KR102382965B1 - Manufacturing method of alginic acid nanoparticle and alginic acid nanoparticle prepared by the method - Google Patents

Abstract

The present invention relates to a method for preparing cross-linked alginic acid nanoparticles, comprising irradiating an electron beam to an aqueous alginic acid solution to form intermolecular or intramolecular crosslinks of alginic acid.

Description

Method for synthesizing alginic acid nanoparticles and alginic acid nanoparticles prepared by this method

The present invention relates to a method for preparing nanoparticles that are biocompatible and pure alginic acid crosslinked form without a crosslinking agent, the nanoparticles thus prepared, and the utilization of these nanoparticles.

Alginic acid is a polysaccharide with a carboxylic acid group. It is a biopolymer contained in seaweeds such as kelp, has very low toxicity to the human body, and forms a protective film in the digestive tract to prevent damage to the digestive tract and digestive tract by gastric acid. Although it is widely used in combination with antacids, studies on the possibility of its use as a tumor diagnostic agent and therapeutic agent have not been actively conducted so far.

Nanoparticles such as hydrogels of pure alginic acid that can be used in a living body and are biocompatible will be essential for their use as contrast agents and in vivo use. Usually, hydrogels are crosslinked by adding chemicals such as a crosslinking agent and/or curing agent to a polymer material. method has been manufactured.

However, since the crosslinking agent and/or curing agent itself used in the crosslinking reaction is harmful to the living body, there will be a problem that may cause harmful effects when a hydrogel prepared using such a crosslinking agent and/or curing agent is used in the living body, When a crosslinking agent and/or curing agent is used, the residual crosslinking agent and/or curing agent in the hydrogel must be removed after the hydrogel is manufactured, so there will be a problem in that the manufacturing process is complicated and the cost is increased.

On the other hand, a method for producing nanoparticles by irradiating an electron beam to an aqueous polysaccharide solution has been provided (Korean Patent No. 10-1893549), but nanoparticles are not generated only when the concentration of the polysaccharide aqueous solution is high and the intensity of the electron beam is above a certain level. There was a limit to

Accordingly, the present invention provides a novel method for preparing biocompatible and pure alginic acid nanoparticles or nanogels, recognizing the problems and needs of the prior art.

The method of the present invention provides a method for forming crosslinked alginic acid nanoparticles in an aqueous solution rather than an organic solvent, more specifically in an acidic aqueous solution, without a separate crosslinking agent, and the crosslinked alginic acid nanoparticles are specifically loaded with an anticancer agent high rate, suggesting that it may have tumor-selective binding properties. In addition, a method for controlling the size of nanoparticles is provided.

In one aspect, the present invention provides a method for preparing cross-linked alginic acid nanoparticles, comprising irradiating an electron beam to an aqueous alginic acid solution to form intermolecular or intramolecular crosslinks of alginic acid.

In the present invention, the nanoparticles refer to particles having a size of several to several hundred nanometers (nm, a material that is one billionth of a meter). In the present invention, the nanoparticles may be characterized in that the particle size is 1 to 600 nm, but is not limited thereto. In the present invention, the nanoparticle is a concept that also includes a nanogel or a nanohydrogel. Hereinafter, nanoparticles, nanogels, or nanohydrogels are all used to mean nanoparticles according to the present invention.

The alginic acid aqueous solution of the present invention is characterized in that it is neutral in acidity. An acid can be added to the aqueous alginic acid solution for an acidic aqueous solution. Preferably, it may be any one or more of HClO ₄ , nitric acid, and formic acid. As will be described and demonstrated in detail below, when the aqueous solution of alginic acid is acidified, or when an acid is added to the aqueous solution of alginic acid, compared to the case where it is not, high nanoparticle synthesis efficiency is newly provided. It also provides that the size of nanoparticles can be unexpectedly controlled by adjusting the pH and adjusting the concentration of the acid compound added.

