KR20100084023A - A lipid nanosphere enclosing a hydrophobic photo-sensitizer, a process for the preparatrion thereof, and an anti-tumor agent comprising the same - Google Patents

Abstract

PURPOSE: A lipid nano microsphere containing hydrophobic photosensitizer is provided to prevent side effect by fluorescence and to enable diagnosis and treatment by photosensitizer. CONSTITUTION: A lipid nano microsphere containing hydrophobic photosensitizer with saturated lipid and unsaturated lipid by biocompatible surfactant. The hydrophobic photosensitizer shows fluorescence in near infrared ray. The biocompatible surfactant has hydrophobic tumor targeting material which is bound to biocompatible aqueous polymer. The hydrophobic tumor targeting material is folic acid, antibody, cobalamin, vitamin A, vitamin C, or vitamin B12.

Description

A lipid nanosphere enclosing a hydrophobic photo-sensitizer, a process for the preparatrion about, and an anti-tumor agent comprising the same}

The present invention relates to a lipid nanoparticles containing hydrophobic photosensitizer, a method for producing the same, and a tumor therapeutic agent including the same. More specifically, the present invention relates to side effects on sites other than the target due to fluorescence interference caused by nanolipid microsphere formation. The present invention relates to a lipid nanoparticle containing a hydrophobic photosensitizer capable of adding a tumor target and targeting to a tumor target, and a method for producing the lipid nanoparticle, and a tumor therapeutic agent and a tumor diagnostic agent comprising the same. will be.

Photodynamic therapy (PDT) is a method of treating a patient with a photosensitizer that is sensitive to abundant oxygen in the body and externally supplied laser light and irradiating a laser of a specific wavelength at which the photosensitizer responds. to be.

Photosensitizers produce photosensitizers that produce cytotoxic substances such as single molecule oxygen, free radicals, or peroxides by light stimulation, and these reactive oxygen species (ROS) destroy surrounding tissues, It can be used for treatment. The photosensitive agent may be selectively administered only to the affected area by topical administration to the site to be treated or by irradiating the laser to the affected area only. Photosensitizers are also used to diagnose tumors by imaging due to the generation of light of a particular wavelength by laser irradiation.

Conventionally, photosensitizers were administered by direct external application or intravenous injection. However, the photosensitizer is a problem because damage may occur to a normal tissue other than the target tissue in response to the near-infrared rays exposed from the outside. In addition, hydrophobic photosensitizers have been found to bind to proteins first in the blood when injected intravenously and travel inside the cell via receptors located on the cell surface. Thus, in this case, the tissue specificity is simply defined by the hydrophobic character, and the residence time in the tissue or cell also shows a large difference in some cases. This has been pointed out as a factor that reduces the effectiveness of photodynamic diagnosis and treatment and increases the probability of recurrence after treatment. In addition, side effects appear due to the action of the photosensitizer on the normal cells is a problem.

To overcome the problem of tissue specificity of such photosensitizers, antibodies to Epithelial Cell Adhesion Molecule (Ep-CAM) proteins, which are overexpressed in most tumor cells, are conjugated with photosensitizers by coupling reagents. Tissue-specific methods of delivery using antigen-antibody responses have been developed (Korean Patent Publication 2005-0022797). However, this method can not use the photosensitizer without a functional group, and the synthetic material has a chemical structural change that can reduce the phototoxic efficiency to the cell and the cost of using the antibody as described above is disadvantageous .

In addition, even if the photosensitiser is delivered by a tissue-specific method, the normal tissue may be damaged by the fluorescence generated during the delivery process, thereby providing a more tissue-specific delivery system, which can significantly reduce side effects. Development of drug carriers is needed.

Bernadette Pegaz et al. Have developed a method of incorporating a photosensitizer into nanoparticles prepared using only lipid-free polymers (Bernadette Pegaz et al. Encapsulation of porphyrins and chlorins in biodegradable nanoparticles: Journal of Photochemistry and Photobiology B: Biology Volume 80, Issue 1, 1 July 2005, Pages 19-27). Such a method has a problem in that the efficiency of incorporation of the photosensitizer is very low and the effect is low, and there is no target-oriented material, so the directivity to the tumor is low.

