JPS63275522A - Artificial erythrocyte and production thereof - Google Patents
Artificial erythrocyte and production thereofInfo
- Publication number
- JPS63275522A JPS63275522A JP62108589A JP10858987A JPS63275522A JP S63275522 A JPS63275522 A JP S63275522A JP 62108589 A JP62108589 A JP 62108589A JP 10858987 A JP10858987 A JP 10858987A JP S63275522 A JPS63275522 A JP S63275522A
- Authority
- JP
- Japan
- Prior art keywords
- liposome
- ganglioside
- liposomes
- red blood
- gangliosides
- 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
- 238000004519 manufacturing process Methods 0.000 title claims description 9
- 239000003633 blood substitute Substances 0.000 title abstract 2
- 239000002502 liposome Substances 0.000 claims abstract description 48
- 150000002270 gangliosides Chemical class 0.000 claims abstract description 41
- 150000002632 lipids Chemical class 0.000 claims abstract description 32
- 210000004369 blood Anatomy 0.000 claims abstract description 18
- 239000008280 blood Substances 0.000 claims abstract description 18
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims abstract description 12
- JQWAHKMIYCERGA-UHFFFAOYSA-N (2-nonanoyloxy-3-octadeca-9,12-dienoyloxypropoxy)-[2-(trimethylazaniumyl)ethyl]phosphinate Chemical compound CCCCCCCCC(=O)OC(COP([O-])(=O)CC[N+](C)(C)C)COC(=O)CCCCCCCC=CCC=CCCCCC JQWAHKMIYCERGA-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000000203 mixture Substances 0.000 claims abstract description 8
- 235000012000 cholesterol Nutrition 0.000 claims abstract description 6
- 210000003743 erythrocyte Anatomy 0.000 claims description 32
- 230000008034 disappearance Effects 0.000 claims description 10
- 239000000725 suspension Substances 0.000 claims description 8
- 238000007796 conventional method Methods 0.000 claims description 7
- 230000015572 biosynthetic process Effects 0.000 claims 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 abstract description 12
- 102000001554 Hemoglobins Human genes 0.000 abstract description 10
- 108010054147 Hemoglobins Proteins 0.000 abstract description 10
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 abstract description 8
- 239000002904 solvent Substances 0.000 abstract description 6
- 239000000126 substance Substances 0.000 abstract description 4
- 239000007864 aqueous solution Substances 0.000 abstract description 3
- 239000010409 thin film Substances 0.000 abstract description 3
- 210000000170 cell membrane Anatomy 0.000 abstract description 2
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical group O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 abstract 1
- 102000002322 Egg Proteins Human genes 0.000 abstract 1
- 108010000912 Egg Proteins Proteins 0.000 abstract 1
- 244000258044 Solanum gilo Species 0.000 abstract 1
- 235000013345 egg yolk Nutrition 0.000 abstract 1
- 210000002969 egg yolk Anatomy 0.000 abstract 1
- 230000008030 elimination Effects 0.000 abstract 1
- 238000003379 elimination reaction Methods 0.000 abstract 1
- 230000014759 maintenance of location Effects 0.000 abstract 1
- 210000000944 nerve tissue Anatomy 0.000 abstract 1
- 239000008346 aqueous phase Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000002504 physiological saline solution Substances 0.000 description 6
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 4
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 4
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 230000008020 evaporation Effects 0.000 description 3
- 239000000693 micelle Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000012736 aqueous medium Substances 0.000 description 2
- 239000003012 bilayer membrane Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 210000000865 mononuclear phagocyte system Anatomy 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 1
- 239000000592 Artificial Cell Substances 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 1
- 210000004268 dentin Anatomy 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 108010042430 galactose receptor Proteins 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229940113478 lecithin 200 mg Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 125000005629 sialic acid group Chemical group 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Dispersion Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
Abstract
Description
【発明の詳細な説明】
［産業上の利用分野］
本発明は、人工赤血球及びその製造方法に関する。特に
、循環血中での消失速度が減少された人工赤血球及びそ
の製造方法に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an artificial red blood cell and a method for producing the same. In particular, the present invention relates to an artificial red blood cell whose rate of disappearance in circulating blood is reduced and a method for producing the same.
