KR20160041166A - Injectable Hydrogels Derived from Phosphorylated Calcium Alginate Complexes - Google Patents

Abstract

The present invention relates to a method for manufacturing a porous injectable hydrogel including a phosphorylated calcium alginate complex, and to a hydrogel manufactured by the manufacturing method. The injectable hydrogel derived from a phosphorylated calcium alginate complex is manufactured by the manufacturing method of the present invention. The injectable hydrogel is physically stable; provides a microenvironment advantageous to cell growth; and provides excellent effects to cytotoxicity and cell transmission. Therefore, the hydrogel of the present invention can be used in soft bone and soft tissue engineering by providing a microenvironment beneficial to 3D capsulation of cells.

Description

[0002] Injectable Hydrogels Derived from Phosphorylated Calcium Alginate Complexes from Phosphorylated Calcium Alginate Complexes [

The present invention relates to a process for preparing a porous injectable hydrogel comprising phosphorylated calcium alginate (CaPAlg) and a hydrogel prepared by the process.

Since the hydrogel of poly-2-hydroxyethyl methacrylate (PHEMA) has been developed by O. Wichterle et al. (Nature, 1960) in the 1960s, the hydrophilic and biocompatibility of the hydrogel The interest in the application has been steadily increasing.

These hydrogels were initially used mainly in hygienic products based on high absorptivity, but nowadays, by introducing various additional functionalities, it is possible to improve the separation, concentration and stabilization of drug delivery system, embolization, support for tissue engineering, Immunoassays, bioreactors, sensors, chromatographic and cosmetic fillers, and the like, from pharmaceutical applications to industrial applications.

Since the development of calcium alginate hydrogels around 1980, hydrogels for various biomaterials using natural or synthetic polymers have been developed. In particular, biodegradable injectable hydrogels are capable of biologically encapsulating active molecules, Due to its unique properties, which can be administered to the human body without painful surgery due to its excellent formability, it has been actively studied as a transdermal extracellular matrix for drug delivery systems and tissue engineering to regulate the release of bioactive agents.

On the other hand, the alginate is Ca ^{2 +,} Mg ^{2 +,} Ba ^{2 +} 2, such as are known to form a hydrogel by rating keel with a cation (Seliktar, D. et al., Science., 2012). The hydrogel is prepared by a typical external setting technique using calcium salts such as CaCl ₂ and CaSO _4, and the oxygen atoms of the two opposing GG blocks (egg-box model) Thereby forming human cross-linking. Such alginate hydrogels are of importance in biomedical applications due to their biocompatibility and low toxicity.

In particular, the alginate hydrogel as a biomaterial can strengthen its intrinsic properties through chemical modification and introduce new properties. For example, Grøndahl et al. ( Biomacromolecules , 2011) reported that phosphorylated alginate derivatives (PAlg) using the urea / phosphate method, which measures the nucleation and growth induction of hydroxyl apatite, ).

However, it is difficult to form a hydrogel by using phosphorylated alginate as a method by adding Ca ^{2 +} externally, which is generally used. This is thought to be due to molecular weight reduction of the phosphorylated alginate and conformational change due to phosphorylation.

Therefore, the inventors of the present invention have made intensive studies and efforts to produce a hydrogel using phosphorylated alginate. As a result, when a phosphorylated alginate calcium complex (CaPAlg) as a new alginate derivative is mixed with an alginate sodium salt, self- -gelation), and thus the present invention has been completed.

One object of the present invention is to provide a method for preparing a phosphorylated alginate calcium complex (CaPAlg) aqueous solution by adding calcium ion (Ca ^{2 +} ) to an aqueous solution containing phosphorylated alginate; And a second step of mixing the phosphorylated alginate calcium complex aqueous solution and sodium alginate aqueous solution to form a hydrogel, wherein the phosphorylated alginate calcium complex (CaPAlg) binds phosphorylated alginate with calcium ions Wherein the hydrogel is dispersed in an aqueous solution.

Another object of the present invention is to provide an injectable hydrogel prepared by the above-mentioned production method.

Yet another object of the present invention is to provide a porous, injectable hydrogel comprising phosphorylated alginate (CaPAlg) crosslinked with calcium ions (Ca < ^{2 +} >).

According to one aspect of the present invention, there is provided a method for preparing a phosphorylated alginate calcium complex (CaPAlg) aqueous solution by adding calcium ions (Ca ²⁺ ) to an aqueous solution containing phosphorylated alginate; And a second step of mixing the phosphorylated alginate calcium complex aqueous solution and sodium alginate aqueous solution to form a hydrogel, wherein the phosphorylated alginate calcium complex (CaPAlg) binds phosphorylated alginate with calcium ions Wherein the hydrogel is dispersed in an aqueous solution.