In one embodiment of the present invention, the pH of the aqueous alginic acid solution is characterized in that it is acidic. Preferably, the pH of the aqueous alginic acid solution may be 1 to 6, more preferably, may be 1 to 5, even more preferably, may be pH 2 to 4, most preferably pH 3 to 4 can be

The alginic acid aqueous solution is characterized in that the concentration of 0.1 to 2.0% (w / v). Preferably, the aqueous alginic acid solution is 0.1 to 1.5% (w/v), more preferably 0.1 to 1.0% (w/v), even more preferably 0.1 to 0.7% (w/v), most preferably may be at a concentration of 0.1 to 0.5% (w/v).

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 the size of the alginic acid nanoparticles is controlled by changing the irradiation dose of the electron beam, and the size of the alginate nanoparticles is reduced by increasing the irradiation dose of the electron beam.

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

In the present invention, the total irradiation energy intensity of the electron beam may be 0.1 MeV to 5 Mev. Preferably, the total irradiation energy intensity of the electron beam may be 0.5 MeV to 3 Mev, most preferably 0.5 MeV to 2.5 MeV.

It is characterized in that by adjusting the pH of the aqueous solution, the size of the alginic acid nanoparticles is controlled, and the pH of the aqueous solution is lowered to reduce the size of the alginic acid nanoparticles.

By controlling the concentration of alginic acid in the aqueous solution, it is characterized in that the size of the alginic acid nanoparticles is controlled, and the size of the alginic acid nanoparticles is increased by increasing the concentration of alginic acid in the aqueous solution.

The method for providing alginic acid nanoparticles provided by the present invention is characterized in that by irradiating an electron beam to an acidic alginic acid aqueous solution, nanoparticles can be prepared even under conditions in which nanoparticles cannot be prepared in a neutral alginic acid aqueous solution.

In particular, when irradiating an electron beam with a low concentration (for example, 0.5% (w/v) or less) aqueous solution of alginic acid in an acidic aqueous solution condition, it is very easy to control the size of nanoparticles depending on the irradiation dose intensity of the electron beam. There is an advantage in that nanoparticles of a size of

Therefore, the present invention is characterized in that, in order to easily control the size of alginic acid nanoparticles, the alginic acid aqueous solution has a pH of 1 to 5, the concentration of alginic acid is less than 1.0% (w/v), and the electron beam irradiation dose is 2 kGy or more , it may be characterized in that it provides a method for preparing cross-linked alginic acid nanoparticles. Preferably, the alginic acid aqueous solution has a pH of 2 to 4, a concentration of alginic acid of 0.7% (w/v) or less, and an electron beam irradiation dose of 3 kGy or more. can be characterized as Most preferably, it provides a method for producing crosslinked alginic acid nanoparticles, characterized in that the pH is 3 to 4, the concentration of alginic acid is 0.5% (w/v) or less, and the electron beam irradiation dose is 3 kGy or more can do.

In another aspect, it provides intermolecular or intramolecular cross-linked nanoparticles of alginic acid prepared by claim 1.

The nanoparticles are characterized in that they are agglomerated with each other.

The nanoparticles are characterized in that the hydrogel has swelling properties.

The nanoparticles are characterized in that they have tumor selectivity. The nanoparticles are characterized as having selectivity for colorectal cancer or melanoma. The nanoparticles are characterized in that they have a tumor-to-muscle ratio of 30 times or more and a tumor-to-blood ratio of 10 times or more for colorectal cancer, and the nanoparticles have a tumor-to-muscle ratio of 16 times or more and 5 times or more for melanoma. It is characterized as having a tumor-to-blood ratio.

The nanoparticles are characterized in that they have a high anticancer agent loading rate compared to nanoparticles of other polysaccharides. The anticancer agent is characterized in that doxorubicin or cisplatin.

In another aspect, the present invention provides a radioactive isotope, an organic fluorescent material, an inorganic quantum dot, a magnetic resonance imaging contrast agent, a computed tomography contrast agent, a positron tomography contrast agent, an ultrasound contrast agent, a fluorescence contrast agent, and a phase transformation material selected from the group consisting of Provided is a contrast agent comprising the above-described alginic acid nanoparticles labeled with one or more labeling substances.

