JPH0586372B2 - - Google Patents

Description

[Detailed description of the invention]

Liposomes, which are formed when lipids are dispersed in water, have a high approximation as a model of cell membrane structure, and can be used as tissue-directed drug carriers, artificial red blood cells,
It is expected to be actively used as a medical material such as a cell modifier and an oxygen fixation substrate. However, two major drawbacks have been pointed out in actively utilizing liposomes as artificial cells. That is, using naturally derived lipids,
This is due to structural instability due to the fact that it is a molecular assembly due to completely non-covalent interactions, that is, it has weak mechanical strength and lacks the ability to recognize specific cells or specific tissues. The present inventors have discovered that liposomes have these two
After repeated research to overcome this major drawback, by coating the surface of liposomes with naturally derived polysaccharide derivatives,
We succeeded in improving the mechanical strength of liposomes under physiological conditions and at the same time imparting specific organ tropism derived from the polysaccharide structure. Special request 1986
−147587 (Japanese Unexamined Patent Publication 1983-49311), Patent Application 1987-82993
(Japanese Patent Publication No. 58-201711), Patent Application No. 58-106683 (Japanese Patent Application
60-1124), and patent application No. 189746 (Japanese Patent Application No. 1989-1897)
69801) Reference. Based on such conventional knowledge, the present inventors have explored a completely new method of making sugar chains exist as recognition sites on the liposome surface instead of polysaccharide coating on the liposome surface, and improving the mechanical strength of the liposome. . As a result, we used newly developed and synthesized lipids with amide bonds in their molecules as boundary lipids, mixed them with naturally occurring lipids, and further reconstituted glycoproteins or mucoproteins extracted from mammalian tissues to form liposomes. At the same time as strengthening the structure, they succeeded in imparting a cell recognition function using a glycoprotein incorporated into the liposome membrane, leading to the completion of the present invention. As is clear from the above, the purpose of the present invention is to
This is a solution to the above problem, and the present invention discloses a method for producing a novel liposome specified in the present invention in order to achieve the above object. [Structure of the Invention] The present invention will be explained in detail below. (1) Synthesis of DDPC Synthetic lipid DDPC (1, 2
-dimyristoylamide-1,2-deoxyphosphatidylcholine) has already been developed and synthesized by the present inventors (Junzo Sunamoto, Mitsuaki Goto, Synthesis of phospholipid with amide bond and artificial Improving cellular functions, 6th Biomembrane-Drug Interaction Symposium (Kyoto) Abstracts, PP.168
(1983)) synthesized it roughly as follows. Aminating 2,3-dibromopropionic acid,
The generated diaminopropionic acid was protected as a methyl ester. This was reacted with myristoyl chloride to synthesize 2,3-dimylstoylpropionate methyl ester having an amide group in its side chain. This was reduced with lithium borohydride to synthesize 2,3-dimyristoylamidopropan-1-ol. A choline group was introduced into this to form the final target product.

【table】

【table】 ‖

O
(9)
1,2-dimyristoylamide-1,2-
Deoxyphosphatidylcholine
The final product was confirmed by NMR, IR and elemental analysis as shown in Table 1.

