New synthesis of chitosan derivatives and uses thereof

Abstract

The present invention relates to a novel cross-linked chitosan and formulations, compositions and uses thereof. In particular, the present invention relates to nanoparticles useful as active agents and delivery systems for at least one biologically active agent and compositions thereof.

Description

Technical Field

[0001] This invention relates to the preparation of chitosan, particularly to nanoparticles prepared from chitosan, their preparation methods, compositions and uses, especially in the fields of chemistry, biology, medicine and materials science. Background Technology

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

[0003] Chitosan is one of the most promising biopolymer candidates for the fabrication of nanoparticles. On the one hand, it possesses attractive natural properties such as mucosal adhesion, biocompatibility, and low toxicity, enabling it to be biodegraded and form complexes; on the other hand, its highly regular structure and the presence of hydroxyl and amino groups offer promising prospects for selective polymer modification.

[0004] Therefore, these properties of chitosan 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 chitosan has several advantageous properties as an excellent carrier, the use of unmodified chitosan is limited due to its low solubility under physiological conditions. To overcome this limitation, many different chemical modifications of chitosan 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, Chitosan-based biomaterials). Biomaterials, Vol. 2; edited by Jennings, J.A., Bumgardner, J.D.; Woodhead Publishing, Cambridge, UK, 2017; pp. 209–244.

[0006] The structure of chitosan offers the possibility of modification in a variety of ways. To increase hydrophobicity, chitosan amides can be readily prepared with fatty acids, sterol derivatives, uric acid, carboxylic acid derivatives containing imidazole fragments, etc. Alternatively, secondary chitosan amines can be prepared by alkylation using alkyl substituents of various structures, pyridine derivatives, spermidine, etc., or by reductive amination with appropriate Schiff bases. To increase hydrophilicity, chitosan amides can be formed with amino acids (including sulfur-containing amino acids, lactobionic acid, mercaptoacetic acid, etc.). Furthermore, secondary chitosan amines can be obtained using various sugars, polyethyleneimine, and their derivatives. The synthesis of polyethylene glycol derivatives via PEGylation is particularly popular. Chitosan derivatives produced according to these methods have an enhanced ability to form nanoparticles, especially with polyelectrolytes such as nucleic acids, and may possess altered pharmacological properties. (Mao et al., 2010, Advanced Drug Delivery Reviews, 62, 12-27 doi:10.1016 / j.addr.2009.08.004).

[0007] Chitosan nanoparticles can be formed in a largely irreversible manner through chemical crosslinking, which involves catalyzing the formation of covalent bonds using various reagents (Bhattarai et al., 2010, ibid.). Historically, glutaraldehyde and formaldehyde were the earliest and most popular reagents used for this crosslinking. However, these reagents are no longer popular due to the difficulty in removing traces of all these toxic compounds and the slow hydrolysis of the nanoparticles that releases these aldehydes. Genepine has recently gained considerable attention, but this reagent is very expensive and readily polymerizable. Diethyl squaric acid (DES), ethylene glycol diglycidyl ether (EGDE), and blocked diisocyanates react rather slowly, and the reactions mainly occur at high temperatures, which can lead to side reactions. Photoactivated crosslinking agents also exist: functionalized azides, functionalized acrylates, and enzyme activators—phloric acid and activated quinones. For example, Baoqiand et al., Acta Biomaterialia, 22, 1742-7061, or Journal of Nanotechnology in Engineering and Medicine, 6, 041001-6, provide a method for preparing cross-linked chitosan. This method involves reacting chitosan with methacrylic anhydride, followed by photo-initiated free radical polymerization of the chitosan in solution, resulting in a polyacrylamide compound in gel or foam form. El-Sherbiny et al., Polymer Journal, 45, 1, 199-210, 2009, describe a carboxylmethyl derivative of chitosan. In the first step, in an alkaline medium, carboxyl groups are introduced into chitosan through the reaction of primary alcohol groups with chloroacetic acid. In the second step, acryloylglycine polymer is introduced via photo-induced polymerization, yielding chitosan without internal cross-linking. US 5,770,712 describes a method for preparing cross-linked chitosan by combining a chitosan material having unreacted primary amine groups with an excess of a polyfunctional epoxy compound having at least two epoxy groups. The cross-linking reaction of chitosan with 1,4-butanediol diglycidyl ether results in an amino-ethanol cross-linked derivative of chitosan that is poorly soluble at physiological pH (7 or higher).

[0008] Pre-derived chitosan can also be crosslinked by forming a Schiff base with chitosan containing aldehyde groups, forming a disulfide bond in chitosan previously functionalized with a reagent having terminal thiol groups, and Michael addition to chitosan previously treated with functional acrylates in the presence of a weak base.

[0009] All these methods involve the formation of nanoparticles when chitosan is in solution. In some cases, all chemical reactions are carried out in the liquid phase; in others, they are completed at a later stage, such as during or after spray drying or lyophilization. Importantly, in all cases, the method begins and its critical stages are carried out with chitosan in a dissolved state (Jayakumar et al., 2011, ibid.). The requirement for the amount of chitosan to be dissolved significantly limits the synthesis of nanoparticles. This is because chitosan dissolves in water only as a salt in the presence of acid. In this form, for any solution higher than 2% chitosan, the viscosity increases anomalously with increasing concentration, a figure that severely limits the possible implementations of the synthesis and the possibility of obtaining high yields from the reaction apparatus. In particular, the wall effect when mixing viscous solutions leads to significant heterogeneity in reagent concentrations within the reaction vessel, resulting in heterogeneity in the properties of the nanoparticles. Another problem is that highly nucleophilic amino groups are most commonly used for crosslinking, but in solution they should be ionized to keep the chitosan in a dissolved state.

[0010] Therefore, new synthetic routes need to be developed to form chitosan nanoparticles in an efficient and cost-effective manner.

[0011] Invention Summary

[0012] This invention relates to an unexpected discovery of a novel method for producing nanoparticles from chitosan. The advantage of this method is that it avoids the need to dissolve chitosan raw materials, which would require the formation of salts or the use of dilute acid aqueous solutions, or the need to disperse chitosan in solution, which would require emulsification, grinding, or other procedures that could mechanically affect the integrity of the polymer. In particular, this invention relates to a novel method for crosslinking chitosan, specifically through two chemical steps to form new internal chemical bonds between different parts of the chitosan molecule. According to one particular aspect, the insolubility of chitosan leads to a more efficient synthetic conversion rate (e.g., about two orders of magnitude higher than crosslinking methods using dissolved chitosan).

[0013] The resulting product spontaneously forms nanoparticles at the end of the second step of the preparation method, which are suitable for a variety of applications. Furthermore, advantageously, the resulting cross-linked chitosan product exhibits similar bioactivity to standard linear or branched chitosan, but unexpectedly demonstrates some unique advantages in terms of stability and physicochemical properties under a wide range of biorelevant pH or microbial conditions, making it easier to handle.

[0014] In one aspect of the invention, a method for preparing cross-linked chitosan as described herein is provided.

[0015] In another aspect of the invention, there is a novel cross-linked chitosan, particularly a novel cross-linked chitosan that spontaneously forms nanoparticles.

[0016] In another aspect of the invention, a composition is provided comprising at least one cross-linked chitosan as described herein and at least one carrier.

[0017] In another aspect of the invention, there is a pharmaceutical composition comprising at least one cross-linked chitosan as described herein and at least one pharmaceutically acceptable carrier.

[0018] In another aspect of the invention, nanoparticles comprising cross-linked chitosan as described in the invention are involved.

[0019] In another aspect of the invention, a cosmetic composition is disclosed, comprising at least one cross-linked chitosan as described herein.

[0020] In another aspect of the invention, there is a soft tissue filler comprising at least one cross-linked chitosan as described in the invention or a composition thereof as described in the invention.

[0021] In another aspect of the invention, a wound dressing is provided, comprising at least one cross-linked chitosan as described in the present invention.

[0022] In another specific embodiment, a reconstructed tissue is provided, comprising at least one cross-linked chitosan or a composition thereof as described in this invention.

[0023] In another aspect of the invention, there is an agricultural composition comprising at least one cross-linked chitosan as described herein.

[0024] In another aspect of the invention, there is a method for preparing a composition comprising at least one cross-linked chitosan or nanoparticles thereof as described in the present invention (e.g., soft tissue filler, wound dressing, or reconstructed tissue).

[0025] In another aspect of the invention, there is a connection to cross-linked chitosan as described herein for in vivo drug delivery, in vitro cell or biological tissue culture, and tissue engineering applications.

[0026] In another specific embodiment, a cell or biological tissue culture medium is provided, which comprises at least one cross-linked chitosan or a combination thereof as described in this invention.

[0027] In another aspect of the invention, there is a connection to cross-linked chitosan as described herein for the prevention and / or treatment of medical conditions selected from: particularly cardiovascular diseases, such as arrhythmias, especially 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 synovial bursae; eye diseases and injuries, such as dry eye, uveitis, glaucoma, and corneal diseases; connective tissue diseases, such as lupus and polymyositis; skin / mucous membrane diseases or injuries, such as wounds, scars, psoriasis, acne, eczema, rosacea, physical or chemical burns, especially surgical wounds and sunburn, ulcers, and hemorrhoids; periodontal and dental diseases; dura mater injuries, such as dura mater injuries following accidental injury or surgery on the brain and central nervous system; malignant and benign tumors, particularly carcinoma, sarcoma, lymphoma, and melanoma; postoperative complications such as fistulas and infections; and tumors or vascular malformations.

[0028] In another aspect of the invention, the use of cross-linked chitosan as described herein in the preparation of pharmaceutical formulations for the prevention and / or treatment of medical diseases, particularly cardiovascular diseases such as arrhythmias, especially 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 synovial bursae; eye diseases and injuries such as dry eye, uveitis, glaucoma, and corneal diseases; connective tissue diseases such as lupus and polymyositis; skin / mucous membrane diseases or injuries such as wounds, scars, psoriasis, acne, eczema, rosacea, physical or chemical burns, especially surgical wounds 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, particularly carcinoma, sarcoma, lymphoma, and melanoma; postoperative complications such as fistulas and infections; and tumors or vascular malformations.

[0029] In another aspect of the invention, there is a connection to the use of cross-linked chitosan as described herein for the preparation of cells or biological culture media or for the reconstruction of tissues.

[0030] In another aspect of the invention, a method for preparing a drug delivery system for bioactive agents is disclosed.

[0031] In another specific embodiment, a method for identifying cross-linked chitosan obtained according to the method of the present invention is provided.

[0032] In another aspect of the invention, methods for preventing, treating, or improving medical diseases, particularly cardiovascular diseases such as arrhythmias, especially 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 synovial bursae; eye diseases and injuries such as dry eye, uveitis, glaucoma, and corneal diseases; connective tissue diseases such as lupus and polymyositis; skin / mucous membrane diseases or injuries such as wounds, scars, psoriasis, acne, eczema, rosacea, physical or chemical burns, especially surgical wounds and sunburn, ulcers, and hemorrhoids; periodontal and dental diseases; dura mater injuries, such as those following accidental injury or surgery on the brain and central nervous system; malignant and benign tumors, particularly carcinoma, sarcoma, lymphoma, and melanoma; postoperative complications such as fistulas and infections; tumors or vascular malformations, wherein the methods comprise administering to a subject in need an effective amount of at least one cross-linked chitosan or a pharmaceutical preparation thereof as described in the invention.

[0033] In another aspect of the invention, there is a kit comprising at least one cross-linked chitosan or a composition thereof as described in the invention, for example in lyophilized form.

[0034] In another aspect of the invention, a kit is provided for preparing encapsulating materials such as bioactive agents, pharmaceuticals, proteins, antibodies, sugars, nucleic acids or combinations thereof, or nanoparticles for prevention or treatment, comprising at least one cross-linked chitosan or a composition thereof as described in the present invention. Attached Figure Description

[0035] Figure 1 Characterization of the cross-linked chitosan nanoparticles of the present invention is shown (Example 2i). A: Micrograph of stained chitosan (3.2% cross-linked 501 kDa chitosan) as described in Example 3; B: Atomic force microscopy images of the dried films of the cross-linked chitosan nanoparticles of the present invention at high magnification (B3) and low magnification (B4), comparing linear chitosan (B1) and the corresponding cross-linked chitosan nanoparticles of the present invention (B2).

[0036] Figure 2 The internalization of the cross-linked chitosan nanoparticles of the present invention (Example 2i) in mouse leukocytes, as measured by fluorescence microscopy as described in Example 4, is shown. A: Leukocyte boundary; B: Nuclear DNA (Hoechst 33258, strong blue fluorescence); C: endocytosis of FITC-labeled cross-linked chitosan (green fluorescence); D: Mixed blue / green fluorescence (blue: background nonspecific and / or non-nuclear DNA in the cytoplasm; green: endocytosis of FITC-labeled cross-linked chitosan).