Accordingly, the present inventors have studied diligently to overcome the above problems, as a result of preparing hydrophobic photosensitizers into lipid nanoparticles, and introducing tumor target materials in preparing lipid nanoparticles, thereby minimizing side effects caused by the photosensitizers. It has been found that the effect of the agent can be enhanced to complete the present invention.

Accordingly, it is an object of the present invention to provide lipid nanoparticles with an improved effect of the photosensitizer while minimizing the side effects caused by the photosensitizer.

Another object of the present invention is to provide a method for preparing the nano lipid microspheres.

Still another object of the present invention is to provide a tumor therapeutic agent by photodynamic therapy containing the lipid nanoparticles.

Still another object of the present invention is to provide a tumor diagnosis agent by photodynamic diagnosis including the lipid nanoparticles.

In order to achieve the above object, the present invention provides a lipid nano-microspheres containing a hydrophobic photosensitizer, formed by the hydrophobic photosensitizer is emulsified with a biocompatible surfactant with a saturated lipid and an unsaturated lipid.

Lipid nanoparticles according to the present invention is

Preparing a biocompatible surfactant by binding the tumor target material to the biocompatible aqueous polymer;

Dissolving the biocompatible surfactant in water to prepare an aqueous phase;

Dissolving saturated and unsaturated lipids in a volatile organic solvent with a photosensitizer to prepare a lipid phase;

Mixing the aqueous phase and the lipid phase to produce an o / w emulsion;

Filtering the prepared emulsion to remove unincorporated photosensitizers or foreign matter; And

Cooling the filtered emulsion to a temperature below the melting point of the solid lipid to solidify the emulsion particles.

The present invention also provides a therapeutic agent for tumors by photodynamic therapy comprising the lipid nanoparticles according to the present invention.

The present invention also provides a tumor diagnostic agent by photodynamic diagnosis containing the lipid nanoparticles.

Hereinafter, the present invention will be described in more detail.

In the present invention, in order to minimize the side effects due to the fluorescence of the photosensitizer, a method of incorporating the photosensitizer into the lipid nanoparticles was introduced. To incorporate the photosensitizer into the lipid microspheres, lipid nanoparticles were prepared by emulsifying the hydrophobic photosensitizer with saturated lipids and unsaturated lipids with a biocompatible surfactant, and the lipid nanoparticles were obtained by separating them.

Accordingly, the present invention in one aspect,

A hydrophobic photosensitizer is emulsified with a biocompatible surfactant together with saturated and unsaturated lipids to provide a lipid nanoparticle containing an hydrophobic photosensitizer.

The lipid nanoparticles according to the invention have a size of 500 nm or less in diameter.

Hydrophobic photosensitisers can damage normal tissues by the generation of fluorescence in the free state, but when present in the lipid nanoparticles, hydrophobic photosensitisers are physically present in the lipid nanoparticles so that each of the photosensitisers is generated. The fluorescence causes interference with each other, and the generation of reactive oxygen species exhibiting cytotoxicity from the photosensitizer is blocked. Therefore, the lipid nanoparticles according to the present invention can suppress the occurrence of side effects that may occur in normal tissues due to fluorescence until the lipid nanoparticles are administered and destroyed in vivo.

In addition, the lipid nanoparticles are characterized in that the inclusion efficiency of the photosensitizer is increased by including both saturated and unsaturated lipids as lipids. Because saturated lipids are solid at room temperature and unsaturated lipids are liquid at room temperature, the lipid nanoparticulates are divided into solid particles and liquid lipids. Can be formed. As a result, the lipid nanoparticles can provide a space for incorporation of the photosensitizer into the incomplete lattice crystals, thereby increasing the incorporation efficiency of the photosensitizer into the nanoparticles.

The hydrophobic photosensitizer is not particularly limited as long as it is a photosensitizer used for photodynamic diagnosis or treatment, but a fluorescence may be used in the near infrared. As the photosensitizer exhibiting fluorescence in the near infrared ray, any hydrophobic photosensitizer such as pheophorbide a, pyropheophorbide a, tetrabenzoformin, mesotetraphenylformine, or Fe (III) mesoporphyrin chloride It is available. Among them, pheophorbide a is extracted from chlorophyll and has higher generation efficiency of reactive oxygen species and absorbs near-infrared wavelength band than conventional photosensitizers. This material reacts well in the visible range, so work must be done with care not to be affected by light during manufacture.

