KR20120089892A - Preparation of biocompatible polymeric drug delivery system with advanced tumor accumulation - Google Patents

Abstract

PURPOSE: A biocompatible polymer delivery system for drug delivery with the surface modified by glycol chitosan and heparin is provided to enhance blood retention and circulation and to effectively deliver a drug to a target site of cancer tissue. CONSTITUTION: A method for preparing biocompatible polymer nano delivery system comprises: a step of mixing and dissolving polyoxyethylene sorbitan monooleate(Tween80) of chemical formula 1 with drug; a step of adding lecithin lipid of chemical formula 2 to the mixture and mixing; a step of impregnating the secondary mixture to a triblock copolymer solution of chemical formula 3(HO(C_2H_4O)_a(C_3H_6O)_b(C_2H_4O)_cH) to prepare a nano delivery system; and a step of adding glycol chitosan of chemical formula 4 and heparin solution of chemical formula 5 to the nano delivery system to modifier the surface of the nano delivery system.

Description

Preparation method of biocompatible polymer carrier for drug delivery with improved tumor accumulation and nanocarrier {Preparation of biocompatible polymeric drug delivery system with advanced tumor accumulation}

The present invention relates to a biocompatible polymer carrier for drug delivery whose surface is modified with glycol chitosan and heparin and its use.

The incidence of cancer continues to increase, and accordingly, the development of cancer treatment methods and drugs is continuously made. Currently used cancer treatments include surgery to remove only cancer cells by surgery, radiation therapy, and chemotherapy using anticancer drugs showing cytotoxicity. Chemotherapy with anticancer drugs is a widely used method for cancer treatment, but it shows cytotoxicity not only to cancer cells but also to normal cells. Therefore, in the field of chemotherapy using anticancer drugs, researches are continuously conducted to reduce the side effects of anticancer drugs while maximizing cancer treatment effects.

Meanwhile, paclitaxel, which was approved by the US FDA in 1992, has the advantage of superior anticancer activity compared to conventional anticancer drugs, but because it has high hydrophobicity, it is not dispersed in water and used for injection. In order to disperse in a 50:50 mixture of cremophore EL (polyethoxylated oil) and ethanol to cause serious side effects in the human body.

As such, due to the problem that paclitaxel, known as a natural substance having important anticancer activity, has low solubility in water, it has been difficult to administer it as an injection. Therefore, various systems have been proposed to solve the problem and make it injectable. For example, a method of combining human serum albumin (HSA) with paclitaxel, which is biodegradable and has significant affinity for paclitaxel, by sonication, high pressure homogenization and microfluidization in a known manner to form an injectable emulsion Proposed. In U.S. Pat.Nos. 5,439,686, 5,498,421, 5,560,933 and WO 94/18954, VivoRx Pharmaceuticals, Inc. provides an ultrasonication technique that provides an average particle size (MPS) of less than 10 μm. Injectable formulations of microparticles of paclitaxel and human serum albumin prepared using are disclosed. However, the manufacturing method described in the above patents cannot be used on an industrial scale, and furthermore, the microparticles thus obtained have the disadvantage that they have an average particle size too large to be unsuitable and useful for administration to a patient. Thus, in U.S. Pat.Nos. 5,916,596, 6,096,331 and WO 98/14174, WO 99/00113, sterilization of paclitaxel and human serum albumin obtained by reconstitution of lyophilized powder having an average particle size of less than 0.2 μm with sterile 0.9% NaCl aqueous solution Nanoemulsions are disclosed. As described in this patent, nanoemulsions obtained using high pressure homogenization describe that the average particle size is constant with time and that nanoparticle precipitates are not produced and therefore stable. However, the above method still has the disadvantage that it can still be used on an industrial scale by suspending paclitaxel in human serum albumin to produce an injectable preparation.