It may include a ligand compound conjugated to the nanoparticles and a radioactive isotope coordinated to the ligand compound.

The ligand compound is NODA-GA-NH ₂ , It is 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, ^99ν Tc, ¹²³ I, ¹¹¹ In, ⁶⁷ Ga, ¹⁷⁷ Lu, ²⁰¹ Tl, ^117ν Sn, ¹²⁵ I, ¹³¹ I, ¹⁶⁶ Ho, ¹⁸⁸ Re, ⁶⁷ Cu, ⁸⁹ Sr, ⁹⁰ Y, ²²⁵ Ac, ²¹³ Bi, and ²¹¹ At.

In another aspect, the present invention includes a drug carrier comprising the above-described intermolecular or intramolecular cross-linked nanoparticles of alginic acid and a drug loaded in the nanoparticles, wherein the drug is an anticancer agent.

As another aspect, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising a drug delivery system loaded with an anticancer agent in the nanoparticles as an active ingredient.

The type of the anticancer agent is not particularly limited, and may include, for example, a compound, a protein, a peptide, a nucleic acid, and the like. Preferably, the anticancer agent may be doxorubicin or a platinum-based anticancer agent, and most preferably, the anticancer agent may be doxorubicin or cisplatin.

The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives in addition to the drug carrier encapsulating the drug, and the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, Colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, Stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol and talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

In addition, the composition of the present invention may be administered in various oral and parenteral formulations during actual clinical administration, and when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants, diluents or excipients such as surfactants It can be prepared using

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in a drug carrier in which a drug is encapsulated in biocompatible polymer nanoparticles, for example, starch, calcium It may be prepared by mixing carbonate, sucrose, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, and syrups, and various excipients such as wetting agents, sweetening agents, fragrances, and preservatives in addition to commonly used simple diluents such as water and liquid paraffin may be included. .

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

On the other hand, the injection may contain conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, and the like.

The dosage for the human body of the pharmaceutical composition of the present invention may vary depending on the patient's age, weight, sex, dosage form, health status and disease level, and is generally 0.01 - 100 mg/kg/day, preferably 0.1-20 mg/kg/day, and more preferably 5-10 mg/kg/day. In addition, according to the judgment of the doctor or pharmacist, divided administration may be administered at regular intervals.

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

In addition, in the present invention, an optimal electron beam irradiation condition that can be efficiently and reproducibly converted into nanoparticles using only an electron beam has been established. It was possible to uniformly synthesize alginic acid nanoparticles with a size of several hundred nanometers reproducibly under the optimized electron beam conditions, and the possibility of application of alginic acid nanoparticles synthesized through radiolabeling as a tumor diagnostic contrast agent and therapeutic agent was verified.

Alginic acid nanoparticles crosslinked by the method of the present invention have a high specific anticancer drug loading rate and tumor-selective binding properties. In addition, a method for controlling the size of nanoparticles is provided.

