KR20080079421A - Nanoparticle containing lipiodol and contrast media for x-ray computer tomography comprising the same - Google Patents

Abstract

A nanoparticle containing lipiodol is provided to maintain excellent X-ray absorption properties, facilitate targeting and obtain a good computer tomography image by improving pharmacokinetic properties, and reduce cytotoxicity, so that the nanoparticle is useful in contrast media for X-ray computer tomography. A nanoparticle contains a lipiodol core, a biocompatible polymer-crosslinked coating surrounding the core, a cross-linker for forming a crosslink between the biocompatible polymers, which is linked to at least one functional group selected from amine group, carboxyl group, hydroxyl group and thiol group, and has an average diameter of 50-200 nm, wherein the biocompatible polymer is selected from polyethylene oxide/polypropylene oxide/polyethylene oxide block copolymer, polylactic acid-glycolic acid(PLGA) copolymer, PEG(polyethylene glycol)ylated PLGA, poly lactic acid(PLA) and PEGylated PLA. A contrast medium for X-ray computer tomography contains 30-70 wt.% of the nanoparticle containing lipiodol. Further, a weight average molecular weight of the cross-linker is 1000 to 20000 Dalton and a weight average molecular weight of the biocompatible polymers is 1000 to 20000 Dalton.

Description

NANOPARTICLE CONTAINING LIPIODOL AND CONTRAST MEDIA FOR X-RAY COMPUTER TOMOGRAPHY COMPRISING THE SAME}

Figure 1a schematically shows the synthesis of nanoparticles comprising Pluronic / PEG encapsulated Lipiodol according to the present invention, 1b schematically shows that the biomolecules conjugated to the nanoparticles.

F127을 니트로페닐 클로로포르메이트(NPC)로 활성화시키고 PEG-아민을 이용하여 가교시키는 과정을 모식적으로 보여주는 것이다.2 Pluronic

It shows schematically the process of activating F127 with nitrophenyl chloroformate (NPC) and crosslinking with PEG-amine.

3A and 3B are micro-CT images obtained by injecting the nanoparticles of the present invention into the tail vein of the mouse, and FIG. 3A shows the distribution of the nanoparticles injected into the mouse tail vein over time, and FIG. 3B. Shows the shading of organs and major blood vessels formed by nanoparticle injection (b: shadow on the heart 10 minutes after injection, c: shadow on the heart and major blood vessels 4 hours after injection, d: heart and 4 hours after injection) Shading in the lungs, e: shading in the spleen and liver 24 hours after injection).

Figure 4 is a photograph showing the radiopacity of the nanoparticles of the present invention.

5A is a scanning electron microscope (SEM) image of the nanoparticles of the present invention, and FIG. 5B is a transmission electron microscope (TEM) image.

F127 분자들 사이를 가교한 본 발명의 나노입자의 시간에 따른 크기 유지 능력을 에멀젼과 비교하여 보여주는 그래프이고, 6b는 시간에 따른 본 발명의 나노입자의 크기변화를 보여주는 그래프이다.6A is Pluronic with Branched PEG-amines

A graph showing the size retention ability of the nanoparticles of the present invention cross-linked between F127 molecules in comparison with emulsions, and 6b is a graph showing the size change of the nanoparticles of the present invention over time.

FIG. 7 is a graph showing the cytotoxicity of the nanoparticles of the present invention containing Lipiodol compared to the case of containing the same iodine concentration of iopromide as Lipiodol.

Figure 8 is a graph showing the size change with temperature for each Lipiodol content of the nanoparticles of the present invention.

The present invention relates to a nanoparticle comprising a Lipiodol core, a biocompatible polymer film surrounding the Lipiodol core, and a crosslinking agent to form crosslinks between the block copolymers, a method for preparing the same, and a contrast agent for X-ray computed tomography including the same.

Modern computer tomography (CT) is a diagnostic technique commonly used in the current clinical field. Recent developments in micro-CT, Positron-emission tomography (PET) or single photon emission tomography (single) Due to the development of X-ray-CT hybrid systems such as photon emission CT and SPECT, and the development of multi detector technology, the resolution has been remarkably improved, and its importance has increased in the field of clinical diagnosis. have.

With the development of such computed tomography technology, various computerized tomography contrast agents have been developed. Many of these contrast agents are barium sulfate or iodine-based preparations that have excellent X-ray absorption properties. In particular, idine-based preparations are widely used in clinical practice for the purpose of angiography. However, most of them are based on iodinate small molecules, which, as mentioned above, have excellent absorption properties of X-rays, but due to their rapid pharmacokinetic properties in the body when injected into blood vessels, clinical application of contrast agents In this case, it is difficult to obtain a higher quality image because the degree of rendering of the CT image is affected by various pharmacokinetic factors such as the injection amount, the injection concentration, and the injection speed. In addition, due to the viscosity and cytotoxicity of the contrast agent itself and its non-specific distribution and rapid metabolism in the body upon vascular administration, it is impossible to contrast or target microvascular vessels at all.

Accordingly, there is a need for the development of a new contrast agent with improved pharmacokinetic properties while maintaining excellent X-ray absorption properties, easy targeting, high quality computed tomography images, and reduced cytotoxicity.

In order to meet the above requirements, an object of the present invention is to provide a radiopaque nanoparticle comprising a Lipiodol core, a biocompatible polymer film surrounding the Lipiodol core and a crosslinking agent to form crosslinks between the block copolymers.

Still another object of the present invention is to provide a method for preparing the nanoparticles.

Still another object of the present invention is to provide a contrast agent for X-ray computed tomography, comprising the nanoparticles described above, which has reduced cytotoxicity and improved pharmacokinetic properties.

The present inventors have studied to solve the problem of the conventional contrast agent for X-ray computed tomography based on the iodide molecule as described above. As a result, the iodide molecule is encapsulated in a nanoparticle having a predetermined biocompatible coating component. By reducing the cytotoxicity of the iodide molecules and by allowing them to circulate in the bloodstream for a long time, passive targeting by histological properties such as vascular permeability seen in certain lesions such as cancer, The present invention has been completed by developing nanoparticles for X-ray computed tomography imaging that can be actively targeted through conjugation with biomolecules.

