New method of synthesis of intramolecular cross-linking chitosan and derivative

Abstract

This invention relates to a novel cross-linked polyglucosamine sugar, its formulations, components, and uses. Specifically, this invention relates to a plurality of nanoparticles that can be used as active agents, their components, and a delivery system for at least one bioactive agent.

Description

Technical Field

[0001] This invention relates to the preparation of polyglucosamine, particularly to multiple nanoparticles obtained from polyglucosamine, their formulations, compositions and uses, in detail in the fields of chemistry, biology, medicine and materials science. Prior Technology

[0002] Biopolymers are promising materials for carrying various drugs and other active substances due to their biocompatibility, biodegradability, and non-toxicity upon administration. With suitable chemical modification, these polymers can provide better materials for drug delivery systems, where the active substance can be either a low-molecular-weight compound (small molecule) or a high-molecular-weight compound (macromolecule). Nanostructured drug carriers allow the delivery of macromolecular compounds such as nucleic acids and proteins (Advances in Polymer Science. Chitosan for Biomaterials, Vol. I, eds.: R. Jayakumar, M. Prabaharan, RAA Muzzarelli. Springer Heidelberg Dordrecht, London, New York 2011, DOI 10.1007 / 978-3-642-23114-8).

[0003] Polyglucosamine is one of the most promising biopolymer candidates for generating multiple nanoparticles. On the one hand, it possesses remarkable natural properties such as mucosal adhesion, biocompatibility and low toxicity, biodegradability and ability to form complexes; on the other hand, its highly regular structure and the presence of hydroxyl and amino groups offer good prospects for selective modification of this polymer.

[0004] Therefore, these properties of polyglucosamine have attracted attention for the development of drug delivery solutions (Bhattarai et al., 2010, Advanced Drug Delivery Reviews, 62 83-99. doi: 10.1016 / j.addr.2009.07.019).

[0005] Although polyglucosamine exhibits several advantageous characteristics as an excellent carrier, the use of unmodified polyglucosamine is limited by its low solubility under physiological conditions. To overcome this limitation, many different chemical modification methods for polyglucosamines have been developed (Kritchenkov et al., 2017, Russ. Chem. Rev., 86, 231-239. http: / / dx.doi.org / 10.1070 / RCR4636; Chuan et al., 2019, Adv. Colloid Interface Sci., 268, 25-38; Jiang, H.-L.; Xing et al., 2018, Curr. Org. Chem., 22, 668-689. DOI: 10.2174 / 1385272821666170926163544; Layek, B.; Singh, J. 8-Chitosan for DNA and gene therapy. In Chitosan Based Biomaterials Vol. 2; Jennings, JA, Bumgardner, JD. Edited by Woodhead Publishing: Cambridge, UK, 2017; pp. 209-244.

[0006] The structure of polyglucosamines offers the possibility of modification in various ways. To increase hydrophobicity, polyglucosamine glycoamines can be readily prepared from fatty acids, sterol derivatives, urocanic acid, and carboxylic acid derivatives containing imidazole fragments. Alternatively, secondary polyglucosamine glycoamines can be formed by alkyl substituents of various structures, pyridine derivatives, spermidine, and analogs via appropriate Schiff base alkylation or reductive amination. To increase hydrophilicity, polyglucosamine glycoamines can be produced from amino acids including sulfur-containing amino acids, lactobionic acid, thioglycolic acid, etc. Furthermore, secondary amines of polyglucosamines can be obtained from various sugars, polyethyleneimine, and their derivatives. Polyethylene glycol derivatives are particularly commonly synthesized via polyethylene glycolation. Polyglucosamine derivatives prepared according to these methods form multiple nanoparticles, especially with polyelectrolytes such as nucleic acids, exhibiting enhanced ability to form multiple nanoparticles and modified pharmacological properties. (Mao et al., 2010, Advanced Drug Delivery Reviews, 62, 12-27 doi:10.1016 / j.addr.2009.08.004 ).

[0007] Multiple nanoparticles of polyglucosamine can be formed via chemical cross-linking in a substantially irreversible manner, involving the use of various reagents to catalyze the formation of covalent bonds (Bhattarai et al., 2010, cf.). Historically, glutaraldehyde and formaldehyde were the first and most commonly used reagents for this cross-linking, but they are no longer commonly used today due to the difficulty in removing all traces of these toxic compounds and the slow hydrolysis of the multiple nanoparticles that release these aldehydes. Recently, Genepine has received considerable attention, but this reagent is extremely expensive and prone to polymerization itself. Diethyl squarate (DES), ethylene glycol diglycidyl ether (EGDE), and capped diisocyanates react rather slowly, and subsequent reactions are mainly carried out at high temperatures, which may cause side reactions. Photoactivated cross-linking reagents, such as functionalized azides and functionalized acrylates, and enzyme-activated reagents, such as phloretic acid and activated quinones, are also available. For example, Baoqiand et al., 2015, Acta Biomaterialia, 22, 1742-7061, or Journal of Nanotechnology in Engineering and Medicine, 6, 041001-6, provide a method for manufacturing cross-linked polyglucosamines. This method involves reacting polyglucosamines with methacrylic anhydride, followed by photo-initiated free radical polymerization of the polyglucosamines in solution form to produce cross-linked polyglucosamines, thereby generating polyacrylamide compounds in gel or foam form. Carboxymethyl derivatives of polyglucosamines are described in El-sherbiny et al., 2009, European Polymer Journal 45, 1, 199-210, in which, in a first step, carboxyl groups are introduced into the polyglucosamines by reacting a primary alcohol group in the polyglucosamine with monochloroacetic acid in an alkaline medium, and in a second stage, an acrylonitrile-glycine polymer is added via photo-induced polymerization to produce polyglucosamines without internal cross-linking. US 5,770,712 describes a method for manufacturing cross-linked polyglucosamine by combining a polyglucosamine material having an unreacted primary amino group with an excess of a polyfunctional epoxy compound having at least two epoxy groups. The polyglucosamine undergoes a cross-linking reaction with 1,4-butanediol diglycidyl ether to yield amino-ethanol cross-linked derivatives of the polyglucosamine, which exhibit poor solubility at physiological pH values ​​(pH 7 and higher).

[0008] It can also crosslink pre-derived polyglucosamine by forming a Schiff base with polyglucosamine containing aldehyde groups, forming a disulfide bridge with polyglucosamine pre-functionalized with a reagent having terminal thiohydrogen groups, and undergoing a Michael addition reaction with polyglucosamine pre-treated with functional acrylates in the presence of a weak base.

[0009] All such methods involve the formation of multiple nanoparticles from polyglucosamine in solution. In some cases, all chemical reactions are driven to occur in the liquid phase, while in others, these reactions are completed later, such as during or after spray drying or lyophilization. Importantly, in all cases, the method begins and its critical stage is carried out with polyglucosamine in a dissolved state (Jayakumar et al., 2011, cf.). The requirement for the amount of polyglucosamine to be dissolved significantly limits the synthesis of multiple nanoparticles. This is due to the fact that polyglucosamine dissolves in water only in the form of a salt in the presence of acid. In this form, for any solution containing more than 2% polyglucosamine, the viscosity increases anomalously with increasing concentration, a figure that severely limits possible synthetic implementations and the possibility of obtaining high yields from the reaction apparatus. Specifically, the wall effect that occurs when mixing viscous solutions can cause significant non-uniformity of reagent concentration in the reaction vessel, and thus lead to non-uniformity of the properties of the multiple nanoparticles. Another emerging issue is that highly nucleophilic amine groups are most commonly used for cross-linking, but in solution, they should be ionized to keep the polyglucosamine sugars in a dissolved state.

[0010] Therefore, there is a need to develop new synthetic routes for forming multiple nanoparticles of polyglucosamine in an efficient and cost-effective manner. Summary of the Invention

[0011] This invention relates to a novel method for the unexpected production of multiple nanoparticles from polyglucosamine. This method eliminates the need for dissolving the polyglucosamine starting material, which necessitates the formation of salts or the use of dilute acid solutions, or for dispersing the polyglucosamine in solution, which requires emulsification, grinding, or other procedures that could mechanically affect the integrity of the polymer. Specifically, this invention relates to a novel method for crosslinking polyglucosamine, wherein new internal chemical bonds are formed between different portions of the polyglucosamine molecule via two chemical steps. In a particular case, the elimination of the need to dissolve the polyglucosamine results in a more efficient synthetic conversion rate (e.g., approximately two orders of magnitude higher than crosslinking methods using dissolved polyglucosamine).

[0012] The resulting product spontaneously forms multiple nanoparticles at the end of the second step of the preparation method, and these nanoparticles are suitable for a wide range of applications. Furthermore, it is advantageous that the resulting cross-linked polyglucosamine product exhibits similar bioactivity to standard linear or branched polyglucosamines, but unexpectedly displays some unique advantages in terms of stability and physicochemical properties under a wide range of biologically relevant pH or microbial conditions, making it easier to process.

[0013] One aspect of the present invention provides a method for preparing cross-linked polyglucan according to the present invention.

[0014] In another embodiment, the present invention relates to a novel cross-linked polyglucosamine, particularly a novel cross-linked polyglucosamine that spontaneously forms a plurality of nanoparticles.

[0015] Another aspect of the present invention relates to a composition comprising at least one cross-linked polyglucan according to the present invention and at least one carrier.

[0016] Another aspect of the present invention relates to a pharmaceutical composition comprising at least one cross-linked polyglucan according to the present invention and at least one pharmaceutically acceptable carrier.

[0017] Another aspect of the present invention relates to a plurality of nanoparticles comprising a cross-linked polyglucan according to the present invention.

[0018] Another aspect of the present invention relates to a cosmetic composition comprising at least one cross-linked polyglucan according to the present invention.

[0019] Another aspect of the present invention relates to a soft tissue filler comprising at least one cross-linked polyglucan according to the present invention or a composition thereof according to the present invention.

[0020] Another aspect of the present invention relates to a wound dressing comprising at least one cross-linked polyglucan according to the present invention.

[0021] According to another specific embodiment, a reconstructed tissue is provided, comprising at least one cross-linked polyglucan or a composition thereof according to the present invention.

[0022] Another aspect of the present invention relates to an agricultural composition comprising at least one cross-linked polyglucan according to the present invention.

[0023] Another aspect of the present invention relates to a method for preparing a composition (e.g., a soft tissue filler, a wound dressing, or a reconstructive tissue) comprising at least one cross-linked polyglucan or a plurality of nanoparticles thereof according to the present invention.

[0024] Another aspect of the present invention relates to cross-linked polyglucosamine sugars according to the present invention for use in in vivo drug delivery, in vitro cell or biological tissue cultures, and tissue engineering modifications.

[0025] According to another specific embodiment, a cell or biological tissue culture medium is provided, which contains at least one cross-linked polyglucan or a composition thereof according to the present invention.

[0026] Another aspect of the present invention relates to the cross-linked polyglucosamine according to the present invention for the prevention and / or treatment of medical conditions, particularly cardiovascular conditions such as arrhythmias, especially atrial fibrillation and hypertension; joint diseases and joint disorders such as rheumatoid arthritis, osteoarthritis, spondyloarthritis, and traumatic events resulting in damage to cartilage, bone, ligaments, or bursae; eye diseases and injuries such as dry eye syndrome, uveitis, glaucoma, and corneal diseases; connective tissue diseases such as lupus and polymyositis; skin / mucous membrane diseases or injuries such as trauma, scars, psoriasis, acne, eczema, rosacea, physical or chemical burns, especially surgical trauma and sunburn, ulcers, and hemorrhoids; periodontal and dental diseases; dura mater injuries such as accidental injuries or dura mater injuries following brain and central nervous system surgery; malignant and benign tumors, especially carcinomas, sarcomas, lymphomas, and melanomas; postoperative complications such as fistulas and infections; tumors or vascular malformations.

[0027] Another aspect of the present invention relates to the use of the cross-linked polyglucosamine sugar according to the present invention for the preparation of pharmaceutical formulations for the prevention and / or treatment of medical conditions, specifically cardiovascular conditions such as arrhythmias, particularly atrial fibrillation and hypertension; joint diseases and lesions such as rheumatoid arthritis, osteoarthritis, spondyloarthritis, and traumatic events resulting in damage to cartilage, bone, ligaments, or bursae; and eye diseases and injuries such as dry eye syndrome, uveitis, glaucoma, and corneal opacity. Membrane lesions; connective tissue diseases, such as lupus and polymyositis; skin / mucous membrane diseases or injuries, such as trauma, scars, psoriasis, acne, eczema, rosacea, physical or chemical burns, especially surgical trauma and sunburn, ulcers, hemorrhoids; periodontal and dental diseases; dura mater injuries, such as accidental injuries or dura mater injuries after brain and central nervous system surgery; malignant and benign tumors, especially carcinomas, sarcomas, lymphomas and melanomas; postoperative complications, such as fistulas and infections; tumors or vascular malformations.

[0028] Another aspect of the present invention relates to the use of the cross-linked polyglucosamine sugar according to the present invention for the preparation of cells or biological culture media or for the reconstruction of tissues.

[0029] Another aspect of the present invention relates to a method for a drug delivery system for preparing bioactive agents.

[0030] According to another specific embodiment, a method is provided for identifying cross-linked polyglucosamine sugars that can be obtained by the method according to the invention.

[0031] Another aspect of the invention relates to a method for preventing, treating, or improving medical conditions, specifically cardiovascular conditions such as arrhythmias, particularly atrial fibrillation and hypertension; joint diseases and lesions such as rheumatoid arthritis, osteoarthritis, spondylitis, and traumatic events resulting in damage to cartilage, bone, ligaments, or bursae; eye diseases and injuries such as dry eye syndrome, uveitis, glaucoma, and corneal diseases; connective tissue diseases such as lupus and polymyositis; and skin / mucous membrane diseases or injuries, etc. Such as trauma, scars, psoriasis, acne, eczema, rosacea, physical or chemical burns, especially surgical trauma and sunburn, ulcers, hemorrhoids; periodontal and dental diseases; dura mater injuries, such as accidental injuries or dura mater injuries after brain and central nervous system surgery; malignant and benign tumors, especially carcinomas, sarcomas, lymphomas and melanomas; postoperative complications, such as fistulas and infections; tumors or vascular malformations, the method comprising administering to an individual in need an effective amount of at least one cross-linked polyglucosamine sugar or a pharmaceutical formulation thereof according to the invention.

[0032] Another aspect of the present invention relates to a kit comprising at least one cross-linked polyglucan or a composition thereof of the present invention, for example in lyophilized form.

[0033] Another aspect of the present invention relates to a kit for preparing a plurality of nanoparticles comprising at least one cross-linked polyglucosamine sugar or a composition thereof of the present invention, wherein the nanoparticles are used for encapsulating materials, such as bioactive agents, pharmaceutical substances, proteins, antibodies, sugars, nucleic acids or combinations thereof, or for prevention or treatment. Simple Explanation of the Diagram

[0034] [Figure 1] shows the characterization of a plurality of nanoparticles of the cross-linked polyglucosamine of the present invention (Example 2i). A: Micrograph of stained polyglucosamine (3.2% cross-linked 501 kDa polyglucosamine) as described in Example 3; [B]: Comparative linear polyglucosamine (B1) and a plurality of nanoparticles of the cross-linked polyglucosamine of the present invention (B2), and atomic force microscopy examination of the dried film of the plurality of nanoparticles of the cross-linked polyglucosamine of the present invention at high magnification (B3) and low magnification (B4). [Figure 2] shows the internalization of multiple nanoparticles of the cross-linked polyglucosamine of the present invention (Example 2i) in mouse leukocytes as measured by fluorescence microscopy, as described in Example 4. [A]: White blood cell boundary; [B]: Nuclear DNA (Hoechst 33258, strong blue fluorescence); [C]: Endocytinated FITC-labeled cross-linked polyglucosamine (green fluorescence); [D]: Mixed blue / green fluorescence (blue: background nonspecific and / or nonnuclear DNA in the cytoplasm; green: endocytosed FITC-labeled cross-linked glucosamine). [Figure 3] shows a plurality of cross-linked polyglucosamine nanoparticles of the present invention in the cornea of ​​a mouse, as described in Example 4, measured by FITC-FAM fluorescence (Example 2i). [B]) internalization and the retention of standard polyglucosamine (PGA) on the corneal surface Comparison of [A] ). A: Cornea; B: Polyglucosamine (bright green fluorescence). [Figure 4] shows the mass spectrometry analysis of the comparative non-crosslinked standard polyglucosamine (A1: total ion current of positive ions and (A2: ion region of interest (lower m / z ratio)) as described in Example 3; and the crosslinked polyglucosamine of the present invention (B1: total ion current of positive ions and B2 (ion region of interest (lower m / z ratio))). [Figure 5] shows the collision studies of the compound with m / z = 180 at impact energies of 5 V (A) and 15 V (B) and the compound with m / z = 252 at impact energies of 5 V (C) and 15 V (D) as described in Example 3, and the spectra obtained by hydrolysis by ovomycozyme (E). [Figure 6] illustrates the germination process of spring wheat seeds in a germination chamber treated with the cross-linked polyglucosamine of the present invention (0.1% w / w or 0.05% w / w in water) as described in Example 6, compared to the control group. [(A)] (Root emergence was measured 1 day later, germination energy was measured 3 days later, and germination capacity was measured 7 days later) and the percentage increase in aboveground growth of spring wheat seedlings in soil substrate relative to the control group. The effect of [(B)] (treating seeds 7 days before sowing). Implementation

[0035] As used herein, the term "degree of crosslinking" refers to the percentage of the number of functional groups that have been converted into crosslinks or grafts to the total number of functional groups that were originally present on the polyglucosamine.

