KR20200050141A - Oxidation-sensitive liposomes incorporating sulfide compounds - Google Patents

Abstract

The present invention relates to an oxidation-sensitive liposome containing sulfide. Particularly, the oxidation-sensitive liposome containing sulfide includes: an oxidation-sensitive polymer comprising a hydrophilic monomer and a hydrophobic monomer; and dioleoyl phosphatidylethanolamine (DOPE) stabilized with the polymer. More particularly, the hydrophilic monomer is hydroxyethyl acrylate (HEA), and the hydrophobic monomer is sulfur-containing aryl methyl sulfide (AMS). The liposome shows an increase in average hydraulic diameter and more strongly depends on the concentration of an oxidizing agent, when the weight ratio of dioleoyl phosphatidyl ethanol amine based on the oxidation-sensitive polymer is increased.

Description

Oxidation-sensitive liposomes incorporating sulfide compounds}

The present invention relates to oxidation-sensitive liposomes containing sulfides.

Liposomes that emit substances loaded therein by simple diffusion have a problem in that they do not release substances loaded by reacting in a specific environment. Therefore, it is necessary to modify the stimulation-sensitive polymer to the liposome, release the substance loaded in a specific environment, and exert efficacy.

In order to effectively exert the efficacy of substances loaded on liposomes, liposomes must release a large amount of contents under specific conditions of the site of action. For example, in order to deliver the contents to the inside of cells, liposomes should release the contents in the process until reaching the cells, and release the contents after reaching the cells to exhibit the characteristics of releasing the contents in order to increase the efficacy of the contents. Effectively.

On the other hand, stimulation-sensitive liposomes are designed to release the contents of liposomes in response to pH changes, temperature changes, and light irradiation by combining stimuli-sensitive materials with the liposomes, Korea Patent Registration No. 10-1978934 No. 10-2011-7023505, Republic of Korea Patent No. 10-2011-7023505, the contents of the pH-stimulating sensitive liposomes, Republic of Korea Patent No. 10-1378934, the content of light-responsive and temperature-responsive liposomes, Republic of Korea Patent No. 10- No. 1652126 describes a temperature-responsive liposome.

Republic of Korea Patent Office Publication Patent Publication, 10-2017-0041509, drug delivery system for sustained release of pharmaceutical protein and its manufacturing method, published on April 17, 2017 Republic of Korea Patent Office Publication Patent Publication, 10-2018-0088569, Reducing responsive liposome containing hydrophobic disulfide, published August 8, 2018

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As described above, the liposomes that do not release the loaded contents by simple diffusion have a problem of not releasing the loaded contents in response to a specific environment, and the stimulation-sensitive polymers are modified to these liposomes, so that the specific environment To release the contents loaded on the liposomes to exert its efficacy. In particular, in the present invention, it is possible to release the contents loaded in an oxidation condition.

In the present invention, an oxidation-sensitive liposome was developed by preparing a polymer having an oxidation sensitivity with a hydrophilic monomer and a hydrophobic monomer, and stabilizing dioleoylphosphatidylethanolamine (DOPE) as an oxidation-sensitive polymer.

The polymer was prepared by using an hydroxyethyl acrylate (HEA) hydrophilic monomer and an allyl methyl sulfide (AMS) hydrophobic monomer having sulfur.

It relates to a liposome characterized by stabilizing DOPE phospholipids with the oxidation-sensitive polymer and releasing its contents by reacting sensitively to an oxidizing environment. When the liposome is placed in an oxidizing environment, the polymer that stabilizes the liposome is oxidized and escapes from the liposome, and the DOPE liposome is destabilized and releases its contents.

The present invention relates to an oxidation-sensitive liposome (Oxidation-sensitive liposome) prepared using an oxidation-sensitive polymer, when the liposome is placed in an oxidizing environment, the polymer stabilizing phospholipids is oxidized to release the contents loaded by the destabilized liposome It works.

The oxidation-sensitive liposome of the present invention exhibits an effect of releasing contents in a non-oxidizing environment and relatively releasing contents in an oxidizing environment.

When the oxidation-sensitive liposomes are placed in oxidative conditions, the oxidation-sensitive polymer that stabilizes the liposomes is desorbed and the liposomes are destabilized and the contents contained therein are released.

The disease site has more reactive oxygen species than normal cells. Therefore, the drug can be released from a diseased site with an oxidative environment to be used as a drug delivery agent. This can exert its effect in the anticancer drug delivery system market, thereby broadening the market share.

The oxidative sensitive liposome of the present invention has the advantage of being able to effectively exert the efficacy of the active ingredient by being released by the oxidative environment at the disease site rather than by simple diffusion in an environment where the loaded active ingredient is not specified. Is very likely to transfer technology.

