KR101919650B1 - A method of preparing partially uncapped liposome - Google Patents

Abstract

The present invention relates to a method for producing a partially opened liposome, and more particularly, to a method for producing a liposome in which a lipid bilayer is partially opened using high density, superparamagnetic Fe ₃ O ₄ nanoparticles, a custom magnet and a magnetic impeller. The partially opened liposome according to the present invention can be produced by a simple method using superparamagnetic Fe ₃ O ₄ nanoparticles, a magnet and a magnetic impeller. Since the open part naturally closes after a certain period of time, It can be safely and easily sealed, exhibits improved drug encapsulation efficiency than conventional liposomes, and can be used as a drug delivery system since a drug release rate can be controlled by enclosing a substance for improving membrane permeability.

Description

[0001] The present invention relates to a partially opened liposome,

The present invention relates to a method for producing a partially opened liposome, and more particularly, to a method for producing a liposome in which a lipid bilayer is partially opened using high density, superparamagnetic Fe ₃ O ₄ nanoparticles, a custom magnet and a magnetic impeller.

The drug is not administered to the human body as it is relative to the chemical substance but is administered through various routes by operating as a formulation that is convenient for the patient to take or use, or in a form capable of expressing the pharmacological effect efficiently / effectively. The administered drug moves to the appropriate place or is absorbed, and is distributed to each organ in the bloodstream and is discharged from the human body through urination, excretion, and excretion through metabolic processes. The drug efficacy is expressed by the molecules of the drug reaching the action site in the living body, and the drugs reaching the other site usually show many side effects. Thus, in order to effectively treat the drug, it is necessary to concentrate and accurately deliver the drug to the target body part.

Interest in liposomes is increasing as one of these drug delivery vehicles.

Liposomes are spherical particles formed by aggregation of molecules such as phospholipids, which have both hydrophilic and hydrophobic groups in one molecule, according to the interaction between solvent molecules and phospholipid molecules in an aqueous solution or an organic solvent. An artificial structure having fluidity, a hydrophilic space exists inside, and such a hydrophilic space can contain a water-soluble substance, and a structure capable of immobilizing a biomaterial using external positive and negative charges and functional groups of the liposome.

The liposome having such a structure is advantageous in that it is easy to change its structure due to its elasticity, can impart completely novel properties and functions by different structure and components, and is excellent in biocompatibility, degradability and stability. In addition, it can carry biologic substances such as genes, enzymes or proteins in the inside, and has a property to protect the loaded substances against the external environment.

These properties are widely used in pharmaceutical and medical industries such as drug delivery systems, target drugs, immobilization of enzymes, gene transfer, catalyst and protein separation, and cosmetics and food additives.

Over the past few years, liposome-based biological / chemical transporter systems have received much attention and have provided a variety of possibilities in the biomedical and cosmetic fields. In particular, many studies have focused on the development of functional liposomes using lipid or antibody / ligand binding lipids that have undergone a physicochemical modification process for targeted disease treatment and drug delivery control. These studies have also made it possible to produce artificial endoplasmicoids using functional amphipathic polymers similar to those of conventional liposomes. As a result, considerable progress has been made in the development of liposome-based medicinal technology. Particularly, studies on the fluidity of liposome lipid bilayer membranes are expanding to new fields in biomaterials field.

As a conventional method for producing liposomes, a thin film hydration method using phosphatidylcholine, which is an amphipathic molecule, has been widely used. However, the liposome produced by the above method has a low efficiency of encapsulating a polymer drug such as protein, nucleic acid, and the like, and protein deformation occurs even when encapsulated.