Hereinafter, the injectable hydrogel of the present invention will be described in detail.

The inventors of the present invention have found that when preparing a hydrogel containing phosphorylated alginate, a commonly used method of dispersing calcium ions in an alginate solution to form a hydrogel (this method is referred to as an "external setting" technique), calcium ions (Ca ²⁺ ) show a strong affinity for phosphate and do not cross-link with other phosphorylated alginate molecules (PAlg), forming a hydrogel containing phosphorylated alginate It was found that it was difficult to do so. Therefore, the present invention relates to a method for preparing a calcium ion complex by combining a calcium ion as a crosslinking agent with a phosphorylated alginate first and then mixing it with an easily ionizable alginate (this method will hereinafter be referred to as an 'internal diffusion technique'), It is a technical feature that a hydrogel containing a phosphorylated alginate can be formed more easily.

"Alginate" of the present invention is alginate, and alginic acid is a natural polysaccharide and is an important constituent of brown algae such as seaweed and kelp. Alginic acid has excellent biocompatibility, low toxicity and relatively low price. In addition, the alginate solution is a divalent cation: is relatively easy to produce a hydrogel in combination with (such as Ca ^{+ 2).} The contents of D-mannuronic acid and L-glutaric acid, which are constituents of alginic acid, differ depending on the kind of brown algae and have a great influence on the physical properties of alginic acid. Particularly, the molecular structure of alginic acid is a block copolymer of D-mannuronic acid and L-glutaric acid, and since the L-glucuronic acid block forms a hydrogel by binding with the divalent cation, the length of the L- It is an important factor in determining the physical properties of alginic acid hydrogels. The general molecular weights of alginic acid currently used are 30,000-400,000 and the constant values K and a of the Mark-Houwink relation ([]] = KM _v ^a are 2 × 10 ^-3 and 0.97, respectively (0.1 M NaCl solution, 25 ° C. ).

In the first step of the method for producing a hydrogel containing phosphorylated calcium alginate according to the present invention, phosphorylated alginate (PAlg) is dissolved in an aqueous solution, calcium ion (Ca ²⁺ ) is added to the phosphorylated alginate calcium complex (CaPAlg) aqueous solution (Fig. 1).

Herein, the method for producing PAlg may be used without limitation as long as it is used in the art to phosphorylate alginate. For example, alginate having a salt form with a monovalent ion can be prepared by mixing with a phosphate. This is because the alginate precursor should be water-soluble, but in the case of divalent ions, alginate which forms a salt with the monovalent ions is used because it forms a gel when reacted with alginate and becomes insoluble. The monovalent ions include, but are not limited to, H ⁺ , Li ⁺ , Na ⁺ , K ⁺ , and NH ₄ ⁺ . In one embodiment of the present invention, sodium alginate is produced by preparing phosphorylated alginate (PAlg) using the H ₃ PO ₄ / P ₂ O ₅ / Et ₃ PO ₄ /1-hexanol technique, The alginate was dispersed in a phosphoric acid (H ₃ PO ₄ ) solution, and a hexanol solution containing phosphorus pentoxide (P ₂ O ₅ ) and triethyl phosphate (Et ₃ PO ₄ ) was added, Of methanol.

The CaPAlg complex is a combination of phosphorylated alginate and calcium ions, which can be prepared by proton exchange of phosphorylated alginate molecules with calcium ions.

Specifically, the PAlg may be dissolved in distilled water and Ca (OAc) ₂ aqueous solution may be added thereto. It may be below pH 8.

The first step may further include a step of dialyzing the CaPAlg complex aqueous solution to remove inorganic salts.

After the dialysis and lyophilization, the phosphorylated alginate calcium complex is obtained as a white powder.

The second step of the present invention is a step of mixing a CaPAlg composite aqueous solution and a PAlg aqueous solution to form a hydrogel. For example, 1 to 10% by weight of phosphorylated alginate calcium complex aqueous solution and the sodium alginate aqueous solution may be mixed, and in another example, 1 to 3% by weight.

The phosphorylated alginate calcium complex aqueous solution and the sodium alginate aqueous solution may be mixed at a volume ratio of 1: 0.1 to 1:10, for example, in a volume ratio of 1: 0.25 to 1: 4. In another example, they may be mixed in a volume ratio of 1: 0.67 to 1: 1.5.

