Nano-polysaccharide composite particle and preparation method therefor

Abstract

The present application provides a novel nano-polysaccharide composite particle. The nano-polysaccharide composite particle is prepared from one polycationic polysaccharide and two polyanionic polysaccharides by means of electrostatic attraction and a complexation reaction. The nano-polysaccharide composite particle has an excellent active ingredient encapsulation efficiency, so that the nano-polysaccharide composite particle can be uniformly distributed within cells upon uptake.

Description

Nanoparticle polysaccharide composite particles and their preparation methods Technical Field

[0001] This application relates to a method for preparing nano-polysaccharide composite particles, in particular, the nano-polysaccharide composite particles have good encapsulation ability of active ingredients, so that they can be evenly distributed after being taken up by cells, thereby enabling the active ingredients to fully act in the cells. Background Technology

[0002] Nanotechnology has garnered significant attention since the 1980s and has been applied in numerous engineering fields, including electronics, mechanics, biomedicine, and aerospace engineering. Nanotechnology designs and manufactures atomic, molecular, or macromolecular structures by effectively manipulating or modifying the basic structure of materials to enhance, regulate, or alter properties. The U.S. National Nanotechnology Initiative defines it as "the extension of existing technologies at the nanoscale, specifically in the 1 to 100 nanometer dimensions." As fundamental research in nanotechnology becomes increasingly comprehensive, its applications in the biomedical field continue to evolve. Nanobiotechnology, through the integration of methods, techniques, and solutions from other scientific branches such as nanotechnology, materials science, biology, and biochemistry, has emerged as a novel and more specialized field or branch of science. Nanotechnology has applications in everything from disease prevention and diagnosis to treatment. For example, nanomedicine delivery systems utilize nanotechnology to encapsulate active ingredients in nanoparticles, which are then released into the body via intravenous injection. This technology overcomes past limitations, ensuring that active ingredients reach their target sites for release, thus improving drug stability. These research findings have brought remarkable progress and contributions to the medical field. In particular, therapies using nanoparticles have been widely used to treat cancer, diabetes, allergies, infections, and inflammation. Currently, there are various methods for preparing nanomedicine carriers, such as nanoparticles, nanoliposomes, solid lipid nanoparticles, and magnetic drug-carrying nanoparticles. However, previous methods for preparing nanomedicine carriers were relatively complex or had many limitations, resulting in poor encapsulation effects. It is necessary to utilize the properties of nanoparticles to enhance cellular uptake rates and efficiency, improve phagocytic mechanisms, and increase the absorption of active ingredients within cells.

[0003] Polysaccharides are long-chain carbohydrate molecules composed of repeating monosaccharide units linked together by glycosidic bonds. Chitosan, alginate, heparin, hyaluronic acid, and dextran are all polysaccharides, natural polymers with storage and functional structures, and are major components of biological systems (such as the extracellular matrix). Polysaccharides exhibit high stability, biocompatibility, and biodegradability. Therefore, polysaccharides and their derivatives are commonly used in the food, biomedical, and environmental fields. Polysaccharides are classified according to their chemical charge, including cationic (e.g., chitosan), anionic (e.g., alginate, heparin, hyaluronic acid), and nonionic (e.g., amylopectin, dextran). Due to the various derivatized groups on their molecular chains, polysaccharides can be easily modified chemically and biochemically. Most natural polysaccharides possess hydrophilic groups such as hydroxyl, carboxyl, and amino groups, which can affect the polymer charge and may form non-covalent bioadhesion or react with functional molecules. Common methods for preparing polysaccharide nanoparticles mostly involve using a single polysaccharide (e.g., chitosan, alginate, heparin, hyaluronic acid) and one or two crosslinking agents (e.g., tripolyphosphate, calcium chloride) or two polysaccharides (e.g., positively charged chitosan and negatively charged alginate) to form nanoparticles through ionic interactions. Van Bavel et al. used low molecular weight chitosan (75-85% deacetylation) and tripolyphosphate as crosslinking agents, synthesized them through ionogelation, prepared the particles using a handheld homogenizer, and purified them through a 0.1 μm polyethersulfone syringe filter (Van Bavel, Nicolas et al. A Simple Method for Synthesis of Chitosan Nanoparticles with Ionic Gelation and Homogenization Nicolas. Molecules 2023, 28(11), 4328). This method is prone to uncontrolled intramolecular and intermolecular crosslinking between chitosan and tripolyphosphate, leading to nanoparticle aggregation, overgrowth, and high polydispersity. Furthermore, the manual, hand-held homogenization process for nanoparticle fabrication raises concerns about reproducibility issues or uneven mixing due to individual differences. Additionally, using filtration and concentration to collect particles increases the difficulty and time required for mass production. Moreover, the prepared particles, using only one polysaccharide as a carrier material, only possess the auxiliary functionalities of that single polysaccharide.In addition, Thai et al. chose to use chitosan and alginate to prepare nanoparticles via ionogel method for loading the drug lovastatin (Thai, H. et al. Characterization of chitosan / alginate / lovastatin nanoparticles and investigation of their toxic effects in vitro and in vivo. Scientific Reports 2020, 10, 909). However, the preparation process they used was too complicated. At the same time, the use of ultrasound often causes temperature rise, which can destroy the activity of the active ingredient if the coated active ingredient is temperature sensitive. Moreover, the volume of solution that can be operated by ultrasound has its limits, making mass production difficult. Meylina et al. prepared a nanoparticle AM-CS / HA containing the drug α-mangostin and applied it to the treatment of breast cancer. The nanoparticle was mainly prepared by mixing chitosan and hyaluronic acid. It was relatively large in size, with an average particle size of about 304 nanometers (Meylina, L. et al. Hyaluronic Acid-Coated Chitosan Nanoparticles as an Active Targeted Carrier of Alpha Mangostin for Breast Cancer Cells. Polymers 2023, 15, 1025).

[0004] Currently, there is still a desire to develop a nano-polysaccharide composite particle that has a simple preparation process but also has small particle size and good ability to encapsulate active ingredients. Summary of the Invention

[0005] Therefore, this application unexpectedly discovers a nanopolysaccharide composite particle, which is prepared from a polycationic polysaccharide and two polyanionic polysaccharides through electrostatic attraction and misalignment reaction. This nanopolysaccharide composite particle has excellent encapsulation rate of active ingredients, allowing it to be uniformly distributed within cells after uptake.

