Biodegradable injectable gel

Abstract

The present invention relates to a triblock copolymer having a polyethylene glycol-poly(D,L-lactide)-polyethylene glycol skeleton.

Claims

1. A biodegradable injectable gel, comprising: the triblock copolymer having a polyethylene glycol-poly(D,L-lactide)-polyethylene glycol skeleton, comprising a repeating unit represented by Formula I: ##STR00018## wherein n is 10 to 1000, and m is 2 to 100, chitosan, and a self-assembling peptide.

2. The biodegradable injectable gel according to claim 1, wherein the self-assembling peptide is (RADA).sub.4.

3. A crosslinking agent comprising Formula I: ##STR00019## wherein n is 10 to 1000, and m is 2 to 100.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 indicates the .sup.1H-NMR spectrum of a product.

(2) FIG. 2 indicates the .sup.1H-NMR spectrum of a product.

(3) FIG. 3 indicates the results of GPC measurement of a product.

(4) FIG. 4 indicates the .sup.1H-NMR spectrum of a product.

(5) FIG. 5 indicates the .sup.1H-NMR spectrum of a product.

(6) FIG. 6 shows gelation phase diagrams in the case of using PEG 5k and PEG-PLA-PEG (left: chitosan/PEG 5k, right: chitosan/PEG-PLA-PEG).

(7) FIG. 7 indicates the results of measuring frequency dependency in the case of using PEG 5k and PEG-PLA-PEG (left: chitosan/PEG 5k, right: chitosan/PEG-PLA-PEG).

(8) FIG. 8 indicates gelation behavior in the case of using PEG 5k and PEG-PLA-PEG (left: chitosan/PEG 5k, right: chitosan/PEG-PLA-PEG).

(9) FIG. 9 indicates gelation behavior of RADA mixed systems.

(10) FIG. 10 indicates Young's modulus in the case of using PEG 5k and PEG-PLA-PEG (dots) and Young's modulus in RADA mixed systems (diagonal lines).

(11) FIG. 11 indicates degree of swelling in the case of using PEG 5k and PEG-PLA-PEG (dots) and degree of swelling in RADA mixed systems (diagonal lines).

(12) FIG. 12 indicates degradation behavior under acidic conditions in the case of using PEG 5k and PEG-PLA-PEG in the presence or absence of mixing with RADA.

(13) FIG. 13 indicates degradation behavior under physiological conditions in the case of using PEG 5k and PEG-PLA-PEG in the presence or absence of mixing with RADA.

(14) FIG. 14 indicates the results of measuring CD spectra in various hydrogels.

(15) FIG. 15 indicates degradation behavior of chitosan/PEG 5k gel and chitosan/PEG-PLA-PEG gel in the presence of cells.

(16) FIG. 16 indicates mitochondrial activity of various hydrogels as determined by MTT assay.

(17) FIG. 17 indicates GAG production levels (left: amount of GAG produced per day, right: cumulative amount of GAG produced).

(18) FIG. 18 indicates GAG production levels in gels.

(19) FIG. 19 indicates cumulative GAG production levels (in medium and gel).

(20) FIG. 20 indicates the .sup.1H-NMR spectrum of a product.

(21) FIG. 21 indicates the .sup.1H-NMR spectrum of a product.

BEST MODE FOR CARRYING OUT THE INVENTION

Example 1

(22) Although the following provides a detailed explanation of the present invention through examples thereof, the present invention is not limited to these examples. Furthermore, commercially available products were used as is for the reagents used in the following examples unless specifically indicated otherwise. In the following examples, measurement of the molecular weight distribution of polymers obtained in each of the examples was carried out in the manner indicated below.

(23) Number Average Molecular Weight ([Mn], Units: g/mol)

(24) The number average molecular weight (Mn) and weight average molecular weight (Mw) of polymers were measured (solvent: THF, temperature: 40 C., flow rate: 0.35 mL/min) using polystyrene standards having known peak molecular weights by gel permeation chromatography (GPC) calibrated with the polystyrene standards (Tosoh HLC-8320GPC chromatography system manufactured by Tosoh Corp., column configuration: TSK Guard Column Super MP(HZ)-M, TSKgel Super Multipore HZ-M, four columns connected in series).

(25) Molecular Weight Distribution ([Mw/Mn])

(26) Molecular weight distribution was determined as the ratio (Mw/Mn) obtained using the values for weight average molecular weight (Mw) and number average molecular weight (Mn) determined according the method described above.

