KR100996975B1 - Liposome coated with protein to prolong circulation time in bloodstream and preparation method thereof - Google Patents

Abstract

The present invention relates to liposomes modified with proteins that increase the circulation time in the bloodstream and a method for preparing the same. More specifically, the biocompatible proteins are ionically bonded to the surface of the cationic liposomes by electrostatic interaction and bound to the liposome surface. By denaturing the protein to enhance the binding force to liposomes to prevent the adsorption of proteins in plasma when liposomes circulate in the bloodstream to enhance the stability of liposomes in the bloodstream, while increasing the circulation time in the body liposomes and methods for preparing the same It is about.

Proteins, formulas, liposomes, ionic bonds, circulation time

Description

Liposome coated with protein to prolong circulation time in bloodstream and preparation method

The present invention relates to liposomes modified with proteins that increase the circulation time in the bloodstream and a method for preparing the same.

Since liposomes use natural or synthetic phospholipids with the same chemical structure as the phospholipids present in the body as their main components, they have excellent biocompatibility and are widely applied as efficient drug carriers that can deliver drugs through cell fusion or intracellular transfer. [AD Bangham, M. Standish, J. C. Watkis, J. Mol. Biol., 13, 238 (1965). In addition, a highly toxic anticancer drug is encapsulated inside the liposome and delivers the drug to the anticancer cell, thereby reducing the intrinsic toxicity of the drug, thereby preventing side effects of the anticancer drug [D. Needham, M. W. Dewhirst, Adv. Drug Deliver. Rev., 53, 285 (2001). In general, since anticancer drugs have serious side effects that affect not only cancer cells but also normal cells, liposomes are used to efficiently transport these anticancer drugs to cancer tissues, thereby reducing drug toxicity and increasing body circulation time. There are reports that we can do it. [DJA Crommelin, L. van Bloois, Int. J. Pharm., 17, 135 (1983). However, such liposomes are inadequately applied as drug carriers because of structural instability of liposomes due to the adsorption of plasma proteins and the degradation of lipids by enzymes. Therefore, various methods for improving the stability of liposomes have been introduced. In particular, biocompatible polymers are combined on the surface of liposomes to prevent adsorption of plasma proteins and increase the stability and circulation time of liposomes in the blood. A study is underway to make it possible [H. Takeuchi, H. Kojima, H. Yamamoto, Y. Kawashima, J. Control. Release, 75, 83 (2001).

1,2-Distaroylyl- sn -glycero-3-phosphoethanolamine-N- [methoxy- (polyethyleneglycol) -2000] which binds polyethylene glycol to phospholipids in recent liposome development cases (DSPE-mPEG2000) Has been published to prepare structurally stable liposomes. Ceh, M. Winterhalter, PM Frederik, JJ Vallner, DD Lasic, Adv. Drug Deliv. Rev. , 24 , 3356 (1997). The preparation of liposomes incorporating methoxy polyethyleneglycol 2000-lipid complexes according to the prior arts has been a suitable method for improving formulation stability and circulation time [B. Ceh, M. Winterhalter, PM Frederik, JJ Vallner, DD Lasic, Adv. Drug Deliv. Rev. , 24 , 3356 (1997). However, liposomes using methoxy polyethylene glycol 2000 do not degrade by enzymes in vivo and accumulate in cells, causing cell dysfunction and inhibiting the release of drugs at target sites. Boomer, JA, Inerowicz, HD, Zhang, Z., Bergstrand, N., Edwards, K., Kim, JM, Thompson, DH, Langmuir , 19 , 6408 (2003).

US Pat. No. 5,922,350 describes a method for enhancing the stability of liposomes based on phospholipids and the like by adding sugars such as trehalose and sucrose prior to dehydration of the liposomes. However, the patent may be absorbed by the reticulum endothelial system in the body circulation time or spleen when the formulation is not uniformly prepared in the particle size of the formulation is about 100 ~ 500 nm and the particle size of more than 200 nm.

US Pat. No. 7,179,484 describes a method of using proteins to increase the stability of liposomes and to improve circulation time in the bloodstream. However, the patent was modified by using liposomes and albumin for hydrophobic binding, but the albumin having a negative charge and the liposome having a positive charge in the present invention were bound by ionic bonding. It uses stronger ionic bonds than hydrophobic bonds, so it is more stable than liposomes prepared by the patent, and by modifying albumin modified in liposomes, it further enhances the binding force between liposomes and albumin to inhibit the adsorption of plasma proteins in the bloodstream. The stability of the liposomes was improved. The patent also introduces 1,2-distearoyl- sn -glycero-3-phosphoethanolamine-N- [methoxy- (polyethyleneglycol) -2000] (DSPE-mPEG2000) at the time of liposome preparation. Because it is accumulated in the cell rather than degraded by the enzyme when administered in the body can cause cell dysfunction.

U.S. Patent Application No. 2003-748094 describes the formation of a membrane by the addition of an isosmotic agent such as histidine-sucrose buffer solution in the preparation of liposomes to form 1,2-distaroyl- sn -glycero-3-phosphoethanolamine- It is described that the circulation time in the body is improved without introducing N- [methoxy- (polyethylene glycol) -2000] (DSPE-mPEG2000). However, liposome formulations according to the patent are in Only a limited potential for prolonging the circulation time of liposomes in vivo can be shown.

Accordingly, the present inventors modified the protein in liposomes to obtain an equivalent or more effect without introducing polyethylenlycol which may cause cell dysfunction, and enhance the stability of the liposomes by physically and chemically modifying the bound proteins. In addition, it was confirmed that stability in the blood flow was maintained and the circulation time in the body was improved.

