Composition, preparation method therefor, and use thereof

Abstract

Provided is a composition, comprising cellulose and an active substance. The Zeta potential value (positive or negative) or the charge property of the active substance is opposite to that of the cellulose. By controlling the Zeta potential value (positive or negative) or the charge property of the active substance to be opposite to that of the cellulose, the cellulose has appropriate flexibility / rigidity and network structure, and can form strong electrostatic adsorption and / or hydrogen bonding as well as physical encapsulation with the active substance, stabilizing the spatial structure of the active substance and thus achieving a sustained-release effect. The composition can be used in daily chemical products, pharmaceuticals, health care products, special medical products, foods, drinks, etc.

Description

A composition, its preparation method and application Technical Field

[0001] This invention belongs to the field of cosmetics and pharmaceutical technology, specifically relating to a composition, its preparation method, and its application. Background Technology

[0002] Bioactive substances include peptides and collagen. Peptides can be classified into monopeptides, oligopeptides, and polypeptides based on their molecular weight. Collagen is a biological macromolecule, a major component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25%-30% of total protein, and even more than 80% in some organisms. It is a family of proteins with multiple coding genes, capable of forming various collagen molecules.

[0003] Collagen can be divided into four main categories:

[0004] Type 1 fibrous collagen includes types I, II, III, V, XI, XXIV, and XXVII. Types I, II, and III are more abundant in vertebrates, while types V and VI are less abundant but play an auxiliary role in the assembly of types I, II, and III. Types XXIV and XXVII are interrupted when the triple helix domain is short. Types III, V, and XI collagen retain C-terminal peptides and some N-terminal peptide domains after processing.

[0005] Type II triple-helix fibrous collagen (FACIT) includes types IX, XII, XIV, XVI, XIX, and XXII. FACIT collagen does not form collagen, but it can interact with fibrillary collagen to regulate the formation and size of collagen fibers and control collagen synthesis in the extracellular matrix.

[0006] The third type is reticular collagen, which includes types IV, VII, and XXVIII. Type IV collagen forms a fibrous network structure, while type VII collagen assembles into anchoring fibers that connect the epidermis to the dermis.

[0007] The fourth type is discontinuous helical collagen (MACITs), which includes types XIII, XXIII, and XXV. Types XIII, XXIII, and XXV collagen are type II transmembrane proteins, consisting of a hydrophobic transmembrane domain, a short N-terminal cytoplasmic domain, and an extracellular domain composed of three types of collagen.

[0008] In current technologies, most of the active skin ingredients in cosmetics are reducing agents, which are unstable. Many active substances can only remain stable within a specific pH range. If the pH value of a cosmetic exceeds the stable range of the active substances, it may lead to their decomposition or deterioration. Temperature and light are also important factors affecting the stability of active substances. Increased temperature accelerates the rate of chemical reactions, thereby speeding up the degradation of active substances. Ultraviolet radiation can cause photodegradation reactions, leading to the decomposition or deterioration of active substances.

[0009] The molecular structure of an active substance is a key factor determining its activity. For example, the triple helix structure of collagen is the basis of its biological activity; this structure gives collagen its high tensile strength, biodegradability, and cell growth-promoting properties. If collagen loses its triple helix structure, its biological activity disappears. However, there are still some problems and limitations in ensuring the activity of active ingredients in cosmetics and pharmaceuticals. Summary of the Invention

[0010] To solve the above-mentioned technical problems, the present invention provides a composition comprising cellulose and an active substance, wherein the zeta potential or charged properties of the active substance are opposite to those of the zeta potential or charged properties of the cellulose.

[0011] The inventors have discovered that by controlling the zeta potential or charge properties of the active substance to be opposite to those of the cellulose, the cellulose possesses appropriate flexibility / rigidity and a network structure, enabling it to form strong electrostatic adsorption and / or hydrogen bonding and physical encapsulation with the active substance, stabilizing the spatial structure of the active substance, achieving a sustained-release effect, and can be used in daily chemical products, pharmaceuticals, health products, special medical products, food, beverages, etc.

[0012] In some embodiments, the cellulose forms a complex or compound with the active substance.

[0013] In some embodiments, the absolute value of the zeta potential of the cellulose is 15-100 mV, preferably 20-60 mV, and more preferably 30-50 mV.

[0014] In some embodiments, the absolute value of the zeta potential of the active substance is 10-100 mV, preferably 20-50 mV, and more preferably 30-40 mV.

[0015] In some embodiments, the sum of the absolute values ​​of the zeta potentials of the cellulose and the active substance is 25-200 mV, preferably 60-90 mV.

[0016] In some embodiments, the cellulose is nanocellulose with a diameter of less than 100 nanometers, preferably less than 50 nanometers, and more preferably 5-20 nanometers.

[0017] In some embodiments, the cellulose is nanocellulose with an aspect ratio greater than 50, preferably greater than 100, and more preferably greater than 200.

[0018] In some embodiments, the cellulose is modified cellulose, which is modified by converting the hydroxyl groups in the cellulose into groups with positive or negative charges; preferably, the modified cellulose is obtained by replacing the hydroxyl groups in the cellulose with carboxymethyl, carboxyl, phosphoric acid or sulfonic acid groups to obtain negatively charged modified cellulose; or by replacing the hydroxyl groups in the cellulose with quaternary ammonium groups to obtain positively charged modified cellulose.

[0019] In some embodiments, the active substance is selected from proteins, such as keratin, silk fibroin, collagen, or cell growth factors; and peptides, which include polypeptides, oligopeptides, and cyclic peptides according to molecular weight and structure, and signal peptides, neurotransmitter inhibitory peptides, carrier peptides, or enzyme inhibitory peptides according to function.

[0020] In some implementations, the zeta potential of cellulose and active substance is controlled by adjusting their pH values ​​separately before mixing. Then, the two are mixed to form a complex or compound. This allows cellulose and active substance to bind tightly together, and even if the pH value of the formed complex or compound is subsequently adjusted, the tight bond between cellulose and active substance will not be destroyed.

[0021] In some implementations, the zeta potential of cellulose and the active substance is controlled by adjusting the pH value, which is adjusted in the range of 3-11, for example, adjusting the pH of cellulose and the active substance to 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, and 11.

[0022] In some embodiments, the mass ratio of the cellulose to the active substance in the composition is 1:100 to 1:10000, for example, 1:100, 1:500, 1:1000, 1:3000, 1:5000, 1:7000, 1:10000.

[0023] The present invention also provides a composition comprising cellulose and an active substance, wherein the active substance is a non-colloidal active small molecule compound, and the spatial structure of the active substance is stabilized by hydrogen bonding and physical encapsulation between the cellulose and the active substance.

[0024] In some embodiments, the cellulose forms a complex or compound with a non-colloidal active small molecule compound.

[0025] In some embodiments, the cellulose has the definition described above.

[0026] In some embodiments, the absolute value of the zeta potential of the cellulose is 15-100 mV, preferably 20-60 mV, and more preferably 30-50 mV.

[0027] In some embodiments, the cellulose is nanocellulose with a diameter of less than 100 nanometers, preferably less than 50 nanometers, and more preferably 5-20 nanometers.

[0028] In some embodiments, the cellulose is nanocellulose with an aspect ratio greater than 50, preferably greater than 100, and more preferably greater than 200.

[0029] In some embodiments, the cellulose is modified cellulose, which is modified by converting the hydroxyl groups in the cellulose into groups with positive or negative charges; preferably, the modified cellulose is obtained by replacing the hydroxyl groups in the cellulose with carboxymethyl, carboxyl, phosphoric acid or sulfonic acid groups to obtain negatively charged modified cellulose; or by replacing the hydroxyl groups in the cellulose with quaternary ammonium groups to obtain positively charged modified cellulose.

[0030] In some embodiments, the non-colloidal active small molecule compound includes one or more of dasatinib, quercetin, fisetin, epigallocatechin gallate, proanthocyanidin C1, hydroquinone, arbutin, kojic acid, phenylethyl resorcinol, or nicotinamide.