[0036] The term "alkyl," when used alone or in combination with other terms, includes straight-chain or branched C1-C50 alkyl groups, referring to monovalent alkyl groups having 1 to 50 carbon atoms. Examples of such groups include: methyl, ethyl, n-propyl, isopropyl, n-butyl, dibutyl, isobutyl, tributyl, n-pentyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, tetrahydrogeranyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecanyl, n-hexadecyl, n-octadecyl, n-nonadecanyl, and n-eicosyl, and similar groups. Preferably, these groups include C1-C9 alkyl groups, more preferably C1-C6 alkyl groups, and even more preferably C1-C4 alkyl groups. Similarly, these groups refer to monovalent alkyl groups having 1 to 9 carbon atoms, monovalent alkyl groups having 1 to 6 carbon atoms, and monovalent alkyl groups having 1 to 4 carbon atoms, respectively. Specifically, these groups include C1-C6 alkyl groups.

[0037] The term "alkenyl," when used alone or in combination with other terms, includes a straight-chain or branched C2-C50 alkenyl group. It may have any number of double bonds in any available position, and the configuration of the double bonds may be (E) or (Z). This term uses groups such as vinyl, allyl, isopropenyl, 1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-ethyl-1-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 1-octenyl, geranyl, 1-decenyl, 1-tetradecenyl, 1-octadecenyl, 9-octadecenyl, 1-eicosenoyl, and 3,7,11,15-tetramethyl-1-hexadecenyl, and similar groups. Preferably, these groups include C2-C8 alkenyl groups, more preferably C2-C6 alkenyl groups. Among them, vinyl (-CH=CH2), n-2-propenyl (allyl, -CH2CH=CH2), isopropenyl, 1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl and 3-methyl-2-butenyl, and similar groups are particularly preferred.

[0038] The term "alkynyl," when used alone or in combination with other terms, encompasses a straight-chain or branched C2-C50 alkynyl group. It can have any number of carbon atoms in any available position. This term takes, for example, alkynyl, which can have 2 to 50 carbon atoms and, where appropriate, double bonds, such as ethynyl (-C≡CH), 1-propynyl, 2-propynyl (propynyl:-CH₂C₅), etc. [≡] CH), 2-butynyl, 2-penten-4-ynyl and similar groups. Specifically, these groups include C2-C8 ynyl groups, more preferably C2-C6 ynyl groups and similar groups. More preferably, these groups include C2-C6 ynyl groups, which refer to groups having 2 to 6 carbon atoms and having at least 1 or 2 ynyl unsaturated sites.

[0039] The term "aryl" refers to an unsaturated aromatic carbocyclic group having 6 to 14 carbon atoms and a single ring (e.g., phenyl) or multiple condensed rings (e.g., indene, naphthyl). Aryl groups include phenyl, naphthyl, anthracene, phenanthrene, and similar groups.

[0040] The term "C1-C6 alkylaryl" refers to an aryl group having C1-C6 alkyl substituents, including methylphenyl, ethylphenyl and similar groups.

[0041] The term "aryl C1-C6 alkyl" refers to C1-C6 alkyl groups having aryl substituents, including 3-phenylpropyl, benzyl and similar groups.

[0042] The term "heteroaryl" refers to a monocyclic heteroaryl group, or a bicyclic or tricyclic fused heteroaryl group. Specific examples of heteroaryl groups include, where appropriate, substituted pyridyl, pyrroloyl, pyrimidinyl, furanyl, thiophene, imidazole, etc. azole group, iso Azolyl, thiazolyl, isothiazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3- Diazolyl, 1,2,4- Diazolyl, 1,2,5- Diazolyl, 1,3,4- Diazolyl, 1,3,4-trisyl Base, 1,2,3-tri Benzofuryl, [2,3-dihydro]benzofuranyl, isobenzofuranyl, benzothiophenyl, benzotriazolyl, isobenzothiophenyl, indoleyl, isoindoleyl, 3H-indoleyl, benzimidazolyl, imidazo[1,2-a]pyridyl, benzothiazolyl, benzo[2,3-dihydro]benzofuranyl azole, quin α-hydroxyl group, quinazolinyl group, phthaloyl group basalt, quinolone phyllyl, phyllyl, Pyridyl, pyrido[3,4-b]pyridyl, pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl, quinolinyl, isoquinolinyl, tetrazolyl, 5,6,7,8-tetrahydroquinolinyl, 5,6,7,8-tetrahydroisoquinolinyl, purinyl, pteridinyl, carbazoleyl, Benzyl or benzoquinoline.

[0043] The term "C3-C8 cycloalkyl" refers to a saturated carbocyclic group having 3 to 8 carbon atoms and a single ring (e.g., cyclohexyl) or multiple condensed rings (e.g., norcamphenyl). C3-C8 cycloalkyl includes cyclopentyl, cyclohexyl, norcamphenyl, and similar groups.

[0044] The term "heterocyclic alkyl" refers to a C3-C8 cycloalkyl group as defined above, wherein at most three carbon atoms are replaced by heteroatoms selected from the O, S, and NR groups, and R is defined as hydrogen or methyl. Heterocyclic alkyl groups include nitrogen-containing heterocyclic butyl, pyrrolidinyl, piperidinyl, and piperazine. base, Linoyl, tetrahydrofuranyl and similar groups.

[0045] Unless otherwise defined by the definition of individual substituents, the term "substituted" means that a group is substituted by one to five substituents selected from the group consisting of: "C1-C6 alkyl", "C2-C6 alkenyl", "C2-C6 alkynyl", "C3-C8 cycloalkyl", "heterocycloalkyl", "C1-C6 alkylaryl", "C1-C6 alkylheteroaryl", "arylC1-C6 alkyl", "heteroarylC1-C6 alkyl", "C1-C6 alkylcycloalkyl", "C1-C6..." Alkyl heterocyclic alkyl, "amino", "aminosulfonyl", "ammonium", "acetylammonium", "aminocarbonyl", "aryl", "heteroaryl", "sulfinyl", "sulfonyl", "alkoxy", "alkoxycarbonyl", "carbamate", "hydrothio", "halogen", trihalomethyl, cyano, hydroxyl, mercapto, nitro and similar groups.

[0046] The term "medically acceptable" refers to a carrier that contains materials that are not biologically or otherwise undesirable and, in particular, non-toxic.

[0047] The term "carrier" refers to any component present in a pharmaceutical formulation other than an active agent, and therefore includes diluents, binders, lubricants, disintegrants, fillers, colorants, wetting agents or emulsifiers, pH buffers, preservatives and the like.

[0048] The term "bioactive agent" is used to describe any biologically active agent to be incorporated into the grafted polymer composition of the present invention. It may be natural, synthetic, semi-synthetic, or derivatives thereof and may include hydrophobic, hydrophilic, soluble, and insoluble compounds. More precisely, it may be any bioactive agent that can be used to treat and / or prevent and / or diagnose conditions in mammals, such as animals and humans, particularly in humans, in any known therapeutic area, including but not limited to arrhythmias such as atrial fibrillation, hypertension; inflammatory conditions, symptoms, or diseases, particularly autoimmune and non-autoimmune inflammatory conditions, including joint diseases and arthropathy (such as rheumatoid arthritis, osteoarthritis, and spondylitis); and eye diseases (such as dry eye syndrome, acetaminophen, and spondylitis). Uveitis, glaucoma, corneal lesions), connective tissue diseases (such as lupus or polymyositis), skin diseases or injuries (such as trauma, scars, psoriasis, acne, eczema, rosacea, physical or chemical burns, sunburn), periodontal and dental diseases, hemorrhoids, ulcers, diseases suitable for gene therapy, diseases suitable for cell therapy, spinal dura mater repair after brain and central nervous system surgery, neuroplastic surgery, pain, postoperative fistulas and infections, orthopedic diseases, benign and malignant tumors and infections. These bioactive agents can be selected from macromolecular or small molecule compounds, such as peptides, proteins, oligonucleotides and polynucleotides, anti-infectives, antibiotics, antimicrobials, antivirals, antibacterial agents, antifungals, growth factors, enzymes, antigenes, antitumor drugs, anti-inflammatory drugs, anesthetics, antitumor drugs, analgesics, anticoagulants, hemostatic agents, cells and antibodies.

[0049] The term "inflammatory condition or disease" refers to all diseases that involve inflammation and the release of pro-inflammatory cytokines as a major or important disease mechanism, such as inflammatory bowel disease, allergies, asthma, autoimmune diseases, hepatitis, inflammation of other organs, and related diseases.

[0050] The term "dermatitis" or "skin disease" includes skin lesions that present with sore depression and may not have cuts on their surface, such as age-related tissue damage (e.g., wrinkles), trauma, and scarring, such as acne or urticaria scars. These conditions further include trauma, scarring, psoriasis, acne, eczema, and rosacea. The term "trauma" includes any damaged tissue, such as tissue damaged after injury or surgery. Trauma in mammals includes, for example, abrasions, lacerations, concussions, puncture wounds, cuts, surgical wounds, gunshot wounds, heat injuries, chemical wounds, sunburns, bites and punctures, and electric shocks. It further includes chronic skin conditions such as ulcers and other inflammatory skin conditions.

[0051] The term "eye disease or condition" refers to any condition or injury affecting the eye, particularly the cornea. Such conditions include corneal abrasions, corneal irritation, corneal alkali burns, age-related macular degeneration, exophthalmos, cataracts, cytomegalovirus retinitis, color blindness, strabismus, diabetic macular edema, floaters and flashes of light, glaucoma, keratoconus, amblyopia, low vision, ocular hypertension, retinal detachment, blepharospasm, uveitis, keratoconjunctivitis dry (KCS), and dry eye syndrome.

[0052] The term "articular or joint disease" includes osteoarthritis, arthritis pain, rheumatoid arthritis, infection and inflammatory pain, cartilage injury to the knee, and traumatic events that cause damage to the hip joint, bones, ligaments, or bursae.

[0053] The term "reconstructive tissue" refers to biological tissue (endogenous or exogenous) or synthetic or semi-synthetic materials that can be used to repair damaged tissues in the body, such as epidermis, nerves, cartilage or bone tissue, and can be used to construct organoids for biotesting, as described in Takebe et al., 2019, Science, 364, 6444, 956-959, DOI:10.1126 / science.aaw756.

[0054] As used herein, "treatment" and similar expressions generally mean achieving a desired pharmacological and / or physical and / or physiological effect. This effect may be preventative in relation to the prevention or partial prevention of a disease, its symptoms, or condition, and / or therapeutic in relation to the partial or complete cure of a disease, the condition, symptoms, or side effects caused by that disease. As used herein, the term "treatment" covers any treatment of a disease in mammals, particularly humans, and includes: (a) preventing the development of the disease in individuals who may be susceptible but have not yet been diagnosed with it; (b) suppressing the disease, i.e., halting its development; or alleviating the disease, i.e., reducing the disease and / or its symptoms or condition, such as improving or repairing damage.

[0055] As used herein, the term "individual" refers to a mammal. For example, the mammals covered by this invention include humans, primates and domesticated animals such as cattle, sheep, pigs, horses and especially racehorses, laboratory rodents and similar animals.

[0056] The term "efficacy" of a treatment according to the invention can be measured based on changes in response to the use of the invention during the course of a disease. For example, the efficacy of a treatment according to the invention can be measured by the disappearance or reduction of clinical signs or symptoms, or by more favorable measurements of disease biomarkers, such as interleukins, growth factors or other signaling molecules and their receptors, cell surface markers, cell counts, and gene expression profiles. [ Method for preparing polyglucosamine sugar according to the present invention and characterization of the polyglucosamine sugar , ] [ , ]

[0057] A method for preparing cross-linked polyglucosamine sugars is provided based on a specific sample, comprising the following steps: [a)] Provide a polyglucosamine sugar and swell the polyglucosamine sugar in a solvent; [b)] The polyglucosamine sugar is amino-ylated using an acrylic compound of formula (I): [Chemical Formula (I)] R1 is a halogen or any other leaving group that ensures aminolation upon removal, such as 3-hydroxybenzotriazole ester, anhydride (including mixed anhydrides), N-hydroxysuccinimide, pentachlorophenol, 2-nitro-4-sulfonylphenol ester, and other similar leaving groups; R2, R3, and R4 are independently selected from H, substituted alkyl groups (e.g., C1-C6 alkyl), substituted alkenyl groups (e.g., C2-C6 alkenyl), substituted alkynyl groups (e.g., C3-C6 alkynyl), substituted cycloalkyl groups (e.g., C3-C8 cycloalkyl), substituted cycloalkenyl groups (e.g., C4-C8 cycloalkenyl), substituted cycloalkynyl groups (e.g., C5-C8 cycloalkynyl), substituted heterocycloalkyl groups; substituted aryl groups (e.g., substituted phenyl groups), substituted heteroaryl groups, and substituted aryl C1-C6 groups. Alkyl, particularly benzyl; wherein the term "substituted" means that the group is substituted with 1 to 5 substituents selected from the group consisting of: halogen, -COOR', -NR'R'', =O, -OR', -COR', -CONR'R'', -SR', -SO3 R', -SO2 NR'R'', -SOR', -SO2 R', -NO2 or -CN; or R1 with R2 or R1 with R3, or R1 with R4 to form, as appropriate, a substituted 4-24 member aryl, heteroaryl, cycloalkyl or heterocycloalkyl (e.g., 6-24 member aryl, heteroaryl, cycloalkyl or heterocycloalkyl, such as, as appropriate, a substituted 8-24 member aryl, heteroaryl, cycloalkyl or heterocycloalkyl); [c)] make [step] [b)] The acetylation product reacts in the presence of a base (Aza-Michael reaction); [d)] Purification from [step] [c)] The obtained cross-linked polyglucosamine sugar (e.g., purified from salt impurities and / or aprotic solvents).

[0058] According to a particular embodiment, in [step] [a)] and / or The solvents used in [b)] are protic solvents (such as alcohols or water).

[0059] According to a particular embodiment, in the process of [step] [c)] Before, the reaction mixture was washed away when... [step] [a)] and / or [b)] Free acrylic acid is formed when the method is carried out using a protic solvent (such as alcohol or water).

[0060] According to another specific embodiment, the solvent is an aprotic solvent, particularly... [step] [a)] and / or [b)] and / or The same applies to [c)].

[0061] According to a particular embodiment, if in [step] [c)] uses an aprotic solvent, if no further action is taken. [step] [f)] Subsequent vinification.

[0062] According to a particular embodiment, [step] [a)] was performed at room temperature.

[0063] According to a particular embodiment, [step] [b)] The R3 or R4 and R2 groups in the acetylated product will react with the primary amino groups of the glucosamine backbone to form crosslinks between the glucosamine sugars. The reaction of these groups will depend on the specific acrylic compound. For example, for acrylic acid and methacrylic acid, the group reacting with the primary amino groups of the glucosamine backbone is group R3 or R4. However, when the R3 and R4 groups are substituted with halogen atoms, the R2 group will react with the primary amino groups of the glucosamine backbone.

