KR100813224B1 - Thermo-reversible coacervate combination gels for protein delivery - Google Patents

Abstract

A drug delivering gel is provided to be easy to handle in a sol state at room temperature and be rapidly converted into a gel state in according to the temperature increase after being injected in the body to show the continuous releasing pattern of the combined gel for a long time, thereby being useful for delivering a protein drug. A drug delivering gel comprises: a complex coacervate consisting of a positively charged protein and a negatively charged polysaccharide; and a negative thermo-reversible polysaccharide including a salted-out salt, wherein the positively charged protein is selected from the group consisting of gelatin, bovine pancreatic trypsinogen, basic fibroblast growth factor, fibronectin, aggrecan, and casein, the negatively charged polysaccharide is selected from the group consisting of chondrotin sulfate, heparin, heparan sulfate, hyaluronan, dermatan sulfate and keratan sulfate, the salted-out salt is selected from the group consisting of salted-out salts including a Hofmeister series anion such as SO4^2-, HPO4^2-, F^-, Cl^-, Br^-, NO3^-, I^-, ClO4^-, and SCN^-, and the negative thermo-reversible polysaccharide is selected from the group consisting of methyl cellulose, hydroxypropyl methyl cellulose, ethylhydroxyethylcellulose, carrageenan, and scleroglucan. A method for preparing the drug delivering gel comprises the steps of: (a) mixing the positively charged protein with the negatively charged polysaccharide to form the complex coacervate; and (b) mixing the complex coacervate with the negative thermo-reversible polysaccharide including the salted-out salt.

Description

Temperature reversible coacervate combination gel for protein drug delivery {THERMO-REVERSIBLE COACERVATE COMBINATION GELS FOR PROTEIN DELIVERY}

The present invention relates to a gel for drug delivery. More specifically, the present invention relates to a combination gel of complex coacervates and temperature reversible polysaccharides useful for protein drug delivery.

The simplest common drug delivery system is oral, or in the form of an injection or patch. However, as the types of drugs have been diversified, such classic forms of drug delivery systems have been inadequate.

In the case of protein drugs, generally, oral administration such as tablets or surgery is performed. However, in the case of oral administration, the desired protein drug is mostly degraded by intestinal digestive enzymes, and thus the desired effect of the drug is insignificant, and the method through surgery is inefficient in terms of risk and cost.

Moreover, the drug delivery system for the whole body such as oral administration has a big disadvantage in that the drug is delivered at the same concentration to the non-disease site which does not need or adversely affect the drug, thereby showing an undesirable effect.

Therefore, not only the need for a drug delivery system for targeting and treating a specific disease area is rapidly increased, but also optimal drug delivery for a long time release of the drug so as to precisely act on the target site to reduce the hassle or risk of frequent administration. There is a need for a system.

In recent years, in situ-forming systems have been reported for a variety of biological medical uses, including drug delivery and tissue engineering (RL Dunn et al., Biodegradable in-situ forming implants and methods of producing the same, US Patent 4 (1990) 938-763; BO Haglund et al., J. Control.Release 41 (1996) 229-235; and Y. An et al., J. Control.Release 64 (2000) 205-215).

For example, several mechanisms, such as solvent exchange, pH change, ultraviolet (UV) irradiation, ion crosslinking and temperature transition, have made it possible to reach in situ gel formation. Negative temperature sensitive polymers with a Low Critical Solution Temperature (LCST) or temperature-reversible hydrogels exhibiting a reversible sol-gel transition behavior upon heating or cooling can be used to stimulate drug delivery. It is the most commonly studied field of sensitive polymer systems. Although several synthetic polymers have been reported to exhibit temperature-reversible gelling behavior at body temperature and have been used for drug delivery, problems with their biocompatibility and biodegradation remain. .

