WO2007064152A1 - Injectable thermosensitive pluronic hydrogels coupled with bioactive materials for tissue regeneration and preparation mehtod thereof - Google Patents

Injectable thermosensitive pluronic hydrogels coupled with bioactive materials for tissue regeneration and preparation mehtod thereof Download PDF

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WO2007064152A1
WO2007064152A1 PCT/KR2006/005104 KR2006005104W WO2007064152A1 WO 2007064152 A1 WO2007064152 A1 WO 2007064152A1 KR 2006005104 W KR2006005104 W KR 2006005104W WO 2007064152 A1 WO2007064152 A1 WO 2007064152A1
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pluronic
growth factor
hydrogel
biologically active
active material
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PCT/KR2006/005104
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French (fr)
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Dong-Keun Han
Kwang-Duk Ahn
Seung-Hwan Cha
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Korea Institute Of Science And Technology
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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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6903—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to an injectable thermosensitive pluronic derivative hydrogel coupled with a biologically active material for tissue regeneration, and a preparation method thereof.
  • Tissue engineering is a new technology emerged during the progress of science, and is a study which integrates and applies science and technology with basic concepts of life science, engineering, medicine, etc.
  • Tissue engineering is an application study which has an objective to understand the correlation between the structure and the function of tissues of a living body, and to maintain, enhance or restore the functions of a human body by means of artificial tissues which are transplantable into the human body, so as to replace damaged tissues or organs by normal ones or to regenerate the same.
  • Tissue engineering techniques using hydrogels are largely divided into two categories.
  • a necessary tissue is extracted from a patient's body, and cells are separated from the tissue. Then, the cell is proliferated via a culture to a necessary amount, mixed with an injectable hydrogel, and then directly injected into the human body.
  • the cell is cultured in a hydrogel outside the human body and then injected into the human body.
  • the transplanted hydrogel is converted to a gel in the human body due to the body temperature, positioned at a specific site, and supplied with oxygen and nutrition by diffusion of the body fluid. If blood is supplied through a blood vessel which is extended into the human body, the cell is proliferated and differentiated to generate new tissues and organs. Then, the hydrogel is discharged outside the body or decomposed to disappear.
  • a hydrogel and a specific drug are mixed, and the resulting mixture is directly injected into a living body.
  • the mixture is converted to a gel due to the body temperature, and the hydrogel is gradually decomposed, by which the drug is delivered to the body for a long time at a suitable concentration.
  • thermosensitive hydrogels that can be converted to a gel at around the body temperature and that are similar to tissues of a living body. It is required that the hydrogels for regeneration of tissues of the human body be converted to a gel at around the body temperature while maintained as a sol at room temperature, and have an affinity to cells so that the cells can generate tissues having a three-dimensional structure within the hydrogels, and also function as a barrier between transplanted cells and host cells.
  • the representative polymer hydrogels having such thermosensitivity include Pluronic (P. Holmqvist et al., Int. J. Pharm., 194, 103, 2000), poly(N-isopropylacrylamide) (PNIPAAm) (M.
  • Polynipaam has its own toxicity.
  • Other hydrogels are disadvantageous in that they have relatively low mechanical properties, and do not have an affinity to cells sufficient to be used for tissue regeneration.
  • the pluronic hydrogels include F38, F68, F77, F77, F98, F108, F127, etc. beginning with 'F', L31 , L42, L43, L44, L62, L72, L101 , etc. beginning with 'L', and P75, P103, P104, etc. beginning with 'P' (respectively denoting trade names). All of these pluronic hydrogels have a structure of PEO-PPO-PEO, but different ratios or forms from one another. Among them, only F68 (molecular weight: 8,700) and F127 (molecular weight: 12,600) were approved by the U.S. FDA have been used as materials for a living body.
  • Pluronic F127 is a non-toxic copolymer (molecular weight: 12,600) of polyethyleneoxide (PEO)-polypropyleneoxide (PPO)-polyethyleneoxide (PEO) in a molar ratio of 98:68:98, and has a temperature-dependent sol-gel conversion properties, and accordingly, it has been used as a material, in a mixture with other materials, for a living body.
  • PEO polyethyleneoxide
  • PPO polypropyleneoxide
  • PEO polyethyleneoxide
  • thermosensitive pluronic hydrogel for tissue regeneration, to which biologically active materials are coupled, and which has an excellent cell affinity while maintains the thermosensitivity of the conventional pluronic hydrogel itself.
  • the present invention provides an injectable pluronic hydrogel having thermosensitivity and cell affinity by coupling a conventional pluronic polymer with a compound having a polymerizable double bond and a functional group, through which a biologically active material such as a ligand peptide or a growth factor can be introduced, while maintaining the thermosensitivity of the conventional pluronic polymer, followed by directly introducing a biologically active material which can improve cell affinity.
  • a biologically active material such as a ligand peptide or a growth factor
  • the polymerizable double bond can be later used for a polymerization, if desired.
  • the present invention relates to an injectable thermosensitive pluronic hydrogel for tissue regeneration, and a preparation method thereof.
  • the injectable thermosensitive pluronic hydrogel for tissue regeneration has a structure in which a thermosensitive pluronic F127 polymer is coupled with a biologically active material such as a ligand peptide having a cell affinity or a growth factor through methacryloxyethyltrimellitic acid.
  • a biologically active material such as a ligand peptide having a cell affinity or a growth factor through methacryloxyethyltrimellitic acid.
  • the ligand peptide having a cell affinity is at least one selected from the group consisting of Arg-Gly-Asp (RGD), Arg-Glu-Asp-Val (REDV), Leu-Asp-Val (LDV), Tyr-lle-Gly-Ser-Arg (YIGSR), Pro-Asp-Ser-Gly-Arg (PDSGR), Ile-Lys-Val-Ala-Val (IKVAV) and Arg-Asn-lle-Ala-Glu-lle-lle- Lys-Asp-Ala (RNIAEIIKDA).
