intracellular compartments because the hydrogel may be programmed.
  • the load is a therapeutic drug
  • the therapeutic drug may be specifically delivered to diseased tissues based on targeting directed by the nucleic acid sequence or functionality of the complex and/or the nucleic acid sequence or functionality of the linker.
  • the hydrogel may be programmed or manipulated based on the target distribution and location of the load. Consequently, the load may exhibit negligible or very low uptake by healthy cells, while achieving uptake at the target location, such as by diseased cells.
  • a relatively high payload may be achieved using the hydrogel of the present invention.
  • a relatively high payload is desirable because various prior art systems suffer from a relatively low payload.
  • the Dox loading of the hydrogel of the present invention may be about 1500 Dox/bp, while prior art hydrogels generally have a Dox loading of about 0.25 Dox/bp.
  • DNA origami form nanovesicles which can carry a load such as a therapeutic subject, including a chemodrug, therapeutic oligonucleotide and targeting motif, these nanovesicles suffer from low payload and rapid drug leakage because the load can merely adopt groove binding to the DNA origami.
  • the load may be encapsulated at least partially or completely within the hydrogel.
  • the term “encapsulated” or“encapsulation” refers to the ability of the hydrogel to encase or contain the load within at least a part of the hydrogel.
  • the load may be at least partially encapsulated within the hydrogel.
  • the load is a small molecule drug such as Dox
  • the small molecule drug may be encapsulated by the hydrogel.
  • using a relatively low concentration of the complex led to efficient gelation, thereby yielding a hydrogel having a loosely crosslinked framework with a relatively large space for encapsulation of the small molecule drug.
  • the loaded hydrogel i.e. when the hydrogel is used as a carrier of the load
  • the load may cause the load to be selective towards diseased cells, such as but not limited to cancerous cells, and not selective towards healthy cells, such as but not limited to, somatic cells.
  • the biologically active moiety may be connected to the loading branch.
  • the loading branch of the complex may be modified to encode a therapeutic oligonucleotide.
  • the nucleic acid sequence of the loading branch may be encoded so that it is complementary to mRNA.
  • the mRNA is survivin RNA.
  • the loading branch may block the transcription of mRNA and enhance cell apoptosis. Consequently, therapeutic sequences may be programmed onto the scaffold of the hydrogel, and the hydrogel may be used for therapy, such as gene therapy.
  • the hydrogel further comprises a shielding strand of nucleic acid hybridized to the loading branch.
  • a shielding strand of nucleic acid hybridized to the loading branch.
  • non-specific targeting towards other cellular nucleic acids may be reduced or eliminated.
  • the hydrogel may be modified to be pH responsive.
  • the crosslinking branches of the complex may be encoded with a cytosine-rich telomeric sequence while the linker may be encoded with guanine-rich telomeric sequences, so that they can hybridize with each other.
  • the crosslinking branches may break the duplex conformation and fold into a compact i-motif structure and simultaneously, the gel framework may release the load.
  • the hydrogel may comprise of particles that are substantially uniformly spherical in shape and have a substantially uniform size.
  • the hydrogel may be substantially spherical in shape.
  • the hydrogel comprises a particle diameter (or particle size) of about 100 nm to about 800 nm, about 100 nm to about 700 nm, about 200 nm to about 600 nm, about 500 nm to about 800 nm, about 100 nm to about 250 nm, preferably about 100 nm to about 200 nm.
  • the hydrogel may have a relatively narrow size distribution.
  • the particle size of the hydrogel may be controlled by the type of linker used. For instance, when the second segment of the linker of a complex and the second segment of a linker of an adjacent complex are non- palindromic and complementary to each other, the resultant hydrogel may comprise a particle diameter of about 500 nm to about 800 nm. Alternatively, when the second segment of the linker of the complex and the second segment of the linker of the adjacent complex are palindromic, the resultant hydrogel may comprise a particle size of about 100 nm to about 250 nm.
  • the particle size of the hydrogel may be less than about 250 nm or less than about 200 nm, optimal EPR effects may be achieved. Consequently, efficient drug delivery may be achieved. More advantageously, optimal EPR effects may be consistently achieved due to the relatively narrow size distribution.
  • the hydrogel further comprises a cell at least partially or completely encapsulated within the hydrogel.
  • the hydrogel further comprises a liquid.
  • the liquid may comprise water, a buffer, a cell medium or a combination thereof.
  • the liquid may be a solution, such as blood.
  • the buffer may be phosphate buffer saline (PBS).
  • the cell medium may be Dulbecco's Modified Eagle's Medium (DMEM) or GlutaMAXTM DMEM.
  • DMEM Dulbecco's Modified Eagle's Medium
  • GlutaMAXTM DMEM may provide nutrients for a cell to proliferate.
  • the cell comprises a somatic cell or a cancer cell.
  • the somatic cell such as but not limited to a HEK cell, a stem cell, an endothelial cell; or a cancer cell such as but not limited to an A549 cell, or a combination thereof.
  • the plurality of cells may form a small colony and/or a spheroid.
  • a 3D cell culture comprising a hydrogel as described above and a cell.
  • a hydrogel as described above for the proliferation of a cell.
  • the hydrogel of the present invention has a relatively larger space for cells to grow and multiply compared to a 2D cell medium.
  • connecting at least one loading branch to a central moiety comprises solid phase phosphoramidite chemistry.
  • the at least two crosslinking branches are connected to the central moiety after the loading branch is connected to the central moiety.
  • three crosslinking branches are connected to the central moiety.
  • the loading branch and the crosslinking branch are synthesized using an automated nucleic acid synthesizer.
  • the method further comprises modifying the loading branch, the crosslinking branch and/or the linker of the hydrogel.
  • each of these moieties may be modified because the nucleic acid may be modified.