First of all, the present invention

A core consisting of Lipiodol;

A biocompatible polymer film surrounding the core; And

Crosslinking agent to form crosslinks between the block copolymers

It provides a radiopaque nanoparticle comprising a.

Lipiodol is a drug that is widely known as an embolizing agent for necrosis of cancer tissues by blocking blood vessels that supply nutrients to peripheral blood vessels of the hepatic artery, particularly cancer tissues. The content of Lipiodol contained in the nanoparticles of the present invention can be appropriately adjusted in a range such that it exhibits X-ray absorptivity suitable for X-ray computed tomography imaging and no embolic effect while maintaining an appropriate particle size.

In the present invention, the biocompatible polymer constituting the coating is a hydrophilic polymer, which has a relatively high iodine content, but reacts with Lipiodol, which may exhibit an embolic effect upon blood vessel injection, thereby forming a micelle (micelle) and encapsulating Lipiodol. It acts as a blocker to acting as a cytotoxic substance. Since the biocompatible polymer should have an appropriate viscosity to effectively block the cytotoxicity of Lipiodol, the weight average molecular weight is preferably 1,000 to 20,000 Daltons, more preferably 1,000 to 15,000 Daltons.

Biocompatible polymers that can be used in the present invention include polyethylene oxide (PEO) / polypropylene oxide (PPO) / polyethylene oxide (PEO) block copolymer, poly lactic-co-glycolic acid (PLGA) , PEGylated (PLGylation) PLGA, poly lactic acid (PLA), or PEGylated PLA and the like, preferably using a PEO / PPO / PEO block copolymer.

블록 공중합체들을 들 수 있다. Pluronic

블록 공중합체는 PEO-PPO-PEO로 구성된 트리블록 공중합체로, 에틸렌옥사이드(EO)와 프로필렌옥사이드(PO)의 비율에 따라 다양한 형태가 존재하며 친수성의 EO 비율이 낮을수록, 또는 소수성의 PO 비율이 높을수록 소수성이 커지고 HLB(hydrophilic lipophilic balance)는 낮아지는 양상을 보이는 양친성의 블록 공중합체이다. 이러한 Pluronic

블록 공중합체들은 생체적합성 고분자로서 약물 전달을 위한 담체로서의 유용성이 잘 알려져 있으며, 약물을 pluronic 미셀의 코어 내에 봉입시킴으로써 난용성 약물의 가용성을 증가시키거나 대사 안정성의 향상에 기여할 수 있으며, 혈액 내 순환 시간을 늘릴 수 있다는 등의 이점을 갖는 것으로 보고된 바 있다. Pluronic commercialized as an example of the PEO / PPO / PEO block copolymer

Block copolymers. Pluronic

The block copolymer is a triblock copolymer composed of PEO-PPO-PEO, and various forms exist according to the ratio of ethylene oxide (EO) and propylene oxide (PO), and the lower the hydrophilic EO ratio, or the hydrophobic PO ratio Higher amphiphilic block copolymers exhibit higher hydrophobicity and lower hydrophilic lipophilic balance (HLB). These Pluronic

Block copolymers are well known for their usefulness as carriers for drug delivery as biocompatible polymers. By enclosing drugs in the core of pluronic micelles, block copolymers can increase the solubility of poorly soluble drugs or contribute to improved metabolic stability and circulate in the blood. It has been reported to have advantages such as increased time.

블록 공중합체들의 물리 화학적 특성을 아래의 표 1에 나타내었다. Representative Pluronic

The physical and chemical properties of the block copolymers are shown in Table 1 below.

TABLE 1

a: Weight average molecular weight provided by the manufacturer (Wyandotte, MI)

b: average number of EO and PO units calculated using weight average molecular weight

c: HLB value [cloud point provided by manufacturer]

d: Critical Micelle Concentration (CMC) previously calculated using a pyrene probe.

블록 공중합체들의 대표적인 예인 Pluronic

L61, Pluronic

P85, Pluronic

F127의 구조와 분자량을 아래의 그림에 나타내었다.Pluronic

Pluronic, a representative example of block copolymers

L61, Pluronic

P85, Pluronic

The structure and molecular weight of F127 are shown in the figure below.

F127은 높은 HLB 값을 나타내고, 분자량의 12,600 정도로 비교적 크며, 물에서 높은 추출성(extractability)을 나타내기 때문에, Pluronic

F127의 양쪽 EO 말단의 하이드록시기를 활성화시키고 오일을 봉입한 미셀을 형성한 후, 유리 Pluronic 을 제거하기에 적합하기 때문에 본 발명에 유용하게 이용될 수 있다.Pluronic Used in the Spheres of the Invention

F127 is Pluronic because it shows high HLB value, relatively high molecular weight of 12,600 and high extractability in water.

After activating the hydroxyl groups at both EO termini of F127 and forming an oil-sealed micelle, it can be usefully used in the present invention because it is suitable for removing free fluoride.

The crosslinking agent serves to enhance stability by forming crosslinks between the biocompatible polymers and thereby crosslinking. The crosslinking agent may be appropriately selected and used depending on the type of biocompatible polymer used. In one embodiment of the present invention, the weight average molecular weight of the dendrimer structure capable of binding a plurality of functional groups to the terminal of the branched structure, such as a biopolymer such as heparin, polyethyleneimine (PEI), branched PEG as a crosslinking agent Polymers of 1,000 to 20,000 Daltons can be used.

In particular, in drug delivery, PEG plays a role of protecting the drug-encapsulated nanostructures from lymphocyte attack or protein adsorption, thereby increasing the stability and residence time of the drug-encapsulated nanostructures. , Carboxyl group, hydroxyl group, thiol group, etc. It can be used as a multi-branched PEG (muti-branched PEG) having one or more functional groups selected from the group consisting of two, three, six, etc. Forms of PEG are commercially available, and various functional groups can be applied at their ends. In the present invention, the branched PEG having a plurality of branches can be cross-linked the coating layer polymer more effectively because a number of suitable reactors capable of reacting with the functional groups of the micelle coating layer can be applied. In addition, the plurality of branches are provided in a free form in the coating layer even after the cross-linking is formed to extend the circulation time in the blood.