(27) NMR Measurement

(28) Polymer structures were analyzed using an NMR measurement system (Bruker Corp., 40 MHz) by .sup.1H-NMR measurement and .sup.13C-NMR measurement. Furthermore, chemical shifts were based on CDCl.sub.3 (.sup.1H: 7.26 ppm, .sup.13C: 77.1 ppm).

(29) Synthesis of Bi-Terminally Reactive PEG-PLA-PEG

(30) The overall reaction scheme is indicated below.

(31) ##STR00010##

(32) Synthesis of THP-PEG-OH (Mx=2,355)

(33) ##STR00011##

(34) Synthesis of (2)

(35) ##STR00012##

(36) 15.0 g (223 mmol) of ethylene glycol (1) were dissolved in 200 ml of dichloromethane (anhydrous) in an Ar atmosphere followed by adding 424 mg of TsOH.H.sub.2O (2.23 mmol, 1 mol % vs. (1)), slowly dropping in 9.33 g of 3,4-dihydro-2H-pyran (111 mmol, 0.5 eq. vs. (1)) and adding a trace amount of TEA after stirring for 30 minutes at normal temperature. The reaction solution was concentrated and applied to a column (EtOAc/hexane=1/1) to obtain a unilaterally THP-protected form (2) (7.36 g (22%)). Moreover, this was followed by purification by vacuum distillation. .sup.1H-NMR (500 MHz, CDCl.sub.3): 4.58-4.54 (q, 1H, J=2.6 Hz), 3.95-3.51 (m, 6H), 2.92-2.88 (t, 1H, J=5.6 Hz), 1.91-1.75 (m, 6H) (FIG. 20).

(37) Synthesis of (3)

(38) ##STR00013##

(39) 146 mg (1 mmol) of (2) were placed in an atmosphere replaced with Ar {vacuum: 15 minutes+Ar)3} followed by adding 15 ml of THF (anhydrous) in the presence of flowing Ar and dropping 1 mmol of potassium naphthalene into the THF to carry out metalation. Following metalation, 2.5 ml (50 mmol) of ethylene oxide were dropped in in the presence of flowing Ar followed by stirring for 2 days at normal temperature. The reaction solution was purified by re-precipitation with 400 ml of diethyl ether to obtain THP-PEG-OH (3) by freeze-drying (2.2 g (recovery rate: 100%)). GPC number average molecular weight (Mn)=2355, Mw/Mn=1.07, .sup.1H-NMR (500 MHz, CDCl.sub.3): 4.63-4.6 (t, 1H), 3.90-3.37 (m, 208H), 1.89-1.47 (m, 8H) (FIG. 21).

(40) Synthesis of THP-PEG-PLA-OH

(41) ##STR00014##

(42) 2000 mg (0.8493 mmol) of THP-PEG-OH (Mn=2,356, Mw/Mn=1.094), 979.2 mg of dl-lactide (recrystallized) (6.794 mmol, 8 eq. vs. THP-PEG-OH) and 34.40 mg of Sn(Oct).sub.2 (0.0849 mmol, 0.1 eq. vs. THP-PEG-OH) were dissolved in 97.92 mL of toluene (dl-lactide: 10 mg/mL) in an N.sub.2 atmosphere followed by stirring for 48 hours at 120 C. Following the reaction, the reaction solution was concentrated and re-precipitated with diethyl ether. After freeze-drying with benzene, the structure of the compound was analyzed by .sup.1H-NMR (yield: 2531 mg, yield rate: 85.0%, FIG. 1).

(43) Protons were assigned according to FIG. 1. A value of 214.00 was used as the basis for calculated values based on the EO chain-derived peak a of PEG. The number of PLA chains was determined to be 5 based on the value of the PLA-derived peak b.