[0037] Figure 3The diagram shows a comparison of the internalization of the cross-linked chitosan nanoparticles of the present invention (Example 2i) (B) in the mouse cornea by FITC-FAM fluorescence detection as described in Example 4, with that of standard chitosan (A), which remained on the corneal surface. A: Cornea; B: Chitosan (bright green fluorescence).

[0038] Figure 4 The mass spectrometry analysis comparing non-crosslinked standard chitosan (A1: total ion current of positive ions) and (A2: ion region of interest (lower m / z ratio)) as described in Example 3 is shown, and the mass spectrometry analysis of crosslinked chitosan of the present invention (B1: total ion current of positive ions and B2 (ion region of interest (lower m / z ratio))) is shown.

[0039] Figure 5 The collision studies of the compound with m / z = 180 described in Example 3 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), and the spectrum of lysozyme (E) are shown.

[0040] Figure 6 The effects of treatment with the cross-linked chitosan of the present invention (at 0.1% w / w or 0.05% w / w in water) in Example 6, compared with the control, on the germination process of spring wheat seeds in germination chamber (A) (radicle emergence was measured after 1 day, germination capacity after 3 days, and germination ability after 7 days), and on the increase in aboveground growth of spring wheat seeds in soil substrate (B) (percentage increase relative to the control) (seeds treated 7 days before sowing). Detailed Implementation

[0041] As used herein, the term "degree of crosslinking" refers to the number of functional groups that are converted into crosslinks or grafts relative to the total number of functional groups originally present on chitosan, expressed as a percentage.

[0042] The term "alkyl," when used alone or in combination with other terms, includes straight-chain or branched C1-C atoms. 50Alkyl refers to a monovalent alkyl group having 1 to 50 carbon atoms. Examples of this term include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, 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, the alkyl group includes C1-C9 alkyl groups, more preferably C1-C6 alkyl groups, and particularly preferably C1-C4 alkyl groups, which by analogy refer to monovalent alkyl groups having 1-9 carbon atoms, monovalent alkyl groups having 1-6 carbon atoms, and monovalent alkyl groups having 1-4 carbon atoms, respectively. In particular, the alkyl group includes C1-C6 alkyl groups.

[0043] The term "alkenyl," when used alone or in combination with other terms, includes straight-chain or branched C2-C. 50 Alkenyl. It can have any number of double bonds in any possible position, and the configuration of the double bonds can be (E) or (Z). Examples of this term include 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-eicosenoenyl, and 3,7,11,15-tetramethyl-1-hexadecenyl and similar groups. Preferably, the alkenyl group includes C2-C8 alkenyl groups, more preferably C2-C6 alkenyl groups. Particularly preferred are vinyl or vinyl (-CH=CH2), n-2-propenyl (allyl, -CH2CH=CH2), isopropenyl, 1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-methyl-2-butenyl, etc.

[0044] The term "alkynyl", when used alone or in combination with other terms, includes straight-chain or branched C2-C groups. 50 Alkynyl group. It can have any number of triple bonds in any possible position. Examples of this term include alkynyl groups that can have 2-50 carbon atoms and optional double bonds, such as ethynyl (-C). CH), 1-propynyl, 2-propynyl (propynyl: -CH2C) CH), 2-butynyl, 2-penten-4-ynyl, etc. In particular, the ynyl group includes C2-C8 ynyl, more preferably C2-C6 ynyl, etc. Preferably, the ynyl group includes C2-C6 ynyl, which refers to a group having 2 to 6 carbon atoms and having at least 1 or 2 ynyl unsaturated sites.

[0045] The term "aryl" refers to an unsaturated aromatic carbocyclic group with 6 to 14 carbon atoms, having a monocyclic ring (e.g., phenyl) or multiple fused rings (e.g., indene, naphthyl). Aryl groups include phenyl, naphthyl, anthracene, phenanthrene, etc.

[0046] The term "C1-C6 alkyl aryl" refers to aryl groups having C1-C6 alkyl substituents, including methylphenyl, ethylphenyl, etc.

[0047] The term "aryl C1-C6 alkyl" refers to C1-C6 alkyl groups with aryl substituents, including 3-phenylpropyl, benzyl, etc.

[0048] The term "heteroaryl" refers to a monocyclic heteroaryl, or a bicyclic or tricyclic fused-ring heteroaryl. Specific examples of heteroaryl include optionally substituted pyridyl, pyrroloyl, pyrimidinyl, furanyl, thiophene, imidazole, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,3,4-triazinyl, 1,2,3-triazinyl, benzofuranyl, [2,3-dihydro]benzofuranyl, isobenzofuranyl, benzothiophene, benzotriazolyl, and isobenzofuranyl. Benzothiophene, indolyl, isoindolyl, 3H-indolyl, benzimidazolyl, imidazo[1,2-a]pyridyl, benzothiazolyl, benzoxazolyl, quinolinyl, quinazolinyl, phthalazinyl, quinoxalinyl, cinnamyl, naphridyl, 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, pteridyl, carbazole, xanthonyl, or benzoquinolinyl.

[0049] The term "C3-C8 cycloalkyl" refers to a saturated carbocyclic group having 3 to 8 carbon atoms, either a monocyclic (e.g., cyclohexyl) or multiple fused rings (e.g., norbornyl). C3-C8 cycloalkyl includes cyclopentyl, cyclohexyl, norbornyl, and similar groups.

[0050] The term "heterocyclic alkyl" refers to a C3-C8 cycloalkyl group as defined above, wherein up to three carbon atoms are substituted by heteroatoms selected from O, S, and NR, and R is defined as hydrogen or methyl. Heterocyclic alkyl groups include azirrobutyl, pyrrolidinyl, piperidinyl, piperazine, morpholinyl, tetrahydrofuranyl, and similar groups.

[0051] Unless otherwise defined, the term “substituted” means 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 alkylheterocycloalkyl”, “amino”, “aminosulfonyl”, “ammonium”, “acylamino”, “aminocarbonyl”, “aryl”, “heteroaryl”, “sulfinyl”, “sulfonyl”, “alkoxy”, “alkoxycarbonyl”, “carbamate”, “thioalkyl”, “halogen”, trihalomethyl, cyano, hydroxyl, mercapto, nitro and similar groups.

[0052] The term "pharmaceutically acceptable" refers to a carrier made of a material that is not undesirable in biological or other respects and is not particularly toxic.

[0053] The term "carrier" refers to any component present in a pharmaceutical preparation other than the active agent, and therefore includes diluents, binders, lubricants, disintegrants, fillers, colorants, wetting agents or emulsifiers, pH buffers, preservatives, etc.

[0054] The term "bioactive agent" is used to describe any bioactive agent to be incorporated into the graft polymer compositions of the present invention. It may be natural, synthetic, semi-synthetic, or a derivative thereof, and may include hydrophobic, hydrophilic, soluble, and insoluble compounds. More specifically, it can be any bioactive agent 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 cardiac arrhythmias such as atrial fibrillation, hypertension, inflammatory conditions or diseases, particularly autoimmune and non-autoimmune inflammatory diseases, including joint diseases and joint disorders (e.g., rheumatoid arthritis, osteoarthritis, and spondyloarthritis), eye diseases (e.g., dry eye, uveitis, glaucoma, corneal diseases), connective tissue diseases (e.g., lupus or polymyositis), skin diseases or injuries (e.g., wounds, 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, brain and posterior dura mater repair and central nervous system surgery, neuroplasty, pain, postoperative fistulas and infections, traumatic diseases, benign and malignant tumors and infections. Bioactive agents can be selected from macromolecular or small molecule compounds, such as peptides, proteins, oligonucleotides and polynucleotides, anti-infective agents, antibiotics, antimicrobial agents, antiviral drugs, antibacterial agents, antifungal drugs, growth factors, enzymes, antigens, antitumor drugs, anti-inflammatory drugs, anesthetics, analgesics, anticoagulants, hemostatic agents, cells, and antibodies.

[0055] The term "inflammatory condition or disease" refers to all diseases in which inflammation and the release of or pro-inflammatory cytokines are the main or significant disease mechanisms, such as inflammatory bowel disease, allergies, asthma, autoimmune diseases, hepatitis, inflammation of other organs and related diseases.

[0056] The term "dermatitis" or "skin disease" includes skin lesions where the skin surface presents as painful depressions, but not necessarily with cuts, such as age-related tissue damage (e.g., wrinkles), wounds, and scars, such as acne or urticaria scars. These conditions also include wounds, scars, psoriasis, acne, eczema, and rosacea. The term "wound" includes any damaged tissue, such as after trauma or surgery. Wounds in mammals include, for example, cuts, abrasions, contusions, punctures, skin cuts, surgical wounds, gunshot wounds, heat injuries, chemical wounds, sunburns, bites, and electrical burns. It also includes chronic skin conditions such as ulcers and other inflammatory skin diseases.

[0057] The term "eye disease or condition" refers to any condition or injury affecting the eye, particularly the cornea. Such conditions include corneal abrasions, corneal lacerations, corneal alkali burns, age-related macular degeneration, proptosis, cataracts, cytomegalovirus retinitis, color blindness, strabismus, diabetic macular edema, floaters and flashes of light, glaucoma, keratoconus, lazy eye, low vision, ocular hypertension, retinal detachment, blepharospasm, uveitis, keratoconjunctivitis sicca (KCS), and dry eye syndrome.

[0058] The term "joint or joint disease" includes osteoarthritis, arthritis pain, rheumatoid arthritis, infection and inflammatory pain, cartilage damage from a traumatic knee event, and damage to the hip joint, bone, ligaments, or synovial bursa from a traumatic event.

[0059] The term “reconstructed tissue” refers to biological tissue (endogenous or exogenous) or synthetic or semi-synthetic material that can be used to repair damaged tissues in the body, such as epidermis, nerves, cartilage, or bone tissue, and to construct organoids for biological testing, as described in Takebe et al., 2019, Science, 364, 6444, 956-959, DOI: 10.1126 / science.aaw756.

[0060] As used herein, "treatment" generally means achieving a desired pharmacological and / or physical and / or physiological effect. This effect can be preventative in the prevention or partial prevention of a disease, symptom, or condition, and / or therapeutic in the partial or complete cure of a disease, condition, symptom, or side effect attributable to a disease. As used herein, the term "treatment" covers any treatment of a disease in mammals, particularly humans, and includes: (a) preventing the occurrence of the disease in subjects who may be susceptible to the disease but have not yet been diagnosed with it; (b) suppressing the disease, i.e., halting its development; or alleviating the disease, i.e., causing the resolution of the disease and / or its symptoms or condition, such as improvement or repair of damage.

[0061] As used herein, the term "subject" refers to a mammal. For example, mammals covered by this invention include humans, primates, and domesticated animals such as cattle, sheep, pigs, horses, especially racehorses, laboratory rodents, etc.

[0062] The therapeutic efficacy described in this invention can be measured during the course of a disease based on changes in response to the use of the invention. For example, the therapeutic efficacy of the invention can be measured by the disappearance or reduction of clinical signs or symptoms, or by favorable measurements of disease biomarkers, such as cytokines, growth factors or other signaling molecules and their receptors, cell surface markers, cell counts, and gene expression profiles.

[0063] The preparation method and characterization of chitosan of the present invention

[0064] According to a particular aspect of the present invention, a method for preparing cross-linked chitosan is provided, comprising the following steps:

[0065] a) Provide chitosan and swell the chitosan in a solvent;

[0066] b) Acylation of the amino groups of the chitosan with an acrylic compound of formula (I):

[0067]

[0068] Wherein, R1 is a halogen, or any leaving group that can ensure aminoacylation after departure, such as: 3-hydroxybenzotriazole ester, acid anhydrides (including mixed acid anhydrides), N-hydroxysuccinimide, pentachlorophenol, 2-nitro-4-sulfonylphenol ester, and other similar leaving groups; R2, R3, and R4 are independently selected from the group consisting of: H, optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted alkynyl (e.g., C3-C6 alkynyl), optionally substituted cycloalkyl (e.g., C3-C8 alkyl), optionally substituted cycloalkenyl (e.g., C4-C8 cycloalkenyl), optionally substituted cycloalkynyl (e.g., C5-C8 cycloalkynyl), optionally substituted heterocyclic alkyl, optionally substituted aryl (e.g., optionally substituted phenyl) ), optionally substituted heteroaryl and optionally substituted aryl C1-C6 alkyl, particularly benzyl; wherein the term “substituted” means that the group is substituted by 1 to 5 substituents selected from the group consisting of: halogen, -COOR', -NR'R'', =O, -OR', -COR', -CONR'R'', -SR', -SO3R', -SO2NR'R'', -SOR', -SO2R', -NO2 or -CN; or, R1 and R2, or R2 and R3, or R3 and R4 together form optionally substituted 4-24 aryl, heteroaryl, cycloalkyl or heterocycloalkyl (e.g. 6-24 aryl, heteroaryl, cycloalkyl or heterocycloalkyl, such as optionally substituted 8-24 aryl, heteroaryl, cycloalkyl or heterocycloalkyl);

[0069] c) In the presence of a base, react the acylation product of step b) (aza-Michael reaction);

[0070] d) Purify the cross-linked chitosan obtained from step c) (e.g., from salt impurities and / or aprotic solvents).