CaPAlg 40 to 60 mixed at the above volume ratio can provide a micro environment favorable for cell growth because of having an optimal gelation time (about 2 to 10 minutes) and a pore structure having a size of 300 to 400 탆 which can be injected (Experimental Example 4, 7 and FIG. 8), it is possible to provide a more stable hydrogel with less change in modulus change depending on the number of vibrations (Experimental Example 3, FIG. 8 (f)) and, in relation to biocompatibility, Toxicity and cell transfer (Experimental Example 5).

In hydrogels containing alginate, when P is involved (phosphorylated alginate), resistance to enhanced degradation appears. However, hydrogel containing phosphorylated alginate could not be prepared by an external setting technique for preparing a hydrogel by adding calcium ion from the outside. Thus, the present inventors confirmed that a hydrogel containing phosphorylated alginate can be easily prepared by using a CaPAlg complex (internal diffusion technique) prepared in advance (Experimental Example 4 and FIG. 8)

In another aspect, the present invention provides an injectable hydrogel prepared by the above method.

The hydrogel prepared by the above method shows a gelation time depending on the ratio of the CaPAlg complex aqueous solution and the PAlg aqueous solution and shows a gelation time of about 2 to 10 minutes in the case of CaPAlg 40 to 60, (Experimental Example 4 and Fig. 8)

In addition, it has been confirmed that it has a porous morphology and can be used as a transient extracellular matrix for drug delivery systems and tissue engineering (Experimental Example 2 and Fig. 6).

In another aspect, the present invention provides a porous, injectable hydrogel crosslinked with calcium ions (Ca ^< ^{2 + &} gt ^; ) comprising phosphorylated alginate (CaPAlg).

In the present invention, the CaPAlg complex provides a polysaccharide chain having Ca ^{2 +} and PO ^{4 -} ions. Unlike a typical external setting technique, the internal diffusion technique of the present invention uses calcium ions (Ca ^< ^{2 + &} gt ^; ) to increase the calcium ion concentration uniformly on the modified molecules, And it was confirmed by the ICP-OES analysis that the DS value was efficiently supplied into the molecule (Experimental Example 1- (3)).

It was confirmed that the injectable hydrogel of the present invention can produce a hydrogel having an optimal gelation time and mechanical strength by controlling the ratio of sodium alginate and phosphorylated alginate calcium complexes 8 (f)).

The injectable hydrogel derived from the phosphorylated calcium alginate complex produced by the preparation method of the present invention has an optimum gelation time (about 2 to 10 minutes) capable of being injected subcutaneously by injection and a pore structure of 300 to 400 mu m in size Can provide a micro environment favorable for cell growth, has less change in modulus change according to the frequency, is more stable, and has excellent effects on cytotoxicity and cell transfer in short-term cell culture with respect to biocompatibility can do.

In addition, the method of preparing an injectable hydrogel of the present invention can produce phosphorylated alginate under mild reaction conditions, and it is possible to use a minimal amount of Ca (OAc) ₂ and a method of externally adding calcium ions It is possible to simply manufacture a hydrogel having a uniform ion concentration by mixing the NaAlg and CaPAlg by the internal diffusion technique of the phosphorylated alginate hydrogel which could not be produced by the method of the present invention.

Therefore, the hydrogel of the present invention can provide a favorable micro environment for 3D encapsulation of cells, and thus can be usefully used for cartilage and soft tissue engineering.

FIG. 1 shows a process for preparing a phosphorylated alginate calcium complex (CaPAlg) according to an embodiment of the present invention.
Figure 2 shows FT-IR spectra of sodium alginate (NaAlg), phosphorylated alginate (PAlg) and phosphorylated calcium alginate complex (CaPAlg).
Figure 3 shows a thermogram of sodium alginate (NaAlg), phosphorylated alginate (PAlg) and phosphorylated calcium alginate complex (CaPAlg).
Figure 4 shows ¹ H-NMR (D ₂ O) spectra of NaAlg, PAlg and CaPAlg. Here, for each of the G and M units, G _n and M _n represent protons at C n (n = 1 to 5).
Figure 5 shows the characteristic peaks in the ³¹ P-NMR spectrum of the PAlg polymer.
6 shows an illustration of the preparation and formation of injectable hydrogels using CaPAlg (A: red) and NaAlg (B: blue). (The inset shows the liquid form of the precursor solution and the injectable nature of the A + B mixed solution.)
7 shows SEM images (a to d) and DMA graph (e) of CaPAlg hydrogel: (a) CaPAlg20, (b) CaPAlg40, (c) CaPAlg60 and (d) CaPAlg80.
Figures 8 a) to d) show hydrogels prepared from different volume ratios of CaPAlg and NaAlg solutions, and e) hydrogels prepared by an external setting technique: (B), and the gelation time (C). f) shows the DMA of CaPAlg having different composition ratios of NaAlg and CaPAlg.
Figure 9 shows fluorescence images of cells encapsulated in hydrogel CaPAlg50 during a) four hours, b) one day and c) three days of culture. The green dot represents the surviving cell.
Figure 10 shows the viability of early cells for 0-1 days of NaAlg hydrogel and CaPAlg50 hydrogel.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

