KR102671525B1 - Temperature And Oxidation Responsive Active Ingredient Delivery System Comprising Polar Cationic polymer, Non-polar Anionic compound And Gold Nanoparticles, and Pharmaceutical Composition Comprising The Same - Google Patents

Abstract

The temperature- and oxidation-responsive ionic complex of the present invention has amphiphilic properties through ion pairing of a polar cationic polymer and a non-polar anionic compound with a hydrophobic residue, so it has the characteristic of forming a self-assembly. When dispersed in an aqueous solution, the anionic compound is formed. It is manufactured as micelle particles in which hydrophobic residues form the core and chains of cationic polymers form the shell, so it has the characteristic of being used as an active ingredient carrier capable of loading active ingredients.
The temperature- and oxidation-responsive ionic complex of the present invention has the advantage that the solubility of the complex increases when the surrounding temperature rises or is oxidized, thereby releasing the payload, and after cancer cell-specific endocytosis is performed including a cancer cell-specific ligand. Since the payload is released from inside the cell, it has the advantage of showing excellent anticancer effects without causing acute toxicity.

Description

Temperature and oxidation responsive active ingredient delivery system comprising a polar cationic polymer, a non-polar anionic compound and gold nanoparticles, and a pharmaceutical composition comprising the same. And Gold Nanoparticles, and Pharmaceutical Composition Comprising The Same}

The present invention relates to a temperature- and oxidation-responsive active ingredient carrier comprising a polar cationic polymer, a non-polar anionic compound, and gold nanoparticles, and a pharmaceutical composition containing the same.

When amphipathic molecules are dispersed in an aqueous solution, they self-assemble spontaneously according to the second law of thermodynamics (entropy-driven process). The self-assembly structure is determined by the shape of the constituent amphipathic molecules. The shape of an amphipathic molecule is defined by the packing parameter (P) as P=V/LA (where V: the volume of the hydrophobic tail of the amphiphilic molecule, L: the length of the hydrophobic tail, and A: the cross-sectional area of the hydrophilic head). For example, if the P value is close to 1, bilayer vesicles are formed, if the P value is greater than 1, they are formed in a hexagonal phase, and if the P value is less than 1, micelles are formed. ) is formed.

The self-assembly can be used as a drug carrier because it can accommodate hydrophilic drugs or hydrophobic drugs in its structure. Drug carriers require biocompatibility, stealth function, targeting properties, efficient tissue penetration, high drug protection ability, and timely release characteristics. Drug carriers must deliver the drug to the target site without side effects and must be released in a timely manner. A method of obtaining target site specificity and specific release characteristics of a drug carrier includes a method of imparting stimulus responsiveness to the drug delivery carrier. Temperature, pH, electric field, magnetic field, light, ions, enzymes, oxidation, reduction, etc. can be used as stimuli that cause drug release.

A multistimulus-responsive drug delivery carrier refers to a drug delivery carrier that has the property of responding to more than one stimulus. According to recent research, drug carriers that can release their contents in response to temperature and oxidation are being developed.

For example, it has been confirmed that the release of doxorubicin (DOX), an anticancer drug, is promoted under oxidizing conditions and high temperature conditions of the drug carrier; Polyhydroxyethyl acrylate-co-phenyl vinyl sulfide (P(HEA-co-PVS)) was developed as a temperature- and oxidation-responsive amphiphilic copolymer for the preparation of dual-responsive micelles. There is a bar. In addition, oxidation of phenyl vinyl sulfide (PVS) induces an increase in the low critical solution temperature of the copolymer and loss of amphiphilic properties, resulting in de-micellization, thereby promoting oxidation-induced release. It is known. However, the production of dual-reactive polymer micelles is technically difficult because the copolymer, which is a component of the micelle, is synthesized and purified through a complex process.

Patent documents and references mentioned herein are herein incorporated by reference to the same extent as if each individual document was individually and specifically identified by reference.

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In the present invention, polar cationic polymers and non-polar anionic compounds self-assemble to form micelles, so they can be used as transport vehicles by loading active ingredients, and folic acid and gold nanoparticles are introduced into the shell to induce endocytosis specific to cancer cells. A temperature and oxidation-responsive ion complex that not only enables (endocytosis) but also has improved temperature sensitivity, a temperature- and oxidation-responsive active ingredient carrier comprising the temperature and oxidation-responsive ion complex and a hydrophobic active ingredient, and the temperature and oxidation responsiveness. The purpose is to provide a pharmaceutical composition containing an active ingredient carrier.

Other objects and technical features of the present invention are presented in more detail by the following detailed description, claims, and drawings.

The present invention relates to a polar cationic polymer containing an amino group in a repeating unit and the formula 1 below:

[Formula 1]

(In the above formula, Ar is C6-C20 aryl, L2 is C1-C5 alkylene, and x is an integer of 1 to 3.):

It provides an ionic complex containing a non-polar anionic compound represented by.

The polar cationic polymer is polyethylene imine, the non-polar anionic compound is phenylthioacetic acid, and the ionic complex is a molar combination of the amino group of the polar cationic polymer and the carboxyl group of the non-polar anionic compound. It is characterized by a ratio of 2:8 to 5:5.

The ionic complex contains gold nanoparticles and is characterized by being folated.

The present invention provides a temperature- and oxidation-responsive active ingredient carrier comprising the ionic complex and the hydrophobic active ingredient.

The present invention provides a pharmaceutical composition or cosmetic composition containing the above temperature and oxidation-responsive active ingredient carrier.

The temperature and oxidation-responsive ionic complex of the present invention has the characteristic of forming a self-assembly because a polar cationic polymer and a non-polar anionic compound with a hydrophobic residue form an ion pair to form an amphipathic complex, and when dispersed in an aqueous solution, the anionic compound It is manufactured as micelle particles in which hydrophobic residues form the core and chains of cationic polymers form the shell, so it has the characteristic of being used as an active ingredient carrier capable of loading active ingredients.

The temperature- and oxidation-responsive ionic complex of the present invention has the advantage that the solubility of the complex increases when the surrounding temperature rises or is oxidized, thereby releasing the payload, and after cancer cell-specific endocytosis is performed including a cancer cell-specific ligand. Since the payload is released from inside the cell, it has the advantage of showing excellent anticancer effects without causing acute toxicity.

Figure 1 shows the results of analyzing the optical density of the PEI/PTA solution of the present invention.
Figure 2 shows the results of analyzing the change in fluorescence intensity due to interfacial tension and Nile red emission of the PEI solution, PTA solution, and PEI/PTA (3/7) solution of the present invention.
Figure 3 shows PEI, PTA, dry PEI/PTA (3/7), PEI/PTA (3/7) (5mM H ₂ O ₂ ), and PEI/PTA (3/7) (5mM H ₂ O ₂ ) of the present invention. ) shows the FT-IR spectrum analysis results.
Figure 4 shows the hydrogen nuclear magnetic resonance spectrum of the PEI/PTA (a/b) solution of the present invention.
Figure 5 shows the emission profile of Nile Red mounted on the IPSAM (3/7) of the present invention under various temperature conditions.
Figure 6 shows transmission electron microscopy results of IPSAM (3/7), folate IPSAM (3/7), and folate IPSAM (3/7)/GNP of the present invention.
Panel (A) of Figure 7 shows the temperature change profile of the suspension upon near-infrared irradiation of the IPSAM (3/7) of the present invention and the release profile of Nile Red loaded on the IPSAM (3/7).
Figure 8 shows folate IPSAM (3/7), free DOX, folate IPSAM (3/7)/DOX, folate IPSAM (3/7)/GNP, and folate IPSAM (3/) of the present invention. 7) Shows changes in in vitro KB cell activity following near-infrared irradiation of /DOX/GNP.
Figure 9 shows the results of flow cytometry analysis of KB cells treated with free DOX, folate IPSAM(3/7)/DOX, and folate IPSAM(3/7)/DOX/GNP of the present invention.
Figure 10 shows confocal laser scanning microscopy images of KB cells treated with free DOX, folate IPSAM(3/7)/DOX, and folate IPSAM(3/7)/DOX/GNP of the present invention.
Figure 11 shows the mechanism of action of the temperature- and oxidation-responsive active ingredient carrier of the present invention.

The present invention relates to a polar cationic polymer containing an amino group in a repeating unit and the formula 1 below:

[Formula 1]

(In the above formula, Ar is C6-C20 aryl, L2 is C1-C5 alkylene, and x is an integer of 1 to 3.) Non-polar anionic compound represented by: Temperature and oxidation responsiveness including Provides an ionic complex.

The polar cationic polymer refers to a polymer that has polar and cationic properties, and the amino group (amine group) is included in the repeating unit and may be included in the main chain, side chain, or terminal. Cationic polymers containing the amine group include polyethyleneimine (PEI), polyvinylamine, polyamidoamine, polyallylamine, poly-Llysine, and chitosan. , may be one or two or more polymers selected from the group of aminated methylcellulose and aminated ethylcellulose, and is preferably polyethyleneimine (PEI).

