CN116942836A - 一种低毒、高效的细胞治疗用载体的制造方法 - Google Patents
一种低毒、高效的细胞治疗用载体的制造方法 Download PDFInfo
- Publication number
- CN116942836A CN116942836A CN202310884943.4A CN202310884943A CN116942836A CN 116942836 A CN116942836 A CN 116942836A CN 202310884943 A CN202310884943 A CN 202310884943A CN 116942836 A CN116942836 A CN 116942836A
- Authority
- CN
- China
- Prior art keywords
- liposome
- drug delivery
- prepared
- dmtmm
- solution
- 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
- 238000000034 method Methods 0.000 title claims abstract description 32
- 231100000053 low toxicity Toxicity 0.000 title abstract description 8
- 238000002659 cell therapy Methods 0.000 title abstract description 5
- 239000002502 liposome Substances 0.000 claims abstract description 53
- BMTZEAOGFDXDAD-UHFFFAOYSA-M 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-ium;chloride Chemical compound [Cl-].COC1=NC(OC)=NC([N+]2(C)CCOCC2)=N1 BMTZEAOGFDXDAD-UHFFFAOYSA-M 0.000 claims abstract description 27
- 238000012377 drug delivery Methods 0.000 claims abstract description 22
- 238000002360 preparation method Methods 0.000 claims abstract description 13
- 239000003814 drug Substances 0.000 claims abstract description 9
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 6
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 6
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 239000008367 deionised water Substances 0.000 claims description 12
- 229910021641 deionized water Inorganic materials 0.000 claims description 12
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 11
- -1 azide-polyethylene Chemical group 0.000 claims description 11
- MWRBNPKJOOWZPW-CLFAGFIQSA-N dioleoyl phosphatidylethanolamine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-CLFAGFIQSA-N 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- KSXTUUUQYQYKCR-LQDDAWAPSA-M 2,3-bis[[(z)-octadec-9-enoyl]oxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCC(=O)OCC(C[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC KSXTUUUQYQYKCR-LQDDAWAPSA-M 0.000 claims description 5
- 239000002202 Polyethylene glycol Substances 0.000 claims description 3
- 229920001223 polyethylene glycol Polymers 0.000 claims description 3
- 230000009471 action Effects 0.000 claims description 2
- 229910021529 ammonia Inorganic materials 0.000 claims description 2
- 238000006116 polymerization reaction Methods 0.000 claims description 2