In addition, albumin, a biocompatible protein modified into liposomes, is the most abundant serum protein, which accounts for 50 to 55% of the protein produced in the liver and is present in serum, and is a substance that maintains the osmotic pressure of blood. These proteins, such as albumin, are composed of amino acids and consist of disulfide bonds, hydrogen bonds, and hydrophobic bonds by a number of organic amino or carboxyl groups present in the albumin molecule, in addition to the primary structures linked by peptide bonds. In addition, protein folding occurs by the chaffron, a protein cluster present in all cells, forming a three-dimensional conformation of albumin. The folding of proteins is determined by the sequence of amino acids, and there are chaperins that induce the folding of proteins with molecular saffron, which binds to unfolded proteins and reduces protein activity. In addition, albumin is a zwitter ion that has both positive and negative charges, and has an isoelectric point of 5.82. It has strong cationicity at pH 1-4, strong anionicity at pH 6-9, and can be degraded in vivo. It has the property of being denatured by physical action by heat or chemical change such as acid and alkali. In particular, proteins such as albumin are denatured in the form of primary structures in which protein folding is released by breaking hydrogen bonds and hydrophobic bonds that stabilize the intrinsic conformational structure when heat is applied [Drummond, IAS, Steinhardt, RA Exp. Cell Res. 173 , 439 (1987). In addition, the denatured protein has new hydrogen bonds and hydrophobic groups formed by the amide and carbonyl groups of peptide bonds to form an interface, so that free energy is minimized hydrodynamically. Therefore, proteins such as enzyme action, hormone physiology, toxicity, and immunity There is a property that loses its inherent activity (Pelham, H. Nature , 332 , 776 (1988)). Application examples of drug delivery systems such as methods for preparing nanospheres and microspheres for the purpose of delivering water-soluble or poorly soluble drugs using the denatured protein have been studied and reported [Burgess. DJ, Davis. SS, Tomlinson. E., Int. J. Pharm. , 43 , 167 (1988).

U. S. Patent No. 6,749, 868 describes albumin-nanoparticles prepared by combining paclitaxel, a diterpenoid family of poorly soluble drugs, with water-soluble albumin. The patent produces albumin-nanoparticles in which paclitaxel is encapsulated by hydrophobic bonding using a surfactant such as oil with paclitaxel dissolved in an organic solvent and albumin dissolved in water, but the patent is limited to poorly soluble drugs only. Applied. Abraxis Bioscience, Inc. has developed a Abraxane ^® introduction of the prepared formulations using the albumin-modified, based on the information described in the patent as an injection. Abraxane ^® has been reported to improve the body's circulation time when compared to Taxol ^® from Bristol-myers squibb co., Which was solubilized with pure paclitaxel and introduced as an injection. It has been shown to improve stability and blood circulation time in the bloodstream than formulations. However, there have been no reports on the development of liposomes capable of encapsulating not only poorly soluble drugs but also water-soluble drugs. Thus, compared to liposomes that are currently used medically, they significantly improve stability and increase circulation time in tissues other than tumor tissues. It is urgent to develop liposome formulations that can lower the biodistribution of drugs, and to maintain and maintain the effective therapeutic concentration of drugs in the body by targeting them independently and stability to plasma proteins in the bloodstream. It is urgent to develop a formulation that allows the drug to be concentrated.

Accordingly, the present inventors have studied to develop a liposome that is stable in the body by suppressing the adsorption of proteins in the body to solve the above problems, and continuously circulating liposomes, to form a liposome to increase the circulation time in the blood flow Among the lipids, the cationic phospholipid contained a certain amount, and the protein at pH conditions exhibiting anionicity on the surface of the cationic liposome was ionically bound by electrostatic attraction, and the bound biocompatible protein on the liposome surface was denatured to hydrophobic group. By forming the interface exposed on the surface of the protein, the hydrodynamic free energy is minimized, thereby inhibiting the adsorption of the protein in the bloodstream and improving the body circulation rate.

Therefore, the present invention is aimed at stabilizing-internal circulation sustained liposomes in which the protein is modified on the surface of the liposome and then denatured to inhibit the adsorption of the protein in the bloodstream and the body circulation speed is improved.

The present invention is characterized by an increased liposomes with increased circulation time in the bloodstream in which proteins are ion-bonded to the surface of liposomes containing cationic lipids.

In another aspect, the present invention is characterized by an injection containing a liposome modified with the protein.

Stabilized-in vivo circulation sustained liposomes that modify proteins and denature the modified proteins on the surface of liposomes according to the present invention have improved in vivo circulation time and enhanced stability in plasma, as well as plasma proteins, compared to conventional liposomes. Suppresses adsorption of Stabilizing-in vivo circulation sustained liposomes, which inhibit the adsorption of these plasma proteins and improve circulation time in the bloodstream, have an effect of prolonging the retention rate of liposomes containing the drug for a long time in the bloodstream. To improve the efficiency of delivering to the target site in the body has the effect that can continue to show the efficacy than the conventional formulation.

Hereinafter, the present invention will be described in detail.

The present invention prevents the adsorption of proteins in plasma when liposomes circulate in the bloodstream by ionically binding proteins to the surface of cationic liposomes by electrostatic interactions and denaturing proteins bound to liposomes to enhance binding to liposomes. The present invention relates to a method for preparing liposomes, which enhances the stability of liposomes in the bloodstream and increases circulation time in the body.

In the present invention, the protein ion-bound to liposomes can be separated and extracted from various animal tissues and plant tissues, or a recombinant protein can be used, and a protein having a weight average molecular weight of 2,000 to 1,500,000 Da or a derivative thereof is preferable. . For example, simple proteins, such as albumin, globulin, glutenine, prolamine, albuminoid; Complex proteins such as nucleoprotein, glycoprotein, phosphoprotein, lipoprotein, and pigment protein; And induction proteins such as gelatin, preteose, peptone, dipeptide, tripeptide and the like, which are intermediate products from which the protein is hydrolyzed.

