KR970001650B1 - A process for preparing ph-sensitive liposome targetting liver cell - Google Patents

Abstract

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Description

Method for preparing pH-sensitive liposomes that can target hepatocytes

1 is a graph showing the results of measuring the pH sensitivity of liposomes containing galactocerebroside.

Figure 2 (a) is a graph showing the results of measuring the permeability of liposomes with time at 4 ℃.

Figure 2 (b) is a graph showing the results of measuring the permeability of liposomes with time at 37 ℃.

3 is a graph showing the results of measuring the stability of liposomes in plasma.

4 is a graph showing the biodistribution of target liposomes.

The present invention relates to a method for preparing liposomes capable of simultaneously targeting hepatocytes and cancer cells. More specifically, the present invention relates to liposomes and a method for preparing the same that are sensitive to pH and can simultaneously target hepatocytes and cancer cells.

In general, closed vesicles formed by dispersing phospholipids in water are called 'liposomes', and liposomes are widely used in terms of basic science and applied science. Liposomes are composed of bilayers of phospholipids, which are the basic structure of biological membranes. Phospholipids are amphipathic substances, two polar molecules with anionic or amphoteric ions and two unsaturations with about 16 hydrocarbons. Because of its molecular structure with nonpolar fat-soluble chains, phospholipids disperse in water and spontaneously form bilayers.

Liposomes are known in the field of basic science to interact with proteins (Lee, JW and Kim, H., Arch Biochem. Biophys., 297: 354 (1992); Hahn, KH and Kim, H., J. Biochem., 110 : 635 (1991), Topology and Activity of Proteins in Biofilms, Fusion of Biofilms (Yun, CH and Kim, H., J. Biochem., 105: 406 (1989); Kim, J. and Kim , H., Biochemistry, 25: 7867 (1986); Kim, J. and Kim, H., Korean Biochem. J., 18: 403 (1985)) and protein delivery mechanism model system.

On the other hand, the field of applied archeology is used as a model for delivering genetic material to cells in culture in the cosmetic industry, drug delivery, and laboratory conditions (in vitro), and is currently used as a drug delivery system (DDS) of liposomes. As a result, much research is being conducted. The reason why liposomes are widely used in the field of applied science is that they can capture both water-soluble and fat-soluble substances in liposomes, are easy to target to specific tissues, they are easy to size and deform, and there are few toxicity problems due to the use of phospholipids. This is because there is an advantage in targeting a specific tissue. In addition, it is also because there is an advantage that can capture more drugs that can be captured to other drug carriers, and enhance the therapeutic effect.

To date, a number of studies have been conducted on various techniques for targeting specific tissues using liposomes:

The first method is a temperature-sensitive liposome that uses the phase transition temperature of liposomes, which takes advantage of the rapid leakage of the contents contained in liposomes near the phase transition temperature of phospholipids. The liposomes can be targeted by heating local tissues using phospholipids with a phase transition temperature of 2 to 3 ° C. higher than the biological temperature (Yatvin, MB, Magin, RL, Weinstein, JN and Zaharko, DS, Science, 202: 188 (1979): Yatvin, MB, Weinstein, JN, Dennis, WH and Bluemntal, R., Science 202: 1290 (1978)), and this method has many limitations when considering the in vivo situation. have.

The second method is to deliver drugs into cells by fusion between liposomes and cells, and uses a substance that causes various fusions. This method has been pointed out that the fusion substance has toxicity in cells. .

A third approach is to prepare pH-sensitive liposomes to selectively deliver anticancer drugs only to cancer cells. Ellens, H., Bentz, J. and Szoka, FC, Biochemistry, 23: 1532 1984); Nayar, R. and Shvoit, A. J., Biochemistry, 24: 5967 (1985); Stranbinger, R. M., Duzgunes, N. and Papahadjopoulos, D., FEBS Lett., 179: 148 (1985); Connor, J., Yatvin, M. B. and Huang, L., Proc. Natl. Acad. Sci. USA, 81: 1715 (1984); Liu, D. and Huang, L., Biochim. Biophys. Acta, 981: 254 (1989). In addition, to selectively introduce foreign DNA into specific organs, pH-sensitive liposomes or pH-sensitive immunoliposomes have been used [Connor, J. and Huang, L., Cancer Res., 46: 3431 (1986); Collins, D., Maxfield, F. and Huang, L., Biochim. Biophys. Acta, 987: 47 (1989); Wang, C.Y. and Huang, L., Biochemistry, 28: 9805 (1989). In general, pH-sensitive liposomes are stable at neutral pH but fusion occurs at acids (pH 6.5 or less) such as endosomes, causing the substances contained in the liposomes to leak out. Can be delivered into the cytoplasm.

