WO2021017335A1 - 一种黄原胶共聚物纳米胶束的制备方法及其应用 - Google Patents

一种黄原胶共聚物纳米胶束的制备方法及其应用 Download PDF

Info

Publication number
WO2021017335A1
WO2021017335A1 PCT/CN2019/120773 CN2019120773W WO2021017335A1 WO 2021017335 A1 WO2021017335 A1 WO 2021017335A1 CN 2019120773 W CN2019120773 W CN 2019120773W WO 2021017335 A1 WO2021017335 A1 WO 2021017335A1
Authority
WO
WIPO (PCT)
Prior art keywords
xanthan gum
copolymer
nanomicelles
gum copolymer
preparing
Prior art date
Application number
PCT/CN2019/120773
Other languages
English (en)
French (fr)
Inventor
张丽萍
倪才华
刘仁
王刚
Original Assignee
江南大学
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by 江南大学 filed Critical 江南大学
Priority to US16/954,734 priority Critical patent/US11623968B2/en
Publication of WO2021017335A1 publication Critical patent/WO2021017335A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/723—Xanthans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0033—Xanthan, i.e. D-glucose, D-mannose and D-glucuronic acid units, saubstituted with acetate and pyruvate, with a main chain of (beta-1,4)-D-glucose units; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F251/00—Macromolecular compounds obtained by polymerising monomers on to polysaccharides or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the invention relates to a preparation method and application of xanthan gum copolymer nano micelles, and belongs to the technical field of medical polymer material synthesis.
  • Xanthan gum is an extracellular water-soluble polysaccharide secreted by Xanthomonas campestris. Because of its good biocompatibility and biodegradability, it is widely used in the fields of industry, agriculture, medicine, daily chemical industry and so on. In the medical field, xanthan gum is mainly used as a binder, disintegrant, sustained-release agent and controlled-release agent. It is a component of microcapsule drug capsules and plays an important role in controlling drug release. Xanthan gum has suitable viscosity and swelling coefficient, which can prolong the gastric retention time of food to reduce blood lipids. Therefore, it is widely used in oral drug carriers and bio-scaffold gels.
  • Nanoparticles can target specific tissues.
  • Xanthan gum nanomicelles can prolong its circulation time in the body and reduce the capture of macrophages, so that nanoparticles not only have the general characteristics of xanthan gum, but also have surface and interface effects, etc.
  • the general characteristics of nanomaterials are of great significance in the field of high-efficiency, low-toxicity, and controlled drug release.
  • xanthan gum itself has good chemical properties, it still has certain defects when used as an anti-cancer drug carrier.
  • the molecular weight of xanthan gum is too large, the viscosity is too high in aqueous solution, and the modification reaction is difficult.
  • the present invention provides a method for preparing xanthan gum copolymer nanomicelles, which includes the following steps:
  • Step 1 Pretreatment: degrading the xanthan gum with a concentration of 0.5w% to reduce the dynamic viscosity to 110mPa ⁇ s, and then drying, crushing, and sieving to obtain the degraded xanthan gum;
  • Step 2 Preparation of xanthan gum bromide: the xanthan gum product degraded in step 1 is dissolved in N,N-dimethylformamide, and 2-bromoisobutyryl bromide is preliminarily prepared under ice bath and stirring conditions. Dissolved in N,N-dimethylformamide, then added to the above xanthan gum solution, then added triethylamine, reacted at room temperature for 48h, the reaction product was precipitated with ether and filtered, dried at a constant temperature of 30 °C to obtain xanthan gum bromine Substitute
  • Step 3 Preparation of xanthan gum copolymer: dissolve the xanthan gum bromide obtained in step 2 in N,N-dimethylformamide, add diacetone acrylamide, vacuum-fill nitrogen for three cycles, and then add three [2 -(Dimethylamino)ethyl]amine (ME 6 TREN) react with cuprous chloride, the reaction temperature is 55 ⁇ 65°C, the reaction time is 3 ⁇ 5h, the product is precipitated with ether and filtered, and then ether is used Wash the product three times, filter and dry the product at a constant temperature of 30°C to obtain a graft copolymer of xanthan gum and polydiacetone acrylamide, referred to as xanthan gum copolymer; step 4, prepare xanthan gum copolymer nanomicelles: Dissolve the xanthan gum copolymer in step 3 in a polar organic solvent, filter with a 0.4 ⁇ m microporous filter membrane to obtain a polymer mother liquor, and