  • RGD and PDSGR enhance adhesion of almost all cells
  • REDV and LDV enhance proliferation of vascular endotheliocytes
  • YIGSR enhance proliferation of vascular cells
  • IKVAV and RNIAEIIKDA enhance proliferation of nerve cells.
  • the growth factor is at least one selected from the group consisting of a transforming growth factor (TGF- ⁇ ), an insulin-like growth factor (IGF), an epithelia growth factor (EGF), a nerve cell growth factor (NGF), a vascular endothelial growth factor (VEGF), a fibroblast growth factor (FGF), a hepatocyte growth factor (HGF) and a platelet-derived growth factor (PDGF).
  • TGF- ⁇ transforming growth factor
  • IGF insulin-like growth factor
  • EGF epithelia growth factor
  • NEF nerve cell growth factor
  • VEGF vascular endothelial growth factor
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • PDGF platelet-derived growth factor
  • methacryloxyethyltrimellitic acid used for coupling pluronic F127 with a biologically active material is the one derived from 4-methacryloxyethyltrimellitic anhydride (4-META) or 2-methacryloxyethyltritrimellitic anhydride (2-META).
  • 4-META 4-methacryloxyethyltrimellitic anhydride
  • 2-META 2-methacryloxyethyltritrimellitic anhydride
  • the following Formula 1 shows a specific example of an injectable thermosensitive pluronic hydrogel (META-pluronic F127-R) for tissue regeneration according to the present invention, in which pluronic F127 is coupled with a biologically active material through 4-methacryloxyethyltritrimellitic anhydride.
  • a preparation method for the injectable thermosensitive pluronic hydrogel for tissue regeneration according to the present invention comprises:
  • thermosensitive pluronic hydrogel (1) reacting a thermosensitive pluronic hydrogel with methacryloxy- ethyltritrimellitic anhydride at room temperature to obtain methacryloxy- ethyltritrimellitic anhydride-pluronic polymer (META-pluronic F127); and
  • the methacryloxyethyltritrimellitic anhydride may be 4-methacryloxyethyltritrimellitic anhydride (4-META) or 2-methacryloxyethyl- tritrimellitic anhydride (2-META).
  • the 4-META which has been used as a conventional dental adhesive has no toxicity and a relatively excellent mechanical property.
  • 4-META or 2-META has a double bond at its one end, so as to enable polymerization, and an anhydride group at the other end, capable of being converted to a carboxyl group which can be used to couple a biologically active material.
  • step (1) pluronic F127 and META are put into a reactor in a molar ratio of 1 :2.2, the reactor is charged with nitrogen at room temperature, and then a reaction is performed for 20-24 hours, to obtain META-pluronic F127 which is a derivative of pluronic F127.
  • step (2) a biologically active material is added to the META-pluronic
  • F127 prepared in step (1) in a molar ratio of 1 :2.2, and then the resulting mixture is reacted using 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC) or 1 -cyclohexyl-3-(2-morpholinoethyl)carbodiimidemeto-p-toluene sulfonate (CMC), to obtain META-pluronic F127-R, wherein R represents a biologically active material as defined above.
  • EDC 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide
  • CMC 1-morpholinoethylcarbodiimidemeto-p-toluene sulfonate
  • thermosensitive pluronic F127 hydrogel 4-META having a double bond and a carboxyl group is coupled with the conventional thermosensitive pluronic F127 hydrogel, thereby enabling polymerization and introduction of a biologically active material. Furthermore, the introduction of a ligand peptide or growth factor to META-pluronic F127 makes it possible to prepare an injectable thermosensitive hydrogel, a cell affinity of which is improved. Therefore, the pluronic hydrogels of the present invention is useful for regenerating tissues or organs by means of tissue engineering technique.
  • Reaction Scheme 1 shows an example for preparing the injectable thermosensitive pluronic hydrogel according to the present invention, comprising coupling a biologically active material to a derivative of pluronic F127 using 4-methacryloxyethyltritrimellitic anhydride.
  • R represents a biologically active material as defined above.
  • 4-META or 2-META having a double bond to enable polymerization and a carboxyl group is coupled to the conventional thermosensitive pluronic F127 hydrogel to obtain META- pluronic F127, and then a biologically active material such as a ligand peptide or a growth factor is introduced to the obtained META-pluronic F127 hydrogel to obtain an injectable thermosensitive pluronic hydrogel having excellent a cell affinity such as a cell proliferation and a cell differentiation, while maintaining the thermosensitivity of the conventional pluronic hydrogel.
  • the injectable thermosensitive pluronic derivative hydrogel according to the present invention has an excellent cell affinity while maintaining the thermosensitivity of the conventional pluronic hydrogel, and thus, it can be suitably used for the regeneration of artificial tissues or organs through a topical injection using a syringe, without a surgical operation.
  • thermosensitivity of the hydrogel was measured at 15-90 0 C with a tube tilting method, and the size of the micelles was measured with a dynamic light scattering (DLS) while changing temperature.
  • the critical micelle temperature (CMT) was measured at 10-60 0 C with ultraviolet rays.
  • Cell culture experiments were carried out by setting the concentration of the hydrogel according to the present invention to be 20% by weight.
  • tissue cells cartilage cells, vascular cells, nerve cells, vascular endothelial cells, etc.
  • stem cells adipose stem cells, bone marrow stem cells, cord-blood stem cells, muscular stem cells, embryonic stem cells, etc.
  • Pluronic F127 having a weight average molecular weight of about
  • F127 hydrogels was increased by approximately 10nm compared with that of the conventional pluronic F127, and the critical micelle temperature was lowered by approximately 10 0 C depending on its concentration compared with that of the conventional pluronic F127.