本発明の人工赤血球は、血中滞留時間が長いので、極め
て有用である。The artificial red blood cells of the present invention have a long residence time in blood and are therefore extremely useful.
［従来の技術］
リン脂質二分子層膜小胞体であるリポソームは、人工細
胞、薬物運搬体のみならず人工赤血球の分野においても
その応用が期待されている。[Prior Art] Liposomes, which are phospholipid bilayer membrane endoplasmic reticulum, are expected to find application not only in the fields of artificial cells and drug carriers but also in the field of artificial red blood cells.
しかし、リポソームは、血中投与されると生体の細網内
皮系等の免疫細胞により異物として認識され、循環血流
中から速やかに排除されてしまうという問題がある。特
に、人工赤血球として用いる場合には長い血中滞留時間
が必要とされるので、致命的な問題であった。However, there is a problem in that when liposomes are administered into the blood, they are recognized as foreign substances by immune cells such as the reticuloendothelial system of the living body, and are rapidly eliminated from the circulating bloodstream. In particular, when used as artificial red blood cells, a long residence time in the blood is required, which is a fatal problem.
医薬等の運搬体としてのリポソームについては、シアル
酸残基で表面を修飾することによって細網内皮系による
認識を抑制して血中半減期を増大する技術が公知である
（米国特許第４．５０１．７２８号公報）が、人工赤血
球については未だ解決法が見出されていない。Regarding liposomes as carriers of medicines, a technique is known in which the surface of liposomes is modified with sialic acid residues to suppress recognition by the reticuloendothelial system and increase the half-life in the blood (U.S. Patent No. 4. No. 501.728), but no solution has yet been found regarding artificial red blood cells.
［発明が解決しようとする問題点コ
本発明は上記従来のリポソームよりなる人工赤血球の欠
点を解決せんとするものであり、本発明の目的は循環血
中での消失速度が減少された人工赤血球とその製造法を
提供することにある。[Problems to be Solved by the Invention] The present invention aims to solve the above-mentioned drawbacks of the conventional artificial red blood cells made of liposomes, and the purpose of the present invention is to provide artificial red blood cells whose disappearance rate in circulating blood is reduced. and its manufacturing method.
［問題点を解決するための手段］
本発明の目的は、以下の構成により達成することができ
る。[Means for Solving the Problems] The object of the present invention can be achieved by the following configuration.
■） リポソームの脂質層にガングリオシドを含有させ
ることによって循環血中での消失速度が減少された人工
赤血球。■) Artificial red blood cells whose disappearance rate in circulating blood is reduced by including gangliosides in the lipid layer of liposomes.
２）ガングリオシドが、ＧＭＩ、ＧＭ２．０Ｍ３゜ＧＤ
ｌａ、ＧＤ１ｂ、ＧＴ１ｂまたはＣ；Ｑ１ｂである前記
第１項に記載の人工赤血球。2) Ganglioside is GMI, GM2.0M3°GD
The artificial red blood cell according to item 1 above, which is la, GD1b, GT1b, or C;Q1b.
３） リポソーム形成脂質が、卵黄レシチンまたは水素
添加卵黄レシチンとコレステロールとの混合物である前
記第１項または第２項に記載の人工赤血球。3) The artificial red blood cell according to item 1 or 2, wherein the liposome-forming lipid is egg yolk lecithin or a mixture of hydrogenated egg yolk lecithin and cholesterol.
４） リポソーム形成脂質とガングリオシドを予じめ均
一に混合して得られた混合脂質を用いて常法によりリポ
ソームを形成することを特徴とするリポソームの脂質層
にガングリオシドを含有させることによって循環血中で
の消失速度が減少された人工赤血球の製造方法。4) Liposome-forming liposomes are formed by a conventional method using a mixed lipid obtained by uniformly mixing lipids and gangliosides in advance. A method for producing artificial red blood cells whose disappearance rate is reduced.