In an embodiment of the present invention, the biocompatible polymer or the crosslinking agent in the dendrimer form may be bonded to a functional group capable of forming a crosslink, thereby forming a crosslink between the biocompatible polymer through a functional group on the biocompatible polymer or the crosslinking agent.

When the functional group is bonded to the biocompatible polymer, the crosslinking agent may be activated by using an activating material capable of introducing a functional group (crosslinker, cross-linker) capable of bonding with the functional group bonded to the biocompatible polymer at the end of the crosslinking agent. In the case where a functional group is bonded to the crosslinking agent, the biocompatible polymer may be activated using an activating material capable of introducing a crosslinking linker capable of binding to the functional group bonded to the crosslinking agent at the terminal of the biocompatible polymer. In this case, the biocompatible polymers may be crosslinked through a crosslinking linker introduced into the crosslinking agent and a functional group bonded thereto, or crosslinked through a crosslinking linker introduced into the crosslinking agent and the functional group bound to the crosslinking agent.

The functional group bonded to the biocompatible polymer may be any functional group capable of forming a crosslink by combining with the functional group on the activated crosslinker by introducing the crosslinking agent or the crosslinking linker. For example, an amine group, a carboxyl group, a hydroxyl group, a thiol group It may be one or more selected from the group consisting of. In addition, the functional group bonded to the crosslinking agent may be any functional group capable of forming a crosslink by combining with the functional group on the activated biocompatible polymer by introducing the biocompatible polymer or the crosslinking linker, for example, an amine group, a carboxy group, or a hydroxyl group. It may be at least one selected from the group consisting of a period, a thiol group and the like.

As described above, the biocompatible polymer or crosslinking agent may be activated by a suitable activating material. Activation of the biocompatible polymer or crosslinking agent in the present invention means introducing a crosslinking linker capable of reacting with a functional group of a crosslinking agent (in the case of a polymer) or a polymer (in the case of a crosslinking agent) at the end of the polymer or the crosslinking agent. The crosslinking linker introduced at the terminal of the polymer or the crosslinking agent may be appropriately selected and used according to the functional group present in the polymer or the crosslinking agent.

For example, when the amine group is bonded to the biocompatible polymer, the crosslinking agent may be activated by introducing a crosslinking linker that is reactive with the amine group. On the contrary, when the amine group is bonded to the crosslinking agent, the biocompatible polymer is crosslinking reactive with the amine group. The linker may be introduced and activated. Crosslinking linkers reactive with the amine groups include succinimidyl succinate, succinimidyl glutarate, p-nitrophenyl carbonate, nitrophenyl chloroformate (NPC), isocyanates, aldehydes (e.g. propionaldehyde, amide- All amine group reactive functional groups such as propionaldehyde, urethane-propionaldehyde, butylaldehyde and the like.

In addition, when the carboxyl group is bonded to the biocompatible polymer, the crosslinking agent may be activated by introducing a crosslinking linker which is reactive with the carboxyl group. On the contrary, when the carboxyl group is bonded to the crosslinking agent, the biocompatible polymer is crosslinking reactive with the carboxyl group. The linker may be introduced and activated. The crosslinking linker reactive with the carboxyl group may be any carboxyl group reactive functional group such as hydrazide.

In addition, when the hydroxy group is bonded to the biocompatible polymer, the crosslinking agent may be activated by introducing a crosslinking linker reactive with the hydroxy group. In contrast, when the hydroxy group is bonded to the crosslinking agent, the biocompatible polymer is hydroxy. A crosslinked linker reactive with the group may be introduced and activated. The crosslinking linker reactive with the hydroxyl group may be any hydroxyl group reactive functional group such as epoxide, NPC, isocyanate, and the like.

In addition, when a thiol group is bonded to the biocompatible polymer, the crosslinking agent may be activated by introducing a crosslinking linker reactive with a thiol group. On the contrary, when a thiol group is bonded to the crosslinking agent, the biocompatible polymer is crosslinking reactive with a thiol group. The linker may be introduced and activated. The crosslinking linker reactive with the thiol group may be maleimide, vinylsulfone, ortho-pyridyl-disulfide, iodoacetamide, 4- (N-maleimidomethyl) cyclopemic acid carboxylic acid N -hydroxysuccinimide ester (SMCC), All thiol group reactive functional groups such as sulfo-SMCC.

In addition, the cross-linker may be applied together two or more different reactive functional groups. For example, a biocompatible polymer (eg, Pluronic) in which an amine group reactive functional group is introduced by reacting with NCP is reacted with SMCC or sulfo-SMCC which is a bifunctional crosslinking linker to introduce a thiol group reactive functional group, and a thiol at the end. Using a branched PEG having a group, crosslinking between the polymers can be formed by bonding between a thiol reactive functional group on the biocompatible polymer and a thiol group on a crosslinking agent (branched PEG). In addition, after reacting the biocompatible polymer with cystamine dihydrochloride, the disulfide bond is reduced by using DL-dithiotheritol to introduce a thiol group into the biocompatible polymer, and a thiol-reactive functional group The biocompatible polymer (inactivated) without branched PEG introduced or crosslinked linker can be used to form crosslinks in the coating using branched PEG introduced with a hydroxyl group reactive epoxy group.

As such, various functional groups and crosslinking linkers may be used to crosslink the coating polymer. In embodiments of the present invention, as functional groups and crosslinking linkers, it is possible to use NPCs with amine groups which can readily induce reactions by a simple method of pH control.

