KR101300744B1 - Photo-responsive vesicles and preparation method thereof - Google Patents

Abstract

The present invention discloses a photoresponsive vesicle in which a coumarin is mounted on a membrane of the vesicle that accommodates a water-soluble target material therein and which can control the emission of the water-soluble target substance by light irradiation.

Description

Photoresponsive vesicles and preparation method thereof

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a photoresponsive vesicle and a method of manufacturing the same, and more particularly, to a photoresponsive vesicle loaded with coumarin on a vesicle and a method of manufacturing the same.

Decades of stimuli-sensitive liposomes have been actively studied. A stimulus-sensitive liposome is a liposome that releases a drug at a target site or releases contents entrapped in response to changes in pH, temperature, light, or magnetic field.

Target-sensitive immuno liposomes were prepared by modifying the surface of a dioleoylphosphatidylcholine (DOPE) liposome with a hydrophobically modified immunoglobin G fragment (Fab portion) [Advanced Drug]. Delivery Reviews, Volume 3, Issue 3, May-June 1989, Pages 343-389, S. Wright, L. Huang. When an antibody of an immune liposome binds to an antigen of a target cell, the phospholipid bilayer membrane of the liposome becomes disturbed and becomes a hexagonal phase because the antibody diffuses laterally on the surface of the liposome. Therefore, the target material is released from the target site.

pH-sensitive liposomes were prepared by incorporating hemisuccinate (CHEMS), dioleoylphosphatidylethanolamine (DOPE), and the like into the liposome membrane [The Journal of Biological Chemistry, Volulme 272, NO. 4, January 1997, V. A. Slepushkin, S. Simoes, P. Dazin, M. S. Newman, L. S. Guo, M. C. Pedroso de Lima, N. Duzgunes]. The liposome surface was modified with a water-soluble polymer containing methylmethacylic acid to prepare a pH sensitive liposome. [Current Opinion in Colloid & Interface Science, doi: 10.1016 / j.cocis.2010.12.004, JA Zasadzinski, B. Wong, N. Forbes, G. Braun, G. Wu]. Changes in the shape of the polymer chain in a specific pH range add mechanical energy to the liposome membrane to release the entrapped target material.

Meanwhile, temperature-sensitive liposomes were prepared with saturated phosphatidylcholines (PC) [Journal of Controlled Release, Volume 76, Issue 1-2, September 2001, Pages 27-37, P. Chandaroy, A. Sen, SW Hui]. ]. The target substance trapped near the phase transition temperature of the liposome membrane was rapidly released. Temperature sensitive liposomes were prepared by modifying the liposome surface with a temperature-sensitive polymer [Colloids and Surfaces B: Biointerfaces, Volume 24, Issue 1, March 2002, Pages 45-52, J. C. Kim, J. D. Kim]. Thermal contraction of the polymer near the phase transition temperature has been shown to promote release because mechanical energy is applied to the liposome membrane.

Bis- (4-n-butylphenylazo-4'-phenylbutyroyl) -L-α-phosphatidylcholine liposomes exhibit photoresponsive emission by cis-trans photoisomers of bis-azo PCs upon UV radiation . Isomers will interfere with the packing of the liposome bilayer. Liposomes composed of O-nitrobenzyl bound to lipids release the contents of liposomes by irradiated UV. The hydrophilic head of the lipid splits at the hydrophobic tail, causing the membrane to be disturbed and rearranged to induce release. Lipid liposomes with vinyl ether bonds exhibit light sensitivity. Plasmenylcholine is oxidized as lipids are degraded by light-sensitive agents. The membrane becomes unstable as the packing parameters of the lipids change.

On the other hand, coumarin and its derivatives have been applied to the development of photo-responsive drug carriers due to their photo-reactive properties. When irradiated with light of 310nm or more, coumarin double bonds photoreact to form a cyclobutane (cyclobutane) to form a coumarin dimer (dimer). Irradiation of dimers with light of 254 nm or less results in the destruction of the cyclone butane as a monomer [Journal of Polymer Science Part A: Polymer Chemistry, Volume 35, Issue 4, March 1997, Pages 613-623, Y. Chen] , KH Chen].

