Biocompatible nanoparticle and use thereof

Abstract

The present invention relates to a biocompatible nanoparticle and a use thereof and, more specifically, to a biocompatible nanoparticle formed by irradiation an electron beam to an aqueous solution comprising at least one substance selected from the group consisting of a polysaccharide, a derivative thereof and a polyethylene glycol, thereby inducing inter-molecular cross-linking or intra-molecular cross-linking, and to a use of the biocompatible nanoparticle in a drug carrier, a contrast agent, a diagnostic agent or an intestinal adhesion prevention agent or for disease prevention and treatment.

Claims

1. A method for preparing biocompatible nanoparticles, the method comprising: (a) adding to water, at least one material selected from the group consisting of polysaccharides, and a mixture of a polysaccharide and polyethylene glycol to prepare a 0.1% (w/v) to 15% (w/v) solution; and (b) irradiating the solution prepared in step (a) with an electron beam at a dose of 5 to 250 kGy to crosslink the material, wherein the biocompatible nanoparticle is formed exclusively by inter-molecular or intra-molecular crosslinking of at least one selected from the group consisting of a polysaccharide, and a mixture of a polysaccharide and polyethylene glycol, wherein the method excludes the use of a crosslinking agent, a curing agent, an inorganic material, and an organic solvent, wherein the polysaccharide is at least one selected from the group consisting of mannan, -cyclodextrin, -cyclodextrin, -cyclodextrin, fructo-oligosaccharides, isomalto-oligosaccharides, inulin, glycogen, amylose, carboxymethyl dextran, beta-glucan, fucoidan, and chondroitin, wherein a size of the nanoparticle is in a range of 1 to 700 nm.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram showing a method for manufacturing nanoparticles by irradiating a carboxymethyl-dextran aqueous solution with an electron beam.

(2) FIG. 2 illustrates DLS results and TEM image of the nanoparticles generated when a 10% carboxymethyl-dextran aqueous solution was irradiated with an electron beam of 200 kGy.

(3) FIG. 3 illustrates the results of confirming, through DLS, the generation of nanoparticles after electron beam irradiation of carboxymethyl-dextran aqueous solutions with different concentrations.

(4) FIG. 4 illustrates the results of confirming, through DLS, the generation of nanoparticles when a hyaluronic acid aqueous solution was irradiated with an electron beam under various conditions.

(5) FIG. 5 illustrates the results of confirming, through DLS, the generation of nanoparticles when a mannan aqueous solution was irradiated with an electron beam under various conditions.

(6) FIG. 6 illustrates the results of confirming, through DLS, the generation of nanoparticles when a -cyclodextrin aqueous solution was irradiated with an electron beam under various conditions.

(7) FIG. 7 illustrates the results of confirming, through DLS, the generation of nanoparticles when an alginate aqueous solution was irradiated with an electron beam under various conditions.

(8) FIG. 8 illustrates the results of confirming, through DLS, the generation of nanoparticles when a fructo-oligosaccharide aqueous solution was irradiated with an electron beam under various conditions.

(9) FIG. 9 illustrates the results of confirming, through DLS, the generation of nanoparticles when an isomalto-oligosaccharide aqueous solution was irradiated with an electron beam under various conditions.

(10) FIG. 10 illustrates the results of confirming, through DLS, the generation of nanoparticles when a fucoidan aqueous solution was irradiated with an electron beam under various conditions.

(11) FIG. 11 illustrates the results of confirming, through DLS, the generation of nanoparticles when a chitosan aqueous solution was irradiated with an electron beam under various conditions.

(12) FIG. 12 shows views of confirming hydrogel characteristics by comparing differences in amount of water remaining on tube bottoms after centrifugation, according to the concentration of carboxymethyl-dextran nanoparticles.

(13) FIG. 13 is a schematic diagram showing a manufacture procedure of a polyethylene glycol and carboxymethyl-dextran mixed nanoparticle using an electron beam.

(14) FIG. 14 illustrates diagrams of confirming the formation of PEG nanoparticles and the particle size thereof when electron beams with various energy intensities were irradiated.

(15) FIG. 15 shows DLS results of nanoparticles formed by irradiating a mixed aqueous solution of 10% carboxymethyl-dextran and 1% polyethylene glycol with an electron beam.

(16) FIG. 16 is a diagram showing .sup.1H-NMR results of polyethylene glycol and carboxymethyl-dextran mixed nanoparticles.