The non-polar anionic compound is an anionic compound containing a non-polar residue and can be expressed by Formula 1 above. The 'aryl' refers to a hydrocarbon ring radical containing hydrogen, a carbon atom, and at least one aromatic ring, and may have 6 to 20 carbon atoms, preferably 6 to 12 carbon atoms, and the 'alkylene' may mean a molecular or straight-chain alkyl radical having 1 to 10 carbon atoms, preferably 1 to 8 carbon atoms, and more preferably 1 to 6 carbon atoms. Preferably, the non-polar anionic compound of the present invention is phenylthioacetic acid.

The temperature and oxidation-responsive ionic complex of the present invention has the characteristic that the polar cationic polymer and the non-polar anionic compound form self-assembly through ion pairing in an aqueous solution. Preferably, the amino group of the polar cationic polymer and the carboxyl group of the nonpolar anionic compound form self-assembly by ion pairing at a molar ratio of 2:8 to 5:5, and more preferably, the amino group of the polar cationic polymer and the nonpolar anion. The carboxyl groups of the compound form ion pairs at a molar ratio of 3:7 to form self-assembly. Most preferably, polyethyleneimine is used as a polar cationic polymer and phenylthioacetic acid is used as a non-polar anionic compound, and the amino group of polyethyleneimine and the carboxyl group of phenylthioacetic acid are used in a molar ratio of 3:7. Ion pairs form self-assembly.

The temperature and oxidation-responsive ionic complex of the present invention has the characteristic of being amphipathic in that the amino group of the polar cationic polymer and the carboxyl group of the non-polar anionic compound form an ion pair by ionic bonding. As the amphipathicity decreases, the solubility of the complex increases, which means that the temperature and oxidation-responsive ionic complex is decomposed, leading to increased release of the loaded active ingredient.

Since the temperature and oxidation-responsive ionic complex of the present invention is an ion pair self-assembly, it has upper critical solution temperature (UCST) characteristics and its solubility changes depending on temperature. In addition, the temperature and oxidation-responsive ionic complex of the present invention contains a sulfur component in the non-polar anionic compound and is oxidized to sulfoxide and sulfone depending on oxidation conditions, so the amphiphilicity of the ion pair is reduced and the solubility is increased. It changes. In summary, the temperature and oxidation-responsive ionic complex of the present invention changes its solubility when the temperature and oxidation conditions change, thereby controlling the release of the payload.

The temperature and oxidation responsive ionic complex of the present invention is characterized by containing gold nanoparticles. The gold nanoparticles have the characteristic of generating heat by causing surface plasmon resonance when irradiated with near-infrared (NIR) rays. Therefore, introducing gold nanoparticles into the temperature- and oxidation-responsive ionic complex of the present invention has the advantage of easily increasing the surrounding temperature of the complex simply by irradiating near-infrared rays.

The temperature- and oxidation-responsive ionic complex of the present invention is characterized in that it is folated. When the folic acid is introduced into the temperature and oxidation-responsive ion complex, the folic acid and the receptor of the cancer cell specifically bind and are endocytized into the cancer cell, so the payload (active ingredient) is specifically placed inside the cancer cell. It can be released as an enemy. The folic acid is characterized in that it is introduced by binding to a non-polar anionic compound of a temperature and oxidation-responsive ionic complex through a condensation reaction. Preferably, the folic acid is introduced through a condensation reaction in polyethyleneimine. do.

The present invention provides a temperature- and oxidation-responsive active ingredient carrier comprising the temperature- and oxidation-responsive ionic complex and an active ingredient.

The active ingredient may be any one or a mixture thereof selected from the group of anticancer agents, antibiotics, cell membrane dyes, amino acids, photosensitisers, and gene complexes. The active ingredient is preferably an ingredient that can be released from the temperature and oxidation-responsive ionic complex to strengthen the oxidation conditions of the complex, and is not limited by ionicity or hydrophobicity. Preferably, the active ingredient may be an anticancer agent, and more preferably, it is doxorubicin (DOX), which strengthens oxidative conditions within cells.

The present invention provides a pharmaceutical composition comprising the temperature and oxidation responsive active ingredient carrier. The “composition” described in the present invention includes the temperature and oxidation-responsive active ingredient carrier as the active ingredient, as well as inert ingredients such as natural or artificial carriers, labels, or detection agents, or adjuvants, diluents, binders, stabilizers, buffers, and salts. , refers to a combination with active ingredients such as lipophilic solvents and preservatives, and includes pharmaceutically acceptable carriers.

The carriers include pharmaceutical excipients and additional proteins, peptides, amino acids, lipids, and carbohydrates (e.g., monosaccharides; disaccharides; trisaccharides; tetrasaccharides; oligosaccharides; sugars such as alditols, aldonic acids, and esterified sugars). derivatives, polysaccharides, sugar polymers, etc.) may be included individually or in combination, and may be included in an amount of 1 to 99.99% by weight or volume.

Protein excipients may include, but are not limited to, human serum albumin, recombinant human albumin, gelatin, casein, etc.

Representative amino acid components that can act as a buffer include alanine, arginine, glycine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, and aspartame. Not limited.

Carbohydrate excipients include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; Disaccharides such as lactose, sucrose, trehalose, cellobiose, polysaccharides such as raffinose, maltodextrin, textran, starch, and alditols such as mannitol, xylitol, maltitol, lactitol, sorbitol, and myoinositol. possible and is not limited to this.

A pharmaceutical composition containing the temperature- and oxidation-responsive active ingredient carrier of the present invention can be formulated by a person skilled in the art by a known method. The pharmaceutical composition of the present invention can be used parenterally, if necessary, in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection of a suspension, and can be used parenterally in the form of a pharmaceutically acceptable carrier or medium, specifically For example, by appropriately combining with sterilized water, physiological saline, vegetable oil, emulsifiers, suspensions, surfactants, stabilizers, excipients, vehicles, preservatives, binders, etc., and mixing them into the unit dosage form required for generally accepted pharmaceutical practice. It can be formulated. In the above formulation, the amount of active ingredient is meant to obtain an appropriate dose within the indicated range.

When the pharmaceutical composition containing the temperature- and oxidation-responsive active ingredient carrier of the present invention is formulated as a sterile composition for injection, it can be prescribed according to conventional formulation practices using an excipient such as distilled water for injection. As an aqueous solution for injection, an isotonic solution containing physiological saline, glucose, and other auxiliaries, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, can be used, and an appropriate solubilizing auxiliary, such as polyethanol, can be used. Alcohol, propylene glycol, and polyethylene glycol can be used in combination with nonionic surfactants such as polysorbate 80(TM) and HCO-50. In addition, sesame oil and soybean oil can be used as an oily liquid, and as a solubilizing agent, it can be used in combination with benzyl benzoate and benzyl alcohol.

Examples of the injection formulation include intravenous injection, intraarterial injection, selective intraarterial injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intracerebroventricular injection, intracerebral injection, and intramedullary injection. And preferably, the injection formulation is an intravenous injection formulation.

The pharmaceutical composition comprising the temperature- and oxidation-responsive active ingredient carrier of the present invention contains a pharmaceutically effective amount of the active ingredient. Determination of the effective amount can be easily determined by a person skilled in the art based on the information disclosed herein.

In general, the pharmaceutically effective amount is determined by first administering the active ingredient at a low concentration and then gradually increasing the dose until the desired effect is achieved without side effects in the subject. A method for determining the appropriate dosage or administration interval of a pharmaceutical composition containing the temperature- and oxidation-responsive active ingredient carrier of the present invention is described in Goodman and Gilman's The Pharmacological Basis of Therapeutics, Goodman et al., eds., 11th Edition, McGraw. -Hill 2005, and Remington: The Science and Practice of Pharmacy, 20th and 21st Editions, Gennaro and University of the Sciences in Philadelphia, Eds., Lippencott Williams & Wilkins (2003 and 2005).

The method of administering a pharmaceutical composition containing the temperature and oxidation-responsive active ingredient carrier of the present invention includes the type of disease, the patient's age, weight, gender, medical condition, severity of the disease, route of administration, and separately administered drugs. It can be determined by considering various factors. The amount of the pharmaceutical composition containing the temperature- and oxidation-responsive active ingredient carrier of the present invention administered to the patient may be determined by many factors such as the administration method, patient's health condition, body weight, and doctor's prescription, which are known in the art. It is within the scope of knowledge of a person with ordinary knowledge.

The term “about” can be understood as within the range commonly accepted in the art, for example, the average standard deviation range, and may be 50%, 45%, 40%, 35%, 30%, 25%, understood to be within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% It can be.

In addition, it can be further combined with buffering agents such as phosphate buffer and sodium acetate buffer, analgesics such as procaine hydrochloride, stabilizers such as benzyl alcohol, or phenol, and antioxidants. The prepared injection solution is usually filled into an appropriate ampoule. Suspensions and emulsions may contain, as carriers, natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. Suspensions or solutions for intramuscular injection may contain the active compound together with a pharmaceutically acceptable carrier such as sterile water, olive oil, ethyl oleate, glycols and a suitable amount of lidocaine hydrochloride.