[0064] According to another specific embodiment, this crosslinking results in the formation of a plurality of nanoparticles.

[0065] Depending on the specific state, the method of the present invention can be represented by the following scheme 1: [plan] [1] The first step involves providing polyglucosamine sugars that swell in a nonprotic solvent at room temperature. [(A)], where m is an integer between 1 and 12,500 and n is an integer between 1 and 12,500, and then acrylic compounds are used. [(I)] Amination of the polyglucosamine sugar, and then, the resulting amination product. [(B1)] reacts in the presence of a base to produce cross-linked polyglucan. [(B2)], the cross-linked polyglucosamine can then be purified and separated to obtain purified cross-linked polyglucosamine according to the present invention.

[0066] According to a specific embodiment, polyglucosamine can be provided in a swollen state in a protic solvent or even in a dissolved state. However, in this case, hydrolysis of the amide may occur as a side reaction, and this must be taken into account when calculating the reaction load. Additionally, in this case, [step] [c)] Before the azira-Michael reaction stage, washing is required. [step] [b)] The obtained acetylated polyglucosamine is used to remove the hydrolysis products of the acetylation agent.

[0067] According to one specific embodiment, the polyglucosamine system is provided in the absence of anhydrous substances, and the acetylation step is carried out in the absence of anhydrous substances. The presence of anhydrous substances advantageously results in higher yields and avoids the formation of byproducts.

[0068] According to a particularly advantageous embodiment, the acylation step is carried out in the absence of anhydrous substances. In this case, an aprotic solvent can be used as the reaction medium or a supercritical fluid. According to a particular embodiment, the aprotic solvent is a polar aprotic solvent, such as a solvent selected from DMF and DMSO. In a particular embodiment, the supercritical fluid can be a supercritical solvent, such as carbon dioxide, nitric oxide (I), or freon (chloro(bromine)(fluoro) hydrocarbon), which is used to provide the acylation agent to the reaction medium, and then the acylation reaction is carried out after the supercritical solvent is removed from the reaction medium, for example by reducing the pressure below a critical value.

[0069] According to another particularly advantageous approach, the acetylation step is carried out in an anhydrous, aprotic medium.

[0070] According to a specific state, the polar aprotic solvent is selected from dimethylformamide (DMF), dimethylacetamide, acetonitrile (MeCN), N-methylpyrrolidone, dimethyl sulfoxide (DMSO), or mixtures thereof.

[0071] Depending on another specific state, dichloromethane, dichloroethane, chloroform, and other chloro(fluorine) hydrocarbons can also be used as polar aprotic solvents, but this is not suitable for azira-Michael reactions. [step] [c)] In this case, the solvent should be distilled off just before the alkali is supplied. It is desirable to perform such distillation at a temperature not exceeding 60°C, which is suitable for most of the solvents mentioned at atmospheric pressure. If a high-boiling-point solvent is used, distillation must be carried out under reduced pressure.

[0072] According to another specific example, under anhydrous conditions, ethers, esters, and ketones can also be used as solvents for reaction steps a) to c), but the reaction will proceed more slowly in such solvents. For example, diethyl ether, diisopropyl ether, methyl tributyl ether, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, acetone, methyl ethyl ketone, and diethyl ketone can be used.

[0073] According to a specific state of the present invention, glazing [step] [b)] This can be carried out by any of the methods described in the case of acryloxychloroacetylation of glucose sugars (Zhang et al., 2017, Biomacromolecules, 1, 3, 778-786; Bu et al., 2017, Advances, 7, 76, 48166-48175) or by any of the methods described that can be used to acetylate amino groups, such as using i) carbodiimide; ii) azide; iii) mixed anhydride; iv) activated ester and v) other methods described below.

[0074] In step b), a known method of acetylation using carbodiimide to form an intermediate enol ester can be used (WO 2019 / 60740; Hao-Bin et al., 2018, Carbohydrate Polymers, 196, 359-367). If N,N'-dicyclohexylcarbodiimide (DCC) can be used as a condensing agent, then 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride and N-cyclohexyl-N'-(2-N- Methyl p-toluenesulfonate (CAS Registry No.: 2491-17-0) is preferred.

[0075] In step b), known acetylation methods using azides can be used (Honzl et al., 1961, Coll. Czech. Chem. Commun., 26, N. 9, 2333-2344).

[0076] In step b), known yation methods using mixed anhydrides can be used (Wieland et al., 1951, Ann. Chem., 572, N3, 190-194; Belleau et al., 1968, J. Amer. Chem. Soc., 90, N6, 1651-1652; Gorecka et al., 1978, Synthesis, N6, 474-476; Diago-Meseguer et al., 1980, Synthesis, N7, 547-551; Leplawy et al., 1960, Tetrahedron, 11, N1, 39-51). Alkylation can also be achieved using anhydrides, such as maleic anhydride (Liwschitz et al., 1957, Journal of the Chemical Society, 4399; Kang et al., 2014, Bioorganic and Medicinal Chemistry Letters, 2, 10, 2364-2367; US 2016 / 200730; Sanchez et al., 2010, Anna European Journal of Organic Chemistry, 13, 2600-2606).

[0077] In step b), known methods of acylation via the formation of activated amides using activated esters can be used, such as the carbonyl diimidazole method (Paul et al., 1960, J. Amer. Chem. Soc., 82, N 17., 4596-4600), the cyanomethyl ester method (Schwyzer et al., 1955, Helv. Chim. Acta, 38, N 1, 80-83), the phenyl thioester method (Wieland et al., 1951, cf.), the substituted phenyl ester method (Gross et al., 1983, Mayenhofer ed., Moscow: Mir., p. 421), and the ester method using heteroaromatic compounds combined with carbodiimide (Jakubke et al., 1966, A. Chem. Ber., 99, N 8, 2419-2429; Taschner et al., 1965, Ann.). (Chem., 690, 177-181), the ester method using hydroxylamine derivatives combined with the carbodiimide method (Losse et al., 1964, Ann. Chem., 678, 185-190; Nefkens et al., 1961, Amer. Chem. Soc., 83, N 5, 1263; Anderson et al., 1963, Ibid., 85, N 19, 3039; König et al., 1970, Chem. Ber., 103, N 3, 788-798), transesterification (a variation of the ester method) (Sakakibara, 1965, Bull. Chem. Soc. Jap., 38, N 1, 1979-1984; Fujino et al., 1968, Ch. Chem. Pharm. Bull., 16, N 5, 929-932; Gudkov et al., 1978, 48, 9, 2146; Devadas et al., 1979, Ind. J. Chem., B16, N 11, 1026-1027).

[0078] In step b), other known acetylation methods may be used, such as the ketoimine method (Stevens et al., 1958, J. Amer. Soc., 80, N 15, 4069-4071); the acetylene derivative method (Arens, 1955, Rec. Trav. Chim., 74, N 6, 759-770; Gais 1978, Aktivierungsmittel für Peptidsynthesen J. Angew. Chem. Int. Ed, 90(8), 625-626 https: / / doi.org / 10.1002 / ange.19780900808); the method using cyanamide derivatives (Losse et al., 1960, Ann. Chem., 636, 144-149); the method using isocyanamide derivatives. Synthesis by azole salts (Woodwart et al., 1961, J. Amer. Chem. Soc., 83, N 4, 1010-1012); synthesis using amide halides (Bergmann et al., 1936, J. Biol. Chem., 115, N 3, 93-611).

[0079] According to a particular embodiment, the acrylic compound of formula (I) may be an acid, an acid halide, an active ester (e.g., 3-hydroxybenzotriazole ester, N-hydroxysuccinimide, pentachlorophenol, 2-nitro-4-sulfonylphenol ester and esters having other similar leaving groups), an anhydride or a mixture thereof.

[0080] According to a specific embodiment, the acrylic compounds of formula (I) are selected from the group consisting of: (Angelic acid, CAS Registry No.: 565-63-9); (2-Isopropylacrylic acid, CAS Registry No.: 4465-04-7); (2-Methylenedodecanoic acid, CAS Registry No.: 52756-21-5); (4S,6S)-4,6-dimethyl-2-methyleneicosanoic acid; ((Z)-3-Cyclohexylacrylic acid, CAS Registry No.: 673456-32-1); ((E)-3-[4-(2,6-dioxy-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-bicyclo[2.2.2]oct-1-yl]-acrylic acid, CAS Registry No.: 340021-16-1); ((2E)-3-((E)-4-{[2-(4-chlorophenoxy)-2-methylpropionic acid}-1-adamantyl)acrylic acid; (3-(2-Carboxy-4-phenyl-3-styryl-cyclobutyl)acrylic acid, CAS Registry No.: 34271-87-9); (3-Methyl-2-acrylic acid-(1)-cyclopropane-carboxylic acid-(1)-carboxylic acid-(1)-ethyl ester, CAS Registry No.: 91971-88-9); (3t,4t-dicarboxy-cyclobutane-1,2c-bis-(trans-acrylic acid), CAS Registry No.: 55011-62-6); Coronadiene; (CAS Registration No.: 1247-53-6) ((E)-3-(2t-ethoxycarbonyl-6ξ-methoxy-cyclohexane-1r-yl)-acrylic acid, CAS Registry No.: 2960-11-4); cinnamic acid; 4-methylcinnamic acid; p-nitrocinnamic acid; caffeic acid; (3-(furan-2-yl)crotonic acid; 3-(2-furan-yl)acrylic acid, CAS Registry No.: 539-47-9); ((Z)-uric acid, CAS Registry No.: 7699-35-6); (3-(4-pyridinyl)-2-acrylic acid; 3-(pyridin-4-yl)acrylic acid, CAS Registry No.: 5337-79-1); argutinosides AI; ((Z)-2-chloro-but-2-enoic acid, CAS Registry No.: 53993-41-2); ((2Z)-2,3-dichloroprop-2-enoic acid, CAS Registry No.: 3533-68-4); cis-N-tert-butoxycarbonyl dehydrogenated β-alanine (CAS Registry No.: 151292-68-1) (2-Hydroxy-3-mercapto-acrylic acid, CAS Registry No.: 6228-60-0;) Hydroxy(meth)acrylic acid; (Dihydroxyacrylic acid, CAS Registry No.: 2702-94-5); (Reaxys ID 2721331); (CAS Registration No.: 64361-31-5); 3-(2-Carboxymethylene-cyclopentyl)acrylic acid; (CAS Registration No.: 78727-62-5); (Fluorobutylene acid, CAS Registry No.: 2365-87-9); ((E)-2,3-bis(phenoxycarbonylamino)but-2-eneic acid); ((Z)-2,3-bis(phenoxycarbonylamino)but-2-eneic acid); (2E,4E)-2,4-hexadienedia acid (muconic acid), maleic acid; trans-butenedia acid; (cis-Butenidylacetic acid, CAS Registry No.: 5698-52-2); (E)-4-(methylamino)-4-sideoxybut-2-enoic acid, an acrylic acid derivative of glutamic acid ; (2-Methylene-9(Z)-octadecenoic acid, CAS Registry No.: 33780-98-2); (2-Methylene-5-hexenoic acid (2-Methylene-5-hexenoic acid), CAS Registry No.: 73505-05-2); ((E)-6,9-dimethyl-2-methyleneundec-5,9-dienoic acid, CAS Registry No.: 1580541-76-9) (2-(cis-7,8-hexadecenyl)-acrylic acid); ((E)-2-methyleneoctyl-4,7-dienoic acid); (2-Methylene-7-octenoic acid, CAS Registry No.: 127559-93-7); and (2-Methylene-5-decynic acid, CAS Registry No.: 150254-20-9); and acetylated halides (R1 = halogen), especially acrylonitrile chloride; ( ) or methacrylic acid chloride.

[0081] According to another specific embodiment, acrylic compounds are acrylate halides (R1 = halogen), particularly acrylonitrile chloride (...). ) or methacrylic acid chloride.

[0082] According to a specific state, when glazing [step] [b)] When using acrylic compounds as acid halides, it is important to avoid the presence of water in the reaction system because acid halides can undergo undesirable hydrolytic side reactions, producing acrylic acid, which can lead to the carboxyl functionalization of multiple nanoparticles during the Michael addition reaction between free acrylic acid and the amino group of polyglucosamine.

[0083] According to a specific state, in acetylene [step] [b)] uses acrylic acid chlorides (e.g., acetylated chlorides). According to one approach, the use of acid chlorides advantageously induces the formation of hydrogen chloride, which does not interfere with the next reaction step in the presence of excess base and is readily removed as a salt during dialysis.

[0084] It is worth noting that acetylation of amines with carboxylic acid halides produces an equivalent amount of acid, which will form a salt with the unreacted amine and reduce the yield of the method of the present invention. Therefore, during acetylation... [step] [b)] When using acid halides, add an equivalent base, preferably a non-nucleophilic base (e.g., in...). [step] [b)] Triethylamine is used to neutralize this acid.

[0085] According to a specific state, when glazing [step] [b)] When using acrylic acid anhydrides, the method of the present invention further includes performing... [step] [c)] Prior to this, the reaction medium is washed and the formed acid is removed to avoid the presence of water in the reaction system. Specifically, the formed acid can be washed away from the acetylation product with a solvent by means of a filter, or by dialysis against the solvent, or by suspending the acetylation product in the solvent and centrifuging and discarding the supernatant.

[0086] According to another specific example, when acetylation is performed using a condensing agent (e.g., DCC) in the presence of free acid, careful control of the completeness of the reaction is required, and preferably, a washing step is still included as a precaution. For example, when a condensing agent such as DCC is used, byproducts such as dicyclohexylurea are formed during the acetylation reaction, which need to be removed from the reactants before proceeding to the next step to prevent them from participating in the formation of crosslinks to generate the plurality of nanoparticles of the present invention.

[0087] According to another specific embodiment, in [step] In [c)], the base system is sufficient to form an alkaline medium to neutralize the [substance / condition]. [step] [b)] The molar excess of hydrogen halide formed in the process. Typically, the molar excess of the base is at least 10% molar excess (e.g., about 20 to 40% molar excess), which will correspond to relative to [step] [b)] The amino groups of the resulting acetylated product are in excess by at least 1-3 moles.

[0088] According to another specific embodiment, in [step] The cross-linked polyglucan obtained in [c)] can be used in... [step] [d)] In this process, standard techniques are used to purify and remove salt impurities and / or residual aprotic solvents, such as by dialysis or washing with solvents such as water or dichloromethane, methanol, ethanol, acetone, and the like, followed by centrifugation or by using a supercritical solvent, such as carbon dioxide, insoluble in the plurality of nanoparticles formed by the cross-linked polyglucosamine, to remove the aprotic solvent, or by evaporation followed by washing the byproducts from the cross-linked polyglucosamine product with a solvent, for example, through a filter having pores of no more than 40 μm. According to a more specific embodiment, hydrophobic filter elements with fine porosity, such as polypropylene or PTFE, can be advantageously used because their hydrophobicity would create an additional barrier for the plurality of nanoparticles of the hydrophilic polyglucosamine to penetrate them.

[0089] According to another specific embodiment, in [step] The purified cross-linked polyglucan obtained in [d)] can be dried, for example, by another drying method chosen as appropriate. [step] [e)] is dried in the process.

[0090] According to a specific pattern [step] The base system used in [c)] for the Michael addition reaction is essentially a pure nonnucleophilic base, and in particular, it is essentially free of secondary amines.

[0091] According to a specific pattern, Michael's addition [step] [c)] This is carried out in a protic solvent, such as water. In this case, it is necessary to control the degree of saponification, for example by measuring the amount of carboxyl and amino groups formed during the saponification stage. Since the concentration of carboxyl groups is usually 1 to 2 orders of magnitude lower, and the difference in the number of amino groups is within the experimental error range, the accurate method for carboxyl groups will be NMR, IR spectroscopy or acid-base titration, such as those described below: Glazunov et al., 1999, 25(3), 216-219; Brugnerotto et al., 2001, Polymer, 42, 3569-3580 or Kubota et al., 2000, Carbohydrate Research, 324, 268-274. However, when it is desired to... [step] [c)] When obtaining multiple nanoparticles of saponified cross-linked polyglucosamine at the end, or when the acetylation step is planned to be repeated as described below to saturate the multiple nanoparticles with carboxyl functional groups and then achieve cross-linking in excess, it is advantageous to use a protic solvent in this step. In practice, the saponification step is performed when the multiple nanoparticles are saturated with carboxyl functional groups. Next, the acetylation and azira-Michael steps are repeated, followed by saponification. If spontaneous saponification occurs in a protic solvent during the azira-Michael step, this is not disadvantageous, because saponification will occur subsequently regardless, and the fact that partial saponification occurs during the azira-Michael reaction is not a problem.