[0006] In one aspect, this application provides a nano-polysaccharide composite particle, which is prepared by electrostatic attraction and misalignment reaction of a polycationic polysaccharide and two polyanionic polysaccharides; wherein the polycationic polysaccharide is selected from the group consisting of chitosan, polyethyleneimine and polyquaternary ammonium salt polysaccharides; the two polyanionic polysaccharides are different from each other and are selected from the group consisting of hyaluronic acid, sodium alginate, heparin sulfate / heparin, chondroitin sulfate, keratin sulfate, dermatan sulfate, xanthan gum and carrageenan and pectin respectively.

[0007] On the other hand, this application provides a nano-polysaccharide composite particle with a particle size between 20 nanometers and 112 nanometers. It is prepared by electrostatic attraction and misalignment reaction of a polycationic polysaccharide and two polyanionic polysaccharides. The polycationic polysaccharide is selected from the group consisting of chitosan, polyethyleneimine and polyquaternary ammonium salt polysaccharides. The two polyanionic polysaccharides are different from each other, with a molecular weight between 10 kDa and 700 kDa, and are selected from the group consisting of hyaluronic acid, sodium alginate, heparin sulfate / heparin, chondroitin sulfate, keratin sulfate, dermatan sulfate, xanthan gum and carrageenan and pectin.

[0008] On another front, this application provides a nano-polysaccharide composite particle with a particle size between 6000 nm and 9000 nm. It is prepared by electrostatic attraction and misalignment reaction of a polycationic polysaccharide and two polyanionic polysaccharides. The polycationic polysaccharide is selected from the group consisting of chitosan, polyethyleneimine and polyquaternary ammonium salt polysaccharides. The two polyanionic polysaccharides are different from each other, with a molecular weight between 1000 kDa and 20,000 kDa, and are selected from the group consisting of hyaluronic acid, sodium alginate, heparin sulfate / heparin, chondroitin sulfate, keratin sulfate, dermatan sulfate, xanthan gum, carrageenan and pectin.

[0009] According to this application, an active ingredient is encapsulated in the nano-polysaccharide composite particles, so that after being taken up by cells, the active ingredient can be evenly distributed within the cells and has excellent dispersibility.

[0010] According to embodiments of this application, the active ingredient is water-soluble, poorly water-soluble, or non-water-soluble.

[0011] On another front, this application provides a pharmaceutical / food composition, such as a composition of a drug, nutritional product, health care or health food, which is a composition of one or more active ingredients encapsulated by the nano-polysaccharide composite particles of this application and an edible or pharmaceutically acceptable carrier.

[0012] In one embodiment of this application, the active ingredient may be selected from the group consisting of Antrodia camphorata extract, curcumin, resveratrol, lutein, astaxanthin, water-soluble vitamins, and paclitaxel and combinations thereof.

[0013] In a specific embodiment of this application, the active ingredient is an alcoholic extract of Antrodia camphorata.

[0014] Furthermore, this application provides a method for preparing the nano-polysaccharide composite particles of this application, comprising the following steps:

[0015] (1) Prepare a solution containing a polycationic polysaccharide as a first solution; wherein the polycationic polysaccharide is selected from the group consisting of chitosan, polyethyleneimine and polyquaternary ammonium salt polysaccharides;

[0016] (2) Prepare a mixed solution containing two polyanionic polysaccharides as the second solution; wherein the two polyanionic polysaccharides are different from each other and are selected from the group consisting of hyaluronic acid, sodium alginate, heparin sulfate / heparin, chondroitin sulfate, keratin sulfate, dermatan sulfate, xanthan gum, carrageenan and pectin respectively.

[0017] (3) Add the second solution dropwise to the first solution, mix and stir until homogeneous to form a mixed solution;

[0018] (4) Centrifuge and concentrate the mixed solution from step (3) and remove the supernatant to obtain the precipitate;

[0019] as well as

[0020] The precipitate was freeze-dried to form the product.

[0021] According to one embodiment of this application, the preparation method may further include a step of rinsing the product with ultrapure water to improve its purity.

[0022] In one embodiment of this application, in order to prepare the nano-polysaccharide composite particles with a particle size between 20 nanometers and 73 nanometers, a polysaccharide-degrading enzyme is added to the second solution to make the molecular weight of the two polyanionic polysaccharides between 10 kDa and 700 kDa to obtain a third solution. The third step is to add the third solution dropwise to the first solution, mix and stir evenly to form a mixed solution.

[0023] In the embodiments of this application, if the active ingredient to be coated is positively charged, it is added to the first solution when preparing a pharmaceutical or food composition.

[0024] In another embodiment of this application, if the active ingredient to be coated is negatively charged, the active ingredient is added to the second solution when preparing the pharmaceutical or food composition.

[0025] In another embodiment of this application, if the active ingredient to be coated is electrically neutral, it can be added to the first solution or the second solution when preparing a pharmaceutical or food composition.

[0026] In one embodiment of this application, the polycationic polysaccharide is chitosan, and the polyanionic polysaccharide is hyaluronic acid and sodium alginate, respectively.

[0027] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only, and not intended to limit the scope of this application. Attached Figure Description

[0028] The foregoing description of the invention and the following detailed description of this application will be better understood when read in conjunction with the accompanying drawings. Preferred embodiments are shown in the drawings to illustrate this application.

[0029] Figures 1A-1C provide flowcharts for preparing nano-polysaccharide composite particles. Figure 1A is a flowchart for preparing polysaccharide composite particle A; Figure 1B is a flowchart for preparing nano-polysaccharide composite particle B; Figure 1C is a flowchart for preparing nano-polysaccharide composite particle B containing active ingredients.

[0030] Figure 2 shows scanning electron microscope (SEM) images, transmission electron microscope (TEM) images, and particle size distribution diagrams of nanopolysaccharide composite particles B and nanopolysaccharide composite particles B containing active ingredients.

[0031] Figure 3 shows the cell activity test results of nanopolysaccharide composite particles B containing active ingredients at 24 hours and 48 hours of the experiment.