F127)에 활성화 물질로서 니트로페닐 클로로포르메이트 (NPC)를 반응시키면, 상기 블록 공중합체의 친수성 PEO 말단에 위치하는 히드록시기에 NPC기가 결합하며, 미셀이 형성되면 소수성 PPO 부분은 내부로 향하고 친수성 PEO 부분은 외부에 노출되어, 여기에 결합된 NPC도 되에 노출되게 된다. 상기 NPC기는 적절한 조건 (예컨대 pH 9 정도)에서 아민기와 반응하여 공유결합이 가능하므로, 가교제로서 아민기를 갖는 물질과 반응하면, 상기 NPC기와 가교제의 아민기 간 공유 결합이 일어나 가교를 형성하게 되어 미셀의 구조를 안정하게 유지할 수 있게 된다. 상기의 활성화 및 안정화 과정을 아래의 그림에 나타내었다:In one embodiment of the invention, a PEO / PPO / PEO block copolymer (eg, Pluronic

When Nitrophenyl chloroformate (NPC) is reacted with F127) as an activating material, an NPC group is bonded to a hydroxyl group located at the hydrophilic PEO end of the block copolymer, and when a micelle is formed, the hydrophobic PPO portion is directed inward and the hydrophilic PEO portion Is exposed to the outside, and the NPC bound thereto is also exposed to the bottom. Since the NPC group is capable of covalent bonding by reacting with an amine group under appropriate conditions (eg, pH 9), when reacted with a material having an amine group as a crosslinking agent, covalent bonding occurs between the NPC group and the amine group of the crosslinking agent to form a crosslink. The structure of can be kept stable. The activation and stabilization process is shown in the figure below:

In an embodiment of the present invention, the crosslinking agent may be a branched polyethylene glycol branched amine having an amine group at the terminal end.

Nanoparticles of the present invention is also referred to herein as nanocapsules as capsule-type nanoparticles having the core and the film structure as described above. The nanoparticles are micro-sized to allow manual targeting by vascular permeation in tissues with appropriate size conditions and long-term circulation in the bloodstream and increased vascular permeability such as tumors to encapsulate Lipiodol inside to block cytotoxicity. It is good to meet all of the conditions, and the average diameter is preferably 50 to 200 nm, preferably 100 to 150 nm.

In addition, the nanoparticles of the present invention may be conjugated with appropriate biomolecules to enable active targeting to a desired target organ or target tissue, in addition to passive targeting by vascular permeation.

In the present invention, by using a polymer having a dendrimer structure having a plurality of branches as a crosslinking agent, by combining a plurality of functional groups, a functional group present in the free state without being used for crosslinking of the biocompatible polymer among the plurality of functional groups to be bonded It may be conjugated with a molecule. For example, the biomolecules can be conjugated to the nanoparticles via exposed free amine groups of branched polyethylene glycol-amines having amine groups as functional groups used as crosslinking agents. As such, the nanoparticles into which the biomolecules are introduced into the free functional groups are still crosslinked with the polymer of the coating, and thus the overall stability of the nanoparticles is not affected. Biomolecules usable for active targeting in the present invention may be selected from the group consisting of ligands such as antibodies, peptides, receptors, and nucleic acids such as DNA and RNA. The biomolecule may be appropriately selected depending on the target cell, target tissue or target organ. The amount of the biomolecule can be appropriately adjusted according to the distribution and expression of the target material.

In the nanoparticles of the present invention, the particle size varies according to the content ratio of Lipiodol and the biocompatible polymer, and as the weight ratio of the biocompatible polymer / lipiodol increases, the particle exhibits thermally sensitive swelling / shrinkage behavior. Is significantly smaller and cannot form a correct micelle, and the closer the ratio is to 1, the less thermally sensitive swelling / shrinkage behavior (see FIG. 8). In addition, when the biocompatible polymer / lipiodol weight ratio is less than 1, the particle size tends to be rather large. Therefore, in the present invention, in order to form the correct micelle, to maintain the appropriate particle size in the body temperature range, and to ensure the size stability according to the temperature change, the weight ratio of the biocompatible polymer and Lipiodol is 1: 0.5 to 1 (polymer weight: Lipiodol weight), the closer the biocompatible polymer / Lipiodol weight ratio to 1 shows a stable size distribution. In addition, the mixing ratio of the biocompatible polymer and the crosslinking agent can be appropriately adjusted according to the respective functional groups, and is preferably 1: 0.2 to 0.125 (mole number of polymers: mole number of crosslinking agents) on a molar basis.

When the amount of Lipiodol is less than the above range, the X-ray absorption characteristic is lowered, and temperature sensitive swelling / shrinkage phenomenon occurs, making it difficult to secure stability against temperature change. When the amount of Lipiodol is higher than the above range, nanoparticle formation is not performed correctly or centrifugation is performed. When the nanoparticles are recovered, they may remain in an unsealed form due to the nature of the large specific gravity of the Lipiodol, and thus may exhibit toxicity. In addition, if the content of the biocompatible polymer is more than the above range, the Lipiodol content is lowered to lower the Xray absorption characteristics, it is difficult to secure stability against temperature changes, and if less than the above range, the Lipiodol is not sufficiently wrapped and the particles are not formed properly, It is good to set it as said range. In addition, the content of the crosslinking agent is preferably in the above range in order to sufficiently crosslink the biocompatible polymer to enhance stability.

In a preferred embodiment of the present invention, the nanoparticles are extremely cytotoxic and the biocompatible polymer activated with an appropriate crosslinking linker is encapsulated by encapsulating Lipiodol which can cause embolism in blood vessels by amphiphilic properties to form micelles. To impart radiopacity to the inside of the micelle, and stabilize cross-linking of polymers forming the micelle by using a bond between a crosslinking linker exposed on the surface of the radiopaque micelle and a functional group bonded to a dendrimer-type crosslinking agent from the outside. It is a radiopaque encapsulated nanoparticle of stable structure obtained by making it, and is very useful for the imaging purpose for X-ray computed tomography.