- 230000008878 coupling Effects 0.000 abstract description 10
- 238000010168 coupling process Methods 0.000 abstract description 10
- 238000005859 coupling reaction Methods 0.000 abstract description 10
- 229940079593 drug Drugs 0.000 abstract description 7
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 230000008569 process Effects 0.000 abstract description 5
- 239000000243 solution Substances 0.000 description 18
- 238000001890 transfection Methods 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 12
- 108020004999 messenger RNA Proteins 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 239000013067 intermediate product Substances 0.000 description 7
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 6
- 238000001415 gene therapy Methods 0.000 description 6
- 239000007810 chemical reaction solvent Substances 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 150000001718 carbodiimides Chemical class 0.000 description 4
- 230000003833 cell viability Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 3
- 108020004459 Small interfering RNA Proteins 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003431 cross linking reagent Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- LDGWQMRUWMSZIU-LQDDAWAPSA-M 2,3-bis[(z)-octadec-9-enoxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)C)OCCCCCCCC\C=C/CCCCCCCC LDGWQMRUWMSZIU-LQDDAWAPSA-M 0.000 description 2
- 239000012124 Opti-MEM Substances 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000007822 coupling agent Substances 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 239000004971 Cross linker Substances 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 239000004593 Epoxy Chemical class 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 238000011072 cell harvest Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- 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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
-
- 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
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
-
- 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
- A61K47/54—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 an organic compound
- A61K47/545—Heterocyclic compounds
-
- 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
- A61K47/6905—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 colloid or an emulsion