[0032] Figure 4 shows a fluorescence image of the cellular uptake rate of nanopolysaccharide composite particles B containing the active ingredient, analyzed using conjugate focal microscopy.

[0033] Figure 5 shows the toxicity test results of human triple-negative breast cancer cells MDA-MB-231, where * indicates p-value < 0.001 and Δ indicates p-value < 0.001 compared with the negative control group. Detailed Implementation

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0035] As used herein, the singular forms “a”, “an”, and “the” include plural references unless otherwise expressly indicated. Thus, for example, reference to “a sample” includes multiple such samples and their equivalents known to those skilled in the art.

[0036] One objective of this application is to provide a nano-polysaccharide composite particle, which is prepared by electrostatic attraction and misalignment reaction of a polycationic polysaccharide and two polyanionic polysaccharides; wherein the polycationic polysaccharide is selected from the group consisting of chitosan, polyethyleneimine and polyquaternary ammonium salt polysaccharides; the two polyanionic polysaccharides are different from each other and are selected from the group consisting of hyaluronic acid, sodium alginate, heparin sulfate / heparin, chondroitin sulfate, keratin sulfate, dermatan sulfate, xanthan gum and carrageenan and pectin respectively.

[0037] The nano-polysaccharide composite particles prepared according to this application have excellent encapsulation rate of active ingredients, which allows them to be evenly distributed after being taken up by cells. Therefore, the nano-polysaccharide composite particles have advantages such as high encapsulation rate, good dispersibility and good biocompatibility.

[0038] As used herein, the term "polycationic polysaccharide" refers to a polysaccharide composed of polysaccharide chains and structurally containing cationic (positively charged) functional groups, commonly used in medicine, biotechnology, and pharmaceuticals. The positive charge of polycationic polysaccharides allows them to interact with negatively charged cell membranes or biomolecules, making them highly effective in applications such as drug delivery and cell-cell interaction regulation. Common polycations include chitin derivatives such as chitosan, polyethyleneimine, and polyquaternary ammonium salt polysaccharides. The first solution in this application is a polycationic polysaccharide solution prepared with chitosan as a representative component.

[0039] As used herein, the term "polyanionic polysaccharide" refers to a negatively charged polysaccharide molecule containing anionic (negatively charged) functional groups in its structure. These anionic groups (such as carboxyl or sulfate groups) enable polyanionic polysaccharides to interact electrostatically with positively charged molecules (such as certain proteins or drugs). Due to these properties, polyanionic polysaccharides have wide applications in biomedicine, drug delivery, and tissue engineering. In this article, polyanionic polysaccharides are further classified by molecular weight into macromolecular polyanionic polysaccharides, typically with molecular weights between 1000 kDa and 20,000 kDa; and small molecular weight polyanionic polysaccharides, typically with molecular weights between 10 kDa and 700 kDa. Common types of polyanionic polysaccharides include, but are not limited to, hyaluronic acid, sodium alginate, heparin sulfate / heparin, chondroitin sulfate, keratin sulfate, dermatin sulfate, xanthan gum, carrageenan, and pectin. In one representative example of this application, the second solution is a macromolecular polyanionic polysaccharide solution prepared from both hyaluronic acid and sodium alginate. In another representative example of this application, the third solution is a small-molecule polyanionic polysaccharide solution prepared from both hyaluronic acid and sodium alginate with a molecular weight between 10 kDa and 700 kDa.

[0040] As used herein, the term "polysaccharide-degrading enzyme" refers to a class of enzymes that can break down polysaccharides (such as starch, cellulose, chitin, etc.). These enzymes break down polysaccharides into smaller oligosaccharides or monosaccharides by hydrolyzing the bonds in polysaccharide molecules, thus facilitating absorption and utilization by organisms. Because the structure and bonding patterns of each polysaccharide differ, different polysaccharides require specific degrading enzymes. Common polysaccharide-degrading enzymes include, but are not limited to, alginate lyase, hyaluronidase, papain, amylase, glucosylamylase, lactase, sucrase, cellulase, hemicellulase, pectinase, and α-galactosidase. In this application example, the third solution is a solution of small-molecule polyanionic polysaccharides formed by adding a polysaccharide-degrading enzyme to the second solution to decompose hyaluronic acid and sodium alginate.

[0041] As used herein, the term "active ingredient" refers to a specific component that has been proven to improve physiological functions, such as enhancing cellular or individual function or alleviating disease, for example, anti-inflammatory, antioxidant, anti-cancer, hepatoprotective, proliferative, and immunomodulatory effects. This active ingredient includes, but is not limited to, drugs or single chemical components, plant extracts, biological substances, or any active ingredient that improves physiological function. In this application, the nano-polysaccharide complex particles can be used to encapsulate water-soluble, poorly water-soluble, or non-water-soluble active ingredients. Specific examples of this application include, but are not limited to, Antrodia camphorata alcohol extract, curcumin, resveratrol, lutein, astaxanthin, water-soluble vitamins, and paclitaxel.

[0042] According to this application, in the preparation process of coating these active ingredients onto the nano-polysaccharide composite particles of this application, an appropriate solvent can be selected to dissolve them according to the characteristics of these active ingredients. Common solvents include water, dimethyl sulfoxide, methanol, and ethanol. In the example of this application, the active ingredient is an antrodia camphorata alcohol extract, which is a solution of an antrodia camphorata alcohol extract dissolved in dimethyl sulfoxide.

[0043] As used herein, the term "pharmaceutically acceptable carrier" means a carrier, diluent, or excipient that is acceptable in the sense of being compatible with other components of the formulation and harmless to the individual to whom the pharmaceutical composition is intended to be administered. Depending on the requirements of the pharmaceutical formulation, any carrier, diluent, or excipient commonly known or used in the art may be used in this application. The carrier may be a diluent, transporter, excipient, or matrix of the active ingredient. Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The composition may additionally contain lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifiers and suspending agents; preservatives such as methylparaben and propylparaben; sweeteners; and flavoring agents.

[0044] As used in this article, the term "coverage ratio" refers to the percentage of the active ingredient content in the prepared nanopolysaccharide composite particles relative to the total amount of that active ingredient added.

