KR101963951B1 - Nanogel using pH sensitive block copolymer, poly(aspartic acid-graft-imidazole)-poly(ethylene glycol) and preparing method thereof - Google Patents

Abstract

The present invention relates to a nano-gel using a pH-sensitive block copolymer, poly (aspartic acid-graft-imidazole) -polyethylene glycol, and a process for preparing the same. The present invention relates to a poly (aspartic acid-graft-imidazole) PH-sensitive nanogels were prepared through chemical cross-linking between polyethylene glycol and chemically modified bis-N-hydroxysuccinimide (bis-NHS) conjugated polyethylene glycols, (PH between 7.2 and pH 6.5) due to their chemical nature (appropriate pK _a value, wide buffer pH range, nano size, positive zeta potential and morphology) and pH dependent properties Interaction can regulate drug release and increase drug efficacy on target cells through reversible swelling and endosomal destruction.
Therefore, a nanogel using poly (aspartic acid-graft-imidazole) -polyethylene glycol having the above effect can be utilized as a drug delivery composition for tumor treatment.

Description

(pH-sensitive block copolymer, poly (aspartic acid-graft-imidazole) -polyethylene glycol) and a method for preparing the same. glycol and preparing method thereof}

The present invention relates to a pH-sensitive block copolymer, a nanogel using poly (aspartic acid-graft-imidazole) -polyethylene glycol and a process for preparing the same.

In effective cancer therapy, tumor specific drug delivery and anticancer drug controlled release are major challenges in improving therapeutic efficacy and reducing side effects. Various nanotransporters have been developed to overcome the limitations of conventional drugs and effectively deliver drugs to tumor sites, such as micelles, nanoparticles, nanogels, liposomes, and polymer drug conjugates.

Among these nano-carriers, the pH-sensitive drug delivery system is able to successfully inhibit the tumor in vitro / in vivo by regulating the drug release profile according to the tumor pH conditions (tumor in vitro pH: ~ 6.5 to 7.2 or endosomes / lysosomal pH ≤ 6.5) Have been reported as nano-carriers. It has also recently been shown that poly (amino acid) -based nanogels such as poly (L-histidine) based nanogels, poly (L-lysine) based nano gels and poly (L-aspartic acid) It was reported as a nano gel. They exhibit reversible swelling and drug release control by cyclisation of pH. Chemical cross-linking to pH sensitivity provides a unique property of polymeric micelles. They are also known to enhance the efficacy of drugs in acidic tumor environments and inhibit the growth of tumor cells.

Korean Patent Publication No. 10-2012-0054279 (published May 30, 2012)

It is an object of the present invention to provide a pH-sensitive nano-gel comprising a poly (aspartic acid-graft-imidazole) -polyethylene glycol copolymer and polyethylene glycol grafted with bis-N-hydroxysuccinimide, And a method for manufacturing the same.

In order to achieve the above object, the present invention provides a poly (aspartic acid-graft-imidazole) -polyethylene glycol copolymer grafted with imidazole to poly (aspartic acid) -polyethylene glycol and bis- Wherein at least one polyethylene glycol grafted with hydroxy succinimide is a covalent bond.

The present invention also relates to a process for preparing poly (aspartic acid-graft-imidazole) -polyethylene glycol by covalently bonding imidazole to poly (aspartic acid) -polyethylene glycol, A second step of reacting polyethylene glycol with succinic anhydride to produce polyethylene glycol grafted with bis-N-hydroxysuccinimide, and a second step of reacting poly (aspartic acid-graft-imidazole) And a third step of reacting polyethylene glycol with polyethylene glycol having the bis-N-hydroxysuccinimide grafted in the second step to prepare a nanogel, wherein the pH-sensitive nano gel is prepared .

The present invention relates to a process for the chemical crosslinking between poly (aspartic acid-graft-imidazole) -polyethylene glycol and chemically modified bis-N-hydroxysuccinimide (bis-NHS) conjugated polyethylene glycol pH-sensitive nanogels were prepared which had appropriate physico-chemical properties (suitable pK _a values, wide buffering pH range, nano-scale, positive zeta potential and morphology) and pH- pH 6.5), it is possible to regulate drug release and increase drug efficacy on target cells through reversible swelling and endosomal destruction due to electrostatic interactions by the imidization of imidazole groups.