<Preparation of materials>

See sodium carbonate of the invention _{(NaAlg; M w 1.2 ~ 1.9} × 10 5, M / G ratio: 1.56), triethyl phosphate (triethylphosphate), phosphorus pentoxide (phosphorous pentoxide), and 1-hexanol (1-hexanol ) Was purchased from Sigma-Aldrich (USA) and phosphoric acid was purchased from JT Baker (USA). All purchased chemicals were used without further purification.

Manufacturing example  1: phosphorylated Alginate (PAlg)  Produce

NaAlg was phosphorylated by the phosphorylation process of chitosan disclosed by Amaral et al., (J. Biomater. Sci., Polymer Ed., 2005). Specifically, 1 g of sodium alginate powder was dispersed in H ₃ PO ₄ (43 mL) at 37 ° C., phosphorus pentoxide (P ₂ O ₅ , 28 g) and triethyl phosphate (Et ₃ PO ₄ , 38 mL) was added slowly. The reaction mixture was stirred at 37 ° C for 2 days, and then poured into an excess amount of methanol (200 ml) to obtain a white precipitate. The precipitate was filtered using a PTFE filter (0.5 μm pore size, 47 mm diameter), washed three times with methanol (50 mL), and dried in vacuum at room temperature for one day to yield 700 mg pale yellow phosphorylated inform Nat (PAlg) powder was obtained with a yield of 72%.

Manufacturing example  2: phosphorylated Alginate  Calcium complex CaPAlg )

100 mg of the PAlg powder obtained in Preparation Example 1 was dissolved in 100 ml of distilled water, and 1.0 N Ca (OAc) ₂ aqueous solution was gradually added thereto to adjust the pH to 8 or less. Thus, a CaPAlg complex in which calcium ions are bonded to phosphorylated alginate by proton exchange of proton of phosphorylated alginate and proton exchange of calcium ion (Ca ²⁺ ) was prepared (FIG. 1). Then, the solution was dialyzed for 2 days using a dialysis tubing membrane (pore: 12,000 Da) to remove all inorganic salts in the solution. Water was changed every 6 hours and 97 mg of CaPAlg white powder was obtained with a yield of 87% after dialysis and lyophilization.

Example  1: phosphorylated Alginate  Calcium complex CaPAlg )from Hydrogel  Produce

An aqueous solution of 2 wt% NaAlg solution (solution A) and an aqueous solution containing 2 wt% of the CaPAlg composite prepared in Preparation Example 2 (solution B) were separately prepared. Solution A and Solution B were mixed in the proportions shown in Table 1 below and subjected to short vortexing to produce CaPAlg20, CaPAlg40, CaPAlg60 and CaPAlg80.

Entry No Solution A (mL) Solution B (mL) Hydrogel
Code-Name One 0.2 0.8 CaPAlg20 2 0.4 0.6 CaPAlg40 3 0.6 0.4 CaPAlg60 4 0.8 0.2 CaPAlg80

Each mixture was fixed in a PDMS mold (SYLGARD ^® ) until a hydrogel was formed (FIG. 6).

Experimental Example  1: manufactured Alginate  Investigation of the properties of derivatives

FT-IR analysis, TGA analysis, ICP-OES analysis and NMR analysis were carried out in order to confirm the differences in the respective characteristics of NaAlg, PAlg and CaPAlg.

(One) FT - IR  analysis

FT-IR analysis was performed in Perkin-Elmer Spectrum BXII FT-IR spectrometer, IR spectra were recorded with 16 scans with a resolution of 4 cm ^-1 between 4000 to 500cm ^-1.

As a result of the FT-IR analysis, FT-IR spectra of unmodified NaAlg and modified alginate samples (PAlg and CaPAlg) were shown, as shown in FIG. First, the IR spectrum of NaAlg was measured at 1614 and 1416 cm ^-1 (C = O stretching mode (asymmetric and symmetrical) of the carboxylate and 1300-1090 cm ^-1 (CO (H)) and COC Characteristic bands.

On the other hand, in the case of PAlg modified by NaAlg phosphorylation, several new peaks were observed at 1740, 1630, 1244, 1084, 948 and 928 cm ^-1 in the IR spectrum. This confirmed that the 1740 and 1630 cm ^-1 peaks correspond to the C = O stretching mode of the carboxylic acid, so that the carboxylate ions of the sodium alginate were mostly converted to the acid-form.