[0092] According to another specific pattern, Michael's addition [step] [c)] is carried out in the absence of water, which can be achieved by using an aprotic solvent as the reaction medium or by using a base as the solvent.

[0093] According to another specific embodiment, the Michael addition reaction step is carried out in an alkaline medium, such that for each amide bond formed during the amide step, an equivalent secondary amine is formed after the Michael addition reaction, thereby ensuring that at least half of the amines are available for salt formation even if all amines are crosslinked. This can be verified by acid-base titration, IR and NMR spectroscopy, as described below: Glazunov et al., 1999, 25(3), 216-219; Brugnerotto et al., 2001, Polymer, 42, 3569-3580; or Kubota et al., 2000, Carbohydrate Research, 324, 268-274. According to one specific example, intramolecular addition reactions within polyglucosamine macromolecules are significantly superior to intermolecular reactions due to statistical and spatial factors, resulting in high yields of crosslinking.

[0094] Michael's additive reaction [step] [c)] The resulting plurality of nanoparticles can then undergo reduction (e.g., by reaction with complex metal hydrides, diborane, cyanoborohydrides, and other derivatives of borohydrides) to convert the amide group into a secondary amine and increase the likelihood of ionization of the plurality of nanoparticles. The likelihood of ionization of the plurality of nanoparticles depends on the number of amine groups. The more amine groups present, the greater the chance of salt formation and corresponding ionization. The degree of ionization can be assessed by acid-base titration, IR, and NMR spectroscopy.

[0095] According to another state, in [step] [c)] or [step] [d)] obtained cross-linked polyglucan products or [step] The corresponding dried product obtained in [e)] can undergo saponification in a protic solvent under alkaline conditions (e.g., in the presence of an excess of alkaline aqueous solution). [step] [f)] This is to increase the solubility of multiple nanoparticles of cross-linked polyglucosamine over a wider pH range, such as under physiologically weakly alkaline conditions (e.g., approximately pH 7.2-7.3). If in [Step] c) [or] [d)] If the solvent used does not allow for saponification (e.g., DMF), then purification is required. [step] [d)] Saponification is then carried out in a solvent that allows saponification, or by means of... [step] [e)] Afterward, the dried cross-linked polyglucosamine is resuspended in a solvent that allows saponification, such as an ether as a solvent, for saponification.

[0096] For example, in this other [step] In [f)], the alkaline solution is compared to... [step] [b)] The amount of acetic acid used is excessive, such as adding approximately 10 or 100 times the amount of such acetic acid to the solution. [step] [c)] [or] The product obtained in [d)]. As follows: [plan] [2] The general outline shows that cross-linked polyglucosamine sugars [(B2)] amide group hydrolysis product The corresponding carboxyl group in [(B3)] can be used to form the corresponding salt. [plan] [2]

[0097] This causes half of the amino groups in the cross-linked polyglucosamine to regenerate into primary amines, and the other half into secondary amines, which are then alkylated with propionic acid (or substituted propionic acid). For example, the saponification of cross-linked polyglucosamine can be carried out by adding a 1M sodium hydroxide aqueous solution at 50 degrees Celsius and maintaining the solution for one hour. The resulting product... [(B3)] Then it can be done as follows [plan] [3a] and [plan] [3b] The general outline shows that it undergoes re-fermentation. [step] [b)], where, under these conditions, acetylation occurs under the same conditions, wherein primary amino groups can occur ( [plan] [3a]) and secondary amino groups (in [plan] [3b] The re-acylation of the amino groups obtained in stages b, c, or f. Then, the resulting product... [(B4)] Again [step] [c)] The polyglucosamine undergoes a Michael addition reaction to achieve cross-linking of all amino groups and obtain a substantially fully cross-linked polyglucan. [(B5)] . [plan] [3a] [plan] [3b]

[0098] In this case, an inner salt is formed with the carboxyl group in the resulting product, which will be the first synthesis [step] [c)] Half the initial amount of amino groups present at the end. According to a particular state, such products will contain substantially the same number of carboxyl groups, total secondary and tertiary amino groups, and acetaminophen functional groups (excluding the initial acetaminophen glycoside fragment), that is, such products will contain a complete internal salt, with all carboxyl groups balanced by amino groups.

[0099] Although I do not wish to be bound by any theory, it can be considered that if in [step] [a)] The initial polyglucosamine sugar provided contains an amino group, then in [step] [b)] will achieve its complete derivatization, X amino groups are amided by an amide agent and therefore, X amino groups will [step] [c)] Aza-Michael reaction occurs between the acetylated fragment grafted onto the polyglucosamine and the acetylated fragment. [step] [c)] The product containing [step] The amide groups formed in [b) undergo saponification. [step] [f)] After saponification, X primary amino groups are regenerated, and X carboxyl groups are obtained from the acrylic acid used in the saponification process, and from... [step] [c)] The resulting X secondary amino groups are retained. Therefore, after repeated complete derivatization via acetylation, the X regenerated primary amino groups can be further acetylated (in... [step] The secondary amino group obtained in [c)] may be amided, but this is an unlikely procedure. Then, when repeated... [step] [c)] At that time, previously in [step] The secondary amine group obtained in [c)] enters the aza-Michael reaction to form X tertiary amine groups.

[0100] Furthermore, re-saponification regenerates X primary amino groups while retaining X tertiary amino groups, which do not undergo saponification and form X carboxyl groups. Therefore, the total number of carboxyl groups will be 2X, with X primary amino groups and X tertiary amino groups, and the polymer will become electrically neutral and form an inner salt.

[0101] If a third derivatization is performed, only X / 2 of the amide is needed, because only X amino groups can enter these reactions, and all of these amino groups are primary amino groups. Therefore, after the third derivatization steps (steps b) to c) and saponification (step f), only 2.5X carboxyl groups, 1.5X tertiary amino groups, and 0.5X primary amino groups are obtained, and the polymer will become an anionic polymer with an excess of 0.5X carboxyl groups. Therefore, repeating this reaction step will increase the number of carboxyl groups by increments of 0.25, 0.125, 0.0625, 0.03175, etc. The presence of carboxyl groups can be easily determined by infrared spectroscopy, acid-base titration, and mass spectrometry.

[0102] According to a particular embodiment, saponification is selected as appropriate. [step] [f)] The amount of alkali used is related to the first reaction. [step] [c)] [ / d)] [or] [e)] The amount of amide groups to be saponified is equal to the molar amount and at most 10 times the molar excess.

[0103] According to a particular embodiment, [step] [b)] [to] [c)] [(] [or] [d) / e)] At least one of the processes in [)] is carried out after the Michael addition process has ended and a new vinification process is being performed. [step] [b)] [to] [c)] [(] [or] [d) / e)] ) Process before saponification [step] [f)] To be carried out together.

[0104] According to another specific embodiment, [step] [a)] [to] [f)] Approximately 2 to 6 reaction steps.

[0105] According to another specific embodiment, [step] [a)] [to] The reaction process [f)] is repeated once or twice.

[0106] According to a particular embodiment, in [step] [a)] [to] [f)] After each step, the cross-linked polyglucosamine achieves solubility over a wide pH range, and the properties of the natural polyglucosamine can be modified, such as mucotropy and cell penetration. Therefore, these properties can be advantageously adjusted depending on the number of reaction steps performed, and the resulting multiple nanoparticles of the cross-linked polyglucosamine can be used to deliver the compound of interest.

[0107] According to one particular embodiment, in terms of charge, cationic substances (e.g., cationic peptides, such as sequences rich in arginine, ornithine, or lysine) or neutral substances can be delivered by cross-linked polyglucosamine according to the invention.

[0108] According to another specific embodiment, the cross-linked polyglucosamine sugar according to the invention can be used to formulate such compounds to prevent their degradation (e.g., degradation via proteolytic hydrolysis) or to prevent them from binding to undesirable substances (e.g., antibodies or other macromolecules) or to ensure the slow release of soluble components of such compounds.

[0109] According to another specific embodiment, the bioactive material can be first amided. [step] [b)] or First Michael Bonus [step] [c)] It is then integrated into the plurality of nanoparticles formed. According to another specific embodiment, the bioactive material may be covalently linked, adsorbed, immobilized in salt form, or encapsulated within the plurality of cross-linked polyglucosamine nanoparticles of the present invention.

[0110] During the saponification step, the covalent cross-linking of the bioactive agent will be destroyed, but ionic bonds with the bioactive agent can be formed within the salt of the resulting cross-linked polyglucosamine.

[0111] Or, when the application [step] [a)] [to] [f)] In this case, multiple nanoparticles can be impregnated with a bioactive substance resistant to the reagents used in a series of such steps, such as impregnation with clusters of heavy elements to produce multiple radiopaque nanoparticles.

[0112] Alternatively, after synthesizing a plurality of cross-linked polyglucosamine nanoparticles of the present invention, it is preferable to introduce multifunctional, unstable bioactive substances into these nanoparticles by diffusion or by using click chemistry, enzyme, protein, nucleic acid and antibody immobilization methods.

[0113] According to a specific pattern, the integration of bioactive materials within a plurality of nanoparticles of the polyglucosamine of the present invention can be achieved by impregnating them with the desired bioactive material. [step] [b)] or [c)] The product obtained is realized. For example, it can be achieved by performing Michael addition. [step] [c)] Previously, a solution of the bioactive material in an aprotic solvent was added to a solution containing... [step] [b)] Among the products obtained. Or, the product obtained can be derived from... [step] [b)] The obtained acetylated polyglucosamine product was dissolved in a solvent suitable for the conditions of such further synthesis.

[0114] Impregnation can be performed without dissolving the cross-linked polyglucosamine sugar of the present invention.

[0115] Given that the cross-linked polyglucosamine of the present invention is impregnated with a bioactive agent, the bioactive agent / substance can be dissolved in a polar aprotic solvent or any other solvent. If a protic solvent is used, the solvent needs to be removed and the impregnated product dried prior to the azira-Michael reaction. For impregnation, supercritical fluids can be used as described below: Weidner 2018, The Journal of Supercritical Fluids, 134, 220-227 https: / / doi.org / 10.1016 / j.supflu.2017.12.024 ; Duarte et al., 2007, The Journal of Supercritical Fluids, 42 (3), 373-377 https: / / doi.org / 10.1016 / j.supflu.2007.01.007 , thereby providing an extremely high degree and quality of impregnation. If the impregnation of the reagent can be accelerated using the Coulomb method, then electrophoresis methods such as those described in Boccaccini et al., 2010, JRSoc. Interface, 7, S581-S613 https: / / doi.org / 10.1098 / rsif.2010.0156.focus; Pishbin et al., 2013, Acta Biomaterialia, 9(7), 7469-7479, or the "gene gun" method described in Zhao et al., 2012, PLoS One, 7(10): e46765.doi:10.1371 / journal.pone.0046765 can be used. For example, an aqueous solution of a bioactive material such as an antibiotic can be mixed with... [step] [b)] Subsequently, the aqueous solution of the acetylated polyglucosamine was mixed and subjected to evaporation, spray drying, freeze-drying, and other drying methods via azeotropic distillation with water to obtain a dry acetylated polyglucosamine impregnated with bioactive material. Next, the dried material was treated with an excess of a non-nucleophilic base to deionize the amino groups of the polyglucosamine, and then the resulting mixture was subjected to a Michael addition reaction. [step] [c)] .

[0116] According to another sample, the polyglucosamine provided in step a) has an average molecular weight in the range of about 5 to about 2,000 kDa, particularly in the range of about 150 to 2,000 kDa.

[0117] According to another example, the polyglucosamine provided in step a) has a deacetylation degree of about 100% (if in glucosamine form) to about two deacetylated segments per macromolecule, for example, for a polyglucosamine (chitin) with a molecular weight of 300 kDa, the deacetylation degree is about 0.14%.

[0118] Suitable polyglucosamines that can be used as starting materials in the methods of this invention can be from various sources, including natural (animal or fungal) sources, particularly from the shells of crabs and shrimp, insect exoskeletons, higher fungi, and cultures of single-celled mushrooms. Polyglucosamines synthesized chemically or enzymatically can also be used.

[0119] According to another state, make [step] [a)] The provided polyglucosamine swells for about 5 minutes to about 1 day, preferably in aprotic solvents.

[0120] According to another embodiment, the deacetylation step b) is carried out at a temperature of about -70°C (start of reaction) to about +150°C (reaction complete), preferably at a temperature of about +2°C (start of reaction) to about +55°C (reaction complete). The reaction time is about 0.5 hours to 10 days, preferably about 2.5 hours at about +20 to about +30°C, and preferably under ultrasonic treatment as a mixing means.

[0121] According to another perspective, Michael's addition [step] [c)] The reaction is carried out at a temperature ranging from approximately -70°C (start of reaction) to approximately +150°C (complete of reaction), preferably at a temperature ranging from approximately +20°C (start of reaction) to +55°C (complete of reaction). The reaction time is approximately 0.5 hours to 10 days, preferably approximately 15-18 hours at approximately +20°C and approximately 6 hours at approximately +55°C, preferably with ultrasound as a mixing method.

[0122] According to another sample, the drying step of cross-linked polyglucosamine is in [step] [e)] is carried out by freeze drying.

[0123] Depending on the other sample, additional saponification may be selected as appropriate. [step] [f)] The reaction is carried out at a temperature of about 0°C (start of reaction) to about +140°C (complete reaction), preferably at a temperature of about +20°C (start of reaction) to about +55°C (complete reaction). The reaction time is about 0.5 hours to 10 days, preferably about 2-6 hours at about +55°C, and preferably with ultrasound as a mixing method.

[0124] In a specific embodiment, the plurality of nanoparticles of the present invention are generated by the method of the present invention. [step] [b)] and The material obtained in [c)] forms spontaneously. These particles are extremely stable over time. For example, a 2% solution remains stable at room temperature for at least one year and in a dry state for at least several years.

[0125] According to one embodiment of the present invention, a method for preparing a drug delivery system for a bioactive agent is provided, comprising the steps of performing the method of the present invention, wherein the bioactive agent is in [step] [b)]、 [c)]、 [d)] or Add to [e)].

[0126] According to one embodiment of the present invention, a method for preparing a drug delivery system for a bioactive agent is provided, comprising the following steps: - The cross-linked polyglucan of the present invention is provided in a moist or dry state; - Provide the bioactive agent to be delivered; - Dissolve the bioactive agent in a solvent or supercritical fluid; - The cross-linked polyglucosamine of the present invention is introduced into a plurality of nanoparticles by impregnation with a bioactive agent solution or by direct introduction via electrophoresis or electric field acceleration, thereby loading the plurality of nanoparticles with the cross-linked polyglucosamine; - Collect the obtained composition or multiple nanoparticles loaded with bioactive agents (e.g., drugs).

[0127] According to another specific embodiment, a method is provided for identifying cross-linked polyglucosamine sugars obtainable by the method according to the present invention, the method comprising the following steps: - The polyglucosamine to be characterized is provided in a solvent (e.g., dichloromethane or chloroform); - As described herein (e.g., with acetyl chloride or acetic anhydride), the polyglucosamine is glycosylated under vigorous stirring, such as at about 0°C to about 20°C, for about 30 minutes; - Neutralize the reaction medium with a base (e.g., diisopropylethylamine as a non-nucleophilic base); - Evaporate the solvents and wash the resulting neutralized product; - Allow the product to undergo reflux acid hydrolysis (preferably with 28% hydrochloric acid, for about 1 to 3 hours, such as about 2 hours); - Evaporate the reaction mixture and resuspend the hydrolysis products in a weak acid such as acetic acid; - Determine the presence or absence of a product selected from the products according to formula (IIIa) or formula (IIIb). [Chemical Formula (IIIa)]; [Chemical Formula (IIIb)]; Where R2 to R4 are as defined in this paper, and R2' to R4' are as defined in this paper for R2 to R4 respectively, and where the formula is... [(IIIa)] and / or The presence of the product of [(IIIb)] indicates the cross-linked polyglucan obtained by the method according to the present invention.

