KR20080094473A - Anionic lipid nanosphere and preparation method of the same - Google Patents

Abstract

An anionic lipid nanosphere is provided to show increased encapsulation efficiency of an insoluble drug in an aqueous phase by introducing a polyethylene glycol(PEG) containing polyol onto the surface of particles formed from an anionic phospholipid, reduce toxicity of the drug against normal cells and increase in vivo circulation time of the drug by encapsulating a very strongly toxic drug into lipid nanosphere. An anionic lipid nanosphere for encapsulating an insoluble drug such as amphotericin B is characterized in that a PEG containing polymer is coated on the surface of particles formed by an anionic phospholipid. A method for preparing the anionic lipid nanosphere comprises the steps of: (a) mixing 100 parts by weight of lipid prepared by mixing phosphatidylcholine, anionic phospholipd and sterol in a weight ratio of 40-70:5-20:10-40 with 10-30 parts by weight of the polymer containing the PEG and dissolving a mixture in an organic solvent to prepare a lipid-PEG mixture solution; (b) dissolving an insoluble drug in C1-6 linear or branched alcohol to prepare a drug solution; (c) mixing the lipid-PEG mixture solution with the drug solution in a volumetric ratio of 1:1-1:9 to prepare a lipid-PEG-drug mixture solution; (d) dispersing the mixture solution into an aqueous phase in a volumetric ratio of 2:1-10:1 to form lipid nanosphere; and (e) after subjecting the lipid nanosphere solution obtained from the step(d) to distillation at a temperature of 20-50 deg.C under reduced pressure to remove the organic solvent therefrom, filtering it to prepare the anionic lipid nanosphere encapsulating the drug.

Description

Anionic Lipid Nanoparticles and Preparation Method thereof

Figure 1 shows the pharmacokinetic results calculated in Test Example 1.

Figure 2 shows the toxicity test results calculated in Test Example 2.

The present invention relates to anionic lipid nanoparticles having a negative charge on the surface and a method of manufacturing the same, and more particularly, by introducing a polyethylene glycol (PEG) -containing polymer into the surface of the particles formed of anionic phospholipids, Increasing the encapsulation efficiency of poorly soluble drugs and enclosing highly toxic drugs into lipid nanoparticles with high biocompatibility reduces the toxicity of anionic for encapsulating poorly soluble drugs in normal cells and improves circulation time in the body. The present invention relates to an increased lipid nanoparticle and a method for preparing the same.

Ampoterisin B, a polyene antifungal agent, has therapeutic effects against almost all fungal infections. In particular, since amphotericin B has an excellent therapeutic effect against systemic fungal diseases, it can effectively treat serious life-threatening infectious diseases in cancer patients, bone marrow transplant patients, neutrophil reduction patients, immunocompromised patients, or immunodeficiency patients. Ampotericin B is a polyene antifungal agent and antibiotic that binds to ergosterol, a membrane sterol of fungi, and relocates to ion channels, thereby preventing osmotic control of fungal cells and treating them against fungi.

However, amphotericin B is toxic to normal cells and tissues by binding to normal cells cholesterol by the same mechanism as ergosterol upon intravenous injection, and accompanied by side effects such as chills, fever, tissue necrosis and nephrotoxicity. In particular, amphotericin B requires restriction and attention in use because of difficulty in release of urinary tract by hemodialysis, and especially when used in children, the elderly and patients with weak immunity. do.

In addition, amphotericin B is insoluble in water at pH 6-7 and has a very low solubility of 0.1 mg / ml at pH 2 or pH 11, so that salts, micelles, emulsions, nanoparticles, or Soluble in the form of liposomes.

US Pat. No. 4,822,777 prepared amphotericin B in the form of a salt to improve solubility. In the patent, 100-400 nm sized particles were prepared using only amphotericin B and cholesterol sulfate, in which a salt with amphotericin B was formed to improve solubility in aqueous phase. However, the patent is prepared by simply preparing the amphotericin B in the form of salts, solubility was improved, but it is not formed as a formulation, resulting in short circulation time in the body, limited dosage, and toxicity to normal cells.

US Patent No. 5,059,591 uses amphotericin B and cholesterol-polyethyleneglycol (PEG) to reduce the toxicity of amphotericin B. In this patent, amphotericin B is combined with cholesterol-PEG in the form of a salt, wherein the circulation time in the body is increased by using the properties of PEG bound with cholesterol. However, the patent has a problem that the toxicity is greater than the formulated amphotericin B because the formulation of amphotericin B is not made.

