KR20080017161A - A composite comprising polysaccharide-functionalized nanoparticle and hydrogel matrix, a drug delivery system and a bone filler for sustained release comprising the same, and the preparation method thereof - Google Patents

Abstract

A nanoparticle-protein-hydrogel composite is provided to be used for a sustained-release drug delivery system and a sustained-release bone filler, which show the controlled initial release and have significantly increased sustained-releasing effect. A nanoparticle-protein-hydrogel composite comprises a polysaccharide-functionalized nanoparticle including a core consisting of a biodegradable polymer, an outside hydrogel membrane consisting of a biocompatible polymer emulsifying agent, and a polysaccharide physically fixed on the core and the hydrogel membrane; a protein specifically bound to the polysaccharide; and a hydrogel carrier which is a carrier of the nano-particle where the protein is specifically coupled and consists of a biocompatible polymer. A sustained release drug delivery system comprises the polysaccharide-functionalized nanoparticle; at least one protein drug which is specifically coupled to the polysaccharide and is selected from the group consisting of growth faction, chemokine, extracellular substrate protein and anti-thrombin III; and the hydrogel carrier. A sustained release growth factor delivery system comprises the polysaccharide-functionalized nanoparticle; an effective amount of growth factor including BMP or TGF-beta and at least one growth factor selected from the group consisting of VEGF, FGF, and PDGF; and the hydrogel carrier. A sustained release bone filler comprises the sustained release growth factor delivery system. A method for preparing a nanoparticle-protein-hydrogel composite comprises the steps of: (a) dispersing an organic solution by dissolving a biodegradable polymer in a non-cytotoxic organic solvent into an aqueous solution obtain by dissolving a polysaccharide and a biocompatible polymer emulsifying agent to prepare a polysaccharide-functionalized nanoparticle; (b) filling the polysaccharide-functionalized nanoparticle with a protein to prepare the protein filled polysaccharide-functionalized nanoparticle; (c) dispersing the protein filled polysaccharide-functionalized nanoparticle into a biocompatible polymer aqueous solution for preparing a hydrogel carrier; and (d) providing at least one crosslinking factor selected from the group consisting of a crosslinking agent, a crosslinking activating agent, and a physical crosslinking factor to the dispersion obtained from the step(c).

Description

A composite comprising polysaccharide-functionalized nanoparticle and hydrogel matrix, a drug delivery system and a bone filler, including a complex comprising a polysaccharide-functionalized nanoparticle and a hydrogel gel carrier, a sustained-release drug delivery agent comprising the same, a bone filler and a method for preparing the same for sustained release comprising the same, and the preparation method eg}

1 is a graph showing the cumulative amount of BMP released from polysaccharide-functionalized nanoparticles, which is a sustained release bone morphogenetic protein (BMP) delivery agent according to an embodiment of the present invention.

Figure 2 is a graph showing the cumulative release amount (%) of the BMP released from the functional nanoparticle-hydration gel complex is a sustained release BMP delivery agent according to an embodiment of the present invention.

Figure 3 is a photograph showing the appearance of a defect formed in the skull of the rat and a series of procedures for implanting the sustained release bone filler according to an embodiment of the present invention in the defect.

Figure 4 in the case of one embodiment and the comparative example of the present invention, the result of evaluating the bone formation ability through a soft X-ray analysis (A: comparative example, B: one embodiment of the present invention).

5 is a result of comparing the evaluation of bone formation ability through the histological analysis of the defect site in the case of one embodiment and comparative example of the present invention (A: Comparative Example, B: One embodiment of the present invention) ).

The present invention relates to a nanoparticle-protein-hydrating gel complex, a sustained-release drug delivery agent, a sustained-release bone filler and a method for preparing the same, and also the content of polysaccharides per unit mass of the nanoparticles and / or the It relates to a method of controlling the release rate of protein drugs by changing the content of nanoparticles per unit mass.

Bone fillers replace partially missing bones. Instead of just replenishing the missing parts, you need to help new bones grow into those parts of your body. These bone fillers can be used to promote bone formation, replace bone tissue defects, etc. in orthopedic surgeons such as fractures or dislocations of the limbs and vertebrae, disunion or delayed union, and removal of tumors or osteomyelitis. May be used.

Currently commonly used method is to implant the necessary bone form directly to the required part, the graft material is autologous bone, allogeneic bone and xenograft, synthetic bone and the like. First, autologous bone is used by culturing the bones obtained from the patient's body. There is an advantage that can be collected, but [J. Foot Ankle Surg. 1996; 35: 413-7], 1) complications such as limit of donor dose, 2) increase in secondary surgery and operation time at donor site, 3) bone defect and nerve damage at donor site, and the possibility of disease development And prolonged recovery periods [Clin. Orthop. 1996; 329: 300-9, Spine 1995; 20: 1055-60, J. Bone Joint Surg. Br. 1988; 70: 431-4, Br. J. Neurosurg 2000; 14: 476-9, J. South Orthop. Assoc. 2000; 9: 91-7, Spine 2000; 25: 2400-2, J. Bone Joint Surg. Br. 1989; 71-B: 677-80, J. Orthop. Trauma 1989; 3: 192-5. Next, the use of allogeneic bone and xenograft has advantages such as 1) fast operation time and recovery period because no secondary surgical part is needed, and 2) low cost compared to synthetic bone graft [AORN J. 1999; 70: 660-70 , Orthop. Clin. North am. 1999; 30: 685-98], 1) about 2 times the bone induction phase compared to autogenous bone, 2) the absorption of a large amount in the ossification process, 3) the bone quality of the formed bone is not good, and the disadvantage of immune response or risk of infection There is Clin. Orthop. 1972; 18: 19-27, J. Bone Joint Surg. Am. 1983; 65-A: 239-46, J. Appl. Biomater. 1991; 2: 187-208, J. Arthroplasty 2000; 15: 368-71, J. Bone Joint Surg. Br. 2001; 83 (1): 3-8, J. Bone Joint Surg. Br. 1999; 81: 333-5, Orthop. Clin. North am. 1987; 18: 235-9. In addition, synthetic bone uses a substance such as hydroxyapatite, tricalcium phosphate, calcium aluminate, plastic, metal, etc. A) Bone is not well formed in the filler, 2) it has problems of toxicity and biocompatibility, and 3) it is most commonly used as a material used in admixture with autologous bone, but it absorbs a lot, has little effect of bone regeneration, The individual particles are also enclosed and have not shown clinically satisfactory effects [Biomaterials 2000; 21: 2615-21, Clin. Orthop. 1989; 240: 53-62, Orthop. Clin. North am. 1999; 30: 591-8. In addition, these bone grafts have a problem in that they do not maintain their shape or space after the procedure in common.

Recently, new methods for regenerating damaged bone tissue using molecular therapy have been attempted. This accelerates tissue regeneration by supplying functional protein molecules, including growth factors, signaling molecules, transcription factors, and other effectors, to damaged tissue over time. It is an approach to induce early stage activation. In particular, tissue regeneration through growth factor transmission is very important in terms of tissue engineering, and BMP is mainly used as a target growth factor related to bone tissue regeneration. Ultimately, the method using BMP is expected to be a more effective bone regeneration method than autologous bone.

BMP is a bone matrix protein involved in bone formation. Urist [Science 1965; 150: 893-899. At least 15 BMP family members [Science 1988; 242: 1528-1534, which belongs to the transforming growth factor beta (TGF-β) superfamily, which plays a very important role in the formation and regeneration of the skeletal skeleton. In addition, BMP is involved in a variety of cell division, apoptosis, cell migration and differentiation [Genes Dev. 1996; 10 (13): 1580-94], development of limb buds in embryoogenesis [Mech Dev 1997; 69 (1-2): 197-202] and ectopic bone formation (Acta Orthop Scand 1996; 67 (6): 606-10], Differentiation of mesenchymal progenitor cells into osteoblasts or chondrocytes [J. Cell Biochem 1997; 66 (3): 394-403, J. Cell Biol 1998; 140 (2): 409-18, Bone 1998; 23 (3): 223-31, Exp Cell Res 1999; 251 (2): 264-74].