[0128] According to another specific embodiment, in the method for identifying the cross-linked polyglucosamine sugar according to the invention, the presence or absence of the product according to formula (IIIa) can be identified, for example, by mass spectrometry electrospray analysis, wherein, for example, a peak having m / z = 252.077 indicates the presence of the compound according to formula (IIIa), particularly the compound of formula (II). [Chemical Formula (II)]

[0129] The method for identifying cross-linked polyglucosamine sugars obtained by the method according to the present invention is extremely useful because it can distinguish cross-linked polyglucosamine sugars obtained by the method according to the present invention from those obtained by other methods, such as those described in Baoqiand et al., 2015 or El-sherbiny et al., 2009. [According to the present invention, polyglucosamine sugar and its derivative] [Multiple] [Nano particles]

[0130] The cross-linked polyglucosamine sugar according to the invention can be prepared from readily available starting materials using the method of the invention. It should be appreciated that, given typical or preferred experimental conditions (i.e., reaction temperature, time, molar amounts of reagents, solvents, etc.), other experimental conditions may be used unless otherwise stated. Optimal reaction conditions may vary depending on the specific reactants or solvents used, but these conditions can be determined by a person skilled in the art using conventional optimization procedures.

[0131] According to a particular embodiment, a cross-linked polyglucosamine and its plurality of nanoparticles obtained by the method of the present invention are provided.

[0132] According to another specific embodiment, a cross-linked polyglucosamine is provided, characterized by a lower viscosity than the starting polyglucosamine. For example, for a polyglucosamine with 90% deacetylation, 3.2% cross-linking, and a concentration of 0.25%, the viscosity of a solution of a plurality of nanoparticles according to the present invention is three times lower than that of the starting polyglucosamine. For instance, the viscosity of a plurality of nanoparticles of the polyglucosamine of the present invention is 2.4 cSt at 25°C and 1.82 cSt at 37°C, compared to 7.5 cSt at 25°C and 5.4 cSt at 37°C for the corresponding starting polyglucosamine.

[0133] According to a particular embodiment, the pharmaceutically acceptable salts of the cross-linked polyglucosamine sugar of the present invention include, for example, acetates, lactates, succinates, citrates, malonates, fumarates, maleates, malates, and other carboxylic acids, hydroxycarboxylic acids, and their hydrochlorides, phosphates, sulfates, and other inorganic acid salts.

[0134] It should be understood that the molecular weight of the cross-linked polyglucosamine of the present invention can be altered by selecting the starting material, and this selection will depend on the intended function of the plurality of nanoparticles and their formulations. For example, the desired degradation rate of the plurality of nanoparticles, the desired release rate of any bioactive agents incorporated therein, and the condition to be treated will influence the selection of the molecular weight of the polyglucosamine as the starting material.

[0135] In one embodiment, the molecular weight of the cross-linked polyglucosamine of the present invention is between about 2 kDa and 2 MDa.

[0136] In another embodiment, the molecular weight of the cross-linked polyglucosamine sugar of the present invention used in the lubricant / filler composition may be selected between about 2 kDa and 2 MDa, preferably between about 30 kDa and 1,000 kDa.

[0137] In another embodiment, the molecular weight of the cross-linked polyglucosamine of the present invention used or as a drug delivery system may be selected between about 2 kDa and 2 MDa, preferably between about 30 kDa and 1,000 kDa. [ Composition , ] [ , ]

[0138] This invention provides pharmaceutical or therapeutic agents in the form of compositions, or medical devices comprising the same, and methods for treating an individual, preferably a mammalian patient, and most preferably a human patient, who suffers from a medical condition, specifically a cardiovascular condition such as arrhythmia, particularly atrial fibrillation and hypertension; joint disorders and diseases such as rheumatoid arthritis, osteoarthritis, spondylitis, and traumatic events resulting in damage to cartilage, bone, ligaments, or bursae; and eye disorders and injuries such as dry eye syndrome, glaucoma, and glaucoma. Meningitis, glaucoma, corneal lesions; connective tissue diseases, such as lupus and polymyositis; skin / mucous membrane diseases or injuries, such as trauma, scars, psoriasis, acne, eczema, rosacea, physical or chemical burns, especially surgical wounds and sunburn, ulcers, hemorrhoids; periodontal and dental diseases; dura mater injuries, such as accidental injuries or dura mater injuries after brain and central nervous system surgery; malignant and benign tumors, especially carcinomas, sarcomas, lymphomas and melanomas; postoperative complications, such as fistulas and infections; tumors or vascular malformations.

[0139] The present invention provides compositions comprising at least one of the cross-linked polyglucosamine sugars of the present invention that can be used in human, veterinary or agricultural applications.

[0140] In one particular embodiment, the agricultural composition according to the present invention can be used as a plant growth stimulant.

[0141] According to one specific embodiment, the cross-linked polyglucosamine of the present invention can be used to prepare self-assembled structures, molecular machines, delivery mechanisms, electronic components, composite materials, and lubricants.

[0142] In one particular embodiment, the present invention provides a pharmaceutical formulation comprising at least one cross-linked polyglucosamine sugar or a composition thereof of the present invention, which is used as a drug.

[0143] In another specific embodiment, the composition of the present invention is a non-oral formulation.

[0144] In one particular embodiment, the components of the present invention are injectable formulations, such as intra-articular, intra-articular, intra-venous, intra-synovial, intradermal, subdermal, submucosal, interstitial, intracranial, intraocular, intratumoral, intratumoral, intragastric, intestinal, anal, intraperitoneal, and intramuscular formulations.

[0145] In another specific embodiment, the composition of the present invention is an oral formulation.

[0146] In another specific embodiment, the components of the present invention are external formulations.

[0147] In another specific embodiment, the composition of the present invention is an ophthalmic formulation.

[0148] Alternatively, the present invention provides a composition that can be used in another mammal (e.g., a human) for the same purpose as described above.

[0149] The present invention further provides compositions or medical devices that can be used in cosmetics, reconstructive surgery (e.g., tissue reconstruction), cell or biological tissue cultures (e.g., stem cell cultures), materials science (e.g., surface coatings, such as implant coatings, lubricant compositions, coagulant compositions), and diagnostics (e.g., imaging compositions). These compositions further comprise cosmetically acceptable carriers or cell or biological tissue culture nutrients.

[0150] The compositions according to the present invention comprise soft tissue filler compositions, such as dermal and subdermal fillers, comprising at least one cross-linked polyglucosamine of the present invention or a plurality of nanoparticles thereof. Preparation of polyglucosamine-based soft tissue filler compositions can be carried out, for example, as described below: Grant et al., 2018, Tissue Eng Part A., 24(13-14):1091-1098. doi: 10.1089 / ten.TEA.2017.0385. Epub March 20, 2018.

[0151] In a particular embodiment, a plurality of nanoparticles of the cross-linked polyglucosamine of the present invention are provided, having an average size in the range of about 5 and 100 nm.

[0152] Another aspect of the present invention relates to a wound dressing comprising at least one cross-linked polyglucan according to the present invention.

[0153] According to another specific embodiment, the present invention relates to a wound dressing comprising a permeable matrix, the matrix comprising a hydrocolloid, hydrogel, alginate, collagen, cellulose, foam, or fabric.

[0154] The composition of the present invention may further include one or more pharmaceutically acceptable additional ingredients, such as alum, stabilizers, antimicrobial agents, buffers, colorants, flavorings, adjuvants and the like.

[0155] The compositions according to the present invention, and conventionally used adjuvants, carriers, diluents, or excipients, can be formulated into pharmaceutical compositions and can be used in solid form, such as lozenges or filled capsules; or in liquid form, such as solutions, suspensions, ointments, emulsions, elixirs, or in capsules, films, or gels, all of which are intended for oral use. These compositions can also be formulated as dried products reconstituted with water or another suitable mediator prior to use.

[0156] The components of this invention are in the form of liquid formulations, including but not limited to aqueous or oily suspensions, solutions, emulsions, syrups and elixirs.

[0157] These liquid formulations may contain additives, including but not limited to suspending agents, emulsifiers, non-aqueous mediators, and preservatives. Suspension agents include, but are not limited to, sorbitol syrup, methylcellulose, glucose / sugar syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifiers include, but are not limited to, lecithin, dehydrated sorbitol monooleate, and gum arabic. Preservatives include, but are not limited to, methylparaben or propylparaben, and sorbic acid. Dispersants or wetting agents include, but are not limited to, polyethylene glycol, glycerin, bovine serum albumin, Tween®, and Span®.

[0158] Other materials, processing techniques for formulations, and similar content are described in The Science and Practice of Pharmacy (Remington: The Science & Practice of Pharmacy), 22nd edition, 2012, Lloyd, Ed. Allen, Pharmaceutical Press, which is incorporated herein by reference.

[0159] The solid composition of this invention can be in the form of tablets or oral tablets formulated in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients, including but not limited to binders, fillers, lubricants, disintegrants, and wetting agents. Binders include, but are not limited to, syrups, gum arabic, gelatin, sorbitol, tragacanth gum, starch paste, and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, maize starch, calcium phosphate, and sorbitol. Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silicon dioxide. Disintegrants include, but are not limited to, potato starch and sodium glycolate. Wetting agents include, but are not limited to, sodium lauryl sulfate. Tablets can be encapsulated by methods well known in the art.

[0160] The agricultural composition according to the present invention includes plant growth accelerators, seed germination promoters, water and nutrient retention modifiers, insecticide enhancers, prolonged forms and pheromone stabilizers, mucosal adhesive forms of insecticides, acaricides, and anti-mollusc agents.

[0161] According to a particular embodiment, an agricultural composition is provided comprising about 0.001 to about 99% w / w of the cross-linked polyglucosamine sugar of the present invention, for example about 0.05 to about 0.1% (w / w).

[0162] In another specific embodiment, the composition or plurality of nanoparticles of the present invention further comprises a bioactive agent dispersed in or covalently linked to the cross-linked polyglucosamine composition.

[0163] In another specific embodiment, the components or plurality of nanoparticles of the present invention are in the form of a bioactive agent delivery system.

[0164] In one embodiment, the amount of the at least one bioactive substance present, based on the total amount of the cross-linked polyglucosamine composition or nanoparticles of the present invention, may be between about 0.001 and 20 wt%, preferably between about 0.01 and 10 wt%.

[0165] According to another embodiment, the composition of the invention comprises a cell or biological tissue culture medium containing cross-linked polyglucosamine sugar or a composition thereof. This culture medium may further contain cellular nutrients such as glucose, vitamins, growth factors, metal ions, and the like.

[0166] According to another embodiment, the composition of the invention comprises biological tissue (endogenous or exogenous) or synthetic or semi-synthetic material that can be used to repair damaged tissues of the body, such as epidermis, nerves, cartilage or bone tissue.

[0167] In another specific embodiment, a method for preparing a culture medium is provided, comprising the step of mixing the cross-linked polyglucan according to the present invention with cell culture nutrients, such as glucose, vitamins, growth factors, metal ions and the like.

[0168] In another specific embodiment, a method for preparing reconstructed tissue is provided, comprising the step of combining the cross-linked polyglucosamine sugar or a composition thereof according to the present invention with materials, tissues or cells that can be used to repair damaged tissues of the body, such as stem cells or epidermal, nerve, cartilage or bone tissue.

[0169] According to a specific configuration, the components or plurality of nanoparticles of the present invention, after being loaded with bioactive substances or bioactive agents, can be used for dosing and in-situ release of the active ingredient in an injection solution. [ Drug delivery mode , ] [ , ]

[0170] The components of this invention can also be administered by injection, such as subcutaneous injection, intrasynovial injection, intra-arterial injection, intravenous injection, intra-articular injection, intramuscular injection, subdermal injection, submucosal injection, intraocular injection, intracranial injection, intragastric injection, intestinal injection, anal injection, intraperitoneal injection, intratumoral injection, and interstitial injection, in any manner.

[0171] The components of this invention can be administered orally in any manner, including to the mucosal surfaces of the oral cavity, including the gums, floor of the mouth, cheeks, lips, tongue, and teeth.

[0172] The components of this invention can also be applied to the skin, various mucous membranes, or eyes via surface application. [ combination , ] [ , ]

[0173] According to one embodiment, the cross-linked polyglucosamine of the present invention, or a plurality of nanoparticles thereof, or any suitable pharmaceutically acceptable salt thereof, and its pharmaceutical formulations, may be administered alone or in combination with at least one co-agent.

[0174] According to a specific example, the common agents of the present invention include antibiotics, nucleic acids (including gene constructs), antibodies and antibody fragments, toxins, cell inhibitors, antifungal agents, components of chemotherapeutic agents, antihypertensive agents, antiarrhythmic substances, proliferation-activating substances, hormones, cytokines, nitric oxide donors, and hydrogen sulfide donors.

[0175] This invention covers the simultaneous or sequential administration of the cross-linked polyglucosamine of the present invention or its particles, pharmaceutical formulations thereof, and at least one co-administered agent to an individual.

[0176] The cross-linked polyglucosamine sugar or its plurality of nanoparticles or its pharmaceutical formulations according to the present invention, which are administered simultaneously with the common pharmaceutical agent, may have the same or different compositions and may be administered via the same or different routes of administration.

[0177] The dosage administered to an individual, whether as a single or multiple dose, will vary depending on a variety of factors, including pharmacokinetic properties, patient condition and characteristics (sex, age, weight, health status and body size), symptom severity, concurrent treatments, treatment frequency, and desired outcome. [ patient , ] [ , ]

[0178] In one embodiment, the patient according to the invention is an individual suffering from cardiovascular conditions, such as arrhythmia, particularly atrial fibrillation and hypertension.

[0179] In another embodiment, the patient according to the invention is an individual suffering from joint lesions and diseases, such as rheumatoid arthritis, osteoarthritis, spondylitis, and traumatic events resulting in damage to cartilage, bone, ligaments, or bursae.

[0180] In another embodiment, the patient according to the present invention is an individual suffering from eye diseases and injuries, such as dry eye syndrome, uveitis, glaucoma, or corneal disease.

[0181] In another embodiment, the patient according to the invention is an individual suffering from connective tissue diseases such as lupus and polymyositis.

[0182] In another embodiment, the patient according to the invention is an individual suffering from skin / mucous membrane conditions or injuries, such as trauma, scars, psoriasis, acne, eczema, rosacea, physical or chemical burns, especially surgical trauma and sunburn, ulcers, hemorrhoids.

[0183] In another embodiment, the patient according to the present invention is an individual suffering from periodontal and dental diseases.

[0184] In another embodiment, the patient according to the invention is an individual suffering from dural injury, such as accidental injury or dural injury following brain and central nervous system surgery.

[0185] In another embodiment, the patient according to the present invention is an individual suffering from malignant and benign tumors.

[0186] In another embodiment, the patient according to the invention is an individual suffering from postoperative complications, such as fistula and infection.

[0187] In another embodiment, the patient according to the invention is an individual who desires or requires enhancement of body part volume or anatomical remodeling, such as any enhancement / alteration and / or increase in the volume of body parts for aesthetic or therapeutic reasons.

[0188] In another embodiment, the patient according to the invention is an individual suffering from a tumor, vascular malformation, or any other newly arising abnormality in body tissues or organs that causes pain. [ According to the uses of the present invention , ] [ , ]

[0189] According to one particular embodiment, the cross-linked polyglucosamine of the present invention can be used in a variety of applications, such as in vitro cell or biological tissue cultures, as a tissue filler, for cosmetics, tissue and organoid engineering, and materials science applications, because it can spontaneously form multiple nanoparticles.

[0190] Specifically, according to one embodiment, the cross-linked polyglucosamine of the present invention can be used as a carrier for pharmaceutically active ingredients (e.g., as a drug delivery system, as a cell penetration system (e.g., in gene therapy or for use as a non-cell penetration agent), as a solubilizer, and as a trapping agent: encapsulating the ingredient of interest within a plurality of nanoparticles to create a suspension for effective removal from solution, for example, by changing the acidic pH to the alkaline pH).

[0191] According to another embodiment, the cross-linked polyglucosamine of the present invention can be used as a medical implant (e.g., a coating for implants to improve biocompatibility, or a cell-containing regenerative medical implant) or a medical material (e.g., a hemostatic material).

[0192] According to another embodiment, the cross-linked polyglucosamine of the present invention can be used as a matrix for contrast agents (e.g., in standard angiography (CT) and other emerging imaging modalities (e.g., EPR-based, NMR-based and X-ray-based imaging modalities)).