[0045] As used in this article, "acidic aqueous solution" refers to an aqueous solution in which the concentration of hydrogen ions is greater than the concentration of hydroxide ions. It is suitable for dissolving materials such as polysaccharides, including acetic acid, hydrochloric acid and combinations thereof, with acetic acid being preferred.

[0046] As used herein, the term "about" refers to a range of values ​​that vary by ±10% from the specified value, unless otherwise stated. Minor deviations from the exact value are permissible due to experimental error, measurement precision, or other factors common in the relevant fields.

[0047] As used herein, the term "composition" refers to any mixture, formulation, or combination of ingredients designed for a particular purpose. Nanoparticle polysaccharide complexes are formulated into compositions for administration (particularly oral administration) for the purpose of encapsulating active ingredients, comprising nanoparticle polysaccharide complexes, active ingredients, and pharmaceutically acceptable carriers.

[0048] In a specific example of this application, the polycationic polysaccharide is a chitosan, a derivative of chitin, which is one of the most abundant organic substances in nature after cellulose. Chitosan is a high-molecular-weight basic polysaccharide polymer carrying cations, mainly polymerized from N-acetylglucosamine monomers, and is widely found in (A) marine animals: crustaceans, coelenterates, annelids, mollusks, lobsters, shrimp, krill, crabs, and fish scales; (B) insects: scorpions, brachiopods, cockroaches, spiders, beetles, and ants; and (C) microorganisms: green algae, yeasts, fungi (cell walls), Penicillium mycelium, brown algae, Chytridaceae, Ascomycetes, Blastomycetes, spores, and other organisms. Chitin can be deacetylated to form chitosan; generally, the higher the degree of deacetylation, the better the solubility of the resulting chitosan. Chitosan is non-toxic and exhibits high biocompatibility and biodegradability in humans. It is generally believed to have effects such as lowering cholesterol, lowering blood pressure, enhancing immunity, hemostasis, and antibacterial properties. Furthermore, it is readily available and widely used in health foods and antibacterial materials. Therefore, in the method of this application, chitosan is used as a polysaccharide to encapsulate the active ingredient. Its viscosity is approximately 20 cP to 2000 cP, preferably approximately 20 cP to 800 cP, and its degree of deacetylation is approximately 75% to 85%.

[0049] In a specific example of this application, the polyanionic polysaccharide is an alginate, such as sodium alginate, a naturally occurring anionic polymer typically obtained from brown algae. Due to its biocompatibility, low toxicity, relatively low cost, and ability to produce mild gelation by adding divalent or higher cations (e.g., Ca²⁺), it has been extensively studied and used in many biomedical applications. Alginate hydrogels can be prepared through various cross-linking methods, and their structural similarity to the extracellular matrix of living tissue makes them widely applicable in wound healing, delivery of small chemical drugs and bioactive agents such as proteins, and cell transplantation. To prepare this nanopolysaccharide composite particle with a particle size between 20 nm and 73 nm, alginate can be cleaved by alginate lyase to produce smaller molecular weight alginate oligosaccharides (AOS).

[0050] In a specific embodiment of this application, the other polyanionic polysaccharide is hyaluronic acid, a linear polysaccharide composed of a disaccharide (D-glucuronic acid and N-acetyl-D-glucosamine) basic structure, which is highly expressed in the extracellular matrix (ECM), cell surface, and even intracellular space. Hyaluronic acid can interact with various proteins or proteoglycans to organize the ECM and maintain tissue stability. Its unique physical and mechanical properties further contribute to maintaining tissue hydration, mediating solute diffusion in the extracellular space, and lubrication of certain tissues. The various biological functions of hyaluronic acid are manifested through its complex interactions with matrix components and resident cells. Hyaluronic acid can also bind to cell surface receptors and activate multiple signaling pathways, further regulating cell function, tissue development, inflammation, wound healing, and tumor progression and metastasis.

[0051] According to an example of preparing the nano-polysaccharide composite particles according to this application, the method includes dissolving the chitosan in an acidic aqueous solution, causing the NH2 groups on the surface to be protonated to form positively charged NH2+ ions, and then mixing it with a solution containing two polyanionic polysaccharides (e.g., alginate and hyaluronic acid). The NH2+ ions then exhibit electrostatic attraction and a misalignment reaction with the polyanionic polysaccharides after dissociation, thereby generating the nano-polysaccharide composite particles of this application. These polysaccharide composite particles overcome the shortcomings of chitosan, such as poor mechanical durability, structural instability, hydrophobicity, and reduced water stability, while increasing the bioactivity diversity of the material, thus forming a new material with better performance and nanoparticles that are easier to control in terms of drug and protein release.

[0052] According to this application, a method for preparing the nano-polysaccharide composite particles includes preparing a chitosan solution with a concentration of 5 mg / mL to 100 mg / mL as a first solution, preferably with a concentration of 5 mg / mL to 20 mg / mL; and preparing an aqueous solution of sodium alginate with a concentration of 5 mg / mL to 100 mg / mL and an aqueous solution of hyaluronic acid with a concentration of 5 mg / mL to 100 mg / mL, preferably with a concentration of 5 mg / mL to 20 mg / mL, and mixing the two to form a second solution. The second solution is added dropwise to the first solution and mixed and stirred until homogeneous to form a mixed solution. The mixed solution is centrifuged and concentrated, and the supernatant is removed to obtain a precipitate; the precipitate is then freeze-dried to form a product. The average particle size of the obtained product is between 6000 nm and 9000 nm.

[0053] According to this application, a method for preparing nano-polysaccharide composite particles with a particle size between 20 nanometers and 73 nanometers includes: preparing a first solution of a chitosan solution with a concentration of 5 mg / mL to 100 mg / mL, preferably 5 mg / mL to 20 mg / mL; preparing an aqueous solution of sodium alginate with a concentration of 5 mg / mL to 100 mg / mL and an aqueous solution of hyaluronic acid with a concentration of 5 mg / mL to 100 mg / mL, preferably 5 mg / mL to 20 mg / mL; mixing the two solutions and then adding a polysaccharide-degrading enzyme to ensure that the molecular weights of the two polyanionic polysaccharides are between 10 kDa and 700 kDa to obtain a third solution; adding the third solution dropwise to the first solution and mixing and stirring to form a mixed solution; centrifuging and concentrating the mixed solution and removing the supernatant to obtain a precipitate; then freeze-drying the precipitate to form the product. The average particle size of the obtained product is between 20 nanometers and 112 nanometers.

