KR101755680B1 - Polysaccharidic nanogel for protein drug delivery and preparation method thereof - Google Patents

Abstract

The present invention relates to a polysaccharide nanogel for protein drug delivery and a preparation method thereof, and more particularly to a biocompatible polymer and a protein drug which are grafted with 3-diethylaminopropyl (DEAP) To a polysaccharide nanogel for protein drug delivery. The polysaccharide nanogel for protein drug delivery according to the present invention can be biodegradable and biocompatible in vivo by synthesizing a biocompatible polymer and a hydrophobic substance. By using the property of being weakly acidified and positively ionized, And when the pH is changed to physiological pH, the efficiency of encapsulating the protein drug in the drug delivery system can be enhanced by using the hydrophobic property of the synthetic material, and the effect of effectively introducing the nanoparticles into the cell is achieved .

Description

Technical Field [0001] The present invention relates to a polysaccharide nanogel for protein drug delivery and a method for preparing the same,

The present invention relates to a polysaccharide nanogel for protein drug delivery and a method for producing the same, which can produce a high packing efficiency and effective intracellular infusion, which is produced by binding a hydrophobic substance to a biocompatible polymer and sealing the protein drug.

With the development of biotechnology and molecular biology as well as the production of recombinant DNAs, the possibility of using proteins or peptides as therapeutics for clinical applications has been put to practical use, and high-performance protein drugs have been developed using them . However, in the case of drugs using such proteins or peptides, the drug efficacy is very excellent, but the bioavailability is extremely low. Unlike the conventional synthetic drug, the drug can be effectively used only when the original three-dimensional structure is stably maintained. It is difficult to maintain the three-dimensional structure for maintenance. Therefore, research and development in the field of pharmaceutical research have been continuously carried out on a method for effectively transferring a therapeutic protein drug into a cell while maintaining the function of the protein for a long period of time and increasing the body utilization rate at the same time.

Particularly, protein drugs are mostly used in other chemical drugs, and the oral administration method is mainly used, and since the digestive enzymes that are secreted into the digestive tract digest a considerable amount of hydrolysis, the bioavailability is reduced, The amount of the active ingredient is not sufficient to cause side effects. For this reason, it is preferable to deliver the protein drug by injecting instead of oral administration, and even when the protein drug is delivered using the injectable preparation, it has a short half-life in the blood, There is a problem that multiple injections are required. In addition, since the therapeutic selectivity of the therapeutic protein is generally insufficient, it can not selectively transmit to diseased cells in a specific position in the body. Therefore, in order to overcome the above-mentioned problems, a carrier for effectively delivering the drug efficacy in the human body is required. The carrier for delivering such a protein is polyionic complex, nano gel, and the like. Korean Patent Laid-Open Publication No. 10-2007-0099767 has developed a protein transporter in which a protein is encapsulated on the surface of a lipid nucleus. However, since the protein is present on the surface, there is concern about protein stability in the blood. Korean Patent Application No. 10-2009-0125727 Proteins bind to biomolecules through ionic bonds, but protein stability may also be present in the blood because proteins may also be present on the surface.

Accordingly, the present invention provides nanoparticles of protein drug delivery using a polysaccharide polymer having excellent biodegradability or biocompatibility, thereby securing a high encapsulation rate, enhancing in vivo stability through protein encapsulation in the nanogel, The inventors of the present invention have attempted to invent a protein transporter capable of achieving this. In the present invention, a polysaccharide having hydrophilic and hydrophobic groups is used, and a protein carrier called a nano-gel having physicochemical cross-linking at physiological pH is used in order to increase the sealing efficiency. Through this process, the protein can be enclosed at a high sealing efficiency And a method for producing a nanogel that is a nano-level protein drug delivery that can be efficiently introduced into a cell while the protein is protected by a stable structure.

In order to produce a protein drug delivery system capable of effectively penetrating a protein drug having a high molecular weight into cells, the present inventors have found that 3-diethylaminopropyl (3-diethylaminopropyl) phosphate is a hydrophobic substance in the amine group of a biocompatible and biodegradable polymer. DEAP) is introduced into the nanocomposite under weakly acidic conditions to bind it with the protein drug and change it to the physiological pH condition, the hydrophobicity of the DEAP is strengthened, so that the nano-sized particles And the present invention has been completed.

Accordingly, it is an object of the present invention to provide a polysaccharide nanogel for protein drug delivery, in which a protein drug is encapsulated in nanoparticles and infiltrated into cells.

It is another object of the present invention to provide a method for producing a polysaccharide nanogel for protein drug delivery.

In order to achieve the above object, the present invention provides a polysaccharide nanogel for protein drug delivery comprising a biocompatible polymer and a protein drug in which 3-diethylaminopropyl (DEAP) is grafted.

In one embodiment of the present invention, the mixing ratio of the biodegradable polymer grafted with 3-diethylaminopropyl and the protein drug may be 0.1: 1 to 10: 1, more preferably 0.5: 1 to 4: : May be one.

In one embodiment of the present invention, the grafting may be a combination of an amine group of the biocompatible polymer and an isothiocyanate group of the 3-diethylaminopropyl group.

In one embodiment of the present invention, the biocompatible polymer is selected from the group consisting of glycol chitosan, hyaluronic acid, pullulan, curdlan, pectin, starch, Dextran, chitosan, poly-L-lysine and polyaspartic acid.

In one embodiment of the present invention, the biocompatible polymer may be glycol chitosan.

In one embodiment of the present invention, the 3-diethylaminopropyl (DEAP) is present in an amount of from 0.1 to 1, more preferably from 0.5 to 1, and most preferably, 0.75.

In one embodiment of the present invention, the protein drug is selected from the group consisting of bovine serum albumin (BSA), pegfilgrastim, interleukin-6 (IL-6), insulin, human growth hormone (hGH), human serum albumin deaminase Leupirudin, Dornase Alfa, Denileukin diftitox, Bivalirudin, Peginterferon alfa-2a, Sargramostim, Anakinra, Collagenase, Alpha-1-proteinase inhibitor, Pegaspargase, Hyaluronidase, Pegvisomant, Streptokinase, Filgrastim, Coagulation Factor IX, Agalsidase beta, Oxytocin, Enfuvirtide And the like.

In one embodiment of the present invention, the particle size of the nanocomposite may be 50 to 4000 nm.

The present invention also provides a method for producing a biocompatible polymer, comprising: dissolving a biocompatible polymer in a solvent; Adding 3-diethylaminopropyl to the solution in which the biocompatible polymer is dissolved to bind the amine group of the polysaccharide and the isothiocyanate of the 3-diethylaminopropyl group; Dialyzing and lyophilizing the combined and synthesized material; Reacting the synthesized substance with a protein drug in a slightly acidic condition to form a complex; And centrifuging the complex and then allowing the precipitate to react at physiological pH conditions. The present invention also provides a method for producing a polysaccharide nanogel for protein drug delivery.

