KR101928161B1 - Drug carrier for photothermal and photodynamic therapy based on nanoassebly comprising gold nanocluster and fabrication method of the drug carrier - Google Patents

Abstract

According to the present invention, there is provided a drug delivery system for treatment of photothermal and photodynamic therapy based on amphiphilic polymer nanoclusters containing gold nanoclusters and a method for preparing the same, wherein the drug delivery system of the present invention comprises a hydrophilic polymer containing a tertiary amine, And a gold nanocluster disposed on the surface of the nanocomposite, wherein the drug is loaded onto the nanocomposite.

Description

Technical Field [0001] The present invention relates to a drug carrier for photothermal and photodynamic therapy based on amphiphilic polymer nanoclusters containing gold nanoclusters and a method for manufacturing the drug carrier, and a method for manufacturing the drug carrier.

The present invention relates to drug delivery vehicles, and more particularly, to amphiphilic polymer nanoparticle-based tumor targeting drug delivery vehicles and methods of making the same.

Inorganic / inorganic hybrid nanomaterials are attracting much attention in the biomedical field because they have both physical and chemical stability and optical properties of inorganic materials, biodegradability of organic materials, and flexible molecular design features. Among the various hybrid nanomaterials, gold-based hybrid nanomaterials have demonstrated their potential as nanomaterials platforms due to their unique low toxicity and high biocompatibility. In general, gold-based hybrid nanomaterials have been widely used as internal materials in various types of gold nanoparticles having a size of 50 to 200 nm, and can be used as surface materials, peptides, antibodies, therapeutic agents, It is designed to selectively accumulate or maximize the therapeutic effect through the modification.

However, gold nanoparticles themselves have a range of surface plasmon resonance in the range of 500-550 nm in the visible light region, which causes overlapping of the absorption range of hemoglobin in the in vivo tissue, There is a problem that there are many limitations as to the material of the electrode.

KR 101618556 B1 KR 101717966 B1

It is an object of the present invention to provide an amphiphilic polymer nanocomposite.

Another object of the present invention is to provide an amphipathic polymer nanocomposite-based drug delivery system capable of activating photo-thermal and photodynamic functions by light.

Still another object of the present invention is to provide a method for producing the drug delivery vehicle.

An amphiphilic polymer nanoassembly for one purpose of the present invention comprises a hydrophilic polymer comprising a tertiary amine and a hydrophobic material combined with the hydrophilic polymer.

In one embodiment, the hydrophilic polymer is selected from the group consisting of dextran, chitosan, glycol chitosan, hyaluronic acid, poly-L-lysine, polyaspartic acid poly-aspartic acid, and a derivative thereof.

In one embodiment, the hydrophobic material is selected from the group consisting of deoxycholic acid, taurodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid, taurokenodoxycholic acid, may include at least one of stearic acid, taurodeoxycholic acid, stearic acid, oleic acid, polycaprolactone, polypyridyl disulfide methacrylate, and derivatives thereof. have.

The drug delivery vehicle for another purpose of the present invention includes any one of the above-described amphiphilic polymer nanocomposite and gold nanoclusters disposed on the surface of the nanocomposite, and the drug can be loaded on the nanocomposite.

In one embodiment, the amphiphilic polymer nanoparticle is an α-alkyne hyaluronic acid conjugated with 3- (dimethylamino) -1-propylamine (DAA) acid and a DAA-hyaluronic acid-polycaprolactam nano assembly comprising azide-terminated polycaprolactam (PCL-N ₃ ) polymerized with the alpha -alkyne hyaluronic acid.

In one embodiment, the drug may be a photosensitizer.

At this time, the photosensitizer may be a phorphyrins compound, a chlorins compound, a bacteriochlorins compound, a phthalocyanines compound, a naphthalocyanines compound, and a 5-aminolevulinic ester (5-aminoevuline esters) compound.

At this time, the photo sensation agent may be vertephorpin.

In one embodiment, the gold nanoclusters are separated from the nanoclusters at near infrared ray wavelengths, and the nanoclusters in which the gold nanoclusters are separated can release the loaded drug.

In one embodiment, the drug delivery vehicle may be for tumor therapy.

The method for preparing a drug delivery vehicle for another object of the present invention comprises the steps of reacting a hydrophilic polymer and a tertiary amine to prepare a tertiary amine-bonded hydrophilic polymer, reacting the tertiary amine-bonded hydrophilic polymer with a hydrophobic substance, Fabricating a nanocomposite; and reacting the nanocomposite with gold (III) chloride (HA (III) chloride, HAuCl ₄ ) to grow gold nanoclusters on the surface of the nanocomposite.

In one embodiment, the tertiary amine may be 3- (dimethylamino) -1-propylamine, the hydrophilic polymer may be alpha -alkyne hyaluronic acid, and the hydrophobic material may be azide-terminated polycaprolactam.

At this time, in the step of preparing the tertiary amine-bonded hydrophilic polymer,? -Alkyne hyaluronic acid and 3- (dimethylamino) -1-propylamine can be produced by reacting 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide Wherein the reaction is carried out in a solution containing 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride, EDC.HCl and 1-hydroxybenzotriazole (HOBt) 3- (dimethylamino) -1-propylamine may be amide bonded to form DAA-linked hyaluronic acid.

Wherein the DAA-bonded hyaluronic acid and the azide-terminated polycaprolactam are selected from the group consisting of copper (II) sulfate pentahydrate, CuSO ₄ .5H ₂ O ) And sodium ascorbate, and the DAA-conjugated hyaluronic acid and the azide-terminated polycaprolactam may be polymerized to form a DAA-hyaluronic acid-polycaprolactam nanoassembly.

In one embodiment, prior to the step of growing the gold nanoclusters, the method further comprises reacting the nanocomposite with a solution comprising a drug to load the drug into the nanocomposite, wherein the gold nanoclusters are grown Step, the gold nanoclusters can be grown on the drug-loaded nanoclusters.

At this time, the drug may be a photosensitizer.

At this time, the photosensitizer may include at least one of a porphyrin compound, a chlorine compound, a bacteriocin compound, a phthalocyanine compound, a naphthalocyanine compound, and a 5-aminolevulinic ester compound.

At this time, the photosensitizer may be vortexorphin.

In one embodiment, in the step of growing the gold nanoclusters, the gold (III) chloride can be reacted with from 0.05 mol to 0.5 mol of the tertiary amine of the nanoclusters.

According to the present invention, there is provided a drug delivery system for photothermal and photodynamic therapy based on amphiphilic polymer nanocomposite containing gold nanoclusters and a method for producing the same, which is an easy process using a click chemical reaction, It is possible to provide a drug carrier based on an amphiphilic polymer nanocomposite capable of activating photothermal and photodynamic functions. In addition, the drug delivery system of the present invention has excellent particle stability due to the growth of gold nanoclusters on its surface. Therefore, the drug delivery system of the present invention has a stable structure at the pH of a normal tissue in vivo, . On the other hand, in tumor lesions, the structure may collapse by near-infrared rays when irradiated from the outside with near-infrared rays. Meanwhile, since the drug delivery system of the present invention has a tumor targeting property, it can accumulate at a high concentration in a tumor lesion site, and the structure can be collapsed during laser irradiation, so that a tumor can be effectively treated by photothermal and photodynamic therapy in a tumor lesion site.