US Pat. No. 4,981,690 describes a method for preparing liposomes encapsulated with amphotericin B. The patent describes a method for preparing a multi-membrane liposome using phospholipids and cholesterol. However, the patent is a formulation of amphotericin B, but does not contain a substance that induces a sustained biocirculation, so the biocirculation time may be relatively short.

U.S. Patent No. 5,965,156 relates to a method for preparing liposomes encapsulated with amphotericin B. The patent encloses amphotericin B in liposomes and ion-bonds amphotericin B with anionic phosphatidylglycerol in an acidic organic solvent to increase the inclusion rate and reduce body toxicity when formed into a formulation. However, the above patent has a disadvantage in that the use of an acidic solution for the ionic bond of phosphatidylglycerol and amphotericin B promotes the decomposition of amphotericin and increases the loss rate. In addition, the stability of the formulation itself is also weakened due to degradation of the acid catalyst, resulting in rapid release of amphotericin B, thus limiting frequent administration and dosage concentrations.

Therefore, there is a need for a method for preparing a new drug delivery system that can improve the aqueous solubility of amphotericin B, reduce the toxicity of drugs, increase the circulation time in the body, and also enable mass production.

Therefore, the present inventors have conducted research to solve the problems of the patents, as a result of encapsulating a very toxic drug, that is, amphotericin B into lipid nanoparticles having excellent biocompatibility to reduce toxicity to normal cells, as well as The present invention has been completed by developing anionic lipid nanoparticles for encapsulating poorly soluble drugs that have been surface-modified with anions having a sustained release effect by modifying the surface of the lipid nanoparticles with anionic phospholipids and anionic substances, and methods for preparing the same. Was done.

Accordingly, an object of the present invention is to provide anionic lipid nanoparticles for encapsulating poorly soluble drugs in which polyethylene glycol (PEG) -containing polymer is modified on the surface of particles formed of anionic phospholipids.

Another object of the present invention is to provide a method for preparing anionic lipid nanoparticles in which the polyethylene glycol (PEG) -containing polymer is modified.

The present invention is characterized by anionic lipid nanoparticles for encapsulating poorly soluble drugs coated with polyethylene glycol (PEG) -containing polymer on the surface of the particles formed of anionic phospholipid.

In addition, the present invention is to introduce a polyethylene glycol (PEG) -containing polymer on the surface of the lipid nanoparticles formed of anionic phospholipids, the method (A) and the prepared anionic phospholipid And a method of forming an ion complex of polyethylene glycol (PEG) containing an amine at its end and (B) to modify the surface of the lipid nanoparticles.

In particular, the present invention

(A-1) 10 to 30 parts by weight of a polyethylene glycol-containing polymer is mixed with 100 parts by weight of a lipid prepared by mixing phosphatidylcholine, anionic phospholipid and sterols in a weight ratio of 40 to 70: 5 to 20: 10 to 40 Dissolving in an organic solvent to prepare a lipid-PEG mixed solution;

(A-2) dissolving the poorly soluble drug in a straight or branched alcohol having 1 to 6 carbon atoms to prepare a drug solution;

(A-3) mixing the lipid-PEG mixed solution of (A-1) and the drug solution of (A-2) in a volume ratio of 1: 1 to 1: 9 to prepare a lipid-PEG-drug mixed solution ;

(A-4) dispersing the mixed solution of (A-3) in a water phase in a volume ratio of 2: 1 to 1:10 to form lipid nanoparticles; And

(A-5) step of preparing the anionic lipid nanoparticles in which the lipid nanoparticle solution of (A-4) is distilled under reduced pressure at 20 to 50 ° C. to remove an organic solvent, and then filtered and encapsulated a drug of uniform size.

It includes a method for producing anionic lipid nanoparticles for poorly soluble drug encapsulation.