  • META-pluronic F127 prepared in Example 1 was completely dissolved in 2-morpholinoethansulfonic acid (MES) buffer solution in a molar ratio of 1 :15 by weight, and then EDC was added to the resulting solution so as the molar ratio between META-pluronic F127 and the EDC to be 1 :2.1 , thereby activating carboxyl group.
  • MES 2-morpholinoethansulfonic acid
  • RGD was added to the reaction mixture so as the molar ratio between META-pluronic F127 and RGD to be 1 :2.1. Then, the mixture was reacted at room temperature for 24 hours, dialyzed for 3 days, and then freeze-dried for 3 days.
  • the yield of the prepared META-pluronic F127-RGD hydrogesl was more than 90%, and according to the results of test for thermosensitivity, its thermosensitivity was maintained although the sol-gel transition temperature was lower compared with that of the conventional pluronic F127 by approximately 3-4 °C .
  • the prepared META-pluronic F127 hydrogel exhibits the increase of micelle size compared with META-pluronic F127 by approximately 10-15nm depending on its concentration, and the critical micelle temperature was lowered compared with that of META-pluronic F127 by approximately 10-15 ° C .
  • the prepared META-pluronic F127-RGD hydrogel exhibited an improvement of approximately 90% in cell affinity compared with the conventional thermosensitive pluronic F127 hydrogel.
  • META-pluronic F127-YIGSR was prepared in the same manner as described in Example 2 except that RGD and EDC were replaced by YIGSR, a ligand peptide relating to a vascular cell proliferation, and CMC.
  • the yield of the prepared META-pluronic F127-YIGSR hydrogel was more than 90%, and its thermosensitivity was maintained although the sol-gel transition temperature was lower compared with the conventional pluronic F127 by approximately 3-4 0 C .
  • the prepared META-pluronic F127-YIGSR hydrogel exhibited increase of approximately 5-1 Onm in micelle size compared with META-pluronic F127-RGD depending on its concentration, and the critical micelle temperature was lowered compared with that of META-pluronic F127-RGD by approximately 5-10°C depending on its concentration.
  • the prepared META-pluronic F127-YIGSR hydrogel exhibited increase of approximately 5-1 Onm in micelle size compared with META-pluronic F127-RGD depending on its concentration, and the critical micelle temperature was lowered compared with that of META-pluronic F127-RGD by approximately 5-10°C depending on its concentration.
  • META-pluronic F127-YIGSR exhibited an improvement of approximately 80% in cell affinity compared with the conventional pluronic F127.
  • Example 4 META-pluronic F127-IKVAV was prepared in the same manner as described in Example 2 except that IKVAN, a ligand peptide relating to a nerve cell proliferation, instead of RGD, was used.
  • the yield of the prepared META-pluronic F127-IKVAV hydrogel was more than 90%, and its thermosensitivity was maintained although the sol-gel transition temperature was lower compared with the conventional pluronic F127 by approximately 3-5°C .
  • the prepared META-pluronic F127-IKVAV hydrogel exhibited increase of approximately 5-1 Onm in micelle size compared with META-pluronic F127-RGD depending on its concentration, and the critical micelle temperature was lowered compared with that of the conventional pluronic F127-RGD by approximately 5-10 0 C depending on its concentration.
  • the prepared META-pluronic F127-IKVAV hydrogel exhibited an improvement of approximately 90% in cell affinity compared with the conventional pluronic F127 hydrogel.
  • META-pluronic F127-REDV was prepared in the same manner as described in Example 2 except that REDV, a ligand peptide relating to an intravascular cell proliferation, instead of RGD, was used.
  • the yield of the prepared META-pluronic F127-REDV hydrogel was more than 90%, and its thermosensitivity was maintained although the sol-gel transition temperature was lower compared with the conventional pluronic F127 by approximately 2-3 ° C.
  • the prepared META-pluronic F127-REDV hydrogel exhibited increase of approximately 5-1 Onm in micelle size compared with META-pluronic F127-RGD depending on its concentration, and the critical micelle temperature was lowered compared with that of META-pluronic F127-RGD by approximately 5-10 ° C depending on its concentration.
  • the prepared META-pluronic F127-IKVAV hydrogel exhibited an improvement of approximately 80% in cell affinity compared with the conventional pluronic F 127 hydrogel.
  • Example 6 META-pluronic F127-TGF- ⁇ was prepared in the same manner as described in Example 2 except that TGF- ⁇ , a growth factor, was used instead of RGD. As a result of a sol-gel experiment, thermosensitivity of META-pluronic F127-TGF- ⁇ was similar to that of the conventional pluronic F127-hydrogel although the sol-gel transition temperature was lowered compared with the conventional pluronic F127 hydrogel by approximately 2-3 ° C .
  • the prepared META-pluronic F127- TGF- ⁇ exhibited increase of approximately 5-1 Onm in micelle size compared with META-pluronic F127-RGD depending on its concentration, and the critical micelle temperature was lowered compared with that of META-pluronic F127-RGD by approximately 5-1O 0 C depending on its concentration.
  • META-pluronic F127-TGF- ⁇ exhibited an improvement in cell affinity in which its differentiation is approximately 90% higher than that of the conventional pluronic hydrogel.
  • META-pluronic F127-EGF was prepared in the same manner as described in Example 6 except that EGF, a growth factor, was used instead of RGD and CMC was used instead of EDC.
  • EGF a growth factor
  • CMC CMC
  • the prepared META-pluronic F127-EGF exhibited increase of approximately 5-1 Onm in micelle size compared with META-pluronic F127-RGD depending on its concentration, and the critical micelle temperature was lowered compared with that of META-pluronic F127-RGD by approximately 5-1O 0 C depending on its concentration.
  • META-pluronic F127-EGF hydrogel exhibited an improvement in cell affinity in which its differentiation is approximately 80% higher than that of the conventional pluronic hydrogel.