1 is a view for explaining a DAA-hyaluronic acid-polycaprolactam nanoassembly (HyNA) according to an embodiment of the present invention.
FIG. 2 is a view for explaining a DAA-hyaluronic acid-polycaprolactam nanoassembly according to an embodiment of the present invention.
3 is a view for explaining energy-dispersive X-ray photoelectron spectroscopy and UV-Vis spectrum of HyNA.
Figure 4 is a TEM image of HyNA and GNc-HyNA.
5 is a diagram for explaining optical characteristics of GNc-HyNA.
Fig. 6 is a diagram for explaining a change in the scattering intensity of Vp-GNc-HyNA and Vp-HyNA.
Fig. 7 is a view for explaining the photoactive property of Vp-GNc-HyNA.
Fig. 8 is a view for explaining the absorption and release profiles of ex vivo cells of GNc-HyNA and Vp-GNc-HyNA.
9 is a diagram showing in vivo NIRF imaging of GNc-HyNA.
FIG. 10 is a diagram for explaining the fusing (ex vivo) of Vp from GNc-HyNA.
11 is a view for explaining in vivo anticancer activity of Vp-GNc-HyNA of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. The present invention is capable of various modifications and various forms, and specific embodiments are illustrated in the drawings and described in detail in the text. It is to be understood, however, that the invention is not intended to be limited to the particular forms disclosed, but on the contrary, is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise. In the present application, the term " comprises " or " having ", etc. is intended to specify that there is a feature, step, operation, element, part or combination thereof described in the specification, , &Quot; an ", " an ", " an "

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in commonly used dictionaries are to be interpreted as having a meaning consistent with the contextual meaning of the related art and are to be interpreted as either ideal or overly formal in the sense of the present application Do not.

The amphiphilic polymer nanoassembly of the present invention comprises a hydrophilic polymer containing a tertiary amine and a hydrophobic material combined with the hydrophilic polymer.

The hydrophilic polymer of the present invention may be selected from the group consisting of dextran, chitosan, glycol chitosan, hyaluronic acid, poly-L-lysine, polyaspartic acid poly-aspartic acid, and derivatives thereof. In the present invention, a hydrophilic polymer including a tertiary amine may refer to a hydrophilic polymer having a tertiary amine introduced into a hydrophilic polymer, and may also mean a tertiary amine moiety including a hydrophilic polymer itself have.

In addition, the hydrophobic polymer of the present invention may be selected from the group consisting of deoxycholic acid, taurodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid, taurodeoxycholic acid, and may include at least one of acid, stearic acid, oleic acid, polycaprolactone, polypyridyl disulfide methacrylate, and derivatives thereof. .

The drug delivery vehicle of the present invention comprises at least one of the amphiphilic polymer nanoclayers of the present invention and gold nanoclusters disposed on the surface of the nanoclay.

For example, the amphiphilic polymer nanocomposite of the present invention may be prepared by reacting an α-alkyne hyaluronic acid (α-alkyne (DAC)) conjugated with 3- (dimethylamino) -1-propylamine hyaluronic acid-polycaprolactam nanoassembly comprising hyaluronic acid and an azide-terminated polycaprolactam (PCL-N ₃ ) polymerized with the? -alkyne hyaluronic acid. have.

The drug delivery system of the present invention loads drugs into the nano assembly.

The drug may be a photoreactive drug, such as a photosensitizer. For example, the photosensitizer may be selected from the group consisting of a phorphyrins compound, a chlorins compound, a bacteriochlorins compound, a phthalocyanines compound, a naphthalocyanines compound, (5-aminoevuline esters) compound. At this time, the photosensitizer may be a verteporfin which is a hydrophobic photodynamic therapy drug which is a polymeric reducing agent.

The drug delivery system of the present invention is different from other supramolecular gold structures based on gold nanoparticle building blocks in which gold nanoclusters are grown on an amphiphilic polymer nanocomposite and placed on the surface of the nanocomposite. In this case, the gold nanoclusters react with the tertiary amine moiety to grow and the tertiary amine moieties are introduced into the outer layer of the hydrophilic polymer of the nanocomposite. Thus, the gold nanoclusters selectively form a shell Shape, i.e., a gold-shell.

Such a gold-shell form may serve as a diffusion barrier to delay the early release of drug loaded in the blood stream. In addition, the fluorescence and photodynamic properties of the drug delivery vehicle of the present invention can be extinguished due to gold nanoclusters covering the surface of the nanoclay. That is, the gold-shell formed of gold nanoclusters can serve as an optical barrier for protecting photoreactive drugs from external light. That is, the gold-shell can inhibit the early release of the drug under physiological conditions and can inhibit singlet oxygen, and therefore can be used to treat skin, eyes, And may reduce the potential toxicity of the drug to superficial tissues.

In addition, the drug delivery system of the present invention is a nanostructure composed of a hydrophobic material core and a hydrophilic polymer corona. When the hydrophobic drug molecule is loaded, the drug can be encapsulated into a hydrophobic core. Therefore, the drug delivery system of the present invention can have a double protection effect not only by the inorganic gold-shell but also by the amphiphilic polymeric self-assembly, thereby stably protecting the drug carried on the drug delivery system of the present invention, .

In addition, for example, when the drug delivery vehicle of the present invention is a DAA-hyaluronic acid-polycaprolactam nano-assembly, the drug delivery system of the present invention is a hyaluronic acid-based drug delivery system, Lt; RTI ID = 0.0 > CD44 < / RTI > to target tumor cells. That is, the hyaluronic acid of the drug delivery vehicle of the present invention can act as an active targeting ligand that allows CD44-mediated tumor-targeting and permeation into tumor cells. In other words, the drug delivery system of the present invention may be a drug delivery vehicle for treatment of tumors having a target for tumor cells, and may accumulate at a high concentration by penetrating into tumor cells.

The drug delivery system of the present invention can be photoactivated upon light irradiation to deliver drugs and treat lesions. At this time, the light irradiated to the drug delivery system of the present invention may be near-infrared rays and light in a wavelength range in which the drug can be optically activated. Specifically, the drug delivery system of the present invention can perform photothermal treatment by near infrared rays, and can control the activity of a photoreactive drug such as a photosensitizer, which is present inside the drug delivery system, by near infrared rays. More specifically, at near infrared wavelengths, gold nanoclusters on the surface of the nanoclay of the present invention can be photothermally activated by surface plasmon resonance, thereby increasing temperature, . That is, the drug delivery system of the present invention can exhibit photothermal therapy upon examination of near-infrared wavelengths. On the other hand, a gold nanocluster-separated nano-assembly can release a drug, and the temperature elevated by the photothermal effect can accelerate drug release. In addition, the drug can generate singlet oxygen according to photodynamic therapy (PDT) laser irradiation. Therefore, the drug delivery system of the present invention can be used for irradiation with near-infrared (light heat) Thus, the photodynamic effect can be activated without delay.

That is, in other words, the drug delivery system of the present invention is stable in the bloodstream by gold nanoclusters, can not only be targeted to tumor cells, but also accumulates at a high concentration in tumor cells. Also, it is not activated by visible light, and gold nanoclusters can exhibit photothermal therapeutic effects at near-infrared wavelengths and can be separated from nano-assemblies. In addition, the drug can be released from the nanoclusters in which the gold nanoclusters are separated, and when irradiated with light that photoactivates the drug, the drug can be activated to treat the tumor photodynamically. In addition, the drug delivery system of the present invention can treat tumors in both in vitro and in vivo.

For example, when the drug delivery vehicle of the present invention is a DAA-hyaluronic acid-polycaprolactam nanoassembly, the fluorescence and photodynamic effects of the drug delivery system of the present invention may be enhanced by certain cellular enzymes, particularly hyaluronidases and With glutathione, tumor cells can be treated faster. In particular, photothermically boosted drug release can be rapidly degraded by hyaluronic acid enzymes known to be over-expressed in various types of tumor cells such as hyaluronidases and glutathione, Can be increased.