In particular, albumin, a protein used in the present invention, includes animal albumin isolated from eggs, animal serum, milk, liver, and muscle, and vegetable albumin isolated from barley, pea, and castor seeds. The molecular weight of albumin, including the extraction step and used in the present invention, ranges from 2,000 to 1,500,000 Da. As used herein, "albumin" is meant to include the albumin, fragments of the albumin, salts of the albumin or derivatives of the albumin.

In the present invention, by modifying the surface of the liposomes with a biocompatible protein and denature the protein to provide a stable stabilization-internal circulation liposomes with improved blood flow stability and circulation time.

As used herein, "surface modification" means that a biocompatible protein exhibiting anionicity on the surface of a cationic liposome is modified through ionic bonds on the surface of the liposome. In addition, the "stabilization-internal circulation sustained-type" liposomes used in the present invention are liposomes in which biocompatible proteins are modified on the surface of the liposomes and denatured by heat, thereby inhibiting the adsorption of plasma proteins in the blood stream to improve the body circulation time. Means liposomes.

The biocompatible protein preferably contains 0.01 to 100 parts by weight, more preferably 0.5 to 50 parts by weight, based on 1000 parts by weight of the lipid forming the total liposome. If the biocompatible protein is less than the above range, since the amount of the biocompatible protein modified on the surface of the liposome is too small, the liposome may become unstable by adsorption of plasma protein in the blood stream, and the biocompatible protein is within the above range. If more present, the liposome particle size is increased, and thus stability and blood circulation time are not increased in the blood stream. Even if the biocompatible protein contains more than 100 parts by weight of liposomes, the surface area of the liposomes is limited, so the anionic enhancement effect of the liposomes no longer occurs.

The average particle diameter of the modified liposome is 30 to 400 nm, preferably 90 to 110 nm. If the mean particle size of the liposome exceeds the above range, uptake occurs by reticuloendothelial system in the organ, ie liver or spleen, during circulation, and if the liposome is below the above range, the amount of drug delivered to the target site (drug payload) is insufficient.

The lipid constituting the liposome of the present invention as the lipid for forming the liposome is not particularly limited and may be a known lipid. Examples of the lipids include phospholipids, glycolipids, sterols, cationic lipids, and the like, polyglycerol alkyl ethers, polyoxyethylene alkyl ethers, alkyl glycosides, alkyl methyl glucamides, alkyl sucrose esters and dialkyl polyoxyethylenes. Long-chain alkylamines or long-chain fatty acid hydrazides such as amphiphilic block copolymers such as polyoxyethylene-polylactic acid, such as ether and dialkyl polyglycerol ether.

As the phospholipid, for example, phosphatidylcholine (soy phosphatidylcholine, egg yolk phosphatidylcholine, bovine phosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine, etc.), phosphatidylethanolamine Lauroyl phosphatidyl ethanolamine, dimyristoyl phosphatidyl ethanol amine, dipalmitoyl phosphatidyl ethanol amine or distearoyl phosphatidyl ethanol amine, dioleoyl phosphatidyl ethanol amine, etc.), phosphatidylserine (dilauroyl phosphatidyl glycerol, Dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine or distearoylphosphatidylserine, etc.), phosphatidic acid, phosphatidylglycerol (dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol or dis) Thea Ylphosphatidylglycerol, etc.), phosphatidyl inositol (dilauroylphosphatidyl inositol, dimyristoyl phosphatidylinositol, dipalmitoyl phosphatidylinositol or distearoyl phosphatidyl inositol, etc.), risphophosphidyl choline, sphingomyelin, yolk lecithin, soybean lecithin Or one or more of natural or synthetic phospholipids such as hydrogenated phospholipids.

As said glycolipid, glycerol glycolipid, sphingolipid glycolipid, etc. are mentioned, for example. Examples of the glycerol glycolipids include digalactosyldiglycerides (digalactosyldilauroylglycerides, digalactosyldimyristoylglycerides, digalactosyldipalmitoylglycerides or digalactosyldistearoylglycerides, and the like) or galactosyldi Glycerides (galactosyl dilauroyl glyceride, galactosyl dimyristoyl glyceride, galactosyl dipalmitoyl glyceride, galactosyl distearoyl glyceride, etc.) etc. are mentioned. Examples of the sphingoglycolipids include galactosyl selecbroside, lactosyl selecbroside, and gangloside.

In addition, the phospholipid or glycolipid preferably contains 30 to 70 mol% of the total liposome lipid composition.

Examples of the sterols include cholesterol, cholesterol hexasuccinate, 3β- [N- (N '(N', N'-dimethylaminoethane) carbamoyl] cholesterol, ergosterol, lanosterol, and the like. It is preferable to include 20 to 50 mol% of the total liposome lipid composition. If the ratio of sterols is out of the above range, the stability of liposomes is lowered and there is a problem in the encapsulation of the drug.

The liposome forming lipids can be used alone or in combination of two or more thereof.

In addition, although the charge of liposomes bound by the properties of proteins that are anionic at biological pH is not necessarily limited, cationic liposomes are preferred to enhance drug delivery into tumor cell tissue.