[0193] According to another embodiment, the cross-linked polyglucosamine of the present invention can be used as a cell culture (e.g., stem cells), cell culture medium, tissue engineering (such as 3D printing of organoids), for separation / purification techniques, and as a matrix for cell transfection.

[0194] According to another embodiment, the cross-linked polyglucosamine of the present invention can be used as a nano-level lubricant (e.g., after thermal decomposition), a dispersant for colloid preparation, or a coagulant.

[0195] According to another specific embodiment, the use of the cross-linked polyglucosamine sugar of the present invention or the nanoparticles or formulations thereof of the present invention is provided for the preparation of delivery systems, in vitro cell or biological tissue cultures or tissue engineering materials, such as neurosurgical, bone, cartilage, and epidermal reconstructive tissues.

[0196] According to another specific embodiment, the cross-linked polyglucosamine sugar or plurality of nanoparticles or formulations thereof according to the present invention can be used to prevent or treat medical conditions, specifically cardiovascular conditions such as arrhythmias, particularly atrial fibrillation and hypertension; joint diseases and lesions such as rheumatoid arthritis, osteoarthritis, spondyloarthritis, and traumatic events resulting in damage to cartilage, bone, ligaments, or bursae; eye diseases and injuries such as dry eye syndrome, uveitis, glaucoma, and corneal diseases; and connective tissue diseases such as lupus. Sores and polymyositis; skin / mucous membrane conditions or injuries such as trauma, scars, psoriasis, acne, eczema, rosacea, physical or chemical burns, especially surgical wounds and sunburn, ulcers, hemorrhoids; periodontal and dental diseases; dura mater injuries such as accidental injuries or dura mater injuries after brain and central nervous system surgery; malignant and benign tumors, especially carcinomas, sarcomas, lymphomas and melanomas; postoperative complications such as fistulas and infections; tumors or vascular malformations, or for the prevention and / or treatment of tissue degeneration and related conditions.

[0197] According to another specific embodiment, cross-linked polyglucosamine sugar or a plurality of nanoparticles or formulations thereof according to the invention are provided for cosmetic use or for use in cosmetic and reconstructive surgeries.

[0198] According to another specific example, cross-linked polyglucosamine sugar or a plurality of nanoparticles or formulations thereof according to the invention are provided for in vivo drug delivery.

[0199] According to a specific pattern, the use of the nanoparticles according to the present invention as a delivery system for at least one bioactive agent is provided.

[0200] According to a particular embodiment, a composition comprising at least one cross-linked polyglucosamine sugar or a plurality of nanoparticles according to the present invention is provided, which can be used in many medical applications, such as delivery systems for at least one bioactive agent or cell delivery systems in tissue engineering, cell or biological tissue culture systems and similar applications.

[0201] Examples of the present invention will be described in more detail below with reference to the embodiments shown in the figures. [Example]

[0202] The following abbreviations refer to the following definitions: [DMF] (Dimethylformamide); [DMSO] [(] Erjia Yaqi); [MWCO] (Molecular weight cutoff value). [Example] [1] [:] [Synthesis of the Polyglucosamine Sugar of this Invention]

[0203] As described in Scheme 1 above, a plurality of nanoparticles of the polyglucosamine sugar of the present invention are synthesized, wherein the starting materials used are as follows: [ a) , ] [ Provide polyglucosamine sugar and prepare the polyglucosamine sugar in the presence of anhydrous conditions. , ] [ ( , ] [ For example, in aprotic solvents , ] [ ) , ] [ Experiencing expansion steps , ] [ , ]

[0204] In an inert atmosphere (e.g., purged with argon), 450 g of dried polyglucosamine (which can be dried by phosphorus oxidation in vacuum, by moderate heating in vacuum, or by azeotropic distillation with ethanol, acetonitrile, acetone, or any other suitable solvent) is placed in a reactor. This reactor is preferably a two-liter round-bottom flask equipped with a magnetic stirrer and bi-angled nozzles, one nozzle from a Drexel flask for purging with an inert gas (e.g., argon) and the other from a dropping funnel with back pressure for adding reagents.

[0205] Next, 1,400 ml of an aprotic solvent (e.g., DMF) was poured into the reactor and allowed to expand the polyglucosamine sugar at room temperature for about 1 hour.

[0206] When undried polyglucosamine is provided, 1,500 ml of an aprotic solvent (e.g., DMF) is poured into the reactor and the polyglucosamine is allowed to expand at room temperature for 1 hour. Then, 300 ml of the aprotic solvent (e.g., DMF) is distilled off under vacuum, and then 200 ml of DMF is added to the dried polyglucosamine in the reactor and the polyglucosamine is allowed to expand at room temperature for 1 hour.

[0207] Next, cool the reaction medium containing the expanded polyglucosamine sugar to approximately 2-6°C in an ice bath or in a bath connected to a thermostat at approximately 2-6°C. In the same bath, also cool a 250 ml reactor (e.g., a round-bottom flask) containing 100 ml of dry DMF. After cooling, while stirring and avoiding localized overheating, add the cytochrome dropwise to the aprotic solvent (100 ml DMF) while vortexing the solvent. The solution of the cytochrome in DMF can be warmed to approximately room temperature. [ b) , ] [ In the absence of water , ] [ , , ] [ Formula , ] [ (I) , ] [ Acrylic acid compounds aminolate the polyglucosamine sugar. , ] [ : , ] [ , ]

[0208] Under vigorous stirring, add the prepared acrylamide compound solution to the obtained polyglucosamine suspension over approximately 15 minutes, preferably while gently agitating the reactor (e.g., manually). During agitation, stop adding the acrylamide compound, as this allows the polyglucosamine clumps on the flask wall to be washed away with a splashing solvent. After the acrylamide compound solution has been added, wash the dropping funnel with approximately 10-15 ml of a minimal amount of an aprotic solvent (e.g., DMF) and add this solution to the reaction medium. Remove from heat and continue stirring for two hours, occasionally agitating, to allow the reaction medium to warm to room temperature. After stirring, place the flask in an ultrasonic bath for approximately 15 minutes and ultrasonically treat the reaction mixture. During this ultrasonic exposure, the reaction mixture is only slightly heated, not exceeding 30°C. [c)] [ Make steps, ] [ b) , ] [ The aziridine product undergoes a nucleophilic addition reaction in the presence of a non-nucleophilic base. , ] [ ( , ] [ Nitrogen , ] [ - , ] [ Michael's bonus , ] [ ): , ]

[0209] Subsequently, under vigorous stirring and occasional vibration, an excess of a non-nucleophilic base (e.g., triethylamine, diisopropylethylamine) relative to the amount of acrylamide compound used in step b) is added to the reaction medium (e.g., via a dropping funnel) over approximately 15 minutes. The mixture is allowed to mix at room temperature overnight or for approximately 15-18 hours. Next, the reactor is placed in a bath at approximately 55°C and the mixture is stirred for approximately 6 hours. The reactor is removed from the bath and placed in an ultrasonic bath preheated to approximately 55°C for approximately 15 minutes, and the reaction mixture is ultrasonically treated.

[0210] After ultrasonic treatment, the reactor is transferred to a rotary evaporator, and the non-nucleophilic base is distilled off when using a volatile organic non-nucleophilic base. If a non-volatile base is used, the reactor is cooled to room temperature or lower, and an equivalent amount of acid (e.g., hydrochloric acid) is added under vigorous stirring. The pH of the resulting reaction medium is controlled to ensure a neutral reaction medium is obtained. [d)] [ Obtain the cross-linked polyglucan of the present invention , ]

[0211] In step c), a suspension of cross-linked polyglucosamine in a nonprotic solvent (e.g., DMF) containing salt impurities is obtained. The product is then subjected to a purification step, such as by dialysis with deionized water. For example, for this purpose, the reaction solution obtained in step c) is transferred to a dialysis bag, sealed with a clamp, and placed in a container of mixed deionized water on a magnetic stirrer. Dialysis can be monitored in different ways: for example, by measuring the conductivity of the washing solution to monitor salt content, or by monitoring DMF content by UV absorption at wavelengths less than 260 nm. If no significant difference in conductivity or UV absorption is observed after incubating the deionized water and dialysis product together for 20 minutes, the dialysis procedure is considered complete. [e)] [ The cross-linked polyglucosamine of the present invention is obtained in a dry state as appropriate. , ] [ , ]

[0212] Can be used in [step] [d)] The obtained wet product is not dried. However, it may be dried, for example, for formulation / storage or transportation purposes.

[0213] Next, the purified product obtained in step d) is dried. For example, drying can be carried out as follows: the product is initially frozen in a freeze dryer; most of the liquid is absorbed at atmospheric pressure in the presence of phosphoric anhydride or any suitable desiccant (such as molecular sieves, potassium hydroxide, and the like), and then in a vacuum in the presence of phosphoric anhydride or any suitable desiccant or by initial evaporation on a rotary evaporator and repeated azeotropic distillation of water and ethanol, and subsequently vacuum dried with phosphoric anhydride or any suitable desiccant.

[0214] Generally, the yield of the method of this invention is about 90%. Losses are likely related to the entrainment of multiple nanoparticles during drying and during transfer from the reaction vessel to the dialysis bag, since sterility and contamination-free operation are prioritized over yield. [f)] [ Further processing of the cross-linked polyglucosamine sugar of the present invention in a dry state , ]

[0215] Depending on the situation, [step] [c)] [,step] [d)] [or steps] The purified cross-linked polyglucosamine obtained in [e)] can undergo saponification. For this purpose, the cross-linked polyglucosamine obtained in step d) is placed in a reactor under an inert atmosphere (e.g., purged with argon). 1,200 ml of deionized water is poured into a flask and, with vigorous stirring, a 20% molar excess of an alkali solution (e.g., aqueous NaOH) relative to the amount of acrylamide compound used in about 100 ml of deionized water is added over about 15 minutes. Preferably, the flask is gently shaken manually while the addition of alkali is stopped, as this procedure allows polyglucosamine clumps to be washed away by splashed solvent in contact with the upper wall of the reactor. After stirring, the reactor is placed in an ultrasonic bath and the reaction mixture is ultrasonically treated; after the reactants reach about 55°C, ultrasonic treatment is continued at this temperature for another 2 hours. Subsequently, the resulting product can be neutralized with an equivalent amount of acid, dialyzed, or dried without neutralization, and then subjected to at least one cycle after drying. [step] [b)] [to] [c)] ( [or] [d) / e)] . Using more than one loop [step] [b)] [to] [c)] ( [or] [d) / e)] Allows for the saturation of carboxyl functional groups in cross-linked polyglucosamine sugars. [Example] [2] [:] [Examples of synthesizing the cross-linked polyglucan of the present invention]

[0216] Various cross-linked polyglucosamine sugars (CHIs) of the present invention have been synthesized by varying the properties and proportions of the cross-linking agent according to the methods described therein. Examples are presented in Table 1 below.

[0217] In each case, 450 g of polyglucosamine was used as the starting material. Examples 2a-2r and C1, the acrylonitrile compounds were acrylonitrile chloride (CAS Registry No.: 814-68-6). Examples 2t-2al, the acrylonitrile compounds were methacrylonitrile chloride (CAS Registry No.: 920-46-7). The reactions were carried out as described in steps a) to c) of the method of the present invention. Examples 2am-2as, the acrylonitrile compounds were different acrylonitrile chlorides. [Table 1] [CHI] Polyglucosamine [Acrylic compounds] [Crosslinking / ] [Polyglucosamine-] [Key Link] [Theoretical number or crosslinking percentage] [ 2a , ] MW: 11.5 kDa DD: 62% 4.250 g (20% excess relative to polyglucosamine) 1 [ 2b , ] MW: 10.7 kDa DD: 91% 4.570 g (20% excess relative to polyglucosamine) 1 [ 2c , ] Same as 2a 11.278 g 9.8% of the amide / amine groups initially present in the polyglucosamine macromolecules (4.9% in step b) and 4.9% in step c) are generated. [ 2d , ] Same as 2b 12.112 g Same as 2c [ 2e , ] Same as 2a 71.35 g All amino groups [ 2f , ] Same as 2b 112.47 g Same as 2e [ 2g , ] MW: 53.0 kDa DD: 70% 0.922 g (20% excess relative to polyglucosamine) 1 [ 2h , ] MW: 50.3 kDa DD: 91% 0.972 g (20% excess relative to polyglucosamine) 1 [ 2i , ] Same as 2g 3.754 g 3.2% of the amide / amine groups initially present in the polyglucosamine macromolecules (1.6% in step b) and 1.6% in step c) are generated. [ 2j , ] Same as 2h 3.953 g Same as 2i [ 2k , ] Same as 2g 82.12 g Same as 2e [ 2l , ] Same as 2h 112.40 g Same as 2e [ 2m , ] MW: 543.4 kDa DD: 60% 90 mg (20% excess relative to polyglucosamine) 1 [ 2n , ] MW: 501 kDa DD: 93% 98 mg (20% excess relative to polyglucosamine) 1 [2o , ] Same as 2m 4.60 g 4.0% of the amide / amine groups initially present in the polyglucosamine macromolecules (2.0% in step b) and 2.0% in step c) are generated. [ 2p , ] Same as 2n 5.00 g Same as 2o [ 2q , ] Same as 2m 68.72 g Same as 2e [ 2r , ] Same as 2n 115.53 g Same as 2e [ C1 , ] Glucosamine MW: 179.17 g / mol DD: 100% 113.66 g Same as 2e [ 2t , ] Same as 2a 4.908 g (20% excess relative to polyglucosamine) 1 [ 2u , ] Same as 2b 5.275 g (20% excess relative to polyglucosamine) 1 [ 2v , ] Same as 2a 13.025 g Same as 2c [ 2w , ] Same as 2b 13.988 g Same as 2c [ 2x , ] Same as 2a 82.40 g Same as 2e [ 2y , ] Same as 2b 129.89 g Same as 2e [ 2z , ] Same as 2g 1.065 g (20% excess relative to polyglucosamine) 1 [ 2aa , ] Same as 2h 1.122 g (20% excess relative to polyglucosamine) 1 [ 2ab , ] Same as 2g 4.335 g Same as 2i [ 2ac , ] Same as 2h 4.565 g Same as 2i [ 2ae , ] Same as 2g 94.84 g Same as 2e [ 2af , ] Same as 2h 129.81 g Same as 2e [ 2ag , ] Same as 2m 104 mg (20% excess relative to polyglucosamine) 1 [ 2ah , ] Same as 2n 113 mg (20% excess relative to polyglucosamine) 1 [ 2ai , ] Same as 2m 4.233 g Same as 2i [ 2aj , ] Same as 2n 4.591 g Same as 2i [ 2ak , ] Same as 2m 79.37 g Same as 2e [ 2al , ] Same as 2n 133.42 g Same as 2e [ 2am , ] Same as 2n α,β-Dimethylpropenyl chloride (CAS [49651-33-4], MW 118.56 g / mol, 5.207 g) Same as 2i [ 2an , ] Same as 2n 2-Methylenedodecanoic acid chloride (CAS [52756-21-5], MW 230.77 g / mol, 10.135 g) Same as 2i [ 2ao , ] Same as 2n (Z)-Uric acid chloride (CAS [91788-89-5], MW 138.13 g / mol, 6.066 g) Same as 2i [ 2ap , ] Same as 2n (Z)-2-chloro-2-butenyl chloride (CAS [56030-36-5], MW 138.98 g / mol, 6.104 g) Same as 2i [ 2aq , ] Same as 2n Mucoconic acid chloride (CAS [58823-55-5], MW 179.00 g / mol, 3.931 g) Same as 2i [ 2ar , ] Same as 2n (Z)-2-methylhept-2-ene-6-yneylchloro(CAS [313217-54-8], MW 156.61 g / mol, 6.878 g) Same as 2i [ 2as , ] Same as 2n Maleic anhydride (CAS [108-31-6], MW 98.06 g / mol, 4.307 g) Same as 2i [ 2at , ] Same as 2h Same as 2j The initial temperature in steps b) and c) is -70°C and the reaction is carried out at -20°C for 10 days. [ 2au , ] Same as 2h 1 / 1000 serving is the same as 2j, 4 mg The reaction was carried out as described in steps b) and c) of the general method, but with a mass reduction of 1000-fold and in a sealed 5 ml reactor. The initial temperature in steps b) and c) was -70°C, and the reaction was carried out at +150°C for 30 minutes. [ 2av , ] exist [ 2fObtained from , ] The reaction system proceeds as described in step f). Under these conditions, the final amount of amino groups modified with propionic acid corresponds to half the number of amino groups initially present in the polyglucosamine macromolecule. [ 2ax , ] exist [ 2k Obtained from , ] same [ 2av , ] [ 2ay , ] Obtained in 2r same [ 2av , ] [ 2az , ] exist [ 2av Obtained from , ] 56.24 g acrylonitrile chloride The reaction proceeds as described in steps b), c), and f). Under these conditions, 75% of the amino groups initially present in the polyglucosamine macromolecule undergo propionic acid modification. [ 2ba , ] Obtained in 2ax 41.05 g acrylonitrile chloride Same as 2az [ 2bb , ] Obtained in 2ay 57.77 g acrylonitrile chloride Same as 2az [ 2bc , ] Obtained in 2r The reaction was carried out as described in step f), but saponification was initiated at 0°C and continued at this temperature for 10 days, with the amount of alkali and the amount of acrylamide chloride used being equal in moles. Under these conditions, the final amount of amino groups modified with propionic acid corresponds to half of the amino groups initially present in the polyglucosamine macromolecule. [ 2bd , ] Obtained in 2r The reaction was carried out as described in step f), but saponification was started at 0°C and continued for 30 minutes at 140°C and pressure, with the amount of alkali and the amount of acrylamide chloride used being equal in moles. Under these conditions, the final amount of amino groups modified with propionic acid corresponds to half of the amino groups initially present in the polyglucosamine macromolecule. [ 2be , ] Obtained in 2r The reaction system was carried out as described in step f), but saponification was initiated at 0°C and continued at 0°C for 10 days, with the amount of alkali relative to acrylamide chloride being 10 equivalents. Under these conditions, the final amount of amino groups modified with propionic acid corresponds to half of the amino groups initially present in the polyglucosamine macromolecule. [ 2bf , ] Obtained in 2r The reaction was carried out as described in step f), but saponification was initiated at 0°C and continued for 30 minutes at 140°C and pressure, with the amount of alkali being 10 equivalents relative to the acrylamide chloride used. Under these conditions, the final amount of propionic acid-modified amines corresponded to half of the amines initially present in the polyglucosamine macromolecules, and the acetyl groups underwent some saponification. [ 2bg , ] Same as 2a 143 g acrylonitrile chloride The reaction proceeds as described in step b). Under these conditions, all the amino groups initially present in the polyglucosamine macromolecule undergo modification with acrylic acid to form acrylamide containing double bonds. [ 2bh , ] 2000 kDa, DD 10% 3.278 g of acrylamide chloride and LiCl in a 10% solution of N,N-dimethylacetamide as solvent. The reaction proceeds as described in steps b) and c). Same as 2i. [Example] [3] [:] [Characteristics of the plurality of nanoparticles of polyglucosamine in this invention]