  • the mass concentration of the xanthan gum in N,N-dimethylformamide is 1.8% to 2.5%; 2-bromoisobutyryl bromide is in the reaction system The mass concentration in the medium is 1.5% to 2.2%; the weight of the added triethylamine is 40% to 60% of the 2-bromoisobutyryl bromide.
  • the mass of diacetone acrylamide is 1 to 3 times of the bromide of xanthan gum; tris[2-(dimethylamino)ethyl]amine and cuprous chloride
  • the molar ratio of is 1:1.5-2.0; preferably, the reaction temperature is 60°C, and the reaction time is 4.5h.
  • the polar organic solvent in step 4 includes absolute ethanol, isopropanol, tetrahydrofuran, N,N-dimethylformamide, dioxane or dimethyl sulfoxide.
  • Another object of the present invention is to provide an application method of xanthan gum copolymer nanomicelles, the xanthan gum copolymer nanomicelles are freeze-dried into a powder, and then the anticancer drug is formulated with methanol to a concentration of 2 mg /mL solution, add 30mg nanomicelle powder to 8mL methanol solution of anticancer drugs, stir for 5 hours and transfer to dialysis bag, the molecular weight cut-off of dialysis bag is 3500, dialyze in ultrapure water for 18 hours to obtain the load Nanoparticles of anticancer drugs.
  • the anticancer drug includes paclitaxel or 10-hydroxycamptothecin.
  • the living radical polymerization reaction is carried out through clever design. Diacetone acrylamide is grafted and polymerized on xanthan gum. The grafting rate is controllable, the particle size of the nanomicelles is controllable, and the micelles have good regularity.
  • Xanthan gum copolymer nanomicelles have good biocompatibility and are used as anti-cancer drug carriers with stable performance, non-toxicity and low price.
  • Figure 1 Road map of a method for preparing xanthan gum copolymer nanomicelles of the present invention
  • Xanthan gum pretreatment Weigh 1g of xanthan gum and add it to 200mL of distilled water, stir to dissolve completely, prepare a solution with a mass concentration of 0.5w%, and place it in an ultrasonic environment with a power of 200W and an ultrasonic frequency of 20kHz. Degradation for 2.5h; in the degradation process of xanthan gum, use the NDJ-99 rotary viscometer to measure the dynamic viscosity of the modified xanthan gum solution, put the sample solution into a flat-bottom centrifuge tube, select the S05 rotor, and set the rotary viscometer Measure the dynamic viscosity under different conditions. The viscosity measurement conditions are: 25°C, 60r/min.
  • the initial dynamic viscosity of xanthan gum at a concentration of 0.5w% was 490mPa ⁇ s, which dropped to 110mPa ⁇ s after 2.5 hours of ultrasound. Then the product is precipitated with ethanol, then dried at a constant temperature of 30°C, crushed, and sieved with a 100-mesh sieve;
  • xanthan gum copolymer a Redissolve 2 g of the above bromide of xanthan gum in 50 mL of N,N-dimethylformamide, add 2.0 g of diacetone acrylamide (DAA), evacuate and circulate nitrogen for three times, and then add three [2-( ⁇ 0.69 g of methylamino)ethyl]amine and 0.594 g of cuprous chloride were reacted at 60°C for 4.5h. The product was precipitated with ether and filtered, and the product was washed with ether 3 times, filtered and dried at a constant temperature of 30°C. A graft copolymer of xanthan gum and polydiacetone acrylamide is obtained, referred to as xanthan gum copolymer a;
  • the above-mentioned xanthan gum copolymer was prepared into a N,N-dimethylformamide solution with an initial mass fraction of 1%, and filtered with a microporous filter membrane with a pore size of 0.4 ⁇ m to obtain a polymer mother liquor. Ultrapure water is added dropwise to the mother liquor until a micellar solution is formed. The micellar solution is dialyzed in deionized water for 4 days, and the deionized water is changed every 8 hours to obtain xanthan gum copolymer nanomicelle a1.