High molecular weight natural cellulose is often insoluble in aqueous solutions due to intramolecular and intermolecular hydrogen bonding (Gels Handbook, Vol. 4; Osaka, Y., Kanji, K., Eds, Hatsuo Ishida; Academic Press: San Diego, CA (2001). When some hydroxyl groups are substituted with hydrophobic groups such as methyl or hydroxylpropyl, some of the hydrogen bonds are broken down, resulting in soluble, hydrophobically modified cellulose (Guenet, J, Thermoreversible Gelation of Polymers and Biopolymers). , Academic Press: London (1992); and Kabayashi, K. et al., Macromolecules 32 (1999) 7070).

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Methylcellulose (MC) is a hydrophobically modified nonionic cellulose derivative that forms a physically reversible physical gel in aqueous solution. Methylcellulose is an inhomogeneous alternating block copolymer structure consisting of a hydrophobic region with a high degree of substitution and a hydrophilic region with low or no substitution (see Kundu, KK, Polymer. 42 (2001) 2015-2020). . The hydrophobic region locally stabilizes the water structure, and upon heating, the water structure is destroyed to enhance hydrophobic interactions, resulting in gelation. Commercial MCs with a degree of substitution (DS) of 1.4-2.0 undergo sol-gel transitions upon heating at 1.0-2.5% of the MC concentration in water. These phase-transfer properties are widely used in the food and pharmaceutical industries and are used as binders or thickeners.

Cellulase is an enzyme complex that breaks down cellulose into beta-glucose. Most animals, including humans, do not produce these enzymes and thus cannot use the energy of plants independently. However, cellulase is present in a wide variety in fungal and microbial organisms, and can be used as a food supplement for human health and hygiene to hydrolyze and decompose plant polysaccharides. Some fungi secrete enzymes that catalyze the oxidation of cellulose. Peroxidase secretes hydrogen peroxide to attack free radicals in the C ₂ -C ₃ position of the cellulose to form aldehyde cellulose, which is highly reactive and promotes hydrolysis to lower molecular weight fractions. Bacteria also produce carbohydrate nutrients by secreting endogenous (endo-)-and exogenous (exo-)-enzymes that degrade cellulose (Aubert, JP, Beguin, P., Millet, J. Biochemistry and Genetics of Cellulose Degradation. See Academic, New York (1988).

In order to apply MC as an in-situ-forming drug delivery system that gels at body temperature, it is necessary to modify the negative heat-gelling mechanism of MC using additives such as polymers, non-electrolytes or salts, for example. The gelation temperature of MC can be lowered by increasing the concentration of MC, but the viscosity of the solution becomes high, making handling difficult.

A simple way to modify the gelation temperature is to use salting-out salts known to have a significant effect on the phase change of the water soluble polymer (Schott, M .; Royce, AE; Han , SK J Colloid Interface Sci 98 (1984) 196). Anions of salts are known to be much more effective than cations to stabilize nonpolar solutes or hydrophobicity of macromolecules. Hopemeister series (Collins, KD; Washabaugh, MW Q Rev Biophys. 4 (1985) 323) or Lyotropic series (M. Khairy et al., Journal of Polymer Science (2003) 3547-3559) According to the salt concentration of the anion is:

Hopemeister series:

SO _{4 ^{2-> HPO 4 2 -> F}} -> Cl -> Br -> NO3 -> I -> ClO 4 -> SCN -

Lyotropic series:

^{Al 3 +> Ca 2 +>} Mg 2 +> K + = NH 4 +> Na +> Li +, PO 4 3 -> SO 4 2 -> Cl -> NO 3 -

Coacervate formation is a process in which a homogeneous solution of charged macromolecules undergoes liquid-liquid phase separation to produce a polymer rich high density phase. Two macromolecules charged with opposite polarity (or polyelectrolytes or colloids charged with opposite polarity) may undergo complex coacervate formation through electrostatic and additional interactions.
Coacervates have been widely applied for protein purification and drug delivery (Xia, J. and Dubin, PL, Protein-polyelectrolyte complexes.In: Dubin, PL, Bock, J., Davis, R., Schulz, DN, and Thies) , C., Eds.Macromolecular Complexes in Chemistry and Biology.Berlin: Springer-Verlag (1994) 247-271; Poznansky MJ, Juliano RL., Pharmacol Rev 36 (1984) 277-335; and Gombotz WR., Bioconjugate Chem 6 (1995) 332-51).