1 is a conceptual diagram showing that an alginic acid polymer is crosslinked by an electron beam to form alginic acid particles.
2 is a graph showing the size distribution of alginic acid of Example 1 of the present invention.
3 is a bar graph showing the size of the synthesized particles as the concentration of alginic acid increases.
4 is a bar graph showing the particle size according to the concentration of acid and the irradiation dose.
5 is a bar graph showing the particle size according to the concentration of acid, the irradiation dose, and the irradiation energy.
6 is a DLS analysis result of alginic acid nanoparticles of one of the samples of Example 1 of the present invention.
7 is a photograph of a sample obtained in powder form after synthesizing alginate nanoparticles in large quantities and freeze-drying.
8 is a TEM photograph of alginic acid nanoparticles prepared according to Example 1 of the present invention.
9 is a SEM photograph of alginic acid nanoparticles prepared according to Example 1 of the present invention.
10 is a photograph of experimental results showing the hydrogel properties of alginic acid nanoparticles prepared according to Example 1 of the present invention.
11 is a schematic diagram showing a process for preparing a chelate compound by conjugating NODA-GA-NH ₂ , which is a bifunctional chelate, to alginic acid nanoparticles of Example 2 of the present invention.
12 is a schematic diagram showing a process of labeling the alginic acid nanoparticles of Example 2 with Cu-64 of the present invention.
13 is a result of confirming the radiochemical purity of the Cu-64 radiolabeled chelate compound in the alginic acid nanoparticles of Example 2 of the present invention using Radio-TLC.
14 is a result of radio-TLC analysis of the stability of Cu-64 radiolabeled chelate compound in alginic acid nanoparticles of Example 2 in PBS (Phosphate Buffer Saline) and serum (Fetal Bovine Serum) over time by radio-TLC. .
15 is a result of an experiment to confirm the biodistribution of a chelate compound radioactively labeled with Cu-64 to alginic acid nanoparticles of Example 2 of the present invention in normal mice.
16 is a biodistribution confirmation experiment result in a tumor model using colorectal cancer tumor cells (CT26) of alginic acid nanoparticles of Example 2 of the present invention.
17 is a biodistribution confirmation experiment result in a tumor model using skin cancer cells (melanoma/B16F10) of alginic acid nanoparticles of Example 2 of the present invention.
18 is a photograph showing the result of loading of doxorubicin of Example 3 of the present invention.
19 to 21 show the tumor growth inhibitory effect of Example 3 of the present invention.
22 shows a standard curve for cisplatin of Example 4 of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. Since the present invention can have various changes and can have various forms, specific embodiments are illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to the specific disclosed form, it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention. In describing each figure, like reference numerals have been used for like elements.

The terms used in the present application are only used to describe specific embodiments and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present application, terms such as “comprise” or “have” are intended to designate that a feature, step, operation, component, part, or combination thereof described in the specification is present, and includes one or more other features or steps. , it should be understood that it does not preclude the possibility of the existence or addition of an operation, a component, a part, or a combination thereof.

Unless defined otherwise, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Example 1: Preparation of alginic acid nanogels

Alginic acid (140 kDa) was mixed with water to prepare an aqueous alginic acid solution, and an experiment was performed by irradiating an electron beam to the aqueous alginic acid solution, and various concentrations (w/v%) of alginic acid, various pH concentrations, types of acids and various A total of 1,152 samples were prepared under the conditions of the intensity of the electron beam (dose of the electron beam), and of these, 150 nanoparticles were synthesized. It was confirmed that the nanoparticle synthesis was determined according to conditions such as the acidity of the aqueous alginic acid solution, the concentration of alginic acid, and the dose of the electron beam.

Analysis of size distribution of nanoparticles

As shown in Figure 2, the synthesized nanoparticles showed a size of 100nm to 400nm, mainly 200nm.

Synthetic analysis by addition of acid to aqueous solution

The synthesis of nanoparticles according to whether acid was added was analyzed. When the presence or absence and size of nanoparticles in the sample that was tested without adding acid and the concentration of alginic acid and the electron beam irradiation dose were checked by DLS, as shown in Table 1 below, nanoparticles were generally synthesized This was not done, and the tendency to observe several peaks for different particle sizes was confirmed.

However, after the addition of 0.1% acid (HClO ₄ ) (pH 3.5), the presence or absence of particle formation and the size of the sample that was tested while controlling the concentration and electron beam irradiation dose of alginic acid could be checked by DLS as well. As shown in Table 2 below, it was confirmed that the nanoparticles were easily synthesized at an alginic acid concentration of 0.5%, and the particle size tended to decrease as the electron beam irradiation dose increased.

However, after adding 0.2% (pH 2.5) and 0.3% (pH 2.0) of acids, the same experiment was performed using alginic acid at a concentration of 0.5% (w/v), but nanoparticle synthesis was not generally made, and different The tendency to observe several peaks for the size of the particles was confirmed. Accordingly, it was confirmed that it was important to add an appropriate acid concentration during the electron beam irradiation process, and it was confirmed that the appropriate acid concentration was less than 0.2% (pH greater than 2.5).