In addition, other peaks from phosphate groups such as P = O asymmetric stretching, P-O-C (aliphatic) and two P-O (H) vibration modes were clearly observed, confirming the presence of phosphate on the ring.

Unlike sodium alginate, 1,300 cm ^{- 1} peak disappeared in alginate after phosphorylation. This indicates that phosphorylation occurs in the hydroxyl group of the alginate ring.

The FT-IR spectrum of the CaPAlg complex was similar to NaAlg as shown in FIG. 2, but slightly different from that of PAlg. The fact that the characteristic peaks of the functional groups (P = O, POC, and PO (H)) associated with phosphorus (P) at 1244 to 928 cm ^-1 are not significantly changed compared to NaAlg indicates that the phosphate is intact in the alginate ring Lt; / RTI &gt; In particular, broad peaks at 1608 and 1422 cm ^-1 associated with C = O shifted from the 1740 and 1630 cm ^{& lt} ; ^-1 &gt; peaks of PAlg indicate that all the carboxylic acids were converted to carboxylate anions forming CaPAlg complexes.

Therefore, it was confirmed that CaPAlg complex was produced by cation exchange reaction between PAlg and Ca (OAc) ₂ according to Production Example 2.

(2) Thermal weight  analysis( Thermogravimetric 분석 , TGA )

Thermogravimetric analysis (TGA) was performed using SCINCO TGA N-1500 (sample weight 5 ± 0.1 mg, temperature ramp rate 10 ^o C / min, initial temperature room temperature).

The thermal decomposition patterns resulting from the TGA analysis are shown in Fig. The NaAlg sample used as a control shows two stages of thermal degradation after water evaporation. The first step of pyrolysis of the hydrocarbon fraction occurred at 200 ° C. After the slightly reduced residual carbohydrate was pyrolyzed in the range of 250-500 ° C, the second stage of pyrolysis occurred at ~ 600 ° C, maintaining about 20.7% of the residual weight of sodium oxide.

On the other hand, in the case of PAlg, a complex phase of pyrolysis was observed. The first step of pyrolysis of the hydrocarbon fraction obviously takes place at 200 ° C. However, the next step takes place in the range of 300-600 ° C and> 600 ° C due to pyrolysis of additional carbohydrates, dehydration of the phosphate group to phosphorus pentoxide, and evaporation of phosphorus pentoxide. As a result, only 0.5 wt% of the remaining polymorphic polyphosphate is present in vitreous or amorphous form.

Similar to the case of PAlg, several steps of pyrolysis are also observed in the thermogram of CaPAlg. After evaporation of the water, the first stage of pyrolysis of the hydrocarbon fraction is clearly observed at 300 - 600 ° C, the second stage for additional carbohydrate pyrolysis involving dehydration of the phosphate group to phosphorus pentoxide, at 200-300 ° C. Additional weight loss at higher temperatures above 800 ° C may be due to continued dehydration of Ca (OH) ₂ and CaP compounds formed at high temperatures. (For _{example: Ca (OH) 2 → CaO} ; Ca (H 2 PO 4) 2 → CaHPO 4 → Ca 2 (PO 4) 2).

(3) ICP - OES  analysis

ICP-OES analysis was used to determine the DS values of PAlg and CaPAlg samples. Samples were digested with nitric acid and analyzed using a Perkin Elmer Optima 5300dV spectrometer at a forward rate of 1.5 mL / min at a temperature of 24 ° C and a relative humidity of 29 ± 1%. At this time, a Burgener PEEK Mira Mist nebulizer was used and the instrument was performed by averaging three 5 second integration values at 213.62 nm for the PAlg sample and 317.93 nm for the CaPAlg sample. All the samples were measured three times.

Thus, the content of Ca in P and CaPAlg in PAlg was measured by elemental analysis using ICP-OES. All analyzes were performed three times for both samples (100 mg each). The contents (ppm and mol%) of the phosphorus and calcium contained in 1 mole of the cyclic monosaccharide unit are summarized in Table 2.