Composite coacervates can be formed simultaneously when mixing the oppositely charged polyelectrolytes in an aqueous medium. Protein-proteins, protein-polysaccharides, and polysaccharide-polysaccharide combinations have been frequently studied for drug delivery and bio-medical use. For example, microspheres by complex coacervate formation consisting of gelatin A as a polyvalent cation or zwitterion and chondroitin 6-sulfate (CS) as polyanion have been reported to be efficient as an intraarticular delivery system for therapeutic proteins. (Kimberly EB, Arthritis & Rheumatism (1998) 2185-2195). In particular, since polyvalent ions are the main component of the extracellular matrix of cartilage and synovium, microspheres are sensitive to matrix metalloprotease (MMP) induced by proinflammatory cytokines.

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Therefore, while efficiently delivering the protein to the target site mainly for the purpose of treatment, etc., which can compensate for the above disadvantages of the conventional drug delivery system, the protein is continuously released for a long time at the site of human application to maintain the desired effect. There is a need for a drug to protein delivery system.

The inventors have surprisingly found that when co-formulated coacervates formed from two biopolymers charged with opposite charges are co-formulated with negative temperature-reversible polysaccharides, they are present in water-like conditions at room temperature and then injected into the human body. When the gel was formed immediately, it was found that the sustained release of the protein contained in the gel was completed to complete the present invention consisting of a protein delivery gel consisting of a combination coacervate and a temperature-reversible polysaccharide gel and a preparation method thereof.

The present inventors provide, in particular, complex coacervates formed by adding two charged biomacromolecules consisting of natural or therapeutic proteins and polysaccharides as physically combined gels that are not chemically crosslinked. Such complex coacervates are further co-formulated with temperature-reversible polymers to produce injectable and intelligent drug delivery systems. The introduction of an optimized novel delivery system with the two advantages of such complex coacervate formation and temperature reactive gels has proven its value as an effective protein delivery system.

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Hereinafter, the present invention will be described in more detail.

In one aspect of the invention there is provided a drug delivery gel consisting of a combination of a negative temperature reversible polysaccharide containing complex coacervate and salt salts consisting of a positively charged protein and a negatively charged polysaccharide.

The type of drug that is effectively delivered by the present invention is not limited. Any drug that requires delivery in vivo can be delivered using the gel of the invention. Preferably the drug is a protein or genetic preparation for therapeutic use.

Positively charged proteins are any proteins commonly used to produce complex coacervates. Examples of positively charged proteins that can be preferably used in the present invention include gelatin, bovine trypsinogen, egg lysozyme, basic fibroblast growth factor, fibronectin, agrecan, casein and the like.

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Most preferably, the positively charged protein is gelatin.

The negatively charged polysaccharide can be any natural polysaccharide commonly used for the production of complex coacervates. Examples of negatively charged polysaccharides that can be preferably used in the present invention can be used chondroitin sulfate, heparin / heparan sulfate (HS), hyaluronan, dermatan sulfate, keratan sulfate and the like.

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Most preferably, the negatively charged polysaccharide is chondroitin 6-sulfate.

For the formation of the most stable complex coacervate, the compositional ratio between the positively charged protein and the negatively charged polysaccharide can be determined by using any method known to those skilled in the art to determine the ratio of the best degree of complex coacervate formation. Preferably, the composition ratio can be determined by measuring the turbidity of the coacervate forming mixture.

Examples of negative temperature reversible polysaccharides that can be preferably used in the present invention may be methyl cellulose (MC), hydroxypropyl methyl cellulose (HPMC), ethyl hydroxyethyl cellulose (EHEC), carrageenan, scleroglucan and the like. . Preferably, the negative temperature reversible polysaccharide is methyl cellulose.