Analysis by type of acid

In the samples that were subjected to the same experiment using other acids (nitric acid, oxalic acid, formic acid) other than HClO ₄ , as a result, it was confirmed that oxalic acid did not significantly affect the formation of alginic acid nanoparticles through electron beam irradiation. .

However, in the case of nitric acid and formic acid, an appropriate concentration of acid was required for particle formation, but it had an effect on the formation of nanoparticles, and the particle size maintained a tendency to decrease as the amount of electron beam irradiation increased.

Particle size analysis

An experiment using an alginic acid solution was carried out, and it was confirmed that nanoparticles were well formed when an acid was added in the same way. An experiment was conducted to check how the size changes.

As a result, as confirmed in FIG. 3 , it was confirmed that the size of the synthesized particles continued to increase as the concentration of alginic acid increased.

Then, the experiment was conducted with different concentrations of the added acid (0.1%, 0.2%, 0.3%), and as shown in FIG. 4 , when the irradiation dose was low, the particles synthesized as the concentration of the added acid increased It was confirmed that the tendency to increase in size was confirmed, but it was confirmed that when the electron beam was irradiated with a high irradiation dose, it did not significantly affect the particle size formation. In addition, it was confirmed that the particle size tends to decrease as the electron beam irradiation dose increases.

The experiment was conducted even under the condition that the intensity of the electron beam energy was increased to 2.5 MeV, and as shown in FIG. 5 , as in 1 MeV, as the concentration of the added acid increased, the size of the synthesized particles tends to increase. It was confirmed that the particle size tends to decrease as the electron beam irradiation dose increases.

The particle size was measured with DLS of one of the samples synthesized in Example 1, and was 156.8 nm as shown in FIG. 6 .

Alginic acid nanoparticles were synthesized in large quantities, and as shown in the photo of FIG. 7 , a sample was obtained in powder form after the freeze-drying process.

Optical Analysis of Nanoparticles

In the synthesized alginic acid nanoparticles, a study was conducted not only to measure the size of the nanoparticles through DLS, but also to directly check the morphological shape of the actually synthesized nanoparticles through a transmission electron microscope (TEM).

Each nanoparticle was dropped 2-3 drops on a carbon-coated copper grid, and after a dyeing process using uranyl acetate, it was analyzed using TEM after leaving enough time to dry the moisture completely, which is shown in FIG. 8 did It was confirmed that the sharp thorns were mainly in the protruding form, and most of them were agglomerated with each other, resulting in a larger particle size than the DLS result.

A study was conducted to confirm the size and shape of the particles once more using a scanning electron microscope (SEM) instead of TEM using the same alginic acid nanoparticles, which is presented in FIG. 9 . As with the TEM results, it was possible to repeatedly check the agglomeration between the particles, and through these results, it can be confirmed that the alginate nanoparticles have a stronger agglomeration force than other polysaccharide-based nanoparticle samples. there was.

Hydrogel Characterization

An experiment was conducted to check whether the synthesized nanoparticles have the characteristics of a hydrogel that holds water well (swelling), and as a control, pure water and alginic acid not irradiated with an electron beam were used as a control.

After dissolving the same amount of alginic acid nanoparticles and a control group in 500 μL of water, centrifugation was performed using a centrifugal filter with a separation membrane to check the amount of water that fell on the bottom of each tube. The water that fell down the tube was dyed with green ink and then photographed. It is presented in FIG. 10 .

When checking the results, it was confirmed that in the nanoparticle sample obtained by irradiating the electron beam, the amount of water falling to the bottom of the tube was less after the centrifugation process, and more solution was left in the supernatant.

Through these results, it was confirmed that the alginic acid nanoparticles synthesized through electron beam irradiation contained water better than the control group and had the characteristics of a hydrogel.