[0054] This application also provides a pharmaceutical / food composition comprising one or more active ingredients encapsulated by the nano-polysaccharide composite particles of this application, and an edible or pharmaceutically acceptable carrier. The active ingredient is an alcoholic extract of Antrodia camphorata.

[0055] The nano-polysaccharide composite particles of this application differ from known materials. They are prepared from three polysaccharides through electrostatic attraction and misalignment reactions. The resulting nano-polysaccharide composite particles possess excellent encapsulation capabilities for active ingredients, allowing them to be evenly distributed after being taken up by cells, thus fully exerting their effects within the cells. This demonstrates their excellent dispersibility and promotes cell activity and proliferation. Furthermore, they can be used to encapsulate active ingredients with different electrical properties, applicable to water-soluble, low-water-soluble, and even water-insoluble active ingredients. For example, to further reduce particle size, polysaccharide-degrading enzymes can be used to decrease their molecular weight. The process is convenient and allows for the preparation of diverse products.

[0056] This application is further illustrated by the following examples, which are provided for illustrative purposes and not for limitation.

[0057] Example

[0058] Example 1: Preparation method of polysaccharide complex particles A

[0059] First, solutions containing polycationic polysaccharides of different concentrations are provided as the first solution. The preparation method is to dissolve chitosan powder (degree of deacetylation: 75% to 85%, viscosity 20 to 200 cp) in an aqueous acetic acid solution with a weight percentage concentration of 0.5% to 3.5% w / v to form an aqueous chitosan solution with a weight percentage concentration of 5 mg / mL, 10 mg / mL, 15 mg / mL and 20 mg / mL.

[0060] Next, solutions containing two macromolecular polyanionic polysaccharides at different concentrations are provided as the second solution. The preparation method is to dissolve hyaluronic acid and sodium alginate in 5 liters of ultrapure water to form hyaluronic acid or sodium alginate aqueous solutions with weight percentage concentrations of 5 mg / mL, 10 mg / mL, 15 mg / mL and 20 mg / mL. Then, the hyaluronic acid and sodium alginate aqueous solutions are homogenized with a rotary stirrer to form a sodium alginate / hyaluronic acid mixed aqueous solution, which completes the second solution.

[0061] The second solution was added dropwise to the first solution at a constant rate of 200 mL per minute, while simultaneously stirring with a rotary stirrer at 1500 rpm at 25°C for approximately 1.5 hours until homogeneous, thus forming an aqueous solution of polysaccharide complex particles A. This aqueous solution of polysaccharide complex particles A was then centrifuged at approximately 10,000 to 20,000 rpm at a temperature of approximately 0°C to approximately 30°C, and the supernatant was removed.

[0062] The precipitate after centrifugation was retained, and finally freeze-dried for storage. Table 1 shows the experimental concentrations of the first and second solutions, which are experimental groups 1 to 16, respectively. A simplified flowchart for the preparation of polysaccharide complex particles A is shown in Figure 1A.

[0063] Table 1. Experimental concentrations of the first and second solutions

[0064] Example 2: Method for preparing nanopolysaccharide composite particles B

[0065] First, a first solution as in Example 1 is provided, consisting of an aqueous solution of chitosan with weight percentage concentrations of 5 mg / mL, 10 mg / mL, 15 mg / mL, and 20 mg / mL, and a second solution as in Example 1 containing two macromolecular polyanionic polysaccharides.

[0066] Next, a solution containing two small-molecule polyanionic polysaccharides is provided as the third solution. This solution is prepared by adding a polysaccharide-degrading enzyme to the second solution, stirring thoroughly in a rotary mixer, and then carrying out the decomposition reaction. The mixture is stirred at 25°C for approximately 12 hours to decompose the polysaccharides, forming a mixed aqueous solution of small-molecule sodium alginate / hyaluronic acid, thus completing the third solution. The molecular weight of the macromolecular polyanionic polysaccharides is generally between approximately 1000 kDa and 20,000 kDa, while the small-molecule polyanionic polysaccharides formed after enzymatic decomposition are controlled to be between approximately 10 kDa and 700 kDa, preferably between approximately 20 kDa and 500 kDa.

[0067] The third solution was added dropwise to the first solution at a constant rate of 200 mL per minute, while simultaneously stirring with a rotary stirrer at 1500 rpm at 25°C for approximately 1.5 hours until homogeneous, thus forming an aqueous solution of nano-polysaccharide complex particles B. This aqueous solution of nano-polysaccharide complex particles B was then centrifuged at approximately 10,000 to 20,000 rpm at a temperature of approximately 0°C to approximately 30°C. The supernatant was removed, and the precipitate was retained. Finally, the precipitate was freeze-dried and stored. Table 2 shows the experimental concentrations of the first and third solutions, ranging from 17 to 32 for experimental groups. A simplified flowchart of the preparation process for polysaccharide complex particles B is shown in Figure 1B.

[0068] Table 2. Experimental concentrations of the first and third solutions

[0069] Example 3: Preparation method of nano-polysaccharide composite particles B containing active ingredients

[0070] First, a first solution, as in Example 1, is provided.

[0071] The active ingredient, Antrodia camphorata alcohol extract, was weighed using an electronic balance and then dissolved in dimethyl sulfoxide to prepare an Antrodia camphorata alcohol extract solution with a weight percentage concentration of 40,000 mg / mL, forming a fourth solution. This fourth solution was then added dropwise to the first solution at a constant rate of 200 mL per minute while stirring.

[0072] The active ingredient was directly mixed with the first solution and stirred until homogeneous. Then, the third solution prepared as in Example 2 was added dropwise to the homogeneous mixture of the first solution and the active ingredient at a rate of 200 mL per minute. The mixture was centrifuged at approximately 10,000 to 20,000 rpm. Finally, after removing the supernatant, the precipitate was retained. This precipitate can be stored after freeze-drying. The components and amounts used in this example are listed in Table 3 below. A simplified flowchart for preparing nano-polysaccharide composite particles B containing the active ingredient is shown in Figure 1C.