F127를 가교시키지 않고 리피오돌과 혼합하면 수중유 (oil-in-water) 에멀젼 형태를 갖지만, 적절한 가교제에 의하여 가교되면 Pluronic

F127이 피막을 형성하고 리피오돌을 봉입하여 캡슐형태의 나노입자 형태를 갖게 된다. 본 발명과 같은 나노입자는 에멀젼과 비교하여 약물동력학적 특성이 개선되어 안정성이 증진된다는 이점을 갖는다. 도 1b는 본 발명의 나노입자 표면의 유리 아민기에 생체분자가 결합된 것을 보여주는 모식도이다. 도 2는 본 발명의 구체예에 따라, Pluronic

F127를 니트로페닐 클로로포르메이트로 활성화시키고 폴리에틸렌글리콜-아민을 사용하여 가교시키는 과정을 모식적으로 보여준다. 도 4는 본 발명에 따른 나노입자가 들어있는 샘플 바이얼과 증류수가 들어있는 샘플 바이얼을 같이 놓고 컴퓨터 단층촬영하여 X-선 흡수 정도를 비교한 것으로, 검게 보일수록 X-선 투과율이 낮다는 것을 의미한다. 도 4의 왼쪽의 NCs로 표시된 것이 본 발명의 나노입자 영상이고, 오른쪽의 DW로 표시된 것이 증류수의 영상이다. 도 4에서 확인할 수 있는 바와 같이, 본 발명에 따른 나노입자는 방사선 비투과성 성질을 갖는 것을 알 수 있다. Figure 1a schematically shows the synthesis process of the nanoparticles of the present invention. As shown in FIG. 1, Pluronic

When F127 is mixed with Lipiodol without crosslinking, it has an oil-in-water emulsion form, but when crosslinked with a suitable crosslinking agent, Pluronic

F127 forms a film and encapsulates Lipiodol to form a capsule-shaped nanoparticle. Nanoparticles such as the present invention have the advantage that the pharmacokinetic properties are improved compared to the emulsion to improve the stability. Figure 1b is a schematic diagram showing that the biomolecule is bonded to the free amine group on the surface of the nanoparticles of the present invention. 2 is a Pluronic, in accordance with an embodiment of the invention.

The process of activating F127 with nitrophenyl chloroformate and crosslinking with polyethyleneglycol-amine is shown schematically. 4 is a comparison of X-ray absorption by computed tomography with a sample vial containing nanoparticles and a sample vial containing distilled water according to the present invention. Means that. The left side of the NCs shown in Figure 4 is an image of the nanoparticles of the present invention, the right side of the DW shown in the image of distilled water. As can be seen in Figure 4, it can be seen that the nanoparticles according to the present invention has radiopaque properties.

In another aspect, the invention

1) dissolving the biocompatible polymer in an organic solvent,

2) preparing an organic phase by adding Lipiodol to the obtained solution;

3) dissolving a crosslinking agent capable of crosslinking the biocompatible polymer molecule in an aqueous solvent to prepare a weak base water phase;

4) reacting the organic phase obtained in step 2) slowly dropwise with the aqueous phase obtained in step 3);

5) concentrating the obtained reaction product under reduced pressure to remove residual solvent and recovering nanoparticles.

It includes, it provides a method for producing nanoparticles according to the present invention.

In the method, between step 1) and step 2), an active material of the copolymer is added to a solution in which the biocompatible polymer obtained in step 1) is dissolved in an organic solvent to activate the copolymer, and then to be dissolved in an organic solvent. Step 1-1) may further include.

In addition, the method may further comprise the step of reacting by adding a biomolecule for active targeting, between step 4) and step 5).

Lipiodol, a biocompatible polymer, a copolymer active material, a crosslinking agent, and a biomolecule in the method for preparing nanoparticles of the present invention are as described above.

The organic solvent used in steps 1) and 1-1) may be selected from the group consisting of C1 to C5 lower alcohols and methylene chloride, preferably methylene chloride.

The aqueous solvent used in step 3) may be distilled water or distilled water saturated with an organic solvent selected from the group consisting of C1 to C5 lower alcohol and methylene chloride, preferably distilled water saturated with methylene chloride. The solution obtained in step 3) is weakly basic, and preferably has a pH of 8 to 10, preferably about pH 9.

In step 5), it is preferable to reduce the pH of the obtained reaction product to 4 or less and perform vacuum concentration. There is no particular limitation on the method for recovering the nanoparticles, and all conventionally used methods such as centrifugation and the like can be used.

Nanoparticles obtained according to the above method can be used by repeatedly washing the process of dispersion in physiological saline, lyophilized and stored in a suitable dispersion medium such as physiological saline.

In another aspect, the present invention provides a contrast agent for X-ray computed tomography comprising the nanoparticles.

The contrast agent of the present invention may be in the form of a dispersion obtained by dispersing the nanoparticles in a suitable dispersion medium such as physiological saline. The higher the content of the nanoparticles in the contrast medium, a clearer image can be obtained, but if too high affects the osmotic pressure, the higher the range does not affect the osmotic pressure is advantageous. In view of this point, the content of the nanoparticles in the contrast medium of the present invention is preferably in the range of 30 to 70% by weight, preferably 40 to 60% by weight. Since the nanoparticles of the present invention use an amphiphilic polymer as a film component, the nanoparticles have an advantage that the viscosity is relatively low compared to conventional contrast agents when applied as a contrast agent, thereby reducing the inconvenience during injection.

Contrast agent according to the present invention can overcome the rapid pharmacokinetic characteristics and non-specific body distribution of the conventional contrast agent, the advantage that can improve lesion diagnosis efficiency through computed tomography images in clinical diagnosis by computed tomography Has

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are merely illustrative of the present invention, and the scope of the present invention is not limited by these examples.