  • Steps (1), (2) and (4) are the same as in Example 1.
  • step (3) the dosage of diacetone acrylamide is changed to 3 grams to obtain xanthan gum copolymer b.
  • the other methods are the same to obtain xanthan gum copolymer b.
  • Steps (1), (2) and (4) are the same as in Example 1.
  • step (3) the amount of diacetone acrylamide is changed to 4 grams to obtain xanthan gum copolymer c.
  • the other methods are the same to obtain xanthan gum copolymer c.
  • Steps (1), (2) and (4) are the same as in Example 1.
  • step (3) the amount of diacetone acrylamide is changed to 6 grams to obtain xanthan gum copolymer d.
  • the other methods are the same to obtain xanthan gum.
  • xanthan gum copolymers a, b, c, and d prepared in Examples 1 to 4 were dissolved in a tetrahydrofuran solution to prepare a 0.5% uniform solution, and then filtered with a filter membrane with a pore size of 0.4 ⁇ m, using tetrahydrofuran as The mobile phase sends the sample to the instrument for testing.
  • the molecular weight of xanthan gum copolymer was measured by Waters 1525EF high performance liquid chromatograph.
  • Table 1 showed that as the amount of diacetone acrylamide increased, the number average molecular weight increased linearly, and the molecular weight distribution index was less than 2. This proves that the graft polymerization of diacetone acrylamide and xanthan gum belongs to living polymerization, and the molecular weight of the copolymer can be adjusted by controlling the amount of diacetone acrylamide.
  • the zeta potential of the xanthan gum copolymer nanomicelles decreased with the increase of diacetone acrylamide content, because the increase in diacetone acrylamide content caused the relative decrease of negatively charged components in the copolymer, because of the generation of Zeta
  • the potential factor is the carboxyl anion in the xanthan gum molecule.
  • the xanthan gum copolymer nanomicelles into a 0.1wt% aqueous solution, dialyzed for 72h, use deionized water as the dialysis external fluid, change the dialysis external fluid every 3h to remove impurities; then, the nanomicelle solution Drop onto a silicon wafer, dry at 40°C, and spray gold to obtain a sample of xanthan gum nanomicelles.
  • the morphology of the nanomicelles is observed by S-4800 field emission scanning electron microscope. It can be seen from Figure 3 that the morphology of the nanomicelles is in a regular spherical shape and the distribution is relatively uniform.
  • the xanthan gum copolymer nanomicelles a1, b1, c1, and d1 prepared in Examples 1 to 4 were freeze-dried into powder, and then the anticancer drug was formulated into a solution with a concentration of 2 mg/mL in methanol, and 30 mg of nano gel
  • the bundle powder was added to 8 mL of the methanol solution of anti-cancer drugs, stirred for 5 hours, and then transferred to a dialysis bag.
  • the molecular weight cut-off of the dialysis bag was 3500. It was dialyzed in ultrapure water for 18 hours to obtain anti-cancer drug-loaded nanoparticles.
  • Anticancer drugs include paclitaxel or 10-hydroxycamptothecin. From the results in Table 3, it can be seen that the drug loading rate of nanomicelles increases with the increase of diacetone acrylamide content.
  • RPMI-1640 complete medium containing 1.0 mg/mL nanomicelles
  • the positive control group is RPMI-1640 complete medium containing 0.64% phenol by mass
  • negative The control group was RPMI-1640 complete medium, each group set 4 multiple holes in parallel, and continued to place them in the above-mentioned incubator to culture at 37°C, and use an inverted biological microscope to observe the cell growth morphology at different times.
  • a 1 represents the absorbance at 570 nm of the experimental group
  • a 2 represents the absorbance at 570 nm of the negative control group.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Materials Engineering (AREA)
  • Dispersion Chemistry (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Emergency Medicine (AREA)
  • Physics & Mathematics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Nanotechnology (AREA)
  • Optics & Photonics (AREA)
  • Inorganic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)