[0218] The 3.2% cross-linked 501 kDa polyglucosamine prepared as described above ( [Example] [2i] The sample was adsorbed onto a copper grid coated with formvar / carbon, held for 30 seconds, and stained with uranium acetate for 5 seconds. Then, a micrograph of the stained material was obtained using a Jeol 1400 transmission electron microscope and a Veleta camera (SIS, Germany) (Fig. 1A). The micrograph shows that most of the formulation consists of spherical particles with an approximate diameter of 15 nm, similar to the size of small mammalian viruses. Some larger particles, with diameters in the range of 60-80 nm, corresponding to the diameter of medium-sized virus particles, were also observed.

[0219] Additionally, a 1 mm layer was formed by pouring a solution of 2% polyglucosamine in a 1% succinic acid aqueous solution into a Petri dish, and then drying it to constant weight with phosphoric anhydride at room temperature to obtain a standard linear polyglucosamine (MW: 501 kDa, 93% DD) (Bioavanta) and a cross-linked polyglucosamine according to the present invention made from the same polyglucosamine. [Example] [2i] ) dried membranes were then analyzed in semi-contact mode using a Solver Bio atomic force microscope (NT-MDT, Zelenograd, Russia). A three-dimensional image of a 100 μm × 100 μm membrane surface region of a standard polyglucosamine sample (MW: 501 kDa, 93% DD) showed a smooth surface and that the membrane was likely collinearly arranged with the linear polyglucosamine polymer. [picture] [1B1]), and correspondingly [Example] The surface region of the cross-linked polyglucan sample [2i] is uneven, with "peaks" and "valleys", which is formed by the aggregation of spherical structures. [picture] [1B2]). [picture] [1B3] and [B4] The membrane, containing bacterial nanocellulose containing a solution of 1% w / w cross-linked polyglucosamine according to the present invention in water, was examined by atomic force microscopy. AFM measurements were performed in semi-contact mode in air (T=26°C, RH=15%) under standard indoor conditions on a Solver Next scanning probe microscope (NT-MDT, Russia). Optimal pressure (SetPoint) was obtained using a ScanAsyst-AIR AFM probe (Bruker, USA) with a cantilever stiffness of approximately 0.4 N / m, a resonant frequency of approximately 70 kHz, and a nominal radius of curvature of 2 nm (AFM probe sharpness). All measurements were performed at a scan rate of 0.5 Hz and a resolution of 512 × 512 points. Post-processing was performed using ImageAnalysis 3.5 by subtracting tilted planes and removing measurement defects ("adhesion"). The obtained... [picture] [1] [B3] and Analysis of [B4] reveals that the spherical structure is uniformly distributed on the sample surface. [picture] [1] [B3] and [B4] ), with an average spherical diameter of 20 nm, which corresponds to the size of small viruses such as Caliciviridae, Picornaviridae, and Parvoviridae. The presence of the structure observed in AFM was confirmed by data from previous transmission electron microscopy examinations. [Figure] 1 [A]).

[0220] Under an inert atmosphere (e.g., argon), the differences between standard polyglucosamine (negative control) and the cross-linked polyglucosamine of this invention were further investigated by reflux acid hydrolysis. [Example] [2i]) and cross-linked glucose (comparative example C1) (positive control) structural differences, this reflux acid hydrolysis method can accurately analyze low molecular weight substances to identify marker substances formed only in the case of successful cross-linking as follows:

[0221] Under vigorous stirring, 100 mg of each sample was added to a solution of acetyl chloride in 5 ml of dichloromethane cooled to -20°C. The mixture was then heated to 0°C and maintained with vigorous stirring for 30 minutes. Diisopropylethylamine (1 ml) was added, and the mixture was heated to room temperature and maintained with vigorous stirring for another 30 minutes. Subsequently, the resulting hydrochloride was neutralized, allowing for washing with water to remove any interfering substances, and the presence of a non-nucleophilic strong base allowed for as complete acetylation as possible. All solvents were carefully evaporated under vacuum, and the residue was washed three times with 1 ml of water sample. The wash water was discarded, and 10 ml of 28% hydrochloric acid was added to the solid precipitate and refluxed for 2 hours. The reaction mixture was carefully evaporated under vacuum, and the residue (approximately 100 mg) was resuspended in 100 ml of 2% acetic acid aqueous solution. The sample was now ready. Subsequently, under the conditions of this invention, the polymer was completely hydrolyzed to glucosamine sugars and glucosamine sugar derivatives. Therefore, since it has not been found in natural chitin and polyglucosamine... [Mode] [(II)] [The substance] can only be obtained by reacting with the remaining acrylic acid via a azira-Michael reaction, and therefore the detection of this substance can be used to identify cross-linked polyglucosamine sugars prepared according to the method of the present invention.

[0222] If any acrylamide chloride of formula (I) (e.g., any acid chloride of acrylic acid used in the crosslinking step according to the present invention) is used as a crosslinking agent (e.g., for...) [2i] [Example] After preliminary sample preparation (e.g., acetylation, washing), the reflux acid hydrolysis of the resulting polymer not only hydrolyzes the glycosidic bonds between glucosamine units, but also hydrolyzes the acetylglucosamine bonds produced during acetylation with a crosslinking agent (e.g., activated to acetylate substituted or unsubstituted acrylic acid derivatives), thereby forming a glucosamine and propionic acid derivative of formula (IIIa): [Chemical Formula (IIIa)] R2 to R4 are as defined in this application, specifically, if unsubstituted amide-activated acrylic acid is used, they are derivatives of glucosamine and propionic acid of formula (II): [Chemical Formula (II)]

[0223] This substance is hydrolyzed stable and must be present in the mixture when initial cross-linking occurs. If other cross-linking agents are used, similar derivatives (IIIa) are formed. In the case of highly cross-linked reactions, formula ( The detection of derivatives of [IIIb] can be used to identify cross-linked polyglucosamine sugars prepared according to the method of the present invention. [Chemical Formula (IIIb)] R2 to R4 are as defined herein, and R2' to R4' are as defined herein with respect to R2 to R4, wherein R2' to R4' may be the same as each of the corresponding R2 to R4, or may be different when the acrylic compound of formula (I) used in subsequent steps b) to c) of the method of the present invention is different from the compound used in the first step.

[0224] To confirm the presence of crosslinks in the product generated by the method of this invention, the sample solution prepared as described above was used directly in mass spectrometry analysis (Bruker Daltonics TOF 180 mkl / h, tune_low.m, ES pos. scan, N2-4 l / min, 190℃, Nb.=0.4, spectral time 1 second), without prior chromatography. A comparative standard polyglucosamine (…) [picture] [4] [A2]) and the cross-linked polyglucosamine sugar of the present invention ( [picture] [4] In the mass spectrum of the ion region of interest (lower m / z ratio) of [B2], a glucose-specific peak (m / z = 180.073) was observed, reflecting the presence of intact glucose residues. However, for the cross-linked polyglucosamine of this invention, a new peak with m / z = 252.077 was observed. [picture] [4B2]), corresponding to the product of formula (II) produced by alkylation of aminoglucose via propionic acid. In this example, the peak intensity is low, reflecting a low percentage of crosslinking in the sample (e.g., 3.2% crosslinking as determined by reaction condition 2i). This is a characteristic peak confirming successful polyglucosamine crosslinking according to the method of the present invention. It is present in all samples (including comparative glucosamine positive controls) obtained by the method of the present invention under various suitable conditions described in Table 1 above and is not present in all polyglucosamines and chitin tested.

[0225] To determine the attribution of the peaks at m / z = 180 and m / z = 252 to glucosamine and compound (II), collision studies were conducted at different impact energies (AB SCIEX Triple Quad™ 3500) (5V and 15V). In this case, a pretreated, dried residue (hydrolyzed and then evaporated under vacuum) was dissolved in an aqueous solution containing 10% methanol and 0.1% formic acid. When the compound at m / z = 180 fractured at higher impact energies (Sciex instrument), spectra with water losses (-18 m / z, -2*18 m / z, -3*18 m / z) were observed. [picture] [5A] [and] [5B] ). As the collision energy increases, the intensity of the water loss peak also increases. This phenomenon is characteristic of carbohydrates. Similar patterns are observed for ions, i.e., compounds with m / z=252. [picture] [5B] [and] [5C] However, as a derivative of carbohydrates, it is more stable for water loss at the same collision energy. Therefore, the observed peak representing water loss has a lower relative intensity.

[0226] After adding 500 μl of a 10 mg / ml solution of lysozyme in sodium acetate buffer (pH 5.5) to 5 mg of the cross-linked polyglucosamine of the present invention, the 3.2% cross-linked 501 kDa polyglucosamine of the present invention prepared as described above was monitored by mass spectrometry. [Example] [2i] ) stability. The mixture was incubated on a vibrator at 37°C for 7 days until no further changes in the mass spectrum were observed. At this stage, the mass spectrometry revealed the characteristic "comb" pattern of the high molecular weight compound: regularly distributed peaks with regularly increasing intensity at m / z ratios of 955-958, 1023-1024, 1101-1104, and 1193-1196. These peaks correspond to the same fragments with different charge units. For a given mass, the ion charges are +15, +14, +13, and +12. The mass of the major fragment is 14,305 Da. Similar results were obtained for samples incubated for a longer period of time, up to 3 weeks. This demonstrates that the hydrolytic enzymes that cause the hydrolysis of polyglucosamine and its derivatives in egg white lysozyme and warm-blooded animal tissues can only hydrolyze cross-linked polyglucosamine into relatively large fragments with a mass of about 14 kDa. Therefore, this indicates that using higher molecular weight deacetylated polyglucosamine to prepare multiple nanoparticles will result in greater stability against enzymatic hydrolysis.

[0227] Therefore, these data confirm that there is a significant structural difference between the cross-linked polyglucosamine of the present invention, which forms multiple nanoparticles, and the standard polyglucosamine used as the starting material. Furthermore, the detection method based on the substance of formula (II) or related substances exhibits high sensitivity and specificity in detecting multiple nanoparticles obtained by the method of the present invention. [Example] [4] [:] [The Polyglucosamine Sugar of this Invention] [Multiple] [Internalization of nanoparticles into cells]

[0228] Using fluorescence microscopy, the ability of multiple nanoparticles of the polyglucosamine of the present invention, which are intended to be internalized by cells, to penetrate the interior of the mouse cornea was tested using fluorescein isothiocyanate (FITC) and 5-(and 6-)carboxyfluorescein (FAM) labeled polyglucosamine.

[0229] Fluorescently labeled cross-linked polyglucosamines were prepared using an initial standard polyglucosamine with a molecular weight of 501 kDa and a degree of deacetylation of 93%, or 3.2% of the cross-linked polyglucosamine according to the present invention (Example 2p). Pre-cooled DMF (1 ml) in an ice bath was added to 100 mg of each polyglucosamine in an Eppendorf tube, and the suspension was allowed to swell for 1 hour. Next, a solution of 2.85 mg of luciferin-5-isothiocyanate (FITC) or 5-(and 6-)carboxyluciferin succinimide (NHS-luciferin) in 250 μL of DMF was added to an ultrasonic bath filled with ice water and ultrasonically treated. The temperature in the bath was maintained at 0°C for the first twenty minutes, and then the bath was heated to 40°C and reacted at this temperature for 100 minutes. The contents of each tube were transferred to 12-14 kDa dialysis tubes and dialyzed against water. After 20 dialysis cycles against 100 ml of water, 50 mg of succinic acid was added to each dialysis tube to dissolve the polyglucosamine, and the dialysis cycle was repeated. The resulting solution was lyophilized and then resuspended to obtain a 0.25% solution.

[0230] Mouse white blood cells were incubated with this solution at 37°C for 4 hours, stained with a DNA-specific dye (Hoechst 33258), and analyzed by fluorescence microscopy. Fluorescence analysis ( [picture] [2] ) shows that green fluorescence (C) is uniformly distributed throughout the cytoplasm, indicating that the FITC-labeled cross-linked glucosamine is inside the cell and has therefore been internalized (the simple association of the labeled glucosamine with the cell membrane will produce a fluorescent ring at the cell boundary (A)). The blue fluorescence observed outside the nucleus (D) may be caused by the initiation of apoptosis.

[0231] A solution of cross-linked polyglucosamine labeled with the same fluorescein isothiocyanate (FITC) and 5(6)-carboxyfluorescein (FAM) (0.25%) was applied to the eyes of CBA mice (one drop per eye). Six hours later, the animals were euthanized, and their eyes were fixed in formalin and stained as described in Stradleigh et al., 2015, Prog Retin Eye Res., 48:181-202, and analyzed by fluorescence microscopy. It was observed that the standard polyglucosamine preferentially concentrated on the surface of the cornea and sclera. [picture] [3A]), and the multiple nanoparticles (=bright green fluorescence) of the polyglucosamine of the present invention penetrate uniformly into the deep part of the cornea. [picture] [3B]).

[0232] These data confirm that the multiple nanoparticles of the cross-linked polyglucosamine of the present invention can be used to increase the cell penetration ability of active ingredients with weak cell permeability (hydrophilic) or no cell permeability (macromolecule) through endocytosis mediated by multiple nanoparticles to reach intracellular locations and / or through endocytosis transport mediated by multiple nanoparticles to potentially cross cells. [Example] [5] [:] [Biocompatibility of the plurality of nanoparticles of the polyglucosamine in this invention]

[0233] Evaluation of the cross-linked polyglucan of the present invention in in vitro cultures of human bone marrow mesenchymal stem cells (MSCs). [Example] [2i] ) to assess its biocompatibility in order to evaluate its potential impact on the culture and growth of MSCs, which are collected from healthy donors via bone marrow aspiration as described in Bieback et al., 2008, Transfus Med Hemother., 35(4):286-294, doi:10.1159 / 000141567.

