CN115850534B - Hyaluronic acid-cholic acid derivative and synthetic method and application thereof - Google Patents
Hyaluronic acid-cholic acid derivative and synthetic method and application thereof Download PDFInfo
- Publication number
- CN115850534B CN115850534B CN202211626808.1A CN202211626808A CN115850534B CN 115850534 B CN115850534 B CN 115850534B CN 202211626808 A CN202211626808 A CN 202211626808A CN 115850534 B CN115850534 B CN 115850534B
- Authority
- CN
- China
- Prior art keywords
- acid
- hyaluronic acid
- dehydrolithocholic
- micelle
- hyaluronic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000002812 cholic acid derivative Substances 0.000 title claims abstract description 10
- 238000010189 synthetic method Methods 0.000 title description 2
- 239000000693 micelle Substances 0.000 claims abstract description 62
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims abstract description 45
- 229920002674 hyaluronan Polymers 0.000 claims abstract description 40
- 229960003160 hyaluronic acid Drugs 0.000 claims abstract description 40
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims abstract description 38
- JLLBPEUXSDBMIZ-UHFFFAOYSA-N 2,5-dibromobenzene-1,4-dicarbonitrile Chemical compound BrC1=CC(C#N)=C(Br)C=C1C#N JLLBPEUXSDBMIZ-UHFFFAOYSA-N 0.000 claims abstract description 28
- 238000000034 method Methods 0.000 claims abstract description 21
- 229920000642 polymer Polymers 0.000 claims abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000007112 amidation reaction Methods 0.000 claims abstract description 13
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000002994 raw material Substances 0.000 claims abstract description 6
- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 5
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical class C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 claims abstract description 4
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 4
- 239000002245 particle Substances 0.000 claims description 54
- 239000003814 drug Substances 0.000 claims description 16
- 238000011282 treatment Methods 0.000 claims description 11
- 150000001875 compounds Chemical class 0.000 claims description 7
- 238000004108 freeze drying Methods 0.000 claims description 7
- 238000000502 dialysis Methods 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- HTSGKJQDMSTCGS-UHFFFAOYSA-N 1,4-bis(4-chlorophenyl)-2-(4-methylphenyl)sulfonylbutane-1,4-dione Chemical compound C1=CC(C)=CC=C1S(=O)(=O)C(C(=O)C=1C=CC(Cl)=CC=1)CC(=O)C1=CC=C(Cl)C=C1 HTSGKJQDMSTCGS-UHFFFAOYSA-N 0.000 claims description 4
- 238000002604 ultrasonography Methods 0.000 claims 1
- 230000002209 hydrophobic effect Effects 0.000 abstract description 6
- 239000012798 spherical particle Substances 0.000 abstract description 5
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical class C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 238000001308 synthesis method Methods 0.000 abstract description 3
- 241000699670 Mus sp. Species 0.000 description 41
- 210000001072 colon Anatomy 0.000 description 28
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 16
- 229940079593 drug Drugs 0.000 description 10
- 206010061218 Inflammation Diseases 0.000 description 9
- 230000004054 inflammatory process Effects 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 210000000068 Th17 cell Anatomy 0.000 description 8
- 229910052757 nitrogen Inorganic materials 0.000 description 8
- 230000001225 therapeutic effect Effects 0.000 description 8
- 230000037396 body weight Effects 0.000 description 7
- 210000000056 organ Anatomy 0.000 description 7
- 239000004094 surface-active agent Substances 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 210000001541 thymus gland Anatomy 0.000 description 6
- 108020004999 messenger RNA Proteins 0.000 description 5
- 238000010172 mouse model Methods 0.000 description 5
- 239000002105 nanoparticle Substances 0.000 description 5
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 4
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 4
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 4
- 239000003613 bile acid Substances 0.000 description 4
- 230000005540 biological transmission Effects 0.000 description 4
- 230000008045 co-localization Effects 0.000 description 4
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 4
- 210000005087 mononuclear cell Anatomy 0.000 description 4
- 210000004400 mucous membrane Anatomy 0.000 description 4
- 230000035484 reaction time Effects 0.000 description 4
- TVZRAEYQIKYCPH-UHFFFAOYSA-N 3-(trimethylsilyl)propane-1-sulfonic acid Chemical compound C[Si](C)(C)CCCS(O)(=O)=O TVZRAEYQIKYCPH-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000024245 cell differentiation Effects 0.000 description 3
- 239000003651 drinking water Substances 0.000 description 3
- 235000020188 drinking water Nutrition 0.000 description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 description 3
- 230000002757 inflammatory effect Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 101000998146 Homo sapiens Interleukin-17A Proteins 0.000 description 2
- 102100033461 Interleukin-17A Human genes 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 229920001400 block copolymer Polymers 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 230000008951 colonic inflammation Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000000799 fluorescence microscopy Methods 0.000 description 2
