KR101468306B1 - An adhesive nanoparticle comprising catechol groups grafted onto the surface thereof and uses thereof - Google Patents

Abstract

The present invention relates to an adhesive nanoparticle comprising a catechol group; A method for producing the same; A composite in which the adhesive nanoparticles are adhered to a substrate; And a method of producing a complex capable of sustained controlled release of the drug.

Description

An adhesive nanoparticle comprising a catechol group bonded to a surface thereof and an adhesive nanoparticle comprising grafted onto the surface thereof and uses thereof,

The present invention relates to an adhesive nanoparticle comprising a catechol group; A method for producing the same; A composite in which the adhesive nanoparticles are adhered to a substrate; And a method of producing a complex capable of sustained controlled release of the drug.

In dental or orthopedic implants with bone-forming ability, various surface treatment approaches have been used to induce sustained growth factor release. Coating technology of PLGA (poly (D, L-lactide-co-glycolide)) and PDLLA (poly (D, L-lactide)) as biodegradable polymers is used to mount and release growth factors. The following problems exist when various growth factors including BMP-2, TGF-? And IGF-1 are loaded in a polymer coating. First, the coating process requires an organic solvent that can adversely affect protein stability by dip coating. Second, the solvent coating that drops the coating solution onto the surface of the implant and dries can not produce a uniform coating layer having a uniform thickness. Thus, it is difficult to obtain the planned controlled release of the growth factor. Third, the coating can be stripped from the implant surface due to the low interfacial adhesion between the polymer coating and the implant surface. Therefore, there is a need to develop new coating techniques capable of releasing the growth factors in a controlled manner while retaining stable and uniform growth factors.

Of the various delivery systems, nanoparticle-type delivery vehicles are ideal for medical devices because they can mount and release drugs or growth factors in a planned manner due to a clear and uniform nanostructure. Several nanoparticle systems have been reported for surface immobilization on medical devices. The release of low molecular weight drugs can not be used for growth factor delivery when controlled PLGA nanoparticles are attached to metal surfaces via ionic interactions. Ionic interactions can not ensure stable immobilization of nanoparticles on the surface of medical devices. Therefore, there is a demand for development of a nanoparticle-type adhesive carrier capable of safely supporting a high molecular weight growth factor and being stably immobilized on the surface of medical equipment.

The present inventors have succeeded in producing nanoparticles having excellent adhesion to various medical substrates by using various polymers capable of self-assembly by binding a catechol group to the surface of nanoparticles formed by self-assembly of a polymer copolymer, The present inventors have accomplished the present invention by confirming that a drug such as an ionic growth factor is loaded on the bound nanoparticles to continuously release a drug such as a growth factor. The present invention is based on this.

According to a first aspect of the present invention, there is provided a method for preparing a drug, comprising the steps of: preparing a third polymer comprising at least one drug-binding first polymer and at least one second polymer that is more hydrophobic than the first polymer; A second step in which the first polymer forms nanoparticles located in the interior of the second polymer through self-assembly of the third polymer; And a third step of binding a catechol group to the surface of the nanoparticles.

The second aspect of the present invention is a nanoparticle formed by self-assembly of at least one first polymer capable of binding to a drug and a third polymer comprising at least one second polymer having hydrophobicity more hydrophilic than the first polymer, Wherein the first polymer is nanoparticles formed by being positioned on the outside; And a catechol group introduced into the surface of the self-assembled nanoparticles.

A third aspect of the present invention relates to a substrate, comprising: a substrate; And at least one adherent nanoparticle of the second aspect adhered to the substrate via a catecholizing agent.

In a fourth aspect of the present invention, there is provided a method of producing a complex capable of continuously releasing controlled release of a drug, comprising: a) a step of adhering one or more adhesive nanoparticles of the second aspect to a substrate via a catecholizing agent; And a second step of binding the drug to the adhesive nanoparticle.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for preparing nanoparticles by self-assembling a third polymer comprising at least one drug-binding first polymer and at least one second polymer having hydrophobicity higher than that of the first polymer, It is characterized by combining.

At this time, the second polymer, which is hydrophobic in the nanoparticles, can be positioned inside the aqueous solution during self-assembly, and can serve as a support capable of positioning the first polymer capable of binding the drug to the outside. After the first polymer capable of binding to the drug by self-assembly is exposed to the outside, the catechol group is bonded to the surface of the nanoparticles, and the nanoparticles can be easily grafted onto the substrate surface of any shape and / And / or strongly bind the nanoparticles to the substrate, and then the drug can be easily loaded onto the drug-binding first polymer, which is located on the surface of the nanoparticle.

