KR20200087722A - Manufacturing method of carboxymethyl-dextran nanoparticle and carboxymethyl-dextran nanoparticle prepared by the method - Google Patents

Abstract

The present invention relates to a method for preparing crosslinked carboxymethyl-dextran nanoparticles, including the step of forming an intermolecular or intramolecular cross-linking of carboxymethyl-dextran by irradiating an electron beam to a carboxymethyl-dextran solution.

Description

Method for synthesizing carboxymethyl-dextran nanoparticles, and carboxymethyl-dextran nanoparticles prepared by this method TECHNICAL FIELD

The present invention relates to a method for producing nanoparticles having a biocompatible, pure carboxymethyl-dextran crosslinker without a crosslinking agent, and nanoparticles thus prepared, and utilization of such nanoparticles.

Carboxymethyl-dextran is a glucose polymer made by decomposing sugar cane into bacteria, and is widely used as a blood plasma substitute for severe bleeding and shock.

Nanoparticles such as a hydrogel of pure carboxymethyl-dextran, which is biocompatible and is biocompatible, will be essential for use as a contrast agent and in vivo, and converts carboxymethyl-dextran into nanoparticle form through crosslinking Research to utilize and use is still in its infancy, and research to synthesize nanoparticles without using a crosslinking agent has not been conducted.

On the other hand, there has been provided a method for producing nanoparticles by irradiating an electron beam to an aqueous carboxymethyl-dextran solution (Korean Patent No. 10-1893549), but the concentration of the aqueous carboxymethyl-dextran solution is high and the electron beam intensity is at a certain level. There was a limit to the generation of nanoparticles only in the above case.

Accordingly, the present invention recognizes the above-mentioned conventional problems and needs, and provides a novel method for preparing biocompatible and pure carboxymethyl-dextran nanoparticles or nanogels.

The method of the present invention provides a method for forming carboxymethyl-dextran crosslinked nanoparticles in an aqueous solution other than an organic solvent, and more specifically in an acidic aqueous solution without a separate crosslinking agent, and also to control the size of the nanoparticles. It provides a way to do it.

In one aspect, a method for producing cross-linked carboxymethyl-dextran nanoparticles comprising irradiating an electron beam to an aqueous carboxymethyl-dextran solution to form an intermolecular or intramolecular crosslink of carboxymethyl-dextran do.

In the present invention, the nanoparticle refers to a particle having a size of several hundreds to hundreds of nanometers (nm, a material of one billionth of a meter). In the present invention, the nanoparticle may be characterized in that the particle size is 1 to 100 nm, but is not limited thereto. In the present invention, the nanoparticles are concepts including nanogels or nanohydrogels. Hereinafter, nanoparticles, nanogels, or nanohydrogels are all used to mean nanoparticles according to the present invention.

The aqueous solution is characterized in that it does not contain a crosslinking agent and an organic solvent.

After irradiating the electron beam, dialysis and freeze-drying steps are further included.

The aqueous carboxymethyl-dextran solution is characterized in that it is acidic, and for the acidic aqueous solution, HClO ₄ may be additionally included in the solution containing the carboxymethyl-dextran. As described and demonstrated in detail below, it is newly provided that the carboxymethyl-dextran aqueous solution is acidic, or when the acid is added to the carboxymethyl-dextran aqueous solution, the high nanoparticle synthesis efficiency is increased compared to the case where it is not. . It also provides that the size of the nanoparticles can be unexpectedly controlled by adjusting the pH and the concentration of the acid compound.

In the present invention, the pH of the aqueous carboxymethyl-dextran solution may be characterized in that it is 2.0 to 6.0. Preferably, the pH of the aqueous solution of carboxymethyl-dextran may be 2.5 to 5.0.

The aqueous carboxymethyl-dextran solution is characterized in that it has a concentration of 0.1 to 5.0% (w/v). Preferably, the aqueous solution of carboxymethyl-dextran is 0.2 to 4.0% (w/v), more preferably 0.3 to 3.0% (w/v), most preferably 0.5 to 2.0% (w/v) Concentration.