Nanoparticles are one of drug carriers for selective target site delivery of drugs, including anticancer agents, and refers to heterogeneous dispersed particles on a colloid having a large surface area having a particle size of several nm to several hundred nm in diameter. Nanoparticles encapsulated with anticancer agents can be targeted to cancer tissues for cancer treatment without adverse effects on normal tissues without any cancer cell target residues. This targeting effect of the nanoparticles is obtained by the specific pathophysiological characteristics of cancer cells called enhanced permeability and retention (EPR) effects. The malignant neoplasm in the body grows faster than normal tissue, and requires a lot of nutrients and oxygen supply, which causes the rapid production of new blood vessels around cancer cells. In other words, because blood vessels are generated rapidly around cancer cells, these neovascularized blood vessels have large outflow holes sufficiently for the nanoparticles to escape from the blood vessels, so that the nanoparticles accumulate in the cancer tissue.

Therefore, various efforts have been made to develop nanoparticles encapsulated with anticancer agents. However, conventional nanoparticles are mostly manufactured with organic solvents, and thus, side effects in vivo may occur due to the solvent remaining in the nanoparticles. have. In addition, there are various problems as nanoparticles that deliver anticancer agents such as paclitaxel in vivo due to short or poor retention and circulation in vivo.

The present invention has been made to solve the problems described above, an object of the present invention is to provide a drug delivery polymer carrier for excellent biocompatibility and improved accumulation to tumors.

In order to achieve the above object, the present invention is to prepare a nanoparticle without using an organic solvent, the surface of the prepared nanoparticles provides a polymer carrier modified with glycol chitosan and heparin.

Since the polymer carrier according to the present invention is prepared without using an organic solvent, the biocompatibility is excellent, and since the surface is modified with glycol chitosan and heparin, and the accumulation to the target site is improved, the specific drug can be effectively delivered to the target site. useful.

Since the polymer carrier according to the present invention can be applied to cancer and other disease models, it can be used as a therapeutic agent for treating a wide range of diseases in the future.

1 is a schematic diagram representing a biocompatible polymer carrier for drug delivery according to the present invention by modifying glycol chitosan and heparin in a drug-containing nanocarrier.
Figure 2 is a graph showing the particle size distribution of the drug-containing nanocarriers.
3 is a graph showing the particle size distribution of the biocompatible polymer carrier for drug delivery of the present invention in which the glycol chitosan and heparin are modified in the drug-containing nanocarrier.
Figure 4 is a TEM (Transmittance electron microscopy, transmission electron micrograph) of the drug delivery biocompatible polymer carrier prepared according to the present invention.
FIG. 5 is a near-infrared spectroscopy of tumor accumulation of the nano-carrier with time after administration of the drug delivery biocompatible polymer carrier of the present invention containing the near-infrared fluorescent substance and the drug into the tail vein of a tumor-induced animal model mouse. Photo shown and quantified data.
6 is administered intravenously the drug delivery biocompatible polymer carrier of the present invention containing a near-infrared fluorescent substance and a drug intravenously in a tumor-induced animal model rat, after 48 hours to extract the major organs through the distribution of the near-infrared fluorescent substance Photographs and quantified data showing the distribution of the body in the body.

Hereinafter, the present invention will be described in detail.

The present invention provides a biocompatible polymer carrier for drug delivery, wherein the biocompatible polymer carrier for drug delivery is characterized in that its surface is modified with an aqueous solution of glycol chitosan and heparin.

The biocompatible polymer carrier for drug delivery according to the present invention is prepared by modifying the surface of the nanocarrier containing or encapsulating a drug with an aqueous solution of glycol chitosan and heparin, and a specific manufacturing method is as follows.

First, polyoxyethylene sorbitan monooleate (Polyoxyethyelene Sorbitan Monooleate, Tween ^® 80) and a drug to be encapsulated in a nanocarrier are mixed and dissolved, and then lecithin lipid is added, mixed, and impregnated in an aqueous triblock copolymer aqueous solution. Prepare the carrier. And by adding glycol chitosan and heparin aqueous solution sequentially, a biocompatible polymer carrier for drug delivery is prepared by modifying the surface of the nanocarrier. In one embodiment, the method comprising the steps of mixing and dissolving the polyoxyethylene sorbitan monooleate of Formula 1 (Tween ^® 80) and the drug to be delivered to the target site; Adding and mixing the lecithin lipid of Formula 2 to the dissolved drug mixture; Preparing a nanocarrier by impregnating the mixture into an aqueous triblock copolymer of Formula 3 and freeze-drying; And sequentially adding glycol chitosan and heparin to the drug-containing nanocarrier to prepare a biocompatible polymer carrier for drug delivery according to the present invention.

