Novel synthesis method for chitosan derivatives and their use

Abstract

The present invention is directed to novel crosslinked chitosans, their preparation, compositions, and uses. In particular, the present invention relates to nanoparticles and compositions thereof useful as active agents and delivery systems for at least one bioactive agent.

Description

[Technical Field]

[0001] This invention relates particularly to the preparation of chitosan, especially chitosan-derived nanoparticles, their preparation, compositions, and use, in the fields of chemistry, biology, medicine, and materials science. [Background technology]

[0002] Biopolymers are promising materials as carriers for various drugs and other active substances due to their biocompatibility, biodegradability, and non-toxicity upon administration. Suitable chemical modifications can provide better materials for drug delivery systems, although the active substances can be either low molecular weight (small molecules) or high molecular weight (macromolecules). Nanostructured drug carriers enable the delivery of macromolecules such as nucleic acids and proteins (Advances in Polymer Science. Chitosan for Biomaterials Vol. 1. Publishers: 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 creation of nanoparticles. On the one hand, it possesses attractive natural properties such as mucosal adsorption, biocompatibility, and low toxicity, as well as biodegradability and the ability to form complexes. On the other hand, its high structural regularity and the presence of internal hydroxyl and amino groups offer a good outlook for selective polymer modification.

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

[0005] While chitosan exhibits several desirable 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, pp. 231-239, http: / / dx.doi.org / 10.1070 / RCR4636; Chuan et al., 2019, Adv. Colloid Interface Sci., 268, pp. 25-38; Jiang, H.-L.; Xing et al., 2018, Curr. Org. Chem., 22, pp. 668-689, DOI: 10.2174 / 1385272821666170926163544; Layek, B.; Singh, J. 8-Chitosan for DNA and gene therapy. In Chitosan Based Biomaterials, Vol. 2, edited by Jennings, JA and Bumgardner, JD, Woodhead). Publishing, Cambridge, UK, 2017, pp. 209-244).

[0006] The structure of chitosan offers the potential for modification in various ways. To increase hydrophobicity, chitosan amides can be readily prepared from fatty acids, sterol derivatives, urocanic acid, carboxylic acid derivatives containing imidazole fragments, etc. Alternatively, secondary chitosan amines can be formed using alkyl substituents of various structures, pyridine derivatives, spermidine, and other methods such as alkylation or reductive amination via a suitable Schiff base. To increase hydrophilicity, chitosan amides can be created from amino acids, including sulfur-containing amino acids, lactobionic acid, thioglycolic acid, etc. In addition, secondary amines of chitosan can be obtained using various sugars, polyethyleneimines, and their derivatives. Synthesis of polyethylene glycol derivatives by PEGylation is particularly common. Chitosan derivatives produced according to these methods can have their pharmacological properties modified, particularly by improving their ability to form nanoparticles using polymer electrolytes such as nucleic acids (Mao et al., 2010, Advanced Drug Delivery Reviews, 62, pp. 12-27, doi:10.1016 / j.addr.2009.08.004).

[0007] Chitosan nanoparticles can be formed in an essentially irreversible manner via chemical crosslinking, including the formation of covalent bonds using different reagents for catalysis such as crosslinking (Bhattarai et al., 2010, cited above). Historically, glutaraldehyde and formaldehyde were the first and most common agents used in this crosslinking, but they are no longer common today due to the difficulty of removing all trace amounts of their toxic compounds, as well as the slow hydrolysis of nanoparticles that release these aldehydes. Genepine has recently attracted more attention, but this reagent is very expensive and readily polymerizes itself. Diethyl squalate (DES), ethylene glycol diglycidyl ether (EGDE), and blocked diisocyanates react rather slowly, and reactions with these are mainly carried out at high temperatures, which can cause side reactions. Photoactive crosslinking reagents also exist (functional azides, functional acrylates), as well as enzyme activators - phloretic acid and activated quinones. For example, Baoqiand et al., 2015, Acta Biomaterialia, 22, pp. 1742-7061 or Journal of Nanotechnology in Engineering and Medicine, 6, pp. 041001-6, provide a method for producing crosslinked chitosan by reacting chitosan with methacrylic anhydride to obtain a polyacrylamide compound in the form of a gel or foam, and then using photoinitiated radical polymerization of chitosan in solution. Carboxymethyl derivatives of chitosan are described by El-sherbiny et al., 2009, European Polymer Journal, 45, 1, pp. 199-210, in which, in the first step, a carboxyl group is introduced into the chitosan by reacting the primary alcohol group in the chitosan with monochloroacetic acid in an alkaline medium, and in the second step, an acryloylglycine polymer is added via photoinitiated polymerization to obtain chitosan without internal crosslinking. US Patent No. 5,770,712 describes a method for preparing crosslinked chitosan by combining a chitosan material having unreacted primary amine groups with an excess amount of a polyfunctional epoxide compound having at least two epoxide groups.A crosslinking reaction between chitosan and 1,4-butanediol diglycidyl ether yields an amino-ethanol crosslinked derivative of chitosan that has low solubility at physiological pH (above 7).

[0008] It can also be used for crosslinked, pre-derivativeized chitosan via Schiff base formation with chitosan containing an aldehyde group, disulfide crosslinking with chitosan in a reagent pre-functionalized with terminal sulfhydryl groups, and Michael addition with chitosan pre-treated with a functionalized acrylate in the presence of a weak base.

[0009] All of these methods involve the formation of nanoparticles when chitosan is in solution. In some cases, all chemical reactions proceed to completion in the liquid phase, while in others, they are completed during or after the final stage, such as spray drying or freeze-drying. Importantly, in all cases, the process is initiated, and its crucial stage takes place in soluble chitosan (Jayakumar et al., 2011, cited above). The need to dissolve chitosan imposes significant constraints on the synthesis of nanoparticles. This is due to the fact that chitosan is water-soluble only in the form of a salt in the presence of an acid. In this form, viscosity increases anomalously as the concentration of any chitosan solution greater than 2%, but the number of such increases significantly limits the possible implementation of synthesis and the possibility of high throughput from the reactor. In particular, the wall effect when mixing viscous solutions can create significant heterogeneity in the concentration of reagents in the reaction vessel, and consequently, heterogeneity in the properties of the nanoparticles. Another complex issue is that while highly nucleophilic amino groups are most commonly used for crosslinking, they must remain ionized in solution to keep the chitosan soluble.

[0010] Therefore, there is a need to develop novel synthetic routes for forming chitosan nanoparticles in an effective and cost-effective manner. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] US No. 5,770,712

Patent document 2

Patent document 3

Non-licensed literature

[0012]

Non-licensed literature 1

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

[0013] The present invention relates to the unexpected discovery of a novel method for producing nanoparticles from chitosan, which has the advantage of avoiding the need to dissolve a chitosan starting material and thus the need to form salts, or use diluted acidic aqueous solutions, or disperse chitosan in a solution, and thus the need to rely on emulsification, grinding, or other procedures that may have a mechanical effect on the strength of the polymer. In particular, the present invention relates to a novel method for crosslinked chitosan, and more specifically, to forming novel internal chemical bonds between different parts of a chitosan molecule through two chemical steps. According to certain embodiments, the absence of dissolution of chitosan results in a more effective synthesis turnover rate (e.g., about two orders of magnitude higher than crosslinking methods using dissolved chitosan).

[0014] The resulting product spontaneously forms nanoparticles at the end of the second step of the preparation method, and these nanoparticles are suitable for a wide range of applications. Even more advantageously, the resulting crosslinked chitosan product has similar biological activity to that of standard linear or branched chitosan, but unexpectedly exhibits several specific advantages in terms of stability across a wide range of biologically relevant pH or microbiological conditions, and physicochemical properties that allow for easier handling. [Means for solving the problem]

[0015] One aspect of the present invention provides a method for preparing crosslinked chitosan according to the present invention.

[0016] Another aspect of the present invention relates to novel crosslinked chitosans, particularly novel crosslinked chitosans that spontaneously form nanoparticles.

[0017] Another aspect of the present invention relates to a composition comprising at least one crosslinked chitosan according to the present invention and at least one carrier.

[0018] Another aspect of the present invention relates to a pharmaceutical composition comprising at least one crosslinked chitosan according to the present invention and at least one pharmaceutically acceptable carrier.

[0019] Another aspect of the present invention relates to a nanoparticle comprising one crosslinked chitosan according to the present invention.

[0020] Another aspect of the present invention relates to a cosmetic composition comprising at least one crosslinked chitosan according to the present invention.

[0021] Another aspect of the present invention relates to a soft tissue filler comprising at least one crosslinked chitosan according to the present invention or a composition thereof according to the present invention.

[0022] Another aspect of the present invention relates to a wound dressing comprising at least one cross-linked chitosan according to the present invention.

[0023] According to another specific embodiment, a reconstructed tissue comprising at least one crosslinked chitosan or composition thereof according to the present invention is provided.

[0024] Another aspect of the present invention relates to an agricultural composition comprising at least one cross-linked chitosan according to the present invention.

[0025] Another aspect of the present invention relates to a method for preparing a composition (for example, a soft tissue filler, a wound dressing, or a reconstructive tissue) comprising at least one cross-linked chitosan or nanoparticles thereof according to the present invention.

[0026] Another aspect of the present invention relates to crosslinked chitosan according to the present invention for use in in vivo drug delivery, in vitro cell or biological tissue culture, and tissue engineering applications.

[0027] According to another specific embodiment, a culture medium for cells or biological tissues comprising at least one cross-linked chitosan or a composition thereof according to the present invention is provided.

[0028] Another aspect of the present invention relates to cross-linked chitosan according to the present invention for use in the prevention and / or treatment of medical disorders, in particular cardiovascular conditions, such as cardiac arrhythmias, especially atrial fibrillation and hypertension; joint lesions and diseases, such as rheumatoid arthritis, osteoarthritis, spondyloarthritis; and traumatic events resulting in damage to cartilage, bone, ligaments or synovial sacs; eye lesions and injuries, such as dry eye syndrome, uveitis, glaucoma, corneal lesions; connective tissue disorders, such as lupus and polymyositis; skin / mucosal disorders or injuries, such as wounds, scars, psoriasis, acne, eczema, rosacea; burns of a physical or chemical nature, especially surgical wounds and sunburns; ulcers, hemorrhoids; periodontal disease and dental diseases, such as injuries from accidents or dural damage after surgery of the brain and central nervous system; malignant and benign neoplasms, in particular carcinomas, sarcomas, lymphomas and melanomas; postoperative complications, such as fistulas and infections; tumors or vascular malformations.

[0029] Another aspect of the present invention relates to the use of cross-linked chitosan according to the present invention for the preparation of pharmaceutical formulations for the prevention and / or treatment of medical disorders, particularly cardiovascular conditions such as cardiac arrhythmias, particularly atrial fibrillation and hypertension, joint lesions and diseases such as rheumatoid arthritis, osteoarthritis, spondyloarthritis, and traumatic events resulting in damage to cartilage, bone, ligaments or synovial capsules, eye lesions and injuries such as dry eye syndrome, uveitis, glaucoma, corneal lesions, connective tissue disorders such as lupus and polymyositis, skin / mucosal disorders or injuries such as wounds, scars, psoriasis, acne, eczema, rosacea, burns of a physical or chemical nature, particularly surgical wounds and sunburns, ulcers, hemorrhoids, periodontal disease and dental diseases such as injuries from accidents or dural damage after surgery of the brain and central nervous system, malignant and benign neoplasms, particularly carcinomas, sarcomas, lymphomas and melanomas, postoperative complications such as fistulas and infections, tumors or vascular malformations.

[0030] Another aspect of the present invention relates to the use of cross-linked chitosan according to the present invention for the preparation of cell or biological culture media or reconstructed tissue.

[0031] Another aspect of the present invention relates to a method for preparing a drug delivery system for a bioactive agent.

[0032] According to another specific embodiment, a method is provided for identifying cross-linked chitosan that can be obtained from the method according to the present invention.

[0033] Another aspect of the present invention relates to medical disorders, in particular cardiovascular conditions, such as cardiac arrhythmias, especially atrial fibrillation and hypertension; joint lesions and diseases, such as rheumatoid arthritis, osteoarthritis, spondyloarthritis; and traumatic events resulting in damage to cartilage, bone, ligaments or synovial capsules; eye lesions and injuries, such as dry eye syndrome, uveitis, glaucoma, corneal lesions; connective tissue disorders, such as lupus and polymyositis; skin / mucosal disorders or injuries, such as wounds, scars, psoriasis, acne, eczema, rosacea; and physical injuries. The present invention relates to a method for preventing, treating, or improving burns of a chemical nature, particularly surgical wounds and sunburns, ulcers, hemorrhoids, periodontal disease and dental diseases, such as injuries from accidents or dural damage after surgery of the brain and central nervous system, malignant and benign neoplasms, particularly carcinomas, sarcomas, lymphomas and melanomas, postoperative complications, such as fistulas and infections, tumors or vascular malformations, the method comprising the step of administering an effective amount of at least one cross-linked chitosan or a pharmaceutical formulation thereof according to the present invention to a subject in need thereof.

[0034] Another aspect of the present invention relates to a kit comprising, for example, a freeze-dried form, at least one cross-linked chitosan or composition thereof of the present invention.