Examples of cationic lipids having surface charges greater than 0 mV when present at physiological pH in the liposome-forming lipids include, for example, dioctadecyl amidoglyciylspermidine (DOGS) and dimethyldiooctadecylammonium bromide (DDAB). ), La-dioleoyl phosphatidylethanolamine (DOPE), [N- (N, N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), N- [1- (2,3-di Oleyloxy) propyl] -N, N, N-trimethylammonium bromide (DOTMA), 2,3-dioleoyloxy-N- [2- (sperminecarbozamide-O-ethyl] -N, N- Dimethyl-propaneaminium trifluoroacetate (DOSPA), 1- [2- (oleoyloxy) -ethyl] -2-oleyl-3- (2-hydroxyethyl) imidazolinium chloride (DOTIM) , 1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide (DMRIE), 1,2-dimyristoyl-3-dimethylammonium propane (DMDAP), 1,2-dipalmi Saturday-Dimethylarm Propane (DPDAP), 1,2-dilauroyl-3-dimethylammonium propane (DLDAP), 1,2-distearoyl-3-dimethylammonium propane (DSDAP), 1,2-diole Oil-3-dimethylammonium propane (DODAP), 1,2-dimyristyl-3-dimethylammonium propane (DMDAP), 1,2-dipalmityl-3-dimethylammonium propane (DPDAP), 1, 2-dilauryl-3-dimethylammonium propane (DLDAP), 1,2-distearyl-3-dimethylammonium propane (DSDAP), 1,2-diolyl-3-dimethylammonium propane (DODAP ), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dipalmitoyl-3-trimethylammonium propane (DPTAP), 1,2-dilauroyl-3-trimethylammonium Propane (DLTAP), 1,2-distearoyl-3-trimethylammonium propane (DSTAP), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dimyristyl-3- Trimethylammonium propane (DMTAP), 1,2-dipalmityl-3-trimethylammonium propane (DPTAP), 1,2- 1 type selected from dilauryl-3-trimethylammonium propane (DLTAP), 1,2-distearyl-3-trimethylammonium propane (DSTAP), 1,2-dioleyl-3-trimethylammonium propane (DOTAP) Use the above. It is preferred that the cationic lipids comprise 1 to 40 mole percent of liposome-forming total lipids, more preferably 1 to 20 mole percent. If the cationic lipid is out of this range, stability problems such as precipitation of liposomes occur.

In addition, one of the preferred compositions of the present invention using a liposome composition consisting of 30 to 70 mol% of phospholipids or glycolipids, 20 to 50 mol% of sterols and 1 to 40 mol% of cationic lipids in order to be effective in the formation and stability of liposomes will be.

The preparation method of liposomes before modifying the biocompatible protein of the present invention can be prepared by various known techniques. One of the preferred methods for preparing liposomes prior to modifying the biocompatible protein of the present invention is not limited to the reversed-evaporation method described below.

First, the lipid components capable of forming liposomes are mixed. The liposome-forming lipid preferably contains cationic lipids as described above. After dissolving the mixed lipid component in a solvent capable of dissolving lipids such as chloroform, the chloroform is distilled off using an evaporative condenser to form a thin lipid film.

Thereafter, a solution such as ammonium sulfate (Ammonium Sulfate), which may play a role of drug encapsulation due to the difference in concentration, is added to hydrate the lipid membrane to form liposomes. The liposomes are subjected to reduced pressure extrusion to prepare liposomes having an average particle diameter of 30 to 400 nm.

Next, the drug is encapsulated in liposomes. “Entrapping” herein means that the compound is enclosed in an oily space between the liposome lipid bilayers or an aqueous space in the bilayer. The stabilizing-internal circulation sustained liposome of the present invention is capable of encapsulating both hydrophobic drugs and water-soluble drugs, and is commonly used anthracycline-based drugs (doxorubicin, epirubicin, hirubicin, daunorubicin, esorubicin, Rubicin, etc.), alkaloid-based drugs, diterpenoid-based drugs (paclitaxel, docetaxel, etc.) or pharmacologically acceptable salts and acids thereof are advantageous for encapsulation, but are not particularly limited thereto. In particular, anthracycline-based drugs such as doxorubicin have a feature of forming a precipitate by meeting with ammonium sulfate in liposomes to seal the drug. The water-soluble drugs include cyclophosphamide, phosphamide, melphalan, carmustine, romustine, streptozosin, carboplatin, cysplatin, oxaliplatin, dacarbazine, procarbazine, uracilmustine, methotrexate , Pemetrexed, ralcitrexed, fludarabine, mercaptopurine, pentostatin, thioguanine, capecitabine, cytarabine, fluorouracil, daunorubicin, doxorubicin, epirubicin, aidarubicin, mito Xanthrone, Valrubicin, Gemcitabine, Actinomycin, Bleomycin, Mitomycin, Topotecan, Irinotecan, Etoposide, D-Aminolevulinic Acid, Methylaminolevulinate, Erlotinib, Geritinib, Yi Methinib, doxorubicin, epirubicin, hirubicin, daunorubicin, esorubicin, idarubicin, alendronate, pamironate, zoleronate, peptide drugs (contrico Tropine, tetracosactide, tyrotropin, somatrem, mecassermine, sermorelin, desmopressin, leipresin, terripressin, ornipressin, argipressin, democytosine, carvetosin, Gonadorerin, naparerin, histerin, somatostatin, octriotide, lanthanated acetate, garnirerix, celorrerix, pegbisomant, etc.).

The efficiency of encapsulation in liposomes of the drug ranges from 80 to 100%.

The drug-encapsulated liposome, preferably the biocompatible protein of the present invention is dissolved in an aqueous solution of pH 5.8 to 14 above the isoelectric point, and then added dropwise to the cationic liposome encapsulated with the drug and stirred at 20 to 40 ° C. for 1 to 24 hours. . The protein to be modified is preferably 0.01 to 100 parts by weight based on 1000 parts by weight of the total lipid forming liposomes. Biocompatible proteins not bound to the liposomes are removed by centrifugation at 1,000 to 10,000 rpm for 1 to 24 hours using Cenricon ^® having a fractional molecular weight of 10,000 to 200,000.

Next, denaturation is applied to the modified biocompatible protein on the surface of the liposome. The method of denaturing the biocompatible protein may be chemical, physical and thermal denaturation, but is not particularly limited thereto.

Physical denaturation involves denaturing proteins with high pressure, ultraviolet radiation, and X-rays. Chemical denaturation involves protein denaturation with organic solvents, heavy metal salts, urea, and guanidine-HCL. Denatured by adding an inducing agent.

The reason for denaturing the biocompatible protein is to maximize the stability of albumin modified in liposomes and thus increase the stability of liposomes. Preferably, the modified protein is denatured on the surface of the liposome by heating at 60-100 ° C. for 1 minute to 24 hours.