1 is a schematic diagram of an oxidation-sensitive DOPE liposome stabilized with liposomes bound to P (HEA-AMS).
FIG. 2 shows the ¹ H NMR spectrum of P (HEA-BMA) (Panel A) and P (HEA-AMS) (Panel B).
Figure 3 shows the FT-IR spectrum of P (HEA-BMA) (panel A) and P (HEA-AMS) (panel B).
Figure 4 shows the air / water interface tension of P (HEA-BMA) (●) and P (HEA-AMS) (○).
5 is liposome / P (HEA-BMA) (200/1) (A), liposome / P (HEA-BMA) (100/1) (B), liposome / P (HEA-BMA) (50/1) (C), liposome / P (HEA-AMS) (200/1) (D), liposome / P (HEA-AMS) (100/1) (E) and liposome / P (HEA-AMS) (50/1 ) (F) is a transmission electron micrograph.
Figure 6 shows the release profile of calcein dependent on the concentration of hydrogen peroxide, which is mounted on the following liposomes: liposome / P (HEA-BMA) (200/1) (A), liposome / P (HEA) -BMA) (100/1) (B) and liposome / P (HEA-BMA) (50/1) (C) were 0% (●), 1.5% (○), 3.0% (▼) and 6.0%, respectively. . (△).
Figure 7 shows the release profile of calcein dependent on the concentration of hydrogen peroxide, which is mounted on the following liposomes: liposome / P (HEA-AMS) (200/1) (A), liposome / P (HEA) -AMS) (100/1) (B) and liposome / P (HEA-AMS) (50/1) (C) 0% (●), 1.5% (○), 3.0% (▼), and 6.0, respectively % (△).

Hereinafter, an oxidation-sensitive liposome containing sulfide according to the present invention will be described in detail with reference to the accompanying drawings. The drawings introduced below are provided as examples in order to sufficiently transfer the spirit of the present invention to those skilled in the art. Therefore, the present invention is not limited to the drawings presented below and may be embodied in other forms. In addition, the same reference numbers throughout the specification indicate the same components.

At this time, unless there are other definitions in the technical terms and scientific terms used, it has the meanings commonly understood by those of ordinary skill in the art to which this invention belongs, and the subject matter of the present invention in the following description and the accompanying drawings Descriptions of well-known functions and configurations that may unnecessarily obscure are omitted.

In the case where a long English expression and its Korean name must be written, for more convenience of description and convenience of reading, when the English expression and its Korean name are first written, it is written together, and thereafter, it is expressed as an abbreviation of the English expression. For example, dioleoyl phosphatidyl ethanolamine (DOPE) is abbreviated as DOPE below.

Hereinafter, a detailed description of the present invention.

One. Introduction

Lipid (Phospholipid) is an amphipathic molecule, which binds itself in the aqueous phase by the entropy-driven process.

Phosphatidylcholine (PC, phosphatidylcholine) has a rectangular shape, and their packing parameters are about 1, so that they can be combined together as a bilayer vesicle such as a liposome.

Phosphatidylethanolamine (PE) is a cone-shaped compound with a packing parameter greater than 1, which binds to a non-bilayer like an inverted hexagonal phase.

If amphiphilic complementary molecules are inserted between the heads of these PE molecules, liposomes can form.

Poly (N-isopropyl acrylamide) (Poly (N-isopropyl acrylamide)) treated with hydrophobicity was adopted as a stabilizer for DOPE liposome formation.

If the liposome is placed at a temperature higher than the phase-transition temperature of the polymer, the chains of the polymer are reduced, and the double-layer domains that are in a stable state because the chains are covered are also reduced.

This results in deintergration of liposomes, and is further accelerated thermodynamically to release the payload (content) inside.

Cholesteryl hemisuccinate (CHEMS) is used as a complementary molecule for stabilizing DOPE liposomes.

If the liposomal suspension is acidified, the head of the CHEMS (such as the carboxyl group) is not ionized, its effective size is reduced, and the complementary molecule will lose the ability to stabilize the DOPE bilayer, thereby causing the so-called Acidification will result in accelerated release.

Hydrophobically modified immunoglobulin G (HmIgG) treated with hydrophobicity can stabilize the DOPE bilayer, which is used as a targetable drug carrier.

If the liposome comes into contact with a specific location of the target, the HmIgG molecule is supposed to diffuse toward the liposome surface, causing liposome degradation.

Glucose oxidase is immobilized on the surface of DOPE liposomes stabilized by CHEMS, which causes the DOPE liposomes to react with glucose.

Enzymes turn glucose into gluconic acid, acidifying the medium, triggering the release of internal payloads from pH-sensitive liposomes.

The present invention relates to an oxidative sensitive liposome stabilized by DOPE liposome and poly (Hydroxyethyl acrylate-co-Aryl Methyl Sulfide (P (HEA-AMS))) .