The pharmaceutical composition containing the temperature- and oxidation-responsive active ingredient carrier of the present invention may be administered to a patient by intravenous injection (bolus injection) or continuous infusion. The pharmaceutical composition containing the temperature and oxidation-responsive active ingredient carrier of the present invention can be used for 1 hour or less, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 8 hours or more, 12 hours or more, 1 day or more, At least 1 for 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 1 month or more, 3 months or more, 6 months or more It may be administered once, at least twice, at least three times, at least four times, or at least five times, continuously, at regular time intervals, or at time intervals determined by clinical judgment.

Injections may be formulated in ampoule form or in unit dose form in multiple dosage containers. However, those skilled in the art will understand that the dosage of the pharmaceutical composition according to the present invention may vary depending on various factors such as the patient's age, weight, height, gender, general medical condition, and previous treatment history.

The present invention will be described in detail through the following examples.

Example

1. Experimental method

1.1. experiment material

Polyethylene imine (PEI, branched, average molecular weight (MW): 2000), chlorauric acid (HAuCl ₄ 3H ₂ O), doxorubicin (DOX), folic acid (FA), 1-ethyl 3 -(3-dimethylaminopropyl)carbodiimide (1-ethyl3-(3-dimethylaminopropyl)carbodiimide, EDC), dimethyl sulfoxide (DMSO), N-hydroxysuccinimide (NHS) , ethylenediaminetetraacetic acid (EDTA), diamidino-2-phenylindole dihydrochloride (DAPI), formaldehyde, trypsin, trizma Buffer, phosphate-buffered saline (PBS, 10-fold concentration, pH 7.4), dimethylformamide (DMF), and 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide. (3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide, MTT) was purchased from Sigma-Aldrich Co. (St. louis, MO). KB cells were provided by the Korea Cell Line Bank (Seoul, Korea). Hydrogen peroxide (H ₂ O ₂ , 30%) was purchased from Daejung Chemicals & Metals Co. (Siheung, Korea). Phenylthioacetic acid (PTA) and nile red were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Roswell Park Memorial Institute (RPMI) 1640 medium without fetal bovine serum (FBS) was purchased from GibcoTM (Dublin, Ireland).

1.2. Upper critical solution temperature analysis

A PEI solution was prepared by dissolving 14.8 mg, 21.8 mg, or 30.5 mg of PEI in 15 ml of 30mM Trizma buffer (pH 7.0) in a 20 ml vial. Add 135.2 mg, 128.2 mg, or 119.5 mg of PTA to the PEI solution to prepare a PEI/PTA mixture in which the concentration of PEI and PTA is 10 mg/ml and the molar ratio of amino group to carboxyl group is 3:7, 4:6, or 5:5. A solution was prepared. The vial was rolled with a roller mixer at room temperature for 24 hours to mix the PEI/PTA mixed solution. The PEI/PTA mixed solution with the molar ratio of amino group to carboxyl group a:b was called PEI/PTA(a/b) solution, and the PEI/PTA ion pair and PEI/PTA ion contained in the PEI/PTA(a/b) solution were The paired self-assemblies were named IP(a/b) and IPSAM(a/b), respectively.

Put 2.5 ml of PEI/PTA (a/b) solution into a 3 ml cuvette, place it in the constant temperature cuvette holder of the temperature controller (CW-05G, Jeio Tech, Korea), and then increase the temperature from 20℃ to 50℃ at a temperature increase rate of 2℃/min. While heating, the optical density (600 nm) was measured using a UV spectrophotometer (UK 6505 UV/Vis Spectrophotometer, JENWAY, UK).

To confirm the change in optical density due to oxidation of the PEI/PTA (3/7) solution, the H ₂ O ₂ solution was treated so that the final concentrations of H ₂ O ₂ were 0, 0.5, 1, 5, 20, and 100mM. . PEI and PTA were adjusted to a total of 10 mg/ml and mixed by rolling for 4 hours with a roller mixer at room temperature. The PEI/PTA (3/7) solution treated with the XmM H ₂ O ₂ solution was named PEI/PTA (3/7) solution (XmM H ₂ O ₂ ). The optical density of the PEI/PTA (3/7) solution (XmM H ₂ O ₂ ) was measured as described above, and the upper critical slution temperature (UCST) was calculated by connecting the data points in the plateau region. It was defined as the intersection of the tangent line connecting the data points of the decreasing region and the decreasing region.

1.3. Interfacial tension measurements

To investigate the effect of oxidation on the air/water interfacial activity of IP (3/7), the interfacial tension was measured by applying the ring method to PEI/PTA (3/7) solution (XmM H ₂ O ₂ ). PEI/PTA (3/7) solution (XmM H ₂ O ₂ ) was prepared as above, except that the concentrations of PEI and PTA were different. The total concentration of PEI and PTA was 4 mg/ml, and the ratio of amino group:carboxyl group = 3:7, and the type of buffer (Trizma buffer 30mM, pH 7.0) and H ₂ O ₂ concentration (0, 0.5, 1, 5, 20, 100mM) and mixing method (roller mixer, 4 hours) were all the same. The PEI/PTA (3/7) solution (XmM H ₂ O ₂ ) was serially diluted twice with 30mM Trizma buffer (pH 7.0) having the same H ₂ O ₂ concentration. As a control, PEI and PTA were each dissolved in 30mM Trizma buffer pH 7.0 at a concentration of 4 mg/ml and then diluted twice in succession with the same buffer solution to obtain a pure PEI solution and a pure PTA solution. The surface tension of the solution was measured by the ring method using an interfacial tensiometer (DST 60, SEO Co., Suwon, South Korea).

1.4. Critical micelle concentration measurement

PEI/PTA (3/7) solution (XmM H ₂ O ₂ ) was prepared as above, except that the total concentrations of PEI and PTA were different. The concentration of PEI+PTA to determine critical micelle concentration (CMC) was 20 mg/ml, amino group/carboxyl group ratio (3/7), type of buffer (Trizma buffer 30mM, pH 7.0), H ₂ O ₂ concentration (0, 0.5, 1, 20mM) and mixing period (4 hours) were the same.

The PEI/PTA solution (XmM H ₂ O ₂ ) was serially diluted twice with 30mM Trizma buffer (pH 7.0) containing the same H ₂ O ₂ concentration to obtain final concentrations of PEI+PTA of 0, 0.0625, 1.25, 2.5, and 5. , 10, 15, and 20 mg/ml. 10 mg of Nile red was dissolved in 10 ml of methanol in a 20 ml vial to prepare a Nile red solution, and 10 ㎕ of the Nile red solution (1 mg/ml) was dissolved in PEI/PTA solution (XmM) in a 5 ml vial. H ₂ O ₂ ) was added to 1 ml and stored for 12 hours under dark and room temperature conditions. After filtering the mixed solution through a filter (0.45 μm), the fluorescence intensity (613 nm) of Nile Red was measured using an excitation wavelength (556 nm) in a fluorescence spectrophotometer (Hitachi F2500, Hitachi, Japan).

1.5. PEI/PTA ion pairing and oxidation examination by FT-IR spectroscopy

After freeze-drying the PEI/PTA(3/7) solution and the PEI/PTA(3/7) solution (5mM H ₂ O ₂ ), PEI/PTA(3/7) and PEI/PTA(3/7)(5mM H ₂ O ₂ ), PEI, and PTA were each mixed with KBr to prepare a mixed powder and then homogenized with a mortar. The homogenized mixed powder was placed in a mold and compressed using a press, and then an FT-IR spectrum was obtained using a Fourier transform infrared spectrophotometer (FTIR, FT-3000-Excalibur, Varian Inc., CA, USA).

1.6. Analysis of PEI/PTA interaction using 1H NMR spectroscopy

The interaction between PEI and PTA was analyzed using hydrogen nuclear magnetic resonance spectroscopy. The ion binding reaction between PEI and PTA was performed in a buffer solution prepared with D ₂ O, and the analysis was performed 24 hours later. Hydrogen nuclear magnetic resonance spectra were obtained using a Bruker Avance 400MHz spectrometer (Kangwon National University Central Research Institute).

1.7. Temperature and oxidation reaction emissions

IPSAM (3/7) including Nile Red was performed to investigate temperature and oxidation-reactive emissions. Add 1.5 ml of 30mM Trizma buffer (pH7.0, H ₂ O ₂ concentration: 0, 0.5, 1, or 5mM) into a cuvette (3 ml) and then scan the cuvette on a UV spectrophotometer (UK 6505 UV/Vis spectrophotometer, JENWAY). , UK) and the temperature of the buffer was adjusted to 25.5°C, 37°C, or 43°C. 1 ml of IPSAM (3/7) suspension containing Nile red was injected into the cuvette containing the temperature-controlled Trizma buffer, and then measured in real time at an excitation wavelength (556 nm) in a fluorescence spectrophotometer (Hitachi F2500, Hitachi, Japan). The wavelength (613 nm) was measured. The release rate (Release %) of the payload was calculated using the following equation: Release%=(1-Ft/Fo)×100 (where Ft refers to the fluorescence intensity at a specific time and Fo refers to the initial fluorescence intensity) ).