The aqueous solution is characterized in that it does not contain a crosslinking agent and an organic solvent.

After irradiating the electron beam, dialysis and freeze-drying steps are further included.

It is characterized in that it is possible to control the size of the carboxymethyl-dextran nanoparticles by changing the radiation dose of the electron beam, and reducing the size of the carboxymethyl-dextran nanoparticles by increasing the radiation dose of the electron beam.

In the present invention, the electron beam irradiation dose may be characterized in that 2 to 300 kGy. Preferably, the electron beam irradiation dose can be characterized in that 3 to 200kGy.

In the present invention, the total irradiation energy intensity of the electron beam may be characterized in that 0.1MeV to 5.0Mev. Preferably, the total irradiation energy intensity of the electron beam may be 0.5 MeV to 3.0 Mev, most preferably 1.0 MeV to 3.0 MeV.

It is characterized by controlling the pH of the aqueous solution to control the size of the carboxymethyl-dextran nanoparticles, and decreasing the pH of the aqueous solution to increase the size of the carboxymethyl-dextran nanoparticles.

The carboxymethyl-dextran nanoparticles are characterized by having a round spherical shape of 10 nm or less.

The nanoparticles are characterized by being hydrogels with swelling properties.

The nanoparticles are characterized by having tumor selectivity.

As another aspect, the present invention, in the group consisting of radioactive isotopes, organic fluorescent materials, inorganic materials quantum dots, magnetic resonance imaging contrast medium, computed tomography contrast medium, positron tomography contrast medium, ultrasound contrast medium, fluorescent contrast agents and pictoconversion material It provides a contrast agent comprising the above-described carboxymethyl-dextran nanoparticles labeled with at least one selected labeling material.

The nanoparticle may include a ligand compound conjugated to the nanoparticle and a radioactive isotope coordinated to the ligand compound.

The ligand compound is NODA-GA-NH ₂ , Characterized in that at least one of DOTA-GA, DOTA, TETA and NOTA.

The radioactive isotopes are ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ F, ³⁸ K, ⁶² Cu, ⁶⁴ Cu, ⁶⁸ Ga, ⁸² Rb, ¹²⁴ I, ⁸⁹ Zr, ^99m Tc, ¹²³ I, ¹¹¹ In, ⁶⁷ Ga, ¹⁷⁷ Lu, ²⁰¹ Tl, ^117m Sn, ¹²⁵ I, ¹³¹ I, ¹⁶⁶ Ho, ¹⁸⁸ Re, ⁶⁷ Cu, ⁸⁹ Sr, ⁹⁰ Y, ²²⁵ Ac, ²¹³ Bi, and ²¹¹ At.

The present invention provides a method for preparing pure carboxymethyl-dextran nanoparticles in an aqueous solution without a crosslinking agent and an organic solvent.

The present invention was able to uniformly synthesize various nanometer-sized carboxymethyl-dextran nanoparticles reproducibly under optimized electron beam conditions, and as a tumor diagnostic contrast agent and therapeutic agent for carboxymethyl-dextran nanoparticles synthesized through radiolabeling Provide a use.