[Formula 1]

Wherein the sum of w, x, y and z is 20.

[Formula 2]

(3)

HO (C ₂ H ₄ O) _a (C ₃ H ₆ O) _b (C ₂ H ₄ O) _c H

Wherein b is 10 or more, and the sum of a and c is a number such that these terminal portions comprise from 5 to 95% by weight, preferably 20 to 90% by weight of the polymer).

[Formula 4]

In Formula 1, n is an integer of 1000 to 5000.

[Chemical Formula 5]

In the above, the polyoxyethylene sorbitan monooleate (trade name: Tween 80) of Formula 1 is a nonionic surfactant having hydrophilicity and hydrophobicity. Polyoxyethylene sorbitan monooleate is a kind of polyoxyethylene sorbitan, a yellowish liquid with viscosity, and nonionic polyoxyethylene sorbitan is polyoxyethylene sorbitan monolaurate (twin 20), polyoxyethylene sorbate Non-elastic monopalmitate (twin 40), polyoxyethylene sorbitan monostearate (twin 60), polyoxyethylene sorbitan ethyl stearate (twin 65), polyoxyethylene sorbitan monooleate (twin 80), etc. This is commercially available. Among these, polyoxyethylene sorbitan monooleate takes the form of a liquid and is frequently used for solubilizing various poorly soluble drugs. It is also a substance that the US Food and Drug Administration (FDA) has approved for intravenous injection. In preparing the nanocarrier of the present invention, the polyoxyethylene sorbitan monooleate may be used, but is not limited thereto, and any solubilizer generally used for solubilizing poorly soluble drugs may be used without any distinction.

In the above, lecithin of Formula 2 is one of phospholipids containing glycerin and phosphoric acid, and is a main component constituting the biofilm, and is present in egg yolk, soybean oil, liver, brain and the like. In addition, it is one of the important components of the cell membrane composition, there are varieties such as egg yolk lecithin, soybean lecithin, and is not particularly limited in manufacturing the present nano-carrier. Lecithin is administered in the body mostly in the form of injections, so there is no problem when administered to the human body. Many studies on drug carriers using the emulsion method and the solvent-evaporation method have been conducted.However, over-release of drugs due to the degradation of the carrier due to rapid destruction or hydrolysis of the carrier in the blood ( Burst effect is a problem. Barenholz Y., "Liposome application: problems and prospects" Current Opinion in Colloids and Interface See Science , 6, (1), 66-77 (12) (2010) (http://www.ingentaconnect.com/content/els/13590294/2001/00000006/00000001

/art0090;jsessionid=6upjonjosaf83.alexandra)

In the above, the triblock copolymer of Formula 3 is polyoxyethylene-polyoxypropylene

It is a triblock copolymer of polyoxyethylene and dissolved in water. The triblock copolymer is a poloxamer. Polaxamers may be prepared by the methods described in the literature or may use commercially available products. The poloxamers used in the present invention have a molecular weight of 1000 to 16000, and their properties depend on the ratio of the polyoxypropylene block and the polyoxyethylene block, that is, the ratio of a + c and b in the formula (1). Poloxamers are solid at room temperature, soluble in water and ethanol, and commercially available Poloxamers 124, 188, 237 and 407. For example, poloxamer 188 means a poloxamer having a molecular weight of about 8350 as a compound of Formula 1 wherein b is 30 and the sum of a and c is about 75.

In the above, glycol chitosan of the formula (4) is water-soluble chitosan dissolved in water. Chitosan is a basic polysaccharide prepared by deacetylation by treating chitin with a high concentration of alkali, and is known to be superior to other synthetic polymers in terms of cell adsorption capacity, biocompatibility, and biodegradability. In one embodiment of the present invention used glycol chitosan having a molecular weight of 250,000, it is also possible to use a glycol chitosan having a lower molecular weight through molecular weight cleavage.