[0071] In one particular implementation, the solvent used in steps a) and / or b) is a protic solvent (e.g., an alcohol or water).

[0072] In one particular embodiment, if a protic solvent (e.g., an alcohol or water) is used in steps a) and / or b), the free acrylic acid formed is washed away from the reaction mixture before step c).

[0073] In another specific embodiment, the solvent is an aprotic solvent, particularly in steps a) and / or b) and / or c).

[0074] In one particular embodiment, if an aprotic solvent is used in step c), then no further acylation step f is performed.

[0075] In one particular implementation, step a) is performed at room temperature.

[0076] In one specific embodiment, the R3 or R4 and R2 groups in the acylation product of step b) will react with the primary amino group of the glucosamine backbone to form crosslinks between glucosamines. The reacting groups will depend on the specific acrylic compound. For example, for acrylic acid and methacrylic acid, the group reacting with the primary amino group 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 group of the glucosamine backbone.

[0077] In another specific implementation, this crosslinking leads to the formation of nanoparticles.

[0078] In one particular aspect of the invention, the method of the invention is shown in Scheme 1 below:

[0079] Option 1

[0080]

[0081] in,

[0082] Chitosan (A), where m is an integer between 1 and 12,500, and n is an integer between 1 and 12,500, is first provided in a swollen state in an aprotic solvent at room temperature. The amino groups of the chitosan are then acylated with an acrylic compound (I), and the resulting acylated product (B1) is reacted in the presence of a base to obtain cross-linked chitosan (B2), which can then be purified to obtain the purified cross-linked chitosan of this invention.

[0083] In one particular embodiment, chitosan can be provided in a swollen or even dissolved state in a protic solvent, but in this case, the side reaction of acylation hydrolysis may occur, which must be taken into account when calculating the reaction load. Additionally, in this case, the acylated chitosan obtained in step b) needs to be washed to remove the hydrolysis products of the acylation agent before the azirmono-Michael reaction stage in step c).

[0084] In one particular embodiment, chitosan is provided in the absence of water, and the acylation step is carried out in the absence of water. Anhydrous conditions are advantageous for increasing yield and avoiding the formation of byproducts.

[0085] In a particularly advantageous aspect of the invention, the acylation step is carried out in the absence of water. In this case, an aprotic solvent can be used as the reaction medium or a supercritical fluid. In one particular embodiment, the aprotic solvent is a polar aprotic solvent, such as selected from DMF and DMSO. In one particular embodiment, the supercritical fluid can be a supercritical solvent, such as carbon dioxide, nitric oxide (I), or chlorofluorocarbons (chloro(bromine)(fluoro)carbons), 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.

[0086] In a particularly advantageous aspect of the invention, the acylation step is carried out in an anhydrous aprotic medium.

[0087] In one particular aspect of the invention, the polar aprotic solvent is selected from: dimethylformamide (DMF), dimethylacetamide, acetonitrile (MeCN), N-methylpyrrolidone, dimethyl sulfoxide (DMSO), or mixtures thereof.

[0088] In another specific aspect of the invention, dichloromethane, dichloroethane, chloroform, and other chloro(fluoro)carbons can also be used as polar aprotic solvents, but these solvents should be distilled off before the base is provided during the azira-Michael reaction step c). Ideally, the distillation should be carried out at a temperature not exceeding 60°C, which is feasible at atmospheric pressure for most of the mentioned solvents. If high-boiling solvents are used, distillation must be carried out under reduced pressure.

[0089] In another specific aspect of the invention, ethers and esters, ketones, can also be used as solvents for reaction steps a) to c) under anhydrous conditions, but the reaction proceeds more slowly in such solvents. For example, diethyl ether, diisopropyl ether, methyl tert-butyl ether, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, acetone, methyl ethyl ketone, and diethyl ketone can be used.

[0090] In one particular aspect of the invention, the acylation step b) can be performed by any method described in the context of acylation of glucosamine with acrylyl chloride (Zhang et al., 2017, Biomacromolecules, 1, 3, 778–786; Bu et al., 2017, Advances, 7, 76, 48166 – 48175), or described as methods that can be used for aminoacylation, such as using i) carbodiimide; ii) azide; iii) mixed anhydride; iv) ester activation methods and v) other methods described below.

[0091] Known acylation methods using carbodiimide to form intermediate enol esters can be used in step b) (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, 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride and N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide p-toluenesulfonate (CAS Registry No.: 2491-17-0) would be preferred condensing agents.

[0092] Known acylation methods using azides can be used in step b) (Honzl et al., 1961, Coll. Czech. Chem. Commun., 26, N. 9, 2333-2344).

[0093] Known acylation methods using mixed anhydrides can be used for step b) (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). Acylation can also be achieved using internal 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).

[0094] Known acylation methods using activated esters to form activated amides can be used in step b), such as the carbonyl diimidazole method (Paul et al., 1960, Journal of the American Chemical Society, 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, ibid.), the substituted phenyl ester method (Gross et al., 1983, Mayenhofer, ed., Mir. Publishing House, Moscow, p. 421), and the esterification method of heteroaromatic compounds with carbodiimides (Jakubke et al., 1966, Acta Chemica Sinica, 99, N 8, 2419-2429; Taschner et al., 1965, Ann. Chem.). (Chem.), 690, 177-181), Esterification of hydroxylamine derivatives and carbodiimide (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 variant of the esterification method) (Sakakibara, 1965, Bulletin of the Chemical Society of Japan, 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, Journal of Industrial Chemistry (Ind. J. Chem.), B16, N 11, 1026-1027)

[0095] Other known acylation methods can be used in step b), such as the ketoimine method (Stevens et al., 1958, Journal of the American Chemical Society, 80, N 15, 4069-4071); the acetylene derivative method (Arens, 1955, Rec. Trav. Chim., 74, N 6, 759-770; Gais 1978, Activators for Peptide Synthesis, International Edition of Applied Chemistry, 90(8), 625-626 https: / / doi.org / 10.1002 / ange.19780900808); and the method using cyanamide derivatives (Losse et al., 1960, Ann Chemistry). (Chem.), 636, 144-149); Synthesis using isoxazolium salt (Woodwart et al., 1961, J. Amer. Chem. Soc.), 83, N 4, 1010-1012; Synthesis using imine halides (Bergmann et al., 1936, J. Biol. Chem.), 115, N 3, 93-611.

[0096] In one particular embodiment, the acrylic compound of formula (I) may be an acid, an acyl 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.

[0097] In one particular embodiment, the acrylic compounds of formula (I) are selected from the group consisting of:

[0098] (Angelic acid, CAS registration number: 565-63-9); (2-Isopropyl Acrylic Acid CAS Registry Number: 4465-04-7);

[0099] (2-Methylene dodecanoic acid, CAS Registry No.: 52756-21-5); (4S,6S)-4,6-dimethyl-2-methylene docosanoic acid;

[0100] ((Z)-3-cyclohexylacrylic acid, CAS Registry No.: 673456-32-1);

[0101] ((E)-3-[4-(2,6-dioxo-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);

[0102] ((2E)-3-((E)-4-{[2-(4-chlorophenoxy)-2-methylpropionyl]amino}-1-adamantyl)acrylic acid;

[0103] (3-(2-Carboxy-4-phenyl-3-styryl-cyclobutyl)acrylic acid, CAS Registry No.: 34271-87-9);

[0104] (3-Methyl-2-acryloyl-(1)-cyclopropane-carbonyl-(1)-carbonyl-(1)-ethyl ester, CAS Registry No.: 91971-88-9);

[0105] (3t,4t-dicarboxy-cyclobutane-1,2c-di-(trans-acrylic acid), CAS Registry No.: 55011-62-6);

[0106] Crown diene; (CAS Registration No.: 1247-53-6)

[0107] ((E)-3-(2t-ethoxycarbonyl-6ξ-)methoxy-cyclohexyl-1r-yl)-acrylic acid, CAS Registry No.: 2960-11-4); cinnamic acid; 4-methylcinnamic acid; p-nitrocinnamic acid; caffeic acid;

[0108] (3-(furan-2-yl)crotonic acid; 3-(2-furan-yl)acrylic acid, CAS Registry No.: 539-47-9);

[0109] ((Z)-uric acid, CAS Registry No.: 7699-35-6);

[0110] (3-(4-pyridinyl)-2-acrylic acid; 3-(pyridin-4-yl)acrylic acid, CAS Registry No.: 5337-79-1); Arginine glycoside AI;

[0111] ((Z)-2-chlorobut-2-enoic acid, CAS Registry No.: 53993-41-2);

[0112] ((2Z)-2,3-dichloroprop-2-enoic acid, CAS Registry No.: 3533-68-4);

[0113] cis-N-tert-Butoxycarbonyldehydro-β-alanine (CAS Registry No.: 151292-68-1); (2-Hydroxy-3-mercapto-acrylic acid, CAS Registry No.: 6228-60-0;

[0114] Hydroxy(meth)acrylic acid; (Dihydroxyacrylic acid, CAS Registry No.: 2702-94-5);

[0115] (Reaxys ID 2721331); (CAS Registration No.: 64361-31-5);

[0116] 3-(2-Carboxymethylene-cyclopentyl)acrylic acid; (CAS Registration No.: 78727-62-5); (Fluorobutylene acid, CAS Registry No.: 2365-87-9);

[0117] ((E)-2,3-bis(phenoxycarbonylamino)but-2-enediol); ((Z)-2,3-bis(phenoxycarbonylamino)but-2-enediol); (2E,4E)-2,4-hexadienediaic acid (muconic acid), maleic acid; fumaric acid;

[0118] (Maleacetoacetic acid, CAS Registry No.: 5698-52-2); (E)-4-(methylamino)-4-oxobut-2-enoic acid, an acrylic acid derivative of glutamic acid.

[0119] ; (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-methyleneundecyl-5,9-dienoic acid, CAS Registry No.: 1580541-76-9); (2-(cis-7,8-hexadecenyl)-acrylic acid);

[0120] ((E)-2-methylene octa-4,7-dienoic acid); (2-Methylene-7-octanoic acid, CAS Registry No.: 127559-93-7); and

[0121] (2-Methylene-5-decenoic acid, CAS Registry No.: 150254-20-9); and acyl halides (R1 = halogen), especially acryloyl chloride;

[0122] ( ) or methacryloyl chloride.

[0123] In one particular embodiment, the acrylic compound is an acyl halide (R1 = halogen), especially acryloyl chloride ( ) or methacryloyl chloride.

[0124] In one particular aspect of the invention, when the acrylic compound used in acylation step b) is an acyl halide, it is important to avoid the presence of water in the reaction system because an undesirable side reaction of acyl halide hydrolysis can occur, resulting in the formation of acrylic acid. This can lead to the carboxyl functionalization of the nanoparticles during the Michael addition reaction of free acrylic acid with chitosan amino groups.

[0125] In one particular aspect of the invention, the acrylic compound used in acylation step b) is an acyl chloride (e.g., an acyl chloride). In another aspect of the invention, the use of an acyl chloride facilitates the formation of hydrogen chloride, which does not interfere with the next reaction in the presence of excess base and is readily removed as a salt during dialysis.

[0126] It is important to note that the acylation of amines with carboxylic acid halides results in the formation of 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, when using an acyl halide in acylation step b), an equal amount of base, preferably a non-nucleophilic base (e.g., triethylamine in step b), is added to neutralize the acid.

[0127] In one particular aspect of the invention, when the acrylic compound used in acylation step b) is an anhydride, the method further includes the step of washing the reaction medium prior to step c) and removing the formed acid to avoid the presence of water in the reaction system. Specifically, the formed acid can be washed away from the acylation product by passing a solvent through a filter, by dialysis of the solvent, or by suspending the acylation product in a solvent and centrifuging to discard the supernatant.

[0128] In another specific aspect of the invention, when acylation is performed with a free acid using a condensing agent (e.g., DCC), the completeness of the reaction must be carefully controlled, and a washing step is preferably included as a precaution. For example, when a condensing agent such as DCC is used, byproducts such as dicyclohexylurea may form during the acylation reaction, which need to be removed from the reactants before proceeding to the next step to prevent them from participating in the crosslinking that leads to the formation of the nanoparticles of the present invention.

[0129] In another specific embodiment, in step c), the base is used in excess molar amounts to form an alkaline medium to neutralize the hydrogen halide formed in step b). Typically, the amount of base used is at least 10% molar excess (e.g., about 20 to 40% molar excess), which corresponds to at least 1-3 molar excess relative to the amino group of the acylated product obtained in step b).