1 is a conceptual diagram showing that a carboxymethyl-dextran polymer is crosslinked by an electron beam to form carboxymethyl-dextran particles.
2 and 3 are graphs showing the size distribution of carboxymethyl-dextran of Example 1 of the present invention.
4 to 5 are bar graphs showing the type of carboxymethyl-dextran, the concentration of the acid, and the particle size according to the irradiation dose.
6 is a result of DLS analysis of carboxymethyl-dextran nanoparticles in one of the samples of Example 1 of the present invention.
FIG. 7 is a photograph obtained by synthesizing carboxymethyl-dextran nanoparticles in bulk and obtaining a sample in powder form after a lyophilization process.
8 is a TEM photograph of carboxymethyl-dextran nanoparticles prepared according to Example 1 of the present invention.
Figure 9 is a photograph showing the experimental results showing the hydrogel properties of the carboxymethyl-dextran nanoparticles prepared according to Example 1 of the present invention.
10 and 13 are schematic diagrams showing a process of preparing a chelating compound by conjugating a bifunctional chelate to the carboxymethyl-dextran nanoparticles of Example 2 of the present invention.
11 and 14 are schematic diagrams showing the process of labeling carboxymethyl-dextran nanoparticles of Example 2 of the present invention with Cu-64.
12 and 15 show the radiochemical purity results of the radiolabeled chelate compound of Cu-64 in the carboxymethyl-dextran nanoparticles of Example 2 of the present invention.
Figure 16 shows the stability of the radio-labeled chelate compound of Cu-64 in the carboxymethyl-dextran nanoparticles of Example 2 of the present invention in PBS (Phosphate Buffer Saline) and serum (Fetal Bovine Serum) by hourly radio-TLC It is the result of the analysis.
17 to 18 are results of biodistribution confirmation experiments in normal mice of radiolabeled chelate compounds of Cu-64 on the carboxymethyl-dextran nanoparticles of Example 2 of the present invention.
19 to 20 are the results of the biodistribution confirmation experiment in the tumor model of the radiolabeled chelate compound of Cu-64 in the carboxymethyl-dextran nanoparticles of Example 2 of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. The present invention may be variously modified and may have various forms, and specific embodiments will be illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to specific disclosure forms, and it should be understood that all modifications, equivalents, and substitutes included in the spirit and scope of the present invention are included. In describing each drawing, similar reference numerals are used for similar components.

The terms used in the present application are only used to describe specific embodiments and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this application, the terms "include" or "have" are intended to indicate that a feature, step, operation, component, part, or combination thereof described on the specification exists, and one or more other features or steps. It should be understood that it does not preclude the existence or addition possibility of the operation, components, parts or combinations thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. Terms, such as those defined in a commonly used dictionary, should be interpreted as having meanings consistent with meanings in the context of related technologies, and should not be interpreted as ideal or excessively formal meanings unless explicitly defined in the present application. Does not.

Example 1: Preparation of carboxymethyl-dextran nanogels

A carboxymethyl-dextran aqueous solution was prepared by mixing carboxymethyl-dextran in water, and experiments were conducted by irradiating the carboxymethyl-dextran aqueous solution with an electron beam, and various conditions (aqueous acidity, electron beam irradiation intensity, carboxy It was confirmed that 264 samples were synthesized as nanoparticles among a total of 1,488 samples of the concentration of methyl-dextran (w/v%).

Analysis of size distribution of nanoparticles

As can be seen in Figure 2, when analyzing the sample of the synthesized carboxymethyl-dextran, through the DLS results, some showed a larger size corresponding to approximately 20 nm, but generally 3 nm Or it was confirmed that small nanoparticles were formed at 7 nm.

Particle analysis according to acidity of aqueous solution

For the synthesis of nanoparticles, an acid was added to the aqueous solution, the experiment was conducted with different concentrations of the acid, and accordingly, the pH of the irradiated sample was changed. It was also confirmed that the range for the particle size was wider.

Particle analysis according to the molecular weight of carboxymethyl-dextran

In the case of the electron beam irradiation experiment using carboxymethyl-dextran, the experiment was conducted using two types of carboxymethyl-dextran having different molecular weights.

In the electron beam irradiation experiment using a large molecular weight carboxymethyl-dextran (150 kDa), uniform nanoparticles were not well formed, but in the case of a carboxymethyl-dextran of 10-20 kDa with a smaller molecular weight, nanoparticles were well formed. lost.

Particle analysis under various conditions

In addition, experiments were performed under different conditions of the aqueous solution.

In the experiment using carboxymethyl-dextran, when adding acid (HClO ₄ ), it was confirmed that nanoparticles were well formed, and acid synthesis experiments were performed by adding acid.