On the other hand, the present invention in another way

(B-1) dissolving a lipid prepared by mixing phosphatidylcholine, anionic phospholipid and sterols in a weight ratio of 40 to 70: 5 to 20: 10 to 40 in an organic solvent to prepare a lipid mixed solution;

(B-2) dissolving the poorly soluble drug in a straight or branched alcohol having 1 to 6 carbon atoms to prepare a drug solution;

(B-3) mixing the lipid mixture solution of (B-1) and the drug solution of (B-2) in a volume ratio of 1: 1 to 1: 9 to prepare a lipid-drug mixture solution;

(B-4) dispersing the mixed solution of (B-3) in a water phase in a volume ratio of 2: 1 to 1:10 to form lipid nanoparticles;

(B-5) distilling the lipid nanoparticle solution of (B-4) at 20 to 50 ° C. under reduced pressure to remove an organic solvent, and filtering to prepare anionic lipid nanoparticles encapsulating a drug of uniform size. ; And

(B-6) coating with PEG by preparing a lipid-PEG ion complex by mixing the lipid of (B-1) and the amine-terminated PEG in a weight ratio of 100: 10 to 100: 30

It includes a method for producing anionic lipid nanoparticles for poorly soluble drug encapsulation comprising a.

The present invention will be described in more detail as follows.

The present invention introduces a polymer containing polyethylene glycol (PEG) to the surface of the particles formed of anionic phospholipids to anionize the surface to increase the encapsulation efficiency of poorly soluble drugs in the water phase, biocompatible to the highly toxic drugs The present invention relates to anionic lipid nanoparticles for encapsulating poorly soluble drugs and reducing the toxicity of normal cells by encapsulating in the excellent lipid nanoparticles and increasing the body circulation time.

In particular, the preparation of lipid nanoparticles containing the poorly soluble drug of the present invention is characterized by using anionic phospholipid as a lipid forming lipid nanoparticles. The anionic phospholipid is preferably phosphatidyl acid having an alkyl chain having 14 to 18 hydrophobic groups. More preferably, dimyristyl glycerophosphate (DMPA), dipalmitoylglycerophosphate (DPPA), dimyristyl glycerophosphate (DMPG), disteroyl glycerophosphate (DSPA), disteroyl glycerophosphate Poglycerol (DSPG), dipalmitoylglycerophosphoraglycerol (DPPG), dimyristylylglycerophosphoseline (DMPS), dipalmitoylglycerophosphoseline (DPPS) and disteroylglycerophosphoseline (DSPS) It includes one or two or more selected from. When the carbon number of the anionic phospholipid is less than 14, the phase transition temperature is lower than the body temperature, and the stability of the lipid nanoparticles is lowered. When the carbon number is more than 18, the binding strength of the anionic phospholipid is weakened and the encapsulation efficiency of the poorly soluble drug is decreased. It is not preferable because the size of the lipid nanoparticle particles increases. The anionic lipid is preferably contained 5 to 20% by weight relative to the total lipid composition to form lipid nanoparticles.

In the present invention, it is preferable to use phosphatidylcholine or phosphatidylcholine as a lipid for forming lipid nanoparticles, soybean phosphatidylcholine, egg yolk phosphatidylcholine or bovine phospholipid can be used, and more preferably 16 to 18 carbon atoms in the hydrophobic group. It has a phosphorus alkyl chain, For example, dipalmitoyl phosphatidyl choline, distearoyl phosphatidyl choline, etc. can be used. Even when the carbon number is less than 16, it forms a strong bond with the amphiphilic amphotericin B. However, the phase transition temperature is lower than the body temperature, which lowers the stability of the lipid nanoparticles, and when the carbon number exceeds 18, the binding strength with the drug becomes weak. It is not preferable because the efficiency of encapsulation of the drug in the lipid nanoparticles decreases and the size of the lipid nanoparticles increases. The phosphatidylcholine is preferably contained 40 to 70% by weight based on the total lipid composition to form lipid nanoparticles.

In addition, sterols are used as lipids used to form lipid nanoparticles. Preferred sterols include cholesterol, cholesterol hexasuccinate, and 3β- [N- (N ', N'-dimethylaminoethane) carba. Mole] cholesterol, ergosterol, stigmasterol or lanosterol and the like. The sterols are preferably contained in an amount of 10 to 40% by weight based on the total lipid composition forming the lipid nanoparticles.