Example 2: Use of alginate nanogels

As shown in the schematic description of FIG. 11 , an experiment was conducted for bonding to alginic acid nanoparticles using NODA-GA-NH ₂ , a dual functional chelate, and after dissolving the alginic acid nanoparticles in pyridine, tosyl chloride was added one drop. The reaction was carried out overnight by dropping it slowly. Thereafter, after adding NODA-GA-NH ₂ and applying high heat to the reaction for about a day, the alginic acid nanoparticles that were conjugated with NODA-GA-NH ₂ were recovered with a centrifugal filter. Alginic acid nanoparticles conjugated with NODA-GA in powder form were obtained through the freeze-drying process.

Stability analysis of radiolabeled nanoparticles

Similarly, as schematically shown in FIG. 12, radiolabeling was performed using Cu-64, NODA-GA-alginic acid nanoparticles were put in a buffer having a pH of 6.8, and reacted with ⁶⁴ CuCl ₂ at an appropriate temperature and time, followed by a centrifugal filter After purifying Cu-64-labeled alginic acid nanoparticles by using Radio-TLC, radiochemical purity was confirmed.

As a result, as shown in FIG. 13, it was confirmed that the labeling reaction was excellently performed with Cu-64 with a radiochemical purity of almost 100%.

In the case of the stability of radiolabeled nanoparticles, the experiment was conducted in a way to check radiolysis, and radio-TLC analysis was performed over time using PBS (Phosphate Buffer Saline) and serum (Fetal Bovine Serum). to check the stability of

As a result, as shown in the results in FIG. 14 , it was confirmed that high stability was shown for 1 hour and 4 hours, and the same excellent stability of 95% or more was shown in the results for 24 hours, so the stability could be sufficiently maintained in the mouse. could have been predicted.

Biodistribution confirmation experiment in normal mice

The biodistribution test was conducted in normal mice using Cu-64 and labeled-reacted nanoparticles, and the distribution to organs and the route of excretion were confirmed.

In order to utilize the synthesized nanoparticles for tumor contrast agent development research, the experiment was conducted with enough time for the nanoparticles to target the tumor. An experiment was conducted to confirm.

When the results were analyzed by comparing the results for 4 hours and 24 hours, as shown in the results in FIG. 15 , it was confirmed that the amount of radioactivity in the blood gradually decreased as time passed, and the liver showed the highest intake. As time goes by, it can be confirmed that the liver is used as a route to be excreted from the body.

Although the intake level for some heart and lung increased slightly over time, the intake level for other organs was almost the same. wanted to proceed.

Biodistribution confirmation experiment in tumor model

A biodistribution test was performed on the tumor model using Cu-64 and labeled-reacted nanoparticles, and the distribution of each organ was checked 24 hours after injection to confirm the level of uptake of the tumor.

First, a tumor model was prepared using colorectal cancer tumor cells (CT26), and when the tumor grew to an appropriate size of 6-8 mm, a biodistribution confirmation experiment was performed on the tumor model, and as shown in FIG. 16 , the liver It showed a high intake of 2.7% ID/g for tumors and a higher intake than other organs with an intake of 0.73% ID/g for tumors. It was confirmed that the tumor to muscle ratio was 32.8 times, and the tumor to blood ratio was 11.9 times, enabling excellent tumor diagnosis.

In addition, a tumor model was prepared using skin cancer cells (melanoma/B16F10), and when the tumor grew to an appropriate size, the biodistribution confirmation experiment was conducted in the same time as 24 hours later, as confirmed in FIG. , As a result, high uptake was shown for kidney and liver at 4.3%ID/g and 3.4%ID/g, respectively, and high uptake was shown for tumor at 1.7%ID/g. It was confirmed that the tumor to muscle ratio was 16 times, and the tumor to blood ratio was 5.2 times, so that it was possible to excellently diagnose the tumor.

Example 3: Anticancer Efficacy of Alginic Acid Nanogels Loaded with Anticancer Agents

A study was also conducted to utilize it as a tumor treatment, and an experiment was conducted to compare and verify the ability of alginic acid nanoparticles loaded with doxorubicin to be used as a treatment capable of inhibiting tumor growth.