  • the nucleotide sequence may be modified so as to become a stimuli-responsive nucleotide sequence. More advantageously, the method is able to provide a complex with multi-functional branches in a relatively simple and straightforward manner.
  • the method of forming the complex is relatively high yielding. In various embodiments, the yield may be about 80%.
  • a method of forming a hydrogel comprising: mixing a linker with a complex in a liquid; wherein the complex comprises a central moiety and a plurality of branches extending from the central moiety, wherein the plurality of branches comprises at least one loading branch and at least two crosslinking branches; wherein the loading branch comprises a nucleic acid sequence that is capable of connecting to a load; wherein each crosslinking branch comprises a nucleic acid sequence that is complementary to a first segment of the linker; wherein the first segment of the linker comprises a nucleic acid sequence that is complementary to the crosslinking branch and the linker further comprises a second segment of nucleic acid sequence which is capable of connecting to a nucleic acid sequence of a second segment of nucleic acid sequence of a linker of an adjacent complex.
  • the method of forming the hydrogel is relatively high yielding.
  • the yield may be about 84%.
  • the liquid may comprise water, a buffer, a cell medium or a combination thereof.
  • the liquid may be a solution, such as blood.
  • the buffer may be PBS.
  • the cell medium may be DMEM or GlutaMAXTM DMEM.
  • the cell medium may provide nutrients for a cell to proliferate.
  • the method further comprises the step of varying the concentration of the complex.
  • the concentration of the complex In other words, the initial concentration or amount of complex used for forming the hydrogel.
  • the mesh density of the hydrogel structure may be tuned because the concentration of the complex affects the gelation, which in turn, affects the mechanical stiffness of the hydrogel.
  • the pore density increases and pore size decreases when the concentration of the complex increases.
  • the mechanical stiffness of the hydrogel may be tuned by varying the concentration of the complex. It was found that a relatively large window of mechanical stiffness may be obtained with the changes in concentration of the complex as compared to prior art hydrogels, such as those made of X- and Y-shaped DNA. Having a tunable mechanical stiffness may be advantageous because the mechanical stiffness of the hydrogel may be tuned to resemble a wide range of extracellular matrices (ECMs).
  • ECMs extracellular matrices
  • ECMs may exist in varying degrees of stiffness and elasticity, from soft brain tissues to hard bone tissues.
  • the tunable mechanical stiffness of the hydrogel of the present invention allows it to resemble various ECMs, such as but not limited to lung, breast and endothelial cells.
  • the hydrogel of the present invention possesses thixotropic properties, such as reversible thixotropy.
  • the hydrogel has reversibility between gel and solution (sol) status.
  • the hydrogel exhibited rapid gel-sol transition when subjected to an external (physical) force, such as pushing of a syringe needle. Consequently, the hydrogel may be easily handled and casted using a relatively simple and straightforward method compared to prior art hydrogels, particularly prior art DNA hydrogels, thereby reducing the technical requirements of any method used in the present invention.
  • the reversibility between gel and sol status allows gel-sol reversible conversion by manipulation or adjustment of stimuli, such as temperature (heating or cooling) or an external (physical) force.
  • modifications to the loading branch, the crosslinking branch and/or the linker of the hydrogel may lead to a change in the hydrogel such that the reversibility between gel and sol status may be achieved by stimuli such as but not limited to pH, metal ions, exogenous factors (such as presence and/or amount of an enzyme or a small molecule), small molecules, light, heat, cooling, ion strength and magnetism.
  • stimuli such as but not limited to pH, metal ions, exogenous factors (such as presence and/or amount of an enzyme or a small molecule), small molecules, light, heat, cooling, ion strength and magnetism.
  • the enzyme DNase I may be present in an individual (or a host, a subject or a patient) when the hydrogel is administered.
  • the ion strength may be affected by the number, quantity and/or type of metal ion.
  • modifications such as changes to the length, sequence composition, branch number, conjugate functionality, can be used to tune the mechanical properties of the hydrogel.
  • the mechanical strength of the hydrogel made using the complex of the present invention may be enhanced due to the more extensive network formed between each complex. This may lead to a more cost efficient hydrogel compared to prior art hydrogels because a relatively smaller amount such as nanomolar concentrations of the complex would be required to make the hydrogel of the present invention.
  • the hydrogel may show good stability against enzymatic degradation. This may be due to the relatively low gelation concentration and loose framework of the complex in the hydrogel. This may also be due to the organic central moiety.
  • the hydrogel is more resistant to enzymatic hydrolysis than prior art hydrogels such as those made of Y-shaped DNA.
  • the degradation of the hydrogel may be controlled by varying the amount of an enzyme such as but not limited to DNase I.
  • the hydrogel could maintain in the gel state at a concentration of less than 2.5 U/pg of DNase I. Consequently, the hydrogel may be degraded when desired using an appropriate amount of an enzyme.
  • the hydrogel may be easily handled and stored either as a powder or as a concentrated stock solution.
  • the hydrogel may be rapidly swollen or re-suspended into an aqueous solution with negligible or insignificant aggregation and negligible or insignificant size alteration. More advantageously, the hydrogel possesses excellent stability over a long shelf life of about 30 days.
  • the ratio of the complex to the linker is 1 :1 .
  • the ratio of the complex to the linker is
  • a high mechanical stiffness may be achieved when the concentration of the complex to the concentration of the linker is 1 :3 because when there are three crosslinking branches, all the crosslinking branches may be hybridized to the linker.
  • the mixing takes place at room temperature.
  • no thermal annealing or application of heat is required when the complex of the present invention is used as a building block for the hydrogel.
  • prior art hydrogels made of X- and Y-shaped DNA requires extra steps of thermal annealing of short oligonucleotides after DNA synthesis. For instance, heating to near boiling temperature for annealing and fabrication may be required for prior art hydrogels. Also, further purification of these prior art hydrogels may be required if assembly is not adequately efficient, which often happens due to the non-ideal design of the sequences of the short oligonucleotides.