[0031] In some embodiments, the composition further comprises cellulase, preferably the cellulase comprising at least one of endonuclease, exonuclease, and glucosidase.

[0032] In some embodiments, the amount of cellulase added to the composition is 100-500 U / g, based on the oven-dry weight of cellulose.

[0033] In some embodiments, the cellulase in the composition has a filter paper enzyme (FPA) activity of 30-60 U / mg, preferably 40-50 U / mg, such as 45 U / mg.

[0034] In some embodiments, the surface adsorption rate of the cellulose in the composition for the active substance is: 60% or more after 5 hours of adsorption, preferably 70% or more, more preferably 90% or more; or 85% or more after 10 hours of adsorption, preferably 95% or more, more preferably 98% or more.

[0035] The present invention also provides a method for preparing the above composition, the method comprising:

[0036] (S1) Prepare a solution of the active substance;

[0037] (S2) After adding the cellulose solution to (S1), the mixture is stirred; preferably, the mixing is carried out by high-speed vortex oscillation; and it also includes optional steps (S3) and (S4):

[0038] (S3) Prepare cellulase solution and refrigerate for later use;

[0039] (S4) Mix the cellulase solution from step (S3) with the solution of the composition obtained from step (S2); preferably, the mixing is carried out by high-speed vortex oscillation.

[0040] In some embodiments, when the cellulose is modified cellulose, the method for preparing the composition further includes the preparation of the modified cellulose:

[0041] (SS1) converts the hydroxyl groups on the surface of cellulose into groups with positive or negative charges;

[0042] And also includes optional steps (SS2):

[0043] (SS2) High-pressure homogenization treatment performed after step (SS1);

[0044] Preferably, in step (SS1), the hydroxyl groups in cellulose are replaced with carboxymethyl, carboxyl, phosphoric acid, or sulfonic acid groups to obtain cellulose with a negative charge; the hydroxyl groups in cellulose are replaced with quaternary ammonium groups to obtain cellulose with a positive charge.

[0045] In some embodiments, the solution concentration of the active substance in step (S1) is 0.01-25 wt%, preferably 0.1-5 wt%, such as 1.0 wt%.

[0046] In some embodiments, step (S1) further includes adjusting the pH of the solution of the active substance after preparation. The pH is adjusted to a range of 3-11. The reagents used to adjust the pH include acid solutions, alkaline solutions, or buffer solutions. Commonly used acid solutions include hydrochloric acid, sulfuric acid, acetic acid, etc., commonly used alkaline solutions include sodium hydroxide, sodium bicarbonate, concentrated ammonia solution, etc., and commonly used buffer solutions include phosphate buffer, borate buffer, borate buffer, etc.

[0047] In some embodiments, the concentration of the cellulose solution in step (S2) is 0.01-25 wt%, preferably 0.1-5 wt%, such as 1.0 wt%.

[0048] In some embodiments, the volume ratio of the active substance solution to the cellulose solution in step (S2) is 1:0.1 to 100, for example, a volume ratio of 1:1;

[0049] In some embodiments, step (S2) further includes adjusting the pH of the cellulose solution before mixing, with the pH ranged between 3 and 11. The reagents used to adjust the pH include acid solutions, alkaline solutions, or buffer solutions. Commonly used acid solutions include hydrochloric acid, sulfuric acid, acetic acid, etc., commonly used alkaline solutions include sodium hydroxide, sodium bicarbonate, concentrated ammonia solution, etc., and commonly used buffer solutions include phosphate buffer, borate buffer, borate buffer, etc.

[0050] In some embodiments, step (S2) further includes adjusting the pH of the system of the formed complex or compound after mixing, adjusting the pH to a range of 5-11, for example, adjusting the pH to 7.0 or 8.0;

[0051] In some implementations, the high-speed vortex oscillation in step (S4) has a rotational speed of 2000-3000 rpm and an oscillation time of 5-10 minutes.

[0052] The present invention also provides the application of the aforementioned composition in pharmaceuticals, daily chemical products, special medical purpose formula foods, food, beverages, and health products, such as cosmetics or drugs for hair care, skin care, repairing damaged skin, healing wounds, or treating skin diseases.

[0053] The present invention also provides a method for hair care, skin care, repairing damaged skin, healing wounds, or treating skin diseases, the method comprising administering the above composition to a patient in need.

[0054] According to an embodiment of the invention, the patient in need may be a mammal, such as a human, livestock, or pet.

[0055] The present invention also provides a daily chemical product, food, beverage, special medical purpose formula food, health product or medicine, which comprises the aforementioned composition.

[0056] The special medical purpose formula foods, health products or medicines include, for example, complete nutritional formula foods, specific nutritional formula foods for people with specific diseases or medical conditions, protein components, fat components, carbohydrate components, electrolyte formulas, thickening components, liquid formulas and amino acid metabolism disorder formulas that meet some nutritional needs, disease-specific nutritional formulas, oral rehydration products, protein health products, special functional health products, and medicines used to repair damaged skin, heal wounds or treat skin diseases.

[0057] In some embodiments, the cosmetic product further contains cosmetic ingredients, including fragrances, water, oils, surfactants, moisturizers, polyols, polymers, preservatives, colorants, emollients, anti-wrinkle agents, antioxidants, or whitening agents.

[0058] In some implementations, the drug also contains one or more pharmaceutically acceptable excipients.

[0059] In some embodiments, the medicine is applied to the skin and may be formulated as a topical preparation; suitable topical preparations include, but are not limited to, ointments, creams, lotions, pastes, aerosol sprays, rolls, or aerosol foam (mousse) compositions.

[0060] In some implementations, the drug is used in 3D-printed artificial skin to promote the repair or healing of defects or injuries. Beneficial effects

[0061] In the composition provided by this invention, the active substance and cellulose have a matching zeta potential. They are bound together by strong electrostatic adsorption, hydrogen bonding, and physical encapsulation. Alternatively, the uncharged active substance and cellulose are bound together by hydrogen bonding and physical encapsulation to stabilize the spatial structure of the active substance, resulting in a tighter binding and adaptability to active substances with different structures. The composition forms a barrier between the active substance and the protease, protecting it from protease cleavage and thus isolating it from external interference. This provides a new approach and method for protecting peptide-based active substances that are susceptible to protease attack. Furthermore, by separately adjusting the pH values ​​of cellulose and the active substance before mixing, their zeta potentials are controlled. Then, they are mixed to obtain the composition. This allows for a tight binding between cellulose and the active substance, protecting the active substance and preventing subsequent pH changes in different application systems from damaging the structure and activity of the active substance.

[0062] The composition provided by the present invention can also degrade cellulose by adding cellulase, causing the cellulose network structure to collapse and releasing active substances adsorbed on the surface of cellulose or embedded in its network structure.

[0063] Compared with existing recombinant collagen technology, the composition provided by this invention is safer and more effective, with a simple preparation process, low cost, and easy large-scale production. This is of great significance for promoting the industrialization of daily chemical products and pharmaceutical technologies. Attached Figure Description

[0064] Figure 1 shows the relationship between the adsorption rate and adsorption time on the surface of nanocellulose in Example 1;

[0065] Figure 2 shows the relationship between keratin stability and adsorption time after adding nanocellulose in Example 1;

[0066] Figure 3 is a scanning microscope image of the repair of hair cuticles with nanocellulose in Example 2.

[0067] Terminology Definitions and Explanations

[0068] Unless otherwise stated, the definitions of groups and terms recorded in this application specification and claims, including definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, and definitions of specific compounds in the examples, can be arbitrarily combined and combined with each other. Such combinations and combinations of group definitions and compound structures should be understood as being within the scope of this application specification and / or claims.

[0069] [Cellulose]

[0070] The cellulose involved in this invention is a polysaccharide structure composed of hundreds to thousands of glucose units, and is a linear polymer composed of glucose molecules linked by β-1,4-glycosidic bonds.