(17) FIG. 17 is a diagram showing .sup.13C-NMR results of polyethylene glycol and carboxymethyl-dextran mixed nanoparticles.

(18) FIG. 18 is a diagram showing structures in which chelates are conjugated to carboxymethyl-dextran nanoparticles.

(19) FIG. 19 is a diagram showing the Cu-64 labeling TLC results of chelate-conjugated carboxymethyl-dextran nanoparticles.

(20) FIG. 20 is a diagram showing TLC results of Cu-64 labeled carboxymethyl-dextran nanoparticles.

(21) FIG. 21 is a diagram showing a radioactive iodine labeling procedure of a carboxymethyl-dextran nanoparticle using Bolton-Hunter Reagent.

(22) FIG. 22 is a diagram showing the Radio-TLC results of I-131 labeled carboxymethyl-dextran nanoparticles after reaction and purification.

(23) FIG. 23 shows a doxorubicin standard curve and an observation diagram of a doxorubicin-conjugated carboxymethyl-dextran nanoparticle confirmed through centrifugation.

(24) FIG. 24 shows the results of confirming the release rates of doxorubicin in a doxorubicin-conjugated carboxymethyl-dextran nanoparticle (CM-DNP) and a doxorubicin-conjugated polyethylene glycol and carboxymethyl-dextran mixed nanoparticle (PEG-DNP).

(25) FIG. 25 shows the results of evaluating the in vivo antitumor activity of doxorubicin-conjugated nanoparticles (a blank group treated with PBS, a group treat with free doxorubicin (free DOX), and a group treated with doxorubicin-conjugated PEG-DNP (DOX @ PEG-DNP)).

(26) FIG. 26 is a diagram of observation of the body weight change of animals according to the administration of each drug in animal models for evaluating the in vivo antitumor activity of doxorubicin-conjugated nanoparticles (a blank group treated with PBS, a group treat with free doxorubicin (free DOX), and a group treated with doxorubicin-conjugated PEG-DNP (DOX @ PEG-DNP)).

MODE FOR CARRYING OUT THE INVENTION

(27) Hereinafter, the present invention will be described in detail.

(28) However, the following examples are merely for illustrating the present invention, and are not intended to limit the scope of the present invention.

Example 1

Conditions for Electron Beam Irradiation Using Electron Beam Accelerator and Preparation Therefor

(29) Experiments were conducted by applying an electron beam with various dose conditions to a solution to be irradiated. In the experiments, a linear electron beam accelerator was used, and an electron beam with doses of 5 kGy, 10 kGy, 50 kGy, 100 kGy, and 200 kGy was applied to a sample on a conveyor moving at a predetermined rate in a manner of adjusting the electron beam and the irradiation time. The irradiation time of the electron beam and the irradiation dose thereof can be properly adjusted and selected by a person skilled in the art according to the size of nanoparticles to be produced, in consideration of the temperature condition at the time of electron beam irradiation, the sample concentration, the energy intensity of an electron beam to be irradiated, and the like.

Example 2

Preparation of Nanoparticles by Electron Beam Irradiation

(30) 2-1. Carboxymethyl-Dextran Solution and Electron Beam Irradiation Conditions

(31) Nanoparticle synthesis experiments were conducted using carboxymethyl-dextran. Electron beams were applied to samples to be irradiated by using a linear electron beam accelerator while the electron beam irradiation conditions were varied in a manner of adjusting the beam current and irradiation time.

(32) More specifically, carboxymethyl-dextran (molecular weight: 10 kDa) was dissolved in water to prepare solutions with concentrations of 0.1%, 0.5%, 1%, 5%, 10%, and 20% (w/v), and the experiments were conducted while the electron beam irradiation energy dose with respect to the carboxymethyl-dextran solutions was varied to 5 kGy, 10 kGy, 50 kGy, 100 kGy, and 200 kGy. Any additive, such as an organic solvent, a crosslinking agent, or an inorganic material, was not added to the prepared carboxymethyl-dextran solutions.

(33) 2-2. Preparation of Carboxymethyl-Dextran Nanoparticles by Electron Beam Irradiation

(34) Experiments were conducted at various concentrations and various electron beam dose conditions, and as a result of confirming particle sizes of the prepared nanogels through dynamic light scattering (DLS), it could be confirmed that nanoparticles with a uniform size of about 10 nm were synthesized only when an electron beam of 200 kGy was applied to a 10% carboxymethyl dextran solution (FIG. 2).

