KR20210096931A - pH sensitive hyaluronate copolymer and drug delivery system using the same - Google Patents

Abstract

The present invention relates to a pH-sensitive hyaluronic acid random copolymer and a drug delivery system for cancer treatment using the same. It was confirmed that the hyaluronic acid random copolymer (HID) exhibits pH sensitivity and amphiphilicity. In the case of the drug delivery system in which doxorubicin is encapsulated in the HID, a stable nanocarrier is formed at physiological pH (pH 7.4) to protect the drug, but becomes destabilized by protonation of an imidazole group in acidic conditions (pH 6.8 to 6.0) of cancer cells, and then the nanocarrier will disintegrate and release the drug. Thus, the HID may be provided as a pH-dependent drug delivery system that safely protects the drug until HID arrives at a target site, reduces drug toxicity in vivo, and improves drug activity locally at the target site.

Description

pH sensitive hyaluronate copolymer and drug delivery system using same

The present invention relates to a pH-sensitive random copolymer of hyaluronic acid and a drug delivery system for cancer treatment using the same.

Pharmacologically effective drugs do not show excellent effects as expected due to the serious toxicity and low solubility of the drug in actual clinical application. has been actively pursued.

A drug delivery system that can minimize toxicity while improving the therapeutic efficacy of a new drug formulation has been developed. Among these drug carriers, there is a micelle having a thermodynamically stable and uniform spherical structure in a compound having both a hydrophilic moiety and a hydrophobic moiety. Since the compound having a micelle structure has hydrophobicity in the center, various hydrophobic drugs can be encapsulated. Encapsulated hydrophobic drugs have a disadvantage of low solubility in aqueous solution, but when encapsulated in micellar particles, drug solubility in aqueous solution can be improved.

Meanwhile, the pH environment of normal tissues and the body generally maintains pH 7.2 to pH 7.4. However, it has been reported that the cancer or inflammatory disease tissue exhibits a locally low pH, and the pH around the cancer exhibits a lower pH than normal tissues due to the organic acid generated by the active metabolism of cancer cells, and is reported to be about pH 6.8 on average. In addition, when particles are absorbed into cells, endosomes are formed, and the pH of the endosomes is known to be about 6.0 or less.

Therefore, pH-sensitive polymers have been prepared for drug delivery by using a locally low pH in cancer or inflammatory tissues, but in the case of existing pH-sensitive polymers, the pH sensitivity according to the pH change is remarkably low, so they cannot be practically used, or hydrophobic drugs When this is encapsulated, there is a problem that a high therapeutic effect on the disease cannot be expected, and therefore, it is necessary to develop a new pH-sensitive drug delivery system.

As hyaluronic acid (HA), a functional biomaterial, specifically interacts with CD44 overexpressed on cancer cells, studies on cancer targeting using hyaluronic acid have been actively conducted. However, since the pKa (3-4, carboxyl group) of hyaluronic acid is an inappropriate biomaterial to release drugs at tumor pH, its use as a drug delivery agent for anticancer treatment is limited.

Republic of Korea Patent Publication No. 10-2014-0118560 (published on Oct. 8, 2014)

The present invention provides a random copolymer obtained by polymerizing an imidazole group and various aminoalkyl groups on hyaluronic acid, which is inappropriate to release a drug under the tumor pH conditions, and is a novel hyaluronic acid-based base that exhibits sensitivity in the acidic microenvironment of the tumor and releases the drug To provide a drug delivery system of

The present invention provides a pH-sensitive hyaluronic acid random copolymer represented by the following formula (1).

[Formula 1]

In Formula 1,

R ¹ is imidazole-(CH ₂ )n-NH-, n is an integer from 1 to 5, R ² is amino(C12-C20)alkyl,

Wherein x is a hyaluronic acid-graft-monomer is already imidazole (hyaluronic acid- graft -imidazole), y is a monomer of the hyaluronic acid, z is a hyaluronic acid-monomer of dodecylamine (hyaluronic acid- graft -dodecylamine) - graft am.

The present invention provides a drug delivery system comprising a random copolymer of pH-sensitive hyaluronic acid represented by Formula 1 above.

In addition, the present invention is a random copolymer of pH-sensitive hyaluronic acid represented by Formula 1; And it provides a pharmaceutical composition for treating cancer diseases containing the anticancer agent encapsulated in the random copolymer as an active ingredient.

