Patent Application
20210009763
The present invention relates to a hydrogel inclusion complex bound with a physiologically active material by a host-guest interaction through beta-cyclodextrin substituted to thermosensitive poly(phosphazene), and use thereof.
As a biocompatible material to construct neotissues for the purpose of tissue regeneration, numerous kinds of materials based on a bioconjugation technology were utilized such as nanoparticle, decellularized matrix, and hydrogel. Among these materials, hydrogel is an excellent material source for tissue engineering owing to capacities in absorption of huge amounts of water, high biocompatibility, and living tissue resemblance. In detail, synthetic hydrogels such as poly(lactic acid-glycolic acid) derivatives, poly(lactic acid) derivatives, hyaluronic acid, polyurethane acrylates, and many other kinds of biomedical polymers are used for tissue regeneration by employing both physiologically active materials and stem cells.
Efforts have been made to fine-tune biocompatible materials such that they are implanted in a living body and tissue regeneration occurs in a desired direction. The fine-tuned biocompatible materials connote well-controlled materials in both physical and chemical aspects. This fine tuning of biocompatible materials is strongly required in several therapeutic perspectives, for example, an enhanced therapeutic window in drug delivery system, immunotherapy efficacy (to avoid auto-immune response or immunotoxicity), disease specific targeting, and appropriate stem cell stimulations for regenerative engineering. Generally, physical control of biocompatible materials means the mechanical property control and microstructure control, affecting cell characteristics or therapeutic drug release patterns. Chemical control of biocompatible materials means the control of chemical linkage. Specifically, chemical linkage between two molecules is called bio-conjugation (Kalia J, Raines R T. Advances in Bioconjugation., Current organic chemistry14, 138-147 (2010)). Although the bio-conjugation technology offers advantages to biomaterials, including structural stabilization from chemical degradation, an improved body residence time, and reduction of immunogenicity, there still remain issues such as toxicity issues, low reproducibility, and complex synthesis processes. In particular, since high chemoselectivity is resulted from the employment of acrylate, methacrylate, copper (I)-catalyzed azide alkyne cycloaddition (CuAAC), and free radicals, there is a high risk issue for toxicity owing to remnants of such groups (Spicer C D, Pashuck E T, and Stevens M M, Achieving Controlled Biomolecule-Biomaterial Conjugation, Chemical reviews, 118, 7702-7743 (2018)). Also, the low reproducibility prevents the fine tuning of biocompatible materials.
In particular, attempts have been made to control the differentiation of stem cells using hydrogels including physiologically active materials. There are diverse bioactive molecules for proliferation and differentiation of MSCs (mesenchymal stem cells), such as small chemicals, proteins, and peptides. Since administration of MSCs alone makes their differentiation into a desired lineage and their survival in a harsh body environment difficult, the assistance with such hydrogels and bioactive molecules is essential to the ideal tissue regeneration. Recently, various approaches for the tissue regeneration by employing MSCs were explored with several kinds of proteins for the desired MSC differentiation. However, even through physical and chemical controls of stem cells, bioactive molecules, and hydrogels, fine tuning of stem cell differentiation has still not been achieved to a satisfactory level.
Meanwhile, a host-guest interaction forms a non-covalent complex of two or more molecules by unique structural recognition through a molecular self-recognition system. There are many macrocyclic host molecules such as cyclodextrins (CDs), crown ethers, cyclophanes, cryptands, and curcubiturils. Among these host molecules, cyclodextrin is suitable for the tissue engineering or biological applications owing to its low toxicity and low immunogenicity. Famous cyclodextrins mainly utilized in the industrial and research realm are α-, β-, and γ-CD. Most of all, β-CD is a superb molecule owing to its high water solubility post-modification, excellent inner cavity size for guest molecule loading, and relatively low cost. The host-guest interaction using β-CD provides several benefits to biocompatible materials involving a facile supramolecular structure building, and an avoidance of multiple synthesis steps and a complicated purification process during the fabrication process. Over the past few decades, β-CD has been actually employed in various biomedical applications such as drug delivery systems, diagnostic applications, and tissue engineering.
However, there have been no attempts to introduce a physiologically active material using the host-guest interaction into a hydrogel such as thermosensitive poly(phosphazene). Under this circumstance, the present inventors had attempted to introduce the host-guest interaction with the purpose of regulating many kinds of bioactive factors in 3D hydrogel of poly(organophosphazene) bearing β-CD. PPZ has been developed as a biocompatible and thermosensitive hydrogel by the present inventors over the past few decades (see Korean Patent Application Publication Nos. 10-2005-0012533 A and 10-2008-0110472 A, etc.), and therapeutically valuable drugs, such as chemical drugs, proteins, and genes, can be delivered by the same.
The present inventors succeeded in the synthesis of β-CD PPZ, and have studied the stoichiometric control of bioactive guest molecules of β-CD PPZ to overcome the limitations of bio-conjugation systems. They formed various functional guest molecules yielding hydrogels without a further synthesis batch of β-CD PPZ through a diverse combination of the guest molecule ratios in the host, and found that had, fine-tuned differentiation can be achieved by application of these hydrogels, thereby completing the present invention.
An object of the present invention is to provide a hydrogel inclusion complex including thermosensitive poly(phosphazene) to which a plurality of hydrophobic amino acids, hydrophilic polymers, and beta-cyclodextrin (β-cyclodextrin; β-CD) as a host molecule are substituted; and a physiologically active material linked directly or via a linker to one or more molecules, as a guest molecule, selected from the group consisting of adamantine, azobenzene, cholesterol, tert-butyl, cyclohexyl ester, and naphthyl, wherein the guest molecule is conjugated to all or part of the beta-cyclodextrin by inclusion of the guest molecule into the beta-cyclodextrin via a host-guest interaction.
Another object of the present invention is to provide a method of controlling stem cell differentiation, the method including a step of treating a plurality of stem cells with a hydrogel composition including, as active ingredients, thermosensitive poly(phosphazene) to which a plurality of hydrophobic amino acids, hydrophilic polymers, and beta-cyclodextrin are substituted; and a stem cell differentiation regulator linked directly or via a linker to one or more molecules, as a guest molecule, selected from the group consisting of adamantine, azobenzene, cholesterol, tert-butyl, cyclohexyl ester, and naphthyl.
Still another object of the present invention is to provide a method of regenerating a tissue, the method including a step of injecting, into a damaged tissue site, a hydrogel composition including, as active ingredients, thermosensitive poly(phosphazene) to which a plurality of hydrophobic amino acids, hydrophilic polymers, and beta-cyclodextrin are substituted; and a stem cell differentiation regulator linked directly or via a linker to one or more molecules, as a guest molecule, selected from the group consisting of adamantine, azobenzene, cholesterol, tert-butyl, cyclohexyl ester, and naphthyl.
Still another object of the present invention is to provide a method of inhibiting cancer cell proliferation or metastasis, the method including a step of administering, to an individual with a tumor, a hydrogel composition including, as active ingredients, thermosensitive poly(phosphazene) to which a plurality of hydrophobic amino acids, hydrophilic polymers, and beta-cyclodextrin are substituted; and IL-2 linked directly or via a linker to one or more molecules, as a guest molecule, selected from the group consisting of adamantine, azobenzene, cholesterol, tert-butyl, cyclohexyl ester, and naphthyl.
Still another object of the present invention is to provide thermosensitive poly(phosphazene) to which a plurality of hydrophobic amino acids, hydrophilic polymers, and beta-cyclodextrin are substituted.
In
In
Hereinafter, the present invention will be described in more detail.
Meanwhile, each description and embodiment disclosed herein may also be applied to other descriptions and embodiments. That is, all combinations of various elements disclosed herein fall within the scope of the present invention. Further, the scope of the present invention is not limited by the specific description described below.
Further, those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Further, these equivalents should be interpreted to fall within the present invention.