Saturated lipids constituting the lipid nano-microspheres may be used cacao butter, butter, shortening, Laad, palm oil, coconut oil, palm kernel oil, palm oil, or a combination thereof, unsaturated lipids are oleic acid, olive oil, camellia oil, Macadamia nut oil, castor oil, lauric acid, myristic acid, palmitic acid, stearic acid, isostearic acid, almond oil, avocado oil, evening primrose oil, malt oil, jojoba oil, corn oil, rice bran oil, or a combination thereof may be used. It may be, but is not limited thereto. The reason why the saturated lipid and the unsaturated lipid are mixed is used because the unsaturated lipid is added to the crystal structure of the saturated lipid to increase the incorporation efficiency of the photosensitizer. Cacao butter is a saturated fatty acid that is solid at room temperature and has a melting point of 38 ° C. Oleic acid is an unsaturated fatty acid with one double bond. It is liquid at room temperature and has a melting point of 16.2 ℃.

Biocompatible surfactants used for the formation of lipid nanoparticles according to the present invention may be used as a surfactant in which a hydrophobic tumor target material is bound to a biocompatible aqueous polymer. The binding of the biocompatible aqueous polymer and the hydrophobic tumor target material may be achieved by or by applying a chemical binding method well known to those skilled in the art, depending on the structure of the material used, respectively. The binding allows both hydrophilicity by the biocompatible aqueous polymer and hydrophobicity by the hydrophobic tumor target material to appear in one molecule to act as a surfactant.

The biocompatible polymer serves to impart hydrophilicity to the surfactant, and the hydrophobic tumor target material imparts hydrophilicity to the surfactant. The biocompatible polymer is still present on the surface of the lipid nanoparticles even after the preparation of the lipid nanoparticles, so that the hydrophobic tumor target material not only provides hydrophobicity to the surfactant, but also serves to target the lipid nanoparticles to the tumor. To perform.

As the biocompatible polymer, flulan, dextran, chondroitin sulfate, heparin, pluronic, hyaluronic acid, chitosan, or fucoidan may be used, and the hydrophobic tumor target material may be folic acid, antibody, cobalabin, Vitamin A, vitamin C, or vitamin B ₁₂ may be used, but is not limited thereto.

One of the biocompatible polymers, pullulan, is a neutral polysaccharide produced from black yeast (Aureobasidium pullulans (DE BARY) ARN.). Soluble in water, insoluble in alcohols and oils. Compared to other gums, the viscosity is lower but stable to acids, alkalis, heat and the like. In particular, it has a strong adhesive force in addition to the film. There are two types of average molecular weights of 200,000 and 100,000, and the viscosity is 1-2 cps.

Folic acid, one of the tumor target substances, is a chemical formula C ₁₉ H ₁₅ N ₇ O _6, which is pteroyl-L-glutamic acid composed of pterin, p-aminobenzoic acid, and glutamic acid. It is present in nature in the form of a tri or heptaglutamyl peptide.

A reaction scheme of synthesizing a biocompatible surfactant using the pullulan and folic acid is shown in FIG. 1. Such a synthesis method is disclosed in Korean Patent Publication No. 2007-0092438, which is incorporated herein by reference in its entirety.

 In the lipid nanoparticles containing the photosensitizer according to the present invention, the photosensitive agent is surrounded by the lipid nanoparticles so that fluorescence interference occurs, so that the fluorescence is not generated when the lipid nanoparticles are not destroyed. When administered by intravenous injection of lipid nanoparticles, the lipid nanoparticles are not destroyed in the aqueous environment in the blood and are therefore not destroyed and stable until they reach a target site, for example, a tumor site, and are incorporated into the cell. Therefore, the lipid nanoparticles do not generate fluorescence until they reach the target site, thereby preventing side effects caused by fluorescence. When the lipid nanoparticles reach the target site, the lipid nanoparticles incorporated into the cell by pagocytosis and incorporated into the cell are decomposed in the surrounding lipid state, so that the photosensitizer is exposed inside the target cell to release the fluorescence interference. At this time, when the laser is irradiated specific to the photosensitizer, the photosensitizer reacts to generate fluorescence to enable diagnosis or to generate reactive oxygen species to damage target tissues. Therefore, the lipid nanoparticles according to the present invention can make it possible to efficiently diagnose or treat a photosensitizer at a target site without giving side effects due to fluorescence in normal cells.