- 229920000578 graft copolymer Polymers 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 230000009437 off-target effect Effects 0.000 description 2
- 238000002727 particle therapy Methods 0.000 description 2
- 230000000704 physical effect Effects 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000000451 tissue damage Effects 0.000 description 2
- 231100000827 tissue damage Toxicity 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- 102100032912 CD44 antigen Human genes 0.000 description 1
- 206010009900 Colitis ulcerative Diseases 0.000 description 1
- 208000019399 Colonic disease Diseases 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010053159 Organ failure Diseases 0.000 description 1
- 201000006704 Ulcerative Colitis Diseases 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000000436 anus Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000004579 body weight change Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000002717 carbon nanostructure Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000011443 conventional therapy Methods 0.000 description 1
- 239000003246 corticosteroid Substances 0.000 description 1
- 229960001334 corticosteroids Drugs 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 208000010643 digestive system disease Diseases 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 210000004921 distal colon Anatomy 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 210000004051 gastric juice Anatomy 0.000 description 1
- 208000018685 gastrointestinal system disease Diseases 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 239000000367 immunologic factor Substances 0.000 description 1
- 229940088592 immunologic factor Drugs 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 239000002539 nanocarrier Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 238000002428 photodynamic therapy Methods 0.000 description 1
- 208000037821 progressive disease Diseases 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000011076 safety test Methods 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 208000037816 tissue injury Diseases 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 230000009278 visceral effect Effects 0.000 description 1
Abstract
The invention discloses a hyaluronic acid-cholic acid derivative, a synthesis method and application thereof; the method comprises the following steps: (1) synthesizing ethylenediamine hyaluronic acid; (2) Using ethylenediamine hyaluronic acid and cholic acid derivatives as raw materials, and carrying out amidation reaction to obtain a hyaluronic acid-dehydrolithocholic acid polymer; (3) Adding hyaluronic acid-dehydrolithocholic acid polymer into water, and performing ultrasonic treatment to obtain a uniformly distributed micelle spherical particle system. The method can realize chemical connection of the hydrophobic bile acid derivative and the hydrophilic hyaluronic acid, thereby constructing a micelle spherical particle system with a hydrophilic hyaluronic acid shell and a hydrophobic dehydrolithocholic acid inner core in water, and being used for treating inflammatory bowel diseases.
Description
Technical Field
The invention relates to the technical field of preparation of high molecular oral delivery carriers, in particular to a hyaluronic acid-cholic acid derivative, a synthesis method and application thereof, and specifically relates to a method and a product for synthesizing micelles based on hyaluronic acid-linked cholic acid derivative and application thereof in treating Inflammatory Bowel Disease (IBD).
Background
The nanocarrier refers to various nanoparticles in which a drug is dissolved or dispersed, such as liposomes, metals, carbon nanostructures, silica nanoparticles, polymer micelles, and the like. The us Food and Drug Administration (FDA) formally approved nanoparticles for clinical treatment in 1989 have been widely used in the fields of photodynamic therapy, biomedical imaging, gene and drug delivery, tissue engineering, and the like.
The polymer micelle is a novel nano particle which is rapidly developed in recent years, and is synthesized into a block copolymer or a graft copolymer with surface activity by a hydrophilic chain segment and a hydrophobic chain segment, and the block copolymer or the graft copolymer is dissolved in water to form the nano micelle. When the positive adsorption of the surfactant reaches saturation, the surfactant is continuously added, molecules of the surfactant are transferred into the solution, and because of the existence of lipophilic groups, the repulsive force between water molecules and the surfactant molecules is far greater than the attractive force, so that the surfactant molecules are mutually aggregated by depending on Van der Waals force to form particles which are inward in lipophilic groups, outward in hydrophilic groups, stably dispersed in water and have the size of colloid grade. Hydrophobic groups of the surfactant molecules aggregate to form micelle cores, and hydrophilic polar groups form micelle outer layers. Hyaluronic acid has good hydrophilicity and can be used as a hydrophilic group of an indicating active molecule. Meanwhile, CD44, which is highly expressed on the cell surface of the tissue inflammation site, is an important receptor for hyaluronic acid, which can target the inflammation site of human body through the interaction with CD 44.