To achieve the above objects, one aspect of the present invention provides a hydrogel inclusion complex including thermosensitive poly(phosphazene) to which a plurality of hydrophobic amino acids, hydrophilic polymers, and beta-cyclodextrin (β-cyclodextrin; β-CD) as a host molecule are substituted; and a physiologically active material linked directly or via a linker to one or more molecules, as a guest molecule, selected from the group consisting of adamantine, azobenzene, cholesterol, tert-butyl, cyclohexyl ester, and naphthyl, wherein the guest molecule is conjugated to all or part of the beta-cyclodextrin by inclusion of the guest molecule into the beta-cyclodextrin via a host-guest interaction.
As used herein, the ‘hydrogel’ refers to a three-dimensional network structure formed from a polymer present in an aqueous solution, and is usually divided into hydrogels formed by chemical crosslinking via covalent bonds and hydrogels formed by physical crosslinking via physical interactions between molecules. The hydrogel of the present disclosure generally has a basic skeleton of thermosensitive poly(phosphazene), to which a host molecule is substituted, and forms a conjugate with a guest molecule, which is involved in a host-guest interaction with the host molecule and to which a physiologically active material is linked. The hydrogel composition is a biocompatible material injectable into a living body.
As used herein, ‘thermosensitive’ means a property that an aqueous polymer solution maintains a liquid state (sol) at a low temperature, but the aqueous polymer solution is transformed to a gel as the temperature increases. Specifically, it means a property of maintaining a liquid state at room temperature, while being transformed into a gel state at a temperature of 35° C. to 37° C. or higher.
Since the hydrogel of the present disclosure is introduced into the body to form a gel of a three-dimensional structure by body temperature, there are advantages in that it is easy to inject owing to the liquid state, and at the same time, a drug can be slowly released owing to its transition to a gel state. Further, the hydrogel in the gel state may form a niche (microenvironment) suitable for stem cell differentiation. For example, when stem cells are simply injected, they non-specifically spread throughout the body. In contrast, when stem cells are injected in a state mixed with the gel, it not only simply makes the injection easy, such as targeting a desired site thereby allowing the stem cells to be delivered to the desired site and to be retained therein, but also contributes to local delivery to a desired site.
Meanwhile, stem cell is characterized by having i) self-renewal and ii) pluripotency, regulating differentiation of the stem cell is an important factor, especially in tissue regeneration. Further, stem cell differentiation can be regulated by treating stem cells with the hydrogel composition including a stem cell differentiation regulator according to the present invention. In this regard, by regulating the type or ratio of the stem cell differentiation regulator, or both of them, sternness of the stem cells can be maintained, or the stem cells may be regulated to be differentiated to a specific state.
Furthermore, the hydrogel composition of the present invention can be used for tissue regeneration. As described above, since the hydrogel composition including a stem cell differentiation regulator of the present invention can regulate stem cell differentiation, it is possible to regulate stem cell differentiation at the corresponding site and promote regeneration of a desired tissue by injecting the composition to a damaged tissue site. In this regard, the composition may further include stem cells, but is not limited thereto, and the hydrogel composition may exert the effect of prompting tissue regeneration.
As used herein, the ‘poly(phosphazene)’ is substituted with hydrophobic amino acids and hydrophilic polymers such that it exhibits a thermosensitive property. Specifically, a hydrophobic amino acid ester and hydrophilic methoxy-polyethylene glycol having a molecular weight in the range of 350 to 2,500 may be introduced into a linear polymer of dichlorophosphazene, and an amino acid, a peptide, or depsipeptide ester capable of controlling a degradation rate of the polymer may be partially introduced into the polymer. Further, functional groups may be introduced into the phosphazene-based polymer of the present invention, e.g., by directly introducing a substituent with functional groups such as a hydroxyl group, an amide group, an amino group, a thiol group, or a carboxyl group on the side chain into the main chain, or introducing the amino acid ester or peptide ester substituent, wherein the functional group is substituted with a protecting group, into the main chain of the polymer followed by removing the protecting group, or by a method of converting the polymer introduced with the substituent having the functional groups to another functional group. The thermosensitive poly(phosphazene) to be used in the present disclosure may be those known in the art (see Korean Patent Application Publication Nos. 20050012533A and 10-2008-0110472 A, etc.). In this regard, the thermosensitive poly(phosphazene) may include a plurality of hydrophobic amino acids, hydrophilic polymers, and beta-cyclodextrin at a molar ratio of (55 to 80):(5 to 25):(5 to 20), but is not limited thereto.
The hydrophobic amino acid may be any one or more selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, and tryptophan. The hydrophobic amino acid has a structure of NHCH(R1)CO2R2, wherein R1 may be selected from the group consisting of H, CH3, CH2SH, CH(CH3)2, CH2CH(CH3)2, CH(CH3)C2H5, CH2CH2SCH3, CH2C6H5, CH2C6H4OH, and CH2C2NH2C6H4, and R2 may be selected from the group consisting of H, CH3, C2H5, C3H7, C4H9, CH2C6H5, and CH2CHCH2.
The hydrophilic polymer may be polyalkylene glycol having a molecular weight in the range of 550-2,500, and specifically, polyethylene glycol, monomethoxy polyethylene glycol, or a block copolymer of ethylene glycol and propylene glycol. The number of the alkylene repeating unit of polyalkylene glycol may be in a range of 7 to 50.
The type or substituted amount of the hydrophobic amino acids and hydrophilic polymers, and the concentration of poly(phosphazene) and the like may be adjusted to control the gelling temperature, gel strength, and/or biodegradation rate of poly(phosphazene). For example, as the composition of the hydrophobic amino acid increases, the gelling temperature can be lowered, and as the concentration of poly(phosphazene) increases, the gelling temperature becomes lower, and the gel strength increases. Further, as the chain length of the hydrophilic polymer increases, the gel strength increases and the gelling temperature becomes higher.
The physiologically active material-bound hydrogel inclusion complex of the present disclosure may deliver, into a body, the physiologically active material which is linked to a guest molecule via ‘host-guest interation’, The ‘host-guest interaction’ forms a non-covalently bound complex of two or more molecules through unique structural recognition of a molecular self-recognition system. As compared with a conjugate system via covalent bonding, holding bioactive molecules through the host-guest interaction is more simple reproducible. According to Experimental Examples 1 to 4 of the present disclosure, it was found that a specific conjugate was produced. In proportion to the content and/or ratio of the guest molecule prepared before mixing. As described, as a result of using the host-guest interaction, the content and/or ratio of the physiologically active material attached to the surface of poly(phosphazene) may be easily controlled. Furthermore, the physiologically active material linked to the guest molecule may be slowly released by cleavage of the linkage with the host molecule in the body.
As used herein, the term ‘host molecule’ refers to any material that is able to capture the guest molecule, and has a hydrophobic cavity and a hydrophilic surface. Specifically, the physiologically active material-bound hydrogel of the present disclosure includes beta-cyclodextrin as the host molecule. The beta-cyclodextrin is suitable for tissue engineering or biological applications owing to its low toxicity and low immunogenicity.
As used herein, the term ‘guest molecule’ refers to a molecule that is trapped in the cavity of the host molecule. Since the cavity of the host molecule shows hydrophobicity, the guest molecule also shows hydrophobicity, and a material having a size suitable for inclusion in the cavity may be used. For example, the hydrogel inclusion complex of the present disclosure includes beta-cyclodextrin as the host molecule, and thus it is preferable to includes, as the guest molecule, adamantine, azobenzene, cholesterol, tert-butyl, cyclohexyl ester, naphthyl, or a combination thereof, which can specifically interact with the host molecule. In this regard, the guest molecule may be further modified to promote the activity of the physiologically active material.
As used herein, the term ‘physiologically active material’ refers to a material exhibiting activity when injected into a body or a cell, and may be one or more selected from the group consisting of proteins, peptides, vaccines, genes, hormones, anti-cancer drugs, angiogenesis inhibitors, sugars, polyols, sugar-containing polyols, polymer-containing polyols, sugar-containing amino acids, and sugar-containing ions.