The liposomes are used alone or in combination with conventional carriers in the pharmaceutical field for injection (subcutaneous injection, intravenous injection, intramuscular injection, thoracic injection, etc.). Injections prepared using the circulating sustained liposomes of the present invention may exhibit pharmacological activity equivalent to or better than that of the pure drug, even if less than 30% of the pure drug is used.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Preparation of Stabilized In-Circulation Sustained Liposomes

(1) Preparation of Liposomes Modified with Modified Albumin

Lipids that form liposomes: 54.0 mol% La-phosphatidylcholine (HSPC), 39.5 mol% cholesterol (CHOL), and 6.5 mol% 1,2-distearoyl-3-trimethylammonium propane (DSTAP) relative to total lipids And total lipid concentration was 12.8 mg / ml. Subsequently, La-phosphatidylcholine, cholesterol, and 1,2-distearoyl-3-trimethylammonium propane (DSTAP) were dissolved using chloroform, and the solvent was distilled off using a rotary evaporator to thin the lipid. A film was formed. Next, liposomes were prepared by hydrating a 250 mM ammonium sulfate solution at 60 ° C. on the formed lipid membrane. After the hydration process, liposomes were formed at about 100 nm in size using a pressure extruder. Unsealed ammonium sulfate was removed using membrane permeation and doxorubicin was encapsulated inside the liposome using ion gradient loading. Unsealed doxorubicin was then removed using membrane permeation.

100 μg / ml, 400 μg / ml, 800 μg / ml, 1200 μg, respectively, dissolved in phosphate buffer solution (PBS) at pH 7.4 to modify albumin on the surface of cationic liposomes (total lipid concentration of 12.8 mg / ml). / ML, and 1600 ㎍ / ㎖ bovine serum albumin (BSA) aqueous solution was added dropwise to the cationic liposome at a rate of 20 μl / min with stirring for 1 hour at 37 ℃, prepared cationic liposomes and BSA aqueous solution Is finally 1: 1 (v / v). Thereafter, in the purification process, heat modification was performed at 100 ° C. for 30 minutes to prevent adsorption of unmodified albumin and modified albumin to liposomes and to enhance stability of albumin-modified liposomes [Leucuta, SE, Risca, R., Daicoviciu, D., Int. J. Pharm. , 43 , (213) 1988]. The process after the cut-off molecular weight of 100,000 by using Centricon ^® centrifugal separation to the denatured albumin is to eliminate unqualified albumin to stabilize over liposome formula - continuous vivo circulating liposomes were prepared.

(2) Preparation of Liposomes Modified with Albumin

The same liposome-forming lipids as in Example 1- (1) were carried out in the same manner, but the modified albumin was not denatured by heat, but after adding 10% sucrose, the liposome was centrifuged using Centricon ^{® with a} molecular weight of 100,000. Unmodified albumin was removed.

Example 2 Preparation of Liposomes Encapsulated with Water-Soluble Drugs

(1) Preparation of Liposome Encapsulated Alendronate

Lipids that form liposomes: 54.0 mol% La-phosphatidylcholine (HSPC), 39.5 mol% cholesterol (CHOL), and 6.5 mol% 1,2-distearoyl-3-trimethylammonium propane (DSTAP) relative to total lipids And total lipid concentration was 12.8 mg / ml. Subsequently, La-phosphatidylcholine, cholesterol, and 1,2-distearoyl-3-trimethylammonium propane (DSTAP) were dissolved using chloroform, and the solvent was distilled off using a rotary evaporator to thin the lipid. A film was formed.

Next, liposomes were prepared by hydrating 300 mM citric acid solution (pH 3.5) at 60 ° C. on the formed lipid membrane. After the hydration process, liposomes were formed to a size of about 100 nm using a pressure extruder. Unsealed citric acid was removed using membrane permeation, alendronate was encapsulated inside the liposomes using pH gradient loading and unsealed alendronate was removed using membrane permeation.

Liposome prepared to modify albumin on the surface of liposome encapsulated with alendronate was adjusted to pH 7.4 using phosphate buffer (pH 7.4) and then dissolved in phosphate buffer (PBS) at 800 μg / ml The aqueous serum albumin (BSA) solution was added dropwise to liposomes at a rate of 20 μl / min with stirring at 37 ° C. for 1 hour, and the prepared liposomes and BSA aqueous solution were finally 1: 1 (v / v). After purification, heat denaturation was performed at 100 ° C. for 30 minutes to prevent adsorption of unmodified albumin and modified albumin to liposomes and to enhance stability of albumin-modified liposomes [Leucuta, SE , Risca, R., Daicoviciu, D., Int. J. Pharm. , 43 , (213) 1988]. After the above process, centrifugation was performed using Centricon ^{® with a} fraction molecular weight of 100,000 to remove unmodified albumin from liposomes, thereby preparing liposomes encapsulated with alendronate and modified with albumin.

(2) Preparation of liposomes enclosed with octriotide

It was prepared with the same liposome-forming lipid component as in Example 2- (1), and octopiide was encapsulated using the Freeze-thaw method. The freeze process was carried out at -10 ° C, the thaw process was carried out at 60 ° C, and the freeze process and thaw process were carried out five times at five minute intervals for each freeze process and thaw process. Triotide was encapsulated in liposomes.

A 20 μl / min rate of 800 μg / ml Bovine Syrum Albumin (BSA) solution dissolved in phosphate buffer (PBS) prepared to modify albumin on the surface of the encapsulated liposome with octriotide was added to the liposome. The mixture was stirred with stirring at 37 ° C. for 1 hour while being added dropwise thereto, and the prepared liposomes and BSA aqueous solution were finally 1: 1 (v / v). After purification, heat denaturation was performed at 100 ° C. for 30 minutes to prevent adsorption of unmodified albumin and modified albumin to liposomes and to enhance stability of albumin-modified liposomes [Leucuta, SE , Risca, R., Daicoviciu, D., Int. J. Pharm. , 43 , (213) 1988]. After the above process, centrifugation was performed using Centricon ^{® with a} fraction molecular weight of 100,000 to remove unmodified albumin from liposomes, thereby preparing liposomes filled with octriotide and modified albumin.