Since the hydroxyethyl acrylate (HEA) is hydrophilic and the allyl methyl sulfide (AMS) is lipophilic, poly (hydroxyethyl acrylate-co-allyl methyl sulfide) (P (HEA-AMS)) is a parent It is a kind of ionic polymer.

The AMS functions as a hydrophobic anchor, and is hydrophobically inserted into the bilayer of the HEA portion facing the aqueous bulk phase.

Thus, the copolymer can stabilize DOPE liposomes (see A in Figure 1).

When the stabilized DOPE liposome is exposed to oxidizing conditions, methyl sulfide of AMS is oxidized to methyl sulfone, and methyl sulfone becomes difficult to function as a hydrophobic anchor.

As a result, the stabilizing agent by polymerization is removed from the membrane of the liposome, causing the decomposition of the liposome, and the release of a substance loaded therein from the liposome (see FIG. 1B).

2. Materials and methods

2.1. material

1,2-dioleoyl-tin-glycero-3-phosphoethanolamine (DOPE), 2-hydroxyethyl acrylate (HEA), butyl methylacrylate (BMA), N, N-dimethylformamide ( DMF), deoxycholic acid (DOC, sodium salt), calcein, Triton X-100, phosphate buffer solution (PBS), phosphotungstic acid (PTA), Deuterium oxide (D ₂ O), allyl methyl sulfide (AMS), azobisisobutyronitrile (AIBN), phosphorus pentoxide (P ₂ O ₅ ), diethyl ether, hydrogen peroxide were prepared, and Sephadex G- Prepare 100 (SephadexG-100). The remaining reagents are analytical grade.

2.2. Poly (Hydroxyethyl Acrylate -Nose-Allyl methyl Sulfide ) (P ( HEA -co-AMS) and Poly (Hydroxyethyl Acrylate -nose- Butyl methyl Acrylate ) ( Preparation for P (HEA-co-BMA)

Poly (hydroxyethyl acrylate-co-allyl methyl sulfide) (P (HEA-AMS)) and poly (HEA-co-BMA), P (HEA-BMA) were prepared using a free radical reaction.

5 g of HEA was dissolved in 50 ml of DMF in a 3-neck round bottom flask (100 ml).

And the hydrophobic comonomer (hydrophobic comonomer) AMS and BMA were dissolved in a HEA solution, and the molar ratio of HEA to the comonomer was 80:20.

After adding 40 mg of azobisisobutyronitrile (AIBN) to the HEA solution mixed with the comonomer, the reactant was removed under nitrogen gas for 30 minutes.

The monomers were allowed to copolymerize at 75 degrees for 12 hours.

And after standing the flask containing the mixture until it reached room temperature, the copolymer was precipitated using non-solvent (non-solvent) diethyl ether, and the precipitate was reprecipitated (re-precipitation) to purify it. Increased. Subsequently, the precipitate having increased purity was separated through drying and filtration at room temperature.

^{2.3. One} H NMR spectroscopy

The remaining solution and water were removed by incubating the copolymer with P ₂ O ₅ in a vacuum incubator adjusted to 45 degrees.

Each dried copolymer was dissolved in D ₂ O, and the copolymer solution was placed in a 10 mm NMR tube and sealed with a cap.

¹ H NMR spectrum was obtained by scanning the copolymer solution using an NMR spectrometer (400 MHz).

2.4. FT-IR spectroscopy

Each dried copolymer was pulverized using KBr, mortar and pestle. The mixed powder was compressed into a pellet form using a press, and the compressed pellet was scanned with an FT-IR spectrometer.

2.5. Measurement of interfacial tension

The air / water interfacial tension was measured for each copolymer solution having a concentration of 0.01 mg / ml to 1.0 mg / ml. These solutions were serially diluted twice in aqueous copolymer solution (1.0 mg / ml), and surface tension was determined by a ring method of a tension meter.

2.6. Preparation of copolymer-stabilized DOPE liposomes

DOPE liposomes stabilized with P (HEA-BMA) and P (HEA-AMS) were prepared using a sonication and detergent removal method.

First, a dry thin film of DOPE was prepared by removing the organic solvent under reduced pressure with a 150 rpm rotary evaporator from a DOPE solution contained in 2 ml of a 25 ml 3-neck round bottom flask.

Then, 0.02 ml, 0.04 ml, and 0.08 ml were prepared from the copolymer solution (5 mg / ml, PBS (10 mM, pH 7.4)), respectively, and 0.09 ml of DOC solution (2% (w / v) in the same buffer solution) , 1 ml calcein solution was mixed in a 10 ml vial, and a buffer solution was added to make the volume of the mixed solution 2 ml.