The proteins may be selected from the group consisting of exendin-4, erythropoietin (EPO), interferon-alpha, interferon-beta, interferon-gamma, growth hormone (human, pig, cow, etc.), growth hormone releasing factor, nerve growth factor (NGF), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), macrophage-colony stimulating factor (M-CSF), blood clotting factor, insulin, oxytocin, vasopressin, adrenocorticotropic hormone, fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGF-β), nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5, prolactin, luliberin, luteinizing hormone releasing hormone (LHRH), LHRH agonists, LHRH antagonists, somatostatin, glucagon, interleukin-2 (IL-2), interleukin-11 (IL-11), gastrin, tetragastrin, pentagastrin, urogastrone, secretin, calcitonin, enkephalins, endorphins, angiotensins, thyrotropin releasing hormone (TRH), tumor necrosis factor (TNF), tumor necrosis factor related apoptosis inducing ligand (TRAIL), heparinase, bone morphogenic protein (BMP), human atrial natriuretic peptide (hANP), glucagon-like peptide (GLP-1), renin, bradykinin, bacitracins, polymyxins, colistins, tyrocidine, gramicidins, cyclosporins, neurotensin, tachykinin, neuropeptide Y (NPY), peptide YY (PYY), vasoactive intestinal polypeptide (VIP), and pituitary adenylate cyclase-activating polypeptide (PACAP).
The peptides may be biomimetic peptides derived from the natural proteins. For example, the peptides may be selected from the group consisting of collagen 1-derived GFOGFR and DGEA; laminin-derived YIGSR, SIKVAV, IKVAV, IKLLI, LRGDN, and SINNNR; laminin γ1-derived LRE, PDGSR, GTFALRGDNGQ, CFALRGDNP, NPWHSIYITRFG, TWYKIAFQRNRK, KAFDITYVRLKF, and LGTIPG; fibronectin-derived GRGDS, PKRGDL, NGRAHA, GACRGDCLGA(cyclic), IDAPS, REDV, PHSRN, KQAGDV, LDV, WQPPRARI, SPPRRARV, LIGRKK, IWKHKGRDVILKKDVRFYC, KLDAPT, and PRARI; vitronectin-derived CKKQRFRHRNRKG; osteopotin-derived KRSR, FHRRIKA, CGGNGEPRGDTYRAY, SVVYGLR, and ELVTDFPTDLPAT; elastin-derived VPGIG and VGVAPG; collagen 4-derived MNYYSNS and CNYYSNS; thrombospondin-derived CSVTCG, GRGDAC, FQGVLQNVRFVF, AELDVP, and VALDEP; nidogen-1-derived GFRGDGQ and SIGFRGDGQTC; N-cadherin-derived HAV; and TGF-β1-derived PWPLPYL, WGLLDLT, PAERLRS, RNLDGWS, NLSSWI, TLPSNTH, MSAFPFL, SRLGQYI, PFGLPP, TIASTLH, PRAPADV, and ESPLKRQ.
The vaccine may be one or more selected from the group consisting of hepatitis vaccine and the like.
The gene may be one or more selected from the group consisting of small interference RNA (siRNA), plasmid DNA, antisense oligodeoxynucleotide (AS-ODN), and the like.
The hormone may be one or more selected from the group consisting testosterone, estradiol, progesterone, prostaglandins, and their synthetic analogs, and a substance that is modified or shows the same effect.
The anti-cancer drug may be one or more selected from the group consisting of paclitaxel, doxorubicin, 5-fluorouracil, cisplatin, carboplatin, oxaliplatin, tegafur, irinotecan, docetaxel, cyclophosphamide, cemcitabine, ifosfamide, mitomycin C, vincristine, etoposide, methotrexate, topotecan, tamoxifen, vinorelbine, camptothecin, danuorubicin, chlorambucil, bryostatin-1, calicheamicin, mayatansine, levamisole, DNA recombinant interferon alfa-2a, mitoxantrone, nimustine, interferon alfa-2a, doxifluridine, formestane, leuprolide acetate, megestrol acetate, carmofur, teniposide, bleomycin, carmustine, heptaplatin, exemestane, anastrozole, estramustine, capecitabine, goserelin acetate, polysaccharide potassium, medroxyprogesterone acetate, epirubicin, letrozole, pirarubicin, topotecan, altretamine, toremifene citrate, BCNU, taxotere, actinomycin D, anasterozole, belotecan, imatinib, floxuridine, gemcitabine, hydroxyurea, zoledronate, vincristine, flutamide, valrubicin, streptozocin, polyethylene glycol conjugated anti-cancer agent, and their synthetic analogs, and a substance that is modified or shows the same effect.
The angiogenesis inhibitor may be one or more selected from the group consisting of clodronate, 6-deoxy-6-demethyl-4-dedimethylaminotetracycline (COL-3), doxycycline, marimastat, 2-methoxyestradiol, squalamine, SU5164, thalidomide, TNP-470, combretastatin A4, soy isoflavone, enzastaurin, CC 5013 (Revimid; Celgene Corp, Warren, N.J.), celecoxib, ZD 6474, halofuginone hydrobromide, interferon-alpha, bevacizumab, shark cartilage extract (AE-941), interleukin-12, vascular endothelial growth factor-trap (VEFG-trap), cetuximab, rebimastat, matrix metalloproteinase (MMPs) inhibitor (e.g., BMS-275291 (Bristol-Myers Squibb, New York, N.Y.), S-3304), etc.), protein kinase C beta inhibitor (e.g., LY317615), endostatin, vatalanib (PTK787/ZK 222584), sunitinib malate (SU11248), cilengitide (EMD-121974), humanized monoclonal antibody MEDI-522, EOS-200-4, integrin alpha-5-beta-1 antagonist (ATN-161), and their synthetic analogs, and a substance that is modified or shows the same effect.
The sugars may be one or more selected from the group consisting of lactose, glucose, dextran, mannose, sucrose, trehalose, maltose, and ficoll.
The polyols may be one or more selected from the group consisting of innositol, mannitol, and sorbitol.
The sugar-containing polyols may be sucrose-mannitol, glucose-mannitol, or a combination thereof.
The polymer-containing polyols may be one or more selected from the group consisting of trehalose-polyethylene glycol (trehalose-PEG), sucrose-polyethylene glycol (sucrose-PEG), and sucrose-dextran.
The sugar-containing amino acids maybe sorbitol-glycine, sucrose-glycine, or a combination thereof.
The sugar-containing ions may be trehalose-zinc sulfate (trehalose-ZnSO4), maltose-zinc sulfate (maltose-ZnSO4), or a combination thereof.
The physiologically active material may be a stem cell differentiation regulator. In this case, the hydrogel composition may be used in controlling stem cell differentiation when co-cultured with stem cells. The stem cell differentiation regulator is the same as described below.
Alternatively, the physiologically active material may be a material that exhibits a therapeutic effect in a body, but is not limited thereto.
In the hydrogel inclusion complex the present disclosure, the physiologically active material may be connected via a linker. The ‘linker’ (or spacer) provides a gap between the guest molecule and the physiologically active material, making it easy for the physiologically active material of the 3D hydrogel to adhere to a bioactive site. For example, as the linker between the quest molecule and the physiologically active material, polyethylene glycol (PEG), polyetherimide (PEI), or polypropylene glycol (PEG) having a molecular weight of 200 Da to 5,000 Da, or a polypeptide selected from the group consisting of polyglycine, polyhistidine, and poly(RADA) may be used. In non-limiting exemplary embodiment of the present disclosure, PEG of 1.0 kDA was used (Examples 1 and 2).