Inflammatory Bowel Disease (IBD) is a chronic gastrointestinal disease, including crohn's disease and ulcerative colitis. It is also a progressive disease, with symptoms in the form of relapse-remission, low mortality, susceptibility to long-term complications, and severe impairment of the quality of life in IBD patients.
The pathogenesis of IBD involves complex factors including genetic, environmental, host microbial, epithelial and immune factors. Clinical treatments for IBD include corticosteroids, immunomodulators, biologicals and surgery. However, conventional therapies have significant side effects due to off-target effects, including cancer, infection, organ failure, and the like. Thus, there is an urgent need for effective therapeutic strategies to inhibit disease progression.
Bile acids are cholesterol-derived natural surfactants that play a vital role in lipid metabolism and sugar metabolism and are capable of being bacterially mediated in the gut to derivatives with unique chemical structures. Bile acids also affect gut-related inflammation and have the potential to modulate gut mucosal immune cells. Dehydrolithocholic acid is a newly discovered bile acid derivative, can inhibit Th17 cell differentiation, and can be used as a potential medicine for treating IBD. However, bile acids are cytotoxic due to their hydrophobicity, and can damage cell membranes and induce oxidative damage to DNA. Therefore, there is a need to develop a micelle carrier to efficiently deliver bile acids to treat IBD and reduce its side effects.
Disclosure of Invention
The invention provides a hyaluronic acid-cholic acid derivative, a synthesis method and application thereof, wherein the method can realize chemical connection of a hydrophobic bile acid derivative and hydrophilic hyaluronic acid, thereby constructing a micelle spherical particle system with a hydrophilic hyaluronic acid shell and a hydrophobic dehydrolithocholic acid inner core in water, and the micelle spherical particle system is used for treating mouse IBD.
The invention firstly provides a method for synthesizing a hyaluronic acid-dehydrolithocholic acid polymer based on hyaluronic acid-linked cholic acid derivatives, which comprises the following steps:
(1) Using hyaluronic acid and anhydrous ethylenediamine as raw materials, performing amidation reaction, dialyzing, and freeze-drying to obtain a compound I;
(2) And (3) taking the compound I and dehydrolithocholic acid as raw materials, performing amidation reaction, dialyzing, and freeze-drying to obtain a compound II, namely the hyaluronic acid-dehydrolithocholic acid polymer.
In a preferred embodiment of the invention, the operation is carried out at room temperature, except for freeze-drying. Wherein, the structural formula of the compound I (aminated hyaluronic acid) is shown as the formula (a):
the amidation reaction process in step (1) and step (2) is as follows:
preferably, the molecular weight of the hyaluronic acid is 1-1000 kDa. More preferably, the hyaluronic acid has a molecular weight of 50kDa.
Preferably, in the step (1), the amidation reaction time is 1 to 48 hours. More preferably, the amidation reaction time is 24 hours. The mole ratio range of the hyaluronic acid to the anhydrous ethylenediamine is 10:1 to 1:10.
preferably, in the step (2), the amidation reaction time is 1 to 24 hours. More preferably, the amidation reaction time is 12 hours. The molar ratio of dehydrolithocholic acid to compound i is in the range of 10:1 to 1:10.
the invention also provides a preparation method of the hyaluronic acid-dehydrolithocholic acid micelle particles, which is to add the hyaluronic acid-dehydrolithocholic acid polymer prepared by the method into water and carry out ultrasonic treatment to obtain spherical micelle particles which are uniformly distributed. Compound II forms spherical particles in water that are hydrophilic in appearance and hydrophobic in the inner core.
Preferably, the concentration of the compound II in water is 0.01 to 30mg/mL, more preferably 0.3mg/mL. Preferably, the ultrasonic time is 1 to 100 minutes. More preferably 4min.
The invention provides application of the hyaluronic acid-dehydrolithocholic acid micelle particles in preparing a medicament for treating inflammatory colon diseases.
Compared with the prior art, the invention has the following beneficial effects:
(1) The method of the invention synthesizes the product by taking hyaluronic acid and dehydrolithocholic acid as raw materials, and the hyaluronic acid and the dehydrolithocholic acid are both bioactive molecules with biocompatibility, and have safety when being used as medicines in clinical treatment.