Sample P Ca x 10 ⁴ (mg / kg) DS (mol%) x 10 ⁴ (mg / kg) DS (mol%) PAlg 1.5 ± 0.1 12.5 - - CaPAlg 0.31 + 0.02 7.8 7.8 ± 0.1 112.5

On average, the PAlg sample contains 1.25 moles of P element per 10 moles of cyclic monosaccharide unit (CMU) and the CaPAlg sample contains more than 1.13 moles of Ca element per mole of cyclic monosaccharide unit. As a result, each shows a DS value of 12.5 and 112.5 mol%. The DS value is the concentration of phosphate group or calcium ion introduced per alginate subunit, indicating that the CaPAlg complex has a high concentration of calcium ions. The DS value (112.5 mol%) of the Ca ion to the CaPAlg complex was much higher than the DS value (12.5 mol%) of the P element to the PAlg. In theory, functional groups such as carboxylic acid and phosphoric acid in the PAlg molecule can be cation exchanged to form a phosphorylated calcium alginate complex in the presence of Ca (OAc) ₂ , The DS values were calculated assuming that all protons in the carboxylic acid functional group in the molecule were exchanged for calcium ions. Thus, the high DS values obtained indicate that all two cyclic monosaccharide units have calcium ions at the carboxylate functional sites and about 12 cyclic monosaccharide units have calcium ions at the phosphate group sites. As a result, this result shows that all the carboxylate and phosphate functionalities form strong ionic bonds with no distinct gelation with the divalent cations, and that each calcium ion is evenly distributed on the surface of the polymer molecule. These chemical features show that the CaPAlg complex is suitable for the preparation of potentially injectable hydrogels.

(4) NMR  analysis

NMR analysis was performed on ¹ H and ³¹ P nuclei on a Bruker Ultrashield 500 PLUS NMR spectrometer.

¹ H-NMR analysis was carried out on all PAlg and CaPAlg to determine the phosphorylation and Ca ^{2 +} and cationic exchange capable sites. Analysis of the ¹ H-NMR spectrum for unmodified alginate and phosphorylated alginate is well described in the prior art (Coleman, R., et al., Biomacromolecules 2011), unmodified sodium alginate and the invention The major peaks of the phosphate-modified alginic acid prepared in Example 1 were determined based on the document values as shown in Fig. The overall spectra show that the phosphorylation and protonation are carried out with a considerably increased complexity downfield. Grøndahl et al. Reported that the phosphorylation of the M-residue occurs mainly in the C-3 equatorial hydroxyl group and also phosphorylation occurs at the G-residue, but it is difficult to determine the site of phosphorylation. However, as shown in FIG. 4, the CH protons at C-2, -3 and -5 of both M and G units for the PAlg prepared above are C-2, -3 and - 5 was strongly shifted to the down field compared to CH proton. On the other hand, the CH protons of C-1 and -4 migrated weakly. These results show that the phosphorylation of the hydroxyl group and the protonation of all the carboxylate groups at C-5 resulted in a lot of electrical changes at C-2, -3 and -5.

Thus, the H ₃ PO ₄ / P ₂ O ₅ / Et ₃ PO ₄ method can reasonably be expected to have a relatively high degree of phosphorylation increase than any other method.

^A significant change in the ¹ H NMR spectrum was consistent with the result of ICP-OES showing a relatively high DS value of PAlg (mean 12.5 mol%). In addition, as shown in Figure 5, the ³¹ P-NMR spectrum of the tested PAlg of the strong signal at -0.01 ppm and the low intensity signal at -10.83 ppm, also known as pyrophosphate, coincided with the Grøndahl result . No other signals were observed.

As a result, the ¹ H NMR spectrum of CaPAlg obtained after cation exchange of calcium ions and PAlg retained the high complexity of the spectrum seen in PAlg caused by phosphorylation. On the other hand, the overall spectrum was shifted back to the up field similar to that of NaAlg due to the formation of calcium salts. When compared to the spectrum of NaAlg, the protons associated with C-2 and C-3 of both the M and G units of CaPAlg were still weakly transferred to the downfield due to phosphorylation (FIG. 4). On the other hand, C-1, C-4 and C-5 almost returned to the chemical shift of NaAlg. In addition, the general splitting pattern of proton peaks was similar to the pattern of NaAlg, but the fine pattern was still more complex than the pattern of NaAlg due to the presence of phosphate groups (peak comparison in color circle display). It is believed that the networked intermolecular and intermolecular complexes between the phosphate and carboxylate groups that are depenetrated through divalent cations will likely affect the splitting pattern of the proton peaks.

As a result, the phosphorylation of the hydroxyl group at C-2 and C-3 and the phosphorylation of calcium such as phosphoric acid calcium salt and carboxylic acid calcium salt at C-2, C-3 and C- The formation of the salt causes a downfield shift of the positive field at C-2 and C-3 for both the G and M units and splitting of more complex proton peaks.