Therefore, a nanogel using poly (aspartic acid-graft-imidazole) -polyethylene glycol having the above effect can be utilized as a drug delivery composition for tumor treatment.

Figure 1 shows the concept and chemical structure of a pH-sensitive nanogel. (a) shows a method of synthesizing a nanogel by cross-linking P (Asp-g-Im) -PEG using bis-NHS-grafted PEG, wherein the pH- pH dependent reversible swelling. Unlike nanogels, micelles cause irreversible structural degradation by decreasing the pH, and degraded micelles can not return to their original micellar structure due to the absence of crosslinking. (b) shows that the nanogels form a stable core by N-hydroxysuccinimide coupling to the imidazole residue bridges. As the pH decreases, the imidazole groups located in the nanogel core become positive and expand by electrostatic repulsion by positive charge. On the contrary, as the pH increases, the expanded nanogels are shrunk due to the decrease of the electrostatic repulsion force due to the deprotonation of imidazole residues.
Fig. 2 shows the chemical synthesis route of the pH-sensitive nanogel.
Figure 3 shows the pH profile of sodium hydroxide (NaCl, ◆), nanogel 2 (NG 2, ●), and nanogel 4 (NG 4, Δ) in acid-base titration curves.
Figure 4 shows the particle size and distribution of (a) NG 2 and (b) NG 4 at various pH conditions (pH 8.0 to 5.0).
Figure 5 shows the zeta potential of (a) NG 2 and (b) NG 4 at various pH conditions (pH 8.0 to 5.0).
FIG. 6 is a graph showing changes in reversible size of NG 2, NG 4 and polymer micelle at continuous pH changes by dynamic light scattering (DLS) and field emission scanning electron microscopy (FE-SEM).

The inventors of the present invention have found that by using a chemical crosslinking reaction of poly (aspartic acid-graft-imidazole) -polyethylene glycol and chemically modified bis-NHS grafted polyethylene glycol as a pH-sensitive block copolymer, The nanogels have reversible swelling and endosome destruction in the acidic tumor microenvironment with appropriate physico-chemical properties (suitable pK _a value, wide buffering pH range, nano size, positive zeta potential and morphology) To control the release of the target drug and effectively deliver the drug, thus completing the present invention.

The present invention relates to poly (aspartic acid-graft-imidazole) -polyethylene glycol copolymer and bis-N-hydroxysuccinimide grafted with imidazole to poly (aspartic acid) -polyethylene glycol. Wherein at least one of the polyethylene glycols is a covalent bond.

Preferably, the copolymer and the bis-N-hydroxysuccinimide conjugated polyethylene glycol may be combined in a molar ratio of 1: 1, but are not limited thereto.

Preferably, the nanogel forms a micellar structure at pH 7.4 or higher, and the micellar structure collapses at pH 6 to pH 7.2.

Preferably, the average diameter of the nanogels may be 100 to 500 nm, but is not limited thereto.

Preferably, the average zeta potential of the nanogel may be -20 to 10 mV, but is not limited thereto.

Preferably, the average pK _a value of the nanogels may be 5 to 8, but is not limited thereto.

Preferably, the average buffering pH range of the nanogels may be from 4 to 8, but is not limited thereto.

The present invention provides a pH-sensitive nano-gel and a drug or a composition for delivering a physiologically active ingredient, which comprises a drug or a physiologically active ingredient encapsulated in the nano-gel.

Preferably, the physiologically active component may be selected from the group consisting of peptides, proteins, anticancer agents, anti-inflammatory agents, antibiotics, antimicrobial agents, hormones, genes and vaccines.

The present invention relates to a first step of preparing a poly (aspartic acid-graft-imidazole) -polyethylene glycol by covalently bonding imidazole to poly (aspartic acid) -polyethylene glycol, a first step of preparing a bis-carboxylated polyethylene A step of reacting lycoric acid with succinic anhydride to produce polyethylene glycol grafted with bis-N-hydroxysuccinimide, and a second step of producing poly (aspartic acid-graft-imidazole) -polyethylene And a third step of reacting lycool with polyethylene glycol, which is grafted with bis-N-hydroxysuccinimide in the second step, to prepare a nanosized gel.

Preferably, the method further includes a fourth step of sealing the drug or physiologically active ingredient in the nanogel after the third step.