(2) The process of the invention is two-step amidation reaction, the synthesis process is simple, the product has good stability, and the long-term storage is convenient.
(3) The hyaluronic acid has the targeting property of the inflammation part, can furthest reduce the off-target effect of the medicine, minimizes adverse reaction of the hyaluronic acid in the IBD treatment period, improves the concentration of the medicine in the inflammation part, and improves the treatment efficiency.
(4) The delivery system provided by the invention has the advantages of low cost, stable operation, easiness in use, and good application prospect, and the oral administration route is more easily accepted by patients.
Drawings
Figure 1 is a schematic representation of the principle of the present invention for the treatment of IBD based on synthetic micelles of hyaluronic acid-cholic acid derivatives.
FIG. 2 is a transmission electron microscope morphology and physical properties of micelle particles under different treatments in example 1;
wherein, a hyaluronic acid-dehydrolithocholic acid micelle particle transmission electron microscope morphology diagram with the concentration of A.0.3 percent; b.3% concentration hyaluronic acid-dehydrolithocholic acid micelle particle transmission electron microscopy morphology; C. zeta potential of 0.3% concentration hyaluronic acid-dehydrolithocholic acid micelle particles in solutions of different pH; D. particle size of 0.3% concentration hyaluronic acid-dehydrolithocholic acid micelle particles in different pH solutions.
FIG. 3 shows the distribution of micelle particles in different organs of IBD or normal mice and the enrichment of inflammatory colon in example 1;
wherein, A. The micelle particles, dehydrolithocholic acid and hyaluronic acid are in IBD or the living body fluorescence imaging diagram of different organs of normal mice; B. micellar particles, dehydrolithocholic acid, hyaluronic acid in IBD or normal mice, colon fluorescence intensity to all visceral fluorescence intensity ratio; C. colo-fluorescence confocal microscopy of micellar particles, dehydrolithocholic acid, hyaluronic acid in IBD or normal mice. The control is normal mice, the IBD model is IBD mice, HADLA-Cy5.5 is micelle particles connected with Cy5.5, DLA-Cy5.5 is dehydrolithocholic acid connected with Cy5.5, and HA-Cy5.5 is hyaluronic acid connected with Cy5.5.
FIG. 4 is a graph showing the therapeutic recovery of body weight and inflammatory factors in IBD mice by micellar particles in example 1;
wherein, ibd or normal mice change in body weight after being given different drugs; ibd or normal mice after being given different drugs the expression of the colon inflammation-associated pro-inflammatory factor mRNA; ibd or normal mice express anti-inflammatory factor mRNA associated with colonic inflammation after being given different drugs.
Figure 5 is a therapeutic recovery effect of micellar particles on colon length and pathology in IBD mice in example 1;
wherein, ibd or normal mice have a gross image of the colon after being given different drugs; colon length of ibd or normal mice after administration of different drugs; colon tissue section HE staining microscopy of ibd or normal mice after administration of different drugs; ibd or normal mice were scored for colon tissue damage after being given different drugs. The water is normal mouse drinking water, the 2.5% DSS is IBD model mouse drinking water, the PBS is blank control administration, the HA is hyaluronic acid administration, the DLA is dehydrolithocholic acid administration, and the HADLA is hyaluronic acid-dehydrolithocholic acid micelle particles administration.
FIG. 6 shows the inhibition of Th17 cell differentiation by micellar particles in example 1;
wherein, A. Flow chart of Th17 cells in colon lamina propria mononuclear cells of normal control mice; flow chart of Th17 cells in colon lamina propria mononuclear cells of ibd model mice; flow pattern of Th17 cells in colon lamina propria mononuclear cells following administration of hyaluronic acid-dehydrolithocholic acid micelle particles by ibd model-building mice; D. different groups of Th17 cells occupy CD3 + CD4 + T cell ratio; E. relative expression of IL17 mRNA in different groups of colon tissues. H 2 O+PBS is the normal control mice, DSS+PBS is the IBD model mice, DSS+HADLA is the IBD model mice given micelle particle therapy.
FIG. 7 is a safety test of micelle particles for biological treatment in example 1;
wherein, A, the normal mice are given micelle particles or the HE staining microscopic image of the important organ slice after blank control; B. body weight change after administration of micelle particles or blank control in normal mice; C. normal mice were given micellar particles or a blank control followed by thymus in proportion to their own weight.
Wherein PBS is used for normal mice to give blank control, and HADLA is used for normal mice to give hyaluronic acid-dehydrolithocholic acid micelle particles.
