KR102081810B1 - Liposome encapsulated with double anti-cancer drug and method for producing the same - Google Patents

Abstract

The present invention relates to a liposome encapsulated with an anticancer agent and a method for manufacturing the same, and instead of a citric acid buffer used in a conventionally known method when preparing a liposome encapsulated with a hydrophobic anticancer agent and a hydrophilic anticancer agent, contains ammonium sulfate. When the solution is used and the hydrophobic anti-cancer drug to lipid ratio is decreased, the encapsulation efficiency of the hydrophobic anti-cancer agent as well as the hydrophilic anti-cancer agent is relatively high.

Description

Liposome encapsulated with double anti-cancer drug and method for producing the same}

The present invention relates to a liposome encapsulated with an anticancer agent and a method for manufacturing the same.

In effectively treating human life-threatening cancer, a treatment method using EGFR pathway inhibitor and DNA damaging chemotherapeutic agent in combination has been spotlighted as a method for treating intractable cancer. have.

In particular, in the treatment of cancer resistant to DNA-damaging chemotherapeutic agents, the EGFR pathway inhibitor first reaches cancer cells, so that a carcinogenic signal maintained by EGFR signaling inhibits the apoptosis process including capase-8 It is known that the delivery efficiency of cancer can be significantly improved by dynamically rewiring the delivery network and then treating the DNA-damaging chemotherapeutic agent.

Non-Patent Document 1 (Sci Signal. 2014 May 13; 7 (325): ra44.), As a means for the treatment method, uses a citric acid buffer to enclose a hydrophilic anticancer agent in the inner hydrophilic region of the liposome. In addition, although a method for preparing liposomes in which a hydrophobic anticancer agent is encapsulated in a hydrophobic region of a phospholipid bilayer of a liposome is disclosed, when a citric acid buffer solution is used when preparing a liposome encapsulated with an anticancer agent, an EGFR pathway inhibitor (ie, a hydrophobic anticancer agent) and DNA Since the encapsulation efficiency of an injured chemotherapeutic agent (ie, a hydrophilic anti-cancer agent) is not good, there is a problem in that the treatment efficiency of cancer is poor when applied to cancer treatment.

Accordingly, there is a need for a liposome encapsulated with a high encapsulation efficiency and a method for manufacturing the EGFR pathway inhibitor and a DNA-damaging chemotherapeutic agent so that the therapeutic efficiency of cancer can be improved when applied to cancer treatment.

Sci Signal. 2014 May 13; 7 (325): ra44.

An object of one aspect of the present invention is to provide a liposome encapsulated with a high encapsulation efficiency of an EGFR pathway inhibitor and a DNA damage therapeutic agent.

An object of another aspect of the present invention is to provide a method for preparing liposomes encapsulated with high encapsulation efficiency by an EGFR pathway inhibitor and a DNA damage therapeutic agent.

An object of another aspect of the present invention is to provide a method for controlling the encapsulation efficiency of hydrophobic and hydrophilic anticancer agents in liposomes encapsulated with high encapsulation efficiency by EGFR pathway inhibitors and DNA damage therapeutic agents.

In order to achieve the above object,

According to one aspect of the invention,

Liposomes are provided in which an ammonium sulfate and a hydrophilic anticancer agent are encapsulated in the inner hydrophilic region of the liposome, and a hydrophobic anticancer agent is enclosed in the hydrophobic region of the phospholipid bilayer of the liposome.

In addition, according to another aspect of the invention,

Preparing a mixed solution by dissolving a hydrophobic anticancer agent and lipid in a solvent;

Evaporating the solvent of the mixed solution to form a lipid film;

Hydrating the lipid membrane with a solution containing ammonium sulfate to form a liposome encapsulated in the hydrophilic region of the liposome and a hydrophobic anticancer agent in the hydrophobic region of the liposome; And

Containing; by adding a solution containing a hydrophilic anticancer agent, encapsulating a hydrophilic anticancer agent in the hydrophilic region of the liposome;

Methods of making liposomes are provided.

Furthermore, according to another aspect of the invention,

Preparing a mixed solution by dissolving a hydrophobic anticancer agent and lipid in a solvent;

Evaporating the solvent of the mixed solution to form a lipid film;

Hydrating the lipid membrane in a solution containing ammonium sulfate to form a liposome in which an ammonium sulfate is enclosed in a hydrophilic region and a hydrophobic anticancer agent is enclosed in a hydrophobic region; And

Containing; by adding a solution containing a hydrophilic anticancer agent, encapsulating a hydrophilic anticancer agent in the hydrophilic region of the liposome;

A method for controlling the encapsulation efficiency of hydrophobic and hydrophilic anticancer agents in liposomes is provided.

When preparing liposomes encapsulated with a hydrophobic anti-cancer agent and a hydrophilic anti-cancer agent, a solution containing ammonium sulfate is used instead of the citric acid buffer used in a conventionally known method, and a hydrophobic anti-cancer agent to lipid ratio (hydrophobic anti- When reducing the cancer drug to lipid ratio), the encapsulation efficiency of the hydrophobic anticancer agent as well as the hydrophilic anticancer agent is relatively high.

1 shows the encapsulation efficiency of doxorubicin (DOX) encapsulated in liposomes prepared in Examples 1 to 4 and Comparative Example 1.
2 shows the encapsulation efficiency of doxorubicin (DOX) encapsulated in liposomes prepared in Example 1 (group 1), Example 5 (group 2), and Example 6 (group 3).
3 is a drop-casting solution containing the liposome prepared in Example 1 to a carbon-coated copper grid (copper grid), dried in the air at room temperature, 200 kV field emission transmission electron microscope (FE- TEM) (JEM 2100F; JEOL Ltd., Tokyo, Japan).
Figure 4 shows the drug release amount of erlotinib (ERL) and doxorubicin (DOX) in the liposome prepared in Example 1.
Figure 5 shows the drug release rate of erlotinib (ERL) and doxorubicin (DOX) in the liposome prepared in Example 1.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention provides a liposome in which an ammonium sulfate and a hydrophilic anticancer agent are enclosed in a hydrophilic region, and a hydrophobic anticancer agent is enclosed in a hydrophobic region.