In the mixed solution, the concentration of the copolymer was 0.05 mg / ml, 0.1 mg / ml or 0.2 mg / ml, and the concentrations of DOC and calcein were 0.09% and 50 mM, respectively.

DOPE thin film is attached to the inner wall and 2 ml of the mixed solution is poured into a round bottom flask, and stirred by hand to make the phospholipid in the mixed solution float.

The DOPE suspension was sonicated for 20 minutes at room temperature (pulsed for 10 seconds, and then stopped for 10 seconds).

DOPE liposomes containing calcein and copolymer were isolated from uncemented calcein and DOC using gel permeation chromatography.

The mass ratio of DOPE to copolymer is indicated as x / y, and the mixture prepared thereby is indicated as liposome / copolymer name (x / y).

2.7. Characteristics of DOPE liposomes stabilized by copolymer

The structure of the DOPE liposome stabilized by the copolymer was examined by transmission electron microscopy (TEM).

To visualize the bilayer and lamella structures, liposomes were negatively stained.

100 ul of a suspension of DOPE liposomes stabilized by the copolymer was mixed with an equal amount of a phosphotungstic acid solution (2% (w / v)), and the mixture was allowed to dye for 3 hours at room temperature so that the liposomes were dyed. Left unattended.

Some samples of the stained liposomal suspension were poured into a formvar / copper-coated grid and dried overnight at room temperature.

This was observed using a transmission electron microscope.

The fluorescence quenching of calcein mounted on the liposome was determined by Equation 1 below.

[Equation 1]

? Qing (%) = (Ft-Fi) / Ft

In Equation 1, Ft refers to the fluorescence intensity after the liposome is completely stabilized by Triton X100, and Fi is the fluorescence intensity before dissolution.

Fluorescence intensity was measured at 514 nm while excited at 495 nm using a fluorescence spectrophotometer.

In addition, a dynamic light scattering method was used to measure the mean hydrodynamic diameter of the DOPE liposome stabilized by the copolymer.

PBS (10 mM, pH 7.4) was diluted in the liposome suspension to ensure that the light scattering intensity was 50 to 200 kCps.

2.8. Observation of oxidation-sensitive release

Hydrogen peroxide (30% (v / v)) was mixed with PBS (10 mM, pH 7.4), but the mixed concentrations were 0% (v / v), 1.5% (v / v), and 3.0% (v / v), respectively. And 6.0% (v / v).

0.1 ml of the liposome suspension was injected into a 1.9 ml hydrogen peroxide solution of a glass cuvette in a cuvette holder of a fluorescent photometer.

Thereafter, the fluorescence intensity was measured for 60 seconds, and the excitation wavelength and emission wavelength were 495 nm and 514 nm, respectively. Accordingly, the emission degree (%) was calculated through Equation 2 below.

[Equation 2]

Emission% = (Ft-Fi) / (Ff-Fi) x 100

Where Fi is the initial fluorescence intensity of the liposome suspension, Ff is the fluorescence intensity after the liposome is completely dissolved by Triton X100, and Ft is the fluorescence intensity of the liposome suspension at a given time.

3. Results and discussion

^{3.1. One} H NMR spectroscopy

2 shows the 1H NMR spectrum of P (HEA-BMA) and P (HEA-AMS).

In the ¹ H NMR spectrum of P (HEA-BMA), the methyl group of BMA was found at 0.92 to 1.06 ppm, the two methylene groups next to the methyl group were found at 1.35 to 2.14 ppm, and the methyl next to the tertiary carbon. The group showed 1.35 to 2.14 ppm, the vinyl methylene group showed 1.35 to 2.14 ppm, the hydroxy group of HEA showed 4.80 ppm, the methylene group next to the hydroxy group was 3.76 ppm, and the methylene group next to the ester bond was 4.16 ppm. , Messine group was about 2.14ppm, vinyl methylene group was 1.35 ~ 2.14ppm.

The signal of the methyl group of BMA did not overlap with other signals, and so did the signal of the methylene group located next to the hydroxy group of HEA.

When the molar ratio of HEA to BMA was calculated using the area of this signal, it was 100: 3.5.

In the ¹ H NMR spectrum of P (HEA-AMS), the methyl group of AMS was found to be 1.63 to 1.99 ppm, the methylene group next to sulfur was 2.69 ppm, and the mesane group was found to be 2.42 ppm.

The HEA signal of P (HEA-AMS) looked almost the same as the signal of HEA of P (HEA-BMA).

The methylene group of AMS did not overlap with any other signal, as did the methylene group next to the hydroxy group of HEA.

When this signal area was used, the molar ratio of HEA to AMS was calculated as 100: 5.2.

3.2. FT-IR spectroscopy

Figure 3 shows the FT-IR spectrum of P (HEA-BMA) and P (HEA-AMS).