Abstract

一种黄原胶共聚物纳米胶束的制备方法，包含以下步骤：将浓度为0.5w％黄原胶经过降解使得动力粘度下降到110mPa•s，然后干燥、粉碎、过筛后溶于N,N-二甲基甲酰胺中，并加入2-溴代异丁酰溴的N,N-二甲基甲酰胺溶液，再添加少量三乙胺进行反应，以沉淀法得到黄原胶溴代物后溶解于N,N-二甲基甲酰胺，再加入双丙酮丙烯酰胺，无氧条件下，加入三[2-(二甲基氨基)乙基]胺和氯化亚铜进行反应，以沉淀法得到黄原胶共聚物并溶于极性有机溶剂中得到共聚物母液，搅拌下缓慢向共聚物母液中滴加水，直到形成胶束溶液，经透析得到黄原胶共聚物纳米胶束。所得的黄原胶共聚物纳米胶束形态规整性良好，生物相容性好，用作抗癌药物载体性能稳定。

Description

一种黄原胶共聚物纳米胶束的制备方法及其应用 技术领域
本发明涉及一种黄原胶共聚物纳米胶束的制备方法及其应用，属于医用高分子材料合成技术领域。
背景技术
黄原胶是一种由野油菜黄单胞菌分泌的胞外水溶性多糖，因其具有良好的生物相容性和生物降解性，在工农业、医药、日用化工等领域有广泛应用。在医用领域，黄原胶主要作为粘合剂、崩解剂、缓释剂和控释剂，是微胶囊药物囊材中的组分，在控制药物释放方面起重要作用。黄原胶具有适宜的粘度和溶胀系数，可延长食物胃滞留时间以降低血脂。因而被大量应用于口服药物载体和生物支架凝胶。
纳米粒子对特定组织具有靶向性，黄原胶纳米胶束可以延长其在体内循环时间并减少巨噬细胞的捕获，使纳米粒子不仅具有黄原胶的一般特性，还具有表面与界面效应等纳米材料的普遍特征，在高效、低毒、控制药物缓释研究领域具有重大意义。
黄原胶自身虽具有良好的化学特性，但是用作抗癌药物载体上还存在一定的缺陷，黄原胶的分子量太大，在水溶液中粘度太高，改性反应困难。黄原胶疏水改性已经有一些报道，例如通过氯代十六烷与黄原胶的反应，使黄原胶连结部分疏水性基团，或者利用邻苯二甲酸酐的开环反应，将疏水性的苯基与黄原胶相连，但是改性程度不易控制，所得纳米粒子分布较宽。因此目前，需要对研究一种以黄原胶制备的纳米胶束具有规整性良好的形态，用于制备抗癌药物载体，同时还需要研究一种便于控制改性过程、易于操作的制备方法。
发明内容
为了克服上述问题，本发明提供一种黄原胶共聚物纳米胶束的制备方法，包含以下步骤：
步骤1，预处理：将浓度为0.5w％黄原胶经过降解使得动力粘度下降到110mPa·s，然后干燥、粉碎、过筛得到降解后的黄原胶；
步骤2，制备黄原胶溴代物：经步骤1降解后的黄原胶产物溶于N,N-二甲基甲酰胺中，在冰浴和搅拌条件下预先将2-溴代异丁酰溴溶于N,N-二甲基甲酰胺中，然后加入到上述黄原胶溶液中，再添加三乙胺，室温反应48h，反应产物用*** 沉淀并过滤，恒温30℃干燥得到黄原胶溴代物；
步骤3，制备黄原胶共聚物：将步骤2所得黄原胶溴代物溶解于N,N-二甲基甲酰胺，加入双丙酮丙烯酰胺，抽真空-充氮气循环三次，接着加入三[2-(二甲基氨基)乙基]胺(ME 6TREN)和氯化亚铜进行反应，反应温度为55～65℃，反应的时间为3～5h，产物用***沉淀并过滤，再用***洗涤产物3次，过滤并恒温30℃干燥产物，得到黄原胶与聚双丙酮丙烯酰胺的接枝共聚物，简称为黄原胶共聚物；步骤4，制备黄原胶共聚物纳米胶束：将步骤3中黄原胶共聚物溶解在极性有机溶剂中，用孔径为0.4μm的微孔过滤膜过滤，得聚合物母液，搅拌下缓慢向聚合物母液中滴加超纯水，直到形成胶束溶液，将胶束溶液在去离子水中透析4天，隔8个小时换一次去离子水，得到黄原胶共聚物纳米胶束。
在一种实施例中，所述步骤2中，所述黄原胶在N,N-二甲基甲酰胺中的质量浓度为1.8％～2.5％；2-溴代异丁酰溴在反应体系中的质量浓度为1.5％～2.2％；所加三乙胺的重量是2-溴代异丁酰溴的40％～60％。
在一种实施例中，所述步骤3中，双丙酮丙烯酰胺的质量是黄原胶溴代物的1～3倍；三[2-(二甲基氨基)乙基]胺与氯化亚铜的摩尔比1:1.5～2.0；优选地，所述反应温度为60℃，反应时间为4.5h。
在一种实施例中，步骤4中所述极性有机溶剂包括无水乙醇、异丙醇、四氢呋喃、N,N-二甲基甲酰胺、二氧六环或二甲亚砜。