[0234] As described by Bieback et al., 2008, MSCs were isolated by density gradient centrifugation at 1,077 g / ml, washed twice, and grown in DMEM medium containing 10% Mesecult fetal serum, L-glutamic acid, and antibiotics. Flow cytometry was used to verify the purity of the cultures. The cultured MSCs exhibited the following phenotype: CD44+ / CD73+ / CD90+ / CD105+ / CD34 / HLA-DR-, and demonstrated chondrogenic, osteogenic, and adipogenic differentiation capabilities upon the addition of specific media, as described by Ciuffreda et al., 2016, Methods Mol Biol. 2016; 1416:149-58. doi: 10.1007 / 978-1-4939-3584-0_8. This study used four passages of cells.

[0235] A membrane formed from cross-linked polyglucosamine was used to cover the walls of 25 cm² culture dishes. The dishes were dried, washed twice with growth medium, and then inoculated with cells. Untreated culture dishes of the same size were inoculated in a similar manner for comparison. The presence of cross-linked polyglucosamine on the plastic walls of the culture dishes did not interfere with cell attachment or growth. Within 3–4 hours after inoculation, cells diffused and exhibited their characteristic spindle shape. The growth rate was slightly slower in the cross-linked polyglucosamine-treated dishes. Consolidation was achieved in standard culture dishes after 48–56 hours and in cross-linked polyglucosamine-treated dishes after 68–76 hours.

[0236] In summary, the cross-linked polyglucosamine of this invention maintains the growth and proliferation of human bone marrow mesenchymal stem cells, although its efficiency is lower than that under standard conditions. [Example] [6] [:] [Uses of the Polyglucosamine of this Invention in Agricultural Applications]

[0237] The following tests were conducted on the cross-linked polyglucosamine sugar of the present invention. [Example] [2i]) Effectiveness of the invention for spring wheat seeds. Two different concentrations of the cross-linked polyglucosamine of this invention were prepared in water. Seeds were immersed in these suspensions, and depending on the treatment applied, the experiments included the following seed groups: [(A)] Control group (seeds untreated); [(B)] Treatment with 0.1% (w / w) cross-linked polyglucan, [(C)] Treated with 0.05% (w / w) cross-linked polyglucan. [ Assessment of growth-promoting effects in the initial stages of organogenesis , ] [ , ]

[0238] After 24 hours of treatment, seeds were placed on moist substrate (filtered with a cotton layer) in Piper dishes (12 seeds per dish, quadruplicate) under natural light at t = +20-22°C. Germination energy was measured as described by Domin et al., 2020, Sustainability 2020, 12(1), 46, 1 day and 3 days after contact with the moist substrate, and germination rate (number of germinated seeds divided by the total number of tested seeds minus the number of ungerminated seeds) was measured 7 days after contact with the moist substrate. Growth indicators of the initial stage of organ formation were recorded 3 days after contact with the moist substrate (root length of each seedling, total length of the seedling) and 7 days after contact with the moist substrate (number of roots of each seedling, length of the seedling, total mass of roots and shoots of each seedling, and mass of each root).

[0239] like [picture] [6] shows the germination and lower [surface] [2] outlines the positive effects of treatment with the cross-linked polyglucosamine of the present invention at the lowest concentration on seedling development during the initial stage of organogenesis. [Table 2] [Growth Duration] [Growth Indicators] [A] [B] [C] 3 days Number of roots per seedling 2,9 3,0 3,0 Root length of each seedling, cm 7,79±0,27 8,64±0,46 10,20±0,94 Length of individual roots, cm 2,60±0,12 2,86±0,08 3,4±0,11 Length of the bud, cm 1,84±0,09 2,29±0,03 2,69±0,30 7 days Number of roots per seedling 4,41 4.68 4,83 Length of the bud, cm 10.05±0.75 10,11±0,69 11,27±0,26 Air-dried biomass of the roots of each seedling, mg 8,20±0,07 9,60±0,56 9,40±0,20 Air-dried biomass of one root, mg 1,86±0,02 2,06±0,15 1.95±0.08 Air-dried biomass of one seedling, mg 15,13±0,13 17,25±0,19 16,45±0,41

[0240] A higher rate of young root emergence was observed within one day of treatment with cross-linked polyglucosamine (CRP). The strongest effect was observed with low-concentration suspensions (22.9% increase with 0.05% CRP; 14.6% increase with 0.1% CRP; and 75% baseline control). The same trend was observed when germination energy (6.2% increase) and germination capacity were measured (control group = 93.8%). Treatment of seeds with high-concentration CRP suspensions increased germination capacity by 4.1%. In the early stages of organogenesis, CRP increased root growth. After 3 days, the total root length increased by 10.9% and 30.9% in the 0.1% and 0.05% CRP groups, respectively (control group = 7.79 cm). Meanwhile, the most uniform increase was observed in the high-concentration crosporoglucosamine group (uniformity coefficient of 0.1% crosporoglucosamine = 81% (n=45); 0.05% crosporoglucosamine = 78.2% (n=48); control group = 68.9% (n=45)). The same trend was observed when seedling height was measured. If the increase after 0.05% crosporoglucosamine treatment (46.2% increase compared to the control group = 1.84 cm) was significantly higher than the increase after 0.1% crosporoglucosamine treatment (24.4% increase compared to the control group), then the uniformity coefficient of the high-concentration crosporoglucosamine treatment group was higher (0.1% crosporoglucosamine = 75.4%; 0.05% crosporoglucosamine = 72.8%; control group = 61.7%).

[0241] Based on measurements taken 7 days after the start of the experiment, the low concentration of cross-linked polyglucosamine (CRP) treatment showed the strongest ability to enhance root formation (0.05% CRP: 9.5% increase; 0.1% CRP: 6.1% increase; average number of roots per seedling in the control group: 4.41). The same finding was observed regarding the increase in overall seedling length (0.05% CRP: 7.7% increase; control group: 10.05 cm). In contrast, treatment with higher concentrations of CRP suspension resulted in the highest increases in biomass observed for roots (0.1% CRP: 17.1% increase; 0.05% CRP: 14.6% increase) and for the entire seedling (0.1% CRP: 12.9% increase; 0.05% CRP: 8.7% increase). [ Assessment of the impact on the growth and development of spring wheat seedlings in the soil substrate , ] [ , ]

[0242] Seven days prior to sowing, seeds were treated with alkaline chernozem (500 g / plastic container / 12 containers, replicated 4 times), under natural light, at t = 20-22℃, and with controlled soil moisture. Germination energy, germination rate, seedling height, and biomass were measured. Growth parameters during the first 7 days were recorded. For seeds treated with cross-linked glucosamine, especially at higher concentrations, a higher rate of root emergence was observed after 24 hours (control group: 70.8%, relative increase in treated seeds: 27.1% and 23%). See below. [surface] [3] and [picture] [6] As shown, 100% germination was obtained after treatment with high concentration of cross-linked polyglucosamine. [Table 3] index experimental group [A] [B] [C] Germination energy, % 1 day later Young roots appear 70,8 97,9 93,8 2 days later Seedlings that germinate normally % 95,9 93,8 100 3 days later 97,9 100 83,4 germination,% 7 days later 93,8 100 83,4 The length of a normally developing bud (cm) 2 days later 0.71±0.05 1,11±0,05 0.85±0.03 3 days later 2,87±0,13 3,40±0,13 2,92±0,06 7 days later 18.95±0.30 20,48±0,24 19,31±0,28 Air-dried biomass, mg Total seedlings 16.65±0.32 18.95±0.41 20,90±0,37 The root of each seedling 6,93±0,14 8,53±0,23 9,33±0,22

[0243] The higher initial rate of growth stimulation induced by 0.1% crosporoglucosamine treatment (56.3% after 2 days) decreased 3-fold after 3 days (11.6-fold decrease with 0.05% crosporoglucosamine) and 2.3-fold after 7 days. The increase in biomass after 7 days compared to the control group (16.63 mg) was higher in the 0.1% treatment group (14% increase in total biomass and 23.8% increase in roots) and the 0.05% treatment group (25.7% increase in total biomass and 35.1% increase in roots). The 0.05% crosporoglucosamine treatment increased total biomass by 1.8-fold and root biomass by 1.5-fold.

[0244] In summary, these data confirm that treatment with the cross-linked polyglucosamine of the present invention induces a significant growth-stimulating effect in spring wheat germination.

Claims

1. A novel method for synthesizing intramolecularly cross-linked polyglucosamine sugars, comprising the following steps: a) providing a polyglucosamine sugar and swelling the polyglucosamine sugar in a solvent; b) aminoating the polyglucosamine sugar with an acrylic compound of formula (I): [Chemical Formula (I)] R1 is a halogen or a group selected from 3-hydroxybenzotriazole ester, anhydride, N-hydroxysuccinimide, pentachlorophenol, and 2-nitro-4-sulfonylphenol ester; R2, R3, and R4 are independently selected from H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C3-C6 alkynyl, optionally substituted cycloC3-C8 cycloalkyl, optionally substituted C4-C8 cycloalkenyl, optionally substituted C8 cycloalkynyl, optionally substituted heterocyclic alkyl; optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted aryl C1-C6 alkyl; c) subjecting the acetylation product of step b) to an aza-Michael reaction in the presence of a base; and d) purifying the cross-linked polyglucosamine sugar obtained from step c); wherein... The term "substituted" refers to a group being substituted by one to five substituents selected from the group consisting of: C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, heterocycloalkyl, C1-C6 alkylaryl, C1-C6 alkyl heteroaryl, aryl C1-C6 alkyl, heteroaryl C1-C6 alkyl, C1-C6 alkylcycloalkyl, C1-C6 alkyl heterocycloalkyl, amino, aminosulfonyl, ammonium, acetylammonium, aminocarbonyl, aryl, heteroaryl, sulfinyl, sulfonyl, alkoxy, alkoxycarbonyl, carbamate, thioalkyl, halogen, trihalomethyl, cyano, hydroxyl, mercapto, and nitro.

2. A novel method for synthesizing intramolecularly cross-linked polyglucosamine sugars as described in claim 1, wherein R1 is selected from 3-hydroxybenzotriazole ester, anhydride, N-hydroxysuccinimide, pentachlorophenol, and 2-nitro-4-sulfonylphenol ester.

3. A novel method for synthesizing intramolecularly cross-linked polyglucosamine sugars as claimed in claim 1, wherein the method further comprises a drying step of the product obtained in step d).

4. A novel method for synthesizing intramolecularly cross-linked polyglucosamine sugars as claimed in claim 1, wherein the polyglucosamine sugar provided in step a) has a value in the range of about 5 to about 2,000 kDa.

5. A novel method for synthesizing intramolecularly cross-linked polyglucosamine sugars as described in claim 1, wherein R1 is a halogen.

6. A novel method for synthesizing intramolecularly cross-linked polyglucosamine sugars as described in claim 1, wherein the acrylic compound is selected from acrylonitrile chloride and methacrylonitrile chloride.

7. A novel method for synthesizing intramolecularly cross-linked polyglucosamine sugars as claimed in claim 1, wherein the method further includes a saponification step performed under alkaline conditions after step c) or d), and the resulting product may further undergo at least one of steps b) to c) (or d)).

8. A cross-linked polyglucosamine sugar or any medically acceptable salt thereof, which can be obtained by a novel method of synthesizing intramolecularly cross-linked polyglucosamine sugar as claimed in claim 1.

9. A cross-linked polyglucosamine sugar obtainable by a novel method for synthesizing intramolecularly cross-linked polyglucosamine sugars as claimed in claim 1, characterized in that it has a hydrolysis product of formula (IIIa) or formula (IIIb), the hydrolysis product being obtained by acidic hydrolysis of the cross-linked polyglucosamine sugar, wherein R2, R3, and R4 are as defined in claim 1, and R2', R3', and R4' are as defined in R2, R3, and R4. [Formula (IIIa)] [Formula (IIIb)] 10. The cross-linked polyglucosamine as claimed in claim 9, characterized in that, upon undergoing acidic hydrolysis, it has a mass spectrum containing a peak of electrospray positive ions at m / z = 252.077 ± 0.

01.

11. A composition comprising at least one cross-linked polyglucosamine as claimed in claim 8 and at least one carrier.

12. The composition as claimed in claim 11, wherein the composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier, or the composition is a cosmetic composition and the carrier is a cosmetically acceptable carrier.

13. The composition as claimed in claim 11, wherein the composition is a cell or tissue culture medium, the culture medium further comprising cell or tissue nutrients.

14. The composition as described in claim 11, wherein the composition is a reconstructed tissue.

15. The composition as claimed in claim 11, wherein the composition is an agricultural composition.

16. A soft tissue filler, a wound dressing, or a reconstructed tissue comprising at least one cross-linked polyglucosamine sugar or a composition thereof as claimed in any one of claims 8 to 11.

17. A plurality of nanoparticles comprising at least one cross-linked polyglucan according to any one of claims 8 to 11.

18. A cell or biological tissue culture medium comprising at least one cross-linked polyglucan as claimed in any one of claims 8 to 11.

19. Use of any one of claims 8 to 11 of a cross-linked polyglucosamine sugar for the preparation of a cell or biological culture medium or a reconstructed tissue.

20. Use of any cross-linked polyglucosamine sugar or a composition thereof as claimed in any one of claims 8 to 11 in the preparation of a medicament for treating a disease, wherein the disease is selected from the following: cardiovascular conditions, arrhythmias, atrial fibrillation, and hypertension; joint diseases and arthritis, rheumatoid arthritis, osteoarthritis, spondyloarthritis, and traumatic events resulting in damage to cartilage, bone, ligaments, or bursae; eye diseases and injuries, dry eye syndrome, uveitis, glaucoma, and corneal diseases; connective tissue diseases, lupus, and polymyositis; skin / mucous membrane diseases or injuries, trauma, scars, psoriasis, acne, eczema, rosacea, physical or chemical burns, surgical trauma, sunburn, ulcers, and hemorrhoids; periodontal and dental diseases; spinal cord injuries; malignant and benign tumors, carcinomas, sarcomas, lymphomas, and melanomas; fistulas and infections; tumors or vascular malformations.

21. A method for preparing a drug delivery system for a bioactive agent, the method comprising the steps of: - providing a cross-linked polyglucosamine as claimed in claim 8 in a wet or dry state; - providing a bioactive agent to be delivered; - dissolving the bioactive agent in a solvent or a supercritical fluid; - loading a plurality of nanoparticles with the cross-linked polyglucosamine by impregnating the cross-linked polyglucosamine with a solution of the bioactive agent or by directly introducing it into a plurality of nanoparticles by electrophoresis or by acceleration by an electric field; - collecting the obtained composition or plurality of nanoparticles loaded with the bioactive agent.

22. A method for identifying cross-linked polyglucosamine sugars obtainable by any one of claims 1 to 4, the method comprising the steps of: - providing a cross-linked polyglucosamine sugar to be characterized in a solvent; - lactating the cross-linked polyglucosamine sugar under stirring; - neutralizing the reaction medium with a base; - evaporating the solvent and washing the obtained neutralized product; - subjecting the product to a reflux acid hydrolysis; - evaporating the reaction mixture and resuspending the hydrolysis product in a weak acid; - determining the presence or absence of a product selected from products according to formula (IIIa) or according to formula (IIIb) [Chemical Formula (IIIa)]; [Chemical Formula (IIIb)]; Wherein R2 to R4 are as defined in any one of claims 1 to 4 above, R2' to R4' are as defined in R2 to R4 in any one of claims 1 to 4 above, and the presence of one of such products (IIIa) and / or (IIIb) indicates cross-linked polyglucan obtained by a method as described in any one of claims 1 to 4.

23. The identification method as described in claim 22, wherein the determination of the presence or absence of the product according to formula (IIIa) or formula (IIIb) is made by mass spectrometry electrospray analysis.

24. The method for identifying cross-linked polyglucosamine sugar as described in claim 22, wherein the product according to formula (IIIa) has the following chemical formula (II). [Chemical Formula (II)]

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