CN110433295A - 一种食源性纳米粒子蛋白冠的制备方法 - Google Patents
一种食源性纳米粒子蛋白冠的制备方法 Download PDFInfo
- Publication number
- CN110433295A CN110433295A CN201910823281.3A CN201910823281A CN110433295A CN 110433295 A CN110433295 A CN 110433295A CN 201910823281 A CN201910823281 A CN 201910823281A CN 110433295 A CN110433295 A CN 110433295A
- Authority
- CN
- China
- Prior art keywords
- nanoparticle
- food
- borne
- albumen
- preparation
- 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
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 116
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 241000251468 Actinopterygii Species 0.000 claims abstract description 8
- 238000004262 preparative liquid chromatography Methods 0.000 claims abstract description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 23
- 210000002966 serum Anatomy 0.000 claims description 17
- 108010088751 Albumins Proteins 0.000 claims description 15
- 102000009027 Albumins Human genes 0.000 claims description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 239000007864 aqueous solution Substances 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 13
- 239000006481 glucose medium Substances 0.000 claims description 12
- 238000002390 rotary evaporation Methods 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 12
- 241000972773 Aulopiformes Species 0.000 claims description 10
- 239000012141 concentrate Substances 0.000 claims description 10
- 235000019515 salmon Nutrition 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000000287 crude extract Substances 0.000 claims description 7
- 235000019688 fish Nutrition 0.000 claims description 7
- 238000004587 chromatography analysis Methods 0.000 claims description 6
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 6
- 238000002189 fluorescence spectrum Methods 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 5
- 230000005284 excitation Effects 0.000 claims description 5
- 238000012856 packing Methods 0.000 claims description 5
- 239000002245 particle Substances 0.000 claims description 5
- 238000002604 ultrasonography Methods 0.000 claims description 5
- 238000005238 degreasing Methods 0.000 claims description 4
