WO2018209579A1 - Composite scaffold containing drug - Google Patents

Abstract

A composite scaffold containing a drug. The scaffold comprises a porous bone material and a drug release nanocarrier, wherein the drug release nanocarrier contains a bone growth-promoting drug, and is distributed in the porous bone material. The present invention enables a local sustained release of the drug to accelerate bone regeneration at the location of a bone defect.

Description

Drug-containing composite stent Technical field

The present invention relates to a drug-containing composite stent characterized in that a drug-releasing nanocarrier is placed in a porous ceramic aggregate to provide a sustained-release effect of a local drug to accelerate bone at a bone defect. Regeneration ability.

Background technique

Porous calcium biphosphate ceramics, clinically recognized biomedical materials, their porosity and local decomposition are also conducive to new bone ingrowth. It has also been pointed out that such porous biphasic calcium phosphate ceramic scaffolds are most effective for ingrowth of neovascularization and new bone tissue at a pore size of 80-160 μm or 500-1000 μm. However, for cases where non-union and large bone defects cannot heal completely, osteoinductive factors are still required to help promote bone growth to enhance bone healing.

及BMP7

之骨诱导作用生长因子产品。但使用上仍有一些限制，如：(1)无药物载体控制其释放，容易造成短期间浓度太高及药效不持久之虞；(2)蛋白质产品保存不易；(3)价格高昂；和(4)机械强度不足。Simvastatin (SIM), an inhibitor of hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase, is a clinically used hypolipidemic drug. In recent years, some in vivo and in vitro studies have indicated that simvastatin (SIM) has an effect of promoting osteogenesis. In vitro studies in vitro have found that simvastatin can promote the proliferation of osteoblasts and induce the ossification of human bone marrow stem cells. However, many studies have shown that the effect of simvastatin on systemic administration has no consistent effect on promoting osteogenesis, because oral simvastatin is mostly metabolized in the liver, and the amount of drug reaching the bones cannot be effectively promoted. Concentration. Commercially available medicine, such as BMP2

And BMP7

Osteoinductive growth factor products. However, there are still some restrictions on the use, such as: (1) no drug carrier to control its release, which is likely to cause too short a concentration and a long-lasting effect; (2) protein products are not easy to store; (3) high prices; (4) Insufficient mechanical strength.

Traditional artificial replacement bones have only bone conduction function, so most of them are used to treat fractures. Although there is bone healing effect, it often takes a long time or can not effectively achieve osseointegration, resulting in poor healing. Autologous bone grafting helps to heal, but the source of autologous bone is scarce and the bone is taken from the bone. Therefore, at present, in the treatment of patients with bone defects, in addition to the use of alternative bone materials, bone growth drugs are simultaneously administered to improve the bone repair effect, but the bone growth drug does not use the form of a drug carrier, so it will cause short-term drugs. The disadvantage of excessive concentration and prolonged drug efficacy. Therefore, another study has combined the use of a combination of a micron-sized powder carrier containing simvastatin (SIM) and an artificial bone material to a clinically slow release microparticle carrier containing simvastatin (eg, SIM/PLGA-HAp or Is SIM/rhBMP-2/PLGA-HAp), its particle size is about 117,000±25μm, which can control the release of simvastatin, so the drug slowly releases the microparticle carrier and applies it to the fracture after localization. Repair, but in clinical use, patients with bone defects must use artificial bones and slow release of particulate carriers for bone growth. According to the test, it is determined how much medicine is given depending on the area of the bone defect and the position of the placement. Currently, there is no such customized bone material, so before each operation, the artificial bone material must be placed for the estimated dose. This in turn involves how to prepare artificial bone material with such drug content, and also needs to accurately release an effective amount of the drug, which is extremely troublesome for clinicians in clinical practice.

Therefore, how to use the artificial bone material and the release carrier containing the drug for promoting bone growth to improve the convenience of the doctor in clinical use is a problem that the industry is currently trying to solve.

Summary of the invention

The invention overcomes the problem that the physician should clinically give a dose of a drug for promoting bone growth by combining a bioceramic bone graft material with a nanocarrier containing simvastatin to form a medicated medical material. The present invention uses polylactic acid glycolic acid-polyethylene glycol (PLGA-PEG) nanoparticles or microlipids to encapsulate oil-soluble bone growth promoting drugs (such as statin drugs), so that the drug properties are released daily. And is not biologically toxic. The drug-containing nanocarrier is further compounded with the artificial bone graft material to produce a drug-containing aggregate having bone conduction and osteoinduction. The present invention also demonstrates that simvastatin-containing medicinal aggregates can continuously release simvastatin and increase Alzine phosphatase (ALP) activity of D1 cells, which is representative of effective bone growth; In the animal experiment, the drug-containing bone material was implanted in the bone defect to effectively repair the defect of the bone, indicating that the drug-containing bone material has bone repairing ability. Therefore, the medicated aggregate produced by the present invention has the use of a local long-acting sustained-release drug, and because it can be adjusted to be most suitable for drug release concentration, it is advantageous for clinical use by a physician and accelerates the bone regeneration ability of a severely damaged bone site.

The articles "a" or "an" are used herein to describe the components and compositions of the invention. This terminology is merely for convenience of description and the basic idea of the invention. This description is to be construed as inclusive of the singular When used in conjunction with the word "comprising", the term "a" is understood to mean one or more than one.

The term "or" in this document means "and/or".