(35) When the electron beam with 200 kGy was applied to the 20% carboxymethyl dextran solution, several peaks were observed in the DLS measurement, and the measurement was not favorably carried out under other conditions, and thus it was confirmed that nanoparticles were not favorably formed (FIG. 3).

(36) 2-3. Preparation of Nanoparticles Derived from Polysaccharides or Oligosaccharides by Electron Beam Irradiation

(37) In addition to the carboxymethyl-dextran nanoparticles, nanoparticles were manufactured by using other polysaccharides or oligosaccharides.

(38) Experiments were conducted by the same method as in Example 2-1 except that various polysaccharides or oligosaccharides, instead of carboxymethyl-dextran, were dissolved in water, followed by electron beam irradiation, to investigate whether nanoparticles were formed.

(39) (1) Results of Hyaluronic Acid (10 kDa)

(40) Experiments were conducted by irradiating 1%, 5%, and 10% (w/v %) hyaluronic acid solutions with an electron beam of 10 kGy, 50 kGy, 200 kGy.

(41) As a result, it can be seen from FIG. 4 that nanogels were formed with a particle size of 108 nm under conditions of 1% and 50 kGy and a particle size of 380 nm under 5% and 200 kGy. It can be confirmed that under the other conditions, very large-sized particles were generated or several peaks were observed, and thus nanogels with a uniform particle size were not formed.

(42) (2) Results of Mannan (0.7 kDa)

(43) Experiments were conducted by irradiating 1%, 5%, and 10% (w/v %) mannan solutions with an electron beam of 10 kGy, 50 kGy, 200 kGy.

(44) As a result, it can be seen from FIG. 5 that nanogels were formed with a particle size of 162 nm under conditions of 1% and 50 kGy and a particle size of 305 nm under 5% and 200 kGy. It can be confirmed that under the other conditions, very large-sized particles were generated or several peaks were observed, and thus nanogels with a uniform particle size were not formed.

(45) (3) Results of -Cyclodextrin (1.1 kDa)

(46) Experiments were conducted by irradiating 0.1%, 1%, and 5% (w/v %) -cyclodextrin solutions with an electron beam of 10 kGy, 50 kGy, 200 kGy.

(47) As a result, it can be seen from FIG. 6 that nanogels were formed with a particle size of 486 nm under conditions of 1% and 50 kGy. It can be confirmed that under the other conditions, several peaks were observed, and thus nanogels with a uniform particle size were not formed.

(48) (4) Results of Alginate (33 kDa)

(49) Experiments were conducted by irradiating 1%, 5%, and 10% (w/v %) alginate solutions with an electron beam of 10 kGy, 50 kGy, 200 kGy.

(50) As a result, it can be seen from FIG. 7 that nanogels were formed with a particle size of 283 nm under conditions of 1% and 10 kGy, a particle size of 354 nm under conditions of 1% and 50 kGy, a particle size of 283 nm under conditions of 5% & 1% and 200 kGy, a particle size of 302 nm under conditions of 5% and 10 kGy, a particle size of 602 nm under conditions of 5% and 200 kGy, and a particle size of 540 nm under conditions of 10% and 10 kGy.

(51) (5) Results of Fructo-Oligosaccharide

(52) Experiments were conducted by irradiating 1%, 5%, 10%, 20%, 30%, and 40% (w/v %) fructo-oligosaccharide solutions with an electron beam of 10 kGy, 50 kGy, 200 kGy.

(53) As a result, it can be seen from FIG. 8 that nanogels were formed with a particle size of 501 nm under conditions of 1% and 200 kGy. Meanwhile, it can be confirmed that also under conditions of 10% and 10 kGy and 10% and 200 kGy, nanoparticles were formed although the particle size is not uniform. Under the other conditions, the particle size was very large or several peaks were observed, and thus nanoparticles were not normally formed.

(54) (6) Results of Isomalto-Oligosaccharide

(55) Experiments were conducted by irradiating 1%, 5%, 10%, 20%, 30%, and 40% (w/v %) isomalto-oligosaccharide solutions with an electron beam of 10 kGy, 50 kGy, 200 kGy.