First, in a 0.2% HClO ₄ aqueous solution (pH 3.1) conditions, an experiment to check how the size of synthesized particles varies depending on the concentration of carboxymethyl-dextran (0.5%, 1%, 2%, w/v%) When proceeding, as can be seen in Figure 4, even if the concentration of carboxymethyl-dextran is different, it is confirmed that the size of the synthesized particles remains almost the same, and the concentration of carboxymethyl-dextran is nano using an electron beam. It was confirmed that there was no significant effect on the synthesis of particles.

The experiment was conducted while varying the concentration of the acid to be added (0.1%, pH 4.0 / 0.2%, pH 3.0). As can be seen in FIG. 5, the size of the synthesized particles increased as the concentration of the acid to be added increased. The tendency to increase was confirmed, and the tendency of the particle size to decrease as the electron beam dose increased.

Although experiments were conducted while varying the intensity of the electron beam energy (1 MeV, 2.5 MeV), it was confirmed that the difference in the intensity of the energy did not affect the size difference of the formed nanoparticles.

Carboxymethyl-dextran nanoparticles showing these results were synthesized in large quantities, and samples in powder form were obtained after the lyophilization process as shown in FIG. 7.

Optical analysis of nanoparticles

The synthesized carboxymethyl-dextran nanogels were also studied to confirm the size and shape of the particles through TEM. As shown in the photo of FIG. 8, although the size difference between the particles was generated to some extent, most of them were 10nm. It was confirmed that the inner spherical shape was maintained.

Hydrogel Characterization

We conducted an experiment to confirm whether the synthesized nanoparticles have the characteristics of a hydrogel that substantially immerses water (Swelling), and experimented with carboxymethyl-dextran without irradiation with pure water and electron beam as a control. Proceeded.

After dissolving the same amount of carboxymethyl-dextran nanoparticles and the control in 500 μL of water, centrifugation was performed using a centrifugal filter with a separation membrane, and the amount of water dropped on the bottom of each tube was checked. To make it visible, the water dropped under the tube was dyed with green ink and photographed.

When the results were confirmed, as shown in FIG. 9, the amount of water falling to the bottom of the tube after the centrifugation process in the nanoparticle sample obtained by irradiating the electron beam was smaller, and more solution was left in the supernatant part. I could confirm.

Through these results, it was confirmed that the carboxymethyl-dextran nanoparticles synthesized through electron beam irradiation contained water better than the control group and had characteristics as a hydrogel.

Example 2: Utilization of carboxymethyl-dextran nanogels

In order to effectively check whether the synthesized nanogels maintain stability in the body and which organs are distributed to what extent when injected in vivo, and through which pathway, the nanogels are labeled with radioisotopes to track them. In order to do this, a radiolabeling experiment was conducted. As a radioactive isotope, the half-life was 12.7 hours, and the study was conducted using a mid-life nuclide, copper-64 (Cu-64). First, in order to label Cu-64 on a nanogel, Cu ions were well bound to the nanogel. The process of conjugating the dual function chelate was performed. First, a study was conducted to select a dual-functional chelate that can be labeled well with Cu-64 by using the synthesized carboxymethyl-dextran nanogel.

As shown in the schematic diagram of FIG. 10, experiments were performed in which carboxymethyl-dextran nanogels were conjugated using a dual-functional chelator, DOTA-GA-NH ₂ , after dissolving carboxymethyl-dextran nanogels in pyridine, tosyl chloride The drop was slowly dropped, and the reaction was performed overnight. Thereafter, after adding DOTA-GA-NH ₂ of the following Chemical Formula 1, the reaction is performed for about one day with high heat, and then centrifugation of the carboxymethyl-dextran nanogel in which a conjugation reaction with DOTA-GA-NH ₂ is performed It was separated by a filter. DOTA-GA-carboxymethyl-dextran nanogel in the form of pure white powder was obtained through a lyophilization process.

[Formula 1]

Radiolabeling using Cu-64 was conducted, and as shown in the schematic of FIG. 11, DOTA-GA-carboxymethyl-dextran nanogel was added to a buffer having a pH of 6.8 and reacted with ⁶⁴ CuCl ₂ at an established temperature and time. After the reaction was completed, a process obtained by purifying only Cu-64-labeled carboxymethyl-dextran nanogels through a centrifugal filter was used to confirm radiochemical purity using Radio-TLC.