[0035] Another aspect of the present invention relates to a kit for the preparation of nanoparticles for encapsulating materials, such as bioactive agents, active pharmaceutical ingredients, proteins, antibodies, sugars, nucleic acids, or combinations thereof, or for prevention or treatment, comprising at least one cross-linked chitosan or composition thereof of the present invention. [Brief explanation of the drawing]

[0036] [Figure 1A] The crosslinked chitosan nanoparticles of the present invention (Example 2i) are characterized. Micrograph of stained chitosan (3.2% crosslinked 501 kDa chitosan) as described in Example 3. [Figure 1B1]Characterization of the crosslinked chitosan nanoparticles of the present invention (Example 2i) is shown. Atomic force microscope image of a dry film of linear chitosan for comparison. [Figure 1B2] Characterization of the crosslinked chitosan nanoparticles of the present invention (Example 2i) is shown. Atomic force microscope image of the corresponding dry film of the crosslinked chitosan nanoparticles of the present invention. [Figure 1B3] Characterization of the crosslinked chitosan nanoparticles of the present invention (Example 2i) is shown. Atomic force microscope of a dried film of the crosslinked chitosan nanoparticles of the present invention at high magnification. [Figure 1B4] Characterization of the crosslinked chitosan nanoparticles of the present invention (Example 2i) is shown. Atomic force microscope of a dry film of the crosslinked chitosan nanoparticles of the present invention at low magnification. [Figure 2] The image shows the internal migration of the cross-linked chitosan nanoparticles of the present invention (Example 2i) within mouse leukocytes as measured by fluorescence microscopy as described in Example 4. A: Leukocyte boundary; B: Nuclear DNA (Hoechst 33258, strong blue fluorescence); C: Endocytized FITC-labeled cross-linked chitosan (green fluorescence); D: Mixed blue / green fluorescence (blue: nonspecific and / or non-nuclear DNA in the cytoplasmic background; green: endocytized FITC-labeled cross-linked chitosan). [Figure 3A] Example 4 shows the internal migration of standard chitosan remaining on the corneal surface as measured by FITC-FAM fluorescence. A: Cornea, B: Chitosan (light green fluorescence). [Figure 3B] The internal migration of the cross-linked chitosan nanoparticles of the present invention (Example 2i) in the mouse cornea is shown by FITC-FAM fluorescence as described in Example 4. A: Cornea, B: Chitosan (light green fluorescence). [Figure 4A1] This shows the mass spectrometry analysis of a comparative non-crosslinked standard chitosan (total ion current relative to cations) as described in Example 3. [Figure 4A2] This shows the mass spectrometry analysis of a comparative non-crosslinked standard chitosan (in the region of the target ion (low m / z ratio)) as described in Example 3. [Figure 4B1]This shows the mass spectrometry analysis of the cross-linked chitosan (total ion current relative to cations) of the present invention as described in Example 3. [Figure 4B2] This shows the mass spectrometry analysis of the cross-linked chitosan of the present invention (in the region of the target ion (low m / z ratio)) as described in Example 3. [Figure 5A] This shows the spectrum obtained from a collision test of a compound with m / z=180 at an impact energy of 5V, as described in Example 3. [Figure 5B] This shows the spectrum obtained from a collision test of a compound with m / z=180 at an impact energy of 15V, as described in Example 3. [Figure 5C] This shows the spectrum obtained from a collision test of a compound with m / z=252 at an impact energy of 5V, as described in Example 3. [Figure 5D] This shows the spectrum obtained from a collision test of a compound with m / z=252 at an impact energy of 15V, as described in Example 3. [Figure 5E] This shows the spectrum obtained from the hydrolysis of egg lysozyme described in Example 3. [Figure 6A] Compared to the control, the effect of treatment with the cross-linked chitosan of the present invention (at 0.1% w / w or 0.05% w / w in water) on the germination process of spring wheat seeds in a germination chamber is shown as described in Example 6 (radicle development was measured after 1 day, germination energy after 3 days, and germination power after 7 days). [Figure 6B] Compared to the control, the effect of treatment with the cross-linked chitosan of the present invention (at 0.1% w / w or 0.05% w / w in water) is demonstrated in the increased growth of the above-ground parts of spring wheat seedlings in soil substrates (increase %) compared to the control, as described in Example 6 (seeds were treated 7 days before sowing). [Modes for carrying out the invention]

[0037] The term "degree of crosslinking," as used herein, refers to the amount of functional groups that are converted into crosslinking or grafting bonds compared to the total amount of functional groups initially present in chitosan, and is expressed as a percentage.

[0038] The term "alkyl," when used alone or in combination with other terms, refers to a monovalent alkyl group having 1 to 50 carbon atoms (C1-C1). 50 This term includes linear or branched alkyl groups. Examples of such groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-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-pentadecyl, n-hexadecyl, n-octadecyl, n-nonadecyl, and n-eicosanyl. These preferably include C1-C9 alkyl groups, more preferably C1-C6 alkyl groups, and particularly preferably C1-C4 alkyl groups, and similarly 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, they include C1-C6 alkyl groups.

[0039] The term "alkenil," when used alone or in combination with other terms, refers to a linear or branched C2-C chain. 50It contains alkenyls. It can have any number of double bonds at any available position, and the arrangement of the double bonds can be (E) configuration or (Z) configuration. This term is exemplified by groups such as vinyl, allyl, isopropenyl, 1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-ethyl-1-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 1-octenyl, geranyl, 1-decenyl, 1-tetradecenyl, 1-octadecenyl, 9-octadecenyl, 1-eicocenyl, and 3,7,11,15-tetramethyl-1-hexadecenyl. These preferably include C2-C8 alkenyls, more preferably C2-C6 alkenyls. Among these, particularly preferred are vinyl or ethenyl (-CH=CH2), n-2-propenyl (allyl, -CH2CH=CH2), isopropenyl, 1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and 3-methyl-2-butenyl.

[0040] The term "alkynyl," when used alone or in combination with other terms, refers to a linear or branched C2-C chain. 50 This term includes alkynyl groups, which can have any number of triple bonds at any available position. The term is exemplified by groups such as alkynyl groups with 2 to 50 carbon atoms and optionally having double bonds, such as ethynyl (-C≡CH), 1-propynyl, 2-propynyl (propargyl: -CH2C≡CH), 2-butynyl, 2-pentene-4-inyl, etc. In particular, these include C2-C8 alkynyl groups, and more preferably C2-C6 alkynyl groups, etc. Preferably, these include C2-C6 alkynyl groups having 2 to 6 carbon atoms and having at least 1 or 2 alkynyl unsaturated sites.

[0041] The term "aryl" refers to an unsaturated aromatic carbocyclic group consisting of 6 to 14 carbon atoms, having a monocyclic ring (e.g., phenyl) or a polycondensed ring (e.g., indenyl, naphthyl). Aryls include phenyl, naphthyl, anthryl, phenantrenyl, and others.

[0042] The term "C1-C6 alkylaryl" refers to an aryl group having a C1-C6 alkyl substituent, including methylphenyl, ethylphenyl, and others.

[0043] The term "aryl C1-C6 alkyl" refers to C1-C6 alkyl groups having an aryl substituent, including 3-phenylpropanyl, benzyl, etc.

[0044] The term "heteroaryl" refers to a monocyclic heteroaromatic group, or a bicyclic or tricyclic fused heteroaromatic group. Specific examples of heteroaromatic groups include optionally substituted pyridyl, pyrrolyl, pyrimidinyl, furyl, thienyl, imidazolyl, 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, benzofuryl, [2,3-dihydro]benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl, isobenzothi Examples include enyl, indolyl, isoindolyl, 3H-indolyl, benzimidazolyl, imidazo[1,2-a]pyridyl, benzothiazolyl, benzoxazolyl, quinolidinyl, quinazolinyl, phthalazinyl, quinoxalinyl, cinnolinyl, naphthilidinyl, pyrido[3,4-b]pyridyl, pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl, quinolyl, isoquinolyl, tetrazolyl, 5,6,7,8-tetrahydroquinolyl, 5,6,7,8-tetrahydroisoquinolyl, purinyl, pteridinyl, carbazolyl, xanthenyl, or benzoquinolyl.

[0045] The term "C3-C8 cycloalkyl" refers to a saturated carbocyclic group of 3 to 8 carbon atoms having a monocyclic ring (e.g., cyclohexyl) or a polycondensed ring (e.g., norbornyl). C3-C8 cycloalkyls include cyclopentyl, cyclohexyl, norbornyl, etc.

[0046] The term "heterocycloalkyl" refers to C3-C8 cycloalkyl groups as defined above, in which up to three carbon atoms are replaced by heteroatoms selected from the group consisting of O, S, and NR, where R is defined as hydrogen or methyl. Heterocycloalkyls include azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, tetrahydrofuranyl, etc.

[0047] Unless otherwise constrained by the definition of individual substitutions, the term "substituted" refers to a group substituted with 1 to 5 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," "aryl C1-C6 alkyl," "heteroaryl C1-C6 alkyl," "C1-C6 alkylcycloalkyl," "C1-C6 alkylheterocycloalkyl," "amino," "aminosulfonyl," "ammonium," "acylamino," "aminocarbonyl," "aryl," "heteroaryl," "sulfinyl," "sulfonyl," "alkoxy," "alkoxycarbonyl," "carbamate," "sulfanyl," "halogen," trihalomethyl, cyano, hydroxy, mercapto, nitro, etc.

[0048] The term "pharmaceutically acceptable" refers to a carrier made of materials that are not biologically or otherwise undesirable, and in particular, non-toxic.

[0049] 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.

[0050] The term “bioactive agent” is used to describe any bioactive agent incorporated into the graft polymer composition of the present invention. It may be natural, synthetic, semi-synthetic, or derivative thereof, and may include hydrophobic, hydrophilic, soluble, and insoluble compounds. More specifically, it may be any bioactive agent useful for the treatment and / or prevention and / or diagnosis of conditions in any therapeutic area known in mammals such as animals and humans, particularly in humans, including cardiac arrhythmias, e.g., atrial fibrillation, hypertension, inflammatory conditions, disorders, or diseases, particularly autoimmune and non-autoimmune inflammatory conditions including joint lesions and diseases (e.g., rheumatoid arthritis, osteoarthritis, and spondyloarthritis), and ocular lesions (e.g., dry eye syndrome, bud flu). This includes, but is not limited to, dysplasia, glaucoma, corneal lesions, connective tissue disorders (e.g., lupus or polymyositis), skin disorders or injuries (e.g., wounds, scars, psoriasis, acne, eczema, rosacea, burns of a physical or chemical nature, sunburn), periodontal and dental diseases, hemorrhoids, ulcers, diseases suitable for gene therapy, diseases suitable for cell therapy, dural formation and repair after surgery of the brain and central nervous system, neurogenesis, pain, postoperative fistulas and infections, traumatic diseases, benign and malignant neoplasms, and infections. Bioactive agents can be selected from high molecular weight or low molecular weight compounds, such as peptides, proteins, oligonucleotides and polynucleotides, anti-infective agents, antibiotics, antimicrobial agents, antiviral agents, antibacterial agents, antifungal agents, growth factors, enzymes, antigens, antitumor agents, anti-inflammatory agents, anesthetics, antineoplastic agents, analgesics, anticoagulants, hemostatic agents, cells, and antibodies.

[0051] The term "inflammatory disorder or disease" refers to all diseases that involve inflammation and the release or inflammatory cytokines as the primary or significant disease mechanism, such as inflammatory bowel disease, allergies, asthma, autoimmune diseases, hepatitis, inflammation of other organs, and related diseases.

[0052] The term “skin disorder” or “skin disease” includes skin injuries that result in painful depressions on the skin surface that do not require incision of the surface, such as age-related tissue damage (e.g., wrinkles), wounds, and scars such as acne or rubella scars. These disorders further include wounds, scars, psoriasis, acne, eczema, and rosacea. The term “wound” includes, for example, any damaged tissue after trauma or surgery. Wounds in mammals include, for example, abrasions, lacerations, contusions, punctures, cuts, surgical wounds, gunshot wounds, burns, chemical wounds, sunburns, punctures and insect bites, and electrical wounds. It further includes chronic skin disorders such as ulcers and other inflammatory skin conditions.

[0053] The term “ocular lesion or disorder” refers to a disorder or injury affecting the eye, particularly the cornea. Such disorders include corneal abrasions, corneal scratches, corneal alkaline burns, age-related macular degeneration, proptosis, cataracts, cytomegalovirus retinitis, color blindness, strabismus, diabetic macular edema, ocular floaters and ocular flicker, glaucoma, keratoconus, amblyopia, low visual acuity, ocular hypertension, retinal detachment, blepharospasm, uveitis, keratoconjunctivitis sicca (KCS), and dry eye syndrome.

[0054] The term "articular lesion" includes osteoarthritis, arthritis pain, rheumatoid arthritis, pain from infection and inflammation, traumatic events of the knee resulting in cartilage, traumatic events of the hip, and damage to bone, ligaments, or synovial sac.

[0055] The term "reconstructed tissue" refers to biological tissue (endogenous or exogenous), synthetic or semi-synthetic material that is useful for repairing damaged tissues of the body, such as epidermis, nerves, cartilage, or bone tissue, and for constructing organoids for biological tests, as described by Takebe et al., 2019, Science, 364, 6444, pp. 956-959, DOI: 10.1126 / science.aaw756.

[0056] As used herein, “treatment” and “to treat” generally mean obtaining a desired pharmacological and / or physical and / or physiological effect. The effect may be prophylactic in that it prevents or partially prevents a disease, its symptoms or condition, and / or therapeutic in that it partially or completely treats a disease, condition, symptoms, or adverse effects contributing to the disease. As used herein, the term “treatment” encompasses all treatments of diseases in mammals, particularly humans, and includes: (a) preventing the onset of a disease in subjects who are susceptible to the disease but have not yet been diagnosed with it; (b) inhibiting the disease, i.e., stopping its onset; or reducing the disease, i.e., causing regression of the disease and / or its symptoms or condition, such as improvement or treatment of the injury.

[0057] As used herein, the term "subject" refers to mammals. For example, the mammals envisioned by the present invention include humans, primates, and domestic animals such as cattle, sheep, pigs, horses, especially racehorses, and laboratory rodents.

[0058] The term "efficacy" in the treatment according to the present invention can be measured based on changes in the course of the disease in accordance with the use of the present invention. For example, the efficacy of the treatment according to the present invention can be measured by the disappearance or reduction of clinical signs or symptoms, or more preferably by measuring disease biomarkers, such as cytokines, growth factors, or other signaling molecules and their receptors, cell surface markers, cell count, and gene expression profiles.