[0130] In another specific embodiment, the cross-linked chitosan obtained in step c) can be purified in step d) using standard techniques to remove salt impurities and / or residual aprotic solvents, for example, by dialysis or washing with solvents such as water or dichloromethane, methanol, ethanol, acetone, etc., followed by centrifugation or using a supercritical solvent, such as carbon dioxide, insoluble in the formed cross-linked chitosan nanoparticles, or by removing the aprotic solvent through evaporation, followed by washing away byproducts from the cross-linked chitosan product with the solvent through a filter, for example, a filter with a pore size not exceeding 40 μm. In a more specific embodiment of the invention, hydrophobic filter elements with fine porosity, such as polypropylene or PTFE, can be advantageously used because their hydrophobicity creates an additional barrier to the penetration of hydrophilic chitosan nanoparticles.

[0131] In another particular embodiment, the purified cross-linked chitosan obtained in step d) is dried, for example, in an optional further drying step e).

[0132] In one particular embodiment, the base used for the Michael addition reaction in step c) is a substantially pure nonnucleophilic base, and in particular, there is virtually no secondary amine present.

[0133] In a particular embodiment of the invention, the Michael addition step c) is carried out in a protonated solvent, such as water. In this case, the degree of saponification must be controlled, for example by measuring the amount of carboxyl and amino groups formed during the acylation stage. Because the concentration of carboxyl groups is typically one to two orders of magnitude lower, and the difference in the number of amino groups can be within the experimental error range, accurate measurement of carboxyl groups can be achieved by NMR, IR spectroscopy, or acid-base titration, as described in 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, the use of a protonated solvent in this step may be advantageous when it is desirable to obtain saponified crosslinked chitosan nanoparticles at the end of step c), or if the acylation step is planned to be repeated as described below to saturate the nanoparticles with carboxyl functional groups and then achieve excessive crosslinking. In fact, the saponification step occurs when the nanoparticles are saturated with carboxyl functional groups. Next, the acylation and aza-Michael reactions are repeated, followed by saponification. If spontaneous saponification occurs during the aza-Michael step in a protic solvent, this is not disadvantageous, because saponification happens afterward anyway, and the partial reaction that occurs during the aza-Michael reaction is not a problem.

[0134] In another particular aspect of the invention, the Michael 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.

[0135] In another specific embodiment, the Michael addition reaction step is carried out in an alkaline medium, such that for each amide bond formed during the acylation step, an equivalent secondary amine is formed after the Michael addition reaction, thus ensuring that at least half of the amino groups are available for salt formation even if all amino groups are crosslinked. This can be verified by acid-base titration, IR, and NMR spectroscopy, as described, for example, in 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. In a particular aspect of the invention, intramolecular addition reactions within the chitosan macromolecule are significantly superior to intermolecular reactions due to statistical and spatial factors, resulting in high crosslinking yields.

[0136] The nanoparticles obtained after Michael addition reaction step c) can be reduced (e.g., by reacting with complexed metal hydrides, diborane, cyanoborohydrides, and other borohydride derivatives) to convert the amide groups to secondary amines and increase the likelihood of nanoparticle ionization. The likelihood of nanoparticle ionization depends on the number of amino groups. The more amino groups present, the greater the chance of salt formation and individual ionization. The degree of ionization can be assessed by acid-base titration, IR, and NMR spectroscopy.

[0137] In another aspect of the invention, the cross-linked chitosan product obtained in step c) or d), or the corresponding dried product obtained in step e), can be saponified in a protic solvent under alkaline conditions (e.g., in the presence of an excess of alkaline aqueous solution) in step f) to increase the solubility of the cross-linked chitosan nanoparticles over a wider pH range, for example, under physiologically weakly alkaline conditions (e.g., pH approximately 7.2-7.3). If the solvent used in step c) or d) does not permit saponification (e.g., DMF), saponification needs to be carried out after purification step d) in a saponification-permitting solvent, or by resuspending the dried cross-linked chitosan in a solvent that permits saponification after step e), for example, in an ether as a solvent.

[0138] For example, in this further step f), an excess molar amount of an aqueous alkaline solution relative to the acylating agent used in step b) is added to the product obtained in step c) or d), for example, about 10 or 100 times the molar amount of the acylating agent.

[0139] Option 2

[0140]

[0141] This allows half of the amino groups in the cross-linked chitosan to be regenerated as primary amines and the other half as secondary amines, which are now alkylated with propionic acid (or substituted propionic acid). For example, the saponification of the cross-linked chitosan can be carried out by reacting with 1 molar sodium hydroxide aqueous solution at 50°C for 1 hour. The resulting product (B3) can then be subjected to acylation step b) again, as shown in Schemes 3a and 3b below, in which case the acylation occurs under the same conditions, where both the primary amino group (Scheme 3a) and the secondary amino group (the amino group obtained in stages b, c, or f is de novo acylated, Scheme 3b) may be acylated. The resulting product (B4) is then subjected to Michael addition again in step c) to achieve cross-linking of all amino groups and obtain substantially fully cross-linked chitosan (B5).

[0142] Option 3a

[0143]

[0144] Option 3b

[0145]

[0146] In this case, the product obtained will form an inner salt with the carboxyl group, which will be half the initial content of the amino group present at the end of the first synthesis step c). In a particular aspect of the invention, such a product will contain substantially the same number of carboxyl groups, total secondary and tertiary amino groups, and amide functional groups (excluding the initial acetaminoglycoside fragment), that is, such a product will contain a complete inner salt with all carboxyl groups balanced by amino groups.

[0147] While not wishing to be bound by any theory, it can be assumed that if the initial chitosan provided in step a) contains amino groups, then it will be fully derivatized in step b), with X amino groups acylated by an acylating agent, and therefore, X amino groups will undergo an azira-Michael reaction with the acyl fragments grafted onto the chitosan in step c). After the amide groups formed in step b) and present in the product of step c) are saponified in step f), X primary amino groups are regenerated, X carboxyl groups can be obtained from the acrylic acid used for acylation, and the X secondary amino groups generated in step c) remain. Therefore, after repeated thorough derivatization via acylation, the X regenerated primary amino groups can be further acylated (the secondary amino groups obtained in step c) may be acylated, but this is a unlikely process). Then, when step c) is repeated, the secondary amino groups previously obtained in step c) undergo an azira-Michael reaction to form X tertiary amino groups.

[0148] Furthermore, a second saponification process is performed, regenerating X primary amines and X tertiary amines, which are not saponified and form X carboxyl groups. Therefore, the total number of carboxyl groups will be 2X, the number of primary amines will be X, and the number of tertiary amines will be X. The polymer will become electrically neutral and form an inner salt.

[0149] If a complete derivatization is performed a third time, only X / 2 acylation agents are needed because only X amino groups can still 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 becomes anionic with 0.5X excess carboxyl groups. Therefore, each repetition of this reaction step increases the number of carboxyl groups by 0.25, 0.125, 0.0625, 0.03175, etc. The presence of carboxyl groups can be easily confirmed by infrared spectroscopy or acid-base titration and mass spectrometry.

[0150] In one particular embodiment, the amount of alkali used in the optional saponification step f) is equimolar to the amount of the amide groups to be saponified obtained in the first reaction steps c) / d) or e), and is at most 10 times in excess.

[0151] In one particular implementation, at least one set of steps b) to c) (or d) / e)) is performed together with saponification step f) after the completion of Michael addition and before a new acylation step b) to c) (or d) / e)) is performed again.

[0152] In another specific embodiment, approximately 2 to 6 sets of reaction steps a) to f) are performed.

[0153] In another specific embodiment, the reaction process of steps a) to f) is repeated once or twice.

[0154] Following steps a) through f), cross-linked chitosan achieves solubility over a wider pH range, and the properties of natural chitosan can be altered, for example, in terms of viscosity and cell permeability. Therefore, these properties can be advantageously tuned depending on the number of reaction steps performed, and the resulting cross-linked chitosan nanoparticles can be used to deliver compounds of interest.

[0155] In one particular embodiment, cations (e.g., cationic peptides, such as sequences rich in arginine, ornithine, or lysine, or sequences that are neutral in terms of charge) can be delivered via cross-linked chitosan according to the invention.

[0156] In another specific embodiment, cross-linked chitosan as described in this invention can be used to prepare compounds to protect them from degradation (e.g., by protein hydrolysis) or binding to undesirable substances (e.g., antibodies or other macromolecules), or to ensure the slow release of such soluble compounds.

[0157] In another specific embodiment, the bioactive material may be integrated into the formed nanoparticles after the first acylation step b) or the first Michael addition step c). In a further specific embodiment, the bioactive material may be covalently linked, adsorbed, or immobilized in the form of a salt, or encapsulated within the cross-linked chitosan nanoparticles of the present invention.

[0158] When the saponification step is performed, the covalent crosslinking of the bioactive agent will be destroyed, but the ionic bonds with the bioactive agent can be formed in the salt of the resulting crosslinked chitosan.

[0159] Alternatively, when applying steps a) through f), the nanoparticles can be impregnated with a bioactive substance resistant to the reagents used in steps a) through f), for example, by impregnation with heavy element clusters to produce radiopaque nanoparticles.

[0160] Alternatively, after synthesizing the cross-linked chitosan nanoparticles of the present invention, it is preferable to introduce multifunctional, unstable, and bioactive substances into the nanoparticles, or to introduce them using click chemistry, enzyme, protein, nucleic acid, and antibody immobilization methods.

[0161] In one particular aspect of the invention, the integration of bioactive materials into the chitosan nanoparticles of the invention can be achieved by impregnating the product produced in step b) or c) with the desired bioactive material. For example, a solution of the bioactive material in an aprotic solvent can be added to the product produced in step b) prior to the Michael addition step c). Alternatively, the acetylated chitosan product obtained in step b) can be dissolved in a solvent suitable for further synthesis conditions.

[0162] Impregnation can be performed without dissolving the cross-linked chitosan of the present invention.

[0163] For impregnating the cross-linked chitosan of the present invention 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, it needs to be removed and the impregnated product dried prior to the aza-Michael reaction. For impregnation, supercritical fluids can be used, as described in Weidner 2018, The Journal of Supercritical Fluids 134220-227 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, which can provide extremely high degree and quality impregnation. If the impregnation of the reagent can be accelerated by coulometric methods, electrophoresis can also be used, as described in Boccaccini et al., 2010, JR Soc. 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, as described in Zhao et al., 2012, PLoS One, 7(10): e46765. doi: 10.1371 / journal.pone.0046765. For example, an aqueous solution of bioactive substances (such as antibiotics) can be mixed with an aqueous solution of acylated chitosan after step b), and dried by azeotropic distillation of water, spray drying, freeze drying, or other drying methods to obtain dried acylated chitosan impregnated with bioactive substances. Then, the dried material is subjected to an excess of non-nucleophilic base to deionize the amino groups of the chitosan, and the resulting mixture is then subjected to Michael addition reaction step c).

[0164] In another aspect of the invention, the chitosan provided in step a) has an average molecular weight of about 5 to about 2,000 kilodaltons, particularly about 150 to 2,000 kilodaltons.

[0165] In another further aspect of the invention, the chitosan provided in step a) has a degree of deacetylation ranging from about 100% (as in glucosamine) to about two deacetylated segments per macromolecule, for example, for chitosan (chitin) with a molecular weight of 300 kDa, the degree of deacetylation is about 0.14%.

[0166] Suitable chitosans that can be used as starting materials in the methods of this invention can be from a variety of sources, including natural ones (from animals or fungi), particularly from crab and shrimp shells, insect exoskeletons, higher fungi, and cultures of single-celled mushrooms. Chemically or enzymatically synthesized chitosans can also be used.

[0167] In another aspect of the invention, the chitosan provided in step a) is allowed to swell for about 5 minutes to about 1 day, preferably in an aprotic solvent.

[0168] In another further aspect of the invention, the deacetylation step b) is carried out at a temperature of about -70°C (at the start of the reaction) to about +150°C (when the reaction is complete), preferably at a temperature of about +2°C (when the reaction is complete) to about +55°C (when the reaction is complete). The reaction time is about 0.5 hours to 10 days, preferably at about +20 to about +30°C for about 2.5 hours, and preferably under the action of ultrasound as a mixing device.

[0169] In another further aspect of the invention, the Michael addition step c) is carried out at a temperature of about -70°C (at the start of the reaction) to about +150°C (at the end of the reaction), preferably at a temperature of about +20°C (at the start of the reaction) to +55°C (at the end of the reaction). The reaction time is about 0.5 hours to 10 days, preferably about 15-18 hours at about +20°C and about 6 hours at about +55°C, preferably under the action of ultrasound as a mixing means.

[0170] In another further aspect of the invention, in step e), the drying step of cross-linked chitosan is carried out by freeze drying.

[0171] In another further aspect of the invention, the optional additional saponification step f) is carried out at a temperature of about 0°C (at the start of the reaction) to about +140°C (at the end of the reaction), preferably at a temperature of about +20°C (at the start of the reaction) to about +55°C (at the end of the reaction). The reaction time is about 0.5 hours to 10 days, preferably about 2-6 hours at +55°C, and preferably using ultrasonic action as a mixing means.