Therefore, the first polymer forming the nanoparticles may be any polymer capable of binding the drug, and the second polymer forming the nanoparticles may be any polymer if it is more hydrophobic than the first polymer.

In the nanoparticles of the present invention, the second polymer may be located within the nanoparticles due to hydrophobicity, and they may form one or more cores, and the first polymer connected to the second polymer may be hydrophilic, (Outside) of the particle can be formed. In this manner, the nanoparticles may be a kind of core-shell structure including at least one core made of a second polymer, and the remaining region and the surface of the nanoparticle are made of the first polymer.

When the interior of the nanoparticles forms multiple cores, the surface of the nanoparticles may preferably consist primarily of the first polymer. At this time, a part of the first polymer may be located together with the second polymer which is aggregated inside the nanoparticles. FIG. 2 is a structural schematic diagram of nanoparticles in which hyaluronic acid as a first polymer and a third polymer graft-polymerized with PLGA as a second polymer are self-assembled and PLGA forms multiple cores inside the nanoparticles.

On the other hand, in the third polymer, the at least one first polymer and the at least one second polymer may be connected directly or via a linker according to a conventional method.

The third polymer may be a graft copolymer or a block copolymer obtained by polymerizing the first polymer and the second polymer. The third polymer may be a graft copolymer in which the second polymer is linked to the first polymer main chain side chain. Furthermore, the third polymer may further include other monomers or polymers so long as the first polymer capable of binding the drug can be self-assembled so as to position the first polymer.

The second polymer may form a single core or multiple cores within the nanoparticles, and the first polymer surrounds the single core or multiple cores and may be located externally. In the case of nanoparticles having multiple cores, they may be formed by two or more third polymers or by one third polymer having two or more second polymers.

The first polymer preferably has a free carboxylate group or an amine group so that the ionic drug (e.g., a growth factor) can be easily loaded through electrostatic interaction. Particularly, a polysaccharide polymer capable of forming a low zeta potential by having a free carboxyl group is preferable. Non-limiting examples of the first polymer include hyaluronic acid, alginate, heparin, carrageenan, dextran sulfate, carboxymethylcellulose, chitosan and the like. Hyaluronic acid (HA) may be representative.

In the case of hyaluronic acid, alginate, heparin, carrageenan, dextran sulfate and carboxymethylcellulose, which are non-limiting examples of the first polymer of the present invention, anionic polysaccharides are cationic drugs positively charged at physiological pH For example, BMP-2 or IGF-1, via electrostatic interactions.

However, among the non-limiting examples of the first polymer of the present invention, chitosan is a cationic polysaccharide, which can be bound through an electrostatic interaction with an anionic drug which is negatively charged at physiological pH.

The second polymer is preferably a biodegradable polymer which is decomposed in vivo and decomposed into water, carbon dioxide, methane, etc., and non-limiting examples thereof include poly (lactide-co-glycolide) (PLGA), polyglycol Polylactic acid (PCA), lactic acid-caprolactone copolymer (PLCL), biodegradable polyurethane, biodegradable polycarbonate, have. The biodegradable polymer may be a natural or synthetic polymer.

Since the adhesive nanoparticles of the present invention can be applied to various medical implantable medical devices, biodegradable polymers are preferably used, and poly (lactide-co-glycolide) (PLGA) is particularly preferable.

Dopamine derived from a mussel adhesive protein having a catechol group (represented by the following formula (1)) may be used when the catechol unit (Formula 2) is introduced into the nanoparticles.

[Chemical Formula 1]

(2)

In a preferred embodiment of the present invention, the carboxyl group of the first polymer moiety located on the outer surface of the nanoparticle is activated and then reacted with the dopamine having the catechol moiety to connect the catechol moiety to the first polymer on the surface of the nanoparticle .

The carboxyl group activation can be achieved by adding EDC · HCl (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride), NHS (N-hydroxysuccinimide) or a combination thereof to the nanoparticles formed in the second step.

In a preferred embodiment of the present invention, the first polymer is hyaluronic acid and the second polymer is poly (lactide-co-glycolide) (PLGA), and the adhesive nanoparticles are catechol, hyaluronic acid, It may be an adhesive nanoparticle comprising a compound represented by the following formula (3) composed of PLGA as a basic structure.

