JP4669665B2 - Polycation-modified liposome having no cytotoxicity and method for producing the same - Google Patents

Description

The present invention relates to a polycation-modified liposome having no cytotoxicity and a method for producing the same, and more particularly, having high biocompatibility and biodegradability, and used as a gene carrier in addition to medical biomaterials and pharmaceutical fields. The present invention relates to a non-cytotoxic polycation-modified liposome (hereinafter simply referred to as “polycation-modified liposome”) and a method for producing the same.

  Liposomes are bilayer closed vesicles formed by the self-assembly of phospholipids, which are naturally derived surfactants, in water. Liposomes have been actively applied as base materials for pharmaceuticals and cosmetics, but in recent years, attention has been focused on researches on application as gene carriers. That is, since the internal aqueous phase of liposomes is a vesicle isolated from the outside world, the encapsulated substance can be transported to a specific site, and a genetic degradation component typified by a nucleolytic enzyme contained in serum Therefore, it is attracting attention as a gene carrier.

  When introducing a gene into a living body, the liposome can be said to be a safe carrier, but in order to use it specifically as a gene carrier, the gene is first encapsulated in the liposome or the liposome and the gene. Using electrostatic attraction between them, liposomes form a complex with the gene. Next, the fusion of the liposome bilayer membrane and the cell membrane causes the gene to be taken into the cell, and the expression of the gene occurs by reaching the nucleus.

  By the way, it is generally known that the cell surface and the gene are negatively charged. However, when a cationic liposome having a positively charged surface of the liposome is used, the ability to capture the negatively charged gene is strong. Therefore, a stronger liposome-gene complex is formed, and a more preferable gene carrier is obtained. In addition, when such a cationic liposome is used, electrostatic attraction with a negatively charged cell surface works, and membrane fusion between the liposome and the cell is likely to occur.

  Thus, it can be said that introduction of a cationic species into a liposome is extremely useful in using the liposome as a gene carrier. However, many cationic surfactants and cationic phospholipids have strong cytotoxicity, and it has been difficult to apply cationic liposomes into which they have been introduced in vivo.

  Therefore, there is a need to provide cationic liposomes that are free from cytotoxicity problems and can be used as gene carriers, and the provision of such cationic liposomes is an object of the present invention.

  As a result of diligent research to solve the above-mentioned problems, the present inventors have no problem of cytotoxicity by allowing chitosan obtained by deacetylating a cationic polysaccharide, particularly chitin, to act on liposomes. The inventors have found that polycation-modified liposomes can be obtained and completed the present invention.

That is, the present invention provides a cationic polysaccharide on the surface of a liposome, which is a phospholipid comprising amphoteric phospholipid and anionic phospholipid mixed at a molar ratio of 9.5: 0.5 to 4: 6. The present invention provides a polycation-modified liposome obtained by modification with

The present invention also provides a solution of liposomes, which are phospholipids comprising amphoteric phospholipids and anionic phospholipids mixed at a molar ratio of 9.5: 0.5 to 4: 6 , to cationic polysaccharides. The present invention provides a method for producing a polycation-modified liposome, which is dripped in an acidic solution.

Furthermore, the present invention relates to a mixture of amphoteric phospholipids and anionic phospholipids in a molar ratio of 9.5: 0.5 to 4: 6 at a temperature and / or pressure higher than the critical point of carbon dioxide. A method for producing a polycation-modified liposome, characterized in that an aqueous phase containing a substance to be encapsulated in a liposome is added to a mixed fluid containing phospholipid, cationic polysaccharide and carbon dioxide. .

  According to the present invention, a polycation modification in which a liposome bilayer surface is modified with a cationic polysaccharide by mixing a phospholipid liposome aqueous solution such as dipalmitoylphosphatidylcholine (DPPC) and a cationic polysaccharide aqueous solution. Liposomes are prepared.

  In addition, amphoteric phospholipid / anionic phospholipid liposomes with high retention efficiency can be obtained by mixing amphoteric phospholipids such as DPPC with anionic phospholipids such as dipalmitoylphosphatidylglyceryl (DPPG). Moreover, since it has a negative charge, polycation-modified liposomes can be efficiently obtained by electrostatic interaction.

  Furthermore, by applying the supercritical reverse phase evaporation method, polycation-modified liposomes can be prepared in one step.