  • the method of the present invention is relatively easy and straightforward with relatively less number of steps, does not require heating, and accordingly more cost effective.
  • heat-sensitive reagents and/or moieties may be used in the method.
  • a heat-sensitive moiety such as a protein factor may be used during the casting step of the hydrogel, and hence, excellent spatiotemporal control may be achieved, such as for the encapsulation of a load.
  • the gelation process is completed within about 10 minutes, within about 8 minutes, within about 6 minutes, within about 5 minutes, within about 4 minutes, within about 3 minutes, within about 2 minutes. In various embodiments, the gelation process is completed within about 5 minutes or within about 2 minutes. In various embodiments, the gelation process is completed within about 2 minutes when the hydrogel is larger than a nanohydrogel.
  • the time taken for gelation was not significantly affected by varying the concentration of the complex.
  • the gelation process involves connecting the linker to the crosslinking branch of each complex (such as by hybridization), thereby leading to self-assembly of the hydrogel.
  • the concentration of the complex used to form the hydrogel is in the range of about 100 nM to about 200 mM. In various embodiments, the concentration of the complex used to form the hydrogel is in a nanomolar concentration, such as in the range of about 100 nM to about 400 nM, about 100 nM, about 200 nM, about 300 nM or about 400 nM.
  • an ultralow gelation concentration of about 100 nM may be achieved using the hydrogel of the present invention, which corresponds to a weight/volume (w/v) ratio of about 4.1 x 10 6 .
  • the hydrogel of the present invention may be fabricated at an ultra-low gelation concentration, even into nanomolar concentration range.
  • prior art hydrogels made of X- and Y-shaped DNA may require a significantly higher concentration to assemble into a hydrogel.
  • the method further comprises changing the length of the first segment, changing the length of the second segment, changing the specific sequence of the first segment, changing the specific sequence of the second segment or a combination thereof. Consequently, the features or properties of the hydrogel may be varied by changes to the linker, therefore reducing the synthetic demands associated with synthesizing different complexes.
  • the method further comprises using a palindromic sequence or a non-palindromic sequence for the second segment.
  • the second segment has a melting temperature at least 10°C above the working temperature. Consequently, the linker may be thermally stable. For instance, when the second segment has a G-C content of about 40%, the second segment of the linker has a melting temperature about 10°C above the working temperature.
  • the nucleic acid sequence of the loading branch is complementary to mRNA.
  • the mRNA is survivin RNA.
  • the method further comprises hybridizing a shielding strand of nucleic acid to the loading branch.
  • the shielding strand of nucleic acid comprises a nucleic acid strand that is complementary to the loading branch.
  • the loading branch may be an antisense oligonucleotide
  • the shielding strand may be complementary to the antisense oligonucleotide.
  • the hydrogel comprises one (1 ) generation. In various embodiments, the hydrogel extends through at least 2 generations, at least 3 generations, at least 4 generations. In various embodiments, 2 or more generations may be added by using different sequences for the complex and the linker depending on the application. For instance, a person skilled in the art would be able to design the hydrogel such that it is suitable for treatment of a medical condition.
  • the method further comprises loading (or adding) a load to the hydrogel, wherein the load is as described above.
  • loading the load to the hydrogel comprises encapsulating the load at least partially within the hydrogel.
  • loading the load to the hydrogel comprises connecting the load to the loading branch.
  • the method further comprises modifying the hydrogel so that the hydrogel is pH responsive.
  • a pharmaceutical composition comprising a hydrogel as described above and a load.
  • the pharmaceutical composition as described above for use in therapy.
  • the pharmaceutical composition may be suitable for oral, topical, intravenous, subcutaneous or intramuscular administration.
  • a hydrogel as described above or a pharmaceutical composition as described above in the manufacture of a medicament for treating cancer or for gene therapy.
  • the cancer may be lung, breast, cervical or kidney.
  • the cancer may be carcinoma, sarcoma or melanoma.
  • the carcinoma may be basal cell carcinoma, biliary tract cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, CNS cancer, colon and rectum cancer, kidney or renal cell cancer, larynx cancer, liver cancer, small cell lung cancer, non-small cell lung cancer (NSCLC, including adenocarcinoma, giant (or oat) cell carcinoma, and squamous cell carcinoma), oral cavity cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer (including basal cell cancer and squamous cell cancer), stomach cancer, testicular cancer, thyroid cancer, uterine cancer, rectal cancer, cancer of the respiratory system, or cancer of the urinary system.
  • NSCLC non-small cell lung cancer
  • sarcomas include but are not limited to liposarcomas (including myxoid liposarcomas and pleiomorphic liposarcomas), leiomyosarcomas, rhabdomyosarcomas, malignant peripheral nerve sheath tumors (also called malignant
  • Ewing's sarcoma of bone, extraskeletal (i.e., not bone) Ewing's sarcoma, and primitive neuroectodermal tumor
  • Synovial sarcoma angiosarcomas, hemangiosarcomas, lymphangiosarcomas, Kaposi's sarcoma, hemangioendothelioma, desmoid tumor (also called aggressive fibromatosis), dermatofibrosarcoma protuberans (DFSP), malignant fibrous histiocytoma
  • MMH hemangiopericytoma
  • malignant mesenchymoma alveolar soft-part sarcoma
  • epithelioid sarcoma clear cell sarcoma
  • desmoplastic small cell tumor desmoplastic small cell tumor
  • GIST gastrointestinal stromal tumor
  • melanomas are tumors arising from the melanocytic system of the skin and other organs. Examples of melanoma include but are not limited to lentigo maligna melanoma, superficial spreading melanoma, nodular melanoma, and acral lentiginous melanoma.