本发明的另一个目的在于提供一种黄原胶共聚物纳米胶束的应用方法，将所述黄原胶共聚物纳米胶束冷冻干燥成粉末,再将抗癌药物用甲醇配成浓度为2mg/mL的溶液，将30mg纳米胶束粉末加入到8mL抗癌药物的甲醇溶液中，搅拌5小时后转移到透析袋，透析袋的截留分子量为3500，在超纯水中透析18小时，得到负载抗癌药物的纳米颗粒。
在一种实施例中，所述抗癌药物包括紫杉醇或10-羟基喜树碱。
本发明的有益效果：
1.通过巧妙的设计进行活性自由基聚合反应，双丙酮丙烯酰胺在黄原胶上接枝聚合，接枝率可控，纳米胶束的粒径可控，胶束形态规整性良好。
2.黄原胶共聚物纳米胶束有良好的生物相容性，用作抗癌药物载体，性能稳定、无毒性、价格低廉。
附图说明
图1本发明的一种黄原胶共聚物纳米胶束的制备方法路线图
图2黄原胶共聚物的红外光谱图，其中a、b、c、d分别对应实施例1～4中的黄原胶共聚物
图3本发明实施例1所得黄原胶共聚物纳米胶束a1的扫描电镜图
具体实施方式
实施例1
(1)黄原胶预处理：称取1g黄原胶加入到200mL蒸馏水中，搅拌完全溶解，配成质量浓度为0.5w％的溶液，置于功率为200W、超声频率20kHz的超声环境中进行降解2.5h；在黄原胶降解过程中，使用NDJ-99型旋转粘度仪测量改性黄原胶溶液的动力粘度，将样品溶液装入平底离心管，选用S05型转子，设定旋转粘度仪的参数，测量不同条件下的动力粘度，粘度测量条件为：25℃，60r/min。浓度为0.5w％黄原胶的初始动力粘度为490mPa.s,经过2.5小时超声后下降到110mPa·s。然后将产物用乙醇沉淀下来，再经过30℃恒温干燥、粉碎、用100目筛子过筛；
(2)黄原胶溴代物的制备：在250mL三口烧瓶中加入上述预处理后的黄原胶2g，再加入N,N-二甲基甲酰胺(代号：DMF)100mL，在冰浴和搅拌条件下预先将2.3克2-溴代异丁酰溴溶于10mL N,N-二甲基甲酰胺中,然后加入到上述黄原胶溶液中，再添加1.2克三乙胺(代号：TEA)，再室温反应48h，反应产物用***沉淀并过滤，恒温30℃干燥得到黄原胶溴代物；
(3)黄原胶接枝共聚物的制备：
将上述黄原胶溴代物2克重新溶解于N,N-二甲基甲酰胺50mL，加入2.0克双丙酮丙烯酰胺(DAA)，抽真空-充氮气循环三次，接着加入三[2-(二甲基氨基)乙基]胺0.69克和氯化亚铜0.594克，在60℃温度下反应4.5h，产物用***沉淀并过滤，再用***洗涤产物3次，过滤并恒温30℃干燥产物，得到黄原胶与聚双丙酮丙烯酰胺的接枝共聚物，简称为黄原胶共聚物a；
(4)黄原胶共聚物纳米胶束的制备：
将上述黄原胶共聚物配成初始质量分数为1％的N,N-二甲基甲酰胺溶液，用孔径为0.4μm的微孔过滤膜过滤，得聚合物母液，搅拌下缓慢向聚合物母液中滴加超纯水，直到形成胶束溶液，将胶束溶液在去离子水中透析4天，隔8个小时换一次去离子水，得到黄原胶共聚物纳米胶束a1。
黄原胶共聚物纳米胶束a1的扫描电镜图如图3所示。
实施例2
步骤(1)、(2)和(4)同实施例1，在步骤(3)中，改变双丙酮丙烯酰胺的用量为3克，得到黄原胶共聚物b，其余方法相同，得到黄原胶共聚物纳米胶束b1。
实施例3
步骤(1)、(2)和(4)同实施例1，在步骤(3)中，改变双丙酮丙烯酰胺的用量为4克，得到黄原胶共聚物c，其余方法相同，得到黄原胶共聚物纳米胶束c1。
实施例4
步骤(1)、(2)和(4)同实施例1，在步骤(3)中，改变双丙酮丙烯酰胺的用量为6克，得到黄原胶共聚物d，其余方法相同，得到黄原胶共聚物纳米胶束d1。
实施例5 黄原胶共聚物的红外光谱
将实施例1～4中所得黄原胶共聚物a、b、c、d样品干燥，取少许样品和适量KBr固体粉末于玛瑙研钵中，在加热灯下研磨至细粉末并混合均匀；然后压成半透明状薄片，将压片放入傅立叶变换近红外光谱仪上，分别测试4个样品的红外光谱，仪器采用WQF-600N型傅立叶变换红外光谱仪。从附图2看到：在3300cm -1到3500cm -1之间大而宽的吸收峰，显然是黄原胶分子中的羟基和羧基的吸收峰重叠；在1660cm -1为双丙酮丙烯酰胺中酰胺键I区的C＝O吸收峰，在1500cm -1～1560cm -1是酰胺II区的吸收峰；2900cm -1～2950cm -1是甲基、亚甲基的伸缩振动峰。由此说明双丙酮丙烯酰胺接枝聚合到黄原胶上。
实施例6 黄原胶共聚物分子量测定