  The polycation-modified liposome of the present invention is obtained by modifying the surface of a liposome whose main component is a phospholipid with a cationic polysaccharide.

Cationic polysaccharides used to modify liposomes include synthetic cationic cations on polysaccharides such as dextran, cellulose, and β- (1,3) -glucan in addition to natural cationic polysaccharides such as chitosan and polygalactosamine. And those having a group introduced therein. Of these, chitosan has the following formula:
It is a compound represented by these.

  This is obtained by deacetylating chitin, which is a linear natural polymer compound in which N-acetyl-2-amino-2-deoxy-D-glucopyranose is β (1-4) linked For example, it is commercially available as Chitosan 1000 (manufactured by Seikagaku Corporation). This product has various chemical and biological functions such as anticancer effect, blood pressure increase suppression effect, cholesterol reduction effect, etc., but is insoluble in water, alkali, alcohol and organic solvents, and only soluble in weakly acidic solution. . Then, in the weakly acidic solution, the amino group of chitosan is ammoniumated to form a cationic polymer (polycation).

  In the present invention, examples of the method for obtaining a polycation-modified liposome by modifying a liposome with a cationic polysaccharide include the following methods.

(1) A liposome solution (hereinafter referred to as “liposome solution”) whose main component is a phospholipid is prepared by a known method, and this solution is dropped into an acidic solution of a cationic polysaccharide to prepare a polycation. (2) An aqueous phase containing a substance to be encapsulated in a liposome in a mixed fluid containing phospholipid, cationic polysaccharide and carbon dioxide under a temperature and / or pressure higher than the critical point of carbon dioxide After that, the pressure is reduced and carbon dioxide is removed to obtain a polycation-modified liposome.

  In order to obtain a polycation-modified liposome by the first method, it is first necessary to produce a liposome encapsulating an aqueous phase containing a substance to be encapsulated by a known method. Examples of this method include an ultrasonic treatment method, a surfactant removal method, an organic solvent injection method, a freeze-thaw method, a reverse phase evaporation method, and the like. For example, the Bangham method is preferred.

  Examples of the lipid used to form the liposome include dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine ( Phosphatidylcholine such as DOPC), phospholipids such as soybean lecithin (SBL), egg yolk lecithin (EYL), or nonionic amphiphilic molecules are used.

  For the above lipids, if necessary, suitable substances such as cholesterols acting as membrane stabilizing substances such as cholesterol, α-cholestanol, β-cholestanol, cholestane and cholesterol esters, and charged substances such as dicetyl phosphate Can be added.

  Furthermore, by using a mixture of amphoteric phospholipid and anionic phospholipid as the lipid, an anionic liposome can be obtained, and modification with a cationic polysaccharide becomes easier. That is, for example, lipids (amphoteric phospholipids) such as phosphatidylcholine, and phosphatidylethanol such as phosphatidylglyceryl such as dipalmitoylphosphatidylglyceryl (DPPG) and dipalmitoylphosphatidylethanolamine An anionic phospholipid such as phosphatidic acid such as amine or dipalmitoyl phosphatidic acid is used in an appropriate ratio, for example, in a molar ratio of 4: 6 or more, preferably 9.5: 0.5, Particularly preferably, an anionic liposome can be obtained by using at 9: 1 to 7.5: 2.5, and the anionic phospholipid itself has a high dispersion stability and a high retention of inclusions. Is. Since this is itself anionic, it has high reactivity when an acidic aqueous solution of a cationic polysaccharide is allowed to act, and a polycation-modified liposome having more preferable properties can be obtained.

  On the other hand, in the first method, as an example of the cationic polysaccharide acidic solution used for the modification of liposome, chitosan is contained at a concentration of 2 to 2000 ppm, preferably 5 to 200 ppm, and its pH is 3 or less, preferably And aqueous solutions having a pH of 2 to 3. At such a chitosan concentration and pH, chitosan dissolves, and in the solution, the amino group of chitosan is ammoniumated to form a cationic polymer (polycation) suitable for liposome coating.