As a result, as shown in FIG. 12, it was confirmed that the radiochemical purity was 66%.

So, we experimented with conjugation to carboxymethyl-dextran nanogels using NODA-GA-NH ₂ of the formula 2 below, which is a dual-functional chelate that is expected to be able to more effectively label Cu-64 with higher yield. We wanted to confirm the radiochemical purity of Cu-64 labeled.

[Formula 2]

Similarly, the carboxymethyl-dextran nanogel was used, and as shown in the schematic of FIG. 13, the nanogel was first dissolved in pyridine, and then tosyl chloride was slowly dropped dropwise, and the reaction was performed overnight. After adding NODA-GA-NH ₂ and applying a high heat of 80° C. for about one day, the carboxymethyl-dextran nanogels conjugated with NODA-GA-NH ₂ were separated by centrifugal filtration. Did. Finally, NODA-GA-carboxymethyl-dextran nanogel in the form of pure white powder was obtained through lyophilization.

As shown in FIG. 14, a radiolabeling process using Cu-64 was performed, and NODA-GA-carboxymethyl-dextran nanogel was added to a buffer having a pH of 6.8, and reacted with ⁶⁴ CuCl ₂ at an appropriate temperature and time, followed by centrifugal filtration. After undergoing the process of purifying Cu-64-labeled carboxymethyl-dextran nanogel through radio-TLC, radiochemical purity was confirmed.

As a result, as shown in FIG. 15, unlike the result of using DOTA-GA as a dual-functional chelate, the radiochemical purity of carboxymethyl-dextran nanogel conjugated with NODA-GA is almost 100% and very high Cu- It was confirmed that the labeling reaction was excellent with 64.

In the case of the stability of the radiolabeled nanoparticles, the experiment was conducted by confirming radiolysis, and the nanoparticles were analyzed by radio-TLC by time using PBS (Phosphate Buffer Saline) and serum (Fetal Bovine Serum). I wanted to check the stability of the.

As a result, as can be seen in Figure 16, it was confirmed that it shows high stability up to 1 hour and 4 hours, and the results for 24 hours also showed excellent stability equal to or higher than 95%, thus maintaining the stability sufficiently in the mouse. I could predict it.

Biodistribution confirmation experiment in normal mice

Using a carboxymethyl-dextran (CM-dextran) nanogel labeled with Cu-64, a biodistribution confirmation experiment was conducted 24 hours after injection for the same normal mice, and distribution to the organs was confirmed. The discharge route was confirmed in vitro.

As can be seen in Figure 17, it showed the results showing the highest intake similarly to the liver and kidneys, because the particle size is small because it is small 6 nm is discharged quickly outside the body after 24 hours after injection, the amount of radioactivity remaining in the body It was confirmed that it was rather low.

For future experiments using the B16F10 tumor model, the same biodistribution verification experiment was conducted using C57BL/6 mice with black hair.

As can be seen in Figure 18, due to the difference in the type of mouse used, the degree of intake in the liver and kidney showed a slight difference, but it was confirmed that the degree and tendency of the intake distribution for other organs was almost constant. there was.

Next, using a carboxymethyl-dextran nanogel labeled with Cu-64, a biodistribution confirmation experiment was conducted for the tumor model, and the target ability for tumors and the distribution and discharge of organs were confirmed.

First, carboxymethyl-dextran nanogels were subjected to a biodistribution confirmation experiment in a melanoma B16F10 tumor model. As can be seen in Figure 19, although it showed a high intake in the liver and kidneys, it was confirmed that the intake to the tumor was high at 1.0% ID/g. The tumor-to-muscle ratio was 41.2 times, and the tumor-to-blood ratio was 7.4 times, confirming that the tumor was excellent.

Next, a biodistribution confirmation experiment was performed on the tumor model using colorectal tumor cells (CT26), and as shown in FIG. 20, the results showed the highest intake of 8.2% ID/g for the liver, Next, the intake of kidney (1.6% ID/g) and spleen (1.4% ID/g) was high.