Preferably, the third step may be performed by an emulsion-evaporation method.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  1: Preparation of reagent

Poly (ethylene glycol) (PEG, MW 2,000 Da and 4,000 Da), L-aspartic acid-β-benzyl ester, 1,4- Anhydrous 1,4-dioxane, hexylamine (HA), trimethylamine (TEA), 1- (3-aminopropyl) imidazole, N-hexane, methanol, MeOH, N, N-dimethylformamide DMF, cellite, palladium on-charcoal, Pd / C ), N-hydroxysuccinimide (NHS), N, N'-dicyclohexylcarbodiimide (DCC), triethyleneamine (TEA) Dimethylaminopyridine (DMAP), succinic anhydride, DMSO-d ₆ and tetramethylsilane (TMS) were synthesized using Sigma-Aldrich (St. Louis, Mo., USA) ). Tree phosgene (triphosgene) was purchased from moving (Alfa Aesar ^® Johnson Matthey Korea, Seoul) to the alpha, ethanol (ethanol, EtOH), dimethyl sulfoxide (dimethylsulfoxide, DMSO) and dichloromethane (chloromethane, DCM) is Honeywell were purchased from Burdick & Jackson ^{(Honeywell Burdick & Jackson ®, Muskegon} , MI, USA). All other reagents used in the experiments were used as analytical grade. LysoTracker ^® Red DND-99, and fluorescein-DHPE (N- (Fluorescein-5- Thiocarbamoyl) -1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolmine Triethylammonium Salt) is Invitrogen ^(TM Invitrogen, Molecular Probes ^®, Life Techloogies, Thermo Fisher Scientific Inc.).

Example  2 : Poly (Aspartic acid-graft-imidazole) - Polyethylene glycol (P (Asp-g-Im) -PEG) < / RTI >

P (Asp-g-Im) -PEG (MW 6.5-2 KDa) was synthesized by referring to a previously reported article (Macromolecular Research, May 2011, Volume 19, Issue 5, pp 453-460). Briefly, the polymer was synthesized by ring-opening polymerization with [alpha] -amino acid-N-carboxyanhydride (NCA) and was characterized by PEG conjugation, deprotection and imidazole grafting ≪ / RTI > The Asp repeating unit of P (Asp) and the graft of the imidazole ring were determined by ¹ H-NMR analysis. As a result of comparing the ¹ H-NMR peak integration ratio between CH ₃ (δ = 0.88) of HA and α-CH (δ = 4.70) of P (Asp), the Asp repeating unit was measured as 35 and P (Asp) PEG was obtained in a yield of 60.5%. The grafting ratio of 1- (3-aminopropyl) -imidazole was 60% as a result of comparing two peaks at δ = 6.88-7.63 (imidazole group) and δ 4.40 (-CH-, repeating unit).

Example  3: Polyethylene glycol  base Cross-linking agent (bis- NHS  coupled PEG) synthesis

Crosslinking agents were prepared using chemically modified PEG with various molecular weights (2000 and 4000 Da). Bis-carboxylated PEG was prepared by esterification of hydroxy groups with succinic anhydride. Briefly, PEG (1 mmol) with two hydroxy groups was reacted with succinic anhydride (2.4 mmol), DMAP (2 mmol) dissolved in DCM and TEA (1 mL) in DCM (30 mL) / pyridine 2.4 mmol) at room temperature for one day. After the reaction, bis-carboxylated PEG (yield: 93 ± 3 wt.%) Was obtained by precipitation with diethyl ether. Conversion of the PEG from the hydroxy group to the carboxy group was confirmed by ¹ H NMR (隆 = 2.5: -CH ₂ -CH ₂ -, an ethylene group grafted to the PEG end formed by carboxymethylation). Bis-carboxylated PEG (2 mmol) was prepared by using HS (2.4 mmol) and DCC (2.4 mmol) dissolved in DCM (30 mL) for the crosslinking of the imidazole group in P (Asp-g- And activated at room temperature for one day. The dicyclohexylurea (DCU) was then removed by filtration and recrystallized with diethyl ether. Finally, it was dried for two days under vacuum to obtain bis-NHS-grafted PEG.