The dynamic properties of lipid bilayers such as fusion, cleavage and morphological modification of liposomes are generally influenced by various experimental conditions. For example, the cytoskeletal sub-membranous protein [eg, talin] protects the hydrophobic part at the lipid bilayer edge and inhibits the lipid increase, thus allowing temporary opening of the liposome. The function of collapsing lipid bilayer membranes, such as talline proteins, is a significant hazard to living cells, but the temporarily open pores in the liposome endoplasmic reticulum are used as a pathway to encapsulate large and small molecules (proteins and nucleic acids) into liposomes. The production of morphologically modified liposomes developed from conventional lipid bilayer structures can be a means for designing high efficiency drug delivery endoplasmic reticulum.

Therefore, the inventors of the present invention have been studying to produce a liposome carrying not only a low-molecular drug but also a polymer drug in a high packing efficiency without deformation, a solution containing a liposome encapsulating high-density superparamagnetic Fe ₃ O ₄ nanoparticles, The liposome layer is partially opened by stirring using an impeller, and after a certain period of time, the partially opened portion of the liposome is naturally closed, so that the polymer drug having a large molecular weight can be easily sealed without being deformed. The present invention has been completed.

1. Japanese Patent No. 3,902,794

Accordingly, an object of the present invention is to provide a method for producing a liposome capable of supporting a polymer drug by physically partially opening a bilayer of liposome.

Another object of the present invention is to provide a partially opened liposome produced by the above method.

In order to achieve the above-described object of the present invention,

(a) preparing a liposome solution in which superparamagnetic Fe ₃ O ₄ nanoparticles are encapsulated;

(b) attaching a magnet to the magnetic impeller and vigorously stirring to produce a liposome in which the lipid bilayer is partially opened by movement of the super-magnetic Fe ₃ O ₄ nanoparticles; And

(c) separating the leached Fe ₃ O ₄ from the liposome to precipitate.

Also, preferably, the magnet of step (b) may be a rotor having a radius of 25-35 mm and a thickness of 2-5 mm.

Also, preferably, the stirring in the step (b) may be performed at a speed of 500 to 1,500 rpm for 1 to 2 minutes.

Also, preferably, step (c) can be performed by separating the leached Fe ₃ O ₄ from the liposome through another magnet outside the flask.

The present invention also provides a partially opened liposome produced by the above method.

In addition, preferably, the partially-opened liposome can easily encapsulate the protein drug of the polymer without modification.

In addition, the partially opened liposome may be filled with a substance that enhances the permeability of the polymer when the drug is encapsulated, thereby controlling the drug release rate.

Preferably, the membrane permeability enhancing substance may be selected from the group consisting of flockamer 188 among the surfactants of the poloxamer series.

As described above, the partially-opened liposome according to the present invention can be produced by a simple method using the super-magnetic Fe ₃ O ₄ nanoparticles, the magnet and the magnetic impeller, and since the open part naturally closes after a certain period of time, Can be safely and easily encapsulated without modification, exhibits improved drug encapsulation efficiency over existing liposomes, and can seal drug permeation enhancing substances together to control the rate of drug release, which is useful as a drug delivery system have.

1 is a schematic view showing a process for producing a partially-opened liposome according to the present invention.
FIG. 2 is a graph showing the size distribution of the partially opened liposome prepared according to the present invention ((a) Example 1, (b) Example 2, and (c) Comparative Example 1).
FIG. 3 shows a structural model and a liposome-enlarged transmission electron microscope image of the Fe ₃ O ₄ -encapsulated liposome prepared under magnetic shear stress ((I) original liposome (IL); (II) Fe ₃ O ₄ nanoparticle (II) a liposome (TSL) in which Fe ₃ O ₄ nanoparticles are biased in both directions, (III) a liposome (UCL) in which a lipid bilayer is partially opened, (IV) a liposome Liposomes (FUL)).
FIG. 4 is a transmission electron microscopy (TEM) photograph showing the morphological change of the liposome under magnetic shear stress after magnetic stirring at 500 rpm in the preparation of the partially opened liposome according to an embodiment of the present invention.
FIG. 5 is a transmission electron micrograph showing the morphological change of liposome under magnetic shear stress after magnetic stirring at 1500 rpm in the preparation of the partially opened liposome according to an embodiment of the present invention.
6 is a transmission electron micrograph showing the morphological change of the liposome under magnetic shear stress after magnetic stirring at 2500 rpm in the preparation of the partially opened liposome according to an embodiment of the present invention.
FIG. 7 is a transmission electron micrograph showing changes in the liposome shape according to the change of the aqueous solution temperature in the preparation of the partially opened liposome according to an embodiment of the present invention.
FIG. 8 is a transmission electron micrograph showing a change in liposome morphology in which a partial opening portion is closed according to agitation time after opening of a partially opened liposome according to an embodiment of the present invention.
9 is a schematic view of a drug encapsulation process of a partially opened liposome according to an embodiment of the present invention.
10 is a graph showing the protein (drug) filling efficiency of the partially opened liposome according to an embodiment of the present invention.
11 is a graph showing protein (drug) release behavior of a partially opened liposome according to an embodiment of the present invention.
FIG. 12 is a graph showing the denaturation of a drug (insulin) released from a partially-opened liposome according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