[0071] In some embodiments, the absolute value of the cellulose zeta potential is 15-100 mV, preferably 20-60 mV, and more preferably 30-50 mV. For example, under pH 7.0 conditions, the absolute value of the cellulose zeta potential is 30 mV, 32 mV, 34 mV, 36 mV, 38 mV, 39 mV, 40 mV, 41 mV, 42 mV, 43 mV, 44 mV, 45 mV, 46 mV, 47 mV, 48 mV, 49 mV, or 50 mV.

[0072] In some embodiments, the zeta potential of cellulose is controlled by adjusting the pH of the system, wherein the pH is adjusted in the range of 3-11; for example, at pH 7.0, the zeta potential of modified cellulose with a negative charge obtained by replacing the hydroxyl groups in cellulose with carboxymethyl or carboxyl groups is -60 mV to -20 mV, and the zeta potential of the cellulose is -50 mV to -10 mV when the pH is adjusted in the range of 3.0-5.0; at pH 7.0, the zeta potential of modified cellulose with a positive charge obtained by replacing the hydroxyl groups in cellulose with quaternary ammonium groups is +20 mV to +60 mV, and the zeta potential of the cellulose is +10 mV to +50 mV when the pH is adjusted in the range of 8.0-11.0.

[0073] [Nanocellulose]

[0074] The nanocellulose involved in this invention is a nanofiber produced by chemical, physical, or combined methods of treating cellulose. Its diameter is usually between a few nanometers and a few hundred nanometers, and its length can reach from a few hundred nanometers to a few micrometers. It has a high specific surface area and good biocompatibility.

[0075] In some embodiments, the diameter of the nanocellulose is less than 100 nanometers, preferably less than 50 nanometers, and more preferably 5-20 nanometers.

[0076] In some embodiments, the specific surface area of ​​the nanocellulose is greater than 100 m² / g, preferably greater than 200 m² / g, and even more preferably greater than 300 m² / g.

[0077] [Modified cellulose]

[0078] The modified cellulose of the present invention is modified by converting the hydroxyl groups in cellulose into groups with positive or negative charges. For example, the modified cellulose is obtained by replacing the hydroxyl groups in cellulose with carboxymethyl, carboxyl, phosphoric acid, or sulfonic acid groups to obtain negatively charged modified cellulose; and by replacing the hydroxyl groups in cellulose with quaternary ammonium groups to obtain positively charged modified cellulose.

[0079] In some embodiments, 1% to 80% of the hydroxyl groups in the modified cellulose are replaced by carboxymethyl, carboxyl, phosphoric acid, sulfonic acid, or quaternary ammonium groups.

[0080] In some embodiments, the crystallinity of the modified cellulose is less than 90%, preferably less than 85%, and more preferably 60-85%.

[0081] In some embodiments, the surface charge of the modified cellulose is greater than 0.5 mmol / g, for example greater than 1 mmol / g, greater than 1.2 mmol / g, or greater than 1.5 mmol / g.

[0082] [Cellulose with a positive or negative charge]

[0083] The cellulose with positive or negative charge involved in this invention is a cellulose whose surface charge characteristics can be controlled by introducing carboxyl, carboxymethyl, phosphoric acid, sulfonic acid or quaternary ammonium groups through surface modification.

[0084] Cellulose with a negative charge can be obtained by replacing the hydroxyl groups in cellulose with carboxymethyl, carboxyl, phosphoric acid, or sulfonic acid groups; cellulose with a positive charge can be obtained by replacing the hydroxyl groups in cellulose with quaternary ammonium groups.

[0085] [Sources of cellulose raw materials]

[0086] The cellulose raw material sources involved in this invention include at least one of natural cellulose, regenerated cellulose, and bacterial cellulose, such as cellulose extracted from cotton, seaweed, hemp, bamboo, trees, wood, grains, beans, plant straw, and cellulose derived from bacterial fermentation. The raw material sources of the cellulose involved in this invention also include microcrystalline cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, microcrystalline cellulose, and methyl cellulose. Preferably, the raw material source of the cellulose is cellulose fiber with a degree of polymerization of 700-1200, and more preferably cellulose fiber with a degree of polymerization of 800-1000.

[0087] [Active Substances]

[0088] The active substances involved in this invention are selected from proteins, such as keratin (e.g., α-keratin, β-keratin), elastin (e.g., proelastin, etc.), fibrillin (e.g., fibrillin-1 (the main component of microfibrils), fibrillin-2 (a component of elasticity), fibrillin-3 (found in the brain), fibrillin-4 (a component of elasticity), etc.), fibrinogen / fibrin / thrombin (e.g., fibrinogen is converted to fibrin by thrombin during wound healing), fibronectin, laminin, silk fibroin, collagen (e.g., collagen I found in skin, tendons, and bones; collagen II found in cartilage; collagen III found in connective tissue; collagen IV found in extracellular matrix proteins; collagen V found in hair, etc.), and from a wide range of sources. Collagen extracted from sources (e.g., pigs, cattle, humans, fish, or rat tails), such as recombinant human collagen (recombinant collagen type III or XVII), transgenic human tissue collagen, porcine collagen, human placental collagen, bovine collagen, autologous collagen, collagen fibers and human tissue collagen matrix; cell growth factors (fibroblast growth factor, endothelial growth factor, or insulin-like growth factor); peptides, including polypeptides, oligopeptides, and cyclic peptides according to molecular weight and structure, and signal peptides, neurotransmitter inhibitory peptides, carrier peptides, or enzyme inhibitory peptides according to function (oligopeptide-1, tripeptide-1, hexapeptide-1, hexapeptide-3, acetyl oligopeptide, palmitoyl oligopeptide, carnosine, glutathione, or copper peptide); non-colloidal active small molecule compounds, such as dasatinib, quercetin, fisetin, epigallocatechin gallate, proanthocyanidin C1, hydroquinone, arbutin, kojic acid, phenylethyl resorcinol, or nicotinamide.

[0089] In some embodiments, when the active substance has a Zeta potential, the absolute value of the Zeta potential of the active substance is 10-100 mV, preferably 20-50 mV, more preferably 30-40 mV, for example 26 mV, 29 mV, 30 mV, 31 mV, 32 mV, 33 mV, 34 mV, 35 mV, 36 mV, 37 mV, 38 mV, 39 mV, 40 mV.

[0090] In some embodiments, the zeta potential of the active substance is controlled by adjusting the pH of the system, wherein the pH is adjusted in the range of 3-11; for example, collagen has an isoelectric point of 7.5-7.8, and when the pH is adjusted in the range of 5.0-11.0, the zeta potential of collagen is in the range of +45mV to -30mV; copper peptide has an isoelectric point of 10.0, and when the pH is adjusted in the range of 5.0-11.0, the zeta potential of copper peptide is in the range of +35mV to -15mV; fibroblast growth factor has an isoelectric point of 9.6, and when the pH is adjusted in the range of 5.0-11.0, the zeta potential of fibroblast growth factor is in the range of +37mV to -10mV.

[0091] In some embodiments, the active substance is selected from non-colloidal active small molecule compounds, and the ionization or hydrolysis balance of the non-colloidal active small molecule compounds is controlled by adjusting the pH value of the system; for example, nicotinamide solution itself is relatively stable between pH 5 and 7, and adjusting the pH to the range of 5.0 to 7.0 controls the ionization or hydrolysis of nicotinamide and prevents excessive hydrolysis to nicotinic acid, which can cause skin irritation; quercetin is relatively stable between pH 5 and 8, and adjusting the pH to the range of 5.0 to 8.0 controls the aoxidation of quercetin and prevents the loss of quercetin's free radical scavenging activity.

[0092] [Introduction of carboxyl groups to hydroxyl groups on the surface of cellulose]

[0093] The present invention relates to the introduction of carboxyl groups into the hydroxyl groups on the surface of cellulose, including but not limited to one or more of the following methods: TEMPO (tetramethylpiperidine oxide) oxidation, H2O2 oxidation, HIO4 oxidation, and NaClO2 oxidation; more preferably, surface carboxylated nanocellulose prepared by TEMPO oxidation. Further preferably, the TEMPO oxidation method includes dispersing cellulose fibers in an aqueous solution of TEMPO and NaBr, adding NaClO to carry out an oxidation reaction for 6-12 hours, maintaining the system alkalinity by adding an alkaline solution, for example, adding potassium hydroxide, sodium hydroxide, potassium carbonate, or sodium carbonate solution to maintain the pH at 10.0-10.5.