Comparative Example 1: Preparation of Control Liposomes

Lipids that form liposomes were mixed with 60.4 mol% L-a-phosphatidylcholine (HSPC) and 39.6 mol% cholesterol (CHOL) relative to the total lipids and the total lipid concentration was 12.8 mg / ml. Thereafter, after dissolving L-a-phosphatidylcholine and cholesterol using chloroform, the solvent was distilled off using a rotary evaporator to form a thin lipid membrane. Next, liposomes were prepared by hydrating a 250 mM ammonium sulfate solution at 60 ° C. on the formed lipid membrane. After the hydration process, liposomes were formed at about 100 nm in size using a pressure extruder. Unsealed ammonium sulfate was removed using membrane permeation, doxorubicin was encapsulated inside the liposome using ion gradient loading, and unsealed doxorubicin was removed using membrane permeation.

Comparative Example 2: Preparation of Cationic Liposomes

In the same manner as in Comparative Example 1, using liposome lipid component 1,2- distearoyl-3-trimethylammonium propane (DSTAP) 54.0 mol% of La-phosphatidylcholine (HSPC), cholesterol ( CHOL) 39.5 mol%, 1,2-distearoyl-3-trimethylammonium propane (DSTAP) 6.5 mol%, the total lipid concentration was 12.8 mg / ml.

Comparative Example 3: Preparation of Control Liposomes Incorporated with Polyethylene Glycol

The same procedure as in Comparative Example 1, except that 1,2-distearoyl- sn -glycero-3-phosphoethanolamine-N- [methoxy- (polyethylene glycol) -2000] (DSPE- mPEG2000) 57.3 mol% La-phosphatidylcholine (HSPC), 37.5 mol% cholesterol (CHOL), 1,2-distearoyl- sn -glycero -3-phosphoethanolamine-N- [Methoxy- (polyethyleneglycol) -2000] (DSPE-mPEG2000) was mixed at 5.2 mol% and the total lipid concentration was 16 mg / ml.

Comparative Example 4: Preparation of Cationic Liposomes Incorporated with Polyethylene Glycol

In the same manner as in Comparative Example 1, 1,2-distearoyl-3-trimethylammonium propane (DSTAP) and 1,2-distearoyl- sn -glycero-3 in the lipid component constituting the liposome 51.3 mol% La-Phosphatidylcholine (HSPC), 37.4 mol% Cholesterol (CHOL), based on total lipids using -phosphoethanolamine-N- [methoxy- (polyethyleneglycol) -2000] (DSPE-mPEG2000) 6.2 mol%, 2-distearoyl-3-trimethylammonium propane (DSTAP), 1,2-distearoyl- sn -glycero-3-phosphoethanolamine-N- [methoxy- (polyethylene glycol ) -2000] (DSPE-mPEG2000) was mixed at 5.1 mol% and the total lipid concentration was 16 mg / ml.

Test Example 1

In the preparation of the liposomes of Examples 1- (1) and 1- (2), the amount of albumin modified in liposomes is more preferably 100 μg / ml, so that 0.5 to 50 parts by weight relative to 1000 parts by weight of total liposome lipids, The particle size and surface charge of stabilized-internal circulation sustained liposomes prepared using 400 μg / ml, 800 μg / ml, 1200 μg / ml, and 1600 μg / ml BSA aqueous solution were respectively determined by using a light scattering device (ELS-Z, Otsuka, Japan) and Zeta Potentiometer (ELS-Z, Otsuka, Japan) measured in Table 1 [L. Zhu, RA Seburg, EW Tsai, J. Pharm . Biomed . Anal . , 8 (2005)]. In addition, 0.1 ml of protein assay reagent (Protein assay, Bio-Rad Lab., USA) was added to 0.1 ml of the stabilizing-internal circulation sustained-type liposome solution prepared in Test Example 1 using a 96-well plate, and then enzyme immunological activity. Absorbance was measured at 590 nm with a measuring instrument to quantify the amount of albumin modified in liposomes and is shown in Table 1 [MM Bradford J. Anal. Biochem. 72 (1946). The calibration curve of protein amount was prepared by preparing 1 mg / ml BSA solution dissolved in PBS solution.

Test Example 2

The particle size and surface charge of the liposomes prepared in Examples 2- (1) and (2) were measured in the same manner as in Test Example 1, and are shown in Table 2 below. In addition, the encapsulation rate of the drug encapsulated in the liposome was measured by using an ultraviolet spectrometer, and is shown in Table 2 below.

Test Example 3

Particle sizes and surface charges of the liposomes prepared in Comparative Examples 1, 2, 3, and 4 were measured in the same manner as in Test Example 1, and are shown in Table 3 below. All liposomes prepared according to Comparative Example 1 exhibited particle sizes within the range of 100-120 nm. Particularly, Comparative Examples 3 and 4 exhibited about 35 to 50 kPa of PEG bound to the polyethyleneglycol-lipid complex used in the preparation of liposomes, depending on the form, which is about 11 nm ± 0.3 nm, which is about 10 nm larger than Comparative Examples 1 and 2. Particle size is shown [Che, B., Winterhalter, M., Frederick, PM, Vallner, JJ, Lasic, DD Adv . Drug Delivery . Rev. (24) 1997]. In addition, the liposomes prepared according to Comparative Example 1 exhibited neutral surface charges, and Comparative Example 2 exhibited cationicity under the influence of DSTAP lipids introduced during the preparation of liposomes. The liposomes prepared by Comparative Example 3 and Comparative Example 4 exhibited anionic surface charges of about -18.0 ± 7.0 ㎷ due to the influence of DSPE lipids contained in the polyethyleneglycol-lipid derivatives introduced during preparation [Che, B ., Winterhalter, M., Frederick, PM, Vallner, JJ, Lasic, DD Adv . Drug Delivery. Rev. (24) 1997].