In one embodiment of the present invention, 3-diethylaminopropyl is added per 1 unit of the biocompatible polymer in an amount of 1 to 3 moles.

In one embodiment of the present invention, the slightly acidic condition may be in the range of pH 5.0 to 7.0, preferably pH 5.5 to 6.5, more preferably pH 6.0.

In one embodiment of the present invention, the physiological pH conditions may be in the range of pH 7.4 to 8.0, preferably pH 7.4.

The polysaccharide nanogel for protein drug delivery according to the present invention can be biodegradable and biocompatible in vivo by synthesizing a biocompatible polymer and a hydrophobic substance, and can produce a negative charge at a weak acidity The protein drug can be efficiently introduced into the drug carrier by using the property that the synthetic material becomes hydrophobic when the drug is bound to the protein drug having the physiological pH and the nanoparticle can be effectively introduced into the cell.

FIG. 1 shows a process of synthesizing a GCS- g- DEAP / BSA nanogel according to an embodiment of the present invention. (a) shows the chemical structure of GCS- g- DEAP, and (b) shows the synthesis process of GCS- g- DEAP / BSA nanogel.
2 is a graph showing the average particle size of the GCS- g- DEAP / BSA complex according to the mixing ratio of GCS- g- DEAP and BSA at pH 6.0 (5 mM PBS) according to one embodiment of the present invention.
3 is a graph showing the zeta potential of the GCS- g- DEAP / BSA complex according to the mixing ratio of GCS- g- DEAP and BSA at pH 6.0 (5 mM PBS) according to one embodiment of the present invention.
FIG. 4 is a graph showing the average particle size of GCS- g- DEAP / BSA nanogels according to the mixing ratio of GCS- g- DEAP and BSA in pH 7.4 (150 mM PBS) according to an embodiment of the present invention.
Figure 5 is a scanning electron microscope (SEM) image of a GCS- g- DEAP / BSA nanogel (GCS- g- DEAP 0.1 mg / ml, BSA 0.1 mg / ml) in pH 7.4 (150 mM PBS) Emission Scanning Electron Microscope (FE-SEM) image.
Figure 6 shows the release of BSA released from GCS- g- DEAP / BSA nanogel (GCS- g- DEAP 0.1 mg / ml, BSA 0.1 mg / ml) in pH 7.4 (150 mM PBS) Graph.
FIG. 7 is a graph illustrating the effect of the GCS- g- DEAP / BSA nanoparticle (10 μg / ml) and free BSA (10 μg / ml) in human epithelial ovarian cancer HeLa cells at pH 7.4 Fluorescence image of the cell when treated for a period of time.
FIG. 8 is a graph showing the effect of the GCS- g- DEAP / BSA nanoparticle (1 μg / ml) and free BSA (1 μg / ml) in human epithelial ovarian cancer HeLa cells at pH 7.4 Lt; RTI ID = 0.0 > cell / cell < / RTI >
Figure 9 is a block diagram of an in- To confirm the effect in vivo , (a) was incubated with GCS- g- DEAP / BSA nanogel (epuivalent BSA 1 μg / ml) containing Chlorin e6 (Ce6) labeled BSA and free BSA the 1㎍ / ml) administered intravenously to each nude mouse tumor HeLa and a fluorescent image obtained after 30 minutes, (b) is a Ce6 at the change over then labeled GCS- -DEAP g / BSA nanogels administration time It is a fluorescence image obtained at 30 minutes, 9 hours, and 15 hours for viewing.

The terms used in the present invention are defined as follows.

Is a dispersion of colloidal particles in a liquid and the term "gel" means that the colloid solution (sol) is concentrated to a certain concentration or more, Is a semi-solid phase that occurs spontaneously when the temperature of the block copolymer is raised above the gelation temperature of the block copolymer.

"Controlled release" refers to the modulation of the rate and / or amount of a drug or therapeutic agent delivered in accordance with the drug delivery formulations of the invention. The controlled emission may be continuous or discontinuous and / or linear or nonlinear. This may be accomplished using one or more types of polymer compositions, drug loading, excipients or degradation-improving agents, or other modifying agents, alone, in combination, or sequentially to provide the desired effect.

"Drug" means an organic or inorganic compound or substance that has physiological activity and is used or modified for therapeutic use. Proteins, oligonucleotides, DNA, and gene therapy agents are included in the broad definition of drugs.

"Therapeutic agent" refers to any compound or composition that, when administered to an organism (human or non-human animal), elicits the desired pharmacological, immunogenic and / or physiological effect by local and / or systemic action . Thus, the term encompasses compounds or chemicals traditionally considered as biomedicals including drugs, vaccines, and molecules such as proteins, peptides, hormones, nucleic acids, gene constructs and the like. "Therapeutic agent" includes compounds or compositions used in all major therapeutic applications including, but not limited to: anti-infectives such as antibiotics and antiviral agents; Analgesics and analgesic combinations; Topical and common anesthetics; Anorexia nervosa; Anti-arthritic agents; Anti-asthmatics; Anticonvulsants; Antidepressants; Antihistamines; Anti-inflammatory agents; Antistatic agent; Anti-migraine agent; Antineoplastic agents; Anti-itch agents; Antipsychotic agents; fever remedy; Animal festival; Cardiovascular products (including calcium channel blockers, beta-blockers, beta-agonists and antiarrhythmics); Antihypertensive agents; Chemotherapeutic agents; diuretic; Vasodilators; Central nervous system stimulants; Cough and cold products; Decongestants; Diagnostic agent; hormone; Osteogenic stimulators and bone resorption inhibitors; Immunosuppressants; Muscle relaxants; Psychostimulants; sedative; Nerve stabilizers; Proteins, peptides and fragments thereof (naturally occurring, chemically synthesized or recombinantly produced); And hexane molecules (including ribonucleotides (RNA) or deoxyribonucleotides (DNA) comprising two or more nucleotide polymeric forms, double- and single-stranded molecules and supercoiled or condensed molecules, gene constructs, expression vectors, plasmids, Antisense molecules, etc.).

"Peptides ", " polypeptides "," oligopeptides ", and "proteins" can be used equally when referring to peptides or protein drugs and are not limited to specific molecular weights, peptide sequences or lengths,

"Therapeutic effect" means any improvement in a subject, human or animal disease that has been treated according to the method, and includes any prophylactic or preventive effect, or a disease that may be detected by physical examination, , And to obtain any relief in the severity of the symptoms and symptoms of the disease or disorder.

The terms "treating" or "treating ", as used herein, unless otherwise defined with respect to a particular disease or disorder, are intended to encompass all types of diseases, disorders, Preventing a disease, a disease or a disease from occurring in a person; (ii) inhibiting a disease, disorder or disease, i.e., inhibiting its progression; And / or (iii) alleviating the disease, disorder, or disease, i. E. Causing the disease, disorder and / or disease.