(3)

In this formula,

n is an integer from 10 to 100,

m is an integer of 10 to 100,

이다.
R is a hydroxy group or

to be.

In the present invention, the substrate may be made of a material selected from the group consisting of plastic, glass, ceramic, metal and polymer. Preferably a medical implant, such as a bioimplant implant, membrane or stent. A dental implant applicable to a living body, an orthopedic implant, a vascular stent, and a non-vascular stent.

In the present invention, since the adhesive nanoparticles can be easily adhered to a medical substrate composed of various materials, the catechol groups located on the particle surface can be easily adhered to the medical substrate by a simple application or immersion of the adhesive nanoparticle composition. It is possible to easily carry out the surface treatment.

Since the nanoparticles can have hundreds of catechol groups on their surface, the binding strength of the nanoparticles to a metal surface such as Ti can be greatly enhanced through cooperative adhesion of the end catechol.

The nanoparticles of the present invention can be coated on the substrate. The nanoparticles may be coated on the substrate and then loaded on the nanoparticles. Alternatively, the drug-loaded nanoparticles may be coated on the substrate.

As described above, the nanoparticles of the present invention serve as a carrier for introducing a drug into a substrate, and can also serve as a carrier that continuously releases a drug in vivo after insertion into the living body.

The adhesive nanoparticles can be loaded with one or more drugs, such as growth factors, through an externally located first polymer. A plurality of drugs may be loaded on one nanoparticle, where the drugs may be the same or different. In addition, two or more nanoparticles in which different drugs are bound to each nanoparticle can be used in combination.

Non-limiting examples of drugs include, but are not limited to, bisphosphonates such as etidronate, clodronate, palmidronate, alendronate, ibandronate, risedronate, ), Zoledronate, tiludronate, [1-hydroxy-2- (imidazo [1,2-a] pyridin-3- yl) ethylidene] -bisphosphonic acid mono (1-hydroxy-2- (imidazo [1,2-a] pyridin-3-yl) ethylidene] -bisphosphonic acid monohydrate, YH529, icadronate, olpadronate, Neridronate and disodium-1-hydroxy-3- (1-pyrrolidinyl) -propylidene-1-hydroxy-3- (1-pyrrolidinyl) -propylidene-1 , 1-bisphosphonate, EB-1053). In addition to the above-mentioned bisphosphonate-based drugs, paclitaxel, docetaxel, sirolimus, dexamethasone and the like are also available. These drugs may be used alone or in combination of two or more.

In the present invention, a growth factor may be used as a drug. Non-limiting examples of growth factors include bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), platelet-derived growth factors (PDGF), transforming growth factor-beta (TGF-beta), insulin-like growth factors (IGFs), parathyroid hormone And combinations thereof.

In order for the drug-containing nanoparticles to function as an implant, a membrane or a stent for medical use, it is preferable to satisfy the following two preconditions.

First, medical devices such as implants or stents mainly use Ti, which preferably does not have a risk of being adhered to or losing the Ti surface. Second, the nanoparticles are preferably designed to simultaneously bind BMP-2 and IGF-1 which have positive isoelectric points of 8.5 and 8.6 and which are positively charged at physiological pH.

For the first requirement for strong adhesion, the present invention employs a catechol group of dopamine hydrochloride derived from mussel adhesive protein. These catechol compounds can act as strong binders to the coating metals and inorganic surfaces as they form strong covalent and non-covalent interactions with metal or metal oxide substrates.

For the second requirement, the present invention uses an anionic polysaccharide polymer (first polymer) such as hyaluronic acid. For this design, we used a Ti-bonded nanoparticle system that takes as a base structure a PLGA-based compound (Formula 3), a catechol-, anionic hyaluronic acid- and a biodegradable polymer. As a result of introducing the cationic growth factors BMP-2 and IGF-1 into the nanoparticle system, it was confirmed that these double growth factors were well mounted (Experimental Example 4).

Furthermore, it was confirmed that the system continuously released BMP-2 and IGF-1 for several days in a controlled manner (Experimental Example 5). Therefore, the nanoparticle system of the present invention can induce rapid osseointegration by maintaining the original appearance of these carriers on the Ti surface of fine grooves, and can promote osteogenesis and bioactivity in living organisms.

