Amphiphilic alginate-oleic acid polymer and method for producing the same

Abstract

The present invention provides a novel hydrophobically modified sodium alginate synthesized by linking alginate and oleic acid with a spacer. The resulting AGO polymers are amphiphilic, have a clinically applicable molecular size, and possess anticancer activity. The resulting AGO nanoparticles exhibit excellent structural and colloidal stability, and are biocompatible in vitro and in vivo, making them potentially useful in the biomedical field, for example, as drug delivery systems.

Description

[Technical Field]

[0001] The present invention relates to an amphiphilic alginate-oleic acid polymer that can be used as a delivery system for active agents such as drugs or biological substances. [Background technology]

[0002] Nanoparticles have often been proposed for use as a delivery system for one or more biologically active substances or drugs, such as pharmaceuticals, biological materials, or cells. In many cases, incorporating (or "loading") biological activators into nanoparticles has not been easy, either due to the limited amount that can be incorporated or because the process of incorporating the substance (e.g., by diffusion) takes a considerable amount of time. This challenge limits the practical or commercial utility of nanoparticles as a delivery mechanism for biological activators.

[0003] Sodium alginate, a type of biopolymer, is well known for its biocompatibility, non-toxicity, and non-immunogenic properties, and has received FDA approval for specific clinical applications, from wound dressings to injections. Alginates are widely used as medical materials in drug delivery, wound dressings, cell culture, and tissue regeneration. Alginates are commonly used in pharmaceutical applications as thickeners, gel-forming agents, and stabilizers. Oral dosage forms are the most frequently used in current pharmaceutical applications, and the use of alginates in targeted drug delivery has increased rapidly in recent years. There are several ways to use alginates as drug vehicles, such as gelation, in combination with chitosan, prodrugs, and / or amphiphilic modifications. Boontheekul et al. reported that flurbiprofen was released in 1.5 hours from an ionically crosslinked, partially oxidized alginate gel. By forming an ionic complex with chitosan, and due to the opposite charge, such alginate-chitosan complexes exhibit pH dependence, and higher swelling and faster drug release from the particulate system have been observed in a simulated intestinal environment (pH 7.5) compared to a simulated gastric environment (pH 1.2). This special property of pH-dependent swelling makes it possible to passively target the gastrointestinal tract. [4] In further versions to delay release, researchers have increased the ratio of crosslinks and decreased swelling by using a combination of ionic and covalent crosslinks (calcium ions and adipic acid dihydrazide).

[0004] Alginates have been used to regulate the release of hydrophobic substances, making them a fascinating subject of research and attracting considerable attention in recent decades. There, amphiphilic modifications of natural alginates with hydrophilic backbones appear to be more interesting and challenging in the development of advanced biomaterials for nanopharmaceutical applications.

[0005] Sodium alginates with molecular weights generally controlled to be less than 70 kDa have been reported to be metabolized by renal clearance in in vivo studies. Furthermore, numerous reports indicate that a number of hydrophobic substances, including ester groups, vinyl groups, or heterocyclic compounds, are frequently used to chemically or covalently associate with hydroxyl or carboxyl moieties along the alginate skeleton, resulting in the amphiphilicity of modified alginates. This can confer self-assembly capabilities to modified alginates, making them more applicable to drug encapsulation and controlled delivery of hydrophilic and / or hydrophobic drugs, or a combination of both, with improved therapeutic performance.

[0006] Commercially available sodium alginate, which has not undergone any treatment such as hydrolysis or oxidation, is unsuitable as a base material for nanoparticle synthesis due to both its high molecular weight (i.e., >600 kDa) and broad molecular weight distribution. Yang et al. reported a drug carrier (OAAD, octyl-grafted amphiphilic alginate amide derivative) based on untreated sodium alginate. The results showed a large particle size and broad distribution (i.e., 0.8–10 μm). For sodium alginate to be metabolized in the human body, the molecular weight must be lower than the renal clearance threshold (i.e., <70 kDa). It is more important and interesting to consider whether additional biofunctionality, such as controlled cytocompatibility and controlled degradation (by renal metabolism), can be introduced into modified alginates without altering their clinically advantageous properties, such as cell-specific compatibility, non-immunogenicity, and structural stability for end use in medical practice. In the literature, oleic acid is well-known as a naturally occurring functional biomolecule in animals, including humans, and in vegetable oils. Oleic acid is also an FDA-approved substance for use as a pharmaceutical ingredient and dietary supplement. Breast tissue is rich in adipose tissue, providing an environmental environment for the development and growth of mammary epithelial cells. Adipose tissue accumulates triacylglycerols and free fatty acids, including monounsaturated fatty acids and oleic acid. Therefore, selecting oleic acid for AGO gel formulations mimics the mammary microenvironment and makes it a biocompatible material for further clinical and translational applications. The antitumor effects of oleic acid have been widely reported in the literature, for example, the suppression of Her-2 / neu overexpression and intracellular calcium signaling pathways that promote cell proliferation, as discussed by Zeng et al., where oleic acid promoted the growth of non-malignant cells but had the opposite effect on malignant cells. Therefore, based on these important pharmaceutical advantages, the long carbon chain of oleic acid could be an excellent hydrophobic ligand for modifying sodium alginate to form a novel type of amphiphilic nanoparticle with specific multifunctional properties.

[0007] There is still a need for a low-cost and efficient method for producing nanoparticles for delivering biologically active substances or drugs, and for the development of the nanoparticles thus produced. SUMMARY OF THE INVENTION

[0008] Thus, the present invention provides an amphiphilic alginate-oleic acid (AGO) polymer in which the polymer is amphiphilic and has a clinically available molecular size, and a method for producing the polymer.

[0009] In one aspect, the present invention provides an alginate-oleic acid (AGO) polymer in which an alginate and an oleic acid are bonded by a spacer.

[0010] According to the present invention, the AGO polymer is amphiphilic and has a clinically available molecular size or has anti-cancer activity.

[0011] In an example of the present invention, the spacer is a diamine, preferably ethylenediamine or 1,6-diaminohexane. In a preferred embodiment, the spacer is ethylenediamine.

[0012] In an example of the present invention, the AGO polymer has the formula I: [Chemical formula] Formula I and has a structure represented thereby.