The choice of target growth factors and the efficiency of release and delivery of target proteins are the most important factors in tissue regeneration through growth factor delivery. In general, during natural healing of tissue defects, molecules that make up the proliferation and extracellular matrix of cells for tissue regeneration are generated at the edges of the defects, but signal molecules are not continuously supplied. The effect is very temporary and limited [J. Orthop Sports Phys Ther 1998; 28 (4): 192-202. As such, it is very important to develop a sustained-release growth factor delivery system capable of continuously delivering signaling molecules in tissue regeneration through growth factor delivery.

In the past few years, various sustained-release systems have been developed for the local delivery of growth factors, but until now, the ideal system has not been constructed. Recently, a matrix-based drug delivery system has been developed. It is applied to tissue regeneration. In order to develop and apply a support-based system, it is basically a condition such as non-immunogenic, non-toxic, biocompatible, biodegradable, and easily manufactured. Signal molecules must be stabilized in the system, the emission propagation can be controlled, and structural strength may be required. In relation to bone tissue regeneration through BMP delivery, growth factor delivery systems based on various materials have been developed.

The most commonly used mineral materials are hydroxyapatite (HAP, Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ), the major constituent of bone [Spine 1999; 15: 1179-1185, J. Biomed. Mater. Res. 2000; 51: 491-499. In addition, β-tricalcium phosphate (β-TCP, β-Ca ₃ (PO ₄ ) ₂ ), calcium phosphate-based cement (CPC), calcium sulfate, metals and bioglass are included in the inorganic base materials [In Society for Biomaterials, 6th World Biomaterials Congress 2000: p. 1135, J. Orthop Res. 2003; 21 (6): 997-1004, J. Biomed. Mat. Res. 1997; 35: 421-432, US 4596574, 4619655.

When the support is composed only of HAP itself, osteoblasts have excellent cell adhesion and tissue calcium, but due to the strong binding strength between HAP and BMP, bone induction is not good and the defect cannot be completely filled with the support. There is a fragile disadvantage. To compensate for this, the absorption rate of the scaffold was controlled by mixing with β-TCP or collagen, and the porous scaffold was constructed to obtain improved results in bone induction [Spine 1999; 15: 1179-1185, Clin. Orthop. 1988; 234: 250-254, Int. Orthop. (SICOT) 1996; 20: 321-325, J. Med. Dent. Sci. 1997; 44: 63-70, US 5001169, 5352715.

In the case of CPC, it can be manufactured by injection and can fill the bone defects, which improves the disadvantages of the existing system, but deactivates BMP due to the heat generated during the hardening process. This can result in a lessened effect. In addition, it is difficult to analyze the results through radiography because it is a radiopaque material [J. Oral Maxillofac. Surg. 1999; 57: 1122-1126, Biomaterials 2003; 24: 2995-3003.

Collagen (collagen), fibrin (fibrin), alginate (alginate), hyaluronic acid (hyaluronic acid) was applied as a natural polymer material.

In the case of collagen, raw materials are obtained from heterogeneous individuals, but technology for removing immunogenic telopeptides has been developed to minimize foreign body reactions caused by raw materials. One of the materials applied [J. Bone Jt Surg Am 2002; 84-A: 2123-34, Spine 2003; 28: 372-7, J. Bone Jt Surg Br 1999; 81: 710-8, Spine 2002; 27: 2654-61, EP 0206801, US 4394370, 4975527]. However, due to the excessive initial burst of growth factors, the economic burden of filling the support with an excessive amount of expensive BMP [Trends Biotechnol. 2001; 19 (7): 255-265], there is a potential risk of disease metastasis due to excessive growth factor concentrations at the beginning of transplantation [Clin Orthop Relat Res 1990; 260: 263-79, Nat Med 1998; 4: 141-4, Nature 1998; 391: 320-4.

Fibrin is a clinical adhesive called fibrin glue that is used during the blood coagulation process and plays an important role in hemostasis and wound healing. Fibrin hydrogels containing heparin have been developed to introduce sustained release systems in fibrin hydrogels and the composition of sustained release systems has been reported for growth factors with heparin binding sites in the molecule. In this case, heparin was immobilized in the hydrogel by covalently binding a peptide capable of electrostatic interaction with heparin in the hydrogel [J. Control Release 2000; 65 (3): 389-402, US 6468731, 6723344. In general, fibrin hydrogels are not sustained-release systems by themselves, but exceptionally, when the target growth factor is nonglycosylated BMP-2, there is a report that the solubility of the protein in the hydrogels may be low so that they can operate as a sustained-release system [J. Orthop Res 22 (2004) 376-381].

Alginate belonging to the polysaccharide is a non-sulfurized anionic natural polymer material, which is a copolymer of L-glucuronic acid and D-mannuronic acid, which can easily form hydrogels by combining Ca ++ ions, but when forming hydrogels, cells, proteins, and DNA This results in serious loss of biological activity, such as the relatively large pore size in the hydrogel, which causes macromolecules to diffuse easily [Adv Drug Deliv Rev 1998; 31 (3): 267-85, US 6748954].

Hyaluronic acid is a polysaccharide with intrinsic physicochemical and biological properties that specifically recognizes many proteins in the extracellular matrix and stabilizes the extracellular matrix through interaction with proteoglycans [J. Intern Med 1997; 242 (1): 27-33]. It is possible to interact with the cell surface that affects cell behavior and is also involved in cell mobility changes [FEBS Lett 1998; 440 (3): 444-9]. Hyaluronic acid support for BMP local delivery has been reported to utilize chemical crosslinks and to introduce hydrophobic functional groups [J. Control Release 1999; 61 (3): 267-79, J. Biomed Mater Res 1999; 47 (2): 152-69, J. Biomed Mater Res. 2002; 59 (3): 573-84, WO 0128602. The introduction of a double hydrophobic functional group allows the construction of a sustained release system, but has the disadvantage that the protein conformation is unstable at the interface in the support.

In addition to the support made of an inorganic material or a natural polymer material, a support using various synthetic polymer materials has been developed in connection with bone tissue regeneration through BMP transfer. Polyester polymers such as PLGA (poly (DL-lactide-co-glycolide)), PLA (poly (L-lactide)) and PGA (polyglycolide) are the most widely applied. Vet Med Sci 1998; 60 (4): 451-8, Bone 2003; 32 (4): 381-6, J. Bone Joint Surg Am 1999; 81 (12): 1717-29, J. Biomed Mater Res 1999; 46 (1): 51-9, J. Biomed Mater Res 2002; 61 (1): 61-5, J. Biomed Mater Res 2000; 50 (2): 191-8, J. Biomed Mater Res 1999; 45 ( 1): 36-41, US 4186448, 4563489, 5133755] In addition, polyanhydride, polyphosphazene and polypropylene fumarate, polyethylene glycol-PLA, poloxamer, and polyphosphate polymers are used [J. Biomed Mater Res 1990; 24: 901-11, Adv Drug Deliv Rev 2003; 55 (4): 467-82, Clin Orthop 1999; 41 (367suppl.): S118-29, Clin Orthop 1993; 109 (294): 333 -43, Plast Reconstr Surg 2000; 105 (2): 628-37, J. Biomed Mater Res 1997; 34: 95-104, US 4526909.

Synthetic polymer material has the advantage of easy processing, it is possible to control the porosity of the support and to configure a variety of forms and can be degraded by enzymes and cellular action in the body. On the other hand, when degradation, the pH is locally lowered, which affects the surrounding tissues. In the case of polymers with excessive inflammatory reactions and high molecular weights, the degradation rate is slow and chronic inflammation symptoms may be observed. In addition, it is difficult to construct a sustained release system when the decomposition of the polymer in the body is bulk erosion [Biomaterials 2000; 21: 1837-1845, Macromolecules 1987; 20: 2398-403, J. Control Release 1991; 16: 15-26]. When BMP is entrapped in the protein, structural denaturation of the protein occurs.

In order to solve the problems of the prior art as described above, the present invention is a sustained-release drug delivery agent, a sustained-release bone filler and their sustained release effect is significantly enhanced by using a nanoparticle-protein-hydrating gel complex Its purpose is to provide a method of manufacturing. Furthermore, the present invention is not limited to the sustained release behavior of the drug, and also provides a method of controlling the release rate of the protein drug by changing the content of polysaccharides per unit mass of nanoparticles and / or the content of nanoparticles per unit mass of the complex. It is for that purpose.