Detailed Description
The following examples will provide a better understanding of the present invention, but are not limited thereto. The experimental methods in the following examples are conventional methods unless otherwise specified.
EXAMPLE 1 preparation of hyaluronic acid-dehydrolithocholic acid Polymer Using hyaluronic acid and dehydrolithocholic acid
(1) Hyaluronic acid (mw=50 kda,0.190g,0.5mmol,1 eq) was dissolved in 10mL of distilled water, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (mw=191.7, 0.288g,1.5mmol,3 eq) and N-hydroxysuccinimide (mw=115.1, 57.6mg,0.5mmol,1 eq) were added and reacted at room temperature under nitrogen protection for 20min; then anhydrous ethylenediamine (MW=60.1, 48.1mg,0.8mmol,1.6 eq) is added, the reaction is carried out for 24 hours at room temperature under the protection of nitrogen, the reaction is stopped, dialysis and purification are carried out for 48 hours, and freeze drying is carried out for 24 hours, so as to obtain the aminated hyaluronic acid;
(2) Dehydrolithocholic acid (mw=374.56, 0.112g,0.3mmol,1 eq) was dissolved in DMF, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (mw=191.7, 57.5mg,0.3mmol,1 eq) and N-hydroxysuccinimide (mw=115.1, 51.8mg,0.45mmol,1.5 eq) were added and reacted at room temperature under nitrogen protection for 20min; then, aminated hyaluronic acid (MW: 50kDa,0.190g,0.45mmol,1.5 eq) was added, and the reaction was stopped at room temperature under nitrogen protection for 12 hours, DMF was distilled off under reduced pressure, purified by dialysis for 48 hours, and freeze-dried for 24 hours to give hyaluronic acid-dehydrolithocholic acid polymer.
EXAMPLE 2 preparation of hyaluronic acid-dehydrolithocholic acid Polymer Using hyaluronic acid and dehydrolithocholic acid under different conditions from example 1
(1) Hyaluronic acid (mw=20kda, 0.380g,1mmol,2 eq) was dissolved in 10mL distilled water, and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (mw=191.7, 0.288g,1.5mmol,3 eq) and N-hydroxysuccinimide (mw=115.1, 57.6mg,0.5mmol,1 eq) were added and reacted at room temperature under nitrogen protection for 30min; then anhydrous ethylenediamine (MW=60.1, 60.1mg,1mmol,2 eq) is added, the reaction is stopped under the protection of nitrogen at room temperature for 36h, the reaction is stopped, the dialysis purification is performed for 48h, and the freeze drying is performed for 24h, so as to obtain the aminated hyaluronic acid;
(2) Dehydrolithocholic acid (mw=374.56, 0.224g,0.6mmol,2 eq) was dissolved in DMF and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (mw=191.7, 57.5mg,0.3mmol,1 eq) and N-hydroxysuccinimide (mw=115.1, 51.8mg,0.45mmol,1.5 eq) were added and reacted at room temperature under nitrogen protection for 30min; then, aminated hyaluronic acid (MW: 20kDa, 0.255 g,0.6mmol,2 eq) was added, and the reaction was stopped at room temperature under nitrogen protection for 18 hours, DMF was distilled off under reduced pressure, purified by dialysis for 48 hours, and freeze-dried for 24 hours to obtain hyaluronic acid-dehydrolithocholic acid polymer.
EXAMPLE 3 preparation and characterization of hyaluronic acid-dehydrolithocholic acid micelle particles
The hyaluronic acid-dehydrolithocholic acid polymer prepared in example 1 was used to study the morphology and physical properties of micelles under different treatments.
The specific research method is as follows:
(1) Dispersing 3mg or 30mg of hyaluronic acid-dehydrolithocholic acid polymer in water, performing ultrasonic treatment for 4min, and stopping for 5s every 30s to obtain hyaluronic acid-dehydrolithocholic acid micelle particles with concentration of 0.3mg/mL or 3mg/mL. 3mg of hyaluronic acid-dehydrolithocholic acid polymer is dispersed in 10mL of acetic acid solution with pH 5 or 10mL of sodium bicarbonate solution with pH 9, ultrasonic treatment is carried out for 4min, and every 30s is stopped for 5s, so that hyaluronic acid-dehydrolithocholic acid micelle particles with concentration of 0.3mg/mL under different pH environments are obtained.