In the P (HEA-BMA) spectrum, hydroxy groups of HEA were found at 3200-3400 cm-1, methyl groups of HEA and BMA were found at 2850-3000 cm-1, and carbonyl groups of HEA and BMA Was found between 1655 and 1710 cm-1, and methylene groups of HEA and BMA were found between 1350 and 1480 cm-1.

In the P (HEA-AMS) spectrum, most signals were almost the same as P (HEA-BMA), but the signal strength of the carbonyl group was weak. This is because the carbonyl group of BMA is not present in AMS.

3.3. Interface tension measurement

Figure 4 shows the air / water interfacial tension of the P (HEA-MA) and P (HEA-AMS) solutions.

The interfacial tension of the P (HEA-BMA) solution rapidly decreased from 73 dyne / cm to 56 dyne / cm as the concentration increased in the range of 0 to 0.02 mg / ml, and 57 dyne in the concentration range of 0.02 to 0.5 mg / ml. It gradually decreased from / cm to 56 dyne / cm. And no reduction in surface tension was found in the remaining concentration range.

The L-type surface tension profile is a typical form for surface-active agents, and the concentration value at which two tangent lines cross each other is called a critical micellization concentration (CMC).

The CMC of the P (HEA-BMA) solution was estimated to be 0.0156 mg / ml.

BMA is a hydrophobic monomer, and copolymerization with a hydrophilic monomer (such as HEA) creates an amphiphilic and interfacial-active copolymer.

Due to the amphiphilicity of the copolymers, the copolymer chains are located side by side at the air / water interface (HEA lowers the interfacial tension while facing water, BMA facing the air).

The P (HEA-AMS) solution also showed an L-type interfacial tension profile.

The plateau interfacial value was 61 dyne / cm, which was higher than the value of P (HEA-BMA).

This indicates that P (HEA-AMS) is less interface-active than P (HEA-BMA).

^{Through 1} H NMR spectroscopy, the molar content of the hydrophobic monomer (eg, AMS) of (P-HEA-AMS) was about 4.9%, which is more molar than 2.4% of BMA, the hydrophobic monomer of P (HEA-BMA). Showed content.

Nevertheless, P (HEA-AMS) was less interfacial-active.

AMS has methylene methyl sulfide (CH ₃ -S-CH _2- ) located on the vinyl group side, and BMA has a butoxy carbonyl group (CH _3- (CH ₂ ) ₃ -OC = O-) and a methyl group.

As such, AMS seems less hydrophobic than BMA and seems less effective in making the copolymer amphiphilic.

This may explain why the surface activity of P (HEA-AMS) is lower due to P (HEA-BMA).

The CMC of P (HEA-AMS) estimated by the intersection of the two tangents was about 0.025 mg / ml, which was higher than 0.0156 mg / ml of P (HEA-BMA).

This also shows that P (HEA-AMS) is less surface-active and less amphiphilic.

3.4. Characteristics of DOPE liposomes stabilized by copolymer

5 is liposomes / P (HEA-BMA ((200/1), liposomes / P (HEA-BMA) (100/1), liposomes / P (HEA-BMA) (50/1), liposomes / P (HEA) -AMS) (200/1), liposomes / P (HEA-AMS) (100/1) and liposomes / P (HEA-AMS) (50/1) shows the transmission electron microscope photograph.

These liposomes are multi-lamella vesicles regardless of whether the stabilizer is P (HEA-BMA) or P (HEA-AMS).

DOPE has a cone shape because its head (ethanolamine) is much smaller than the tail (dioleoyl group), with a packing parameter greater than one.

When dispersed in an aqueous solution, DOPE molecules are bound in an inverted hexagonal phase.

On the other hand, the amphipathic molecule is a complementary molecule with a very large hydrophilic head and a small hydrophobic tail, allowing it to be bound by a liposomal bilayer, and the hydrophobic tail of these molecules is easily inserted between the tails of DOPE molecules through hydrophobic interaction. The hydrophilic head fills the space between the heads of the DOPE molecule.

P (HEA-BMA) and P (HEA-AMS) function as complementary molecules that stabilize the DOPE liposome bilayer. Specifically, in the copolymer, the HEA portion fills the space of the head portion of the DOPE molecule, whereas it is hydrophobic. The anchor (BMA or AMS) is anchored between the tails. This will be the mechanism by which the copolymer will have DOPE molecules bound to the liposome bilayer.

The quenching degree (%) of calcein loaded in liposomes decreases as the amount of the copolymer increases. For example, calcein loaded with liposome / P (HEA-BMA) (200/1), liposome / P (HEA-BMA (100/1) and liposome / P (HEA-BMA) (50/1)) % Were 55.6%, 51.0% and 46.2%, respectively, and liposome / P (HEA-AMS) (200/1), liposome / P (HEA-AMS) (100/1) and liposome / P (HEA-AMS) In (50/1), the nominal% was 57.7%, 55.6%, and 53.9%, respectively.