The method for preparing a drug delivery vehicle of the present invention comprises the steps of reacting a hydrophilic polymer and a tertiary amine to prepare a tertiary amine-bonded hydrophilic polymer; Reacting a tertiary amine-bonded hydrophilic polymer with a hydrophobic substance to prepare an amphiphilic polymer nanocomposite; And reacting the nanocomposite with gold (III) chloride (HAuCl ₄ ) to grow gold nanoclusters on the surface of the nanocomposite.

Specifically, the method for producing a drug delivery system of the present invention is characterized in that the tertiary amine is 3- (dimethylamino) -1-propylamine, the hydrophilic polymer is alpha -alkyne hyaluronic acid, and the hydrophobic material is azide-terminated polycaprolactam The present invention firstly relates to a method for producing a drug delivery system of the present invention by reacting? -Alkyne hyaluronic acid and 3- (dimethylamino) -1-propylamine to obtain DAA-conjugated hyaluronic acid .

α-Alkyne hyaluronic acid and 3- (dimethylamino) -1-propylamine can be obtained by reacting 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloric acid, EDC · HCl) and 1-hydroxybenzotriazole (HOBt), wherein α-alkyne hyaluronic acid and 3- (dimethylamino) -1-propylamine Can be amide bonded.

The DAA-conjugated hyaluronic acid is then reacted with an azide-terminated polycaprolactam to produce a DAA-hyaluronic acid-polycaprolactam nanoassembly.

Alkylated hyaluronic acid and azide-modified polycaprolactam can undergo click chemistry to form nano-assemblies. Specifically, the DAA-conjugated hyaluronic acid and the azide-terminated polycaprolactam include copper (II) sulfate pentahydrate, CuSO ₄ .5H ₂ O, and sodium ascorbate. , And DAA-bonded hyaluronic acid and azide-terminated polycaprolactam can be polymerized.

Next, gold nanoclusters are grown on the surface of the nano assembly by reacting the nanocomposite with gold (III) chloride (HA (III) chloride, HAuCl ₄ ) to prepare the drug delivery system of the present invention.

At this time, the nanocomposite includes a DAA-bonded hyaluronic acid corona in which 3- (dimethylamino) -1-propylamine, which is a tertiary amine, is amide-bonded with alkaline hyaluronic acid so that gold can be grown, and hyaluronic acid Gold can grow along the main chain. Also, as described above, the gold nanoclusters can increase the particle stability of the drug carrier in vivo, and can realize an effect of photodynamic treatment due to the photo-thermal effect by near-infrared rays and the collapse of the structure.

In other words, the drug delivery system of the present invention can grow gold nanoclusters in a hydrophilic polymer-based amphiphilic polymer nanocomposite containing a hydrophobic substance to minimize the excellent particle stability in the body and the release of the encapsulated photoreactive drug, The gold nanoclusters are disintegrated from the nanocomposite by the near-infrared rays introduced in the nanocomposite, and the photoreactive drug contained in the nanoclusters can be selectively released.

In addition, when the hydrophilic polymer of the present invention is hyaluronic acid, for example, the hydrophilic polymer hyaluronic acid constituting the nanoconjugate accumulates in the tumor tissue due to its excellent tumor-like property, and the nano-assembly collapses due to the near- Reactive drugs may be released.

In other words, the gold nanoclusters grown on the nanocomposite can improve the stability of the drug delivery system of the present invention and can be used as a therapeutic agent capable of selectively treating the tumor tissue with light / photodynamic therapy by near infrared rays.

According to the present invention, nanoparticles can be prepared in an aqueous solution in the manufacturing process, and the reaction can be carried out at room temperature, and gold can be easily grown on the nanoconjugate without the use of an additional surfactant and the use of an organic solvent . The present invention also relates to a process for the preparation of gold nanoparticles comprising the steps of maintaining a high temperature reaction environment or using additional low molecular weight reducing agents such as sodium citrate or sodium borohydride, A hyaluronan-polycaprolactone (Hy-PCL) conjugate is used as the polymeric nano-conjugate, for example, a hyaluronic acid-polycaprolactone (Hy-PCL) conjugate.

That is, since the present invention does not use a surfactant or an organic solvent such as cetyl trimethylammonium bromide which can induce toxicity in vivo to grow gold nanoclusters, it can be stable against toxicity in the body , Supramolecular self-assemblies that extend the surface plasmon resonance range to the near-infrared region using amphiphilic polymer nanoparticles, without using additional low molecular weight reductants to maintain the reaction for the formation of gold nanoclusters, assemblies.

Hereinafter, the drug delivery system of the present invention, a method for producing the same, and a tumor treatment method using the same will be described in detail with reference to specific examples.

DAA-hyaluronic acid-polypolycaprolactam nanoassembly (HyNA)

In order to prepare the DAA-hyaluronic acid-polycaprolactam nanoassembly according to one embodiment of the present invention, first, α-alkyne hyaluronic acid (a-alkyne Hy) which is hyaluronic acid modified with an alkyne was prepared . 640 mg of sodium hyaluronate and propargylamine were dissolved in 0.1 M borate buffer solution of pH 8.5 containing 0.4 M chloride and then 330 mg of sodium cyanoborohydride (sodium cyanoborohydride) was slowly added dropwise and stirred at 50 ° C for 5 days. Thereafter, the reaction solution was dialyzed for 3 days to remove unreacted materials, and then freeze-dried to produce? -Alkyne hyaluronic acid.

Then, a-alkyne hyaluronic acid (200 mg, 0.527 mmol) was added to 50 mL of distilled water with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride carbodiimide hydrochloride (EDC.HCl) (163.94 mg, 1.056 mmol), 1-hydroxybenzotriazole (HOBt) (161.70 mg, 1.056 mmol) and 3- (dimethylamino) Amine (3- (dimethylamino) -1-propylamine) (DAA) (21.56 mg, 0.264 mmol). The pH of the solution was adjusted to 6.8 and stirred at ambient temperature for 24 hours. The resultant mixture was purified by dialysis with distilled water for 2 days and lyophilized to prepare DAA-linked? -Alkyne hyaluronic acid (hereinafter referred to as DAA-linked? -Alkyne Hy).

Then, the Hy- b -PCL copolymer combined DAA- was synthesized by Huisgen's 1,3- dipolar addition cyclization reaction (1,3-dipolar cycloaddition reaction). Alkyne DA (200 mg, 0.015 mmol) and azide-terminated polycaprolactam (PCL-N ₃ , MW = 5774 g / mol) (64 mg, 0.01 mmol) Dissolved in deionized water, and degassed with dimethylformamide (DMF). The solution was then treated with copper (II) sulfate pentahydrate (CuSO ₄ .5H ₂ O) (10.60 mg, 0.042 mmol) and sodium ascorbate (8.41 mg, 0.042 mmol) ≪ / RTI > The mixed solution was then stirred for 2 days at 45 DEG C and dialyzed with water to remove excess hyaluronic acid. Then, lyophilization was carried out to prepare DAA-hyaluronic acid-polycaprolactam nanoassembly (hereinafter referred to as HyNA) based on hyaluronic acid.

HyNA according to one embodiment of the present invention was dissolve in DMSO- D ₆ / D ₂ O and then synthesized using a 500 MHz nuclear magnetic resonance analyzer. The results are shown in Fig.

1 is a view for explaining a DAA-hyaluronic acid-polycaprolactam nanoassembly (HyNA) according to an embodiment of the present invention.

(A) of FIG. 1 is a diagram for explaining the HyNA synthesis reaction formula, wherein (A) of "A" represents an α-alkyne Hy, a (B) It represents a functionalized polycaprolactone (PCL-N ₃₎ and (D) combined DAA- Hy- b -PCL (HyNA). &Quot; B " in Fig. 1 is a diagram for explaining ¹ H-NMR of HyNA.