In the above, heparin of Formula 5 consists of sulfated linear polysaccharides, mainly composed of uronic acid (uronic acid). Heparin is a mixture of various structures and has various properties such as antithrombogenicity, inflammation inhibition, angiogenic cancer growth inhibition and immunosuppression. Heparin is a solid at room temperature and soluble in water. Heparin, which is used as an anticoagulant, is also used to prevent the thrombosis that can occur in cancer patients. In addition, it is possible to inhibit the formation of fibrin by interfering with the process of the coagulation promoting enzyme around the tumor cells, and to prevent abnormal hemostatic mechanisms. In this regard, fibrin formation around cancer cells is generally difficult to be affected by the immune system by forming a thick shell, but when heparin inhibits fibrin formation, tumor cells become more accessible from natural killer cells. , Cytokines, and the like, or by controlling their activity, can induce the destruction or death of tumor cells by immune system action. In an embodiment of the present invention, heparin having a molecular weight of about 20,000 is used, but is not limited thereto, and heparin and heparin derivatives having lower molecular weights may also be used.

In the above, the mixing ratio of triblock copolymer to polyoxyethylene sorbitan monooleate is 2: 8 to 99: 1, preferably 5: 5 to 9: 1. If the ratio of triblock copolymer (polyoxamer) to polyoxyethylene sorbitan monooleate (Tween 80) is out of the above range, there is a disadvantage in that the yield in preparing the nanocarrier is decreased or the drug release is increased rapidly.

The biocompatible polymer nanocarrier for drug delivery according to the present invention prepared by the above method is useful because it can effectively deliver a poorly soluble drug to a target site.

Currently, anticancer agents such as Taxol, which are commercially available, are poorly soluble and are used as a dissolving agent in Cremophor EL, a solubilizer, to dissolve in water for injection. In the case of the nanocarrier according to the present invention, a polymer micelle containing lipids is used. Since the drug can be contained inside, it is possible to dissolve the nanocarrier containing dozens to hundred times the drug in the water for injection compared to the solubility (1 µg / ml) of the conventional paclitaxel. In addition, since the lecithin lipid core is wrapped in a polymer membrane by Pluronic F-68, it is possible to control the drug release rate. Therefore, the poorly soluble drug is enhanced solubility and free control of drug release behavior in vivo by the nano-carrier according to the present invention.

The average size of the biocompatible polymer carrier for drug delivery according to the present invention is 1 μm to 10 μm, preferably 500 nm to 5 μm, more preferably 100 nm to 2 μm, and the particle size distribution is uniform. In FIG. 2 and FIG. 3, the particle size distribution of the drug-contained nanocarrier and the biocompatible polymer carrier for drug delivery in which the nanocarrier was modified with a glycol chitosan and an aqueous solution of haparin were analyzed by a particle size analyzer. The results are shown. From the data of FIG. 2 and FIG. 3, it can be seen that the particle size of the nanocarrier was increased by the modification to glycol chitosan and heparin aqueous solution.

The biocompatible polymer carriers according to the present invention contain drugs. The drug that may be contained is not particularly limited and may be used as long as it is a drug to be delivered in vivo as a biopharmaceutical formulation. When encapsulating a drug including a mixture of polyoxyethylene sorbitan monooleate, lecithin lipid, and poloxamer, the nanocarrier may be prepared using poloxamer. In the case of preparing nanocarriers with poloxamer, the drug is preferred since it is easily entrapped in the nanocarriers and shows an excellent encapsulation rate. On the other hand, in the polymer carrier according to the present invention can be encapsulated together with the drug and a fluorescent substance which can confirm the accumulation to the target site. In one embodiment, in the polymer carrier, GN-A, a natural anticancer agent, and Cy 5.5, a fluorescent substance, may be encapsulated together. In addition to the cyanine-based fluorescent material, a radioisotope, a quantum dot, or an MRI contrast agent capable of confirming the location of nanoparticles and the like without adversely affecting the living body may be used without limitation.