According to the present invention, it was confirmed that a hyaluronic acid random copolymer (HID) obtained by polymerizing an imidazole group and a dodecylamine group in hydrophilic hyaluronic acid exhibits pH sensitivity and amphiphilicity. While it protects the drug by forming a stable nanocarrier at physiological pH (pH 7.4), in acidic conditions (pH 6.8 to 6.0) of cancer cells, it is destabilized by protonation of the imidazole group and the nanocarrier collapses to release the drug. As confirmed, the HID can be provided as a pH-dependent drug delivery system that safely protects the drug until it arrives at the target site, reduces drug toxicity in vivo, and improves drug activity locally at the target site.

Figure 1 (a) is a schematic diagram showing the chemical synthesis process of HID, Figure 1 (b) is a schematic diagram showing the concept of pH-sensitive HID in an acidic tumor environment.
Fig. 2 is a result of 1H-NMR analysis, Fig. 2 (a) is a structural analysis result of HID, Fig. 2 (b) is a structural analysis result of HA-graft polymerization-imidazole, and Fig. 2 (c) is a HA- This is the result of structural analysis of graft polymerization-dodecylamine.
3 is a result of confirming the pH profile of NaCl, HA and HID through acid-base titration.
Figure 4 is the result of confirming the pH sensitivity of the HID nanocarrier (nanocarrier, HN), Figure 4 (a) is the result of confirming the pH-dependent particle size and zeta potential of HN, Figure 4 (b) is the pH-dependent CMC of HID This is the result of checking the value.
5 is an FE-SEM result confirming the shape of HN at each pH condition and a DLS result confirming a particle size distribution, FIG. 5 (a) is a HN result at pH 7.4, and FIG. It is the result.
6(a) is the result of confirming the cumulative DOX release from doxorubicin-loaded HID nanocarriers (DHNs) at pH 7.4 and pH 6.5, and FIG. 6(b) shows the converted structure and particle size distribution of DHNs at pH 7.4. The confirmed FE-SEM and DLS analysis results, and FIG. 6(c) is the FE-SEM and DLS analysis results confirming the converted structure and particle size distribution of DHNs at pH 6.5.
7 is a result of analyzing the cell viability of Hep3B cells treated with HID, DOX·HCl and DHNs.

Hereinafter, the present invention will be described in more detail.

As hyaluronic acid (HA), a functional biomaterial, specifically interacts with CD44 overexpressed on cancer cells, cancer targeting studies using hyaluronic acid have been actively conducted, but despite high functionality, HA pKa (3-4 , carboxyl group) is a biomaterial that is inappropriate to release a drug at tumor pH, so to solve the above problem, the present inventors have polymerized an imidazole group showing pH sensitivity to hyaluronic acid under EDC / NHS and an aminoalkyl group showing hydrophobicity. The present invention was completed by preparing a random ronic acid polymer.

The present invention may provide a pH-sensitive hyaluronic acid random copolymer represented by the following formula (1).

[Formula 1]

In Formula 1,

R ¹ is imidazole-(CH ₂ )n-NH-, n is an integer from 1 to 5, R ² is amino(C12-C20)alkyl,

Wherein x is a hyaluronic acid-graft-monomer is already imidazole (hyaluronic acid- graft -imidazole), y is a monomer of the hyaluronic acid, z is a hyaluronic acid-monomer of dodecylamine (hyaluronic acid- graft -dodecylamine) - graft am.

More specifically, the random copolymer of hyaluronic acid is hydrophilic hyaluronic acid (y); a pH-sensitive region (x) formed by bonding an imidazole group to a carboxyl group of hyaluronic acid; and a hydrophobic region (z) formed by bonding an aminoalkyl group to a carboxyl group of hyaluronic acid, and may exhibit pH sensitivity and amphiphilicity.

The hyaluronic acid random copolymer may have a molar ratio of x:y:z of 30-40:40-50:10-20, and more preferably a molar ratio of 40:45:15, but is not limited thereto. does not

The random copolymer of hyaluronic acid may be micelled by self-assembly in a neutral condition of pH 7.0 to pH 7.5, and the micelles may be disintegrated in an acidic condition of pH 6.0 to pH 6.8.

The micelles may have an average diameter of 120 nm to 190 nm.

The present invention may provide a drug delivery system comprising a random copolymer of pH-sensitive hyaluronic acid represented by Formula 1 above.

The drug delivery system may be one in which micelles are disintegrated in the acidic microenvironment of the tumor, pH 6.0 to pH 6.8, to release the drug.

In addition, the present invention is a pH-sensitive hyaluronic acid random copolymer represented by the following formula (1); And it can provide a pharmaceutical composition for treating cancer diseases containing the anticancer agent encapsulated in the random copolymer as an active ingredient.