Referring to FIG. 1, it can be confirmed that both Hy and PCL exist in the ¹ H-NMR spectrum. Specifically, in the ¹ H-NMR spectrum, the characteristic peaks of the methyl and methylene groups of PCL at 0.8-1.1 ppm, the methyl group at the C2 position of N-acetylglucosamine of HA, and the methylene group of DAA at 2.2 ppm It can be confirmed that it exists. This means that an amphiphilic block copolymer was successfully synthesized by the coupling of azide-functionalized PCL with DAA-linked? -Alkyne Hy through the click chemistry according to the present invention. That is, it can be confirmed that the DAA-hyaluronic acid-polycaprolactam nano assembly was produced. Also, it can be confirmed that DAA, which is selected as a reducing moiety for the formation of gold nanoclusters, binds to the backbone of hyaluronic acid through amide bond formation.

The critical micelle concentration (CMC) of HyNA was then evaluated using a fluorescence microscope in the presence of pyrene molecules. Pyrene fluorescence spectra were obtained using a fluorescence spectrophotometer (FluoroMatye FS-2, SINCO Co., Ltd., Seoul, Korea). The results are shown in Fig.

FIG. 2 is a view for explaining a DAA-hyaluronic acid-polycaprolactam nanoassembly according to an embodiment of the present invention.

Referring to FIG. 2, it can be seen that Hy-PCL bonds self-assemble above a critical micelle concentration of 72 μg / ml, which is significantly lower than in low molecular weight detergents. That is, the degree of substitution (DS) defined by the number of DAA derivatives per 100 Hy repeating unit is 31%, and due to its amphiphilic nature, the DAA-modified Hy-PCL bond immediately forms a micellar self- assembly And Hy-PCL bonds can self-assemble above a critical micelle concentration of 72 μg / ml.

Gold nano-cluster grown nano-assembly (GNc-HyNA)

(III) chloride trihydrate (HAuCl ₄ .3H ₂ O) aqueous solutions (hereinafter referred to as gold (III) chloride trihydrate) in various molar ratios were prepared in order to form gold nanoclusters on HyNA, Solution). At this time, the supply molar ratio of HAuCl ₄ to the tertiary amine of HyNA was adjusted to the range of 0.05 to 0.5. 2 mg / mL gold stock solutions of various molar ratios were added to HyNA solution in deionized water, respectively. Thereafter, the reaction solution was stirred at room temperature and dark room for one day, and then centrifuged at 15,000 g for 30 minutes to remove unreacted gold. Then, freeze-drying was performed to prepare a nanoclay (hereinafter referred to as GNc-HyNA) in which gold nanoclusters were grown.

Energy-dispersive X-ray photoelectron spectroscopy of GNc-HyNA was performed using JEOL JEM-2100F equipped with an EDX Genesis Series γ-TEM at 200 kV. / Vis spectra were confirmed. The results are shown in Fig.

3 is a view for explaining energy-dispersive X-ray photoelectron spectroscopy and UV-Vis spectrum of HyNA.

FIG. 3A is a diagram for explaining energy-dispersive X-ray photoelectron spectroscopy of GNc-HyNA, and FIG. 3B is a diagram for explaining the UV / Vis spectrum of GNc-HyNA.

Referring to FIG. 3, the energy-dispersive X-ray photoelectron spectroscopy of GNc-HyNA reveals a distinct peak for gold. This implies successful anchoring of gold nanoclusters to HyNA. In addition, it can be confirmed that a remarkable peak for gold appears in HyNA even by UV / Vis spectroscopy. That is, according to the present invention, it can be confirmed that gold nanoclusters are bound to HyNA.

Also, the shapes of HyNA and GNc-HyNA were confirmed by HR-TEM (JEOL-2100F, Tokyo, Japan) operated at an acceleration voltage of 200 kV. For TEM images, HyNA and GNc-HyNA were treated with 0.5% uranyl acetate for negative staining, dispersed in deionized water (1 mg / mL), 200-mesh ) Was dropped on a copper grid. The results are shown in Fig.

Figure 4 is a TEM image of HyNA and GNc-HyNA.

4 (A) shows photographs of GNc-HyNA at various concentrations of Au-seed, and (B) TEM images of HyNA and GNc-HyNA prepared with Au-seeds at 0.2, 0.5 and 0.9 μM concentrations . The scale bar is 100 nm.

4, TEM images of HyNAs and GNc-HyNAs indicate that both HyNA and GNc-HyNA are spherical and that GNc-HyNA is more dense and non-uniform (Fig. 4) compared to gold- edges and a more compact shape. This means that gold nanoclusts have been stabilized with tertiary amines of DAA groups, and that the formation of gold nanoclusters in HyNAs can also modulate the physicochemical properties of GNc-HyNAs.

To be more specific, the size and surface zeta potential of HyNa and Gnc-HyNA were confirmed using a Zetasizer Nano ZS90 (Malvern Instruments, Worcestershire, UK) at 25 ° C. The results are shown in Table 1.

Sample Size (nm) ^a ξ ^b (mV) HyNA 204.05 ± 5.44 -26.4 ± 0.87 GNc-HyNA 0.2 μM 198.57 ± 1.44 -19.5 + 0.46 GNc-HyNA 0.5 μM 187.31 + - 2.93 -17.2 ± 0.31 GNc-HyNA 0.9 μM 150.27 ± 3.10 -16.8 ± 0.65

^a Measured by mechanical optical scattering in PBS

^b Measured by zeta potential analyzer

Referring to Table 1 above with reference to FIG. 4, HyNA and GNc-HyNA hydrodynamic diameters were measured at 204.0 ± 5.4 and 150.2 ± 3.1 nm, respectively, and the decrease in diameters after binding of gold nanoclusters to HyNAs Can be attributed to the physical crosslinking of the gold nanoclusters. That is, gold-bound HyNA can form dense nanostructures.

Therefore, it can be confirmed that HyNA having the gold nanoclusters grown according to the present invention has a dense structure and exhibits excellent particle stability.

The optical properties of GNc-HyNAs were then confirmed. The GNc-HyNA solution was exposed to the PTT laser at a voltage density of 1.5 W / cm ² and the temperature change was measured. The results are shown in Fig.

5 is a diagram for explaining optical characteristics of GNc-HyNA.

FIG. 5A is a view for explaining synthesis steps and light heat effects of GNc-HyNA according to 808 nm PTT laser irradiation, and FIG. 5B is a TEM image of GNc-HyNA after PTT laser irradiation. The scale bar is 100 nm. (C) shows photothermographic images of GNc-HyNA and HyNA under PTT laser irradiation, (D) shows the thermal heating curve of GNc-HyNA at different Au-seed concentrations under PTT laser irradiation, (0.9 [mu] M) at different concentrations (0.1, 0.5, and 1 mg / ml) under the irradiation of 690 nm PTT or 690 nm PDT laser.

Referring to FIG. 5, it can be seen that the optical properties of GNc-HyNA can be adjusted according to various concentrations of HAuCl ₄ added to HyNA. Specifically, when a 0.5 μM or lower amount of Au-seed is added to HyNA, the surface plasmon resonance peak is observed at about 550 nm, but when 0.9 μM or more of Au-seed is added, due to aggregation of Au nanoparticles It can be seen that a new broad peak concentrated at 780 nm appears with a range between 650 nm and 900 nm.

In addition, the photothermal effect of GNc-HyNA can be confirmed that GNc-HyNA added with 0.2 μM or 0.5 μM Au-seed exhibits poor light heat effect, and the temperature of the solution is higher than that of their surface plasmon resonance (SPR) Are found in the range of 450 nm to 600 nm, it can be seen that they slightly increased to 39.1 캜 or 33.9 캜, respectively. However, no significant absorbance was detected in the near infrared (NIR) region.