[ Example ]

Example  1: Activation of Biocompatible Polymers

F127 (Mw 12500, BASF, Wyandotte, MI) 10g을 80 ℃에서 감압 건조 후, 증류된 메틸렌클로라이드 30ml에 용해시켰다. 얻어진 용액에 증류된 트리에틸아민 300ul과 니트로페닐 클로로포름 (NPC) 1.3g을 차례로 첨가하여 실온에서 24시간동안 반응시켜, Pluronic

F127을 활성화시켰다. 미반응 NPC를 제거하기 위하여, 상기 반응 용액을 분별깔때기로 옮기고, 5.5M NaCl로 포화된 증류수로 2회 세척하고, 유기상만을 취하였다. 황산나트륨을 이용하여 6시간 내지 하룻밤동안 반응시켜 상기 유기상으로부터 남은 물을 제거하고, 감압 건조하여 활성화된 Pluronic

F127만을 수집하였다 (수율 88%). 상기 수집된 활성화된 Pluronic

F127는 질소를 충진한 용기에 저온 보관하였다가 나노입자 제조에 사용하였다.Pluronic

10 g of F127 (Mw 12500, BASF, Wyandotte, MI) was dried under reduced pressure at 80 ° C., and then dissolved in 30 ml of distilled methylene chloride. To the obtained solution was added 300ul of distilled triethylamine and 1.3g of nitrophenyl chloroform (NPC) in order, followed by reaction at room temperature for 24 hours.

F127 was activated. To remove unreacted NPC, the reaction solution was transferred to a separatory funnel, washed twice with distilled water saturated with 5.5 M NaCl, and only the organic phase was taken. 6 hours to overnight reaction using sodium sulfate to remove the remaining water from the organic phase, and dried under reduced pressure to activate the activated Pluronic

Only F127 was collected (yield 88%). Activated Pluronic Collected

F127 was stored at low temperature in a nitrogen-filled container and used to prepare nanoparticles.

Example  2: Preparation of Nanoparticles

F127 50mg을 메틸렌클로라이드 200ul에 녹인 후, 얻어진 용액에 리피오돌 (Lipiodol Ultra Fluid;Guerbet; France) 50mg을 첨가하여 얻어진 결과물을 유기상으로 하였다. 분지형 폴리에틸렌글리콜-아민 (6 arm, Mw 12,000; sunbio, Korea) 10mg을 메틸렌클로라이드로 포화된 증류수 2ml에 녹인 후, 1M NaOH를 가하여 pH 9정도의 약염기상태의 용액을 얻어서, 이를 수상으로 하였다.Activated Pluronic Collected

50 mg of F127 was dissolved in 200ul of methylene chloride, and then 50 mg of Lipiodol (Lipiodol Ultra Fluid; Guerbet; France) was added to the obtained solution to obtain an organic phase. 10 mg of branched polyethylene glycol-amine (6 arm, Mw 12,000; sunbio, Korea) was dissolved in 2 ml of distilled water saturated with methylene chloride, and then 1M NaOH was added to obtain a weakly basic solution having a pH of about 9, which was used as an aqueous phase.

The organic phase obtained above was sonicated for at least 3 minutes while slowly falling into the aqueous phase. After the completion of the sonication, 1M HCl was added to the obtained solution to reduce the pH to 4 or less, and the residual organic solvent was removed using a rotary depressurizer (buch. Retavafor-r200), and a high speed centrifuge (SORVALL) was used. Only nanoparticles were recovered.

SEM and TEM images of the recovered nanoparticles are shown in FIGS. 5A and 5B, respectively. Figure 5a shows a SEM image of the nanoparticles, the size of the obtained nanoparticles ranged from about 100 to 200 nm, it can be seen that it has a stable and constant spherical structure. Figure 5b shows a TEM image of the nanoparticles, the high electron density Lipiodol appears as a black dot, it can be seen that the Lipiodol is evenly distributed in the nanoparticles.

The nanoparticles obtained above were dispersed in physiological saline and dialyzed for about 2 days, and then lyophilized (EYELA FDU2100; Japan) and stored in a nitrogen-filled state at 0 ° C or less until used.

Example  3: Preparation of Nanoparticles Combined with Biomolecules

F127 8uM과 다중 분지형 PEG-아민 (6 arm, Mw 12000; sunbio) 1uM를 사용하고, Pluronic

F127과 리피오돌은 동일 중량으로 사용하고, Pluronic

F127과 PEG의 몰비를 8:1로 하여, 상기 실시예 2의 방법으로 나노입자를 제조하였다. 상기 얻어진 나노입자를 증류수에 분산시키고, 설포-SMCC(15 uM)와 25℃에서 1일간 반응시킨 후, 초원심분리 방법으로 나노입자만을 회수 하였다. Pluronic activated with NPC obtained in Example 1

Pluronic using F127 8uM and 1uM of multi-branched PEG-amine (6 arm, Mw 12000; sunbio)

F127 and Lipiodol are used in the same weight, and Pluronic

Nanoparticles were prepared by the method of Example 2 with a molar ratio of F127 to PEG of 8: 1. The obtained nanoparticles were dispersed in distilled water, and reacted with sulfo-SMCC (15 uM) at 25 ° C. for 1 day, and only nanoparticles were recovered by ultracentrifugation.

Cetuximab 항체 (Merck) (6.65nmol)를 소듐 보레이트 버퍼 (pH=8; Supplemented with 0.1 mM EDTA)에서 2-이미노티올란(Traut' reagent; Pierce)과 40:1의 몰비(Cetuximab 몰수: Traut' reagent 몰수)로 1시간 동안 반응 시켜 티올화시켰다. Cetuximab 항체는, 종양세포의 성장을 자극하며, 화학 요법이나 방사선 요법 후의 암세포의 생존을 도우며, 종양세포 아팝토시스를 억제하고, 혈관생성을 촉진하고 종양세포의 전이성을 증진시키는 EGFR(Epidermal Growth Factor Receptor)를 표적화하는 IgG1 키메릭 모노클로날 항체로서 암세포에서 발현 되는 증식 신호 전달 체계인 티로신 키나아제와 종양세포의 EGFR 결합을 억제하고, 항원-항체 복합체를 형성하여 면역세포로 하여금 종양세포의 EGFR을 제거하여 암세포의 비정상적인 성장을 막아주는 역할을 한다.CERBITUX

Cetuximab antibody (Merck) (6.65nmol) was added to 2-iminothiolane (Traut 'reagent; Pierce) and 40: 1 molar ratio (Cetuximab mole: Traut' reagent) in sodium borate buffer (pH = 8; Supplemented with 0.1 mM EDTA). Molol) for 1 hour to thiolated. Cetuximab antibodies stimulate the growth of tumor cells, help the survival of cancer cells after chemotherapy or radiation therapy, inhibit tumor cell apoptosis, promote angiogenesis, and promote tumor cell metastasis. IgG1 chimeric monoclonal antibody targeting Receptor) inhibits EGFR binding between tyrosine kinase, a proliferative signal transduction system expressed in cancer cells, and tumor cells, and forms an antigen-antibody complex to allow immune cells to suppress EGFR of tumor cells. Eliminates abnormal growth of cancer cells by removing it.