５）常法により製造されたリポソームの懸濁液にガング
リオシドを添加することを特徴とするリポソームの脂質
層にガングリオシドを含有させることによって循環血中
での消失速度が減少された人工赤血球の製造方法。5) A method for producing artificial red blood cells whose disappearance rate in circulating blood is reduced by including gangliosides in the lipid layer of liposomes, which comprises adding gangliosides to a suspension of liposomes produced by a conventional method. .
リポソームは、脂質人工膜の一種であって脂質の二分子
層膜よりなる小胞体である。本発明において使用される
リポソームは、特に、人工赤血球のリポソームであり、
その内水相にヘモグロビンを含有する。Liposomes are a type of artificial lipid membrane and are endoplasmic reticulum consisting of a bilayer membrane of lipids. The liposomes used in the present invention are especially liposomes of artificial red blood cells,
It contains hemoglobin in its internal aqueous phase.
本発明において使用されるリポソームを形成する脂質は
、天然のリン歯質、合成の脂質類縁体等、リポソーム（
またはベシクル）を形成し得るものであれば特に限定さ
れるものではないが、卵黄レシチンまたは水素添加卵黄
レシチンとコレステロールとの混合物が好ましい。Lipids forming the liposomes used in the present invention include natural phosphorus dentin, synthetic lipid analogs, etc.
Although there are no particular limitations on the material as long as it can form vesicles), egg yolk lecithin or a mixture of hydrogenated egg yolk lecithin and cholesterol is preferred.
本発明で使用されるガングリオシドは、シアル酸を含有
する糖脂質群をいい、神経組織の細胞膜に存在する。本
発明で使用されるのは、中でもモノ、ジ、トリおよびテ
トラシアル酸付加ガングリオシドであって、５ｖｅｎｎ
ｅｒｈａｌ塵の命名によればＧＭｌ、ＧＭ２，０Ｍ３．
ＧＤｌａ、ＧＤ１ｂ。Gangliosides used in the present invention refer to a group of glycolipids containing sialic acid, and are present in the cell membranes of nervous tissues. Among those used in the present invention are mono-, di-, tri- and tetrasialylated gangliosides, including 5venn
According to the nomenclature of erhal dust, GMl, GM2, 0M3.
GDla, GD1b.
ＧＴ１ｂ、ＧＱ１ｂである。Ｇはガングリオシドを、Ｍ
、Ｄ、Ｔ及びＱはそれぞれモノシア口、ジシアロ、トリ
ジアロ及びテトラシア口を意味する。GT1b and GQ1b. G is ganglioside, M
, D, T and Q mean monosialo, disialo, tridialo and tetrasialo, respectively.
本発明においては上記ガングリオシドの中の１種または
２種以上の混合物を用いる。一般には、糖末端にガラク
トースを有するガングリオシド例えばＧＭｌ、ＧＭ２．
ＧＤ１ｂは、ガラクトースレセプターを有する食細胞の
食作用を受は易くなる可能性がある。従って末端がシア
ル酸である０Ｍ３．ＧＤｌａ、ＧＴ１ｂが望ましい。In the present invention, one type or a mixture of two or more of the above gangliosides is used. Generally, gangliosides having galactose at the sugar end, such as GMl, GM2.
GD1b may be more susceptible to phagocytosis by phagocytes having galactose receptors. Therefore, 0M3. whose terminal end is sialic acid. GDla and GT1b are preferable.