(56) As a result, it can be seen from FIG. 9 that nanogels were formed with a particle size of 542 nm under conditions of 5% and 50 kGy. Meanwhile, it can be confirmed that also under conditions of 5% and 10 kGy and 10% and 200 kGy, nanoparticles were formed although the particle size is not uniform. Under the other conditions, the particle size was very large or several peaks were observed, and thus nanoparticles were not normally formed.

(57) (7) Results of Fucoidan

(58) Experiments were conducted by irradiating 0.5%, 1%, and 5% (w/v %) fucoidan solutions with an electron beam of 10 kGy, 50 kGy, 200 kGy.

(59) As a result, it can be seen from FIG. 10 that nanogels were formed with a particle size of 100 nm under conditions of 1% and 200 kGy. It can be confirmed that under the other conditions, several peaks were observed, and thus nanogels with a uniform particle size were not formed.

(60) (8) Results of Chitosan (5 kDa)

(61) Experiments were conducted by irradiating 0.5%, 1%, and 5% (w/v %) chitosan solutions with an electron beam of 10 kGy, 50 kGy, 200 kGy.

(62) As a result, it can be seen from FIG. 11 that nanogels were formed with a particle size of 172 nm under conditions of 0.5% and 200 kGy. Meanwhile, it can be confirmed that also under conditions of 0.1% and 50 kGy, 0.1% and 100 kGy, 0.5% and 10 kGy, 1% and 10 kGy, and 1% and 200 kGy, nanoparticles were formed although the particle size was not uniform. Under the other conditions, the particle size was very large or several peaks were observed, and thus nanoparticles were not normally formed.

Example 3

Evaluation of Physical Properties of Carboxymethyl-Dextran Nanoparticles (CM-DNP)Characteristics of Gel

(63) The samples irradiated with the electron beam were dialyzed using a dialysis membrane tube with a size of 3.5-6 kDa while water containing NaCl was exchanged two times a day for 5 days. It was investigated through DLS whether the particle size was changed, but it could be confirmed that the particle size did not change during the dialysis procedure. Thereafter, the samples were freeze-dried to calculate the yield of nanoparticles formed through crosslinking by the irradiated electron beam. A yield of about 43% was obtained and a total of 8.2 g of nanoparticles were obtained.

(64) Experiments were conducted, by using nanoparticles manufactured according to the examples above, to investigate whether these nanoparticles show gel characteristics. After 0 mg, 100 mg, 200 mg, and 300 mg of CM-DNP were dissolved in 400 uL of water, centrifugation using a centrifugal filter (YM-3) with a 3 kDa membrane was repeatedly conducted three times at 13000 rpm for 30 min. It was investigated how the degree of water swelling varies according to the concentration of CM-DNP. As a result, it could be confirmed that the amount of water falling to the bottom of the tube after centrifugation was different depending on the concentration of CM-DNP, and the higher the concentration of CM-DNP, the lower the amount of water. It can be confirmed from these results that the synthesized CM-DNP nanoparticles had gel characteristics (FIG. 12).

Example 4

Synthesis of Carboxymethyl-Dextran Nanoparticles (CM-DNP) Mixed with Polyethylene Glycol (PEG) Through Electron Beam Irradiation

(65) It was planned to study the effect of organic molecules, which have already been used in clinical trials, on the formation of dextran nanoparticles and physical properties thereof by adding the organic molecules, in addition to dextran, while polyethylene glycol (PEG) already widely used in clinical trials was used as the organic molecules to be added (FIG. 13).

(66) 4-1. Generation of Polyethylene Glycol (PEG) Nanoparticles by Electron Beam Irradiation

(67) First, experiments for investigating whether PEG itself induces crosslinking at the time of electron beam irradiation were conducted, and experiments was conducted by electron beam irradiation at several energy intensities with varying PEG concentrations. In the experiments, PEG of 6 kDa was used. The PEG was dissolved in water to prepare solutions with concentrations of 1%, 5%, and 10% (w/v), and the electron beam was applied with energy intensities of 10 kGy, 30 kGy, 50 kGy, 100 kGy, and 200 kGy. When the energy intensity increased to 30 kGy or higher, the colors of the samples became turbid and the tendency of bulk gel formation was confirmed. When the concentration of PEG was low, the tendency that a larger sized nanogel was produced was confirmed (FIG. 14).