(A) preparing a liposome solution encapsulating superparamagnetic Fe ₃ O ₄ nanoparticles;

(b) attaching a magnet to the magnetic impeller and vigorously stirring to produce a liposome in which the lipid bilayer is partially opened by movement of the super-magnetic Fe ₃ O ₄ nanoparticles; And

(c) separating the leached Fe ₃ O ₄ from the liposome to precipitate.

The method for producing the partially opened liposome according to the present invention will be described in more detail with reference to FIG.

One. Super magnetic Fe ₃ O ₄  Encapsulated nanoparticles Liposome  Dispersion manufacturing

In the method for producing a partially opened liposome according to the present invention, a method commonly used in the art may be used for producing the liposome dispersion liquid containing the super-magnetic Fe ₃ O ₄ nanoparticles enclosed therein, Membrane rehydration methods have been used but are not limited thereto.

Specifically, after the phospholipid and the superparamagnetic Fe ₃ O ₄ nanoparticles are dissolved in an organic solvent, the solvent is evaporated to form a membrane, and then a buffer solution such as PBS is added, followed by ultrasonication to rehydrate the supernatant Fe ₃ O ₄ A liposome dispersion in which nanoparticles are encapsulated can be prepared.

Examples of the phospholipid include phosphatidylcholine (lecithin), soybean lecithin, lysolecithin, sphingomyelin, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, diphosphatidylglycerol, Cardiolipin, natural phospholipids selected from plasmarogens; Hydrogenation products of natural phospholipids; Dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylserine, eleostearoylphosphatidylcholine, eleostearoylphosphatidylethanolamine, and Eelestearoylphosphatidylethanolamine and diesterylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylserine, Synthesis of osteoyl phosphatidylserine Lipids; Derivatives of synthetic lipids; And a mixture of fatty acids obtained by hydrolysis of synthetic lipids can be used. Preferably, phosphatidylcholine (PC) can be used.

The Fe ₃ O ₄ nanoparticles can be prepared by using an organic chemical reaction which is commercially available or commonly used in the art. For example, after reacting iron (III) acetylacetonate, 1,2-hexadecanediol, oleic acid and oleylamine in benzyl ether at 200 ° C. for 2 hours, the temperature is raised to 300 ° C. and further reaction is carried out for 1 hour Fe ₃ O ₄ nanoparticles can be produced.

At this time, together phospholipids and Fe ₃ O mixing ratio of the ₄ nano-particles is preferably Fe ₃ O ₄ nanoparticles 2 to 40 parts by weight based on the phospholipid 100 parts by weight, if Fe ₃ O ₄ nano-back particles added amount is less than 2 parts by weight of the It is difficult for the liposome to partially open due to the magnetic stirring, and if it exceeds 40 parts by weight, the preparation of the liposome particles may not be easy.