Nevertheless, the average diameter of liposomes increased as the amount of copolymer increased. For example, the hydrodynamic mean diameter of liposome / P (HEA-BMA) (200/1), liposome / P (HEA-BMA) (100/1) and liposome / P (HEA-BMA) (50/1) is 163nm, 273nm and 283nm, respectively. In addition, liposomes / P (HEA-AMS) (200/1), liposomes / P (HEA-BMA) (100/1) and liposomes / P (HEA-BMA) (50/1) are about 169nm, 176nm and 262nm It was.

As the amount of the copolymer increases, the amount of the formed DOPE bilayer increases, because the copolymer stabilizes the DOPE bilayer.

When the amount of the bilayer increases, the number of liposome particles increases or the number of bilayers per one liposome particle increases.

Since the average diameter is proportional to the amount of the copolymer, the increase in the number of bilayers per particle can occur more easily than the increase in the number of liposome particles.

In fact, it can be seen that the higher the amount of the copolymer, the larger the diameter, and the larger the number of double layers per liposome particle (see FIG. 5).

3.5. Observation of oxidation-sensitive release

6 shows the release profile of the hydrogen peroxide concentration dependent calcein.

The calcein is mounted on liposomes / P (HEA-BMA) (200/1), liposomes / P (HEA-BMA) (100/1) and liposomes / P (HEA-BMA) (50/1).

The release rate (%) of the fluorescent dye mounted on the liposome / P (HEA-BMA) (200/1) was not significantly increased with time lapse regardless of the concentration of hydrogen peroxide. , It was not very strongly dependent on the concentration of hydrogen peroxide.

For example, when the concentrations of hydrogen peroxide were 0%, 1.5%, 3.0%, and 6.0%, the maximum emission (%) was about 0.2%, 3.4%, 5.8%, and 7.8%, respectively.

As the hydrogen peroxide concentration increased, the release rate (%) showed a very small increase, probably because hydrogen peroxide induced the oxidation of DOPE molecules.

On the other hand, there are two double bonds on the phospholipid tail, which are easily affected by oxidation, for example, immediately after oxidation triggered by hydrogen peroxide, the double bond of DOPE is broken, and the hydrophobic chain (oleoyl group) is shortened, DOPE This results in destabilization of the bilayer and dye release through it.

P (HEA-BMA) is not affected by the concentration-dependent release of hydrogen peroxide because there are no oxidation-sensitive groups.

Hydrogen peroxide concentration dependent release profile and release rate (%) of calcein loaded with liposomes / P (HEA-BMA) (100/1) and liposomes / P (HEA-BMA) (50/1) are liposomes (200: It is almost the same as the release profile and% release for the dye mounted on 1).

This soon suggests that the copolymer cannot have a significant effect on the hydrogen peroxide concentration dependent release, in fact there are no oxidation-sensitive groups in the copolymer.

Figure 7 is a liposome / P (HEA-AMS (200/1), liposome / P (HEA-AMS) (100/1) and liposome / P (HEA-AMS) (50/1) of the hydrogen peroxide of calcein Concentration-dependent release profiles are shown.

Liposomes / P (HEA-AMS (200/1) did not show remarkable release results even when hydrogen peroxide was not included in the release medium during 60 seconds of the release experiment.

This indicates that the liposome is stable in a buffer solution without oxidizing agent.

When the oxidizing agent was set to 1.5%, the liposome released the contents quickly loaded for the first few seconds, and then the contents were released very slowly. In particular, the emission rate was 43% at 3 seconds, and gradually increased to 50.8% for the remaining 57 seconds.

The hydrophobic chain of the copolymer (methylene methyl sulfide) is able to anchor between DOPE molecules through hydrophobic interactions, while the hydrophilic HEA portion is directed towards the aqueous bulk phase.

This is thought to be the mechanism by which DOPE liposomes are stabilized by the copolymer.

When the copolymer is exposed to oxidizing conditions, sulfide is oxidized to sulfone. And as soon as it is oxidized, the hydrophobic anchor becomes hydrophilic, which makes it difficult to maintain anchoring in the liposome bilayer, and the copolymer is removed from the liposome bilayer. This causes the degradation of liposomes, which is a trigger for the release of the contents mounted on the liposomes.

When the concentration of hydrogen peroxide was 3.0%, the release pattern was similar to that when the concentration of hydrogen peroxide was 1.5%. However, it showed higher release at higher concentrations during the release time in the release experiment. For example, the maximum release rate at 3.0% hydrogen peroxide was about 78.0%, which was much higher than the maximum release rate obtained at 1.5% hydrogen peroxide, about 50.8%.