[0059] Method for preparing chitosan according to the present invention and its characterization According to a particular embodiment, a method for preparing cross-linked chitosan, comprising the following steps: a) A step of preparing chitosan and swelling the chitosan in a solvent; b) The amino group of the chitosan is given formula (I):

[0060] [ka]

[0061] [wherein R1 is a halogen or any other leaving group that ensures acylation of the amino group upon removal, e.g., 3-hydroxybenzotriazole ester, anhydride (including mixed anhydride), N-hydroxysuccinimide, pentachlorophenol, 2-nitro-4-sulfophenol ester, and other similar leaving groups; R2, R3, and R4 are 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 cycloalkyl), optionally substituted cycloalkenyl (e.g., C4-C8 cycloalkenyl), optionally substituted cycloalkynyl (e.g., C5-C8 cycloalkynyl), optionally substituted heterocycloalkyl; [Optionally substituted aryls (e.g., optionally substituted phenyls), optionally substituted heteroaryls, and optionally substituted aryl C1-C6 alkyls, particularly benzyls, independently selected; the term "substituted" refers to a group substituted with 1 to 5 substituents selected from the group consisting of halogens, -COOR', -NR'R", =O, -OR', -COR', -CONR'R", -SR', -SO3R', -SO2NR'R", -SOR', -SO2R', -NO2, or -CN; or R1 and R2, or R1 and R3, or R1 and R4 together form optionally substituted 4-24 memberned aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups (e.g., 6-24 memberned aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups, e.g., optionally substituted 8-24 memberned aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups)] A process of acylation with an acrylic compound; c) Reacting the acylation product from step b) in the presence of a base (Azachaïc reaction); d) A step of purifying the crosslinked chitosan obtained from step c) (e.g., from salt impurities and / or aprotic solvents). A method is provided that includes this.

[0062] According to a particular embodiment, the solvent used in steps a) and / or b) is a protic solvent (e.g., alcohol or water).

[0063] According to a particular embodiment, if a protic solvent (e.g., alcohol or water) is used in steps a) and / or b) to carry out the process, the free acrylic acid formed is washed off from the reaction mixture before step c) is carried out.

[0064] According to another specific embodiment, the solvent is an aprotic solvent, particularly in steps a) and / or b) and / or c).

[0065] According to certain embodiments, if an aproton solvent is used in step c), the subsequent acylation step f) is not performed.

[0066] According to a particular embodiment, step a) is carried out at room temperature.

[0067] According to certain embodiments, not only the R3 or R4 group of the acylated in step b), but also the R2 group reacts with the primary amino group of the glucosamine skeleton to form crosslinks between glucosamine molecules. The reacting group depends on the specific acrylic compound. For example, with respect to acrylic acid and methacrylic acid, the group that reacts with the primary amino group of the glucosamine skeleton is R3 or R4. However, if the R3 and R4 groups are replaced by halogen atoms, the R2 group reacts with the primary amino group of the glucosamine skeleton.

[0068] According to a further specific embodiment, this crosslinking results in the formation of nanoparticles.

[0069] According to a particular embodiment, the method of the present invention is as follows: Scheme 1:

[0070] [ka]

[0071] This can be systematized under the following: Here, chitosan (A) (wherein m is an integer in the range of 1 to 12,500, and n is an integer in the range of 1 to 12,500) is first brought into a swollen state in an aprotic solvent at room temperature, then the amino group of the chitosan is acylated with an acrylic compound (I), and then the resulting acylation product (B1) is reacted in the presence of a base to obtain crosslinked chitosan (B2), which is then isolated by purification to obtain purified crosslinked chitosan according to the present invention.

[0072] According to certain embodiments, chitosan can be obtained in a swollen state or in a soluble state in a protic solvent, but in this case, a side reaction of hydrolysis of the acylating agent may occur, which must be taken into consideration when calculating the reaction load. In addition, in this case, the acylated chitosan obtained in step b) must be washed to remove the hydrolysis product of the acylating agent before the Azachaeich reaction step in step c).

[0073] According to certain embodiments, chitosan is produced in the absence of water, and the acylation process is carried out in the absence of water. The absence of water is advantageous in that it results in a higher yield and avoids the formation of byproducts.

[0074] In certain advantageous embodiments, 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 supercritical fluid. In certain embodiments, the aprotic solvent is a polar aprotic solvent, such as selected from DMF and DMSO. In certain embodiments, the supercritical fluid may be a supercritical solvent such as carbon dioxide, nitric oxide (I), or Freon (chloro(bromo)(fluoro) hydrocarbon) used to supply the acylation agent to the reaction medium, and the acylation reaction is then carried out after removing the supercritical solvent from the reaction medium, for example by reducing the pressure to below a critical value.

[0075] Furthermore, according to a particular advantageous embodiment, the acylation step is carried out in an anhydrous aprotic medium.

[0076] According to a particular embodiment, the polar aprotic solvent is selected from dimethylformamide (DMF), dimethylacetamide, acetonitrile (MeCN), N-methylpyrrolidone, dimethyl sulfoxide (DMSO), or a mixture thereof.

[0077] In another specific embodiment, dichloromethane, dichloroethane, chloroform, and other chloro(fluoro) hydrocarbons can also be used as polar aprotic solvents, but when carrying out step c) of the Azachaimhaek reaction, these solvents must be removed by distillation immediately before providing the base. Such distillation is preferably carried out at a temperature not exceeding 60°C, and many of the solvents mentioned can be carried out at ambient pressure. If high-boiling point solvents are used, distillation must be carried out under reduced pressure.

[0078] In another specific embodiment, ethers and esters and ketones can also be used as solvents in reaction steps a) to c) under anhydrous conditions, although 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.

[0079] According to a particular aspect of the present invention, the acylation step b) can be carried out by any method described for the acylation of glucosamine with acryloyl chloride (Zhang et al., 2017, Biomacromolecules, 1, 3, pp. 778-786; Bu et al., 2017, Advances, 7, 76, pp. 48166-48175), or by any method described as useful for acylation of an amino group, such as using i) carbodiimide; ii) azide; iii) mixed anhydride; iv) activated ester; and v) other methods described above.

[0080] Known acylation methods using carbodiimides, including the formation of an intermediate enol ester, can be used in step b) (WO 2019 / 60740; Hao-Bin et al., 2018, Carbohydrate Polymers, 196, pp. 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 methyl p-toluenesulfonate (CAS registry number: 2491-17-0) are preferred.

[0081] Known acylation methods using azides can be used in step b) (Honzl et al., 1961, Coll. Czech. Chem. Commun., 26, N. 9, pp. 2333-2344).

[0082] Known acylation methods using mixed anhydrides can be used in step b) (Wieland et al., 1951, Ann. Chem., 572, N3, pp. 190-194; Belleau et al., 1968, J. Amer. Chem. Soc., 90, N6, pp. 1651-1652; Gorecka et al., 1978, Synthesis, N6, pp. 474-476; Diago-Meseguer et al., 1980, Synthesis, N7, pp. 547-551; Leplawy et al., 1960, Tetrahedron, 11, N1, pp. 39-51). The use of internal anhydrides, such as maleic anhydride, also enables acylation (Liwschitz et al., 1957, Journal of the Chemical Society, p. 4399; Kang et al., 2014, Bioorganic and Medicinal Chemistry Letters, 2, 10, pp. 2364-2367; US 2016 / 200730; Sanchez et al., 2010, Anna European Journal of Organic Chemistry, 13, pp. 2600-2606).

[0083] Known acylation methods using activated esters via the formation of activated amides, e.g., the carbonyl diimidazole method (Paul et al., 1960, J. Amer. Chem. Soc., 82, N17, pp. 4596-4600), the cyanomethyl ester method (Schwyzer et al., 1955, Helv. Chim. Acta, 38, N1, pp. 80-83), the thiophenyl ester method (Wieland et al., 1951, previously mentioned), the substituted phenyl ester method (Gross et al., 1983, Mayenhofer, editors. Moscow: Mir., p. 421), and esterification methods with heteroaromatic compounds in combination with the carbodiimide method (Jakubke et al., 1966, A. Chem. Ber., 99, N8, pp. 2419-2429; Taschner et al., 1965, Ann. Chem., 690, pp. 177-181), esterification method with hydroxylamine derivatives using the carbodiimide method (Losse et al., 1964, Ann. Chem., 678, pp. 185-190; Nefkens et al., 1961, Amer. Chem. Soc., 83, N5, p. 1263; Anderson et al., 1963, Ibid, 85, N19, p. 3039; Konig et al., 1970, Chem. Ber., 103, N3, pp. 788-798), transesterification method (variation of the esterification method) (Sakakibara, 1965, Bull. Chem. Soc. Jap., 38, N1, pp. 1979-1984; Fujino et al., 1968, Ch. Chem. Pharm. Bull., 16, N5, pp. 929-932; Gudkov et al., 1978, 48, 9, pp. 2146; Devadas et al., 1979, Ind. J. Chem., B16, N11, pp. 1026-1027) can be used in step b).

[0084] Other known acylation methods, e.g., the ketenimine method (Stevens et al., 1958, J. Amer. Soc., 80, N15, pp. 4069-4071); the acetylene derivative method (Arens, 1955, Rec. Trav. Chim., 74, N6, pp. 759-770; Gais, 1978, Aktivierungsmittel fur Peptidsynthesen J. Angew. Chem. Int. Ed, 90(8), pp. 625-626, https: / / doi.org / 10.1002 / ange.19780900808); and the method using cyanamide derivatives (Losse et al., 1960, Ann. Chem., 636, pp. 144-149). A synthesis method using isoxazolium salt (Woodwart et al., 1961, J. Amer. Chem. Soc., 83, N4, pp. 1010-1012); a synthesis method using halide midomyl (Bergmann et al., 1936, J. Biol. Chem., 115, N3, pp. 93-611) can be used in step b).

[0085] According to certain embodiments, the acrylic compound of formula (I) may be an acid, an acid halide, an active ester (e.g., 3-hydroxybenzotriazole ester, N-hydroxysuccinimide, pentachlorophenol, 2-nitro-4-sulfophenol ester, and other similar esters having a leaving group), an anhydride, or a mixture thereof.

[0086] According to a particular embodiment, the acrylic compound of formula (I) is selected from the following group:

[0087] [ka]

[0088] (Angelic acid, CAS Registry Number: 565-63-9);

[0089] [ka]

[0090] (2-Isopropylacrylic acid, CAS Registry Number: 4465-04-7);

[0091] [ka]

[0092] (2-Methylenedodecanoic acid, CAS Registry Number: 52756-21-5);

[0093] [ka]

[0094] (4S,6S)-4,6-dimethyl-2-methylenedocosanoic acid;

[0095] [ka]

[0096] ((Z)-3-cyclohexylpropenoic acid, CAS registry number: 673456-32-1);

[0097] [ka]

[0098] ((E)-3-[4-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purine-8-yl)-bicyclo[2.2.2]octa-1-yl]acrylic acid, CAS registry number: 340021-16-1);

[0099] [ka]

[0100] ((2E)-3-((E)-4-{[2-(4-chlorophenoxy)-2-methylpropanoyl]amino}-1-adamantyl)acrylic acid;

[0101] [ka]

[0102] (3-(2-carboxy-4-phenyl-3-styryl-cyclobutyl)-acrylic acid, CAS registry number: 34271-87-9);

[0103] [ka]

[0104] (3-methyl-2-acrylic acid (acrylsaeure)-(1)-cyclopropane-carboxylic acid (carbonsaeure)-(1)-carboxylic acid-(1)-ethyl ester, CAS registry number: 91971-88-9);

[0105] [ka]

[0106] (3t,4t-Dicarboxycyclobutane-1,2c-di-(trans-acrylic acid), CAS Registry Number: 55011-62-6);

[0107] [ka]

[0108] Coronadiene;

[0109] [ka]

[0110] (CAS Registry Number: 1247-53-6)

[0111] [ka]

[0112] ((E)-3-(2t-ethoxycarbonyl-6ξ-methoxycyclohexane-1r-yl)-acrylic acid, CAS registry number: 2960-11-4); cinnamic acid; 4-methylcinnamic acid; p-nitrocinnamic acid; caffeic acid;

[0113] [ka]

[0114] (3-(flu-2-yl)crotonic acid; 3-(2-furyl)acrylic acid, CAS registry number: 539-47-9);

[0115] [ka]

[0116] ((Z)-Urocanic acid, CAS Registry Number: 7699-35-6);

[0117] [ka]

[0118] (3-(4-pyridinyl)-2-propenoic acid; 3-(pyridin-4-yl)acrylic acid, CAS registry number: 5337-79-1); argutinoside AI;

[0119] [ka]

[0120] ((Z)-2-chlorobuta-2-enoic acid, CAS registry number: 53993-41-2);

[0121] [ka]

[0122] ((2Z)-2,3-dichloropropa-2-enoic acid, CAS Registry Number: 3533-68-4);

[0123] [ka]

[0124] cis N-tert-butyloxycarbonyldehydroβ-alanine, CAS Registry Number: 151292-68-1);

[0125] [ka]

[0126] (2-Hydroxy-3-mercaptoacrylic acid, CAS registry number: 6228-60-0;

[0127] [ka]

[0128] Hydroxy(meth)acrylic acid;

[0129] [ka]

[0130] (Dihydroxyacrylic acid, CAS registry number: 2702-94-5);

[0131] [ka]

[0132] (Reaxys ID 2721331);

[0133] [ka]

[0134] (CAS Registry Number: 64361-31-5);

[0135] [ka]

[0136] 3-(2-carboxymethylene-cyclopentyl)-acrylic acid;

[0137] [ka]

[0138] (CAS Registry Number: 78727-62-5);

[0139] [ka]

[0140] (Fluorobutenic acid, CAS registry number: 2365-87-9);

[0141] [ka]

[0142] ((E)-2,3-bis(phenoxycarbonylamino)buta-2-enediolic acid);

[0143] [ka]

[0144] ((Z)-2,3-bis(phenoxycarbonylamino)buta-2-enediolic acid);

[0145] [ka]

[0146] (2E,4E)-2,4-Hexadienediolic acid (muconic acid), maleic acid; fumaric acid;

[0147] [ka]

[0148] (Maleilacetoacetic acid, CAS registry number: 5698-52-2); (E)-4-(methylamino)-4-oxobuta-2-enoic acid, acrylic derivative of glutamic acid

[0149] [ka]

[0150] (2-Methylene-9(Z)-Octadenoic Acid, CAS Registry Number: 33780-98-2);

[0151] [ka]

[0152] (2-Methylene-5-hexenoic acid (2-methylenehex-5-enoic acid), CAS Registry Number: 73505-05-2);

[0153] [ka]

[0154] ((E)-6,9-dimethyl-2-methylideneundeca-5,9-dienoic acid, CAS Registry Number: 1580541-76-9);

[0155] [ka]

[0156] (2-(cis-7,8-hexadecenyl)-acrylic acid);

[0157] [ka]

[0158] ((E)-2-methyleneocta-4,7-dienoic acid);

[0159] [ka]

[0160] (2-methylene-7-octic acid, CAS registry number: 127559-93-7); and

[0161] [ka]

[0162] (2-Methylene-5-decynic acid, CAS registry number: 150254-20-9); and acyl halides (R1=halogen), especially acryloyl chloride;

[0163] [ka]

[0164] Alternatively, methacryloyl chloride.