Cetuximab 항체가 콘주게이션된 나노입자만을 회수하였다.Centricon YM-30 (Amicon) was used to remove the remaining reagents, and the antibody was concentrated and dissolved in 50ul of 0.01M HEPES buffer (pH = 7), and the thiolated antibody and the nanoparticles had a molar ratio of 1: 3 (thiol). Molarized antibody molar number: maleimide mole number) was reacted by stirring at room temperature for 1 day. Then, perform ultracentrifugation again to CERBITUX

Only nanoparticles to which the Cetuximab antibody was conjugated were recovered.

Experimental Example  1: Micro-CT Application Experiment

The dispersion obtained by dispersing 2 g of the nanoparticles obtained in Example 2 in 5 ml of physiological saline was sonicated for about 30 seconds and then used as a contrast agent for X-ray computed tomography.

200ul / head of the dispersion was injected into the tail vein of the mouse (Male BALB / c nude mouse, 20 ~ 25g), and after 0, 10 minutes, 4 hours, 24 hours and 72 hours, respectively, micro-CT was performed on the whole body. The vascular structure was contrasted. The mouse model is a model produced by itself with reference to "A conpat spect / ct system for small animal imagin; Key jo hong, IEEE Transactions on nuclear science, vol 53, No. Oct 2006).

Figure 3a shows the results of tracking the distribution of the body over time of the nanoparticles injected into the mouse tail vein thus obtained. As shown in FIG. 3A, the organs and main blood vessels were distributed evenly in the body until 4 hours after the injection, compared to before the nanoparticle injection, and accumulated in the spleen and liver after 24 hours. It showed an aspect. It is thought that the nanoparticles are treated by lymphocytes and accumulate in the spleen and liver areas where lymphocytes are mainly distributed. After 72 hours, the spleen and liver disappear completely. Figure 3b shows the shading of organs and major blood vessels formed due to nanoparticle injection, b is the shadow occurring in the heart 10 minutes after the injection, c is the shadow occurring in the heart and each major blood vessel 4 hours after the injection. , d is the shadow of the heart and lung 4 hours after the injection, e is the shadow of the spleen and liver 24 hours after the injection.

These results show that the contrast agent according to the present invention is distributed for a long time in the body for more than 4 hours, when the contrast agent using the conventional iodide molecule is injected into the tail vein of the mouse due to the metabolic rate which is much higher than that of humans and tested simultaneously. This is a very meaningful result considering that it is difficult to see the shadows generated by CT scan without sacrificing the object.

Experimental Example  2: stability experiment

In order to confirm the enhanced stability of the copolymer-crosslinked nanoparticles by the crosslinking agent according to the present invention, the dispersion of the nanoparticles prepared in Example 2 and the particles containing no crosslinking agent were measured to measure the stability of the emulsion. By comparison.

The emulsion used as a comparative example in this Experimental Example was prepared in the same manner as in Example 2 except that no branched polyethylene glycol-amine was used.

100 g of the nanoparticles prepared in Example 2 of the present invention was dispersed in 2 ml of physiological saline and the particle size of the emulsion was measured according to time and is shown in FIG. 6a. As shown in Figure 6a, while the nanoparticles of the present invention maintain a constant size for more than 72 hours, the emulsion is agglomerated from the beginning of the measurement it can be seen that the particle size increases to be larger than five times the initial within 24 hours. Therefore, it can be seen that the nanoparticles according to the present invention exhibit excellent stability in the dispersion state.

In addition, the particle size of the nanoparticles according to Example 2 of the present invention was measured at different temperatures, and the results are shown in 6b. As can be seen in Figure 6b, it can be seen that the nanoparticles according to the present invention maintain size stability at various temperature ranges.

Experimental Example  3: Cytotoxicity Experiment

A549 cell line (ATCC; American Type Culture Collection) was incubated in 10% FBS (fetal bovine serum) (GIBCO) conditions in RPMI1640 medium (GIBCO). After inoculating 50000 cells per well into a 96-well plate, after treatment with the free form of iopromide (Schering Korea) and nanoparticles prepared in Example 2 of the present invention After incubation for 20 hours at 37 ℃, washed with 1x PBS (phosphate buffered saline, pH 7.6). The throughputs of iopromide and Lipiodol were 0.1, 1, 3, 5, 7, 10, 30, 50, 70, 100, 300, 500, and 700 Mm, respectively, based on the iodine content. Absorbance values at 690 nm and 450 nm were measured using a 2,3-bis [2-Methoxy-4-nitro-5-sulfophenyl] -2Htetrazolium-5-carboxyanilide inner salt (XTT) assay (Sigma Aldrich) method. Pro, Tecan AG) and normalized to assess each cytotoxicity.

The result obtained above is shown in FIG. As shown in Figure 7, it can be seen that the nanoparticles of the present invention is low in cytotoxicity at the same iodine concentration as compared to the case of using the iopromide.

As described above, the present invention encapsulates materials with high iodine concentrations into nanocapsules to improve the rapid pharmacokinetic properties and nonspecific body distribution of conventional contrast agents, allowing nanocapsules to exist for a long time in the bloodstream. In addition, by minimizing the influence of CT on the CT image due to the pharmacokinetic properties of the conventional contrast agent, a higher quality CT image can be obtained, and it can be used as a microvascular contrast agent that is difficult to apply as a conventional contrast agent. In addition, the effect of actively targeting the lesions by modifying the biological material on functional groups scattered on the branched PEG-amine ends of the contrast medium, including passive targeting by pathophysiological characteristics (such as high vascular permeability) of lesions such as cancer Can be.