The present invention provides a drug-containing composite scaffold comprising a porous aggregate and a drug-releasing nanocarrier, wherein the drug-releasing nanocarrier comprises a drug for promoting bone growth and is distributed in the porous aggregate, Wherein the drug release nanocarrier is an amphiphilic carrier.

The porous aggregate of the present invention belongs to a biomedical material and has good biocompatibility. In one embodiment, the constituent material of the porous aggregate comprises hydroxyapatite (HAp), a β-tricalcium phosphate (β-TCP), and a hydroxyapatite- Hydroxypatite tricalcium phosphate (HATCP), alpha-tricalcium phosphate (α-TCP), bioactive glass ceramic, calcium monosulfate, bone cement Or a combination thereof. In a preferred embodiment, the porous aggregate is a porous aggregate comprising a dual phase phosphate. In another embodiment, the dual phase phosphate is monohydroxyapatite-tricalcium phosphate (HATCP). In a preferred embodiment, the biphasic phosphate is one Hydroxyapatite-β-tricalcium phosphate (HAp-β-TCP).

In another embodiment, the porous aggregate is a porous ceramic aggregate. In a preferred embodiment, the porous ceramic aggregate is a porous ceramic aggregate prepared by using a warm water gel as a template. In a more preferred embodiment, the method for preparing a porous ceramic aggregate prepared by using a temperature sensitive water gel as a template comprises the steps of: (a) synthesizing a nitrogen-isopropylacrylamide-methacrylic acid polymer ( Poly(N-isopropylacrylamide-co-methacrylic acid); (b) mixing hydroxyapatite or calcium phosphate with a dispersing agent, wherein the dispersing agent is polyacrylic acid (PAA), polymethacrylic acid Polymethacrylic acid (PMA) or polyvinyl-alcohol (PVA); (c) mixing the nitrogen-isopropyl acrylamide-methacrylic acid polymer of step (a) with water to obtain a colloidal solution; (d) mixing the colloidal solution of step (c) with the product of step (b) to obtain a mixture; (e) adding the polymer particles to the mixture of step (d) and stirring to obtain a slurry, wherein the polymer a polyethylene having a particle volume of 5% to 20% of the total volume of the mixture of step (d); (f) filling the slurry of step (e) into the template tank; and (g) the step (f) The template tank filled with the slurry is placed in a ceramic crucible and then sent to a high temperature furnace for sintering at a high temperature to obtain a porous ceramic. The preparation method of the porous ceramic aggregate prepared by using the temperature sensitive water gel as a template can be referred to the patent of No. 8940203 of the US Patent Publication No. No. I411595 of the Taiwan Patent Publication No. I411595 or Yin- The literature of Chih Fu (Preparation of porous bioceramics using reverse thermo-responsive hydrogels in combination with rhBMP-2carriers: In Vitro and In Vivo evaluation, Journal of the Mechanical Behavior of Biomedical Materials, 27: 64-76, 2013). The contents of the literature and the contents of the literature are incorporated in the present invention.

In one embodiment, the porous aggregate has a porosity of 15-85%. In a preferred embodiment, the porous aggregate has a porosity of 20-75%. In a more preferred embodiment, the porous aggregate has a porosity of 40-75%. In another embodiment, the porous aggregate has a pore size of from 5 to 1000 μm. In a preferred embodiment, the porous aggregate has a pore size of 50-800 μm. In a more preferred embodiment, the porous aggregate has a pore size of from 100 to 600 μm.

The porous aggregate of the present invention has the effect of bone conduction. As used herein, "bone conduction" refers to the growth of new bone that is implanted into a substrate (e.g., an aggregate) that is tolerated or strengthened on its surface or its pores, channels, or other internal voids. When a graft material or a graft matrix (such as an aggregate) can be used as a scaffold for new bone growth, it is considered to have "osteoconductivity." The porous aggregate of the present invention can be used as a skeleton to diffuse osteoblasts (osteoblasts) at the position of the main bone defect in repair and to generate new bone. Further, the porous aggregate of the present invention can be designed into a specific shape depending on the shape of the bone to be repaired or the desired shape of the specific bone defect. According to a specific embodiment, the porous aggregate is composed of a porous block. Suitable shapes for the porous aggregate include a sphere, a sheet, a cuboid, a wedge, a dome, a cylinder, or a combination thereof.

The drug-releasing nanocarrier comprising the drug for promoting bone growth of the present invention needs to be placed in the porous aggregate, that is, the drug-releasing nanocarrier containing the drug for promoting bone growth is distributed in the porous aggregate. Therefore, the drug-releasing nanocarrier promotes the bone growth drug by locally long-acting sustained release, and enhances the effect of the porous aggregate on bone healing. In this article "Promoting bone growth drugs" includes, but is not limited to, drugs having a repair process of bone tissue that accelerates or promotes fractures, osteotomy, bone lengthening surgery, and bone graft surgery. The period of bone tissue repair is shortened by the action of the drug for promoting bone growth, or the bone strength after repair is restored to the pre-fracture level of the bone defect. Therefore, the drug for promoting bone growth is a drug that promotes bone growth and maintains bone quality. As used herein, the term "promoting" refers to an increase in activity, response, or other biological parameter.