【table】

[Table] (2) Extraction and purification method of human and pig glycofurin Human glycofurin is obtained from human type A red blood cell concentrate, and pig glycofurin is obtained by first obtaining red blood cell ghosts from pig whole blood (Arch.Biochem.
Biophys. 100, 119-130 (1963)), and further isolated glycophorin (Science, 174, 1247-1248
(1981)). The isolated protein was developed by SDS-acrylamide gel electrophoresis and PAS-stained, and the average molecular weight was found to be approximately 70,000. (3) Extraction and purification method of sphingomyelin (SM) Sphingomyelin is extracted from pig brain (Tohoku J. Exp. Med., 98, 87-47 (1969). J.
Biol.Chem., 226, 497-509 (1957). The isolated lipid was confirmed to be a single substance using TLC spot film (Tokyo Kasei).
Developing solvent chloroform: methanol: water = 65:
At 25:4, Rf=0.12. (4) Preparation of Egg PC (Egg Yolk Lecithin) Egg yolk lecithin was obtained by separating egg yolk from fresh chicken eggs and purifying it by a method already established by the present inventors. Reconstituted liposomes with various composition ratios were formed using the lipids and glycoproteins prepared as described above, and achievement of the objective was evaluated by several methods described below. The effects of the invention will be explained in detail below. [Effect of the invention] Experimental example 1: Evaluation of membrane barrier ability A water-soluble fluorescent probe, 5,6-carboxyfluorescein (CF, Eastman Kodak), undergoes concentration quenching at high concentrations (Biochim. Biophys. Acta,
551, 295 (1977)). Taking advantage of this property, 200mM
A CF aqueous solution was encapsulated in the internal aqueous phase, and the increase in fluorescence intensity (It) depending on the leaked CF was tracked. Finally, the film was destroyed by adding Triton X-100 and the total fluorescence intensity ( _I∞ ) was determined. Fluorescence measurements were performed with a Hitachi spectrophotometer 650-IS. The CF outflow rate was calculated using the following formula. I ₀ is the fluorescence intensity at time zero. Efflux rate of CF (%) = I _t −I ₀ /I _∞ −I ₀ ×100 In order to examine the effect of adding glycofurin to Egg PC or Egg PC/DDPC mixed liposomes, the following three types of liposomes were prepared. a Egg PC: DDPC = 25 mg: 15 mg b Egg PC: Glycophorin = 30 mg: 0.6 mg c Egg PC: DDPC = Glycophorin =
25mg: 15mg: 0.8mg Dissolve a) in chloroform, b) in chloroform:methanol:water = 3:1.5:0.12, or c) in chloroform:methanol:water = 3:1.5:0.16, and form a thin film under reduced pressure. Created. to this
200mM CF, 20mM Tris-HCl, 200mM NaCl
4 ml of buffer solution (PH8.6) was added to swell it, and the thin film was peeled off using a vortex mixer. This was irradiated with ultrasound for 20 minutes at 0°C under a nitrogen stream (Tomy Seiko Co.)
Ltd.UR-200P, 25W). This was fractionated using a Sepharose 4B column (φ18×500mM) equilibrated with 20mM Tris-HCl and 200mM NaCl buffer (PH8.6). The phospholipid concentrations of MLV and SUV were each determined using a Wako phosphorus determination kit and adjusted to 1.3×10 ⁻⁴ M. Figure 1 shows the results of natural outflow of CF in the SUV. Adding DDPC to EggPC-only liposomes
By adding 40wt%, CF outflow was suppressed, and by adding glycophorin to this, the barrier ability was further improved. Experimental Example 2: Evaluation of membrane fluidity Steady-state fluorescence depolarization value (p
- value), the fluidity of the membrane was evaluated. Fluorescent probes include 1,6-diphenylhexatriene (DPH, Aldrich) and N-dansylhexadecylamine (DSHA, synthesized according to the literature (Proc. Nat. Acad. Sci. USA, 67, 579).
(1979), Colloid Interface Sci., V, 111
(1976)) and were used as probes for the inside of the membrane in the hydrophobic region and the membrane surface near the lipid head, respectively. For fluorescence polarization measurements, a Union Giken fluorescence depolarizer FS-501S was used, and p-value analysis was performed by connecting a sword microcomputer and M200 mark. The measurement temperature was adjusted using a Komatsu-Yamato Coolnics model CTR-120. The p-value was calculated using the following formula. P-value I _v −C _f I _H /I _v +C _f I _H I _v = Vertical component fluorescence intensity C _f = Correction factor I _H = Horizontal component fluorescence intensity DDPC/DMPC, SM/DMPC and DDPC/
A thin film was formed by mixing DMPC/glycophorin in an appropriate ratio. DMPC is dimylstolphosphatidylcholine (Sigma). to this
20mM Tris-HCl, 200mM NaCl (PH8.6) 4ml
The cells were swollen with water and irradiated with ultrasound for 15 minutes under a N ₂ stream.
After diluting this solution to 1.3×10 ^-3 M, add a methanol solution of DPH or DSHA (1.
×10 ^-3 M) was added to give a concentration of 1.3 × 10 ^-7 M, and ultrasonic irradiation was performed for 1 minute, and the mixture was left to stand for 30 minutes or more before use in the experiment. The effect of adding DDPC to DMPC liposomes is not observed inside the membrane, but on the membrane surface it causes a decrease in fluidity in both the gel state and liquid crystal state. This is thought to be because the hydrogen bond band of DDPC is located near the lipid head (Figure 2). On the other hand, in SM, fluidity increased in the gel state inside the membrane and decreased in the liquid crystal state. In addition, the fluidity of the membrane surface was reduced in any state (third
figure). When DMPC/DDPC mixed glucophorin is added, the fluidity increases up to a certain concentration of glycophorin, but the fluidity decreases after a certain concentration (Figure 4). This is because the abundance of glycophorin in liposomes is lower than its boundary concentration.
DDPC tends to gather around glycofurin, apparently improving its fluidity, but as the concentration of glycofurin increases, does the protein reduce the fluidity of the membrane, or does the probe and protein increase the fluidity of the membrane? It is considered that the p-value appears to be high because of a decrease in the p-value, or because the movement of the probe is suppressed due to the interaction between the probe and the protein. Experimental Example 3: Uptake into phagocytes Alveolar macrophages were collected from guinea pigs by bronchotomy. Monocytes and neutrophils were isolated from heparinized type A blood of healthy adults by the dextran precipitation method and the fluoricol-cholein method. The cells are
Suspended in RPMI (10% HI-FCS), 1.5-2 x ¹⁰⁶
Adjusted to cell/ml. After creating a thin film from 30 mg of Egg PC or Egg PC: DDPC: Glycophorin = 19 mg: 11 mg: 0.6 mg, large unilamellar liposomes (LUV) were created by the reverse phase method using 200 mM CF, 100 mM phosphate buffer. To remove CF that was not trapped in the liposomes, the cells were washed by centrifugation at 350 x G for 40 minutes twice. Egg PC-only liposomes were coated with CHM-200-1.7, which was 1/5 the weight of the lipid. CHM−200−1.7
is mannan, a polysaccharide with a molecular weight of 200,000, modified with 1.7 cholesterol groups per 100 mannose. 0.95 ml of the above cell suspension and the liposome suspension were placed in a plastic tube (10 ml) and cultured with rotation at 37° C. for 30 minutes. This was centrifuged at 450×G for 10 minutes to separate the cells and unphagocytosed liposomes. In the case of CF-encapsulated liposomes, this washing was repeated. Evaluation of cellular internalization efficiency was performed by the following method. FACS-1S to check whether CF-encapsulated liposomes have migrated to cultured cells and emitted light.
(Fluorescence Activated Cell Sorter, Belton
Dickinson). As a result, the number of cells with significant fluorescence intensity among all cells was determined as a ratio.