2. Partial opening Liposomal  Produce

In the method of manufacturing a partially-opened liposome according to the present invention, the magnet is attached to the magnetic impeller and then vigorously stirred to partially open the phospholipid by the migration of the super-magnetic Fe ₃ O ₄ nanoparticles .

Specifically, Fe ₃ O ₄ encapsulated within the liposome will migrate to a specific location under strong magnetic and magnetic shear stresses, resulting in pores in the lipid bilayer.

At this time, the shape and size of the magnet to be used are not particularly limited, but it is preferable that the magnet has a radius of 25-35 mm and a thickness of 2-5 mm so that stirring can be performed well.

However, if the relatively stronger magnetic shear stress persists after opening, the lipid bilayers of the liposomes may undergo morphological collapse. As a result, it was found that the stirring was preferably carried out at a speed of 500 to 1,500 rpm for 1 to 2 minutes (see FIGS. 4-6), and in particular, When the intensity of the magnetic stirring was increased to 1,500 rpm, the fraction of the partially opened liposome was increased to 61% (see FIG. 5), confirming that it was the optimum condition for preparing a partially opened liposome having a hole of about 25 nm .

The partially opened region thus formed is closed naturally after a certain period of time (see FIG. 8), so that the polymer protein can be safely and easily sealed without being deformed.

3. Leaked Fe ₃ O ₄ To In liposomes  Separation and precipitation step

In the method for preparing a partially-opened liposome according to the present invention, the partially-opened liposome prepared above is placed on the upper layer of the liposome dispersion, and the amount of Fe ₃ O ₄ Nanoparticles can be separated from the liposomes through other magnets outside the flask. For example, neodymium rare-earth magnets (10 mm in radius, 10 mm in thickness) are attached to the bottom of the flask to form partially open liposomes and leaked Fe ₃ O ₄ can be easily separated and precipitated.

Such a partially-opened liposome manufacturing method is simple and can be applied to mass production.

The present invention also provides a partially opened liposome produced by the above method.

The partially opened liposomes of the present invention are produced using the above-described production method, and the common description of the two inventions is omitted in order to avoid the excessive complexity of the specification according to the repetitive description.

The partially opened liposome according to the present invention can be prepared by a simple method using superparamagnetic iron nanoparticles, a magnet and a magnetic impeller, and since a portion that is opened after a certain period of time is naturally closed (see FIG. 8) (See FIG. 12), exhibits improved drug encapsulation efficiency (see FIG. 10), and encapsulates materials for enhancing membrane permeability to control the rate of drug release (see FIG. See FIG. 11), and can be usefully used as a drug delivery vehicle.

At this time, the membrane permeability enhancing substance may be selected from the group consisting of poloxamers 188, 407, and 127.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

Example 1 Preparation of partially opened liposomes

<1-1> Substance

(BSA), human recombinant insulin, Poloxamer 188, iron (III) acetylacetonate, 1,2-hexa-biphenylylcarboxylate, Decane diol, oleic acid, oleylamine, sodium azide, and benzyl ether were purchased from Sigma-aldrich (USA), and the BCA protein assay kit was Pierce (USA).

<1-2> Fe ₃ O ₄ Manufacture of nanoparticles

After reacting iron (III) acetylacetonate, 1,2-hexadecanediol, oleic acid and oleylamine in benzyl ether at 200 ° C. for 2 hours, the temperature was raised to 300 ° C. and reacted for 1 hour, nm Fe ₃ O ₄ nanoparticles were prepared.