In the present invention, a salt salt is added to the negative temperature reversible polysaccharide in order to lower the gelation temperature. Examples of salt salts that can be preferably used in the present invention include ammonium sulfate, sodium dodecyl sulfate, glycerophosphate, sodium carbonate, sodium perchlorate, sulfuric acid, sodium bicarbonate and the like. The type of salt salt used may be selected according to the type and properties of the polysaccharide. Most preferred is ammonium sulfate in the present invention.

In another aspect of the invention, a drug delivery method comprising mixing a positively charged protein and a negatively charged polysaccharide to form a complex coacervate, and mixing the complex coacervate with a negative temperature reversible polysaccharide containing a salt salt. Provided is a method for preparing a combination gel.

The formation conditions of the composite coacervate and the formation of the combination gel of the composite coacervate and the negative temperature reversible polysaccharide may be used using any method well known in the art for appropriately selecting and mixing the temperature and the buffer according to the component selection.

Those skilled in the art can vary the conditions according to the specific components and form the most stable coacervates.

In another embodiment of the present invention, the method comprises mixing a positively charged protein with a negatively charged polysaccharide to form a complex coacervate, mixing the complex coacervate with a negative temperature reversible polysaccharide containing a salt salt, and the desired drug. It provides a method for producing a combination gel for drug delivery, characterized in that the mixing.

Using an optimized novel drug delivery system consisting of the complex coacervates and temperature-reversible polymer gels according to the invention, it is possible to effectively deliver protein drugs into the body.

In addition, the temperature reversible composite coacervate gel prepared according to the method of the present invention may be easily prepared because the composite coacervate and the temperature reversible polymer are physically combined without chemically crosslinking and have excellent biodegradability and biocompatibility, and a simple reaction process. have.

In addition, the delivery system according to the present invention has advantages in terms of ease of administration and an aqueous environment free of toxic organic solvents.

The biodegradable and temperature-reversible injectable physical gels of the present invention were prepared by forming complex coacervates between two charged biomacromolecules and then coforming with negative temperature sensitive polysaccharides containing salt salts.

The biodegradable and temperature-reversible gel of the present invention thus prepared formed a spherical depot shortly after subcutaneous injection into the rat, and the form and firmness of the depot were firmly maintained for 1 week.

The combination of complex coacervate and temperature-reversible gel demonstrated a synergistic effect on release rate of protein and in situ gel depot formation. The gel shows a pattern in which the model protein is released continuously over 25 days after the initial start of release. For sustained protein release, a stable complex coacervate with a polyelectrolyte charged according to the isoelectric point (pI) was provided and formulated simultaneously with the negative temperature sensitive polysaccharide (MC gel in this example) containing salt salts. It became.

An optimized novel in situ gel depot system of the present invention has been developed which has two advantages of complex coacervate formation and temperature reactivity. Such systems have proven valuable for effective protein drug delivery in terms of ease of administration, an aqueous environment free of toxic organic solvents, and simplicity of preparation.

1. Complex coacervate formation between GA and CS

A simple method for assessing polyionic complex formation (composite coacervate) between gelatin and chondroitin 6-sulfate (CS) at pH 7.4, by measuring the turbidity of the mixed solution by absorbance at 450 nm. The optimum ratio between gelatin and CS was found to form a stable complex coacervate.

Figure 1 shows turbidity titration curves for gelatin and CS mixtures of various concentrations in PBS (pH7.4) at 25 ℃. When cationic GA was used for the titration, the mixed solution had a maximum turbidity at a CS / GA weight ratio of 0.1, which increased with time of complex formation. However, as expected, anionic GB did not form coacervate with CS at pH 7.4.

Bungenberg de Jong has reported a phase separation phenomenon, in which two charged molecules interact and form a biopolymer phase with a weak solvent (Kimberly EB, Arthritis & Rheumatism ( 1998) 2185-2195). The main interaction between the biopolymers is the electrostatic dependence of pH and ion concentration values, and the prominent factor affecting composite coacervates is the charge.