[0013] In the present invention, the AGO polymer has self-organizing behavior to form nanoparticles in an aqueous solution, and it has been found that the AGO polymer has anti-cancer activity by virtue of the alginate.

[0014] In the present invention, the AGO polymer has self-organizing behavior in an aqueous solution and forms nanoparticles.

[0015] As a result, AGO nanoparticles can be formed by the self-assembly of AGO polymers and possess excellent structural stability, colloidal stability, and biocompatibility in vitro and in vivo.

[0016] According to the present invention, AGO polymers can form AGO nanoparticles that can be used as biomedical materials for multifunctional applications, such as delivery systems for active agents including but not limited to drugs or biological agents or substances (e.g., peptides, proteins, antibodies, serum products, vaccines, multiple cells or stem cells).

[0017] In the present invention, one, two, or more types of activators can be encapsulated in AGO nanoparticles, and therefore, the present invention also provides two-drug nanoparticles or multiple-drug nanoparticles.

[0018] In another embodiment, the present invention is a method for producing an AGO polymer, (1) Mix oleic acid (OA) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC-HCl) in dichloromethane (DCM), then mix this with ethylenediamine in DCM to obtain a reaction mixture; react the reaction mixture with brine (NH4Cl(aq)) to obtain a product, extract the aqueous phase of the product with DCM to collect the organic phase of the product, dry it over anhydrous magnesium sulfate, concentrate it under reduced pressure to produce a crude product, wash the crude product with diethyl ether, filter it to obtain modified OA; and (2) Prepare a solution by dissolving sodium alginate in water, adjust the pH of the solution to 3-4 using HCl, and slowly add an aqueous solution of EDC-HCl while adjusting the pH to 3-4 to complete the reaction and obtain the product, dialyze the product against distilled water, freeze-dry and purify to obtain the AGO polymer, step This provides a method that includes [something].

[0019] It should be understood that both the general description above and the detailed description below are for illustrative and illustrative purposes only and do not limit the invention. [Brief explanation of the drawing]

[0020] The above summary and the following detailed description of the present invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, preferred embodiments at present are shown in the drawings.

[0021] The drawings are as follows:

[0022] [Figure 1] Figure 1 shows the 1H-NMR spectrum of alginate-mOA(AGO) nanoparticles according to the present invention.

[0023] [Figure 2] Figure 2 shows the FT-IR spectra of alginate-mOA(AGO) nanoparticles with different NEDC-HCl / Nhexuronic ratios in the ranges of 0.2, 0.4, 0.6, 0.8, and 1.0, indicated by the inserted symbols.

[0024] [Figure 3] Figure 3 shows the change in I373 / I385 values ​​of alginate-mOA(AGO) at different ratios of NEDC-HCl / Nhexuronic, which are shown as different degrees of substitution.

[0025] [Figure 4] Figure 4 shows the change in critical micelle concentration (CMC) values ​​for AGO nanoparticles with different degrees of substitution (DS).

[0026] [Figure 5] Figure 5 shows the nanostructure morphology of AGO nanoparticles with different NEDC-HCl / Nhexuronic ratios, as examined by a field emission scanning electron microscope. Figure 5(A) shows 0.4AGO, (B) shows 0.6AGO, (C) shows 0.8AGO, and (D) shows 1.0AGO.

[0027] [Figure 6A] Figure 6A shows the cytotoxicity of AGO nanoparticles, i.e., the cell viability (%) against SK-BR-3 for the control, 24hrSA, and different AGO nanoparticles: 0.4AGO, 0.6AGO, 0.8AGO, and 1.0AGO. [Figure 6B] Figure 6B shows the cytotoxicity of AGO nanoparticles, i.e., the cell viability (%) against MDA-MB-231 for the control, 24hrSA, and different AGO nanoparticles: 0.4AGO, 0.6AGO, 0.8AGO, and 1.0AGO. [Figure 6C] Figure 6C shows the cytotoxicity of AGO nanoparticles, i.e., the cell viability (%) against H184B5F5 / M10 for the control, 24hrSA, and different AGO nanoparticles: 0.4AGO, 0.6AGO, 0.8AGO, and 1.0AGO.

[0028] [Figure 7] Figure 7 shows the histopathological findings of a toxicity study involving intravenous injection of AGO nanoparticles in ICR mice, including two groups: one treated with 0.25 wt% AGO nanoparticles and the other with 0.5 wt% AGO nanoparticles. No significant lesions were observed in the heart (A), kidneys (B), liver (c), lungs (D), or spleen (E) in the group treated with 0.25 wt% AGO nanoparticles, and no significant lesions were observed in the heart (F), kidneys (G), liver (H), lungs (I), or spleen (J) in the group treated with 0.5 wt% AGO nanoparticles (H&E staining; 400×).

[0029] [Figure 8]Figure 8 shows the histopathological findings of a toxicity study involving intravenous injection of AGO nanoparticles in ICR mice, including two groups: one treated with 0.8 wt% AGO nanoparticles and the other with 1.0 wt% AGO nanoparticles. No significant lesions were observed in the heart (A), kidneys (B), liver (c), lungs (D), or spleen (E) in the group treated with 0.8 wt% AGO nanoparticles, and no significant lesions were observed in the heart (F), kidneys (G), liver (H), lungs (I), or spleen (J) in the group treated with 1.0 wt% AGO nanoparticles (H&E staining, 400×). [Modes for carrying out the invention]

[0030] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this invention pertains.

[0031] The present invention provides a novel polymer capable of forming nanoparticles with biofunctional properties such as controlled cytocompatibility and controlled degradation (by renal metabolism), which can be introduced into modified alginates (alginates) without altering clinically advantageous properties, such as structural stability, non-immunogenicity, and cell-specific compatibility for end use in medical practice.

[0032] This invention provides an alginate-oleic acid polymer, also known as "AGO macromolecule," which is composed of alginate and oleic acid bonded together by a spacer.