<1-3> Partial opening Liposomal  Produce

Phosphatidylcholine (20 mg) and the previously prepared Fe ₃ O ₄ nanoparticles (0.5 mg) were dissolved in chloroform (5 ml) in a round bottom flask. The solvent in the round bottom flask was then evaporated using a rotary evaporator (EYELA, N-1000, Fisher Scientific Inc., USA) to allow the phosphatidylcholine and Fe ₃ O ₄ nanoparticles to be coated in film form on the inner surface of the flask . Next, PBS (pH 7.4, 20 ml) was added to the phosphatidylcholine and Fe ₃ O ₄ nanoparticles coated on the flask at room temperature (25 ° C) and rehydrated by ultrasonic treatment (60 Hz, 5 minutes) to obtain Fe ₃ O ₄ nanoparticles To form a phosphatidylcholine liposome encapsulated therein. The Fe ₃ O ₄ inclusion efficiency was measured using a Jobin-Yvon Ultima-C inductively-coupled plasma-atomic emission spectrometer (JY-Ultima-2, France) It was found that it was efficient and well done.

Next, the obtained liposome was agitated at a low speed using a magnet having a radius of 30 mm and a thickness of 2 mm and a magnetic impeller and attached to the magnet, and then the magnetic impeller was vigorously agitated at 25 rpm at 500 rpm for 1 minute, Partially opened liposomes were prepared by entrapping Fe ₃ O ₄ to a specific position under strong magnetic and magnetic shear stress to form holes in the lipid bilayer.

Immediately after magnetic stirring of the liposome aqueous solution, leaking Fe ₃ O ₄ was separated from the liposome by depositing neodymium rare-earth magnets (10 mm in radius, 10 mm in thickness) on the bottom surface of the outer surface of the flask.

&Lt; Example 2 >

The partially opened liposomes were prepared in the same manner as in Example 1, except that the magnetic impeller was agitated vigorously for 1 minute at 1500 rpm.

&Lt; Comparative Example 1 &

The partially opened liposomes were prepared in the same manner as in Example 1, except that the magnetic impeller was stirred vigorously at 2500 rpm for 1 minute.

&Lt; Comparative Example 2 > Preparation of liposome not containing Fe ₃ O ₄ nanoparticles

The liposomes were prepared in the same manner as in Example 1 except that Fe3O4 nanoparticles were not contained.

<Analysis>

1. Particle size

The size distribution of the partially opened liposomes (1 mg / ml, PBS) prepared in Example 1-2 and Comparative Example 1 was measured using a 633 nm wavelength helium-neon laser, a fixed dispersion angle 90 ° Zetasizer 3000 (Malvern Instruments, USA) The results are shown in Fig.

As shown in Fig. 2, it was confirmed that the diameter of each liposome was 60 nm or less.

2. Liposomal  Morphological change

The morphological change of the liposome under magnetic shear stress was observed by transmission electron microscopy (TEM) and is shown in FIG. 3 to FIG.

Figure 3 is a schematic representation of liposome morphology. Under the magnetic shear stress and magnetic stirring, (I) the Fe ₃ O ₄ nanoparticles in the liposomes (IL) are oriented in the direction of one-sided liposome (OSL) or (II ') two-sided liposome: TSL). In addition, if the shear stress is continued to a specific part, the Fe ₃ O ₄ nanoparticles may leak from the liposome and exhibit a partially unclipped liposome (UCL) in the lipid bilayer (III). On the other hand, when several open sites are formed in the liposome, the basic form of the liposome is collapsed (Ⅳ) to form a full uncapped liposome (FUL).

4 to 6 are diagrams showing the morphological change of the liposome under magnetic shear stress according to magnetic stirring intensity.

As shown in FIG. 4, the result after magnetic stirring at 500 rpm in Example 1 was (IL), (II) OSL or TSL, (III) UCL and (IV) FUL were 9, 13, 42 and 36% , And the size of the partially opened hole of the UCL was confirmed to be about 22 nm.

As shown in FIG. 5, when the magnetic stirring intensity was increased to 1,500 rpm in Example 2, the yield fraction of UCL was increased to 61%, and the pore size was formed to be about 25 nm.

As shown in FIG. 6, in Comparative Example 1, when the magnetic stirring intensity was increased to 2,500 rpm, the fraction of the UCL obtained decreased to 12% and the fraction of IL and FUL was increased. This result is presumed to be due to the nature of the lipid which is supposed to be rapidly restored to the original liposome form.