1.8. Preparation of folic acid PEI

Folated PEI was prepared using a known condensation reaction. A folic acid solution was prepared by dissolving 0.11 g of folic acid in 5 ml of DMSO in a 10 ml vial. 0.042 g of EDC and 0.025 g of NHS were dissolved in the folic acid solution and stirred at room temperature for 3 hours to activate the carboxyl group. A PEI solution was prepared by dissolving 1 g of PEI in 25 ml of distilled water in a 50 ml vial. The activated folic acid solution was slowly added to the PEI solution and stirred overnight at room temperature to react, and the reaction mixture was dialyzed against distilled water using a dialysis membrane (MWCO 1,000) for 3 days. The synthesized folic acid PEI was dissolved in D2O to obtain a hydrogen nuclear magnetic resonance spectrum (Bruker Avance 400MHz, Kangwon National University Central Research Institute).

1.9. Preparation of folic acid IPSAM containing gold nanoparticles

For temperature and oxidation reactive release analysis, folate IPSAM (3/7) was prepared so that the total mass of PEI and folate PEI was 2% (w/w) of the total content. The total concentration of folate PEI, PEI, and PTA was 20 mg/ml, the amino group/carboxyl group ratio (molar ratio) was about 3/7, and 30mM Trizma buffer (pH 7.0) was used. Gold nanoparticles (GNPs) were introduced into folate IPSAM (3/7) through an in-situ synthesis method. HAuCl ₄ was dissolved in the IPSAM suspension to a concentration of 0.5mM and left at room temperature for 24 hours to form GNPs and be introduced into the folate-oxidized IPSAM (3/7). IPSAM(3/7) where GNP was introduced was referred to as IPSAM(3/7)/GNP.

1.10. transmission electron microscope

IPSAM (3/7) suspension, folate IPSAM (3/7) suspension, and folate IPSAM (3/7)/GNP suspension were mixed in equal parts with uranyl acetate (1.2% (w/v)) at room temperature. It was left in dark conditions for 3 hours. A Formvar/copper-coated grid was immersed in the suspension, and IPSAM (3/7), folate IPSAM (3/7), and folate IPSAM (3/7)/GNP nanoparticles were added to the surface. It was allowed to deposit and then dried at room temperature. TEM images, selected area electron diffraction (SAED) patterns, and high-resolution TEM (HR-TEM) of GNPs were obtained using a transmission electron microscope (LEO-912AB OMEGA, LEO, Germany).

1.11. Release of payload from folate IPSAM/GNPs

Payloads (Nile Red or DOX) were loaded onto folic acid IPSAM(3/7)/GNP. Nile red was used to analyze stimulated release following near-infrared irradiation, and DOX was used to analyze anticancer efficacy in vitro. Put 10㎕ of Nile Red solution (1㎎/㎖, methanol) or DOX solution (10㎎/㎖, DMSO) and 1㎖ of folate IPSAM (3/7)/GNP suspension (20mg/ml) into a 5㎖ vial. It was kept in the dark at room temperature for 12 hours. The mixture of DOX solution and folate IPSAM (3/7)/GNP suspension was dialyzed against 30mM Trizma buffer (pH 7.4) for 24 hours using a dialysis membrane (MWCO 3000) to remove excess DOX that was not loaded. After the dialysis, the DOX-loaded folate IPSAM(3/7)/GNP suspension was freeze-dried. To confirm the exact loading amount (%) of DOX, 5 mg of the freeze-dried DOX-loaded folate IPSAM (3/7)/GNP was dissolved in 1 ml of DMF and ultracentrifuged at 22,000 rpm for 30 minutes to obtain a supernatant. The supernatant was analyzed with a fluorescence spectrophotometer (Hitachi F2500, Hitachi, Japan) to analyze the fluorescence intensity of DOX. The excitation and emission wavelengths of the fluorescence spectrophotometric analysis were 485 nm and 550 nm, respectively. The amount of DOX was determined through a calibration curve. The specific DOX payload (%) was defined as the mass percent of DOX loaded into the IPSAM. DOX-loaded folate IPSAM(3/7)/GNP was named folate IPSAM(3/7)/DOX/GNP.

1.12. Zeta potential measurements

IPSAM(3/7), folate IPSAM(3/7), folate IPSAM(3/7)/DOX, folate IPSAM(3/7)/GNP and folate IPSAM(3/7)/DOX/GNP To analyze the surface potential of nanoparticles, zeta potential was measured. The above IPSAM (3/7), folate IPSAM (3/7), folate IPSAM (3/7)/DOX, folate IPSAM (3/7)/GNP or folate IPSAM (3/7)/DOX/ The suspension of GNPs was diluted with 30mM Trizma buffer (pH 7.0) so that the light scattering intensity was 50 to 100 Kcps. Zeta potential was measured at room temperature (20-23°C) using a dynamic light scattering instrument (ZetaPlus 90, Brookhaven Instrument Co., U.S.A.).

1.13. Near-infrared stimulated emission analysis

10 ml of suspension of Nile Red IPSAM (3/7) containing GNP or Nile Red IPSAM (3/7) without GNP was added to a test tube (1 cm in diameter and 10 cm in length). The total concentration of PEI and PTA was set to 20 mg/ml, and the concentration of GNP was set to 0.5mM. After insulating the test tube with Styrofoam, near-infrared rays (2W, 808 nm) were irradiated using a light source 22 cm away from the surface of the suspension, and the temperature of the suspension was measured at regular time intervals to obtain a suspension (0.6 ml). The obtained suspension sample was ultracentrifuged at 22,000 rpm for 10 minutes, then the supernatant was obtained, the fluorescence intensity of Nile red was measured, and the release rate (%) of Nile red was determined. As a control, the release rate (%) of a suspension of Nile Red IPSAM (3/7) with GNPs or Nile Red IPSAM (3/7) without GNPs without near-infrared irradiation was used.

1.14. Anticancer efficacy evaluation

The folate PSALM (3/7)/DOX suspension or folate PSALM (3/7)/DOX/GNP suspension was diluted with 10mM PBS (pH 7.4) so that the DOX concentration was 10 μg/ml. As positive controls, folate IPSAM (3/7) and folate IPSAM (3/7)/GNP suspension were used, and the concentrations of IPSAM were folate IPSAM (3/7)/DOX and folate IPSAM (3/7)/ It was adjusted in the same way as the DOX/GNP suspension. As another positive control, 10 μg/ml free DOX solution (10mM PBS, pH 7.4) was used, and as a negative control, 10mM PBS (pH 7.4) was used. 1x10 ⁴ cells/ml KB cells (200 ㎕) were placed in a 96-well plate and cultured in a CO ₂ incubator at 37°C for 24 hours. After removing the medium, RPMI 1640 culture medium (180 μl) without FBS and the sample (20 μl) were added to each well and cultured for another 12 hours under the same conditions. Near-infrared rays were irradiated for 1 hour at a distance of 30 cm from the 96-well plate, and cultured for an additional 12 hours. After incubation, the medium was removed, the cells were washed with 10mM PBS (pH 7.4), and 5 mg/ml MTT reagent (20 μl) was added and cultured for 4 hours. After centrifugation, the supernatant from each well was removed, and DMSO (200 ㎕) was added to dissolve formazan formed in living cells. The absorbance (540 nm) of formazan present in the wells was analyzed in a microplate reader (N10588, Thermo Fisher Scientific, USA). Cell viability was expressed as a percentage by comparing the formazan absorbance obtained with the test sample or control to the formazan absorbance obtained with the buffer solution.

1.15. Interaction between DOX-loaded folate IPSAM and cancer cells

3x10 ⁴ cells/ml KB cell suspension (1 ml) was placed in a 12-well plate and cultured in a CO ₂ incubator at 37°C for 24 hours. After incubation, the medium was removed and the cells were washed with 10mM PBS (pH 7.4), followed by Mire-prepared FBS-free RPMI 1640 culture medium (0.5 ml) and test sample (0.5 ml, free DOX solution, folic acid IPSAM (3/7)) /DOX and folic acid (IPSAM (3/7)/DOX/GNP) mixture was added to each well and then cultured in a CO ₂ incubator at 37°C for an additional 4 hours. After incubation, the medium was removed, the cells were washed with 10mM PBS (pH 7.4), and the cells were separated by adding trypsin/EDTA solution. The cell suspension was placed in a tube and centrifuged to remove the supernatant. The cells precipitated in the centrifuged tube were prepared as a suspension by adding 10mM PBS (400㎕, pH 7.4, 4℃) and used a flow cytometer (fluorescence-activated cell sorting, FACS, Calibur, Becton Dickinson, USA; Kangwon National University). The fluorescence intensity of DOX (560 nm) was measured while exciting at 470 nm. The interaction between free DOX or DOX-loaded IPSAM and KB cells was analyzed using confocal laser scanning microscopy (CLSM). After culturing the cells under the same conditions as above, the cells were cultured to interact with free DOX or DOX-loaded IPSAM, and then the cells were fixed using a formaldehyde solution (2.5% (v/v)). The fixed cells were washed with 10mM PBS (pH 7.4), then DAPI stained, and fluorescence images were taken with a confocal laser scanning microscope (LSM 880 with Airyscan, Carl Zeiss; Kangwon National University Central Research Institute).