According to one aspect of the invention, (1) a polysaccharide comprising a core made of biodegradable polymer, ② an external hydrogel film made of a biocompatible polymer emulsifier, and ③ a polysaccharide physically fixed to the core and the hydrogel gel film Functionalized nanoparticles; (2) a protein that specifically binds to the polysaccharide; And (3) a nanoparticle-protein-hydrogel complex comprising a hydrogel carrier made of a biocompatible polymer as a carrier of nanoparticles to which the protein is specifically bound.

According to another aspect of the present invention, the present invention relates to a sustained-release drug delivery agent comprising an effective amount of at least one protein drug selected from the complex and growth factor, chemokine, extracellular matrix protein, and antithrombin III. In particular, the present invention includes BMP or transforming growth factor (hereinafter, TGF-beta) among the protein drugs, in addition to vascular endothelial growth factor (VEGF), fibroblasts The present invention relates to a sustained-release growth factor delivery agent for growth factors involved in bone formation, selected from fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF). .

According to another aspect of the invention, the present invention relates to a sustained release bone filler comprising a sustained release growth factor delivery agent according to various embodiments of the present invention.

According to one preferred embodiment of the above aspect, the sustained release growth factor transfer agent of the present invention is (1) ① poly (D, L-lactide- co -glycolide) (hereinafter 'PLGA'), poly (lactic acid), Consisting of one or more biodegradable polymers selected from poly (glycolic acid), poly (ε-caprolactone), poly (δ-valerolactone), poly (β-hydroxybutyrate) or poly (β-hydroxyvalrate) Core, ② a hydrogel film made of one or more biocompatible polymer emulsifiers selected from polyethylene glycol ethers of poloxamer, poloxamine, poly (vinyl alcohol), or alkyl alcohol, and ③ on the core and the hydration gel film. Polysaccharide-functionalized nanoparticles comprising at least one polysaccharide selected from physically immobilized heparin, alginate, hyaluronic acid and chitosan; And (2) an effective amount of growth factor specifically binding to the polysaccharide, including BMP or TGF-beta, in addition to growth factors selected from VEGF, FGF, and PDGF; And (3) poly (ethylene glycol), poloxamer, poly (organophosphazene), oligo (poly (ethylene glycol) fumarate), collagen, gelatin as carriers of nanoparticles to which the growth factors are specifically bound. It is preferable to include a hydrogel gel made of at least one biocompatible polymer selected from fibrin, hyaluronic acid and alginate.

According to another preferred embodiment, the sustained release bone filler of the present invention is (i) autologous bone, allogeneic bone, xenograft; (ii) It is preferable to further include HAP, tricalcium phosphate, calcium aluminate, β-TCP, CPC, calcium sulfate, and bioglass in terms of promoting bone formation. In addition, (iii) it is more desirable to add cell adhesion protein in terms of enhancing the intimacy of bone cells to the bone graft in the early stage of transplantation and to reduce the objection to foreign substances, and finally to hydrolyze the hydrogel so that only human tissue remains. It is preferable to further include a degradable peptide linker in terms of acceleration. The sustained release bone filler of the present invention can be used for the treatment or prevention of one or more diseases selected from osteoporosis, fracture, fracture dislocation, nonunion, delayed union, bone defect, alveolar bone defect.

In the present invention, "biodegradable polymer" means a polymer that can decompose when exposed to a physiological solution having a pH of 6-8, preferably can be decomposed by body fluids or microorganisms in vivo. It means a polymer.

Representative examples of biodegradable polymers usable in the present invention include poly (lactide- co -glycolide), poly (lactic acid), poly (glycolic acid), poly (ε-caprolactone), and poly (δ-baler of formula 1 Rockactone), poly (β-hydroxybutyrate), poly (β-hydroxyvalerate), or a combination thereof, but is not limited thereto. In particular, polysaccharide is added to an aqueous solution of an emulsifier to which polysaccharide is added to be functionalized as a polysaccharide. It is not limited to the polymer illustrated above as long as it is enough to manufacture a nanoparticle. However, it is preferable to use poly (lactide- co -glycolide), which is approved by the US FDA as non-toxic in vivo.

In the present invention, the term "combination" of oligomers or polymers includes not only the molten or liquid mixtures of each polymer listed, but also all kinds of copolymers thereof, and the term "combination" of monomers is By a monomer means a homooligomer or a combination of homopolymers (homopolymers).

If the molecular weight of the biodegradable polymer is less than 5,000 to form nanoparticles, the aggregation of molecules themselves is difficult, resulting in a decrease in yield and unstable fixation of polysaccharides in the particles. Thus, the biodegradable polymer has a weight average molecular weight of 5,000-100,000. More preferably 10,000-20,000.

In the present invention, "biocompatibility" refers to tissue compatibility and blood compatibility that does not necrosis or coagulate the tissue in contact with living tissue or blood, and "biocompatible polymer" It means a polymer having such biocompatibility.

In the present invention, "biocompatible polymer emulsifier" refers to a polymer having biocompatibility and having an emulsifying property to mix two or more separated phases.

Representative examples of biocompatible polymer emulsifiers that can be used in the present invention may include, but are not limited to, polyethylene glycol ethers of poloxamer, poloxamine, poly (vinyl alcohol), alkyl alcohols, or combinations thereof. Among them, it is preferable to use a poloxamer approved by the US FDA as being non-toxic in vivo.

The biocompatible polymer emulsifier of the present invention preferably has a weight average molecular weight of 5,000 to 100,000, more preferably 10,000 to 20,000, and the hydrophilic portion is selected to constitute 60-80% of the above, and is unstable of the nanoparticles if it is outside the above range. Difficulties may arise in yield reduction due to dispersion and in the immobilization of functional polysaccharides.

In the present invention, the term "polysaccharide-functionalized nanoparticles" or "functional nanoparticles" or the terms interpreted as being used in the same sense, as described in the preparation of the present invention, will be physically bonded to the protein through the polysaccharides. Means the nanoparticles imparted functionality.

On the other hand, in the present invention, "a hydrogel support made of a biocompatible polymer", "biocompatible polymer hydrogel support", "biocompatible polymer for producing a hydrogel support" and terms that are interpreted as being used in the same sense, The present invention is not limited to the biocompatible polymers exemplified in the present invention, and all kinds of biocompatible polymers conventionally used as hydrogel gel carriers can be used, regardless of whether they are synthetic polymers or natural polymers.

Among them, representative examples of the biocompatible polymer for preparing a hydrogel gel carrier which can be used in the present invention include poly (ethylene glycol), poloxamer, poly (organophosphazene), oligo (poly (ethylene glycol) fumarate) Polymers; And natural polymers such as collagen, gelatin, fibrin, hyaluronic acid, alginate; And / or combinations thereof.

In particular, in the following examples, as a biocompatible polymer for preparing a hydrogel carrier, fibrin has a cell binding site and a sugar aminoglycan binding site in the polymer, is easy to manufacture a hydrogel, and its clinical application as a tissue bonding and hemostatic agent. Although the experiment was carried out using the present invention, the present invention is not limited thereto, and the present invention can achieve the desired effect of the sustained release effect and the drug release control effect, and may be used regardless of its kind as long as it has biocompatibility.

In the present invention, the hydrogel carrier made of the biocompatible polymer acts as a carrier for bone tissue to grow as well as a filler to fill the space implanted at the beginning of transplantation. In addition, the protein or protein drug filled in the complex is primarily to form a specific bond with the polysaccharide in the nanoparticles to stabilize, and furthermore, the hydrogel carrier of the present invention can be additionally stabilized in the hydrogel carrier Some may be in charge.

In the present invention, the "physically immobilized" to the core and / or the hydrogel film means that the polysaccharide is "secured by a method other than 'chemical bonding through a chemical reaction'" and thus adsorption and aggregation In addition to physical fixation such as coheison, entanglement, entrapment, etc., non-chemical fixation generated by electrical interactions such as hydrogen bonds or van der Waals bonds acting alone or in combination with the physical fixation. The concept includes.

In the present invention, since the polysaccharide is physically fixed to the core and / or the hydrogel film and does not change the structure and physical properties through a separate chemical reaction, if the respective components have biocompatibility, the composite according to the present invention, Sustained release drug delivery systems and sustained release bone fillers also have the advantage of being biocompatible.