本発明の人工赤血球を製造するには、リポソーム形成脂
質およびガングリオシドをともに溶解し得る溶媒中に両
者を均一に溶解させる。リポソーム形成脂質に対するガ
ングリオシドのモル比は、０．５％乃至１０％１ｆｆｉ
比で１％乃至２０％）であり、２％乃至５％（ｆｆｉ量
比で４％乃至１０％）が望ましい。人工赤血球中のガン
グリオシドの含有量は、上記範囲未満である場合にはシ
アル酸による異物認識抑制の効果が発現されに（く、上
記範囲を越える場合には、ガングリオシドの界面活性作
用によってリポソームが不安定となり易い。上記のリポ
ソーム形成脂質およびガングリオシドをともに溶解し得
る溶媒には例えばクロロホルムとメタノール、ジクロロ
メタンとメタノールの混合溶媒などがあり、中でもクロ
ロホルム−メタノールの２：１（体積比）の混合物は望
ましい。To produce the artificial red blood cells of the present invention, liposome-forming lipids and gangliosides are uniformly dissolved in a solvent that can dissolve both. The molar ratio of ganglioside to liposome-forming lipids ranges from 0.5% to 10%1ffi.
1% to 20% in terms of ratio), and preferably 2% to 5% (4% to 10% in terms of ffi amount ratio). If the content of gangliosides in artificial red blood cells is less than the above range, the effect of suppressing foreign body recognition by sialic acid will not be expressed (if it exceeds the above range, liposomes will be damaged by the surfactant action of gangliosides). Easily stabilized. Examples of solvents that can dissolve both the liposome-forming lipid and ganglioside include mixed solvents of chloroform and methanol, and dichloromethane and methanol, among which a 2:1 (volume ratio) mixture of chloroform and methanol is preferred. .
上記のように溶解した後、溶媒を蒸発除去し、得られた
混合脂質薄膜にヘモグロビンの１０〜６０％水溶液を加
えて振盪、超音波照射またはフレンチプレス処理する。After dissolving as described above, the solvent is removed by evaporation, and a 10-60% aqueous solution of hemoglobin is added to the obtained mixed lipid thin film, followed by shaking, ultrasonic irradiation, or French press treatment.
この方法によれば、ガングリオシド脂質部分がリポソー
ム脂質層中に固定され、その糖鎖部分がリポソームの内
水相及び外水相表面に露出した構造のガングリオシド含
有リポソームが得られる。According to this method, a ganglioside-containing liposome with a structure in which the ganglioside lipid moiety is fixed in the liposome lipid layer and the sugar chain moiety thereof is exposed on the inner and outer aqueous phase surfaces of the liposome can be obtained.
また本発明の人工赤血球を製造するには、ヘモグロビン
をリポソーム内水相に含有するリポソーム（水溶性及び
脂溶性薬物等を含有するものであってもよい）を常法に
より形成させた後に、得られたリポソームの緩衝液等水
性媒体中の懸濁液中にガングリオシドを上記と同様の量
で添加し、撹拌することによりガングリオシドをリポソ
ームに含有させてもよい。ガングリオシドは水性媒体中
でミセルを形成して分散しているが、ここにリポソーム
が共存すれば疎水的相互作用によってガンクリオシドの
脂質部分がリポソームの脂’Ｅｌ中に取り込まれる。ま
たこの方法によれば、ガングリオシドの糖鎖部分がリポ
ソームの外水相側表面のみに露出した人工赤血球を得る
ことができる。Furthermore, in order to produce the artificial red blood cells of the present invention, liposomes containing hemoglobin in the liposome aqueous phase (which may contain water-soluble and fat-soluble drugs, etc.) are formed by a conventional method, and then the obtained The ganglioside may be incorporated into the liposome by adding ganglioside in the same amount as above to a suspension of the obtained liposome in an aqueous medium such as a buffer solution and stirring. Gangliosides form micelles and are dispersed in an aqueous medium, and if liposomes coexist in these micelles, the lipid moiety of gangliosides is incorporated into the lipid 'El' of the liposomes due to hydrophobic interactions. Furthermore, according to this method, it is possible to obtain artificial red blood cells in which the sugar chain moiety of ganglioside is exposed only on the outer aqueous phase surface of the liposome.
以下本発明を実施例及び比較例によって詳しく説明する
。The present invention will be explained in detail below with reference to Examples and Comparative Examples.