2. Results

2.1. Upper critical solution temperature analysis results

Panel (A) of Figure 1 shows the optical measurements of the PEI/PTA (5/5) solution, PEI/PTA (4/6) solution, and PEI/PTA (3/7) solution of the present invention in the temperature range of 20 to 50°C. Shows the results of density analysis.

It was confirmed that the optical density of the PEI/PTA (5/5) solution began to decrease around 23°C and reached 0 at 36°C. Since PTA contains a carboxyl group and PEI contains an amino group, PTA and PEI can form an ionic bond. The formed PEI/PTA ion pair (IP(5/5)) is amphiphilic and surface-active because PTA is a non-polar molecule and PEI is a polar molecule. There is a high possibility of having it. The reason for the high optical density at low temperatures is believed to be because ion pairs self-assemble due to their amphipathic properties. When ion pairs are dispersed in an aqueous solution, the phenyl groups of PTA bind to each other through hydrophobic interactions, forming a core, and the PEI chains form a shell surrounding the core, thereby forming micelle particles.

Visible light is scattered by the formed IPSAM (IPSAM(5/5)), so high optical density can be generated. As the temperature of the ion pair solution increases, the solubility of PTA increases and the hydrophobic attraction between the phenyl groups of the PTA molecule weakens, making IPSAM easier to decompose. The temperature-induced decomposition of IPSAM as described above is believed to be the main cause of the decrease in optical density as temperature increases.

The optical density decreases significantly with increasing temperature, and begins to decrease particularly rapidly around 23°C. Therefore, the ion pair (IP(5/5)) of the PEI/PTA(5/5) solution is judged to exhibit upper critical slution temperature (UCST) behavior, and the ion pair (IP(5/5)) of the PEI/PTA(5/5) solution UCST is judged to be approximately 23℃. The optical density of the PEI/PTA (4/6) solution decreased slightly while increasing from 25°C to 31°C and decreased sharply after 31°C. Therefore, it is believed that the ion pair of PEI/PTA(4/6) solution (IP(4/6)) exhibits UCST around 31°C. The optical density profile of the PEI/PTA (3/7) solution was found to decrease slightly and then rapidly decrease in the range of 27 to 37°C, similar to the above. Therefore, the ion pair of PEI/PTA(3/7) solution (IP(3/7)) was analyzed to exhibit UCST at about 37°C.

UCST was confirmed to be proportional to the content of PTA. As the PTA content increases, the hydrophobic attraction between the phenyl groups of the PTA molecule acts more extensively, so more heat energy is required to decompose IPSAM. Since the UCST of IP(3/7) is similar to body temperature of 37℃, IP(3/7) and IPSAM(3/7) were selected and additional experiments were conducted.

Panel (B) of Figure 1 shows the results of heating the PEI/PTA (3/7) solution (0mM, 0.5mM, 1mM, 5mM, 20mM and 100mM H ₂ O ₂ ) of the present invention at a temperature range of 20 to 50°C. shows. First, it was confirmed that the optical density of the PEI/PTA solution (0mM H ₂ O ₂ ) gradually decreased as the temperature increased from 25 to 37°C, and rapidly decreased after 37°C. Therefore, the UCST of IP (3/7) without H ₂ O ₂ treatment is judged to be around 37°C. The temperature-dependent optical density profile of the PEI/PTA (3/7) solution (0.5mM H ₂ O ₂ ) was found to be similar to that of the PEI/PTA (3/7) solution (0mM H ₂ O ₂ ). However, the temperature at which the optical density of the PEI/PTA (3/7) solution (0mM H ₂ O ₂ ) began to rapidly decrease is the temperature at which the optical density of the PEI/PTA (3/7) solution (0.5mM H ₂ O ₂ ) began to decrease. It was about 4℃ lower than that of

The sulfide of PTA can be oxidized by H ₂ O ₂ to form sulfoxide and sulfone. When PTA is oxidized, its polarity and solubility in water increase, making it easily soluble even at lower temperatures. For this reason, the UCST of IP (3/7) treated with 1mM H ₂ O ₂ solution was judged to be lower than that of IP (3/7) not treated with H ₂ O ₂ . It was confirmed that the optical density of PEI/PTA (3/7) solutions treated at different concentrations of 1, 5, and 20mM H ₂ O ₂ changed as the temperature increased, similar to the above. As a result of the analysis of the present invention, PEI/PTA (3/7) solution (1mM H ₂ O ₂ ), PEI/PTA (3/7) solution (5mM H ₂ O ₂ ), and PEI/PTA (3/7) solution ( The UCST of IP (3/7) contained in 20mM H ₂ O ₂ ) was approximately 31°C, 27°C, and 23°C, a result consistent with the above prediction. In contrast, the optical density of the PEI/PTA (3/7) solution (100mM H ₂ O ₂ ) was close to 0 over the entire temperature range. The above results are believed to be because the UCST of IP (3/7) treated with 100mM H ₂ O ₂ is much lower than 20°C.

In summary, the UCST of IP(3/7) decreases as the H ₂ O ₂ concentration increases.

2.2. Interfacial tension analysis results

Panel (A) of Figure 2 shows PEI solution (0, 0.5, 1, 5, 20, or 100mM H ₂ O ₂ ), PTA solution (0, 0.5, 1, 5, 20, or 100 mM H ₂ O ₂ ), and PEI/ Shows the results of analyzing the interfacial tension of PTA (3/7) solutions (0, 0.5, 1, 5, 20, or 100mM H ₂ O ₂ ).

In the case of the PEI solution, there was little change in the interfacial tension, but the concentration was confirmed to have increased to 4 mg/ml. PEI is a water-soluble polymer and has low amphiphilic properties, showing little surface activity. It was confirmed that the interfacial tension of the PTA solution slightly decreased from 73 to 68 dyne/cm, while the concentration increased to 4 mg/ml. PTA has a hydrophobic phenyl group and a hydrophilic carboxyl group, so it can have a certain degree of surface activity. The interfacial tension of the PEI/PTA mixed solution without H ₂ O ₂ treatment decreased to 46 dyne/cm in the saturated state, while the concentration of PEI and PTA increased to 4 mg/ml. A decrease in interfacial tension in a saturating manner is a typical pattern for surfactants.

PTA contains a carboxyl group and PEI contains an amino group, so PTA and PEI can form an ionic bond. The resulting PEI/PTA ion pair (IP(3/7)) is likely to have amphipathic properties and surface activity because PTA is a non-polar molecule and PEI is a polar molecule. The above results explain why the interfacial tension of the PTA/PEI mixed solution effectively decreases as the concentration increases. The interfacial tension of H ₂ O ₂ treated PEI/PTA solutions, e.g. PEI/PTA(3/7) solutions (0.5, 1, 5, 20, or 100mM H ₂ O ₂ ), was reduced in a similar manner as above. However, it was confirmed that the concentrations of PEI and PTA increased to 4 mg/ml. However, the degree of reduction in interfacial tension was confirmed to be lower than that of the PEI/PTA mixed solution without H ₂ O ₂ treatment and was confirmed to be inversely proportional to the concentration of H ₂ O ₂ . For example, when the concentrations of H ₂ O ₂ are 0mM, 0.5mM, 1mM, 5mM, 20mM and 100mM, the interfacial tension at 4mg/ml is 46dyne/cm, 49.5dyne/cm, 51.5dyne/cm and 54.2, respectively. dyne/cm, 56 dyne/cm, and 64 dyne/cm, which means that the surface activity of IP (3/7) decreases as the concentration of H ₂ O ₂ increases.

In summary, when the concentration of H ₂ O ₂ is high, PTA can be more easily oxidized, so the amphipathicity and interfacial activity of IP (3/7) become lower.

2.3. Critical micelle concentration analysis

Panel (B) of Figure 2 shows Nile red contained in PEI/PTA (3/7) solution (0, 0.5, 1, 20mM H ₂ O ₂ ) according to the change in concentration of PEI and PTA of the present invention. shows the change in fluorescence intensity.

The fluorescence intensity was confirmed to be weaker in a relatively low concentration range, and even if the concentration increased to some extent, the fluorescence intensity did not increase any further and was confirmed to be almost constant. The above results mean that Nile red exists only in polar aqueous solutions in a low concentration range and does not exist in non-polar environments.