[0172] In a particular embodiment, the nanoparticles of the present invention are spontaneously formed from the material obtained under steps b) and c) of the method of the present invention. These particles are very stable over time. For example, a 2% solution is stable for at least one year at room temperature and for at least several years in a dry state.

[0173] In one aspect of the invention, a method for preparing a drug delivery system for a bioactive agent is provided, comprising the steps of carrying out the method of the invention, wherein the bioactive agent is added in step b), c), d), or e).

[0174] In one aspect of the present invention, a method for preparing a drug delivery system for a bioactive agent is provided, comprising the following steps:

[0175] - The cross-linked chitosan of the present invention is provided in a wet or dry state;

[0176] - Provide bioactive agents to be delivered;

[0177] - Dissolve the bioactive agent in a solvent or supercritical fluid;

[0178] - The cross-linked chitosan nanoparticles of the present invention are loaded by impregnating cross-linked chitosan with a bioactive agent solution or by directly introducing it into nanoparticles by electrophoresis or electric field acceleration.

[0179] - Collect compositions or nanoparticles thus loaded with bioactive agents (e.g., drugs).

[0180] In another specific embodiment, a method for identifying cross-linked chitosan obtained from the method according to the invention is provided, the method comprising the steps of:

[0181] - Provide chitosan characterized in solvents such as dichloromethane or chloroform;

[0182] - Under vigorous stirring, at a temperature of about 0°C to about 20°C, for example, for about 30 minutes, the chitosan as described herein is acylated (e.g., using acetyl chloride or acetic anhydride).

[0183] - Neutralize the reaction medium with a base (e.g., diisopropylethylamine as a nonnucleophilic base);

[0184] - Evaporate the solvent and wash the resulting neutralized product;

[0185] - subject the product to reflux acid hydrolysis (e.g., with preferably 28% hydrochloric acid for about 1 to 3 hours, for example about 2 hours).

[0186] - Evaporate the reaction mixture and resuspend the hydrolysis products in a weak acid, such as acetic acid;

[0187] - Determine whether a product selected from formula (IIIa) or (IIIb) exists.

[0188]

[0189] (IIIa); (IIIb);

[0190] R2 to R4 are defined in this paper, R 2' To R 4'The definitions are as described in R2 to R4 herein, wherein the presence of the product of formula (IIIa) and / or (IIIb) indicates that the cross-linked chitosan was obtained by the method according to the invention.

[0191] In one particular embodiment, in the method for identifying cross-linked chitosan as described in the present invention, the presence or absence of the product (IIIa) can be determined, for example, by mass spectrometry electrospray analysis, wherein a peak at m / z = 252.077 indicates the presence of compound (IIIa), especially compound (II).

[0192]

[0193] (II).

[0194] The method for identifying cross-linked chitosan obtained by the method of the present invention is very useful because it allows cross-linked chitosan obtained by the method according to the present invention to be distinguished from cross-linked chitosan obtained by other methods, such as those described by Baoqiand et al., 2015, ibid. or El-sherbiny et al., 2009, ibid.

[0195] Chitosan and its nanoparticles as described in this invention

[0196] The cross-linked chitosan of the present invention can be prepared from readily available starting materials according to the method of the present invention. It should be understood 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 such conditions can be determined by those skilled in the art using conventional optimization procedures.

[0197] In one specific embodiment, cross-linked chitosan and its nanoparticles obtained by the method of the present invention are provided.

[0198] In another specific embodiment, a cross-linked chitosan is provided, having a lower viscosity than the starting chitosan. For example, for chitosan with a degree of deacetylation of 90%, a degree of cross-linking of 3.2%, and a concentration of 0.25%, the viscosity of the nanoparticle solution of the present invention is three times lower than that of the starting chitosan. For instance, the viscosity of the chitosan nanoparticles of the present invention is 2.4 cSt at 25°C and 1.82 cSt at 37°C, while the corresponding viscosity of the starting chitosan is 7.5 cSt at 25°C and 5.4 cSt at 37°C.

[0199] In one specific embodiment, the pharmaceutically acceptable salts of the cross-linked chitosan of the present invention include, for example, acetates, lactates, succinates, citrates, malonates, fumarates, maleates, malates, and other carboxylic acids and hydroxycarboxylates, as well as inorganic acid salts such as hydrochlorides, phosphates, and sulfates.

[0200] It should be understood that the molecular weight of the cross-linked chitosan of the present invention can be adjusted by the selection of the starting material, and the selection will depend on the intended function of the nanoparticles of the present invention and their formulation. For example, the desired degradation rate of the nanoparticle composition, the desired release rate of the bioactive agent optionally incorporated therein, and the treatment conditions to be treated will affect the selection of the molecular weight of the chitosan as the starting material.

[0201] In one embodiment, the molecular weight of the cross-linked chitosan of the present invention is between about 2 kDa and 2 MDa.

[0202] In another embodiment, the molecular weight of the cross-linked chitosan of the present invention used in the lubricating / filler composition can be selected between about 2 kDa and 2 MDa, preferably between about 30 kDa and 1,000 kDa.

[0203] In another embodiment, the molecular weight of the cross-linked chitosan of the present invention used in 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.

[0204] Composition

[0205] This invention provides a medicament or therapeutic agent in the form of a composition, or a medical device comprising the same, and a method for treating a subject, preferably a mammalian patient, and most preferably a human patient, who suffers from a medical condition, particularly cardiovascular disease, such as arrhythmias, especially atrial fibrillation and hypertension; joint disorders and diseases, such as rheumatoid arthritis, osteoarthritis, spondyloarthritis, and traumatic events resulting in damage to cartilage, bone, ligaments, or synovial bursae; and eye disorders and injuries, such as dry eye, pterygium, etc. Uveitis, glaucoma, corneal lesions; connective tissue diseases such as lupus and polymyositis; skin / mucous membrane diseases or injuries such as wounds, 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 following brain and central nervous system surgery; malignant and benign tumors, carcinomas, sarcomas, lymphomas, and melanomas; postoperative complications such as fistulas and infections; tumors or vascular malformations.

[0206] The present invention provides compositions comprising at least one cross-linked chitosan or a composition thereof, which may be used for human, veterinary or agricultural purposes.

[0207] In one particular embodiment, the agricultural composition of the present invention can be used as a plant growth inducer.

[0208] In one particular embodiment, the cross-linked chitosan of the present invention can be used to prepare self-assembled structures, molecular machines, delivery mechanisms, electronics, composite materials, and lubricants.

[0209] In one particular embodiment, the present invention provides a pharmaceutical formulation comprising at least one cross-linked chitosan or a composition thereof of the present invention, for use as a drug.

[0210] In another specific embodiment, the composition of the present invention is a parenteral preparation.

[0211] In one particular embodiment, the composition of the present invention is an injectable formulation, such as an intra-articular, intra-arterial, intravenous, intra-synovial, intradermal, subcutaneous, submucosal, interstitial, intracranial, intraocular, intratumoral, intragastric, intraintestinal, anal, intraperitoneal, and intramuscular formulation.

[0212] In another specific embodiment, the composition of the present invention is an oral formulation.

[0213] In another specific embodiment, the composition of the present invention is a topical formulation.

[0214] In another specific embodiment, the composition of the present invention is an ophthalmic preparation.

[0215] 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.

[0216] The present invention further provides compositions or medical devices for use in cosmetic procedures, reconstructive surgery (e.g., tissue reconstruction), cell or biological tissue culture (e.g., stem cell culture), materials science (e.g., surface coatings, such as implant coatings, lubricant compositions, flocculant compositions), and diagnostics (e.g., imaging compositions). These compositions also respectively comprise cosmetically acceptable carriers or cell or biological tissue culture nutrients.

[0217] The compositions of the present invention comprise soft tissue filler compositions, such as dermal and subcutaneous fillers, comprising at least one cross-linked chitosan or nanoparticles thereof of the present invention. Chitosan-based soft tissue filler compositions can be prepared, for example, by the method described in Grant et al., 2018, Tissue Engineering Part A, 24(13-14):1091-1098. doi:10.1089 / ten.TEA.2017.0385. Epub March 20, 2018.

[0218] In a particular embodiment, cross-linked chitosan nanoparticles of the present invention are provided, having an average size ranging from about 5 to 100 nm.

[0219] Another aspect of the invention relates to a wound dressing comprising at least one cross-linked chitosan according to the invention.

[0220] In another specific aspect of the invention, the invention relates to a wound dressing comprising a permeable matrix, the matrix comprising hydrocolloids, hydrogels, alginates, collagen, cellulose, foams, or fabrics.

[0221] The compositions of the present invention may also contain one or more pharmaceutically acceptable additional ingredients, such as alum, stabilizers, antimicrobial agents, buffers, colorants, flavoring agents, adjuvants, and the like.

[0222] The compositions according to the invention, together with conventionally used adjuvants, carriers, diluents, or excipients, can be placed in the form of a pharmaceutical composition and can be used as a solid, such as tablets or filled capsules, or as a liquid, such as a solution, suspension, ointment, emulsion, elixir, or capsule, film, or gel filled with the same, all for oral administration. The compositions can also be formulated as dry products, reconstituted with water or another suitable carrier prior to use.

[0223] The compositions of the present invention as liquid formulations include, but are not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs.

[0224] This liquid formulation may contain additives, including but not limited to suspending agents, emulsifiers, non-aqueous carriers, and preservatives. Suspension agents include, but are not limited to, sorbitol syrup, methylcellulose, glucose / syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, and hydrogenated edible oils. 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®.

[0225] Other materials and formulation processing techniques are listed in The Science and Practice of Pharmacy (Remington: The Science & Practice of Pharmacy), 22nd edition, 2012, edited by Lloyd, and are incorporated herein by reference.

[0226] The solid compositions of the present invention may be in the form of tablets or lozenges 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 mucilage, and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, corn starch, calcium phosphate, and sorbitol. Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica. Disintegrants include, but are not limited to, potato starch and sodium glycolate starch. Wetting agents include, but are not limited to, sodium dodecyl sulfate. Tablets may be coated according to methods well known in the art.

[0227] The agricultural compositions according to the present invention comprise plant growth promoters, seed germination enhancers, moisture and nutrient retainers, insecticide enhancers, elongation formulations and pheromone stabilizers, insecticide adhesives, arachnids, and molluscicides.

[0228] In one embodiment, an agricultural composition is provided comprising about 0.001 to about 99% w / w (e.g., about 0.05 to about 0.1% (w / w)) of the cross-linked chitosan of the present invention.

[0229] In another specific embodiment, the composition or nanoparticles of the present invention further comprise a bioactive agent dispersed in or covalently linked to the cross-linked chitosan composition.

[0230] In another specific embodiment, the composition or nanoparticles of the present invention are in the form of a bioactive agent delivery system.

[0231] In one embodiment, based on the total amount of the cross-linked chitosan composition or nanoparticles of the present invention, at least one bioactive substance may be present in an amount of about 0.001 to 20 wt%, preferably about 0.01 to 10 wt%.

[0232] In another aspect of the invention, the composition comprises a cell or biological tissue culture medium containing cross-linked chitosan or a combination thereof. The culture medium may also contain cell nutrients such as glucose, vitamins, growth factors, metal ions, etc.

[0233] In another aspect of the invention, the compositions of the invention comprise biological tissue (endogenous or exogenous) or synthetic or semi-synthetic materials that can be used to repair damaged tissues of the body, such as epidermis, nerves, cartilage, or bone tissue.

[0234] In another specific embodiment, a method for preparing a culture medium is provided, the method comprising the step of mixing the cross-linked chitosan of the present invention with cell culture nutrients, such as glucose, vitamins, growth factors, metal ions and the like.

[0235] In another specific embodiment, a method for preparing reconstructed tissue is provided, comprising the step of combining the cross-linked chitosan or a composition thereof of the present invention with a material, tissue or cell that can be used to repair damaged tissues of the body such as stem cells or epidermis, nerves, cartilage or bone tissue.

[0236] In one particular aspect of the invention, once loaded with a bioactive substance or reagent, the composition or nanoparticles of the invention can be used for administration and in situ release of the said active ingredient injection.

[0237] Application method

[0238] The compositions of the present invention can also be administered by injection in any manner, 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, intraintestinal injection, anal injection, intraperitoneal injection, intratumoral injection, and interstitial injection.

[0239] The compositions of the present 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.

[0240] The compositions of the present invention can also be applied topically to the skin, various mucous membranes, or eyes.

[0241] combination

[0242] In one aspect of the invention, the cross-linked chitosan or its nanoparticles or any suitable pharmaceutically acceptable form thereof and its pharmaceutical formulations may be administered alone or in combination with at least one adjuvant.