Photo-responsive polymeric micells were prepared using the photochemical properties of coumarins as described above [Macromolecules, Volume 40, Issue 4, January 2007, Pages 790-792, J. Jiang, B. Qi, M. Lepage, Y. Zhao. A polymer micelle was prepared by dispersing an amphiphilic polymer composed of a hydrophilic block and a coumarin-conjugated hydrophobic block in a polar solvent (a mixed solvent of tetrahydrofuran and dichloromethane). Then, the polymer micelles were irradiated with light having a wavelength of 365 nm to dimerize coumarin in micelle cores to crosslink micelle cores. As a result of irradiating light of 254 nm wavelength to the polymer micelles prepared as described above, the release of the hydrophobic target substance entrapped in the core was promoted. This phenomenon has been explained because coumarin dimers are photo-cleavage with monomers, resulting in a low crosslinking density of cores, resulting in loosening of cores.

Since the crosslinking density of the hydrophobic core is controlled by photo-irradiaition, the polymer micelles can control only the fat-soluble target substance contained in the hydrophobic core in emission. Moreover, photo-controlled release of fat-soluble targets was demonstrated in the oil phase (mixture of tetrahydrofuran and dichloromethane) and not in the water phase. The reason why it is demonstrated in the oil phase rather than the aqueous phase is that even if the crosslinking density of micelle cores can be photo-controlled, the fat-soluble target substance entrapped in the hydrophobic core cannot be released into the aqueous phase. In addition, the polymer micelle may contain only a limited amount of the hydrophilic target material in the hydrophilic corona shell due to the structure of the hydrophobic core / hydrophilic corona shell. Due to its loose structure, it is quickly released and exhausted. In addition, since the hydrophilic corona shell portion is not bonded to the photo-reactive material, light-controlled emission is impossible even if the hydrophilic target material is included in the corona.

Accordingly, an object of the present invention is to provide a photoresponsive vesicle which can control the emission of a water-soluble target material by photo-irradiation using coumarin in order to solve the problems of the prior art.

In order to achieve the above object, the present invention provides a light response characterized in that the coumarin is mounted on the membrane of the vesicle to accommodate the water-soluble target material therein so that the emission of the water-soluble target substance can be controlled by light irradiation. Provide a St. Bicicle.

In addition, the present invention comprises the steps of (A) dissolving coumarin and amphiphilic molecules in an organic solvent, and then evaporating the organic solvent to form a dry mixed film of coumarin / amphiphilic molecules; (B) hydrating by adding an aqueous phase in which a water-soluble target substance is dissolved in the dry mixed membrane, and then homogenizing; And (C) removing the unloaded coumarin and the water-soluble target substance. It provides a method for producing a photoresponsive vesicle comprising a. For example, first, coumarin and amphiphilic components are dissolved together in an organic solvent, and the solution is placed in a large container such as a round flask, and then the organic solvent is evaporated to prepare a dry mixed membrane of coumarin / amphiphilic molecules. The dry mixed membrane is added with an aqueous phase in which the target substance is dissolved to swell (hydrate) / homogenize the dry mixed membrane, and finally, coumarin and water-soluble target substance which are not mounted in the vesicle are subjected to column chromatography, thereby loading the coumarin on the membrane. Photoreactive vesicles can be prepared.

In the photoreactive vesicle of the present invention, when vesicle film is disturbed while coumarin dimerization when light of wavelength of 365 nm or more is irradiated, the coumarin dimer is monomeric when light of wavelength 254 nm or less is irradiated. Since the membrane is disturbed while being separated by), it is possible to control the release of the water-soluble target substance from becyclo by irradiation of light (irradiation).

As coumarin of the present invention, coumarin, 7-hydroxy coumarin, 7-hydroxy coumarin, carboxylic coumarin, carboxylic coumarin, 7-acetoxy coumarin and the like can be used.

In addition, as a material constituting the vesicle of the present invention, amphiphilic molecules having a packing parameter of about 1 may be used. As such amphiphilic molecules, phosphatidylcholine, phosphatidylethanolamine, or the like may be used. Can be.