The higher the concentration of hydrogen peroxide, the more easily the sulfide copolymer oxidizes, and the DOPE liposomes become more widely destabilized, showing a higher release (%).

On the other hand, when the concentration of hydrogen peroxide was 6.0%, the release percentage was about 78.4%, which was almost the same as that observed when the concentration of hydrogen peroxide was 3.0%.

This is because the degree of oxidation of the sulfide copolymer was already high enough to decompose liposomes when the concentration of hydrogen peroxide was 3.0%.

The concentration of the hydrogen peroxide concentration-dependent release profile of the dye mounted on liposome / P (HEA-AMS) (100/1) was similar to that of liposome / P (HEA-AMS) (200/1).

Liposomes / P (HEA-AMS) (100/1) showed no specific release phenomenon when the release medium had no hydrogen peroxide. However, when the release medium contained an oxidizing agent, it showed the trigger of the release phenomenon (trigger trigger). For example, when the concentrations of the oxidizing agents were 0%, 1.5%, 3.0% and 6.0%, respectively, the maximum emissivity was about 0.5%, 60.6%, 79.3% and 80.2%, respectively.

In the case of liposome / P (HEA-AMS) (200/1), the release rate at the concentration of the oxidizing agent at 3.0% was higher than that observed at the concentration of the oxidizing agent at 1.5%, but at 3.0% and 6.0%. There was no significant difference in the degree of release observed.

This suggests that oxidation at 3.0% is already sufficient to cause liposome degradation.

At 1.5%, the liposomes / P (HEA-AMS) (100/1) showed higher release than the liposomes / P (HEA-AMS) (200/1).

Liposome / P (HEA-AMS) (100/1) is more easily combined with more sulfide copolymers than liposome / P (HEA-AMS) (200/1), making it easier to influence the instability caused by oxidation. Would say

The concentration of the hydrogen peroxide concentration-dependent release profile of the dye mounted on liposome / P (HEA-AMS) (50/1) was similar to that of the dye loaded on other liposomes.

When the concentrations of hydrogen peroxide were 0%, 1.5%, 3.0% and 6.0%, the maximum release rates were 0.6%, 77.2%, 83.2% and 83.7%, respectively.

As with the other liposomes, there was no significant difference in concentrations between 3.0% and 6.0%.

Even when the concentration was 1.5%, no significant difference was found compared to when the release degree was 3.0% and 6.0%. Moreover, liposomes / P (HEA-AMS) (50/1) showed a higher release degree compared to other liposomes, especially at 1.5%.

This showed that liposome / P (HEA-AMS) (50/1) was the most sensitive to hydrogen peroxide among the various liposomes tested.

This is because liposome / P (HEA-AMS) (50/1) binds more sulfide copolymers than the other two types of liposomes, so the amount of copolymers to be oxidized is much higher and it is more easily destabilized under oxidizing conditions. .

This makes liposomes / P (HEA-AMS) (50/1) more sensitive to oxidizing agents than liposomes / P (HEA-AMS) (200/1) and liposomes / P (HEA-AMS) (100/1). Is to give a reason.

4. conclusion

A novel oxidation-sensitive liposome was developed by making P (HEA-AMS) stabilize the DOPE bilayer. Control DOPE liposomes were prepared using P (HEA-AMS). The monomers were copolymerized to obtain a copolymer by free radical polymerization reaction. By 1H NMR spectroscopy, the molar ratio of HEA to AMS in P (HEA-AMS) and the molar ratio of HEA to BMA in P (HEA-BMA) were 100: 5.2 and 100: 3.5, respectively. This copolymerization was reconfirmed by FT-IR spectroscopy.

As a result of measuring the air / water interfacial tension, it was confirmed that the copolymer was surface-active and amphiphilic. In addition, a sound wave treatment and detergent removal method were used to prepare a stabilized DOPE liposome by the copolymer, and through a transmission electron microscope. It was found to be a multi-lamellar vesicle.

Regardless of the molar ratio of DOPE to copolymer, the 60-second release degree of calcein sealed in DOPE liposomes stabilized by P (HEA-BMA) increased slightly (when hydrogen peroxide increased from 0 to 6%). This is probably because the tail of the DOPE is oxidized by a shortened double bond.

On the other hand, hydrogen peroxide had a great influence on the release degree of the sealed dye in the DOPE liposome stabilized by P (HEA-AMS). For example, when the concentration of the oxidizing agent is 0%, 1.5%, 3.0% and 6.0%, the release degree of the sealed dye in the DOPE liposome stabilized by P (HEA-AMS) (200/1) is 0.5%, 50.8%, respectively. 78.0% and 78.4%. This is because the hydrophobic anchor of the copolymer (such as the methylene methyl sulfide group) was oxidized and was likely to become hydrophilic, since it would have been removed from the DOPE bilayer, resulting in degradation of liposomes.