In a specific embodiment, the bone growth promoting drug has an osteoinductive effect. As used herein, the term "osteoinductive action" refers to the process of stimulating osteoprogenitor cells to differentiate into osteoblasts, which in turn begin to form new bone. A chemical or biological composition is said to have "osteoinductive" when it stimulates primitive, undifferentiated, and multifunctional cells to become osteoblastic cell lines. Therefore, the drug for promoting bone growth can induce or stimulate the production of osteoinductive growth factors. In another embodiment, the osteoinductive growth factor comprises, but is not limited to, bone morphogenetic protein, osteocalcin, osteogenin or alkaline phosphatase (ALP), and the like. . The osteoinductive growth factor can actively promote or induce the differentiation and growth of bone cells to effectively shorten the healing of bone defects.

Moreover, in another embodiment, the bone promoting growth promoting drug induces angiogenesis. In a preferred embodiment, the induced angiogenic line increases the production of an angiogenic protein. In a more preferred embodiment, the angiogenic protein comprises a von Willebrand factor (vWF).

In another embodiment, the bone growth promoting drug is an oil soluble drug that promotes bone growth. In a preferred embodiment, the oil soluble bone growth promoting drug comprises a statin drug. In a more preferred embodiment, the statin comprises simvastatin.

The drug releases a nanocarrier, that is, a drug carrier, which has the effects of reducing side effects, improving drug tolerance, improving the convenience of administration, and maintaining a long therapeutic duration. The ideal drug carrier must be biodegradable, small size, good stability, high loading capacity, prolonged circulation in the body, and substantial accumulation of lesions. As used herein, "amphoteric carrier" means a carrier composed of amphiphilic molecules. In one embodiment, the amphiphilic carrier is a liposome, a polymeric micelle or a dendrimer. Among them, the microlipid system is formed by amphiphilic molecules, and the liposome is a vesicle formed by a phospholipid bilayer membrane. The most representative one is lecithin and phosphatidylcholine. In addition, cholesterol is another important liposome. Composition. The polymer microcell is a nanosphere having a core-shell shape, and the polymer microcell is composed of an amphoteric polyester segment with a hydrophilic segment at one end. The other end is a lipophilic segment, and the lipophilic segment in the amphoteric polymer segment is combined in the aqueous phase by van der Waals to form a core hydrophobic region, which can serve as a storage tank for fat-soluble drugs, hydrophilic The sexual segments are located outside of the hydrophobic core to increase the structural stability of the polymer micelles in the aqueous phase; therefore, the polymers self-assemble to form nano-sized micelles. Therefore, through the uniqueness and size effect of nano-cell structure, nano-cells are used as drug carriers to control drug release, in the fields of drug delivery and gene therapy. Great potential for application. At present, a polymer microcapsule containing a drug is prepared by dissolving, trapping, or adhering to a substrate, or coating a drug with a substrate to form nanoparticles, nanospheres, nanocapsules, and the like.

In one embodiment, the drug release nanocarrier is a polymeric microcell. In a preferred embodiment, the polymeric microcells are composed of an amphoteric polymer. In a more preferred embodiment, the amphoteric polymer is selected from the group consisting of polyesters, polyanhydrides, and polyethers. The polyester, polyanhydride, and polyether according to the present invention are not particularly limited, and any of those having biocompatibility and biodegradability can be used in the present invention. In particular, biological materials that have been approved for use in human or animal body are preferred materials for the present invention because they have been tested by biosafety and the like.

The term "polyester" as used herein includes polycaprolactone (PCL), polyvalerolactone (PVL), polypropiolactone (PPL), polybutyrolactone (PBL), Poly(lactide-co-glycolide; PLGA), polylactic acid (PLA), polyglycolide (PGA), poly(isobutylcyanoacrylate) ; PIBCA), polyisophthalic acid (PIPA), poly-1,4-phenylene dipropionic acid (PPDA), poly(mandelic acid); PMDA ), poly(propylene fumarate; PPF), poly(ortho ester); POE), or a combination thereof.

The term "polyanhydride" as used herein includes poly(sebacic anhydride; PSA), poly-(bis(p-carboxyphenoxy)propane anhydride; PCPPA), Poly-carboxyphenoxypropane-co-sebacic acid (PCP), poly-carboxyphenoxypropane-co-sebacic acid (pPP) -SA)), poly-carboxyphenoxypropane-co-isophthalic acid (p(CPP-IPA)), copolymer of poly-fatty acid binary and sebacic acid (poly(fatty acid dimmer-co-sebacic acid); p(FAD-SA)), or a combination of the above.

The term "polyether" as used herein includes poly(ethylene glycol); PEG, poly(propylene glycol; PPG), poly(butylene glycol); PBG. Or a combination of the above.

In one embodiment, the amphoteric polymer is a polylactic acid glycolic acid (PLGA-PEG).

The term "carrier" as used in the present invention means a carrier substance which can carry an active substance such as a drug. In one embodiment, the drug release nanocarrier is a sustained release drug release nanocarrier. Therefore, the release of the nanocarrier by the drug controls the slow release of the drug to effectively promote bone growth. The term "slow release" or "slow release" as used in the present invention means that the active substance (e.g., drug) is gradually released at a steady rate for a certain period of time. The slow release or sustained release state allows the active substance to maintain a certain concentration in the body or blood of an individual (such as an animal, a human) for a period of time.

The invention also provides a method for treating a bone defect, comprising: providing a drug-containing composite stent with a bone a defective body, wherein the drug-containing composite stent is implanted into a site of the bone defect, wherein the drug-containing composite stent comprises a porous aggregate and a drug-releasing nanocarrier, wherein the drug releases the nanocarrier A drug for promoting bone growth is included and distributed in the porous aggregate.