Test Example 4 Pharmacokinetic Experiment

Example 1- (1) Sample 3 produced by the stabilization of the present invention the body circulation persisted liposome SD rats rats in pure doxorubicin injections of Co. Boryeong's Adriamycin ^® to observe in vivo circulation time of, Doxil ^® of Alza, a liposome formulation using phospholipids, and stabilizing-internally sustained liposomes of Sample 3 modified with 147 μg of albumin prepared according to Example 1- (1) of the present invention were administered via the tail vein. The blood concentration was measured by measuring the concentration of the drug encapsulated inside the liposome. Pharmacokinetic results were calculated as the measured concentration of the drug, and the body circulation time of the drug was confirmed through the calculated pharmacokinetic results. The concentration of the drug in the liposome was measured by diluting the blood sampled with the heparin solution by centrifugation and diluting the supernatant with methanol to prepare the solution at a wavelength of 234 nm using an ultraviolet spectrophotometer. The absorbance of was measured [T. Hu et al J. pharmaceutical and biomedical analysis. 43 (2007).

The pharmacokinetic results calculated in the above experiments are shown in Table 4 and FIG. 1.

AUC (Area Under the Curve) in Table 4 is the area for the concentration of residence, Mean Residence Time (MRT) is the residence time of the drug in the blood stream, C _max is the largest concentration of the drug in the blood stream, t _1/2 is the blood flow Drug half-life, and CL (Clearance) is the rate of drug loss in the bloodstream.

As shown in Table 4 and Figure 1, the injection using a liposome modified with the protein of Sample 3 of Example 1- (1) according to the present invention has a half-life of 30.0 hours, retention time 45.7 hours, The area was 1931.7 hr * µg / ml and the drug loss rate was 2.5 hours. However, the half-life of Adriamycin ^® was 6.5 hours and the half-life of Doxil ^® was 36.3 hours. The increase in half-life suggests a retention time of 50% or more of the drug in the bloodstream that can maintain the effective concentration of the drug, and shows a significantly longer circulatory time compared to Adriamycin ^® and the Doxil ^® liposome formulation. Equivalent or more body circulation time can be observed. As a result, by binding the albumin, a plasma protein, on the surface of liposomes, the body circulation time of liposomes can be increased more than the pure doxorubicin drug, and the body circulation time of the liposomes that is equivalent to the conventional doxorubicin-encapsulated liposome formulation is Prove the fact

Test Example 5 Comparative Experiment of Stability of Liposomes in Plasma

In order to measure the stability of the liposome particles in plasma, the liposomes prepared in Samples 3, Comparative Examples 1, 2, 3, and 4 of Example 1- (1) and SD rat plasma solution were 1: 3 (v / v) and then change the liposome particle size change over time at 37 ℃ and the results are shown in Figure 2 below. As shown in FIG. 2, the particle size of the liposome modified with albumin, a plasma protein according to the present invention of Example 1, was observed in the plasma at about 120 nm after 48 hours. However, the liposomes prepared in Comparative Examples 1 and 2 gradually increased in size due to opsonization action by plasma proteins, and especially the cationic liposomes of Comparative Example 3 suddenly mixed with plasma proteins. An increase in size was observed. In addition, the liposomes prepared in Comparative Examples 3 and 4 have increased particle sizes from about 140 nm to 170 nm due to the introduction of hydrophilic polymer polyethylene glycol. Therefore, it can be said that liposomes modified with albumin and modified with albumin are stable in plasma.

Test Example 6 Comparison of Protein Adsorption of Liposomes in Plasma

In order to evaluate the protein adsorption prevention efficiency of Example 1, the liposome solution modified with albumin, a plasma protein of Example 1- (1), and the liposomes prepared in Comparative Examples 1, 2, 3, and 4 Each sample was mixed with rat plasma solution 1: 3 (v / v), and 1 ml of the sample was taken at a predetermined time while stirring at 37 ° C. The protein adsorbed on the liposome is shown in FIG. 3. The collected sample was centrifuged at 4000 rpm for 1 hour using Centricon ^® with a molecular weight of 100,000 to obtain only the liposome to which the protein was adsorbed, and the process of purifying the liposome was repeated twice. Since the protein adsorbed liposomes were measured by absorbance at 590 nm using an enzyme immunoassay to determine the protein adsorbed on liposomes. As shown in FIG. 3, the liposomes prepared in Comparative Examples 1 and 2 were found to adsorb 1 mg or more of plasma proteins after 48 hours, and in particular, the cationic liposome prepared by Comparative Example 2 was about 8 mg of protein adsorption. The amount was measured. The stabilization-in vivo circulating sustained liposome according to the present invention of Example 1- (1) measured protein adsorption amount of about 400 μg after 48 hours, thereby significantly inhibiting protein adsorption than liposomes prepared by Comparative Example 1 and Comparative Example 2. When compared with Comparative Example 3 and Comparative Example 4 in which a polyethylene glycol-lipid derivative was introduced, it was shown that the plasma protein adsorption was inhibited. Thus, denatured albumin modified on the surface of liposomes can be said to prevent the adsorption of plasma proteins in the blood.

1 is a graph showing the pharmacokinetic results calculated in Test Example 4 [■; Adriamycin ^® , ●; Doxil ^® , ▲; Injection using sample 3 of Example 1- (1)].

FIG. 2 is a graph illustrating changes in particle size of liposomes in plasma calculated in Test Example 5 [●; Comparative Example 1, ▲; Comparative Example 2, ▼; Comparative Example 3, ◀; Comparative example 4, ■; Sample 3, ▶ of Example 1- (1); Sample 3 of Example 1- (2)].