  The modification of the liposome with the cationic polysaccharide acidic solution described above is carried out by dropping the liposome prepared as described above into the cationic polysaccharide acidic solution under stirring and mixing well, and then, in a cool dark place for about 12 hours. And solid-liquid separation, followed by redispersion in purified water or buffer. Among these, the step of standing or redispersing in a cool and dark place may be repeated. In the modification of the liposome with the acidic solution of the cationic polysaccharide, the lipid forming the liposome and the cationic polysaccharide such as chitosan are in a weight ratio of 1: 0.000005 to 1: 0.005, Is preferably 1: 0.0001 to 1: 0008, and more preferably 1: 0.0003 to 1: 0.0006 (in the case of anionic liposomes in which amphoteric phospholipids and anionic phospholipids are mixed) Has a weight ratio of a lipid forming a liposome to a cationic polysaccharide such as chitosan of 1: 0.000001-3: 0.0025, 1: 0.000002-2: 00167, particularly 1: 0.0020 to 1: 0.0035).

  The second method uses a device capable of achieving the supercritical condition of carbon dioxide, and puts a mixture containing phospholipid, cationic polysaccharide and carbon dioxide into the cell portion of this device, and the carbon dioxide has a criticality of carbon dioxide. It is produced by applying a pressure and temperature above a point to form a supercritical fluid, adding an aqueous phase containing a substance to be encapsulated in liposomes, and then depressurizing to remove carbon dioxide.

  As an example of an apparatus used for this supercritical reverse phase evaporation method, there is an apparatus whose structure is shown in FIG. In the figure, 1 is a variable pressure cell, 2 is a piston, 3 is a stirring bar, 4 is a carbon dioxide cylinder, 5 is a screw pump, 6 is a vacuum pump, 7 is a pressure gauge, 8 is a pump for liquid chromatography, 9 Is a liquid reservoir and 10 is an electronic scale.

  In the apparatus of FIG. 1, first, liposome constituents such as phospholipids and cationic polysaccharides are put into the space of the variable volume pressure cell 1 in which air is sucked by the vacuum pump 6, and then from the carbon dioxide cylinder 4, Carbon dioxide is injected through the pump 5. At this time, the stirrer 3 is placed in the space of the variable volume pressure cell 1, whereby the liposome constituent components and carbon dioxide are stirred.

  Furthermore, by compressing the space of the cell 1 by the piston 2 in the variable volume pressure cell 1, the internal pressure increases and reaches the supercriticality of carbon dioxide. Specifically, it becomes a supercritical state at a temperature of about 60 ° C. and a pressure of 300 bar. In this state, carbon dioxide changes from a gas to a supercritical fluid and dissolves the liposome constituents.

  In the space of the volume variable pressure cell 1 in such a state, an aqueous phase containing a substance to be encapsulated in the liposome from the liquid reservoir 9 via the liquid chromatography pump 8 (hereinafter referred to as “encapsulated liquid”). ) Is added.

  Then, after confirming that a predetermined amount of the encapsulating liquid has been encapsulated by the electronic balance 10, the pressure is lowered, and carbon dioxide is released from the space of the volume variable pressure cell 1, thereby obtaining a polycation-modified liposome liquid. be able to.

  Examples of the lipid used in obtaining the polycation-modified liposome by the second method include those used in the first method, and it is more preferable to use a mixture of amphoteric phospholipid and anionic phospholipid. It is the same. The mixing ratio of the amphoteric phospholipid and the anionic phospholipid in the second method is 4: 6 or more, more preferably 9.5: 0.5, and particularly 9: 1 to 7. 5: 2.5 is preferable.

  Further, the cationic polysaccharide used in the second method can be used in a solid state, not an acidic solution. This is because, in the supercritical reverse phase evaporation method, carbon dioxide is saturated and dissolved in the encapsulated liquid added under supercritical conditions, so that the pH becomes slightly acidic around 3.0. Therefore, in the state where supercritical carbon dioxide and water coexist, cationic polysaccharides such as chitosan are considered to dissolve in water. Furthermore, since the pH of the encapsulated liquid (water) becomes neutral by discharging carbon dioxide, cationic polysaccharides such as chitosan dissolved in water are placed on the liposome surface by the discharge operation of carbon dioxide. This is thought to be due to precipitation.