[Table] Uptake of CF-encapsulated liposomes into neutrophils depends on the concentration of liposomes. than LUV
Increased uptake was observed as CHM-LUV.
In contrast, the uptake of glucophorin-LUV was significantly suppressed (Table 2). Approx.
“ ¹⁴ C” of 0.1μCi/μmol lipid - DPPC
(Phpsphatidyl-choline-L-α-dipalmitoyl
−［choline−methyl［ ¹⁴ C''−, New England
Nuclear) was added, and LUVs were created in the same manner as above using 0.1M PBS. In experiments using radioisotope-labeled liposomes, mixed silicone oil was used for centrifugation. ¹⁴ C-labeled liposomes are phagosomes and cells are ACS
(Amersham) and radioactivity was measured using a liquid scintillation counter (Aloka). The results are summarized in Figure 5. In cell internalization experiments using ¹⁴ C-labeled liposomes, it was possible to roughly estimate the number of liposomes that migrated into single cells. The number of liposomes shown in Table 2 is based on a liposome size of 1080 Å.
Calculations were made assuming that the bilayer is doubled.
In monocytes and neutrophils, the amount of human glucoforin liposomes phagocytosed by LUVs is reduced. In contrast, butaglycophorin
LUVs tended to have more phagosomes than human glucophorin, suggesting that the sugar chains of human glycophorin are recognized and exert an influence on human cells. On the other hand, in guinea pig alveolar macrophages, compared to normal LUV, glycoprotein-containing LUV
The phagosome efficiency has improved, and it is thought that the difference in species may have an effect on uptake. Experimental example 4; Blood coagulation experiment Activated partial thromboplastin time
(APTT)) and prothrombin time (prothrombin
time (PT)). A coagulation time measuring device (KC-4: Amelung) was used. Egg PC: DDPC Glycophorin = 500mg:
They were added at a ratio of 50mg:50mg and dissolved in chloroform:methanol:water=3:1.5:0.1 to form a thin film. This was swollen with 10 ml of physiological saline, the thin film was peeled off, and probe-type ultrasonic irradiation was performed for 15 minutes. In vitro experiment Blood collected from male Wistar rats (300-350 g) was separated by centrifugation to obtain platelet-poor plasma (PPP). For APTT, APTT reagent (Bratelin Plus Activator: Warner-Lambert)
After incubating 100 μl at 37°C for 3 minutes, add 30 μl of liposomes or 0.9% saline and 70 μl of PPP, incubate for another 3 minutes, and add 25 mM
100 μl of CaCl ₂ was added and the clotting time was measured. For PT, 70 μl of PPP and 30 μl of liposome or saline
After incubating at 37℃ for 3 minutes, add PT reagent (Simprasautin: Warner-Lambert).
200 μl was added and the clotting time was measured. In in vitro experiments, liposomes showed a tendency to prolong APTT, but not significantly, and had no effect on PT (Table 3).