It is known that Nile Red does not fluoresce in a polar environment. When the concentration of PEI+PTA increases and reaches a certain value, the fluorescence intensity begins to increase, which means that a non-polar environment is formed at that concentration. Since IP(3/7) is amphipathic, it can form micelles at a certain concentration (critical micelle concentration, CMC), and in the formed micelles, the phenyl group of PTA forms a micelle core, and the core is nonpolar. environment can be provided. Nile Red easily dissolves in the non-polar core of micelles and thus exhibits strong fluorescence intensity. Therefore, the concentration at which fluorescence begins to increase can be judged as the critical micelle concentration (CMC) of the sample. The CMC corresponds to the concentration at the intersection of a straight line connecting data points in the fluorescence constant range and a straight line connecting data points in the fluorescence increase range. CMC obtained using the intersection of two straight lines is about 4.70 mg/ml, 6.59 mg/ml, and 9.40 mg/ml when the H ₂ O ₂ concentration of the PEI/PTA (3/7) solution is 0mM, 0.5mM, and 1mM, respectively. It is confirmed that it is. When the H ₂ O ₂ concentration was 20mM, there were only 1 or 2 data points corresponding to the range in which fluorescence increased, so it was difficult to draw a straight line and analyze it, but the fluorescence value of Nile Red was between log values 1 and 1.2. As it began to increase, it was possible to calculate that the CMC was equivalent to 10 mg/ml.

In summary, as the H ₂ O ₂ concentration in the PEI/PTA (3/7) solution increases, the CMC increases, and as the H ₂ O ₂ concentration increases, the oxidation degree of PTA increases and nonpolarity decreases. It has been done. Therefore, it is believed that more IP (3/7) is needed to form micelles after oxidation.

2.4. PEI/PTA ion pairing and oxidation analysis by FT-IR spectroscopy

Panel (A) of Figure 3 shows the FT-IR spectrum results of PEI, PTA, and dry PEI/PTA (3/7) of the present invention. The amino group of PEI was confirmed as a strong and wide peak around 3300 cm ^-1 , and the carbonyl group of PTA carboxyl group was confirmed as a strong and sharp peak around 1700 cm ^-1 . In the spectrum of PEI/PTA (3/7), the signal of the amino group almost disappeared, and the intensity of the signal of the carbonyl group of the carboxyl group was also confirmed to be significantly lowered, and the signal of carboxylate (COO ^- ) appeared around 1573 to 1406 cm ^-1. It was confirmed that The above result means that the amino group of PEI and the carboxyl group of PTA are ionized, and that PEI can interact with PTA to form an ion pair.

Panel (B) of Figure 3 shows the FT-IR spectra of PEI/PTA(3/7) (5mM H ₂ O ₂ ) and PEI/PTA(3/7) (5mM H ₂ O ₂ ). Sulfoxide formed due to H ₂ O ₂ treatment is found at 1034 cm ^-1 , and sulfone is found at 1271 cm ^-1 , which means that PTA is easily oxidized by H ₂ O ₂ .

2.5. PEI/PTA ion pair analysis using hydrogen nuclear magnetic resonance spectroscopy

Panel (A) of Figure 4 shows the hydrogen nuclear magnetic resonance spectrum of the PEI/PTA (a/b) solution of the present invention. It was confirmed that the aromatic proton of PTA moved upward from 7.42ppm to 7.27ppm, and the methylene proton moved upward from 3.75ppm to 3.65ppm. As the PTA content increased, the upward shift and width change of the peak became more obvious, indicating that a strong hydrophobic interaction occurred between the phenyl groups of PTA. Additionally, it was observed that the protons of PEI moved downward from 2.65 ppm to 3.08 ppm, which is believed to be due to the ionic interaction between PEI and PTA and the hydrophobic interaction between PTA molecules.

2.6. Temperature and oxidation reaction release analysis

Panel (A) of Figure 5 shows the emission profile of Nile Red mounted on the IPSAM (3/7) of the present invention at 25.5°C.

In the case of IPSAM (3/7) with a H ₂ O ₂ concentration of 0mM, the release rate of Nile red was almost 0%. The UCST of IP(3/7) in PEI/PTA(3/7) solution was found to be about 37°C when the H ₂ O ₂ concentration was 0mM (see panel (B) of Figure 1). Therefore, since the experimental conditions are 25.5°C, which is much lower than UCST, it is judged that the integrity of IPSAM (3/7) is maintained and no Nile red is emitted at all. In addition, the IPSAM (3/7) was confirmed to have almost no release of payload under all H ₂ O ₂ concentration conditions at a temperature of 25.5°C.

Panel (B) of Figure 5 shows the emission profile of Nile Red mounted on the IPSAM (3/7) of the present invention at 37°C. When the H ₂ O ₂ concentration was 0mM, about 9.6% was released for the first 2 hours, and it was confirmed that the release of Nile Red remained almost constant thereafter. The UCST of IP(3/7) in PEI/PTA(3/7) solution (0mM H ₂ O ₂ ) was approximately 37°C (see panel (B) of Figure 1). Therefore, even under conditions where the H ₂ O ₂ concentration is 0mM, IPSAM (3/7) decomposes to some extent at 37°C and significant emissions occur. This is believed to be because the experimental temperature is almost the same as UCST (about 37°C). do.

In the case of IPSAM (3/7) with a H ₂ O ₂ concentration of 0.5mM, the degree of release was confirmed to increase in a saturated manner over time, as in the case of a H ₂ O ₂ concentration of 0mM. However, the maximum release rate was approximately 30.4%, approximately 20% higher than that observed at 0mM. The UCST of IP (3/7) was found to be about 33°C at 0.5mM, and the temperature of the release medium (37°C) was higher than the UCST (see panel (B) of Figure 1). In fact, when the H ₂ O ₂ concentration was 0.5mM, the optical density at 37°C decreased by about 45%, confirming that IPSAM (3/7) was significantly decomposed (see panel (B) of Figure 1).

The optical density should be proportional to the amount of IPSAM (3/7), and the emission rate is closely related to the degree of reduction of the optical density. The release rate is believed to increase in proportion to the H ₂ O ₂ concentration.

For example, when the H ₂ O ₂ concentration was 1mM and 5mM, the maximum release rates were found to be 54.4% and 69.9%, respectively. Since UCST decreases as H ₂ O ₂ concentration increases, it is believed that oxidation-induced decomposition of IPSAM (3/7) will increase in proportion to H ₂ O ₂ concentration. In fact, when the H ₂ O ₂ concentration was 1 and 5mM, the optical density decreased by about 72% and 89% at 37°C, respectively.

Panel (A) of Figure 5 shows the emission profile of Nile Red mounted on the IPSAM (3/7) of the present invention at 43°C.

The release rate of Nile Red observed at 43°C increased in a saturated manner over time, similar to that observed at 37°C, and increased in proportion to the H ₂ O ₂ concentration. For example, when H ₂ O ₂ concentrations were 0mM, 0.5mM, 1mM, and 5mM, the release rates were about 50.4%, 72%, 88%, and 92%, respectively. As explained above, IPSAM is believed to be more susceptible to oxidation-induced degradation at higher H ₂ O ₂ concentrations because UCST decreases with increasing H ₂ O ₂ concentration (see panel (B) of Figure 1 ). At all H ₂ O ₂ concentrations, the payload release was found to be extensive in the order of 43°C > 37°C > 25.5°C.

IPSAM(3/7) is likely to decompose more easily at higher temperatures due to the UCST behavior of IP(3/7). In fact, the optical density decreased by about 62%, 89%, 96%, and 99% when the H ₂ O ₂ concentration was 0mM, 0.5mM, 1mM, and 5mM, respectively, which means that IPSAM(3/7) was lower than 43°C. This means that it dissolves easily at lower temperatures.

The above results explain why the release of the payload was accelerated due to the increase in temperature. In the case of body temperature conditions, the release is carried out in response to the H ₂ O ₂ concentration. However, the emission sensitivity of the payload mounted on IPSAM (3/7) to H ₂ O ₂ concentration is not considered high enough to operate in vivo because the amount of oxidizing agent that can induce oxidation in vivo is released. This is because it is much lower than the minimum H ₂ O ₂ concentration that induces about 0.5mM moisture.

According to the results of the present invention, it is judged that it is better to release the payload using a temperature reaction rather than an oxidation reaction because the temperature around the IPSAM (3/7) can be easily increased through external stimuli such as near-infrared rays. Because you can.

In the present invention, gold nanoparticles were introduced into the IPSAM (3/7). Additionally, PEI was foxidized to allow IPSAM to penetrate cancer cells through receptor-mediated endocytosis.