実施例 １
水素添加卵黄レシチン２００ ｔａｇ、コレステロール
１００ｍｇ及び牛脳から採取された精製ガングリオシド
（Ｇ Ｄ １ ａ）２０ＩＩｇをクロロホルム−メタノ
ール混合物（体積比２ ： １） ２０ｍ１中に溶解し
、エバポレーションによって有機溶媒を除去した。この
混合脂質薄膜に螢光色素カルボキシフルオレセインを２
０ｄの濃度で含有する３０％ヘモグロビン水溶液（１）
１１７．４）２０ｍｌを加えて振盪し、脂質を分散させ
た後、２５０ｋｇ／ｃ−の圧力でフレンチプレス処理を
５回繰り返した。得られたフレンチプレス処理液を生理
食塩水により１０倍に希釈して遠心分離処理（１７，０
００ｒｐｉ ３０９） Ｌ、沈澱リポソームを生理食塩
水４０ｍ１により遠心洗浄を２回繰り返した。得られた
沈澱リポソームを生理食塩水１０ｍ１中に分散させてヘ
モグロビンとカルボキシフルオレセインを内水相に保持
したガングリオシド修飾リポソームからなる人工赤血球
の懸濁液を得た。Example 1 200 tags of hydrogenated egg yolk lecithin, 100 mg of cholesterol, and 20 II g of purified ganglioside (G D 1 a) collected from bovine brain were dissolved in 20 ml of a chloroform-methanol mixture (volume ratio 2:1), and organic matter was extracted by evaporation. Solvent was removed. Two fluorescent dyes, carboxyfluorescein, were added to this mixed lipid thin film.
30% hemoglobin aqueous solution containing at a concentration of 0d (1)
117.4) After adding 20 ml and shaking to disperse the lipids, the French press treatment was repeated 5 times at a pressure of 250 kg/c-. The resulting French press treatment solution was diluted 10 times with physiological saline and centrifuged (17,0
00 rpi 309) L, the precipitated liposomes were washed twice by centrifugation with 40 ml of physiological saline. The resulting precipitated liposomes were dispersed in 10 ml of physiological saline to obtain a suspension of artificial red blood cells consisting of ganglioside-modified liposomes retaining hemoglobin and carboxyfluorescein in the internal aqueous phase.
この懸濁液を脂質量で２ｈ＋ｇ／）ｃｇ体重の投与量と
して体重２７０ｇのラットに静脈注射した後、経時的に
０．２ｍｌずつ採血した。各０．２ｍｌの血液に蒸留水
１．８ｍｌ及びイソプロピルアルコール２ｍｌを加え、
赤血球及びリポソームを破壊してヘモグロビンを変性さ
せた。遠心分離によって変性ヘモグロビン沈澱分を除去
して、上清中の螢光量を測定することにより人工赤血球
の血中半減期を求めたところ１．５時間であった。This suspension was intravenously injected into a rat weighing 270 g in a lipid amount of 2 h+g/)cg body weight, and then 0.2 ml of blood was collected over time. Add 1.8 ml of distilled water and 2 ml of isopropyl alcohol to each 0.2 ml of blood,
Hemoglobin was denatured by destroying red blood cells and liposomes. The half-life of the artificial red blood cells in the blood was determined to be 1.5 hours by removing the denatured hemoglobin precipitate by centrifugation and measuring the amount of fluorescence in the supernatant.
実施例 ２
水素添加卵黄レシチン２００ ｍｇとコレステロール１
００鳳ｇをクロロホルム２０ｍ１中に溶解し、エバポレ
ーション後、実施例１と同様のヘモグロビン溶液２０ｍ
１を加えて同様にフレンチプレス処理した。Example 2 Hydrogenated egg yolk lecithin 200 mg and cholesterol 1
00g was dissolved in 20ml of chloroform, and after evaporation, 20ml of the same hemoglobin solution as in Example 1 was added.
1 was added and subjected to the same French press treatment.