The fourth method is liposomes that are sensitive to temperature and pH, which uses liposomes that have the characteristics of temperature-sensitive liposomes and those of pH-sensitive liposomes. These liposomes simply compensate for the disadvantages of pH-sensitive liposomes. can do.

On the other hand, galactose is known to have a specific affinity for hepatocytes, and a breakthrough biography has been prepared for target studies on hepatocytes [Spanjer, H. H. and Scherphof, G. L., Biochim. Biophys. Acta, 734: 40 (1983); Grosse, E., Kieda, C. and Nicolau, C., Biochim. Biophys. Acta, 805: 354 (1984); Spanjer, H. H., van Derkel, T. J. C., Scherphof, G. L. and Kempen, H. J. M., Biochim. Biophys. Acta, 816: 396 (1985)].

Accordingly, the inventors of the present invention have repeatedly studied the results, and hepatic cells and cancer table containing lipids containing galactose residue in all pH-sensitive compositions, that is, lipids such as galactoserebroside and lactocerebroside. Liposomes were prepared that can target simultaneously.

Accordingly, an object of the present invention is to prepare a liposome capable of simultaneously targeting hepatocytes and cancer cells by attaching galactose residues to all pH-sensitive compositions.

In the present invention, in order to target liver tissue using galactose residues, 8-10 mol% of galactocerebroside or lactocerebroside is added to the pH-sensitive liposome composition to pH. The sensitivity change and the ability to target hepatocytes were investigated. Since cancer cells can be targeted with a liposome that is sensitive to pH, and galactose residues can be contained in these compositions, the liver tissue can be targeted by the composition of liposomes containing 8 to 10 mol% of galactose residues in the pH-sensitive liposome composition. Targeting cancer cells at the same time, these compositions are a good model for liver cancer treatment. Therefore, by capturing anticancer drugs in liposomes consisting of these compositions can be delivered to cancer cells of liver tissue selectively to reduce the side effects of anticancer drugs, can increase the therapeutic effect. In addition, liposomes that can target tissues can be used to target other tissues and expect the same effect, which can be a good model for not only liver cancer but also other early cancer treatments, and can be used as a model for delivering external DNA to liver tissues. It is possible.

Abbreviations and source words used to describe the invention are as follows:

* CTL: Cytotoxic-T lymphocytes

DPPA: dipalmitoylphosphatidic acid

DOPA: Dioleylphosphatidic acid

* PE: phosphatidyl ethanolamine

* DOPE: dioleoyl phosphatidyl ethanolami

ne)

DPG: dipalmitoyl-sn-glycerol

DOG: Dioleyl-sn-glycerol

* MLV: multilamellar vesicle

* SUV: small unilamellar vesicle

FTV: freezing-thawing vesicles

* SPLV: Stable pulrilamellar vesicle

* BPB: bromophenol blue

ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt

* DPX: p-xylene bis (pyridinium) bromide (p-xylene bis (pyridinium) bro

mide)

* POPE: phosphatidylethanolamine-β-oleyl-γ-palmitoyl (phosphatidylethanolamine-β-oleoyl-γ-palmitoyl)

Hereinafter, various methods introduced in the present invention will be described.

Method for preparing liposomes

(1) MLV Manufacturing

Phospholipids were dissolved in an organic solvent in a glass vial, and nitrogen gas was blown to form a thin film in the vial, and a buffer solution (pH 8.0) in which the drug, fluorescent substance or peptide to be captured was dissolved, was added thereto. After sufficiently hydrated at 37 to 40 ° C. near the phase transition temperature of, the mixture was vigorously stirred to prepare a superposed multilayer membrane liposome (MLV).

(2) manufacturing of SUVs and FTVs

The prepared MLVs are made of small monolayer membrane liposomes (SUVs) using a sonicator. The SUV thus prepared is left at room temperature for 2 to 12 hours, then rapidly frozen with liquid nitrogen, and then thawed slowly at room temperature. This process is repeated 3-4 times to prepare freeze-thaw liposomes (FTV).