[0165] Furthermore, according to certain embodiments, the acrylic compound is an acyl halide (R1=halogen), particularly acryloyl chloride.

[0166] [ka]

[0167] Alternatively, it is methacryloyl chloride.

[0168] In certain embodiments, when the acrylic compound used in acylation step b) is an acid halide, it is important to avoid the presence of water in the reaction system, as this can lead to an undesirable side process of hydrolysis of the acid halide resulting in acrylic acid, which can result in the functionalization of nanoparticles by carboxyl groups during the Michael addition reaction of free acrylic acid to the amino group of chitosan.

[0169] In a particular embodiment, the acrylic compound used in the acylation step b) is an acid chloride (e.g., acyl chloride). In one embodiment, the use of an acid chloride is advantageous because it results in the formation of hydrogen chloride, which does not interfere with the next reaction step in the presence of excess base and is readily removed in the form of a salt during dialysis.

[0170] It is important to note that the acylation of an amine with a carboxylic acid halide results in the formation of one equivalent of acid, which forms a salt with the unreacted amine and reduces the yield of the method of the present invention. Therefore, when an acid halide is used in step b), the addition of an equivalent amount of base, preferably a non-nucleophilic base (e.g., triethylamine), is used in step b) to neutralize the acid.

[0171] In particular embodiments, if the acrylic compound used in step b) is an acid anhydride, the method of the present invention further includes a step of washing the reaction medium and removing the formed acid to avoid the presence of water in the reaction system before proceeding to step c). In particular, the formed acid can be washed away from the acylation product containing the solvent by passing it through a filter, by dialyzing it against the solvent, or by suspending the acylation product in the solvent and centrifuging it while discarding the supernatant.

[0172] In another specific embodiment, when a condensing agent (e.g., DCC) is used for acylation with a free acid, the completion of the reaction must be carefully controlled, and nevertheless, it is preferable to include a washing step as a precautionary measure. For example, when a condensing agent such as DCC is used, by-products such as dicyclohexylurea are formed during the acylation reaction, and these need to be removed from the reactants before the next step in order to prevent them from being included in the crosslinks formed that result in the nanoparticles of the present invention.

[0173] According to another specific embodiment, in step c), a base is used in a molar excess sufficient to form an alkaline medium that neutralizes the hydrogen halide formed in step b). Typically, the molar excess of the base is at least 10% molar excess (e.g., about 20–40% molar excess), which corresponds to at least 1–3 molar excess relative to the amino groups of the acylation product obtained in step b).

[0174] According to another specific embodiment, the crosslinked chitosan obtained in step c) can be purified in step d) by removing the aprotic solvent by standard techniques, such as dialysis, or by washing with water or a solvent such as methylene chloride, methanol, ethanol, or acetone, followed by centrifugation or a process using a supercritical solvent such as carbon dioxide in which the crosslinked chitosan nanoparticles are insoluble, or by evaporation, and then washing off by-products from the crosslinked chitosan product with a solvent through a filter, for example a filter having pores of 40 μm or less. According to a more specific embodiment, finely porous hydrophobic filter elements, such as polypropylene or PTFE, can be advantageously used because their hydrophobicity further hinders the penetration of hydrophilic chitosan nanoparticles through them.

[0175] According to another specific embodiment, the purified cross-linked chitosan obtained in step d) can be dried in step e), for example, an optional further drying step.

[0176] According to certain embodiments, the base used in step c) for the Michael addition reaction is essentially a pure non-nucleophilic base and, in particular, essentially does not involve the presence of a secondary amine.

[0177] In certain embodiments, the Michael addition step c) is carried out in a protic solvent such as water. In this case, it is necessary to control the degree of saponification by measuring, for example, the amounts of both the carboxyl groups formed in the acylation step and the amino groups formed. Precise methods for carboxyl groups are NMR, IR spectroscopy, or acid-base titration, as described in Glazunov et al., 1999, 25(3), pp. 216-219; Brugnerotto et al., 2001, Polymer, 42, pp. 3569-3580; or Kubota et al., 2000, Carbohydrate Research, 324, pp. 268-274, since their concentration is usually one to two orders of magnitude smaller and the difference in the number of amino groups can be within the range of experimental error. However, a protic solvent in this step may be advantageous if it is desired to obtain saponified crosslinked chitosan nanoparticles at the end of step c), or if it is planned to repeat the acylation step as described below to saturate the nanoparticles with carboxyl functional groups and then achieve excess crosslinking. In fact, if the nanoparticles are saturated with carboxyl functional groups, the saponification step is performed. Next, the acylation and Azachaech steps are repeated, followed by saponification. If spontaneous saponification occurs in a protic solvent during the Azachaech step, this is not disadvantageous, as the fact that saponification will be performed later anyway and occurs partially during the Azachaech reaction is not a problem.

[0178] In another specific embodiment, 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.

[0179] According to another specific embodiment, for each amide bond formed during the acylation step, 1 equivalent of a secondary amine is formed after the Michael addition reaction, so that even in the case of cross-linking of all amino groups, at least half of the amino groups remain available for salt formation, and thus it is carried out in an alkaline medium. This can be confirmed by acid-base titration, IR, and NMR spectroscopy as described in Glazunov et al., 1999, 25(3), pp. 216 - 219; Brugnerotto et al., 2001, Polymer, 42, pp. 3569 - 3580, or Kubota et al., 2000, Carbohydrate Research, 324, pp. 268 - 274. According to a specific aspect, the intramolecular addition reaction within the chitosan polymer is significantly favored for the intermolecular reaction due to statistical and steric factors, thereby obtaining a high yield in cross-linking.

[0180] The nanoparticles obtained after the Michael addition reaction step c) can be reduced (for example, by reacting with other derivatives of complex metal hydrides, diborane, cyanoborohydride, and borohydride) to convert the amide groups to secondary amines and increase the degree of ionization of the nanoparticles. The degree of ionization of the nanoparticles depends on the number of amino groups. The more amino groups are present, the greater the chance of salt formation and the resulting ionization. The degree of ionization can be evaluated by acid-base titration, IR, and NMR spectroscopy.

[0181] According to a further aspect, the crosslinked chitosan product obtained in step c) or step 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 an aqueous base) to enhance the solubility of the crosslinked chitosan nanoparticles under a wide range of pH, for example, under physiological weak alkaline conditions (e.g., at a pH of about 7.2 - 7.3). If saponification cannot be carried out with the solvent (e.g., DMF) used in step c) or d), saponification needs to be carried out after the purification step d) in a solvent that enables saponification, or by resuspending the dried crosslinked chitosan in a solvent that enables saponification after step e), such as an ether as the solvent.

[0182] For example, in this further step f), an aqueous base is added to the product obtained in step c) or d) in a molar excess compared to the amount of the acylating agent used in step b), for example, about 10 times or 100 times the molar amount of such an acylating agent. When systematized in the following Scheme 2, the amide groups of the crosslinked chitosan (B2) are hydrolyzed to the corresponding carboxyl groups with the product (B3) available for the formation of the corresponding salts.

[0183]

Chemical Structure

[0184] This allows for the regeneration of half of the amino groups of the crosslinked chitosan as primary amines, and the other half as secondary amines, which are then alkylated with propionic acid (or substituted propionic acid). For example, saponification of crosslinked chitosan can be carried out by adding it to 1 mole of aqueous solution of sodium hydroxide at 50°C for 1 hour. The resulting product (B3) can then be subjected to acylation step b) again, as systematized in schemes 3a and 3b below, in which case the acylation occurs under the same conditions that allow for acylation of both primary amino groups (scheme 3a) and secondary amino groups (novel acylation of amino groups obtained in steps b, c, or f, scheme 3b). The resulting product (B4) can then be subjected to the Michael addition reaction again in step c) to achieve crosslinking of all amino groups and obtain essentially complete crosslinked chitosan (B5).

[0185] [ka]

[0186] [ka]

[0187] In this case, the internal salt in the resulting product is formed of carboxyl groups, which is half the initial content of amino groups present at the end of the first synthesis step c). According to a particular embodiment, such a product essentially contains the same number of carboxyl groups, all secondary and tertiary amino groups, and amide functional groups (except for the first acetylaminoglycoside fragment), i.e., such a product contains a complete internal salt and all carboxyl groups in balance with the amino groups.

[0188] While not intending to be limited to any particular theory, if an amino group is present in the initial chitosan obtained in step a), it can be considered that its complete derivation is reached in step b), where the amino group of X is acylated with the acylating agent, and thus the amino group of X undergoes an Azachaech reaction with the acyl fragment grafted onto the chitosan in step c). After saponification of the amide group formed in step b) present in the product of step c) following saponification step f), the primary amino group of X is regenerated, the carboxyl group of X is obtained from the acrylic acid used in acylation, and the secondary amino group of X obtained from step c) remains. Thus, if complete derivation by acylation is repeated, the regenerated primary amino group of X can be further acylated (the secondary amino group obtained in step c) may also be acylated, but this is an unlikely process). Then, if step c) is repeated, the secondary amino group obtained earlier in step c) undergoes an Azachaech reaction to form the tertiary amino group of X.

[0189] Furthermore, by performing saponification a second time, the primary amino group of X is regenerated once more, while the unsaponified tertiary amino group of X remains, forming the carboxyl group of X. Therefore, the total number of carboxyl groups is 2X (primary amino group X, tertiary amino group X), the polymer becomes electrically neutral, and an internal salt is formed.

[0190] If the complete induction is performed three times, only the amino groups of X remain capable of undergoing these reactions, and since all of these amino groups are primary, only X / 2 of the acylating agent is required. Therefore, after the third induction sequence (steps b)-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 an excess of 0.5X carboxyl groups. Thus, repeating this reaction sequence results in an increase in the number of carboxyl groups by units such as 0.25, 0.125, 0.0625, 0.03175 each time. The presence of carboxyl groups can be easily confirmed by infrared or acid-base titration, and can also be confirmed by mass spectrometry.

[0191] According to a particular embodiment, the amount of alkali used in any saponification step f) is equimolar to the amide group obtained in the first reaction step c) / d) or e) being saponified, and is a molar excess of up to 10 times.

[0192] According to a particular embodiment, at least one sequence of steps b) to c) (or d) / e)) is performed in saponification step f) as completion of Michael addition before a new sequence of acylation steps b) to c) (or d) / e)) is performed again.

[0193] Furthermore, according to a particular embodiment, approximately 2 to 6 reaction sequences of steps a) to f) are performed.

[0194] According to another specific embodiment, the reaction sequence of steps a) to f) is repeated once or twice.

[0195] According to certain embodiments, after each sequence of steps a) to f), the solubility of the crosslinked chitosan is obtained over a wide pH range, and the properties of the natural chitosan can be modified, for example, in terms of mucotropy and cell penetration. Thus, these properties can be advantageously tuned by the number of reaction sequences performed, and the resulting crosslinked chitosan nanoparticles may be useful for the delivery of the compound of interest.

[0196] According to certain embodiments, cationic substances (for example, cationic peptides, such as arginine, ornithine, or lysine-rich sequences, or substances neutral in terms of charge) can be delivered by the crosslinked chitosan according to the present invention.

[0197] According to another specific embodiment, the crosslinked chitosan according to the present invention can be used to formulate these compounds in order to protect them from degradation (e.g., protein degradation) or binding of undesirable substances (e.g., antibodies or other polymers), or to ensure sustained release of solubility for these compounds.

[0198] According to another specific embodiment, the biologically active material can be incorporated into the formed nanoparticles after either the first acylation step b) or the first Michael addition step c). According to a more specific embodiment, the biologically active material can either covalently bond, or adsorb or be fixed in the form of a salt, or be encapsulated within the crosslinked chitosan nanoparticles of the present invention.

[0199] When performing the saponification step, the covalent crosslinking of the biologically active agent is disrupted, but the ionic bond with the biologically active agent can be formed within the salt of the formed crosslinked chitosan.

[0200] Alternatively, when creating radiopaque nanoparticles by applying the sequence of steps a) - f) with a cluster of heavy elements or the like, the nanoparticles can be impregnated with a biologically active substance that is resistant to the reagents used in these steps.

[0201] Furthermore, alternatively, a multifunctional and labile biologically active substance is preferably introduced by dispersion into the crosslinked chitosan nanoparticles of the present invention after its synthesis, or by using click chemistry methods, enzyme, protein, nucleic acid, and antibody immobilization methods.

[0202] According to a specific aspect, incorporating the biologically active material into the chitosan nanoparticles of the present invention can be achieved by impregnating the product obtained from step b) or c) with the desired biologically active material. For example, a solution of the biologically active material in an aprotic solvent can be added to the product obtained from step b) before performing the Michael addition step c). Alternatively, the acetylated chitosan product obtained from step b) can be dissolved in a solvent suitable for further synthesis conditions.

[0203] The impregnation can be carried out without dissolving the crosslinked chitosan of the present invention.