[Formula 1]

In Formula 1,

R ¹ is imidazole-(CH ₂ )n-NH-, n is an integer from 1 to 5, R ² is amino(C12-C20)alkyl,

Wherein x is a hyaluronic acid-graft-monomer is already imidazole (hyaluronic acid- graft -imidazole), y is a monomer of the hyaluronic acid, z is a hyaluronic acid-monomer of dodecylamine (hyaluronic acid- graft -dodecylamine) - graft am.

The hyaluronic acid may specifically interact with CD44 overexpressed cancer cells to target cancer cells.

The pharmaceutical composition may be one in which the micelle is disintegrated in the acidic microenvironment of the tumor, pH 6.0 to pH 6.8, to release the anticancer agent.

The anticancer agent may be selected from the group consisting of doxorubicin, paclitaxel, and docetaxel.

The cancer disease may be selected from the group consisting of lung cancer, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, breast cancer, liver cancer, skin cancer, stomach cancer, esophageal cancer, testicular cancer, kidney cancer, colorectal cancer and rectal cancer, It is not limited thereto.

In addition, the present invention may provide a pharmaceutical composition for the treatment of cancer diseases comprising the drug carrier.

According to an embodiment of the present invention and FIG. 1, it was confirmed that the hyaluronic acid random copolymer (HID) obtained by polymerizing an imidazole group and a dodecylamine group in the hyaluronic acid exhibits pH sensitivity and amphiphilicity, and doxorubicin to the HID. In the case of a drug carrier encapsulated with a nanocarrier, it forms a stable nanocarrier at physiological pH (pH 7.4) to protect the drug, whereas in acidic conditions of cancer cells (pH 6.8 to 6.0), the nanocarrier is destabilized by protonation of the imidazole group. As it is confirmed that it decays and releases the drug, the HID safely protects the drug until it arrives at the target site, thereby reducing drug toxicity in vivo and improving drug activity locally at the target site. Provided as a pH-dependent drug carrier and anticancer agent can be

In one embodiment of the present invention, the pharmaceutical composition for the treatment of cancer diseases is an injection, granule, powder, tablet, pill, capsule, suppository, gel, suspension, emulsion, drop or liquid according to a conventional method. Any one formulation selected from may be used.

In another embodiment of the present invention, suitable carriers, excipients, disintegrants, sweeteners, coating agents, swelling agents, lubricants, flavoring agents, antioxidants, buffers, bacteriostats, It may further include one or more additives selected from the group consisting of diluents, dispersants, surfactants, binders and lubricants.

Specifically, carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules. agent and the like, and such a solid preparation may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives in addition to commonly used simple diluents such as water and liquid paraffin may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base material for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

According to an embodiment of the present invention, the pharmaceutical composition is administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered to the subject.

The preferred dosage of the drug carrier may vary depending on the condition and weight of the subject, the type and extent of the disease, the drug form, the route and duration of administration, and may be appropriately selected by those skilled in the art. According to an embodiment of the present invention, although not limited thereto, the daily dose may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or may be administered in several divided doses, thereby not limiting the scope of the present invention.

In the present invention, the 'subject' may be a mammal including a human, but is not limited to these examples.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Substance

Sodium hyaluronate (Sodium hyaluronate, HA, weight-average molecular weights 480kDa) was purchased from SK bioland (Seoul, Republic of Korea).

1-(3-aminopropyl)-imidazole (1-(3-aminopropyl)-imidazole; IM), dodecylamine, pyrene, N-hydroxysuccinimide (NHS) , N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; EDC-HCl), triethylamine (TEA), anhydrous 1,4-dioxane (anhydrous 1,4-dioxane), (N,N-dimethylformamide; DMF) and D2O-d6 were purchased from Sigma-Aldrich (St. Louis, MO, US).

Ethanol (EtOH) and acetonitrile (ACN) were ^{purchased from Honeywell Burdick & Jackson ®} (Muskegon, MI, US), and doxubicin-hydrochloride (DOX-HCl) was obtained from Boryung Co. (Seoul, Republic of Korea). Docetaxel was purchased from Samyang Bio Pharmaceutical® (Daejeon, South Korea). All compounds were used as analytical grade.

For cell culture, human breast cancer Hep3B cells were obtained from Korean Cell Line Bank (KCLB, Seoul, Republic of Korea), and DMEM medium, fetal bovine serum (FBS), penicillin and streptomycin were added to Welgene (Seoul, Republic of Korea), and Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Tokyo, Japan) and used.