[Table] In vitro reagents Liposomes or physiological saline were administered through the tail vein of male Wistar rats (300-350 g), and 1 hour later, blood was collected from the abdominal aorta to obtain PPP. PPP was 100 μl for both APTT and PT, and the clotting time was measured according to the method for in vitro experiments. Even in in vitro experiments
APTT and PT were not different from controls (Table 4).

[Table] From the above, it was found that this liposome had almost no effect on the blood coagulation system. As is clear from the detailed experimental results above,
The present invention has achieved the objectives of strengthening the structure of liposomes and imparting cell recognition functions. That is, by using a lipid with an amide bond in the molecule called DDPC, it is possible to form a hydrogen bond band in the liposome membrane (see the DSC results in Figure 6), and the primary structure of the liposome is strengthened. It was done. In addition, by reconstituting the glycoprotein glycofurin, the membrane fluidity of liposomes is suppressed, and the mechanical strength of the membrane is further increased, allowing water-soluble fluorescent probes encapsulated in liposomes to escape from the liposomes. This was revealed through flow control and fluidity measurements using fluorescence depolarization method. On the other hand, with respect to cell recognition function, isotope labeling and isotope labeling have shown that reconstituted liposomes containing glycoproteins reduce the efficiency of phagocytosis by phagocytic cells rather than simple liposomes that do not contain glycoproteins.
It was carried out quantitatively by FACS method. In other words, we succeeded in adding an exclusion recognition function. This represents a major development in the use of the liposome of the present invention as a medical material. For example, when trying to use liposomes as artificial red blood cells, it is absolutely necessary to avoid liposomes administered into a living body from being recognized as foreign substances by a group of phagocytic cells and being phagocytosed and losing their oxygen carrying ability. This problem has been solved by the liposome of the present invention. When trying to use the liposome of the present invention as an artificial red blood cell or administering it by intravenous injection, one of the major issues is how it acts on the blood coagulation system. As shown by the evaluation, the liposome of the present invention was shown to be almost harmless in the blood coagulation system. As described above, the liposome of the present invention can be used as an artificial cell in a wide range of fields of pharmaceutical materials and biotechnology, such as tissue-directed drug carriers, artificial red blood cells, cell media, and cell modification. It is something that expands.

[Brief explanation of the drawing]

Figure 1 is a graph showing the results of natural outflow of CF in an SUV. FIG. 2 is a graph showing the effect of adding DDPC. FIG. 3 is a graph showing the effect of adding SM. FIG. 4 is a graph showing the effect of adding glycophorin. FIG. 5 is a graph showing the phagosome efficiency of liposomes. FIG. 6 is a graph showing the DSC curve of liposomes.

Claims (1)

[Scope of Claims] 1. A method for producing liposomes, characterized in that the liposome membrane is composed of lipids, cholesterol, and glycoproteins. 2. The method for producing a liposome according to claim 1, wherein the lipid is a naturally occurring phosphatidylcholine and a synthetic phosphatidylcholine having an amide bond. 3. Production of the liposome according to claim 1 or 2, wherein the glycoprotein is at least one selected from glycofurin extracted from mammalian red blood cell membranes and mucoprotein extracted from mammalian secretions. Law. 4. The method for producing liposomes according to claim 1 or 2, wherein the amount of glycoprotein is 0.01-0.1 part by weight per 1 part by weight of phosphatidylcholine. 5 Claim 1: 0 to 10 parts by weight of naturally occurring phosphatidylcholine per 1 part by weight of synthetic phosphatidylcholine containing an amide compound.
4. A method for producing a liposome according to item 2, item 3, or item 4.