  • the cancer may be a solid tumor lymphoma.
  • solid tumor lymphoma examples include but are not limited to Hodgkin's lymphoma, Non- Hodgkin’s lymphoma, and B cell lymphoma.
  • the cancer may be bone cancer, brain cancer, breast cancer, colorectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, cancer of the head and neck, gastric cancer, intra-epithelial neoplasm, melanoma neuroblastoma, Non-Hodgkin’s lymphoma, non-small cell lung cancer, prostate cancer, retinoblastoma, or rhabdomyosarcoma.
  • a method of delivering a load to a patient wherein the method comprises administering to a patient, a therapeutically effective amount of a hydrogel as described above.
  • the hydrogel further comprises a load as described above.
  • the method further comprises adding an amount of an enzyme, wherein the enzyme is preferably DNase I.
  • the hydrogel may be treated with DNase I with an adequately high concentration of DNase I or an adequately long incubation time, thereby converting the hydrogel to an aqueous solution.
  • the complex and hydrogel comprises DNA, which is biodegradable due to the existence of nucleases
  • the presence of an enzyme can lead to the degradation of the complex and hydrogel into degraded products (such as nucleotides), which are naturally occurring metabolites in the body, thereby rendering it non toxic and biocompatible. Consequently, the hydrogel of the present invention is desirable as a biomaterial.
  • the presence of the enzyme provides versatility and controllability to the biomaterial. Furthermore, if a load were encapsulated within the hydrogel, the amount of the load released may be controlled using the enzyme. In particular, rate of release of the load may be achieved by varying the amount of the enzyme. For instance and as illustrated in Example 6, D-DNG exhibited slow drug release upon enzymatic digestion.
  • the hydrogel of the present invention possesses various features that make it suitable for a wide range of applications.
  • the hydrogel of the present invention showed tunable mechanical stiffness and high thixotropic response, high biocompatibility, biodegradability and biosafety.
  • the hydrogel has been demonstrated to be highly promising for fabricating micrometer- and nanometer- size vessels as delivery platforms for small molecule drugs, stimuli- responsive therapeutics and gene therapy. It would be understood by a skilled person that the delivery of new therapeutics, such as by gene therapy, immune therapy and hormone therapy, would also be possible using the hydrogel of the present invention.
  • the complex and/or hydrogel of the present invention has versatile applications such as biological, biomedical, biotechnological, therapeutic applications.
  • the hydrogel was shown to be suitable for 3D cell culture. 3D cell cultures are widely used in applications in the research fields of biology, stem cell and tissue engineering and regeneration, as well as drug screening in pharmaceutic industrial revenues. It was also illustrated in the Examples that the hydrogel may be a delivery vesicle for a small molecule drug, a cancerous cell targeting and anti-proliferation aptamer, a mRNA interference antisense oligonucleotide, pFI-responsive drug administration and fluorescent probe for bio-imaging.
  • the hydrogel may be used in tissue engineering and regeneration, such as but not limited to bioartificial connective tissue and organs, or in wound care such as but not limited to dissolvable wound dressing.
  • the complex and/or hydrogel of the present invention may be applied to the environmental sciences, such as water purification, water quality monitoring, toxin and pollutant detection.
  • Dendritic oligonucleotides were synthesized by DNA solid phase synthesis using a DNA synthesizer (Bioautomation Mermade 4) with a standard phosphoramidite DNA synthesis protocol (Wu, J.; Meng, Z.; Lu, Y.; Shao, F., Efficient Long-Range Hole Transport Through G-Quadruplexes. Chemistry- A European Journal 2017, 23 (56), 13980-13985).
  • a tetraalkyl-linker (long trebler phosphoramidite, catalog number 10-1925-90) was used to synthesize dendritic DNA according to the protocol.
  • the cleavage and deprotection process was performed in 33% ammonium hydroxide solution at 37 °C.
  • Dimethoxytrityl (DMT)-on products were purified by a Shimadzu high performance liquid chromatography (HPLC) equipped with a reverse phase column (microsorb 100-5 C18 Dynamax column, 250 mm c 10.0 mm).
  • DMT -on dendritic DNA was eluted or flushed out at around 47% ACN, while non-modified oligonucleotide was eluted out at 30% ACN, and both were dried using a lyophilizer.
  • the resultant DMT-on oligonucleotides were incubated with 80% acetic acid at room temperature for 15 min and dried by the lyophilizer again.
  • DMT-off oligonucleotides were further purified by HPLC using the same method as outlined above ( Figure 1 A).
  • the resultant DNA was characterized by matrix-assisted laser desorption/ionization (MALDI)-TOF (time-of-flight mass spectrometer) ( Figure 1 B).
  • MALDI matrix-assisted laser desorption/ionization
  • Different units (0.2 U, 0.5 U, 1 U, 2 U, 5 U, respectively) of the enzyme, DNase I was added into the DDH solution, mixed thoroughly using a pipette and incubated at 37°C for 1 hour (h).
  • 1 mI_ of 0.5 M ethylenediaminetetraacetic acid (EDTA) i.e. to a final concentration of 5 mM was added to stop the reaction.
  • the resulting mixture was vortexed and then placed upside down on a bench.
  • DMEM Modified Eagle’s Medium
  • the mixture (S-D) was heated to 95°C and slowly cooled down to room temperature. 200 mM of S-D was stored in 4°C for further usage.
  • the mixture was quickly pipetted before the aptamer modified C-DDH1 was casted.