2. Actin-Based Titration

Titration plots of HID, HA and NaCl (control) were obtained using a potentiometric method. The polymer powder and NaCl were dissolved in 10 mL of distilled water (0.0108 mmol/ml, number of moles of -COOH), and the pH was adjusted to 12 with 1 N NaOH. The solution was titrated with 0.5 N HCl and a pH profile was obtained. Three titrations were performed to indicate the average value.

3. Preparation of hyaluronic acid random copolymer (HID) nanocarriers (HID nanocarriers; HNs)

A random hyaluronic acid copolymer (HID, 10 mg) was dissolved in 10 ml of ethanol. The organic layer was removed from a round-bottom flask using a rotary evaporator (EYELA n-1000, Tokyo, Japan) to produce a thin film.

For nanocarrier preparation, the film was rehydrated with phosphate-buffered saline (PBS) solution. Thereafter, each solution was sonicated for 10 minutes using a probe-type ultrasonicator (VCV-505, Sonics & Materials, CT, US). The pH value of the nanocarrier solution was adjusted with PBS (pH 6.0-7.4).

4. Critical micelle concentration (CMC) analysis

For the determination of the critical micelle concentration (CMC), fluorescence was measured with excitation light (343 nm) and emission light (384 nm) using a Scinco FS-2 fluorescence spectrometer (Seoul, Republic of Korea) equipped with a polarizer.

HN samples were prepared at various pH values in PBS ranging from 6.0-7.4, and ^{a fluorescent probe pyrene at a concentration of 6.0 × 10 -7} M was mixed and stirred at room temperature overnight. All measurements were performed at room temperature in an air-equilibrated solution.

The CMC value was determined by plotting the ratio of I1 (intensity of first peak) to I3 (intensity of the third peak) of the emission spectrum profile against the log10 value of the nanocarrier concentration, and the CMC value as the intersection of the low polymer concentration in the plot was defined.

5. Particle Size and Zeta Potential Measurements

The efficient hydrodynamic diameter (Deff.) of the nanocarriers and the zeta potential of HID were measured by photon correlation method using Zetasizer Nano-ZS (Malvern Instruments, UK) equipped with Multi Angle Sizing Option (BI-MAS). .

HNs were prepared under different pH conditions (0.01 wt%) by diluting the stock solution 20-fold using PBS solution (pH 6.0-7.4).

Prior to measurement, the HN solution was incubated at room temperature for 6 hours (n = 3).

With software provided by the manufacturer, Dff. and zeta potential values were calculated; Each sample was measured three times to calculate the average value for each parameter (n = 3).

6. Confirmation of morphology of HID nanocarriers (HNs)

Nanocarriers were incubated for at least 12 hours at pH 7.4 and 6.5. The diluted nano-E carrier solution (0.1 mg/mL) was placed on a glass slide and dried in vacuo. The morphology of HNs was confirmed using a field emission scanning electron microscope (FE-SEM, Sigma, Carl Zeiss, Germany).

7. Preparation of HID nanocarriers (DHNs) loaded with doxorubicin (DOX)

To prepare doxorubicin-supported HID nanocarriers (DHNs), DOX and HID were dissolved in 10 mL EtOH and prepared by a thin layer method. Before loading DOX into the HID nanocarrier, DOX.HCl was added to EtOH together with TEA (2 mol) and stirred overnight to obtain a doxorubicin salt.

To prepare DHN, the organic solution was removed using a rotary evaporator (model n-1000, EYELA, Tokyo, Japan) to form a thin film in each round-bottom flask.

The film solution rehydrated with PBS solution (pH 7.4, 0.15 M) was sonicated for 10 min to generate DHN. The unencapsulated drug was removed by passing the nanocarrier solution through a 0.45 μm membrane filter. Drug loading capacity and efficiency were calculated using the following formula.

Drug-loading capacity (%) = (Weight of drugs in nanocarriers) / (Weight of total nanocarriers) × 100

Drug-loading efficiency (%) = (Weight of drugs in nanocarriers) / (Weight of drug initially added to formulation) × 100

The content of doxorubicin supported on HN was measured at 481 nm UV absorbance using a UV-1200 Spectrophotometer (Labentech, Incheon, Republic of Korea) after dissolving in water: PBS solution (90: 10 vol%).

8. Preparation of HID nanocarriers (DTHNs) loaded with docetaxel (DTX)

To prepare docetaxel-supported HID nanocarriers (DTHNs), DTX and HID were dissolved in 10 mL EtOH and prepared by a thin layer method.

To prepare DTHN, the organic solution was removed using a rotary evaporator (model n-1000, EYELA, Tokyo, Japan) to form a thin film in each round-bottom flask.