- 238000000605 extraction Methods 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 239000003960 organic solvent Substances 0.000 claims description 4
- 239000008280 blood Substances 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 3
- NEHMKBQYUWJMIP-UHFFFAOYSA-N chloromethane Chemical class ClC NEHMKBQYUWJMIP-UHFFFAOYSA-N 0.000 claims description 2
- 238000011177 media preparation Methods 0.000 claims description 2
- 239000000047 product Substances 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 239000012071 phase Substances 0.000 claims 4
- 238000001704 evaporation Methods 0.000 claims 2
- 230000008020 evaporation Effects 0.000 claims 2
- 238000004458 analytical method Methods 0.000 claims 1
- 238000004108 freeze drying Methods 0.000 claims 1
- 239000007791 liquid phase Substances 0.000 claims 1
- 235000013305 food Nutrition 0.000 abstract description 10
- 230000001988 toxicity Effects 0.000 abstract description 6
- 231100000419 toxicity Toxicity 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 238000011160 research Methods 0.000 abstract description 4
- 239000002086 nanomaterial Substances 0.000 abstract description 3
- 108091006905 Human Serum Albumin Proteins 0.000 abstract description 2
- 102000008100 Human Serum Albumin Human genes 0.000 abstract description 2
- 229940079593 drug Drugs 0.000 abstract description 2
- 150000003384 small molecules Chemical class 0.000 abstract description 2
- 238000000638 solvent extraction Methods 0.000 abstract 2
- 238000000622 liquid--liquid extraction Methods 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 239000011852 carbon nanoparticle Substances 0.000 description 13
- 238000000034 method Methods 0.000 description 9
- 210000003292 kidney cell Anatomy 0.000 description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 239000012894 fetal calf serum Substances 0.000 description 4
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 230000006907 apoptotic process Effects 0.000 description 3