取实施例1～4制得的黄原胶共聚物a、b、c、d样品分别溶于四氢呋喃溶液中，配置成0.5％均匀溶液，然后用孔径为0.4μm的滤膜过滤，以四氢呋喃为流动相，将样品送入仪器检测。采用Waters 1525EF型高效液相色谱仪测定黄原胶共聚物的分子量，测定结果(表1)表明，随着双丙酮丙烯酰胺用量的增加，数均分子量呈线性升高，分子量分布指数小于2。由此证明双丙酮丙烯酰胺与黄原胶的接枝聚合属于活性聚合，可以过控制双丙酮丙烯酰胺的用量，对共聚物的分子量进行调节。
表1 黄原胶共聚物的制备及其分子量
Figure PCTCN2019120773-appb-000001
实施例7 黄原胶共聚物纳米胶束的Zeta电位测试
以实施例1～4制得的黄原胶共聚物纳米胶束a1、b1、c1、d1配置溶液，用0.1M的氢氧化钠和0.1M盐酸调节纳米胶束溶液的pH值到7.4，采用ZetaPALS型Zeta电位及纳米粒度分析仪对纳米胶束的粒径和Zeta电位进行测定，测试温度为25℃。结果见表2，由此观察到：随着双丙酮丙烯酰胺用量的增加，纳米胶束的粒径增大，这是因为当聚双丙酮丙烯酰胺在共聚物中的含量增加时，疏水性成分增加，更加容易团聚成较大的颗粒。同时发现：黄原胶共聚物纳米胶束的Zeta电位随着双丙酮丙烯酰胺含量增加而下降，是因为随着双丙酮丙烯酰胺含量增加导致共聚物中负电荷组分的相对减少，因为产生Zeta电位的因素是黄原胶分子中的羧基负离子。
表2 黄原胶共聚物纳米胶束的性能
样品编号 实施例 直径(nm) Zeta电位(mV)
a1 1 281 -27.7
b1 2 298 -26.2
c1 3 325 -25.8
d1 4 350 -23.6
实施例8 黄原胶共聚物纳米胶束的形貌观察
首先，将黄原胶共聚物纳米胶束配成0.1wt％的水溶液，透析72h，以去离子水为透析外液，每隔3h换一次透析外液，去除杂质；然后，将纳米胶束溶液滴在硅片上，40℃下烘干，喷金后得到黄原胶纳米胶束样品.采用S-4800型场发射扫描电子显微镜观察纳米胶束的形貌。从附图3看到：纳米胶束的形貌呈规整球型，分布比较均一。
实施例9 载药黄原胶共聚物纳米胶束的制备
将实施例1～4制得的黄原胶共聚物纳米胶束a1、b1、c1、d1冷冻干燥成粉末,再将抗癌药物用甲醇配成浓度为2mg/mL的溶液，将30mg纳米胶束粉末加入到8mL抗癌药物的甲醇溶液中，搅拌5小时后转移到透析袋，透析袋的截留分子量为3500，在超纯水中透析18小时，得到负载抗癌药物的纳米颗粒，所述抗癌药物包含紫杉醇或10-羟基喜树碱，从表3结果看出：纳米胶束的载药率随着双丙酮丙烯酰胺含量增加而提高。
表3 黄原胶共聚物纳米胶束的载药率(％)
样品编号 紫杉醇 10-羟基喜树碱
a1 10.5 11.4
b1 11.9 12.7
c1 12.1 13.6
d1 13.6 14.8
实施例10 黄原胶共聚物纳米胶束的生物相容性检测
用RPMI-1640完全培养基将HeLa细胞稀释成6×10 4个/mL细胞密度并接种于96孔板，每孔接种100μL，待细胞贴壁后弃培养基，实验组采用4个纳米胶束样品a1、b1、c1、d1，分别加入100μL含纳米胶束浓度为1.0mg/mL的RPMI-1640完全培养基；阳性对照组为含质量浓度为0.64％苯酚的RPMI-1640完全培养基、阴性对照组为RPMI-1640完全培养基，每组平行设置4个复孔，继续放置上述培养箱中37℃培养，采用倒置生物显微镜观察不同时间内细胞生长形貌。
将96孔板放置在培养箱中培养24h后取出，每孔加入10μL MTT(5.0mg/mL)继续37℃培养4h后取出，每孔加入150μL的DMSO，将其放置培养箱中15min以溶解其生成的结晶紫，在570nm处采用酶标仪测定其吸光度A，按下面公式计算细胞存活率：
细胞存活率(％)＝A 1/A 2×100
其中A 1表示实验组570nm处的吸光度；A 2表示阴性对照组570nm处的吸光度。结果发现：HeLa细胞在37℃下与纳米胶束样品a1、b1、c1、d1分别孵育28h后，其细胞存活率分别为：95.8％、93.6％、94.3％和94.7％，可以判定纳米胶束无细胞毒性。
以上实施例仅用以说明本发明的技术方案而非限制，本领域的技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。