実施例１と同様に遠心洗浄した後、得られた沈澱リポソ
ームを、精製ガングリオシドＧＤ１ａｌＯ■をミセルと
して溶存する生理食塩水１０ｍ１中に懸濁させた。この
懸濁液を３７℃で３０分間撹拌してガングリオシドをリ
ポソーム表面に導入した。遠心分離処理（１７，００ｏ
ｒｐｍ、３０分間）によりリポソームを沈澱させ、得ら
れたリポソーム沈澱を生理食塩水４０ｍ１によって遠心
洗浄を２回行ないリポソームに導入されなかったガング
リオシドを除去した。最終的に得られた沈澱リポソーム
を生理食塩水１０ｍ１中に分散させ、ヘモグロビンとカ
ルボキシフルオレセインを内水相に保持したガングリオ
シド修飾リポソームからなる人工赤血球の懸濁液を得た
。After centrifugal washing in the same manner as in Example 1, the precipitated liposomes obtained were suspended in 10 ml of physiological saline in which purified ganglioside GD1alO was dissolved as micelles. This suspension was stirred at 37° C. for 30 minutes to introduce ganglioside onto the liposome surface. Centrifugation treatment (17,00o
rpm for 30 minutes), and the resulting liposome precipitate was centrifugally washed twice with 40 ml of physiological saline to remove gangliosides that were not introduced into the liposomes. The finally obtained precipitated liposomes were dispersed in 10 ml of physiological saline to obtain a suspension of artificial red blood cells consisting of ganglioside-modified liposomes retaining hemoglobin and carboxyfluorescein in the internal aqueous phase.
これについて実施例１と同様の動物実験を行なったとこ
ろ人工赤血球の血中半減期は１．５時間であった。Regarding this, animal experiments similar to those in Example 1 were conducted, and the half-life of the artificial red blood cells in blood was 1.5 hours.
比較例
実施例１のＧＤｌａのかわりにガングリオシドと同様の
負荷電脂質であるジパルミトイルホスファチジン酸２０
ｍｇを用いて、実施例１と同様の実験を行なったところ
、人工赤血球の半減期は１０分以下であった。Comparative Example Instead of GDla in Example 1, dipalmitoylphosphatidic acid 20, which is a negatively charged lipid similar to ganglioside, was used.
When an experiment similar to that in Example 1 was conducted using 1 mg, the half-life of the artificial red blood cells was 10 minutes or less.
［発明の効果コ
以上詳しく説明したように、本発明によればリポソーム
の脂質層にガングリオシドを含有させることによって循
環血中での消失速度が減少された人工赤血球が提供され
る。[Effects of the Invention] As explained in detail above, the present invention provides artificial red blood cells whose disappearance rate in circulating blood is reduced by incorporating gangliosides into the lipid layer of liposomes.
このガングリオシドのシアル酸を含む糖鎖部分がリポソ
ームの表面に露出していることによって、生体の免疫細
胞による異物としての認識が抑制される。その結果、循
環血中での消失速度が従来のリポソームに比べて著しく
遅く、滞留時間は長くなる。従って、特に長い血中滞留
時間が不可欠である人工赤血球として有用性が高い。Since the sugar chain moiety containing sialic acid of this ganglioside is exposed on the surface of the liposome, recognition as a foreign substance by the body's immune cells is suppressed. As a result, the rate of disappearance in the circulation is significantly slower than that of conventional liposomes, resulting in longer residence times. Therefore, it is particularly useful as an artificial red blood cell in which a long blood residence time is essential.
本発明の人工赤血球の製造方法は、リポソーム形成脂質
とガングリオシドを予じめ均一に混合した混合脂質から
通常の方法に従ってリポソームを形成させる方法及び通
常の方法に従ってリポソームを形成させた後にその懸濁
液にガングリオシドを添加する方法であるので、従来知
られているリポソームの応用技術のいずれの例にも広（
適用することができる。The method for producing artificial red blood cells of the present invention includes a method of forming liposomes according to a conventional method from a mixed lipid in which liposome-forming lipids and gangliosides are uniformly mixed in advance, and a method of forming liposomes according to a conventional method and then producing a suspension of the liposomes. Since this is a method of adding gangliosides to
Can be applied.