- -1 amino, carboxyl Chemical group 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000017074 necrotic cell death Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241000272525 Anas platyrhynchos Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010021703 Indifference Diseases 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- 238000001241 arc-discharge method Methods 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229960004756 ethanol Drugs 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000021060 food property Nutrition 0.000 description 1
- 238000001027 hydrothermal synthesis Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 230000001699 photocatalysis Effects 0.000 description 1
- 238000007146 photocatalysis Methods 0.000 description 1
- 238000005424 photoluminescence Methods 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 230000005641 tunneling Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
-
- 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
- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
-
- 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
- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6923—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Ceramic Engineering (AREA)
- Inorganic Chemistry (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种食源性纳米粒子蛋白冠制备方法,属于新型纳米材料蛋白冠作为分子载体载运技术领域。本发明以烤鱼为原料,经溶剂提取、液液萃取及半制备液相色谱纯化等步骤制备得到食品源纳米粒子,所得纳米粒子可与人血清蛋白相互作用形成食源性纳米粒子蛋白冠,所述蛋白冠具有降低纳米粒子毒性的效应。基于本发明制备的食源性纳米粒子蛋白冠的性质,其可作为载体在药物,食品功能因子,营养物质小分子等众多生物医药方向具有优异的应用,为研究食品热加工中食源性纳米粒子蛋白冠的载运功能提供理论基础。
Description
技术领域
本发明涉及新型食源性纳米材料蛋白冠制备及生物医药应用技术领域,更具体地说,涉及一种食源性纳米粒子蛋白冠的制备方法。
背景技术
烤制食品具有诱人的色泽和鲜美的口感,是人们日常生活中的一道美味菜肴,千百年来一直深受消费者的喜爱。近年来研究者报道了在烤制食品中发现了碳纳米粒子,如在烤面包、烤牛肉、烤鸭以及烤羊肉串等涉及到热加工的食物,这些碳纳米粒子多是无定形态,粒径在1~20nm左右。碳纳米粒子具有一系列优异的物理及化学性质,如表面效应、量子尺寸效应、量子隧道效应、良好的水溶性、发光稳定性等。因此,碳纳米粒子在生物成像、生物传感、药物载体及光催化等众多领域表现出良好的应用前景,引起了人们的广泛关注,使其研究得到了飞速发展。
碳纳米粒子表面存在丰富的带电荷官能团,如氨基,羧基等,这使其可以通过共价接枝或者静电相互作用与蛋白质结合,其表面吸附的一层或多层蛋白所组成的结构称为蛋白冠,蛋白冠的形成取决于纳米材料的大小、化学成分和表面基团。碳纳米粒子的吸附会对某些蛋白分子本身的结构甚至是功能造成一定影响。更重要地,碳纳米粒子吸附蛋白后常改变碳纳米粒子原有的粒径、分散性及表面电荷等,降低碳纳米粒子本省的毒性和改变生物相容性。
目前制备蛋白冠的碳纳米粒子一般采用水热合成法、电弧放电法合成法、高温高压法、电化学扫描法以及有机物热解法等方法制得,这些方法制备的碳纳米粒子具有抑制细胞增长的风险,为碳纳米粒子的应用带来了一定的挑战。
发明内容
本发明针对烤制食品中产生的纳米粒子的安全性存在风险等问题,提出了一种食源性纳米粒子蛋白冠制备方法。本研究采用食源性纳米粒子与人血清白蛋白相互作用形成蛋白冠,有效地减低食源性纳米粒子本身的毒性,能够作为载体应用于生物医药领域,可以为评价食源性纳米粒子形成蛋白冠的安全性提供理论依据。
为了达到上述目的,本发明提供一种食源性纳米粒子蛋白冠制备方法,包括如下步骤:
S1:浸提:取烤鱼,浸泡于无水乙醇中,700~900w超声1~4h,静置1~2h,取上清液;所述烤鱼和无水乙醇的重量体积比为1:1~1:5g/mL;
S2、去除有机溶剂:将步骤S1所述上清液置于60~80℃旋转蒸发至所述上清液体积的1/8~1/10,得混合液;
S3、脱脂:向步骤S2所述混合液中加入其1~3倍体积的三氯甲烷水溶液(v水/v三氯甲烷=2:1);使用分液漏斗充分摇匀分液,除去下层三氯甲烷层,得到上层水溶性纳米粒子粗提液;
S4:纯化:将步骤S3所述纳米粒子粗提液置于60~80℃、旋转蒸发至所述纳米粒子粗提液原体积的1/8~1/10,得纳米粒子浓缩液;将所述纳米粒子浓缩液过半制备液相色谱层析,检测器为荧光检测器,荧光光谱激发波长为380nm,发射波长为400~500nm;收集荧光部分,真空冷冻干燥,得到食源性纳米粒子;
S5:食源性纳米粒子蛋白冠制备:采用无血清高糖培养基配制纳米粒子溶液和血清蛋白溶液;取所述纳米粒子溶液和所述血清蛋白溶液混匀,静置10~30min,所得混合液即为食源性纳米粒子蛋白冠;所述血清蛋白与纳米粒子摩尔比为1:90~1:110;其中,所述纳米粒子的分子量为1056m/z。
优选方式下,步骤S1所述烤鱼为烤三文鱼,其制备方法为:取三文鱼置于180℃~220℃烤制30~60min;
优选方式下,步骤S2所述旋转蒸发的转速为50~70rpm。
优选方式下,步骤S4所述半制备液相色谱条件为:ODS-BP色谱柱,ODS-BP填料粒径为10μm,色谱柱内径为20mm,长度为300mm,流动相为体积分数为5%~20%(v/v)甲醇水溶液,所述纳米粒子浓缩液的上样量为5~10mL,流速为16~20mL/min,洗脱时间为90~150min。
优选方式下,步骤S4所述旋转蒸发的转速为50~70rpm;所述真空冷冻干燥的参数为:-50~60℃,真空度为1~10Pa,干燥10~24h。
优选方式下,步骤S5所述血清蛋白为人血清白蛋白。
优选方式下,所述食源性纳米粒子蛋白冠制备方法,包括如下步骤:
S1、浸提:500g三文鱼经过200℃烤制50min,将烤制的三文鱼浸泡于1500mL的无水乙醇中,800w超声浸提2h,静置1h,取上清液;
S2、去除有机溶剂:将步骤S1所述上清液置于70℃、60rpm旋转蒸发至150mL,得混合液;
S3、脱脂:向步骤S2所述混合液中加入三氯甲烷水溶液300mL,通过分液漏斗充分震荡,去除下层三氯甲烷,得到水相纳米粒子粗提液30mL;所述三氯甲烷水溶液由水和三氯甲烷按体积比为2:1混合而成;
S4:纯化:将步骤S3所述水相纳米粒子粗提溶液于70℃,60rpm旋转蒸发,浓缩至3mL,得纳米粒子浓缩液;经过依利特半制备液相色谱进行纯化,ODS-BP色谱柱,填料粒径为10μm,色谱柱内径为20mm,长度为300mm,流动相为体积分数10%(v/v)的甲醇水溶液,上样品为5mL纳米粒子浓缩液,流速为18mL/min,洗脱时间为120min,荧光光谱激发波长为380nm,发射波长为400~500nm,收集荧光部分,在-50℃,真空度为10Pa下冷冻干燥24h,得到食源性纳米粒子;
S5:食源性纳米粒子蛋白冠制备:取步骤S4所述食源性纳米粒子,采用无血清高糖培养基配制成食源性纳米粒子浓度为1mol/L的纳米粒子溶液;取人血清白蛋白,使用无血清高糖培养基配置成人血清白蛋白浓度为1×10-5mol/L的人血清白蛋溶液;取所述纳米粒子溶液2μl加入到2ml所述血清蛋白溶液中,混匀,静置20min,所得混合液即为食源性纳米粒子蛋白冠;其中,所述血清蛋白与纳米粒子摩尔比为1:100,所述纳米粒子的分子量为1056m/z。
本发明的有益效果是:
本发明提供了一种食源性纳米粒子蛋白冠制备方法,本发明通过烤鱼的方法获得食源性纳米粒子,由于纳米粒子表面存在丰富的官能团,可以和血清蛋白形成蛋白冠,降低食源性纳米粒子本身的毒性,所述蛋白冠可以作为载体应用于生物医药领域。
本发明中纳米粒子来源于食品热加工过程中产生的纳米粒子,具有安全性高,水溶性好,生物相容性好等优点。本发明首次采用食源性纳米粒子与蛋白质相互作用形成蛋白冠,从而降低纳米粒子的毒性,将为食源性碳纳米粒子蛋白冠作为载体在生物医药领域的应用打下基础。
综上所述,本发明制备的食源性纳米粒子蛋白冠可作为载体,在药物、食品功能因子、营养物质小分子等众多生物医药方向具有优异的应用,为研究食品热加工中食源性纳米粒子蛋白冠的载运功能提供理论基础。
附图说明:
图1为本发明实施例1中制备的食源性纳米粒子的透射电镜图;
图2为本发明实施例1中制备的食源性纳米粒子的粒径分布图;
图3为本发明实施例1中制备的人血清白蛋白和蛋白冠的荧光光谱图;
图4为本发明实施例1中大鼠肾细胞在无血清高糖培养基配制的食源性纳米粒子浓度为1×10-3mol/L的纳米粒子溶液孵育12h的相位对比图;
图5为大鼠肾细胞在本发明实施例1制备的1×10-3mol/L的食源性纳米粒子蛋白冠孵育12h的相位对比图;
图6为本发明实施例1中大鼠肾细胞在无血清高糖培养基配制的食源性纳米粒子浓度为1×10-3mol/L的纳米粒子溶液孵育12h的细胞凋亡检测结果;