In addition, when preparing the vesicle loaded with coumarin, the weight ratio of coumarin and amphiphilic molecules is preferably 1/1000 to 1/2, more preferably 1: 500 to 1/3, most preferably 1 : 100 to 1: 5. In the lower ratio range, the amount of coumarin is so small that the photoreactivity of the coumarin does not sufficiently fluidize the liposome membrane and consequently the target substance is not released by light irradiation. In the range of the ratio higher than the above range, the amount of coumarin is so large that precipitation occurs in the process of hydrating / homogenizing the coumarin / amphiphilic dry film.

In addition, the concentration of the target substance in the aqueous phase used to hydrate the coumarin / amphiphilic dry mixed membrane cannot be accurately expressed in numerical values because the concentration of the drug is different depending on the type, but the lowest concentration and the highest concentration at which the drug concentration is shown. Choose a concentration between. As the target component, sodium fusidate, neomycin sulfate, allantoin (alllantoin), heparin, extratum cepae and the like can be used.

According to the present invention, a photo-responsive vesicle can be prepared by loading coumarin in a vesicle. This photoreactive vesicle of the present invention can control the emission of the water-soluble target material from the becyclo when light of a specific wavelength range is irradiated.

1A is the FT-IR spectrum of 7-hydroxy coumarin, and FIG. 1B is the FT-IR spectrum of acetoxy coumarin.
2 is a ¹ H NMR spectrum of acetoxy coumarin.
3 is a transmission electron micrograph of liposomes with ratios of acetoxy coumarin to phospholipids 0: 1 (a) and 1: 8 (b), respectively.
4 is a graph showing the degree of dimerization of acetoxy coumarin according to the cycle light irradiation.
5 is a graph showing the degree of dimerization of acetoxy coumarin mounted on liposomes according to cycle light irradiation.
Figure 6a is a transmission electron micrograph of liposomes containing no acetoxy coumarin taken before irradiation, Figure 6b is a 80 nm light for 365 minutes (400 W) in a suspension of liposomes containing no acetoxy coumarin Transmission electron micrograph of liposome taken after irradiation and subsequent 254 nm (6W) light for 10 minutes.
Figure 7a is a transmission electron micrograph of liposomes containing acetoxy coumarin taken before light irradiation, Figure 7b is irradiated with 365 nm (400 W) light to the suspension of liposomes containing acetoxy coumarin for 80 minutes Transmission electron micrograph of liposomes containing acetoxy coumarin taken after successive irradiation of 254 nm (6W) light for 10 minutes.
FIG. 8 shows the results of observing the emission (%) of the fluorescent substance (carboxy fluorescein) by irradiating 254 nm light to a suspension of liposomes containing acetoxy coumarin dimer.

Hereinafter, the content of the present invention will be described in more detail in the following examples, but the scope of the present invention is not limited only to the following examples, and includes modifications of equivalent technical ideas.

Example  One : Acetoxy  Coumarin (7- acetoxy coumarin   synthesis

50 g of 7-hydroxy coumarin, 38 g of sodium acetate, and 180 ml of acetic anhydride were put together in a 250 ml three-neck flask. After adding 2-3 drops of pyridine to the mixture, the reaction proceeded under reflux at 144 ° C. for 7 hours. The reaction mixture was cooled to room temperature and washed with ethanol and cooling water. After filtration using a filtration membrane, the cake was then recrystallized in ethanol.

1A is the FT-IR spectrum of 7-hydroxy coumarin, and FIG. 1B is the FT-IR spectrum of acetoxy coumarin.

In Figure 7 a spectrum of 1a-hydroxy-coumarin -OH stretching signal was observed at 3116.1 cm ^-1, C = O signal 1703.5 cm ^-1 and 1677.1 cm ^- was observed at ^1, C = C signals 1407.8 cm ^- Observed in the range of ¹ to 1564.7 cm ⁻¹ , and CO signal was observed at 832.7 cm ⁻¹ .

In the spectrum of acetoxy coumarin of FIG. 1b, the -OH signal was almost disappeared, and the C = O signal, C = C signal, and CO signal were observed at the same position as in the 7-hydroxy coumarin spectrum. Acetoxy C = O signals of the ethoxy group is 1735.9 cm ^- was observed in ^one -COC- signal of the acetoxy group is 1184.2 cm ^- was observed in the ^first. From this, it was confirmed that acetoxy coumarin was synthesized.