(68) 4-2. Preparation of Nanoparticles of PEG and Carboxymethyl-Dextran Mixture by Electron Beam Irradiation

(69) It could be directly confirmed that crosslinking occurred when the PEG solution was irradiated with the electron beam. Experiments were conducted to investigate whether crosslinking was favorably done when a mixture of PEG and carboxymethyl dextran was irradiated with an electron beam and whether nanoparticles having different sizes and physical properties from the nanoparticles manufactured using only carboxymethyl dextran were manufactured.

(70) It was investigated whether nanoparticles were formed, by preparing dextran and PEG with different concentrations of each other, followed by electron beam irradiation with 10 kGy, 50 kGy, and 200 kGy, and then measuring the size of the samples using DLS.

(71) The formation of nanogels were confirmed in the electron beam irradiation using 10% carboxymethyl dextran and 1% PEG unlike the other conditions. Bulky gels were formed under the other conditions (results not shown). The results of measuring the size of samples by DLS were 63 nm at 10 kGy, 50 nm at 50 kGy, and 9 nm at 200 kGy, and thus it could be confirmed that the size of nanoparticles showed a tendency to decrease as the irradiated energy increases (FIG. 15).

(72) 4-3. Yield of PEG and Carboxymethyl-Dextran Mixed Nanoparticles (PEG-DNP)

(73) As described above, the nanoparticles were well formed even when the mixture of 1% PEG and 10% carboxymethyl-dextran were irradiated with an electron beam at energy densities of 10, 50, and 200 kGy. Then, in order to investigate the yield of formation of nanoparticles, the samples were dialyzed using a dialysis membrane tube with a size of 3.5-5 kDa while water containing NaCl was exchanged two times a day for days, and then the respective samples were freeze-dried to calculate the yield of nanoparticles manufactured through crosslinking by electron beam irradiation, and as a result, all the samples under the respective energy conditions showed a yield of 24-36%.

(74) Experiments were conducted wherein a much larger amount of PEG-carboxymethyl-dextran nanoparticles (PEG-DNP) were synthesized at the 200 kGy electron beam irradiation condition under which the nanoparticles with a size of 9-10 nm were generated. As a result, a size of about 10 nm was reproducibly shown, and since the experiments were massively conducted, the amount lost in each step was reduced, leading to a yield of about 52%, higher than before, and a total of 2 g of nanoparticles.

(75) 4-4. Analysis of PEG-DNP Structure

(76) In order to investigate whether the production of PEG and CM-DNP mixed nanoparticles by electron beam irradiation results from the formation of nanoparticles by crosslinking of only PEG through electron beam irradiation (that is, in order to investigate whether the produced nanoparticles were formed by inter-molecular crosslinking of PEG and carboxymethyl-dextran), experiments to investigate whether both PEG and dextran were present in the produced nanoparticles and whether peaks of PEG or dextran could be confirmed were conducted by performing NMR analysis of CM-DNP obtained in example 4-3 above.

(77) In order to perform analysis using .sup.1H-NMR, the samples were dissolved in D.sub.2O and analyzed. As a result, the peaks of dextran together with the peaks of PEG could be observed, indicating that the produced nanoparticles were obtained by crosslinking of PEG and dextran together (FIG. 16).

(78) The results of .sup.13C-NMR analysis also confirmed the peaks of dextran together with the peaks of PEG, again indicating that the produced nanoparticles were obtained by crosslinking of PEG and dextran together (FIG. 17).

Example 5

Chelate Conjugation of Carboxymethyl-Dextran Nanoparticle and Cu-64 Radioactive Labeling

(79) Experiments were conducted wherein various chelates were conjugated to the synthesized CM-DNP, followed by radioactive labeling with Cu-64. In order to conduct labeling experiments using Cu-64 on nanoparticles, chelates were first conjugated to the nanoparticles. Three types of chelates, DOTA-Bn-p-NH.sub.2, DOTA-GA-NH.sub.2, and TE2A-NH.sub.2, were used.

(80) CM-DNP (100 mg) was placed in a round-bottomed flask, and then dissolved by addition of DMSO (5 mL), and thereafter, carbodiimidazole (CDI, 10 mg) was added, and then the reaction was conducted with stirring at 40 C. for 2 hours under nitrogen atmosphere. After the reaction, it was confirmed using TLC whether unreacted CDI remains with proper stationary phase and mobile phase conditions (C-18, CH.sub.2Cl.sub.2:MeOH=10:1). When the CDI was completely reacted, three kinds of chelates were added respectively, followed by reaction at 60 C. for 16 hours. Thereafter, dialysis was conducted for purification, and then the respective samples were freeze-dried, thereby obtaining dextran nanoparticles conjugated with chelates of approximately 35-45 mg (FIG. 18).