  • META-pluronic F127-NGF was prepared in the same manner as described in Example 6 except that NGF, a growth factor, was used instead of RGD.
  • NGF a growth factor
  • the thermosensitivity of the obtained META-pluronic F127-NGF was similar to that of the conventional pluronic F127 hydrogel although it was lowered compared with the conventional pluronic F127 hydrogel by approximately 2-3 0 C .
  • the META-pluronic F127-NGF exhibited increase of approximately 5-1 Onm in micelle size compared with META- pluronic F127-RGD depending on its concentration, and the critical micelle temperature was lowered compared with that of META-pluronic F127-RGD by approximately 5-10 0 C depending on its concentration.
  • META-pluronic F127-NGF exhibited an improvement in cell affinity in which its differentiation is approximately 90% higher than that of the conventional pluronic hydrogel.
  • META-pluronic F127-VEGF was prepared in the same manner as described in Example 6 except that VEGF, a growth factor, was used instead of RGD.
  • VEGF a growth factor
  • the thermosensitivity of the obtained META-pluronic F127-VEGF was similar to that of the conventional pluronic F127 hydrogel although it was lowered compared with the conventional pluronic F127 hydrogel by approximately 2-3 °C.
  • the prepared META-pluronic F127-VEGF exhibited increase of approximately 5-1 Onm in micelle size compared with META- pluronic F127-RGD, and the critical micelle temperature was lowered than META-pluronic F127-RGD by approximately 5-10 °C .
  • META-pluronic F127-VEGF exhibited an improvement in cell affinity in which its differentiation was approximately 80% higher than that of the conventional pluronic hydrogel.
  • the pluronic hydrogel according to the present invention exhibits the increase of cell affinity to the specific cells or cell differentiation into the specific cells by approximately 80-90% compared the conventional pluronic F127 hydrogel, while it maintains the thermosensitivity of the conventional fluronic hydrogel.
  • the puronic hydrogel according to the present invention exhibits increase in micelle size compared with the conventional pluronic F127 hydrogel by approximately 10-15nm depending on its concentration, and the critical micelle temperature was lowered than the conventional pluronic F127 hydrogel by approximately 10-15 ° C depending on its concentration.

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Abstract

An injectable thermosensitive pluronic derivative hydrogel coupled with a biologically active material such as a ligand peptide having a cell affinity or a growth factor for tissue regeneration, and a preparation method thereof, comprising: coupling a pluronic derivative with methacryloxyethyltritrimellitic anhydride having a double bond and a functional group; and introducing a ligand peptide having a cell affinity or a growth factor to the resultant.

Description

INJECTABLE THERMOSENSITIVE PLURONIC HYDROGELS COUPLED WITH BIOACTIVE MATERIALS FOR TISSUE REGENERATION
AND PREPARATION METHOD THEREOF
TECHNICAL FIELD
The present invention relates to an injectable thermosensitive pluronic derivative hydrogel coupled with a biologically active material for tissue regeneration, and a preparation method thereof.
BACKGROUND ART
Tissue engineering is a new technology emerged during the progress of science, and is a study which integrates and applies science and technology with basic concepts of life science, engineering, medicine, etc.
Tissue engineering is an application study which has an objective to understand the correlation between the structure and the function of tissues of a living body, and to maintain, enhance or restore the functions of a human body by means of artificial tissues which are transplantable into the human body, so as to replace damaged tissues or organs by normal ones or to regenerate the same.
Tissue engineering techniques using hydrogels are largely divided into two categories. In one technique, a necessary tissue is extracted from a patient's body, and cells are separated from the tissue. Then, the cell is proliferated via a culture to a necessary amount, mixed with an injectable hydrogel, and then directly injected into the human body. Alternatively, the cell is cultured in a hydrogel outside the human body and then injected into the human body. In this technique, the transplanted hydrogel is converted to a gel in the human body due to the body temperature, positioned at a specific site, and supplied with oxygen and nutrition by diffusion of the body fluid. If blood is supplied through a blood vessel which is extended into the human body, the cell is proliferated and differentiated to generate new tissues and organs. Then, the hydrogel is discharged outside the body or decomposed to disappear.
In the other technique, a hydrogel and a specific drug are mixed, and the resulting mixture is directly injected into a living body. The mixture is converted to a gel due to the body temperature, and the hydrogel is gradually decomposed, by which the drug is delivered to the body for a long time at a suitable concentration.
For studying tissue engineering, it is important to prepare thermosensitive hydrogels that can be converted to a gel at around the body temperature and that are similar to tissues of a living body. It is required that the hydrogels for regeneration of tissues of the human body be converted to a gel at around the body temperature while maintained as a sol at room temperature, and have an affinity to cells so that the cells can generate tissues having a three-dimensional structure within the hydrogels, and also function as a barrier between transplanted cells and host cells. The representative polymer hydrogels having such thermosensitivity include Pluronic (P. Holmqvist et al., Int. J. Pharm., 194, 103, 2000), poly(N-isopropylacrylamide) (PNIPAAm) (M. Harmon et al., Macromolecules, 36, 1 , 2003), hyaluronic acid (HA) (M. Ogiso et al., J. Biomed. Mater. Res., 39, 3, 1998), linear polyethyleneglycol (PEG)-polylactic acid/glycolic acid copolymer (PLGA)- polyethyleneglycol (PEG) (B. Jeong et al., J. Biomed. Mater. Res., 50, 2, 2000), linear polyethlyeneglycol (PEG)-polylactic acid (PLA)- polyethyleneglycol (PEG), star-shaped polylactic acid (PLA)- polyethyleneglycol (PEG), star-shaped poly-ε-caprolactone (PCL)- polyethyleneglycol (PEG) (S. Zhao et al., J. Func. Polym., 15, 1 , 2002) etc. Among them, only hyaluronic acid, and F127 and F68 of Pluronics are approved by the U.S. FDA as materials that can be injected to the human body.