The volume and weight of bones in the human body are the largest part. The most important function is to assist the body in exercising and supporting the body structure. Bone defects often occur in clinical treatment, mainly due to defects caused by severe fractures, defects caused by unhealed fractures, bone defects after osteomyelitis, postoperative defects of bone tumors, collapse of vertebral bodies, and reconstruction of artificial joints Acetabular defects and so on. At present, bone grafting is still a common treatment in clinical practice. Accordingly, the present invention is a bone graft for treating bone defects with a drug-containing composite stent.

In this context, the term "bone defect" includes a defect in any part of the bone that needs to be restored to the original bone, regardless of the cause of the defect, for example, caused by surgery, caused by a tumor, caused by an ulcer, caused by an implant, or fractured. All caused by bone defects or bone tissue defects. In one embodiment, the bone defect is a fracture, or a bone graft site or an implant site. The drug-containing composite stent of the present invention is applied to a site of a bone defect in a body, and is not particularly limited in the case of a patient suffering from bone tissue other than bone formed by ossification of the skull, such as a skull bone. For bones other than bones formed by ossification in the membrane, any part of the bone can be applied. It should be suitable for the head (eye, humerus, mandible) and body trunk (rib, hip, cervical, and thoracic vertebrae). , lumbar vertebrae, humerus, coccyx), upper limbs (scapula, clavicle, humerus, elbow, humerus, ulna, scaphoid, hookbone, metacarpal, phalanx), lower extremity (femoral, thigh, tibia, tibia, foot, tibia) , scaphoid, tibia). Further, the damage form of the bone tissue is not particularly limited, and for example, treatment of bone healing, fracture (bone fracture, crack, comminuted fracture, complicated fracture, etc.) cut by osteotomy or bone extension surgery.

The term "subject" as used herein refers to an animal. In a preferred embodiment, the system refers to a mammal. In a more preferred embodiment, the system refers to a human.

In one embodiment, the constituent material of the porous aggregate comprises hydroxyapatite (HAp), a β-tricalcium phosphate (β-TCP), and a hydroxyapatite- Hydroxypatite tricalcium phosphate (HATCP), alpha-tricalcium phosphate (α-TCP), bioactive glass ceramic, calcium monosulfate, bone cement Or a combination thereof. In a preferred embodiment, the porous aggregate is a porous aggregate comprising a dual phase phosphate. In another embodiment, the dual phase phosphate is monohydroxyapatite-tricalcium phosphate (HATCP). In a preferred embodiment, the dual phase phosphate is monohydroxyapatite-[beta]-tricalcium phosphate (HAp-[beta]-TCP).

In another embodiment, the porous aggregate is a porous ceramic aggregate. In a preferred embodiment, the porous ceramic aggregate is a porous ceramic aggregate prepared by using a warm water gel as a template.

In one embodiment, the porous aggregate has a porosity of 15-85%. In a preferred embodiment, the porous aggregate has a porosity of 20-75%. In a more preferred embodiment, the porous aggregate has a porosity of 40-75%. In another embodiment, the porous aggregate has a pore size of from 5 to 1000 μm. In a preferred embodiment, the porous aggregate has a pore size of 50-800 μm. In a more preferred embodiment, the porous aggregate has a pore size of from 100 to 600 μm.

In a specific embodiment, the bone growth promoting drug has an osteoinductive effect. Thus, the drug-containing composite scaffold of the present invention promotes the formation of mature bone of normal morphology at the site of the bone defect to be treated. Mature bone is any type of bone, whether cortical or trabecular, mineralized than the immature or cartilage bone of the new model. A morphologically normal bone is a histologically examined normal bone (ie, a bone composed of endochondral bone or membrane-like bone, including the medullary cavity with osteoblasts and osteoclasts). This is in contrast to the formation of osteophytes with fibrous stroma seen in the first stage of fracture repair. Therefore, the term "osteoinduction" can be understood not only as the acceleration of bone regeneration seen during fracture, but also as stimulating bone formation to its normal form. In a preferred embodiment, the bone growth promoting drug induces or stimulates the production of osteoinductive growth factors. In a more preferred embodiment, the osteoinductive growth factor comprises a bone morphogenetic protein, ostercalcin, osteogenin or alkaline phosphatase (ALP).

Moreover, in another embodiment, the bone promoting growth promoting drug induces angiogenesis. In a preferred embodiment, the induced angiogenic system increases the production of an angiogenic protein. In a more preferred embodiment, the angiogenic protein comprises a von Willebrand factor (vWF).

In one embodiment, the bone growth promoting drug is an oil soluble drug that promotes bone growth. In a preferred embodiment, the oil soluble bone growth promoting drug comprises a statin drug. In a more preferred embodiment, the statin comprises simvastatin.

In another embodiment, the drug release nanocarrier is a liposome, a polymeric micelle, or a dendrimer. In a preferred embodiment, the drug-releasing nanocarrier is a polymeric microcell. In a more preferred embodiment, the polymeric microcells are selected from the group consisting of polyesters, polyanhydrides, and polyethers.

In another embodiment, the polymeric microcells are comprised of an amphoteric polymer. In a preferred embodiment, the amphoteric polymer is a polylactic acid glycolic acid (PLGA-PEG).