In the present invention, in order to modify the surface of the lipid nanoparticles encapsulated with the drug, a PEG-containing polymer is introduced into the lipid nanoparticles and used. To this end, in the present invention, in the lipid nanoparticle-forming lipid, polyethylene glycol (PEG) -containing polymer is disteroylglycero phosphoethanolamine methyloxyethylene glycol (DSPE-mPEG), polyoxyethylene sorbitan monopalmitate (tween) and One or more selected from polyethylene polypropylene glycol (poloxamer) is used, and the polymer is preferably added in an amount of 10 to 30 parts by weight based on 100 parts by weight of the total lipid. If the content of polyethylene glycol (PEG) -containing polymer is out of the lower limit, the surface of the lipid nanoparticles is not sufficiently modified with polyethylene glycol (PEG), and even if it is outside the upper limit, the surface area of the lipid nanoparticles is limited, so that the higher concentration. Does not modify the surface of lipid nanoparticles. The introduction of PEG results in the formation of an ionic complex of anionic groups on the surface of the lipid nanoparticles with an amine group at the end of the PEG, or PEG is introduced through a lipophilic bond between the PEG-containing lipid and other lipid-soluble phospholipids and cholesterol. PEG is coated on the particle surface.

In addition, the term "drug" refers to a poorly soluble drug that was not easily encapsulated in a drug carrier, etc., but amphotericin B is typically used in the present invention, but is not limited thereto.

In the present invention, the surface of the lipid nanoparticles is modified by anion or polyethylene glycol (PEG) to provide a "in vivo circulation sustained-type" lipid nanoparticles showing stability in the blood. As used herein, "surface modification" means coated with a combination or ionic bond with an anion or polyethylene glycol (PEG) having the ability to increase biocirculating function. In addition, as used herein, “in-vivo sustained” lipid nanoparticles refers to a formulation that remains for 24 hours or more in the blood stream after administration, unlike a conventional formulation that disappears from the blood stream within several hours after administration.

The average particle diameter of the lipid nanoparticles encapsulated with amphotericin B of the present invention and whose surface is modified with anions is 50 to 300 nm, preferably 100 to 150 nm. If the average particle diameter of the lipid nanoparticles exceeds the above range, uptake occurs by reticuloendothelial system in the organ, ie liver or spleen, during circulation, and if less than the above range, the amount of drug delivered to the target site. (drug payload) is insufficient.

Hereinafter, the method of preparing a lipid nanoparticles in which a drug is encapsulated and the surface is modified with anions will be described step by step.

First, the method (A) which mix | blends a polyethyleneglycol (PEG) polymer containing lipid with a lipid is as follows.

In the step (A-1), phosphatidylcholine, anionic phospholipids and sterols are mixed at a weight ratio of 40 to 70: 5 to 20: 10 to 40 to prepare a lipid. In this case, when the phosphatidylcholine used is less than the above range, the formation of lipid nanoparticles is not easy, and when exceeded, the stability of the lipid nanoparticles is reduced. If the content of the anionic phospholipid is outside the lower limit, there is a problem such as an increase in the size of the lipid nanoparticles in the lipid nanoparticles, agglomeration between the lipid nanoparticles, and, if the anionic phospholipid is out of the upper limit, agglomeration of the lipid nanoparticles with PEG There is a problem of increase in particle size. In addition, when the content of cholesterol is out of the lower limit, there is a problem that the encapsulation efficiency of the drug is reduced, and when it is out of the upper limit, there is a problem that the stability of the lipid nanoparticles is reduced. In addition, sterols may be prepared in the above range by increasing the weight ratio of the drug as the weight ratio increases, but the particle size increases above the above range.

10 to 30 parts by weight of a polyethylene glycol-containing polymer is mixed with respect to 100 parts by weight of the lipid prepared above, and dissolved in an organic solvent to prepare a lipid-PEG mixed solution.

In the case of the PEG-containing polymer, the lower limit of the above range is not easy to coat the PEG, and when the upper limit is out of the upper limit, the size of the particles is excessively increased to prepare lipid nanoparticles in the above range. The lipid nanoparticle-forming lipid is dissolved in an organic solvent capable of dissolving the lipid, such as chloroform, methanol, toluene, and the like.

In step (A-2), a poorly soluble drug is dissolved in a linear or branched alcohol having 1 to 6 carbon atoms to prepare a drug solution. Examples of the linear or branched alcohol having 1 to 6 carbon atoms include methanol, ethanol, propanol, butanol, isobutanol, isopropanol, and the like. It is preferable to dissolve the poorly soluble drug in the alcohol at 0.1 ~ 1 mg / ㎖. When the concentration of the poorly soluble drug, preferably amphotericin B, is out of the lower limit, the drug encapsulation concentration is lowered, and when the concentration is out of the upper limit, the drug is not completely dissolved in alcohol. In addition, when the drug is used other organic solvents such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF) and the like, there is a problem that the removal of the solvent is not easy and the biocompatibility is reduced.