Example  4 : Nano Gel  Produce

Nanogels were prepared using modified emulsification-evaporation. Briefly, bis-NHS grafted PEG dissolved in dichloromethane (12 μmol / 3 mL) was added to an acidic aqueous solution of P (Asp-g-Im) -PEG (12 μmol / 30 mL, pH 4.0) Ultrasonography was performed in an ice-bath for 30 minutes. The ratio between the crosslinking agent and the polymer was fixed at 1. The white emulsion was evaporated in vacuo to give a clear solution. Nano gel suspension was dialyzed for 48 hours at 25 ℃ using a hydrochloric acid (or sodium hydroxide) -Na ₂ B ₄ O ₇ buffer solution (pH 8.2, 1 mM) and SpectraPore 6 K-8 KDa membrane bag (bag). Thereafter, the resultant was lyophilized to obtain a powdery nano gel at a yield of 86 ± 6%.

To make the nano-gel in a liquid state, the nano-gel powder state DMSO and hydrochloric acid (or sodium hydroxide) -Na ₂ B ₄ O ₇ buffer solution (pH 8.2, 1mM) (9 : 1) into a mixture of hydrochloric acid (or sodium hydroxide ) Na ₂ B ₄ O ₇ buffer (pH 8.2, 1 mM) at 25 ° C for 48 hours. Then, the solution was collected in a dialysis bag and used for the experiment. The chemical synthesis route of the nanogel is shown in FIG.

Two nanogels were synthesized using PEG grafted with bis-NHS having a molecular weight of 2000 and 4000 Da, respectively, and NG 2 and NG 4, respectively, and the following physical properties studies were carried out.

Example  5: Acid-base titration experiment

Titration plots of nanogels (NG 2, NG 4) and sodium chloride (NaCl, control) were measured using a potentiometric method. The powdered nanogel or sodium chloride was dissolved in deionized water (2 mmol / L) and the pH was adjusted to 11 using 1 N sodium hydroxide. The solution was then titrated with 0.5 N hydrochloric acid to obtain a pH profile. The titration was repeated three times and the mean value was plotted.

The acid - base titration experiments were performed because the pK _a value and the buffer buffer capacity were highly related to the pH sensitivity of the nanogel. As a result, referring to FIG. 3, it was confirmed that the nanogel 2 and the nanogel 4 had a proper pK _a value and a wide buffering pH range. The pK _a value of Nanogel 2 was about 6.88, the buffer pH range was 7.0 to 5.3, the pK _a value of Nanogel 4 was about 6.99 and the buffer pH range was 7.3 to 5.5. It was confirmed that the nanogel is similar to the pK _a value of P (Asp-g-Im) -PEG (pKa: 6.5).

The pK _a value of the nanogels with positively charged group and negatively charged group was 6.9, indicating that the partially positively charged imidazole group of grafted P (Asp-g-Im) had a hydrophobicity of the deprotected carboxy group of P (Asp) While keeping it neutralized. P (Asp-g-Im) -PEG is reduced by the positivity of the imidazole ring of grafted P (Asp-g-Im) and corresponding reduction of the carboxy group charged with P (Asp) It becomes a positive charge. At pH 5.0 or less, all carboxy and imidazole groups of P (Asp-g-Im) -PEG were positive and showed hydrophilicity. In particular, nanogel polymer electrolyte copolymers having a pI value of 6.9 or less can be suitably utilized to target acidic pH environments of tumors.

Previous studies have shown that P (Asp-g-Im) -PEG has a broad buffer pH range due to the imidazole group (pH 7.5-5.7). Referring to FIG. 3, it can be confirmed that the nanogel has a similar buffering pH range (NG 2: pH 7.0 to 5.3, NG 4: pH 7.3 to 5.5) due to structural similarity. The high polymer buffer capacity results in excellent osmotic effects in endosomes and increases the release of nanogels from endosomes and endosomes.

To analyze the cross-linking, the pH buffer region was analyzed in terms of molarities of protons reacting with carboxy and imidazole groups. The number of carboxy and imidazole groups was determined by the proton concentration of the hydrochloride. As two imidazole groups form a bridge, 2 μmol of nanogel has 42 μmol imidazole groups. As a result of the acid-base titration test of the nano gel, c.a. of the imidazole group was confirmed to be 40 μmol. Next, an experiment was conducted to analyze the physicochemical properties to confirm the crosslinking reaction.