[0033] In one embodiment of the present invention, the AGO polymer can be produced by commonly used or standard methods. For example, ethylenediamine is used as a spacer to link hydrolyzed sodium alginate and oleic acid to produce a novel amphiphilic sodium alginate-modified oleic acid (mOA) polymer linked by two peptide bonds. The peptide-bonded oleic acid can then be cleaved and released from the modified alginate by the enzymatic hydrolysis of the fatty acid amide in mammals. The oleic acid-modified alginate nanoparticles can function as a material for forming therapeutic amphiphilic nanoparticles used as carriers for active agents such as drugs or biological agents.

[0034] According to the present invention, AGO polymers provide anticancer activity derived from alginates. Such novel amphiphilic AGO nanoparticles can form micelle structures that enable drug loading during self-assembly in an aqueous environment, and are expected to facilitate subsequent biomedical applications.

[0035] Alginate Alginates are unbranched anionic polysaccharides consisting of β-D-mannuronic acid (M) and C-5 epimer α-L-guluronic acid (G) linked by a 1→4 bond. [ka]

[0036] Such anionic polysaccharide copolymers are extracted from natural brown algae such as Macrocystis pyrifera, Laminaria hyperborea, and Ascophyllum nodosum, and are also exopolysaccharides of bacteria such as Pseudomonas and Azotobacter.

[0037] Alginates can be produced by any conventional method. For example, alginates can be produced from algal sources. The gel-like alginates in the intracellular matrix of natural brown algae contain sodium, calcium, magnesium, strontium, and barium ions, and their counterion composition is determined by ion exchange equilibrium with seawater. The procedure for extracting alginates from algae is shown below. [ka]

[0038] First, counterions are removed from the natural alginate gel by proton exchange using a 0.1-0.2 M inorganic acid. This process yields alginic acid. In the second step, the insoluble alginic acid is dissolved by neutralization with an alkali such as sodium hydroxide or sodium carbonate to form sodium alginate. Subsequently, particulate matter such as ash, dust, and other insoluble impurities contained in natural algae are removed by separation processes such as sieving, suspension, centrifugation, and filtration. There are three known methods for the final step in the production of alginate: (i) precipitation with alcohol, calcium chloride, or inorganic acid, (ii) free-flow electrophoresis, and (iii) chemical extraction with alginate barium gel. After step (i), the alginate still contains some mitogens and cytotoxic impurities and is not suitable for biomedical applications. Both steps (ii) and (iii) can effectively solve this problem, but step (ii) is not cost-effective and takes time to apply to large-scale commercial production. In process (iii), Ba 2+ It plays an important role. After forming the alginate barium gel, first, mitogens and cytotoxic impurities were eluted from the alginate barium beads by treatment with various chemicals, followed by ethanol extraction, and then the pure alginate beads were dissolved in an alkaline solution. 2+ The solution was then dialyzed to remove the reagents, and finally ethanol was added to precipitate sodium alginate.

[0039] Sodium alginate Sodium alginate, a type of biopolymer, is well known for its biocompatibility, non-toxicity, and non-immunogenicity. Alginates are widely used as medical materials for substance delivery, wound dressings, cell culture, and tissue regeneration. In pharmaceutical applications, alginates are commonly used as thickeners, gel-forming agents, and stabilizers. Oral dosage forms are the most frequently used in current pharmaceutical applications. Alginates form ionic complexes with chitosan due to their opposite charge. Such ionic complexes exhibit pH dependence, as evidenced by the observation of higher swelling and faster drug release from the particulate system in a simulated intestinal environment (pH 7.5) compared to a simulated gastric environment (pH 1.2). This unique property of pH-dependent swelling makes it possible to passively target the gastrointestinal tract.

[0040] Oleic acid Oleic acid (OA) is a type of monounsaturated fatty acid and has the following chemical structure. [ka]

[0041] Oleic acid (OA) is a monounsaturated fatty acid that is naturally present in various animal fats and oils. Its pharmacodynamic effects and mechanisms of action are debatable. Previous studies have reported that OA reduces low-density lipoprotein (LDL) concentration and oxidation, and reduces the risk of cardiovascular disease (CVD). Oleic acid has also been reported to have antitumor activity, mainly due to its ability to induce apoptosis and inhibit cell proliferation.

[0042] The antitumor effect of OA is primarily due to its ability to induce apoptosis and suppress cell proliferation, particularly its action of inhibiting the overexpression of Her-2 / neu and limiting tumor growth.

[0043] Manufacturing of Alginate-mOA Nanoparticles (AGO Nanoparticles) According to the present invention, a modified alginate is obtained using sodium alginate, and then modified alginate-mOA(AGO) nanoparticles are synthesized.

[0044] In one example of the present invention, an alginate is reacted with an aqueous solution of acetic acid. The reaction mixture is neutralized, then dialyzed against distilled water to remove low molecular weight impurities; the precipitate is separated by centrifugation; and then freeze-dried.

[0045] Next, modified oleic acid (mOA) is synthesized by the following method: Oleic acid and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (referred to as EDC-HCl) are dissolved together in dichloromethane (referred to as DCM), and then mixed with ethylenediamine (1.34 mL) in a DCM solution. The reaction mixture is reacted with triethylamine to obtain a crude product, and this crude product is dissolved in brine (NH4Cl (aq) The mixture is combined with the organic phase, the aqueous phase containing the product is extracted with DCM, the organic phase is collected, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to remove the DCM. Next, the crude product is added to diethyl ether, sonicated in an ultrasonic bath to remove residual oleic acid, and the precipitate is collected using a pump filter. Finally, the diethyl ether is removed under reduced pressure. A pure white powder of modified oleic acid (mOA) is obtained. The synthesis scheme for modified oleic acid (mOA) is shown below. [ka]

[0046] Sodium alginate (0.5g) was dissolved in water to a concentration of 3.0% by weight. The pH of the solution was adjusted to 3.4 using 0.4M HCl. Next, an aqueous solution of EDC-HCl was slowly added to the system, and the pH of the reaction mixture was maintained at 3.4 by adding 0.4M HCl. To produce AGO with different degrees of substitution (DS), the amount of EDC-HCl (N EDC-HCl / N hexuronicThe values ​​for 0.2, 0.4, 0.6, 0.8, and 1.0 were 0.096g, 0.857g, 0.288g, 0.383g, and 0.479g, respectively. After 5 minutes of reaction, the moOA(N amine / N hexuronic (1.05, 0.857 g) was added, and the mixture was uniformly stirred at 35°C for 24 hours. After the reaction was complete, low molecular weight impurities were removed by dialyzing with distilled water for 3 days; the unreacted precipitate, which was moOA, was separated by centrifugation (9000 rpm, 15 min); and then freeze-dried. After freeze-drying, residual organic impurities were removed by Soxhlet extraction with acetone for 3 days. The acetone was removed using a vacuum system. Subsequently, a pure pale yellow powder of novel amphiphilic molecules, referred to as AGO nanoparticles, was collected.