Claims (1)
ことによって循環血中での消失速度が減少された人工赤
血球。 ２）ガングリオシドが、ＧＭ１、ＧＭ２、ＧＭ３、ＧＤ
１ａ、ＧＤ１ｂ、ＧＴ１ｂまたはＧＱ１ｂである特許請
求の範囲第１項に記載の人工赤血球。 ３）リポソーム形成脂質が、卵黄レシチンまたは水素添
加卵黄レシチンとコレステロールとの混合物である特許
請求の範囲第１項または第２項に記載の人工赤血球。 ４）リポソーム形成脂質とガングリオシドを予め均一に
混合して得られた混合脂質を用いて常法によりリポソー
ムを形成することを特徴とするリポソームの脂質層にガ
ングリオシドを含有させることによって循環血中での消
失速度が減少された人工赤血球の製造方法。 ５）常法により製造されたリポソームの懸濁液にガング
リオシドを添加することを特徴とするリポソームの脂質
層にガングリオシドを含有させることによって循環血中
での消失速度が減少された人工赤血球の製造方法。[Scope of Claims] 1) An artificial red blood cell whose disappearance rate in circulating blood is reduced by including ganglioside in the lipid layer of the liposome. 2) Gangliosides are GM1, GM2, GM3, and GD.
The artificial red blood cell according to claim 1, which is 1a, GD1b, GT1b, or GQ1b. 3) The artificial red blood cell according to claim 1 or 2, wherein the liposome-forming lipid is egg yolk lecithin or a mixture of hydrogenated egg yolk lecithin and cholesterol. 4) Liposome formation Liposomes are formed by a conventional method using a mixed lipid obtained by uniformly mixing lipids and gangliosides in advance.By incorporating gangliosides into the lipid layer of the liposomes, it is possible to increase A method for producing artificial red blood cells with a reduced rate of disappearance. 5) A method for producing artificial red blood cells whose disappearance rate in circulating blood is reduced by including gangliosides in the lipid layer of liposomes, which comprises adding gangliosides to a suspension of liposomes produced by a conventional method. .
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62108589A JPS63275522A (en) | 1987-05-01 | 1987-05-01 | Artificial erythrocyte and production thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62108589A JPS63275522A (en) | 1987-05-01 | 1987-05-01 | Artificial erythrocyte and production thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS63275522A true JPS63275522A (en) | 1988-11-14 |
Family
ID=14488638
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62108589A Pending JPS63275522A (en) | 1987-05-01 | 1987-05-01 | Artificial erythrocyte and production thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63275522A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009104649A1 (en) * | 2008-02-22 | 2009-08-27 | 片山化学工業株式会社 | Synthetic glycolipid-containing liposomes |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57146710A (en) * | 1981-03-07 | 1982-09-10 | Kunio Yagi | Blood brain-barrier permeating ribosome-like microcapsule |
| JPS61274739A (en) * | 1985-04-04 | 1986-12-04 | フア−スト．ミシシツピ−．コ−ポレ−シヨン | Phospholipid vesicle and method for treating the same |
| JPH02151718A (en) * | 1988-12-05 | 1990-06-11 | Yamaha Corp | Rotary encoder |
-
1987
- 1987-05-01 JP JP62108589A patent/JPS63275522A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57146710A (en) * | 1981-03-07 | 1982-09-10 | Kunio Yagi | Blood brain-barrier permeating ribosome-like microcapsule |
| JPS61274739A (en) * | 1985-04-04 | 1986-12-04 | フア−スト．ミシシツピ−．コ−ポレ−シヨン | Phospholipid vesicle and method for treating the same |
| JPH02151718A (en) * | 1988-12-05 | 1990-06-11 | Yamaha Corp | Rotary encoder |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009104649A1 (en) * | 2008-02-22 | 2009-08-27 | 片山化学工業株式会社 | Synthetic glycolipid-containing liposomes |
| JP5465542B2 (en) * | 2008-02-22 | 2014-04-09 | 片山化学工業株式会社 | Synthetic glycolipid-containing liposome |
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