图7为大鼠肾细胞在本发明实施例1制备的1×10-3mol/L的食源性纳米粒子蛋白冠孵育12h的细胞凋亡检测结果。
具体实施方式
以下通过具体实施例对发明作进一步说明,而不是对本发明的限制。
下述实施例使用的人血清白蛋白为北京索莱宝科技有限公司,货号A8230;所述青霉素-链霉素为Hycone公司,货号SV30010;所述无血清高糖培养基为BI公司,货号06-1055-57-1ACS。
实施例1:
500g三文鱼经过200℃烤制50min,将烤制的三文鱼浸泡于1500mL的无水乙醇中,800w超声浸提2h,静置1h,取上清液;将所述上清液置于70℃、60rpm旋转蒸发至150mL,得混合液;向所述混合液中加入三氯甲烷水溶液300mL复溶(所述三氯甲烷水溶液中,水和三氯甲烷的体积比为2:1),通过分液漏斗充分震荡,然后去除三氯甲烷,得到水相纳米粒子粗提液30mL;将所述水相纳米粒子粗提溶液于70℃,60rpm旋转蒸发,浓缩至3mL,得纳米粒子浓缩液;经过依利特半制备液相色谱进行纯化,ODS-BP色谱柱,填料粒径为10μm,色谱柱内径为20mm,长度为300mm,流动相为体积分数10%(v/v)甲醇水溶液,上样品为5mL纳米粒子浓缩液,流速为18mL/min,洗脱时间为120min,荧光光谱激发波长为380nm,发射波长为400~500nm,收集荧光部分,在-50℃,真空度为10Pa下冷冻干燥24h,得到食源性纳米粒子;
采用无血清高糖培养基(BI公司,货号06-1055-57-1ACS),分别配制成人血清白蛋白浓度为1×10-5mol/L的人血清白蛋溶液和食源性纳米粒子浓度为1mol/L的纳米粒子溶液;取所述1mol/L纳米粒子溶液2μL,加入到所述2mL1×10-5mol/L人血清白蛋白溶液中(所述人血清白蛋白与纳米粒子摩尔比1:100,纳米粒子终浓度为1×10-3mol/L),充分混匀,静置20min,所得混合液即为食源性纳米粒子蛋白冠。
取1mg本实施例制备的食源性纳米粒子配成1mg/mL水溶液,滴到超薄碳膜中,采用透射电镜观察纳米粒子的形貌,如图1所示,纳米粒子呈现球状,粒径分布在1.5~3.9nm,平均粒径大小为2.67±0.36nm(图2)。
如图3所示,1×10-5mol/L的人血清白蛋白与本实施例制备的蛋白冠比较,荧光强度明显降低,最大发射波长由338nm变为350nm,说明纳米粒子与人血清白蛋白结合形成蛋白冠。
采用无血清高糖培养基配制食源性纳米粒子浓度为1×10-3mol/L的纳米粒子溶液;将大鼠肾细胞以每孔1×105个细胞接种于12孔板中(每孔加1mL含1%(v/v)青霉素-链霉素和10%(v/v)胎牛血清的高糖培养基),并在含有5%CO2的培养箱中37℃培养24h,弃去原培养基(即含1%(v/v)青霉素-链霉素和10%(v/v)胎牛血清的高糖培养基);加入1mL无血清高糖培养基配制1×10-3mol/L的纳米粒子溶液,并在含有5%CO2的培养箱中37℃培养12h(图4)后,球形细胞的个数明显增加,表明纳米粒子促进了细胞黏附性丧失和造成部分细胞损伤。然而,将大鼠肾细胞以每孔1×105个细胞接种于12孔板中(每孔加1mL含1%(v/v)青霉素-链霉素和10%(v/v)胎牛血清的高糖培养基),并在含有5%CO2的培养箱中37℃培养24h,弃去原培养基(即含1%(v/v)青霉素-链霉素和10%(v/v)胎牛血清的高糖培养基),加入1mL本实施例制备的食源性纳米粒子蛋白冠溶液,并在含有5%CO2的培养箱中37℃孵育12h(图5),球形细胞数量显著减少,可能是因为人血清白蛋白与纳米粒子形成蛋白冠后减缓了纳米粒子的毒性。
进一步通过流式细胞术对细胞凋亡分析,选择488nm和535nm俩通道进行检测,验证蛋白冠是否对纳米粒子减轻了细胞毒性。如图6所示,在含有1×10-3mol/L纳米粒子的无血清高糖培养基中,大鼠肾细胞细胞存活率为81.35%,晚期凋亡率为11.25%,坏死率为6.36%;而本实施例制备的食源性纳米粒子蛋白冠处理的活细胞比例为90.74%,晚期凋亡率为4.60%,坏死率为3.91%(图7),明显低于单独纳米粒子处理的凋亡率,这说明本发明制备的食源性纳米粒子蛋白冠在减轻纳米粒子的细胞毒性方面具有明显的作用。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (8)
1.一种食源性纳米粒子蛋白冠制备方法,其特征在于,包括步骤:
S1:浸提:取烤鱼,浸泡于无水乙醇中,700~900w超声1~4h,静置1~2h,取上清液;所述烤鱼和无水乙醇的重量体积比为1:1~1:5g/mL;
S2、去除有机溶剂:将步骤S1所述上清液置于60~80℃旋转蒸发至所述上清液体积的1/8~1/10,得混合液;
S3、脱脂:向步骤S2所述混合液中加入其1~3倍体积的三氯甲烷水溶液,摇匀分液,除去下层三氯甲烷层,得到上层水溶性纳米粒子粗提液;其中,所述三氯甲烷水溶液由水和三氯甲烷按体积比2:1混合而成;