In the present invention, the protein or protein drug and the like "specifically bound" to the nanoparticles, specifically polysaccharides means that the protein or protein drug is bound based on the specific structure of the polysaccharides present in the nanoparticles, It means binding based on complementary chemical structures such as specific binding of receptor-ligand or specific binding of antigen-antibody. Such specific binding includes both covalent and non-covalent bonds, and is a concept that in particular includes the interaction between polysaccharide-proteins that maintain the three-dimensional structure of the protein, increase biological activity and inhibit hydrolysis to stabilize the protein.

In addition, the specific binding should be separated from the minimum binding force of the complex of the present invention to the extent that can be maintained in a relatively stable state in vivo, and must have a degree of separable binding force that can exhibit a slow release effect. It will be apparent in view of the object of the present invention. Those skilled in the art to which the present invention pertains can clearly understand it based on the prior art such as "Eur. J. Biochem. 1996; 237: 295-302" or "Biochem Soc Trans. 2006; 34: 458-6". It means. The interaction between the protein drug and the polysaccharide is to greatly reduce the initial release of the protein drug serves to enhance the slow release effect.

In the present invention, the 'polysaccharide' is specifically bound to various proteins or peptides such as growth factors, chemokines, extracellular matrix proteins, antithrombin III, etc., thereby maintaining the three-dimensional structure of the protein and increasing biological activity. Any polysaccharide that can serve to stabilize the protein by inhibiting hydrolysis can be used. However, the molecular weight of the polysaccharide preferably has a molecular weight of 3,000-100,000, more preferably 8,000-15,000 in terms of stability of the nanoparticles obtained in the composite and the core and the hydrogel film in the composite.

Representative examples of the polysaccharide that can be used in the present invention may include, but are not limited to, heparin, alginate, hyaluronic acid, chitosan, or a combination thereof. Among them, it is preferable to use heparin, an anionic polysaccharide approved by the US FDA as non-toxic in vivo.

'Protein drugs' used in the present invention include all proteins and polypeptides capable of specific binding to polysaccharides, in particular growth factors such as BMP, VEGF, FGF, PDGF, etc., chemokines, extracellular matrix proteins, and antithrombin III At least one protein selected from (Antithrombin III) is preferred. According to a preferred embodiment, one or more BMPs selected from among BMP-2, BMP-4, BMP-6, BMP-7, BMP-8, and BMP-9, which exhibit an osteoinductive effect, may be used.

In the sustained-release growth factor delivery agent and sustained-release bone filler of the present invention, the polysaccharide preferably contains 2-100 μg per 1 mg of the nanoparticles, the monodispersed nanoparticles are obtained when out of the range This is because a difficult problem and a problem of decreasing the slow release effect of the nanoparticles may occur. In addition, it is preferable to include 0.01-5 μg of the growth factor per mg of the nanoparticles in consideration of the release trend and the locally effective amount of the drug.

The nanoparticles present in the sustained release drug delivery system and sustained release bone filler according to an embodiment of the present invention are preferably 400 nm or less in diameter in that the sterilization process of the final product can be easily processed using a sterilization filter. In addition, the surface charge of the nanoparticles is determined according to the protein of interest and is preferably +20 mV or more and -40 mV or less in consideration of effective filling. In addition, the polydispersity index is particularly preferably less than 0.1, since it is generally regarded as a nanoparticle having a stable monodisperse distribution when the polydispersity index is less than 0.1. In addition, the sustained release growth factor delivery agent according to an embodiment of the present invention has an elastic modulus (G ′) observed after the hydrogel is completely formed to allow normal survival, proliferation and differentiation of cells included in the complex. It is desirable to have a value in the range of 200-20,000 Pa, which can be controlled by changing the concentration of the biocompatible polymer aqueous solution and the degree of crosslinking factor described below.

The present invention also discloses a method for preparing the complex, sustained release drug delivery agent, and sustained release bone filler.

According to one aspect according to this, (a) ① dissolving the biodegradable polymer in an organic solvent that is not cytotoxic at low concentrations to obtain an organic solution, ② dissolving a polysaccharide and a biocompatible polymer emulsifier in water to obtain an aqueous solution, And ③ preparing the polysaccharide-functionalized nanoparticles comprising adding and dispersing the organic solution to the aqueous solution. (b) preparing a polysaccharide-functionalized nanoparticle filled with protein by filling the protein with the polysaccharide-functionalized nanoparticle prepared above; (c) dispersing the polysaccharide-functionalized nanoparticles filled with the protein prepared above in an aqueous biocompatible polymer solution for preparing a hydrogel gel carrier; And (d) providing the at least one crosslinking agent selected from a crosslinking agent, a crosslinking activator, and a physical crosslinking factor to the dispersion prepared in step (c) to crosslink the biocompatible polymer for preparing the hydrogel carrier. It relates to a method for producing a hydrogel gel composite.

According to another aspect according to the present invention, the method for producing a sustained-release drug delivery agent comprising the step of filling an effective amount of at least one protein drug selected from growth factor, chemokine, extracellular matrix protein, antithrombin III It is about. In particular, the present invention relates to a method for preparing a sustained-release growth factor delivery agent comprising the step of filling an effective amount of at least one growth factor selected from BMP, VEGF, FGF, PDGF among the protein drugs.

According to yet another aspect, the present invention relates to a method for producing a sustained release bone filler comprising the step of forming a sustained release drug delivery agent according to various embodiments of the present invention using a mold to be suitable for a defect in bone or alveolar bone will be.

According to a preferred embodiment of the above aspect, the composite production method of the present invention (a) ① poly (D, L-lactide- co -glycolide), poly (lactic acid), poly (glycolic acid), poly (ε Dissolve one or more biodegradable polymers selected from -caprolactone), poly (δ-valerolactone), poly (β-hydroxybutyrate) or poly (β-hydroxyvalerate) at low concentrations with no cytotoxic organic solvents. (I) at least one polysaccharide selected from heparin, alginate, hyaluronic acid, chitosan and (ii) poloxamer, poloxamine, poly (vinyl alcohol), or alkyl alcohol Dissolving at least one biocompatible polymer emulsifier selected from polyethylene glycol ethers in water to obtain an aqueous solution, and (3) adding and dispersing the organic solution to the aqueous solution. Preparing a functionalized nanoparticle; (b) preparing polysaccharide-functionalized nanoparticles filled with growth factors by filling an effective amount of the growth factor selected from VEGF, FGF, and PDGF containing BMP or TGF-beta in the polysaccharide-functionalized nanoparticles prepared above; step; (c) biological materials for preparing at least one hydrogel carrier selected from poly (ethylene glycol), poloxamer, poly (organophosphazene), oligo (poly (ethylene glycol) fumarate), collagen, gelatin, fibrin, hyaluronic acid and alginate Dispersing the polysaccharide-functionalized nanoparticles filled with the growth factor prepared above in an aqueous solution of a compatible polymer; And (d) a crosslinking agent selected from glutaraldehyde, diepoxide and carbodiimide in the dispersion prepared in step (c); Crosslinking agents selected from thrombin, coagulation factor XIII, and mixtures thereof; It is preferable to include crosslinking the biocompatible polymer for preparing a hydrogel gel carrier by providing at least one crosslinking factor selected from physical crosslinking factors selected from temperature, pH, and specific intermolecular specific interactions.

First, in the step (a), the organic solution of step ① is preferably in the concentration of 0.5-2.0% (w / v) in terms of minimizing the loss due to the aggregation of the biodegradable polymer during the nanoparticle manufacturing step ; The aqueous solution of step ② is preferably a concentration of the biocompatible polymer emulsifier is 0.01-5% (w / v) in consideration of the thickness of the hydrogel film formed on the outside of the particles, the viscosity of the aqueous solution for efficient particle formation.

The organic solvent in which the biodegradable polymer is dissolved is then dispersed in an aqueous solution in which a biocompatible polymer emulsifier is added to form a polysaccharide-functionalized nanoparticle, wherein the amount of polysaccharide added to the aqueous solution in which the biocompatible polymer emulsifier is dissolved is In consideration of the polydispersity and yield of the nanoparticles, it is desirable to maintain 10% or less of the biocompatible polymer emulsifier mass in the aqueous solution.

In addition, the content ratio of the organic solution and the aqueous solution in step ③ does not require a special limitation, but considering the residual of the organic solvent in the nanoparticles and the resulting cytotoxicity, the volume of the organic solution to 10% or less of the aqueous solution volume It is preferable.

In addition, the step (b) includes (b ') redispersing the polysaccharide-functionalized nanoparticles in a dispersion medium and (b' ') adding the growth factor aqueous solution to the redispersant to fill an effective amount. More preferably.