2 is a ¹ H NMR spectrum of acetoxy coumarin. Signals of aromatic protons were observed in the range of 6.4 ppm-7.7 ppm and methyl group of the acetoxy group was observed at 2.3 ppm. The ratio of methyl proton signal area to aromatic proton signal area was 4.9 / 3. This ratio approximates 5/3 of the theoretical ratio, indicating that acetoxy coumarin was successfully synthesized.

Example  2 : Acetoxy  Preparation of Liposomes Containing Coumarins

In this example, liposomes loaded with acetoxy coumarin were prepared by using film hydration [International Journal of Biological Macromolecules, Volume 47, Issue 5, December 2010, Pages 635-639, YJ Hong, JC Kim]. .

0.14 ml of egg phosphatidylcholine (EPC) solution (100 mg / ml) dissolved in chloroform and acetoxy coumarin monomer solution (6 mg / ml) dissolved in chloroform are mixed in a 25 ml round bottom flask. It was. At this time, the ratio of acetoxy coumarin to EPC was 0: 1, 1:20, 1:10, 1: 8, 1: 4, 1: 1. The solvent was evaporated in a rotary evaporator under reduced pressure to obtain a dry mixed film consisting of EPC and acetoxy coumarin. The dry film was then hydrated with 2 ml of carboxy fluorescein (CF) solution dissolved in HEPES buffer (pH 8.0) to disperse the film from the flask wall into the solution. Then, the dispersion was ultrasonically ground at room temperature for 20 minutes in a bath sonicator (Sonics & Materials, USA) to homogenize the dispersion. The liposome membrane was then left to stand at 5 ° C. for about 12 hours to anneal. In order to remove the fluorescent material not encapsulated in liposomes, the liposome suspension was passed through a Sephadex G-100 column (1.8 cm> 40 cm) to finally prepare a liposome loaded with acetoxy coumarin and fluorescent dye.

The degree of fluorescence quenching of the fluorescent dye encapsulated in liposomes was determined using the following equation after adjusting the phospholipid (EPC) concentration to 0.07%.

Where F _i is the fluorescence of the liposome suspension measured after removal of the fluorescent dye not included in the liposome, and F _f is the fluorescence of the solution measured after dissolving the liposome containing the fluorescent dye with triton X-100. Fluorescence intensity was measured at room temperature, excitation at 492 nm and fluorescence at 517 nm.

In addition, the average diameter of liposomes was measured by a particle size analyzer (ZetaPlus 90, Brookhaven Instrument Co., USA). The liposome suspension was diluted with buffer so that the light scattering intensity of the liposome suspension was 40 Kcps -100 Kcps.

Table below Figure 1 shows the quenching of the fluorescent dye encapsulated in liposomes and the average size of liposomes.

EPC For ATC Rain of 0: 1 1:20 1:10 1: 8 1: 4 1: 2 1: 1 Quench (%) 71 69 83 55 59 56 72 Average diameter  ( nm ) 181 192 116 137 214 155 159

According to Table 1, when the ratio of acetoxy coumarin to phospholipid (EPC) is 0: 1, 1:20, 1: 8, 1: 4, the quenching (%) is about 71%, 69%, 55% , 59%. Quenching (%) is a measure of the degree to which liposomes are formed, which means that the higher the quenching (%), the higher the yield of liposomes. Therefore, as the ratio of acetoxy coumarin to phospholipid (EPC) increases, it was found that acetoxy coumarin reduces the degree of formation of liposomes. Acetoxy coumarin is insoluble in water and thus can hydrophobicly interact with the hydrophobic tail of phospholipids. In addition to loading on liposome membranes, acetoxy coumarins can form mixed micelles with phospholipids (EPC). It may be. As the ratio of acetoxy coumarin increased, it was determined that the fluorescent quenching of the fluorescent dye was lowered. In addition, when the ratio of acetoxy coumarin to phospholipid (EPC) is 1: 2 and 1: 1, a homogeneous suspension was rarely obtained, and instead a heterogeneous suspension containing clusters was obtained. Therefore, in order to prepare liposomes containing acetoxy coumarin in high yield, it was found that the ratio of phospholipid (EPC) to acetoxy coumarin does not exceed 1: 4.