(44) Synthesis of THP-PEG-PLA-PEG-THP

(45) ##STR00015##

(46) 2000 mg (0.7366 mmol) of THP-PEG-PLA-OH and 539.9 mg of DMAP (4.419 mmol, 3.0 eq. vs. THP-PEG-PLA-OH) were dissolved in 30 mL of CH.sub.2Cl.sub.2 in an N.sub.2 atmosphere followed by the addition of 411 L of TEA (2.946 mmol, 2 eq. vs. THP-PEG-PLA-OH) and stirring for 10 minutes while cooling with ice (0 C.). After mixing in 67.41 mg of adipoyl chloride (0.3683 mmol, 0.5 eq. vs. THP-PEG-PLA-OH) dissolved in 20 mL of CH.sub.2Cl.sub.2, the mixture was stirred for 48 hours at room temperature. Following the reaction, the product was washed with 1 N HCl and Milli-Q water and this procedure was repeated twice. Moreover, after adding MgSO.sub.4 and concentrating, the product was freeze-dried with benzene. The structure of the resulting compound was analyzed by .sup.1H-NMR and GPC (yield: 1619 mg, yield rate: 79.3%, FIG. 2).

(47) Protons were assigned according to FIG. 2. A value of 428.00 was used as the basis for calculated values based on the EO chain-derived peak a of PEG assuming that THP-PEG-PLA-OH was completely bound to both ends of the adipoyl chloride. The number of PLA chains was determined to be 10 based on the value of the PLA-derived peak b. In addition, GPC measurement results are shown in FIG. 3.

(48) As a result of GPC measurement, unimodal elution peaks shifted to the high molecular weight side in the order of THP-PEG-OH, THP-PEG-PLA-OH and THP-PEG-PLA-PEG-THP. On the basis of these results, THP-PEG-PLA-PEG-THP was confirmed to have been synthesized.

(49) Synthesis of OH-PEG-PLA-PEG-OH

(50) ##STR00016##

(51) 1500 mg (0.2707 mmol) of THP-PEG-PLA-PEG-THP and 68.03 mg of pyridinium para-toluenesulfonate (PPTS, 0.2707 mmol, 1.0 eq. vs. THP-PEG-PLA-PEG-THP) were dissolved in 20 mL of MeOH in an N.sub.2 atmosphere followed by stirring for 7 hours at room temperature. Following the reaction, the reaction solution was concentrated and re-precipitated with diethyl ether. After freeze-drying with benzene, the structure of the resulting compound was analyzed by .sup.1H-NMR (yield: 1385 mg, yield rate: 95.2%, FIG. 4).

(52) Protons were assigned according to FIG. 4. A value of 428.00 was used as the basis for calculated values based on the EO chain-derived peak a of PEG. The THP protecting groups were confirmed to have been removed since the peak derived from THP was no longer observed.

(53) Synthesis of NHS-PEG-PLA-PEG-NHS

(54) ##STR00017##

(55) 1200 mg (0.2232 mmol) of OH-PEG-PLA-PEG-OH were dissolved in 50 mL of CH.sub.2Cl.sub.2 in an N.sub.2 atmosphere. Separate from this, 285.9 mg (1.116 mmol) of di(N-succinimidyl) carbonate (DSC) were dissolved in 20 mL of CH.sub.3CN at 60 C. over the course of 30 minutes. After mixing the 20 mL of the DSC solution with the 50 mL of the PEG solution, 200 L of pyridine were added followed by stirring for 48 hours at room temperature. Following the reaction, the reaction solution was filtered and concentrated followed by re-precipitating with diethyl ether. After freeze-drying with benzene, the structure of the resulting compound was analyzed by .sup.1H-NMR (yield: 1142 mg, yield rate: 90.1%, FIG. 5).

(56) Protons were assigned according to FIG. 5. A value of 428.00 was used as the basis for calculated values based on the EO chain-derived peak a of PEG. The rate of conversion to NHS was determined to be 99.3% based on the NHS-derived peak.

(57) Preparation of Chitosan/PEG-PLA-PEG/RADA Gel and

(58) Evaluation of Properties

(59) Preparation of 300 L of Chitosan/PEG (2.0/1.0 wt %) Gel

(60) 150 L of PBS were added to 100 L of 6.0% by weight chitosan prepared using PBS (150 mM, pH 7.4). 50 L of 6.0% by weight bi-terminated NHS-PEG prepared using PBS (150 mM, pH 7.4) were then added thereto.

(61) Preparation of 300 L of Chitosan/PEG/RADA16 (2.0/1.0/0.25 wt %)

(62) 75 L of PBS (300 mM, pH 7.4) were added to 100 L of 6.0% by weight chitosan prepared using PBS (150 mM, pH 7.4). 50 L of 6.0% by weight bi-terminated NHS-PEG prepared using PBS (150 mM, pH 7.4) were then added thereto followed by promptly adding 75 L of a 1.0% by weight aqueous solution of RADA16.