In the step (b '), the polysaccharide-functionalized nanoparticle redispersion is preferably at least 25% (w / v) in consideration of the volume and strength of the final implant, in the step (b' ') The growth factor solution is preferably prepared using at least one solvent selected from PBS (phosphate buffered saline), PB (phosphate buffer), Tris, Hepes (Hepes) buffer in terms of stability of the protein conformation In addition, the concentration is preferably 0.01-0.5% (w / v) in consideration of the volume and strength of the final implant.

In addition, in the step (c), the preparation of the hydrogel gel carrier may include (i) poly (ethylene glycol), poloxamer, poly (organophosphazene), oligo (poly) as described in step (c). (Ethylene glycol) fumarate), collagen, gelatin, fibrin, hyaluronic acid, alginate polysaccharides-functionalized nanoparticles filled with the growth factor prepared above may be dispersed in an aqueous solution of a biocompatible polymer for preparing a hydrogel carrier. However, the present invention is not limited thereto, and (ii) the nanoparticles may be dispersed in an aqueous solution of the monomers or oligomers of the biocompatible polymers listed above, and then the monomers or oligomers may be polymerized and / or crosslinked.

In addition, in step (d), the degree of provision of chemical and physical crosslinking factors such as a crosslinking agent, a crosslinking agent, and a temperature, pH, and specific interaction between molecules for crosslinking the biocompatible polymer for preparing a hydrogel gel carrier is included in the complex. It is desirable to adjust the observed elastic modulus to have a value in the range of 200-20,000 Pa after the hydrogel is completely formed to allow normal survival, proliferation and differentiation of the cells.

In addition, the method for producing a sustained release bone filler according to an embodiment of the present invention (e) bone or alveolar bone formed by one or more diseases selected from osteoporosis, fracture, fracture dislocation, nonunion, delayed union, bone defect, alveolar bone defect It is preferable to include the step of forming the growth factor transfer agent using a mold to suit the defects of the.

In addition, in performing step (e), before and / or after performing step (e) and / or at the same time as performing step (e), (f) ① autologous bone, allogeneic bone, xenograft, and ② HAP which have eliminated cells Preferably, the method further comprises adding at least one component selected from among tricalcium phosphate, calcium aluminate, β-TCP, CPC, calcium sulfate, bioglass, and ③ cell adhesion protein, degradable peptide linker.

In the present invention, "an organic solvent having no cytotoxicity at low concentration" means an organic solvent which is reported to have no cytotoxicity at low concentration that can remain in nanoparticles. Preferably, but not limited to, dimethylsulfoxide (hereinafter referred to as 'DMSO') or tetraglycol, which is reported to be non-cytotoxic at concentrations below 10% (w / w).

In addition, in the present invention, 'water' as the solvent of the 'aqueous solution' may be used as long as it shows in vivo compatibility without exhibiting any particular toxicity, and the like, and is not particularly limited to distilled water.

In addition, the dispersion method used in the present invention, such as by adding the organic solution to the aqueous solution to disperse, can be used as long as the dispersion method used in the art.

In the present invention, "effective amount" means a minimum amount by which a protein drug according to various embodiments of the present invention can achieve the desired therapeutic or prophylactic effect by a conventional assay method, and in particular osteoporosis To improve bone formation in bone defects remaining after removal of fractures or fractures of the limbs and vertebrae of the orthopedic surgeon, dislocation or fracture of the spine, nonunion or delayed union, tumor or osteomyelitis, replacement of bone tissue defects or alveolar bone defects in dentistry It means the least amount that can have the effect of regenerating or inducing bone to improve or treat it.

Such effective amount is determined by those skilled in the art to which the present invention pertains, the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the age, weight, general state of health and sex of the patient. And various factors including diet, time of administration, route of administration and rate of composition, duration of treatment, drug used concurrently.

Furthermore, the present invention not only provides a sustained release drug delivery agent as described above and a method for preparing the same, but also provides a method of controlling the release rate of protein drugs by varying the release behavior as needed.

In particular, in the above-described method for preparing a sustained-release drug delivery agent, (A) (i) in step (a) ② to change the concentration of the polysaccharide in the aqueous solution, or (ii) (a) ③ Changing the content of the polysaccharides per unit mass of the nanoparticles by changing the mixing ratio of the organic solution and the aqueous solution in a step; (B) the content of nanoparticles per unit mass of the complex is changed by changing the concentration ratio of the polysaccharide-functionalized nanoparticles and the biocompatible polymer for preparing the hydrogel gel carrier in the aqueous hydrogel polymer preparation for preparing the hydrogel gel carrier of step (c). How to change; Alternatively, the release rate of the protein drug may be controlled by performing the combination of the above (A) and (B) methods.

In addition, the following examples do not describe specific experimental procedures for controlling the drug release rate, but those skilled in the art to which the present invention pertains can easily and easily determine the concentration of polysaccharides based on the description in the above paragraph of the present invention. Alternatively, the content of polysaccharides per unit mass of nanoparticles may be changed by changing the mixing ratio of the organic solution and the aqueous solution, and the content of nanoparticles per unit mass of the composite may be changed by changing the concentration ratio of the functional nanoparticles and the biocompatible polymer for preparing a hydrogel carrier. It is obvious that this could change.

Example

Hereinafter, the content of the present invention will be described in detail through examples. However, the following examples are provided to explain the contents of the present invention and do not limit the scope of the present invention.

A. Step 1: Nanoparticle Manufacturing

1. Comparative Production Example: Preparation of Nanoparticles Having a Hydrophilized Hydrogel Film

Nanoparticles were prepared by dissolving 40 mg PLGA completely in 2 mL of dimethylsulfoxide and slowly adding 30 mL of a 5% aqueous solution of poloxamer. Polaxamer and dimethyl sulfoxide that did not participate in the particle formation in the nanoparticle solution were removed by high-speed centrifugation to separate the supernatant, and the recovered nanoparticles were redispersed in distilled water or PBS (phosphate buffered saline, pH 7.4) solution. It became.

2. Preparation Example 1-5: Preparation of Heparin-functionalized Nanoparticles

Nanoparticles were prepared by slowly adding 2 mL of dimethylsulfoxide in which 40 mg of PLGA was dissolved in 30 mL of a 5% aqueous solution of poloxamer containing 10, 30, 60, 120, and 240 mg of heparin, respectively. Excess heparin, poloxamer and dimethyl sulfoxide in the nanoparticle solution were removed by high-speed centrifugation to separate the supernatant and redispersed in the nanoparticles recovered using distilled water or PBS solution.

3. Experimental Example: Observation of particle size, surface charge, component mass ratio, and polydispersity index of heparin-functionalized nanoparticles

The size of the nanoparticles prepared by the dynamic light scattering method was measured using ELS-8000 manufactured by Otsuka Electronics Co., and the surface charge of the particles was measured by the electrophoretic light scattering method.

As the amount of heparin in the aqueous solution of poloxamer increased, the surface charge of the synthesized nanoparticles increased from -26.0 ± 1.1 to -44.4 ± 1.2 mV and the size was changed from 123.1 ± 2.0 to 188.1 ± 3.9. Since heparin is a strong negatively charged material, a surface charge that increases to a negative value indicates that more heparin is present on the surface of the resulting particles.

To obtain the mass ratio of each component constituting the particles, the nanoparticle solution was first freeze-dried to calculate the dry weight of the nanoparticles. Anti-factor Xa analysis of particles (C. Chauvierre et al., Biomaterials 25 (2004) 3081-3086) was used to calculate the amount of heparin in the heparin present in the particle surface layer, and the particles were dissolved to collect the whole heparin. Anti-factor Xa analysis was used to calculate the total amount of heparin in the particles. Finally, the mass ratio of each component was determined by calculating the ratio of PLGA and poloxamer in the particles by ¹ H NMR analysis (Table 1).

As shown in Table 1, heparin was physically immobilized in PLGA nanoparticles because most of the physically immobilized heparin was present in the particle surface layer, and heparin and poloxamer that were not strongly bound in the prepared particles were removed by high-speed centrifugation. It can be seen that it is immobilized by and is mostly in the particle surface layer composed of poloxamer. The poloxamer forming the hydration gel layer is thought to be stabilized during the preparation of the particles by the attraction of the hydrophobic portion of the poloxamer and also the hydrophobic PLGA, and the attraction of the carboxyl group of heparin to the polyethylene glycol portion, the hydrophilic portion of the poloxamer. It is believed that heparin is immobilized on the surface layer during particle formation.