In the step (A-3), the lipid-PEG mixed solution of (A-1) and the drug solution of (A-2) are mixed at a volume ratio of 1: 1 to 1: 9 to prepare a lipid-PEG-drug mixed solution. do. If the ratio of the solution (A-1) exceeds 50 (v / v)%, the concentration of the drug is reduced, and if it is less than 10 (v / v)%, the amount of phospholipid is excessively reduced to form a formulation. There is a difficulty.

In the step (A-4), the lipid-PEG-drug mixed solution of (A-3) is dispersed in a volume ratio of 2: 1 to 1:10 in an aqueous phase, more preferably 1: 1 to 1: 3 To form lipid nanoparticles. As the aqueous phase, sugar solutions such as distilled water, phosphate buffer solution, saline solution, sucrose solution, maltose solution and mannitol solution, buffer solution or isotonic solution may be used. When the aqueous phase is less than the volume ratio range, the dispersed lipid nanoparticles aggregate to increase the particle size of the final lipid nanoparticles, and when the aqueous phase exceeds the volume ratio, the solution is diluted to add an unnecessary concentration process. There is a case to do it. In addition, it is preferable to disperse the lipid-PEG-drug mixed solution of step (A-3) in water using a syringe at a rate of 1-5 ml / min in tip sonication to form lipid nanoparticles. Do. When the rate of dispersion exceeds the above range, the size of the particles is increased, and even if less than the above range, it is difficult to form particles below a certain size, so the rate of dispersion in the above range is preferable.

In the step (A-5), the lipid nanoparticle solution of (A-4) is distilled under reduced pressure at 20 to 50 ° C. to remove the organic solvent, and filtered and the lipid nanoparticles having PEG functional groups encapsulating a drug of uniform size. To prepare. When the distillation under reduced pressure is less than 20 ℃ the removal time increases and it is difficult to completely remove the organic solvent, if the temperature exceeds 50 ℃ above the temperature because the formation of lipid nanoparticles and the drug can be denatured It is desirable to maintain. In addition, in order to completely remove the organic solvent, it is preferable to remove 0.5 to 5 times the volume of the organic solvent together with the organic solvent during the vacuum distillation. The purified lipid nanoparticle solution is prepared as a lipid nanoparticle solution having a uniform size distribution between 0.1 ~ 0.5 ㎛ using an injection molding machine. At this time, the filter membrane used in the injection molding machine is suitable for the pore size of 0.1 ~ 0.5 ㎛ the same as the particle size of the lipid nanoparticles. When the pore size exceeds 0.5 μm and the lipid nanoparticles exceed 0.5 μm, the intravenous injection causes capillary or reticulum endothelial cells to shorten the circulation time, which is not preferable. Filtration in this semipermeable membrane causes a problem that the yield is drastically reduced.

Additionally, dialysis, gel permeation chromatography, or a high pressure filter may be used to remove unmodified polyethylene glycol (PEG) from the lipid nanoparticles. More preferably, gel permeation chromatography is preferably used to simultaneously remove phospholipids and drugs that are not formed with lipid nanoparticles.

On the other hand, another method for producing anionic lipid nanoparticles according to the present invention will be described in detail as a method (B) for forming an ion complex of polyethylene glycol (PEG) containing an amine group at the end of the lipid as follows.

In step (B-1), phosphatidylcholine, anionic phospholipids and sterols are mixed in a weight ratio of 40 to 70: 5 to 20: 10 to 40 in the same manner as in (A-1) to prepare a lipid.

In the step (B-2), a poorly soluble drug is dissolved in a linear or branched alcohol having 1 to 6 carbon atoms in the same manner as in (A-2) to prepare a drug solution.

In the step (B-3), the lipid-drug is mixed by mixing the lipid mixture solution of (B-1) and the drug solution of (B-2) in a volume ratio of 1: 1 to 1: 9 in the same manner as in (A-3). Prepare a mixed solution.

In step (B-4), the mixed solution of (B-3) is dispersed in a water phase in a volume ratio of 2: 1 to 1:10 in the same manner as in (A-4), more preferably 1: 1 to 1 Dispersion is carried out in a volume ratio of 3: 3 to form lipid nanoparticles.

In step (B-5), the lipid nanoparticle solution of (B-4) is distilled under reduced pressure at 20 to 50 ° C. in the same manner as in (A-5) to remove the organic solvent, and filtered to obtain a drug of uniform size. Encapsulated anionic lipid nanoparticles were prepared.