"Subject" or "patient" means any single entity that requires treatment, including human, cow, dog, guinea pig, rabbit, chicken, In addition, any subject who participates in a clinical study test that does not show any disease clinical findings, or who participates in epidemiological studies or used as a control group is included. In one embodiment of the present invention, the present invention was applied to humans.

"Tissue or cell sample" refers to a collection of similar cells obtained from a subject or tissue of a patient. The source of the tissue or cell sample may be a solid tissue from fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; Blood or any blood components; It may be a cell at any point in the pregnancy or development of the subject. Tissue samples can also be primary or cultured cells or cell lines.

Optionally, tissue or cell samples are obtained from primary or metastatic tumors. Tissue samples may include compounds that are not mixed with the tissue by nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. For purposes of the present invention, a "section" of a tissue sample refers to a portion or piece of tissue sample, eg, a thin slice of tissue or cell that has been cut from a tissue sample. It will be appreciated that multiple sections of a tissue sample may be taken for analysis in accordance with the present invention if the present invention is understood to include methods in which the same section of tissue sample is analyzed both at the morphological and molecular levels, It is understood that it can do.

"Cancer "," tumor ", or "malignant" refers to or represents the physiological condition of a mammal that is generally characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma and sarcoma.

An "effective amount" is an appropriate amount that affects a beneficial or desired clinical or biochemical outcome. An effective amount may be administered one or more times. For purposes of the present invention, an effective amount of an inhibitor compound is an amount sufficient to temporarily alleviate, ameliorate, stabilize, reverse, slow down, or delay the progression of a disease state. If the recipient animal is capable of enduring the administration of the composition, or the administration of the composition to the animal is suitable, the composition will be "pharmaceutically or physiologically acceptable ". If the dose administered is physiologically significant, it can be said that the formulation is administered in a "therapeutically effective amount ". The formulation is physiologically relevant if the presence of the formulation results in a physiologically detectable change in the recipient.

The terms "about "," substantially ", etc. used to the extent that they are used herein are intended to be taken to mean an approximation of, or approximation to, the numerical values of manufacturing and material tolerances inherent in the meanings mentioned, Accurate or absolute numbers are used to help prevent unauthorized exploitation by unauthorized intruders of the referenced disclosure.

Hereinafter, the present invention will be described in detail.

The present invention relates to a polysaccharide nanogel for protein drug delivery, which enhances the efficiency of encapsulation of a protein drug and enables effective intracellular inflow.

In the present invention, a biocompatible polymer and a hydrophobic substance are synthesized through a simple organic chemical synthesis for protein drug delivery, and a physiochemical ionic bond and a hydrophobic bond are used to bind the synthesized substance and a protein drug, , A nano-level protein drug delivery polysaccharide nanogel that can be effectively introduced into cells was prepared.

Particularly, in a weakly acidic condition, a biocompatible polymer having a hydrophobic substance is positively charged and a protein is negatively charged, so that a biocompatible polymer and a protein drug are bound to each other through ionic bonding, When the environment changes, a portion of the synthetic material becomes hydrophobic, so that the protein drug is encapsulated in the nanoparticles by hydrophobic bonding. This is characterized by the ability of the protein drug to enter the cell safely and effectively by preventing rapid degradation of the protein drug in the in vivo environment.

Accordingly, the present invention provides a protein drug delivery nanogel comprising a biocompatible polymer and a protein drug, more specifically, 3-diethylaminopropyl (DEAP) grafted.

Here, it is preferable that the biodegradable polymer and the protein drug grafted with DEAP are mixed with the polymer: protein drug at a mixing ratio (weight%) of 0.1: 1 to 10: 1, more preferably 0.5: 1 to 4: Mix ratio can be used.

The polysaccharide nanogel for protein drug delivery according to the present invention may be prepared by organically binding a hydrophobic substance using a polymer having excellent biodegradability and biocompatibility, physically chemically bonding the protein drug to the protein drug through ionic bonding, To the inside of the nanoparticle.

In the polysaccharide nanogel of the present invention, the hydrophobic block polymer is characterized by having a block copolymer structure composed of a hydrophobic region having a high degree of substitution and a hydrophilic region having a low degree of substitution or no substitution in order to form a physical gel . The hydrophobic region locally stabilizes the water structure and, upon heating, destroys the water structure and enhances the hydrophobic interaction, resulting in a gelation phenomenon.

Amphiphilic polymers with both hydrophilic and hydrophobic properties form micelles or self-aggregates through interaction between hydrophobic blocks to stabilize interfacial energy in aqueous solution. The size, distribution, rheological properties, and thermodynamic stability of polymeric colloidal particles formed by amphiphilic polymers are known to vary depending on the degree of hydrophilicity and hydrophobicity.

In the present invention, the natural polymer is biodegradable and has biodegradability and biocompatibility.

"Biodegradable" means that the block copolymer can be chemically degraded in the body to form a non-toxic compound. The rate of degradation is the same or different from the drug release rate. And "biocompatibility" refers to the ability to interact with the body without undesirable subsequent effects.

The polymers used in the present invention include, but are not limited to, hyaluronic acid, pullulan, curdlan, pectin, heparin, starch, dextran, dextran, chitosan, poly-L-lysine and polyaspartic acid, and derivatives thereof. Chitosan may be preferably used, and more preferably, chitosan Chitosan derivatives such as glycol chitosan (GCS) can be used.

Chitosan is a compound that is obtained by deacetylation of chitin, which is a natural polymer material rich in natural substances after cellulose in the natural world. The chitosan includes 2-amino-2-deoxy-β-D-glucopyranose It is a polysaccharide composed of 2-amino-2-deoxy-β-D-glucopyranose. Unlike other polysaccharides present in the natural world, chitosan contains primary amine in the main chain and thus has very unique properties. It is applied in various fields such as environment, agriculture, medicine, etc. Especially, it has excellent biocompatibility and biodegradability Gene and drug delivery system, scaffold for tissue engineering, injection type hydrogel, and the like.

The chitosan exhibits sol-gel transfer characteristics at a specific pH. The chitosan exhibits gelation at a pH of about 7.0 to 7.4 similar to that of the body. When the chitosan is solubilized below the above range, the chitosan shows clogging The gel can be formed safely in the body without occurrence.

In the present invention, 3-diethylaminopropyl (DEAP) having the following general formula (1) may be used as the hydrophobic substance to be bonded to the biocompatible polymer.