Experimental Example  2 : CaPAlg  And NaAlg According to the ratio of Hydrogel  Pore structure analysis

The microporous structure of the CaPAlg hydrogel was analyzed using a scanning electron microscope (SEM) at a voltage of 20 kV in a reduced-pressure freeze-dried hydrogel sample (Hitachi S-4300). SEM analysis was performed twice each.

The microporous structure of the alginate hydrogel exhibited highly interconnected pores crosslinked by calcium ions as shown in Fig. Depending on the amount of CaPAlg increased to 20 to 80 v / v%, the hydrogel has several important characteristic trends including i) reduction in pore size, ii) decrease in regularity of pore shape, and iii) increase in hardness of pore walls Lt; / RTI &gt; In fact, CaPAlg20 is NaAlg and CaCl ₂ Similar to the pore structure formed by Due to their size and soft properties (average pore size in length ~ 800 μm) they exhibit very folded and compressed pore structure. Other hydrogels show less compacted pore structure due to their small size (average pore size in the range of 320-400 μm in diameter). The increased hardness of the pore wall is believed to be the result of an increased content of CaPAlg in the hydrogel. The increased irregularity of the pore morphology for increasing CaPAlg concentration may be due to the irregular participation of the polymeric counteranion (P-modified alginate) in the network process with the pure alginate molecule. As a result of the SEM analysis, in particular, the hydrogel CaPAlg 40-60 has a pore structure with an optimum gelation time (about 2-10 minutes) and a diameter of 300-400 μm, thereby providing a favorable micro environment for cell growth in the field of tissue engineering can do.

Experimental Example  3: Dynamic Mechanical Analysis Dynamic mechanical 분석 , DMA )

Dynamic mechanical analysis was performed to investigate the viscoelastic behavior of hydrogels with different composition ratios of NaAlg and CaPAlg, and this dynamic mechanical analysis (DMA) was performed in the tensile compression mode and the compression rod- -plate configuration) was performed on a dynamic mechanical analyzer (DMA 25N, 01dB-Metravib, France). The dynamic compression stress cycle of the increasing frequency of the sample was measured in the range of 0.5 to 10 Hz for 10 minutes at room temperature. Three replicate samples were measured to obtain an average value of storage modulus (E ') and loss modulus (E ") for each condition.

As a result, the modulus of CaPAlg20 significantly increased as the frequency increased (Fig. 8 (f)). The modulus change for CaPAlg40 and CaPAlg60 was minimal over a broad range of frequencies representing a higher content of CaPAlg which significantly contributes to crosslinking to provide a more stable hydrogel. 8 (a) - (e), the optimum content of NaAlg and CaPAlg for hydrogel formation was found to be CaPAlg 60%.

Experimental Example  4 : Gel time  Measure

The gelation time for the hydrogel prepared according to Example 1 was measured using a well-known vial vial inversion test (or the flow test). The gelling time is determined by the point where the mixture of solution A and solution B no longer flows when the vial is inverted. Three replicate samples were measured at room temperature and their average values were obtained.

The gel time for each sample and the molopoly morphology before and after lyophilization are shown in FIG. CaPAlg20 and CaPAlg80 exhibit a relatively longer gel time due to the lack of a normal alginate skeleton containing the G block required for the lack of Ca ions and the coupling of two opposing GG blocks (egg-box model) Respectively> 40 minutes and> 30 minutes, FIGS. 8 (a) and 8 (d)). The hydrogels of CaPAlg40 and CaPAlg60 gelled in 2-10 minutes and obtained optimal results (Fig. 8 (b) and (c)) well fixed to the PDMS mold. The optimum gelling time can be obtained by well controlling the concentrations of both components (Solution A and Solution B). In the preparation of CaPAlg50 hydrogel (1 mL of 2 wt% CaPAlg solution + 1 mL of 2 wt% NaAlg solution), the amount of Ca ions supplied internally was assumed to be about 1.56 mg (0.04 mmol) in 38.44 mg of alginate component do. To measure the gelation potential using such molar amounts as NaAlg (38.44 mg / 1.5 mL) and CaCl ₂ (0.04 mmol / 0.5 mL or 4.44 mg / 0.5 mL) contained in the CaPAlg50 hydrogel, (Conventional method) was attempted, but no gelation was observed at all (Fig. 8 (e)). It is believed that at least 17.32 mg (0.156 mmol, CaCl _2, about 4 times more than in the case of CaPAlg) is needed to obtain optimal hydrogel formation by external supply of calcium ions. However, in this case, it is inappropriate to inject under the skin through the syringe because the gelation time is too short. The amount of subcutaneously injectable Ca ions from CaPAlg 40-60 and the optimal gelation time and the improved gelation results and improved results are due to (i) different routes of Ca ion supply (internal versus external), (ii) (Iii) the different mechanisms of Ca ion transfer from the carboxylate (and / or phosphate) functional groups of CaPAlg to the carboxylate functional groups of the NaAlg molecule, and (iv) the function of the normal alginate molecules Could be caused by the direct participation of the polymeric counter ion of CaPAlg with phosphate and carboxylate in networking.