[0094] [Introduction of carboxymethyl groups to hydroxyl groups on cellulose surface]

[0095] The present invention relates to the introduction of carboxymethyl groups into the hydroxyl groups on the surface of cellulose, including but not limited to alkalization-etherification methods. More preferably, the alkalization is carried out using an aqueous solution of an organic or inorganic base. Examples of such organic or inorganic bases include sodium hydroxide, potassium hydroxide, calcium hydroxide, carbon carbonate, potassium carbonate, triethylamine, and ammonia. The etherification is carried out using sodium chloroacetate or chloroacetic acid, for example, using a methanol or ethanol solution of sodium chloroacetate. The alkalization is carried out at 10-50°C for 10-60 minutes, preferably at 25°C for 30 minutes; the etherification is carried out at 50-100°C for 30-150 minutes, preferably at 60-80°C for 60-120 minutes.

[0096] [Introduction of quaternary ammonium groups into cellulose surface hydroxyl groups]

[0097] The present invention relates to the introduction of quaternary ammonium groups into the hydroxyl groups on the surface of cellulose, including but not limited to substitution reactions between cellulose and a quaternizing agent under alkaline conditions. More preferably, the quaternizing agent is selected from one or more of 3-chloro-2-hydroxypropyltrimethylammonium chloride, 2,3-epoxypropyltrimethylammonium chloride, 3-chloro-2-hydroxypropyltrimethylammonium bromide, and 2,3-epoxypropyltrimethylammonium bromide. Even more preferably, the reaction temperature for introducing the quaternary ammonium groups is 50-100°C, such as 65°C.

[0098] [Introduction of phosphate groups to hydroxyl groups on the cellulose surface]

[0099] The present invention relates to the introduction of phosphate groups into the hydroxyl groups on the surface of cellulose, including but not limited to reactions with the hydroxyl groups on the surface of cellulose using a phosphorylation reagent. More preferably, the phosphorylation reagent includes diammonium hydrogen phosphate or a mixture of diammonium hydrogen phosphate and urea. The method includes, for example, impregnating cellulose with a phosphorylation reagent (at a mass ratio of cellulose:diammonium hydrogen phosphate:urea of ​​1:0.8:3.7) for 1 hour, followed by further reaction in an oven at 170°C for 30 minutes.

[0100] [Introduction of sulfonic acid groups to hydroxyl groups on the cellulose surface]

[0101] The present invention relates to the introduction of sulfonic acid groups into the hydroxyl groups on the cellulose surface, including but not limited to reactions with the hydroxyl groups on the cellulose surface via a sulfonating agent. More preferably, the reaction includes hydrolyzing the amorphous regions of the cellulose fibers at 45°C using 64% sulfuric acid, while simultaneously introducing sulfonic acid groups onto the surface of the crystalline regions of the cellulose via an esterification reaction for 60 minutes.

[0102] [Cellulose Pretreatment and Posttreatment]

[0103] Prior to introducing carboxyl, carboxymethyl, phosphoric acid, sulfonic acid, or quaternary ammonium groups, the method of the present invention optionally includes a pretreatment step of cellulose. Preferably, the pretreatment includes pretreating cellulose with an alkali and an acid. More preferably, cellulose is dispersed in an alkali solution, heated, washed with water until neutral, then dispersed in an acid solution, heated, and washed with water until neutral.

[0104] Preferably, in the step of pretreating cellulose, the alkali includes organic and inorganic alkalis, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, carbon carbonate, potassium carbonate, triethylamine, ammonia, etc.; the acid includes organic and inorganic acids, such as hydrochloric acid, sulfuric acid, phosphoric acid, formic acid, acetic acid, citric acid, etc. When pretreating cellulose with alkali and acid, an alkali solution or acid solution is added to the cellulose and the slurry concentration is adjusted to 0.1-10 wt%, preferably 2.5 wt%.

[0105] Preferably, in the step of pretreating cellulose, the heating treatment temperature is 100-150℃ and the time is 10-60 minutes; preferably, the heating treatment temperature is 120-130℃ and the time is 30 minutes.

[0106] After the reaction involving the introduction of carboxyl, carboxymethyl, phosphoric acid, sulfonic acid, or quaternary ammonium groups is completed, the method of the present invention optionally includes a post-treatment step. Preferably, the post-treatment step includes adjusting the pH to neutral, washing the reaction product with water, and filtering until the conductivity of the filtrate is less than 50 μS / cm, preferably less than 5 μS / cm.

[0107] [High-pressure homogenization treatment]

[0108] The high-pressure homogenization process described in this invention refers to the process of uniformly mixing the components in a substance by applying high pressure to form a homogeneous system. The high-pressure homogenization process of this invention may include adjusting the concentration of the cellulose fibers obtained in step (S1) to 1 wt% with water, pre-shearing and dispersing them using a disperser at 2000 rpm for 5 minutes, and then performing high-pressure homogenization at a pressure of 200-1200 bar.

[0109] [The sum of the absolute values ​​of the zeta potentials of cellulose and active substances]

[0110] The sum of the absolute values ​​of the zeta potentials of cellulose and the active substance involved in this invention refers to the summation of the absolute values ​​of their zeta potentials when cellulose and the active substance carry opposite charges.

[0111] [cellulase]

[0112] The cellulase involved in this invention refers to an enzyme capable of degrading cellulose to produce glucose, including at least one of endonucleases, exonucleases, and glucosidases, such as a complex cellulase derived from Trichoderma reesei.

[0113] [The flexibility / rigidity of cellulose]

[0114] The cellulose flexibility involved in this invention refers to cellulose with a crystallinity of 60-85%, an aspect ratio higher than 250, or an absolute value of Zeta potential higher than 40mV, which indicates strong cellulose flexibility.

[0115] The cellulose rigidity involved in this invention refers to cellulose with a crystallinity of 85% or more, an aspect ratio of less than 150, or an absolute value of zeta potential of less than 30mV, which has strong rigidity.

[0116] [Daily Chemical Products]

[0117] The daily chemical products involved in this invention refer to chemical products manufactured using certain chemicals or natural products as raw materials, related to people's daily lives, and intended to clean and beautify people's homes and meet the health, cleanliness, beauty, and comfort needs of animals or pets. According to their uses, they can be divided into toiletries, household goods, kitchen and bathroom supplies, decorative items, cosmetics, and animal or pet care products. According to industry practice, they can be divided into cosmetics (such as skincare cosmetics, beauty cosmetics, cleaning cosmetics, and hair care cosmetics), detergents (such as soaps, laundry detergents, and cleaning agents), oral care products (such as toothpaste and mouthwash), fragrances, deodorants, insect repellents and pest control products, and animal or pet care products (such as pet-specific bathing and cleaning products, oral care products, insect repellent and flea control products, and skin care products). Detailed Implementation

[0118] The technical solutions of this disclosure will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of this disclosure and should not be construed as limiting the scope of protection of this disclosure. All technologies implemented based on the above content of this disclosure are covered within the scope of protection intended by this disclosure.

[0119] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0120] Unless otherwise specified, % in the following preparation examples and embodiments refers to mass percentage.

[0121] Method for testing the Zeta potential of modified nanocellulose: Take the prepared modified nanocellulose, dilute it with ultrapure water to 0.05% solid content, and use a Zeta potential analyzer to detect the Zeta potential of the modified nanocellulose.

[0122] Method for testing the zeta potential of active substances: Prepare an active substance solution with a solid content of 0.01% using ultrapure water, and detect its zeta potential using a zeta potential analyzer.

[0123] Crystallinity test method: Take the prepared modified nanocellulose, freeze-dry it at -50℃ and 0.01mbar for 72 hours, and use an X-ray diffractometer to scan at 5-40 degrees to analyze the X-ray diffraction pattern of the sample and calculate the crystallinity of the sample.