Example  6: Measurement of particle size and zeta potential

The effective hydrodynamic diameters (D _eff ) and size distributions of the nanogels were analyzed by photon correlation spectroscopy using a Zetasizer Nano-ZS (Malvern Instruments, Malvern, UK). Size measurements were performed at 90 ° scatter angle and in a thermostatic cell. The available fluid diameter of the particles was calculated using software provided by the manufacturer. Nano-gel samples at various pH conditions (0.01 wt%) were prepared by diluting 10-fold with phosphate buffered saline (PBS) or citrate-phosphate buffer (pH 8.0 to 5.0). Before measurement, the nanogel samples were reacted at room temperature for 3 hours.

To confirm the pH-dependent reversible expansion / contraction of the nanogel, the pH value of the nanogel sample solution was adjusted from pH 7.4 to pH 6.5 (or pH 6.5 to pH 6.4) using 0.1 N hydrochloric acid or sodium hydroxide, Before measuring the size, they were reacted for 12 hours or more at each pH condition.

The zeta potential of the nanogel solution (0.1 mg / mL) having various pH values (pH 8.0 to 5.0, ionic strength 0.15) was measured using a Zetasizer Nano-ZS. Nano-gel samples at various pH conditions (0.01 wt%) were prepared by diluting 10-fold with phosphate-buffered saline or citric acid-phosphate buffer (pH 8.0-5.0). Before measurement, the nanogel samples were reacted at room temperature for 3 hours.

The pH dependent expansion of the nanogel was analyzed by dynamic light scattering (DLS). As a result, referring to FIG. 3, it was confirmed that the particle size at pH 7.4 was smaller than 200 nm and had a narrow size distribution (NG 2: c.a. 168 nm, NG 4: c.a.

Referring to FIG. 4, it was confirmed that the nanogel expands gradually as the pH is decreased to increase the particle size (NG 2: c.a. 549 nm, NG 4: c.a. 484 nm, pH 5.0). This indicates the expansion of the nanogel by imidazole biocatalysis located in the nanogel core at acidic pH conditions. In the case of polymerized micelles, as the pH decreases, the two imidazole groups become charged, and the decrease in hydrophobicity of the core causes instability and the particle size is ca. To 280 nm. Due to the similarity of the structure, the electrostatic repulsion causes the expansion of the nanogel by pH reduction.

In addition, the nanogel showed a decrease in size at pH 7.4 than pH 8.0. Similar particle size changes due to pH were observed in (Asp-g-Im) -PEG micelles. The particle size of the polymerized micelle decreased slightly at pH 7.0, indicating that the positively charged imidazole group of the P (Asp-g-Im) block electrostatically interacts with the ionized carboxy group of P (Asp-g-Im) To form a solid core of the nanogel.

Referring to FIG. 5, the pH sensitivity of the nanogel was confirmed by zeta potential measurement. At pH 7.4, the zeta potentials of Nanogel 2 and Nanogel 4 are due to the imidazole and ionized carboxy groups in the P (Asp-g-Im) block, respectively. -15.4 mV and c.a. -5.9 mV. At pH 7.0, the zeta potential of each particle was c.a. -8.8 mV and c.a. But increased slightly at -4.68 mV. Thus, it can be seen that a partially positively charged imidazole group neutralizes the carboxy group at the negative charge.

At pH 4.0, the zeta potential of the nanogels gradually increased to ca + 3.8 and ca + 2.0 mV, respectively. When lower than physiological pH (<pK _{a in the} imidazole group), imidazolium and carboxylate cause electrostatic interactions. The carboxy group is positive by positive charge of the nanoparticles at pH 4.0 (<pK _a of the carboxy group of the P (Asp) block). Nano gel 4 showed higher zeta potential than nano gel 2 at pH 7 and pH 8. When the pH was lower than 6.5, two nanogels showed similar zeta potential.

Example  7: Morphometric measurement

To observe the morphology of the nanogel, a solution of nanogel (0.1 mg / mL or 0.01 wt%) was prepared by injection into a glass slide and dried under vacuum. Before drying in vacuum, the nanogel sample was diluted with phosphate buffered saline (pH 7.4 or 6.5) and the nanogel solution was allowed to react at room temperature for 3 hours. The morphology was observed using a field emission scanning electron microscope (FE-SEM, SIGMA, Carl Zeiss, Germany).

Referring to FIGS. 6 and 7, reversible swelling due to continuous pH changes between pH 7.4 and 6.5 was analyzed by dynamic light scattering and field emission scanning electron microscopy. It was confirmed that the nano gel expands as the pH decreases due to the protonation and decarboxylation of the imidazole and carboxy groups of P (Asp-g-Im) -PEG, and contracts as the pH increases.

The particle size of Nanogel 2 is c.a. At 168 nm (pH 7.4) c.a. 302 nm (pH 6.5), and c.a. 134 nm (pH 7.4). Similarly, the particle size of Nanogel 4 is c.a. At 144 nm (pH 7.4) c.a. 295 nm (pH 6.5), and c.a. 127 nm (pH 7.4). Nano Gel 2 and Nano Gel 4 exhibited reversible swelling and contraction which swelled by pH reduction and returned to their original particle size at pH 7.4. Contrary to the nanogels, the particle size of the polymerized micelles increased when the pH was decreased from 7.4 to 6.5, and when the pH increased from 6.5 to 7.4, c.a. The irreversible particle change was further increased by 484 nm.

In addition, the reversible size change between pH 7.4 and pH 6.5 was visualized by field emission scanning electron microscopy. The dynamic light scattering measurements revealed that the nanogels expanded and contracted by cyclic pH changes while retaining their spherical shape.

Cross-linking between pH-sensitive polymers causes reversible swelling and prevents degradation of the structure. Reversible swelling of the nanogel enables controlled drug release due to cyclic pH changes. Referring to FIG. 1, the nanogel formed a spherical particle structure at pH 7.4, which is physiological pH, and reversibly expanded and contracted by the protonation and depolarization of imidazole residues by pH. Thus, nanogels can be used as nanocarriers by regulating drug release due to pH-dependent reversible expansion and contraction.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims (12)

At least one poly (aspartic acid-graft-imidazole) -polyethylene glycol copolymer grafted with imidazole to poly (aspartic acid) -polyethylene glycol and at least one poly Wherein the pH-sensitive nanogel comprises polyethylene glycol and is formed through crosslinking by covalent bonding between imidazole and bis-N-hydroxysuccinimide. The pH-sensitive nanogel of claim 1, wherein the copolymer and the polyethylene glycol are combined in a molar ratio of 1: 1. The pH-sensitive nanogel according to claim 1, wherein the nanogel forms a nanogel structure at a pH of 7.4 or more, and the nanogel structure collapses at a pH of 6 to 7.2. The pH-sensitive nanogel according to claim 1, wherein the average diameter of the nanogels is 50 to 500 nm. The pH-sensitive nanogel according to claim 1, wherein the average zeta potential of the nanogel is -20 to 10 mV. The pH-sensitive nanogel according to claim 1, wherein the average pK _a value of the nanogel is 5 to 8. The pH-sensitive nanogel according to claim 1, wherein the nanogel has an average buffering pH range of 4 to 8. A composition for delivering a drug or a physiologically active ingredient comprising the pH-sensitive nano-gel of claim 1 and a drug or physiologically active ingredient encapsulated in the nano-gel. The composition for delivering a drug or a physiologically active ingredient according to claim 8, wherein the physiologically active ingredient is selected from the group consisting of peptides, proteins, anticancer agents, anti-inflammatory agents, antibiotics, antibacterial agents, hormones, genes and vaccines. A first step of preparing poly (aspartic acid-graft-imidazole) -polyethylene glycol by covalently bonding imidazole to poly (aspartic acid) -polyethylene glycol;
A second step of reacting bis-carboxylated polyethylene glycol with succinic anhydride to prepare polyethylene glycol grafted with bis-N-hydroxysuccinimide; And
The poly (aspartic acid-graft-imidazole) -polyethylene glycol of the first step is reacted with the polyethylene glycol having the bis-N-hydroxysuccinimide grafted with the second step to prepare a nanogel And a third step,
Wherein the imidazole is formed through crosslinking by covalent bonding between imidazole and bis-N-hydroxysuccinimide. The method of claim 10, further comprising a fourth step of sealing the drug or the bioactive component in the nanogel after the third step. The method of claim 10, wherein the third step is performed by an emulsion-evaporation method.

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