- A61K47/6911—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 colloid or an emulsion the form being a liposome
-
- 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
- A61K9/127—Liposomes
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dispersion Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Medicinal Preparation (AREA)
Abstract
本发明提供一种低毒、高效的细胞治疗用载体的制造方法,具体涉及提供DMTMM在制备药物递送脂质体中的应用,一种药物递送脂质体的制备方法,前述的制备方法制备得到的药物递送脂质体,前述的药物递送脂质体在制备核酸药物中的应用。本发明提供的制备方法制备得到的脂质体毒性低且与生物分子偶联的获取率高。本方法安全性高、获得量大、工艺难度低,适于推广应用。
Description
技术领域
本发明属于生物医药领域,具体涉及一种低毒、高效的细胞治疗用载体及其制造方法和应用。
背景技术
基因治疗是将人的正常基因或有治疗作用的基因通过一定方式导入人体靶细胞以纠正基因的缺陷或者发挥治疗作用,从而达到治疗疾病目的的生物医学高技术。目前利用“表达某种基因”、“表达某种蛋白”进行治疗主要通过质粒DNA、mRNA的导入来实现,利用“抑制某种基因”进行治疗则主要通过siRNA或微小RNA来实现。基于siRNA、mRNA的基因治疗手段具有其他类型核酸药物所没有的独特优势,然而mRNA和siRNA开发的共同难点就是如何将其有效地递送到靶部位的细胞中去。
脂质体以其灵活的物理化学和生物物理性质,作为一种重要的潜在的关键药物输送系统而继续被研究。脂质体偶联药物分子数量是治疗的关键,增加脂质体偶联药物分子数量,进而提高药物分子的表达量,从而达到提高治疗的效果的目的。
通过脂质体载药的基因治疗在细胞治疗中是非常重要的,而它的安全性高,毒性低是最重要的。同时还需要其获得率高,制备容易。
目前作为基因治疗用递送系统(如脂质体、LNP等)的生产方式缺点明显,导致相关基因治疗的安全性风险大,传统方法包括两种(一步法、两步法),其不足如下:
1)以碳二亚胺(EDC)为交联剂的一步法,原理是首先与羧基反应形成中间产物,中间产物与氨基反应,然后脱去中间产物,实现羧基与氨基的偶联。
2)以碳二亚胺(EDC)与N-羟基琥珀酰亚胺(NHS)为交联剂的两步法。原理是首先与羧基反应,然后与NHS反应形成中间产物,中间产物与氨基反应,然后脱去中间产物,实现羧基与氨基的偶联。
上述两种方法对pH要求较高,偶联效率较低,值得一提的是,传统方法所使用的反应溶剂(醇类、醛类、环氧类)等具有较大的生物毒性,残留基团多且杂,对产品带来较大风险,废弃物处理给企业、环境带来较大的压力。在临床上往往伴随着不良的副作用,用这种方法制备的脂质体,能实现更加的安全及低毒性。
发明内容
本发明的目的在于提供一种低毒性的脂质体与生物分子偶联的高获取率的方法。本方法安全性高、获得量大、工艺难度低,适于推广应用。
为达到上述目的,本方法所采用的技术方案如下:
本发明的第一个目的是提供DMTMM在制备药物递送脂质体中的应用。
本发明的第二个目的是提供一种药物递送脂质体的制备方法,所述制备方法包括以下步骤:
(1)DMTMM溶液配制:取DMTMM(4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐)溶解于去离子水中,配置成1-2M的DMTMM溶液;
(2)脂质体的制备:取氯化三甲基-2,3-二油烯氧基丙基铵(DOTMA)与二油酰基磷脂酰乙醇胺(DOPE)混合制备脂质体;
(3)十二烷基硫酸钠溶液配制:取十二烷基硫酸钠溶解于去离子水中,配置成0.1-0.5%的溶液;
(4)NH2-PEG-N3溶液配制:取NH2-PEG-N3(叠氮-聚乙二醇-氨)溶解于去离子水中,配置成0.1-1M的溶液。
(5)脂质体与叠氮-聚乙二醇-氨基结合:将步骤(2)制备的脂质体与与步骤(3)配制的十二烷基硫酸钠溶液混合后,与步骤(4)制备的叠氮-聚乙二醇-氨基在步骤(1)配制的DMTMM溶液的作用下结合生成所述药物递送脂质体。
进一步的,步骤(2)所述氯化三甲基-2,3-二油烯氧基丙基铵与二油酰基磷脂酰乙醇胺的摩尔比为1:1-1:3。
进一步的,步骤(4)中所述叠氮-聚乙二醇-氨基中聚乙二醇的聚合度为10~23。
进一步的,步骤(5)中所述步骤(2)制备的脂质体与叠氮-聚乙二醇-氨基的摩尔比为1:1-1:3。
进一步的,步骤(5)中所述步骤(2)制备的脂质体与步骤(1)配制的DMTMM溶液的体积比为1:1-1:5。
进一步的,步骤(5)中脂质体与十二烷基硫酸钠溶液的体积比为1:1。
进一步的,步骤(2)及步骤(5)中的混合操着均为过夜搅拌混合。
本发明的第三个目的是提供采用前述的制备方法制备得到的药物递送脂质体。
本发明的第四个目的是提供前述的药物递送脂质体在制备核酸药物中的应用。
进一步的,将所述药物递送脂质体搭载核酸。
有益效果
DMTMM作为酰胺化,酯化,苷化和磷酸化反应中的高效缩合剂使用。相对于一些常有缩合剂,这种DMTMM具有更高的选择性。在DMTMM参与的反应,可以以水或者醇作为反应溶剂;相对于羟基,DMTMM对羧基选择性非常高。