  • MTT Assay [00245] The cells in hydrogels were incubated for 24 hours, 36 hours, 48 hours, 60 hours, 72 hours and 96 hours, respectively. After incubation, 3-(4,5- dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution (1.2 mM) was added and further incubated for 4 hours. 50 mI_ of sodium dodecyl sulphate (SDS)-hydrochloric acid (HCI) solution (10 ml_ of 0.01 M HCI to one tube containing 1 gram (gm) of SDS) was added and mixed by a pipette. The cells were further incubated at 37°C for 4 hours and then submitted for the absorbance measurement. The absorbance at 570 nm was measured by a Tecan Infinite M200 microplate reader.
  • SDS sodium dodecyl sulphate
  • HCI hydroochloric acid
  • Live/Dead Cell Staining Kit (0451 1 , Sigma-Aldrich) was used to determine the viability of the cells.
  • the kit contains Calcein-AM which stains the viable cells to generate green fluorescence (excitation wavelength at 490 nm, emission wavelength at 515nm), and Propidium Iodide (PI, excitation wavelength at 535nm, emission wavelength at 617nm) which stains the dead cells to emit red fluorescence.
  • 10 pL of solution A (Calcein-AM) and 5pL of solution B (PI) was added to 5mL PBS to prepare the assay solution. Firstly, the cell media of the sample was removed.
  • Cell viability was calculated by counting the cells using the Imaris (Bitplane, Northern Ireland) spot detection function. The relative proportion of live cells (green fluorescent) and dead cells (red fluorescent) are used to determine the cell viability. The data is presented as mean values ⁇ standard deviation.
  • Example 1 Design and Synthesis of a Dendritic DNA Molecule as a Building Block of a Hydrogel
  • a dendritic DNA molecule comprising a focal core and four single strands of nucleic acid (such as DNA) connected to the focal core, wherein the focal core is tetra alkyl-ethylene glycol was designed as a starting material to fabricate a DNA hydrogel via self-assembly ( Figure 2).
  • One of the single strands of DNA is a loading branch, which is capable of encoding or loading exogenous subjects and functionalities to the hydrogel.
  • the loading branch is connected to the focal core at the 5’-end using solid phase phosphoramidite chemistry.
  • D comprises a dendritic DNA architecture with multi-functional branches.
  • the synthesis of D was conducted using an automated DNA synthesizer as described above and the product was purified by conventional methods of DNA purification, such as polyacrylamide gel electrophoresis (PAGE) or FIPLC as described above.
  • PAGE polyacrylamide gel electrophoresis
  • FIPLC FIPLC
  • L L
  • first segment is a 15 nucleotide (nt) sequence complementary to the gelation branches of D
  • second segment is a 12nt palindromic sequence to hybridize another L ( Figure 3).
  • crosslinking D with L could allow precise tuning of the mesh density of the hydrogel microstructure.
  • the time taken for gelation was not significantly affected because the gelation of DDH was achieved rapidly within minutes at room temperature for all the experiments conducted.
  • properties of DDH can be tuned by changes to the first segment and/or the second segment of L, such as length of the first segment and/or the second segment and/or specific sequence of the first segment and/or the second segment, without varying D.
  • this may reduce the synthetic demands associated with synthesizing various D.
  • Example 3 Characterization of DDH: Mechanical properties, Morphology and Enzymatic Resistance [00261] The DDH synthesized in Example 2 was characterized, as elaborated below.
  • X- and Y-shaped DNA often required a concentration of 1 imM and above to assemble into a hydrogel (Xing, Y.; Cheng, E.; Yang, Y.; Chen, P.; Zhang, T.; Sun, Y.; Yang, Z.; Liu, D., Adv. Mater. 2011 , 23 (9), 1 1 17-1 121 ).
  • [D] 200 mM
  • the resultant DDH was adequately stiff to hold or maintain its shape.
  • the resultant DDH could be placed on a surface and maintain its shape while on the surface ( Figure 7A).
  • the mechanical strength of DDH can be easily tuned by the concentration of D ([D]).
  • concentration of D [D]
  • Various concentrations of D were used to investigate the effect of [D] on the self-assembly of D to obtain DDH. It was found that G’ of DDH enhanced from 0.6 Pa to 1943.2 Pa when [D] was increased from 100 nM to 200 mM (see Figures 6A and 6D).
  • having such a stiffness level makes the DDH of the present invention resemble a wide range of ECMs, e.g., the lung, breast and endothelial cells (Greenleaf, J. F.; Fatemi, M.; Insana, M., Annu. Rev. Biomed. Eng. 2003, 5, 57-78).
  • the thixotropic property of the DDH can result in easy-casting and handling.
  • the DDH of the present invention was shown to have tunable mechanical properties and also shown to be robust with rapid thixotropic behavior. 3.2. Morphology of DDH
  • the porous morphology of the DDH of the present invention may advantageously facilitate efficient movement (or traffic) of nutrients and gas molecules, and allow a relatively large space for cell proliferation and potentially the formation of cell clusters and spheroids.
  • DDH showed better stability against enzymatic hydrolysis than a Y-shaped DNA hydrogel (B Xiang, K Fie, R Zhu, Z Liu, S Zeng, Y Fluang, Z Nie, S Yao, ACS Appl. Mater. Interfaces 2016, 8, 22801 -22807).
  • DDH Upon increasing the amount of DNase I to 2.5 U/pg, DDH was converted to a solution, presumably due to the cleavage of DNA networks. As such, this shows that DDH can be made biodegradable by varying the amount of DNase I to an adequately high concentration. Consequently, the desirably high enzymatic resistance indicates that DDH can have adequate stability for applications such as serving as a scaffold to facilitate 3D cell proliferation at an initial stage. At the same time, DDH can be degraded when desired, such as to yield more space in the newly formed ECM at a later stage of cell growth. In other words, the internal structure of DDH can be easily tuned by changes to the environment, such as, presence and/or amount of a nuclease.
  • Example 2 The suitability of DDH synthesized in Example 2 as a scaffold to reconstitute the 3D environment of ECMs was studied by using DDH to facilitate 3D cell proliferation.