Another aspect of the present invention provides a hydrogel composition for controlling stem cell differentiation, the hydrogel composition including, as active ingredients, thermosensitive poly(phosphazene) to which a plurality of hydrophobic amino acids, hydrophilic polymers, and beta-cyclodextrin are substituted; and a stem cell differentiation regulator linked directly or via a linker to one or more molecules, as a guest molecule, selected from the group consisting of adamantine, azobenzene, cholesterol, tert-butyl, cyclohexyl ester, and naphthyl.
The terms used herein are the same as described above.
Further, the present invention provides use of the hydrogel composition in controlling stem cell differentiation, the hydrogel composition including, as active ingredients, thermosensitive poly(phosphazene) to which a plurality of hydrophobic amino acids, hydrophilic polymers, and beta-cyclodextrin are substituted; and a stem cell differentiation regulator linked directly or via a linker to one or more molecules, as a guest molecule, selected from the group consisting of adamantine, azobenzene, cholesterol, tert-butyl, cyclohexyl ester, and naphthyl.
As used herein, the term ‘stem cells’ collectively refers to undifferentiated cells at a stage prior to differentiation into cells constituting each tissue, and stem cells differentiate into specialized cells under specific differentiation stimulation (environment). Unlike differentiated cells of which cell division is stopped, stem cells have the characteristic of proliferation (expansion) because they are able to produce the same cells as themselves by cell division (self-renewal), and stem cells also have plasticity in differentiation, because they differentiate into specialized cells when differentiation stimulation is applied, and may also differentiate into different cells under different environment or different differentiation stimulation. These stem cells may classified into embryonic stem cells and adult stem cells according to their origin, and in the present invention, adult stem cells are preferably used, rather than embryonic stem cells which have many biological, ethical, and legal problems and are limited in clinical applications. Among adult stem cells, mesenchymal stem cells (MSCs) rarely present in adult tissues such as bone marrow and adipose tissue may be used.
In the present disclosure, the use in controlling stem cell differentiation means a use in controlling stem cells to induce differentiation of stem cells to specialized cells such as chondrocytes, osteocytes, neurocytes, neuroblasts, muscle cells, adipocytes, etc.
The hydrogel composition for controlling stem cell differentiation of the present disclosure may be directly implanted to a disease site of an individual, together with stem cells, and may be cultured in vitro. For implantation, both a non-surgical treatment of using a catheter and a surgical treatment of injecting after incision of a disease site are possible.
As used herein, the ‘stem cell differentiation regulator’ refers to a chemical substance, a protein, or a peptide that influences stem cell differentiation. In particular, a material that exists in an extracellular matrix (ECM) and controls cell proliferation and differentiation may correspond thereto. Non-limiting examples thereof may include proteins such as BMP, N-cadherin, insulin-like growth factor (IGF), fibroblast growth factor (FGF), and transforming growth factor β (TGF-β). Use of natural proteins generates problems related to stability and a financial pressure, and thus biomimetic peptides derived therefrom may be used.
The stem cell differentiation regulator of the present disclosure may be a peptide including arginine-lysine-aspartic acid (RGD). In the hydrogel composition, when a ratio of the number of moles of RGD to the number of moles of the host molecule is in a range of 50% to 100%, the hydrogel composition may be a composition for inducing chondrocyte and/or osteocyte differentiation. Specifically, the hydrogel composition may induce differentiation in an early hypertrophic stage. When the ratio of the number of moles of RGD to the number of moles of the host molecule is in a range of 0% to 25%, the hydrogel composition may be a composition for inducing adipocyte differentiation.
In this regard, in non-limiting exemplary embodiments of the present disclosure, among ECM molecule candidates, arginine-lysine-aspartic acid (RGD) which is an adhesive and developmentally effective peptide to MSC was selected as the stem cell differentiation regulator in the present invention (Example 1). RGD is a tri-peptide involved in MSC recognition, attachment, survival, and differentiation, and is a major binding site within fibronectin. Therefore, possessing of RGD in 3D hydrogel is necessary for survival and differentiation of MSC. RGD stimulation to MSC leads to various differentiation, such as osteocytes, chondrocytes, and adipocytes. According to Experimental Example 3 of the present disclosure, it was found that the fate of MSC may be controlled only by controlling the amount of Ad-RGD (see
Further, in a situation where synthesis of hydrogel by controlling the concentration of various physiologically active materials present in ECM is required to attempt several synthesis batches, bone/cartilage/fat production could be induced by controlling the concentration of Ad-RGD as desired using host-guest interaction in the present disclosure (
Meanwhile, the stem cell differentiation regulator may be a peptide including CESPLKRQ and a peptide including CLRAHAVDIN. Here, the molar ratio of the peptide including CESPLKRQ and the peptide including CLRAHAVDIN may be in a range of 4:6 to 6:4. In this case, the hydrogel composition may be a composition for inducing differentiation into chondrocytes.
In this regard, TGF-β with a molecular weight of 25 kDa exists in the site of embryonic bone and cartilage development and has a critical role tor the intracellular signaling cascade facilitating cartilage-specific gene expression. In particular, TGF-β1 regulates MAPK including p38, extracellular signal regulated kinase 1 (ERK 1), and c-Jun N-terminal kinase (JNK) as chondrogenesis controllers. In non-limiting exemplary embodiments of the present disclosure, CESPLKRQ was used, which is the most reactive binding site to TGF-β1 receptors in the whole peptide sequences of TGF-β1.
Next, a natural protein scarce for mimicking as a peptide is N-cadherin significant for cell to cell interaction and chondrogenesis. N-cadherin has a molecular weight of about 99.7 kDa. In the recent decade, the His-Ala-Val (HAV) motif inducing MSC chondrogenesis and mimicking N-cadherin action was highlighted in many studies. Although the sequence of HAV alone is enough to induce MSC chondrogenesis, an extended HAV sequence such as CLRAHAVDIN was shown to be more excellent as an effective motif. Hence, in non-limiting exemplary embodiments of the present disclosure, this extended HAV peptide sequence was chosen. Consequently, the present inventors chose a couple of peptide sequences derived from natural proteins such as TGF-β1 mimicking CESPLKRQ and N-cadherin mimicking CLRAHAVDIN under the stoichiometric peptide ratio control by a host-guest interaction. These peptides could affect MSCs to induce successive reaction of MSC with the mechanism of cell to cell interaction and intracellular signaling of mitogen activated protein kinase (MAPK), respectively.
In Experimental Example 6 of the present disclosure, the highest chondrogenic gene and protein expression levels were observed in T50 and H50 (see
Still another aspect of the present invention provides a hydrogel composition for inhibiting cancer cell proliferation or metastasis, the hydrogel composition including, as active ingredients, thermosensitive poly(phosphazene) to which a plurality of hydrophobic amino acids, hydrophilic polymers, and beta-cyclodextrin are substituted; and IL-2 linked directly or via a linker to one or more molecules, as a guest molecule, selected from the group consisting of adamantine, azobenzene, cholesterol, tert-butyl, cyclohexyl ester, and naphthyl.
Additionally, still another aspect of the present invention provides use of a hydrogel composition in inhibiting cancer cell proliferation or metastasis, the hydrogel composition including, as active ingredients, thermosensitive poly(phosphazene) to which a plurality of hydrophobic amino acids, hydrophilic polymers, and beta-cyclodextrin are substituted; and IL-2 linked directly or via a linker to one or more molecules, as a guest molecule, selected from the group consisting of adamantine, azobenzene, cholesterol, tert-butyl, cyclohexyl ester, and naphthyl.
In this regard, the hydrogel composition may be administered directly to or near the tumor, but is not limited thereto.
Meanwhile, the hydrogel inclusion complex of the present invention may be prepared by a process including a first step of preparing thermosensitive poly(phosphazene) to which a plurality of hydrophobic amino acids, hydrophilic polymers, and host molecules are substituted; a second step of preparing a physiologically active material linked to a guest molecule via a linker; and a third step of mixing the poly(phosphazene) with the physiologically active material.