[0243] In one particular aspect of the invention, the co-agent of the invention includes antibiotics, nucleic acids including gene constructs, antibodies and antibody fragments, toxins, cell inhibitors, antifungal agents, chemotherapeutic drug components, antihypertensive agents, antiarrhythmic substances, proliferation activators, hormones, cytokines, nitric oxide donors, and hydrogen sulfide donors.

[0244] The present invention includes applying the cross-linked chitosan or its particles and pharmaceutical formulations thereof to an individual simultaneously or sequentially with the at least one adjuvant.

[0245] The cross-linked chitosan or its nanoparticles of the present invention, or the pharmaceutical formulation thereof administered simultaneously with the adjuvant, can be administered in the same or different compositions and via the same or different routes of administration.

[0246] The dosage given to an individual 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 and body type), symptom severity, concurrent treatment, treatment frequency and desired effect.

[0247] patient

[0248] In one embodiment, the patient of the present invention is a subject suffering from cardiovascular disease, such as arrhythmia, particularly atrial fibrillation and hypertension.

[0249] In another embodiment, the patient of the present invention is a subject suffering from joint lesions and joint diseases, such as rheumatoid arthritis, osteoarthritis, spondyloarthritis, and traumatic events resulting in damage to cartilage, bone, ligaments, or synovial bursae.

[0250] In another embodiment, the patient according to the invention is a subject suffering from ocular lesions and injuries, such as dry eye, uveitis, glaucoma, or corneal lesions.

[0251] In another embodiment, the patient of the present invention is a subject suffering from a connective tissue disease, such as lupus and polymyositis.

[0252] In another embodiment, the patient of the present invention is a subject suffering from skin / mucous membrane diseases or injuries, such as wounds, scars, psoriasis, acne, eczema, rosacea, physical or chemical burns, especially surgical wounds and sunburn, ulcers, and hemorrhoids.

[0253] In another embodiment, the patient of the present invention is a subject suffering from periodontal and dental diseases.

[0254] In another embodiment, the patient of the present invention is a subject suffering from dura mater injury, such as accidental injury or dura mater injury following brain and central nervous system surgery.

[0255] In another embodiment, the patient of the present invention is a subject suffering from malignant and benign tumors.

[0256] In another embodiment, the patient of the present invention is a subject suffering from postoperative complications, such as fistulas and infections.

[0257] In another further embodiment, the patient of the present invention is a subject who desires or requires enhancement or anatomical reshaping of body parts, such as any enhancement / modification and / or increase in body part volume for aesthetic or therapeutic reasons.

[0258] In another further embodiment, the patient of the present invention is a subject suffering from a tumor, vascular malformation, or any other newly emerging physical tissue or organ abnormality that causes pain.

[0259] As described in this invention

[0260] In one particular embodiment, due to its ability to spontaneously form nanoparticles, the cross-linked chitosan of the present invention can be used in a variety of applications, such as in vitro cell or biological tissue culture, as a tissue filler, for cosmetics, tissue and organoid engineering, and materials science applications.

[0261] In particular, in one aspect of the invention, the cross-linked chitosan of the invention can be used as a carrier of a pharmaceutical active ingredient (e.g., as a drug delivery system, as a cell-penetrating system (e.g. in gene therapy or as a non-cell-penetrating agent), as a solubilizer, as a trapping agent: encapsulating the ingredient of interest in nanoparticles to generate a suspension for efficient removal from solution, for example by switching from an acidic to an alkaline pH).

[0262] In another aspect of the invention, the cross-linked chitosan of the invention can be used as a medical implant (e.g., an implant coating for improving biocompatibility, a cell-containing regenerative medical implant) or a material (e.g., a hemostatic material).

[0263] In another aspect of the invention, the cross-linked chitosan of the invention can be used as a substrate for imaging agents (e.g., for standard angiography (CT) and other emerging imaging modalities (e.g., EPR-based, NMR-based and X-ray-based)).

[0264] In another aspect of the invention, the cross-linked chitosan of the present invention can be used as a substrate for cell culture (e.g., stem cells), cell culture medium, tissue engineering (e.g., 3D printing of organoids), for separation / purification techniques, and for cell transfection.

[0265] In another aspect of the invention, the cross-linked chitosan of the invention can be used as a nanoscale lubricant (e.g., after pyrolysis), a dispersant for colloidal formulations, or as a flocculant.

[0266] In another specific embodiment, the use of the cross-linked chitosan of the present invention or the nanoparticles of the present invention or their formulations thereof is provided for the preparation of delivery systems, in vitro cell or biological tissue cultures or tissue engineering materials, such as for neurosurgical, bone, cartilage, or epidermal reconstructive tissues.

[0267] In another specific embodiment, the cross-linked chitosan or nanoparticles or formulations thereof of the present invention may be used for the prevention or treatment of medical diseases, particularly cardiovascular diseases such as arrhythmias, especially 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 synovial bursae; eye diseases and injuries such as dry eye, uveitis, glaucoma, and corneal diseases; connective tissue diseases such as lupus and polymyositis; skin / mucous membrane diseases or injuries such as wounds, scars, psoriasis, acne, eczema, rosacea, physical or chemical burns, especially surgical wounds and sunburn, ulcers, and hemorrhoids; periodontal and dental diseases; dura mater injuries such as those following accidental injury or surgery on the brain and central nervous system; malignant and benign tumors, 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 diseases.

[0268] In another further aspect of the invention, cross-linked chitosan or nanoparticles or formulations thereof are provided for use in cosmetics or for cosmetic and reconstructive surgeries.

[0269] In another further aspect of the invention, the cross-linked chitosan or nanoparticles or formulations thereof are provided for use in in vivo drug delivery.

[0270] In one particular aspect of the invention, the use of the nanoparticles according to the invention as a delivery system for at least one bioactive agent is provided.

[0271] A composition comprising at least one cross-linked chitosan or nanoparticle according to the invention is provided, which can be used in a variety of medical applications, such as for delivery systems of at least one bioactive agent or cell delivery systems in tissue engineering, cell or biological tissue culture systems, etc.

[0272] Embodiments of the present invention will be described in more detail below, and will be described with reference to the embodiments illustrated in the accompanying drawings.

[0273] Example

[0274] The following are the definitions respectively:

[0275] DMF (dimethylformamide); DMSO (dimethyl sulfoxide); MWCO (molecular weight cutoff).

[0276] Example 1: Synthesis of chitosan of the present invention

[0277] The chitosan nanoparticles of the present invention are synthesized as described in Scheme 1 above, wherein the starting materials used are as follows:

[0278] a) Providing chitosan and subjecting the chitosan to a swelling step in the absence of water (e.g., in an aprotic solvent).

[0279] 450 g of dried chitosan (which can be dried in a vacuum on phosphorus oxide, moderately heated in a vacuum, or azeotropically distilled with ethanol, acetonitrile, acetone, or any other suitable solvent) is placed in a reactor under an inert atmosphere (e.g., purged with argon). The reactor is preferably a 2-liter round-bottom flask equipped with a magnetic stirrer, a bi-angled nozzle with a nozzle from a Drexel flask inserted for purging with an inert gas (e.g., argon), and a dropping funnel with back pressure inserted for adding reagents.

[0280] Then pour 1,400 ml of an aprotic solvent (e.g., DMF) into the reactor and allow the chitosan to swell at room temperature for about 1 hour.

[0281] If undried chitosan is provided, pour 1,500 ml of aprotic solvent (e.g., DMF) into the reactor and allow the chitosan to swell at room temperature for 1 hour. Then, vacuum distill off 300 ml of the aprotic solvent (e.g., DMF) and add 200 ml of DMF to the dried chitosan in the reactor, allowing the chitosan to swell at room temperature for 1 hour.

[0282] The reaction medium containing swollen chitosan is then cooled in an ice bath to approximately 2–6°C, or in a thermostat connected to an approximately 2–6°C thermostat. In the same bath, a 250 ml reactor (e.g., a round-bottom flask) containing 100 ml of dry DMF is cooled. After cooling, the acylation agent is added dropwise to the aprotic solvent (100 ml DMF) while stirring and avoiding localized overheating, and while swirling the solvent. The solution of the acylation agent in DMF can be warmed to approximately room temperature.

[0283] b) In the absence of water, acylate the amino group of the chitosan with an acrylic acid compound of formula (I):

[0284] Under vigorous stirring, the acryloyl compound solution prepared above is added to the chitosan suspension obtained above over approximately 15 minutes, preferably while gently shaking the reactor (e.g., manually). The addition of the acryloyl compound is then stopped, as this allows the chitosan clumps to be washed off the upper wall of the flask with a solvent spray. After the addition of the acryloyl compound solution is complete, the dropping funnel is washed with a minimal amount (10-15 ml) of an aprotic solvent (e.g., DMF), and this solution is added to the reaction medium. The mixture is removed, cooled, and stirred for another two hours, occasionally shaking, to allow the reaction medium to warm to room temperature. After stirring, the flask is placed in an ultrasonic bath for approximately 15 minutes to sonicate the reaction mixture. During this sonication period, the reaction mixture is only slightly heated to no more than 30°C.

[0285] c) Allow the acylated product from step b) to undergo a nucleophilic addition reaction (aza-Michael addition) in the presence of a non-nucleophilic base:

[0286] Subsequently, under vigorous stirring and periodic shaking, an excess of a non-nucleophilic base (e.g., triethylamine, diisopropylethylamine) compared to the amount of the acryloyl compound used in step b) is added to the reaction medium (e.g., via a dropping funnel) over approximately 15 minutes. The mixture is then mixed at room temperature overnight or for approximately 15–18 hours. The reactor is then 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 sonicated.

[0287] After ultrasonic treatment, the reactor is transferred to a rotary evaporator. If a volatile organic non-nucleophilic base is used, the non-nucleophilic base is distilled off. If a non-volatile base is used, the reactor is cooled to room temperature or lower, and an equal volume of acid (e.g., hydrochloric acid) is added with vigorous stirring. The pH of the resulting reaction medium is controlled to ensure a neutral reaction medium is obtained.

[0288] d) Obtaining the cross-linked chitosan of the present invention

[0289] In step c), cross-linked chitosan is obtained in a suspension of a protic solvent (e.g., DMF) and salt impurities. The product is then purified, for example by dialysis with deionized water. For this purpose, the reaction solution obtained in step c) is transferred to a dialysis bag, clamped, and placed in a container, where it is mixed with deionized water on a magnetic stirrer. Dialysis can be monitored in various ways: for example, by measuring the conductivity of the washing solution to monitor salt content, or by monitoring DMF content via ultraviolet absorption at wavelengths less than 260 nm. If, after 20 minutes of mixing deionized water with the dialysis product, there is no significant difference in either conductivity or ultraviolet absorption, the dialysis process is considered complete.

[0290] e) Optionally, obtain the cross-linked chitosan of the present invention in a dried state.

[0291] The wet product obtained in step d) can be used without drying it. However, it can also be dried, for example, for formulation / storage or transportation.

[0292] After obtaining the purified product in step d), it is dried. For example, drying can be carried out by the following steps: initially freezing the product in a freeze dryer; first absorbing most of the liquid at atmospheric pressure in the presence of phosphoric anhydride or any suitable desiccant (such as molecular sieves, potassium hydroxide, etc.), then performing initial evaporation in a vacuum in the presence of phosphoric anhydride or any suitable desiccant, or on a rotary evaporator, and repeating azeotropic distillation of water and ethanol, followed by vacuum drying on phosphoric anhydride or any suitable desiccant.

[0293] Typically, the yield of the method of this invention is approximately 90%. Losses are most likely related to the entrainment of nanoparticles during the drying process and during the transfer from the reaction vessel and dialysis bag, since sterility and contamination-free conditions are prioritized over yield.

[0294] f) Further processing of the cross-linked chitosan of the present invention in a dry state

[0295] Optionally, the purified cross-linked chitosan obtained in steps c), d), or e) can be saponified. For this purpose, the cross-linked chitosan 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 the flask, and an alkaline solution (e.g., an aqueous solution of NaOH) in excess of 20% molar relative to the amount of acryloyl compound used in about 100 ml of deionized water is added over about 15 minutes with vigorous stirring. It is recommended to gently shake the flask manually as you stop adding the alkali, as this process washes away any chitosan clumps sprayed onto the upper wall of the reactor with the solvent. After stirring, the reactor is placed in an ultrasonic bath to sonicate the reaction mixture; sonication is continued at this temperature for another 2 hours after the reactants reach about 55°C. The resulting product can then be neutralized with an equal amount of acid, dialyzed, or dried without neutralization, and after drying, at least one cycle from b) to c) (or d) / e) is repeated. Using a set of steps b) to c)(or d) / e) in more than one cycle can increase the saturation of cross-linked chitosan with carboxyl functional groups.

[0296] Example 2: Synthesis of Cross-linked Chitosan of the Present Invention

[0297] Various cross-linked chitosans (CHI) of the present invention are synthesized by the methods described herein, with variations in the properties and proportions of the cross-linking agents. Examples are shown in Table 1 below.