On the other hand, the temperature of the 0.9 μM solution of GNc-HyNA increased to 74.1 ° C when given its strong SPR peak in the near infrared (NIR) region. At a concentration of GNc-HyNA increased from 0.1 to 1 mg / mL, the temperature of the solution increased from 44 to 74 ° C after 10 minutes of PTT laser irradiation, whereas the temperature of pure water exposed to HyNA solution or PTT laser under PDT laser irradiation Could be neglected.

Therefore, it can be confirmed that HyNA and GNc-HyNA grown with gold nanoclusters according to the present invention can be used as photo-thermally activatable photochemistry, and it can be confirmed that broad red-shifted SPR spectra and remarkable temperature rise Indicating that GNc-HyNA with a 0.9-mu M Au-seed would be most suitable for NIR-based light-heat applications.

(Vp-HyNA) and a vortex-loaded nanoassembly (Vp-GNc-HyNA) grown with gold nanoclusters

In order to confirm the drug loading characteristics of HyNA of the present invention, a nanoassembly (Vp-HyNA) carrying Verteporfin (Vp) as a drug was prepared. 100 mg of HyNA was dissolved in 10 mL of DMF and 1: 1 co-solvent of deionized water, and then a solution of 11 mg of vertephrine dissolved in 1 mL of the co-solvent was slowly added dropwise. At the same time, ultrasonication was performed for 3 minutes and then dialysis was performed for 12 hours. Thereafter, the reaction mixture solution was filtered with a dialysis and filter, and freeze-dried to prepare a nano-assembly (hereinafter referred to as Vp-HyNA) containing black powder of verteoporphin.

Next, except for using Vp-HyNA, a substantially same method as that of the above-mentioned GNc-HyNA was performed to prepare a gold nanoparticle-grown berteloporpin-containing nanoassembly (hereinafter referred to as Vp-GNc-HyNA) .

Vp-HyNA and Vp-GNc-HyNA were dissolved in a 1: 1 (v / v) DMSO / DIW solution, respectively, and the Vp concentration was measured using a fluorescence spectrophotometer at 700 nm Fluorescence was measured and estimated. The results are shown in Table 2.

Sample Diameter (nm) ^a Vp supply amount ^b Loading efficiency (%) ^c Loading content (%) ^c Vp-HyNA 237.16 + - 2.46 10 90 9.0 Vp-GNc-HyNA 195.42 ± 1.73 10 87 8.7

^a Measured by DLS in PBS

The weight ratio of Vp to the ^b nano-conjugate

^c Measured by UV / Vis spectroscopy

Referring to Table 2, when 10 wt% of Vp is added, it can be confirmed that 90% and 87% of the added Vp are encapsulated into Vp-HyNA and Vp-GNc-HyNA, respectively.

In order to investigate the structural stability of Vp-GNc-HyNA, a dynamic light scattering analyzer (DLS) instrument was used with PBS containing 50% fetal bovine serum (FBS). 10 mg of Vp-GNc-HyNA was dispersed in 10 mL of PBS containing 50% fetal bovine serum at 37 ° C, and the scattering degree was varied and the results were confirmed with DLS over time. Vp-GNc-HyNA was dispersed using a trajectory stirrer, and the degree of scattering of light was measured at a specified time. In addition, the stability evaluation of Vp-HyNA was carried out under the above-described conditions. The relative changes in scattering intensity with time of Vp-GNc-HyNA and Vp-HyNA are shown in Fig.

Fig. 6 is a diagram for explaining a change in the scattering intensity of Vp-GNc-HyNA and Vp-HyNA.

Referring to FIG. 6, Vp-GNc-HyNA maintained 63% of the initial scattering intensity for 9 hours. However, the scattering intensity of Vp-GNc-HyNA decreased steeply to 26% of the initial stage. This means that the gold nanoclusters introduced into the hyaluronic acid nanocomposite effectively enhance the particle stability of the nanocomposite, indicating that Vp-GNc-HyNA can effectively maintain their structural strength in the physiological environment. That is, it can be confirmed that Vp-GNc-HyNA can firmly protect the drug molecules. This is one of the most important requirements for drug delivery systems to efficiently deliver drugs to their target sites under physiological conditions and therefore the structural stability of the Vp-GNc-HyNA of the present invention is sufficient for use as a drug delivery vehicle Can be confirmed.

Next, the photophysical and photochemical properties of Vp-GNc-HyNA were confirmed.

First, the fluorescence intensity of Vp-GNc-HyNA was confirmed by using a fluorescence spectrophotometer, and then the Vp release behavior from Vp-GNc-HyNA was evaluated using a dialysis method. Vp-GNc-HyNA was dissolved in PBS solution (1 mL, 1 g / L, pH 7.4) containing glutathione (GSH) and hyaluronidase (Hyal) and dialyzation membrane (MW blocking = 3,500) Were exposed to an 808-nm PTT laser (Changchun New Industries Optoelectronics Technology Co., Ltd., Changchun, China) and compared with Vp-GNc-HyNA not exposed as a control. The solution was shaken at a rate of 100 rpm at 37 ° C, and the solution released at a fixed time was transferred to a laboratory tube and replaced with a fresh solution. The amount of released Vp was quantified by measuring the Vp absorbance at 690 nm. The absorbance standard curve was prepared by dissolving free Vp in dimethylsulfoxide (DMSO) solvent and measuring the absorbance thereof.

In addition, to analyze the ability of Vp-GNc-HyNA to generate singlet oxygen through photodynamic effects, nano-conjugates of aqueous solutions were irradiated with a 690 nm PDT laser, And was monitored using p-nitrosodimethylaniline (RNO) as an oxygen sensor. To quantitatively evaluate the production of singlet oxygen from Vp, 200 μl of Vp-GNc-HyNA (100 μg / ml of Vp) in a 1% DMSO solution of distilled water was added to 5.58 mg of L-histidine and The resulting solution was exposed to a 690-nm laser (Shanghai Dream Laser Technology Co., Ltd., Shanghai, China) or a PTT laser. Then, the absorbance at 405 nm, which is the RNO wavelength band, was measured using a UV-Vis spectroscope. The RNO concentration was recorded as a function of time under various conditions. The results are shown in Fig.

Fig. 7 is a view for explaining the photoactive property of Vp-GNc-HyNA.

7A is a schematic diagram for explaining the Vp emission mechanism by the sequential PTT / PDT laser processing from Vp-GNc-HyNA, FIG. 7B is a schematic view for explaining Vp, Vp-HyNA and Vp before and after the 808 nm PTT laser irradiation, (1 mg / ml), and (C) shows the fluorescence intensity of Vp-GNc-HyNA (1 mg / ml) in the presence or absence of PTT laser irradiation in vitro release profiles. (D) shows singlet oxygen evolution from Vp-HyNA and Vp-GNc-HyNA (100 μg / ml of Vp) in the presence of PTT laser and / or PDT laser irradiation, Denotes standard deviations (n = 5). Asterisks (*) represent statistically significant differences (* p <0.05) calculated via one-way ANOVA.

Referring to FIG. 7, when Vp is loaded into GNc-HyNA, its intrinsic fluorescence signals are highly extinction due to the strong surface plasmon effect of gold nanoclusters. The remarkable extinction effect of Vp-GNc-HyNA was not limited to the fluorescence signal but also the oxygen production of Vp-GNc-HyNA was significantly reduced.

7C, it can be confirmed that Vp is emitted from Vp-GNc-HyNA upon irradiation with the PTT laser, and the release rate of Vp from Vp-GNc-HyNA is slowed down under physiological conditions . On the other hand, it can be seen that the pure vortexorphin is 30.8% released in 48 hours, whereas the Vp release from Vp-GNc-HyNA is rapidly increased to 73% over 48 hours.

Referring to FIG. 7 (D), it can be confirmed that singlet oxygen generation from Vp is delayed by the self-extinction effect when densely aggregated in the micelle core, and gold nanoclusts of GNc-HyNA It can be confirmed that the energy transfer to the nanoclusters inhibits the singlet oxygen production of Vp. When Vp molecules are encapsulated by GNc-HyNA, PDT laser treatment can effectively prevent singlet oxygen from being generated. The PDT quenching phenomenon of Vp-GNc-HyNA not only shows stable Vp-loading in nano-assemblies, but also implies the possibility of minimizing the nonspecific toxicity of PDT in patients who have received PDT treatment with sunlight exposure. That is, it can be confirmed that Vp-GNc-HyNA of the present invention can be used as a stable and effective photoactivatable drug delivery vehicle.

In order for Vp-GNc-HyNA to be an effective nanomedicine platform, it must not produce reactive oxygen species in the bloodstream or normal organs, and effectively treats singlet oxygen in disease sites such as tumor cells . Referring to FIG. 7, as described above, pure VP in PBS was only 30.8% released within 48 hours, but Vp emission from Vp-GNc-HyNA was dramatically accelerated under PTT laser irradiation. More than 73% of the encapsulated Vp was released under exposure to the 48-hour PTT laser, and sequential irradiation of PTT / PDT lasers with Vp-GNc-HyNA significantly increased production of singlet oxygen compared to single PDT laser treatment Can be confirmed. This indicates that the gold-shell of Vp-GNc-HyNA quenched the fluorescence signals and suppressed singlet oxygen production from Vp, but under sequential exposure to PTT / PDT lasers, with fast Vp emission, Oxygen production, resulting in increased PDT efficacy.

That is, in general, the drug delivery system according to the present invention can exhibit excellent particle stability in the bloodstream, does not generate toxic reactive oxygen species, effectively inhibits the fluorescence signal and singlet oxygen in the tumor cells by PTT / PDT laser irradiation Can be generated. Therefore, it can be confirmed that the drug delivery system according to the present invention can be used as a nano drug platform having excellent performance.

On the other hand, to confirm the tumor cell targeting property of HyNA of the present invention, the cell uptake of GNc-HyNA was examined under a focusing microscope. First, GNc-HyNA (30 mg) was dispersed in a phosphate buffer (4 mg / ml, 10 mM, pH 6.8) and stirred in the presence of EDC (1.2 mg) and HOBt (0.9 mg) for 12 hours. The solution was dialyzed against distilled water for 2 days and lyophilized to give a NIR dye, Cy5.5 mono-hydrazide, chemically bonded to the carboxyl group of the hyaluronic acid backbone via amide formation Cy5.5-HyNA and Cy5.5-GNc-HyNA were prepared.

Then, the absorption and emission profiles of the in vitro cells by enzymes and laser treatment were confirmed. MDA-MB-231 and NIH-3T3 cells were cultured in RPMI medium (Gibco, Grand Island, NY) containing 10% FBS and 1% penicillin-streptomycin at 37 ° C in 5% CO ₂ atmosphere. Cells (1 × 10 ⁵ cells / well) were then incubated in 6-wells (1 × 10 ⁵ cells / well) to confirm cell uptake and behavior of Cy5.5-labeled (Cy5.5-HyNA) or Vp- Plates were plated on gelatin-coated cover slips and stained with Cy5.5-labeled GNc-HyNA (100 μg / ml), Vp and Vp-GNc-HyNA (30 μg / Gt; C &lt; / RTI &gt; in a CO ₂ incubator. For competitive inhibition studies, cells were pre-exposed for 1 hour in hyaluronic acid-containing medium with 2 mL of serum removed. The cells were then washed twice with DPBS and fixed with 4% paraformaldehyde solution. After cells were stained with 4,6-diamidino-2-phenylindole (DAPI), the cells were observed with a photomicroscopic laser microscope (Zeiss LSM 700, Carl Zeiss, Oberkochen, Germany). The absorption behavior of the gold nanoclusters was visualized using an IX81-ZDC focus drift compensating microscope (Olympus, Tokyo, Japan). The results are shown in Fig.

Fig. 8 is a view for explaining the absorption and release profiles of ex vivo cells of GNc-HyNA and Vp-GNc-HyNA.

Figure 8 (A) is confocal microscopy images of MDA-MB-231 cells cultured for 3 hours with Cy5.5-GNc-HyNA (100 μg / ml) (B) shows intracellular internalization of Vp-GNc-HyNA as MDA-MB-231 cells after 3 hours of treatment. (C) shows the in vitro Vp release behavior from Vp-GNc-HyNA (1 mg / ml) in the presence or absence of PTT laser treatment and enzyme, and (D) -GNc-HyNA (100 [mu] g / ml of Vp). (E) represents the cell viability of MDA-MB-231 cells cultured for 6 hours at different concentrations of Vp-GNc-HyNA in the presence of laser treatment, and error bars in the graph are standard (N = 5). Figures 8 (F) and 8 (G) show the cell viability of MDA-MB-231 cultured in GNc-HyNA and Vp-GNc-HyNA, respectively, in the dark room conditions without laser treatment. Asterisks (*) represent statistically significant differences (* p <0.05) calculated via one-way ANOVA.

Referring to FIG. 8 (A), strong fluorescence signals from GNc-HyNA were observed in the cytosol of MDA-MB-231 cells, but only faint signals were detected in NIT-3T3 cells have. This is because the HyNA hyaluronic acid has a targeting property against the Hy receptor, CD44, which is expressed on the cancer cell surface. Therefore, strong fluorescence signals are detected in MDA-MB-231 cancer cells overexpressing CD44, and NIH-3T3 Which means that a faint fluorescence signal is detected in cancer cells.

On the other hand, in order to block the CD44 receptor, it can be confirmed that when MDA-MB-231 cells were pretreated with an excess of free hyaluronic acid molecules, cellular uptake of GNc-HyNA was also significantly suppressed. This means that the uptake of GNc-HyNA into tumor cells is mainly due to active interactions between hyaluronic acid and CD44 receptors in the cells.

In addition, this indicates that despite the presence of gold nanoclusters on the surface of HyNA, the Hy polymeric chain is still exposed to the surface of GNc-HyNA and the negative surface charge and CD44-mediated GNc-HyNA Endocytosis of the endoplasmic reticulum.

That is, it can be confirmed that the hyaluronic acid nano-assembly according to the present invention is capable of targeting tumor receptor-specific tumor cells because it can target the over-expressed Hy receptor, CD44, in various types of tumor cells.

8B, visualization of intracellular Vp or gold nanoclusters delivered by Vp-GNc-HyNA revealed that Vp-GNc-HyNA was associated with MDA-MB-231 cells , Fluorescence signals from prominent Vp, and dark field signals from gold nanoclusters were observed in the cell matrix of the cells. Strong fluorescence signals from Vp in cells indicate the release of Vp molecules from GNc-HyNA. Intracellular Vp release may be due primarily to the enzymatic degradation of the Hy backbone by intracellular enzyme hyaluronidase, which is known to be over-expressed in a variety of cancer cell lines. This also allows for the substitution of strong Au-S interactions (~ 40 kcal / mol) between gold nanoclusters and intracellular glutathione (GSH) molecules with Au-N interactions (~8 kcal / mol) , It can be attributed to the dissociation of the gold-shell from Vp-GNc-HyNA.

Referring to FIG. 8 (C), in order to explain the intracellular drug release mechanism, the drug release profile was monitored in the presence of two intracellular enzymes, GSH and hyaluronidase (Hyal), GSH and Hyal Vp-GNc-HyNA culture significantly accelerated Vp release. In addition, in PBS, it was confirmed that Vp of 20% or less was released from Vp-GNc-HyNA within 6 hours, while that of GSH and Hyal was released by 50% or more. In addition, more than 80% of Vp was released when Vp-GNc-HyNA was associated with GSH and Hyal and subsequently exposed to the PTT laser, thus, in the presence of GSH and Hyal with sequential PTT and PDT laser treatment , And the singlet oxygen production rate was significantly higher than that of the other groups.

Referring to Figure 8 (D), the singlet oxygen production from Vp-GNc-HyNA (Hyal + GSH + PTT + PDT) was 2.6 and 1.7 times higher than single laser or dual laser treatments, Vp-GNc-HyNA was degraded by intracellular GSH and Hyal and completely separated by PTT laser irradiation, which resulted in a dramatic increase in singlet oxygen production from Vp-GNc-HyNA following PDT laser irradiation, Indicating that the release is promoted.

8 (E) to 8 (G), cytotoxicity of Vp-GNc-HyNA in MDA-MB-231 cells was confirmed. As a result, as expected, GNc-HyNA or Vp- HyNA did not induce clear cytotoxicity without laser irradiation. However, sequential cytotoxicity was induced in cells exposed to PDT, PTT or sequential PDT / PTT laser treatments. Vp-GNc-HyNA, which was treated sequentially with PTT and PDT laser, It can be confirmed that it induces stronger cytotoxicity than Vp-GNc-HyNA or PDT laser alone under PTT. This indicates that the Vp-GNc-HyNA and sequential PTT / PDT laser irradiation treatments resulted in the highest anticancer efficacy among the treatment groups.

That is, cytotoxicity was also increased as the concentration of vertefopin in the nano-conjugate increased, and it was confirmed that PTT and PDT laser irradiation showed stronger cytotoxicity than PTT laser irradiation or PDT laser irradiation alone have.

Therefore, it can be confirmed that the drug delivery system of the present invention can exhibit photothermal and photodynamic effects by PTT and PDT laser irradiation, and it is preferable to treat both PTT and PDT lasers.

The in vivo distribution of GNc-HyNA was then confirmed. First, a dispersion of 1 x 10 ⁷ MDA-MB-231 cells in 100 μl of saline was injected into the subcutaneous tissue of allogeneic or xenografted MDA-MB-231 tumor-bearing mice. When tumors grew to a volume of about 150-200 mm ³ , 200 μl of Cy5.5 labeled GNc-HyNA (1 mg / ml) solution was injected into the mice via the tail vein (n = 3 per group) . In order to observe the long-term distribution of the nano-conjugate, 24-hour after the sample injection, the nude mice of each group were dissected and the major organs and cancer And the fluorescence was confirmed using an eXplore Optix system. Time-dependent biological distribution and accumulation profiles of the nanoparticles were monitored by the eXplore Optix system (ART Advanced Research Technologies, Inc., Montreal, Canada). All data were calculated using the region of interest (ROI) function of the Analysis Workstation software (ART Advanced Research Technologies, Inc., Montreal, Canada) and the values were calculated using standard deviations of at least 3 animals Expressed as an average. The results are shown in Fig.

9 is a diagram showing in vivo NIRF imaging of GNc-HyNA.

9 (A) shows subcutaneous MDA-MB-231 tumor models and (D) shows time-lapse fluorescence imaging of GNc-HyNA (1 mg / ml) 24 h after injection with allograft tumor models And tangential long-term imaging. (B) shows the fluorescence intensity of Cy5.5-GNc-HyNA according to the time function in the tumor or whole body in subcutaneous tumor models or (E) allograft tumor models, and (C) (F) Quantification of fluorescence intensity in major organs and tumors of allograft tumor models. (G) shows thermal images of MDA-MB-231 tumor-bearing mice at 9 hours after GNc-HyNA (1 mg / ml) and PBS injection under PTT laser irradiation and (H) subcutaneous tumor models It shows the long-term distribution of Au after the intravenous injection of GNc-HyNA. (1 mg / ml) of GNc-HyNA in tumor (I) tumor.

Referring to FIG. 9, the fluorescence signals of GNc-HyNA were found throughout the body 1 hour after injection, and the signals gradually decreased over time. That is, after 24 hours of injection, weak fluorescence signals were found in the body. On the other hand, fluorescence signals stronger than those seen in the whole body were detected at the tumor sites 24 hours after injection, and fluorescence signals gradually increased to reach the highest level at 9 hours (Fig. 9 (B) and (E)). This means that strong fluorescence intensities in tumors are steadily maintained for 24 hours, as opposed to a rapid decrease in fluorescence intensity throughout the body. These results indicate that GNc-HyNA can be circulated in the bloodstream and can rapidly target tumors. That is, GNc-HyNA is easily released from the body, and it can be confirmed that it can stay in tumor tissues for a long time due to lymphatic drainage in tumor tissues.

On the other hand, to evaluate the relative organ and tumor distribution of GNc-HyNA, major organs of subcutaneous xenograft and allogeneic breast tumor rats were collected on day 2 after injection, (See Figures 9 (C) and (F)) and ex vivo organ images show that GNc-HyNA can accumulate at high levels in tumors, liver and kidney tissues, It is possible to confirm that tumor accumulation is greater than other organs.

Gold accumulated in major organs was quantitated by ICP-MS at different time points (see Figure 9 (H)), and GNc-HyNA accumulates significantly in day 1 in tumors, liver and spleen can confirm. In addition, more than 25.1% of the injected gold was found in tumor tissues, and 36.1% and 16.9% were detected in liver and spleen tissues, respectively. Compared with the gold content of the first day, accumulated gold content rapidly decreased in the organs over time. More than 69.8% and 50.2% of the accumulated gold in the liver and spleen tissues were removed on day 7, 86.1% and 92.9% were removed on day 14, and 56.1% or more of the accumulated gold in the tumor tissues were found in 7 Remained in the first day. On the other hand, 20.4% remained at 14 days, which means the extended maintenance effect of the nanoassemblies of the present invention in tumor tissues.

This confirmed the tissue distribution of PDT gene Vp delivered to GNc-HyNA by detecting the fluorescence signal of Vp in both tumors and organs. The results are shown in Fig.

FIG. 10 is a diagram for explaining the fusing (ex vivo) of Vp from GNc-HyNA.

Fig. 10 (A) shows transflective images of Vp from GNc-HyNA after 24 hours of injection in major organs and tumors, and (B) fluorescence intensity in major organs and tumors.

Referring to FIG. 10 with FIG. 9, consistent with the biological distribution of GNc-HyNA, the strongest Vp signals were detected in tumors as compared to other organs, and significant Vp accumulation was also detected in liver and kidney . As discussed above, the non-specific accumulation of GNc-HyNA is due to the fact that Vp-GNc-HyNA is non-toxic without photo-activation steps by PTT and / or PDT laser irradiation, Can be used as a potential therapeutic regimen with excellent tumor targeting in combination with radiation. That is, it can be confirmed that the drug delivery system of the present invention can be used as a drug delivery system for treating tumors.

On the other hand, FIG. Still referring to 9, GNc-HyNA PBS according to for 15 minutes as a result of examining the photo-thermal characteristic (Fig. 9 (G) and (refer to I)), the laser power density 1.5 W / cm ² in a living body PTT laser irradiation The local temperature of the tumor portion of the control was not significantly changed, which means that the low voltage NIR laser is harmless to organs. However, the local tumor temperature of rats treated with Vp-GNc-HyNA and PTT lasers increased rapidly to 60 ° C, confirming that this exceeds the damage threshold required for tumor therapy. In other words, the group of the experimental group injected with Vp-GNc-HyNA showed that the temperature around the cancer increased to 60 ° C after the irradiation of the photothermal laser, showing sufficient effect for cancer treatment.