S4:纯化:将步骤S3所述纳米粒子粗提液置于60~80℃、旋转蒸发至所述纳米粒子粗提液原体积的1/8~1/10,得纳米粒子浓缩液;将所述纳米粒子浓缩液过半制备液相色谱层析,检测器为荧光检测器,荧光光谱激发波长为380nm,发射波长为400~500nm;收集荧光部分,真空冷冻干燥,得到食源性纳米粒子;
S5:食源性纳米粒子蛋白冠制备:采用无血清高糖培养基配制纳米粒子溶液和血清蛋白溶液;按血清蛋白与食源性纳米粒子摩尔比1:90~1:110,取所述纳米粒子溶液和所述血清蛋白溶液混匀,静置10~30min,所得混合液即为食源性纳米粒子蛋白冠。
2.根据权利要求1所述食源性纳米粒子蛋白冠制备方法,其特征在于,步骤S1所述烤鱼为烤三文鱼,其制备方法为:取三文鱼置于180℃~220℃烤制30~60min。
3.根据权利要求1所述食源性纳米粒子蛋白冠制备方法,其特征在于,步骤S2所述旋转蒸发的转速为50~70rpm。
4.根据权利要求1所述食源性纳米粒子蛋白冠制备方法,其特征在于,步骤S4所述半制备液相色谱条件为:ODS-BP色谱柱,ODS-BP填料粒径为10μm,色谱柱内径为20mm,长度为300mm,流动相为体积分数5%~20%(v/v)的甲醇水溶液,所述纳米粒子浓缩液的上样量为5~10mL,流速为16~20mL/min,洗脱时间为90~150min。
5.根据权利要求1所述食源性纳米粒子蛋白冠制备方法,其特征在于,步骤S4所述旋转蒸发的转速为50~70rpm。
6.根据权利要求1所述食源性纳米粒子蛋白冠制备方法,其特征在于,步骤S4所述真空冷冻干燥的参数为:-50~60℃,真空度为1~10Pa,干燥10~24h。
7.根据权利要求1所述食源性纳米粒子蛋白冠制备方法,其特征在于,步骤S5所述血清蛋白为人血清白蛋白。
8.根据权利要求1所述食源性纳米粒子蛋白冠制备方法,其特征在于,包括步骤:
S1、浸提:500g三文鱼200℃烤制50min,将烤制的三文鱼浸泡于1500mL无水乙醇中,800w超声浸提2h,静置1h,取上清液;
S2、去除有机溶剂:将步骤S1所述上清液置于70℃、60rpm旋转蒸发至150mL,得混合液;
S3、脱脂:向步骤S2所述混合液中加入三氯甲烷水溶液300mL,通过分液漏斗震荡,去除下层三氯甲烷,得到水相纳米粒子粗提液30mL;所述三氯甲烷水溶液由水和三氯甲烷按体积比为2:1混合而成;
S4:纯化:将步骤S3所述水相纳米粒子粗提溶液于70℃,60rpm旋转蒸发至3mL,得纳米粒子浓缩液;使用依利特半制备液相色谱进行纯化,ODS-BP色谱柱,ODS-BP填料粒径为10μm,色谱柱内径为20mm,长度为300mm,流动相为体积分数10%(v/v)的甲醇水溶液,上样品为5mL所述纳米粒子浓缩液,流速为18mL/min,洗脱时间为120min,荧光光谱激发波长为380nm,发射波长为400~500nm,收集荧光部分,在-50℃,真空度10Pa干燥24h,得到食源性纳米粒子;
S5:食源性纳米粒子蛋白冠制备:取步骤S4所述食源性纳米粒子,采用无血清高糖培养基配制成食源性纳米粒子浓度为1mol/L的纳米粒子溶液;取人血清白蛋白,使用无血清高糖培养基配置成人血清白蛋白浓度为1×10-5mol/L的人血清白蛋溶液;取所述纳米粒子溶液2μl加入到2ml所述血清蛋白溶液中,混匀,静置20min,所得混合液即为食源性纳米粒子蛋白冠。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910823281.3A CN110433295A (zh) | 2019-09-02 | 2019-09-02 | 一种食源性纳米粒子蛋白冠的制备方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910823281.3A CN110433295A (zh) | 2019-09-02 | 2019-09-02 | 一种食源性纳米粒子蛋白冠的制备方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN110433295A true CN110433295A (zh) | 2019-11-12 |
Family
ID=68438753
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910823281.3A Pending CN110433295A (zh) | 2019-09-02 | 2019-09-02 | 一种食源性纳米粒子蛋白冠的制备方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110433295A (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016015522A1 (zh) * | 2014-07-31 | 2016-02-04 | 天津派格生物技术有限公司 | 脂肪酸结合型白蛋白-药物纳米粒子冻干制剂及制备方法 |
CN108273071A (zh) * | 2018-01-26 | 2018-07-13 | 大连工业大学 | 一种食源性荧光纳米粒及其制备方法和应用 |