3 is a graph showing the amount of protein adsorbed on liposomes in the plasma produced in Test Example 6 [▨; Comparative Example 1, i; Comparative Example 2, □; Comparative Example 3, i; Comparative Example 4, ■; Sample 3 of Example 1- (1)].

Claims (14)

Albumin is ion-bonded to the liposome surface including a cationic lipid, and the albumin is liposome with increased circulation time in the blood stream, characterized in that the heat is denatured for 1 minute to 24 hours at 60 ~ 100 ℃. delete The phospholipid of claim 1, wherein the phospholipid forming the liposome is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, risphophosphatidylcholine, sphingomyelin, egg yolk lecithin, soybean lecithin and hydrogenated phospholipids. Liposomes, characterized in that one or more. The method of claim 1, wherein the liposome-forming glycolipid is at least one selected from digalactosyl diglycerides, galactosyl diglycerides, galactosyl selebrosides, lactosyl selebrosides and hepaglosides. Liposomes. The method of claim 1, wherein the sterols forming the liposome include cholesterol, cholesterol hexasuccinate, 3β- [N- (N ', N'-dimethylaminoethane) carbamoyl] cholesterol, ergosterol or lanosterol. Liposomes, characterized in that at least one selected. The method of claim 1, wherein the cationic lipid is dioctadecyl amidoglyciylspermidine (DOGS), dimethyldiooctadecylammonium bromide (DDAB), La-dioleoyl phosphatidylethanolamine (DOPE), [ N- (N, N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium bromide (DOTMA), 2,3-dioleoyloxy-N- [2- (sperminecarbozamide-O-ethyl] -N, N-dimethyl-propaneaminium trifluoroacetate (DOSPA), 1- [2- (Oleoyloxy) -ethyl] -2-oleyl-3- (2-hydroxyethyl) imidazolinium chloride (DOTIM), 1,2-dimyristyloxypropyl-3-dimethyl- Hydroxy ethyl ammonium bromide (DMRIE), 1,2-dimyristoyl-3-dimethylammonium propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium propane (DPDAP), 1,2- Dilauroyl-3-dimethylammonium propane (DLDAP), 1,2-diste Royl-3-dimethylammonium propane (DSDAP), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-dimyristyl-3-dimethylammonium propane (DMDAP), 1 , 2-dipalmityl-3-dimethylammonium propane (DPDAP), 1,2-dilauryl-3-dimethylammonium propane (DLDAP), 1,2-distearyl-3-dimethylammonium propane ( DSDAP), 1,2-dioleyl-3-dimethylammonium propane (DODAP), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dipalmitoyl-3-trimethyl Ammonium propane (DPTAP), 1,2-dilauroyl-3-trimethylammonium propane (DLTAP), 1,2-distearoyl-3-trimethylammonium propane (DSTAP), 1,2-dioleoyl- 3-trimethylammonium propane (DOTAP), 1,2-dimyristyl-3-trimethylammonium propane (DMTAP), 1,2-dipalmityl-3-trimethylammonium propane (DPTAP), 1,2-dilauryl 3-trimethylammonium propane (DLTAP), 1,2-distearyl-3-trimethylammonium Ropan (DSTAP), liposomes, characterized in that at least one member selected from the group consisting of 1,2-oleyl-3-trimethylammonium propane (DOTAP). delete delete The liposome according to claim 1, wherein the liposome has an average particle diameter of 30 to 400 nm. According to claim 1, wherein the albumin liposomes, characterized in that 0.01 to 100 parts by weight based on 1000 parts by weight of the lipid forming the liposomes. The liposome according to claim 1, wherein the liposome is composed of 30 to 70 mol% of phospholipids or glycolipids, 20 to 50 mol% of sterols and 1 to 40 mol% of cationic lipids, based on the total liposome-forming lipid. Injectable drug, characterized in that the drug is encapsulated in any one of the liposomes selected from claim 1, 3 to 6 and 9 to 11. The method of claim 12, wherein the drug is cyclophosphamide, phosphamide, melphalan, carmustine, lomustine, streptozosin, carboplatin, cisplatin, oxaliplatin, dacarbazine, procarbazine, uracilmu Stain, methotrexate, pemetrexed, ralcitrexed, fludarabine, mercaptopurine, pentostatin, thioguanine, capecitabine, cytarabine, fluorouracil, daunorubicin, doxorubicin, epirubicin, Idarubicin, mitoxanthrone, varubicin, gemcitabine, actinomycin, bleomycin, mitomycin, topotecan, irinotecan, etoposide, D-aminolevulinic acid, methylaminolevulinate, erlotinib, Geritinib, Imetinib, Doxorubicin, Epirubicin, Hyrubicin, Daunorubicin, Esorubicin, Idarubicin, Alendronate, Pamideronate, Zoledronate, Contricotropine, Tetracosatide, Tyrotropin, Somatrem, Mecassermine, Sermorelin, Desmopressin, Leipresin, Terlippressin, Ornipressin, Argipressin, Demoxitocin, Carvetosin, Konadore Injectable drug, characterized in that selected from lean, naparerin, histerin, somatostatin, octriotide, olantate acetate, garnirerix, setlorrex and pegbisomant. 1) mixing a phospholipid or glycolipid 30 to 70 mol%, sterols 20 to 50 mol% and cationic lipids 1 to 40 mol% relative to the lipid forming the total liposomes to prepare a drug-containing cationic liposome, 2) dissolving albumin in an aqueous solution of pH 5.8 to 14, and then dropwise adding the cationic liposome to the mixture at 20 to 40 ° C. for 1 to 24 hours, and 3) thermally denaturing the modified albumin on the surface of the liposome at 60 to 100 ° C. for 1 minute to 24 hours Method for producing a liposome with increased circulation time in the blood stream, characterized in that consisting of.

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