[0204] From the perspective of impregnating crosslinked chitosan with a biologically active agent according to the present invention, the biologically active agent / substance can be dissolved in a polar aprotic solvent or any other. When using a protic solvent, it is desirable to remove it and dry the impregnated product before the Azachaekami reaction. For impregnation, supercritical fluids can be used as described in Weidner, 2018, The Journal of Supercritical Fluids, 134, pp. 220-227, https: / / doi.org / 10.1016 / j.supflu.2017.12.024; Duarte et al., 2007, The Journal of Supercritical Fluids, 42(3), pp. 373-377, https: / / doi.org / 10.1016 / j.supflu.2007.01.007, which results in a very high degree of high-quality impregnation. Furthermore, if drug impregnation can be accelerated by Coulomb's method, it is possible to use electrophoresis as described by 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), pp. 7469~7479, or the "gene gun" method as described by Zhao et al., 2012, PLoS One, 7(10): e46765. doi: 10.1371 / journal.pone.0046765. For example, an aqueous solution of a biologically active material such as an antibiotic can be mixed with an aqueous solution of acylated chitosan after step b), and the acylated chitosan impregnated with the biologically active substance in a dry state can be obtained by evaporation by azeotropic distillation of water, spray drying, freeze-drying, and other drying methods. The dried material is then subjected to an excess amount of a non-nucleophilic base to deionize the amino groups of the chitosan, and the resulting mixture is then subjected to a Michael addition reaction step c).

[0205] In a further embodiment, the chitosan obtained in step a) has an average molecular weight in the range of about 5 to about 2,000 kilodaltons, and particularly about 150 to 2,000 kilodaltons.

[0206] In another further embodiment, the chitosan obtained in step a) has a degree of deacetylation of deacetylated fragments ranging from about 100% (as glucosamine) to about 2% per polymer molecule, for example, chitosan (chitin) with a molecular weight of 300 kDa has a degree of deacetylation of about 0.14%.

[0207] Suitable chitosans that can be used as starting materials in the method of the present invention may come from a variety of sources, including natural (animal or fungal), particularly those derived from crab and shrimp shells, insect exoskeletons, higher fungi, and single-celled mushroom cultures. Chemically or enzymatically synthesized chitosans can also be used.

[0208] In a further embodiment, the chitosan obtained in step a) is preferably swollen in an aprotic solvent for about 5 minutes to about 1 day.

[0209] In another further embodiment, the deacetylation step b) is carried out at a temperature of about -70°C (start of reaction) to about +150°C (completion of reaction), preferably about +2°C (start of reaction) to about +55°C (completion of reaction). The reaction time is about 0.5 hours to 10 days, preferably about 2.5 hours, at about +20 to about +30°C, preferably under sonication as a mixing method.

[0210] In another further embodiment, the Michael addition step c) is carried out at a temperature of about -70°C (start of reaction) to about +150°C (completion of reaction), preferably about +20°C (start of reaction) to +55°C (completion of reaction). The reaction time is about 0.5 hours to 10 days at about +20°C, preferably about 15 to 18 hours, and about 6 hours at about +55°C, preferably under sonication as a mixing method.

[0211] In another further embodiment, the drying of the cross-linked chitosan is carried out by freeze-drying in step e).

[0212] In another further embodiment, any further saponification step f) is carried out at a temperature of about 0°C (start of reaction) to about +140°C (completion of reaction), preferably about +20°C (start of reaction) to about +55°C (completion of reaction). The reaction time is about 0.5 hours to 10 days, preferably about 2 to 6 hours, at +55°C, preferably under ultrasonic action as a mixing method.

[0213] In certain embodiments, the nanoparticles of the present invention are spontaneously formed from the material obtained in steps b) and c) of the method of the present invention. These particles are very stable over time. For example, a 2% solution at room temperature is stable in a dry state for at least one year and at least several years.

[0214] According to one aspect of the present invention, a method is provided for preparing a drug delivery system for a bioactive agent, comprising the step of performing the method of the present invention, wherein the bioactive agent is added in step b), c), d), or e).

[0215] According to one aspect of the present invention, a method for preparing a drug delivery system for a bioactive agent, comprising the following steps: - A step of preparing the crosslinked chitosan of the present invention in a wet or dry state; - The process of preparing the bioactive agent to be delivered; - A step of dissolving a bioactive agent in a solvent or supercritical fluid; - A step of filling the cross-linked chitosan nanoparticles of the present invention by impregnating the cross-linked chitosan in a solution of a bioactive agent, directly introducing it into nanoparticles by electrophoresis, or accelerating it with an electric field; - A step of collecting the thus obtained composition or nanoparticles filled with a bioactive agent (e.g., a drug). A method is provided that includes this.

[0216] According to another specific embodiment, a method for identifying cross-linked chitosan obtainable by a method according to the present invention, comprising the following steps: - A step of preparing chitosan characterized in a solvent (e.g., methylene chloride or chloroform); - A step of acylating the chitosan described herein (for example, as acetyl chloride or acetic anhydride) while vigorously stirring at, for example, about 30 minutes at a temperature of about 0°C to about 20°C; - A step of neutralizing the reaction medium with a base (for example, diisopropylethylamine as a non-nucleophilic base); - A step of evaporating the solvent and washing the resulting neutralized product; - A step of subjecting the product to reflux acid hydrolysis (for example, preferably in 28% hydrochloric acid for about 1 to 3 hours, e.g., about 2 hours); - A step of evaporating the reaction mixture and resuspending the hydrolyzed product in a weak acid such as acetic acid; - Product according to formula (IIIa) or product according to formula (IIIb)

[0217] [ka]

[0218] [In the formula, R2 to R4 are defined herein, R 2' ~R 4' These are defined herein as R2 to R4, respectively. A step of determining the presence or absence of a product selected from, wherein the presence of the product of formula (IIIa) and / or formula (IIIb) is an indicator of crosslinked chitosan obtained by the method according to the present invention. A method is provided that includes this.

[0219] According to another specific embodiment, in a method for identifying crosslinked chitosan according to the present invention, the presence or absence of the product according to formula (IIIa) is performed, for example, by electrospray analysis of mass spectrometry, and for example, the peak at m / z = 252.077 is an indicator of the presence of the compound according to formula (IIIa) of formula (II).

[0220] [ka]

[0221] The method for identifying cross-linked chitosan obtained by the method according to the present invention is extremely useful because it makes it possible to distinguish the cross-linked chitosan obtained by the method according to the present invention from cross-linked chitosan obtained by other methods, such as those described by Baoqiand et al., 2015, previously mentioned, or by El-sherbiny et al., 2009, previously mentioned.

[0222] Chitosan and its nanoparticles according to the present invention The cross-linked chitosan according to the present invention can be prepared from readily available starting materials by the method of the present invention. Given typical or preferred experimental conditions (i.e., reaction temperature, time, number of moles of reagent, solvent, etc.), it will be understood that other experimental conditions may also be used unless otherwise stated. Optimal reaction conditions may vary depending on the specific reactants or solvent used, but such conditions can be determined by those skilled in the art using routine optimization procedures.

[0223] According to a specific embodiment, crosslinked chitosan and its nanoparticles obtained by the method of the present invention are provided.

[0224] According to another specific embodiment, a crosslinked chitosan is provided that is characterized by lower viscosity than the starting chitosan. For example, with a concentration of about 90% deacetylation, 3.2% crosslinking, and 0.25% chitosan, a solution of nanoparticles according to the present invention has one-third the viscosity of the starting chitosan, for example, 2.4 cSt at 25°C and 1.82 cSt at 37°C for the chitosan nanoparticles of the present invention, compared to 7.5 cSt at 25°C and 5.4 cSt at 37°C for the corresponding starting chitosan.

[0225] According to certain embodiments, pharmaceutically acceptable salts of the crosslinked chitosan of the present invention include salts of acetate, lactate, succinate, citrate, malonate, fumarate, maleate, malic acid, as well as salts of other carboxylic acids, hydroxycarboxylic acids (in addition to their hydrochlorides), phosphates, sulfates, and other inorganic acids.

[0226] It should be understood that the molecular weight of the crosslinked chitosan of the present invention can be adjusted by the selection of the starting material, and that this selection depends on the intended function of the nanoparticles and their formulations of the present invention. For example, the degradation rate of the desired nanoparticle composition, the desired release rate of any bioactive agent optionally incorporated therein, and the therapeutic state to be treated will influence the selection of molecular weight for the chitosan as the starting material.

[0227] In one embodiment, the molecular weight of the crosslinked chitosan of the present invention is between approximately 2 kDa and 2 MDa.

[0228] In a further embodiment, the molecular weight of the crosslinked chitosan of the present invention for use in lubricant / filler compositions can be selected from about 2 kDa to 2 MDa, preferably from about 30 kDa to 1,000 kDa.

[0229] In another embodiment, the molecular weight of the crosslinked chitosan of the present invention for use or as a drug delivery system can be selected from about 2 kDa to 2 MDa, preferably from about 30 kDa to 1,000 kDa.

[0230] composition The present invention relates to pharmaceutical or therapeutic agents as compositions, or medical devices containing the same, and to medical disorders, particularly cardiovascular conditions such as cardiac arrhythmias, particularly atrial fibrillation and hypertension, joint lesions and diseases such as rheumatoid arthritis, osteoarthritis, spondyloarthritis, and traumatic events resulting in damage to cartilage, bone, ligaments or synovial capsules, eye lesions and injuries such as dry eye syndrome, uveitis, glaucoma, corneal lesions, connective tissue disorders such as lupus and polymyositis, skin / mucosal disorders or injuries such as wounds, The present invention provides a method for treating subjects, preferably mammalian patients, most preferably human patients, who are suffering from scars, psoriasis, acne, eczema, rosacea, burns of a physical or chemical nature, especially surgical wounds and sunburns, ulcers, hemorrhoids, periodontal disease and dental diseases, for example, dural damage after accidental injuries or surgery of the brain and central nervous system, malignant and benign neoplasms, carcinomas, sarcomas, lymphomas and melanomas, postoperative complications, for example, fistulas and infections, tumors or vascular malformations, fistulas and infections and postoperative complications such as tumors or vascular malformations.

[0231] The present invention provides compositions, at least one cross-linked chitosan of the present invention, or compositions thereof, that are useful for human, veterinary, or agricultural use.

[0232] In certain embodiments, the agricultural composition according to the present invention is useful as a plant growth inducer.

[0233] According to certain embodiments, the crosslinked chitosan of the present invention can be used in the preparation of self-assembled structures, molecular machines, delivery mechanisms, electronics, composite materials, and lubricants.

[0234] In certain embodiments, the present invention provides a pharmaceutical formulation comprising at least one cross-linked chitosan or composition thereof for use as a pharmaceutical.

[0235] In another specific embodiment, the composition of the present invention is a parenteral formulation.

[0236] In certain embodiments, the compositions of the present invention are injectable formulations such as intra-articular, intra-arteriole, intra-vein, intra-sacral, intradermal, subcutaneous, submucosal, interstitial, intracranial, intraocular, intratumoral, intragastric, intestinal, anal, intraperitoneal, and intramuscular formulations.

[0237] In another specific embodiment, the composition of the present invention is an oral formulation.

[0238] In another specific embodiment, the composition of the present invention is a topical formulation.

[0239] In another specific embodiment, the composition of the present invention is an ophthalmic formulation.

[0240] Alternatively, the present invention provides a composition that can be used in another mammal for the same use as described above, in other words, in humans.

[0241] The present invention further provides compositions or medical devices useful for applications in cosmetics, 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). Each of these compositions further comprises a carrier acceptable for cosmetic use, or a culture nutrient for cells or biological tissues.

[0242] The compositions according to the present invention include soft tissue filler compositions, such as transdermal and subcutaneous fillers, comprising at least one cross-linked chitosan or nanoparticles thereof. The preparation of chitosan-based soft tissue filler compositions can be carried out, for example, as described in Grant et al., 2018, Tissue Eng Part A, 24(13-14):1091-1098, doi: 10.1089 / ten.TEA.2017.0385. Epub March 20, 2018.

[0243] In certain embodiments, the crosslinked chitosan nanoparticles of the present invention are provided, having an average size in the range of approximately 5 to 100 nm.

[0244] Another aspect of the present invention relates to a wound dressing comprising at least one cross-linked chitosan according to the present invention.

[0245] In a more specific embodiment, the present invention relates to a wound dressing that impregnates a matrix comprising a hydrophilic colloid, hydrogel, alginate, collagen, cellulose, foam, or cloth.

[0246] The composition of the present invention may further contain one or more pharmaceutically acceptable additional components, such as alum, stabilizers, antibacterial agents, buffers, colorants, flavoring agents, and auxiliary agents.

[0247] The compositions according to the present invention can be put into the form of pharmaceutical compositions together with conventionally used auxiliaries, carriers, diluents, or excipients, and can be used for oral applications as solids such as tablets or filled capsules, or liquids such as solutions, suspensions, ointments, emulsions, or elixirs, or as capsules, films, or gels filled with these. The compositions can also be formulated as dried products for reconstitution with water or another suitable vehicle before use.

[0248] The compositions of the present invention as liquid formulations include, but are not limited to, aqueous or oily suspensions, liquids, emulsions, syrups, and elixirs.

[0249] Such liquid preparations may contain additives, but are not limited to, suspending agents, emulsifiers, non-aqueous vehicles, and preservatives. Suspending agents include, but are not limited to, sorbitol syrups, methylcellulose, glucose / sugar syrups, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gels, and hydrogenated edible fats. Emulsifiers include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid. Dispersing or wetting agents include, but are not limited to, poly(ethylene glycol), glycerol, bovine serum albumin, Tween®, and Span®.

[0250] Further information regarding materials and formulation techniques is provided in The Science and Practice of Pharmacy (Remington: The Science & Practice of Pharmacy), 22nd edition, 2012, Lloyd, Ed. Allen, Pharmaceutical Press, which is incorporated herein by reference.