The DEAP block has a tertiary amine and can exhibit sensitivity to the pH of the body. In particular, the DEAP block has sensitivity at a weak acidic pH of 6.8. Due to the tertiary amine group, the solubility in water varies depending on the pH, so that the gel can be formed or the sol state can be maintained according to the pH change. The tertiary amine groups are chemically non-reactive and typically exhibit reduced toxicity in normal tissues relative to secondary or primary amine groups. Also, unlike primary or secondary amine groups, tertiary amines are not converted to acyl derivatives coupled with carboxylic acids of biological proteins.

In the present invention, the organic bonding between the biocompatible polymer and the hydrophobic substance DEAP is achieved by reacting the free amine group of the biocompatible polymer having excellent reactivity with the isothiocyanate of DEAP. Particularly, in order to biodegrade the polymer in the human body and completely discharge it to the outside, a DEAP component is subjected to a coupling reaction, whereby a nanogel capable of biodegradation in the human body and being able to be completely discharged from the body can be produced.

In addition, when a substance and a protein drug synthesized by chemically bonding a biocompatible polymer and a hydrophobic substance are bound to each other through ionic bonding under weakly acidic conditions, and when the environment is changed at physiological pH conditions in this state, the hydrophobic binding is strengthened, And is enclosed in the particles to form a protein drug delivery system.

In the present invention, a protein drug which can be encapsulated in the hydrophobic substance and the polymer synthesized may be any protein having a negatively charged negative acidity. The protein having a negative charge may include bovine serum albumin (BSA) , Pegfilgrastim, Interleukin-6 (IL-6), Insulin, Human growth hormone (hGH), Human serum albumin (HSA), Asparaginase and Adenosine deaminase Leupirudin, Dornase Alfa, Denileukin diftitox, Bivalirudin, Peginterferon alfa-2a, Sargramostim, , Collagenase, Alpha-1-proteinase inhibitor, Pegaspargase, Hyaluronidase, Pegvisomant, Streptokinase, Filgrastim, Coagulation Factor IX, Agalsidase beta, Oxytocin and Enfuvirtide.

In one embodiment of the invention glycol chitosan: by the grafting (grafting) of 3-diethylaminopropyl (DEAP) group in (glycol chitosan GCS) were prepared GCS- g -DEAP, so synthesized GCS- g -DEAP the sealed by the BSA and allowed to react at weakly acidic (pH 6.0) conditions to bind the BSA to induce an ionic bond, by changing it to a physiological pH (pH 7.4) to enhance the hydrophobicity of the nanoparticle DEAP GCS- -DEAP g / BSA Nanogels were prepared (see Fig. 1).

In the protein gel delivery nanogel according to the present invention, the degree of substitution of DEAP, which is a hydrophobic substance per primary amine of the biocompatible polymer, is preferably in the range of 0.1 to 1, and most preferably the degree of substitution of DEAP is 0.75. This is because an optimal nanogel for encapsulating the drug can be formed within the range of degree of substitution.

The protein drug delivery nanogel of the present invention is self-organized in an aqueous solution. Due to the amphiphilic nature of the hydrophobic group of the DEAP group and the hydrophilic group of the biocompatible polymer, a nanogel of a spherical self assembly can be formed in the aqueous system .

Generally, self-assembly forms a spherical aggregate in which amphiphilic molecules having both hydrophilic and hydrophobic associations form in an aqueous solution. The hydrophilic group is collected inside the spherical aggregate and the hydrophobic group is collected inside. Particularly, in the present invention, the conjugate of a biocompatible polymer and a hydrophobic substance binds to a protein drug having a negative charge at a weak acidity using a property of being ionically positively charged at a weak acidity. When the protein drug binds to a negative charge at a physiological pH, , Which can increase the rate of encapsulation of the protein drug, and further, the introduction of nanotransporter into the cell is effectively performed.

In addition, the particle size of the protein drug delivery nanogel may range from 50 to 4000 nm, and the size is suitable for the nanocomposite to migrate to the target cell. The particle size of the nanogel may be slightly different depending on the mixing ratio of the synthesized polymer and the protein drug having pharmacological activity.

In one embodiment of the present invention, the particle size variation of the GCS- g- DEAP / BSA complex prepared by ion-binding at a weakly acidic condition was examined. As the weight ratio of GCS- g- DEAP increases, (See FIG. 2).

In addition, in one embodiment of the present invention, the zeta potential of the GCS- g- DEAP / BSA complex was observed to change. In general, the zeta potential is a function of the repulsive force between particles or the electrostatic dispersion It is used as an important factor in control. The zeta potential of the GCS- g- DEAP / BSA complex of the present invention varies from -6.0 mV to +5.2 mV as the weight ratio of GCS- g- DEAP increases at pH 6.0 (see FIG. 3). That is, this means that the negative zeta potential generated from BSA is increased by increasing the degree of ionic bonding between particles as the weight ratio of GCS- g- DEAP increases.

In another embodiment of the present invention, particle size changes of the GCS- g- DEAP / BSA nanogel prepared by reacting the GCS- g- DEAP / BSA complex at physiological pH conditions (pH 7.4) To about 4000 nm, as compared to the GCS- g- DEAP / BSA complex (see Fig. 4). In addition, it was confirmed that the morphological image obtained from the FE-SEM shows an irregular shape on the GCS- g- DEAP / BSA nanogel (see FIG. 5).

Therefore, the drug delivery method using the nanocomposite polymer is a drug delivery system that sufficiently shows the selectivity to the target cell and significantly reduces the toxicity to the normal cells and continuously releases the drug for a long period of time, and treats a serious disease such as cancer It can be used in the study of a new concept of therapy.

Further, the present invention provides a method for producing a nanocomposite for protein drug delivery according to the present invention.

The proteinaceous drug delivery nanocomposite of the present invention can be prepared by using methods apparent to those skilled in the art, but preferably includes the steps of: (a) dissolving the biocompatible polymer in a solvent; (b) adding 3-diethylaminopropyl to the solution in which the biocompatible polymer is dissolved to bind the amine group of the polysaccharide and the isothiocyanate of the 3-diethylaminopropyl group; (c) dialyzing and lyophilizing the combined and synthesized substance; (d) reacting the synthesized substance with a protein drug in a slightly acidic condition to form a complex; And (e) centrifuging the complex and then allowing the precipitate to react again at physiological pH conditions.

In the method for producing a polysaccharide nanogel for delivering a protein drug according to the present invention, the solvent capable of dissolving the biocompatible polymer may be any solvent that can dissolve the biocompatible polymer, Dimethyl sulfoxide (DMSO) may be used.

Also, 3-diethylaminopropyl (DEAP) added to the solution in which the biocompatible polymer is dissolved may be added in an amount of 1 to 3 moles per 1 unit of the biocompatible polymer, and triethylamine triethylamine, TEA), pyridine and the like are added to chemically bond the amine group of the biocompatible polymer to the isothiocyanate group of 3-diethylaminopropyl.