In the present disclosure, it was confirmed that the hydrogel composition for optimized chondrogenic differentiation of MSCs may be prepared without a further synthesis process after stoichiometrically controlling Ad-TGF and Ad-HAV, based on the host-guest interaction (
Although some hydrogels were prepared with some Ad-peptide combinations for the illustrative purpose in the present disclosure, more Ad-peptide ratio combinations and sequences may also be used to easily prepare optimized niche for differentiation of stem cells to a desired state. Eventually, this technology provides a platform system by switching guest molecules or ratios to manufacture ideal 3D biomedical constructs. Accordingly, the hydrogel composition of the present disclosure, in which the kind, ratio, and sequences of the physiologically active material suitable for stem cell differentiation are controlled, may be easily prepared.
In this context, based on the composition of ECM known to be required for differentiation of stem cells to a specific state, known information about the kind and ratio of the physiologically active materials may be obtained, before the second step. Based on this information, the physiologically active material of the second step may be prepared according to the above information and ratio. Accordingly, the prepared hydrogel composition may be finely controlled such that it includes the physiologically active material according to the kind and ratio.
In the preparation method, when the number of moles of the physiologically active material is larger than the number of moles of the host molecule, a large amount of the physiologically active material is not bound, and thus the number of moles of the physiologically active material is preferably smaller than the number of moles of the host molecule.
Still another aspect of the present invention provides thermosensitive poly(phosphazene) to which a plurality of hydrophobic amino acids, hydrophilic polymers, and beta-cyclodextrin are substituted.
In this regard, the beta-cyclodextrin is characterized in that it is linked to the main chain of poly(phosphazene) via a hydroxyl group at C6 position of C1-6 alkylene diamine, poly(C1-6 alkylene diamine), n-amino-n-oxoalkanoic acid (wherein n is an integer of 2 to 6), thiol, carboxylate, C2-6 hydroxyalkyl m-amino-m-oxoalkanoic acid (wherein m is an integer of 2 to 6), or cyano-amino-C1-4 alkylthio-C1-6 alkane as a linker. By including the beta-cyclodextrin linked via the linker of a suitable length having a predetermined functional group as described above, the influence on the gelling temperature of the thermosensitive poly(phosphazene) according to the substitution may be controlled to provide a thermosensitive gel which is gelled at a temperature near the be temperature.
Hereinafter, the present invention will be described in more detail with reference to the following Examples. However, these Examples are for illustrative purposes only, and the scope of the present invention is not intended to be limited thereby.
Materials
Hexachlorocyclotriphosphazene (Aldrich) was purified by sublimation at under vacuum (about 0.1 mmHg). Poly(dichlorophosphazene) was prepared according to a known method (Sohn, Y. S. et al., Macromolecules 1995, 28 (22), 7566-7568). It was prepared from hexachlorocyclotriphosphazene using aluminum chloride (AlCl3) as a catalyst at 250° C. for 5 hr. L-Isoleucine ethyl ester hydrochloride (IleOEt.HCl) was prepared from L-isoleucine (Aldrich) according to a known method. α-Amino-ω-methoxy-poly(ethylene glycol) (AMPEG) with a molecular weight of 750 Da was prepared according to a known method (Loccufier, J.; Crommen at. al., Die Makromolekulare Chemie, Rapid Communications 1991, 12 (3), 159-165). Tetrahydrofuran (THF) and triethylamine (TEA) (Junsei Chemical Co., Ltd.) were purified under the dry nitrogen atmosphere by refluxing at the boiling point over sodium metal/benzophenone (Acros) and barium oxide (Acros). β-Cyclodextrin purchased from Aldrich was used without further purification. Mono-6-OTs-βCD and mono-6-diethylamino-βCD (NH2-βCD) were synthesized according to a known method (Liu, Y.-Y.; Fan et. al., Macromolecular Bioscience 2003, 3 (12), 715-719). Acetonitirile (ACN), ethanol amine (AEtOH), 4-(dimethylamino) pyridine (DMAP), isobutyl chloroformate (IBCF) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were obtained from Aldrich. Dichloromethane (DCM) with an extra pure quality was purchased from Daejung chemical company (Korea) and used without further purification.
(1) Synthesis of Acid PPZ
An acid polymer was synthesized as elucidated below. IleOEt.HCl (7.36 g, 0.03 mmol) suspended in dried THF (200 mL) containing TEA (16.54 mL, 0.12 mmol) was slowly added to poly(dichlorophosphazene) (3.00 g, 0.03 mmol) dissolved in dried THF (100 mL). The reaction mixture was stirred at the dry ice with acetone bath for 12 hr, and then kept stirring up to room temperature for 36 hr. To this mixture, AEtOH (2-aminoethanol, 0.46 ml, 0.008 mmol) dissolved in dried THF (20 mL) containing TEA (2.16 mL, 0.02 mmol), and AMPEG750 (7.57 g, 0.01 mmol) dissolved in dried THF (50 mL) containing TEA (4.92 g, 0.04 mmol) were gradually added, and the reaction mixture was stirred at room temperature for 24 hr, and then at 40° C. to 50° C. for 24 hr. The polymer reaction mixture was purified according to a known method. Briefly, the reaction mixture was filtered. After the filtrate was concentrated, it was poured into n-hexane to obtain a precipitate, which was re-precipitated in the same solvent system. The polymer product was dialyzed with a dialysis membrane (MW 12,000-14,000 cutoff) against methanol for 3 days, and distilled at 4° C. for 4 days. The finally dialyzed solution was subsequently freeze-dried to obtain poly(organophosphazene) carrying AEtOH. Finally, the carboxylic acid-terminated polymer was obtained by following reactions. A solution of a polymer carrying AEtOH (5.84 g, 0.01 mmol) in distilled THF (200 mL) was stirred at room temperature under nitrogen atmosphere. To this polymer solution, each solution of glutaric anhydride (3.18 g, 0.02 mmol) and 4-(dimethylamino) pyridine (DMAP) (3.41 g, 0.02 mmol) in distilled THF (50 mL) was added. The reaction mixture was stirred at 40° C. for 24 hr. After stirring, the reaction mixture was filtered and concentrated. The polymer product was purified by dialysis in methanol for 3 days and then by dialysis in distilled water at 4° C. for 3 days. The dialyzed solution was freeze-dried to obtain poly(organophosphazene) carrying glutaric acid. A schematic illustration of the synthesis is shown in
(2) Synthesis of β-CD PPZ
An acid polymer (3.70 g, 6.77 mmol) was dissolved in ACN (100 mL) with stirring. Then, DMAP (2.48 g, 20.31 mmol), EDC (3.15 g, 20.31 mmol), and TEA (2.83 mL, 20.31 mmol) were added to the fully dissolved polymer solution. NH2-βCD (0.54 g, 10.16 mmol) was dissolved in deionized water (10 mL). To activate the carboxyl group of the polymer, the deionized water solution of NH2-βCD was added to the polymer solution 30 min after the addition of DMAP, EDC, and TEA. The reaction mixture was stirred at room temperature for 48 hr, and then evaporated under reduced pressure to collect the polymer. The collected polymer product was dissolved in methanol. The polymer product was dialyzed with a dialysis membrane (MW 12,000-14,000 cutoff) against methanol for 3 days and against deionized water at 4° C. for 4 days, and the finally dialyzed solution was subsequently freeze-dried. A schematic illustration of the synthesis is shown in
In the synthesized β-CD PPZ, the specific evidence for β-CD conjugation was observed with an anomeric proton peak of β-CD at 4.03 ppm after synthesis of β-CD PPZ (C of
(3) Synthesis of Adamantane-PEG-MeAc
A schematic illustration of the synthesis is shown in
Ad-PEG-NH2 (1 g, 0.3 mmol) was dissolved in DMC. Subsequently, methacryloyl chloride (0.97 mL, 9.3.mmol) and TEA (1.38 mL, 9.95 mmol) were added to the Ad-PEG-NH2 solution. The reaction was performed at 50° C. for 1 day. The resulting solution was purified by evaporation under reduced pressure, dialysis with MWCO 1 kDa, and lyophilization. The NMR value of the prepared Ad-PEG-MeAc is shown in
(4) Synthesis of Adamantane-PEG-RGD (Ad-RGD)
A schematic illustration of the synthesis is shown in
(5) Preparation of Conjugate in which β-CD PPZ and Ad-RGD are Mixed
Conjugates were prepared by mixing the prepared β-CD PPZ and Ad-RGD. The prepared conjugates included the Ad-RGD molecule at a concentration of 0%, 25%, 50%, or 100%, based on the number of host molecules present in the β-CD PPZ hydrogel. Ad-RGD 100, 50, and 25 mean that guest molecules are inserted to the host molecules of β-CD polymer through 100%, 50%, 25% host-guest interaction, respectively. Exact concentrations of Ad-RGD 100, 50, and 25 are described in Table 1 below.