In step (B-6), the lipid and the amine terminal group-PEG of (B-1) are mixed at a weight ratio of 100: 10 to 100: 30, but adjusted to pH 1 to 4 and 10 to 30 minutes at 40 to 65 ° C. The heat is applied to form an ion complex of lipids and PEG on the surface of the anionic lipid nanoparticles to form lipid nanoparticles coated with PEG on the surface of the lipid nanoparticles. That is, PEG is coated on the surface of the particle while an anionic group existing on the surface of the lipid particle forms an ionic complex between the amine groups of the PEG terminal.

When the pH is in an acidic condition, it is to facilitate the formation of an ionic complex, and when the reaction temperature is lower than 50 ° C., the reaction takes a long time, and when the temperature exceeds 65 ° C., lipid nanoparticle stability may be problematic. In particular, PEG (amine terminal group-PEG) containing an amine group at the terminal is specifically amino polyethylene glycol, diamino polyethylene oxide, amino (polyethylene glycol) methyl, and others polyethylene glycol or polyethylene oxide. And a substance having amines at the terminal.

Additionally, dialysis, gel permeation chromatography, or a high pressure filter may be used to remove the unmodified amine end group-PEG in the lipid nanoparticles. More preferably, gel permeation chromatography is preferably used to simultaneously remove phospholipids and drugs that are not formed with lipid nanoparticles.

The anionic lipid nanoparticles prepared in this way can improve the encapsulation rate of poorly soluble drugs in the water phase, while introducing PEG-containing polymers on the surface of the lipid nanoparticles and modifying the surface of the lipid nanoparticles, thereby increasing the circulation time in the body. It is expected to be very useful for the solubilization of various poorly soluble drugs including Shin B.

Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.

Preparation Example 1

60 mg of phosphatidylcholine (PC) and cholesterol (CHOL) selected from dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC) ) 20 mg was dissolved in 2 ml of chloroform to prepare a lipid mixture solution.

Ampoterisin B (AmB) was dissolved in methanol at a concentration of 0.5 mg / ml, and 2 ml of the lipid mixture solution and 8 ml of amphotericin B solution were mixed to prepare 10 ml of the AmB-lipid mixed solution.

10 mL of the AmB-lipid mixed solution was dispersed in 20 mL of distilled water at a rate of 2 mL / min while tip sonication using a syringe to form lipid nanoparticles, and then the total amount of the solution at 35 ° C. using a pressure distillation unit. 10 ml of the organic solvent and 10 ml of distilled water were removed until it became 10 ml, and the size distribution of the particles was uniformed by passing through a 0.2 µm semipermeable membrane using an extruder.

The particle size of the lipid nanoparticles prepared by the above process was measured by a particle analyzer (Electrophoretic light scattering spectrophotometer, ELS-Z, Otsuka Electronics, Japan) and are shown in Table 1 below.

Preparation Example 2 Preparation of Lipid Nanoparticles Containing Anionic Phospholipids

In preparing lipid nanoparticles in the same manner as in Preparation Example 1, in order to increase the encapsulation rate of Sample 3 having a lipid nanoparticle particle of less than 150 nm and Tg of 41 ° C. to 90% or more, Phosphorous dipalmitoylglycerophosphate (DPPA) and dysteroylglycerophosphoglycerol (DSPG) were added to prepare anionic lipid nanoparticles, respectively.

Changes in particle size, zeta potential, and encapsulation efficiency of lipid nanoparticles with DPPA and DSPG contents were measured and shown in Tables 2 and 3, respectively.

Preparation Example 3: Preparation of Lipid Nanoparticles Coated with Polyethylene Glycol (A)

To prepare lipid nanoparticles in the same manner as in Sample 8 of Preparation Example 2, DSPE-mPEG2000 solution was added at 0 to 80 parts by weight to 100 parts by weight of the lipid mixture solution of Sample 8 to coat PEG. The size change of the prepared particles is shown in Table 4 below.

Preparation Example 4 Preparation of Lipid Nanoparticles Coated with Polyethylene Glycol (B)

To prepare lipid nanoparticles in the same manner as in Sample 8, an amine terminal group-PEG (mPEG-NH ₂ 2000) solution was added to the lipid of Sample 8 at a weight ratio of 100: 0 to 80, and pH 2 After the alignment, heat was applied at 55 ° C. for 20 minutes to form an ionic complex of lipid and PEG, and the surface of the lipid nanoparticles was coated with PEG. The size change of the prepared particles is shown in Table 5 below.