(3) manufacture of SPLV

Stable multilayer membrane liposomes (SPLV) are prepared by dissolving phospholipids in ether solution, adding a buffer solution, pulverizing ultrasonically to form a stable emulsion, and then depressurizing stepwise to remove ether.

Measurement of pH Sensitivity

12.5 mM 8-aminonaphthalene-1,3,6-trisulfonic acid bisodium salt (ANTS), 45 mM p-xylenebis (pyridinium) bromide (DPX), 68 mM NaCl and 10 mM HEPES buffer (pH) in liposomes 8.0) and uncaptured ANTS and DPX were passed through Sephadex G-50 column (1 × 20 cm) to collect only liposome solution and used for the experiment. Phospholipid concentrations in liposomes are measured by the method of Vaskovsky (Vaskovsky, V. E., Kostetsky, E. Y. and Vasendin, I. M., J. Chromatogr., 114: 129 (1975)).

In the pH sensitivity experiment, the intensity of fluorescence after passing through the column is called '0% leakage' and the intensity of fluorescence obtained after destroying the liposome solution with Triton X-100 is '100%'. Leak ', concentrated liposome (30μM) in 2ml of the buffer solution having each pH value (7.5, 6.9, 6.4, 5.9, 5.4, 4.9 and 4.5) and reacted for 30 minutes at room temperature and 37 ℃, PH sensitivity is measured by observing the intensity of fluorescence shown. In this case, fluorescence is measured at an excitation wavelength of 360 nm and an emission wavelength of 545 nm using a spectrofluorometer (FP-777, JASCO, Japan).

Measurement of permeability

Capture ANTS / DPX in liposomes, and measure the permeability by observing the intensity of fluorescence over time by measuring the amount of ANTS / DPX exiting the liposomes while keeping them at 4 ° C and 37 ° C in HEPES buffer at pH 7.4. .

Interaction with Plasma

In order to examine the stability of liposome solution in vivo, human plasma was extracted and 3 times the amount of liposome was added to liposome solution and the amount of ANTS / DPX released from liposome was measured by spectrofluorometer (JASCO, FP-777). , JAPAN) to observe by time and measure. At this time, the amount of ANTS released from the liposome at 37 ° C. in the presence of plasma is calculated by comparing the intensity of fluorescence at 0 hours.

Agenda target

A target liposome was prepared by containing 0.1-0.2 mol% of phospholipids labeled with 8 mol% of galactoserebroside and fluorescein isothiocyanate (FITC) in the composition of pH-sensitive liposomes. In vivo distribution of liposomes is examined by injection into the tail vein of the mouse. Two hours after injecting liposomes, each organ was excised and homogenized, and the amount of FITC in each organ was measured using a spectrofluorometer (JASCO FP-777) (Ex: 485, Em: 520 nm).

Hereinafter, the present invention will be described in more detail with reference to Examples.

These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples according to the gist of the present invention.

Example 1

In order to target using galactose residues, 8 to 10 mol% of galactocerebroside was added to the above-described pH-sensitive liposome composition to investigate the change in sensitivity according to pH and the ability to target hepatocytes. PE / DPPA; POPE / CHOH, DOG and DPPA; Various amphoteric substances such as DOPE / DPG and CHOH are mixed in a molar ratio of 6: 4 to 8: 2, preferably 7: 3, and captured into each liposome of FTV, SPLV and MLV prepared according to the liposome preparation method described above. The amount of material to be measured was compared and compared. As a result, liposomes made of FTV showed the best stability and capture, and the pH sensitivity was measured with FTV.

Although DPPA reduced the content to 20%, the sensitivity to pH was slightly decreased, but similar results were obtained.When DOPA was used, it showed a lot of leakage even at neutral pH, causing stability problems. Supplemented. Even when using DPG or DOG instead of DPPA, it was confirmed that the degree of leakage increases with the decrease in pH, but DOG showed a lot of leakage even near the neutral pH, so there was a problem in stability like DOPA. When CHOH was used, there was almost no leakage at neutral pH, and a rapid leakage was observed near the pH such as cancer cells. In addition, when galactoserebroside is present, the sensitivity to pH is good near pH 6.5, but falls below that when galactoserebroside is not present. However, in the case of containing CHOH, the pH sensitivity increases when galactoserebroside is present. Therefore, when exposed to conditions such as cancer cells, pH 6.5 to 6.0 and 37 ° C., many leaks may occur and target cancer cells. It was supposed to be. Therefore, it was possible to point out that POPE / DPPA, POPE / CHOH or DOPE / CHOH, etc. that showed excellent results as a result of pH sensitivity measurement.