For example, in one embodiment of the present invention, GCS- g- DEAP was first synthesized using glycol chitosan as a biocompatible polymer and 3-diethylaminopropyl (DEAP) as a hydrophobic material. The GCS- -DEAP g as shown in Figure 1 of the synthesis process, glycol chitosan (GCS) free amine groups are 3-diethylaminopropyl (DEAP) isothiocyanate group (isothiocyanate) reacting with the amine group of the 3-diethylaminopropyl (DEAP), which can be reacted at room temperature for 5 to 7 days.

Next, the hydrophobic substance-bound polymer material formed in the organic solvent can be obtained in a powder state through dialysis and freeze-drying.

Next, the synthetic material thus obtained is ionized by hydrogenation of tertiary amine of 3-diethylaminopropyl (DEAP) under weakly acidic conditions to have a positive charge, and ionic bonding with a protein drug having negative charge in the same environment To form a complex. At this time, the slightly acidic condition is preferably in the range of pH 5.0 to 7.0. More preferably in the range of pH 5.5 to 6.5, and most preferably in the range of pH 6.0.

In addition, the synthesized substance and the protein having pharmacological activity may be mixed with the synthesized substance: the protein having pharmacological activity at a mixing ratio (% by weight) of 0.1: 1 to 10: 1. More preferably in a mixing ratio of 0.5: 1 to 4: 1.

Finally, after centrifuging the thus obtained complex, the precipitate is reacted again at pH 7.4 to 8.0, more preferably at pH 7.4, and the 3-diethylaminopropyl (DEAP) moiety is dehydrogenated to become hydrophobic By their hydrophobic binding, the size of the particles is reduced and a nano-unit protein drug delivery nanogel can be produced.

Therefore, in the present invention, when the therapeutic protein is encapsulated in the polymer through two steps of ionic bonding and hydrophobic bonding, it can have a high encapsulating efficiency. Since nanoparticles are formed at physiological pH, It can be delivered in a stable state into the cell.

Meanwhile, the present invention provides a protein drug carrier having a pharmacologically active protein drug encapsulated in the nanocomposite synthesized in the present invention. The drug delivery system also has a nanoparticle size.

Drug Delivery System is a technology that minimizes side effects by controlling the concentration and position of drugs introduced into the body and effectively transfers drugs for treatment of disease to the treatment site. It changes the pharmacokinetics of existing drugs to improve efficacy and effectiveness And the drug formulation technique which is desired to be maximized. These drug delivery systems have been intensively developed in recent years in the rapid development of micro-technology or nanotechnology to deliver pharmaceutical ingredients or formulations into cells using nanoscale carriers.

The nanoparticles are non-uniformly dispersed particles of a colloidal phase having a large surface area ranging from several nanometers to several hundreds of nanometers in size. When the nanoparticles are injected into the human body, they are transferred through various methods such as injection, oral administration and skin, The distribution of the drug is slightly different depending on the characteristics of the nanoparticles.

Meanwhile, the protein drug carrier according to the present invention has a negative charged protein drug sealed therein to form a self-assembled spherical shape. At this time, the particle size of the nanogels of the present invention may be 50 to 4000 nm, but the size of the nanogels may vary depending on the mixing ratio of the synthesized polymer and the pharmacologically active protein drug.

Since the protein drug delivery system according to the present invention has a nano-sized form as described above, it has an advantage that it can be easily, quickly, and deeply inserted into a target site, particularly a target cell.

In addition, the protein drug carrier of the present invention may comprise one or more drugs or therapeutic agents for short-term therapeutic effect or treatment, wherein the drugs or therapeutic agents are prepared by dissolving the natural polymer and the synthetic polymer polymer in a solution before, May then be added to the polymer used to make the gel. Preferably, the drug or therapeutic agent is added prior to dissolution to promote a more uniform dispersion or dissolution of the drug or therapeutic agent.

A variety of techniques are known for incorporating drugs or therapeutic agents into gels (polymers), including but not limited to spray drying, solvent evaporation, phase separation, rapid freezing and solvent extraction.

The drug or therapeutic agent is included in the gel in an amount of about 0.1 to about 100% by weight, preferably about 1 to about 50% by weight, and more preferably about 10 to about 30% by weight. However, the drug or therapeutic agent may be contained in an amount of 0.01 to 95% by weight of the gel. The amount or concentration of the drug or therapeutic agent contained in the gel will depend on the rate of absorption inactivation and release of the drug or therapeutic agent as well as the delivery rate of the polymer in the gel.

The drug carrier according to the present invention prepared by the above method is a liquid at room temperature. Immediately after injection into the subject, it becomes a gel due to body temperature. The drug or therapeutic agent contained within the nanocomposite will diffuse into the extracellular matrix of the subject and will be released in a controlled manner at the target site.

In the present invention, as the drug which can be encapsulated in the synthesized polymer, any component having a positive charge and having a positive charge can be used. Preferably, a protein having a negative charge can be used.

In another aspect, the present invention provides a controlled drug release pharmaceutical composition comprising a plurality of drugs or therapeutic agents in a bioerodable, biocompatible nanogel, wherein the composition drug or therapeutic agent is introduced into a patient in need thereof A method of treating a disease, disorder, or condition, including a disease or condition, and a method of producing the system of the present invention.

The drug delivery system of the present invention can be formulated into bioabsorbable, biodegradable, and biocompatible. By bioabsorbable is meant that the polymer can disappear from the initial application in the body, without decomposition or degradation of the dispersed polymer molecules. Biodegradable means that the polymer can be disrupted or degraded in the body by hydrolysis or enzymatic degradation. Biocompatibility means that all of the components are non-toxic in the body.

The Therapeutic Drug Delivery System of the Present Invention The drug delivery system of the present invention is a system for delivering a drug contained in the drug system to a person or other mammal having a therapeutically effective disease state or condition by injection or other means By coating the tissue surface of the body, or by coating the surface of the implantable device), but it is particularly preferred that the composition be delivered parenterally. "Parenteral" means intramuscular, intraperitoneal, intravesical, subcutaneous, intravenous, and intraarterial. Therefore, the composition of the present invention can be formulated into injection form representatively.

The injectable compositions of the present invention may be injected or injected into the body of a human or other mammal by any suitable method, preferably by injection through a hypodermic needle.

For example, it can be administered by intravenous, intravenous, genitourinary, subcutaneous, intramuscular, subcutaneous, intracranial, intrathecal, intrapleural, or other bodily fluids or possible space by injection or other means. Diagnosis, or surgery, through the catheter or syringe, into the joint, for example, into the joint during arthroscopy, into the urogenital tract, into the pulmonary, into the lining or into the pleura, or into any cavity or space within the body It can be introduced during the intervention procedure.

Further, according to the drug delivery system of the present invention, the drug or therapeutic agent can be released in a controlled manner to the target site of the subject.