Claims (9)

  1. 一种黄原胶共聚物纳米胶束的制备方法，包含以下步骤：
    步骤1，预处理：将浓度为0.5w％黄原胶经过降解使得动力粘度下降到110mPa·s，然后干燥、粉碎、过筛得到降解后的黄原胶；
    步骤2，制备黄原胶溴代物：经步骤1降解后的黄原胶产物溶于N,N-二甲基甲酰胺中，在冰浴和搅拌条件下预先将2-溴代异丁酰溴溶于N,N-二甲基甲酰胺中，然后加入到上述黄原胶溶液中，再添加三乙胺，室温反应48h，反应产物用***沉淀并过滤，恒温30℃干燥得到黄原胶溴代物；
    步骤3，制备黄原胶共聚物：将步骤2所得黄原胶溴代物溶解于N,N-二甲基甲酰胺，加入双丙酮丙烯酰胺，抽真空-充氮气循环三次，接着加入三[2-(二甲基氨基)乙基]胺和氯化亚铜进行反应，反应的温度为55～65℃，反应的时间为3～5h，产物用***沉淀并过滤，再用***洗涤产物3次，过滤并恒温30℃干燥产物，得到黄原胶与聚双丙酮丙烯酰胺的接枝共聚物，简称为黄原胶共聚物；
    步骤4，制备黄原胶共聚物纳米胶束：将步骤3中黄原胶共聚物溶解在极性有机溶剂中，用孔径为0.4μm的微孔过滤膜过滤，得聚合物母液，搅拌下缓慢向聚合物母液中滴加超纯水，直到形成胶束溶液，将胶束溶液在去离子水中透析4天，隔8个小时换一次去离子水，得到黄原胶共聚物纳米胶束。
  2. 如权利要求1所述一种黄原胶共聚物纳米胶束的制备方法，其特征是在步骤2)中，黄原胶在N,N-二甲基甲酰胺中的质量浓度为1.8％～2.5％。
  3. 如权利要求1所述一种黄原胶共聚物纳米胶束的制备方法，其特征是在步骤2)中，2-溴代异丁酰溴在反应体系中的质量浓度为1.5％～2.2％。
  4. 如权利要求1所述一种黄原胶共聚物纳米胶束的制备方法，其特征是在步骤2)中，所加三乙胺的重量是2-溴代异丁酰溴的40％～60％。
  5. 如权利要求1所述一种黄原胶共聚物纳米胶束的制备方法，其特征是在步骤3)中，双丙酮丙烯酰胺的质量是黄原胶溴代物的1～3倍。
  6. 如权利要求1所述一种黄原胶共聚物纳米胶束的制备方法，其特征是在步骤3)中，三[2-(二甲基氨基)乙基]胺与氯化亚铜的摩尔比1:1.5～2.0，优选地，所述反应温度为60℃，反应时间为4.5h。
  7. 如权利要求1所述一种黄原胶共聚物纳米胶束的制备方法，其特征在于，步骤4中所述极性有机溶剂包括无水乙醇、异丙醇、四氢呋喃、N,N-二甲基甲酰胺、二氧六环或二甲亚 砜。
  8. 一种黄原胶共聚物纳米胶束的应用，其特征在于，按权利要求1～7任一项所述一种黄原胶共聚物纳米胶束的制备方法得到纳米胶束，将所述纳米胶束冷冻干燥成粉末，再将抗癌药物用甲醇配成浓度为2mg/mL的溶液，将30mg纳米胶束粉末加入到8mL抗癌药物的甲醇溶液中，搅拌5小时后转移到透析袋，透析袋的截留分子量为3500，在超纯水中透析18小时，得到负载抗癌药物的纳米颗粒。
  9. 根据权利要求8所述的一种黄原胶共聚物纳米胶束的应用，其特征在于，所述抗癌药物包括紫杉醇或10-羟基喜树碱。
PCT/CN2019/120773 2019-07-31 2019-11-26 一种黄原胶共聚物纳米胶束的制备方法及其应用 WO2021017335A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/954,734 US11623968B2 (en) 2019-07-31 2019-11-26 Method for preparation of xanthan gum copolymer nanomicelles

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910700200.0 2019-07-31
CN201910700200.0A CN110302155B (zh) 2019-07-31 2019-07-31 一种黄原胶共聚物纳米胶束的制备方法及其应用

Publications (1)

Publication Number Publication Date
WO2021017335A1 true WO2021017335A1 (zh) 2021-02-04

Family

ID=68082552

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/120773 WO2021017335A1 (zh) 2019-07-31 2019-11-26 一种黄原胶共聚物纳米胶束的制备方法及其应用

Country Status (3)