More specifically, MDA-MB-231 tumor-bearing mice were prepared to evaluate the photodynamic and photothermal effects of Vp-GNc-HyNA. A suspension of 1 x 10 ⁷ MDA-MB-231 cancer cells dispersed in physiological saline (100 μl) was subcutaneously injected into the thymus nude mice. Rats were divided into 6 groups: (1) saline, (2) laser (PTT and PDT laser treatment), (3) PDT laser treated Vp at 5 mg / kg, (4) PDT laser treated Vp-GNc -HyNA, and (5) PTT laser and PDT laser treated Vp-GNc-HyNA. When the tumor volume reached 200 mm ³ , each sample was injected via tail vein injection at day 0. Thereafter, PTT treated (5 min, 808 nm, 1.5 W / cm 2), PDT treatment (20 min, 690 nm, 0.5 W / cm 2), and the initial PTT and a (PTT / PDT) sequence of PDT treatment then Was performed 9 hours after injection. Tumor volume was observed once a day for 20 days. Tumor volume was calculated as ^{[a × b 2/2]} , where a is the largest, b is the smallest diameter. Values are expressed as the mean of the standard deviation for each group of five animals. The results are shown in Fig.

11 is a view for explaining in vivo anticancer activity of Vp-GNc-HyNA of the present invention.

11 (A) is a graph showing the results of various treatments (saline, laser (PTT + PDT), Vp (PDT ) And 5. Vp-GNc-HyNA (PDT, PTT and PTT + PDT)) relative to the tumor volume of the allogeneic MDA-MB-231 tumor model. (B) shows the survival curves of the mice after various treatments. (C) represent representative photographs of MDA-MB-231 tumor-bearing mice after various treatments. Error bars represent the standard deviation of 5 rats per group. An asterisk (*) indicates statistically significant differences (* p <0.05) calculated by the one-way ANOVA test.

11, it can be seen that the group irradiated with (1) saline, (2) PTT and PDT laser irradiation increased 8 times more than the volume of cancer for the first time after 20 days, (3) , The number of cancer cells was 6 times higher than the volume of cancer for the first time. This indicates that injecting only verteporin does not significantly affect the growth of cancer, indicating that the low dose of free Vp is not sufficient to effectively inhibit tumor growth.

In addition, (4) Vp-GNc-HyNA irradiated with a PDT laser shows that the growth rate of cancer is remarkably decreased. However, Vp-GNc-HyNA irradiated with PDT laser can be confirmed to have developed cancer after treatment. (5) Vp-GNc-HyNA irradiated with PTT laser also inhibited the growth of cancer, but it did not completely eliminate the cancer, and the wound was also completely treated after 50 days. On the other hand, (6) a group of Vp-GNc-HyNA that sequentially examined PTT and PDT lasers showed that the cancer was completely removed and that the skin was completely regenerated after the treatment.

The group treated with PDT laser after (1) saline, (2) PTT, PDT laser irradiation, and (3) Vp injection did not survive for 33, 35, Dogs died or had cancer recurrence between 30 and 40 days after treatment, whereas 40% and 80% of Vp-GNc-HyNA treated with PDT or PTT laser survived at 50 days, respectively , PTT and PDT treated groups did not show any deaths or relapsed rats after 50 days of treatment.

Therefore, the drug delivery system of the present invention can be used for the treatment of tumors, and it can be confirmed that PTT and PDT lasers exhibit excellent anticancer effects together with PTT and PDT lasers. However, PTT and PDT lasers It can be confirmed that it is most preferable to investigate.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention as defined by the following claims. It can be understood that it is possible.

Claims (19)

An amphiphilic polymer nanoassembly comprising a hydrophilic polymer comprising a tertiary amine and a hydrophobic material associated with the hydrophilic polymer; And
And a gold nanocluster disposed on the surface of the nanocoil assembly,
The drug is loaded on the nano assembly and the drug is released upon irradiation with near infrared rays,
Wherein the hydrophilic polymer is an α-alkyne hyaluronic acid alkenylated with an alkyne compound having 3 carbon atoms, and the hydrophobic polymer is a hydrophobic Characterized in that the substance is an azide-terminated polycaprolactam (PCL-N ₃ ).
Drug delivery.
delete delete delete delete The method according to claim 1,
Characterized in that the drug is a photosensitizer.
Drug delivery.
The method according to claim 6,
The photosensitizer may be selected from the group consisting of a phorphyrins compound, a chlorins compound, a bacteriochlorins compound, a phthalocyanines compound, a naphthalocyanines compound, and a 5- 5-aminoevuline esters) compound.
Drug delivery.
The method according to claim 6,
Characterized in that the photo-sensory agent is vertephorpin.
Drug delivery.
The method according to claim 1,
Wherein the gold nanoclusters are separated from the nanocomposite at a near infrared ray wavelength,
Wherein the gold nanoclusters separate nanoassemblies release the loaded drug.
Drug delivery.
The method according to claim 1,
Wherein said drug delivery vehicle is a drug delivery for tumor therapy.
Drug delivery.
A step of reacting the hydrophilic polymer and the tertiary amine to prepare a tertiary amine-bonded hydrophilic polymer;
Reacting a tertiary amine-bonded hydrophilic polymer with a hydrophobic substance to prepare an amphiphilic polymer nanocomposite; And
Reacting the nanocomposite with gold (III) chloride (HAuCl ₄ ) to grow gold nanoclusters on the surface of the nanocomposite,
Wherein the hydrophilic polymer is an α-alkyne hyaluronic acid alkenylated with an alkyne compound having 3 carbon atoms, and the hydrophobic substance is an azide-terminated polycaprolactam Features,
A method for manufacturing a drug delivery system.
delete 12. The method of claim 11,
In the step of preparing the tertiary amine-bonded hydrophilic polymer,
α-Alkyne hyaluronic acid and 3- (dimethylamino) -1-propylamine can be obtained by reacting 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride, EDC · HCl) and 1-hydroxybenzotriazole (HOBt)
characterized in that alpha -alkyne hyaluronic acid and 3- (dimethylamino) -1-propylamine are amide-bonded to form DAA-linked hyaluronic acid.
A method for manufacturing a drug delivery system.
12. The method of claim 11,
In the step of preparing the amphiphilic polymer nanocomposite,
The DAA-conjugated hyaluronic acid and the azide-terminated polycaprolactam are prepared from a solution comprising copper (II) sulfate pentahydrate (CuSO ₄ .5H ₂ O) and sodium ascorbate Lt; / RTI &
Wherein the DAA-conjugated hyaluronic acid and the azide-terminated polycaprolactam are polymerized to form a DAA-hyaluronic acid-polycaprolactam nanoassembly.
A method for manufacturing a drug delivery system.
12. The method of claim 11,
Before the step of growing the gold nanoclusters,
Further comprising the step of reacting the nanocomposite with a solution comprising a drug to load the drug into the nanocomposite,
In the step of growing the gold nanoclusters,
Lt; RTI ID = 0.0 &gt; nanoclusters &lt; / RTI &
A method for manufacturing a drug delivery system.
16. The method of claim 15,
Wherein the drug is a photosensitizer.
A method for manufacturing a drug delivery system.
17. The method of claim 16,
Wherein the photosensitizer is at least one of a porphyrin compound, a chlorine compound, a bacteriocin compound, a phthalocyanine compound, a naphthalocyanine compound and a 5-aminolevulinic ester compound.
A method for manufacturing a drug delivery system.
17. The method of claim 16,
&Lt; / RTI &gt; wherein said photosensitizer is &lt; RTI ID = 0.0 &gt;
A method for manufacturing a drug delivery system.
12. The method of claim 11,
In the step of growing the gold nanoclusters,
Wherein the gold (III) chloride is reacted in an amount of 0.05 mol to 0.5 mol relative to the tertiary amine of the nanocomposite.
A method for manufacturing a drug delivery system.

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