FIG. 2 shows gel formation time between GA and CS coacervates measured by viscosity and rheometer. For the coacervate mixture formed of GA and CS, the viscosity rose sharply at 25 ° C. However, when the mixture was heated to 37 ° C., the viscosity was significantly reduced and the coacervate gel was converted to the sol state. Since gelatin is a positive heat-sensitive biopolymer that exhibits a decrease in viscosity with increasing temperature, the protein chain mobility of the gelatin increased at 37 ° C., leading to coacervate gel breakdown between GA and CS. To this result, the rest of the experiments were carried out using this method.

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2. Preparation of Negative Temperature-Reversible Methylcellulose (MC) Gel

In order to apply complex coacervates having such properties to drug delivery systems, especially in situ forming gels, the complex must be stable and able to form instant viscous gels at body temperature. MC gels were tested in injectable formulations with incorporating complex coacervates. At room temperature, the MC aqueous solution is in a clear sol state. When heated to about 50 ° C., the solution becomes an opaque gel. For convenience, the MC gelling temperature can be lowered by adjusting the concentration or using additives such as polymers, polymer electrolytes or salts, for example. Increasing the MC concentration to lower the gelling temperature below the body temperature limits the viscosity and makes handling difficult, which is undesirable.

For the purpose of lowering the gelling temperature, the addition of a salt salt such as AS is a simple and effective way to lower the gelling temperature of the aqueous MC solution.

3 shows the gelation temperature of a 2 wt% MC solution as a function of AS concentration. As the AS concentration rises, the gelation temperature drops rapidly, and the gelation temperatures are 37 ° C and 29 ° C, respectively, when MC is 5% and 9%.

2.2. Optimization of Temperature-Reversible Gels Containing Complex Coacervates

To prepare a temperature-reversible gel containing a GA-CS complex coacervate, various proportions of GA and CS mixtures were added to a 2% by weight MC solution containing 9% AS, gelled from the sol state at temperatures near body temperature. Denatured.

Since commercially available GA has a nonuniform molecular weight distribution, it is difficult to form a gel that is stable at a temperature near body temperature. For this purpose, high molecular weight GA was prepared by ethanol dissolution which was used to prepare a composite coacervate containing MC gel.

4A shows turbidity measurement titration curves of high molecular weight GA (HMw GA) and CS at various concentrations in PBS at pH 7.4. When titrated with high molecular weight GA (HMw GA), the mixed solution shows maximum turbidity, which rises with time of complex formation.

In FIG. 4B, the turbidity rises rapidly as a stable complex coacervate of HMw GA / CS is formed at an appropriate weight ratio 1. Significantly, turbidity was higher than coacervate when MC was mixed with coacervate, demonstrating the synergistic effect of MC on complex coacervate formation.

The concentrations of the various gel formulations with and without coacervate were compared at 25 ° C and 37 ° C. The MC and AS mixed solution associated with the optimum ratio of CS / GA coacervate was sol at 25 ° C. and stable gel at 37 ° C. However, the gel concentration of coacervate was lower than that of normal gels, presumably due to the increased elasticity by gelatin.

3 in vitro release studies

3.1. FITC-BSA Release Test

As shown in FIG. 7, the release profile of the model protein FITC-BSA from the gel suggested a sustained release pattern over 25 days from the initial minimal release. There was no significant difference in release rates among gels containing various ratios of CS and GA, but release rates from combination gels containing coacervate were slightly faster than conventional MC and AS gels. It was foreseen. However, it should be noted that the release characteristics from the combination gel containing coacervate show a long-lasting pattern and the gel becomes more resilient to reduce pain and inflammation after subcutaneous injection. After about 30 days of the release test, the gel is converted into a clear solution.

3.2. Gelatin A Release Test

FITC-BSA release and viscosity results suggest that the incorporation of GA and CS coacervates lowers the viscosity of the MC gel and increases FITC-BSA release compared to normal MC gels, so that the release rate of the model protein is a stable complex coacervate. It can be hypothesized that is formed together with the charged polymer electrolyte and may be delayed when added to the MC gel concomitantly.