At this time, the hydrophilic anti-cancer agent (DNA-damaging chemotherapeutic agent) is doxorubicin (doxorubicin, DOX), daunorubicin (daunorubicin), arugomycin (arugomycin), epirubicin (epirubicin), idarubicin (idarubicin) or dyne Cells containing anthracycline DNA inserts containing ene-dyene antibiotics such as dynemicin, camptothecin, irinotecan or topotecan DNA enzymes such as toxic quinolone alkaloids topoisomerase I inhibitors, and DNA alkylating agents such as carmustine (BCNU) or cisplatin (cis-platin).

In addition, the hydrophobic anti-cancer agent (EGFR pathway inhibitor) is a compound targeting EGFR as erlotinib (ERL), gefitinib, lapatinib, trastuzumab or other compound; A compound targeting multiple receptor tyrosine kinases as sunitinib or other compound; Compounds targeting Abl kinase and PDGF receptors as imatinib or other compounds; A compound targeting B-raf as sorafenib or other compound; Lenvatinib or other compound that targets the FGF receptor and the PDGF receptor; Bevacizumab or other compound that targets the VEGF receptor; BMS-345541 or another compound that targets IKB / IKK; A compound that targets mTOR as torin, rapamycin or other compound; BEZ-235 or another compound that targets PI3K and mTOR; Compounds targeting MAPK / ERK kinase as cobimetinib, trametinib, PD98059 or other compounds; SB202190 or a compound targeting p38 MAPK as a related compound; Compounds targeting PI3K as Wortmannin, LY294002 or other compounds; A compound targeting p21 activation kinases as PF-03758309, FRAX486 or other compound; A compound that inhibits the FGF receptor as ponatinib, nintedanib, or other compound; And SP600125 or other compounds that inhibit JNK kinases.

Another aspect of the invention,

Preparing a mixed solution by dissolving a hydrophobic anticancer agent and lipid in a solvent;

Evaporating the solvent of the mixed solution to form a lipid film;

Hydrating the lipid membrane with a solution containing ammonium sulfate to form a liposome in which an ammonium sulfate is enclosed in a hydrophilic region and a hydrophobic anticancer agent is enclosed in a hydrophobic region; And

It provides a method for producing a liposome comprising; adding a solution containing a hydrophilic anticancer agent, encapsulating a hydrophilic anticancer agent in the hydrophilic region of the liposome.

Hereinafter, a method of manufacturing the liposome will be described in detail step by step.

Step 1: Hydrophobic anticancer agent and lipid in solvent Dissolve  Steps to prepare a mixed solution

In the method for preparing liposomes provided in another aspect of the present invention, step 1 is a step of preparing a mixed solution by dissolving a hydrophobic anticancer agent and a lipid in a solvent.

At this time, the solvent can be used without limitation as long as it can dissolve the hydrophobic anti-cancer agent and lipids. In some embodiments, methanol, ethanol, propanol, butanol, chloroform, etc. can be used alone or in combination.

In addition, the lipid may be used without limitation as long as it is known as a lipid used for liposome production. In some embodiments, 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2) -Distearoyl-sn-glycero-3-phosphocholine (DSPC), hydrogenated L-α-phosphatidylcholine (soybean) [L-α-Phosphatidylcholine, hydrogenated (Soy), HSPC], 1,2-palmitoyl-sn-glycero- 3-phosphocholine (1,2-Dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [meth Thoxy (polyethylene glycol) -2000] {1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000]. DSPE-mPEG}, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- (1'-rac-glycerol) (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- (1'-rac-glycerol), POPG) and 1,2-dioleoyl-sn-glycero-3-phospho- (1'-rac-glycerol) [1,2-Dioleoyl-sn-glycero-3 -phospho- (1'-rac-glycerol)].

The hydrophobic anti-cancer agent is the same as described above, and thus is omitted to avoid overlapping explanations.

In addition, the hydrophobic anti-cancer drug to lipid weight ratio (hydrophobic anti-cancer drug to lipid weight ratio) may range from 0.1 to 0.01, may range from 0.08 to 0.01, and ranges from 0.06 to 0.01 It may be, it may be in the range of 0.1 to 0.02, it may be in the range of 0.1 to 0.04, it may be in the range of 0.1 to 0.06, it may be in the range of 0.07 to 0.05, it may be 0.06. When the hydrophobic anti-cancer drug to lipid ratio is greater than 0.1, there is a problem in the structural stability of the liposome, and when it is less than 0.01, there is a problem in that an excessive amount of lipid is used.

Step 2: evaporating the solvent of the mixed solution, Lipid membrane  Forming step

In the method for preparing liposomes provided in another aspect of the present invention, step 2 is a step of evaporating the solvent of the mixed solution to form a lipid film.

At this time, a known drying method can be used to remove most of the solvent. Some specific examples of temperature and time as drying conditions for removing the solvent are as follows.

The drying temperature may range from 10 to 70 ° C, range from 20 to 70 ° C, range from 30 to 70 ° C, range from 40 to 70 ° C, range from 10 to 60 ° C, and range from 10 to 10 ° C It may be in the range of 50 to 50 ° C, may be in the range of 10 to 40 ° C, may be in the range of 20 to 60 ° C, may be in the range of 30 to 50 ° C, and may be in the range of 40 ° C. When the drying temperature is less than 10 ° C, there is a problem that it takes a long time to dry the solvent, and when the drying temperature is higher than 70 ° C, there is a problem of unnecessary use of excess energy and denaturation of the sample.

The drying time may range from 1 to 40 hours, may range from 5 to 40 hours, may range from 10 to 40 hours, may range from 15 to 40 hours, may range from 20 to 40 hours, and may range from 25 to 25 hours. It may range from 40 to 40 hours, may range from 1 to 35 hours, may range from 1 to 30 hours, may range from 1 to 25 hours, may range from 15 to 35 hours, may range from 20 to 30 hours And can be performed throughout the day. When the drying time is less than 1 hour, there is a problem that sufficient drying of the solvent is impossible, and when the drying time is more than 40 hours, there is a problem of drying for an unnecessarily long time.