  • the human embryonic kidney (FIEK) 293 cell line which is derived from FIEK cells was chosen as an example of a somatic cell line, while the A549 cell line which is derived from A549 cells was chosen as an example of a cancerous cell line.
  • C-DDH1 which is a dendritic DNA hydrogel formed or fabricated by directly mixing D and L in DMEM cell medium, was synthesized under ambient conditions. In other words, DMEM cell medium was gelated by D and L to form C-DDH1. DMEM cell medium was added because it is an enhanced supplementary formulation that can provide nutrients to cells.
  • the G7G” of C- DDH1 was determined to be about 1 1 and confirmed that it was in gel state ( Figure 8B).
  • C-DDH1 was a biocompatible material to maintain high cell viability.
  • C-DDH1 can offer a larger 3D space for cell proliferation, thereby providing adequate space for cells to grow and multiply, compared to the 2D cell medium, wherein cells would expand on the surface of the culture dish.
  • DNA hydrogels no cell proliferation was studied. This may be because of a condensed framework from the high gelation concentration of the DNA building blocks, which could not allow adequate space for cells to grow and multiply.
  • An aptamer (S) is an example of a hybridizing agent which may be regarded as a regulatory agent, which can specifically bind to A549 cells and consequently induce cell death (Kang, H.-W.; Tabata, Y.; Ikada, Y., Biomaterials 1999, 20 (14), 1339-1344). S was self-assembled into DDH via pre-casting hybridization to the loading branch of D, thereby forming S-D. Subsequently, S-DDH1, which is DDH loaded with various concentration of S, was prepared by adding free D, S-D at a ratio corresponding to the desired concentration of S, and L to the cell medium solution at room temperature.
  • Both 3D cultured FIEK 293 cells and A549 cells showed a spherical shape. Consequently, the 3D cell culture comprising C-DDH1 was found to have tissue-like cell morphology. In contrast, 2D cultured cells appeared flattened and stretched, presumably due to attachment of the cells to the dish surface. Moreover, both types of cells (i.e. FIEK 293 cells and A549 cells) showed small cell colonies (as indicated by the boxed areas in Figure 1 1 B, and Figure 12D) after culturing for 48 hours in C-DDH1. Both types of cell morphologies (i.e. spherical shape and small cell colonies) are seldom achieved in 2D cell culture or other DNA hydrogels (Urn, S. FI.; Lee, J. B.; Park, N.; Kwon, S. Y.; Umbach, C. C.; Luo, D., Nat. Mater.2006, 5 (10), 797-801 ).
  • a GlutaMAXTM DMEM cell medium was gelated by D and L to form C-DDH2 ( Figure 13). Extra sugar was supplied to the cell culture in this hydrogel medium via encapsulation of sugar molecules into the hydrogel. As such, the effect of an external subject, such as a sugar molecule, which is encapsulated into the hydrogel on cell morphology was investigated. [00285] It was found that A549 cells cultured in C-DDH2 achieved comparable proliferation as those in C-DDH1 ( Figure 10A). More remarkably, A549 cells formed cell spheroids with a diameter above 100 pm over 72 hours ( Figure 12C, 12F and Figure 14). The cell spheroids were imaged at different plateau.
  • 3D reconstituted images of spheroids in Figure 12 and Figure 15 showed that the cells were densely packed inside the spheroids and the morphology of the cells were not as spherical as those obtained in C-DDH1.
  • the slight deformation of cell shape suggests that the neighbouring cells actively interact in the spheroids, instead of taking part in nonspecific aggregation.
  • Figure 16 shows the merged fluorescent image of the 2D cell culture in solution medium after 72 hours and 3D cell culture in hydrogel medium after 96 hours.
  • the live/dead fluorescent assays indicate that the viability of the A549 cells in the 2D cell culture, 3D cell culture, and spheroids was 97.2 ⁇ 2.4%, 99.4 ⁇ 1.7%, and 99.6 ⁇ 1.4%. Therefore, the DDH of the present invention is suitable for 3D cell culture applications as it is highly biocompatible and biosafe to cell lines over the entire culture span having a time scale of days.
  • a main criteria for a cell culture medium is the ability to repeatedly culture generations of cells into the fresh cell culture medium. As such, efforts were made to develop a protocol to re-culture cells from a DNA hydrogel in accordance with embodiments of the present invention to fresh hydrogel medium. A549 cells were chosen as the exemplary cells.
  • First (1 st ) generation cells were harvested in a small amount of hydrogel medium (10 pL) and were placed into a second (2 nd ) generation cell culture medium containing D and L. It was found that the fresh hydrogel medium was isothermally formed and encapsulated the cultured 1 st generation cells within seconds. As shown in Figure 18, more A549 cells proliferated in the DNA hydrogel medium across the entire fresh hydrogel medium after the cells are re-cultured into the fresh hydrogel medium. Furthermore, the 2 nd generation cells showed similar spherical morphology as that of the 1 st generation cells. Notably, the cell viability of the 2 nd generation cell culture medium remained as high as the 1 st generation cell culture medium and a high cell viability of 98.7 ⁇ 1.8% was achieved.
  • cells such as A549 cells can be harvested from an old hydrogel medium and re-planted into a fresh hydrogel medium without compromising the viability. It thus follows that these results indicate the ability of the DNA hydrogel of the present invention to repeatedly culture generations of cells. Materials and Methods for Examples 5 to 9
  • Dendritic DNA was synthesized by following literature protocol of DNA solid phase synthesis on a DNA synthesizer (Bioautomation Mermade 4) (Wu, J.; Meng, Z.; Lu, Y.; Shao, F., Efficient Long-Range Hole Transport Through G-Quadruplexes. Chemistry - A European Journal 2017, 23 (56), 13980-13985).