Tumors also showed higher intake than other organs by ingesting 1.48% ID/g, Tumor to muscle ratio was 13.2 times, and Tumor to blood ratio was 3.7 times, superior to tumors. It was confirmed that can diagnose.

Claims (15)

Comprising forming an intermolecular or intramolecular crosslinking of carboxymethyl-dextran by irradiating an electron beam to an aqueous solution of carboxymethyl-dextran,
Method for preparing crosslinked carboxymethyl-dextran nanoparticles. According to claim 1,
The aqueous solution of carboxymethyl-dextran is characterized in that it is acidic,
Method for preparing crosslinked carboxymethyl-dextran nanoparticles. According to claim 2,
The alginate aqueous solution is characterized in that the pH is 2 to 6,
Method for preparing crosslinked carboxymethyl-dextran nanoparticles. According to claim 1,
HClO ₄ is further included in the solution containing carboxymethyl-dextran,
Method for preparing crosslinked carboxymethyl-dextran nanoparticles. According to claim 1,
The carboxymethyl-dextran aqueous solution is characterized in that the concentration of 0.1 to 3% (w / v),
Method for preparing crosslinked carboxymethyl-dextran nanoparticles. According to claim 1,
The electron beam is characterized in that irradiated with an irradiation amount of 2 to 300kGy,
Method for preparing crosslinked carboxymethyl-dextran nanoparticles. According to claim 1,
Characterized in that by controlling the size of the carboxymethyl-dextran nanoparticles by changing the irradiation dose of the additive electron beam,
Method for preparing crosslinked carboxymethyl-dextran nanoparticles. According to claim 1,
Characterized in that, by increasing the irradiation dose of the electron beam, the size of the carboxymethyl-dextran nanoparticles is reduced,
Method for preparing crosslinked carboxymethyl-dextran nanoparticles. According to claim 1,
Characterized in that, by controlling the pH of the aqueous solution, to control the size of the carboxymethyl-dextran nanoparticles,
Method for preparing crosslinked carboxymethyl-dextran nanoparticles. Nanoparticles formed only by intermolecular or intramolecular crosslinking of carboxymethyl-dextran prepared by claim 1, characterized in that the nanoparticles have a round spherical shape of 10 nm or less,
Carboxymethyl-dextran nanoparticles. The method of claim 10,
The nanoparticles are characterized by having tumor selectivity,
Carboxymethyl-dextran nanoparticles. Radioactive isotopes, organic fluorescent materials, inorganic materials, quantum dots, magnetic resonance imaging contrast agents, computed tomography contrast agents, positron tomography contrast agents, ultrasound contrast agents, fluorescent contrast agents, and agents labeled with one or more labeling materials selected from the group consisting of pictographs A contrast agent and a therapeutic agent comprising the carboxymethyl-dextran nanoparticles of claim 7 or 8. The method of claim 12,
A ligand compound conjugated to the nanoparticle; And
Containing a radioactive isotope coordinated to the ligand compound,
Contrast and therapeutic agents. The method of claim 13,
The ligand compound is NODA-GA-NH ₂ , Characterized in that at least one of DOTA-GA, DOTA, TETA and NOTA,
Contrast and therapeutic agents. The method of claim 13,
The radioactive isotopes are ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ F, ³⁸ K, ⁶² Cu, ⁶⁴ Cu, ⁶⁸ Ga, ⁸² Rb, ¹²⁴ I, ⁸⁹ Zr, ^99m Tc, ¹²³ I, ¹¹¹ In, ⁶⁷ Ga, Characterized in that at least one of ¹⁷⁷ Lu, ²⁰¹ Tl, ^117m Sn, ¹²⁵ I, ¹³¹ I, ¹⁶⁶ Ho, ¹⁸⁸ Re, ⁶⁷ Cu, ⁸⁹ Sr, ⁹⁰ Y, ²²⁵ Ac, ²¹³ Bi, and ²¹¹ At,
Contrast and therapeutic agents.

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