The adhesive nanoparticles of the present invention have excellent adhesion to the surface of the base material due to the presence of a catechol group on the surface thereof. The adhesive nanoparticles of the present invention can mount drugs such as ionic growth factors through the anionic first polymer and induce sustained controlled release of the drug And has an effect of promoting osteogenesis and bioactivity in an organism through the use of multiple growth factors.

1 is a ¹ H NMR spectrum for confirming the structure of a catechol-containing nanoparticle according to an embodiment of the present invention.
Figure 2 schematically shows the appearance of the adhesive nanoparticles of the catechol-hyaluronic acid-PLGA structure for loading / releasing of BMP-2 and IGF-1.
FIG. 3 shows the average diameter, zeta potential and shape of the adhesive nanoparticles according to an embodiment of the present invention.
Figure 4 is an Fe-SEM image showing the appearance of surface-immobilized nanoparticles on a Ti substrate.
Figure 5 is an XPS survey spectrum of unmodified Ti and nanoparticle-immobilized Ti surfaces.
Figure 6 shows the water contact angle on unmodified Ti and nanoparticle-immobilized Ti substrate.
Figure 7 shows the FITC-labeled BMP-2 and TRITC-labeled IGF-1 applied to the unmodified Ti substrate and the nanoparticle-immobilized Ti substrate, and the IX71 fluorescence microscope image of the FITC fluorescence and TRITC fluorescence thereof And a combination image thereof.
Figure 8 shows the in vitro controlled release profile of growth factors BMP-2 and IGF-1 by applying FITC-labeled BMP-2 and TRITC-labeled IGF-1 on nanoparticle-immobilized Ti substrate.

Hereinafter, the present invention will be described in more detail by way of examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example  One: PLGA ( Poly (D, L- lactide - co - glycolide )) And hyaluronic acid

0.2 g of desalted hyaluronic acid and 1.0 g of dmPEG (dimethyl ether of poly (ethyleneglycol)) were mixed and the mixture was lyophilized. The whitish hyaluronic acid / dmPEG complex mixture was added to a 12 ml solution of anhydrous dimethyl sulfoxide (DMSO), dissolved at 80 ° C for 2 hours, and cooled at room temperature. To activate the carboxyl group of hyaluronic acid, 206 mg (1 mmol) of DCC (dicyclohexylcarbodiimide) and 122 mg (1 mmol) of 4-dimethylaminopyridine (DMAP) were added to the mixed solution. After stirring for 2 hours, a 6 ml solution of DMSO in which 0.26 g of PLGA was dissolved was slowly added to the mixed solution to prepare a reaction mixture. The reaction mixture was stirred at room temperature for 2 days. The reaction mixture was then dialyzed against anhydrous DMSO for one day using a membrane (cut-off molecular weight (DMSO): 10,000 Da), separated for 3 days against distilled water, lyophilized, (HA-PLGA copolymer) (yield: 90%) was obtained.

Example  2: Kate Colegiga Combined HA - PLGA  Manufacture of nanoparticles

Catechol-hyaluronic acid-PLGA polymers and nanoparticles were synthesized through a bond between a free carboxylate group of HA-PLGA copolymer and a primary amine group of dopamine.

The HA-PLGA copolymer self-assembled nanoparticles were prepared by stirring an aqueous solution (pH 5.5) of the HA-PLGA copolymer prepared in Example 1 at room temperature for 12 hours. 9.6 mg (0.05 mmol) of EDC · HCl (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride) and 5.8 mg (0.05 mmol) of N-hydroxysuccinimide were added to activate the free carboxyl groups on the surfaces of the nanoparticles Was added to the solution in which the nanoparticles were formed. After 2 hours, 6.2 mg (0.033 mmol) of dopamine hydrochloride was mixed with the solution and stirred at room temperature for 12 hours. Then, the solution was separated using a membrane (MWCO: 10,000 Da) for 2 days and lyophilized to finally obtain HA-PLGA nanoparticles bound with catechol groups (yield: 95%).

Experimental Example  1: Identification of nanoparticle structure

The chemical structure of the nanoparticles prepared in Example 2 was analyzed by ¹ H NMR spectroscopy (400 MHz, Varian, USA). The nanoparticles were dissolved in 1 ml of heavy water (D ₂ O) and 1 ml of DMSO-d ₆ , and 10 mg of each sample for ¹ H NMR measurement was prepared. The ratio of catechol incorporated was calculated by UV-Visible spectrometry (Shimadzu, Japan). The nanoparticles (Catechol-HA-PLGA) were dissolved in DMSO at a concentration of 5 g / l and the spectrum was measured at 260-360 nm.