  • N DOX ([. DOX] 0 * 10 mL- [DOX] out * V out )/([bp] * V in ) [00300] wherein V out and V in are the solution volume outside and inside the Amicon filter, and [bp] is the base pair concentration of DNG solution inside the Amicon filter, which is determined by the original OD of DNG.
  • the cell medium was replaced by PBS before the confocal images were taken. All images were collected on a confocal microscope (Zeiss LSM 800) with a 60c oil immersion objective. Excitation wavelength and emission filters: fluorescein (FAM), 488nm laser excitation, 520 ⁇ 20 nm emission. LysoTracker® Deep Red, 633nm laser excitation, 668 ⁇ 20 nm emission. DOX and tetra-methylrhodamine (TAMRA), 488 nm laser excitation, 580 ⁇ 20 nm emission.
  • FAM fluorescein
  • TAMRA tetra-methylrhodamine
  • FRET Fluorescent Resonance Energy Transfer
  • the fluorescence of DNG solutions was measured as described above.
  • SNP single nucleotide polymorphism
  • the florescence spectra were collected as described above.
  • the ratio of fluorescent intensity at 520 nm versus 580 nm was calculated after the fluorescence of TAMRA signal was corrected by subtracting the fluorescence of FAM at 580 nm.
  • A549 cells (10 6 ) were seeded in 6-well plates (Thermo Fisher
  • PCR Polymerase Chain Reaction
  • Ct survivin Ct GADPH are PCR threshold circle number for survivin and GADPFI mRNA in cells. The standard deviation for this data was calculated from three independent experiments.
  • a glow-discharge grid (Glow discharger EMS 100, Electron Microscopy Sciences) was placed on the wet part of the filter paper.
  • the grids were dried under air at ambient temperature overnight.
  • TEM images were collected by using a JEM-1400 (JEOL) transmission electron microscopy operated at 100 kV.
  • the size of the DNA nanohydrogel was analysed by the software DigitalMicrograph by averaging more than 300 nanoparticles.
  • a dendritic DNA molecule (D1) with four oligonucleotide strands connected to a tetra alkyl-ethylene glycol linkage was designed to encode two distinctive sequences for either crosslinking (or gelation) or loading functions ( Figure 20).
  • the loading branch is at first synthesized from the 3’-end followed by a focal core. Sequentially, one crosslinking branch is further extended from each of the three glycol linkages. Both the tetrahedral geometry of the focal carbon and the electrostatic repulsion of the DNA strands endow D1 with an inherent 3D conformation.
  • the size of the hydrogel may be controlled by varying the ratio of D1/L1. It thus follows that the size of the hydrogel may be controlled by varying the amount of D1 and the amount of L1. Subsequent screenings were all conducted at a stoichiometric ratio (1 :1 ) of D1/L1 because of the uniformity observed in the resultant hydrogel.
  • the 10 nt segment at the 3’-end of L2 is complementary with that of L3 and both L2 and L3 have the same guanine- cytosine content (G-C %) as L1 to maintain the thermal stability.
  • G-C % guanine- cytosine content
  • the difference in diameters observed between the non-palindromic linkers and the palindromic linkers may be because a palindromic linker such as L1 , which possesses a palindromic segment, can self-anneal to efficiently connect to D1 , as well as offer an end closing effect by forming a hairpin structure. Consequently, further crosslinking to form a larger nanohydrogel may be prevented or limited.
  • the size of the nanohydrogel may be controlled by changing the sequence of the linker, which leads to changes in the annealing fashion and accordingly, the size of the nanohydrogel.
  • DNG of the present invention can be rapidly swollen and re suspended back to aqueous solution. Furthermore, DNG showed nearly no aggregation (or insignificant aggregation) and no size alteration (or negligible size alteration). Notably, DNG was found to be stable when the nanohydrogel solution was purified and concentrated simultaneously by centrifuge. Consequently, DNG solution can be concentrated one hundred times to prepare the stock solution. Furthermore, DNG was robust enough to resist fragmentation and aggregation upon dehydration and physical sheering force. Consequently, these results demonstrate that the nanohydrogel of the present invention can be easily handled and stored either as a powder or as a highly concentrated aqueous solution.
  • the nanohydrogels were found to possess excellent aqueous stability over a long shelf time (or shelf life).
  • DLS monitored the size of DNG in an aqueous buffer, such as 1X PBS, over a bench standing time of 0 (0D), 1 (1 D), 7 (7D) and 30 (30D) days.
  • a longer standing time was allowed, such as 7 days or 30 days, DNG showed no aggregation and narrower dispersion in size ( Figure 24B). Consequently, DNG was shown to have a standing time of at least 7 days or at least 30 days.
  • the nanohydrogel of the present invention shows high stability against dissociation, aggregation, and polymorphism in aqueous buffer over a long standing time of at least 30 days. Furthermore, the nanohydrogel can be easily handled and stored either as a powder or as a highly concentrated aqueous solution.
  • Example 6 Encapsulation and Enzymatic Responsive Release of Small Molecule Drugs (Delivery Module 1)
  • DNG DOX-containing nanohydrogel
  • [DOX] concentration of DOX solution
  • L1 was modified using a cyanine dye, Cy5, so as to form a Cy5-labelled L1 (i.e. Cy5- L1 , Figure 21 ).
  • L1 was modified using fluorescein (FAM) dye so as to form a FAM-labelled L1 (i.e. FAM-L1 , Figure 21 ).
  • FAM fluorescein
  • Cy5-DNG was assembled using a stoichiometric equivalence of D1 and Cy5-L1 and 100 nM of D1.
  • D-DNG can selectively deliver DOX to cancerous cells such as A549 cells, while preventing or minimizing uptake of DOX by healthy somatic cells, such as HEK 293 cells.