Step 3: Ammonium Sulfate  With the containing solution Lipid membrane  Hydrate to ammonium in the hydrophilic region Sulfate Being sealed  Hydrophobic anticancer drugs in the hydrophobic area Sealed  Steps to form liposomes

In the method for preparing liposomes provided in another aspect of the present invention, step 3 hydrates the lipid membrane with a solution containing ammonium sulfate, thereby forming a liposome in which a hydrophilic region is filled with ammonium sulfate and a hydrophobic anticancer agent is loaded in a hydrophobic region. It is a step.

At this time, the solution containing ammonium sulfate may be used an aqueous solution of ammonium sulfate, but is not limited thereto. This step corresponds to a lipid film-hydration method.

The concentration of ammonium sulfate in the solution can be used without limitation under the range that can lead to doxorubicin (DOX) to the hydrophilic region of liposomes in the steps described below, and in some embodiments, it may be in the range of 50 to 650 mM, 100 To 650 mM concentration range, 150 to 650 mM concentration range, 200 to 650 mM concentration range, 250 to 650 mM concentration range, 50 to 600 mM concentration range, 50 To 550 mM concentration range, 50 to 500 mM concentration range, 50 to 450 mM concentration range, 50 to 400 mM concentration range, 150 to 550 mM concentration range, 250 To 400 mM concentration.

When the concentration of ammonium sulfate in the solution is less than 50 mM, there is an insufficient amount of ammonium sulfate to lead doxorubicin (DOX) to the hydrophilic region of liposomes in the steps described below, resulting in a decrease in the sealing efficiency of doxorubicin (DOX). In addition, when the concentration of ammonium sulfate in the solution is more than 650 mM, an unnecessary excess of ammonium sulfate is used, and a sample is wasted.

Step 4: Adding a solution containing a hydrophilic anti-cancer agent, Liposome  Enclosing a hydrophilic anticancer agent in a hydrophilic region

In the method for preparing liposomes provided in another aspect of the present invention, step 4 is a step of adding a solution containing a hydrophilic anticancer agent to enclose a hydrophilic anticancer agent in the hydrophilic region of the liposome.

However, prior to adding the solution containing the hydrophilic anticancer agent, the step of adjusting the diameter of the liposome is first performed by dispersing the solution containing the liposome in which the ammonium sulfate is enclosed in the hydrophilic region and the hydrophobic anticancer agent is enclosed in the hydrophobic region. In this case, the dispersion may use a known dispersion method without limitation, and ultrasonic treatment may be used as one specific example.

Dispersion conditions for controlling the diameter of the liposomes are as follows.

The dispersion temperature may range from 10 to 120 ° C, range from 20 to 120 ° C, range from 30 to 120 ° C, range from 40 to 120 ° C, range from 50 to 120 ° C, and range from 60 to 60 It may be in the range of 120 to 120 ℃, may be in the range of 65 to 120 ℃, may be in the range of 10 to 110 ℃, may be in the range of 10 to 100 ℃, may be in the range of 10 to 90 ℃, may be in the range of 10 to 80 ℃ Can be, can range from 10 to 70 ° C, can range from 10 to 65 ° C, can range from 20 to 110 ° C, can range from 30 to 100 ° C, can range from 40 to 90 ° C, can range from 50 to It may be in the range of 80 ℃, may be in the range of 60 to 70 ℃, may be 65 ℃.

If the dispersion temperature is less than 10 ° C, there is a problem that a desired liposome diameter cannot be obtained, and if the dispersion temperature is more than 120 ° C, unnecessary energy is used.

The dispersion time may range from 1 minute to 20 minutes, may range from 2 minutes to 20 minutes, may range from 3 minutes to 20 minutes, may range from 4 minutes to 20 minutes, and ranges from 5 minutes to 20 minutes. Can be, can range from 1 minute to 18 minutes, can range from 1 minute to 16 minutes, can range from 1 minute to 14 minutes, can range from 1 minute to 12 minutes, range from 1 minute to 10 minutes Can be, can range from 1 minute to 8 minutes, can range from 1 minute to 6 minutes, can range from 1 minute to 5 minutes, can range from 2 minutes to 18 minutes, range from 3 minutes to 15 minutes It may be, may range from 4 minutes to 12 minutes, may range from 5 minutes to 10 minutes.

If the dispersion time is less than 1 minute, there is a problem that the diameter of the liposome becomes larger than necessary, and if the dispersion time is more than 20 minutes, the diameter of the liposome becomes smaller than necessary.

As the dispersion time increases, the diameter of the liposome decreases, and the diameter range of the liposome provided in one aspect may be 200 nm or less, 50 to 200 nm, 60 to 200 nm, and 70 to 200 nm May be 80 to 200 nm, may be 90 to 200 nm, may be 100 to 200 nm, may be 110 to 200 nm, may be 120 to 200 nm, may be 130 to 200 nm , May be 140 to 200 nm, may be 50 to 190 nm, may be 50 to 180 nm, may be 50 to 170 nm, may be 50 to 160 nm, may be 50 to 150 nm, 50 To 140 nm, 50 to 200 nm, 70 to 190 nm, 90 to 180 nm, 110 to 170 nm, and 140 nm.

When the liposome diameter is less than 50 nm, there is a small amount of the anti-cancer agent that is enclosed, and thus, when applied to the treatment of cancer, there is a problem that the treatment efficiency is poor, and when the liposome diameter is more than 200 nm, the liposome is cancer by the enhanced permeability and retention (EPR) effect There is a problem that the effect of accumulating in the tissue decreases.

The solution containing the hydrophilic anticancer agent may be used without limitation as long as it is mixed with a solvent capable of dissolving the hydrophilic anticancer agent, and in one embodiment, it may be a solution containing an aqueous sodium chloride solution.