[0047] In accordance with this invention, AGO nanoparticles were characterized and tested. It was found that AGO nanoparticles are structurally stable, biocompatible, and possess excellent cytocompatibility and biological safety.

[0048] The present invention is further illustrated by the following embodiments, which are not limiting and are provided for demonstrative purposes. [Examples]

[0049] 1. Methods and Materials 1.1 Decomposition by hydrolysis under acidic conditions 5 g of sodium alginate powder was first dissolved in 45 mL of 1 M acetic acid and uniformly stirred at 85°C for 2, 6, 24, and 48 hours, respectively. After the reaction was complete, the reaction mixture was cooled to room temperature and neutralized with 5 M sodium hydroxide; dialyzed against distilled water for 2 days to remove low molecular weight impurities; the precipitate was separated by centrifugation (9000 rpm, 15 min) and then freeze-dried. Finally, four types of sodium alginate with different molecular weights were obtained in a high yield of 90%.

[0050] 1.2 Characterization of low molecular weight sodium alginate 1 H nuclear magnetic resonance (1 H-NMR) 1 1H nuclear magnetic resonance ( 1 1H-NMR) was used to identify the chemical structure of the compound. The purpose of this experiment was to confirm the effect of hydrolysis of sodium alginate over different time periods. 50 mg of non-hydrolyzed sodium alginate and sodium alginate hydrolyzed for different times were separately dissolved in 1 mL of heavy water and placed in NMR tubes for each composition. The NMR spectra of the above samples were recorded using an America VARIAN 300 MHz NMR spectrometer.

[0051] Fourier transform infrared spectroscopy (FTIR) To observe the changes in the functional groups of non-hydrolyzed sodium alginate and sodium alginate with different hydrolysis times, measurements were taken using Fourier transform infrared spectroscopy (FTIR). For further investigation, a small amount of each sodium alginate powder was placed directly on an attenuated total reflection (ATR) FTIR accessory (Quest ATR S / N U54913, Specac). Subsequently, the FTIR spectra of five sodium alginate compositions (one non-hydrolyzed sample and four hydrolyzed samples) were recorded using a Unican Mattson Mod 7000 FT-IR (at a resolution of 4 cm -1 −1 and 32 scans).

[0052] Gel permeation chromatography (GPC) The molecular weights (M wThe changes in the polydispersity index (PDI) and ) were measured by JASCO gel permeation chromatography (PU-4180 RHPLC pump, RI-4030 refractive index detector). The molecular size profile of sodium alginate was determined using two TSKgelSuperMultiporePW-M columns (dimensions 6 × 150 mm) connected in series in an oven (40°C). Each type of sodium alginate was dissolved in 0.069 M PBS / 0.005 M NaCl as the eluate. The flow rate was 0.5 mL / min. SIGMA-ALDRICH dextran standard was used in the column (M w (5, 12, 50, 150, 270, 410, 670 kDa). The injection volume was 20 μL each time. w The PDI was calculated using the following formula.

number

[0053] Oleic acid and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (referred to as EDC-HCl) were dissolved together in dichloromethane (referred to as DCM) (80 mL) and mixed uniformly at 4°C for 30 minutes. After 30 minutes, another vial was prepared containing a small amount of ethylenediamine in DCM (20 mL). Subsequently, the contents of the vial containing ethylenediamine were mixed with the contents of the first vial, which had been mixed at 4°C for 30 minutes, and triethylamine (3.4 mL) was added. The mixture was then mixed uniformly at room temperature for 48 hours. After the reaction was complete, the crude product was dissolved in brine (NH4Cl). (aq) The mixture was then combined with [another substance]. The aqueous phase was extracted with DCM, the organic phase was collected, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to remove the DCM. Next, diethyl ether was added to the resulting crude product, and it was sonicated in an ultrasonic bath for 1.5 hours to remove any remaining oleic acid. The precipitate was collected using a pump filter. Finally, the diethyl ether was removed under reduced pressure. A pure white powder of modified oleic acid (mOA) was then obtained.

[0054] Sodium alginate was dissolved in water to a concentration of 3.0% by weight. The pH of the solution was adjusted to 3.4 using 0.4 M HCl. Next, an aqueous solution of EDC-HCl was slowly added to the system, and the pH of the reaction mixture was maintained at 3.4 by adding 0.4 M HCl. To produce AGO with different degrees of substitution (DS), the amount of EDC-HCl (N EDC-HCl / N hexuronic The values ​​for 0.2, 0.4, 0.6, 0.8, and 1.0 were 0.096g, 0.857g, 0.288g, 0.383g, and 0.479g, respectively. After 5 minutes of reaction, the moOA(N amine / N hexuronic (=1.05) was added, and the mixture was uniformly stirred at 35°C for 24 hours. After the reaction was complete, low molecular weight impurities were removed by dialyzing with distilled water for 3 days; the precipitate, which was unreacted moOA, was separated by centrifugation (9000 rpm, 15 minutes); and then freeze-dried. After freeze-drying, residual organic impurities were removed by Soxhlet extraction with acetone for 3 days. The acetone was removed using a vacuum system. Subsequently, a pure pale yellow powder was collected as the resulting AGO powder.

[0055] 1.3 Characterization of Modified Oleic Acid and Alginate-mOA Nanoparticles 1 H nuclear magnetic resonance ( 1 (H-NMR) Samples were prepared by dissolving 50 mg each of mOA and AGO powder in 1 mL of deuterium chloroform / deuterium water, and each was placed in an NMR tube. The chemical structures of AGO and mOA were determined as follows: 1 H nuclear magnetic resonance ( 1 They were identified by 1H-NMR.