2.7. Hydrogen nuclear magnetic resonance analysis of folic acid PEI

The hydrogen nuclear magnetic resonance spectrum of folic acid PEI was analyzed. As a result of the analysis, the ethylene group of PEI was found between 2.40 and 2.85 ppm, and in relation to the signal of folic acid, the methylene proton near the amide bond was found at 2.05 and 2.15 ppm, and the methylene proton adjacent to the secondary amino group was found at 4.3 ppm. Methine protons near the carboxyl group were identified at 4.6ppm, phenyl protons were identified at 6.83 and 7.73 ppm, amide protons were identified at 8.5ppm, and protons of the primary amino group were identified at 8.6ppm.

The molar ratio of folic acid to the ethylene imine unit of PEI was calculated using the integral value of the ethylene proton of PEI and the methine proton peak of folic acid, and was found to be 1:0.022. The molar ratio of PEI to folic acid introduced into PEI was confirmed to be about 1:0.92, which is believed to be because the average molecular weight of PEI is 1,800.

2.8. Transmission electron microscopy analysis results

Figure 6 shows transmission electron microscopy results of IPSAM (3/7), folate IPSAM (3/7), and folate IPSAM (3/7)/GNP of the present invention.

IPSAM (3/7) was confirmed to have a nearly circular shape and a diameter of 30 to 40 nm. The IP(3/7) of the present invention is amphipathic and surface active and self-assembles into spherical micelles in aqueous solution by an entropy-driven process. Folated IPSAM (3/7) was confirmed to be similar in shape and size to IPSAM (3/7), and only 2% of PEI was folate-oxidized, showing little change in physical properties.

Gold nanoparticles (GNPs) are identified as small black dots (see panels (C) and (D) of Figure 6). Gold ions can be reduced to GNPs by a reducing agent. The nitrogen atom of the PEI amino group of the present invention has a lone pair of electrons, so it can act as a reducing agent for PEI, so there was no need to add an additional reducing agent for the experiment. GNPs had a diameter of less than 9 nm and were mostly found to be bound to IPSAM. As a result of high-resolution transmission electron microscopy analysis, the lattice spacing was confirmed to be 0.24 nm, and the sharp diffraction spots detected in the SAED pattern of the particle were confirmed to indicate the pure crystalline characteristics of GNP.

It is known that GNP can form a coordination bond with a compound having a lone pair of electrons. Therefore, it is believed that the GNP is bound to IPSAM through a coordination bond with PEI due to the lone electron pair of the nitrogen atom of PEI.

2.9. Loaded with folate IPSAM/GNP

The correction for DOX dissolved in DMF is as follows: Y=1050.5X+20.4 (R ² =0.9998).

The X refers to the DOX concentration (㎍/ml) and the Y refers to the fluorescence intensity. The amount of DOX loaded into IPSAM (3/7) was approximately 91% of the DOX added to the IPSAM suspension, and the specific loading was calculated to be 0.455%. IPSAM can be classified as a type of hydrophobic core-rich micelle because it contains PEI and PTA in a mass ratio of PEI (1.97 mg PEI/ml):PTA (18.03 mg PTA/ml) = 1:9.16. In the present invention, DOX was added to the IPSAM suspension and loaded at a mass ratio of DOX:PTA=1:91.6, and it is believed that the DOX is easily loaded into the core mainly composed of PTA.

2.10. Zeta potential measurements

IPSAM(3/7), folate IPSAM(3/7), folate IPSAM(3/7)/DOX, folate IPSAM(3/7)/GNP and folate IPSAM(3/7)/DOX/GNP The zeta potentials were found to be +49.3mV, +49.7mV, +46.5mV, +21.2mV, and +24.3mV, respectively. The PEI chains contained in IPSAM are positively charged and polar, which may lead them to the aqueous bulk phase. Cationic polymer chains have a great influence on the surface potential, resulting in a strong positive zeta potential. The zeta potential of folate IPSAM(3/7) was found to be very similar to that of IPSAM(3/7). According to nuclear magnetic resonance analysis, it was confirmed that one molecule of folic acid was attached to one molecule of PEI. As a result of introducing folic acid PEI into IPSAM, it was confirmed that the total content of PEI + folic acid PEI was only 2% (w/w) of the mass of IPSAM. Additionally, the folate residue of folate PEI was found to contain an amino group, which means that folate PEI has little effect on the zeta potential of IPSAM. The zeta potential of folate IPSAM(3/7)/DOX was found to be slightly lower than that of folate IPSAM(3/7).

DOX is a cationic anticancer agent and is loaded in small amounts in IPSAM. In detail, the content of DOX specifically loaded into IPSAM is only 0.455%. Therefore, it is believed that the loaded DOX will not have a significant effect on the zeta potential of the positively charged IPSAM.

The zeta potential of folate IPSAM(3/7)/GNP was found to be much lower than that of folate IPSAM(3/7). GNPs are negatively charged nanoparticles that neutralize the positive charge of IPSAM, thereby reducing the zeta potential. The zeta potential of folate IPSAM(3/7)/DOX/GNP was slightly higher than that of folate IPSAM(3/7)/GNP, probably because DOX, which has a positive charge, was loaded.

2.11. Near-infrared stimulated emission analysis

Panel (A) of Figure 7 shows the temperature change profile of the suspension upon near-infrared irradiation for IPSAM (3/7) of the present invention.

When IPSAM (IPSAM(3/7)), which does not contain GNPs, was irradiated with near-infrared rays, the temperature of the suspension increased from 25°C to approximately 34°C in 13 minutes, and there was no significant increase thereafter. It is believed that light energy is dispersed and increases the suspension temperature. When IPSAM(3/7)/GNP was irradiated with near-infrared rays, the temperature of the suspension rose to 48°C in 33 minutes and there was no significant increase thereafter. When the GNP is irradiated with near-infrared rays, it generates surface plasmon resonance and generates heat. In contrast, when near-infrared rays were not irradiated, the temperature of the suspension did not change regardless of the inclusion of GNP.

Panel (B) of Figure 7 shows the emission profile of Nile Red mounted on IPSAM (3/7) due to near-infrared irradiation of the present invention.

In the absence of near-infrared irradiation, Nile red emission from IPSAM was not confirmed for 1 hour regardless of whether GNP was included. In contrast, when irradiated with near-infrared rays, IPSAM without GNP did not show significant Nile red emission for the first 10 minutes, but showed Nile red emission rates of 5% and 7.5% after 40 and 60 minutes, respectively. When near-infrared rays are irradiated to an IPSAM suspension without GNPs under near-infrared irradiation, the temperature of the suspension increases to 33°C for the first 10 minutes. However, sufficient release did not occur within the 10-minute period. When near-infrared rays were irradiated for 15 minutes, the temperature reached a maximum of 34°C and did not change after that (see panel (A) of FIG. 7). In the range of 25 to 35°C, the optical density of the PSAM suspension showed only a slight decrease with increasing temperature (see panel (A) of Figure 1). The partial decomposition of the IPSAM explains the release of the payload that occurred after 10 minutes of near-infrared irradiation.

When IPSAM(3/7)/GNP is irradiated with near-infrared rays, the emission rate (%) of the payload rapidly improves. In detail, the release rate of IPSAM(3/7)/GNP increased by about 0.5% for the first 5 minutes after near-infrared irradiation and increased significantly thereafter, with the release rate rapidly increasing to about 32% at 60 minutes. The temperature of the IPSAM(3/7)/GNP suspension increased to about 37°C within 5 minutes of near-infrared irradiation, to about 43°C after 10 minutes, and then increased further to reach the maximum temperature of 48°C (Figure 7 (see panel (A)).

The IPSAM(3/7)/GNP suspension temperature reached 37°C, the UCST of IP(3/7), in 5 minutes of near-infrared irradiation, but release of the payload took longer (panel (A) of Figure 7) and Figure 5). The above results explain why the release rate of IPSAM (3/7) was very low during the first 5 minutes (see panel (B) of Figure 7). In summary, the payload emission rate of IPSAM (3/7) is proportional to temperature, so when the temperature increases by irradiating near-infrared rays, the improvement in emission rate accelerates.

2.12. In-vitro anticancer efficacy analysis

Figure 8 shows folate IPSAM (3/7), free DOX, folate IPSAM (3/7)/DOX, folate IPSAM (3/7)/GNP, and folate IPSAM (3/) of the present invention. 7) Shows changes in in vitro KB cell activity following near-infrared irradiation of /DOX/GNP.

It was confirmed that folate IPSAM (3/7) had no significant effect on the survival rate of cancer cells (KB cells) regardless of near-infrared irradiation. The above results mean that folate IPSAM or near-infrared irradiation had no effect on the activity of cancer cells. Free DOX, a well-known anti-cancer agent, showed significant efficacy in inhibiting cancer cell growth, but no difference in anti-cancer effect was observed depending on near-infrared irradiation. Folated IPSAM(3/7)/DOX showed higher cancer cell growth inhibition efficacy than free DOX. Folate receptors are known to be overexpressed in cancer cells (KB cells). Therefore, folic acid-incorporated IPSAM can easily penetrate into cancer cells through receptor-mediated endocytosis. The above results explain why folate IPSAM(3/7)/DOX shows higher cancer cell growth inhibition efficacy than free DOX. Like the anticancer effects of folate IPSAM(3/7) and free DOX, the cancer cell growth inhibitory effect of folate IPSAM(3/7)/DOX was also almost unaffected by near-infrared irradiation.