In one embodiment, the nanocomposite is used to provide site-specific release of the drug or therapeutic agent to the subject. In another embodiment, the pH-sensitive nanogel comprises one or more drugs or therapeutic agents that can be administered to the subject, such that the drug or therapeutic agent is released by diffusion from the nanogel and / or degradation of the nanogel.

At this time, the dosage of the composition will vary depending on the type of disease and the severity of the disease or disorder being treated. It should further be understood that for any particular subject, the particular dosage regimen should be adjusted over time according to the needs of the individual and the professional judgment of the person administering the composition or administering the administration. In vivo administration can be based on in vitro release studies in cell culture or in vivo animal models

The rate of release of a drug or therapeutic agent is dependent on many factors, in particular, on the rate of degradation of the biodegradable polymer that constitutes the gel. It is also dependent on the particle size of the drug or therapeutic agent. Thus, by adjusting the factors discussed above, dissociation, diffusion and controlled release can be varied in a wide variety of ranges. For example, emissions may be designed to occur over time, days, and months.

As described above, the nanocomposite for protein drug delivery according to the present invention can be biodegradable and biocompatible in vivo by synthesizing a biocompatible polymer and a hydrophobic substance, and is capable of ionizing at a weak acidity to a positively charged state , It is possible to increase the efficiency of protein encapsulation in the drug delivery system by using a property of binding to a protein having a weak acidity at a weak acidity and a hydrophobic nature of a synthetic material when the pH is changed to a physiological pH, It is effective.

Hereinafter, the present invention will be described in detail with reference to the following embodiments and accompanying drawings. The following examples are intended to illustrate the present invention more specifically, but the protection scope of the present invention is by no means limited to the examples described in the following examples. It is apparent that the scope of the present invention covers not only the following embodiments, but also matters which can easily be deduced therefrom by a person skilled in the art.

Material preparation

First, the following materials were prepared to prepare nano-sized protein drug carriers using chitosan derivatives.

DMSO, Glycol chitosan (GCS, Mw = 250 kDa, deacetylation degree = 82.7%), 3-diethylaminopropyl isothiocyanate (DEAP), pyridine, dimethylsulfoxide , Triethylamine (TEA), bovine serum albumin (BSA), n-propyl galate, glycerol, fluorescein isothiocyanate (FITC), N Nd-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (USA), chlorin e6 (Ce6) Were purchased from Frontier Scientific Inc (USA).

Dulbecco's Modified Eagle's Medium (DMEM), TrisHCl (pH 8.4), 0.25% (w / v) trypsin / 0.03% (w / v) EDTA solution, fetal bovine serum (FBS), penicillin- Inc. (Korea).

< Example  1>

GCS - g - DEAP Synthesis of

First, 0.01 mM of glycol chitosan (GCS) was dissolved in 20 ml of DMSO, followed by reaction with 1 ml of triethylamine (TEA) and 0.1 ml of pyridine at room temperature for 7 days to graft DEAP 2 mM to GCS to obtain GCS- g- DEAP . After the reaction, the solution was transferred to a pre-swollen dialysis membrane tube (Spectra / Por MWCO 8K) and dialyzed against deionized water to remove unreacted DEAP and the solution was lyophilized.

Subsequently, the degree of substitution (defined as the number of DEAP blocks per primary amine of DS, GCS) was calculated from the number of DEAP blocks derived from δ 1.0 (-CH ₃ , DEAP block) and δ 2.73 (-CH-, ¹ &gt; H-NMR (DMSO-d6 with TMS) peak using the introduction ratio of the peaks.

As a result, in the case of GCS- g- DEAP synthesized by the above method, it was confirmed that the number of DEAP blocks substituted by primary amine groups of glycol chitosan analyzed by ¹ H-NMR was 0.75.

< Example  2>

GCS - g - DEAP / BSA  Manufacture of Composites

GAP- g- DEAP and BSA prepared in Example 1 were mixed at various mass ratios, i.e., GCS- g -DEAP: BSA = 0.5: 1, 1: 1, 2: 1, 4: PBS solution (pH 6.0), and the solution was mixed at 600 rpm for 2 hours.

< Example  3>

GCS - g - DEAP / BSA Nanogel  Produce

To separate the GCS- g- DEAP / BSA complex prepared in Example 2 above, the complex solution was centrifuged at 15,000 rpm for 5 minutes at 0 ° C. BSA loading performance of the supernatant was analyzed by HPLC (Agilent 1100 series, USA).

After centrifuging, the supernatant was discarded and the remaining precipitate was rinsed with PBS (5 mM, pH 6.0) solution and redispersed in 150 mM PBS solution (pH 7.4) to self-assemble hydrophobic DEAP and hydrophilic GCS ) Finally, GCS- g- DEAP / BSA nanogel was obtained. To determine the final BSA loading performance, the nanogel solution was centrifuged again at 25,000 rpm for 5 minutes at 0 ° C and the BSA concentration of the unmixed supernatant was analyzed by HPLC.

As a result, the BSA loading efficiency of GCS- g- DEAP / BSA nanogels was about 80 ± 2%.

< Example  4>

GCS - g - DEAP / BSA  Complex properties

In order to examine the physicochemical properties of the GCS- g- DEAP / BSA complex prepared in Example 2, the particle size distribution and the zeta potential were measured as follows.

<4-1> Particle size distribution

In order to investigate the physicochemical properties of the GCS- g- DEAP / BSA complexes, a Zetasizer 3000 (Malvern Instruments, USA) equipped with a He-Ne laser beam was used at a wavelength of 633 nm and a fixed scattering angle of 90 ° , And the particle size distribution of the GCS- g- DEAP / BSA complex (pH 6.0) was measured in PBS solution.

As a result, as shown in FIG. 2, the average particle size of -DEAP GCS- g / BSA conjugate GCS- g -DEAP: was changed according to the weight ratio of BSA, GCS- g -DEAP: BSA = 0.5: 1 be when The average particle size was about 250 nm, and when GCS- g- DEAP: BSA = 4: 1, the average particle size was about 5.2 μm. These results show that the degree of ionic bond between particles increases with increasing the weight ratio of GCS- g- DEAP.

<4-2> Zeta potential ( zeta potential ) Measure

The zeta potential change of the GCS- g- DEAP / BSA complex was measured using a Zetasizer 3000. Prior to testing, the GCS- g- DEAP / BSA complex was stabilized at room temperature for 1 hour.

As a result, the zeta potential, GCS- -DEAP g / BSA conjugate as shown in Fig. 3 was changed from -6.1mV as the weight ratio of GCS- g -DEAP increased to + 5.2mV. These results indicate that the negative zeta potential from BSA at pH 6.0 is offset by increasing the weight ratio of GCS- g- DEAP.