Table 1 shows the mass ratios of the components constituting the nanoparticles prepared by Comparative Preparation Example, Preparation Example 2, and Preparation 4.

Mass ratio of each component in nanoparticles Heparin in aqueous solution (mg) PLGA (mg) Polaxamer (mg) Heparin (mg) in particle surface layer Total Heparin in Particles (mg) 0 36.8 ± 1.6 (72.7%) 13.8 ± 0.6 (27.3%) 0.0 (0.0) 0.0 (0.0) 30 34.7 ± 0.9 (70.6%) 13.3 ± 0.4 (27.0%) 0.94 ± 0.04 (1.9%) 1.20 ± 0.04 (2.4%) 120 29.0 ± 1.8 (66.9%) 12.3 ± 0.8 (28.4%) 1.71 ± 0.09 (4.0%) 2.01 ± 0.10 (4.7%)

As shown in Table 1, as the amount of heparin dissolved in the aqueous solution of poloxamer increased, the amount of heparin present in the hydrogel on the particle surface also increased. However, in consideration of the polydispersity index and the yield of the nanoparticles, the amount of heparin added in the poloxamer aqueous solution is preferably maintained at 120 mg or less. In particular, nanoparticles containing 0, 2.4, and 4.7 wt% of heparin were prepared for nanoparticles prepared under the conditions of 0, 30, and 120 mg of heparin in aqueous solution of poloxamer.

B. Step 2: Preparation of Protein Filled Nanoparticles

Comparative Preparation Example: Preparation of Nanoparticles Filled with Model Protein Lysozyme

The nanoparticles prepared in the first step comparative preparation were separated by high-speed centrifugation and redispersed in PBS solution, and then charged with 1 mg of lysozyme, a model protein.

2. Preparation Example 1-2 Preparation of Model Protein Lysozyme-Filled Heparin-Functionalized Nanoparticles

The nanoparticles prepared in step 1 of Preparation Examples 2 and 4 were separated by high-speed centrifugation and redispersed in PBS solution, and then charged with 1 mg of lysozyme, a model protein.

3. Preparation Example 3-4: Preparation of VEGF-Filled Heparin-Functionalized Nanoparticles

VEGF was filled in the same manner as in the second step Preparation Example 1-2 using the nanoparticles prepared in Step 1 Preparation Example 4. 15.6 ng and 156 ng of VEGF were filled per mg of nanoparticle, respectively.

4. Preparation Example 5 : BMP Filled  Preparation of Heparin-functionalized Nanoparticles

Using the nanoparticles prepared in the first step Preparation Example 4, the excess heparin, poloxamer and DMSO present in the nanoparticle solution was removed by separating the supernatant after high-speed centrifugation. Then, after redispersing the recovered nanoparticles using 40 μL of PBS, BMP-2 (Peprotech, cat # 120-02) was charged, and 156 ng of BMP per mg of nanoparticles was mixed and gently stirred. Incubation was performed for one day at < RTI ID = 0.0 >

5. Experimental Example 2: In vitro observation of model protein lysozyme sustained release and stabilizing effect

In order to confirm the sustained release and stabilizing effect of the protein drug for the nanoparticles filled with the drug prepared in Comparative Example 2, Preparation Example 1-2, the release pattern was measured under the following in vitro conditions.

After lysozyme-filled nanoparticle dispersion was put into dialysis tube (MWCO 500k), the released lysozyme was recovered using excess PBS solution satisfying the infinite dilution condition and the amount was determined using the Micro BCA protein assay. Quantification was carried out using. The PBS solution used for lysozyme recovery was replaced with fresh PBS solution daily and the samples were stored at 4 ° C. until protein quantification.

In the case of nanoparticles without heparin, two-thirds of the filling amount was released within three days, and the sustained release form of the protein was observed as the amount of immobilized heparin in the nanoparticles increased. On the other hand, nanoparticles having a heparin content of 4.7 wt% were released for up to 19 days without initial drug release.

In addition, the biological activity of the lysozyme released from the nanoparticles functionalized with heparin was measured, and it was confirmed that the protein filled by heparin immobilized in the hydrogel membrane of the nanoparticles was stabilized.

6. Experimental Example 2: In vitro observation of the sustained release effect of BMP

For the drug-filled nanoparticles prepared in Preparation Step 5, the release pattern of BMP was measured under in vitro conditions as follows. After the BMP-filled nanoparticle dispersion solution was placed in a dialysis tube (MWCO 500k), the released BMP was recovered using an excess PBS solution satisfying infinite dilution conditions, and the amount of BMP recovered was ELISA (enzyme-linked immunosorbent). Quantification using the assay). The PBS solution was replaced with fresh solution every day and the recovered samples were stored at −30 ° C. or lower until protein quantification.

When 156 ng of BMP was charged per 1 mg of nanoparticles with a heparin content of 4.7% by weight, no initial release was observed and 24% of BMP filled up to 15 days was continuously released (FIG. 1). This confirmed the applicability of heparin-functionalized nanoparticles for sustained-release BMP delivery.

C. Step 3: Preparation of BMP Filled Heparin-Functionalized Complex (Drug Delivery Agent)

Example 1 Preparation of BMP Filled Heparin-Functionalized Complex (Drug Delivery Agent)

After redispersing the nanoparticles recovered in Preparation Step 4, 26 μL of the final dispersion obtained after filling with BMP was uniformly mixed with 70 μL of a fibrinogen solution having a concentration of 7-12% (w / v). Nanoparticle hydrogel composite was prepared by further mixing thrombin solution, a crosslinking activator, in the same volume with fibrinogen solution.

The fibrinogen solution was prepared by dissolving fibrinogen (71.5-126.5 mg), blood coagulation factor XIII (44-88 U) and aprotinin (1,100 KIU) as a main component, and lyophilized fibrinogen in an aqueous solution containing the remaining components. The final concentration was adjusted with 65-115 mg fibrinogen, 40-80 U blood coagulation factor, and 1000 KIU aprotinin per mL of aqueous solution.

The thrombin solution is a calcium chloride solution in which thrombin (400-600 IU) is dissolved and prepared by dissolving lyophilized thrombin in an aqueous solution containing the remaining components. The final concentration is 0.6% of 1.2 mL of 400-600 IU of thrombin. (w / v) adjusted to dissolve in calcium chloride solution. In addition, the two solutions were finally mixed and then incubated at room temperature for 30 minutes.

2. Experimental Example 1: In vitro observation of the sustained release effect of BMP

For the complex (drug delivery agent) prepared in Example 1 was measured the release pattern of BMP under in vitro conditions as follows.

After preparing a BMP-filled heparin-functionalized complex and placing it in a release container, the released BMP was recovered using an excess PBS solution satisfying infinite dilution conditions, and the amount of recovered BMP was determined by an enzyme-linked immunosorbent assay (ELISA). Quantification was carried out using. The PBS solution was replaced with fresh solution every day and the recovered samples were stored at −30 ° C. or lower until protein quantification.

As prepared in Example 1, when 156 ng of BMP was charged per 1 mg of nanoparticles with a heparin content of 4.7% by weight, no initial release was observed and 23% of the BMP filled up to 15 days was continuously released. (Figure 2). This confirmed the applicability of the heparin-functionalized complex for sustained release BMP delivery.

D. Step 4: Preparation of BMP Filled Heparin-Functional Bone Filler

Example 1 Preparation of Bone Filler Comprising Protein Filled Heparin-Functionalized Complex

The composite prepared in Example 1 of step 3 was molded in a mold, and in particular, the system was prepared in a disk shape having a diameter of 8 mm and a height of 2.1 mm to apply to a rat skull defect model.

2. Experimental Example and Comparative Experimental Example: Evaluation of bone formation ability in vivo for sustained release BMP bone filler and hydrogel carrier

As an experimental example, in vivo bone formation ability of the sustained-release BMP bone filler was evaluated using a rat skull defect model. The bone filler prepared in Example 1 was implanted into 12 bone defects of mice (FIG. 3).

In addition, as a comparative example, transplantation was performed for 12 bone defects of mice under the same conditions, but only a hydrogel carrier was implanted instead of the bone filler. However, through preliminary experiments, it was determined that bleeding problems caused by skull defects could delay bone formation.