[0298] In each case, 450 g of chitosan was used as the starting material. In Examples 2a-2r and C1, the acryloyl compound was acryloyl chloride (CAS Registry No.: 814-68-6). In Examples 2t-2al, the acryloyl compound was methacryloyl chloride (CAS Registry No.: 920-46-7). The reaction was carried out according to steps a) to c) of the method of the present invention. In Examples 2am-2as, the acryloyl compounds were different acryloyl chlorides.

[0299] Table 1

[0300]

[0301] Example 3: Characterization of the chitosan nanoparticles of the present invention

[0302] The 3.2% crosslinked 501 kDa chitosan sample (Example 2i) prepared as described above was adsorbed onto a formvar / carbon-coated copper grid for 30 seconds and stained with uranyl acetate for 5 seconds. Micrographs of the stained material were then obtained using a Jem 1400 transmission electron microscope (Jeol) and a Veleta camera (SIS, Germany). Figure 1 (A) This indicates that the formulation is mostly composed of spherical particles with a diameter of approximately 15 nm, similar in size to small mammalian viruses. Some larger particles, with diameters in the range of 60-80 nm, corresponding to medium-sized viral particles, were also observed.

[0303] Furthermore, by pouring a solution of 2% chitosan in a 1% succinic acid aqueous solution into a petri dish, standard linear chitosan (molecular weight: 501 kDa, 93% DD) (Bioavanta) and dry films of cross-linked chitosan according to the invention made from the same chitosan (Example 2i) were obtained, forming 1 mm layers. These films were then dried to constant weight in a vacuum at room temperature with phosphoric anhydride, and analyzed in semi-contact mode using a Solver Bio atomic force microscope (NT-MDT, Zelenokre, Russia). Three-dimensional characterization of the 100 μm x 100 μm film surface area of ​​the standard chitosan sample (molecular weight: 501 kDa, 93% DD) showed a smooth surface, with the linear chitosan polymers possibly collinearly aligned (Fig. 1B1), while the corresponding cross-linked chitosan sample of Example 2i had an uneven surface area with "peaks" and "valleys" formed by the aggregation of spherical structures (Fig. 1B2). Figure 1B3 and B4 were visualized by atomic force microscopy, comprising bacterial nanocellulose containing 1% w / w of the cross-linked chitosan aqueous solution of this invention. AFM measurements were performed in semi-contact mode on a Solver Next scanning probe microscope (NT-MDT, Russia) under standard indoor air conditions (T = 26 °C, RH = 15%). Optimal pressure (SetPoint) was determined using a ScanAsyst-AIR AFM probe (Bruker, USA) with a beam cantilever stiffness of ~0.4 N / m, a resonant frequency of ~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 in the ImageAnalysis 3.5 program by subtracting bevels and removing measurement defects (“sticking”). The obtained... Figure 1 Analysis of B3 and B4 showed that the spherical structure was uniformly distributed on the sample surface. Figure 1 B3 and B4), the average diameter of the spheres is 20 nm, corresponding to the size of small viruses, such as those in the families Caliciviridae, Picornaviridae, and Parvoviridae. Early transmission electron microscopy data confirmed the presence of the structures observed in AFM (Figure 1A).

[0304] The structural differences between standard chitosan (negative control) and the cross-linked chitosan (Example 2i) and cross-linked glucosamine (Comparative Example C1) (positive control) of the present invention were further investigated by reflux acid hydrolysis under an inert atmosphere (e.g., argon). This method allows for accurate analysis of low molecular weight substances to identify labeled substances formed only under the following conditions of successful cross-linking:

[0305] Under vigorous stirring, the sample (100 mg per serving) was added to 1 ml of a dichloromethane solution of acetyl chloride cooled to -20°C (5 ml). The mixture was then heated to 0°C and reacted under vigorous stirring for 30 minutes. Diisopropylethylamine (1 ml) was added, and the mixture was heated to room temperature and reacted under vigorous stirring for another 30 minutes. Subsequently, neutralization of the resulting hydrochloride allowed for washing with water to remove any interfering substances, and the presence of a non-nucleophilic strong base ensured that acetylation proceeded as fully as possible. All solvents were evaporated under vacuum, and the residue was washed three times with 1 ml of water. The wash water was discarded, and 10 ml of 28% hydrochloric acid was added to the solid precipitate, and the mixture was 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. Under the current conditions, the polymer was completely hydrolyzed to glucosamine and glucosamine derivatives. Therefore, since no substance of formula (II) has been found in natural chitin and chitosan, it can only be obtained by reacting with the remaining acrylic acid via an aza-Michael addition reaction. Thus, the detection of this substance can be used to identify cross-linked chitosan prepared by the method according to the present invention.

[0306] If any acryloyl chloride of formula (I) (e.g., any acryloyl chloride of acrylic acid used in the crosslinking step of the present invention) is used as a crosslinking agent (e.g., for Example 2i), after preliminary sample preparation (e.g., acetylation, washing), the reflux acid hydrolysis of the resulting polymer leads not only to the hydrolysis of the glycosidic bonds between glucosamine units, but also to the hydrolysis of the amide bonds of acetylglucosamine and the amide bonds generated in the acylation process with the crosslinking agent (e.g., activation of acylated or unsubstituted acrylic acid derivatives), resulting in the formation of the following derivatives of glucosamine and propionic acid of formula (IIIa):

[0307]

[0308] (IIIa)

[0309] Wherein R2 to R4 are as defined in this application, and in particular, if unsubstituted acylated-activated acrylic acid is used, the following derivatives of formula (II) glucosamine and propionic acid are used:

[0310]

[0311] (II)

[0312] This substance is hydrolyzed stable and must be present in the mixture if initial cross-linking occurs. If other cross-linking agents are used, similar derivatives (IIIa) will be formed. In cases of high cross-linking, the detection of derivatives of formula (IIIb) can be used to identify cross-linked chitosan prepared by the method according to the invention.

[0313]

[0314] (IIIb)

[0315] R2 to R4 are defined in this paper, R 2' To R 4' The definitions are as shown in this paper for R2 to R4, where R 2' To R 4' Each can be the same as each corresponding R2 to R4, or if the acrylic compound of formula (I) used in subsequent steps b) to c) of the method of the invention is different from those used in the first set of steps, then R 2' To R 4' Each of them is different from the corresponding R2 to R4.

[0316] To demonstrate the presence of crosslinks in the product generated by the method of the present invention, the sample solution prepared as described above was directly used for mass spectrometry analysis (Bruker Daltonics TOF 180 mkl / h, tune_low.m, ES pos. scan, N2-4 l / min, 190 C, Nb.=0.4, spectral time 1 s) without prior chromatographic analysis. This was compared with standard chitosan (… Figure 4 A2) and the cross-linked chitosan of the present invention ( Figure 4 In the mass spectrum of the ion of interest (lower m / z ratio) region of B2), a glucosamine-specific peak (m / z = 180.073) was observed, reflecting the presence of intact glucosamine residues. However, for the cross-linked chitosan of the present invention, a new peak of m / z = 252.077 appeared ( Figure 4 B2), corresponding to the product of formula (II) produced by alkylation of glucosamine with propionic acid. In this example, the peak intensity is low, reflecting a low percentage of crosslinking in the sample (3.2% crosslinking as determined by reaction condition 2i). This characteristic peak supports successful crosslinking of chitosan in the method according to the invention. It is present in all samples produced by the method of the invention under the various suitable conditions described in Table 1 above (including the comparative glucosamine positive control), and is absent in all tested chitosan and chitin.

[0317] To confirm that the peaks at m / z = 180 and m / z = 252 belong to glucosamine and compound (II), collision studies were conducted at different impact energies (AB SCIEX Triple Quad™ 3500) (5V and 15V). In this case, the pretreated dried residue (after hydrolysis, the reaction mixture was vacuum evaporated) was dissolved in an aqueous solution containing 10% methanol and 0.1% formic acid. After the compound at m / z = 180 was fragmented at higher impact energies (Sciex instrument), dehydration spectra (-18 m / z, -2*18 m / z, -3*18 m / z) were observed. Figure 5 (Figures A and 5B). The intensity of the water loss peak increases with increasing collision energy. This phenomenon is characteristic of carbohydrates. Similar patterns were observed for the ion of the compound at m / z = 252 (Figures 5B and 5C). However, as a derivative of carbohydrates, it may be more stable in terms of water loss at the same collision energy. Therefore, the observed peak representing water loss has a relatively lower intensity.

[0318] After adding 500 μl of a solution of lysozyme at a concentration of 10 mg / mL in sodium acetate buffer (pH 5.5) to 5 mg of the cross-linked chitosan of the present invention, the stability of the 3.2% cross-linked 501 kDa chitosan of the present invention (Example 2i) as described above was detected by mass spectrometry. The mixture was incubated on a shaker 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: peaks regularly distributed at m / z ratios of 955–958, 1023–1024, 1101–1104, and 1193–1196 with regularly increasing intensities. The peaks corresponded to the same fragments with different charge units. For a given mass, the ionic charges were +15, +14, +13, and +12. The mass of the major fragment was 14,305 Da. Similar results were obtained for samples incubated for longer periods (up to 3 weeks). This indicates that the hydrolytic enzyme responsible for hydrolyzing egg white lysozyme and chitosan and its derivatives in warm-blooded animal tissues can only hydrolyze cross-linked chitosan into relatively large fragments with a mass of approximately 14 kDa. Therefore, this suggests that larger molecular weight deacetylated chitosan is more stable for enzymatic hydrolysis when used in the preparation of nanoparticles.

[0319] Therefore, these data demonstrate a high degree of difference between the cross-linked chitosan of the present invention that forms nanoparticles and the standard chitosan used as a starting material. Furthermore, the method for detecting substances based on formula (II) or related substances exhibits high sensitivity and specificity in detecting nanoparticles obtained by the method of the present invention.

[0320] Example 4: Cellular internalization of chitosan nanoparticles of the present invention

[0321] The ability of the chitosan nanoparticles of the present invention to be internalized by cells has been tested by fluorescence microscopy as follows: chitosan labeled with fluorescein isothiocyanate (FITC) and 5-(and 6-)carboxyfluorescein (FAM) penetrated into the cornea of ​​mice.

[0322] Fluorescein-labeled cross-linked chitosan was prepared using standard initial chitosan with a molecular weight of 501 kDa and a degree of deacetylation of 93%, or 3.2% of the cross-linked chitosan of the present invention (Example 2p). Pre-chilled DMF (1 ml) in an ice bath was added to both chitosans (100 mg) in an Eppendorf tube, and the suspension was allowed to swell for 1 hour. Then, a solution of 2.85 mg of fluorescein-5-isothiocyanate (FITC) or 5-(and 6-)carboxyfluorescein succinimide (NHS-fluorescein) in 250 μL of DMF was added to an ultrasonic bath filled with ice water and sonicated. The temperature in the bath was maintained at 0°C for the first twenty minutes, then the bath was heated to 40°C, and the reaction was carried out at this temperature for 100 minutes. The contents of the tube were transferred to 12–14 kDa dialysis tubes and dialyzed with water. After 20 dialysis cycles with 100 ml of water, 50 mg of succinic acid was added to each dialysis tube to dissolve the chitosan, and the dialysis cycle was repeated. The resulting solution was lyophilized and subsequently resuspended to produce a 0.25% solution.

[0323] Mouse leukocytes were incubated with these solutions at 37°C for 4 hours, stained with a DNA-specific dye (Hoechst 33258), and analyzed by fluorescence microscopy. Fluorescence analysis (Figure 2) showed that green fluorescence (C) was uniform throughout the cytoplasm, indicating that the FITC-labeled cross-linked chitosan was intracellular and therefore had been endocytosed (simple binding of labeled chitosan to the cell membrane produces a fluorescent ring at the cell boundary (A)). The blue fluorescence (D) observed outside the cell nucleus may be due to the initiation of apoptosis.

[0324] The same solutions (0.25%) labeled with fluorescein isothiocyanate (FITC) and 5(6)-carboxyfluorescein (FAM) were applied to the eyes of CBA mice (one drop per eye). Six hours later, the animals were sacrificed, and the eyes were fixed in formalin and stained using the method described in Stradleigh et al., 2015, Progress in Retinology and Ophthalmology, 48:181-202, and analyzed by fluorescence microscopy. It was observed that while standard chitosan preferentially concentrated on the corneal and scleral surfaces (Fig. 3A), the chitosan nanoparticles of this invention (bright green fluorescence) penetrated the cornea uniformly (Fig. 3B).

[0325] These data indicate that the cross-linked chitosan nanoparticles of the present invention can be used to reach intracellular locations through nanoparticle-mediated endocytosis and / or potentially penetrate cells through nanoparticle-mediated endocytosis, thereby improving the cell penetration ability of active ingredients with poor cell permeability (hydrophilic) or cell non-permeability (macromolecules) to intracellular locations.