[0073] Table 3. Experimental concentrations of the first, third, and fourth solutions.

[0074] According to all the preparation methods in Examples 1-3, the precipitate obtained in the centrifugation step can be washed with ultrapure water and then centrifuged again, and repeated two or more times as needed to maximize the purity of the obtained particles. Finally, if necessary, the obtained nano-polysaccharide composite particles can also be dispersed with ultrapure water and stored in the form of a dispersion for later use.

[0075] Furthermore, the reaction temperature and time must be carefully controlled during the mixing of different solutions. The temperature should be controlled between approximately 10°C and 80°C, preferably between 20°C and 40°C. If the reaction temperature is too low or the reaction time is too short, the reaction rate may be too slow or incomplete, resulting in low yield and poor coating of the nano-polysaccharide composite particles. If the reaction temperature is too high, it may adversely affect the efficacy of the active ingredients. The reaction time should be controlled between approximately 0.1 hours and approximately 3 hours, preferably between approximately 0.5 hours and approximately 1.5 hours. If the reaction time is too long, it will cause over-reaction of the polyanionic polysaccharides, leading to larger particle sizes and increased particle aggregation in the nano-polysaccharide composite particles.

[0076] Example 4: Particle Size Analysis

[0077] Materials and Methods

[0078] This material and method are applicable to polysaccharide composite particles A, nano-polysaccharide composite particles B, and nano-polysaccharide composite particles B containing active ingredients. First, the sample is placed in an ultrasonic vibrator and vibrated for 2 hours to uniformly disperse the particles. Then, an appropriate amount of sample is dropped onto a silicon chip and dried in an oven. The surface morphology of the sample is then observed using a field emission scanning electron microscope (JSM-7800F, JEOL, Japan). Alternatively, the sample is placed in an ultrasonic vibrator and vibrated for 2 hours to uniformly disperse the particles. An appropriate amount of sample is then dropped onto a copper grid and dried in an oven. The particle size and structure of the sample are then observed using a transmission electron microscope (JEM-1400F, JEOL, Japan).

[0079] result

[0080] Polysaccharide complex particles A

[0081] As shown in Table 4, particle size analysis revealed that the polysaccharide complex particles A could only be composed of specific weight percentages of the first and second solutions. According to the experimental results, among all experimental groups 1-16, only experimental groups 1, 2, and 7 did not exhibit aggregation or clumping, and polysaccharide complex particles A were successfully prepared.

[0082] Table 4. Particle size analysis results of polysaccharide complex particles A

[0083] Nanoparticle polysaccharide composite particles B

[0084] As shown in Table 5, particle size analysis revealed that the product, nanopolysaccharide composite particles B, could be composed of specific weight percentages of the first and third solutions. According to the experimental results, no aggregation or agglomeration was observed in any of the experimental groups 17-32, and nanopolysaccharide composite particles B were successfully prepared. The average particle size was approximately between 20 and 73 nanometers, as shown in Figure 2. The top row shows the scanning electron microscope and transmission electron microscope images and particle size distribution diagram of the nanopolysaccharide composite particles B.

[0085] Table 5. Particle size analysis results of nanopolysaccharide composite particles B

[0086] Nanoparticles containing active ingredients (polysaccharide complex particles B)

[0087] As shown in Table 6, particle size analysis revealed that the product could be composed of a first solution, a third solution, and a fourth solution in a specific ratio. According to the experimental results, no aggregation or clumping was observed in any of the experimental groups 33-48, and nano-polysaccharide composite particles B containing *Antrodia camphorata* alcohol extract were successfully prepared, with particle sizes ranging from approximately 20 nm to 112 nm. Figure 2 shows the scanning electron microscope and transmission electron microscope images and particle size distribution diagrams of the nano-polysaccharide composite particles B containing *Antrodia camphorata* alcohol extract.

[0088] Table 6. Particle size analysis results of nanopolysaccharide composite particles B containing active ingredients.

[0089] Based on the comprehensive particle size analysis data, the nanopolysaccharide composite particles of this application exhibit excellent particle size control and a uniform spherical particle structure, with a minimum particle size of approximately 20 nanometers. Furthermore, as the concentration of the second solution increases, the particle size also increases. In addition, because chitosan molecules carry a positive charge on their surface, while undigested polyanionic polysaccharides have extremely long molecular chains and a large number of negative charges on their surface, when undigested polyanionic polysaccharides combine with chitosan, extremely strong intermolecular forces are generated, leading to agglomeration or aggregation and preventing the formation of nanostructures. Therefore, by treating polyanionic polysaccharides with polysaccharide-degrading enzymes, their molecular chains are shortened, their fluidity is improved, and the amount of negative charge on each molecular chain is reduced, forming small-molecule polyanionic polysaccharides. This allows for a more uniform reaction with chitosan, resulting in more uniform nanopolysaccharide composite particles.

[0090] Example 5: Calculation of the encapsulation rate of active ingredients

[0091] The stock solution of the active ingredient, *Antrodia camphorata* alcohol extract, was diluted to 10 ppm, 20 ppm, 40 ppm, 80 ppm, 100 ppm, and 200 ppm, respectively. The absorbance of these samples was measured at the wavelength (298 nm) of the *Antrodia camphorata* alcohol extract using a UV-Vis spectrometer (UV-1700, SHIMADZU, Japan). After calculating the calibration curve, the absorbance of the supernatant formed during the preparation process of the *Antrodia camphorata* alcohol extract-containing nanopolysaccharide composite particles B in Example 3 was measured at the same wavelength to calculate and analyze the content of uncoated active ingredients. The coating rate was calculated using the following formula:

[0092] Encapsulation rate % = [(Total amount of active ingredient added - content of unencapsulated active ingredient) / Total amount of active ingredient added] × 100

[0093] As shown in Table 7, the nanopolysaccharide composite particles of this application exhibit excellent coating efficiency, with the best reaching approximately 98.02%.