[0056] Fourier transform infrared spectroscopy (FT-IR) The functional groups in the moOA and AGO powders were measured using the FTIR method described in the examples below.

[0057] Elemental analysis (EA) 20 mg of each AGO (24hr, 0.4, 0.6, 0.8, 1.0) was placed in a vial and then sent to the National Taiwan University Measurement Center. The degree of substitution (DS) of SAO can be calculated using the following formula reported by Yang et al., depending on the nitrogen (N) content in the SAO measured by elemental analysis.

[37]

number

[0058] Fluorescence spectrophotometer (FL) The critical micelle concentrations (CMCs) of amphiphilic AGO nanoparticles of different DS in distilled water were investigated using a fluorescence spectrophotometer (FL-2700) with pyrene as the probe. The following steps describe the sample preparation of each AGO nanoparticle of different DS. First, a pyrene-containing acetone solution (1.0 × 10⁻⁶) was prepared. -4 M) was added dropwise to 15 vials. Simultaneously, 15 different concentrations of AGO solutions were prepared by dissolving AGO nanoparticles in distilled water (concentrations (wt%): 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125, 0.00390625, 0.001953125, 0.0009765625, 0.0004882813, 0.0002441407, 0.0001220704, 0.0000610352). After evaporating acetone under vacuum, the 15 different concentrations of AGO nanoparticle solutions were added to each vial. The mixtures were then stirred at room temperature for 24 hours, strictly shielded from light before the experiment. The probe was excited at 343 nm, and the emission spectrum was recorded in the 300–500 nm range with a 1.0 second integration. The apertures of the excitation slit and emission slit were 5 nm and 5 nm, respectively. The PMT voltage was 400 V. The response time was 0.04 seconds. The scan speed was 60 nm / min.

[0059] Dynamic light scattering (DLS) The average particle size and particle size distribution of AGO nanoparticles from different DS were measured by dynamic light scattering (DLS, BI-200SM Goniometer DLS, Brookhaven Inc., Holtsville, NY). Samples were prepared by dissolving AGO nanoparticles from different DS in distilled water and stirring at room temperature for 24 hours before loading into cuvettes.

[0060] Zeta potential The zeta potentials (Beckman Coulter, Inc., USA) of AGO nanoparticles from different DS were estimated using a laser Doppler velocometer (Beckman Coulter, Inc., USA). Before loading the solution into the flow cell for zeta potential measurement, samples of AGO nanoparticles from different DS were prepared by dissolving them in distilled water and stirring at room temperature for 24 hours.

[0061] 1.4 Cell Culture and In Vitro Cytotoxicity SKBR-3 cells and MDA-MB-231 cells, as well as HER-2-positive and triple-negative breast cancer cells, were treated with AGO for 24 hours. H184B5F5 / M10 (human mammary epithelial cells, derived from BCRC; BCRC number: 60197), MDA-MB-231 (human mammary epithelial carcinoma cells, derived from ATCC; ATCC number: CRM-HTB-26) TM ), SK-BR-3 (human breast epithelial carcinoma cells, derived from ATCC; ATCC number: HTB-30 TM The following was used in the cytotoxicity test. Cell viability was determined by MTT.

[0062] 2. Results When hydrolysis is extended over different time periods, the resulting sodium alginate has a molecular weight (M) as shown in Table 1. w) and a decrease in the polydispersity index (PDI) are observed, suggesting a narrower molecular weight distribution due to intensive hydrolysis, indicating that the molecular weight distribution is narrowed by hydrolysis. The average MW can be reduced to a range of approximately 23,000–35,000 24–48 hours after hydrolysis, and therefore this can be metabolized by renal clearance in the kidney (well below the threshold of 60 kDa) and is available for clinical use. Hydrolyzed sodium alginate of various molecular weights is also available. 1 As confirmed by structural analysis using 1H-NMR and FTIR spectra, the molecular structure and functional groups were identical.

[0063] Table 1. Characterization of sodium alginate with different hydrolysis times. [Table 1]

[0064] To further modify it with hydrophobic oleic acid, sodium alginate hydrolyzed for 24 hours was used as a model matrix for the following studies, given its moderate hydrolysis time (the rapid increase in viscosity of the hydrolyzed solution after 24 hours does not clearly suggest further treatment) and its molecular weight and distribution suitable for medical applications.

[0065] 3.1 Formation of Modified Oleic Acid (mOA) The chemical structure of modified oleic acid is, 1Confirmed by 1H nuclear magnetic resonance spectroscopy, a weak peak at 6.12 ppm represents a proton on the amide group (-NH-C=O-) marked "a"; a peak at 5.32 ppm represents a proton on the ethylene group (-CH=CH-) marked "b"; a central peak at 3.37 ppm represents a proton on the amine group (-NH2) marked "c"; and a peak at 2.12–2.17 ppm represents a proton on the carbon near the amide group (-NH-C=O-CH2) marked "d". It was shown that the peaks in the 1.98–2.00 ppm range represent the proton on the carbon near the ethylene group (-CH2-CH=CH-CH2-), marked "e"; the peak in the 0.84–0.88 ppm range represents the proton at the terminal (-CH3) of the oleic acid group, marked "f"; and the peak in the 1.25–1.59 ppm range represents the proton on the other carbons (-CH2-CH2-CH2-, and NH2-CH2, -CH2-NH2-), marked "g". Notably, the broad peak in the 10.0–10.7 ppm range representing the proton on the carboxylic acid (-COOH) disappeared, and the peak in the 2.30–2.35 ppm range representing the proton on the carbon near the carbonyl group (-C=O-CH2-) reappeared at 2.12–2.17 ppm after the reaction. The proton peak on the carboxylic acid disappeared, which is because the amide group replaced the carboxylic acid group. The shift in the proton peak on the carbon near the carbonyl group is due to the stronger electron-withdrawing ability of the carboxylic acid than that of the amide group, resulting in a different shielding effect. The different chemical shifts indicate that the proton peak on the carbon near the amide group is at a higher magnetic field than the proton peak on the carbon near the carboxylic acid group. This clearly shows that moOA was synthesized successfully.