In addition, the use of CaPAlg to produce an injectable alginate-based hydrogel showed easy gelation without significant change in the volume of the hydrogel prepared. This may be an important feature for cell encapsulation because the encapsulated cells may be free from the physical stresses that can lead to volume changes during the gelling action of the hydrogel.

Experimental Example  5: In vitro cell-encapsulated Hydrogel  Cell test

To assess the applicability of injectable CaPAlg hydrogels for use as cell encapsulating materials, the viability of MC3T3 encapsulated by hydrogel was measured by fluorescence microscopy and MTS analysis using the LIVE / DEAD Viability / Cytotoxicity Kit ( Invitrogen, Carlsbad, Calif.). MC3T3 subcultured at a cell density of 1 x 10 &lt; ⁶ &gt; was encapsulated in 1 ml of hydrogel and incubated for 7 days. All procedures were carried out in a laminated lamellar flow hood.

The medium was completely replaced every 2 days during the incubation. After the pre-defined incubation time, the cell-encapsulated hydrogel was transferred to a new plate and washed three times with PBS solution. Viability and morphology of the cells after cell staining were measured. To analyze the encapsulation efficiency of the hydrogel, cell viability after encapsulation was measured by direct cell-counting based on Live / Dead staining. During the incubation, the metabolic activity of the cells was measured using an MTS assay kit. After incubation, the hydrogel was placed in a 24-well plate and the MTS solution was added to each hydrogel. After incubation for 4 hours at 37 [deg.] C, colorimetric measurements were performed in a micro reader with optical density reading at 490nm. All cell culture experiments were performed in triplicate and were compared using one-way ANOVA at a significance level of p <0.0006 in performing statistical analysis.

Biocompatibility of injectable hydrogel CaPAlg50, with an intermediate composition of CaPAlg 40-60, was assessed through cell morphology, viability monitoring and metabolic activity of the cells encapsulated in the hydrogel. For this, 50 mM CaCl ₂ The biocompatibility of the NaAlg hydrogels prepared using the solutions was compared as a control. Figure 9 shows fluorescence images of the encapsulated cells in the hydrogel during a 3 day incubation period. Green dots indicate surviving cells. Through fluorescence images, the present inventors confirmed the cell dispersion, cell morphology and viability in the hydrogel. Figure 10 quantifies the viability of early cells for 0-1 days in Figure 9a). As shown in FIG. 9 a) and FIG. 10, after 4 hours of encapsulation (day 0), round shaped cells with uniform dispersion were observed in all hydrogels and the cells of CaPAlg 50 hydrogel compared to the control (NaAlg) There was no significant difference in viability (Fig. 9).

Claims (8)

A first step of preparing a phosphorylated alginate calcium (CaPAlg) complex aqueous solution by adding calcium ions (Ca ²⁺ ) to an aqueous solution containing phosphorylated alginate; And
And a second step of mixing the phosphorylated alginate calcium (CaPAlg) complex aqueous solution and sodium alginate (NaAlg) aqueous solution to form a hydrogel, wherein the phosphorylated alginate calcium (CaPAlg) complex is phosphorylated alginate PAlg) and calcium ions are combined with each other.
The method according to claim 1,
Wherein the phosphorylated alginate of the first step is prepared by mixing sodium alginate with a solution containing phosphate.
The method according to claim 1,
Wherein the phosphorylated calcium alginate complex aqueous solution has a pH of 7 to 8.
The method according to claim 1,
Wherein said first step further comprises the step of dialysis to remove inorganic salts from an aqueous solution of phosphorylated alginate calcium complex (CaPAlg).
The method according to claim 1,
Wherein the second step comprises mixing 1 to 3 weight% phosphorylated alginate calcium complex aqueous solution and 1 to 3 weight% sodium alginate aqueous solution.
The method according to claim 1,
Wherein the third step comprises mixing the phosphorylated calcium alginate complex aqueous solution and the sodium alginate aqueous solution at a volume ratio of 1: 0.67 to 1: 1.5.
An injectable hydrogel prepared by the method of any one of claims 1 to 6.
A porous, injectable hydrogel comprising phosphorylated alginate (CaPAlg) and crosslinked with calcium ions (Ca &lt; ^{2 +} &gt;).

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