Meanwhile, folate-oxidized IPSAM(3/7)/GNP had little effect on cell viability in the absence of near-infrared irradiation, but significantly inhibited cell growth under near-infrared irradiation. GNPs can absorb near-infrared rays and dissipate them as heat through surface plasmon resonance. The heat generated by the GNPs can inhibit cell growth. Like folate IPSAM(3/7)/DOX, folate IPSAM(3/7)/DOX/GNP was found to have significant efficacy in inhibiting cancer cell growth due to the anticancer properties of DOX. It was confirmed that the activity of cancer cells was significantly reduced when treated with folate IPSAM (3/7)/DOX/GNP while irradiating near-infrared rays. In summary, the folate-oxidized IPSAM(3/7)/DOX/GNP of the present invention not only has the effect of inhibiting cell growth by a photothermal effect under near-infrared irradiated conditions, but also decomposes IPSAM due to the heat, thereby causing As a result, the payload DOX is released and reduces the activity of cancer cells.

2.13. Interaction between DOX-loaded folate IPSAM and cancer cells

Figure 9 shows the results of flow cytometry analysis of KB cells treated with free DOX, folate IPSAM(3/7)/DOX, and folate IPSAM(3/7)/DOX/GNP of the present invention.

KB cells treated with free DOX were confirmed to exhibit relatively low fluorescence intensity (GMFI=364.3), and since the fluorescence was caused by DOX, it was determined that the anticancer drug (DOX) was delivered into the cells.

It is believed that the transfer of free DOX is through simple diffusion. In contrast, cancer cells treated with folate IPSAM(3/7)/DOX showed much stronger fluorescence intensity (GMFI=1701.3) than cancer cells treated with free DOX (GMFI=364.3). Folate receptors are known to be overexpressed in KB cells. Therefore, it is believed that folate IPSAM(3/7)/DOX is internalized into KB cells through folate receptor-mediated endocytosis, generating strong fluorescence intensity. KB cells treated with folate IPSAM(3/7)/DOX/GNP were also found to exhibit strong fluorescence intensity due to ligand-receptor interaction and receptor-mediated endocytosis.

The GMFI of KB cells treated with folate IPSAM(3/7)/DOX/GNP was 1403.9, which was slightly lower than that of KB cells treated with folate IPSAM(3/7)/DOX (GMFI=1701.3), which was slightly lower than that of DOX fluorescence. It is believed that this is because some of it was absorbed by GNP.

In addition to receptor-mediated endocytosis, electrostatic interactions between IPSAM and cancer cells are believed to promote internalization of nanoparticles into cells. In detail, the positive zeta potential of IPSAM (folate IPSAM(3/7)/DOX/GNP=+24.3mV, folate IPSAM(3/7)/DOX=+46.5mV) is associated with IPSAM through electrostatic interaction. It is believed that it can promote adhesion and entry into cells.

According to the results of flow cytometry, the amount of DOX delivered to cancer cells by folic acid nanoparticles was confirmed to be much greater than that of free DOX. The above results explain why DOX loaded into folate IPSAM shows higher anticancer efficacy than free DOX (see Figure 8).

Figure 10 shows confocal laser scanning microscopy (CLSM) images of KB cells treated with free DOX, folate IPSAM(3/7)/DOX, and folate IPSAM(3/7)/DOX/GNP of the present invention.

Nuclei stained with DAPI are confirmed in blue in the image of KB cells treated with 10mM PBS (pH 7.4). DOX fluorescence is displayed in red, and DOX fluorescence was found not only around the nucleus but also within the nucleus. The above results indicate that DOX was absorbed by cells, delivered to the nucleus, and inserted into the double helix of the DNA strand, which inhibited protein biosynthesis and induced the death of cancer cells.

The DOX fluorescence intensity of cancer cells treated with folate IPSAM(3/7)/DOX was found to be much stronger than that of cancer cells treated with free DOX. Folic oxidation of the present invention is believed to play an important role in improving the intensity of DOX fluorescence through endocytosis. Cancer cells treated with folate IPSAM(3/7)/DOX/GNP showed strong DOX fluorescence at a similar level to cancer cells treated with folate IPSAM(3/7)/DOX. When a ligand such as folic acid is introduced into IPSAM, receptor-mediated endocytosis is induced, allowing the IPSAM to easily penetrate into cancer cells. Additionally, along with receptor-mediated endocytosis, electrostatic interactions between IPSAM and cancer cells are also believed to have promoted the endocytosis of IPSAM.

The results of the FACS analysis and the CLSM analysis of the present invention were consistent with the results of the in vitro anticancer efficacy analysis of the present invention (see Figure 8), which means that the greater the cellular uptake of DOX, the higher the anticancer effect (Figures 8 and 9 , and 10).

IPSAM(3/7) releases its payload when oxidized within the cell. However, it is believed that the ROS inherent in the cells are insufficient to induce the release of DOX loaded on IPSAM. The reason is that the oxidation sensitivity of the IPSAM is not sufficient (see Figure 5). DOX is known to generate oxygen free radicals within cells, and it is believed that this can also oxidize IPSAM that has penetrated into cells. The above results show that IPSAM (folate IPSAM (3/7)/DOX and folate IPSAM (3/7)/DOX/GNP), which does not contain GNP and is an anticancer drug (DOX) wrapped in nanoparticles, penetrates into the cells. This explains why DOX can exert anticancer activity (see Figure 8).

In summary, when IPSAM (3/7) containing GNP is irradiated with near-infrared rays, the decomposition of IPSAM is promoted due to an increase in temperature, which promotes the release of DOX, leading to higher anticancer activity (see Figure 8).

3. Conclusion

PEI of the present invention can form ion pairs with PTA, and the ion pairs can self-assemble into nanoparticles, i.e. IPSAM, in aqueous solution due to its amphipathic and surface active properties.

The IPSAM showed UCST behavior, and in detail, in the case of IPSAM (IPSAM (3/7)) with an amino group: carboxyl group ratio of 3:7, it showed a UCST of about 37°C at pH 7.0, and as the H2O2 concentration increased, the treatment improved. The amount of IPSAM (3/7) used was reduced.

The IPSAM of the present invention showed the characteristic of releasing the payload in response to oxidation and temperature. As the H ₂ O ₂ concentration increased, the release rate of the payload, Nile Red, increased significantly.

When the IPSAM of the present invention is oxidized, the interfacial activity of the ion pair (IP(3/7)) decreases and the CMC increases. Therefore, the UCST of IPSAM (3/7) decreases, which leads to the decomposition of IPSAM and release of the payload.

When heat is applied to the IPSAM of the present invention and the temperature rises above UCST, the IPSAM decomposes, leading to the release of intense payload.

The emission of Nile Red mounted on IPSAM containing GNP increases rapidly due to near-infrared irradiation.

The heat generated by the surface plasmon resonance of GNPs can decompose IPSAM and induce enhanced emission.

DOX loaded into folate IPSAM (3/7) was found to have significantly higher in vitro anticancer activity against KB cells than free DOX.

DOX loaded in folate IPSAM showed a higher rate of internalization into cancer cells than free DOX, as demonstrated by FACS and CLSM.

When IPSAM containing GNP is irradiated with near-infrared rays, its anticancer activity increases because the decomposition of IPSAM is promoted and the release of DOX is promoted.

IPSAM of the present invention is the first drug delivery system developed and has the characteristic of releasing the payload in response to oxidation, heat, and near-infrared irradiation.

The specific embodiments described in this specification are meant to represent preferred embodiments or examples of the present invention, and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that modifications and other uses of the present invention do not depart from the scope of the invention as set forth in the claims herein.

Claims (7)

A polar cationic polymer containing an amino group in the repeating unit and the following formula 1:
[Formula 1]

(In the above formula, Ar is C6-C20 aryl, L2 is C1-C5 alkylene, and x is an integer of 1 to 3.):
A non-polar anionic compound represented by ; and a temperature- and oxidation-responsive ionic complex containing gold nanoparticles,
A temperature and oxidation responsive ionic complex, wherein the polar cationic polymer is polyethylene imine and the non-polar anionic compound is phenylthioacetic acid.
delete The temperature and oxidation responsive ion complex according to claim 1, wherein the temperature and oxidation responsive ion complex has a molar ratio of the amino group of the polar cationic polymer and the carboxyl group of the non-polar anionic compound of 2:8 to 5:5. Complex.
delete The temperature and oxidation responsive ionic complex according to claim 1, wherein the ionic complex is folated.
A temperature- and oxidation-responsive active ingredient carrier comprising the temperature- and oxidation-responsive ion complex of any one of claims 1, 3, and 5 and an active ingredient.
A pharmaceutical composition comprising the temperature- and oxidation-responsive active ingredient carrier of claim 6.