[0066] The functional groups of mOA were identified using FT-IR analysis. A comparison of the FT-IR spectra of mOA and oleic acid revealed differences in the chemical structure of OA before and after modification. (3400 cm⁻¹) -1 The broadband peak centered around 3297cm² represents the NH stretching vibration of primary amines. -1 The peak near the moderate signal represents the NH stretching vibration of the secondary amide, while the peak at 2971 cm² represents the NH stretching vibration of the secondary amide.-1 The peak near the strong signal is due to CH stretching vibration. 1640cm -1 The peak near the strong signal represents the C=O stretching vibration of the amide, at 1600 cm. -1 The peak near the strong signal represents the NH bending vibration of the amide. The peak at 1708cm represents the C=O stretching vibration of the carboxylic acid group of oleic acid. -1 The strong signal was observed to disappear. Therefore, the spectral information demonstrates that the modification of oleic acid with ethylenediamine was successful.

[0067] 3.2 Synthesis and Characterization of Alginate-mOA Nanoparticles 1 The chemical structure of AGO was confirmed using 1H nuclear magnetic resonance. The spectrum is shown in Figure 1. The peak at 0.91–0.96 ppm represents the proton at the terminal (-CH3) of oleic acid, marked as c; the peak at 2.74 ppm represents the proton on the carbon (-CH2-CH2-), marked as b; the peaks at 2.93–3.09 ppm represent the protons on the carbon (-CH2-CH=CH-CH2-) near the ethylene group (-CH2-CH=CH-CH2-), marked as a, and the proton on the carbon (-NH-C=O-CH2-) near the amide group; and the peak at 3.5–4.5 ppm represents the proton of the pyranose ring. The marks a, b, and c confirm that the modified oleic acid was successfully bonded to sodium alginate. On the other hand, the signal intensity of the marked peaks clearly increased with the amount of EDC-HCl reacted. The reason is that all the marked peaks originate from oleic acid, and the degree of substitution of oleic acid can increase with increasing the amount of EDC-HCl reacted. The results suggest that the synthesis of AGO was successful.

[0068] As shown in Figure 2, FT-IR analysis revealed that N EDC-HCl / N hexuronicThe chemical structures of alginate-mOA(AGO) with different ratios were revealed, and each functional group or chemical bond was marked. Compared with the functional groups specific to sodium alginate in Figure S2, the difference between the two compounds is that the amide group represents C=ONH at 1704cm². -1 It was a nearby peak. C=OO - Signal (1595cm) -1 ) overlaps with 1561cm -1 The nearby peak represents NH bending oscillations; 1241 cm -1 The peaks in the vicinity represent the C-NH of the amide group containing the new functional group (-NH-CO-) formed by the AGO molecule. Therefore, it was demonstrated that the novel compound alginate-mOA(AGO) was successfully synthesized.

[0069] Different amounts of EDC(N EDC-HCl / N hexuronic The oleic acid content bonded to AGO is determined from the reaction (as a ratio). The formation of peptide bonds is the most obvious difference between the modified version, i.e., AGO, and SA after 24 hours of hydrolysis. The nitrogen atom content to which peptide bonds contribute can be quantified by elemental analysis. The degree of substitution was then calculated using the following formula: where M1 represents the molecular weight of the modified monomer; M2 represents the molecular weight of the unmodified (original) monomer; and M3 represents the molecular weight of the nitrogen atom.

number

[0070] The results are shown in Table 2. The degree of substitution (DS) increases with increasing amounts (and ratios) of EDC-HCl. These results demonstrate that it is feasible to appropriately control the degree of AGO substitution using a predetermined modification scheme with specific amounts of EDC-HCl.

[0071] Table 2. N in the ranges of 0.2, 0.4, 0.6, 0.8, and 1.0 (where a stepwise increase in DS can be measured and observed). EDC-HCl / N hexuronicDegree of alginate-mOA (AGO) substitution in different ratios [Table 2]

[0072] 3.3 Critical micelle concentration (CMC) of AGO nanoparticles The critical micelle concentration (CMC) is a characteristic feature of the self-assembly behavior ability of amphiphilic molecules, in particular, to form distinct aggregate structures while dispersed in an aqueous environment. The minimum concentration required for micelle formation, i.e., the CMC, is determined using a fluorescence spectrophotometer with pyrene as a probe, by measuring the intensity ratio (I) of AGO nanoparticles with different degrees of substitution. 372 / I 385 ) can be detected and determined. I corresponds to the logarithmic concentration of AGO with different degrees of substitution. 372 / I 385 The interception of the gradient change of the curve formed by represents the CMC of each AGO composition. Figure 3 shows I 372 / I 385 The relationship between the logarithmic concentrations of AGOs with different degrees of substitution is shown, and Table 3 summarizes the CMC values ​​corresponding to AGOs with different degrees of substitution.

[0073] The CMC value decreased with increasing substitution, dropping to a minimum of 0.008 wt% for AGO with 58.8% DS, represented as 0.8AGO. However, when the DS reached 66.4%, represented as 1.0AGO, the CMC further increased to 0.018%. This finding clearly demonstrates the existence of an optimal balance between hydrophilicity and hydrophobicity in AGO nanoparticles, revealing that the amphiphilicity of 0.8AGO is thermodynamically most feasible and allows for aggregate formation at the lowest concentration. From the relationship between substitution degree and critical micelle concentration shown in Figure 4, modification of sodium alginate with excess oleic acid (DS = ~66.4%) results in excessive hydrophobicity, disrupting the amphiphilic equilibrium and leading to a rapid increase in CMC. As a result, at 0.2AGO, the lowest DS was 16.4%, while the highest CMC value was measured at 0.125 wt%. This means that the AGO concentration required to form aggregate structures through self-assembly is more than 15 times higher compared to 0.8AGO, and the highest AGO concentration is required to form aggregates in an aqueous environment, which is thermodynamically disadvantageous.