As described, they were ultimately prepared in order to control the contents of stem cell adhesive moieties (i.e., RGD) in the 3D hydrogel space.
(6) Characterization of β-CD PPZ/Ad-RGD
The structures of the prepared polymers were estimated by measuring 1H NMR (Bruker avance III 400 MHz Fourier transform mode (DMSO-d6 and CDCl3). The viscosity of the aqueous polymer solutions was assessed on a Brookfield RVDV-III+ viscometer between 5° C. and 70° C. under a fixed shear rate of 0.1. The measurements were carried out with a set spindle speed of 0.2 rpm and with a heating rate of 0.33° C./min.
Spatial information was obtained from 2D-NMR (NOESY) with a 1:1 to 1:0 molar mixture of β-CD and adamantine containing RGD dissolved in DMSO-d6. 2D-NMR spectra were recorded on a DD2 600 MHz FT NMR (Agilent Technologies), and are shown in
In this example, it was hypothesized that binding affinity between β-CD and Ad-RGD would induce the well-controlled guest molecule amount through the Ad-RGDs inserted at different amounts with one another. First of all, β-CD PPZ and β-CD PPZ+Ad-RGD gave different results from measurement of dynamic light scattering (DLS). The average particle size of β-CD PPZ and β-CD PPZ+Ad-RGD in an aqueous environment were 121.3±26.2 nm and 180.3±32.4 nm, respectively. As Ad-RGDs were included in β-CD PPZ-, the practical size of aqueous particles of β-CD PPZ+Ad-RGD was increased.
To elucidate the direct evidence of the host-guest interaction, the integration values of 2D-NOESY of β-CD PPZ and Ad-RGD was measured with the increase of guest molecules. First of all, in the 2D-NOESY results, the inclusion complex was confirmed at cross peaks composed of Ad protons of methylene (Ha, Hc), methane (Hb), and β-CD inner cavity protons of H-5 (A of
The viscosity change between acid PPZ and β-CD conjugate was observed. Acid PPZ Tmax (temperature at which the solution reaches its maximum viscosity) was 48.8° C. which is above the body temperature. The Tmax of β-CD PPZ, in which hydrophilic carboxylic acid groups in acid PPZ were substituted with only β-CD possessing another hydrophilic moiety (i.e., OH group), was decreased to approximately 36.8° C. (A of
Gelation after the preparation of the mixture of β-CD PPZ and Ad-RGD was examined. As a result, in a condition where the temperature is lower than the body temperature (4° C.), the β-CD PPZ where Ad-RGD 0, Ad-RGD 50, and Ad-RGD 100 were added showed an aqueous state (B of
(1) Cell Culture Method
Mouse mesenchymal stem cells (mMSCs) were purchased from Cyagen Biosciences Inc. mMSCs were cultured with Dulbecco's Modified Eagle's medium (DMEM) (Gibco BRL, Grand Island, N.Y.) containing 1% penicillin-streptomycin (Sigma-aldrich, USA) and 10% fetal bovine serum (FBS) (Welgene, Korea) in dishes at 37° C. in a humidified atmosphere of 5% CO2 and 95% air.
(2) Live/Dead Cell Viability Assay of Mesenchymal Stem cells & measurement of CCK-8 cultured with 3D hydrogel niche
Harvested mMSCs (passage 7, 5×105 cells) were suspended in 0.1 mL of 10 wt % prepared hydrogel. mMSC/hydrogel mixtures were incubated in a 24-well culture plate with a cell insert.
At day 0, day 1, day 3, and day 7, the cell medium was removed, and calcein AM/ethidium homodimer-1 (Live/dead cell viability assay kit, Thermo Fisher Scientific Inc.) dissolved in a DPBS solution was used to perform a live/dead cell viability assay of mesenchymal stem cells. All images were obtained by a confocal microscope (Zeiss LSM 800, DE) in a 3D state.
For CCK-8 assay, on day 7, all of the 3D hydrogels including mMSCs were destroyed with media. The cultured hydrogel was moved to a 96-well culture plate (SPL life sciences, KR), and 10 μL of CCK-8 (Dojindo Molecular Technology, Inc. JP) solution was added to each well. CCK-8 solution containing the hydrogel was placed in a cell incubator in a humidified atmosphere at 37° C., 5% CO2 for 2 hr. After incubation, the absorbance was measured using a microplate reader (BIO-RAD, Hercules, Calif., USA) at a wavelength of 450 nm.
(3) Gene Assay of Mesenchymal Stem Cells Cultured with 3D Hydrogel Niche (RT-PCR)
An RNA extract was prepared by using Trizol (Invitrogen, Carlsbad, Calif.). After samples were treated with DNase (Invitrogen), 1 mg of total RNA was used for cDNA synthesis (Superscript First-strand synthesis system, GibcoBRL, Life Technologies). In brief, a reverse transcription reaction was carried out in a 20 mL mixture (1 RT buffer, 1.25 mM MgCl2, 5 mM DTT, 2.5 g random hexamer, 0.5 mM each of dATP, dCTP, dGTP, and dTTP, and 50 U of Superscript II enzyme). After the reverse transcription reaction, RNA was degraded by 2 U of Escherichia coli RNase H. PCR was performed in 50 mL of a reaction buffer containing 2 U of Takara Taq, 1× PCR buffer, 0.8 mM dNTP mixture, and 100 pmol of specific primers. Standard PCR conditions were as follows: at 95° C. for 3 min, followed by cycles of denaturation at 95° C. for 5 sec, annealing at 60° C. for 34 sec, and extension at 72° C. for 1 min. Oligonucleotides used as primers are described in Table 2. The gene expression values were normalized against the housekeeping gene of β-actin.
(4) Experimental Salts
A simple mixing mediated RGD concentration controllable system was designed to manufacture a stem cell 3D niche based on a host-guest interaction. The main integrin-binding domain, RGD, is abundant in ECM proteins such as fibronectin, vitronectin, fibrinogen, osteoponin, and bone sialoprotein. The manufactured 3D hydrogel stem cell niche was regulated by the amount of guest molecule, Ad-RGD. MSC survival rate was evaluated in recent studies with 3D scaffold of several synthetic materials, and the results showed 50% or higher viability within specific conditions.
The results of evaluating the survival rates of MSCs in the stem cell culture system are shown in C of
Conjugates in which Ad-RGDs of 0%, 25%, 50%, and 100% based on the number of moles of β-CD in the β-CD PPZ hydrogel were added were fabricated according to Example 1, and the conjugates were added to MSCs.
With regard to MSC differentiation, when Ad-RGD 50 and 100 were used, high binding possibility between Ad-RGD and α5β1 integrin of MSCs resulted in enhanced gene expression of osteogenic factors (ALP and collagen I) and chondrogenic factors (aggrecan and collagen II) (A, B, D, and E of
On the other hand, adipogenesis-related factors (C/EBPα and PPARγ) were expressed highly in the low level or in the absence of Ad-RGDS such as RGD 0 and RGD 25 (C and F of
From these results, a schematic illustration of MSC differentiation activity of the conjugate of β-CD PPZ and Ad-RGD of the present invention is shown in
(1) Synthesis of PPZ
β-CD PPZ was prepared in the same manner as described in (1) and (2) of Example 1.