To test this hypothesis, the release rate of GA as a model protein from the MC combination gel containing GA / CS coacervate was measured. As shown in FIG. 6, the release rate of GA from an MC combination gel containing coacervate at a CS / GA ratio of 0.1 is slower than a normal MC gel and MC containing coacervate at a non-optimum ratio. Much slower than combination gels. Reduced release rates suggest a synergistic effect of complex coacervates and temperature-reversible gels on protein controlled release.

4. In vivo gel depot formation

As shown in FIG. 8, various MC gel formulations using a 26GX gauge syringe [(a) MC + AS, (b) MC + AS + GA, and (c) MC + AS + CS / GA (= 0.1). )] Was injected subcutaneously in the rat's back and the formation of spherical depots was observed immediately. The depot's morphology and firmness remained robust with the MC combination gel containing coacervate (MC + AS + CS / GA (= 0.1)).

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are not intended to limit the scope of the invention.

<Example>

Materials and reagents

Methylcellulose (MC, M _n = 86,000, viscosity = 4,000 cps), gelatin type A (GA, PI = 8.6, from pig skin derived from acid-treated tissue (Bloom No. 300)), gelatin type B ( GB, PI = 4.7, from bovine hides derived from acid-treated tissue (Bloom No. 300), chondroitin 6-sulfate (CS, derived from shark cartilage tissue), fluorine Bovine serum albumin (FITC-BSA) conjugated with lecein isothiocyanate, and ammonium sulfate (AS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other compounds were of analytical grade.

Example  1. Composite Coacervate  Formation and turbidity ( turbidity ) Measure

1.1. Preparation of High Molecular Weight Gelatin Solution

Ethanol high molecular weight gelatin solution was prepared according to the modified ethanol desolvation method. GA was dissolved in PBS at 50 ° C., and the same amount of ethanol was added to volatilize the solvent to precipitate high molecular weight gelatin. The supernatant was decanted and the precipitate was redissolved in PBS at 50 ° C.

1.2. Complex coacervate formation and turbidity measurement

GA or 4 wt% high molecular weight GA was dissolved in PBS at pH 7.4, 50 ° C. for 1 hour. CS in PBS at pH 7.4 was mixed with the gelatin solution in various weight ratios (CS / GA = 0.002, 0.01, 0.02, 0.1, 0.2 and 1.0). The increase in turbidity of the mixture solution was measured by absorbance at 450 nm (DU 730, Life science UV / Vis spectrophotometer, Beckman Coulter, Fullerton, CA, USA).

Example  2. Temperature Reversible Composite Coacervate  Combination Gel  Produce

2.1. Preparation of Methyl Cellulose Aqueous Solution

MC powder was dispersed in about half the required amount of phosphate buffered saline (PBS) preheated to 90 ° C. and stirred until completely wet to prepare a pure 0.3-2.5 wt% MC solution. The remaining portion of cold PBS was added and the mixture was slowly stirred in an ice cold bath for 30 minutes to produce a colorless clear solution. The solution was shaken and frozen.

2.2 Preparation of Thermoreversible Composite Coacervate Combination Gel

The gelation temperature of a 0.3-2.5 wt% MC aqueous solution was measured by a tube inverting method while raising the temperature by 0.5 ° C. at 5 minute intervals over a 25-60 ° C. temperature range. Salted salt agonist AS was added to the MC solution to determine significant gelatin gelation temperature in the same manner.

To prepare the combination gel, a composite coacervate consisting of cationic GA and anionic CS was added to the AS containing MC solution. The mixed solution was vortexed for 5-10 minutes at room temperature and incubated in a 37 ° C. water bath.

Example  3. Gel Formation and Viscosity Measurement

Using pulsatile blood flow meters (Bohlin, Malvern, UK), gel formation and viscosity measurements at various temperatures were performed by cone and plate geometry methods within the temperature range of 0-60 ° C. Samples (500 μl) were placed between two 20 mm parallel plates separated at 500 μm intervals.