[0251] 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 are not limited to, binders, fillers, lubricants, disintegrants, and wetting agents. Binders include, but are not limited to, syrups, acacia, gelatin, sorbitol, tragacanth, starch mucilage, and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugars, 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 starch glycolate. Wetting agents include, but are not limited to, sodium lauryl sulfate. Tablets may be coated by methods well known in the art.

[0252] The agricultural compositions according to the present invention include plant growth promoters, seed germination enhancers, moisturizing and nutrient retention preparations, pest control enhancers, long-acting pheromone stabilizers, mucosal adsorbent insecticides, arachnocides, and antimollusk preparations.

[0253] According to certain embodiments, agricultural compositions are provided that contain approximately 0.001 to approximately 99% w / w of the cross-linked chitosan of the present invention, for example, approximately 0.05 to approximately 0.1% (w / w).

[0254] In another specific embodiment, the composition or nanoparticles of the present invention further comprises a bioactive agent, which is either dispersed in the crosslinked chitosan composition or covalently bonded to the crosslinked chitosan composition.

[0255] In another specific embodiment, the composition or nanoparticles of the present invention are in the form of a bioactive agent delivery system.

[0256] In one embodiment, 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%, based on the total amount of the cross-linked chitosan composition or nanoparticles of the present invention.

[0257] In another embodiment, the composition according to the present invention comprises a culture medium for cells or biological tissues containing cross-linked chitosan or a composition thereof. This culture medium may further contain cellular nutrients such as glucose, vitamins, growth factors, and metal ions.

[0258] In another embodiment, the compositions according to the present invention include biological tissues (endogenous or exogenous) or synthetic or semi-synthetic materials useful for repairing damaged tissues of the body, such as epidermis, nerves, cartilage, or bone tissue.

[0259] In another specific embodiment, a method for preparing a culture medium is provided, comprising the step of mixing the cross-linked chitosan according to the present invention with cell culture nutrients such as glucose, vitamins, growth factors, and metal ions.

[0260] In another specific embodiment, a method for preparing reconstructed tissue is provided, comprising the step of combining cross-linked chitosan or a composition thereof according to the present invention with stem cells or materials, tissues, or cells useful for repairing damaged tissue of the body, such as epidermis, nerves, cartilage, or bone tissue.

[0261] In certain embodiments, the compositions or nanoparticles of the present invention, when filled with a bioactive substance or drug, may be useful for the injection and in situ release of the active ingredient.

[0262] Mode of administration The compositions of the present invention can also be administered by any form of injection, such as subcutaneous injection, intrabursal injection, intraarteriole injection, intravenous injection, intraarticular injection, intramuscular injection, subcutaneous injection, submucosal injection, intraocular injection, intracranial injection, intragastric injection, intraintestinal injection, intraanal injection, intraperitoneal injection, intratumoral injection, and intrainterstitial injection.

[0263] The composition of the present invention can be administered in any manner via an oral route, including the mucosal surface of the oral cavity including the gums, the floor of the mouth, the cheeks, lips, tongue, and teeth.

[0264] The compositions of the present invention can also be administered topically to the skin, various mucous membranes, or eyes.

[0265] combination According to one embodiment, the cross-linked chitosan or its nanoparticles, or any suitable pharmaceutically acceptable salt thereof, and the pharmaceutical formulation thereof, can be administered alone or in combination with at least one combination agent.

[0266] According to a particular embodiment, the combination agent according to the present invention includes antibiotics, nucleic acids including gene constructs, antibodies and antibody fragments, toxins, cell proliferation inhibitors, antifungal agents, components of chemotherapeutic agents, antihypertensive agents, antiarrhythmic substances, growth activators, hormones, cytokines, nitric oxide donors, and hydrogen sulfide donors.

[0267] The present invention includes administering the cross-linked chitosan or its particles, and the pharmaceutical formulation thereof, to an individual simultaneously with or in conjunction with the at least one combination agent.

[0268] The cross-linked chitosan or its nanoparticles, or the pharmaceutical formulation thereof, according to the present invention, administered simultaneously with the aforementioned concomitant agent, may be administered using the same or different compositions and the same or different routes of administration.

[0269] The dosage administered to an individual, whether as a single dose or multiple doses, varies depending on various factors, including pharmacokinetic properties, the patient's condition and characteristics (sex, age, weight, health, and size), the severity of symptoms, concomitant treatments, frequency of treatment, and the desired effect.

[0270] patient In one embodiment, the patient according to the present invention is a subject suffering from a cardiovascular condition, such as cardiac arrhythmias, particularly atrial fibrillation and hypertension.

[0271] In another embodiment, the patient according to 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 sac.

[0272] In another embodiment, the patient according to the present invention is a subject suffering from eye lesions and injuries, such as dry eye syndrome, uveitis, glaucoma, or corneal lesions.

[0273] In another embodiment, the patient according to the present invention is a subject suffering from connective tissue disorders, such as lupus and polymyositis.

[0274] In another embodiment, the patient according to the present invention is a subject suffering from skin / mucosal disorders or injuries, such as wounds, scars, psoriasis, acne, eczema, rosacea, burns of a physical or chemical nature, especially surgical wounds, sunburn, ulcers, and hemorrhoids.

[0275] In another embodiment, the patient according to the present invention is a subject suffering from periodontal disease and dental diseases.

[0276] In another embodiment, the patient according to the present invention is, for example, a person suffering from an injury resulting from an accident or a dural injury following surgery on the brain and central nervous system.

[0277] In another embodiment, the patient according to the present invention is a subject suffering from malignant and benign neoplasms.

[0278] In another embodiment, the patient according to the present invention is a subject suffering from postoperative complications such as fistula and infection.

[0279] In another further embodiment, the patient according to the present invention is a subject who desires or needs an improvement in the volume or anatomical reshaping of a body part, for example, any improvement / modification and / or increase in volume of a body part for aesthetic or therapeutic reasons.

[0280] In another further embodiment, the patient according to the present invention is a subject suffering from a tumor, a vascular malformation, or any other newly occurring physical tissue or organ abnormality that causes pain.

[0281] Use according to the present invention According to certain embodiments, the cross-linked chitosan of the present invention is useful for a variety of applications, such as in vitro cell or biological tissue culture, as a tissue filler, in cosmetics, tissue engineering and organoid engineering, and in materials science applications, due to its ability to spontaneously form nanoparticles.

[0282] In particular, according to one embodiment, the cross-linked chitosan of the present invention can be used as a carrier for pharmaceutical active ingredients (for example, as a drug delivery system, as a cell penetration system (for example in gene therapy or non-cell penetration agents), as a solubilizer, and as a capture agent (for example, encapsulation of the desired ingredient in nanoparticles to create a suspension for effective removal from solution by changing the pH from acid to alkali)).

[0283] In another embodiment, the cross-linked chitosan of the present invention can be used as a medical implant (e.g., a coating for an implant to improve biocompatibility, or a regenerative medicine implant containing cells) or a material (e.g., a hemostatic material).

[0284] According to another embodiment, the cross-linked chitosan of the present invention can be used as a substrate for contrast agents (for example, used in standard angiography (CT) and other imaging methods (e.g., EPR, NMR, and X-ray systems)).

[0285] According to another embodiment, the cross-linked chitosan of the present invention can be used as a substrate for cell culture (e.g., stem cells), cell culture media, tissue engineering such as 3D printing of organoids, separation / purification techniques, and cell transfection.

[0286] In another embodiment, the crosslinked chitosan of the present invention can be used as a nano-sized lubricant (e.g., after thermal decomposition), a dispersant for colloid preparation, or a flocculant.

[0287] According to another specific embodiment, the use of cross-linked chitosan or nanoparticles or formulations thereof according to the present invention is provided for delivery systems, in vitro culture of cells or biological tissues, or tissue engineering materials, such as for preparing reconstructive tissues for neurosurgical, bone, cartilage, or epidermis.

[0288] According to another specific embodiment, the cross-linked chitosan or nanoparticles or formulations thereof according to the present invention may treat medical disorders, particularly cardiovascular conditions such as cardiac arrhythmias, especially atrial fibrillation and hypertension, joint lesions and diseases such as rheumatoid arthritis, osteoarthritis, spondyloarthritis, and traumatic events resulting in damage to cartilage, bone, ligaments or synovial capsules, eye lesions and injuries such as dry eye syndrome, uveitis, glaucoma, corneal lesions, connective tissue disorders such as lupus and polymyositis, and skin / mucosal disorders. It is also useful for the prevention or treatment of injuries, such as wounds, scars, psoriasis, acne, eczema, rosacea, burns of a physical or chemical nature, especially surgical wounds and sunburns, ulcers, hemorrhoids, periodontal disease and dental diseases, such as injuries from accidents or dural damage after surgery of the brain and central nervous system, malignant and benign neoplasms, especially carcinomas, sarcomas, lymphomas and melanomas, postoperative complications, such as fistulas and infections, tumors or vascular malformations, or for the prevention and / or treatment of tissue degeneration and related disorders.

[0289] According to another, more specific embodiment, cross-linked chitosan or nanoparticles or formulations thereof are provided for cosmetic use or for aesthetic and reconstructive surgery.

[0290] According to another, more specific embodiment, cross-linked chitosan or nanoparticles or formulations thereof are provided for use in in vivo drug delivery.

[0291] In certain embodiments, the use of nanoparticles according to the present invention as a delivery system for at least one bioactive agent is provided.

[0292] According to certain embodiments, there is a composition comprising at least one cross-linked chitosan or nanoparticles according to the present invention that is useful in many medical applications, such as a delivery system for at least one bioactive agent, or a cell delivery system in tissue engineering, or a cell or biological tissue culture system.

[0293] The embodiments illustrated in this invention will be described below in more detail with reference to the embodiments shown in the drawings. [Examples]

[0294] The following abbreviations refer to the following definitions: DMF (dimethylformamide); DMSO (dimethyl sulfoxide); MWCO (molecular weight cutoff).

[0295] (Example 1) Synthesis of Chitosan according to the present invention The chitosan nanoparticles of the present invention were synthesized as described in Scheme 1 above, using the following starting materials:

[0296] a) Prepare chitosan and swell the chitosan in the absence of water (for example, in an aprotic solvent). 450 g of dried chitosan (which can be dried by azeotropic distillation with ethanol, acetonitrile, acetone, or any other suitable solvent under vacuum over phosphorus oxide, under vacuum at moderate heat) is placed in the reactor under an inert atmosphere (e.g., purged with argon). The reactor is preferably a 2-liter round-bottom flask equipped with an electromagnetic stirrer, a two-horn nozzle connected to a nozzle from a Drexel flask for purging with an inert gas (e.g., argon), and a back-pressurized dropping funnel for adding reagents.

[0297] Next, 1,400 ml of an aprotic solvent (e.g., DMF) was poured into the reactor, and the chitosan was allowed to swell at room temperature for about 1 hour.

[0298] To prepare undried chitosan, 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, remove 300 ml of aprotic solvent (e.g., DMF) under reduced pressure, and then add 200 ml of DMF to the dried chitosan in the reactor and allow the chitosan to swell at room temperature for 1 hour.

[0299] Next, the reaction medium containing the swollen chitosan is cooled to approximately 2-6°C in an ice bath, or in a bath connected to a thermostat at approximately 2-6°C. In the same bath, a 250 ml reactor (e.g., a round-bottom flask) containing 100 ml of dry DMF is also cooled. After cooling, the acylation agent is added dropwise to the aprotic solvent (100 ml of DMF) while stirring and swirling the solvent to avoid localized overheating. The solution of the acylation reagent in DMF can be heated to approximately room temperature at most.

[0300] b) Acylate the amino group of the chitosan with an acrylic compound of formula (I) in the absence of water: The acryloyl compound solution prepared above is added to the chitosan suspension obtained above, while vigorously stirring for about 15 minutes, preferably while gently shaking the reactor (e.g., manually), and stopping the addition of the acryloyl compound as it is possible to wash away the chitosan clumps by spraying the solvent from the top wall of the flask. After the addition of the acryloyl compound solution is complete, the dropping funnel is washed with a minimum volume of aprotic solvent (e.g., DMF) on the order of 10-15 ml, and this solvent is added to the reaction medium. Cooling is stopped, and stirring is continued for another 2 hours with occasional shaking, allowing the reaction medium to be heated to room temperature. After stirring, the flask is placed in an ultrasonic bath for about 15 minutes, and the reaction mixture is subjected to ultrasonic treatment. During the ultrasonic exposure time, only light heating of the reaction mixture to below 30°C is performed.

[0301] c) The acylation product from step b) is subjected to a nucleophilic addition reaction (azamichaeus addition) in the presence of a non-nucleophilic base: Subsequently, a non-nucleophilic base (e.g., triethylamine, diisopropylethylamine) is added to the reaction medium in excess of the amount of acryloyl compound used in step b) by vigorously stirring for about 15 minutes and shaking periodically (e.g., through a dropping funnel). The mixture is continued to be mixed overnight or for about 15-18 hours at room temperature. The reactor is then placed in a bath at about 55°C and the mixture is stirred for about 6 hours. The reactor is removed from the bath and the reaction mixture is ultrasonically treated by placing it in an ultrasonic bath preheated to about 55°C for about 15 minutes.

[0302] After sonication, transfer the reactor to a rotary evaporator and, if using volatile organic non-nucleophilic bases, distill off the non-nucleophilic bases. If using non-volatile bases, cool the reactor to room temperature or low temperature and add an equal volume of acid (e.g., hydrochloric acid) while vigorously stirring. Control the pH of the resulting reaction medium to ensure a neutral reaction medium is achieved.

[0303] d) Obtain the crosslinked chitosan of the present invention Crosslinked chitosan is obtained in step c) in a suspension in an aprotic solvent (e.g., DMF) containing salt impurities. The product is then purified, for example, by dialysis with deionized water. For this purpose, for example, the reaction solution obtained in step c) is transferred to a dialysis bag, sealed with a fastener, and placed in a container while mixing with deionized water using an electromagnetic stirrer. Dialysis can be monitored in various ways: for example, by monitoring the salt content by measuring the conductivity of the washing solution, or by monitoring the DMF content by UV absorption at wavelengths less than 260 nm. After incubation with the product dialyzed with deionized water for 20 minutes, if there is no significant difference in either electrical conductivity or UV absorption, the dialysis process can be considered complete.