< Example  5>

GCS - g - DEAP / BSA Nano-gel  characteristic

In order to examine the physicochemical properties of the self-assembled GCS- g- DEAP / BSA nanogel in the aqueous solution prepared in Example 4, the following changes in particle size distribution and morphology were measured Respectively.

<5-1> Particle size distribution

G- DEAP / BSA nanogels at a wavelength of 633 nm and a fixed scattering angle of 90 degrees using a Zetasizer 3000 (Malvern Instruments, USA) equipped with a He-Ne laser beam at a pH of 7.4 PBS (150 mM) solution.

As a result, as shown in Fig. 4, at pH 6.0, hydrophobic interaction between deprotonated DEAP blocks around BSA occurred at pH 7.4 instead of electrical interaction of protonated DEAP block to BSA at pH 6.0, resulting in GCS- g- DEAP / BSA nanogels have a particle size of 100-150 nm which is lower than that of the GCS- g- DEAP / BSA complex.

<5-2> Morphological observation

g- DEAP / BSA nanogel (GCS- g- DEAP 0.1 mg / ml, BSA 0.1 mg / ml, GCS- g- DEAP: BSA = 1: 1 (wt%)) in pH 7.4 (150 mM PBS) The morphology was confirmed using a Field Emission Scanning Electron Microscope (FE-SEM, Hitachi s-4800, Japan). At this time, the FE-SEM sample was prepared by casting a nanogel diluted in PBS 150 mM on a slide glass at pH 7.4, and drying it in a vacuum. The morphology of the sample was imaged by FE-SEM.

As a result, as shown in Fig. 5, the GCS- g- DEAP / BSA nanogel showed an irregular shape.

<5-3> BSA  Emission test

G- DEAP / BSA nanogel (GCS- g- DEAP 0.1 mg / ml, BSA 0.1 mg / ml, GCS- g- DEAP: BSA = 1: 1 (wt.%)) In 150 mM PBS ) Was measured using a spectrofluorometer (Shimadzu RF-5301PC, Japan).

That is, a dialysis membrane tube (Spectra / Por MWCO 100K) containing a nano-gel solution (equivalent BSA 1 mg / ml) was immersed in a vial containing 10 ml of a 150 mM PBS solution (pH 7.4) (120 rev./min). The concentration of BSA released from GCS- g- DEAP / BSA nanogels was analyzed by HPLC and the BSA emission pattern was expressed as a function of time according to zero-order kinetics.

As a result, as shown in Fig. 6, 82% of the BSA was gradually released at pH 7.4 for 48 hours.

< Example  6>

Protein transfer efficiency test

<6-1> Cell culture

Human epithelial carcinoma HeLa cells (Korean Cell Line Bank) were cultured in a wet incubator in DMEM / PBS medium containing 1% penicillin-streptomycin and 10% FBS at 37 ° C and 5% CO ₂ air condition Lt; / RTI &gt; Prior to testing, the cells growing in a single layer were obtained by trypsinization with (1 x ¹⁰⁶ cells / ml) 0.25% (w / v) trypsin / 0.03% (w / v) EDTA solution . HeLa cells attached to DMEM medium (200 [mu] l) were seeded in 6-well plates and cultured for 24 hours before in vitro cell testing.

<6-2> Fluorescence microscopy analysis

Different amounts of BSA accumulation in HeLa cells (1 x 10 ³ cells / ml) by GCS- g- DEAP / BSA nanogel (equivalent BSA 10 μg / ml) or free BSA (10 μg / ml) (? ex 494 nm and? em 520 nm, E-SCOPE 1500F). To observe the distribution of BSA in HeLa cells, FITC (15 mg) was labeled with BSA (150 mg) for 1 hour at room temperature under a 5 mM borate buffer (pH 7.4), and dialyzed bags (Spectra / Por MWCO 15K) And lyophilization was performed.

Cells treated with GCS- g- DEAP / BSA nanogel or free BSA were incubated for 1 hour, then the cells were washed three times with PBS (pH 7.4) and incubated with 1% formaldehyde in PBS for 10 minutes at room temperature Lt; / RTI &gt; The cover slip was then mounted on a microscope slide with a dot of anti-fade mounting media (5% N-propyl gallate, 47.5% glycerol and 47.5% Tris-HCl, pH 8.4) to reduce fluorescence photobleaching .

The fluorescence image of HeLa cells treated with GCS- g- DEAP / BSA nanogel or free BSA at pH 7.4 is shown in FIG. 7, and the BSA concentration in the cells was observed through the green fluorescence intensity of the FIFC-bound BSA .

As a result, as shown in Fig. 7, it was confirmed that fluorescence intensity of HeLa cells treated with GCS- g- DEAP / BSA nanogel was stronger than that of free BSA treatment.

<6-3> Flow cell  analysis

Cellular uptake of BSA was confirmed using a FACSCalibur ^™ Flow Cytometer (Becton Dickinson, USA). BSA was labeled with FITC for analysis.

First, HeLa cells (1 × 10 ³ cells / ml) were dispensed into 6-well plates in DMEM medium. Next, HeLa cells were treated with GCS- g- DEAP / BSA nanogel (equivalent BSA 1 μg / ml) or free BSA (1 μg / ml) for 1 hour at pH 7.4, Washed once and trypsinized using a 0.25% (w / v) trypsin / 0.03% (w / v) EDTA solution. The cell solution thus obtained was fixed with 1% formaldehyde and analyzed by Flow Cytometer.

As a result, as shown in FIG. 8, free BSA showed inefficient intracellular uptake ability whereas GCS- g- DEAP / BSA nano-gel increased intracellular uptake by pinocytosis .

< Example  7>

in vivo in GCS - g - DEAP / BSA Effect of

&Lt; 7-1 > Animal care )

In In vivo experiments, female nude mice (BALB / c nu / nu mice, Institute of Medical Science, Tokyo) were used at 4-6 weeks of age and nude mice were used at the Institutional Animal Care and Use Committee of IACUC ) In accordance with the approved protocol guidelines.

<7-2> in vivo  Fluorescence image analysis

The inventors have GCS- -DEAP g / BSA In order to evaluate the pharmaceutical application of the nano-gel, using a BALB / c nu / nu female mice model the HeLa cancer cells implanted in vivo efficacy was tested.

in To obtain a vivo fluorescence image, the fluorescent dye, chlorine e6 (Ce6), was bound to BSA as follows. To the 250 mg of BSA was added dropwise a solution of the fluorescent dye Ce6 (25 mg) preactivated with DCC (25 mg) and NHS (30 mg) in DMSO (1 ml) at room temperature under PBS (100 ml) I poured it. The solution was filtered to remove dicyclohexylurea (DCU) and then lyophilized by immersion in a dialysis bag (Spectra / Por MWCO 1K).