The experiment was carried out by loading doxorubicin, which is widely used as an anticancer agent, into the alginic acid nanoparticles prepared in Examples, and alginic acid nanoparticles 1mg/mL and doxorubicin (1mg/mL) were added and stirred at room temperature for 1 hour. The reaction proceeded in this way.

After the reaction, when centrifugation was performed at 10,000 rpm for 30 minutes, the alginic acid nanoparticles loaded with doxorubicin form a pellet on the bottom, and the supernatant and the pellet were separated.

By measuring the absorption spectrum of the separated supernatant, the amount of doxorubicin that is not bound to the nanoparticles can be confirmed. As a method to check the amount of doxorubicin remaining in the supernatant by substituting it into the standard curve for doxorubicin in As a result of confirming how much doxorubicin was loaded into the particles, it was confirmed that almost all of the loading efficiency (LE) (%) was 98.5%, and there was hardly any red doxorubicin color left in the supernatant as shown in FIG. 18 .

Performance analysis as an anticancer drug

As a tumor model used in the experiment, a breast cancer mouse tumor model (EMT6 tumor model) was used, and after confirming that a tumor occurred, it was used in the experiment. A control group treated with PBS, a Doxorubicin group treated with doxorubicin, and a group treated with doxorubicin-loaded alginic acid nanoparticles (Dox@Algi-NP) were randomly selected and started the experiment with a mouse tumor model. . The number of mice corresponding to each group was 3, and each was treated with a mouse tumor model through intravenous injection twice at intervals of 3 days, and the amount of doxorubicin treated at this time was constant at 200 μg.

The size of the tumor and the weight of the mouse in the tumor model were checked over 24 days, and when the results were checked and compared, the group treated with DOX@Algi-NP showed the most inhibition of tumor growth and the tumor size was 2000 cm. ³ , which was clearly smaller than the other two groups. It is confirmed in FIG. 19 .

When the weight of the removed tumor was measured, as shown in FIG. 20 , in the group treated with DOX@Algi-NP, the growth of the tumor was most inhibited, and it was confirmed that the weight of the tumor was clearly lower than that of the other two groups. did

In addition, it was confirmed that the weight of the tumor model in each group was not rapidly reduced or changed, as shown in FIG. 21 , and through these results, DOX@Algi-NP certainly does not cause great toxicity to the tumor model and It was confirmed that growth may be inhibited.

Example 4: Anticancer drug loading capacity of alginate nanogels

In addition to doxorubicin, an experiment in which another anticancer agent called Ciplatin was loaded was conducted. For the loading of ciplatin, Aquated cisplatin([Pt(NH ₃ ) ₂ (H ₂ O) ₂ ] ²⁺ ) was prepared. Ciplatin (10 mg, 1 equiv) and silver nitrate (11 mg, 2 equiv) were added to 10 mL of distilled water and reacted at 35 °C for 24 hours while blocking light. During the reaction, a pale white precipitate (Silver chloride) was formed, and then the precipitate was removed by centrifugation at 10,000 rpm, and only Aquated cisplatin ([Pt(NH ₃ ) ₂ (H ₂ O) ₂ ] ²⁺ ) was obtained to obtain nano alginate. used to load particles.

After dissolving 5 mg of alginic acid nanoparticles, the reaction was carried out while stirring at 35 °C for 30 minutes. Then, after adding 1 mg of the obtained Aquated cisplatin, the reaction was performed at 35°C for 24 hours in a state of blocking light, and centrifugation was performed using a centrifugal filter with a separation membrane to remove unreacted Aquated cisplatin.

OPD (ortho-Phenylenediamine) reacts with cisplatin to form an OPD-Pt complex and exhibits high absorbance at a wavelength of 705 nm. After reacting for a while, absorbance was measured at a wavelength of 705 nm.

By substituting the value into a standard curve for cisplatin at a wavelength of 705 nm prepared in advance, the amount of cisplatin in the finally obtained sample was confirmed. It is confirmed in FIG. 22 .