[0074] Table 3. Different ratios of N dissolved in ddH2O EDC-HCl / N hexuronic Key physicochemical properties of alginate-mOA (AGO), including those shown in the "Sample Name" column (AGO concentration fixed at 0.05 wt%). [Table 3]

[0075] The CMC of AGO with different DSs was determined, and the particle size of the resulting AGO was investigated by dynamic light scattering. As shown in Table 3, a 0.05% AGO concentration was used for all AGO compositions and 24-hrSA (meaning sodium alginate treated with hydrolysis for 24 hours without further OA modification). There were no signs of particle induction in either the 0.2AGO or 24-hrSA samples, suggesting that neither 0.2AGO nor 24-hrSA formed particle entities when dispersed in aqueous solution. The CMC of 0.2AGO was 0.125%, which is much higher than the 0.05% concentration used to prepare the sample solutions, while SA is a simple linear nanoparticle without hydrophobic interactions that induce a self-assembly mechanism to form aggregate structures. Therefore, DLS appears to support this explanation. In contrast, the remaining AGO compositions with a CMC content far lower than 0.05% showed a clear and strong signal forming particle entities with particle sizes distributed between 200–600 nm or an average of 300–450 nm. This demonstrates the presence of self-assembly capability conferred to the AGO nanoparticles synthesized in this study, as previously mentioned.

[0076] 3.3 Zeta potential of AGO nanoparticles The zeta potential of a solidified entity is fundamentally influenced by the surface properties of the entity, the pH value, the presence of electrolytes, and the concentration of electrolytes in the given solution. Here, the same solution composition and conditions used in DLS measurements were prepared. The zeta potentials of the obtained AGO compositions are also shown in Table 3, compared to 24-hrSA molecules. All samples showed a negative charge, clearly caused by the carboxylic acid groups along the natural sodium alginate skeleton. The zeta potential decreases with the degree of substitution, as the amount of carboxylate groups in AGO decreases as the substitution of oleic acid for the carboxylate groups of AGO increases. The zeta potential is also an indicator of the stability of the colloidal solution system. The absolute values ​​of the zeta potentials of all AGO compositions were well above 20 mV, suggesting that the AGO colloidal nanoparticles have sufficient dispersion stability in solution.

[0077] Considering the physicochemical properties shown in Table 3, it is strongly suggested that the AGO nanoparticles designed and fabricated in this study could be medically useful in biomedical applications such as drug delivery nanosystems.

[0078] 3.4 Nanostructure Morphology Figures 5a, 5b, 5c, and 5d show the nanoparticle morphology of AGO nanoparticles, symbolized by compositions of 0.4AGO, 0.6AGO, 0.8AGO, and 1.0AGO, respectively. The particles exhibit an average size of 300–500 nm in diameter, which appears to be similar to that determined by the aforementioned DLS. All AGO nanoparticles (0.4, 0.6, 0.8, and 1.0) exhibit a spherical shape, suggesting direct evidence as a result of the self-assembly described above, where an energetically favorable structure develops for AGO nanoparticles dissolved in aqueous solution, a phenomenon commonly observed in many amphiphilic molecules disclosed in the literature. Such a spherical morphology could also be applied to the cell delivery of anticancer drugs via the bloodstream.

[0079] 4. In vitro cytotoxicity To evaluate the cytotoxicity of AGO nanoparticles with different compositions (DS), AGO samples were applied for 24 hours to two highly malignant breast cancer cell lines, SKBR-3 and MDA-MB-231, and human mammary epithelial cells, H184B5F5 / M10. SKBR-3 and MDA-MB-231 cells are designated as HER-2-positive and triple-negative breast cancer cells, respectively. Cell viability was determined by MTT. As shown in Figure 6, the toxicity of AGO nanoparticles to these two cancer cells was minimal. In both cells, various AGO formulations (0.4, 0.6, 0.8, 1.0 AGO) with increasing concentrations in the range of 0–0.125 mg / mL showed little to no toxicity to either cell line. However, in H184B5F5 / M10 cells, the remaining compositions showed no toxicity to cell viability, with the exception of the 0.8 AGO and 1.0 AGO compositions, which exhibited some inhibitory behavior. Cytotoxicity was clearly concentration-dependent, and within the scope of this study, AGO appears to be cytocompatible with both normal and cancer cells. Furthermore, these results may indicate the formation of AGO with biological digestive enzyme resistance. When AGO formulations were incubated with cells and culture media, no effects on breast cancer mediated by released free oleic acid were observed. This suggests further investigation to evaluate in vivo toxicity.

[0080] 5. In vivo evaluation Pathological changes induced by intravenous injection of AGO nanoparticles in mice were evaluated. In this study, 20 male and 20 female ICR mice, 7 weeks old, were divided into four groups receiving doses of 0.25%, 0.5%, 0.8%, and 1% by weight of AGO in PBS-buffered aqueous solution.

[0081] Each group consisted of five male and five female mice, and a single dose was administered to the test animals via tail vein injection (iv). On day 14, all mice that had not died before the end of the study period were sacrificed. The heart, kidney, lung, liver, and spleen were collected and submitted for histopathological evaluation. Microscopic examination of these section samples is shown in Figures 7a and 7b for the 0.25% / 0.5% and 0.8% / 1.0% dose groups, respectively. Histopathological evaluation revealed no significant lesions in the heart, kidney, liver, lung, or spleen in ICR mice for 0.25%, 0.5%, 0.8%, or 1% by weight AGO nanoparticles in aqueous PBS buffer administered via intravenous (iv) injection. These results further confirm the biocompatibility and biosafety of AGO nanoparticles in animals and recommend further evaluation for future anti-cancer therapies.

[0082] 6. Use of AGO amphiphilic polymers to encapsulate a single drug. A manufactured oleic acid-modified sodium alginate (AGO polymer), an amphiphilic polymer used for encapsulating hydrophobic drugs, is disclosed. Using curcumin, a highly hydrophobic drug (water-insoluble), 100 μL (curcumin dissolved at 4 mg / mL in DMSO) was mixed with 1 mL of ddH2O, and 0.5 mg of AGO powder was added to prepare a mixture. The mixture was gently mixed for 12 hours to form a clear solution. The final solution was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was removed. The obtained solid samples were characterized, the structural morphology of the manufactured drug-loaded AGO material was examined using a scanning electron microscope, the surface charge of the curcumin-loaded AGO nanoparticles was determined by examining the zeta potential (Table 4), and the curcumin encapsulation efficiency of the AGO polymer was analyzed using high-performance liquid chromatography (Table 5). For control, curcumin was encapsulated using another amphiphilic polysaccharide described in US8,263,130B2, namely carboxymethyl-hexanoyl chitosan (CHC) (control group). The results are shown in Table 2. The encapsulation efficiency was significantly lower at 65.2% compared to AGO.