[0094] Table 7. Coating rate of nano-polysaccharide complex particles B containing Antrodia camphorata alcohol extract

[0095] In summary, the nanopolysaccharide composite particles prepared according to the method of this application exhibit excellent encapsulation efficiency, the preparation process has been greatly simplified, and the drug production cost can be reduced.

[0096] Example 6: Cell viability assay

[0097] 3T3 fibroblasts were seeded into 96-well culture dishes (1×10⁴ cells / well) and placed in an incubator. After culturing the cells at 37°C and 5% CO₂ for 24 hours, the cells were treated with nano-polysaccharide complex particles B containing Antrodia camphorata alcohol extract. Different concentrations of the active ingredient were administered to each group (5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, and 100 μg / mL). Cells without the active ingredient served as a control group. After culturing for 24 and 48 hours, the cells were washed twice with phosphate-buffered saline (PBS) at a concentration equal to the control. Cell viability was then measured using a CCK-8 assay kit (Dojindo Molecular Technologies, Kumamoto, Japan). The control group was treated with ultrapure water. 110 μL of assay reagent was then added to each well for CCK-8 testing. The 96-well dishes containing the test cells were then incubated at 37°C for another 2 hours. To prevent nanoparticle interference with this assay, after a 2-hour reaction time, the test reagents in the 96-well plate were transferred to a new 96-well plate, while the deposited nanoparticles and cells remained in the initial culture plate. The optical density (OD) of each well was measured at a single wavelength of 450 nm using an ELISA reader, with each sample repeated six times.

[0098] As shown in Figure 3, the cell viability assay results showed that treatment with nano-polysaccharide complex particles B containing the active ingredient (taking experimental group 38 in Example 3 as an example) significantly enhanced the proliferation capacity of 3T3 fibroblasts. Specifically, after administration of 5 to 20 micrograms per milliliter of nano-polysaccharide complex particles B containing the active ingredient, the cell viability ratio was observed to increase by approximately 2.3 times compared to the control group. Since nano-polysaccharide complex particles B containing the active ingredient have many advantages, including polysaccharide protection, increased water solubility, and uniform particle size, the cell viability treated with these particles was significantly higher than that of the control group, indicating its ability to promote cell proliferation.

[0099] Example 7: Analysis of the uptake rate of nanopolysaccharide composite particles

[0100] First, to synthesize the fluorescent nanopolysaccharide nanoparticle conjugate, nanopolysaccharide nanoparticles B containing the active ingredient need to be dissolved in 7-Methoxycoumarin-3-carboxylic Acid N-Succinimidyl Ester (N7M3C) (Tokyo Chemical Industry, Japan) at a ratio of 1:100. One mL of nanopolysaccharide nanoparticles B containing Antrodia camphorata alcohol extract is centrifuged at 3800 rpm for 10 minutes, followed by the addition of 0.1 M N7M3C (10 μL). To avoid fluorescence loss, the reaction is allowed to proceed for 2 hours at room temperature and in the dark. After the reaction is complete, the sample is centrifuged at 3800 rpm for 10 minutes to remove unreacted N7M3C solvent, thus obtaining the N7M3C-fluorescent nanopolysaccharide nanoparticle conjugate.

[0101] 3T3 cells were seeded into 35 mm culture dishes (10 × 10⁴ cells) and cultured for 24 hours in Durbeco Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS). Different volumes (5 μL, 10 μL, 15 μL, and 20 μL) of N7M3C-fluorescent nanopolysaccharide nanoparticle conjugates were added and the cells were cultured for 10 minutes. After washing twice with PBS, the culture dishes were transferred to a conjugate focal microscope (OLYMPSU, IXplore Pro, Japan) for cell observation, imaging, and analysis.

[0102] As shown in Figure 4, the cell uptake results show that after treatment with nanopolysaccharide composite particles B containing active ingredients (taking experimental group 38 of Example 3 as an example), the cells can effectively exhibit blue fluorescence within ten minutes. This means that the cells can effectively take up the N7M3C-fluorescent nanopolysaccharide composite particle conjugate into the cells and distribute it evenly, so that the active ingredients it encapsulates can fully act in the cells. This experiment proves that the nanopolysaccharide composite particles B containing active ingredients have excellent dispersibility in cells.

[0103] Example 8: Breast Cancer Cell Toxicity Test

[0104] The final cell count was calculated based on a standard curve, which was created according to the specific cell count. First, the actual cell count in the standard cell suspension was counted using a cell counting chamber, and then cells were seeded into well plates for culture. Next, cells were serially diluted with culture medium to create eight cell concentration gradients, each with six replicates. Finally, after seeding, cells were cultured for 2-4 hours to allow adherence, and then a CCK-8 assay kit (Dojindo Molecular Technologies, Kumamoto, Japan) was added. Cell viability was assessed according to the CCK-8 assay kit manual, and the optical density (OD value) was measured. A standard curve was then constructed based on the experimental results.

[0105] Human triple-negative breast cancer cells MDA-MB-231 were seeded in 96-well plates (1×10⁴ cells / well) and incubated at 37°C with 5% CO₂ for 24 hours before cell assays. The experimental groups included (1) a control group treated with ultrapure water; (2) a negative control group treated with *Antrodia camphorata* alcohol extract; (3) nano-polysaccharide complex particles B containing *Antrodia camphorata* alcohol extract; and (4) nano-polysaccharide complex particles B, all with a concentration of 21.467 μg / mL. Cells were cultured for 48 and 72 hours after treatment according to the above groups. At the experimental endpoint, cells were washed twice with 1X PBS, and cell viability was assessed according to the CCK-8 kit manual. Cells containing the assay reagents were then cultured at 37°C for another 2 hours. To prevent nanoparticle interference with this assay, after the reaction was complete, the reagents from the 96-well plate were transferred to a new 96-well plate, while the deposited nanoparticles and cells remained in the initial culture plate. The optical density (OD value) of each well was measured using an ELISA reader at a single wavelength of 450 nm. Each sample was repeated six times.