Animals were sacrificed at 4 weeks and soft x-rays were taken (30 KVP, 1.5 mA, distance: 52 cm, exposure: 90 seconds). Bone formation was observed in all 12 animals of Experimental Example 2, but some specimens did not completely regenerate the skull defects, but significantly better bone formation compared to the comparative example (Fig. 4).

In addition, bone formation was observed through MT staining after deliming using EDTA for histological analysis. In the regenerated bone tissue photograph of Experimental Example 2, collagen and osteoblast isolated from the inside of collagen, bone marrow cells, osteoblasts and osteoblasts (osteoclast) at the border of collagen and bone marrow cells Complete bone formation could be observed, and in the comparative experiments, some bone formations resulted from the bone defect margins, but fell significantly compared to Experimental Example 2 (FIG. 5). From the above results, the bone formation ability of the bone filler of the present invention was confirmed.

As seen above, the sustained release drug delivery agent and the sustained release bone filler comprising the complex according to the present invention are simpler in the manufacturing process of the system than the conventional bone formation induction method, and dramatically reduce the economic burden and bone formation effect of the patient In addition to having the advantage of improving, the nanoparticle-hydration gel complex system can be applied to other growth factor delivery systems, which are important in tissue engineering, and can be easily applied to restoration of other tissues. The bone filler using the sustained release BMP delivery system can be widely applied in orthopedics, plastic surgery, dentistry, etc. The advantage of this system is that the market size is much larger than the existing medical market. It is expected to occur.

Claims (20)