(2) Synthesis of Ad-PEG-MeAc
Ad-PEG-MeAc was prepared in the same manner as described in (3) of Example 1.
(3) Synthesis of Ad-TGF
The prepared Ad-PEG-MeAC (M.W. 1302.6 Da, 200 mg, 0.15 mmol) was dissolved in an aqueous solution of which pH was adjusted to 10.0. Tris(2-carboxyethyl)phosphine (TCEP) (44.0 mg, 0.15 mmol) and TGF-β1 mimic peptide (CESPLKRQ, 960.12 Da, 442.2 mg, 0.46 mmol) were simultaneously added to the aqueous solution. The reaction solution was purged in N2 atmosphere for 10 min. The reaction was performed for 2 hr at room temperature. After reaction, purification was carried out with dialysis and lyophilization. A schematic illustration of the synthesis is shown in
(4) Synthesis of Ad-HAV
The prepared Ad-PEG-MeAc (M.W. 1302.6 Da, 300 mg, 0.23 mmol) was dissolved in an aqueous solution of which pH was adjusted to 10.0. TCEP (66.0 mg, 0.23 mmol) and N-cadherin mimic peptide (CLRAHAVDIN, 1,111.29 da, 767.8 mg) were simultaneously added to the aqueous solution. The reaction solution was purged in N2 atmosphere for 10 min. The reaction was performed for 2 hr at rooms temperature. After reaction, purification was carried out with dialysis and lyophilization. A schematic illustration of the synthesis is shown in
(5) Preparation of Conjugates by Mixing β-CD PPZ with Ad-TGF and Ad-HAV
The prepared β-CD PPZ was mixed with Ad-TGF and Ad-HAV in the ratio of the following Table 3 to prepare conjugates.
(6) Characterization of β-CD PPZ/Ad-TGF and(or) HAV
The structures of the prepared β-CD PPZ, Ad-HAV, and Ad-TGF were estimated by measuring 1H NMR (Bruker avance III 400 MHz Fourier transform mode with DMSO-d6 and CDCl3) (
The viscosity of the aqueous polymer solutions was assessed on a Brookfield RVDV-III+ viscometer between 5° C. and 70° C. at a fixed shear rate of 0.1. The measurements were carried out with a set spindle speed of 0.2 rpm and with a heating rate of 0.33° C./min (A of
Spatial information was obtained from 2D-NMR (NOESY) with a 1:1 molar mixture of β-CD and adamantine containing peptides dissolved in D2O. A2D-NMR spectra were recorded on DD2 600 MHz FT NMR (Agilent Technologies) (C of
To verify the host-guest interaction between β-CD PPZ and Ad-peptides, 2D NOESY spectra in an aqueous state with D2O under the same conditions of the prepared conjugate hydrogels were measured. 2D NMR is the strongest method of observing the intermolecular interactions and/or the configuration of an inclusion complex. Cross peaks in 2D-NOESY could be obtained from nuclei resonance connections that are spatially closer than a coupled bond. Ha, c, and Hb (δ 0.6 ppm to 1.3 ppm) of adamantane and H-5′ (δ 3.4 ppm to 3.5 ppm) of β-CD inner cavity, which are cross peaks in 2D-NOESY spectra involved in host-guest interactions, were fairly elucidated in C of
Further, to identify whether Ad-peptides were stoichiometrically and accurately inserted into the host molecules, dynamic light scattering (DLS) measurement was performed by mixing host molecules and guest molecules such that the ratio of host molecule:guest molecule was 1:1, 1:1.2, and 0:1. Above all, the increased. hydrodynamic diameters were observed in the conjugates in which the ratio of host molecule:guest molecule was 1:1 (C of
Meanwhile, whether the conjugates in which Ad-TGF and Ad-HAV were initially bound at different contents in β-CD PPZ could be maintained was examined using an in vivo imaging system (IVIS) with live animals. In the molecular tails of Ad-TGF and Ad-HAV, rhodamine (Rho) and fluorescein isothiocyanate (FITC) were linked in a conjugation process, respectively. Then, these fluorescence expressing guest molecules were injected along with β-CD PPZ in MSCs post-inclusion complex fabrication. Since β-CD in PPZ was released to the outside owing to biodegradability or dissolution of PPZ, and the Ad-peptide signals containing fluorescence became smaller over time. Furthermore, the stoichiometric control patterns of Ad-TGF/Ad-HAV were observed to employ different guest molecules for 21 days (A of
Following the synthesis of host and guest molecules, the rheological measurement and the visualization of thermosensitive sol gel transition with β-CD PPZ incorporating T100 H0 and T0 H100 were performed. Even when Ad-TGF 100 and Ad-HAV 100 were incorporates into β-CD PPZ, hydrogels of the prepared conjugates also showed the gelation properties and enough viscosity in the body temperature (
(1) Measurement Method of Biocompatibility
mMSCs (mouse mesenchymal stem cells) and NIH3T3 (mouse fibroblast cells) with a constant density (1×104 cells/well) were seeded in a 96-well tissue culture plate (SPL, Korea). Each cell line was incubated for 24 hr with the well dissolved β-CD PPZ (concentration: 0 μg/mL to 20,000 μg/mL). After incubation, the medium used was discarded and cells were washed once with DMEM and fresh PBS. After adding a fresh medium (200 mL/well), 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazoliumbromide (MTT) solution (100 mg/well) was added to the cells followed by incubation for 4 hr at 37° C. in a humidified atmosphere of 5% CO2. The formed formazan crystals were solubilized by incubating the cells with DMSO. The absorbance of the solution was measured at 570 nm using a microplate reader (Bio-Tek Instruments, USA). The cell viability (%) was calculated from [ab]test/[ab]control×100%.
(2) In Vivo Tissue Generation Test
All of the experiments with live mice were accomplished in compliance with the relevant laws and institutional guidelines of Institutional Animal Care and Use Committee (IACUC) in Korea Institute of Science and Technology (KIST). IACUC approved the experiment (approval number: 2017-092). Balb/c nude mice (4 weeks old, 20-25 g, female) were purchased from Nara Bio INC. (Gyeonggi-do, Korea). Nude mice were anaesthetized with 3% isoflurane in the balanced oxygen and nitrogen. MSCs/β-CD PPZ (10 wt %)/0% to 100% of Ad-TGF (T, adamantane-PEG1000-CESPLKRQ, 2,248.70 Da) and Ad-HAV (H, adamantane-PEG1000-CLRAHAVDIN, 2,399.87 Da) were injected into subcutaneous pockets in mice on their right sides lateral to the dorsal midline using a syringe with a 31 gauge needle. Each mouse received a 100 μL injection containing 2×106 cells mixed with 10 wt % of β-CD PPZ. All the tissues were collected 4 weeks post injection, and were used for histological examinations and gene analyses.
(3) Histological and Immunohistological Analyses
All the collected tissues were embedded in paraffin, and sectioned with a microtome (6 μm in thickness). For histological evaluation, tissue sections were deparaffinized, rehydrated, and stained with H&E, safranin-O, Von kossa, and. immunohistochemistry. For immunohistochemical staining, the sectioned tissues were incubated at 4° C. overnight with primary antibodies of anti-aggrecan (1:500, Abcam, ab3778) and anti-collagen II (1:500, Abcam, ab34712). After washing three times, the slides were incubated with appropriate secondary antibodies conjugated to fluorescent dyes, such as goat anti-mouse IgG (TRICT, Abcam, ab6786) and goat anti-rabbit IgG (Alexa 488, Abcam, ab150077). Images were captured using a confocal laser scanning microscope (Zeiss) and a bright imaging microscope (Zeiss).