The results are shown in Fig.

1, it was confirmed that nanoparticles having the structure of catechol-hyaluronic acid-PLGA were synthesized. In addition, PLGA bound to hyaluronic acid is 4.69%, and the ratio of catechol groups bound is 5.51%.

Further, as shown in Fig. 2, nanoparticles form polymer nanoparticles in the form of multicore-shells with three distinct, distinct domains consisting of a substrate-adhesive catechol moiety, a hyaluronic acid shell and a PLGA multi-core.

Experimental Example  2: Identification of physical properties of nanoparticles

The average particle size and size distribution of the nanoparticles prepared in Example 2 were calculated through dynamic light scattering (BrookHAVEN Instruments Cooperation, New York, USA).

The vertically polarized He-Ne laser (632.8 nm) light was scattered and the resulting scattered light was analyzed at a 90 degree angle. The mean particle diameter (d) of the nanoparticles was calculated by the Stokes-Einstein equation and the polydispersity factor was calculated by the cumulant method. The zeta potential (ζ) of the nanoparticles was measured by a particle size analyzer (90 PLUS).

As a result, it was confirmed that the average particle size of the adhesive nanoparticles was 137.1 nm. Furthermore, it was confirmed that the zeta potential (ζ) was a negative value (-23.71) due to the anionic free carboxyl group of hyaluronic acid and the polydispersity index was 0.180 (FIG. 3). In particular, these anionic hyaluronic acid domains could be used as binding sites for BMP-2 and IGF-1 positively charged at physiological pH (isoelectric points of BMP-2 and IGF-1 are 8.5 and 8.6, respectively) It was expected.

Meanwhile, the nanoparticles prepared in Example 2 were observed through a transmission electron microscope (TEM. CM30, Philips, CA). An aqueous solution of 1 g / l of nanoparticles was prepared and the droplets were placed on a carbon-coated 200-mesh cooper grid. After 5 minutes, the excess amount of the aqueous solution was removed using a filter paper. TEM images (negatively stained with 2% uranyl acetate) were obtained under a voltage of 200 kV.

As a result of the investigation, it was confirmed that spherical uniform nanoparticles were formed (FIG. 3).

Example  3: Ti On the substrate  Immobilization

And etched for 5 minutes 2% hydrofluoric acid, it was prepared in spherical Ti base material having a surface area of 2 cm ² and washed with acetone, isopropyl alcohol and distilled water. The spherical Ti base was then dried under vacuum for 12 hours. 1 g / l, 2 g / l and 5 g / l of the nanoparticle aqueous solutions prepared in Example 2 were prepared and the Ti base material was immersed in the nanoparticle aqueous solution. Each of the Ti-based samples was immobilized on the surface of the Ti substrate through light stirring for 24 hours. After the immobilization step, each of the samples was washed three times with distilled water and lyophilized.

Experimental Example  3: Ti On the substrate  Immobilization investigation

The immobilization density of the nanoparticles immobilized on the surface of the Ti substrate of Example 3 and the surface morphology of the Ti substrate were analyzed through a scanning electron microscope (FE-SEM, S-4200, Hitachi, Tokyo, Japan).

As shown in FIG. 4, it was confirmed that the adhesive nanoparticles were stably fixed on the Ti surface through the field emission scanning electron microscope (Fe-SEM). And we could control the immobilization density by controlling the concentration of nanoparticles. Specifically, when the concentration of the nanoparticles was 2 g / l, the density was 1.13 × 10 ⁸ / cm ² , and when the concentration was 5 g / l, the density increased to 1.36 × 10 ⁸ / cm ² .

The immobilization of the nanoparticles on the Ti surface was also examined by X-ray photoelectron spectroscopy (XPS, ESCALAB MK II, V.G. Scientific Co., UK) and contact angle measurement (Surface Electro Optics, PHOENIX 150).

Figure 5 shows the XPS survey spectrum of unmodified Ti-based (Bare Ti) and nanoparticle-immobilized Ti-based. The original Ti surface consists essentially of Ti and O originating from the oxides of the original surface and contains a small amount of C derived from the hydrocarbon contaminants. Fixation of catechol-hyaluronic acid-PLGA nanoparticles was confirmed by a significant increase in the C mole ratio. As the concentration of nanoparticles used for immobilization increased, the Ti atomic ratio decreased, whereas the surface C atomic ratio increased from 37.72% to 48.82%. In addition, the presence of N almost unrecognized on the unmodified Ti substrate was observed, and the N atom ratio was increased in proportion to the nanoparticle concentration similarly to the increasing tendency of the C signal. In addition, high-resolution XPS spectral analysis showed a good agreement with the chemical structure of the immobilized nanoparticles.