By using the biocompatible polymer carrier for drug delivery according to the present invention, it is possible to prepare a polymer carrier that can not only have a desired particle size and distribution, but also can effectively deliver various drugs and biologics in vivo, inexpensively and simply.

Since the biocompatible polymer carrier for drug delivery according to the present invention does not contain an organic solvent, side effects due to residual toxicity of the organic solvent when used in vivo can be prevented. This is because the polymer carrier according to the present invention is a poloxamer nanocarrier prepared using poloxamer, and no organic solvent is used during the manufacturing process.

Hereinafter, the present invention will be described in more detail with examples. The following description is merely illustrative of the technical idea of the present invention, and those skilled in the art to which the present invention pertains will be able to various modifications and variations without departing from the essential characteristics of the present invention. Therefore, the embodiments disclosed in the present invention are for describing the technical idea of the present invention, and the scope of the technical idea of the present invention is not limited by these embodiments. The scope of protection of the present invention should be construed according to the following claims, and all technical ideas falling within the scope of the same shall be construed as falling within the scope of the present invention.

Example 1

20% by weight of lecithin lipid and 5% by weight of trehalose (freeze-drying protector) were added to the distilled water and dispersed, and the lipid particles were pulverized using ultrasonic waves. In this case, lecithin lipid particles having a size of about 100 nm are produced, and the polyoxyethylene sorbitan monooleate (trade name Tween 80) is dispersed by adding the same amount as the lecithin lipid, and then the lipid particles are pulverized using ultrasonic waves. A final aqueous solution of lecithin lipid was prepared to contain. In this case, lecithin lipid particles of about 30 nm size are produced.

GN-A (glionitrin-A), a natural anticancer drug, which is a model drug, is dissolved in polyoxyethylene sorbitan monooleate, and then a solution of lecithin lipid prepared above is added to prepare a mixed solution.

On the other hand, 35% by weight of aqueous solution of Polaxamer 188 (polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer, trade name Floronic F-68) was prepared. The model drug and the lecithin lipid mixture prepared above were impregnated in the prepared solution of Polaxamer 188, and then lyophilized to prepare a nanocarrier containing GN-A, a natural anticancer agent.

Example 2

The solidified mixture prepared by the same procedure as in Example 1 was dissolved by adding 10 ml of 1% by weight and 5% by weight of distilled water, and then 1 ml of an aqueous solution of 0.025% by weight glycol chitosan and 0.0625% by weight heparin was sequentially added. And freeze-dried to modify the surface of the nanocarrier to obtain a biocompatible polymer carrier for drug delivery, which improved tumor targeting. The shape of the nanocarrier thus obtained was confirmed using a transmission electron microscope, shown in FIG.

Example 3

Example 1 Except for dissolving GN-A, a natural anticancer agent, and Cy 5.5, a fluorescent substance, together with polyoxyethylene sorbitan monooleate in order to confirm tumor accumulation in a tumor-derived animal model. It was prepared by the same procedure as in and 2. The nanocarriers prepared in this way were administered to the tail vein of the mouse, and the body distributions of the mice were examined over time. Glycol chitosan and heparin-unmodified nanocarriers also had the characteristics of accumulating into tumors and higher tumor accumulation when the surface of the nanocarriers was modified. It was confirmed by the image and quantitative fluorescence amount having sex, it is shown in FIG.

As a result of extracting and checking the main organs of the animals after 24 hours to confirm the organ distribution behavior, it was confirmed that a significant amount of nanocarriers were accumulated in the tumor compared to other organs, and it is shown in FIG. 5.

Example 4

The same procedure was repeated as in Examples 1 and 2 except adding 0.042 g of Paclitaxel.

A poloxamer nanocarrier containing paclitaxel was obtained, and its inclusion amount was analyzed by HPLC (high performance liquid chromatography). The inclusion rate of paclitaxel was more than 98%. The drug release form from the nanoparticles thus obtained is shown in FIG. 3.