Country Link
US (1) US11623968B2 (zh)
CN (1) CN110302155B (zh)
WO (1) WO2021017335A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110302155B (zh) 2019-07-31 2021-09-21 江南大学 一种黄原胶共聚物纳米胶束的制备方法及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090017120A1 (en) * 2007-03-23 2009-01-15 Humco Holding Group, Inc. Phase stable lecithin organogel composition
CN103002885A (zh) * 2010-05-13 2013-03-27 莫诺索尔克斯有限公司 用于递送活性成分的膜组合物
CN103083222A (zh) * 2011-10-28 2013-05-08 江南大学 一锅法制备三组分聚合物胶束
CN104188926A (zh) * 2006-10-20 2014-12-10 索尔瓦药物有限公司 化学物质的胶束纳米颗粒
CN105658633A (zh) * 2013-09-03 2016-06-08 新加坡科技研究局 用于生物医药应用的聚合物-类黄酮缀合物和水凝胶
CN107308112A (zh) * 2017-07-24 2017-11-03 江南大学 一种改性黄原胶纳米胶束的制备方法
CN110302155A (zh) * 2019-07-31 2019-10-08 江南大学 一种黄原胶共聚物纳米胶束的制备方法及其应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014130846A1 (en) * 2013-02-22 2014-08-28 Seungpyo Hong Transdermal drug delivery using amphiphilic dendron-coil micelles
CN107098988B (zh) * 2017-05-03 2019-05-17 江南大学 一种黄原胶纳米微凝胶的制备方法
CN107312126B (zh) * 2017-07-05 2019-10-18 江南大学 一种接枝改性黄原胶纳米微凝胶的制备方法
CN109265695B (zh) * 2018-08-24 2020-08-04 江南大学 一种增稠改性黄原胶的制备方法及应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104188926A (zh) * 2006-10-20 2014-12-10 索尔瓦药物有限公司 化学物质的胶束纳米颗粒
US20090017120A1 (en) * 2007-03-23 2009-01-15 Humco Holding Group, Inc. Phase stable lecithin organogel composition
CN103002885A (zh) * 2010-05-13 2013-03-27 莫诺索尔克斯有限公司 用于递送活性成分的膜组合物
CN103083222A (zh) * 2011-10-28 2013-05-08 江南大学 一锅法制备三组分聚合物胶束
CN105658633A (zh) * 2013-09-03 2016-06-08 新加坡科技研究局 用于生物医药应用的聚合物-类黄酮缀合物和水凝胶
CN107308112A (zh) * 2017-07-24 2017-11-03 江南大学 一种改性黄原胶纳米胶束的制备方法
CN110302155A (zh) * 2019-07-31 2019-10-08 江南大学 一种黄原胶共聚物纳米胶束的制备方法及其应用

Also Published As

Publication number Publication date
CN110302155A (zh) 2019-10-08
US11623968B2 (en) 2023-04-11
CN110302155B (zh) 2021-09-21
US20210032375A1 (en) 2021-02-04

Similar Documents

Publication Publication Date Title
Dharmalingam et al. Fabrication, characterization and drug loading efficiency of citric acid crosslinked NaCMC-HPMC hydrogel films for wound healing drug delivery applications
Ooi et al. Cellulose nanocrystals extracted from rice husks as a reinforcing material in gelatin hydrogels for use in controlled drug delivery systems
WO2017166903A1 (zh) 一种改性海藻酸钠栓塞微球的制备方法
Saboktakin et al. pH-sensitive starch hydrogels via free radical graft copolymerization, synthesis and properties
Li et al. Chitosan modified metal–organic frameworks as a promising carrier for oral drug delivery
Sun et al. Glucose-and temperature-responsive core–shell microgels for controlled insulin release
WO2017107486A1 (zh) 可还原降解超支化聚合物纳米胶束及其制备方法和应用
WO2016155051A1 (zh) 一种可还原降解聚两性离子纳米胶束及其制备方法
Wang et al. In situ formation of chitosan-cyclodextrin nanospheres for drug delivery
Pooresmaeil et al. Folic acid-modified photoluminescent dialdehyde carboxymethyl cellulose crosslinked bionanogels for pH-controlled and tumor-targeted co-drug delivery
Abbasi et al. Folic acid-decorated alginate nanoparticles loaded hydrogel for the oral delivery of diferourylmethane in colorectal cancer
Wei et al. Preparation and characterization of starch-cellulose interpenetrating network hydrogels based on sequential Diels-Alder click reaction and photopolymerization
CN114191408A (zh) 一种酪蛋白-姜黄素载药纳米粒的制备方法和应用
Michailova et al. Nanoparticles formed from PNIPAM-g-PEO copolymers in the presence of indomethacin
WO2021017335A1 (zh) 一种黄原胶共聚物纳米胶束的制备方法及其应用
Pooresmaeil et al. Development of the new pH-driven carrier from alginate/carboxymethyl starch bio-coated co-drugs@ COF-OH for controlled and concomitant colon cancer treatment
CN108503718B (zh) 一种羟烷基淀粉偶联物及其制备方法和应用
Ma et al. UV cross-linked redox-responsive hydrogels for co-delivery of hydrophilic and hydrophobic drugs
Wang et al. Self-assembled micelles of N-phthaloylchitosan-g-poly (N-vinylcaprolactam) for temperature-triggered non-steroidal anti-inflammatory drug delivery
Das et al. Controlled and targeted delivery of diclofenac sodium to the intestine from pH-Responsive chitosan/poly (vinyl alcohol) interpenetrating polymeric network hydrogels
CN109400830B (zh) 一种pH可解离轻度交联聚合物纳米材料及其制备方法和应用
CN116687869A (zh) 一种双载体的氟比洛芬酯纳米载药材料及其制备方法
Saboktakin et al. Synthesis and characterization of biodegradable chitosan beads as nano-carriers for local delivery of satranidazole
CN114524932A (zh) 双亲性三嵌段聚氨基酸共聚物、中间体、制备及应用
CN113081976A (zh) 一种基于mmp-2酶敏感的纳米制剂及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19939608

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19939608

Country of ref document: EP

Kind code of ref document: A1