Another aspect of the invention,

Preparing a mixed solution by dissolving a hydrophobic anticancer agent and lipid in a solvent;

Evaporating the solvent of the mixed solution to form a lipid film;

Hydrating the lipid membrane with a solution containing ammonium sulfate to form a liposome in which an ammonium sulfate is enclosed in a hydrophilic region and a hydrophobic anticancer agent is enclosed in a hydrophobic region; And

Containing; by adding a solution containing a hydrophilic anticancer agent, encapsulating a hydrophilic anticancer agent in the hydrophilic region of the liposome;

It provides a method for controlling the encapsulation efficiency of hydrophobic and hydrophilic anticancer agents in liposomes.

Most steps of the method for controlling the encapsulation efficiency of the hydrophobic anti-cancer agent and the hydrophilic anti-cancer agent in the liposome provided in another aspect of the present invention are the same as the method for preparing the liposome provided in another aspect of the present invention described above.

However, by adjusting the amount of the hydrophobic anti-cancer agent used in the production of liposomes, the amount of the hydrophilic anti-cancer agent, the amount of lipids, and the like, a liposome having an anticancer agent encapsulation efficiency that is expected to have an optimal treatment effect for cancer patients is provided, thereby improving cancer treatment efficiency. Can increase

When preparing liposomes encapsulated with a hydrophobic anti-cancer agent and a hydrophilic anti-cancer agent, a solution containing ammonium sulfate is used instead of the citric acid buffer used in a conventionally known method, and a hydrophobic anti-cancer agent to lipid ratio (hydrophobic anti- When reducing the cancer drug to lipid ratio), the encapsulation efficiency of the hydrophobic anticancer agent as well as the hydrophilic anticancer agent is relatively high.

To demonstrate this effect,

The encapsulation efficiency (EE) of doxorubicin (DOX) or erlotinib (ERL) encapsulated in liposomes prepared in Examples 1 to 4 and Comparative Example 1 was measured, and as a result, doxorubicin (DOX) was used using an aqueous solution of ammonium sulfate. ) And the sealing efficiency of doxorubicin (DOX) of Examples 1 to 4, which are liposomes encapsulated with erlotinib (ERL), was confirmed to be at least about 90%, but with citric acid buffer, doxorubicin (DOX) and It was confirmed that the encapsulation efficiency of doxorubicin (DOX) of Comparative Example 1, a liposome encapsulated with erlotinib (ERL), was only about 17% (see FIG. 1 of Experimental Example 1).

In addition, the encapsulation efficiency of doxorubicin (DOX) and erlotinib (ERL) in the liposomes prepared in Example 1 (group 1), Example 5 (group 2), and Example 6 (group 3) was measured in <Experiment Example 1>. As a result of measuring in the same manner as the method, the encapsulation efficiency of doxorubicin (DOX) in the liposomes prepared in Example 1 (group 1), Example 5 (group 2), and Example 6 (group 3) is about 90% or more. It was confirmed (see FIG. 2 of Experimental Example 2).

Furthermore, after drop-casting the solution containing the liposome prepared in Example 1 onto a carbon-coated copper grid, and drying it in the air at room temperature, a 200 kV field emission transmission electron microscope (FE-TEM) ) (JEM 2100F; JEOL Ltd., Tokyo, Japan) was used to confirm the morphology of the liposomes, and the liposomes prepared in Example 1 exhibited a spherical shape, in particular, an inner compartment of a nano-liposome having a high crystalline lattice, It was confirmed that the interface between the outermost layers having a low crystal or amorphous structure was present (see FIG. 3 of Experimental Example 3).

In addition, as a result of evaluating drug release over time in the anti-cancer agent in the liposome prepared in Example 1, erlotinib (ERL) is released in a relatively large amount compared to doxorubicin (DOX) as the drug release experiment time elapses, The release rate of erlotinib (ERL) was significantly increased at the beginning of the drug release experiment and then gradually decreased, and the release rate of doxorubicin (DOX) gradually increased with time (FIG. 4 and FIG. 4 of Experimental Example 4). 5).

Furthermore, the EE of doxorubicin (DOX) or erlotinib (ERL) encapsulated in the liposomes prepared in Examples 7 to 10 and Comparative Example 2 were compared, and as a result, all manufacturing conditions except the loading method of doxorubicin (DOX) When comparing the same Example 7 and Comparative Example 2, it was confirmed that the sealing rate of doxorubicin (DOX) was significantly increased from 24% to 79% due to the difference in the loading method of doxorubicin (DOX), as well as using ammonium sulfate. Even if the method of encapsulating doxorubicin (DOX) as a hydrophilic region in the liposome is a known technique, when comparing Example 7 and Comparative Example 2, the effect of increasing the encapsulation rate of erlotinib (ERL), a hydrophobic anticancer agent, is also increased by 5%. When it was confirmed that all the manufacturing conditions are the same, except for erlotinib (ERL) to lipid ratio (ERL to lipid ratio), comparing Examples 9 to 10 and Comparative Example 2, erlotinib (ERL) to lipid ratio ( It was confirmed that the encapsulation rate of erlotinib (ERL) increased from 17% to 41% and 52% according to a decrease in ERL to lipid ratio (see Table 1 in Experimental Example 5).

Hereinafter, it will be described in detail by examples and experimental examples.

However, the following examples and experimental examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples and experimental examples.

< Example  1> Ammonium Sulfate  Hydrophilic anti-cancer agent and hydrophobic using aqueous solution term Preparation of cancer-encapsulated liposomes 1 (group 1)

Liposomes encapsulating hydrophilic and hydrophobic anticancer agents were prepared through the following steps.

Step 1: Hydrophobic anticancer agent and lipid in solvent Dissolve  Prepare a mixed solution

1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-Distearoyl-sn-glycero-3-phosphocholine, DSPC), cholesterol and 1-palmitoyl-2-oleoyl-sn -Glycero-3-phospho- (1'-rac-glycerol) (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- (1'-rac-glycerol), POPG) in a weight ratio of 27: A mixed lipid containing 20: 3 (w: w: w) was prepared. The hydrophobic anticancer agent erlotinib was mixed with the prepared mixed lipid in a weight ratio of 3:50 (w: w). The mixture was dissolved in 9 mL of a mixed solvent (chloroform: methanol = 2: 1 (v / v)) to prepare a mixed solution.