(2) Different concentrations of hyaluronic acid-dehydrolithocholic acid micelle particle morphology were observed with a JEM-1400 transmission electron microscope (JEOL).
(3) The particle size and zeta potential of hyaluronic acid-dehydrolithocholic acid micelle particles under different pH environments were measured with a ZS90 nm particle size potentiometric analyzer (Malvern).
The measurement results are shown in FIG. 2, and the transmission electron microscope result shows that the hyaluronic acid-dehydrolithocholic acid micelle particles with the concentration of 3mg/mL tend to be aggregated, and the hyaluronic acid-dehydrolithocholic acid micelle particles with the concentration of 0.3mg/mL are more uniformly dispersed in water. The nano particle size potential analysis result shows that compared with a neutral environment, hyaluronic acid-dehydrolithocholic acid micelle particles are easier to agglomerate in an acidic environment, and the hyaluronic acid-dehydrolithocholic acid micelle particles are more stable in distribution in an alkaline environment, so that the hyaluronic acid-dehydrolithocholic acid micelle particles are not easy to be absorbed when passing through gastric juice in the acidic environment, and are easier to be absorbed in the alkaline intestinal environment.
EXAMPLE 4 colon targeting, therapeutic Effect and side Effect study of mouse IBD
The hyaluronic acid-dehydrolithocholic acid micelle prepared in example 1 was used as a control, and colon targeting, therapeutic effect and side effect of mouse IBD were studied by using hyaluronic acid and dehydrolithocholic acid not linked with hyaluronic acid as controls.
The specific method comprises the following steps:
(1) Mice were subjected to IBD modeling using 2.5% dss and normal mice were used as controls. And respectively connecting hyaluronic acid-dehydrolithocholic acid micelle, hyaluronic acid or dehydrolithocholic acid with Cy5.5 fluorescent dye, respectively orally administering 3mg/kg of hyaluronic acid-dehydrolithocholic acid micelle, hyaluronic acid or dehydrolithocholic acid with Cy5.5 connection to IBD or normal mice, taking all important organs after 7 hours, performing in vitro fluorescence imaging to observe the condition of the medicine enriched in colon, taking sections at the tail ends of the colon, and performing ice cutting to observe tissue fluorescence co-localization.
(2) From the modeling day, 3mg/kg of hyaluronic acid-dehydrolithocholic acid micelle, hyaluronic acid or dehydrolithocholic acid are respectively orally administered to IBD or normal mice every 1 day, daily body weight of the mice is recorded, and a body weight curve is produced; euthanizing the treated mice on day 9 of molding, collecting the mouse colon, and measuring the colon length from the ileocecum to the anus; taking 0.5cm from the tail end of the colon, performing paraffin section and HE staining, and scoring the tissue injury; the 0.5cm tissue from the end of the colon was collected for fluorescent quantitative PCR detection of inflammation-associated mRNA.
(3) For normal mice given ordinary drinking water, IBD model mice and IBD model mice given micelle particle therapy, the colon of the mice was taken on day 9 of model making, the colon lamina propria mononuclear cells were isolated, th17 cells were detected by flow cytometry (Beckman Coulter), and the differentiation differences of different groups of Th17 cells were compared; another 0.5cm of tissue from the distal colon was collected for fluorescent quantitative PCR detection of Th 17-associated IL17 mRNA.
(4) PBS or hyaluronic acid-dehydrolithocholic acid micelle particles are given to a normal mouse every other day, the weight of the mouse is recorded every day, and a weight curve is produced; taking blood from the orbit on the 9 th day, and measuring blood routine and blood biochemical indexes; euthanizing the treated mice, collecting important organs, slicing and HE staining, and observing organ tissue damage; the thymus of the mice is taken, the thymus weight is measured, the ratio of the thymus of the mice to the body weight of the mice is calculated, and whether the thymus is reduced or not is evaluated.
FIG. 1 is a schematic representation of self-assembly of hyaluronic acid-dehydrolithocholic acid polymers in water into micellar particles and their use in the treatment of IBD.
As shown in figure 3, both hyaluronic acid-dehydrolithocholic acid micelle particles and hyaluronic acid were enriched in the colon of IBD mice to a higher extent than dehydrolithocholic acid, whereas no significant increase in the enrichment of hyaluronic acid-dehydrolithocholic acid micelle particles was seen in the colon of normal mice. Immunofluorescence co-localization showed that both hyaluronic acid-dehydrolithocholic acid micelle particles and hyaluronic acid showed a binding with colon CD3 + Co-localization of T cell infiltration at sites of inflammation and inflammation epithelium was present, whereas co-localization with anchored sites was not found with administration of dehydrolithocholic acid or micelle particles in the colon of normal mice. The above results demonstrate that hyaluronic acid-dehydrolithocholic acid micelle particles are able to target the colonic inflammation site of IBD mice, during which hyaluronic acid interacts with the inflammation site to play an important role.