Example  4. In vivo release

The release properties of coacervate formed GA and FITC-BSA from the combination gels were measured at 37 ° C. Combination gels were prepared from 4 wt% high molecular weight GA and 0.4 wt% CS and co-formulated with 2 wt% MC containing 9 wt% AS. FITC-BSA was added to the aqueous gel solution. Sample vials were placed in a 37 ° C. water bath to form gels, covered with pre-warmed PBS at pH 7.4 and vibrated at 150 rpm. At predetermined time intervals, the medium was released and drained and replaced with fresh buffer. Protein concentration was measured with a UV spectrophotometer.

Example  5. In vivo experiment

Sprague-Dawley rats (200-250 g, SLC, Tokyo, Japan) were anesthetized by intramuscular administration (90 mg / kg) of ketamine hydrochloride (90 mg / kg) and xylazine hydrochloride (5 mh / kg). All handling and care of these laboratory animals is published in the National Institute of Health, "The Guide for the Care and Use of Laboratory Animals" (NIH Publication 85-23, 1985). Revised).

Samples (0.5 ml) of various formulations ((a) MC + AS, (b) MC + AS + GA, and (c) MC + AS + CS / GA (0.1)) were sterilized in a 26G X 1/2 gauge Subcutaneous injections were performed subcutaneously on the back of rats and recovered after injection.

1 is a graph showing the titration for measuring turbidity of gelatin type A and mixtures between type B and CS. Figure 1a is a graphical representation of the gelatin A, Figure 1b using gelatin B to measure the turbidity of the gelatin and CS mixture at various concentrations in PBS (pH7.4) at 25 ℃. (●: 6 hours, ○: 12 hours, ▼: 24 hours)

Figure 2 is a graph measuring the viscosity and gel formation time by a blood flow meter at 25 ℃ and 37 ℃. Viscosity and gel formation time are storage coefficients representing the elastic accumulation of energy when strain is recovered in an elastic solid as an elastic component, and loss coefficients representing the loss of energy (loss) through permanent deformation of the flow. Indicates.

3 shows the effect of AS concentration on MC solution gelling temperature. It is a graph showing the gelation temperature of 2% MC solution as a function of AS concentration.

Figure 4 is a graph measuring the turbidity over time when forming complex coacervate by high molecular weight gelatin and CS. Figure 4a shows the turbidity of the high molecular weight gelatin and CS coacervate mixtures at various concentrations in PBS (pH7.4) over time (•: 10 min, ○: 30 min, ▼: 1 h, Δ: 3 h, :: 6 h, : 12h, ◆: 1d, ◇: 2d), and FIG. 4B is a barometric measurement bar graph of the combination of composite coacervate and temperature sensitive gel. (1: HMw GA, 2: HMw GA / AS, 3: MC / AS, 4: HMw GA / MC, 5: HMw GA / MC / AS, 6: CS / HMw GA (1), 7: CS / HMw GA (1) / AS, 8: CS / HMw GA (1) / MC / AS, 9: CS / HMw GA (0.1), 10: CS / HMw GA (0.1) / AS, 11: CS / HMw GA ( 0.1) / MC / AS, 12: CS / HMw GA (0.01), 13: CS / HMw GA (0.01) / AS, 14: CS / HMw GA (0.01) / MC / AS)

5 is a graph showing viscosity and gel formation time as a function of temperature. The viscosity of various gel formulations with and without coacervate at 25 ° C. and 37 ° C. is shown. Figure 5a is a viscosity of the gel formulation consisting of MC / AS Figure 5b is a gel formulation consisting of CS / HMw GA / MC / AS (0.4% HMw GA, CS / HMw GA (1), 2% MC, 9% AS) The viscosity of

6 is a graph showing the results of testing gelatin release. The release rate of GA from the MC combination gel containing coacervate at an optimal ratio of 0.1 between GA and CS is compared with the release rate from the MC gel. (●: HMw GA / MC / AS, ○: CS / HMw GA (2) / MC / AS, ▼: CS / HMw GA (1) / MC / AS)