(1) a core made of a biodegradable polymer,     ② an external hydrogel film made of a biocompatible polymer emulsifier, and     A polysaccharide-functionalized nanoparticle comprising a polysaccharide physically fixed to the core and the hydrogel film; (2) a protein that specifically binds to the polysaccharide; And (3) A nanoparticle-protein-hydrogel complex comprising a hydrogel carrier made of a biocompatible polymer as a carrier of nanoparticles to which the protein is specifically bound. (1) a core made of a biodegradable polymer,     ② an external hydrogel film made of a biocompatible polymer emulsifier, and     A polysaccharide-functionalized nanoparticle comprising a polysaccharide physically fixed to the core and the hydrogel film; (2) an effective amount of protein drug that specifically binds to the polysaccharide, the drug drug selected from among growth factors, chemokines, extracellular matrix proteins and antithrombin III; And (3) A sustained release drug delivery agent comprising a hydrogel carrier made of a biocompatible polymer as a carrier of nanoparticles to which the growth factor is specifically bound. (1) ① poly (D, L-lactide- co -glycolide), poly (lactic acid), poly (glycolic acid), poly (ε-caprolactone), poly (δ-valerolactone), poly (β -Hydroxybutyrate) or a core consisting of at least one biodegradable polymer selected from poly (β-hydroxyvalerate),     ② a hydrogel gel film composed of one or more biocompatible polymer emulsifiers selected from polyethylene glycol ethers of poloxamer, poloxamine, poly (vinyl alcohol), or alkyl alcohol, and     (3) polysaccharide-functionalized nanoparticles comprising at least one polysaccharide selected from heparin, alginate, hyaluronic acid, and chitosan that are physically fixed to the core and the hydration gel film; And (2) an effective amount of growth factor specifically binding to the polysaccharide, including BMP or TGF-beta, in addition to growth factors selected from VEGF, FGF, PDGF; And (3) Poly (ethylene glycol), poloxamer, poly (organophosphazene), oligo (poly (ethylene glycol) fumarate), collagen, gelatin, as carriers of nanoparticles to which the growth factors are specifically bound A sustained release growth factor delivery agent comprising a hydrogel gel carrier composed of at least one biocompatible polymer selected from fibrin, hyaluronic acid and alginate. The biodegradable polymer of claim 3, wherein the biodegradable polymer is poly (D, L-lactide-co-glycolide), the biocompatible polymer emulsifier is poloxamer, the polysaccharide is heparin, and the growth factor is BMP-2. , BMP-4, BMP-6, BMP-7, BMP-8, BMP-9 and one or more selected from BMP or TGF-beta, in addition to the growth factor selected from VEGF, FGF, PDGF, the hydrogel gel carrier Sustained release growth factor delivery agent, characterized in that the fibrin. The method of claim 4, wherein the biodegradable polymer and the biocompatible polymer emulsifier have a weight average molecular weight of 5,000-100,000; The polysaccharide and the growth factor include 2-100 μg and 0.01-5 μg, respectively, per mg of the nanoparticle; The nanoparticles have a diameter of 400 nm or less, a surface charge of +20 mV or more or -40 mV or less, and a polydispersity index of less than 0.1; The sustained release growth factor transfer agent is a sustained release growth factor transfer agent, characterized in that it has an elastic modulus of 200-20,000 Pa. A sustained release bone filler comprising a sustained release growth factor transfer agent according to any one of claims 3 to 5. The sustained-release bone filler according to claim 6, wherein the sustained-release bone filler is for the treatment or prevention of one or more diseases selected from osteoporosis, fracture, fracture dislocation, nonunion, delayed union, bone defect, and alveolar bone defect. The method of claim 7, wherein the sustained-release bone fillers are (i) autologous bone, allogeneic bone, xenograft; (ii) HAP, tricalcium phosphate, calcium aluminate, β-TCP, CPC, calcium sulfate, bioglass; And (iii) one or more components selected from cell adhesion proteins and degradable peptide linkers. (a) ① dissolving a biodegradable polymer in an organic solvent having no cytotoxicity at low concentration to obtain an organic solution, ② dissolving a polysaccharide and a biocompatible polymer emulsifier in water to obtain an aqueous solution, and ③ the organic solution Preparing a polysaccharide-functionalized nanoparticle comprising adding and dispersing in an aqueous solution; (b) preparing a polysaccharide-functionalized nanoparticle filled with protein by filling the protein with the polysaccharide-functionalized nanoparticle prepared above; (c) dispersing the polysaccharide-functionalized nanoparticles filled with the protein prepared above in an aqueous biocompatible polymer solution for preparing a hydrogel gel carrier; And (d) providing the dispersion prepared in step (c) to at least one crosslinking agent selected from a crosslinking agent, a crosslinking agent, and a physical crosslinking agent to crosslink the biocompatible polymer for preparing the hydrogel gel carrier. Method for preparing a hydrogel gel composite. (a) ① dissolving a biodegradable polymer in an organic solvent having no cytotoxicity at low concentration to obtain an organic solution, ② dissolving a polysaccharide and a biocompatible polymer emulsifier in water to obtain an aqueous solution, and ③ the organic solution Preparing a polysaccharide-functionalized nanoparticle comprising adding and dispersing in an aqueous solution; (b) preparing a polysaccharide-functionalized nanoparticle filled with protein drug by filling an effective amount of at least one protein drug selected from growth factor, chemokine, extracellular matrix protein, and antithrombin III to the polysaccharide-functionalized nanoparticle prepared above step; (c) dispersing the polysaccharide-functionalized nanoparticles filled with the protein prepared above in an aqueous biocompatible polymer solution for preparing a hydrogel gel carrier; And (d) sustained-release drug delivery agent comprising the step of crosslinking the biocompatible polymer for preparing a hydrogel carrier by providing at least one crosslinking agent selected from a crosslinking agent, a crosslinking activator, and a physical crosslinking factor in the dispersion prepared in step (c). Manufacturing method. (a) ① poly (D, L-lactide- co -glycolide), poly (lactic acid), poly (glycolic acid), poly (ε-caprolactone), poly (δ-valerolactone), poly (β Dissolving at least one biodegradable polymer selected from -hydroxybutyrate) or poly (β-hydroxyvalerate) in an organic solvent having low cytotoxicity at low concentrations to obtain an organic solution,     (I) at least one polysaccharide selected from heparin, alginate, hyaluronic acid, chitosan and (ii) at least one selected from polyethylene glycol ethers of poloxamer, poloxamine, poly (vinyl alcohol), or alkyl alcohols. Dissolving the biocompatible polymer emulsifier in water to obtain an aqueous solution, and     ③ preparing a polysaccharide-functionalized nanoparticle comprising the step of adding the organic solution to the aqueous solution to disperse; (b) preparing polysaccharide-functionalized nanoparticles filled with growth factors by filling an effective amount of the growth factor selected from VEGF, FGF, and PDGF containing BMP or TGF-beta in the polysaccharide-functionalized nanoparticles prepared above; step; (c) biological materials for preparing at least one hydrogel carrier selected from poly (ethylene glycol), poloxamer, poly (organophosphazene), oligo (poly (ethylene glycol) fumarate), collagen, gelatin, fibrin, hyaluronic acid and alginate Dispersing the polysaccharide-functionalized nanoparticles filled with the growth factor prepared above in an aqueous solution of a compatible polymer; And (d) a crosslinking agent selected from glutaraldehyde, diepoxide and carbodiimide in the dispersion prepared in step (c); Crosslinking agents selected from thrombin, coagulation factor XIII, and mixtures thereof; A method of preparing a sustained-release growth factor transfer agent comprising crosslinking a biocompatible polymer for preparing a hydrogel gel carrier by providing at least one crosslinking factor selected from physical crosslinking factors selected from temperature, pH, and interaction. 12. The method of claim 11, wherein step (b) comprises (b ') redispersing the polysaccharide-functionalized nanoparticles in a dispersion medium and (b' ') adding the growth factor solution to the redispersion solution. Method for producing a sustained release growth factor delivery agent, characterized in that. The method according to claim 12, wherein in the step (a), the organic solution of step ① has a concentration of 0.5-2.0% (w / v); The aqueous solution of step ② has a concentration of 0.01-5% (w / v), and the polysaccharide is used in an amount of 10% by weight or less of the amount of the biocompatible polymer emulsifier; The organic solution of step ③ is used in less than 10% by volume of the aqueous solution; In the step (b '), the polysaccharide-functionalized nanoparticle redispersion has a concentration of at least 25% (w / v); The solution of the growth factor in step (b '') is prepared using one or more solvents selected from PBS, PB, Tris, Hepes buffer, characterized in that the concentration is determined in the range of 0.01-0.5% (w / v) Method for producing a sustained release growth factor delivery agent to. The biodegradable polymer of claim 13, wherein the biodegradable polymer is poly (D, L-lactide-co-glycolide), the biocompatible polymer emulsifier is poloxamer, the polysaccharide is heparin, and the growth factor is BMP-2. , BMP-4, BMP-6, BMP-7, BMP-8, BMP-9, at least one selected from BMP or TGF-beta, in addition to the growth factor selected from VEGF, FGF, PDGF, the hydrogel gel carrier Method for producing a sustained release growth factor delivery agent, characterized in that the fibrin. The method of claim 14, wherein the biodegradable polymer, the biocompatible polymer emulsifier and the polysaccharide have a weight average molecular weight of 5,000-100,000, 5,000-100,000 and 3,000-100,000, respectively; The polysaccharide and the growth factor include 2-100 μg and 0.01-5 μg, respectively, per mg of the nanoparticle; The nanoparticles have a diameter of 400 nm or less, a surface charge of +20 mV or more or -40 mV or less, and a polydispersity index of less than 0.1; The sustained release growth factor transfer agent is a method for producing a sustained release growth factor transfer agent, characterized in that it has an elastic modulus of 200-20,000 Pa. Preparing a sustained release growth factor delivery agent according to claim 11; And (e) molding the growth factor delivery agent with a mold to be suitable for a defect in a bone or alveolar bone formed due to one or more diseases selected from porosity, fracture, fracture dislocation, nonunion, delayed union, bone defect, and alveolar bone defect. Method for producing a sustained release bone filler comprising the step. In the method for preparing a bone filler according to claim 16, the step (e) comprises ① autologous bone, allogeneic bone, xenograft, ② HAP, tricalcium phosphate, calcium aluminate, β-TCP, CPC, calcium sulfate, bioglass , And ③ The method of producing a sustained release bone filler, characterized in that it comprises the step of adding one or more components selected from cell adhesion protein, degradable peptide linker. In the method of controlling the release rate of protein drugs, the method of controlling the release rate of protein drugs (a) ① dissolving a biodegradable polymer in an organic solvent having no cytotoxicity at low concentration to obtain an organic solution, ② dissolving a polysaccharide and a biocompatible polymer emulsifier in water to obtain an aqueous solution, and ③ the organic solution Preparing a polysaccharide-functionalized nanoparticle comprising adding and dispersing in an aqueous solution; (b) preparing polysaccharide-functionalized nanoparticles filled with protein drugs by filling the polysaccharide-functionalized nanoparticles prepared above with at least one protein drug selected from growth factor, chemokine, extracellular matrix protein and antithrombin III. step; (c) dispersing the polysaccharide-functionalized nanoparticles filled with the protein prepared above in an aqueous biocompatible polymer solution for preparing a hydrogel gel carrier; And (d) providing the at least one crosslinking agent selected from a crosslinking agent, a crosslinking agent, and a physical crosslinking factor to the dispersion prepared in step (c) to crosslink the biocompatible polymer for preparing a hydrogel carrier by nano In preparing the particle-protein-hydrogel complex, (A) (i) changing the concentration of the polysaccharide in the aqueous solution in step (a) ②, or (ii) changing the mixing ratio of the organic solution and the aqueous solution in step (a) ③, the nanoparticles Changing the content of polysaccharides per unit mass; (B) the content of the nanoparticles per unit mass of the composite is changed by changing the concentration ratio of the polysaccharide-functionalized nanoparticles and the biocompatible polymer for preparing the hydrogel gel carrier in the aqueous hydrogel polymer preparation for preparing the hydrogel gel carrier of step (c). How to change; or A method of controlling the release rate of protein drug by carrying out the combination of the method (A) and (B). In the method of controlling the release rate of protein drugs, the method of controlling the release rate of protein drugs (a) ① poly (D, L-lactide- co -glycolide), poly (lactic acid), poly (glycolic acid), poly (ε-caprolactone), poly (δ-valerolactone), poly (β Dissolving at least one biodegradable polymer selected from -hydroxybutyrate) or poly (β-hydroxyvalerate) in an organic solvent having low cytotoxicity at low concentrations to obtain an organic solution,     (I) at least one polysaccharide selected from heparin, alginate, hyaluronic acid, chitosan and (ii) at least one selected from polyethylene glycol ethers of poloxamer, poloxamine, poly (vinyl alcohol), or alkyl alcohols. Dissolving the biocompatible polymer emulsifier in water to obtain an aqueous solution, and     ③ preparing a polysaccharide-functionalized nanoparticle comprising the step of adding the organic solution to the aqueous solution to disperse; (b) preparing polysaccharide-functionalized nanoparticles filled with growth factors by filling an effective amount of the growth factor selected from VEGF, FGF, and PDGF containing BMP or TGF-beta in the polysaccharide-functionalized nanoparticles prepared above; step; (c) biological materials for preparing at least one hydrogel carrier selected from poly (ethylene glycol), poloxamer, poly (organophosphazene), oligo (poly (ethylene glycol) fumarate), collagen, gelatin, fibrin, hyaluronic acid and alginate Dispersing the polysaccharide-functionalized nanoparticles filled with the growth factor prepared above in an aqueous solution of a compatible polymer; And (d) a crosslinking agent selected from glutaraldehyde, diepoxide and carbodiimide in the dispersion prepared in step (c); Crosslinking agents selected from thrombin, coagulation factor XIII, and mixtures thereof; To prepare a nanoparticle-protein-hydride gel complex by a preparation method comprising the step of crosslinking the biocompatible polymer for preparing a hydrogel carrier by providing at least one crosslinking factor selected from physical crosslinking factors selected from temperature, pH, and interaction. In (A) (i) changing the concentration of the polysaccharide in the aqueous solution in step (a) ②, or (ii) changing the mixing ratio of the organic solution and the aqueous solution in step (a) ③, the nanoparticles Changing the content of polysaccharides per unit mass; (B) in the biocompatible polymer aqueous solution for preparing a hydrogel gel carrier of step (c), by changing the concentration ratio of the polysaccharide-functionalized nanoparticles and the biocompatible polymer for producing a hydrogel gel carrier, the content of nanoparticles per unit mass of the complex is increased. How to change; or A method of controlling the release rate of protein drug by carrying out the combination of the method (A) and (B). The method of claim 19, wherein the protein drug is a growth factor selected from BMP, VEGF, bGFG, FGF, PDGF; Chemokines; Extracellular matrix protein; And at least one drug selected from antithrombin III.

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