[0083] It was revealed that curcumin can be encapsulated very efficiently in AGO nanoparticles, and the resulting drug-supported nanoparticles have a negatively charged surface on a nanometer scale, confirming their colloidal stability for practical use.

[0084] Table 4. Particle size and zeta potential of AGO and curcumin-supported AGO nanoparticles. [Table 4]

[0085] Table 5. Curcumin encapsulation efficiency in AGO nanoparticles and carboxymethyl-hexanoyl chitosan (CHC) nanoparticles as a comparison group. [Table 5]

[0086] 7. Use of AGO amphiphilic polymers for encapsulating two types of drugs simultaneously. A manufactured oleic acid-modified sodium alginate (AGO polymer), an amphiphilic polymer used for encapsulating hydrophobic drugs, is disclosed. Using two highly hydrophobic drugs (water-insoluble), curcumin and paclitaxel, 100 μL (curcumin at 4 mg / mL and paclitaxel at 0.8 mg / mL simultaneously dissolved in DMSO) was mixed with 1 mL of ddH2O, and 0.5 mg of AGO powder was added to prepare a mixture. The mixture was gently mixed for 12 hours to form a clear solution. The final solution was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was removed. The obtained solid samples were characterized, the structural morphology of the manufactured drug-loaded AGO material was examined using a scanning electron microscope, the surface charge of curcumin-loaded AGO nanoparticles was determined by examining the zeta potential (Table 6), and the encapsulation efficiency of curcumin by the AGO polymer was analyzed using high-performance liquid chromatography (Table 7).

[0087] It was revealed that both curcumin and paclitaxel can be very efficiently encapsulated simultaneously in AGO nanoparticles, and the resulting drug-supported nanoparticles have a negatively charged surface at the nanometer level, confirming their colloidal stability for practical use.

[0088] Table 6. Particle size and zeta potential of AGO and two drug-carrying AGO nanoparticles (curcumin / paclitaxel). [Table 6]

[0089] Table 7. Simultaneous encapsulation efficiency of curcumin and paclitaxel in AGO nanoparticles [Table 7]

[0090] conclusion We successfully synthesized a new type of amphiphilic alginate-based nanoparticle (i.e., alginate-mOA, or AGO) by hydrolysis to various molecular weights and chemical bonding with modified oleic acid (mOA). The resulting AGO nanoparticles with different degrees of substitution (DS) were obtained with N25, 0.2, 0.4, 0.6, 0.8, and 1.0. EDC-HCl / N hexuronicUsing predetermined ratios, the mixture was precisely manipulated to obtain DS in the ranges of 16.4%, 29.6%, 39.4%, 58.8%, and 66.4%, respectively. The AGO nanoparticles exhibited self-assembly behavior in aqueous solution, yielding spherical nanoparticles with an average size of 300–500 nm in diameter. The critical micelle concentration (CMC) of AGO was measured, achieving a minimum CMC of 0.008%, suggesting the structural stability of AGO nanoparticles for medical applications, particularly in blood circulation. The colloidal stability of AGO nanoparticles in aqueous solution was further demonstrated by the strong negative potential of AGO nanoparticles with varying degrees of substitution. The biocompatibility of AGO was evaluated using two types of cancer cells and one type of normal cell, followed by in vivo studies, all demonstrating excellent cytocompatibility and biosafety. This study clearly demonstrates the success of designing and synthesizing a novel type of alginate-based AGO nanoparticle that is self-organizing, structurally stable, and biocompatible. Its minimal molecular weight suggests that it will be metabolized via renal clearance when clinically deployed, thus confirming its potential use in biomedical fields, such as drug delivery applications.

[0091] Furthermore, the oleic acid-modified sodium alginate (AGO) disclosed in this invention demonstrates excellent drug encapsulation capabilities for one or more water-insoluble drugs. Experimental observations have also demonstrated its benefits in the following ways: (1) reducing the cytotoxicity that may be exerted by these highly potent pharmaceutical components for purposes such as anticancer, antiproliferative, and anti-inflammatory treatments; (2) increasing the water solubility of these highly water-insoluble drugs to enhance bioavailability during treatment; (3) synergistically enhancing therapeutic performance through dual therapy for treating difficult-to-treat diseases such as metastatic solid tumors; (4) forming stable colloidal doses over short to long storage periods for clinical use as needed; and (5) providing potential versatility for subsequent novel dosage forms for specific delivery.

[0092] This specification includes many specific descriptions, which should not be construed as limitations on the scope of the invention or the claims, but rather as descriptions of features specific to particular embodiments or examples of the invention. Certain features described herein in the context of other embodiments or examples may also be implemented in combination in one embodiment.

Claims

1. Alginate-oleic acid (AGO) polymer, in which alginate and oleic acid are bonded together by a spacer, Here, The molecular weight of AGO polymers is 23,000 to 35,000 g / mol. The degree of substitution of AGO polymers is 16.4% to 66.4%, and The AGO polymer is given by formula I: 【Chemistry 1】 Equation I Structure and Formula II: 【Chemistry 2】 Formula II A method for producing an AGO polymer having the following structure, (1) Oleic acid (OA) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC-HCl) are mixed in dichloromethane (DCM), and then mixed with ethylenediamine in DCM to obtain a mixture; (2) The mixture is reacted with brine to obtain a product, the aqueous phase of the product is extracted with DCM to collect the organic phase of the product, and the product is dried; (3) The product is concentrated under reduced pressure to obtain crude modified OA (mOA), and the residual OA is removed to obtain mOA; (4) Dissolve sodium alginate in water to prepare a solution, adjust the pH of the solution to 3-4, and then add an aqueous solution of EDC-HCl while maintaining the pH at 3-4; (5) Add mOA to the solution and react; and (6) Dialysis of the obtained aqueous solution with distilled water, freeze-dry the dialyzed solution to obtain an AGO polymer. A method that includes a process.

2. The method according to claim 1, wherein in step (5), the reaction is carried out by stirring at 35°C for 24 hours.

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