[0124] Diameter and aspect ratio testing methods: The prepared modified nanocellulose was diluted with ultrapure water to a solid content of 0.01%, ultrasonically dispersed for 10 minutes, dropped onto a copper mesh, and air-dried. Morphology images of the modified nanocellulose were then captured using a transmission electron microscope at magnification of 10,000-20,000. The length and diameter of the modified nanofibers were measured and statistically analyzed using ImageJ software, and the aspect ratio was calculated.

[0125] Specific surface area testing method: Take 1% of modified nanocellulose sample and place it in a centrifuge tube, immerse it in liquid nitrogen for rapid freezing, transfer it to -50 degrees Celsius, freeze dry the sample under vacuum of 0.05 mbar for 72 hours, and use a specific surface area analyzer to detect the specific surface area of ​​the sample.

[0126] Test method for surface charge of modified cellulose:

[0127] Take 5 g of 1% modified nanocellulose, dilute to 0.1% with deionized water, add 2 mL of 0.1 M HCl and 1 mL of 50 mM sodium chloride solution, and stir at room temperature for 30 minutes. Titrate with 0.1 M NaOH and monitor the conductivity and pH of the suspension until pH 11. Calculate the charge of anionic groups on the cellulose surface (including introduced carboxyl, carboxymethyl, and sulfonic acid groups) using the titration curve.

[0128] Take 5 g of 1% modified nanocellulose, dilute it to 0.1% with deionized water, titrate it with 0.01 mg NO3 and monitor the conductivity of the suspension. Titrate until the conductivity value continues to increase steadily. Calculate the charge of the cationic quaternary ammonium salt groups on the cellulose surface through the titration curve.

[0129] Adsorption rate test method:

[0130] Weigh out the active substance to be adsorbed and dissolve or disperse it in deionized water. For active substances with characteristic ultraviolet absorption wavelengths, use an ultraviolet spectrophotometer to establish a working curve between the concentration of the active substance and the absorbance value. For active substances without characteristic ultraviolet absorption, use high performance liquid chromatography or the corresponding enzyme-linked immunosorbent assay kit to establish a working curve between the concentration of the active substance and the detection response value.

[0131] Weigh out cellulose, adjust the solid content to 0.1% with deionized water, add the active substance, shake to mix, incubate for a certain time, centrifuge at 10,000 rpm for 15 minutes, and filter through a 0.2 μm filter membrane. Detect the concentration of free active substance using a UV spectrophotometer, high-performance liquid chromatography, or a corresponding enzyme-linked immunosorbent assay (ELISA) kit, and then calculate the content of active substance adsorbed on the cellulose surface.

[0132] Establishment of absorbance standard curve:

[0133] (1) Dissolve keratin in deionized water to form a keratin solution with a concentration of 0.01%, and determine the maximum absorption wavelength by full-wavelength scanning;

[0134] (2) Gradually dilute the keratin solution and measure the absorbance at the maximum absorption wavelength to establish a concentration-absorbance standard curve.

[0135] Preparation Example 1: Preparation of TJ-300:

[0136] Cellulose fibers with a degree of polymerization of 800-1000, a cellulose alpha content >90%, and a whiteness >85% ISO were dispersed using 0.1M NaOH solution. The slurry concentration was maintained at 2.5%. The slurry was heated at 121°C for 30 minutes, washed with sterile water, and filtered until the pH of the filtrate was neutral. Subsequently, the slurry was dispersed using 0.3M HCl solution, the slurry concentration was adjusted to 2.5%, heated at 121°C for 30 minutes, washed with sterile water, and filtered until the pH of the filtrate was neutral.

[0137] Weigh out 5.0±0.1 g of pretreated cellulose fibers (dry weight), mix thoroughly with 15±0.1 g of 30% NaOH alkaline solution at 25℃, and impregnate for 30 min. After impregnation, add 7±0.1 g of sodium chloroacetate dispersion (dispersed in 100 mL of 95% ethanol and preheated to 70℃), and stir at 500 rpm for 30-120 min in a constant temperature water bath at 60-80℃. After the reaction, adjust the pH to neutral with glacial acetic acid, wash the reaction product repeatedly with ultrapure water, and filter using an ultrafiltration system until the conductivity of the filtrate is less than 5 μS / cm. Adjust the concentration of the cellulose fibers to 1% with sterile water and homogenize sequentially at 200, 400, 600, 800, 1000, and 1200 bar in a sterile room. Store at 4℃ for later use.

[0138] In this preparation example, by controlling the etherification reaction temperature and time (60℃, 30 min; 70℃, 100 min; 80℃, 120 min), the prepared TJ-300 exhibits high flexibility. The characterization results obtained through testing and calculation are shown in Table 1.

[0139] Table 1: Characterization results of TJ-300 prepared at different etherification reaction temperatures and times

[0140] Preparation Example 2: Preparation of TJ-321T

[0141] Cellulose fibers with a degree of polymerization of 800-1000, a cellulose alpha content >90%, and a whiteness >85% ISO were dispersed using 0.1M NaOH solution. The slurry concentration was maintained at 2.5%. The slurry was heated at 121°C for 30 minutes, washed with sterile water, and filtered until the pH of the filtrate was neutral. Subsequently, the slurry was dispersed using 0.3M HCl solution, the slurry concentration was adjusted to 2.5%, heated at 121°C for 30 minutes, washed with sterile water, and filtered until the pH of the filtrate was neutral.

[0142] Weigh out 10.0±0.1 g of pretreated cellulose fibers (octane-dry weight) and disperse them in a solution containing 1.6±0.1 g TEMPO and 1.0±0.1 g NaBr at 25°C. Stir continuously at 500 rpm and add 75 ml of 10% NaClO dropwise to initiate the oxidation reaction for 6-12 hours. During the reaction, monitor the pH of the suspension and maintain it at 10.0 by adding 0.5 M NaOH solution dropwise. After the reaction, adjust the pH to neutral with 1.0 M HCl. Wash the reaction product repeatedly with ultrapure water and filter it using an ultrafiltration system until the conductivity of the filtrate is less than 5 μS / cm. Adjust the concentration of the oxidized cellulose fibers to 1% with sterile water and homogenize them sequentially at 200, 400, 600, 800, 1000, and 1200 bar in a sterile environment. Store the homogenized fibers at 4°C for later use.

[0143] The TJ-321T prepared in this example, obtained by adjusting the TEMPO catalytic oxidation time (6h; 8h; 12h), exhibits moderate flexibility. The characterization results obtained through testing and calculation are shown in Table 2.

[0144] Table 2: Characterization results of TJ-321T prepared under different TEMPO catalytic oxidation times

[0145] Preparation Example 3: Preparation of QCNF

[0146] Cellulose fibers with a degree of polymerization of 800-1000, a cellulose alpha content >90%, and a whiteness >85% ISO were dispersed using 0.1M NaOH solution. The slurry concentration was maintained at 2.5%. The slurry was heated at 121°C for 30 minutes, washed with sterile water, and filtered until the pH of the filtrate was neutral. Subsequently, the slurry was dispersed using 0.3M HCl solution, the slurry concentration was adjusted to 2.5%, heated at 121°C for 30 minutes, washed with sterile water, and filtered until the pH of the filtrate was neutral.

[0147] Weigh out 15.0 ± 0.1 g of pretreated cellulose fibers (octane-dry weight) and disperse them in 200 mL of 5% NaOH solution at room temperature, stirring and soaking for 30 min. After soaking, add 7.5 g to 10 g of 2,3-epoxypropyltrimethylammonium chloride, heat to 65 °C, and stir at 500 rpm for 1-8 hours. After the reaction, adjust the pH to neutral with 1.0 M HCl, wash the reaction product repeatedly with ultrapure water, and filter using an ultrafiltration system until the conductivity of the filtrate is less than 5 μS / cm. Adjust the concentration of the reacted cellulose fibers to 1% with sterile water and homogenize them sequentially at 200, 400, 600, 800, 1000, and 1200 bar in a sterile room, storing them in a 4 °C refrigerator for later use.