As shown in fig. 4 and 5, hyaluronic acid-dehydrolithocholic acid micelle particles exhibited optimal efficacy in treating IBD compared to hyaluronic acid or dehydrolithocholic acid alone, as demonstrated by decreasing body weight loss, maintaining colon length, protecting colon tissue integrity, decreasing pro-inflammatory cytokine expression, and increasing anti-inflammatory cytokine expression. Although dehydrolithocholic acid shows a certain therapeutic effect, the therapeutic effect of dehydrolithocholic acid is also obviously different from that of micelle particles.
As shown in figure 6, hyaluronic acid-dehydrolithocholic acid micelle particles were able to significantly inhibit Th17 cell differentiation in colon tissue of IBD mice, demonstrating that the therapeutic mechanism of action of micelle particles on IBD was dependent on the action of dehydrolithocholic acid.
As shown in fig. 7, the hyaluronic acid-dehydrolithocholic acid micelle particles did not cause damage to important organs in normal mice, and the change in body weight during administration was not significantly different from that of the control group, and there was no thymus inhibition, demonstrating the oral safety of the hyaluronic acid-dehydrolithocholic acid micelle particles.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of the invention should be assessed as that of the appended claims.
Claims (9)
1. A method for synthesizing a hyaluronic acid-dehydrolithocholic acid polymer based on hyaluronic acid-linked cholic acid derivatives, characterized by comprising the steps of:
(1) The hyaluronic acid and the anhydrous ethylenediamine are used as raw materials, and the molar ratio range of the hyaluronic acid to the anhydrous ethylenediamine is 10:1 to 1:10, performing amidation reaction, dialysis and freeze drying to obtain a compound I;
(2) The compound I and the dehydrolithocholic acid are used as raw materials, and the molar ratio range of the dehydrolithocholic acid to the compound I is 10:1 to 1:10, performing amidation reaction, dialysis and freeze drying to obtain the compound II, namely the hyaluronic acid-dehydrolithocholic acid polymer.
2. The method of claim 1, wherein the hyaluronic acid has a molecular weight of 1 to 1000KDa.
3. The method according to claim 1, wherein in the step (1), the amidation reaction is carried out for 2 to 48 hours.
4. The method according to claim 1, wherein in the step (2), the amidation reaction is performed for 1 to 24 hours.
5. A method for preparing hyaluronic acid-dehydrolithocholic acid micelle particles, which is characterized in that hyaluronic acid-dehydrolithocholic acid polymer prepared by the method of any one of claims 1-4 is added into water, the concentration of the hyaluronic acid-dehydrolithocholic acid polymer in the water is 0.01-30.0 mg/mL, and evenly distributed spherical micelle particles are obtained by ultrasonic treatment.