[0304] e) To optionally obtain the crosslinked chitosan of the present invention in a dry state. It is possible to use the wet product obtained in step d) without drying. However, it may also be dried, for example, for formulation / storage or transport.

[0305] Next, the purified product obtained in step d) is dried. For example, drying can be carried out in a freeze-dryer by first pre-freezing the product in the presence of an anhydrous phosphoric acid or any suitable desiccant such as molecular sieve or potassium hydroxide at ambient pressure to absorb much of the liquid, and then in the presence of an anhydrous phosphoric acid or any suitable desiccant under vacuum, or by pre-evaporating in a rotary evaporator, repeatedly azeotropically distilling the water with ethanol, and then vacuum drying with an anhydrous phosphoric acid or any suitable desiccant.

[0306] Generally, the yield of the method of the present invention is approximately 90%. Since sterility and freedom from contamination are higher priorities than yield, losses are likely to be related to droplet entrainment of nanoparticles during drying, as well as losses during transfer from the reaction vessel and dialysis bag.

[0307] f) Further cross-linked chitosan of the present invention in a dry state Optionally, the purified cross-linked chitosan obtained in step c), step d), or step e) can be saponified. For this purpose, the cross-linked chitosan obtained in step d) is placed in the 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., NaOH in water) in a molar quantity approximately 20% more than the amount of acryloyl compound used in the deionized water is added for about 15 minutes with vigorous stirring. It is desirable to gently shake the flask by hand while stopping the addition of alkali, as this procedure allows for the washing and removal of the chitosan clumps obtained by solvent spraying from the top wall of the reactor. After stirring, the reactor is placed in an ultrasonic bath, and the reaction mixture is ultrasonically treated until a reaction clump reaches approximately 55°C, after which the ultrasonic treatment is continued at this temperature for a further 2 hours. The resulting product can then be neutralized with an equal amount of acid, or dialyzed or dried without neutralization. After drying, at least one cycle of steps b) to c) (or d) / e)) is repeated. Using a sequence of two or more cycles of steps b) to c) (or d) / e)) allows for increased saturation of the carboxyl functional group of the crosslinked chitosan.

[0308] (Example 2) Example of the synthesis of crosslinked chitosan according to the present invention Various crosslinked chitosans (CHI) of the present invention are synthesized by the processes described herein, while varying the properties and proportions of the crosslinking agent. Examples are shown in Table 1 below.

[0309] In each case, 450 grams of chitosan is used as the starting material. In Examples 2a-2r and C1, the acryloyl compound is acryloyl chloride (CAS registry number: 814-68-6). In Examples 2t-2al, the acryloyl compound is methacryloyl chloride (CAS registry number: 920-46-7). The reaction is carried out as described in steps a)-c) of the method of the present invention. In Examples 2am-2as, the acryloyl compound is a different acryloyl chloride.

[0310] [Table 1A]

[0311] [Table 1B]

[0312] [Table 1C]

[0313] [Table 1D]

[0314] [Table 1E]

[0315] (Example 3) Characterization of chitosan nanoparticles of the present invention A sample of 3.2% crosslinked 501kDa chitosan (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 Jem1400 transmission electron microscope (Jeol) and a Veleta camera (SIS, Germany) (Figure 1A), which shows that the bulk of the preparation consists of spherical particles with a diameter of approximately 15 nm, similar in size to small mammalian viruses. Some larger particles can also be observed, which have a diameter in the 60-80 nm range, corresponding to medium-sized virus particles.

[0316] Furthermore, dried films of standard linear chitosan (MW: 501 kDa, 93% DD) (Bioavanta) and the crosslinked chitosan according to the present invention (Example 2i) prepared from the same chitosan were obtained by pouring a 2% chitosan solution in a 1% succinic acid aqueous solution into a Petri dish, thereby forming a 1 mm layer, and then drying it under vacuum at room temperature on anhydrous phosphoric acid to a constant mass. These films were then analyzed in semi-contact mode using a Solver Bio atomic force microscope (NT-MDT, Zelenograd, Russia). Three-dimensional visualization of the surface area of ​​a 100 μm × 100 μm film of a standard chitosan sample (MW: 501 kDa, 93% DD) shows that the surface is smooth with linearly arranged chitosan polymers (Figure 1B1), while the surface area of ​​the corresponding cross-linked chitosan sample of Example 2i, formed by spherical structural aggregates, is undulating with "peaks" and "valleys" (Figure 1B2). Figures 1B3 and 1B4 visualize the atomic force microscope film containing bacterial nanocellulose that absorbed a 1% w / w solution of the cross-linked chitosan according to the present invention in water. AFM measurements were performed using a Solver Next scanning probe microscope (NT-MDT, Russia) in semi-contact mode under standard room conditions in air (T=26°C, RH=15%). A ScanAsyst-AIR AFM probe (Bruker, USA) with a beam-cantilever stiffness of approximately 0.4 N / m, a resonant frequency of approximately 70 kHz, and a nominal bending radius (AFM probe sharpness) of 2 nm was used at the optimal pressure (setpoint). All measurements were performed at a scanning speed of 0.5 Hz and a resolution of 512 × 512 points. Subsequent processing was performed using the ImageAnalysis 3.5 program by subtracting slopes and removing measurement defects ("Adhesion"). Analysis of the resulting Figures 1B3 and 1B4 revealed a uniformly distributed spherical structure on the sample surface with an average spherical diameter of 20 nm (Figures 1B3 and 1B4), which corresponds to the size of small viruses such as those of the Caliciviridae, Picornaviridae, and Parvoviridae families.The presence of the structure observed by AFM is confirmed by previous transmission electron microscope data (Figure 1A).

[0317] The structural differences between standard chitosan (negative control) and the cross-linked chitosan of the present invention (Example 2i) and cross-linked glucosamine (Comparative Example C1) (positive control) were further investigated under an inert atmosphere (e.g., argon) by reflux acid hydrolysis, which allows for accurate analysis of low molecular weight substances in order to identify marker substances formed only in the case of successful cross-linking, as described below.

[0318] Add the sample (100 mg each) to a solution of 1 ml of acetyl chloride in 5 ml of methylene chloride, stirring vigorously, and cool to -20°C. Then heat the mixture to 0°C and maintain vigorous stirring for 30 minutes. Add diisopropylethylamine (1 ml), warm the mixture to room temperature, and maintain vigorous stirring for another 30 minutes. Neutralization of the resulting hydrochloride salt allows for subsequent washing with water of any substances interfering with the analysis, and the presence of a non-nucleophilic strong base allows the acetylation to be carried out as completely as possible. Carefully evaporate all solvents under vacuum and wash the residue three times with a 1 ml water sample. Discard the washing water and add 10 ml of 28% hydrochloric acid to the solid precipitate and reflux for 2 hours. Carefully evaporate the reaction mixture under vacuum and resuspend the residue (approximately 100 mg) in 100 ml of 2% aqueous acetic acid solution. At this point, prepare the sample. Complete hydrolysis of the polymer to glucosamine and glucosamine derivatives then occurs under these conditions. Therefore, since the substance of formula (II) is not found in natural chitin and chitosan, and can only be obtained by the reaction of Azamechic with the remainder of acrylic acid, the detection of such a substance can be used to identify crosslinked chitosan produced by the method according to the present invention.

[0319] When any acryloyl chloride of formula (I) (e.g., any acrylic acid chloride used in the crosslinking process according to the present invention) is used as a crosslinking agent (e.g., for Example 2i), reflux acid hydrolysis of the resulting polymer after preliminary preparation of the sample (e.g., acetylation, washing) yields not only hydrolysis of the glycosidic bonds between glucosamine units, but also amide bonds created during acylation with acetylglucosamine and the crosslinking agent (e.g., activated for acylated substituted or unsubstituted acrylic acid derivatives), but formula (IIIa):

[0320] [ka]

[0321] (In the formula, R2 to R4 are defined herein.) The following derivatives of glucosamine and propionic acid, in particular, formula (II):

[0322] [ka]

[0323] This results in the formation of the following derivatives of glucosamine and propionic acid, but this is when using unsubstituted acylated activated acrylic acid.

[0324] This substance is hydrolyzable and should be present in the mixture if crosslinking occurs first. A similar derivative (IIIa) is formed when other crosslinking reagents are used. When a high degree of crosslinking is supported, the detection of the derivative of formula (IIIb) can be used to identify the crosslinked chitosan prepared by the method according to the present invention.

[0325] [ka]

[0326] (In the formula, R2 to R4 are as defined herein, R2' ~R 4' is defined herein as R2 to R4 respectively, and R 2' ~R 4' is identical to the corresponding R2 to R4 respectively, or different if the acrylic acid compound of formula (I) used in the next sequence of steps b) to c) in the method of the present invention is different from that used in the first sequence)

[0327] To support the presence of crosslinking in the product obtained by the method of the present invention, the sample solution prepared as described above is used directly without prior chromatography by mass spectrometry (Bruker Daltonics TOF 180mkl / h, tune_low.m, ES pos. scan, N2 - 4l / min, 190 °C, Nb.=0.4, spectrum time 1 second). In the mass spectra in the region of the ions of interest (low m / z ratio) for both the comparative standard chitosan (Figure A2) and the crosslinked chitosan of the present invention (Figure B2), a peak specific to glucosamine was observed at (m / z = 180.073), reflecting the presence of a complete residue of aminoglucose. However, for the crosslinked chitosan of the present invention, a new peak was seen at m / z = 252.077 (Figure B2), corresponding to the product of formula (II) obtained from the alkylation of aminoglucose with propionic acid. In this example, the peak intensity was low, reflecting the low percentage of crosslinking of the sample (3.2% crosslinking determined by reaction condition 2i). This is a characteristic peak supporting the successful crosslinking of chitosan by the method of the present invention. This is present in all samples obtained from the method of the present invention carried out under various suitable conditions as described in Table 1 above (including the positive control of glucosamine compared), and is not present in all chitosan and chitin tested.

[0328] To confirm the assignment of the peaks at m / z=180 and m / z=252 to glucosamine and compound (II), impact tests were performed at different impact energies (AB SCIEX Triple Quad™ 3500) (5V and 15V). In this case, the pre-treated dried residue (after hydrolysis, the reaction mixture was evaporated under vacuum) was dissolved in an aqueous solution containing 10% methanol and 0.1% formic acid. During fragmentation of the compound at m / z=180 at higher impact energies (Sciex equipment), spectra of water loss (-18m / z, -2*18m / z, -3*18m / z) were observed (Figures 5A and 5B). As the impact energy increased, the intensity of the water loss peak also increased. This phenomenon is characteristic of carbohydrates. Similar images are observed for the ion of the compound at m / z=252 (Figures 5B and 5C). However, as a derivative of a carbohydrate, this may be more stable with respect to water loss at the same impact energy. Therefore, the observed relative intensity of the peak representing water loss is low.

[0329] The stability of the 3.2% crosslinked 501 kDa chitosan of the present invention (Example 2i), prepared as described above, was monitored by mass spectrometry after adding 5 mg of the present invention to 500 μl of a solution of egg lysozyme at a concentration of 10 mg per ml in sodium acetate buffer (pH 5.5). The mixture was incubated in a shaker at 37°C for 7 days until no further changes in the mass spectrum were observed. At this stage, the mass spectrum revealed a characteristic "comb" pattern of the high molecular weight compound: regularly scattered peaks with regularly increasing intensity at the 955-958, 1023-1024, 1101-1104, and 1193-1196 m / z ratios. The peaks correspond to the same fragments with different units of charge. For a given mass, the ionic charges are +15, +14, +13, and +12. The mass of the main fragment is 14,305 Da. Similar results were obtained with samples incubated for extended periods, up to three weeks. This demonstrates that the hydrolytic enzymes involved in the hydrolysis of chitosan and its derivatives in both egg white lysozyme and warm-blooded animal tissues can hydrolyze cross-linked chitosan only against larger fragments of approximately 14 kDa in mass. Therefore, this suggests that using deacetylated chitosan with a larger molecular weight for nanoparticle preparation would make it more stable against enzymatic hydrolysis.

[0330] Therefore, these data support the significant structural differences between the crosslinked chitosan of the present invention forming nanoparticles and the standard chitosan used as a starting material. Furthermore, methods based on the detection of the substance of formula (II) or related substances are highly sensitive and specific for the detection of nanoparticles obtained by the method of the present invention.

[0331] (Example 4) Intracellular transfer of chitosan nanoparticles according to the present invention The ability of the chitosan nanoparticles of the present invention to move into cells was tested by fluorescence microscopy using the penetration of fluorescein isothiocyanate (FITC) and 5-(and 6-)carboxyfluorescein (FAM)-labeled chitosan within the mouse cornea, as follows:

[0332] Fluorescein-labeled crosslinked chitosan was prepared using either standard initial chitosan with a molecular weight of 501 kDa and a degree of 93% deacetylation, or 3.2% crosslinked chitosan according to the present invention (Example 2p). 1 ml of DMF, pre-cooled in an ice bath, was added to both the chitosan (100 mg) in an Eppendorf tube and the suspension, which had been allowed to swell for 1 hour. Next, a solution of 2.85 mg of fluorescein-5-isothiocyanate (FITC) or 5-(and 6-)carboxyfluorescein succinimimidyl ester (NHS-fluorescein) in 250 μL of DMF was added to an ultrasonic bath filled with ice water and subjected to sonication. The bath temperature was maintained at 0°C for the first 20 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 a 12-14 kDa dialysis tube and dialyzed against water. After 20 dialysis procedures in 100 ml of water, 50 mg of succinic acid was added to each dialysis tube to dissolve the chitosan, and the dialysis procedure was repeated. The resulting solution was freeze-dried and then resuspended to obtain a 0.25% solution.