As a result of comparing the amount of cisplatin used in the reaction with the amount of cisplatin remaining in the final sample in this way, it was confirmed that the amount of cisplatin was loaded in the alginate nanoparticles. .

Claims (20)

In the method for producing crosslinked alginic acid nanoparticles comprising irradiating an electron beam to an aqueous alginic acid solution to form intermolecular or intramolecular crosslinks of alginic acid, by adjusting the pH of the aqueous solution to an acidic level, A method for producing cross-linked alginic acid nanoparticles, characterized in that the size is controlled. delete According to claim 1,
The alginic acid aqueous solution is characterized in that the pH is 1 to 6,
A method for preparing cross-linked alginic acid nanoparticles. According to claim 1,
HClO ₄ In the alginic acid aqueous solution, further comprising any one or more of nitric acid and formic acid,
A method for preparing cross-linked alginic acid nanoparticles. According to claim 1,
The alginic acid aqueous solution is characterized in that the concentration of 0.1 to 2% (w / v),
A method for preparing cross-linked alginic acid nanoparticles. According to claim 1,
The electron beam is characterized in that it is irradiated with an irradiation amount of 2 to 300 kGy,
A method for preparing cross-linked alginic acid nanoparticles. According to claim 1,
Characterized in controlling the size of alginate nanoparticles by changing the irradiation dose of the electron beam,
A method for preparing cross-linked alginic acid nanoparticles. According to claim 1,
Characterized in reducing the size of the alginic acid nanoparticles by increasing the irradiation dose of the electron beam,
A method for preparing cross-linked alginic acid nanoparticles. 9. The method of claim 8,
The method is characterized in that the alginic acid aqueous solution has a pH of 1 to 6, a concentration of less than 1.0% (w/v), and an electron beam irradiation dose of 2 kGy or more,
A method for preparing cross-linked alginic acid nanoparticles. delete According to claim 1,
Characterized in that by lowering the pH of the aqueous solution to increase the size of the alginic acid nanoparticles,
A method for preparing cross-linked alginic acid nanoparticles. According to claim 1,
Characterized in that by increasing the concentration of alginic acid in the aqueous solution to increase the size of the alginic acid nanoparticles,
A method for preparing cross-linked alginic acid nanoparticles. A nanoparticle formed only by intermolecular or intramolecular crosslinking of alginic acid prepared according to claim 1, wherein the nanoparticles have tumor selectivity, alginic acid nanoparticles. Agents labeled with one or more labeling substances selected from the group consisting of radioactive isotopes, organic fluorescent substances, inorganic quantum dots, magnetic resonance imaging contrast agents, computed tomography contrast agents, positron tomography contrast agents, ultrasound contrast agents, fluorescence contrast agents, and phase transformation materials A contrast agent comprising the alginic acid nanoparticles of claim 13. 15. The method of claim 14,
a ligand compound conjugated to the nanoparticles; and
Containing a radioactive isotope coordinated to the ligand compound,
Youngjae Cho. 16. The method of claim 15,
The ligand compound is NODA-GA-NH ₂ , Characterized in that at least one of DOTA-GA, DOTA, TETA and NOTA,
Youngjae Cho. 16. The method of claim 15,
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, ^99ν Tc, ¹²³ I, ¹¹¹ In, ⁶⁷ Ga, ¹⁷⁷ Lu, ²⁰¹ Tl, ^117ν Sn, ¹²⁵ I, ¹³¹ I, ¹⁶⁶ Ho, ¹⁸⁸ Re, ⁶⁷ Cu, ⁸⁹ Sr, ⁹⁰ Y, ²²⁵ Ac, ²¹³ Bi, and ²¹¹ At. Intermolecular or intramolecular cross-linked nanoparticles of alginic acid prepared according to claim 1; and a drug loaded into the nanoparticles,
drug carrier. 19. The method of claim 18,
The drug is characterized in that it is an anticancer agent,
drug carrier. A pharmaceutical composition for preventing or treating cancer comprising the drug delivery system loaded with the anticancer agent on the nanoparticles of claim 13 as an active ingredient.

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