Therefore, in the production of the partially opened liposome according to the present invention, the magnetic stirring intensity is preferably set to less than 2,500 rpm.

On the other hand, in Comparative Example 2, the liposome not containing Fe ₃ O ₄ did not have an open site even under magnetic shear stress.

Therefore, it can be seen that the incorporation of Fe ₃ O ₄ nanoparticles is very important in the production of the partially opened liposome according to the present invention.

<Experimental Example 1> Morphological change of liposome according to temperature

The liposome morphology was observed when the liposome was vigorously agitated using a custom magnetic impeller (1,500 rpm, 1 minute) and the aqueous solution temperature was changed from 2 占 폚 to 40 占 폚. The results are shown in Fig.

As shown in Fig. 7, the shape of the liposome obtained at 40 DEG C remained largely unformed (IL). It is presumed that the liposome is partially opened at 40 캜 but the structure of the opened liposome is restored quickly.

However, at low temperatures, such as in a 2 &lt; 0 &gt; C environment, the rigidity of lipids increases and there is a limit to the recovery of the open structure of the liposomes and thus the structural collapse of the liposomes is expected to accelerate.

From the above results it can be expected that the partially opened liposomes (UCL) according to the present invention exist between structurally damaged un-recovered liposomes and damaged liposomes (recovered liposomes) .

EXPERIMENTAL EXAMPLE 2 Observation of open partial recovery of partially opened liposome

The following experiment was conducted to confirm whether the open portion of the partially opened liposome (UCL) of the present invention was restored.

Specifically, the partial-opened liposome (UCL) prepared in Example 1 was stirred at a low speed (30 rpm) for 1 to 2 hours at 37 ° C in an aqueous solution, and the structural change of the liposome was observed by TEM.

As shown in FIG. 8, after one hour of agitation, the partial opening of the UCL recovered to return to the present liposome form (IL). After 2 hours of agitation, the liposomes coagulated.

Also at 25 ℃, the partial opening structure of UCL was completely restored within 2 hours.

Therefore, the partially opened liposome according to the present invention can be conveniently used as a drug delivery vehicle because it can easily enclose a high molecular weight substance such as proteins and nucleic acids because the open lipid bilayer part is restored naturally.

&Lt; Example 3 > Preparation of liposome with increased membrane permeability

Poloxamer 188, a hydrophilic surfactant, was added when the open portion of the UCL was recovered while stirring the partially opened liposome (UCL) prepared in Example 1 at 37 ° C in an aqueous solution at a low speed (30 rpm) for 1 to 2 hours, Liposomes with increased permeability were prepared. These liposomes were named poloxamer-capped liposomes (PCL).

Experimental Example 3 Measurement of Drug Loading Efficiency

As shown in the schematic diagram of FIG. 9, 50 mg of UCL and PCL liposomes prepared in Examples 1 and 3, respectively, were dispersed in PBS (pH 7.4, 20 ml) together with bovine serum albumin or 10 mg of insulin as a protein (drug) The protein was sealed in the liposome by stirring at low speed (30 rpm) for 2 hours. The concentration of bovine serum albumin (and insulin) remaining in the supernatant of each liposome aqueous solution prepared beforehand was measured using a BSA protein assay kit to measure the protein inclusion rate. Specifically, the supernatant was centrifuged at 20,000 rpm for 10 minutes, and the concentration of each protein was measured.

The protein encapsulation efficiency was defined as the mass% of the protein encapsulated inside the liposome versus the mass of the protein administered for the initial encapsulation.

As a control group, insulin-encapsulated liposomes prepared by the conventional membrane rehydration method were prepared and the protein encapsulation efficiency was measured. Specifically, an insulin-sealed liposome was prepared by administering PBS dissolved in insulin (10 mg) to a flask whose inner surface was coated with lipid and treating the liposome at 60 Hz for 5 minutes.