  In the second method, the amount of the cationic polysaccharide such as chitosan added to the mixed fluid is preferably 2 to 55 ppm, more preferably 10 to 40 ppm, and the lipid forming the liposome and the cationic polysaccharide such as chitosan The weight ratio is preferably 1: 0.000005 to 1: 0.005, more preferably 1: 0.0001 to 1: 0008, and particularly preferably 1: 0.0003 to 1: 0.0006 ( In the case of an anionic liposome in which an amphoteric phospholipid and an anionic phospholipid are mixed, the weight ratio of the lipid forming the liposome to the cationic polysaccharide is from 1: 0.001 to 1: 0.007, Is preferably 1: 0.0023 to 1: 0065, and more preferably 1: 0.0015 to 1: 0.006).

  In the polycation-modified liposome thus obtained, the cationic polysaccharide used for cationization is highly safe while having various chemical and biological functions. It is used as a gene carrier that carries this to the target cell. In particular, when an anionic liposome mixed with amphoteric phospholipid and anionic phospholipid is used, the bilayer surface of the liposome is positively charged, the inclusion rate of DNA having negative charge is improved, and the negative charge is reduced. It is expected that the fusion with the cell membrane will be likely to occur.

  In addition, the present invention is not limited to this, and can also be used to transport physiologically active substances such as pharmaceuticals to target cells, and can be used as new pharmaceuticals.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in more detail, this invention is not restrict | limited at all by these Examples.

Example 1
Production of polycation-modified liposome (1):
(1) Liposomes were prepared by the following method according to the Bangham method. The phospholipid that is the main component of the liposome is L-α-dipalmitoylphosphatidylcholine (L-α-dipalmitoylphosphatidylcholine), which is a saturated phospholipid having both 16 alkyl chains consisting of only saturated bonds. α-dipalmitoyl phosphatidylcholine; DPPC, purity 99.6%, manufactured by NOF Corporation was used.

  Specifically, in 1 ml of chloroform as a solvent, DPPC in an amount of 0.05 mmol as a lipid is taken, and 5 ml of distilled water for injection (Otsuka Pharmaceutical ( Co., Ltd.) was added, and external force was applied by stirring to obtain liposomes.

(2) The chitosan-modified liposome was prepared according to the following procedure. First, liposomes prepared by the above (1) were dropped into various aqueous compositions in a chitosan aqueous solution (concentration: 20 ppm) adjusted to pH 3.0 using hydrochloric acid (manufactured by Wako Pure Chemical Industries, Ltd.), and about 500 rpm The mixture was stirred by stirring. This was stabilized by standing for 12 hours in a cool and dark place. After removing the supernatant by centrifugation at 3000 rpm, the same amount of phosphate buffered physiological saline (dulbeccolic acid buffered physiological saline powder (manufactured by Wako Pure Chemical Industries, Ltd., composition; sodium chloride, potassium chloride) An isotonic solution (pH 7.4) / PBS prepared by dissolving disodium hydrogen phosphate (anhydrous diphosphate, anhydrous potassium phosphate) in distilled water for injection is added, and the solution is reconstituted with a vortex mixer. Dispersed. This was again allowed to stand in a cool dark place for 12 hours. Similarly, the supernatant was removed, and PBS was added and redispersed. This process was repeated three times, and finally dispersed with distilled water for injection to prepare chitosan-modified liposomes.

(3) FIG. 2 shows the relationship between the ratio of the chitosan solution in the total volume of the liposome solution and the chitosan solution (hereinafter referred to as “chitosan ratio”) and the measurement result of the ζ potential of the obtained chitosan-modified DPPC liposome. From this result, it was found that the ζ potential of DPPC liposome alone was about −4 mV, but this was largely positively charged by modifying chitosan. From the visual observation of the prepared chitosan-modified DPPC liposome aqueous solution, the static dispersion stability is the best when the chitosan ratio at which the ζ potential is maximum is 0.2 (the ratio of the liposome solution to the chitosan solution is 8: 2). Met. However, in the region where the chitosan aqueous solution was richer, the ζ potential on the liposome surface decreased again. This is considered to indicate that chitosan modification to the liposome surface does not occur under conditions where chitosan is excessive.
This shows that the mixing ratio of the DPPC liposome aqueous solution and the chitosan aqueous solution in obtaining chitosan-modified DPPC liposomes should be a ratio that maximizes the ζ potential.