Polynipaam has its own toxicity. Other hydrogels are disadvantageous in that they have relatively low mechanical properties, and do not have an affinity to cells sufficient to be used for tissue regeneration.
The pluronic hydrogels include F38, F68, F77, F77, F98, F108, F127, etc. beginning with 'F', L31 , L42, L43, L44, L62, L72, L101 , etc. beginning with 'L', and P75, P103, P104, etc. beginning with 'P' (respectively denoting trade names). All of these pluronic hydrogels have a structure of PEO-PPO-PEO, but different ratios or forms from one another. Among them, only F68 (molecular weight: 8,700) and F127 (molecular weight: 12,600) were approved by the U.S. FDA have been used as materials for a living body.
Pluronic F127 is a non-toxic copolymer (molecular weight: 12,600) of polyethyleneoxide (PEO)-polypropyleneoxide (PPO)-polyethyleneoxide (PEO) in a molar ratio of 98:68:98, and has a temperature-dependent sol-gel conversion properties, and accordingly, it has been used as a material, in a mixture with other materials, for a living body. However, there has been no example to synthesize a derivative by chemically coupling biologically active materials with pluronic F127.
DETAILED DESCRIPTION OF THE INVENTION
TECHNICAL PROBLEM TO BE SOLVED
Therefore, it is an object of the present invention to provide an injectable thermosensitive pluronic hydrogel for tissue regeneration, to which biologically active materials are coupled, and which has an excellent cell affinity while maintains the thermosensitivity of the conventional pluronic hydrogel itself.
TECHNICAL SOLUTION
To achieve the above object, the present invention provides an injectable pluronic hydrogel having thermosensitivity and cell affinity by coupling a conventional pluronic polymer with a compound having a polymerizable double bond and a functional group, through which a biologically active material such as a ligand peptide or a growth factor can be introduced, while maintaining the thermosensitivity of the conventional pluronic polymer, followed by directly introducing a biologically active material which can improve cell affinity. Herein, the polymerizable double bond can be later used for a polymerization, if desired.
Therefore, the present invention relates to an injectable thermosensitive pluronic hydrogel for tissue regeneration, and a preparation method thereof.
The injectable thermosensitive pluronic hydrogel for tissue regeneration according to the present invention has a structure in which a thermosensitive pluronic F127 polymer is coupled with a biologically active material such as a ligand peptide having a cell affinity or a growth factor through methacryloxyethyltrimellitic acid.
The ligand peptide having a cell affinity is at least one selected from the group consisting of Arg-Gly-Asp (RGD), Arg-Glu-Asp-Val (REDV), Leu-Asp-Val (LDV), Tyr-lle-Gly-Ser-Arg (YIGSR), Pro-Asp-Ser-Gly-Arg (PDSGR), Ile-Lys-Val-Ala-Val (IKVAV) and Arg-Asn-lle-Ala-Glu-lle-lle- Lys-Asp-Ala (RNIAEIIKDA). It has been known that RGD and PDSGR enhance adhesion of almost all cells, REDV and LDV enhance proliferation of vascular endotheliocytes, YIGSR enhance proliferation of vascular cells, and IKVAV and RNIAEIIKDA enhance proliferation of nerve cells.
The growth factor is at least one selected from the group consisting of a transforming growth factor (TGF-β), an insulin-like growth factor (IGF), an epithelia growth factor (EGF), a nerve cell growth factor (NGF), a vascular endothelial growth factor (VEGF), a fibroblast growth factor (FGF), a hepatocyte growth factor (HGF) and a platelet-derived growth factor (PDGF).
In the injectable thermosensitive pluronic hydrogel for tissue regeneration according to the present invention, methacryloxyethyltrimellitic acid used for coupling pluronic F127 with a biologically active material is the one derived from 4-methacryloxyethyltrimellitic anhydride (4-META) or 2-methacryloxyethyltritrimellitic anhydride (2-META). The following Formula 1 shows a specific example of an injectable thermosensitive pluronic hydrogel (META-pluronic F127-R) for tissue regeneration according to the present invention, in which pluronic F127 is coupled with a biologically active material through 4-methacryloxyethyltritrimellitic anhydride.
Formula 1 :
Figure imgf000007_0001
wherein -PEO-PPO-PEO- represents pluronic F127, and R represents the biologically active material as defined above.
A preparation method for the injectable thermosensitive pluronic hydrogel for tissue regeneration according to the present invention comprises:
(1) reacting a thermosensitive pluronic hydrogel with methacryloxy- ethyltritrimellitic anhydride at room temperature to obtain methacryloxy- ethyltritrimellitic anhydride-pluronic polymer (META-pluronic F127); and
(2) coupling the obtained methacryloxyethyltritrimellitic anhydride-pluronic polymer with a ligand peptide having a cell affinity or a growth factor to obtain a methacryloxyethyltritrimellitic anhydride-pluronic derivative hydrogel to which a biologically active material is coupled.
In step (1), the methacryloxyethyltritrimellitic anhydride may be 4-methacryloxyethyltritrimellitic anhydride (4-META) or 2-methacryloxyethyl- tritrimellitic anhydride (2-META). The 4-META which has been used as a conventional dental adhesive has no toxicity and a relatively excellent mechanical property. 4-META or 2-META has a double bond at its one end, so as to enable polymerization, and an anhydride group at the other end, capable of being converted to a carboxyl group which can be used to couple a biologically active material.