[0148] The QCNF obtained in this preparation example by adjusting the amount of etherifying agent and reaction time (7.5 g, 1 h; 8.5 g, 6 h; 10 g, 8 h) exhibits certain rigidity (+15 / +46 mV) or flexibility (+79 mV). The characterization results obtained through testing and calculation are shown in Table 3.

[0149] Table 3: Characterization results of QCNF prepared under different amounts of etherifying agent and reaction times

[0150] Example 1: Adsorption and stability of keratin on the surface of nanocellulose

[0151] (1) Select TJ-300 (Zeta potential -49mV at pH 7.0), TJ-321T (Zeta potential -47mV at pH 7.0), and QCNF (Zeta potential +15 / +46 / +79mV at pH 7.0) nanocellulose prepared in Preparation Examples 1-3, adjust the solid content to 0.1%, add keratin solution (10mg / mL, Zeta potential -26mV at pH 7.0), wherein the volume ratio of keratin solution to nanocellulose prepared in Preparation Examples 1-3 is 1:1, shake to mix, and incubate at 37 degrees Celsius for 0-24 hours.

[0152] (2) Samples were taken at six time points: 0h, 2h, 4h, 6h, 8h, 12h, and 24h and centrifuged (10000rpm, 15min) and filtered through a 0.2μm filter membrane.

[0153] (3) Set an equal volume of deionized water as a control, measure the absorbance of the filtrate, and calculate the adsorption rate of keratin on the surface of nanocellulose based on the concentration-absorbance curve. The relationship between the adsorption rate of keratin on the surface of TJ-300, TJ-321T, and QCNF nanocellulose and the adsorption time is shown in Figure 1.

[0154] (4) Add keratinase (50 IU / g) to the nanocellulose-keratin complex that has been adsorbed and stabilized in (1) for 24 hours, and incubate at 37 degrees Celsius for 0 to 24 hours.

[0155] (5) Samples were taken at six time points: 0h, 2h, 4h, 6h, 8h, 12h, and 24h. The keratin in the samples was quantified using a keratin ELISA kit. The keratin content in the sample at time point 0h was used as a control. The stability of keratin after the addition of nanocellulose was calculated. The results are shown in Figure 2.

[0156] Example 2: Preparation of keratin-cellulose nanocomposite and its effect on repairing hair cuticles

[0157] (1) Collect adult female hair, wash it and soak it in 5% ammonia water, incubate it at 50°C for 2 hours, take it out and wash it to prepare simulated hair with broken cuticles.

[0158] (2) Take 1% concentration of TJ-300 (Zeta potential -49mV at pH 7.0), TJ-321T (Zeta potential -47mV at pH 7.0), and QCNF (Zeta potential +15mV / +46mV / +79mV at pH 7.0) nanocellulose prepared in Preparation Examples 1-3 respectively, mix them with 1% concentration of keratin (Zeta potential -26mV at pH 7.0) aqueous solution (modified nanocellulose and keratin are mixed in equal volumes), and treat with high-speed vortex oscillation (2000-3000rpm) for 5-10 minutes to obtain a solution containing keratin-nanocellulose complex. Immerse the damaged hair with cracked cuticles in it (10 hairs 10cm long are immersed in 10mL of the mixture), incubate at 37℃ for 8 hours, take it out and wash it to obtain the repaired hair.

[0159] (3) After drying, the images were taken using a scanning electron microscope and the results are shown in Figure 3.

[0160] Example 3: Preparation of collagen-cellulose nanocomplex and experiment on the stabilizing and protecting collagen by cellulose nanocomplex.

[0161] (1) Prepare collagen with a concentration of 1.0wt%, including recombinant collagen type III (Zeta potential -29mV at pH 7.0) and type XVII (Zeta potential -31mV at pH 7.0), and store at 4℃ for later use.

[0162] (2) The collagen was diluted with pure water and 1% QCNF (Zeta potential +46mV at pH 7.0) nanocellulose obtained in Preparation Example 1 to an effective concentration of 0.1% in the solution, and incubated at 25°C and 60°C for 1 hour respectively, and then cooled to room temperature.

[0163] (3) Use a circular dichroism chromatograph to scan the wavelength range of 190-260nm and scan at a speed of 1nm / s to record the ellipticity. Record the maximum positive and negative absorption peaks of the ellipticity of each sample. The results are shown in Table 4 (Col-III represents type III recombinant collagen, Col-XVII represents type XVII recombinant collagen, Col-III-QCNF represents type III recombinant collagen complex with QCNF, and Col-XVII-QCNF represents type XVII recombinant collagen complex with QCNF).

[0164] Table 4: Maximum positive and negative absorption peaks of sample ellipticity

[0165] According to Table 4, at 25℃, the circular dichroism ellipticity of type III and XVII recombinant collagen (Col-III, 25℃ and Col-XVII, 25℃) showed maximum positive absorption peaks at 224-225 nm and 220 nm, respectively, and maximum negative absorption peaks at 192-194 nm and 197-198 nm, respectively. These absorption peaks confirm that type III and XVII recombinant collagen possess a triple-helix structure. After incubation at 60℃ for 1 hour, the maximum positive absorption peaks of the circular dichroism ellipticity of type III and XVII recombinant collagen (Col-III, 60℃ and Col-XVII, 60℃) disappeared, indicating that the triple-helix structure of the collagen was disrupted. After incubating type III and type XVII recombinant collagen with QCNF complexes (Col-III-QCNF and Col-XVII-QCNF) at 60°C for 1 hour, the maximum positive absorption peak at 220 nm and the maximum negative absorption peak at 197 nm of the circular dichroism chromatogram were retained, indicating that the collagen did not lose its triple helix structure under the protection of QCNF.

[0166] Example 4: Moisturizing effect test

[0167] Test samples: The 1% TJ-300 (Zeta potential -49mV under pH 7.0) obtained in Preparation Example 1 was diluted with pure water to concentrations of 10%, 25% and 50%, with 5% glycerol and 0.5% hyaluronic acid as positive controls and water as a blank control.

[0168] Test Method: Before testing, the sample was placed in a desiccator with a supersaturated ammonium sulfate solution to maintain a constant humidity. Medical 3M tape was used to partially simulate the skin surface environment. The 3M tape was first applied to a 5cm x 5cm glass plate, and the saturated ammonium sulfate was separated by a porous partition. The glass plate with the tape was placed on the porous partition, the desiccator was closed, and the plate was left to stand for 20 minutes. The weight of the glass plate containing the 3M tape was then measured. The concentration of 2 mg / cm³ was accurately measured. 2 The sample was applied to 3M tape and then spread evenly. The applied sample was immediately placed in a constant humidity desiccator and left to stand for 1 h, 2 h, 4 h, 8 h, 24 h, and 72 h. At each time point, the weight of the glass plate containing the 3M tape and the sample was accurately weighed once. The moisture retention rate of the sample was calculated by calculating the degree of moisture retention. The results are shown in Table 5.

[0169] Table 5. Average moisturizing rate of different samples at different time points.

[0170] Test Results: Over time, the moisturizing rate of all samples showed a decreasing trend. Specifically, at 1h and 2h, the 50% concentration of TJ-300 exhibited the best moisturizing effect, followed by the 25% concentration, which was superior to 0.5% glycerin. However, at 4h, the best moisturizing effect shifted to the 5% glycerin sample, with a moisturizing rate of 30.00%. At this point, the moisturizing effects of the 25% and 50% concentrations of TJ-300 were slightly inferior to 5% glycerin, but still significantly better than 0.5% HA. After 4h, 5% glycerin continued to maintain its best moisturizing effect. Meanwhile, the differences in moisturizing effects between the 25% and 50% concentrations of TJ-300 and 0.5% HA gradually narrowed, indicating that these two concentrations of TJ-300 possess a certain degree of stability and competitiveness in long-term moisturizing performance.

[0171] Conclusion: TJ-300 tested in this study exhibits excellent water-retention performance for short-term hydration at high concentrations (50% and 25%), while its long-lasting hydrating effect at low concentrations (10%) shows no significant difference compared to the control sample 0.5% HA. This provides valuable data support and theoretical basis for the development and optimization of related moisturizing products.