Reference example 1
Production of anionic liposomes:
In DPPC, an anionic phospholipid, L-α-dipalmitoyl phosphatidylglycerol (DPPG), which is a saturated chain having a chain length of 16 for both two alkyl chains and has a negative charge on the hydrophilic head. , 99.8% purity, manufactured by Nippon Oil & Fats Co., Ltd.) were mixed at various molar ratios to obtain lipids, and then anionic liposomes were prepared in the same manner as in Example 1 (1). The relationship between the ratio of DPPG to the total lipid (hereinafter referred to as “anionic lipid ratio”) and the appearance after standing for 24 hours after preparation was examined, and the results are shown in FIG.

  As is apparent from the results, DPPC alone has a low static dispersion stability, and two-phase separation due to aggregation and sedimentation was observed. On the other hand, it was found that by mixing DPPG with DPPC, the stationary dispersion stability of the liposome aqueous solution was dramatically improved. This is probably because the amphoteric phospholipid DPPC and the anionic DPPG were mixed, and as a result, the bilayer surface of the liposome was negatively charged. Then, electrostatic repulsion occurred between the negatively charged liposome surfaces, suggesting that aggregation of the liposomes was suppressed.

  The standing dispersion stability of the aqueous solution prepared with a DPPG molar fraction of 0.1 to 0.6 was very good, and this dispersed state was maintained even after 6 months from the adjustment. On the other hand, when the DPPG molar fraction was 0.7 or more, the appearance of the DPPC / DPPG liposome became almost colorless and transparent. Moreover, the aqueous solution prepared by adjusting the molar fraction of DPPG to 0.7 to 1.0 showed viscosity. This suggested that liposomes do not form in regions rich in DPPG.

Reference example 2
Retention efficiency test for anionic liposomes:
Based on the result of Reference Example 1, in order to confirm the formation of liposomes, the retention efficiency was measured for the DPPC / DPPG two-component aqueous solution. In this DPPC / DPPG two-component aqueous solution, D (+)-glucose was added to the aqueous solution, and the relationship between the anion lipid ratio and the retention efficiency was examined from the content in the DPPC / DPPG two-component liposome. The retention efficiency (%) was expressed as a value obtained by dividing glucose in the aqueous phase of the liposome by the amount of glucose in the entire system and multiplying this by 100. The result is shown in FIG.

  As is clear from this figure, the retention efficiency greatly changed by adding DPPG to DPPC. In particular, the retention efficiency was maximized when DPPG was added at an anionic lipid ratio (DPPG molar fraction) of 0.2, and the retention efficiency was about 8 times higher than that of a DPPC-only liposome. As in the past, multilamellar liposomes were formed in the DPPC single system, but the addition of DPPG suggested the formation of single membrane liposomes. It is considered that electrostatic repulsion occurs between the layers of the bilayer membrane in the formation process of the liposome by mixing the charged phospholipid.

  In addition, when the anionic lipid ratio was 0.7 or more, the retention efficiency was almost 0, and it was confirmed that no molecular assembly having an aqueous phase was formed. The appearance is almost colorless and transparent, and since the solution has viscosity, it is considered that string-like micelles are formed. As the molar fraction of DPPG increases, it is suggested that the morphology changes to DPPC multilamellar liposomes, DPPC / DPPG monolayer liposomes, DPPC / DPPG mixed micelles, DPPG micelles with DPPC solubilized, and DPPG micelles It was done.

Example 2
Production of polycation-modified liposome (2):
A mixture of DPPC and DPPG at 8: 2 was used as a lipid, and an anionic liposome solution was prepared in the same manner as in Example 1 (1). Subsequently, this liposome solution was dropped into the chitosan solution so as to have various ratios as in Example 1 (2) to obtain polycation-modified liposomes. The relationship between each polycation modified liposome chitosan ratio and ζ potential is shown in FIG.

  The ζ potential of DPPC / DPPG liposome alone was about −25 mV, but it was found that it was positively charged by modifying chitosan. Compared with chitosan modification to DPPC liposomes, DPPC / DPPG liposomes are more positively charged. By mixing DPPC, an anionic phospholipid, with DPPC, chitosan modification, a polycation, is modified. It turns out that it is performed more efficiently. This is thought to be because chitosan modification is likely to occur by applying a negative charge to the liposome bilayer surface. However, in the region where an excessive amount of chitosan aqueous solution coexists, it was suggested that chitosan could not be efficiently modified on the liposome surface and chitosan modification on the liposome surface did not occur.