The protein inclusion efficiency of each liposome is shown in Fig.

As shown in FIG. 10, when the UCL and PCL prepared according to the present invention were stirred with the protein (bovine serum albumin: BSA) for 2 hours, the encapsulation efficiency was found to be 30% or more, When a high molecular weight substance such as BSA was encapsulated in a common liposome by the same method, even when stirring was carried out for 4 hours, a poor sealing efficiency of less than 5% was exhibited.

As a result of inserting the human recombinant insulin, the UCL and PCL prepared according to the present invention showed an insulin sealing efficiency of about 40-50% for 2 hours of stirring.

Therefore, it can be understood that the partially opened liposome according to the present invention can seal not only low molecular weight but also high molecular weight drug with excellent sealing efficiency.

<Experimental Example 4> Measurement of drug release behavior

In order to investigate the effect of the partially opened liposome according to the present invention on the drug release, the liposome preparation solution (1 mg / ml, pH 7.4, 0.01% sodium azide) containing BSA and insulin in Experimental Example 3 was applied to the dialysis membrane / por MWCO 100K). After the dialysis membrane was sealed, it was placed inside a vial containing high purity PBS (10 ml, 150 mM), and the release tendency was confirmed over time at 37 ° C water bath (100 rev./min). The external solution of the dialysis membrane was extracted at the planned time (0 to 24 hours), and the concentration of the released protein was analyzed using a BSA protein analysis kit, and the extracted external solution was replaced with a fresh solution.

The results are shown in Fig.

As shown in Fig. 11, protein release was found to accelerate protein release in both PCL and bovine serum albumin. It is expected that hydrophilic poloxamer 188 penetrates into the open pores of the UCL and induces a change in the state of liposome membrane permeability.

Experimental Example 5 Evaluation of stability of released insulin

In the partially opened liposome according to the present invention, the protein drug may undergo structural modification at the time of encapsulation.

Specifically, the denaturation of insulin released for 4 hours in each of the liposomes prepared in Examples and Comparative Examples was measured using a circular polarization dichromatism measuring device (J-815 CD spectrometer, Jasco International, UK) Respectively.

As shown in FIG. 12, the α-helical structure of the insulin in liposomes prepared by the conventional method (ultrasonication using a membrane rehydration method (PBS dissolved with insulin) was significantly reduced due to strong ultrasonic treatment, The partially opened liposome was found to be released without deformation of the drug.

Therefore, it can be seen that the partially opened liposome according to the present invention can safely seal the protein drug into the UCL.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (8)

(a) preparing a liposome solution in which superparamagnetic Fe ₃ O ₄ nanoparticles are encapsulated;
(b) attaching a magnet to the magnetic impeller and vigorously stirring to produce a liposome in which the lipid bilayer is partially opened by movement of the super-magnetic Fe ₃ O ₄ nanoparticles; And
(c) separating the leached Fe ₃ O ₄ from the liposome to precipitate,
Characterized in that the liposome is formed of phosphatidylcholine.
A method for producing a partially opened liposome. The method according to claim 1,
Wherein the magnet of step (b) is a rotary blade having a radius of 25-35 mm and a thickness of 2-5 mm. The method according to claim 1,
Wherein the stirring of step (b) is performed at a speed of 500 to 1,500 rpm for 1 to 2 minutes. The method according to claim 1,
Wherein the step (c) is performed by separating the leaking Fe ₃ O ₄ from the liposome through another magnet outside the flask. A partially opened liposome prepared by the method of any one of claims 1 to 4. 6. The method of claim 5,
Wherein the partially opened liposome easily encapsulates the protein drug of the polymer without any modification. 6. The method of claim 5,
Wherein the partially opened liposome is a substance that enhances membrane permeability upon encapsulation of a polymer drug, and a surfactant of a poloxamer series is enclosed together to adjust a drug release rate. 8. The method of claim 7,
Wherein the surfactant of the poloxamer series is Poloxamer 188.

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