In step (1), pluronic F127 and META are put into a reactor in a molar ratio of 1 :2.2, the reactor is charged with nitrogen at room temperature, and then a reaction is performed for 20-24 hours, to obtain META-pluronic F127 which is a derivative of pluronic F127. In step (2), a biologically active material is added to the META-pluronic
F127 prepared in step (1) in a molar ratio of 1 :2.2, and then the resulting mixture is reacted using 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC) or 1 -cyclohexyl-3-(2-morpholinoethyl)carbodiimidemeto-p-toluene sulfonate (CMC), to obtain META-pluronic F127-R, wherein R represents a biologically active material as defined above.
In the present invention, 4-META having a double bond and a carboxyl group is coupled with the conventional thermosensitive pluronic F127 hydrogel, thereby enabling polymerization and introduction of a biologically active material. Furthermore, the introduction of a ligand peptide or growth factor to META-pluronic F127 makes it possible to prepare an injectable thermosensitive hydrogel, a cell affinity of which is improved. Therefore, the pluronic hydrogels of the present invention is useful for regenerating tissues or organs by means of tissue engineering technique. The following Reaction Scheme 1 shows an example for preparing the injectable thermosensitive pluronic hydrogel according to the present invention, comprising coupling a biologically active material to a derivative of pluronic F127 using 4-methacryloxyethyltritrimellitic anhydride.
Reaction Scheme 1 :
Figure imgf000010_0001
Pluronic F127 4-META
Figure imgf000010_0002
META-Pluronic F127
Figure imgf000010_0003
Figure imgf000010_0004
META-Pluronic F127-R {R: Bioactive materials)
wherein R represents a biologically active material as defined above.
EFFECTS OF THE INVENTION
According to the present invention, 4-META or 2-META having a double bond to enable polymerization and a carboxyl group is coupled to the conventional thermosensitive pluronic F127 hydrogel to obtain META- pluronic F127, and then a biologically active material such as a ligand peptide or a growth factor is introduced to the obtained META-pluronic F127 hydrogel to obtain an injectable thermosensitive pluronic hydrogel having excellent a cell affinity such as a cell proliferation and a cell differentiation, while maintaining the thermosensitivity of the conventional pluronic hydrogel. The injectable thermosensitive pluronic derivative hydrogel according to the present invention has an excellent cell affinity while maintaining the thermosensitivity of the conventional pluronic hydrogel, and thus, it can be suitably used for the regeneration of artificial tissues or organs through a topical injection using a syringe, without a surgical operation.
EXAMPLES
Hereinafter, the present invention will be illustrated in more detail with reference to the following examples. The following examples are provided merely to illustrate, not to limit the scope of the present invention thereto.
In the following examples, the thermosensitivity of the hydrogel was measured at 15-900C with a tube tilting method, and the size of the micelles was measured with a dynamic light scattering (DLS) while changing temperature. The critical micelle temperature (CMT) was measured at 10-600C with ultraviolet rays. Cell culture experiments were carried out by setting the concentration of the hydrogel according to the present invention to be 20% by weight. After culturing segmented tissue cells (cartilage cells, vascular cells, nerve cells, vascular endothelial cells, etc.) or stem cells (adipose stem cells, bone marrow stem cells, cord-blood stem cells, muscular stem cells, embryonic stem cells, etc.) were cultured, and cell proliferation and differentiation were observed, so as to evaluate an affinity to cells. Example 1
Pluronic F127 having a weight average molecular weight of about
12,700 and 4-META were mixed in a molar ratio of 1 :2.2, and the resulting mixture was completely dissolved in toluene in a ratio of 1 :4 by weight. After charging the reactor with nitrogen gas in order to prevent contact with moisture, and the inlet of the reactor was sealed. Pyridine was added to the reactor using a syringe while stirring so as to have a ratio of 1 :25 by volume with the mixture. The resulting mixture was reacted at room temperature for 20-24 hours with stirring and then was poured into 1000ml of cold ether to precipitate.
The yield of the obtained META-pluronic F127 hydrogel was more than
90%, and according to the results of a sol-gel experiment, it was discovered that its thermosensitivity was maintained although the sol-gel transition temperature was lowered by approximately 2-30C compared with the conventional pluronic F127. The micelle size of the obtained META-pluronic
F127 hydrogels was increased by approximately 10nm compared with that of the conventional pluronic F127, and the critical micelle temperature was lowered by approximately 100C depending on its concentration compared with that of the conventional pluronic F127.
Example 2
META-pluronic F127 prepared in Example 1 was completely dissolved in 2-morpholinoethansulfonic acid (MES) buffer solution in a molar ratio of 1 :15 by weight, and then EDC was added to the resulting solution so as the molar ratio between META-pluronic F127 and the EDC to be 1 :2.1 , thereby activating carboxyl group. After stirring the mixture for 2 hours, RGD was added to the reaction mixture so as the molar ratio between META-pluronic F127 and RGD to be 1 :2.1. Then, the mixture was reacted at room temperature for 24 hours, dialyzed for 3 days, and then freeze-dried for 3 days.
The yield of the prepared META-pluronic F127-RGD hydrogesl was more than 90%, and according to the results of test for thermosensitivity, its thermosensitivity was maintained although the sol-gel transition temperature was lower compared with that of the conventional pluronic F127 by approximately 3-4 °C . The prepared META-pluronic F127 hydrogel exhibits the increase of micelle size compared with META-pluronic F127 by approximately 10-15nm depending on its concentration, and the critical micelle temperature was lowered compared with that of META-pluronic F127 by approximately 10-15°C .
As a result of the test for adhesion to bone marrow cells, the prepared META-pluronic F127-RGD hydrogel exhibited an improvement of approximately 90% in cell affinity compared with the conventional thermosensitive pluronic F127 hydrogel.