[0326] Example 5: Biocompatibility of the chitosan nanoparticles of the present invention

[0327] The biocompatibility of the cross-linked chitosan of the present invention (Example 2i) was evaluated in in vitro cultures of human bone marrow mesenchymal stem cells (MSCs) collected from healthy donors by bone marrow aspiration, as described in Bieback et al., 2008, Transfus Med Hemother., 35(4): 286–294, doi: 10.1159 / 000141567, to assess its potential impact on the culture and growth of MSCs.

[0328] As described above by Bieback et al. (2008), MSCs were isolated by density gradient centrifugation at 1,077 g / ml, washed twice, and grown in DMEM growth medium containing 10% Mesecult fetal serum, L-glutamine, and antibiotics. The purity of the cultures was verified using flow cytometry. The cultured MSCs exhibited the following phenotype: CD44+ / CD73+ / CD90+ / CD105+ / CD34- / HLA-DR-, and demonstrated chondrogenic, osteogenic, and adipogenic capabilities upon the addition of specific media, as described in 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.

[0329] Cover 25 cm with cross-linked chitosan film 2 The culture dishes were dried, washed twice with growth medium, and then inoculated with cells. Untreated culture dishes of the same size were also inoculated as controls. The presence of cross-linked chitosan on the plastic walls of the culture dishes did not interfere with cell attachment and growth. Within 3–4 hours after inoculation, cells diffused and exhibited their characteristic spindle-shaped shape. Growth was slightly slower in cross-linked chitosan-treated culture dishes. Confluence was achieved after 48–56 hours in standard culture dishes and 68–76 hours in cross-linked chitosan-treated culture dishes.

[0330] In summary, the cross-linked chitosan of the present invention can maintain the growth and proliferation of human bone marrow mesenchymal stem cells, although its efficiency is lower than that under standard conditions.

[0331] Example 6: Uses of the chitosan of the present invention in agricultural applications

[0332] The effectiveness of the cross-linked chitosan of the present invention was tested on spring wheat seeds as follows (Example 2i). Suspensions of the cross-linked chitosan of the present invention in water were prepared at two different concentrations, and seeds were immersed in the suspensions. Each experiment included the following seed groups, depending on the treatment applied:

[0333] (A) Control (seeds not treated);

[0334] (B) Treatment with 0.1% (w / w) cross-linked chitosan,

[0335] (C) Treat with 0.05% (w / w) cross-linked chitosan.

[0336] Assessment of growth-promoting effects in the early stages of organogenesis

[0337] After 24 hours of treatment, the seeds were placed on moist substrate (filter with cotton layer) in petri dishes (12 seeds per dish, quadruplicate), under natural light, t = +20-22°C. Germination energy was determined as described by Domin et al., Sustainability 2020, 12(1), 46, and was measured after 1 and 3 days of exposure to moist substrate, and germination capacity (number of germinated seeds divided by total number of test seeds minus number of empty seeds) was determined after 7 days of exposure to moist substrate. Growth indicators at the early stage of organ formation were recorded after 3 days of exposure to moist substrate (root length per plant, total plant length) and 7 days (number of roots per plant, plant length, total mass of roots and shoots per plant, mass per root).

[0338] The positive effects of treatment with the cross-linked chitosan of the present invention have been demonstrated at the lowest concentrations, such as... Figure 6 Seedling development during the initial stages of germination and organogenesis are shown in Table 2 below:

[0339] Table 2

[0340]

[0341] Higher radicle emergence rates were observed within one day of treatment with cross-linked chitosan. The strongest effect was observed with low-concentration suspensions (0.05% cross-linked chitosan increased radicle emergence by 22.9%; 0.1% cross-linked chitosan increased radicle emergence by 14.6%, baseline control was 75%). The same trend was observed when measuring germination energy (increase of 6.2%) and germination capacity (control = 93.8%). Treatment of seeds with high-concentration cross-linked chitosan suspensions increased germination capacity by 4.1%. In the early stages of organogenesis, cross-linked chitosan led to increased root growth. After 3 days, the total root length increased by 10.9% and 30.9% in the 0.1% cross-linked chitosan group and the 0.05% cross-linked chitosan group, respectively (control = 7.79 cm). Meanwhile, the most uniform increase was observed in the high-concentration cross-linked chitosan group (0.1% cross-linked chitosan, uniformity coefficient = 81% (n = 45); 0.05% cross-linked chitosan = 78.2% (n = 48); control = 68.9% (n = 45)). The same trend was observed when measuring seedling height. If the increase after 0.05% cross-linked chitosan treatment (46.2% higher than control = 1.84 cm) was more significant than the increase after 0.1% cross-linked chitosan treatment (24.4% higher than control), then the uniformity coefficient of the high-concentration cross-linked chitosan treatment group was higher (0.1% cross-linked chitosan = 75.4%; 0.05% cross-linked chitosan = 72.8%, control = 61.7%).

[0342] Based on measurements taken 7 days after the start of the experiment, low-concentration cross-linked chitosan treatment had the greatest promoting effect on root formation (0.05% cross-linked chitosan: increase of 9.5%; 0.1% cross-linked chitosan: increase of 6.1%, control group: average number of roots per seedling: 4.41). The same was true for the increase in overall seedling length (0.05% cross-linked chitosan: increase of 7.7%; control: 10.05 cm). Conversely, the highest increase in biomass was observed after treatment with a more concentrated cross-linked chitosan suspension in both the roots (0.1% cross-linked chitosan: increase of 17.1%; 0.05% cross-linked chitosan: increase of 14.6%) and the entire seedling (0.1% cross-linked chitosan: increase of 12.9%; 0.05% cross-linked chitosan: increase of 8.7%).

[0343] Evaluation of the impact of soil substrate on the growth and development of spring wheat seedlings

[0344] Seeds were treated 7 days prior to sowing (alkaline black calcareous soil, 500 g / 12 plastic pots, replicate 4 times, natural light, t = 20-22℃, controlled soil moisture). Germination energy, germination capacity, height, and biomass of seedlings were measured. Growth parameters were recorded during the first 7 days. For seeds treated with cross-linked chitosan, a higher radicle emergence rate was observed after 24 hours, particularly for higher concentrations (control: 70.8%, relative increase for treated seeds: 27.1% and 23%). See Table 3 below. Figure 6 As shown, a germination rate of 100% was obtained after treatment with high concentration of cross-linked chitosan.

[0345] Table 3

[0346]

[0347] The high initial growth stimulation generated by 0.1% cross-linked chitosan treatment (56.3% after 2 days) decreased by 3-fold after 3 days (11.6-fold reduction with 0.05% cross-linked chitosan) and by 2.3-fold after 7 days. Compared with the control group (16.63 mg), the increase in biomass after 7 days was significantly higher in both the 0.1% treatment group (14% higher total biomass, 23.8% higher root biomass) and the 0.05% treatment group (25.7% higher total biomass, 35.1% higher root biomass). The 0.05% cross-linked chitosan treatment resulted in a 1.8-fold increase in total biomass and a 1.5-fold increase in root biomass.

[0348] In summary, these data demonstrate that treatment with the cross-linked chitosan of this invention has a significant growth-stimulating effect on the germination of spring wheat.

Claims

1. A method for preparing cross-linked chitosan, characterized in that, Including the following steps: a) Provide chitosan and swell the chitosan in a solvent; b) Acylate the amino group of the chitosan with an acrylic acid compound of formula (I), Wherein, R1 is selected from the group consisting of: halogens, -OH, 3-hydroxybenzotriazole esters, acid anhydrides, N-hydroxysuccinimide, pentachlorophenol, and 2-nitro-4-sulfonylphenol esters; R2, R3, and R4 are independently selected from the group consisting of: H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted aryl C1-C6 alkyl; wherein the term "substituted" means that the group is replaced by 1 to 5 groups selected from the group consisting of: Substituents in the group: 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, acylamino, aminocarbonyl, aryl, heteroaryl, sulfinyl, sulfonyl, alkoxy, alkoxycarbonyl, carbamate, thioalkyl, halogen, trihalomethyl, cyano, hydroxyl, mercapto, nitro; c) In the presence of a base, the acylation product of step b) undergoes an azira-Michael reaction; d) Purify the cross-linked chitosan obtained from step c).

2. The method as described in claim 1, characterized in that, The method further includes a drying step e) of the product from step d).

3. The method as described in claim 1, characterized in that, The chitosan provided in step a) has an average molecular weight of 5 to 2,000 kilodaltons.

4. The method as described in claim 1 or 2, characterized in that, R1 is a halogen.

5. The method as described in claim 1 or 2, characterized in that, The acrylic compound is selected from the group consisting of acryloyl chloride and methacryloyl chloride.

6. The method as described in claim 1 or 2, characterized in that, The method further includes a saponification step f) under alkaline conditions after step c) or d), and the resulting product may be further subjected to at least one set of steps b) to c) or d).

7. A cross-linked chitosan or a pharmaceutically acceptable salt thereof, characterized in that, The cross-linked chitosan is obtained by the method as described in claim 1 or 2.

8. The cross-linked chitosan or a pharmaceutically acceptable salt thereof as described in claim 7, characterized in that, The mass spectrum of the cross-linked chitosan after reflux acid hydrolysis includes a peak of electrospray positive ions at m / z = 252.077 ± 0.

01.

9. A composition, characterized in that, It comprises at least one cross-linked chitosan as described in claim 7 or 8 and at least one carrier.

10. The composition according to claim 9, characterized in that, The composition is a pharmaceutical composition, and the carrier is a pharmaceutically acceptable carrier.

11. The composition according to claim 9, characterized in that, The composition is a cell or tissue culture medium, which further contains cell or tissue nutrients.

12. The composition according to claim 9, characterized in that, The composition is for reconstructing tissue.

13. The composition according to claim 9, characterized in that, The composition is an agricultural composition.

14. The composition according to claim 9, characterized in that, The composition is a cosmetic composition, and the carrier is a cosmetically acceptable carrier.

15. A soft tissue filler, wound dressing, or reconstructed tissue, characterized in that, It contains at least one cross-linked chitosan as described in claim 7 or 8, or a composition thereof.

16. A nanoparticle, characterized in that, It contains at least one cross-linked chitosan as described in claim 7 or 8.

17. A culture medium for cells or biological tissues, characterized in that, It contains at least one cross-linked chitosan as described in claim 7 or 8.

18. Use of the cross-linked chitosan as described in claim 7 or 8 in the preparation of cells or biological culture media, or in the reconstruction of tissues.

19. The cross-linked chitosan or a pharmaceutically acceptable salt thereof as described in claim 7 or 8, characterized in that, Used for the prevention and / or treatment of diseases or conditions selected from the group consisting of: arrhythmias, atrial fibrillation and hypertension; rheumatoid arthritis, osteoarthritis, spondyloarthritis, and traumatic events resulting in damage to cartilage, bone, ligaments or synovial bursae; Dry eye, uveitis, glaucoma, corneal lesions; lupus and polymyositis; wounds, scars, psoriasis, acne, eczema, rosacea, physical or chemical burns, surgical wounds and sunburn, ulcers, hemorrhoids; periodontal and dental diseases; dura mater injury following accidental injury or surgery on the brain and central nervous system; cancer, sarcoma, lymphoma and melanoma; fistulas and infections; tumors or vascular malformations.

20. A method for preparing a drug delivery system for a bioactive agent, characterized in that, Including the following steps: - Provide the cross-linked chitosan as described in claim 7 or 8, in a wet or dry state; - Provide bioactive agents to be delivered; - Dissolve the bioactive agent in a solvent or supercritical fluid; - The cross-linked chitosan nanoparticles are loaded by impregnating the cross-linked chitosan with a bioactive agent solution or by directly introducing it into the nanoparticles by electrophoresis or electric field acceleration. - Collect the resulting composition or nanoparticles loaded with bioactive agents.

21. A reagent kit, characterized in that, It contains at least one cross-linked chitosan as described in claim 7 or 8, or a composition thereof.

22. A method for identifying cross-linked chitosan obtained by the method as described in claim 1 or 2, characterized in that, Including the following steps: - Provides chitosan characterized in solvent; - The chitosan was acylated under vigorous stirring; - Neutralize the reaction medium with alkali; - Evaporate the solvent and wash the resulting neutralized product; - The product is subjected to reflux acid hydrolysis; - Evaporate the reaction mixture and resuspend the hydrolysis products in a weak acid; - Determine whether a product selected from those described in formula (IIIa) or (IIIb) exists. (IIIa) (IIIb) Wherein, R2 to R4 are defined as in any of the preceding claims, R 2’ To R 4’ The definitions are as described in any one of the preceding claims for R2 to R4, wherein the presence of the product of formula (IIIa) and / or formula (IIIb) indicates that the cross-linked chitosan is obtained by the method as described in claim 1 or 2.

23. The method as described in claim 22, characterized in that, The presence of compound (II) was determined by mass spectrometry electrospray ionization analysis. (II).

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