Test Example 1 In Vitro Circulation Time Confirmation Experiment

A fungizone from Bristol Myers-Squibb with sodipium dioxycholate for solubilizing the injection of amphotericin B in SD rat rats to observe the circulating time of lipid nanoparticles, Nexstar Pharma made with liposome formulation using lipid Ambisome  Samples 8, 14 and 18 were administered through the tail vein and blood was collected by time to measure the concentration of the drug encapsulated in the lipid nanoparticles, and the pharmacokinetic results were calculated as the measured concentration of the drug. The pharmacokinetic results were used to confirm the circulation time of the drug. The concentration of the drug inside the lipid nanoparticles was diluted with heparin solution by diluting the blood collected by time, and the supernatant was diluted with a solvent dissolved in methanol at a concentration of 0.5 ㎍ / ml of 1-amino 4-nitronaphthalene after centrifugation. The solution to be measured was prepared, and the absorbance was measured using this solution. The absorbance was measured using an ultraviolet spectrophotometer and the wavelength of the drug was measured at 408 nm.

Pharmacokinetic results calculated in the above experiment are shown in FIG. 1.

As shown in FIG. 1, the lipid nanoparticles coated with PEG of Sample 14 of Preparation Example 3 and Sample 18 of Preparation Example 4 according to the present invention showed a significantly longer body circulation time compared to Fungizone, and after injection 3 Ambisome by time  Longer body circulation time can be observed. As a result, injections using the anionic lipid nanoparticles of the sample 14 and the sample 18 and the lipid-coated nanoparticles coated with PEG have the best circulation time in the body and can increase the circulation time in the body than the conventional formulation of amphoteracin B. Prove it is.

Test Example 2: Toxicity Test

MTT experiments were performed using 293 cells, human kidney cells, for the toxicity test of lipid nanoparticles. 293 cells were incubated for 24 hours at 37 ° C. in a carbon dioxide incubator in 96-well plates at a concentration of 1 × 10 ⁴ cells / ml, and then amphotericin B was added to each well plate at 6.25, 12.5, 25, 50 and 100 μg. Fungizone, Sample 8, Sample 14, and Sample 18 were added at a concentration of / ml and incubated in a carbon dioxide incubator for 12 hours. MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) reagent was added to incubate at 37 ° C. for 4 hours in a carbon dioxide incubator to produce formazan crystals. After removing 200 μg / ml of solution from each well plate, formazan crystals were dissolved by adding 150 μg / ml of dimethylsulfoxide solution. The well plates containing the dissolved samples were measured at 590 nm by enzyme immunoassay (ELISA).

Toxicity test results calculated in the above experiment is shown in FIG.

As shown in FIG. 2, the lipid nanoparticles of the sample 14 and the sample 18 according to the present invention can be seen that the overall cell viability compared to the Fungizone. This may be due to the low toxicity of amphotericin B, which is formulated with biocompatible phospholipids, cholesterol and the same PEG. This proves that cytotoxicity can be lowered by encapsulating amphotericin B in lipid nanoparticles as a biocompatible material.

As described above, the present invention is to introduce a PEG-containing polymer on the surface of the lipid nanoparticles to modify the surface of the lipid nanoparticles in order to improve the sealing rate of lipid nanoparticles containing poorly soluble drugs in the water phase and increase the body circulation time The present invention relates to a lipid nanoparticle containing a drug having an increase in circulation time and a method for preparing the same, which is very useful for a novel solubilization method of a poorly water-soluble drug as a water-soluble injection, and for reducing toxicity and increasing circulation time of amphotericin B preparations. I am expected to.

Claims (16)