[0333] Mouse leukocytes were incubated in these solutions for 4 hours at 37°C, stained with a DNA-specific dye (Hoechst 33258), and analyzed by fluorescence microscopy. Fluorescence analysis (Figure 2) shows that green fluorescence (C) is uniform throughout the cytoplasm, indicating that FITC-labeled cross-linked chitosan is endocytotic because it is located inside the cell (simple association of labeled chitosan with the cell membrane creates a fluorescent ring at the cell boundary (A)). Blue fluorescence observed outside the nucleus (D) may be triggered by the initiation of apoptosis.

[0334] The same solution (0.25%) labeled with fluorescein isothiocyanate (FITC) and 5(6)-carboxyfluorescein (FAM) was applied to the eyes of CBA mice (one drop per eye). After 6 hours, the experimental animals were euthanized, the eyes were fixed in formalin, stained as described in Stradleigh et al., 2015, Prog Retin Eye Res., 48: pp. 181-202, and analyzed by fluorescence microscopy. It was observed that standard chitosan preferentially concentrated on the surface of the cornea and sclera (Figure 3A), while the chitosan nanoparticles of the present invention (bright green fluorescence) penetrated deeply into the cornea in a uniform manner (Figure 3B).

[0335] These data support the idea that the cross-linked chitosan nanoparticles of the present invention are useful for enhancing the cell permeability of active ingredients that are either poorly permeable (hydrophilic) or impermeable (large molecules) to intracellular locations via nanoparticle-mediated endocytosis and / or transcellular via nanoparticle-mediated transcytosis.

[0336] (Example 5) Biocompatibility of Chitosan Nanoparticles of the Present Invention The biocompatibility of the cross-linked chitosan of the present invention (Example 2i) was evaluated in vitro by culturing human bone marrow MSCs collected from healthy donors via bone marrow aspiration, as described in Bieback et al., 2008, Transfus Med Hemother., 35(4): 286-294, doi: 10.1159 / 000141567, in order to assess its potential effects on the culture and growth of mesenchymal stem cells (MSCs).

[0337] MSCs were isolated via density gradient centrifugation at 1,077 g / ml as described by Bieback et al. in 2008, followed by two washes and growth in DMEM growth medium containing 10% Mesecult fetal serum, l-glutamine, and antibodies. Culture purity was verified using flow cytometry. The cultured MSCs exhibited the following phenotype: CD44+ / CD73+ / CD90+ / CD105+ / CD34- / HLA-DR-, accompanied by demonstrated ability to differentiate into chondrogenic, osteogenic, and adipogenic forms, upon the addition of specific media as described by Ciuffreda et al. in 2016, Methods Mol Biol. 2016;1416:149-58, doi: 10.1007 / 978-1-4939-3584-0_8. This study utilized cells passed through four passages.

[0338] 25cm 2 The walls of the culture dishes were covered with a film of cross-linked chitosan, the dishes were washed and dried twice with growth medium, and cells were seeded. Untreated culture dishes of the same size were similarly seeded for comparison. The presence of cross-linked chitosan on the plastic walls of the culture dishes did not interfere with cell adhesion or growth. Within 3-4 hours after inoculation, the cells spread and adopted their characteristic spindle shape. The growth rate was slightly slower in the cross-linked chitosan-treated dishes. Dense growth was achieved after 48-56 hours in standard culture dishes and after 68-76 hours in the cross-linked chitosan-treated culture dishes.

[0339] In conclusion, the cross-linked chitosan of the present invention sustains the growth and proliferation of human bone marrow mesenchymal stem cells, although at a lower efficiency than under standard conditions.

[0340] (Example 6) Use of the present invention of chitosan for agricultural application The usefulness of the cross-linked chitosan of the present invention (Example 2i) was tested on spring wheat seeds as follows. Aqueous suspensions of the cross-linked chitosan of the present invention were prepared at two different concentrations, and seeds were immersed in the suspensions. Each experiment included the following groups of seeds depending on the treatment applied: (A) Control (no seed treatment); (B) Treatment with 0.1% (w / w) cross-linked chitosan. (C) Treated with 0.05% (w / w) cross-linked chitosan.

[0341] Evaluation of growth-promoting effects in the early stages of organ formation After a 24-hour treatment, the seeds were placed in Petri dishes (12 seeds per dish, 4 rows) on a moist substrate (filter with a cotton layer) under natural light at t=+20~22°C. Germination energy was determined as described in Domin et al., 2020, Sustainability 2020, 12(1), p. 46, and measured 1 and 3 days after contact with the moist substrate. Germination power (number of germinated seeds divided by the total number of tested seeds minus empty seeds) was determined 7 days after contact with the moist substrate. Growth indicators in the early stages of organogenesis were recorded 3 days after contact with moisture (root length of each seedling, total seedling length) and 7 days after contact with moisture (number of roots per seedling, seedling length, total mass of roots and shoots per seedling, and mass of each root).

[0342] The positive effects of treatment with the cross-linked chitosan of the present invention were demonstrated at the lowest concentration, as shown in Figure 6 for germination and in Table 2 below for seedling growth in the early stages of organ formation.

[0343] [Table 2]

[0344] A higher rate of radicle development was observed within 1 day of cross-linked chitosan treatment. A stronger effect was observed with lower concentrations of the suspension (22.9% increase with 0.05% cross-linked chitosan; 14.6% increase with 0.1% cross-linked chitosan, compared with 75% baseline control). The same trend was observed when germination energy (6.2% increase) and germination capacity (control = 93.8%) were measured. Treatment of seeds with a high concentration of cross-linked chitosan suspension increased germination capacity by 4.1%. In the early stages of organogenesis, cross-linked chitosan resulted in increased root growth. After 3 days, 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). Simultaneously, a nearly uniform increase was observed in the high-concentration cross-linked chitosan group (equilibrium coefficient for 0.1% cross-linked chitosan = 81% (n=45); 0.05% cross-linked chitosan = 78.2% (n=48); control = 68.9% (n=45)). The same trend was observed when seedling height was measured. When the increase after treatment with 0.05% cross-linked chitosan (46.2% higher than the control = 1.84 cm) was significantly greater than the increase after treatment with 0.1% cross-linked chitosan (24.4% higher than the control), the equilibrium coefficient was higher than that of the high-concentration cross-linked chitosan treatment group (0.1% cross-linked chitosan = 75.4%; 0.05% cross-linked chitosan = 72.8%, control = 61.7%).

[0345] Based on measurements taken 7 days after the start of the experiment, low-concentration cross-linked chitosan treatment resulted in the greatest improvement in root formation (0.05% cross-linked chitosan: 9.5% increase; 0.1% cross-linked chitosan: 6.1% increase, mean number of roots in the control group: 4.41 per seedling). The same was observed in the increase in total length of the seedlings (0.05% cross-linked chitosan: 7.7% increase; control: 10.05 cm). In contrast, the greatest increase in biomass was observed after treatment with higher concentrations of cross-linked chitosan suspension, both for roots (0.1% cross-linked chitosan: 17.1% increase; 0.05% cross-linked chitosan: 14.6% increase) and for the entire seedling (0.1% cross-linked chitosan: 12.9% increase; 0.05% cross-linked chitosan: 8.7% increase).

[0346] Evaluation of the effects of soil substrates on the growth and development of spring wheat seedlings. Seeds were treated 7 days before sowing (alkaline black soil, 500 grams per plastic pot / 12 pots, 4 repeats, natural light, t=20-22°C, controlled soil moisture). Germination energy, germination rate, seedling height, and biomass were measured. Growth parameters were recorded for the first 7 days. A higher percentage of radicle development was observed after 24 hours for seeds treated with cross-linked chitosan, particularly at high concentrations (control: 70.8%, relative increase compared to treated seeds: 27.1% and 23%). 100% germination was achieved after high-concentration cross-linked chitosan treatment, as shown in Table 3 and Figure 6 below.

[0347] [Table 3]

[0348] The high rate of early growth stimulation obtained from 0.1% cross-linked chitosan treatment (56.3% at 2 days) decreased threefold at 3 days (cross-linked chitosan, 0.05%, 11.6 times) and 2.3 times at 7 days. Compared to the control (16.63 mg), the increase in biomass at 7 days was higher in both the 0.1% treatment group (14% higher for total biomass and 23.8% higher for root biomass) and the 0.05% treatment group (25.7% higher for total biomass and 35.1% higher for root biomass). Treatment with 0.05% cross-linked chitosan resulted in a 1.8-fold increase in total biomass and a 1.5-fold increase in root biomass.

[0349] In short, these data support the finding that treatment with the cross-linked chitosan of the present invention resulted in a significant growth stimulus during the germination of spring wheat.

Claims

1. A method for preparing cross-linked chitosan, comprising the following steps: a) A step of preparing chitosan and swelling the chitosan in a solvent; b) The amino group of the chitosan is given formula (I): 【Chemistry 1】 [wherein, R 1 is selected from the group consisting of halogen and acid anhydride; R 2 , R 3 , and R 4 are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl; substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted arylC 1 -C 6 alkyl; the term "substituted" means a group substituted with one substituent selected from the group consisting of C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 1 -C 6 alkylaryl, C 1 -C 6 alkylheteroaryl, arylC 1 -C 6 alkyl, and heteroarylC 1 -C 6 alkyl] A process of acylation with an acrylic compound; c) Reacting the acylation product from step b) in the presence of a base (Azachaïc reaction); d) A step to purify the cross-linked chitosan obtained from step c). Methods that include...

2. The method according to claim 1, further comprising step e) drying the product from step d).

3. The method according to claim 1, wherein the chitosan prepared in step a) has an average molecular weight in the range of 5 to 2,000 kilodaltons.

4. R 1 The method according to any one of claims 1 to 3, wherein the material is a halogen.

5. The method according to any one of claims 1 to 4, wherein the acrylic compound is selected from acryloyl chloride and methacryloyl chloride.

6. The method according to any one of claims 1 to 5, further comprising a saponification step f) after step c) or d) under alkaline conditions, wherein the obtained product can be further subjected to at least one sequence of steps b) to c) (or d)).

7. It is cross-linked chitosan, 【Chemistry 2】 [In the formula, R2 to R4 are defined in claim 1, and R2' to R4' are defined as R2 to R4 respectively in claim 1.] A crosslinked chitosan characterized in that the hydrolysis product represented by formula (IIIa) or formula (IIIb) is obtained by acid hydrolysis of the crosslinked chitosan.

8. The crosslinked chitosan according to claim 7, characterized in that, when subjected to acid hydrolysis, the spectral pattern obtained by mass spectrometry using electrospray cations shows a peak at m / z = 252.077 ± 0.

01.

9. A composition comprising at least one crosslinked chitosan and at least one carrier as described in claim 7 or 8.

10. The composition according to claim 9, wherein the pharmaceutical composition is a pharmaceutically acceptable carrier, or the cosmetic composition is a cosmetic-use acceptable carrier.

11. The composition according to claim 9, which is a cell or tissue culture medium further containing nutrients for cells or tissues.

12. The composition according to claim 9, which is a reconstructed tissue.

13. The composition according to claim 9, which is an agricultural composition.

14. A soft tissue filler, wound dressing, or reconstructive tissue comprising at least one cross-linked chitosan or composition thereof as described in claim 7 or 8.

15. Nanoparticles comprising one crosslinked chitosan by at least one crosslinked chitosan according to claim 7 or 8.

16. A culture medium for cells or biological tissues comprising at least one cross-linked chitosan as described in claim 7 or 8.

17. Use of the cross-linked chitosan according to claim 7 or 8 for the preparation of cell or biological culture media or reconstructed tissue.

18. A pharmaceutical composition for the prevention and / or treatment of a disease or disorder selected from cardiovascular conditions, joint lesions and diseases, osteoarthritis, spondyloarthritis, and traumatic events resulting in damage to cartilage, bone, ligaments or synovial sacs, eye lesions and injuries, connective tissue disorders, skin / mucosal disorders or injuries, ulcers, hemorrhoids, periodontal and dental diseases, dural damage, malignant and benign neoplasms, postoperative complications, tumors or vascular malformations, comprising the cross-linked chitosan described in claim 7 or 8.

19. The pharmaceutical composition according to claim 18 for the prevention and / or treatment of diseases or disorders selected from cardiac arrhythmias, atrial fibrillation, hypertension, rheumatoid arthritis, dry eye syndrome, uveitis, glaucoma, corneal lesions, lupus, polymyositis, wounds, scars, psoriasis, acne, eczema, rosacea, burns of a physical or chemical nature, sunburn, carcinoma, sarcoma, lymphoma, melanoma, fistula and postoperative infection.

20. A method for preparing a drug delivery system for a bioactive agent: - A step of preparing the crosslinked chitosan according to claim 7 or 8 in a wet or dry state; - The process of preparing the bioactive agent to be delivered; - A step of dissolving a bioactive agent in a solvent or supercritical fluid; - A step of filling the cross-linked chitosan nanoparticles of the present invention by impregnating the cross-linked chitosan with a solution of a bioactive agent, directly introducing it into nanoparticles by electrophoresis, or accelerating it with an electric field; - A process of collecting compositions or nanoparticles filled with bioactive agents. Methods that include...

21. A method for identifying crosslinked chitosan that can be obtained by the method described in any one of claims 1 to 6: - Steps to prepare chitosan in a solvent; - A step of acylating the chitosan while stirring; - A step of neutralizing the reaction medium with a base; - A step of evaporating the solvent and washing the resulting neutralized product; - A step in which the product is subjected to reflux acid hydrolysis; - A step of evaporating the reaction mixture and resuspending the hydrolyzed product in a weak acid; - Product according to formula (IIIa) or product according to formula (IIIb) 【Transformation 3】 [In the formula, R 2 ~R 4 is defined in claim 1, R 2' ~R 4' In claim 1, R 2 ~R 4 [Defined as] The presence or absence of a product selected from the above is determined, and the presence of such products of formula (IIIa) and / or formula (IIIb) is an indicator of crosslinked chitosan obtained by the method of any one of claims 1 to 6. Methods that include...

22. The method according to claim 21, wherein formula (II) 【Chemistry 4】 The presence or absence of the product according to equation (IIIa) is determined by electrospray analysis of mass spectrometry.

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