Step 2: evaporating the solvent of the mixed solution, Lipid membrane  formation

The mixed solution prepared in step 1 was placed in a round-bottom flask, and a solvent was evaporated at 40 ° C. under vacuum using a rotary evaporator (Rotavapor R-210; BUCHI Labortechnik AG, Flawil, Switzerland), to form a lipid membrane. (lipid membrane) was formed. The lipid membrane was left under vacuum for one day to remove all residual solvent. Through this process, the erlotinib molecule penetrates into the lipid membrane due to hydrophobic attraction. As a result, the erlotinib molecule is naturally encapsulated in the lipid bilayer (ie, hydrophobic region) of the liposome, which is generated when a subsequent hydration process is performed.

Step 3: Ammonium Sulfate  As an aqueous solution Lipid membrane  Hydrate to ammonium in the hydrophilic region Sulfate Being sealed  Hydrophobic anticancer drugs in the hydrophobic area Sealed  Forming liposomes

In order to hydrate the lipid membrane containing the erlotinib molecule prepared in step 2 above, 8 mL of an aqueous solution of ammonium sulfate (AS) at a concentration of 250 mM was added to a round-bottom flask on which the lipid membrane was formed. The round-bottom flask was rotated and kept at 65 ° C. for 30 minutes. The suspension containing liposomes produced by hydration of the lipid membrane was sonicated at 65 ° C. for 5 minutes using a bath sonicator (Branson 3510E-DTH; Branson, Danbury, CT, USA). In order to adjust the diameter of the liposome, the suspension containing the liposome was sonicated using a probe sonicator, and specific conditions are as follows: 5 seconds on and 2 seconds off under magnetic stirring. , Sonicated in an ice bath with a pulse with 20% amplitude and energy of 36 J per pulse.

Dialysis tubing cellulose membrane, 14,000 molecular weight cut-off (MWCO); Sigma-Aldrich Corp., St. Louis, MO, USA to remove residual ammonium sulfate present in the suspension and not encapsulated in liposomes ) And PBS (phosphate buffered saline, pH 7.4), dialyzed for one day.

Step 4: Adding a solution containing a hydrophilic anti-cancer agent, Liposome  Encapsulating hydrophilic anticancer agent in hydrophilic area

Doxorubicin hydrochloride was dissolved in an aqueous 0.9% NaCl solution at 65 ° C (1.5 mg / mL). This solution was added to the 8 mL solution containing liposomes in which ammonium sulfate was enclosed in the hydrophilic area, and erlotinib was encapsulated in the hydrophobic area, where the dialysis of step 3 was completed, by 2 mL. Using a bath sonicator (bath sonicator, Branson 3510E-DTH; Branson, Danbury, CT, USA), ultrasonication was performed at room temperature (about 25 ° C) to induce encapsulation of doxorubicin, a hydrophilic anticancer agent, into the hydrophilic region of liposomes. Dialysis tubing cellulose membrane, 14,000 molecular weight cut-off (MWCO); Sigma-Aldrich Corp., St. to remove doxorubicin that is not encapsulated in liposomes on suspension containing liposomes that have been sealed up to Louis, MO, USA) and PBS (phosphate buffered saline, pH 7.4) were used for dialysis for one day.

Thus, a liposome was prepared in which an ammonium sulfate and a hydrophilic anticancer agent were enclosed in a hydrophilic region, and a hydrophobic anticancer agent was enclosed in a hydrophobic region.

As a result of measuring the average diameter of the liposomes prepared in Example 1 using a particle size analyzer (ELS-Z; Otsuka Electonics Co., Ltd, Osaka, Japan), it was confirmed that the average diameter was about 140 nm. .

< Example  2> Ammonium Sulfate  Hydrophilic anti-cancer agent and hydrophobic using aqueous solution term Preparation of cancer-encapsulated liposomes 2

In step 3 of <Example 1>, except for using an aqueous solution containing ammonium sulfate (AS) at a concentration of 300 mM, instead of using an aqueous solution containing ammonium sulfate (AS) at a concentration of 250 mM,

By performing the same steps as in <Example 1>, a liposome was prepared in which an ammonium sulfate and a hydrophilic anticancer agent were enclosed in a hydrophilic region, and a hydrophobic anticancer agent was enclosed in a hydrophobic region.

As a result of measuring the average diameter of the liposomes prepared in Example 2 using a particle size analyzer (ELS-Z; Otsuka Electonics Co., Ltd, Osaka, Japan), it was confirmed that the average diameter was about 170 nm. .

< Example  3> Ammonium Sulfate  Hydrophilic anti-cancer agent and hydrophobic using aqueous solution term Preparation of cancer-encapsulated liposomes 3

In step 3 of <Example 1>, except for using an aqueous solution containing ammonium sulfate (AS) at a concentration of 350 mM, instead of using an aqueous solution containing ammonium sulfate (AS) at a concentration of 250 mM,

By performing the same steps as in <Example 1>, a liposome was prepared in which an ammonium sulfate and a hydrophilic anticancer agent were enclosed in a hydrophilic region, and a hydrophobic anticancer agent was enclosed in a hydrophobic region.

As a result of measuring the average diameter of the liposomes prepared in Example 3 using a particle size analyzer (ELS-Z; Otsuka Electonics Co., Ltd, Osaka, Japan), it was confirmed that the average diameter was about 160 nm. .

< Example  4> Ammonium Sulfate  Hydrophilic anti-cancer agent and hydrophobic using aqueous solution term Preparation of cancer-encapsulated liposomes 4

In step 3 of <Example 1>, except for using an aqueous solution containing ammonium sulfate (AS) at a concentration of 400 mM, instead of using an aqueous solution containing ammonium sulfate (AS) at a concentration of 250 mM,

By performing the same steps as in <Example 1>, a liposome was prepared in which an ammonium sulfate and a hydrophilic anticancer agent were enclosed in a hydrophilic region, and a hydrophobic anticancer agent was enclosed in a hydrophobic region.