Example 3
Production of polycation-modified liposome (3):
(1) Polycation-modified liposomes were produced using the supercritical reverse phase evaporation method. In this supercritical reverse phase evaporation method (hereinafter referred to as “scCO ₂ RPE method”), an apparatus as shown in FIG. 1 is used, and a predetermined amount of DPPC (0.3 wt% with respect to carbon dioxide) is placed in a cell 1 of this apparatus. ) And 1 ml of ethanol and a predetermined amount of solid chitosan (chitosan 1000 (manufactured by Seikagaku Corporation)) were sealed, and 11.87 g of CO ₂ was introduced. This was heated and pressurized to 60 ° C. and 91.7 bar. Next, CO ₂ was introduced into the rear part of the cell, and the liposome preparation conditions were set to 60 ° C. and 200 bar. In this state, 4.8 ml of the liquid to be retained (liquid to be retained in the liposome) was slowly injected (0.10 ml / min) with a liquid chromatography pump and sufficiently stirred, and then CO ₂ in the cell was removed. By discharging, a uniform polycation-modified liposome aqueous solution could be obtained inside the cell. In addition, stirring inside the cell was performed with a magnetic stirrer.

(2) About the obtained polycation modification liposome, the relationship between the zeta potential and the addition amount of chitosan was examined. The result is shown in FIG.

As shown in this result, the ζ potential of DPPC liposome alone without addition of chitosan was about −4 mV, which was about the same as that prepared by the Bangham method, but it was greatly positively charged by modifying chitosan. did. This indicates that the scCO ₂ RPE method can be applied to the preparation of polycation-modified liposomes. However, it was found that the ζ potential on the surface of the liposome bilayer did not depend on the amount of chitosan added, and that the maximum value was obtained when the amount of chitosan added was 20 ppm. Also from the visual observation of the prepared solution, when the chitosan addition amount is 50 ppm or more, chitosan not dissolved in the liposome solution can be confirmed, and the solubility of chitosan in pH 3.0 water in which carbon dioxide is saturated and dissolved is about 40 ppm. I found out.

Reference example 3
Retention efficiency test for anionic liposomes:
The same as in Example 3 except that, by scCO ₂ RPE method, DPPC and DPPG mixed in various compositions are used as lipids, solid chitosan is not used, and D (+)-glucose is added to the liquid to be retained. Thus, an anionic liposome was prepared. About the obtained anionic liposome, the result of having examined the relationship between anion lipid ratio and retention efficiency is shown in FIG.

  From this result, as in the case of the bangham method, it is shown that the retention efficiency is greatly changed by adding DPPG to DPPC, and the retention efficiency is maximized when DPPG having a molar fraction of 0.2 is added. It was. Thus, the composition with the maximum retention efficiency does not depend on the preparation method, the anionic lipid ratio is 0.2 (DPPC to DPPG ratio is 8: 2), and DPPC and DPPG are It is considered that an ideal mixed bilayer film is formed. Moreover, the maximum value of the retention efficiency was the same value in any of the preparation methods.

Example 4
Production of polycation-modified liposome (4):
Polycation-modified liposomes were produced by the scCO ₂ RPE method in the same manner as in Example 3 except that a mixture of DPPC and DPPG at 8: 2 was used as a lipid. FIG. 8 shows the relationship between the amount of solid chitosan added and the measurement result of the ζ potential obtained for the obtained polycation-modified liposome.

  As is clear from this result, the ζ potential in DPPC / DPPG liposomes to which chitosan was not added was about −25 mV, the same as in the Bangham method. It was found that DPPC / DPPG liposomes can also be modified by adding chitosan and modifying the liposome bilayer membrane in the same manner as DPPC liposomes. The DPPC / DPPG mixed system was more positively charged than DPPC, as was the case with the Bangham method. The reason why the ζ potential showed the maximum value was that the chitosan concentration was 30 ppm. Therefore, it is considered that the modification of chitosan is more likely to occur because the liposome bilayer surface is negatively charged.

  In the polycation-modified liposome of the present invention, since cationic polysaccharides such as chitosan used for cationization have various chemical and biological functions and are highly safe, It is used as a gene carrier that encapsulates and transports it to the target cells.