For the full-scale experiment in vivo In animal models, HeLa cancer cells were suspended in PBS (pH 7.4, ion strength: 0.15) medium and injected subcutaneously into female nude mice at 1 × 10 ⁶ cells. Then, when the tumor volume become 20mm ^3, the Ce6 the HeLa tumor labeled GCS- -DEAP g / BSA nanogels (equivalent BSA 1 mg / kg body ) or free BSA (1 mg / kg body ) of the cover Ce6 And injected through the tail vein of a nude mouse. A fluorescent image of a nude mouse was measured using a 12-bit CCD camera (Image Station 4000 MM; Kodak, New Haven, CT) equipped with a Cmount lens and a long wavelength transmission filter (600-700 nm; Omega Optical, USA). At this time, fluorescent images were observed for 15 hours.

As a result, as shown in Figure 9, in various cases for vivo fluorescence intensity.

GAG- g- DEAP / BSA nanogels were intravenously administered to nude mice and fluorescence images were obtained 30 minutes later. As a result, GCS- g- DEAP / BSA nanogels (equivalent BSA 1 mg / Kg body) showed a strong fluorescence signal at the tumor site as compared to the result of administration of free BSA (1 mg / Kg body), indicating that GCS- g- DEAP / BSA nanogel It is thought that it is because it is accumulated high. The enhanced permeation and retention (EPR) effect of GCS- g- DEAP / BSA nanogels may contribute to increasing the level of BSA in tumors.

Further, FIG. 9 (b) further shows that the GCS- g- DEAP / BSA nanogel emits a fluorescent signal that continuously lasts for 15 hours at the tumor site. When conjugated to specific target moieties, the system carrying this protein may have great potential for effective treatment of various disease states.

In addition, as shown in Fig. 9 (b), the GCS- g- DEAP / BSA nanogel was shown to emit a continuous fluorescence signal at the tumor site for 15 hours.

Finally, GCS- g- DEAP / BSA nanogels according to the present invention have an optimal capacity for protein encapsulation and a clear protein transport capacity in the body, and the GCS- g- DEAP / BSA nanogel It can be efficiently used to transport a high molecular weight protein to a target site.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (12)

Dissolving the biocompatible polymer in a solvent (step 1);
3-diethylaminopropyl isothiocyanate (DEAP) is added to a solution in which the biocompatible polymer is dissolved to bond the amine group of the biocompatible polymer to the isothiocyanate group of the 3-diethylaminopropyl isothiocyanate (Step 2);
Dialyzing and lyophilizing the combined and synthesized material to prepare a nanogel formed by the DEAP core region and the biocompatible polymer shell region (Step 3);
The nanoparticles obtained in step 3 and the protein drug negatively charged at pH 5.5-6.5 were reacted in an aqueous solution of pH 5.5 to 6.5 to make the DEAP core region positively charged in a weakly acidic environment of pH 5.5-6.5, Inducing the protein drug to be negatively charged to form an ion-bound complex of a protein drug negatively charged in the positively charged DEAP core region (step 4); And
The complex obtained in the step 4 was centrifuged and the precipitate was reacted again in an aqueous solution of pH 7.4 to 8.0 to neutralize the charge of the DEAP core region of the nanogel to induce hydrophobic binding to prepare a nanogel containing the protein drug (Step 5);
&Lt; / RTI &gt; Protein drug delivery nanogels prepared with:
(A protein drug negatively charged at pH 5.5-6.5 of step 4 above,
(BSA), Pegfilgrastim, IL-6, insulin, human growth hormone (hGH), human serum albumin (HSA), asparaginase, Adenosine deaminase, Lepirudin, Dornase Alfa, Denileukin diftitox, Bivalirudin, Peginterferon alfa-2a (Peginterferon alfa-2a Pegaspargase, Hyaluronidase, Pegvisomant, Streptokinase (Streptokinase), Streptococcus pneumoniae (Streptococcus pneumoniae, Streptococcus pneumoniae, Staphylococcus aureus, Streptokinase, Filgrastim, Coagulation Factor IX, Agalsidase beta, Oxytocin, and Enfuvirtide). The method according to claim 1,
Wherein the mixing ratio of the nano-gel and the protein drug obtained in the step 3 is 0.1: 1 to 10: 1. delete The method according to claim 1,
The biocompatible polymer may be selected from the group consisting of glycol chitosan, hyaluronic acid, pullulan, curdlan, pectin, starch, dextran, chitosan, , Poly-L-lysine, and polyaspartic acid. 5. The method of claim 4,
Wherein the biocompatible polymer is glycol chitosan. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt; The method according to claim 1,
Wherein the 3-diethylaminopropyl isothiocyanate is substituted with 0.1 to 1 per primary amine of the biocompatible polymer. delete The method according to claim 1,
Wherein the particle size of the protein drug delivery nanogel is 100 to 4000 nm. Dissolving the biocompatible polymer in a solvent (step 1);
3-diethylaminopropyl isothiocyanate (DEAP) is added to a solution in which the biocompatible polymer is dissolved to bond the amine group of the biocompatible polymer to the isothiocyanate group of the 3-diethylaminopropyl isothiocyanate (Step 2);
Dialyzing and lyophilizing the combined and synthesized material to prepare a nanogel formed by the DEAP core region and the biocompatible polymer shell region (Step 3);
The nanoparticles obtained in step 3 and the protein drug negatively charged at pH 5.5-6.5 were reacted in an aqueous solution of pH 5.5 to 6.5 to make the DEAP core region positively charged in a weakly acidic environment of pH 5.5-6.5, Inducing the protein drug to be negatively charged to form an ion-bound complex of a protein drug negatively charged in the positively charged DEAP core region (step 4); And
The complex obtained in the step 4 was centrifuged and the precipitate was reacted again in an aqueous solution of pH 7.4 to 8.0 to neutralize the charge of the DEAP core region of the nanogel to induce hydrophobic binding to prepare a nanogel containing the protein drug (Step 5)
The protein drug negatively charged at pH 5.5-6.5 is selected from the group consisting of BSA, Pegfilgrastim, IL-6, insulin, hGH, (HSA), asparaginase, adenosine deaminase, Lepirudin, Dornase Alfa, Denileukin diftitox, Bivalirudin, Peginterferon alfa-2a, Sargramostim, Anakinra, Collagenase, Pegaspargase, Hyaluronidase, and Peginterferon alfa- Pegvisomant, Streptokinase, Filgrastim, Coagulation Factor IX, Agalsidase beta, Oxytocin, and Enfuvirtide. ) &Lt; / RTI &gt; Method for producing a nano-gels for protein drug delivery to a. 10. The method of claim 9,
The method for producing a protein drug delivery nanogel according to claim 1, wherein 3 to 6 mol of 3-diethylaminopropyl isothiocyanate is added per 1 unit of the biocompatible polymer. delete delete

Images