[0172] Example 5: Compound Cellulase Slow-Release Experiment

[0173] (1) Prepare a 1.0 wt% recombinant human collagen solution (Zeta potential of +35 mV and pH of 7.0 at pH 7.0) and a complex cellulase solution (the complex cellulase is a commercial complex cellulase purchased from Shanghai Yuanye Biotechnology Co., Ltd., containing endonuclease, exonuclease, glucosidase, and filter paper enzyme activity FPA of 45 U / mg protein) and store at 4℃ for later use.

[0174] (2) The collagen solution prepared in (1) is mixed with 1% TJ-300 (Zeta potential -49mV under pH 7.0) nanocellulose obtained in Preparation Example 1 or 1% QCNF (Zeta potential +46mV under pH 7.0) nanocellulose obtained in Preparation Example 3 at a volume ratio of 1:100 (pH 7.0). After mixing, the mixture is treated by high-speed vortex oscillation (2000-3000rpm) for 5-10 minutes to obtain the collagen-nanocellulose complex, which is then stored at 4℃ for later use.

[0175] (3) Take the compound cellulase solution prepared in (1) and add it to the collagen-nanocellulose complex prepared in (2) according to the enzyme dosage of 100-500 U / g of dry cellulose. After mixing, vortex at high speed (2000-3000 rpm) for 1 minute to obtain the collagen-loaded nanocellulose degradation complex. Store at 4℃ for later use.

[0176] (4) The complex prepared in (3) was loaded into a 96-well plate and placed in a 37°C constant temperature incubator to monitor the degradation of the complex and the release of collagen. Samples were taken at different degradation time points (0, 1, 2, 4, 8, 16, 24 h), centrifuged at 5000 rpm, and the supernatant was collected. The amount of collagen released was quantitatively detected by enzyme-linked immunosorbent assay (ELISA), and the amount of free glucose produced by cellulase degradation of TJ-300 and QCNF was detected by DNS colorimetric method.

[0177] (5) Take the complex prepared in (3) and put it into a 50 mL centrifuge tube. Place it in a 37°C constant temperature incubator to carry out complex degradation and monitor the complex viscosity. Take out the sample at different degradation time points (0, 1, 2, 4, 8, 16, 24 h) and cool it to room temperature. Use a rotational viscometer with a #4 rotor at 12 rpm to detect the viscosity of the complex and characterize the enzymatic degradation of the nanocellulose gel network.

[0178] The results of the enzyme degradation test are shown in Tables 6 and 7.

[0179] Table 6. Enzymatic degradation test results of the collagen-loaded TJ-300 nanocellulose degradation complex.

[0180] The isoelectric point (PI) of recombinant human collagen is 8.0-9.0. Under neutral conditions, the zeta potential of recombinant human collagen is +35mV. It can adsorb onto the negatively charged surface of TJ-300 (Zeta potential -49mV). The sum of the absolute values ​​of the zeta potentials of TJ-300 and recombinant human collagen is 84mV.

[0181] According to Table 6, with the increase of cellulase dosage and degradation time, the cumulative release of collagen and glucose increased, while the viscosity of the complex decreased. When the cellulase dosage reached 500 U / g, the cumulative release of collagen exceeded 50% and the cumulative release of glucose exceeded 10 mg after a degradation time of more than 4 hours, indicating that the nanocellulose network structure collapsed and could effectively release collagen adsorbed on the surface or embedded in the network structure.

[0182] Table 7. Enzymatic degradation test results of the collagen-loaded QCNF nanocellulose degradation complex.

[0183] At pH 7.0, the zeta potentials of QCNF and recombinant human collagen were +46 mV and +35 mV, respectively. Due to the identical surface charge, electrostatic repulsion hindered collagen adsorption on the QCNF surface. Collagen existed in free form in the solution (approximately 27%), while some was embedded in the QCNF network through hydrogen bonding and physical encapsulation. Some collagen or QCNF formed flocs with the added negatively charged cellulase (zeta potential -28 mV), resulting in a lower overall viscosity of the complex and reduced cellulase degradation ability.

[0184] Example 6: Preparation and Free Radical Scavenging Detection of Quercetin-Cellulose Nanocomplex

[0185] TJ-300 prepared in Preparation Example 1 (Zeta potential -49 mV at pH 7.0) was selected, and the solid content was adjusted to 0.1%, pH 7.0. Quercetin (1.0 mg / mL) was added, with a quercetin to TJ-300 volume ratio of 1:1. The mixture was shaken and incubated at 37°C (100 rpm) for 24 hours. The pH was adjusted to 8.0 using 0.1 M NaOH, and incubation was continued at 37°C (100 rpm) for another 24 hours. Using an equal concentration of quercetin (0.5 mg / mL) without TJ-300 nanocellulose as a control, the free radical scavenging activity of quercetin and its nanocellulose complex was detected using a superoxide anion radical assay kit.

[0186] The tests showed that after adjusting the pH of quercetin solution (0.5 mg / mL) to 8.0 with 0.1 M NaOH and incubating at 37°C for 24 hours, the superoxide anion radical scavenging activity of the complex decreased from the initial 83% to 16%. After co-incubating with TJ-300 for 24 hours, the superoxide anion radical scavenging activity of the complex decreased from the initial 87% to 79%. Under the same conditions, the superoxide anion radical scavenging activity of TJ-300 remained stable at 5-6%.

[0187] Example 7: Single cellulase sustained-release experiment

[0188] This embodiment refers to Example 5, the only difference being: in step (1), the composite cellulase is replaced by single cellulases of endonuclease, exonuclease and glucosidase respectively; in step (2), 1% TJ-300 (Zeta potential -49mV under pH 7.0) nanocellulose obtained in Preparation Example 1 is mixed with collagen solution; in step (3), enzyme is added to the collagen-nanocellulose complex prepared in step (2) at an amount of 500U / g of dry cellulose; the cumulative release of collagen, the cumulative release of glucose and the viscosity of the complex are detected after 24 hours; the remaining steps are the same as in Example 5.

[0189] The test results showed that after adding endonucleases, exonucleases, and glucosidase to hydrolyze the TJ-300 and collagen complex for 24 hours, the cumulative collagen release, cumulative glucose release, and complex viscosity were as follows: endonucleases: 53%, 3.5 mg, 9920 mP s; exonucleases: 16%, 0.52 mg, 13010 mP s; glucosidase: 8%, 0.22 mg, 14370 mP s.

[0190] Example 8: Wound Healing Experiment

[0191] Two ml of the type III recombinant collagen and QCNF complex (Col-III-QCNF) from Example 3 was spread evenly in a 6-well plate. A 20mm × 20mm piece of medical gauze was cut, immersed in Col-III-QCNF, and incubated at 4°C for 24 hours. After incubation, it was placed in a sterile petri dish and sterilized by UV irradiation for 15 minutes; this was the experimental group. QCNF nanocellulose-impregnated gauze prepared under the same conditions served as a blank control, untreated medical gauze as a negative control, and commercial collagen wound dressing (3M Promogran Prisma) as a positive control. The dressing was applied to a full-thickness skin defect (8mm in diameter) on the back of SPF-grade SD rats (20 male SPF-grade SD rats, 5-6 weeks old, weighing approximately 220-240 grams, and divided into 4 groups) to evaluate the material's effect on promoting skin wound healing. The dressings were changed and the wound diameter was measured on postoperative days 3, 7, 14 and 21. The wound healing rate was calculated and the results are shown in Table 8.

[0192] Table 8: Wound healing rate (%) of gauze impregnated with collagen-cellulose nanoparticles (QCNF) complex

[0193] Example 9: Preparation of fibronectin-cellulose nanocomplex and the effect of pH on the complex

[0194] (1) Prepare a 1 mg / ml fibronectin solution using a 50 mM Tris buffer solution at pH 7.0 (fibronectin was purchased from Sigma, with an isoelectric point (PI) of 5.5 and a Zeta potential of -30 mV at pH 7.0). Adjust the pH to 4.5 using 0.1 M HCl; at this pH, the Zeta potential of fibronectin is +25 mV.