  In addition, the present invention is not limited to this, and can also be used to transport physiologically active substances such as pharmaceuticals to target cells, and can be used as new pharmaceuticals.

The drawing which shows a structure of an example of the apparatus used by the supercritical reverse phase evaporation method. Drawing which shows the relationship between the mixing ratio of a liposome solution-chitosan acidic solution, and the measurement result of the zeta potential of the obtained chitosan modified DPPC liposome. Drawing (photograph) showing the appearance after production of anionic liposomes obtained from amphoteric and anionic lipids. Drawing which shows the result of having investigated retention efficiency about the anionic liposome obtained from amphoteric and anionic lipid. Drawing which shows a zeta potential measurement result about the polycation modification liposome obtained from amphoteric and anionic lipid. Drawing showing the relationship between the zeta potential and the amount of chitosan added for polycation-modified liposomes using the supercritical reverse phase evaporation method. Drawing which shows the result of having investigated the retention efficiency about the anionic liposome obtained from the amphoteric and anionic lipid using the supercritical reverse phase evaporation method. The figure which showed the relationship between the addition amount of a solid chitosan, and the measurement result of (zeta) potential about the polycation modification liposome obtained from the amphoteric and anionic lipid using the supercritical reverse phase evaporation method.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ...... Volume variable pressure | voltage resistant cell 2 ...... Piston 3 ...... Stirrer 4 ...... Carbon dioxide cylinder 5 ...... Screw pump 6 ...... Vacuum pump 7 ...... Pressure gauge 8 ...... Liquid chromatography pump 9 ...... Liquid No. 10… Electronic scale

Claims (12)

The surface of the liposome, which is a phospholipid consisting of amphoteric phospholipid and anionic phospholipid mixed in a molar ratio of 9.5: 0.5 to 4: 6, is modified with a cationic polysaccharide. Polycation-modified liposome without cytotoxicity . 2. The non-cytotoxic polycation-modified liposome according to claim 1, wherein the amphoteric phospholipid is phosphatidylcholine and the anionic phospholipid is phosphatidylglycerol, phosphatidylethanolamine or phosphatidic acid. The non- cytotoxic polycation-modified liposome according to claim 1 or 2, wherein the cationic polysaccharide is chitosan. A solution of liposomes, which are phospholipids comprising amphoteric phospholipids and anionic phospholipids mixed in a molar ratio of 9.5: 0.5 to 4: 6, in an acidic solution of a cationic polysaccharide. A method for producing a polycation-modified liposome having no cytotoxicity , characterized by being dripped. The method for producing a non-cytotoxic polycation-modified liposome according to claim 4, wherein the acidic solution of the cationic polysaccharide is on the acidic side of pH 3. The liposome solution and the acidic solution of the cationic polysaccharide are used in a weight ratio of phospholipid and cationic polysaccharide contained in the solution of 1: 0.000005 to 1: 0.005. 5. A method for producing a polycation-modified liposome having no cytotoxicity according to 5. The cell according to any one of claims 4 to 6, wherein the amphoteric phospholipid is phosphatidylcholine and the anionic phospholipid is phosphatidylglycerol, phosphatidylethanolamine or phosphatidic acid. A method for producing non-toxic polycation-modified liposomes. The method for producing a non-cytotoxic polycation-modified liposome according to any one of claims 4 to 7, wherein the cationic polysaccharide is chitosan. A phospholipid comprising a mixture of amphoteric phospholipids and anionic phospholipids in a molar ratio of 9.5: 0.5 to 4: 6 at a temperature and / or pressure higher than the critical point of carbon dioxide; A method for producing a non-cytotoxic polycation-modified liposome, which comprises adding an aqueous phase containing a substance to be encapsulated in a liposome to a mixed fluid containing a cationic polysaccharide and carbon dioxide. 10. Production of non-cytotoxic polycation-modified liposome according to claim 9, wherein the amphoteric phospholipid is phosphatidylcholine and the anionic phospholipid is phosphatidylglycerol, phosphatidylethanolamine or phosphatidic acid. Law. The method for producing a non-cytotoxic polycation-modified liposome according to claim 9 or 10, wherein the concentration of the cationic polysaccharide in the mixed fluid is 2 to 55 ppm. The method for producing a non-cytotoxic polycation-modified liposome according to any one of claims 19 to 11, wherein the cationic polysaccharide is chitosan.