CN108929683A (zh) * | 2018-07-28 | 2018-12-04 | 大连工业大学 | 食源性纳米粒子的制备方法 |
-
2019
- 2019-09-02 CN CN201910823281.3A patent/CN110433295A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016015522A1 (zh) * | 2014-07-31 | 2016-02-04 | 天津派格生物技术有限公司 | 脂肪酸结合型白蛋白-药物纳米粒子冻干制剂及制备方法 |
CN108273071A (zh) * | 2018-01-26 | 2018-07-13 | 大连工业大学 | 一种食源性荧光纳米粒及其制备方法和应用 |
CN108929683A (zh) * | 2018-07-28 | 2018-12-04 | 大连工业大学 | 食源性纳米粒子的制备方法 |
Non-Patent Citations (2)
Title |
---|
RONGGANG LIU ET AL.: "Nanocorona Formation between Foodborne Nanoparticles Extracted from Roast Squid and Human Serum Albumin", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
张长平等: "纳米材料表面蛋白冠的形成、鉴定及生物学效应研究进展", 《功能材料》 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN109303922B (zh) | 一种刺梨多糖功能化纳米硒复合物及其制备方法与在降糖药物中的应用 | |
WO2014176900A1 (zh) | 皂苷纳米胶束及其制备方法、应用和药物组合物 | |
CN103705940A (zh) | 一种天然活性药物-多糖靶向复合物的制备及其抗肿瘤的应用 | |
CN107802646A (zh) | 一种肿瘤治疗药物 | |
US10835552B2 (en) | Method for preparing linseed polysaccharide having antiviral activity and immunological activity, and use of the linseed polysaccharide | |
CN101411879B (zh) | SiO2/Au核壳结构纳米材料-生物蛋白药物复合物及其制备方法 | |
CN108372307A (zh) | 一种纳米金的制备方法、纳米金及应用 | |
CN104761655B (zh) | 一种从海鲜菇下脚料中提取海鲜菇多糖的方法 | |
CN110433295A (zh) | 一种食源性纳米粒子蛋白冠的制备方法 | |
JP5583259B1 (ja) | コラーゲン産生作用を有する新規な誘導体及びその製造方法 | |
CN102000133A (zh) | 一种中药抗氧化制剂的制备方法 | |
CN105125419B (zh) | 一种聚乙二醇修饰的维生素e脂质体面膜及其制备方法 | |
CN103976958B (zh) | 一种金纳米颗粒在制备抗凝血剂或抗血小板剂中的应用 | |
CN102000132B (zh) | 一种中药制剂的制备方法及其制备抗氧化药物的用途 | |
CN115192708A (zh) | 负载抗肿瘤药物的纳米复合材料、纳米载药体系及制备与应用 | |
CN101974248A (zh) | 一种茶色素组分的分离方法 | |
CN108653209A (zh) | 一种新型天然茶树花衍生脂质纳米囊泡的制备及其应用 | |
CN100471504C (zh) | 纳米级螯合型珍珠制品及其生产工艺和应用 | |
JP2018154566A (ja) | エキス輸送作用を呈する環状ペプチド誘導体 | |
CN108969580B (zh) | 蓝布正总鞣质的制备方法及应用 | |
TW202222301A (zh) | 發酵薑黃素微脂體之製法 | |
CN107279965B (zh) | 一种猴头菇体外高效抗氧化组分及其提取方法与应用 | |
KR101065443B1 (ko) | 마황의 피부 노화억제 활성을 위한 식용소재 나노입자화 방법 | |
CN115633783B (zh) | 一种多糖纳米剂型的制备方法和应用 | |
CN114796002B (zh) | 活性成分的纳米搭载、透皮递送系统及其制备方法和应用 |
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 | ||
WD01 | Invention patent application deemed withdrawn after publication | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20191112 |