Thus, the drug-containing composite stent of the present invention can form a controlled drug release implant at or near the location of a fracture, bone injury or bone defect. In one embodiment, the drug release nanocarrier is a sustained release drug release nanocarrier. The release of the nanocarrier by the drug controls the slow release of an effective dose of the drug for promoting bone growth to effectively and stably promote bone growth at the bone defect. As used herein, the term "effective dose" is a therapeutic dose that prevents, reduces, prevents, or reverses the development of a symptom of a body under certain conditions, or partially, completely relieves the individual's existence in a particular condition when it begins treatment. Symptoms.

According to the bone defect pattern of the New Zealand white rabbit of the present invention (φ: 3.5 to 3.6 mm, L: 10 mm), the bone defect is about For 0.384 cubic centimeters (cc), and 160 μl of SIM/PP (containing 10 μg simvastatin), the size of bone defects per cubic centimeter (cc) in the individual should be converted to 416.7 μl SIM/PP (containing 26 μg simvastatin) nano drug carrier. . Therefore, depending on the size of the bone defect of the individual, the effective dose of the drug for promoting bone growth per cubic centimeter (cc) of bone defect corresponding to the drug release nanocarrier is greater than 20 μg, preferably greater than 26 μg.

In the present invention, the effective amount of the drug for promoting bone growth released by the drug releasing nanocarrier exceeds 0.5 μM or more. In one embodiment, the drug releases the nanocarrier for a sustained release period of more than 10 days. The drug releases the nanocarrier for a sustained release period of more than 7 days. In a more preferred embodiment, the drug releases the nanocarrier for a sustained release period of more than 3 days. Further, the drug-releasing nanocarriers have an effective dose of the drug for promoting bone growth released on days 1 and 2 after administration of more than 0.5 μM.

The drug-containing composite stent provided by the present invention has the following advantages when compared with other prior art technologies:

In the treatment of bone defects, traditional artificial bones may not heal or encounter large bone defects and cannot completely heal. In this case, bone-inducing factors are needed to promote bone growth to fill the bone defects that cannot be healed. However, clinicians must rely on experience to determine how much bone-promoting drug is administered to the bone defect area. Therefore, clinical treatment may be inefficient. Therefore, the invention promotes the bone growth drug coated with the nano carrier, so that it can be directly integrated into and distributed in the artificial bone material; and the nano carrier slowly releases the characteristics of the drug for promoting bone growth, so the artificial bone material is implanted in the defect. After that, it can accurately release the therapeutic range of bone growth and control the release rate, improve the effect of bone defect treatment, achieve effective drug reduction, and do not need to additionally evaluate how much additional drug dose to be given by the clinician, just as before Artificial bone implants can be performed at the bone defect.

Therefore, the drug-containing composite stent of the present invention has the use of a local long-acting sustained-release drug, and is advantageous for clinical use because it can adjust the most suitable drug release concentration. Therefore, the drug-containing composite stent of the present invention not only has a function of promoting bone formation, but also has no disadvantage that proteinaceous drugs are difficult to store.

DRAWINGS

Figure 1 is a structural diagram of a polylactic acid glycol-polyethylene glycol nano drug carrier containing simvastatin. SIM: Simvastatin; and PP: polylactic acid glycolic acid-polyethylene glycol (PLGA-PEG).

Figure 2 is a structural diagram of the liposome nanoparticles encapsulating simvastatin. SIM: Simvastatin.

3A is a daily simvastatin release amount of a porous bioceramic containing a simvastatin-coated PLGA-PEG nanoparticle; and FIG. 3B is a porous organism containing a simvastatin-coated PLGA-PEG nanoparticle. Cumulative release rate of ceramic simvastatin;

80 μL Sim-PP/BC: Porous bio-ceramic containing 80 μL of simvastatin-coated PLGA-PEG nanoparticles Porcelain; 160 μL Sim-PP/BC: Porous bioceramic containing 160 μL of simvastatin-coated PLGA-PEG nanoparticles.

Figure 4 is a graph showing the effect of porous bioceramics containing simvastatin-coated PLGA-PEG nanoparticles on osteogenic formation of D1 cell lines. ALP: alkaline phosphatase. Control group: medium only; simvastatin group: 0.5 μM simvastatin (SIM) in the medium; and simvastatin release group: PLGA-PEG nanoparticles containing simvastatin on the first day of addition The simvastatin (SIM) release solution of the porous bioceramic is in the medium. The ALP activities on days 3 and 5 of each group were compared, **: p < 0.01; ***: p < 0.001.

Figure 5A shows the results of X-ray tracking of the defect pattern of New Zealand white rabbits; Figure 5B shows the results of H&E staining of the defect pattern of New Zealand white rabbits; Figure 5Bi shows the quantitative results of H&E staining of the defect pattern of New Zealand white rabbits, The H&E staining results in Figure 5Bi were quantified by imaging software to calculate the new bone formation; and Figure 5C is the result of immunohistochemical staining of vWF in the New Zealand white rabbit defect model, in which the color of vWF was Brown, 100X image is 10 times magnified in red frame in 10X image; BC group (3 in number): treated with bioceramics (BC); BC+80μL SIM/PP NPs (3 in number) : treatment with 80 μL of PLGA-PEG nanoparticles coated with simvastatin (5 μg of simvastatin); and BC + 160 μL of SIM/PP NPs (3 in number): containing 160 μL of coated sin Bioceramic treatment of statin PLGA-PEG nanoparticles (10 μg simvastatin). Scale bar: 10000 μm.