As a result of measuring the average diameter of the liposomes prepared in Example 4 using a particle size analyzer (ELS-Z; Otsuka Electonics Co., Ltd, Osaka, Japan), it was confirmed that the average diameter was about 150 nm. .

< Example  5> Ammonium Sulfate  Hydrophilic anti-cancer agent and hydrophobic using aqueous solution term Preparation of cancer-encapsulated liposomes 5 (group 2)

In step 3 of <Example 1>, immediately before performing dialysis to remove residual ammonium sulfate which is not encapsulated in liposomes and is present in the suspension, except that it further has a stabilization time of 30 minutes at room temperature. and,

By performing the same steps as in <Example 1>, a liposome was prepared in which an ammonium sulfate and a hydrophilic anticancer agent were enclosed in a hydrophilic region, and a hydrophobic anticancer agent was enclosed in a hydrophobic region.

As a result of measuring the average diameter of the liposomes prepared in Example 5 using a particle size analyzer (ELS-Z; Otsuka Electonics Co., Ltd, Osaka, Japan), it was confirmed that the average diameter was about 160 nm. .

< Example  6> Ammonium Sulfate  Hydrophilic anti-cancer agent and hydrophobic using aqueous solution term Preparation of cancer-encapsulated liposomes 6 (group 3)

In step 3 of <Example 1>, except that the ultrasonic treatment time is performed for 15 minutes,

By performing the same steps as in <Example 1>, a liposome was prepared in which an ammonium sulfate and a hydrophilic anticancer agent were enclosed in a hydrophilic region, and a hydrophobic anticancer agent was enclosed in a hydrophobic region.

As a result of measuring the average diameter of the liposomes prepared in Example 6 using a particle size analyzer (ELS-Z; Otsuka Electonics Co., Ltd, Osaka, Japan), it was confirmed that the average diameter was about 150 nm. .

< Example  7> Ammonium Sulfate  Hydrophilic anti-cancer agent and hydrophobic using aqueous solution term Preparation of cancer-encapsulated liposomes 7

In step 1 of <Example 1>, 30 mg of DSPC and 20 mg of cholesterol were used as lipids; Except using ammonium sulfate concentration of 350 mM,

By performing the same steps as in <Example 1>, a liposome was prepared in which an ammonium sulfate and a hydrophilic anticancer agent were enclosed in a hydrophilic region, and a hydrophobic anticancer agent was enclosed in a hydrophobic region.

As a result of measuring the average diameter of the liposomes prepared in Example 7 using a particle size analyzer (ELS-Z; Otsuka Electonics Co., Ltd, Osaka, Japan), it was confirmed that the average diameter was about 155 nm. .

< Example  8> Ammonium Sulfate  Hydrophilic anti-cancer agent and hydrophobic using aqueous solution term Preparation of cancer-encapsulated liposomes 8

In step 1 of <Example 1>, except that 27 mg of DSPC, 20 mg of cholesterol, 3 mg of DOPG were used as lipids, and ammonium sulfate concentration was used at 350 mM,

By performing the same steps as in <Example 1>, a liposome was prepared in which an ammonium sulfate and a hydrophilic anticancer agent were enclosed in a hydrophilic region, and a hydrophobic anticancer agent was enclosed in a hydrophobic region.

As a result of measuring the average diameter of the liposomes prepared in Example 8 using a particle size analyzer (ELS-Z; Otsuka Electonics Co., Ltd, Osaka, Japan), it was confirmed that the average diameter was about 160 nm. .

< Example  9> Ammonium Sulfate  Hydrophilic anti-cancer agent and hydrophobic using aqueous solution term Preparation of cancer-encapsulated liposomes 9

In step 1 of <Example 1>, except that 36 mg of DSPC and 24 mg of cholesterol were used as lipids, and ammonium sulfate concentration was used at 350 mM,

By performing the same steps as in <Example 1>, a liposome was prepared in which an ammonium sulfate and a hydrophilic anticancer agent were enclosed in a hydrophilic region, and a hydrophobic anticancer agent was enclosed in a hydrophobic region.

As a result of measuring the average diameter of the liposomes prepared in Example 9 using a particle size analyzer (ELS-Z; Otsuka Electonics Co., Ltd, Osaka, Japan), it was confirmed that the average diameter was about 165 nm. .

< Example  10> Ammonium Sulfate  Hydrophilic anti-cancer agent and hydrophobic using aqueous solution term Preparation of cancer-encapsulated liposomes 10

In step 1 of <Example 1>, except that the DSPC 45 mg, cholesterol 30 mg, and ammonium sulfate concentration of 350 mM as a lipid,

By performing the same steps as in <Example 1>, a liposome was prepared in which an ammonium sulfate and a hydrophilic anticancer agent were enclosed in a hydrophilic region, and a hydrophobic anticancer agent was enclosed in a hydrophobic region.

As a result of measuring the average diameter of the liposomes prepared in Example 10 using a particle size analyzer (ELS-Z; Otsuka Electonics Co., Ltd, Osaka, Japan), it was confirmed that the average diameter was about 160 nm. .

< Comparative example  1> containing citric acid Buffer solution  Used, anticancer and hydrophobic Anticancer Preparation of encapsulated liposomes 1

In step 3 of <Example 1>, except for using an aqueous solution of ammonium sulfate (AS), instead of using a 300 mM citric acid buffer (pH 3.9),

By performing the same steps as in <Example 1>, a liposome was prepared in which an ammonium sulfate and a hydrophilic anticancer agent were enclosed in a hydrophilic region, and a hydrophobic anticancer agent was enclosed in a hydrophobic region.

However, in the case of <Comparative Example 1> using a citric acid buffer solution, before performing step 4 of <Example 1>, the solution of the solution to form a pH gradient between the inside and outside of the liposome is additionally performed. The pH was adjusted to about 6.5, and a sodium bicarbonate buffer of 300 mM was used as a sample for pH adjustment.