On the other hand, when the ratio of acetoxy coumarin to phospholipid was 0: 1, 1:20, 1: 8, 1: 4, the average size of liposomes was 181 nm, 192 nm, 137 nm, and 214 nm. Since the size of liposomes is sensitive to hydration temperature, ultrasonic pulverization strength, and the location of the flask in the bath of the ultrasonic pulverizer, it is difficult to produce them reproducibly [Phrmarceutical Manufcaturing Handbook: Production and Processes editied S.C. Gad, John Wiley & Sons, Inc., New Jersey, 2008, Page 456, S. G. Antimisiaris, P. Kallinteri, D. G. Fatouros. Therefore, it was judged that the change in size was due to the low reproducibility and was not closely related to the change in the ratio of acetoxy coumarin. In addition, when classified in terms of average size, it can be seen that liposomes can be classified into multi-lamellar vesicles.

3 is a transmission electron micrograph (TEM) of liposomes with ratios of acetoxy coumarin to phospholipid (EPC) 0: 1 and 1: 8. On the photo, the size of liposome was about 100 nm to 200 nm and multi-lamellar structures were observed.

Example  3: Acetoxy  On coumarin and liposomes Included Acetoxy  Coumarin Dimerization tube The

In this example, acetoxy coumarin was dissolved in ethanol and the solution was diluted with distilled water to a concentration of 0.02 mg / ml. Cyclic dimerization of acetoxy coumarin by irradiating the solution with light at 365 nm (400W) for 80 minutes and periodically at 254 nm (6W) for 10 minutes. and de-dimerization).

On the other hand, the periodic dimerization / dedimerization of acetoxy coumarin mounted on the liposome was observed while irradiating in the same manner as the above light irradiation conditions. At this time, the concentration of acetoxy coumarin mounted on the liposome was the same as the concentration of acetoxy coumarin not encapsulated in the liposome.

The degree of dimerization was determined by the following formula.

The absorbance at 310 nm is the absorbance of the acetoxy coumarin monomer. Irradiation with 365 nm of light reduces the absorbance of the solution at 310 nm because the acetoxy coumarin monomer becomes a dimer and the amount of monomer decreases.

Figure 4 shows the degree of dimerization of acetoxy coumarin according to the cycle light irradiation, the degree of dimerization increased to 66% when irradiated with light (400W) of 365nm wavelength for 80 minutes, followed by the light (6W) of 254nm wavelength in succession When irradiated for 10 minutes, the degree of dimerization was reduced to 27.5%. When the light of 365 nm wavelength and 254nm wavelength were alternately irradiated, the increase and decrease of the degree of dimerization were repeated.

Figure 5 shows the degree of dimerization of acetoxy coumarin mounted on liposomes according to the cycle light irradiation, the degree of dimerization increased to 57% when irradiated with light (400W) of 365nm wavelength for 80 minutes, followed by 254nm wavelength The amount of dimerization decreased by 38% when irradiated with light (6W) for 10 minutes. When the light of 365 nm wavelength and 254nm wavelength were alternately irradiated, the increase and decrease of the degree of dimerization were repeated.

From this, it was found that not only acetoxy coumarin not loaded on liposomes but also acetoxy coumarin loaded on liposomes repeat dimerization / dedimerization by periodic light irradiation.

Example  4: Observation of Light Stability of Liposomes

In this example, the safety of liposomes against periodic light irradiation was evaluated by observing liposome size and shape changes.

Cyclic irradiation was performed on a 5m liposome suspension contained in a vial. That is, a periodic irradiation was performed in which light of λ = 365 nm (400W) wavelength was irradiated for 80 minutes and light of λ = 254 nm (6W) wavelength for 10 minutes. Transmission electron micrographs of liposomes without acetoxy coumarin and liposomes with acetoxy coumarin (acetoxy coumarin: phospholipid = 1: 8) to observe the effect of periodic irradiation on the changes in the size and shape of liposomes. Was taken before and after light irradiation.

Figure 6a is a transmission electron microscope photograph of liposomes containing no acetoxy coumarin taken before light irradiation, a multi-layered vesicle of 100nm-200nm was observed. 6B is a transmission electron micrograph of a liposome taken after a suspension of liposomes containing no acetoxy coumarin for 80 minutes with 365 nm (400 W) of light followed by 10 minutes of 254 nm (6W) of light. The vesicles of the structure were observed and the size was slightly increased than before the irradiation of light, but there was no significant change.