Example 3
META-pluronic F127-YIGSR was prepared in the same manner as described in Example 2 except that RGD and EDC were replaced by YIGSR, a ligand peptide relating to a vascular cell proliferation, and CMC. The yield of the prepared META-pluronic F127-YIGSR hydrogel was more than 90%, and its thermosensitivity was maintained although the sol-gel transition temperature was lower compared with the conventional pluronic F127 by approximately 3-40C . The prepared META-pluronic F127-YIGSR hydrogel exhibited increase of approximately 5-1 Onm in micelle size compared with META-pluronic F127-RGD depending on its concentration, and the critical micelle temperature was lowered compared with that of META-pluronic F127-RGD by approximately 5-10°C depending on its concentration. As a result of the test for proliferation of vascular cells, the prepared
META-pluronic F127-YIGSR exhibited an improvement of approximately 80% in cell affinity compared with the conventional pluronic F127.
Example 4 META-pluronic F127-IKVAV was prepared in the same manner as described in Example 2 except that IKVAN, a ligand peptide relating to a nerve cell proliferation, instead of RGD, was used. The yield of the prepared META-pluronic F127-IKVAV hydrogel was more than 90%, and its thermosensitivity was maintained although the sol-gel transition temperature was lower compared with the conventional pluronic F127 by approximately 3-5°C . The prepared META-pluronic F127-IKVAV hydrogel exhibited increase of approximately 5-1 Onm in micelle size compared with META-pluronic F127-RGD depending on its concentration, and the critical micelle temperature was lowered compared with that of the conventional pluronic F127-RGD by approximately 5-100C depending on its concentration.
As a result of the test for proliferation of nerve cells, the prepared META-pluronic F127-IKVAV hydrogel exhibited an improvement of approximately 90% in cell affinity compared with the conventional pluronic F127 hydrogel.
Example 5
META-pluronic F127-REDV was prepared in the same manner as described in Example 2 except that REDV, a ligand peptide relating to an intravascular cell proliferation, instead of RGD, was used. The yield of the prepared META-pluronic F127-REDV hydrogel was more than 90%, and its thermosensitivity was maintained although the sol-gel transition temperature was lower compared with the conventional pluronic F127 by approximately 2-3 °C. The prepared META-pluronic F127-REDV hydrogel exhibited increase of approximately 5-1 Onm in micelle size compared with META-pluronic F127-RGD depending on its concentration, and the critical micelle temperature was lowered compared with that of META-pluronic F127-RGD by approximately 5-10°C depending on its concentration.
As a result of the test for proliferation of intravascular cells, the prepared META-pluronic F127-IKVAV hydrogel exhibited an improvement of approximately 80% in cell affinity compared with the conventional pluronic F 127 hydrogel.
Example 6 META-pluronic F127-TGF-β was prepared in the same manner as described in Example 2 except that TGF-β, a growth factor, was used instead of RGD. As a result of a sol-gel experiment, thermosensitivity of META-pluronic F127-TGF-β was similar to that of the conventional pluronic F127-hydrogel although the sol-gel transition temperature was lowered compared with the conventional pluronic F127 hydrogel by approximately 2-3°C . The prepared META-pluronic F127- TGF-β exhibited increase of approximately 5-1 Onm in micelle size compared with META-pluronic F127-RGD depending on its concentration, and the critical micelle temperature was lowered compared with that of META-pluronic F127-RGD by approximately 5-1O0C depending on its concentration.
As a result of an experiment for the differentiation of chondrocyte using fat stem cells, META-pluronic F127-TGF-β exhibited an improvement in cell affinity in which its differentiation is approximately 90% higher than that of the conventional pluronic hydrogel.
Example 7
META-pluronic F127-EGF was prepared in the same manner as described in Example 6 except that EGF, a growth factor, was used instead of RGD and CMC was used instead of EDC. As a result of a sol-gel experiment, the thermosensitivity of META-pluronic F127-EGF was similar to that of the conventional pluronic F127 hydrogel although it was lowered compared with the conventional pluronic F127 hydrogel by approximately 2-30C. The prepared META-pluronic F127-EGF exhibited increase of approximately 5-1 Onm in micelle size compared with META-pluronic F127-RGD depending on its concentration, and the critical micelle temperature was lowered compared with that of META-pluronic F127-RGD by approximately 5-1O0C depending on its concentration. As a result of an experiment for the differentiation of osteocyte using cord-blood stem cells, META-pluronic F127-EGF hydrogel exhibited an improvement in cell affinity in which its differentiation is approximately 80% higher than that of the conventional pluronic hydrogel.
Example 8
META-pluronic F127-NGF was prepared in the same manner as described in Example 6 except that NGF, a growth factor, was used instead of RGD. As a result of a sol-gel experiment, the thermosensitivity of the obtained META-pluronic F127-NGF was similar to that of the conventional pluronic F127 hydrogel although it was lowered compared with the conventional pluronic F127 hydrogel by approximately 2-30C . The META-pluronic F127-NGF exhibited increase of approximately 5-1 Onm in micelle size compared with META- pluronic F127-RGD depending on its concentration, and the critical micelle temperature was lowered compared with that of META-pluronic F127-RGD by approximately 5-100C depending on its concentration.
As a result of an experiment for the differentiation of nerve cells using bone marrow stem cells, META-pluronic F127-NGF exhibited an improvement in cell affinity in which its differentiation is approximately 90% higher than that of the conventional pluronic hydrogel.
Example 9
META-pluronic F127-VEGF was prepared in the same manner as described in Example 6 except that VEGF, a growth factor, was used instead of RGD. As a result of a sol-gel experiment, the thermosensitivity of the obtained META-pluronic F127-VEGF was similar to that of the conventional pluronic F127 hydrogel although it was lowered compared with the conventional pluronic F127 hydrogel by approximately 2-3 °C. The prepared META-pluronic F127-VEGF exhibited increase of approximately 5-1 Onm in micelle size compared with META- pluronic F127-RGD, and the critical micelle temperature was lowered than META-pluronic F127-RGD by approximately 5-10 °C .
As a result of an experiment for the differentiation of vascular endothelial cells using embryonic stem cells, META-pluronic F127-VEGF exhibited an improvement in cell affinity in which its differentiation was approximately 80% higher than that of the conventional pluronic hydrogel.
As described above, the pluronic hydrogel according to the present invention exhibits the increase of cell affinity to the specific cells or cell differentiation into the specific cells by approximately 80-90% compared the conventional pluronic F127 hydrogel, while it maintains the thermosensitivity of the conventional fluronic hydrogel. The puronic hydrogel according to the present invention exhibits increase in micelle size compared with the conventional pluronic F127 hydrogel by approximately 10-15nm depending on its concentration, and the critical micelle temperature was lowered than the conventional pluronic F127 hydrogel by approximately 10-15 °C depending on its concentration.
It will also be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

Claims

1. An injectable thermosensitive pluronic hydrogel for tissue regeneration in which a biologically active material is coupled with a thermosensitive pluronic polymer through methacryloxyethyltrimellitic acid.
2. The injectable thermosensitive pluronic hydrogel for tissue regeneration according to claim 1 , wherein the methacryloxyethyltrimellitic acid is 4-methacryloxyethyltrimellitic acid or 2-methacryloxyethyltrimellitic acid.
3. The injectable thermosensitive pluronic derivative hydrogel for tissue regeneration according to claim 2, having a structure represented by the following Formula 1 : Formula 1 :
Figure imgf000020_0001
wherein R represents a biologically active material.
4. The injectable thermosensitive pluronic derivative hydrogel for tissue regeneration according to claim 1 or 3, wherein the biologically active material is a ligand peptide having a cell affinity or a growth factor.
5. The injectable thermosensitive pluronic derivative hydrogel for tissue regeneration according to claim 4, wherein the ligand peptide having a cell affinity is at least one selected from the group consisting of Arg-Gly-Asp (RGD), Arg-Glu-Asp-Val (REDV), Leu-Asp-Val (LDV), Tyr-lle-Gly-Ser-Arg (YIGSR), Pro-Asp-Ser-Gly-Arg (PDSGR), Ile-Lys-Val-Ala-Val (IKVAV) and Arg-Asn-lle-Ala-Glu-lle-lle- Lys-Asp-Ala (RNIAEIIKDA).
6. The injectable thermosensitive pluronic derivative hydrogel for tissue regeneration according to claim 4, wherein the growth factor is at least one selected from the group consisting of a transforming growth factor (TGF-β), an insulin-like growth factor (IGF), an epithelia growth factor (EGF), a nerve cell growth factor (NGF), a vascular endothelial growth factor (VEGF), a fibroblast growth factor (FGF), a hepatocyte growth factor (HGF) and a platelet-derived growth factor (PDGF).
7. A preparation method for an injectable thermosensitive pluronic derivative hydrogel for tissue regeneration, comprising:
(1) reacting a thermosensitive pluronic polymer with methacryloxyethyltritrimellitic anhydride at room temperature to obtainin a methacryloxyethyltrimellitic acid-pluronic polymer; and
(2) coupling the methacryloxyethyltrimellitic acid-pluronic polymer obtained in step (1 ) with a ligand peptide having a cell affinity or a growth factor to obtain a methacryloxyethyltrimellitic acid-pluronic hydrogel to which a biologically active material is coupled.
8. The method according to claim 7, wherein methacryloxyethyltritrimellitic anhydride is 4-methacryloxyethyltritrimellitic anhydride or 2-methacryloxyethyltritrimellitic anhydride.
9. The method according to claim 7, wherein in step (1), the pluronic polymer is reacted with methacryloxyethyltritrimellitic anhydride in a ratio of 1 :2.2 by weight.
10. The method according to claim 7, wherein in Step (1), toluene is used as a solvent.
11. The method according to claim 7, wherein in step (2), the methacryloxyethyltrimellitic acid-pluronic polymer is reacted with a biologically active material in a molar ratio of 1 :2.2.
12. The method according to claim 7, wherein the coupling of step (2) is carried out in an aqueous solution using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide or 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimidemeto-p- toluene sulfonate.
13. The method according to claim 7, wherein in step (2), the methacryloxyethyltrimellitic acid-pluronic polymer is coupled with a biologically active material using a 2-morpholinoethanesulfonic acid buffer solution in an amount that the weight ratio of the methacryloxyethyltrimellitic acid-pluronic polymer : the 2-morpholinoethanesulfonic acid buffer is 1 : 15.
14. The method according to claim 13, wherein the methacryloxyethyltrimellitic acid-pluronic polymer is coupled with the biologically active material in a molar ratio of 1 :2.1.
15. The method according to claim 7, wherein in step (2), the biologically active material is used in an amount of 2.1 times of mole number of the methacryloxyethyltrimellitic acid-pluronic polymer.
16. The method according to claim 7, wherein the biologically active material is a ligand peptide having a cell affinity or a growth factor.
17. The method according to claim 16, wherein one or more of the ligand peptide having a cell affinity is at least one selected from the group consisting of Arg-Gly-Asp (RGD), Arg-Glu-Asp-Val (REDV), Leu-Asp-Val (LDV), Tyr-lle-Gly-Ser-Arg (YIGSR), Pro-Asp-Ser-Gly-Arg (PDSGR), Ile-Lys-Val-Ala-Val (IKVAV) and Arg-Asn-lle-Ala-Glu-lle-lle- Lys-Asp-Ala (RNIAEIIKDA).
18. The method according to claim 16, wherein the growth factor is at least one selected from the group consisting of a transforming growth factor (TGF-β), an insulin-like growth factor (IGF), an epithelia growth factor (EGF), a nerve cell growth factor (NGF), a vascular endothelial growth factor (VEGF), a fibroblast growth factor (FGF), a hepatocyte growth factor (HGF) and a platelet-derived growth factor (PDGF).
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