As shown in Fig. 6, the contact angle change also confirmed the immobilization of the nanoparticles on the Ti substrate. Before nanoparticle immobilization, the static water contact angle of the Ti substrate was

68.47 占 4.87 占. After immobilization of nanoparticles (1 g / ℓ), the contact angle decreased by 57.61 ± 0.93 ° and further decreased to 22.87 ± 1.56 ° as the concentration of nanoparticles increased. This indicates that the surface hydrophilicity of the Ti substrate is increased due to the immobilized nanoparticles. An anionic hyaluronic acid shell is considered to be a major factor for the reduction of the contact angle.

Example  4: Double ( dual ) Mounting of Growth Factor

(50 ng of BMP-2, 50 ng of IGF-1 or 200 ng of BMP-2 or 200 ng of BMP-2 per ¹ unit of substrate area (cm ² )), which is a combination of BMP-2 and IGF- The Ti substrate immobilized with the nanoparticles of Example 3 was immersed in a saline solution (PBS, pH 7.4) solution for 24 hours with a slight stirring. It was then washed three times with distilled water and lyophilized to complete.

Experimental Example  4: mounted double growth factor ( BMP -2 and IGF -1) visual confirmation

In this experiment, whether or not BMP-2 and IGF-1 were double-loaded (or conjugated) with immobilized nanoparticles was investigated using green fluorescent protein FITC (BMP-2 label) and red fluorescent protein TRITC (BMP-2 label) Respectively.

The nanoparticles have an anionic hyaluronic acid (HA) shell, which is capable of binding positively charged BMP-2 and IGF-1 through electrostatic interactions. Green fluorescent protein (FITC-labeled BMP-2) was used to visualize BMP-2 binding to the nanoparticle phase and red fluorescent protein (TRITC-labeled IGF-1) was used to visualize IGF- Respectively. FITC aqueous solution (0.3 μg FITC dissolved in 300 μl sodium carbonate solution, pH 9.0) was mixed with BMP-2 solution (20 μg BMP-2 dissolved in 700 μl sodium carbonate solution) for 2 hours at room temperature. FITC-labeled BMP-2 was filtered (separated) using a membrane (MWCO: 3,500) for 24 hours and lyophilized. TRITC-labeled IGF-1 was prepared by synthesizing the same amount in the same manner as above. All of the above procedures proceeded in the dark room in order not to lose the fluorescence activity of FITC and TRITC.

The Ti substrate on which the nanoparticles of Example 3 were immobilized and the other (unmodified) Ti substrate were immersed in an aqueous solution of FITC-labeled BMP-2 and TRITC-labeled IGF-1 (100 ng of FITC-labeled BMP- 100 ng of TRITC-labeled IGF-1 was dissolved in 1 ml of phosphate buffered saline (PBS)) for 24 hours at a temperature of 4 ° C under mild agitation. Then, it was washed three times with distilled water and lyophilized. Fluorescence microscopy images were observed using the green and red filters of the IX71 fluorescence microscope (Olympus Optical Co., Tokyo, Japan).

Figure 7 shows that BMP-2 and IGF-1 are not effectively bound on the unmodified Ti surface. However, on the nanoparticle-immobilized surface, strong FITC fluorescence indicating the efficient loading of BMP-2 on the nanoparticles and strong TRITC fluorescence indicating the efficient loading of IGF-1 were observed, and furthermore, IGF-1 was found to be double-loaded well.

Experimental Example  5: Double growth factor Ti  Control emission from surface

In vitro release profiles of growth factors BMP-2 and IGF-1 were measured at pH 7.4 (nanoparticle concentration of 2 cm ² and 2 g / l of surface area) containing nanoparticle-Ti substrates containing fluorescent protein labeled growth factors Of phosphoric acid buffered saline.

The nanoparticle-immobilized Ti substrate prepared in Example 4 in which BMP-2 and IGF-1 were mounted was immersed in 3 ml of phosphate buffered saline and equilibrated by stirring at 150 rpm at 37 캜. The emission medium was completely replaced at the pre-determined time interval, and all samples were stored at -20 ° C during the experiment. The media supernatants for released BMP-2 and IGF-1 were analyzed according to conventional methods, using an ELISA diagnostic kit.