(4) Gene Assay Measured in Artificial Tissue
An RNA extract was prepared by using Trizol (Invitrogen, Carlsbad, Calif.). After tissues were treated with DNase (Invitrogen), 1 mg of RNA was used for cDNA synthesis (Superscript First-strand synthesis system, GibcoBRL, Life Technologies). In brief, a reverse transcription reaction was carried out in a 20 mL mixture (1 RT buffer, 1.25 mM MgCl2, 5 mM DTT, 2.5 g of random hexamer, 0.5 mM each of dATP, dCTP, dGTP, and dTTP, and 50 U of Superscript II enzyme) at 42′C. After the reverse transcription reaction, RNA was degraded by 2 U of Escherichia coli RNase H. PCR was performed in a 50 mL reaction buffer containing 2 U of Takara Taq, 1×PCR buffer, 0.8 mM dNTP mixture, and specific primers of 100 μmol. Standard PCR conditions were as follows: at 95° C. for 3 min, followed by cycles of denaturation at 95° C. for 5 sec, annealing at 60° C. for 34 sec, and extension at 72° C. for 1 min. Oligonucleotides used as primers are as described in Table 4 below. The gene expression values were normalized against the housekeeping gene of β actin.
(5) Experimental Results
According to (1) described above, an in vitro cytotoxicity test was performed using MTT assay in both cell lines of MSCs and NIH3T3. Although each cell line was treated with a high concentration (10,000 μg/mL) of a β-CD PPZ polymer solution, the cell viabilities of MSCs and NIH3T3 still remained high to be above 30%. Therefore, this polymer was regarded as appropriate for the net study of in-vivo animal test.
In the in-vivo tissue generation test, the biocompatibility in the body administered with β-CD PPZ/ratio-controlled Ad-peptides/MSCs was confirmed using H&E staining. According to the histological results from H&E staining, any foreign body response such as a generation of foreign body giant cells and any toxicity were not observed in all of the groups treated with β-CD PPZ/ratio-controlled Ad-peptides (A of
Moreover, MSC maintenance in living bodies were measured to examine whether local MSC maintenance enhances their therapeutic efficiency. Hence, MSCs capable of expressing green fluorescent protein (GFP) were encompassed with the inclusion of β-CD PPZ/Ad-peptide conjugate to confirm whether these MSCs are maintained in living animals. Locally injected MSCs were maintained in their injection sites for a period of 21 days. However, the significantly low maintenance of MSC fluorescence was monitored in the T0 H0 group in which both a receptor mediated MSC attachment and a cell to cell interaction available factor were excluded (B of
To evaluate exact MSC chondrogenesis levels in the flexible and different guest molecule contents, an analysis was performed with the typical chondrogenesis markers of type II collagen (Col II) and aggrecan (Agg). Based on the output, MSC chondrogenic differentiation with flexibility of guest molecules was shown in results of safranin-O staining. All of the groups, except the T0 H0 group, showed chondrogenic differentiation. Based on the results of safranin-O staining, the accurate chondrogenesis consequences were evaluated using the measurement of gene expression levels and immunohistochemistry. Neo-tissues resulting from the locally and subcutaneously injection of MSCs encompassed by β-CD PPZ and Ad-peptides were extracted from all of the experiment groups. Agg which is a cartilage-specific proteoglycan core protein was significantly synthesized and expressed in the all Ad-TGF and/or Ad-HAV incorporating groups. In particular, the highest Agg-specific fluorescence and gene expression levels were observed in T50 H50, compared to T100 H0, T75 H25, T25 H75, and T0 H100 groups (B and C of
Osteogenesis is a single continuous development process including cartilage formation as a precursor. In detail, even if hypertrophic chondrocytes are differentiated to osteocytes under specific stimulations such as runt-related transcription factor 2 (Run×2) and osterix, Ad-peptides used in the present disclosure should prevent any further process of chondrocytes to osteogenic termination. Inhibition of osteogenesis is important because the incorrect differentiation product at the terminal MSCs during the chondrogenesis process. Generally, Run×2 is considered as a typical and key transcription factor for osteogenesis. Hence, Run×2 was selected as a gene assay marker for detecting a further progress of osteogenesis. Furthermore, production of calcium, which is a a final product of osteogenesis, was confirmed via Von kossa staining. As a result, there was no calcium dot (A of
From these results, a schematic illustration of MSC differentiation activity of β-CD PPZ/ratio-controlled Ad-TGF/Ad-HAV conjugate of the present invention is shown in
(1) Synthesis of Host Molecule, β-CD PPZ
β-CD PPZ was prepared in the same manner as described in (1) and (2) of Example 1.
(2) Synthesis of Ad-PEG-MeAc
Ad-PEG-MeAc was prepared in the same manner as described in (3) of Example 1.
(3) Synthesis of Ad-IL2
1) Method 1 (via Click Chemistry)
The prepared Ad-PEG-MeAc (0.09 mg, 0.00007 mmol) was dissolved in an aqueous solution of which pH was adjusted to 10.0. TCEP (0.2 mg, 0.0007 mmol) and rhIL-2 (1 mg) were added simultaneously to the aqueous solution. The reaction solution was purged in N2 atmosphere for 10 min. The reaction was performed for 2 hr at room temperature. After reaction, purification was carried out with dialysis and lyophilization. A schematic illustration of the synthesis is shown in
2) Method 2 (via EDC Chemistry)
The prepared Ad-FEG-MeAC (0.09 mg, 0.00007 mmol) was dissolved in an aqueous solution with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) (0.28 mg, 0.0015 mmol). rhIL-2 (1 mg) was dissolved in an aqueous solution of Ad-PEG-NH2+EDC. The reaction solution was purged in N2 atmosphere for 10 min. The reaction was performed for 2 hr at room temperature. After reaction, purification was carried out with dialysis and lyophilization. A schematic illustration of the synthesis is shown in
(4) Characterization of β-CD PPZ/Ad-IL2
As a result of electrophoresis of a ladder, IL-2, and Ad-IL2 spreading on a polyacrylamide gel according to their molecular weight, the molecular weight of Ad-IL2 was found to be higher than that of IL-2.
(1) Experimental Method
5.0×105 cells of B16 melanoma cell line was injected into B6 mice (0 day, n=5). When the size of the cancer tissue reached 100 mm3 or larger, β-CD PPZ+Ad-IL2 complex and PBS alone were prepared and directly injected into the cancer tissues of each mouse, respectively. The size of the mouse cancer tissue was measured for a total of 12 days, and mean values thereof were calculated.
(2) Experimental Result
In the control PBS-injected group, it was confirmed that the size of the cancer tissue grew uncontrollably. During the experimental period, 2 mice out of 5 mice in the PBS group died due to hyperplasia of the cancer tissue. In contrast, in the group injected with β-CD PPZ+Ad-IL2 complex, all of the mice survived and the size of cancer tissue was also significantly well-suppressed, compared to the control group.
Based on the above description, it will be understood by those skilled in the art that the present disclosure may be implemented in a different specific form without changing the technical spirit or essential characteristics thereof. Therefore, it should be understood that the above embodiment is not limitative, but illustrative in all aspects. The scope of the disclosure is defined by the appended claims rather than by the description preceding them, and therefore all changes and modifications that fall within metes and bounds of the claims, or equivalents of such metes and bounds are therefore intended to be embraced by the claims.
A hydrogel composition of the present disclosure is prepared by linking a physiologically active material to thermosensitive poly(phosphazene) via a host-guest interaction. When injected into a living body, the hydrogel composition slowly releases the physiologically active material and provides favorable conditions for stem cell differentiation. In addition, the hydrogel composition, in which the kind, ratio, and sequence of the physiologically active material are controlled to be suitable for stem cell differentiation, may be prepared with high reproducibility.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2019-0073071 | Jun 2019 | KR | national |