采用DMTMM作为高效交联剂,相比于传统的EDC/NHS体系活性更高,可接受水或醇类作为反应溶剂,对pH要求低,偶联效率高,残留基团少,生物惰性(毒性小、抑制细胞粘附与免疫反应)。DMTMM作为高效交联剂,在大规模生产中的应用场景少,传统制造企业的保守策略导致相关技术在商业化中的研发投入少。本发明提供一套成熟、可行的高效、低毒性的基因治疗药物的生产方法。
附图说明
图1细胞转染前后细胞对比;
图2细胞转染后收获细胞活率对比;
图3对照组与转染组蛋白A表达量对比图;
图4不同添加物培养细胞图片;
图5不同添加物培养细胞收获活率;
图6不同溶剂下不同偶联剂偶联成功的收获率。
具体实施方式
实施例1药物递送脂质体的制备及转染
(1)DMTMM试剂配制:用电子天平称取3g的DMTMM加入50ml的离心管内,加入8ml的去离子水溶解,最后用去离子水定容至10ml,所述DMTMM溶液的摩尔浓度为1.09M。
(2)脂质体的制备:DOTMA(氯化三甲基-2,3-二油烯氧基丙基铵)与二油酰基磷脂酰乙醇胺(DOPE)按摩尔比1:1混合(每组各取5M进行1:1混合)。
(3)十二烷基硫酸钠溶液配制:取十二烷基硫酸钠0.25g加入至50ml离心管内,加入30ml离子水溶解,最后用去离子水定容至50ml,配置成0.5%的溶液;
(4)NH2-PEG-N3溶液配制:用电子天平称取0.4g的NH2-PEG-N3加入5ml的离心管内,加入1ml去离子水溶解,最后用去离子水定容至4ml,所述NH2-PEG-N3溶液的摩尔浓度为0.1M。
(5)脂质体与叠氮-聚乙二醇-氨基结合:取一只5ml的离心管,加入50μL以上(2)制备的脂质体,加入50μL浓度为0.5%十二烷基硫酸钠溶液(由去离子水配制)用枪头轻轻混合,再在悬浮液中加入50ul的DMTMM和1.25ml的0.1M的NH2-PEG-N3(分子量1000),在800-1000转速的摇床中摇晃2-3h,反应得到聚乙二醇化的脂质体。
(6)带有目标mRNA(能表达目标蛋白A:具有治疗作用)的脂质体的制备:取3ug的DBCO-mRNA,用Opti-MEM稀释至375ul于0.5ml的离心管内;再取18ul以上(5)制备的聚乙二醇化的脂质体,用Opti-MEM稀释至375ul于另一只0.5ml的离心管内。用枪头轻轻吹打混合以上2种稀释液共750ul于一只1.5ml的离心管内,室温静置5-10min备用。
(7)MSC细胞转染及目标蛋白A检测:准备汇合度60%-80%的MSC细胞6孔板1块,取以上(6)制备的试剂750ul,加入准备好的6孔板内,每个孔245ul,共3个孔,此3孔记为转染组(转染此方法制备得到的带目标mRNA的脂质体),分别标记组1,组2,组3。另外3个孔不做处理,此3孔记为对照组,分别标记组1,组2,组3。取3孔加入常规制备的带目标mRNA的脂质体进行转染,此3孔记为常规转染组,分别标记组1,组2,组3。将2块6孔板放入37℃培养箱5%CO2条件下培养48h观察及收获,收获时转染组细胞活率与对照组差别不大均在92-95%左右,常规转染组细胞活率在87-89%左右,相对较低,如图2所示。转染培养48h后的细胞与转染前的细胞状态差别不大,常规转染组转染培养48h后的细胞相对于转染前状态稍差,说明了用此方法制备的带有目标mRNA的脂质体具有安全性。如图1所示(转染后48h图中细胞表现为带目标mRNA的脂质体转染成功)。检测目标mRNA表达的蛋白A表达量,相较于对照组与常规转染组,转染组所表达的目标蛋白A大大增加,如图3所示,说明此方法制备的脂质体大大的增加了目标mRNA的携带量。
实施例2药物递送脂质体的安全性验证
分别用DMEM/F-12完全培养基在37℃及5%CO2条件下培养MSC细胞,正常培养下不做任何处理为对照组,相对于对照组内添加DMTMM,相对于对照组内添加DMTMM制备的脂质体共3组,每组做3组平行分别为组1,组2,组3,在培养过程的第3天观察细胞状态如图4所示,3组不同培养下细胞形态差别不大。培养相同时间(第3天)收获细胞,细胞活率没有明显差别,如图5所示。显示本发明采用的DMTMM制备的药物递送脂质体生物相容性好。
实施例3不同溶剂下不同偶联剂偶联成功的收获率验证
以含羧基基团的有机物为原料,在醇溶液或水溶液作为反应溶剂条件下,各分3组分别加入DMTMM,DCC,EDC在常温下过夜搅拌,各组偶联收获率在0-98%左右,如图6所示,说明在醇溶液及水溶液作为反应溶剂条件下DMTMM的偶联收获率最高。
Claims (10)
1.DMTMM在制备药物递送脂质体中的应用。
2.一种药物递送脂质体的制备方法,其特征在于,所述制备方法包括以下步骤:
(1)DMTMM溶液配制:取DMTMM溶解于去离子水中,配置成1-2M的DMTMM溶液;
(2)脂质体的制备:取氯化三甲基-2,3-二油烯氧基丙基铵与二油酰基磷脂酰乙醇胺混合制备脂质体;
(3)十二烷基硫酸钠溶液配制:取十二烷基硫酸钠溶解于去离子水中,配置成0.1-0.5%的溶液;
(4)叠氮-聚乙二醇-氨(NH2-PEG-N3)溶液配制:取NH2-PEG-N3溶解于去离子水中,配置成0.1-1M的溶液。
(5)脂质体与叠氮-聚乙二醇-氨基结合:将步骤(2)制备的脂质体与步骤(3)配制的十二烷基硫酸钠溶液混合后,与步骤(4)制备的叠氮-聚乙二醇-氨基在步骤(1)配制的DMTMM溶液的作用下结合生成所述药物递送脂质体。
3.根据权利要求2所述的药物递送脂质体的制备方法,其特征在于,步骤(2)所述氯化三甲基-2,3-二油烯氧基丙基铵与二油酰基磷脂酰乙醇胺的摩尔比为1:1-1:3。
4.根据权利要求2所述的药物递送脂质体的制备方法,其特征在于,步骤(4)中所述叠氮-聚乙二醇-氨基中聚乙二醇的聚合度为10~23。