Claims (20)

A core consisting of Lipiodol; A film in which the biocompatible polymer surrounding the core is crosslinked; And It includes a cross-linking agent to form cross-linking between the biocompatible polymer, The biocompatible polymer or crosslinking agent is bound to at least one functional group selected from the group consisting of amine groups, carboxyl groups, hydroxyl groups and thiol groups, Having an average particle diameter of 50 to 200 nm, Radiopaque nanoparticles. The method of claim 1, The biocompatible polymer has a polyethylene oxide / polypropylene oxide / polyethylene oxide block copolymer, a polylactic acid-glycolic acid (PLGA) copolymer, a PEGylated PLGA, a polylactic acid (PLA), and a weight average molecular weight of 1,000 to 20,000 Daltons. At least one selected from the group consisting of PEGylated PLA, nanoparticles. The method of claim 1, The crosslinking agent has a dendrimer structure having a weight average molecular weight of 1,000 to 20,000 Daltons, and at least one selected from the group consisting of polyethylene glycol (PEG), polyethyleneimine (PEI), and heparin. The method of claim 1, The weight ratio of the biocompatible polymer to Lipiodol is 1: 0.5 to 1 (biocompatible polymer weight: Lipiodol weight), and the molar ratio of the biocompatible polymer to the crosslinking agent is 1: 0.2 to 0.125 (mole number of biocompatible polymer: mole number of crosslinking agent). particle. The method of claim 1, One of the biocompatible polymer and the crosslinking agent is combined with an amine group, and the other is an amine group reactive functional group introduced therein. The method of claim 5, The amine group reactive functional group is at least one selected from the group consisting of succinimidyl succinate, succinimidyl glutarate, p-nitrophenyl carbonate, nitrophenyl chloroformate (NPC), isocyanate, and aldehydes Phosphorus Nanoparticles. The method of claim 1, Any one of the biocompatible polymer and the crosslinking agent is combined with a carboxyl group, and the other is a nanoparticle having a carboxyl group reactive functional group introduced thereto. The method of claim 7, wherein Wherein said carboxyl group is a hydrazide nanoparticle. The method of claim 1, One of the biocompatible polymer and the crosslinking agent is bound to a hydroxyl group, and the other is a nanoparticle having a hydroxyl group reactive functional group introduced therein. The method of claim 9, The hydroxyl group reactive functional group is one or more nanoparticles selected from the group consisting of epoxide, nitrophenyl chloroformate, and isocyanate. The method of claim 1, One of the biocompatible polymer and the crosslinking agent is bound to a thiol group, and the other is a nanoparticle having a thiol group reactive functional group introduced therein. The method of claim 11, The thiol group reactive functional groups are maleimide, vinylsulfone, ortho-pyridyl-disulfide, iodoacetamide, 4- (N-maleimidomethyl) cyclopemic acidcarboxylic acid N-hydroxysuccinimide ester (SMCC), and sulfo -Nanoparticles of at least one selected from the group consisting of SMCC. The method according to any one of claims 1 to 12, Nanoparticles characterized in that conjugated with a biomolecule selected from the group consisting of antibodies, peptides, ligands, receptors, and nucleic acids. 1) PEGylation of polyethylene oxide / polypropylene oxide / polyethylene oxide block copolymer, polylactic acid-glycolic acid copolymer, polylactic acid, and the polylactic acid-glycolic acid copolymer or polylactic acid having a weight average molecular weight of 1,000 to 20,000 Dalton Dissolving at least one polymer selected from the group consisting of forms in an organic solvent selected from the group consisting of C1 to C5 lower alcohols and methylene chloride, 2) preparing an organic phase by adding Lipiodol to the obtained solution; 3) A dendrimer structure having a weight average molecular weight of 1,000 to 20,000 Daltons, wherein at least one crosslinking agent is selected from the group consisting of polyethylene glycol (PEG), polyethyleneimine (PEI), and heparin in distilled water, or C1 to C5 lower alcohol and methylene chloride. Preparing an aqueous phase having a pH of 8 to 10 by dissolving in distilled water saturated with an organic solvent selected from the group consisting of; 4) reacting the organic phase obtained in step 2) slowly dropwise with the aqueous phase obtained in step 3); 5) concentrating the obtained reaction product under reduced pressure to remove residual solvent and recovering nanoparticles. Including, The biocompatible polymer or crosslinking agent is bound to at least one functional group selected from the group consisting of amine groups, carboxyl groups, hydroxyl groups and thiol groups, Method for producing a nanoparticle according to any one of claims 1 to 4. The method of claim 14, The crosslinking agent is bound to at least one functional group selected from the group consisting of an amine group, a carboxy group, a hydroxyl group and a thiol group, Between step 1) and step 2), a step of introducing at least one selected from the group consisting of an amine group reactive functional group, a carboxyl group reactive group, a hydroxy group reactive functional group and a thiol reactive functional group to the polymer obtained in step 1) Manufacturing method comprising the. The method of claim 14, The group biocompatible polymer is bound to at least one functional group selected from the group consisting of an amine group, a carboxyl group, a hydroxyl group and a thiol group, Between step 3) and step 4), the step of introducing at least one member selected from the group consisting of an amine group reactive functional group, a carboxyl group reactive group, a hydroxy group reactive functional group and a thiol reactive functional group to the crosslinking agent obtained in step 3) Manufacturing method comprising the. The method of claim 14, Conjugating between step 4) and step 5) by adding a biomolecule selected from the group consisting of antibodies, peptides, ligands, receptors, and nucleic acids. A contrast agent for X-ray computed tomography, comprising the nanoparticles according to any one of claims 1 to 12. The method of claim 18, Contrast agent for X-ray computed tomography having a content of the nanoparticles 30 to 70% by weight. The method of claim 18, The nanoparticles are conjugated with a biomolecule selected from the group consisting of antibodies, peptides, ligands, receptors, and nucleic acids to enable active targeting, contrast agent for X-ray computed tomography.

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