Example 5

The same procedure as in Examples 1 and 2 was repeated except adding 0.042 g of docetaxel.

Poloxamer nanoparticulates doped with docetaxel were obtained and their inclusion amounts were analyzed by HPLC (high performance liquid chromatography). The inclusion rate of docetaxel was 98% or more.

As described above, the biocompatible polymer carrier for drug delivery according to the present invention prepared by modifying the surface with an aqueous solution of glycol chitosan and heparin effectively contains various synthetic drugs or biologics encapsulated in nanocarriers as target sites in vivo. I can deliver it. When the nanocarrier according to the present invention is used, it is easy to deliver the poorly soluble drug in vivo, and since the organic solvent is not used in the manufacturing process, the nanocarrier according to the present invention is free from residual substances due to the organic solvent and no toxicity. It has the advantage of high stability at.

Claims (13)

Mixing and dissolving a polyoxyethylene sorbitan monooleate (Polyoxyethyelene Sorbitan Monooleate, Tween ^® 80) and a drug of Formula 1,
Adding and mixing the lecithin lipid of Formula 2 to the dissolved drug mixture;
Preparing a nanocarrier by impregnating the mixture into an aqueous triblock copolymer solution of Formula 3, and
Modifying the surface of the nanocarrier by sequentially adding glycol chitosan of Formula 4 and heparin aqueous solution of Formula 5 to the drug-containing nanocarrier
A method of producing a biocompatible polymer nano-carrier comprising a.
[Formula 1]

Wherein the sum of w, x, y and z is 20.
(2)

(3)
HO (C ₂ H ₄ O) _a (C ₃ H ₆ O) _b (C ₂ H ₄ O) _c H
Wherein b is 10 or more, and the sum of a and c is a number such that these terminal portions comprise from 5 to 95% by weight, preferably 20 to 90% by weight of the polymer).
[Chemical Formula 4]

In Formula 1, n is an integer of 1000 to 5000.
[Chemical Formula 5]

The method of claim 1, wherein the triblock copolymer of Chemical Formula 3 is a triblock copolymer of polyoxyethylene-polyoxypropylene-polyoxyethylene and dissolved in water. The method of claim 1, wherein the triblock copolymer is a poloxamer. The method of claim 1, wherein the mixing ratio of the triblock copolymer to lecithin is 5: 5 to 9: 1. A biocompatible polymer nanocarrier, surface modified with glycol chitosan and heparin, prepared by the method of any one of claims 1 to 4. The biocompatible polymer nanocarrier according to claim 5, wherein the nanocarrier modified with glycol chitosan and heparin has an increased accumulation in tumors compared to a carrier not modified with glycol chitosan and heparin. The biocompatible polymer nanocarrier of claim 5, wherein the average particle size of the nanocarrier is 100 nm to 400 nm. The biocompatible polymer nanocarrier of claim 5, wherein the nanocarrier comprises a synthetic drug or a biologic to deliver a drug. The biocompatible polymer nanocarrier of claim 5, wherein the nanocarrier does not include an organic solvent. Polyoxyethylene sorbitan monooleate; Lecithin lipids; triblock copolymers; Glycol chitosan; Heparin; And a nanoparticle having an average particle size of 100 nm to 400 nm, including an anticancer agent. A nanocarrier composed of polyoxyethylene sorbitan monooleate and a triblock copolymer, wherein the nanocarrier is a surface modified with glycol chitosan and heparin. The biocompatible polymer nanocarrier of claim 11, wherein the nanocarrier further comprises a fluorescent substance, a radioisotope, a quantum dot, or an MRI contrast agent. The method of claim 11, wherein the nano-carrier is squamous cell carcinoma, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, breast cancer, liver cancer, skin cancer, esophageal cancer, testicular cancer, kidney cancer, colon cancer, rectal cancer, stomach cancer, A biocompatible polymer nanocarrier for drug delivery containing a drug for the treatment of a disease selected from the group consisting of kidney cancer, bladder cancer, ovarian cancer, cholangiocarcinoma and gallbladder cancer.

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