Anionic lipid nanoparticles for embedding poorly soluble drugs, characterized in that the polyethylene glycol (PEG) -containing polymer is coated on the surface of the particles formed of anionic phospholipid. The lipid nanoparticles of claim 1, wherein the anionic phospholipid is contained in an amount of 5 to 20% by weight based on the total lipid composition forming the lipid nanoparticles.        The lipid nanoparticles of claim 1, wherein the lipid nanoparticles are coated while an ionic complex between the PEG and the lipid nanoparticles containing the amine group is formed at the end thereof.       According to claim 1, wherein the poorly soluble drug is lipid nanoparticles, characterized in that amphotericin B. According to claim 1, wherein the anionic phospholipid is a lipid nanoparticles, characterized in that the phosphatidyl acid having an alkyl chain having a hydrophobic group of 14 to 18 carbon atoms The method of claim 1, wherein the anionic phospholipid is dimyristylylglycerophosphate (DMPA), dipalmitoylglycerophosphate (DPPA), dimyristylylglycerophosphate (DMPG), dissteroylglycerophosphate (DSPA) , Disteroylglycerophosphoglycerol (DSPG), dipalmitoylglycerophosphoraglycerol (DPPG), dimyristylglycerophosphoseline (DMPS), dipalmitoylglycerophosphoseline (DPPS) and dissteroyl Lipid nanoparticles comprising one or two or more selected from glycerophosphoserine (DSPS). The method of claim 1, wherein the polyethylene glycol (PEG) -containing polymer is disteroylglycerophosphoethanolamine methyloxyethylene glycol (DSPE-mPEG), polyoxyethylene sorbitan monopalmitate (tween), polyethylene glycol And polyethylene polypropylene glycol (poloxamer). Lipid nanoparticles, characterized in that one or two or more selected from. 1) 10 to 30 parts by weight of a polyethylene glycol-containing polymer is mixed with 100 parts by weight of a lipid prepared by mixing phosphatidylcholine, anionic phospholipid and sterols in a weight ratio of 40 to 70: 5 to 20: 10 to 40. Dissolving to prepare a lipid-PEG mixed solution; 2) dissolving the poorly soluble drug in a straight or branched alcohol having 1 to 6 carbon atoms to prepare a drug solution; 3) mixing the lipid-PEG mixed solution of 1) and the drug solution of (A-2) at a volume ratio of 1: 1 to 1: 9 to prepare a lipid-PEG-drug mixed solution; 4) dispersing the mixed solution of 3) in a water phase in a volume ratio of 2: 1 to 1:10 to form lipid nanoparticles; And 5) distilling the lipid nanoparticle solution of 4) under reduced pressure at 20 to 50 ° C. to remove the organic solvent, and filtering to prepare anionic lipid nanoparticles encapsulated with a uniform size drug. Method for producing anionic lipid nanoparticles for poorly soluble drug encapsulation comprising a. 1) preparing a lipid mixture solution by dissolving a lipid prepared by mixing phosphatidylcholine, anionic phospholipid and sterols at a weight ratio of 40 to 70: 5 to 20: 10 to 40 in an organic solvent; 2) preparing a drug solution by dissolving the poorly soluble drug in a straight or branched alcohol having 1 to 6 carbon atoms; 3) preparing a lipid-drug mixed solution by mixing the lipid mixed solution of 1) and the drug solution of 2) in a volume ratio of 1: 1 to 1: 9; 4) dispersing the mixed solution of 3) in a water phase in a volume ratio of 2: 1 to 1:10 to form lipid nanoparticles; 5) distilling the lipid nanoparticle solution of 4) at 20 to 50 ° C. under reduced pressure to remove the organic solvent, and filtering to prepare anionic lipid nanoparticles encapsulating a drug of uniform size; And 6) mixing the lipid of 1) with the amine-terminated PEG in a weight ratio of 100: 10 to 100: 30 to prepare a lipid-PEG ion complex to coat with PEG Method for producing anionic lipid nanoparticles for poorly soluble drug encapsulation comprising a. 10. The method according to claim 8 or 9, wherein the drug is amphotericin B. 10. The method of claim 8 or 9, wherein in step 2) the drug is dissolved in a linear or branched alcohol having 1 to 6 carbon atoms at a concentration of 0.1 to 1 mg / ml. . 10. The method according to claim 8 or 9, wherein the mixed solution of step 3) is dispersed using a syringe at a rate of 1-5 ml / min using tip sonication in step 4). Method for preparing lipid nanoparticles. 10. The method according to claim 8 or 9, wherein the step 5) removes 0.5 to 5 times the volume of the organic solvent together with the organic solvent. 10. The method for preparing lipid nanoparticles according to claim 8 or 9, wherein the pore size is filtered using a filtration membrane having a pore size of 0.1 to 0.5 µm in step 5) in order to uniformize the size distribution of the particles. 10. The method of claim 9, wherein in step 6), the pH is set to 1 to 4 to form an ion complex.       10. The method of claim 9, wherein in step 6), the reaction temperature is 40 to 65 ° C to form an ion complex.

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