(81) It was investigated whether the chelate-conjugated nanoparticles as above were favorably radio-labeled with Cu-64.

(82) After 10 g of carboxymethyl-dextran nanoparticles conjugated with three kinds of chelates were added to 100 L of 0.1 M NH.sub.4OAc (pH 6.8), Cu-64 was added thereto, followed by reaction at 60 C. for 1 hour. When it was investigated, by using ITLC as a stationary phase and 50 mM EDTA as a mobile phase, whether Cu-64 labeling were favorably achieved, the labeling was confirmed to be done by 25% for DOTA-Bn-p-NH.sub.2-conjugated dextran nanoparticles, 47% for DOTA-GA-NH.sub.2-conjugated dextran nanoparticles, and 27% for TE2A-NH.sub.2-conjugated dextran nanoparticles (FIG. 19).

(83) Then, centrifugation was repeatedly conducted five times by using a centrifugal filter (YM-10 filter) with a 10 kDa porous membrane, and then a purification procedure of removing unlabeled free Cu-64 and separating only labeled nanoparticles was conducted, and thereafter, it was confirmed through TLC that free Cu-64 was completely removed in the separated nanoparticles (FIG. 20).

Example 6

Labeling of Carboxymethyl-Dextran Nanoparticles Using Radioactive Iodine

(84) The development of CM-DNP-based nuclear medicine imaging contrast agents through radionuclide labeling was conducted using radioactive iodine in addition to Cu-64. Radioiodine labeling was conducted by using a Bolton-Hunter reagent, which is a prosthetic group widely utilized. As the radioactive iodine, I-131, which has a half-life of 8 days and a relatively low price, was purchased for the experiments (FIG. 21).

(85) The labeling experiments were conducted using CM-DNP having a size of 10 nm. After the Bolton-Hunter reagent (1.1 g/L in DMSO) was reacted with I-131 and Chloramine-T (10 g/L in D.W) for 1 min, the reaction was stopped using sodium metabisulfite, Sep-PAK C18 was used to separate the labeled Bolton-Hunter reagent by water and ethanol. The separated Bolton-Hunter reagent and carboxymethyl-dextran nanoparticles were subjected to conjugation for 30 minutes using 0.1 M Na.sub.2B.sub.4O.sub.7 (pH 9.2), and then the labeled nanoparticles were separated using PD-10 column (FIG. 22).

(86) The labeling yield results after the reaction and the purification procedure using PD-10 column were investigated using radio-TLC. The labeling yield was 43%, and free I-131 was removed through the PD-10 column, thereby favorably separating only the labeled nanoparticles.

Example 7

Synthesis of Doxorubicin-Conjugated Carboxymethyl-Dextran-Based Nanoparticles

(87) 7-1. Conjugation of Doxorubicin to Nanoparticles

(88) Experiments wherein doxorubicin widely used as an anticancer drug was conjugated to a dextran nanoparticle were conducted. The experiments were first conducted using CM-DNP. After 15 mg of CM-DNP with a size of 10 nm was dissolved in 3 mL of DW, the pH was adjusted to 8 using NaOH, and then a reaction was carried out by adding doxorubicin (1 mg/mL), blocking light, and stirring the mixture at room temperature overnight. After the reaction, centrifugation was carried out at 16,000 g for 90 min, so that doxorubicin-conjugated dextran nanoparticles formed pellets on the bottom. The supernatant and the pellets were separated (FIG. 23).

(89) The amount of doxorubicin not conjugated to nanoparticles could be confirmed by measuring the absorption spectrum of the separated supernatant. The absorbance was determined at a wavelength of 481 nm, which indicates the absorption maximum for doxorubicin, and the measurement value were put into the previously prepared standard curve for doxorubicin at a wavelength of 481 nm to investigate the amount of doxorubicin remaining in the supernatant. The amount of doxorubicin used in the reaction and the amount of doxorubicin remaining in the supernatant were put into the formula below to investigate the amount of doxorubicin loaded in CM-DNP. As a result, it was confirmed that the loading efficiency (LE, %) was 78.9%.