[0195] (2) Take the 1% concentration of TJ-300 prepared in Preparation Example 1 (Zeta potential -49mV under pH 7.0 conditions), and adjust the pH to 4.5 using 0.1M HCl. Under this pH condition, the Zeta potential of TJ-300 is -37mV.

[0196] (3) Mix the fibronectin solution with TJ-300 at a mass ratio of 1:100 and vortex at high speed (2000-3000 rpm) for 5-10 minutes to obtain a solution containing the fibronectin-cellulose nanocomplex. Adjust the pH of the complex solution to 7.0 using 0.1M NaOH.

[0197] (4) Take the solution of fibronectin-nanocellulose complex prepared in step (3), centrifuge at 10000 rpm for 10 minutes, take the supernatant, and detect the content of free fibronectin.

[0198] (5) Take the unadjusted fibronectin solution (Zeta potential -30mV at pH 7.0) and TJ-300 (Zeta potential -49mV at pH 7.0) from steps (1) and (2), and perform the steps (3) and (4) to detect the free fibronectin content.

[0199] The test results showed that adjusting the pH of the system to below the isoelectric point of fibronectin before preparing the fibronectin-cellulose nanocomplex caused the zeta potential of fibronectin to change from negative to positive, thereby enhancing its binding with negatively charged nanocellulose. After the two formed the complex, adjusting the pH of the system back to neutral resulted in only a small amount (<5%) of fibronectin remaining free in the supernatant. Conversely, when the pH of the system was not adjusted before preparing the fibronectin-cellulose nanocomplex, the nanocellulose in the resulting complex only provided physical coating protection, and the binding was loose, with more than 35% of fibronectin remaining free in the supernatant.

[0200] The above description provides an exemplary account of the implementation methods of the technical solution disclosed herein. It should be understood that the scope of protection of this disclosure is not limited to the above-described embodiments. Any modifications, equivalent substitutions, or improvements made by those skilled in the art within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this application.

Claims

1. A composition, characterized in that, The composition comprises cellulose and an active substance, the active substance having a zeta potential or charge property opposite to that of the cellulose.

2. The composition according to claim 1, characterized in that, The cellulose forms a complex or compound with the active substance.

3. The composition according to any one of claims 1-2, characterized in that, The absolute value of the cellulose Zeta potential is 15-100mV, preferably 20-60mV, and more preferably 30-50mV; Alternatively, the absolute value of the Zeta potential of the active substance is 10-100mV, preferably 20-50mV, and more preferably 30-40mV.

4. The composition according to any one of claims 1-3, characterized in that, The sum of the absolute values ​​of the zeta potentials of the cellulose and the active substance is 25-200 mV, preferably 60-90 mV.

5. The composition according to any one of claims 1-4, characterized in that, The cellulose is nanocellulose, with a diameter of less than 100 nanometers, preferably less than 50 nanometers, and more preferably 5-20 nanometers.

6. The composition according to any one of claims 1-5, characterized in that, The cellulose is nanocellulose with an aspect ratio greater than 50, preferably greater than 100, and more preferably greater than 200.

7. The composition according to any one of claims 1-6, characterized in that, The cellulose includes modified cellulose, which is obtained by converting hydroxyl groups in cellulose into groups having positive or negative charges.

8. The composition according to claim 7, characterized in that, The modified cellulose is obtained by replacing the hydroxyl groups in cellulose with carboxymethyl, carboxyl, phosphoric acid or sulfonic acid groups to obtain a negatively charged modified cellulose; or by replacing the hydroxyl groups in cellulose with quaternary ammonium groups to obtain a positively charged modified cellulose.

9. The composition according to any one of claims 1-8, characterized in that, The active substances are selected from proteins, such as keratin, silk fibroin, collagen, or cell growth factors; and peptides, which include polypeptides, oligopeptides, and cyclic peptides according to molecular weight and structure, and signal peptides, neurotransmitter inhibitory peptides, carrier peptides, or enzyme inhibitory peptides according to function.

10. The composition according to any one of claims 1-9, characterized in that, The zeta potential of cellulose and active substances in the composition is controlled by adjusting the pH of the system, for example, by adjusting the pH in the range of 3-11.

11. A composition, characterized in that, The composition comprises cellulose and an active substance, wherein the active substance is a non-colloidal active small molecule compound, and the spatial structure of the active substance is stabilized by hydrogen bonding and physical encapsulation between the cellulose and the active substance; preferably, the cellulose forms a complex or compound with the non-colloidal active small molecule compound.

12. The composition according to claim 11, characterized in that, The cellulose thereon is the cellulose as defined in any one of claims 3-8; Alternatively, the non-colloidal active small molecule compound includes one or more of dasatinib, quercetin, fisetin, epigallocatechin gallate, proanthocyanidin C1, hydroquinone, arbutin, kojic acid, phenylethyl resorcinol, or nicotinamide.

13. The composition according to any one of claims 1-12, characterized in that, The composition further comprises cellulase, preferably the cellulase comprising at least one of endonuclease, exonuclease, and glucosidase.

14. The composition according to claim 13, characterized in that, The cellulase has one or both of the following conditions: (1) The amount of cellulase added is 100-500 U / g, based on the oven-dry weight of cellulose; (2) The cellulase has a filter paper enzyme (FPA) activity of 30-60 U / mg, preferably 40-50 U / mg.

15. The composition according to any one of claims 1-14, characterized in that, The surface adsorption rate of cellulose for the active substance in the composition is: 60% or more after 5 hours of adsorption, preferably 70% or more, more preferably 90% or more; or 85% or more after 10 hours of adsorption, preferably 95% or more, more preferably 98% or more.

16. A method for preparing the composition according to any one of claims 1-15, the method comprising: (S1) Prepare a solution of the active substance; (S2) Add cellulose solution to (S1) and mix. The preferred method for mixing is high-speed vortex oscillation; And also includes optional steps (S3) and (S4): (S3) Prepare cellulase solution and refrigerate for later use; (S4) Mix the cellulase solution from step (S3) with the solution of the composition obtained from step (S2); preferably, the mixing is carried out by high-speed vortex oscillation.

17. The method according to claim 16, characterized in that, The cellulose is modified cellulose, and the preparation method of the modified cellulose includes: (SS1) The hydroxyl groups on the surface of cellulose are converted into groups with positive or negative charges; And also includes optional steps (SS2): (SS2) High-pressure homogenization is performed after step (SS1); Preferably, in step (SS1), the hydroxyl groups in cellulose are replaced with carboxymethyl, carboxyl, phosphoric acid, or sulfonic acid groups to obtain cellulose with a negative charge; or the hydroxyl groups in cellulose are replaced with quaternary ammonium groups to obtain cellulose with a positive charge.

18. The method according to claim 16 or 17, characterized in that, In step (S1), the concentration of the active substance in the solution is 0.01-25 wt%, preferably 0.1-5 wt%, such as 1.0 wt%. Alternatively, step (S1) may further include adjusting the pH of the solution of the active substance after preparation, for example, adjusting the pH range to between 3 and 11. Alternatively, step (S2) may further include adjusting the pH of the cellulose solution before mixing, for example, adjusting the pH to a range of 3-11; Alternatively, step (S2) may further include adjusting the pH of the system of the formed complex or compound after mixing, adjusting the pH to the range of 5-11, such as adjusting the pH to 7.0 or 8.0; Alternatively, the high-speed vortex oscillation described in step (S4) has a rotation speed of 2000-3000 rpm and an oscillation time of 5-10 minutes.

19. The use of the composition according to any one of claims 1-15 in daily chemical products, food, beverages, special medical purpose formula foods, health products or pharmaceutical preparations, such as in hair care, skin care, cosmetics or drugs for repairing damaged skin, healing wounds, and treating skin diseases.

20. A method for hair care, skin care, repairing damaged skin, healing wounds, or treating skin diseases, said method comprising administering to a patient in need the composition of any one of claims 1-15.

21. A daily chemical product, food, beverage, special medical purpose formula food, health product or medicine, comprising the composition according to any one of claims 1-15.

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