Figure 7a is a transmission electron microscope photograph of liposomes containing acetoxy coumarin taken before light irradiation, a multi-layered vesicle of 100nm-200nm was observed. FIG. 7B shows the transmission electrons of liposomes containing acetoxy coumarin, photographed after irradiating 365 nm (400 W) light for 80 minutes to a suspension of liposomes containing acetoxy coumarin, followed by 10 minutes of 254 nm (6 W) light. The micrograph shows a vesicle with a multi-layered structure and the size was significantly increased than before irradiation.

According to Example 3, the acetoxy coumarin was dimerized by the light of 365 nm wavelength and dedimerized by the light of 254 nm even when it was mounted on the liposome. This dimerization and dedimerization was thought to significantly increase the size of liposomes because the liposome membrane was fluidized and the liposomes were fused.

Example  5: emission from liposomes upon light irradiation

In this example, the release of the target substance upon light irradiation of liposomes loaded with acetoxy coumarin dimer was observed.

First, liposomes equipped with acetoxy coumarin dimer were prepared using the film hydration method as in Example 2. 25 ml round bottom flask with 0.14 ml of egg phosphatidylcholine (EPC) solution (100 mg / ml) dissolved in chloroform and 0.875 ml of acetoxy coumarin dimer solution (2 mg / ml) dissolved in chloroform And mixed so that the weight ratio of acetoxy coumarin dimer to phospholipid (EPC) was 1: 8. Thereafter, a mixed dry film was prepared in the same manner as in Example 2, the mixed dry film was hydrated, the suspension was homogenized, and the non-inclusion fluorescent dye was separated to prepare a liposome loaded with acetoxy coumarin dimer.

In order to observe the emission of fluorescent dyes from liposomes by light irradiation, liposome suspension containing coumarin dimer was irradiated with 254 nm of light and the emission (%) was determined using the following formula.

Where F _t is the fluorescent of the liposome suspension after t hours of light irradiation, F _t is the fluorescent of the liposome suspension before light irradiation to the liposome suspension, and F _f is the fluorescent of the solution after the liposome is dissolved with triton X-100. Fluorescence intensity was measured at room temperature, excitation at 492 nm and fluorescence at 517 nm.

8 is a result of observing the emission (%) by irradiating 254 nm light to the liposome suspension containing acetoxy coumarin dimer. The fluorescent material increased up to 19% for 100 minutes by light irradiation, which caused the liposomal membrane to undergo fluid stress and fluidized during the process of photolysis of the coumarin dimer on the liposome to form a monomer, resulting in a packing defect in the liposome membrane. ) Was generated to emit fluorescent material. This is a result consistent with the result that the coumarin dimer mounted in the liposome confirmed by FIG. 5 turns into a monomer by irradiation of 254 nm light.

Claims (7)

Becyclosor containing water-soluble target substance inside,
Amphiphilic molecules constituting the vesicle are at least one of phosphatidylcholine or phosphatidylethanolamine,
Coumarin is mounted on the membrane of the vesicle to control the emission of the water-soluble target substance by light irradiation,
Optical response vesicles, characterized in that the weight ratio of the coumarin and the amphipathic molecule is 1/1000 ~ 1/2. The method of claim 1,
The coumarin is a photoresponsive vesicle, characterized in that at least one selected from the group consisting of coumarin, 7-hydroxy coumarin, carboxyl coumarin and acetoxy coumarin. delete (A) dissolving coumarin and amphiphilic molecules in an organic solvent, and then evaporating the organic solvent to form a dry mixed film of coumarin / amphiphilic molecules;
(B) hydrating by adding an aqueous phase in which a water-soluble target substance is dissolved in the dry mixed membrane, and then homogenizing; And
(C) removing unloaded coumarin and the water-soluble target substance;
The amphipathic molecule is at least one of phosphatidylcholine or phosphatidylethanolamine,
The weight ratio of the coumarin and the amphipathic molecules is 1/1000 ~ 1/2 method for producing a photoresponsive vesicle. delete 5. The method of claim 4,
The coumarin is a method of producing a photoresponsive vesicle, characterized in that at least one selected from the group consisting of coumarin, 7-hydroxy coumarin, carboxyl coumarin and acetoxy coumarin. delete

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