(90) Experiments wherein doxorubicin was also conjugated to PEG-DNP were conducted by the same method as above, and it was confirmed that LE (%) was lower, 56.1%. Therefore, experiments were conducted to find conditions that increase LE (%) of doxorubicin. When the amount of PEG-DNP used to react with 1 mg/mL of doxorubicin was reduced from 3 mg/mL to 0.5 mg/mL, it was confirmed that LE (%) increased to 72.7%. Here, it was confirmed that there was no significant difference in LE (%) even if the reaction time was reduced to 45 minutes, and thus the reaction time could be shortened.

(91) 7-2. Drug Release Rate from Doxorubicin-Conjugated Nanoparticles

(92) Efflux experiments for investigating the drug release rate by using doxorubicin-conjugated carboxymethyl-dextran nanoparticles (CM-DNP) and PEG and carboxymethyl-dextran mixed nanoparticles (PEG-DNP) were conducted. In order to investigate the release rate of doxorubicin in PBS with pH 7.4 by using 10 mg of nanoparticles, samples were obtained at various time points, followed by centrifugation, and then the amount of doxorubicin present in the supernatant was investigated, and thus the amount of doxorubicin released from the nanoparticles was investigated. As a result, it was confirmed that 9.16% of doxorubicin was released out of CM-DNP over 6 days, and 18.4% of doxorubicin, which is conjugated to nanoparticles, was significantly slowly and continuously released out of PEG-DNP (FIG. 24).

(93) 7-3. Tumor Growth Inhibitory Effect of Doxorubicin-Conjugated PEG-DNP

(94) Experiments were conducted to compare and investigate the ability of doxorubicin-conjugated dextran-based nanoparticles whether the nanoparticles could be used as a therapeutic agent capable of inhibiting tumor growth. Colorectal cancer mouse tumor models (CT26 tumor model) were used as tumor models in the experiments. 510.sup.6 cells were injected into the flank of mice, and after 10 days, the tumors were visually recognized, and the mice were used for the experiments. The experiments were started by dividing the animal models into a blank group treated with PBS, a group treated with free doxorubicin (free DOX), and a group treated with doxorubicin-conjugated PEG-DNP (DOX@PEG-DNP)). The number of mice corresponding to each group was three, and each administration material was administered to the mouse tumor models via intravenous injection three times at intervals of two days. Here, the amounts of doxorubicin administered to the group treated with free doxorubicin and the group treated with doxorubicin-conjugated PEG-DNP (DOX@PEG-DNP) were 200 g.

(95) The size of tumors and the weight of mice in the tumor model were checked over 33 days, and the measurement results were monitored and compared. As a result, it could be confirmed that the tumor growth was most strongly inhibited and the tumor size was 3000 mm.sup.3 or less in the group treated with DOX@PEG-DNP, and the above tumor size was definitely smaller than those in the other two groups. In the group treated with free doxorubicin, the tumor growth rate of only one of three mice was not inhibited and was similar to the tumor growth rate in the blank group treated with PBS, and the remaining two tumor models excluding the above tumor model surely inhibited the tumor growth rate compared with the blank group treated with PBS. In the case of the tumor models, the body weight was not rapidly reduced even in spite of the treatment with free doxorubicin or DOX@PEG-DNP. As time passed, there was a slight difference in body weight among the groups, but this was thought to be due to the difference of tumor size in the respective groups. It could be confirmed through these results that DOX@PEG-DNP did not certainly exhibit great toxicity on the tumor models compared with free doxorubicin and could inhibit tumor growth.

INDUSTRIAL APPLICABILITY

(96) The biocompatible nanoparticles of the present invention are manufactured by inducing inter-molecular or intra-molecular crosslinking of a polysaccharide or a derivative thereof through an electron beam, so there is no a concern of occurrence of toxic problems in the human body due to the incorporation of an organic solvent or a crosslinking agent, and a separate purification process is not needed during the manufacturing procedure of the nanoparticles, and thus the nanoparticles can be massively produced with merely electron beam irradiation for a short time, leading to very excellent productivity. Furthermore, the nanoparticles of the present invention are very useful in that the nanoparticles can be utilized in various fields, such as a drug delivery system, a pharmaceutical composition, a contrast agent composition, or an adhesion barrier, and thus are highly industrially applicable.