  • FIEK 293 cells While no apparent toxicity was observed on FIEK 293 cells, the cell viability of FIEK 293 remained nearly 100% at 50 mM of DOX in D-DNG. Slow drug release and efficient subcellular delivery by D-DNG could account for the improved therapeutic effects observed via control of DOX availability and/or distribution in cancerous cells, and consequently minimizing drug efflux.
  • Example 7 Specific Targeting of Cancerous Cell Lines via Aptamer (Delivery Module 2)
  • DNG surface of DNG was modified using an aptamer. Furthermore and as mentioned above, the stock solution of DNG comprising 100 nM of dendritic DNA and stoichiometric equivalents of palindromic linker DNA was used.
  • DNG could achieve specific targeting towards cancer cells via surface decoration by a DNA aptamer, A, to form a modified DNG (i.e. A-DNG) ( Figure 21 and Figure 28A).
  • A was designed to contain an aptamer sequence specifically targeting A549 cells at the 5’-end, and a complementary sequence to the loading branch of D1 at the 3’-end (Tan, W.; Donovan, M. J.; Jiang, J., Aptamers from Cell-Based Selection for Bioanalytical Applications Chem Rev 2013, 1 13 (4), 2842-2862). Subsequently, A was loaded onto DNG via hybridization to form the nanohydrogel, A-DNG.
  • the distinctive tuning on cell cytotoxicity is consistent with that of cell uptake and suggests that the targeting effects further impact the potency of DNG-loaded chemodrugs towards different diseased cells.
  • the selectivity of A-DNG on cancerous cells indicates that DNG could interact with the unique protein factors on the cell surface to achieve disease-specific targeting instead of merely relying on EPR effects due to the size of the nanohydrogel to achieve delivery specificity.
  • Example 8 Antisense Oligonucleotide Delivery for Gene Therapy (Delivery Module 3)
  • the bi-entity branches i.e. presence of at least one loading branch and at least one gelation branch
  • the dendritic DNA molecule such as D1
  • AO antisense oligonucleotides
  • the AO sequence is an undetachable component from the nanohydrogel scaffold, it was postulated that survivin mRNA could be wound onto the nanohydrogel upon hybridization, and encapsulated into the nanohydrogel, which could prevent mRNA from disassociation and block the downstream transcription significantly more efficiently.
  • Equal stoichiometric amounts of D2, L1 and O were thermally annealed to assemble the nanohydrogel G-DNG by following the protocol optimized for DNG. It was found that G-DNG showed similar hydrodynamic diameters (197 ⁇ 21 nm) as DNG ( Figure 30B). As such, this indicates that modification of the loading branch still led to a resultant nanohydrogel having a similar diameter.
  • a synthetic DNA target (SV) was used as a mimic of survivin mRNA to investigate the selectivity and sensitivity of G-DNG via a FRET assay. Accordingly, the 5’-end of the linker L1 and the 3’-end of the shielding strands O were labelled with a FRET pair (i.e. FAM and TAMRA), thereby forming FAM- L1 and TAMRA-O, respectively ( Figure 21 ).
  • the nanohydrogel WT-DNG was assembled using D2, FAM-L1 and TAMRA-0 by following the method of the present invention.
  • S1 and SNP-1 to SNP-4 were synthesized and 200 nM of each target was separately titrated to WT-DNG.
  • S1 was designed to comprise a scramble sequence
  • SNP-1 , SNP-2, SNP-3 and SNP-4 were designed to comprise a single nucleotide polymorphism strand ( Figure 21 ). It was found that all of the non-wild type targets showed significantly less binding to WT-DNG ( Figure 33C).
  • S1 induced only a 2.3-fold increase of F520/F580, while F520/F580 showed no more than 47 in the presence of either one of the four SNP targets.
  • the selectivity of WT-DNG for SNP was observed to improve when the position of the single nucleotide mutation was changed.
  • nanohydrogel of the present invention was shown in Example 6 to have high therapeutic potency via slow release of a chemodrug, such as DOX, stimulus-initiated release of a drug is highly desirable since it would allow control of the time and location of drug administration.
  • a stimuli responsive vesicle was designed by using a pH responsive DNG (pH-DNG).
  • cytosine (C)-rich telomeric sequence can undergo robust reversible conformation transfer between duplex DNA and quadruplex i-motif
  • the linker L4 was designed to have a palindromic 10 nt sequence at the 3’-end for gelating to the gelation branch of D3. Furthermore, equal amounts of D3 and L4 were assembled to form pH-DNG in accordance with the method used to synthesize DNG.
  • pH-DNG showed similar uniform size and spherical shape as compared to DNG, albeit having a slightly larger average size of about 260 nm ( Figure 35B).
  • encapsulation of DOX using pH-DNG achieved a similarly high payload of about 120 DOX molecules per bp ( Figure 35C).
  • pH-DNG could hold up to 80% of the drug molecules for at least 24 hours at neutral pH of 7.4 ( Figure 34B), while an acute (or fast) drug release could be induced when the pH dropped to 6.2.

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Abstract

La présente invention concerne un complexe comprenant une partie centrale et un grand nombre de branches s'étendant à partir de la partie centrale, le grand nombre de branches comprenant au moins une branche de chargement et au moins trois branches de réticulation, la branche de chargement comprenant une séquence d'acide nucléique susceptible de se connecter à une première charge, et chaque branche de réticulation comprenant une séquence d'acide nucléique susceptible de se connecter à un lieur. La présente invention concerne également un hydrogel comprenant le complexe, une culture cellulaire 3D, une composition pharmaceutique, des méthodes et des utilisations de ces dernières.
PCT/SG2019/050096 2018-02-22 2019-02-20 Complexe, hydrogel et méthode Ceased WO2019164450A1 (fr)

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