The higher the molar ratio of DOPE compared to the copolymer, the more sensitive release occurs, and the oxidation-sensitive liposomes researched and developed in the present invention can be used as a drug delivery system capable of sensitively releasing internal substances under oxidizing conditions.

As described above, the contents of the present invention have been looked at, and based on this, solutions for solving the problems according to the present invention are described.

One means according to the invention is an oxidation sensitive liposome containing sulfide.

Preferably, the oxidation-sensitive liposome includes an oxidation-sensitive polymer composed of a hydrophilic monomer and a hydrophobic monomer; And a diolyl phosphatidyl ethanolamine (DOPE) stabilized with the polymer; is an oxidation-sensitive liposome containing sulfide.

More preferably, the hydrophilic monomer is hydroxyethyl acrylate (hydroxyethyl acrylate, HEA), the hydrophobic monomer is sulfur, and allyl methyl sulfide (Aryl methyl sulfide, AMS), characterized in that the oxidation-sensitive liposome containing sulfide to be.

These liposomes are characterized by oxidizing the oxidative sensitive polymer when released under oxidizing conditions, escaping from the liposomes, and causing the liposomes to become unstable and to release the loaded contents.

The oxidation-sensitive polymer is produced by a free radical reaction.

The oxidation-sensitive polymer is characterized by being surface-active and amphipathic. In addition, the oxidation-sensitive liposomes are prepared by sonication and detergent removal methods.

On the other hand, when a fluorescent dye (dye) is mounted in the oxidation-sensitive liposome, the quenching degree is 46.2% to 57.7%.

And when the mass ratio of the dioleyl phosphatidyl ethanolamine compared to the oxidation-sensitive polymer increases from 200: 1 to 50: 1, the mean hydrodynamic diameter increases.

On the other hand, when a dye is mounted in the oxidation-sensitive liposome, the release degree of the dye depends on the concentration of the oxidizing agent (for example, a liposome prepared with a mass ratio of DOPE to 200: 1 compared to P (HEA-AMS)). In, the 60-second release rate (%) of the dye was 0.5%, 50.8%, 78.0% and 78.4%, respectively, when the concentration of the oxidizing agent was 0%, 1.5%, 3.0% and 6.0%. The larger the mass ratio of dioleyl phosphatidyl ethanolamine compared to sensitive polymers, the more sensitive it is to oxidizing agents.

Claims (11)

Oxidative sensitive liposomes containing sulfides. The method of claim 1, wherein the oxidation-sensitive liposome,
(a) an oxidation-sensitive polymer composed of a hydrophilic monomer and a hydrophobic monomer; And
(b) Dioleyl phosphatidyl ethanolamine stabilized with the polymer (dioleoyl phosphatidyl ethanolamine, DOPE); oxidation-sensitive liposomes containing sulfide, characterized in that it comprises a. According to claim 2, wherein the hydrophilic monomer is hydroxyethyl acrylate (hydroxyethyl Acrylate, HEA), the hydrophobic monomer is sulfur and sulfuric acid containing sulfide, characterized in that allyl methyl sulfide (Aryl methyl sulfide, AMS) Sensitive liposomes. The oxidation-sensitive liposome containing sulfide according to claim 2, wherein the oxidation-sensitive polymer is oxidized and released from the liposome when subjected to oxidizing conditions, and the liposome becomes unstable and releases the loaded contents. 3. The oxidation-sensitive liposome containing sulfide according to claim 2, wherein the oxidation-sensitive polymer is produced by a free radical reaction. The oxidation-sensitive liposome containing sulfide according to claim 2, wherein the oxidation-sensitive polymer is surface-active and amphipathic. [3] The oxidation-sensitive liposome of claim 2, wherein the oxidation-sensitive liposome is produced by sonication and detergent removal method. The oxidation-sensitive liposome containing sulfide according to claim 2, wherein when the fluorescent dye is mounted in the oxidation-sensitive liposome, the degree of quenching is 46.2% to 57.7%. The method according to claim 2, wherein the average hydrodynamic diameter increases when the mass ratio of the dioleyl phosphatidyl ethanolamine compared to the oxidation-sensitive polymer increases from 200: 1 to 50: 1. An oxidation-sensitive liposome containing sulfide. 3. The oxidation-sensitive liposome of claim 2, wherein when the dye is mounted in the oxidation-sensitive liposome, the degree of release of the dye depends on the concentration of the oxidizing agent. The oxidation-sensitive liposome containing sulfide according to claim 10, wherein the liposome reacts more sensitively to an oxidizing agent as the mass ratio of dioleyl phosphatidyl ethanolamine to the oxidation-sensitive polymer is larger.

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