5.根据权利要求2所述的药物递送脂质体的制备方法,其特征在于,步骤(5)中所述步骤(2)制备的脂质体与叠氮-聚乙二醇-氨基的摩尔比为1:1-1:3。
6.根据权利要求2所述的药物递送脂质体的制备方法,其特征在于,步骤(5)中所述步骤(2)制备的脂质体与步骤(1)配制的DMTMM溶液的体积比为1:1-1:5。
7.根据权利要求2所述的药物递送脂质体的制备方法,其特征在于,步骤(5)中脂质体与十二烷基硫酸钠溶液的体积比为1:1。
8.采用权利要求2所述的制备方法制备得到的药物递送脂质体。
9.权利要求8所述的药物递送脂质体在制备核酸药物中的应用。
10.根据权利要求9所述的应用,其特征在于,将所述药物递送脂质体搭载核酸。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310884943.4A CN116942836A (zh) | 2023-07-19 | 2023-07-19 | 一种低毒、高效的细胞治疗用载体的制造方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310884943.4A CN116942836A (zh) | 2023-07-19 | 2023-07-19 | 一种低毒、高效的细胞治疗用载体的制造方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116942836A true CN116942836A (zh) | 2023-10-27 |
Family
ID=88454189
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310884943.4A Pending CN116942836A (zh) | 2023-07-19 | 2023-07-19 | 一种低毒、高效的细胞治疗用载体的制造方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116942836A (zh) |
-
2023
- 2023-07-19 CN CN202310884943.4A patent/CN116942836A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7829657B2 (en) | Polycationically charged polymer and the use of the same as a carrier for nucleic acid | |
JP2024507482A (ja) | イオン化可能な脂質分子、その製造方法及び脂質ナノ粒子の製造における応用 | |
CN110746599B (zh) | 具有高效基因递送能力的UV光响应性超支化聚β-氨基酯及其制备方法与应用 | |
CN113999846B (zh) | 一种抑制afap1-as1表达的干扰rna及在增加乳腺癌放疗敏感性中的应用 | |
CN111718494A (zh) | 具有高效基因递送能力的还原响应性超支化聚β-氨基酯及其制备方法与应用 | |
CN112142972A (zh) | 修饰的聚乙烯亚胺衍生物及其合成方法和应用 | |
CN102464801B (zh) | 一种阳离子聚合物及其制备方法和用途 | |
CN114904003B (zh) | 可电离的阳离子脂质类似物材料在作为核酸药物递送载体或转染试剂中的应用 | |
CN109157514B (zh) | 一种以脂肪酸为膜材的阳离子脂质体及其制备方法和应用 | |
US9970002B2 (en) | Compositions and methods for functional nucleic acid delivery | |
CN109988780B (zh) | 基于甲基丙烯酸缩水甘油酯的高性能基因载体及其应用 | |
CN116942836A (zh) | 一种低毒、高效的细胞治疗用载体的制造方法 | |
CN109550057B (zh) | 主动靶向型基因递送纳米粒及其制备方法和应用 | |
CN106188223B (zh) | 一种含有二肽类脂质阳离子的化合物及其制备方法与应用 | |
CN116574070A (zh) | 一种多尾型可电离脂质及其制备方法与应用 | |
CN115521220B (zh) | 长链烷基酯胺类化合物及其制备方法和在核酸递送方面的应用 | |
CN116510027A (zh) | 一种基于“多氨基-多环氧”开环反应构建的多羟基支化阳离子聚合物在构建疫苗递送系统上应用 | |
CN106086069B (zh) | 一种转基因复合物及其制备方法与应用 | |
CN111944851A (zh) | 一种纳米尺度siRNA递送系统 | |
CN112336702B (zh) | 一种药物载体及核酸类药物制剂 | |
CN104497168B (zh) | 一种壳聚糖氨基烷基化衍生物 | |
CN111655758B (zh) | 核酸投递用聚合物 | |
CN112843246B (zh) | 含cbx3的l-精氨基氨酸化聚胺类聚合物基因载体的制备方法及其应用 | |
CN113755528B (zh) | 软骨靶向肽修饰的两亲性高分子聚合物基因载体及其制备方法和应用 | |
CN114874422B (zh) | 一种聚烷基胺、其合成方法、颗粒及用途 |
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 |