Figure 6A is an image of a Micro-CT trace of a rat skull defect. Fig. 6B is an analysis result of bone regeneration in a defect of a rat skull defect. Control group (quantity 3): no bioceramics; BC group (quantity 3): only bioceramics; BC+2.5 SIM-PP group (quantity 3): using 2.5 μg of coated simvastatin Bioceramics of PLGA-PEG (simvastatin/PLGA-PEG) nanoparticles; and BC+5SIM-PP group (quantity 3): PLGA-PEG (simvastatin/PLGA-PEG) nanometer containing 5 μg of coated simvastatin Bioceramics of particles.

7A is a daily simvastatin release amount of a porous ceramic containing simvastatin/Liposome nanoparticles; and FIG. 7B is a porous ceramic containing simvastatin-containing microlipid nanoparticles. Simultaneous cumulative release of simvastatin. Lipo 40 μL: Porous ceramic containing 40 μL of simvastatin/Liposome nanoparticles of simvastatin; Lipo 80 μL: Porous ceramic containing 80 μL of simvastatin/Liposome nanoparticles of simvastatin.

detailed description

The technical solutions of the present invention are further illustrated by the following specific examples, which are not intended to limit the scope of the present invention. Some non-essential modifications and adaptations made by others in accordance with the teachings of the present invention are still within the scope of the present invention.

1. Methods and materials

A. Preparation of simvastatin-containing polylactic acid glycolic acid-polyethylene glycol nano drug carrier

Poly(lactic-co-glycolic acid; PLGA) and polyethylene glycol (PEG) are polymerized at a ratio of 1:10 or 2:10 to form a linear polylactic acid glycolic acid. - Polyethylene glycol (PLGA-PEG; PP) copolymer, the chemical structure of the PP is as follows:

Me: methyl

The linear polylactic acid glycol-polyethylene glycol (PP) copolymer is coated with simvastatin (SIM) by oil-in-water (O/W) to form nanoparticles (NPs). Its structure is shown in Figure 1. The dialyzed simvastatin-coated PLGA-PEG nanoparticles (Simvastatin/PLGA-PEG nanoparticles; SIM/PPNPs) were further subjected to high performance liquid chromatography (HPLC) for entrapment efficiency (EE%). With the analysis of the loading content (LC%), the calculation formulas of EE% and LC% are shown in the following formulas (1) and (2):

PP: poly(lactic-co-glycolic acid-polyethylene glycol)

The conditions of the optimal simvastatin drug for polylactic acid glycol-polyethylene glycol (PP) were determined by the above formulas of EE% and LC%. The present invention takes the form of a fixed material weight (25 mg/batch) to take into consideration the conditions of the optimum or maximum coverage and load ratio. In the present invention, the drug-coated nanoparticles are dialyzed and purified by HPLC, and the coating ratio is 35%.

B. Preparation of porous two-phase bioceramic scaffold

B-1. Temperature sensitive colloid preparation

25 g of N-isopropylacrylamide and 200 μl of Methacrylic Acid were successively added to a 500 ml round bottom flask containing 125 ml of water to completely dissolve; and 0.25 g of the initiator was sequentially added. Ammonium persulfate (APS) and catalyst tetramethylethylenediamine (TEM, TEMED) were placed in a 500 ml round bottom flask and stirred for 24 hours. Dialysis to facilitate the removal of N-isopropyl acrylamide and methacrylic acid monomer to achieve a purification effect. The purified liquid is frozen to liquid nitrogen and sent to a freeze dryer, and the water is removed by sublimation to obtain a product of a temperature-sensitive colloid having a chemical formula of poly(N-isopropylacrylamide-co-Metharylic acid (p) (NIPAAM-co-MAA)), its structural formula is as follows:

B-2. Preparation of porous bioceramics

Mixing hydroxyap-atide (HAp) and β-tricalcium phosphate (β-TCP) mixed bioceramic powder with the above-mentioned warm water gel into a centrifugal mixer for porosity The mixing of bioceramic slurry (HAp/β-TCP/p(NIPAAM-co-MAA)slurry) to achieve uniform and stable process requirements, HAp/β-TCP phase structure ratio can be prepared according to requirements. In the range of 7/3, the porosity can be in the range of 20 to 75%, and the pore size is in the range of 5 to 500 μm. Further, the pressure-resistant sample of the porous bioceramic was made into a porous ingot-shaped scaffold material having a size of φ6 mm × h12 mm in accordance with ASTM F451-99a specification to have a compressive strength of > 5 MPa or more.

For the above steps, refer to the literature of Yin-Chih Fu (Preparation of porous bioceramics using reversethermo-responsive hydrogels in combination with rhBMP-2carriers: In Vitro and In Vivoevaluation, Journal of the Mechanical Behavior of Biomedical Materials, 27: 64-76, 2013). .

C. Drug release calculation of PLGA-PEG nanoparticles coated with simvastatin combined with porous ceramics

In the present invention, viranistat-coated PLGA-PEG nanoparticles (SIM/PP NPs) are dropped into a porous bioceramic at 20 μL each time and placed in a draft cabinet to be air-dried to prepare a medicated ceramic. Therefore, the experimental group of the present invention separately drops 80 and 160 μL of simvastatin-coated PLGA-PEG nanoparticles into a porous bioceramic, each of which obtains 5 medicated ceramics, and then puts 3 mL of phosphate buffering physiologically. In saline (PBS), release at 60 ° C for 24 hours at 37 ° C, each time 2 mL was taken and 2 mL of PBS was added, and after 7 days of continuous collection, simvastatin (SIM) was detected by high performance liquid chromatography (HPLC). The amount released.