Figure 8 shows that BMP-2 and IGF-1 are released over 28 days without noticeable burst release. As a result, it was found that the amount of BMP-2 and IGF-1 released was controlled and continuously released.

Furthermore, it was predicted that even a single drug, for example, a nanoparticle-immobilized Ti substrate on which BMP-2 alone was loaded would exhibit the above-described release profile.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (19)

A first step of preparing a third polymer comprising at least one drug-binding first polymer and at least one second polymer that is hydrophobic more than the first polymer;
A second step in which the first polymer forms nanoparticles located in the interior of the second polymer through self-assembly of the third polymer; And
And a third step of binding a catechol group to the surface of the nanoparticles,
Wherein the first polymer is an anionic polysaccharide-based polymer, hyaluronic acid,
A method for producing adhesive nanoparticles.
2. The method of claim 1, wherein the second polymer in the second step forms a single core or multiple cores within the nanoparticles, wherein the first polymer surrounds the single core or multiple cores and is located externally.
2. The method of claim 1, wherein the catechol is attached to a first polymer located on an outer surface of the nanoparticle.
A nanoparticle formed by self-assembly of at least one first polymer capable of binding to a drug and at least one second polymer comprising at least one second polymer more hydrophobic than the first polymer, wherein the first polymer has a ; And a catechol group introduced into the surface of the self-assembled nanoparticles,
Wherein the first polymer is an anionic polysaccharide polymer, hyaluronic acid.
5. The adhesive nanoparticle of claim 4, wherein the second polymer forms a single core or multiple cores within the nanoparticles, and wherein the first polymer surrounds and is external to the single core or multiple cores.
5. The adhesive nanoparticle of claim 4, wherein the catechol group is incorporated into a first polymer located on an outer surface of the nanoparticle.
5. The adhesive nanoparticle of claim 4, wherein the first polymer is an ionic polymer having a charge so as to be ionically bondable with the drug.
delete 5. The method of claim 4, wherein the second polymer is selected from the group consisting of poly (lactide-co-glycolide) (PLGA), polyglycolic acid (PGA), polylactic acid (PLA), poly-epsilon -caprolactone (PCL) Wherein the biodegradable polymer is a biodegradable polymer selected from the group consisting of caprolactone copolymer (PLCL), biodegradable polyurethane, and biodegradable polycarbonate.
5. The adhesive nanoparticle according to claim 4, wherein the third polymer is a graft copolymer in which the second polymer is linked to the first polymer main chain side-by-side.
5. The adhesive nanoparticle of claim 4, wherein the first polymer is hyaluronic acid and the second polymer is PLGA.
5. The adhesive nanoparticle of claim 4, wherein the adhesive nanoparticles are loaded with a drug.
13. The method of claim 12, wherein the drug is selected from the group consisting of an osteogenic growth factor, a fibroblast growth factor, a platelet derived growth factor, a transforming growth factor-beta, an insulin-like growth factor, a parathyroid hormone, .
13. The adhesive nanoparticle of claim 12, wherein the drug is a combination of growth factors BMP-2 and IGF-1.
13. The method of claim 12, wherein the drug is selected from the group consisting of etidronate, claudronate, palmyrronate, alendronate, ibandronate, risedronate, zoledronate, tyluronate, [1- (Imidazo [1,2-a] pyridin-3-yl) ethylidene] -bisphosphonic acid monohydrate, icadronate, olefadronate, neridronate, disodium- 1-pyrrolidinyl) -propylidene-1,1-bisphosphonate, paclitaxel, docetaxel, sylolimus, dexamethasone and combinations thereof.
materials; And at least one adhesive nanoparticle according to any one of claims 4 to 7 and 9 to 15 adhered to the substrate via a catecholizing agent.
The composite according to claim 16, wherein the substrate is made of a material selected from the group consisting of plastic, glass, ceramic, metal and polymer.
17. The composite of claim 16, wherein the substrate is a bioimplant implant, membrane or stent.
A method of producing a complex capable of sustained controlled release of a drug,
A) a step of adhering one or more adhesive nanoparticles of any one of claims 4 to 7 and 9 to 11 to a substrate via a catechol group; And
And a step (b) of binding the drug to the adhesive nanoparticle.

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