Example 2

Liposomes were prepared in the same manner as in Example 1 except for adding lactocerebroside instead of galactoserebroside.

Example 3

While storing the liposomes prepared in Example 1 at 4 ° C and 37 ° C, the amount of ANTS / DPX escaping from the liposomes was measured over time. As a result, when stored at 4 ℃ can be stored for a long time (second (a)), but at 37 ℃ showed a very fast leak, depending on the phospholipid composition of liposomes showed a difference in the exit rate (second ( b) degrees). The use of DOPA and DOG resulted in very rapid leaks compared to other compositions, as shown by pH sensitivity.

In the case of phospholipids, the use of POPE was the most stable, and the use of DPPA, DPG, CHOH as the amphoteric substance added to the phospholipids was excellent. Therefore, the composition having the highest stability may be referred to as POPE / CHOH or POPE / DPG.

Example 4

As a result of examining the stability of liposomes in the presence of plasma, the use of DPG and CHOH having a double-chain structure was superior to that of liposomes using fatty acids as amphoteric substances (see FIG. 3). This is because general fatty acids are absorbed by albumin present in plasma. When DPG and CHOH having a double-chain structure are used, albumin almost does not occur, and thus it can be maintained in plasma. As shown in Figure 3, the most stable in liposomes using CHOH when comparing the leakage after 24 hours in the presence of plasma.

The same results as in the measurement of permeability were obtained for the stability in the presence of plasma, but the stability was poor in liposomes with high pH sensitivity. It was pointed out that the composition of POPE / DPG and POPE / CHOH was the best in stability in plasma.

Example 5

Hepatocyte targetability was measured in vitro and in vivo in the liposomes prepared in Example 1. In these experiments, the target liposomes were absorbed much more when compared to the extent of absorption using hepatocytes in vitro. In vivo experiments showed that the organs were excised into the tail vein of the mouse 2 hours after injection, and compared to other tissues. In the case of hepatocytes, more than 70% were found, about 10% in plasma and about 15% in kidney [Ref. 4]. Therefore, it can be seen that the target composition liposomes containing 8 to 10 mol% galactoserebroside in the pH-sensitive liposome composition are new dual target liposomes that are sensitive to pH and can target liver tissue.

As described and demonstrated in detail above, when the composition of all liposomes sensitive to pH] contains 8 to 10 mol% galactose cerbroside or lactocerebroside, it causes a lot of leakage in the environment such as cancer cells, A dual target liposome was prepared that could be targeted to cancer tissue of hepatocytes. These liposomes will be a good model for treating cancer of other tissues by targeting other tissues as well as liver cancer.

Claims (6)

(Iii) one selected from the group consisting of phosphatidylethanolamine-β-oleyl-γ-palmitoyl (POPE) and (ii) dipalmitoylphosphatidyl acid (DPPA) and cholesterol hemisuccinate (CHOH) A method of preparing a liposome capable of simultaneously targeting hepatocytes and cancer cells by mixing the compound at a molar ratio of 6: 4 to 8: 2, and further mixing lipids containing 8 to 10 mol% of galactose groups thereto. The method of claim 1, wherein the lipid containing a galactose group is galactosereveloside or lactocerebroside. The method of claim 1, wherein the liposome is one selected from the group consisting of multilayer membrane liposomes (MLV), small monolayer membrane liposomes (SPLV), and freeze-thaw liposomes (FTV). Phosphatidylethanolamine- [beta] -oleol- [gamma] -palmitoyl (POPE) and cholesterol hemisuccinate (CHOH) were mixed at a molar ratio of 6: 4 to 8: 2 with respect to 8 to 10 mol% of galactose groups A method for producing a liposome capable of simultaneously targeting the liver cells and cancer cells by further mixing the containing lipids. 5. The method of claim 4, wherein the lipid containing galactose group is galactosereveloside or lactocerebroside. The method of claim 4, wherein the liposome is one selected from the group consisting of multilayer membrane liposomes (MLV), small monolayer membrane liposomes (SPLV), and freeze-thaw liposomes (FTV).