< Comparative example  2> containing citric acid Buffer solution  Used, anticancer and hydrophobic Anticancer Preparation of encapsulated liposomes 2

In <Example 7>, instead of using an aqueous solution of ammonium sulfate (AS), using a 300 mM citric acid buffer solution (pH 3.9),

By performing the same steps as in <Example 7>, a liposome was prepared in which an ammonium sulfate and a hydrophilic anticancer agent were enclosed in a hydrophilic region, and a hydrophobic anticancer agent was enclosed in a hydrophobic region.

However, in the case of <Comparative Example 2> using a citric acid buffer solution, before performing step 4 of <Example 7>, the solution of the solution to form a pH gradient between the inside and outside of the liposome is additionally performed. The pH was adjusted to about 6.5, and a sodium bicarbonate buffer of 300 mM was used as a sample for pH adjustment.

< Experimental Example  1> Example  1 to 4, Comparative example  Manufactured in 1 Liposome  Anticancer drug Encapsulation efficiency (Encapsulation efficiency, EE ) evaluation

The EE of doxorubicin (DOX) or erlotinib (ERL) encapsulated in liposomes prepared in Examples 1 to 4 and Comparative Example 1 was calculated through Equation 1 below.

[Equation 1]

In Equation 1,

C _f represents the amount of doxorubicin (DOX) or erlotinib (ERL) that breaks the liposome by treating the prepared liposome with 10% Triton-X 100. The absorbance of erlotinib (ERL) was measured at 345 nm using a UV-Vis spectrometer (DU-800 spectrophotometer; Backman Coulter Inc., Fullerton, CA, USA); The fluorescence intensity of doxorubicin (DOX) is measured at excitation and emission wavelengths of 495 nm and 590 nm, respectively, using a spectrofluorometer (SFM-25; Tegimenta AG, Rotkreuz, Switzerland).

C _i represents the amount of doxorubicin (DOX) or erlotinib (ERL) used or added during liposome preparation.

The encapsulation efficiency of doxorubicin (DOX) calculated through Equation 1 is shown in FIG. 1.

1 shows the encapsulation efficiency of doxorubicin (DOX) encapsulated in liposomes prepared in Examples 1 to 4 and Comparative Example 1.

As shown in Figure 1,

It can be seen that the encapsulation efficiency of doxorubicin (DOX) of Examples 1 to 4, which is a liposome encapsulated with doxorubicin (DOX) using an aqueous solution of ammonium sulfate, is about 90% or more,

It was confirmed that the encapsulation efficiency of the anticancer agent of Comparative Example 1, which is a liposome in which doxorubicin (DOX) was sealed using a citric acid buffer, was about 17%.

That is, compared with the conventional method, a pH-gradient (pH-gradient) method using a citric acid buffer (citric acid buffer) compared to doxorubicin (DOX) is sealed, compared to doxorubicin (DOX) using an aqueous solution of ammonium sulfate. In this case, it can be seen that the sealing efficiency of doxorubicin (DOX) is significantly improved.

< Experimental Example  2> Example  1 (group 1), Example  5 (group 2), Example  Encapsulation efficiency of doxorubicin (DOX) in liposomes prepared in 6 (group 3), EE ) evaluation

The encapsulation efficiency of doxorubicin (DOX) in liposomes prepared in Example 1 (group 1), Example 5 (group 2), and Example 6 (group 3) was measured by performing the same method as in Experimental Example 1 above. Did. The results are shown in FIG. 2.

2 shows the encapsulation efficiency of doxorubicin (DOX) encapsulated in liposomes prepared in Example 1 (group 1), Example 5 (group 2), and Example 6 (group 3).

As shown in Figure 2,

It can be seen that the encapsulation efficiency of doxorubicin (DOX) in the liposomes prepared in Example 1 (group 1), Example 5 (group 2), and Example 6 (group 3) is about 90% or more.

However, in the case of Example 6 (group 3), when the ultrasonic treatment time was increased to 15 minutes, it was found that the encapsulation efficiency slightly decreased compared to Example 1 (group 1) in which the ultrasonic treatment was shortened to 5 minutes. .

In the case of Example 5 (group 2), compared with Example 1 (group 1), it is a case that has a stabilization time for 30 minutes before dialysis, but it can be seen that the encapsulation efficiency slightly increased.

< Experimental Example  3> Morphology evaluation

The solution containing the liposome prepared in Example 1 was drop-cast onto a carbon-coated copper grid, dried in air at room temperature, and then subjected to a field emission transmission electron microscope (FE-TEM) of 200 kV ( JEM 2100F; JEOL Ltd., Tokyo, Japan) was used to confirm the morphology of liposomes. The results are shown in FIG. 3.

3 is a drop-casting solution containing the liposome prepared in Example 1 to a carbon-coated copper grid (copper grid), dried in the air at room temperature, 200 kV field emission transmission electron microscope (FE- TEM) (JEM 2100F; JEOL Ltd., Tokyo, Japan).

As shown in Figure 3 (A), it can be seen that the liposome prepared in Example 1 exhibits a spherical shape. 3 (B) is an enlarged observation of one liposome, and it can be seen that the liposome prepared in Example 1 shows a spherical shape. 3 (C) is observed at a high resolution, and it is possible to confirm the interface between the inner compartment of the nano-liposome having a high crystalline lattice and the outermost part having a low crystalline or amorphous structure. FIG. 3 (D) shows a selected area electron diffraction (SAED) pattern of FIG. 3 (C), and the crystalline lattice of FIG. 3 (C) can be confirmed.

< Experimental Example  4> Differential drug release evaluation

In order to evaluate the drug release of the anti-cancer agent in the liposome prepared in Example 1, the following experiment was performed.

The suspension containing the liposome prepared in Example 1 was charged into a dialysis cassette (10,000 MWCO, Slide-A-Lyser Dialysis Cassette G2; Thermo Scientific Corp., Rockford, IL, USA). The dialysis cassette was placed in PBS (pH 7.4) and stirred at 37 ° C. The amount of anticancer agent released at each predetermined elapsed time point was calculated by the following Equation 2.

[Equation 2]

In Equation 2,

D _t is the concentration of the anticancer agent remaining in the liposomes during the drug release experiment;

D _i is the concentration of the anticancer agent encapsulated in liposomes prior to drug release experiments.