D. New Zealand white rabbit tibia defect model test

In the New Zealand white rabbit tibia defect test, the present invention was divided into three experimental groups: (1) BC group: treated with bioceramics (BC); (2) BC + 80 μL SIM/PP NPs group: containing 80 μL Bioceramic treatment of simvastatin-coated PLGA-PEG (simvastatin/PLGA-PEG) nanoparticles; and (3) BC+160 μL SIM/PPNPs group: PLGA-PEG containing 160 μL of simvastatin coated ( Simvastatin/PLGA-PEG) Bioceramic treatment of nanoparticles. Each group was tested with 3 New Zealand white rabbits. The New Zealand white rabbit's right forelimb tibia was cut off by 1 cm with a drill bit, treated with a bioceramic placement defect, followed by X-rays for 10 weeks every 2 weeks. After sacrifice, the tibia was removed for decalcification, paraffin embedding and tissue sectioning. H&E staining and immunohistochemical staining were used to detect the new bone and angiogenesis factor vWF (Von Willebrand factor). In addition, the new bone formation rate is calculated from the new bone (red stained portion)/deficient area in the defect area.

E. Rat skull bone defect model test

(150mg/kg体重)进行腹腔注射以进行全身麻醉。以牙科电动钻进行外科手术，于大鼠的头盖骨产生直径5mm的严重骨缺损(critical size defect)。再以含辛伐他汀(SIM)之纳米药物载体复合多孔性生物陶瓷支架置入大鼠头盖骨的骨缺损处后，进行缝合。本发明将大鼠分为四组(每组各3只)，分别是(1)控制组：不用任何生物陶瓷；(2)BC组：仅使用生物陶瓷；(3)BC+2.5SIM-PP组：使用含有2.5μg的包覆辛伐他汀的PLGA-PEG(simvastatin/PLGA-PEG)纳米粒子的生物陶瓷；以及(4)BC+5SIM-PP：使用含有5μg的包覆辛伐他汀的PLGA-PEG(simvastatin/PLGA-PEG)纳米粒子的生物陶瓷。大鼠头骨缝合后，每4周以微计算器断层扫描技术(micro computed tomography；Micro-CT)进行追踪持续8周，并分别于植入生物陶瓷后第0、4、8周将动物进行安乐死，将头盖骨取出，进行脱钙、包埋和切片以及H&E组织染色以评估骨质新生能力。The severe bone defect pattern of the rat skull is based on SD rats (Sprague-Dawley rats; nine weeks old)

(150 mg/kg body weight) was intraperitoneally injected for general anesthesia. Surgical operation with a dental electric drill produced a critical size defect of 5 mm in diameter in the skull of the rat. The nano-drug carrier composite porous bioceramic scaffold containing simvastatin (SIM) was placed in the bone defect of the rat skull and sutured. The invention divides the rats into four groups (3 in each group), which are (1) control group: no bioceramics are used; (2) BC group: only bioceramics; (3) BC+2.5 SIM-PP Group: bioceramics containing 2.5 μg of PLGA-PEG (simvastatin/PLGA-PEG) nanoparticles coated with simvastatin; and (4) BC+5SIM-PP: using PLGA containing 5 μg of coated simvastatin Bioceramics of PEG (simvastatin/PLGA-PEG) nanoparticles. After the rat skull was sutured, it was tracked by micro computed tomography (Micro-CT) every 4 weeks for 8 weeks, and the animals were euthanized at 0, 4, and 8 weeks after implantation of bioceramics. The skull was removed for decalcification, embedding and sectioning and H&E tissue staining to assess bone regeneration.

F. Microlipid nanoparticles coated with simvastatin

Simvastatin was coated with dipalmitoyl-phosphatidyl-choline (DPPC) to prepare liposome (Liposome; Lipo) nanoparticles, and its structure is shown in Fig. 2. 10 mg of DPPC and 1 mg of simvastatin were placed in a reaction flask, dissolved in chloroform, and vacuum-dried. After adding pure water, the mixture was shaken back by mechanical force, and finally the particle size was adjusted to about 200 nm by the granulator. The yield of 87-93% gives simvastatin/Liposome nanoparticles containing simvastatin.

G. Drug release calculation of microlipid nanoparticles coated with simvastatin combined with porous ceramics

The simvastatin/Liposome nanoparticles containing simvastatin were instilled into bioceramics at 40 μL (Lipo 40 μL) or 80 μL (Lipo 80 μL), placed in a wind exhaust cabinet, and air-dried to prepare a medicated ceramic. Three medicated ceramics containing 40 μL (Lipo 40 μL) and 80 μL (Lipo 80 μL) were placed in 2.5 mL PBS and released at 60 ° C for 24 hours at 37 ° C. Each time 2 mL was taken and 2 mL of PBS was added for 7 consecutive doses. After days, by HPLC The amount of simvastatin was measured.