6. The method of claim 5, wherein the ultrasound time is 1 to 100 minutes.
7. A hyaluronic acid-dehydrolithocholic acid polymer prepared by the method of any of claims 1-4.
8. A hyaluronic acid-dehydrolithocholic acid micelle particle prepared by the method of claim 5.
9. Use of hyaluronic acid-dehydrolithocholic acid micelle particles according to claim 8 for the preparation of a medicament for the treatment of inflammatory bowel disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211626808.1A CN115850534B (en) | 2022-12-16 | 2022-12-16 | Hyaluronic acid-cholic acid derivative and synthetic method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211626808.1A CN115850534B (en) | 2022-12-16 | 2022-12-16 | Hyaluronic acid-cholic acid derivative and synthetic method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115850534A CN115850534A (en) | 2023-03-28 |
| CN115850534B true CN115850534B (en) | 2024-03-12 |
Family
ID=85673783
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211626808.1A Active CN115850534B (en) | 2022-12-16 | 2022-12-16 | Hyaluronic acid-cholic acid derivative and synthetic method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115850534B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104367556A (en) * | 2014-11-14 | 2015-02-25 | 郑州大学 | Preparation method and application of hyaluronic acid nitrate deoxycholic acid polymer micelle capable of providing nitric oxide |
| CN104603156A (en) * | 2012-09-05 | 2015-05-06 | 中外制药株式会社 | Hyaluronic acid derivative having amino acid and steryl group introduced thereinto |
-
2022
- 2022-12-16 CN CN202211626808.1A patent/CN115850534B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104603156A (en) * | 2012-09-05 | 2015-05-06 | 中外制药株式会社 | Hyaluronic acid derivative having amino acid and steryl group introduced thereinto |
| CN104367556A (en) * | 2014-11-14 | 2015-02-25 | 郑州大学 | Preparation method and application of hyaluronic acid nitrate deoxycholic acid polymer micelle capable of providing nitric oxide |
Non-Patent Citations (2)
| Title |
|---|
| Ki Young Choi等.Self-assembled hyaluronic acid nanoparticles as a potential drug carrier for cancer therapy: synthesis, characterization, and in vivo biodistribution.《Journal of Materials Chemistry》.2009,第19卷4102-4107. * |
| Saiyu Hang等.Bile acid metabolites control TH17 and Treg cell differentiation.《Nature》.2019,第576卷143-164. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115850534A (en) | 2023-03-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gou et al. | Multi-bioresponsive silk fibroin-based nanoparticles with on-demand cytoplasmic drug release capacity for CD44-targeted alleviation of ulcerative colitis | |
| Chung et al. | In vivo biodistribution and clearance of peptide amphiphile micelles | |
| CN101401941B (en) | Preparation and uses of tumor-targeted carrier material RGD-fatty alcohol series of compounds | |
| CN111848830A (en) | Application of fluorine-containing compound modified chitosan as drug carrier and preparation method thereof | |
| Bai et al. | Reduction-active polymeric prodrug micelles based on α-cyclodextrin polyrotaxanes for triggered drug release and enhanced cancer therapy | |
| CN112516109B (en) | Mesenchymal stem cell-based fused cancer cell membrane bionic nanoparticle and preparation method thereof | |
| CN113679670B (en) | Vesicle nano-drug carrying chloroquine compound, and preparation method and application thereof | |
| CN115998889A (en) | Carbon quantum dots for diagnostic analysis and drug delivery | |
| WO2021203959A1 (en) | Heparin nano drug carrier system for loading amino anti-tumor drug, and preparation method therefor | |
| CN113663079B (en) | Carrier-free self-assembly nano particle and preparation method and application thereof | |
| CN115850534B (en) | Hyaluronic acid-cholic acid derivative and synthetic method and application thereof | |
| CN113855813A (en) | Preparation method and application of ROS (reactive oxygen species) response marine fucoidin nano-carrier based on Fenton reaction and AIE (immune-induced emission) effect | |
| CN112402378A (en) | ROS (reactive oxygen species) -responsive dexamethasone liposome containing selenium bond as well as preparation method and application thereof | |
| Jeon et al. | pH-Responsive hyaluronic acid-based nanocarrier for treatment of rheumatoid arthritis | |
| CN108126209B (en) | Nano drug delivery system for time-sequence release of curcumin and chemotherapeutic drugs and application thereof | |
| CN102250205A (en) | Preparation method of fatty acyl-RGD (Arg Gly Asp) induced docetaxel target liposome and antitumor activity thereof | |
| KR101323102B1 (en) | Nanoparticles formed by encapsulating an anticancer drug into glycolchitosan-cholanic acid complex and a process for the preparation thereof | |
| CN111973572A (en) | Manganese-based dendritic macromolecular composite nanomaterial, and preparation method and application thereof | |
| Liang et al. | Fluorinated hyaluronates endow oral nanoparticles with mucus penetration and colonic macrophage targeting properties | |
| CN114788874B (en) | Nanometer micelle coated with baicalein and application thereof | |
| CN115154422B (en) | CD44 targeting and ROS response nano micelle pharmaceutical composition, and preparation method and application thereof | |
| CN114887076B (en) | Mixed triblock micelle with chemotherapy-immune function and preparation method and application thereof | |
| CN117045619B (en) | Liposome-polymer nanoparticle loaded with melittin oncolytic peptide and adenosine A2AR receptor inhibitor, and preparation and application thereof | |
| CN114276390B (en) | Dithiocarbamate derivative nano-drug for anti-tumor drug delivery, and preparation method and application thereof | |
| CN114306340B (en) | Preparation method and application of cholic acid-quaternized chitosan oligosaccharide-ES 2 peptide/camptothecin conjugate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |