CN116854831A - Quaternary ammonium salt hydroxyethyl starch-curcumin prodrug as anticancer medicine and its prepn process - Google Patents
Quaternary ammonium salt hydroxyethyl starch-curcumin prodrug as anticancer medicine and its prepn process Download PDFInfo
- Publication number
- CN116854831A CN116854831A CN202310748028.2A CN202310748028A CN116854831A CN 116854831 A CN116854831 A CN 116854831A CN 202310748028 A CN202310748028 A CN 202310748028A CN 116854831 A CN116854831 A CN 116854831A
- Authority
- CN
- China
- Prior art keywords
- quaternary ammonium
- hydroxyethyl starch
- ammonium salt
- curcumin
- prodrug
- 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.)
- Pending
Links
- 229940109262 curcumin Drugs 0.000 title claims abstract description 113
- 239000004148 curcumin Substances 0.000 title claims abstract description 96
- 229940002612 prodrug Drugs 0.000 title claims abstract description 71
- 239000000651 prodrug Substances 0.000 title claims abstract description 71
- 238000000034 method Methods 0.000 title claims description 11
- 239000003814 drug Substances 0.000 title abstract description 23
- 230000008569 process Effects 0.000 title description 3
- 230000001093 anti-cancer Effects 0.000 title description 2
- VFLDPWHFBUODDF-FCXRPNKRSA-N curcumin Chemical compound C1=C(O)C(OC)=CC(\C=C\C(=O)CC(=O)\C=C\C=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-FCXRPNKRSA-N 0.000 claims abstract description 102
- 235000012754 curcumin Nutrition 0.000 claims abstract description 51
- VFLDPWHFBUODDF-UHFFFAOYSA-N diferuloylmethane Natural products C1=C(O)C(OC)=CC(C=CC(=O)CC(=O)C=CC=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-UHFFFAOYSA-N 0.000 claims abstract description 51
- 229920001612 Hydroxyethyl starch Polymers 0.000 claims abstract description 33
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 33
- 229940041181 antineoplastic drug Drugs 0.000 claims abstract description 33
- 229940050526 hydroxyethylstarch Drugs 0.000 claims abstract description 33
- 150000003242 quaternary ammonium salts Chemical class 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000003490 Thiodipropionic acid Substances 0.000 claims abstract description 15
- ODJQKYXPKWQWNK-UHFFFAOYSA-N 3,3'-Thiobispropanoic acid Chemical compound OC(=O)CCSCCC(O)=O ODJQKYXPKWQWNK-UHFFFAOYSA-N 0.000 claims abstract description 12
- 235000019303 thiodipropionic acid Nutrition 0.000 claims abstract description 12
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 238000001556 precipitation Methods 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 49
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 42
- 238000006243 chemical reaction Methods 0.000 claims description 42
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 36
- 239000000126 substance Substances 0.000 claims description 25
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 24
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 20
- 239000007864 aqueous solution Substances 0.000 claims description 18
- 239000012295 chemical reaction liquid Substances 0.000 claims description 18
- 239000007787 solid Substances 0.000 claims description 17
- 239000002244 precipitate Substances 0.000 claims description 16
- 239000000843 powder Substances 0.000 claims description 15
- PUVAFTRIIUSGLK-UHFFFAOYSA-M trimethyl(oxiran-2-ylmethyl)azanium;chloride Chemical compound [Cl-].C[N+](C)(C)CC1CO1 PUVAFTRIIUSGLK-UHFFFAOYSA-M 0.000 claims description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- 229910052757 nitrogen Inorganic materials 0.000 claims description 12
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 12
- 239000012498 ultrapure water Substances 0.000 claims description 12
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 10
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 10
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 claims description 9
- 238000004108 freeze drying Methods 0.000 claims description 9
- 229960000583 acetic acid Drugs 0.000 claims description 6
- 239000012362 glacial acetic acid Substances 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- 238000006467 substitution reaction Methods 0.000 claims description 5
- 238000005886 esterification reaction Methods 0.000 claims description 4
- 238000007710 freezing Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 206010028980 Neoplasm Diseases 0.000 abstract description 26
- 239000000693 micelle Substances 0.000 abstract description 26
- 229940079593 drug Drugs 0.000 abstract description 21
- 230000009471 action Effects 0.000 abstract description 6
- 230000002438 mitochondrial effect Effects 0.000 abstract description 5
- 210000004881 tumor cell Anatomy 0.000 abstract description 5
- 230000002209 hydrophobic effect Effects 0.000 abstract description 4
- 125000001453 quaternary ammonium group Chemical group 0.000 abstract description 4
- 230000008685 targeting Effects 0.000 abstract description 4
- 230000004044 response Effects 0.000 abstract description 3
- 230000000259 anti-tumor effect Effects 0.000 abstract description 2
- 238000002425 crystallisation Methods 0.000 abstract description 2
- 230000008025 crystallization Effects 0.000 abstract description 2
- 229920000642 polymer Polymers 0.000 abstract description 2
- 239000002861 polymer material Substances 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 26
- 239000002609 medium Substances 0.000 description 10
- 206010009944 Colon cancer Diseases 0.000 description 9
- 208000029742 colonic neoplasm Diseases 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 238000011534 incubation Methods 0.000 description 9
- 239000001257 hydrogen Substances 0.000 description 8
- 229910052739 hydrogen Inorganic materials 0.000 description 8
- 235000019441 ethanol Nutrition 0.000 description 6
- -1 polyphenol compound Chemical class 0.000 description 6
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 5
- 238000005481 NMR spectroscopy Methods 0.000 description 5
- 210000003470 mitochondria Anatomy 0.000 description 5
- 230000035515 penetration Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 230000012202 endocytosis Effects 0.000 description 4
- 238000012869 ethanol precipitation Methods 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 238000012377 drug delivery Methods 0.000 description 3
- 238000002329 infrared spectrum Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- IKEOZQLIVHGQLJ-UHFFFAOYSA-M mitoTracker Red Chemical compound [Cl-].C1=CC(CCl)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 IKEOZQLIVHGQLJ-UHFFFAOYSA-M 0.000 description 3
- 239000002105 nanoparticle Substances 0.000 description 3
- 230000004043 responsiveness Effects 0.000 description 3
- 108010087230 Sincalide Proteins 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 230000007480 spreading Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 229920000945 Amylopectin Polymers 0.000 description 1
- 235000003392 Curcuma domestica Nutrition 0.000 description 1
- 244000008991 Curcuma longa Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- VBVAVBCYMYWNOU-UHFFFAOYSA-N coumarin 6 Chemical compound C1=CC=C2SC(C3=CC4=CC=C(C=C4OC3=O)N(CC)CC)=NC2=C1 VBVAVBCYMYWNOU-UHFFFAOYSA-N 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 235000003373 curcuma longa Nutrition 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000431 effect on proliferation Effects 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000002121 endocytic effect Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 229960002949 fluorouracil Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 210000001700 mitochondrial membrane Anatomy 0.000 description 1
- 239000002159 nanocrystal Substances 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000003058 plasma substitute Substances 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 239000003642 reactive oxygen metabolite Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000001839 systemic circulation Effects 0.000 description 1
- 150000003568 thioethers Chemical class 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011277 treatment modality Methods 0.000 description 1
- 235000013976 turmeric Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B31/00—Preparation of derivatives of starch
- C08B31/08—Ethers
- C08B31/12—Ethers having alkyl or cycloalkyl radicals substituted by heteroatoms, e.g. hydroxyalkyl or carboxyalkyl starch
- C08B31/125—Ethers having alkyl or cycloalkyl radicals substituted by heteroatoms, e.g. hydroxyalkyl or carboxyalkyl starch having a substituent containing at least one nitrogen atom, e.g. cationic starch
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention discloses an anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug and a preparation method thereof, belonging to the field of biomedical high polymer materials. According to the invention, quaternary ammonium salt hydroxyethyl starch is used as a polymer skeleton, and hydrophobic curcumin and hydrophilic quaternary ammonium salt hydroxyethyl starch are coupled through thiodipropionic acid to prepare the curcumin prodrug capable of self-assembling to form nano micelle. The obtained curcumin prodrug can self-assemble in water to form nano micelle, and solves the problems of poor solubility and easy crystallization precipitation of curcumin in water. Under the action of ROS, thioether bonds in curcumin prodrug micelle can be rapidly broken and curcumin is released, so that the drug is released in response to the tumor part. Meanwhile, under the action of surface quaternary ammonium root, curcumin prodrug micelle can be rapidly phagocytized by tumor cells and has mitochondrial targeting delivery capability. The curcumin prodrug micelle designed by the invention can obviously improve the anti-tumor effect of curcumin and has good application prospect.
Description
Technical Field
The invention belongs to the field of biomedical high polymer materials, and in particular relates to an anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug and a preparation method thereof.
Background
Colon cancer constitutes a serious threat to human health due to its high morbidity and mortality. Current methods of treatment of colon cancer include chemotherapy, radiation therapy, surgical excision, immunotherapy, etc., with chemotherapy being the most common treatment modality. However, chemical drugs such as 5-fluorouracil and the like have poor water solubility, and are distributed throughout the body along with blood after intravenous administration, and the drugs are randomly distributed in normal tissues and cancer tissues, so that the curative effect of the drugs is reduced and a series of toxic and side effects are caused. Curcumin is a polyphenol compound extracted from turmeric, and has remarkable inhibition effect on proliferation and metastasis of colon cancer cells. However, curcumin is hardly soluble in water and is easily metabolized in the systemic circulation to be inactivated, and its clinical application is very limited. Therefore, the drug delivery system of the chemical drug is established, the solubility and the stability of the drug are improved, the accumulation and the permeation of the drug at the tumor position are enhanced, the responsive release of the drug in the tumor microenvironment is realized, and the drug becomes a hot spot and a difficult point of research.
With the discovery of high permeation long retention effects (EPR effects), nanoparticle drug delivery systems such as nanocrystals, prodrug micelles, emulsions, and the like have been rapidly developed. The prodrug micelle formed by coupling a hydrophilic polymer and a hydrophobic chemical drug through a response type connecting arm has attracted wide attention due to the nano-scale particle size distribution, longer blood circulation time and tumor microenvironment responsiveness. Hydroxyethyl starch (HES) is a natural polysaccharide derivative formed by glucose cycloethylation in amylopectin, has good biocompatibility, and can be clinically used as a plasma expander. HES has a large number of hydroxyl functionalities and can be used as the hydrophilic backbone for prodrugs. Meanwhile, the thioether bond has good reactive oxygen species responsiveness and can be used as a responsive connecting arm of a prodrug.
The ideal drug delivery system not only needs to effectively deliver the drug to the tumor site, but also is more important to complete the efficient transportation of tumor cells and the accurate intracellular delivery. Researches show that the surface modification of the nano micelle by positive charge can effectively improve the endocytosis efficiency of tumor cells. Meanwhile, the positive charge nanoparticle also shows good tumor deep penetration and subcellular precise delivery capability. However, most of the surface modifying groups commonly used at present are lipophilic cations, which are easy to cause protein adsorption and immune clearance in the circulation process. Hydrophilic quaternary ammonium radical has good membrane binding capacity, is expected to be applied to surface modification of prodrug micelle, and improves the curative effect of curcumin.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide an anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug and a preparation method thereof. The invention can improve the solubility and stability of curcumin, enhance the deep penetration capacity, endocytic efficiency and mitochondrial targeting of micelle, and provide reference for clinical application of curcumin.
The specific technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides an anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug having the chemical structure of formula (I):
wherein R is H or CH 2 CH 2 OH。
In a second aspect, the invention provides a preparation method of an anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug, which comprises the following steps:
s1: dissolving hydroxyethyl starch in a sodium hydroxide aqueous solution, and reacting with 2, 3-epoxypropyl trimethyl ammonium chloride under heating to generate quaternary ammonium salt hydroxyethyl starch with a chemical structure shown in a formula (II);
the formula (II) is specifically as follows:
wherein R is H or CH 2 CH 2 OH；
S2: the generated quaternary ammonium salt hydroxyethyl starch and thiodipropionic acid are catalyzed by 4-dimethylaminopyridine and dicyclohexylcarbodiimide to generate quaternary ammonium salt hydroxyethyl starch-thiodipropionic acid monoester with a chemical structure shown in a formula (III);
the formula (III) is specifically as follows:
wherein R is H or CH 2 CH 2 OH；
S3: in the presence of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole, carrying out esterification reaction on curcumin and the quaternary ammonium salt hydroxyethyl starch-thiodipropionic monoester generated in the step S2 to generate an anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug with a chemical structure shown in a formula (I);
the formula (I) is specifically as follows:
wherein R is H or CH 2 CH 2 OH。
Preferably, the hydroxyethyl starch has a molecular weight of 130kDa and a degree of substitution of 0.4.
Preferably, the step S1 specifically includes the following steps:
s11: 7.2g of hydroxyethyl starch is dissolved in 45mL of sodium hydroxide aqueous solution, and the mixture is heated and reacted for 0.5 to 2 hours at the temperature of 45 ℃; the mass concentration of the sodium hydroxide aqueous solution is 0.05-0.1%;
s12: dropwise adding 5.28mL of 2, 3-epoxypropyl trimethyl ammonium chloride aqueous solution into the reaction solution obtained in the step S11, and heating at 45 ℃ for reaction for 24 hours; the mass concentration of the 2, 3-epoxypropyl trimethyl ammonium chloride aqueous solution is 15-30%;
s13: regulating the pH value of the reaction solution obtained in the step S12 to 7 by glacial acetic acid, precipitating with alcohol, and collecting precipitate; redissolving the precipitate in water, and dialyzing with ultrapure water for 24-72 h; and then freezing the obtained dialysate for 24 hours at the temperature of minus 80 ℃, and freeze-drying to obtain solid powder, thus obtaining the quaternary ammonium salt hydroxyethyl starch with the chemical structure of formula (II).
Further, the alcohol precipitation is carried out by utilizing absolute ethyl alcohol.
Preferably, the step S2 specifically includes the following steps:
s21: 1-2 g of thiodipropionic acid and 0.1-0.3 g of 4-dimethylaminopyridine are dissolved in 15mL of dimethyl sulfoxide to obtain a first solution; dissolving 0.5-1.5 g of dicyclohexylcarbodiimide in dimethyl sulfoxide to obtain a second solution; dropwise adding the second solution into the first solution, and reacting for 0.5-2 h at room temperature;
s22: adding 1g of quaternary ammonium salt hydroxyethyl starch into the reaction liquid obtained in the step S21, and reacting for 48 hours at room temperature under the protection of nitrogen;
s23: and (2) dialyzing the reaction solution obtained in the step (S22) in dimethyl sulfoxide for 48 hours, wherein the obtained dialysate is the quaternary ammonium salt hydroxyethyl starch-thiodipropionic monoester with the chemical structure shown in the formula (III).
Preferably, the step S3 specifically includes the following steps:
s31: 0.2 to 0.4g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 0.1 to 0.2g of 1-hydroxybenzotriazole are dissolved in a quaternary ammonium salt hydroxyethyl starch-thiodipropionic monoester water solution together, and react for 0.5 to 2 hours at room temperature;
s32: adding 0.03-0.25 g of curcumin into the reaction liquid obtained in the step S31, and carrying out light-shielding reaction for 24-48 h under the protection of nitrogen; and then dialyzing the obtained reaction liquid in ultrapure water for 48 hours, and then freeze-drying the reaction liquid into solid powder to obtain the anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug with the chemical structure shown in the formula (I).
Compared with the prior art, the invention has the following beneficial effects:
the invention takes biodegradable QHES as a hydrophilic skeleton, takes thioether bond responsive to tumor microenvironment as a connecting arm, and couples hydrophobic curcumin to the QHES through ester bond. The QHES-S-CUR provided by the invention can self-assemble in water to form nano-scale micelle, and accumulation at a tumor part is realized by means of high-permeability long retention effect. The prodrug micelle is positively charged through quaternary ammonium surface modification, so that deep penetration of tumor sites, phagocytosis of tumor cells on the prodrug micelle and mitochondria targeted delivery are promoted. Thioether linkages in the prodrug can be cleaved under active oxygen stimulation of the tumor microenvironment. Thereby effecting ROS-responsive release of the drug. The quaternary ammonium salt hydroxyethyl starch-curcumin prodrug of the anticancer drug provided by the invention has obvious tumor inhibiting effect on colon cancer tumor models, and has the potential of being applied to clinical treatment of colon cancer.
Drawings
FIG. 1 is a synthetic route diagram of a quaternary ammonium salt hydroxyethyl starch-curcumin prodrug.
FIG. 2 is a Fourier infrared spectrum of the quaternary ammonium salt hydroxyethyl starch-curcumin prepared in example 1.
FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of the quaternary ammonium salt hydroxyethyl starch-curcumin prepared in example 1.
FIG. 4 is a graph showing the drug release profile of the quaternary ammonium salt hydroxyethyl starch-curcumin prepared in example 1.
FIG. 5 shows the results of an in vitro cytotoxicity test of the quaternary ammonium salt hydroxyethyl starch-curcumin prepared in example 1.
FIG. 6 shows endocytosis of the quaternary ammonium salt hydroxyethyl starch-curcumin prepared in example 1.
FIG. 7 shows subcellular distribution of the quaternary ammonium salt hydroxyethyl starch-curcumin prepared in example 1.
FIG. 8 shows the three-dimensional tumor sphere penetration of the quaternary ammonium salt hydroxyethyl starch-curcumin prepared in example 1.
Detailed Description
The invention is further illustrated and described below with reference to the drawings and detailed description. . The technical features of the embodiments of the invention can be combined correspondingly on the premise of no mutual conflict.
The invention provides an anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug, which has a chemical structure shown in a formula (I):
wherein R is H or CH 2 CH 2 OH。
As shown in fig. 1, the preparation method of the quaternary ammonium salt hydroxyethyl starch-curcumin prodrug of the anticancer drug provided by the invention specifically comprises the following steps:
s1: hydroxyethyl starch (HES) is dissolved in aqueous sodium hydroxide solution and reacted with 2, 3-epoxypropyl trimethyl ammonium chloride under heating to produce quaternary ammonium salt hydroxyethyl starch (QHES) having the chemical structure of formula (II).
The formula (II) is specifically as follows:
wherein R is H or CH 2 CH 2 OH。
In this example, hydroxyethyl starch (HES) has a molecular weight of 130kDa and a degree of substitution of 0.4. The step is preferably performed in the following manner, but it should be noted that the amount, reaction conditions, and the like of the following preferred steps can be adjusted according to actual needs, and all the steps belong to the protection scope of the present invention, and the preferred steps are specifically as follows:
s11: 7.2g of hydroxyethyl starch is dissolved in 45mL of sodium hydroxide aqueous solution, and the mixture is heated and reacted for 0.5 to 2 hours at the temperature of 45 ℃; the mass concentration of the sodium hydroxide aqueous solution is 0.05-0.1%;
s12: dropwise adding 5.28mL of 2, 3-epoxypropyl trimethyl ammonium chloride aqueous solution into the reaction solution obtained in the step S11, and heating at 45 ℃ for reaction for 24 hours; the mass concentration of the 2, 3-epoxypropyl trimethyl ammonium chloride aqueous solution is 15-30%;
s13: regulating the pH value of the reaction solution obtained in the step S12 to 7 by glacial acetic acid, precipitating with alcohol, and collecting precipitate; redissolving the precipitate in water, and dialyzing with ultrapure water for 24-72 h; and then freezing the obtained dialysate for 24 hours at the temperature of minus 80 ℃, and freeze-drying to obtain solid powder, thus obtaining the quaternary ammonium salt hydroxyethyl starch with the chemical structure of formula (II).
In the step, the ethanol precipitation is carried out by slowly pouring the filtrate into absolute ethyl alcohol, fully stirring, standing for 0.5h, and discarding the liquid after precipitate is formed.
S2: the generated quaternary ammonium salt hydroxyethyl starch and thiodipropionic acid are catalyzed by 4-Dimethylaminopyridine (DMAP) and Dicyclohexylcarbodiimide (DCC) to generate quaternary ammonium salt hydroxyethyl starch-thiodipropionic acid monoester (QHES-S-COOH) with the chemical structure shown in the formula (III).
The formula (III) is specifically as follows:
wherein R is H or CH 2 CH 2 OH。
The step is preferably performed in the following manner, but it should be noted that the amount, reaction conditions, and the like of the following preferred steps can be adjusted according to actual needs, and all the steps belong to the protection scope of the present invention, and the preferred steps are specifically as follows:
s21: 1-2 g of thiodipropionic acid and 0.1-0.3 g of 4-dimethylaminopyridine are dissolved in 15mL of dimethyl sulfoxide to obtain a first solution; dissolving 0.5-1.5 g of dicyclohexylcarbodiimide in dimethyl sulfoxide to obtain a second solution; dropwise adding the second solution into the first solution, and reacting for 0.5-2 h at room temperature;
among them, dimethyl sulfoxide is commercially available with a purity of 99.8%. The dimethyl sulfoxide used in each of the following steps is the same as the above dimethyl sulfoxide unless otherwise specified.
S22: adding 1g of quaternary ammonium salt hydroxyethyl starch into the reaction liquid obtained in the step S21, and reacting for 48 hours at room temperature under the protection of nitrogen;
s23: and (2) dialyzing the reaction solution obtained in the step (S22) in dimethyl sulfoxide for 48 hours, wherein the obtained dialysate is the quaternary ammonium salt hydroxyethyl starch-thiodipropionic monoester with the chemical structure shown in the formula (III).
S3: esterification reaction is carried out on curcumin and quaternary ammonium salt hydroxyethyl starch-thiodipropionic monoester generated in the step S2 in the presence of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCl) and 1-hydroxybenzotriazole (HOBt) to generate anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug (QHES-S-CUR) with the chemical structure of formula (I).
The formula (I) is specifically as follows:
wherein R is H or CH 2 CH 2 OH。
The step is preferably performed in the following manner, but it should be noted that the amount, reaction conditions, and the like of the following preferred steps can be adjusted according to actual needs, and all the steps belong to the protection scope of the present invention, and the preferred steps are specifically as follows:
s31: 0.2 to 0.4g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 0.1 to 0.2g of 1-hydroxybenzotriazole are dissolved in a quaternary ammonium salt hydroxyethyl starch-thiodipropionic monoester water solution together, and react for 0.5 to 2 hours at room temperature;
s32: adding 0.03-0.25 g of curcumin into the reaction liquid obtained in the step S31, and carrying out light-shielding reaction for 24-48 h under the protection of nitrogen; and then dialyzing the obtained reaction liquid in ultrapure water for 48 hours, and then freeze-drying the reaction liquid into solid powder to obtain the anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug with the chemical structure shown in the formula (I).
In the anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug prepared by the method, the substitution degree of quaternary ammonium is 0.13, and the mass content of curcumin is 2.5-15.0%.
The following specific examples are set forth to illustrate the present invention in detail, but the present invention is not limited to the following examples.
Example 1
The preparation method of the quaternary ammonium salt hydroxyethyl starch-curcumin prodrug as an anticancer drug comprises the following specific steps:
(1) 7.2g of HES was dissolved in 45mL of aqueous sodium hydroxide solution having a concentration of 0.07%. The reaction was heated at 45℃for 0.5h.
(2) 5.28mL of 2, 3-epoxypropyl trimethyl ammonium chloride aqueous solution was added dropwise to the reaction solution obtained in step (1), and the concentration of 2, 3-epoxypropyl trimethyl ammonium chloride was 25%. The reaction was heated at 45℃for 24h.
(3) After the pH of the reaction solution obtained in the step (2) was adjusted to 7 with glacial acetic acid, the reaction solution was subjected to alcohol precipitation and the precipitate was collected. The precipitate was redissolved in water and dialyzed against ultrapure water for 48h. The dialysate was frozen at-80 ℃ for 24 hours and then freeze-dried to a solid powder.
In the step, the ethanol precipitation is carried out by slowly pouring the filtrate into absolute ethyl alcohol, fully stirring, standing for 0.5h, and discarding the liquid after precipitate is formed.
(4) 1.682g of thiodipropionic acid and 0.232g of DMAP were dissolved in 15mL of dimethyl sulfoxide. 1.03g of DCC was dissolved in dimethyl sulfoxide, and the solution was added dropwise to the reaction mixture to react at room temperature for 0.5h.
(5) And (3) adding 1g of QHES solid powder obtained in the step (3) into the reaction liquid obtained in the step (4), and reacting for 48 hours at room temperature under the protection of nitrogen.
(6) Dialyzing the reaction solution obtained in the step (5) in dimethyl sulfoxide for 48 hours, wherein the obtained dialysate is QHES-S-COOH.
(7) 0.23g of EDCl and 0.168g of HOBt were co-dissolved in the dialysate obtained in (6), and reacted at room temperature for 0.5h.
(8) And (3) adding 0.147g of curcumin into the reaction liquid obtained in the step (7), and carrying out light-shielding reaction for 24 hours under the protection of nitrogen. The resulting reaction solution was dialyzed in ultrapure water for 48 hours, followed by freeze-drying to a solid powder.
Example 2
The preparation method of the quaternary ammonium salt hydroxyethyl starch-curcumin prodrug as an anticancer drug comprises the following specific steps:
(1) 7.2g of HES was dissolved in 45mL of aqueous sodium hydroxide solution having a concentration of 0.07%. The reaction was heated at 45℃for 0.5h.
(2) 5.28mL of 2, 3-epoxypropyl trimethyl ammonium chloride aqueous solution was added dropwise to the reaction solution obtained in step (1), and the concentration of 2, 3-epoxypropyl trimethyl ammonium chloride was 25%. The reaction was heated at 45℃for 24h.
(3) After the pH of the reaction solution obtained in the step (2) was adjusted to 7 with glacial acetic acid, the reaction solution was subjected to alcohol precipitation and the precipitate was collected. The precipitate was redissolved in water and dialyzed against ultrapure water for 48h. The dialysate was frozen at-80 ℃ for 24 hours and then freeze-dried to a solid powder.
In the step, the ethanol precipitation is carried out by slowly pouring the filtrate into absolute ethyl alcohol, fully stirring, standing for 0.5h, and discarding the liquid after precipitate is formed.
(4) 0.841g of thiodipropionic acid and 0.116g of DMAP are dissolved in 15mL of dimethyl sulfoxide. 0.515g of DCC was dissolved in dimethyl sulfoxide, and the solution was added dropwise thereto, followed by reaction at room temperature for 0.5h.
(5) And (3) adding 1g of QHES solid powder obtained in the step (3) into the reaction liquid obtained in the step (4), and reacting for 48 hours at room temperature under the protection of nitrogen.
(6) Dialyzing the reaction solution obtained in the step (5) in dimethyl sulfoxide for 48 hours, wherein the obtained dialysate is QHES-S-COOH.
(7) 0.23g of EDCl and 0.168g of HOBt were co-dissolved in the dialysate obtained in (6), and reacted at room temperature for 0.5h.
(8) 0.0735g of curcumin is added into the reaction liquid obtained in the step (7) and is reacted for 24 hours in a dark place under the protection of nitrogen. The resulting reaction solution was dialyzed in ultrapure water for 48 hours, followed by freeze-drying to a solid powder.
Example 3
The preparation method of the quaternary ammonium salt hydroxyethyl starch-curcumin prodrug as an anticancer drug comprises the following specific steps:
(1) 7.2g of HES was dissolved in 45mL of aqueous sodium hydroxide solution having a concentration of 0.07%. The reaction was heated at 45℃for 0.5h.
(2) 5.28mL of 2, 3-epoxypropyl trimethyl ammonium chloride aqueous solution was added dropwise to the reaction solution obtained in step (1), and the concentration of 2, 3-epoxypropyl trimethyl ammonium chloride was 25%. The reaction was heated at 45℃for 24h.
(3) After the pH of the reaction solution obtained in the step (2) was adjusted to 7 with glacial acetic acid, the reaction solution was subjected to alcohol precipitation and the precipitate was collected. The precipitate was redissolved in water and dialyzed against ultrapure water for 48h. The dialysate was frozen at-80 ℃ for 24 hours and then freeze-dried to a solid powder.
In the step, the ethanol precipitation is carried out by slowly pouring the filtrate into absolute ethyl alcohol, fully stirring, and standing for 0.5h. After a precipitate had formed, the liquid was discarded.
(4) 1.682g of thiodipropionic acid and 0.232g of DMAP were dissolved in 15mL of dimethyl sulfoxide. 1.03g of DCC was dissolved in dimethyl sulfoxide, and the solution was added dropwise to the reaction mixture to react at room temperature for 0.5h.
(5) And (3) adding 1g of QHES solid powder obtained in the step (3) into the reaction liquid obtained in the step (4), and reacting for 48 hours at room temperature under the protection of nitrogen.
(6) Dialyzing the reaction solution obtained in the step (5) in dimethyl sulfoxide for 48 hours, wherein the obtained dialysate is QHES-S-COOH.
(7) 0.23g of EDCl and 0.168g of HOBt were co-dissolved in the dialysate obtained in (6), and reacted at room temperature for 0.5h.
(8) 0.0367g of curcumin is added into the reaction liquid obtained in the step (7), and the reaction is carried out for 24 hours in a dark place under the protection of nitrogen. The resulting reaction solution was dialyzed in ultrapure water for 48 hours, followed by freeze-drying to a solid powder.
The solid state characterization is carried out on the quaternary ammonium salt hydroxyethyl starch-curcumin prodrug of the anticancer drug prepared in the embodiment 1, and the solid state characterization is specifically as follows:
the chemical structure of QHES-S-CUR prepared in example 1 was confirmed using Fourier infrared spectroscopy and nuclear magnetic resonance hydrogen spectroscopy. FIGS. 2 and 3 are Fourier infrared spectra and nuclear magnetic resonance hydrogen spectra of QHES-S-CUR prepared in example 1.
As shown in FIG. 2, curcumin is at 1510cm -1 The infrared absorption peak is shown, which is attributed to c=o stretching vibration. Compared with curcumin, QHES-S-CUR is 1731cm -1 A new infrared absorption peak occurs, which is attributed to the stretching vibration of the ester bond c=o, at 1024cm -1 The absorption peak at the site is attributed to C-O-C vibration of QHES at 1514cm -1 The absorbance peak at c=o vibration attributed to curcumin confirmed that curcumin was covalently linked to QHES by an ester linkage.
As shown in FIG. 3, the characteristic peak of the nuclear magnetic resonance hydrogen spectrum of QHES-S-CUR at 3.15ppm was attributed to methyl hydrogen in the quaternary ammonium radical. Meanwhile, a set of characteristic peaks corresponding to hydroxyl hydrogen in QHES are shown at 5.06-5.59 ppm. The characteristic peak at 2.50-2.68ppm is attributed to methylene hydrogen among thiodipropionic acid. Characteristic peaks at 6.60-7.50ppm are ascribed to aromatic hydrogens of curcumin. Fourier infrared spectrum and nuclear magnetic resonance hydrogen spectrum confirm that the curcumin prodrug prepared in example 1 has no error in structure.
For the anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug prepared in example 1, drug release behavior study was performed, specifically as follows:
to determine the release behavior of curcumin prodrugs in simulated tumor microenvironments, two different simulated release vehicles were formulated, respectively, (a) phosphate buffer at pH 7.4; (B) phosphate buffer pH 7.4 containing 10mM hydrogen peroxide. About 5mg of the curcumin prodrug prepared in example 1 was dissolved in 1mL of a release medium and sealed in a dialysis bag having a molecular weight cutoff of 3500 Da. The dialysis bag was immersed in 9mL of release medium and placed in a water bath shaker for drug release (37 ℃,100 rpm). At set time intervals, 1mL of release medium outside the dialysis bag was removed and 1mL of fresh release medium was replenished to maintain the volume of the release system constant. The drug content of the released medium was determined by measuring absorbance at 425nm with an ultraviolet spectrophotometer. Three groups of drug release experiments were performed in parallel for each release medium. The release of the curcumin prodrug in the three media is shown in figure 4.
As shown in fig. 4, the curcumin prodrug showed a faster release behavior in the hydrogen peroxide-containing buffer compared to drug release in the pH 7.4 release medium, accumulating drug release over 72h to 71.50±11.07%. The prepared anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug is proved to have better responsiveness to active oxygen in tumor microenvironment.
In vitro cytotoxicity tests were performed on the anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug prepared in example 1, and the specific steps are as follows:
curcumin and curcumin prodrug in vitro tumor cytotoxicity was determined by CCK-8 method. Spread into 10 wells per 96 well plate 4 The volume of the cell culture medium of colon cancer cells of mice was 100. Mu.L. After the cells are completely attached, each timeThe equivalent curcumin concentration of 5 μg/mL, 10 μg/mL, 25 μg/mL or 50 μg/mL drug solution prepared by adding culture medium into the wells. After incubation for 24h in a carbon dioxide incubator at 37℃respectively, 20. Mu.L of CCK-8 reagent was added to each well, incubation was continued for 4h, and absorbance at 450nm was measured using a microplate reader.
As shown in fig. 5, the anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug shows a remarkable inhibition effect on colon cancer cells of mice. When the equivalent concentration of curcumin in the quaternary ammonium salt hydroxyethyl starch-curcumin prodrug of the anticancer drug is 50 mug/mL, the activity of colon cancer cells is only 20.78+/-4.04% after 24 hours of incubation. In-vitro tumor cytotoxicity experiments prove that the quaternary ammonium salt hydroxyethyl starch-curcumin prodrug of the anticancer drug has good tumor inhibition effect.
For the anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug prepared in example 1, endocytosis condition research is performed, and the specific steps are as follows:
collecting colon cancer cells of mice in logarithmic phase, making into cell suspension, spreading on 24-well plate, adding 1×10 cells into each well 5 Cell suspensions of individual cells (1 mL). After incubation in a carbon dioxide incubator until the cells are completely adherent, coumarin-6 labeled QHES-S-CUR prodrug micelle solution (curcumin equivalent concentration 10. Mu.g/mL) was added to each well. After incubation for 0.5, 1, 2 or 6h, the culture medium in each well was discarded and washed 3 times with phosphate buffer solution. Endocytosis of the prodrug micelles was observed with an inverted fluorescence microscope.
As shown in fig. 6, the fluorescence intensity in the cell matrix was significantly enhanced and the fluorescence distribution in the cell matrix was widened with the extension of the incubation time. After the incubation time is prolonged to 2h and 6h, fluorescence distribution exists in the whole cell, which indicates that QHES-S-CUR can be endocytosed into the cell by CT-26 cells in a shorter time and gradually transported into the cell from the edge of the cell membrane.
Aiming at the anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug prepared in the embodiment 1, subcellular distribution condition research is carried out, and the specific steps are as follows:
collecting CT-26 cells in logarithmic growth phase, making into cell suspension, and spreading on 1In a 2-well plate, a plate containing 2X 10 is added to each well 5 Cell suspensions of individual cells (1 mL). After the cells are attached, DMEM medium (curcumin equivalent concentration is 5 mug/mL) containing the C-6 labeled QHES-S-CUR prodrug micelle is added into each hole, and the cells are incubated for 0.5 or 2 hours. Subsequently, the mitochondrial fluorescent dye MitoTracker Red was added, incubated for 0.5h, and the mitochondria of CT-26 were labeled as Red fluorescence. The medium containing the fluorescent dye was discarded, and after washing the cells 3 times with PBS, subcellular distribution of the QHES-S-CUR prodrug micelles was observed with an inverted fluorescence microscope. The excitation and emission wavelengths of C-6 were 497nm and 523nm,MitoTracker Red nm, respectively, and 579nm and 599nm, respectively.
As shown in FIG. 7, the C-6 labeled QHES-S-CUR exhibited green fluorescence, and the MitoTracker Red labeled mitochondria exhibited Red fluorescence, which when overlapped exhibited yellow fluorescence. When the incubation time of the prodrug micelle and the cells is 0.5h, a small amount of yellow fluorescence distribution exists in CT-26 cells, which indicates that partial QHES-S-CUR prodrug micelle is delivered into mitochondria of the cells in a targeted manner. After the incubation time is prolonged to 2 hours, the cells show stronger yellow fluorescence, and the fluorescence of QHES-S-CUR and the mitochondrial fluorescence are overlapped greatly. Along with the extension of the action time, after QHES-S-CUR enters the cell, electrostatic interaction can be generated with electronegative mitochondrial membranes under the action of surface quaternary ammonium salt, so that prodrug micelle targeting enters mitochondria is promoted.
For the anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug prepared in example 1, three-dimensional tumor sphere penetration condition research is carried out, and the specific steps are as follows:
after incubating the C-6 labeled QHES-S-CUR prodrug micelle solution with the tumor spheres for 3h, the medium was discarded. After 3 washes with PBS, tumor balls were transferred to confocal dishes and observed under a laser confocal microscope.
As shown in FIG. 8, after the QHES-S-CUR acts on the tumor sphere for 3 hours, the tumor sphere has stronger green fluorescence, a slice at the center presents a circle of thicker aperture, and the inner core also has certain fluorescence distribution, which indicates that the QHES-S-CUR can be swallowed by cells and transported into the tumor sphere in a shorter time.
According to the invention, quaternary ammonium salt hydroxyethyl starch is used as a polymer skeleton, and hydrophobic curcumin and hydrophilic quaternary ammonium salt hydroxyethyl starch are coupled through thiodipropionic acid to prepare the curcumin prodrug capable of self-assembling to form nano micelle. The curcumin prodrug can be prepared through two-step esterification reaction, and the preparation process is simple. The obtained curcumin prodrug can self-assemble in aqueous solution to form nano micelle, and solves the problems of poor solubility and easy crystallization precipitation of curcumin in water. Under the action of ROS, thioether bonds in curcumin prodrug micelle can be rapidly broken and curcumin is released, so that the drug is released in response to the tumor part. Meanwhile, under the action of surface quaternary ammonium root, curcumin prodrug micelle can be rapidly phagocytized by tumor cells and has mitochondrial targeting delivery capability. The curcumin prodrug micelle designed by the invention can obviously improve the anti-tumor effect of curcumin and has good application prospect.
The above embodiment is only a preferred embodiment of the present invention, but it is not intended to limit the present invention. Various changes and modifications may be made by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present invention. Therefore, all the technical schemes obtained by adopting the equivalent substitution or equivalent transformation are within the protection scope of the invention.
Claims (7)
1. An anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug characterized in that it has a chemical structure of formula (I):
wherein R is H or CH 2 CH 2 OH。
2. The preparation method of the quaternary ammonium salt hydroxyethyl starch-curcumin prodrug of the anticancer drug is characterized by comprising the following steps:
s1: dissolving hydroxyethyl starch in a sodium hydroxide aqueous solution, and reacting with 2, 3-epoxypropyl trimethyl ammonium chloride under heating to generate quaternary ammonium salt hydroxyethyl starch with a chemical structure shown in a formula (II);
the formula (II) is specifically as follows:
wherein R is H or CH 2 CH 2 OH；
S2: the generated quaternary ammonium salt hydroxyethyl starch and thiodipropionic acid are catalyzed by 4-dimethylaminopyridine and dicyclohexylcarbodiimide to generate quaternary ammonium salt hydroxyethyl starch-thiodipropionic acid monoester with a chemical structure shown in a formula (III);
the formula (III) is specifically as follows:
wherein R is H or CH 2 CH 2 OH；
S3: in the presence of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole, carrying out esterification reaction on curcumin and the quaternary ammonium salt hydroxyethyl starch-thiodipropionic monoester generated in the step S2 to generate an anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug with a chemical structure shown in a formula (I);
the formula (I) is specifically as follows:
wherein R is H or CH 2 CH 2 OH。
3. The method for preparing a quaternary ammonium salt hydroxyethyl starch-curcumin prodrug as claimed in claim 2, wherein the molecular weight of the hydroxyethyl starch is 130kDa and the substitution degree is 0.4.
4. The method for preparing the quaternary ammonium salt hydroxyethyl starch-curcumin prodrug as an anticancer drug according to claim 2, wherein the step S1 is specifically as follows:
s11: 7.2g of hydroxyethyl starch is dissolved in 45mL of sodium hydroxide aqueous solution, and the mixture is heated and reacted for 0.5 to 2 hours at the temperature of 45 ℃; the mass concentration of the sodium hydroxide aqueous solution is 0.05-0.1%;
s12: dropwise adding 5.28mL of 2, 3-epoxypropyl trimethyl ammonium chloride aqueous solution into the reaction solution obtained in the step S11, and heating at 45 ℃ for reaction for 24 hours; the mass concentration of the 2, 3-epoxypropyl trimethyl ammonium chloride aqueous solution is 15-30%;
s13: regulating the pH value of the reaction solution obtained in the step S12 to 7 by glacial acetic acid, precipitating with alcohol, and collecting precipitate; redissolving the precipitate in water, and dialyzing with ultrapure water for 24-72 h; and then freezing the obtained dialysate for 24 hours at the temperature of minus 80 ℃, and freeze-drying to obtain solid powder, thus obtaining the quaternary ammonium salt hydroxyethyl starch with the chemical structure of formula (II).
5. The method for preparing a quaternary ammonium salt hydroxyethyl starch-curcumin prodrug as claimed in claim 4, wherein the alcohol precipitation is performed by absolute ethanol.
6. The method for preparing the quaternary ammonium salt hydroxyethyl starch-curcumin prodrug as an anticancer drug according to claim 2, wherein the step S2 is specifically as follows:
s21: 1-2 g of thiodipropionic acid and 0.1-0.3 g of 4-dimethylaminopyridine are dissolved in 15mL of dimethyl sulfoxide to obtain a first solution; dissolving 0.5-1.5 g of dicyclohexylcarbodiimide in dimethyl sulfoxide to obtain a second solution; dropwise adding the second solution into the first solution, and reacting for 0.5-2 h at room temperature;
s22: adding 1g of quaternary ammonium salt hydroxyethyl starch into the reaction liquid obtained in the step S21, and reacting for 48 hours at room temperature under the protection of nitrogen;
s23: and (2) dialyzing the reaction solution obtained in the step (S22) in dimethyl sulfoxide for 48 hours, wherein the obtained dialysate is the quaternary ammonium salt hydroxyethyl starch-thiodipropionic monoester with the chemical structure shown in the formula (III).
7. The method for preparing the quaternary ammonium salt hydroxyethyl starch-curcumin prodrug as an anticancer drug according to claim 2, wherein the step S3 is specifically as follows:
s31: 0.2 to 0.4g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 0.1 to 0.2g of 1-hydroxybenzotriazole are dissolved in a quaternary ammonium salt hydroxyethyl starch-thiodipropionic monoester water solution together, and react for 0.5 to 2 hours at room temperature;
s32: adding 0.03-0.25 g of curcumin into the reaction liquid obtained in the step S31, and carrying out light-shielding reaction for 24-48 h under the protection of nitrogen; and then dialyzing the obtained reaction liquid in ultrapure water for 48 hours, and then freeze-drying the reaction liquid into solid powder to obtain the anticancer drug quaternary ammonium salt hydroxyethyl starch-curcumin prodrug with the chemical structure shown in the formula (I).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310748028.2A CN116854831A (en) | 2023-06-25 | 2023-06-25 | Quaternary ammonium salt hydroxyethyl starch-curcumin prodrug as anticancer medicine and its prepn process |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310748028.2A CN116854831A (en) | 2023-06-25 | 2023-06-25 | Quaternary ammonium salt hydroxyethyl starch-curcumin prodrug as anticancer medicine and its prepn process |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116854831A true CN116854831A (en) | 2023-10-10 |
Family
ID=88229578
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310748028.2A Pending CN116854831A (en) | 2023-06-25 | 2023-06-25 | Quaternary ammonium salt hydroxyethyl starch-curcumin prodrug as anticancer medicine and its prepn process |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116854831A (en) |
-
2023
- 2023-06-25 CN CN202310748028.2A patent/CN116854831A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9999678B2 (en) | C6-C18-acylated derivative of hyaluronic acid and method of preparation thereof | |
| Liu et al. | Hyperbranched polyphosphates: synthesis, functionalization and biomedical applications | |
| CN107669632B (en) | Drug carrier, micelle, drug preparation, preparation method and application thereof | |
| Hu et al. | Targeting and anti-tumor effect of folic acid-labeled polymer–doxorubicin conjugates with pH-sensitive hydrazone linker | |
| Cui et al. | “Stealth” dendrimers with encapsulation of indocyanine green for photothermal and photodynamic therapy of cancer | |
| CN103611165B (en) | Hyaluronic acid-cyclodextrin-diamantane (obsolete) polyethylene glycol carrier and its preparation method and application | |
| CN111870579B (en) | Tumor-targeted nano micelle, preparation method and application of nano micelle as drug carrier | |
| Wang et al. | Diselenide-crosslinked carboxymethyl chitosan nanoparticles for doxorubicin delivery: preparation and in vivo evaluation | |
| Zhou et al. | One-pot synthesis of acid-degradable polyphosphazene prodrugs for efficient tumor chemotherapy | |
| CN111053911A (en) | Reduction response type cross-linking agent and preparation and application of cross-linked hydroxyl drug molecule thereof | |
| Shirani et al. | Redox responsive polymeric micelles of gellan gum/abietic acid for targeted delivery of ribociclib | |
| CN113321812B (en) | Polylactic acid-hydroxyethyl starch-folic acid macromolecular compound, drug delivery system, preparation method and application thereof | |
| CN113633785A (en) | Preparation method and application of intelligent responsive shell-core polyelectrolyte nanogel | |
| CN116854831A (en) | Quaternary ammonium salt hydroxyethyl starch-curcumin prodrug as anticancer medicine and its prepn process | |
| KR101323102B1 (en) | Nanoparticles formed by encapsulating an anticancer drug into glycolchitosan-cholanic acid complex and a process for the preparation thereof | |
| CN104758244A (en) | Nanogel, preparation method of nanogel and anti-tumor nanogel drug loading system and preparation method of anti-tumor nanogel drug loading system | |
| CN112386705A (en) | Gold nanoparticle based on hyaluronic acid modification, preparation method thereof and application of gold nanoparticle as nano-drug carrier | |
| CN113912841A (en) | PH and Redox dual-response diblock amphiphilic polymer prodrug and preparation method thereof | |
| CN108721636B (en) | Drug delivery material with dual responsiveness connected by diselenide bond and preparation method and application thereof | |
| CN116726196A (en) | Amphiphilic curcumin prodrug micelle and preparation method thereof | |
| CN116874621A (en) | Oxidation/pH dual-response curcumin prodrug and preparation method thereof | |
| CN102552930B (en) | Water-soluble paclitaxel derivative with cell targeting effect and preparation thereof | |
| CN110339368B (en) | Preparation method of reduction-responsive targeting polyethylene glycol-polycarbonate maytansine prodrug micelle | |
| CN115590879B (en) | Platinum drug lipid derivative and application thereof | |
| CN114870031B (en) | CD44 targeted taxane nanocrystal and preparation method and application thereof |
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 |