DTHN was generated by sonicating the film solution rehydrated with PBS solution (pH 7.4, 0.15 M) for 10 min. The unencapsulated drug was removed by passing the nanocarrier solution through a 0.45 μm membrane filter. Drug loading capacity and efficiency were calculated using the following formula.

Drug-loading capacity (%) = (Weight of drugs in nanocarriers) / (Weight of total nanocarriers) × 100

Drug-loading efficiency (%) = (Weight of drugs in nanocarriers) / (Weight of drug initially added to formulation) × 100

The concentration of docetaxel was determined using high-performance liquid chromatography (Agilent 1200 series, Agilent Technologies Santa Clara, CA, USA).

A reversed-phase C18 column (150×4.5 mm id., pore size 5 μm; Micro solv Tech. Corp., Eatontown, NJ, USA) was used, and it was composed of a mixture of ACN and distilled water (55:45, v/v%). It was transferred isocratic using the mobile phase. When the sample was transported by a pump with a flow rate of 1 mL/min, the retention time of docetaxel was 4.5 minutes, and the column temperature was maintained at 25°C.

The column effluent was detected at 230 nm and the concentration of docetaxel was calculated using a linear calibration curve of a standard docetaxel solution.

9. In vitro drug release

To confirm drug release, DOX.HCl or DHNs solution (80 μg of DOX) was placed in triplicate Spectra/Por dialysis membrane bags (MWCO 6-8 kDa), and each sample was incubated in PBS with pH 7.4 and 6.5. In order to maintain the sink conditions, the mixture was stirred at 37° C. and 100 rpm in a circulating constant temperature water bath (Nexus Tech., Seoul, Republic of Korea).

The outer phase of the dialysis membrane bag was withdrawn for analysis of drug concentration and replaced with fresh PBS at predetermined time intervals to maintain sink conditions.

The concentration of DOX was measured by the above method with a UV Spectrophotometer.

10. Confirmation of in vitro anticancer effect

Hep3B cells were inoculated into the growth medium at a vertical density of ^{8×10 3} per well of a 96-well plate 24 hours before cytotoxicity was confirmed. Immediately before use, DOX·HCl and DHNs were prepared in DMEM medium. The medium was removed from the 96-well plate, different concentrations of DOX were added, and incubated for 48 hours.

CCK-8 assay was performed to confirm cell viability.

Briefly, 10 µL of CCK solution in fresh medium (90 µL) was added to each well and the plate was incubated for an additional 3 h. The absorbance of each well was checked with a Flexstation three microplate reader (Molecular Devices, Sunnyvale, CA, US) at a wavelength of 450 nm.

<Example 1> Hyaluronic acid-polymerization-(imidazole-dodecylamine) (HID) synthesis

1. hyaluronic acid-polymerization-(imidazole-dodecylamine) [hyaluronic acid-graft-(imidazole-dodecylamine), [(h- g -I) _x -(h) _y -(h- g -D) _z ] _ran , HID] synthesis

In order to provide a nanocarrier suitable for anticancer treatment, hyaluronic acid-polymerization-(imidazole-dodecylamine) [Hyaluronic acid-graft-(imidazole-dodecylamine), (h- g- I) _x -(h) _y -(h- g -D) _z ] _ran , HID] was synthesized.

1-(3-aminopropyl)-imidazole (IM) chemically binds to the backbone of hyaluronic acid (HA) by amide formation in the presence of binding reagents, EDC HCl and NHS, to form hyaluronic acid (HA) with pH sensitivity transformed.

After dissolving HA (MW 480 kDa, 500 mg, 1.35 mmol (the number of moles of -COOH)) in distilled water at a concentration of 5 mg/ml, to activate the carboxyl group of HA, EDC (776 mg, 4.05 mmol) and NHS (466 mg, 4.05 mmol) was added. After stirring at room temperature for 12 hours, IM (5.4 mmol) and dodecylamine (1.35 mmol) dissolved in ethanol were slowly added, followed by stirring at 65° C. for 2 days.

To prepare hyaluronic acid-polymerized-imidazole (hyaluronic acid-graft-imidazole; HI) and hyaluronic acid-polymerized-dodecylamine (HD), IM dissolved in ethanol (5.4 mmol) and dodecylamine (5.4 mmol) were added slowly and stirred at 65° C. for 2 days.

The reaction mixture was dialyzed against water:ethanol (1:1 v/v) for 1 day using a dialysis membrane (molecular weight cut off (MWCO) 12000-14000 Da), and dialyzed with water for 2 days to remove by-products After removal, freeze-drying was performed to obtain a dried HA derivative.

The structural characteristics of the synthesized polymers such as HID, HI and HD ^{were confirmed using 1} H-NMR 600 MHz spectrometer (Delta, JEOL, MA, US).

2. Check HID

A random copolymer was prepared by polymerizing the carboxyl group of hyaluronic acid with an imidazole ring, which is a pH-sensitive moiety, in order to release the drug into the tumor tissue in an acidic environment as described above.

In the previous report, the imidazole ring exhibited 6.8 pKa and was confirmed to exhibit pH reactivity in the cancer microenvironment. In addition, since hyaluronic acid is a hydrophilic polymer, dodecylamine is bound to act as an amphiphilic property as a hydrophobic moiety.

A polymer was synthesized by amine coupling reaction of the carboxyl group of hyaluronic acid using an EDC/NHS compound. As a result, a polymer was obtained in a yield of 93.6%.

Attached through comparison of ^{three 1} H-NMR peaks, such as δ 7.1 - 7.5 (imidazole group), δ 0.75 (-CH ₃ -, the last portion of dodecylamine ) and δ 4.34 (-CH-, the backbone of hyaluronic acid) The amounts of imidazole groups and dodecylamine groups were confirmed.

As a result, as shown in FIG. 2, the polymerization ratio was confirmed to be 45% of imidazole groups and 15% of dodecylamine groups.

Two other polymers were further synthesized and structural analysis was performed by comparison with HID.

The amounts of polymerized imidazole groups and dodecylamine groups were confirmed through δ 7.1 to 7.5 (imidazole group) and δ 0.75 (-CH ₃ ^{-, dodecylamine) of 1 H-NMR peak.} The polymerization rate of the imidazole ring in HI was found to be 55%, whereas the polymerization rate of dodecylamine in HD was found to be 40%.

<Example 2> Confirmation of pH profile of HID

Since the pKa and buffering capacity are closely related to the pH sensitivity of the polymer, the pH profile was investigated by acid-base titration. As a result, as shown in FIG. 3 , the pKa of HID was about 6.84, and it was confirmed that the buffer pH range was between 6.32 and 8.23.

It has been reported that pH-sensitive polymers exhibiting a pKa value of about 6.84 and broad buffering capacity are suitable materials for converting drug release profiles in the acidic environment of solid tumors. did not show

In order to clearly confirm the pH profile of HID, ^{the substitution ratio (Graft ratio, GR) of imidazole groups on 1} H-NMR and the number of moles of imidazole groups reacted to protons of hydrochloride were compared.

As a result of confirming the number of imidazole groups through measurement of the proton concentration of hydrochloride, 108 μmol of carboxyl groups in 40 mg of HID by ¹ H-NMR analysis based on GR as shown in FIG. 2 were imidazole moieties and dodecylamine These were 45% and 15% polymerized, respectively. In addition, considering that 45 μmol of imidazole was protonated as shown in FIG. 3, the GR of HID was confirmed to be 42% by acid titration, and it was confirmed that the result was similar to that of NMR analysis.

<Example 3> pH-sensitivity (sensitivity) confirmation of HNs

Based on the pH profile of HID, we confirmed the pH sensitivity of HNs to confirm their functionality in the acidic tumor microenvironment.

As a result of confirming the pH-dependent particle size of HN using DLS, the particle diameter was smaller than 200 nm at pH 7.4, as shown in FIG. 4a, and showed a narrow size distribution.

In addition, the average diameter of the micelles at pH 7.4 was 151.1 nm, and exhibited a narrow size distribution and a PDI value of 0.20. At pH 6.5 and 6, HNs was disintegrated by protonation of the imidazole group, resulting in an increase in particle diameter to 376 nm and 465 nm, respectively. was found to increase.

In addition, the pH sensitivity of the nanocarriers was confirmed by measuring the zeta potential.

As a result, it was confirmed that the zeta potential of HNs at pH 7.4 was about -15.1 mV as shown in FIG. 4a. It was confirmed that when the pH was decreased below physiological pH, the proton of the imidazole group increased and the zeta potential of the particle rapidly increased and approached a nearly neutral charge of about -3.0 mV at pH 6.5.

Meanwhile, pH dependence of HN formation was confirmed by performing CMC analysis using a fluorescence analyzer in the presence of pyrene, a fluorescent hydrophobic probe.

As a result of confirming the formation of micelles through CMC measurement of HID at different pH conditions, CMC was found to be 1.6 and 10.9 μg/mL, respectively, in neutral conditions of pH 7.4 and pH 7.0, as shown in FIG. 3b. Compared with a low molecular weight surfactant (e.g., sodium dodecyl sulfate; 2.0 mg/mL at pH 7.4), it was confirmed that HID had a very low CMC value.

From the above results, stability as a nanocarrier for drug delivery was confirmed, and inhibition of drug loss from micelles at a concentration below CMC by rapid dilution after injection was confirmed.

Therefore, HIDs can self-generate into nanocarriers under physiological pH conditions.

As the pH decreased, the imidazole group of HID became protonated, leading to a change in the micropolarity of the micelle center and an increase in CMC. At pH 6.8, the CMC of HID significantly increased to 49.5 μg/mL, which is because micelles became unstable as the HID became more hydrophilic in a hydrophobic state due to the increase in protons of the imidazole group, and pH 6.5 through FE-SEM Collapse of micelles was confirmed in

Referring to FIG. 5 , HID, which produced nano-sized particles at pH 7.4, was disrupted at pH 6.5, and at pH 7.4, the nanocarriers showed a narrow unimodal size distribution (PDI: 0.20), whereas at pH 6.5, micellar particles The size distribution showed two peaks (PDI: 0.45).

From the above results, it was confirmed that the nanocarriers were disintegrated by the repulsive force between the positive charges due to the protonation of the imidazole group.

<Example 4> Confirmation of the converted drug release profile of DHNs at acidic pH

To confirm the high functionality of HID as a pH-sensitive nanocarrier, doxorubicin (DOX) and docetaxel (DTX) were used as model drugs.

DOX and DTX are drugs that show strong effects on many cancers, including breast cancer, prostate cancer, lung cancer, and stomach cancer, but their pharmaceutical applications are limited because they are very hydrophobic drugs and show low solubility.

Therefore, the solubilization and targeted delivery of DOX and DTX nanocarriers can overcome the above problems and improve the pharmacological effect.

In order to confirm the drug loading profile of DHNs and the formation of nano-sized particles, the characteristics of DHNs were analyzed. Referring to Table 1, nanocarriers were prepared with target loading contents of 10, 20 and 30 wt%. As the target loading concentration increased, it was confirmed that the particle size was kept below 200 nm, but the loading efficiency decreased.

In addition, the results related to the drug loading profile of DTHNs and the formation of nanoparticles were confirmed. As a result, as shown in Table 1, it showed a similar tendency to DHNs, and when prepared with a target loading content of 10w%, the particle size was about 200nm.

To determine the drug release profile of DHNs, DOX-HCl and DHNs were incubated in PBS (pH 7.4 and pH 6.5) under sink conditions.

As a result, as shown in FIG. 6a, DOX-HCl was widely released at a similar rate in both pH conditions, and it was confirmed that the sink condition was sufficient to determine the drug release profile.

On the other hand, DHNs showed a pH-dependent drug release, which showed a release of less than 40% for more than 48 hours at pH 7.4, but at pH 6.5, a structural change was observed due to protonation of the imidazole group on the HID, resulting in a DOX of about 70%. was released

In addition, as a result of confirming the morphology of DHN by FE-SEM at pH 7.4 and 6.5, it was confirmed that the structural transformation of the nanocarrier was induced as the pH decreased as shown in FIGS. 6b and 6c.

At pH 7.4, DHNs exhibited a more spherical structure than HN due to the hydrophobic interaction between DOX of HID and 'imidazole and dodecylamine'. The protonation of nanocarriers decreased the loading capacity of the nanocarriers and induced good DOX release. The shifted drug release profile is due to electrostatic repulsion among positive charges, which induced a conformational shift.

From the above results, it was confirmed that DHNs effectively inhibited the pH-dependent release of DOX at physiological pH (pH 7.4). Therefore, such inhibition can reduce systemic toxicity during blood circulation by minimizing drug loss in the bloodstream before DHNs reach the tumor site, and the tumor's pHex (tumor extracellular pH; pH 6.5 to 7.2) and pHen (endosomal pH; At pH 6.5 or lower), it induces DOX release from DHNs, enabling more effective antitumor activity.

Nanocarriers
(loaded drug) Target
Content
(wrt %) loading efficiency
(wt%) Loading content
(wt%) Average Size
( D _eff , nm) PDI DHNs
(DOX) 10 88.0 8.9 145.0 ± 2.9 0.26 ± 0.01 20 72.2 15.3 151.7 ± 2.1 0.21 ± 0.09 30 68.1 22.6 180.9 ± 2.7 0.25 ± 0.07 DTHNs
(DTX) 10 76.5 7.8 213.0 ± 5.0 0.19 ± 0.02

<Example 5> Confirmation of antitumor effect of DHNs

In order to confirm the antitumor effect of DHNs, the cell viability of Hep3B cells treated with DOX·HCl and DHNs was evaluated to confirm the activity against cancer treatment.

As a result, as shown in FIG. 7 , HID did not show toxicity at any concentration, and DHNs (0.54 μg/ml) showed 1.8-fold cytotoxicity compared to _{the IC 50 value of DOX·HCl (1.00 μg/ml).}

This result is due to the increased loading capacity of HID by the interaction between HA and CD44, because intracellular drug distribution and endosomal destruction were induced by the proton sponge effect.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (13)

A random copolymer of pH-sensitive hyaluronic acid represented by the following formula (1):
[Formula 1]

In Formula 1,
R ¹ is imidazole-(CH ₂ )n-NH-, n is an integer from 1 to 5, R ² is amino(C12-C20)alkyl,
wherein x is a monomer of hyaluronic acid-graft-imidazole, y is a monomer of hyaluronic acid, and z is a monomer of hyaluronic acid-graft-dodecylamine. Lim. The method according to claim 1, wherein the hyaluronic acid random copolymer is hydrophilic hyaluronic acid (y); a pH-sensitive region (x) formed by bonding an imidazole group to a carboxyl group of hyaluronic acid; and a hydrophobic region (z) formed by bonding an aminoalkyl group to a carboxyl group of hyaluronic acid, and pH-sensitive hyaluronic acid random copolymer, characterized in that it exhibits pH sensitivity and amphiphilicity. The method according to claim 1, wherein the random copolymer of hyaluronic acid is pH-sensitive hyaluronic acid random copolymer, characterized in that x: y: z is 30-40: 40-50: 10-20 molar ratio consisting of. The method according to claim 1, wherein the hyaluronic acid random copolymer is pH-sensitive hyaluronic acid random copolymer, characterized in that x:y:z is 40:45:15 molar ratio consisting of. The method according to claim 1, wherein the random copolymer of hyaluronic acid is micellarized by self-assembly in neutral conditions of pH 7.0 to pH 7.5, and micelles are disintegrated in acidic conditions of pH 6.0 to pH 6.8 pH-sensitive hyaluronic acid ronic acid random copolymer. The method according to claim 5, wherein the micelles are pH-sensitive hyaluronic acid random copolymer, characterized in that it exhibits an average diameter of 120 nm to 190 nm. A drug delivery system comprising a random copolymer of pH-sensitive hyaluronic acid represented by the following formula (1).
[Formula 1]

In Formula 1,
R ¹ is imidazole-(CH ₂ )n-NH-, n is an integer from 1 to 5, R ² is amino(C12-C20)alkyl,
wherein x is a monomer of hyaluronic acid-graft-imidazole, y is a monomer of hyaluronic acid, and z is a monomer of hyaluronic acid-graft-dodecylamine. Lim. The drug delivery system according to claim 7, wherein the micelle is disintegrated in the acidic microenvironment of the tumor, pH 6.0 to pH 6.8, to release the drug. pH-sensitive hyaluronic acid random copolymer represented by the following formula (1); and
A pharmaceutical composition for treating cancer diseases containing an anticancer agent encapsulated in the random copolymer as an active ingredient.
[Formula 1]

In Formula 1,
R ¹ is imidazole-(CH ₂ )n-NH-, n is an integer from 1 to 5, R ² is amino(C12-C20)alkyl,
wherein x is a monomer of hyaluronic acid-graft-imidazole, y is a monomer of hyaluronic acid, and z is a monomer of hyaluronic acid-graft-dodecylamine. Lim. The pharmaceutical composition for the treatment of cancer diseases according to claim 9, wherein the hyaluronic acid specifically interacts with the CD44 overexpressed cancer cells to target the cancer cells. The pharmaceutical composition for the treatment of cancer diseases according to claim 9, wherein the micelle is disintegrated in the acidic microenvironment of the tumor, pH 6.0 to pH 6.8, to release the anticancer agent. The pharmaceutical composition according to claim 9, wherein the anticancer agent is selected from the group consisting of doxorubicin, paclitaxel, and docetaxel. 10. The method of claim 9, wherein the cancer disease is lung cancer, uterine cancer, cervical cancer, prostate cancer, head and neck cancer, pancreatic cancer, brain tumor, breast cancer, liver cancer, skin cancer, stomach cancer, esophageal cancer, testicular cancer, kidney cancer, colon cancer and rectal cancer from the group consisting of A pharmaceutical composition for the treatment of cancer diseases, characterized in that selected.

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