[0106] As shown in Figure 5, compared with group (2), group (3) significantly inhibited the number of MDA-MB-231 cells at 72 hours of the experiment, with an effect of approximately 5.33 times; while group (4) showed no statistical difference from the control group. The experimental results show that Antrodia camphorata fruiting body extract, as an anti-cancer active ingredient, is difficult to be taken up by cells and act on them due to its poor water solubility. However, after being coated with nano-polysaccharide complex particles B, due to the protective effect of the polysaccharides, the improvement of water solubility, excellent dispersibility and size uniformity, the alcoholic extract containing Antrodia camphorata showed a significant effect in resisting the growth of cancer cells, while the nano-polysaccharide complex particles B did not have cytotoxicity.

[0107] Although this specification contains many specific details, these details should not be construed as limiting the scope of this application or the patent application, but rather as descriptions of features characteristic of particular instances or examples of this application. Certain features described in the text of individual instances or examples in this specification may also be implemented in combination in a single instance.

Claims

1. A nano-polysaccharide composite particle, which is prepared by electrostatic attraction and misalignment reaction of a polycationic polysaccharide and two polyanionic polysaccharides; wherein the polycationic polysaccharide is selected from the group consisting of chitosan, polyethyleneimine and polyquaternary ammonium salt polysaccharides; the two polyanionic polysaccharides are different from each other and are selected from the group consisting of hyaluronic acid, sodium alginate, heparin sulfate / heparin, chondroitin sulfate, keratin sulfate, dermatan sulfate, xanthan gum and carrageenan and pectin respectively.

2. A nano-polysaccharide composite particle with a particle size of approximately 20 nanometers to 112 nanometers, which is prepared by electrostatic attraction and misalignment reaction of a polycationic polysaccharide and two polyanionic polysaccharides; wherein the polycationic polysaccharide is selected from the group consisting of chitosan, polyethyleneimine and polyquaternary ammonium salt polysaccharides; the two polyanionic polysaccharides are different from each other, with molecular weights between 10 kDa and 700 kDa, and are selected from the group consisting of hyaluronic acid, sodium alginate, heparin sulfate / heparin, chondroitin sulfate, keratin sulfate, dermatan sulfate, xanthan gum and carrageenan and pectin.

3. A nano-polysaccharide composite particle with a particle size between 6000 nm and 9000 nm, which is prepared by electrostatic attraction and misalignment reaction of a polycationic polysaccharide and two polyanionic polysaccharides; wherein the polycationic polysaccharide is selected from the group consisting of chitosan, polyethyleneimine and polyquaternary ammonium salt polysaccharides; the two polyanionic polysaccharides are different from each other, with a molecular weight between 1000 kDa and 20,000 kDa, and are selected from the group consisting of hyaluronic acid, sodium alginate, heparin sulfate / heparin, chondroitin sulfate, keratin sulfate, dermatan sulfate, xanthan gum and carrageenan and pectin.

4. Polysaccharide composite particles as requested in items 1, 2 or 3, wherein the polycationic polysaccharide is chitosan, and the two polyanionic polysaccharides are hyaluronic acid and sodium alginate, respectively.

5. The nanopolysaccharide complex particles as claimed in claims 1, 2, or 3, wherein the nanopolysaccharide complex particles are used to encapsulate an active ingredient, so that after being taken up by cells, the active ingredient can be uniformly distributed within the cells. It has excellent dispersibility.

6. Nanoparticles of polysaccharide complex as claimed in items 1, 2 or 3, wherein the active ingredient is water-soluble, poorly water-soluble or non-water-soluble.

7. A pharmaceutical or food composition comprising one or more active ingredients and a pharmaceutically acceptable carrier encapsulated in nanopolysaccharide composite particles as defined in claims 1, 2 or 3.

8. The composition of claim 7, wherein the composition is a pharmaceutical, nutritional, health or health food product.

9. The composition of claim 7, wherein the active ingredient is selected from the group consisting of Antrodia camphorata extract, curcumin, resveratrol, lutein, astaxanthin, water-soluble vitamins, and paclitaxel and combinations thereof.

10. The composition of claim 9, wherein the active ingredient is an alcoholic extract of Antrodia camphorata.

11. A method for preparing nanopolysaccharide composite particles as claimed in claims 1, 2, or 3, comprising: (1) Prepare a solution containing a polycationic polysaccharide as a first solution; wherein the polycationic polysaccharide is selected from the group consisting of chitosan, polyethyleneimine and polyquaternary ammonium salt polysaccharides; (2) Prepare a mixed solution containing two polyanionic polysaccharides as the second solution; wherein the two polyanionic polysaccharides are different from each other and are selected from the group consisting of hyaluronic acid, sodium alginate, heparin sulfate / heparin, chondroitin sulfate, keratin sulfate, dermatan sulfate, xanthan gum, carrageenan and pectin respectively. (3) Add the second solution dropwise to the first solution, mix and stir until homogeneous to form a mixed solution; (4) Centrifuge and concentrate the mixed solution from step (3) and remove the supernatant to obtain a precipitate; and (5) The precipitate was freeze-dried to form a product.

12. A method for preparing nanopolysaccharide composite particles as claimed in claim 2, comprising: (1) Prepare a solution containing a polycationic polysaccharide as a first solution; wherein the polycationic polysaccharide is selected from the group consisting of chitosan, polyethyleneimine and polyquaternary ammonium salt polysaccharides; (2) A mixed solution containing two polyanionic polysaccharides is prepared as a second solution; a polysaccharide-degrading enzyme is then added to the second solution to make the molecular weight of the two polyanionic polysaccharides between 10 kDa and 700 kDa to obtain a third solution; wherein the two polyanionic polysaccharides are different from each other and are selected from the group consisting of hyaluronic acid, sodium alginate, heparin sulfate / heparin, chondroitin sulfate, keratin sulfate, dermatan sulfate, xanthan gum, carrageenan and pectin respectively; (3) Add the third solution dropwise to the first solution, mix and stir until homogeneous to form a mixed solution; (4) Centrifuge and concentrate the mixed solution from step (3) and remove the supernatant to obtain a precipitate; and (5) The precipitate was freeze-dried to form a product.

13. The method of claim 11 or 12, further comprising a step of rinsing the product with ultrapure water to improve its purity.

14. The method of claim 11 or 12, wherein the polycationic polysaccharide is chitosan, and the two polyanionic polysaccharides are hyaluronic acid and sodium alginate, respectively.

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