(63) [Preparation of Gelation Phase Diagrams]

(64) Experimental Procedure

(65) Gelation of the solutions was evaluated using a tilting test after allowing to stand undisturbed for 20 minutes. Gelation phase diagrams were prepared after observing the gelation behavior of each solution. The target concentrations of chitosan/PEG-PLA-PEG gels were prepared by suitably modifying the mixing ratio of chitosan, PEG-PLA-PEG and PBS. The final concentration of PBS in all gels was adjusted to 150 mM by suitably using PBS having different ion concentrations. The gelation phase diagrams are shown in FIG. 6.

(66) Chitosan/PEG-PLA-PEG was confirmed to gel over a high concentration range in comparison with that of PEG 5k. This is thought to be caused by a decrease in solvation due to containing PLA and resulted in a reduction in reactivity. In addition, PEG-PLA-PEG is known to only form a physically crosslinked gel by temperature phase transition (Sol-Gel transition) at high concentrations in the manner of 30% by weight at room temperature (T. Mukose, et al., Macromol. Biosci., 2014, 4, 361-367). Since the gelation of this system occurs over an extremely low concentration range, gelation was suggested to have occurred based on the formation of chemical crosslinks between molecular chains.

(67) Measurement of Frequency Dependency

(68) Gel having a volume of 300 L was molded into the shape of a disk having a diameter of 15 mm. After allowing to swell for 24 hours (4 C.) in PBS (150 mM, pH 7.4), the swollen gel was placed on a mounting stand. Parallel plates were placed in close proximity so as to apply a load of 0.5 N. During frequency measurement, shear strain stress was set to 1% (=) and oscillation frequency was measured over a range of 0.1 Hz to 100 Hz (FIG. 7). Storage modulus (G) was higher than loss modulus (G) in both cases and typical gel properties were confirmed.

(69) Observation of Gelation Behavior

(70) Gelation behavior was evaluated by preparing a gel precursor solution having a volume of 210 L on a rheometer stand using a method similar to a typical gel preparation method, followed immediately by beginning measurement of viscoelasticity. The measurement frequency was 1 Hz and load was measured at 1 Pa. Furthermore, gelation of RADA was observed by adding 52.5 L of a 1.0% by weight aqueous solution of RADA16 to 157.5 L of PBS (200 mM, pH 4.7) (FIG. 8).

(71) In both cases, G increased rapidly after the passage of a prescribed amount of time and a well-defined gel point was observed (G>G). In addition, a decrease in the gelation rate was observed in the case of using PEG-PLA-PEG. This is due to a decrease in solvation due to containing PLA and resulted in a reduction in the reaction rate.

(72) Next, the results of observing gelation behavior in the RADA mixed systems are shown (FIG. 9). In the case of the RADA16 peptide gel, G was higher than G at the start of measurement, suggesting that gelation occurred immediately as a result of mixing with PBS. In addition, the value of G increased considerably in comparison with the case of each gel alone as a result of mixing in RADA. Moreover, G changed in multiple stages with the passage of time, and the inflection points thereof exhibited a high correlation with the gelation time of chitosan/PEG-PLA-PEG. On the basis of these results, chitosan/PEG/RADA16 and chitosan/PEG-PLA-PEG/RADA16 were suggested to have formed a network of RADA16 followed by the formation of an interpenetrating polymer network (IPN) in which chitosan/PEG and chitosan/PEG-PLA-PEG formed crosslinks there between.

(73) Measurement of Mechanical Strength

(74) Gel having a volume of 300 L was molded into the shape of a disk having a diameter of 15 mm. After allowing to swell for 24 hours (4 C.) in PBS (150 mM, pH 7.4), the swollen gel was placed on a mounting stand.

(75) After setting the frequency to a constant frequency of 1 Hz, mechanical strength was measured by applying a pressure ranging from 1 Pa to 3000 Pa to each gel sample. The point at which loss modulus G exceeded storage modulus G was defined as the breaking point of the gel and a stress-strain curve was prepared on the basis thereof. Young's modulus was calculated by approximating the initial slope of the resulting stress-strain curve to be a straight line (FIG. 10).

(76) Mechanical strength of the chitosan/PEG-PLA-PEG gel was lower than that of the chitosan/PEG 5k gel. This is thought to be due to a decrease in reactivity caused by introduction of PLA, which in turn caused a decrease in mechanical strength.

(77) In addition, in both cases, mechanical strength improved as a result of containing peptide. This is due to compounding of the gel network, suggesting the formation of an IPN structure.

(78) Measurement of Degree of Swelling

(79) After preparing gel having a volume of 300 L and allowing to swell for 48 hours (4 C.) in PBS (150 mM, pH 7.4), the weight of the gel after swelling was measured. After freeze-drying each sample for 24 hours, the weight of the gel was measured again. The degree of swelling Q0 was calculated using the equation indicated below (FIG. 11).
Q0=(W.sub.sW.sub.d)/W.sub.d
W.sub.s:Weight of swollen gel,W.sub.d:Weight of Dry Gel

(80) The degree of swelling of the chitosan/PEG-PLA-PEG gel was higher than that of the chitosan/PEG 5k gel. This is thought to be due to a decrease in reactivity caused by introduction of PLA, which in turn caused the degree of swelling to increase. In addition, in both cases, the degree of swelling decreased significantly as a result of containing peptide. This suggests an increase in network density based on the formation of an IPN structure.

(81) Evaluation of Degradation Behavior

(82) A degradation test conducted regarding the following:

(83) acid-accelerated degradation test (acetic acid, room temperature conditions, and

(84) experimental procedure.

(85) Gels having a volume of 300 L were respectively prepared and allowed to swell for 48 hours (4 C.) in 1 mL of PBS (150 mM, pH 7.4). After the gels had swollen, the PBS was removed followed by the addition of 1 mL of CH.sub.3COOH and allowing to stand undisturbed under room temperature conditions. After a prescribed amount of time had elapsed, the samples were washed three times using PBS followed by measuring the swollen weight of the gels. After freeze-drying each sample for 24 hours, the dry weight of the gels was measured. Degree of swelling Q and weight loss were calculated using the equations indicated below. The solutions were replaced once a day (FIG. 2).
Q=(W.sub.sW.sub.d)/W.sub.d
W.sub.s:Weight of swollen gel,W.sub.d:Weight of dry gel
Weight loss (%)=(W.sub.d0W.sub.d)/W.sub.d0100
W.sub.d0=initial dry weight(day 0)

(86) The gel using PEG 5k did not exhibit degradation behavior. On the other hand, in the gel using PEG-PLA-PEG, remarkable degradation behavior was confirmed regardless of the presence or absence of RADA. Degradation behavior based on hydrolysis of PLA was confirmed to be demonstrated in the case of using PEG-PLA-PEG. Degradation Test under Physiological Conditions (PBS (150 mM, pH 7.4), 37 C.)

(87) Gels having a volume of 300 L were respectively prepared and allowed to swell for 48 hours (4 C.) in 1 mL of PBS (150 mM, pH 7.4). After allowing the gels to swell, the PBS was removed followed by the addition of 1 mL of fresh PBS and allowing to stand under conditions of 37 C. After a prescribed amount of time had elapsed, the samples were washed three times using PBS followed by measurement of the swollen weight of the gels. After freeze-drying each sample for 24 hours, the dry weight of the gels was measured. Degree of swelling Q and weight loss were calculated using the equations indicated below. The solutions were replaced every 3 days (FIG. 13).
Q=(W.sub.sW.sub.d)/W.sub.d
W.sub.s:Weight of swollen gel,W.sub.d:Weight of dry gel
Weight loss (%)=(W.sub.d0W.sub.d)/W.sub.d0100
W.sub.d0=initial dry weight(day 0)

(88) Increases in weight loss accompanying outflow of non-crosslinked chains was confirmed in the initial stages for PEG-PLA-PEG and RADA. In the case of using PEG-PLA-PEG, gradual degradation behavior was confirmed regardless of the presence or absence of RADA. Degradation rate accelerated in comparison with the case of using PEG 5k, and degradation behavior accompanying hydrolysis of PLA was similarly confirmed even under physiological conditions.

(89) Measurement of Circular Dichroism (DC) Spectrum

(90) Samples having a volume of 90 L were respectively prepared and coated onto a quartz cell having an optical path length of 0.1 mm. Measurement conditions were as indicated below.

(91) Measurement wavelength: 300 nm to 205 nm

(92) Data interval: 0.5 nm

(93) Scanning speed: 200 nm/min

(94) No. of scans: 3

(95) Response time: 2.0 s

(96) Bandwidth: 1.0 nm

(97) Measurement temperature: 20 C.

(98) RADA is known to exhibit a negative Cotton effect based on a sheet structure in the vicinity of 220 nm of the CD spectrum. Since negative Cotton effects were similarly obtained in the gels containing peptide, the fiber structure of the peptide was determined to be stably retained within the gel.

(99) Cell Culturing of Chitosan/PEG-PLA-PEG/RADA Gel

(100) (1) A 4% by weight chitosan solution (150 mM PBS) was prepared using chitosan (carboxymethyl chitosan, Koyo Chemical Co., Ltd.) preliminarily subjected to UV sterilization treatment for 20 minutes.

(101) (2) Subconfluent bovine chondrocytes (P1) preliminarily cultured in an incubator (37 C., 5% CO.sub.2) were detached from the plate by treating with trypsin followed by centrifuging (1,500 rpm, 5 minutes) and removing the supernatant.

(102) (3) 10 mL of DMEM medium were added followed by measuring the number of cells.

(103) (4) After centrifuging (1,500 rpm, 5 minutes) and removing the supernatant, a cell suspension was prepared using a 4% by weight chitosan solution.

(104) (5) 25 L of the cell suspension were added to a 1.5 mL sampling tube.

(105) (6) A 4% by weight PEG solution (in 300 mM PBS) was separately prepared using NHS-PEG-PLA-PEG-NHS subjected to UV treatment for 20 minutes.

(106) (7) 12.5 L of the 4% by weight PEG solution of (6) were added to the cell suspension of (5) followed by the addition and mixing of 12.5 L of a 1.0% by weight RADA16 solution immediately after pipetting. Furthermore, in the case of using chitosan/PEG-PLA-PEG (2.0% by weight/1.0% by weight), an equal volume of 150 mM PBS was added instead of the RADA16 solution.

(107) (8) After allowing to gel for 10 minutes, 500 L of DMEM (10% FBS, 2% Pen-Strep) were added to the upper portion of the gel followed by culturing in an incubator (37 C., 5% CO.sub.2). Samples of the medium were recovered and 500 L of fresh DMEM were added every two to three days. The recovered medium samples were stored at 80 C.

(108) Gel Compositions:

(109) 1. Chitosan/PEG 5k=2.0% by weight/1.0% by weight

(110) 2. Chitosan/PEG-PLA-PEG=2.0% by weight/1.0% by weight

(111) 3. Chitosan/PEG 5k/RADA16=2.0% by weight/1.0% by weight/0.25% by weight 4. Chitosan/PEG-PLA-PEG/RADA16=2.0% by weight/1.0% by weight/0.25% by weight

(112) Culturing Conditions:

(113) Cell count: 5.010.sup.5 cells

(114) Cell density: 1.010.sup.7 cells/ml

(115) Gel volume: 50 L

(116) Media volume: 500 L

(117) Evaluation of Effect of Chitosan/PEG-PLA-PEG/RADA

(118) Gel on Chondrocyte Function

(119) Evaluation of Degradation Behavior (in Presence of Cells)

(120) Chitosan/PEG 5k gel and chitosan/PEG-PLA-PEG gel having a volume of 300 L were respectively prepared using the same method as described above followed by the addition of 1 mL of DMEM and allowing to stand undisturbed under conditions of 37 C. and 5% CO.sub.2. After a prescribed amount of time had elapsed, the samples were washed three times using PBS and each sample was freeze-dried for 24 hours followed by measuring the dry weight of the gel. Weight loss was calculated using the equation indicated below. The medium was replaced every two to three days.
Weight loss (%)=(W.sub.d0W.sub.d)/W.sub.d0100
W.sub.d0=initial dry weight(day 0)

(121) Culturing Conditions:

(122) Cell count: 3.010.sup.6 cells

(123) Cell density: 1.010.sup.7 cells/ml

(124) Gel volume: 300 L

(125) Media volume: 1000 L

(126) In the presence of cells, the chitosan/PEG 5k gel did not exhibit prominent degradation behavior through day 40, while the chitosan/PEG-PLA-PEG gel exhibited gradual degradation behavior over time. Thus, the chitosan/PEG-PLA-PEG gel was suggested to exhibit similar degradation behavior attributable to PLA in the presence of cells as well. In addition, in the chitosan/PEG 5k gel starting on day 40, crosslinked chains were cleaved as the cells spread, and this gel is thought to have exhibited prominent degradation behavior as a result thereof.

(127) MTT Assay

(128) (1) 450 L of DMEM and 50 L of 5 mg/mL MTT reagent were added when the sample medium was replaced.

(129) (2) The cells were incubated for 24 hours (37 C., 5% CO.sub.2).

(130) (3) The supernatant was removed and the cells were disrupted followed by adding 500 L of MTT extraction reagent (2-propanol:1 M HCl=24 vol %:1 vol %) and shaking for 24 hours at 37 C.

(131) (4) Following centrifugation (1,500 rpm, 5 minutes), the supernatant was added to a 96-well plate at 100 L/well followed by measuring absorbance at 570 nm (FIG. 16).

(132) Mitochondrial activity as determined with the MTT assay decreased in all of the hydrogels during the early stage of culturing. Subsequently, although activity remained low in gel not mixed with RADA, a remarkable increase in activity was observed in gel mixed with RADA. This is thought to be due to an improvement in cell activity as a result of the peptide fiber structure mimicking the environment in the body. In addition, cell activity improved significantly in the case of using PEG-PLA-PEG. This is thought to be due to the effect of imparting degradability. In addition, a decrease in activity was confirmed during the latter stage of culturing in the case of using PEG-PLA-PEG. This suggests that the gel degraded and released cells.

(133) Dimethylmethylene Blue (DMMB) Assay

(134) (1) 4 mg of DMMB were dissolved in 1.25 mL of ethanol followed by the addition of 0.75 mL of formic acid and 6.4 mL of a 1.0 M NaOH solution and bringing to a final volume of 250 mL with Milli-Q water to prepare a DMMB solution.

(135) (2) The DMMB solution was added to a 96-well plate at 125 L/well.

(136) (3) A calibration curve working solution of chondroitin sulfate solution (in PBS) and sample (melted gel diluted two-fold with medium) were accurately pipetted into each well at 20 L/well.

(137) (4) Absorbance at 570 nm was measured (FIGS. 17 to 19).

(138) When the prepared chitosan/PEG-PLA-PEG/RADA16 gel was compared with the chitosan/PEG 5k/RADA16 gel, although the amount of GAG produced in the medium and the amount GAG produced in the gel indicated roughly the same levels of GAG production during the early stage of culturing, in the latter stage of culturing, the amount of GAG produced by the chitosan/PEG-PLA-PEG/RADA16 gel was higher. The chitosan/PEG-PLA-PEG/RADA16 gel was confirmed to demonstrate gradual degradation behavior under the culturing conditions, and GAG production is thought to have improved as a result of imparting degradability to the gel.

(139) A polymerization initiator or polymerization stimulator (such as UV light or gamma rays and the like) is required in the case of using PEG-PLA-PEG having polymerizable functional groups on both ends (polymerizable functional group-PEG-PLA-PEG-polymerizable functional group) or in the case of using as a crosslinking material for gelation.

(140) In this case, there are considerable disadvantages, such as (1) a considerable decrease in cell viability following the gelation reaction due to phototoxicity or a toxic signal such as a polymerization initiator degradation product, or (2) difficulty in ensuring safety at the clinical stage due to increased risk to cells of genetic abnormalities or malignant transformation attributable to irradiation with light (and mainly ultraviolet light) (Williams, et al., Biomaterials, 2005, 26, 1211-1218; Liu, et al., Adv. Mater., 2014, 26, 3912-3917; Cui, et al., Biomacromolecules, 2013, 14, 1904-1912).

(141) On the other hand, succinimide-PEG-PLA-PEG-succinimide differs from physically crosslinked gels using PEG-PLA-PEG or PLA-PEG-PLA in that it is able to crosslink at extremely low temperatures. Thus, since it can be handled as a cell mixture having low viscoelasticity, it can be used as an injectable gel administered to the body, and therefore is highly compatible with clinical treatment.

(142) In addition, since the reaction between protein molecule amino acid residues and succinimides having safe degradation products (by-products) is used as the driving force behind the gelation reaction, and since gelation proceeds simply by mixing, another third stimulating factor is not required.

(143) As a result, the reaction has extremely low toxicity that enables cell viability following the gelation reaction to be maintained at 100% relative to prior to crosslinking.

INDUSTRIAL APPLICABILITY

(144) The triblock copolymer of the present invention is extremely useful for the production of a biodegradable interpenetrating polymer network (IPN).