The absorbance of erlotinib (ERL) was measured at 345 nm using a UV-Vis spectrometer (DU-800 spectrophotometer; Backman Coulter Inc., Fullerton, CA, USA), and the fluorescence intensity of doxorubicin (DOX) was spectrofluorometer (SFM- 25; Tegimenta AG, Rotkreuz, Switzerland) at excitation and emission wavelengths of 495 nm and 590 nm, respectively.

The results are shown in FIGS. 4 and 5.

Figure 4 shows the drug release amount of erlotinib (ERL) and doxorubicin (DOX) in the liposome prepared in Example 1.

Figure 5 shows the drug release rate of erlotinib (ERL) and doxorubicin (DOX) in the liposome prepared in Example 1.

As shown in FIG. 4, it can be seen that erlotinib (ERL) is released in a relatively large amount compared to doxorubicin (DOX) as the drug release experiment time elapses.

As shown in FIG. 5, it can be seen that the release rate of erlotinib (ERL) significantly increased and then decreased gradually at the beginning of the drug release experiment, and the release rate of doxorubicin (DOX) gradually increased with time.

< Experimental Example  5> Example  7 to 10, Comparative example  Manufactured in 2 Liposome  Anticancer drug Encapsulation efficiency (Encapsulation efficiency, EE ) evaluation

The EE of doxorubicin (DOX) or erlotinib (ERL) encapsulated in liposomes prepared in Examples 7 to 10 and Comparative Example 2 was evaluated, and the evaluation method was performed in the same manner as in <Experimental Example 1>.

The lipid composition used in Examples 7 to 10 and Comparative Example 2, the doxorubicin loading method, and the drug encapsulation rate are summarized in Table 1 below.

[Table 1]

As shown in Table 1 above,

When Example 7 and Comparative Example 2 in which all the manufacturing conditions were the same except for the loading method of doxorubicin (DOX) were compared, the loading rate of doxorubicin (DOX) was 24% to 79% due to the difference in the loading method of doxorubicin (DOX). It can be seen that it increases significantly.

In addition, even if a method of encapsulating doxorubicin (DOX) into a hydrophilic region in a liposome using ammonium sulfate is a known technique, when comparing Example 7 and Comparative Example 2, the encapsulation rate of erlotinib (ERL), a hydrophobic anticancer agent, is compared. It can be seen that the effect is also increased, except for erlotinib (ERL) to lipid ratio (ERL to lipid ratio), when all the manufacturing conditions are the same Examples 9 to 10, Comparative Example 2, hydrophobic anticancer agent to lipid It was confirmed that the encapsulation rate of erlotinib (ERL) increased from 17% to 41% and 52% as the ratio (hydrophobic anti-cancer drug to lipid ratio) decreased.

Claims (12)

Preparing a mixed solution by dissolving a hydrophobic anticancer agent and lipid in a solvent;
Evaporating the solvent of the mixed solution to form a lipid film;
Hydrating the lipid membrane with a solution containing ammonium sulfate to form a liposome in which an ammonium sulfate is enclosed in a hydrophilic region and a hydrophobic anticancer agent is enclosed in a hydrophobic region; And
Containing; by adding a solution containing a hydrophilic anticancer agent, encapsulating a hydrophilic anticancer agent in the hydrophilic region of the liposome;
In the production method of liposomes,

The hydrophobic anti-cancer agent is erlotinib;
The lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-Distearoyl-sn-glycero-3-phosphocholine, DSPC), cholesterol and 1-palmitoyl-2-ole Contains oil-sn-glycero-3-phospho- (1'-rac-glycerol) (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- (1'-rac-glycerol), POPG) and;
The hydrophilic anti-cancer agent is doxorubicin; And

Characterized in that the diameter of the liposome prepared is 140 to 200 nm,
Method of manufacturing liposomes.
delete delete delete delete According to claim 1,
The solvent is characterized in that at least one member selected from the group consisting of methanol, ethanol, propanol, butanol and chloroform,
Method of manufacturing liposomes.
According to claim 1,
The solution containing ammonium sulfate is characterized in that it is an aqueous solution of ammonium sulfate,
Method of manufacturing liposomes.
According to claim 1,
Before adding a solution containing a hydrophilic anti-cancer agent,
Dispersing a solution containing a liposome in which an ammonium sulfate is encapsulated in a hydrophilic region and a hydrophobic anticancer agent is encapsulated in a hydrophobic region, further comprising adjusting the diameter of the liposome,
Method of manufacturing liposomes.
The method of claim 8,
Characterized in that the dispersion is performed by processing ultrasonic waves,
Method of manufacturing liposomes.
According to claim 1,
Characterized in that the diameter of the liposome prepared is 140 to 170 nm,
Method of manufacturing liposomes.
According to claim 1,
The solution containing the hydrophilic anti-cancer agent is characterized in that it comprises an aqueous sodium chloride solution,
Method of manufacturing liposomes.
Preparing a mixed solution by dissolving a hydrophobic anticancer agent and lipid in a solvent;
Evaporating the solvent of the mixed solution to form a lipid film;
Hydrating the lipid membrane in a solution containing ammonium sulfate to form a liposome in which an ammonium sulfate is enclosed in a hydrophilic region and a hydrophobic anticancer agent is enclosed in a hydrophobic region; And
Containing; by adding a solution containing a hydrophilic anticancer agent, encapsulating a hydrophilic anticancer agent in the hydrophilic region of the liposome;
In the method for controlling the encapsulation efficiency of the hydrophobic anti-cancer agent and the hydrophilic anti-cancer agent in the liposome,

The hydrophobic anti-cancer agent is erlotinib;
The lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-Distearoyl-sn-glycero-3-phosphocholine, DSPC), cholesterol and 1-palmitoyl-2-ole Contains oil-sn-glycero-3-phospho- (1'-rac-glycerol) (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- (1'-rac-glycerol), POPG) and;
The hydrophilic anti-cancer agent is doxorubicin; And
Characterized in that the diameter of the liposome is 140 to 200 nm,
A method for controlling the encapsulation efficiency of hydrophobic and hydrophilic anticancer agents in liposomes.

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