Figure 7 shows the results of the release test of the negative thermosensitive gel containing HMw GA-CS coacetate. 4% gelatin was used. (△: HMw GA / MC / AS, CS / HMw GA (1) / MC / AS, ▼ CS / HMw GA (0.5) / MC / AS, ○ CS / HMw GA (0.1) / MC / AS, ■ CS / HMw GA (0.05) / MC / AS, □ CS / HMw GA (0.01) / MC / AS, ◆ MC / AS)

Release of FITC-BSA protein from gels, suggesting 25-day sustained release pattern

FIG. 8 is a subcutaneous injection of various MC gel formulations (a) MC / AS, (b) MC / AS / GA, (c) MC / AS / CS / HMw GA (1)) which are thermoreversible composite coacervate gels. This is a photograph of spherical depot formation observed.

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Claims (17)

A drug delivery gel comprising a combination of (a) a complex coacervate consisting of a positively charged protein and a negatively charged polysaccharide, and (b) a negative temperature reversible polysaccharide containing salt salts, wherein the positively charged protein is gelatin, Selected from the group consisting of bovine and trypsinogen, egg lysozyme, basic fibroblast growth factor, fibronectin, agrecan and casein, the negatively charged polysaccharides include chondroitin sulfate, heparin, heparan sulfate, hyaluronan, der dermatan sulfate, and Ke is selected from the group consisting of sulfate, rattan, the salting out salt is _{^{SO 4 2-, HPO 4 2-,}} F -, Cl -, Br -, NO3 -, I -, ClO 4 - and SCN ^- selected from Is selected from salt salts comprising Hofmeister series anions, wherein the negative temperature reversible polysaccharides are methyl cellulose, hydroxyprop Methyl cellulose, ethyl hydroxyethyl cellulose, carrageenan and Klee's gel for drug delivery, characterized in that the cellulose derivative is selected from consisting of a glucan. The gel of claim 1 wherein the drug is a protein preparation. The gel according to claim 1 or 2, wherein the positively charged protein is gelatin. The gel according to claim 1 or 2, wherein the negatively charged polysaccharide is chondroitin 6-sulfate. delete The gel according to claim 1 or 2, wherein the cellulose derivative is methylcellulose. The gel according to claim 1 or 2, wherein the salt salt is ammonium sulfate. (a) mixing a positively charged protein and a negatively charged polysaccharide to form a complex coacervate; (b) a method for preparing a drug delivery gel comprising mixing the complex coacervate of step (a) with a negative temperature reversible polysaccharide containing a salt salt, wherein the positively charged protein is gelatin, small isy trypsinogen , Egg lysozyme, basic fibroblast growth factor, fibronectin, agrecan and casein, wherein the negatively charged polysaccharides are chondroitin sulfate, heparin, heparan sulfate, hyaluronan, dermatan sulfate and keratan is selected from the group consisting of sulfate, the salting out salt is _{^{SO 4 2-, HPO 4 2-,}} F -, Cl -, Br -, NO3 -, I -, ClO 4 - and SCN ^- Hope Meister series anion selected from And the negative temperature reversible polysaccharides are selected from methyl cellulose, hydroxypropyl methyl cellulose, ethyl hydroxy. A method for producing a drug delivery gel, characterized in that it is selected from cellulose derivatives consisting of tilcellulose, carrageenan and scleroglucan. 10. The method of claim 8, wherein the desired drug is mixed after step (b). 10. The method of claim 8 or 9, wherein the drug is a protein. 10. The method of claim 8 or 9, wherein the positively charged protein is gelatin. 10. The method of claim 8 or 9, wherein the negatively charged polysaccharide is chondroitin 6-sulfate. delete 10. The method of claim 8 or 9, wherein the cellulose derivative is methylcellulose. 10. The method of claim 8 or 9, wherein the salt salt is ammonium sulfate. delete delete

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