2. Experimental results

A. Drug release rate of PLGA-PEG nanoparticles coated with simvastatin combined with porous ceramics

The experimental group was instilled with 80 and 160 μL of simvastatin-coated PLGA-PEG (simvastatin/PLGA-PEG) nanoparticles in bioceramics to form medicated ceramics, and the coverage ratio of PLGA-PEG nanoparticles was At 35%, it can be calculated that the amount of simvastatin encapsulated by 80 and 160 μL of bioceramics containing simvastatin-coated PLGA-PEG nanoparticles is 5 and 10 μg, respectively. Previous studies have indicated that the effective concentration of simvastatin in cytogenesis and rat animal experiments to promote osteogenesis is 0.5 μM. Therefore, the present invention found that 160 μL of simvastatin-coated PLGA-PEG nanoparticles were in the first three days. It can release simvastatin (greater than 0.5μM) to the effective concentration (as shown in Figure 3A), and 160μL of simvastatin-coated PLGA-PEG nanoparticles can release more than 80% on the 5th day ( As shown in Figure 3B).

In addition, in order to carry out in vitro experiments to evaluate the osteogenic effect of porous bioceramics containing simvastatin-coated PLGA-PEG nanoparticles on D1 cell lines, the experimental components were divided into three groups: (1) Control group: medium only (2) Simvastatin group: 0.5 μM simvastatin (SIM) was added to the medium; and (3) Simvastatin-release group: PLGA-PEG nanoparticles containing simvastatin coated on the first day were added. The simvastatin release solution of the porous bioceramic is in the medium. Three groups of medium were treated with D1 cell line, and the third and fifth day culture medium was collected to detect alkaline phosphatase (ALP) activity (osteogenesis effect). From the results of Figure 4, it was found that the ALP activity of the simvastatin-releasing group was significantly higher than that of the control group. This result shows that the medicated ceramic does have an osteogenic effect on the D1 cell line.

B. Therapeutic effect of New Zealand white rabbit tibia defect

In the treatment experiment of the New Zealand white rabbit tibia defect, the results of X-ray tracking (as shown in Figure 5A) and H&E staining (as shown in Figure 5Bi) can be found that nano-coated bioceramics can improve bone growth. The effect; and in the immunostaining results, the vWF of the high concentration nanoparticle ceramic (as shown in Fig. 5C, the color of the vWF is brown) is better than that of the non-medicated ceramic. It is confirmed by the above results that the medicated ceramics have the effect of repairing the bone defect, and it can be seen that the bone growth effect of the BC+160 μL SIM/PP NPs group is superior to the other two groups.

In addition, the H&E tissue staining results (Fig. 5Bi) of each group were quantified by image software (ImageJ) to calculate new bone formation (%). From the results of the new bone formation rate of Fig. 5Bii, it can be seen that the bone growth of the nano drug-coated ceramics significantly promotes the ceramics not coated with the nano drug.

C. Therapeutic effect of rat skull defect

The rats with less blood flow were simulated by the skullcap defect model. The three groups were evaluated by Micro-CT tracking and tissue staining for the assessment of bone regeneration after 0, 4 and 8 weeks after implantation. As shown by the results of 6B, the two groups of BC+2.5SIM-PP and BC+5SIM-PP did have the effect of promoting the repair of the skull defect.

D. Calculation of the release amount of simvastatin encapsulated by microlipid nanoparticles

From the results of Fig. 7A, it can be found that the Lipo 40μL group containing the drug-containing ceramics and the Lipo 80μL group can be released in the first 2 days. Effective concentration of simvastatin (SIM) (over 0.5 μM). However, as shown in FIG. 7B, the sustained release ability of the medicated ceramic Lipo 40 μL group and the Lipo 80 μL group was smaller than that of the bioceramics containing the simvastatin-coated PLGA-PEG nanoparticles.

Suitable descriptions of the invention may be implemented in elements or limitations not specifically disclosed herein. The terminology that has been used for description is not limiting. There is no difference in the expression and description of the use of these terms and any equivalents, but it is to be understood that the scope of the invention may be modified. Therefore, the present invention has been described with reference to the embodiments and other aspects, and the modifications and variations of the present invention are considered to be within the scope of the present invention.

Claims (10)

A drug-containing composite scaffold comprising a porous aggregate and a drug-releasing nanocarrier, wherein the drug-releasing nanocarrier comprises a drug for promoting bone growth and is distributed in the porous aggregate, wherein the drug The nanocarrier is released as an amphiphilic carrier. The drug-containing composite stent according to claim 1, wherein the porous aggregate is a porous ceramic aggregate. The drug-containing composite stent according to claim 1, wherein the porous aggregate comprises monohydroxyapatite, a beta-tricalcium phosphate, monohydroxyapatite-tricalcium phosphate, and a-tricalcium phosphate. , a bioactive glass ceramic, calcium monosulfate, a bone cement or a combination thereof. The drug-containing composite stent according to claim 1, wherein the porous aggregate has a porosity of 15 to 85%. The drug-containing composite stent according to claim 1, wherein the porous aggregate has a pore size of from 5 to 1000 μm. The drug-containing composite stent according to claim 1, wherein the bone growth promoting drug is an oil-soluble bone growth promoting drug. The drug-containing composite stent according to claim 6, wherein the oil-soluble promoting bone growth drug comprises a statin. The drug-containing composite scaffold of claim 7, wherein the statin comprises a simvastatin. The drug-containing composite stent according to claim 1, wherein the drug-releasing nanocarrier is a liposome, a polymer microcell or a dendrimer. The drug-containing composite scaffold according to claim 9, wherein the drug-releasing nanocarrier is a polymer microcell.

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