In another aspect of the present invention,

Preparing a biocompatible surfactant by binding the tumor target material to the biocompatible aqueous polymer;

Dissolving the biocompatible surfactant in water to prepare an aqueous phase;

Dissolving saturated and unsaturated lipids in a volatile organic solvent with a photosensitizer to prepare a lipid phase;

Mixing the aqueous phase and the lipid phase to produce an o / w emulsion;

Filtering the prepared emulsion to remove unincorporated photosensitizers or foreign matter; And

Cooling the filtered emulsion to a temperature below the melting point of the solid lipid to solidify the emulsion particles,

It provides a method for producing lipid nanoparticles according to the present invention.

In order to prepare the biocompatible surfactant, a method of binding a tumor target material to the biocompatible aqueous polymer is well known in the art of organic synthesis, and according to the structures of the biocompatible aqueous polymer and the tumor target material using synthetic methods, It can be performed as appropriate. The ratio of combining the biocompatible aqueous polymer and the tumor target material should be such that the prepared biocompatible surfactant is dissolved in water.

For example, in order to prepare a biocompatible surfactant by combining pullulan and folic acid, it may be bound to -OH of pullulan using a catalyst that activates the carboxyl group (-COOH) of folic acid. At this time, it is preferable to dissolve folic acid having a small molecular weight in pullulan and drop it drop by drop. The pullulan / folate combination thus made can serve to enhance the hydrophobicity of folic acid and increase the target orientation to cancer cells.

After the biocompatible surfactant is prepared, the biocompatible surfactant is dissolved in water to prepare an aqueous phase. Biocompatible surfactants that are soluble in an appropriate amount of water can be prepared by placing it in water, preferably distilled water, and stirring to make a clear solution. If the concentration of biocompatible surfactant is high, the size of the nanoparticles formed afterwards tends to be small, and if too thick, the surfactant itself aggregates to form micelles, so that the size of the lipid nanoparticles is rather large. It is preferable to use. The concentration of the biocompatible surfactant can be appropriately determined according to the type of the surfactant, the type of the lipid used, and the like through self-experimentation, which is commonly known in the pharmaceutical field. For example, when using a surfactant of pullulan-folic acid, it can be used in the range of 1-15 mg / mL.

In the step of preparing the lipid phase, the saturated and unsaturated lipids are dissolved in an organic solvent with a photosensitizer in an appropriate ratio to prepare a lipid phase. The saturated lipids and unsaturated lipids may be used in ratios of 10 to 90% with respect to the total lipid weight, respectively. If the ratio of unsaturated lipids is high, there is a problem that a sticky substance remains in the recovery step after manufacturing the lipid nanoparticles, and if the ratio of saturated lipids is high, the photosensing agent may leak to the surface due to solidification during storage. . As the volatile organic solvent, it is preferable to use an organic solvent having a high volatility, since the organic solvent should be removed after preparing the emulsion for the preparation of the lipid nanoparticles. Acetone, methylene chloride, acetonitrile, toluene, ethanol, or a combination thereof may be used as the volatile organic solvent, and since it should be removed later, it is preferable to use the smallest amount possible.

When the aqueous phase and the lipid phase are prepared, a step of preparing an o / w emulsion is performed by mixing the aqueous phase and the lipid phase. It is preferable that the lipid phase is added dropwise to the aqueous phase so that the interface is not increased. The temperature at the time of mixing should be at or above the melting point of the saturated lipids so as not to change into a solid phase before the saturated lipids are emulsified. In order to prepare the emulsion, the mixture of the aqueous phase and the lipid phase may be ultrasonically applied using an ultrasonic machine to disperse the lipid particles to a certain size. When the lipid particles are finely divided by ultrasonic waves, the biocompatible surfactants present in the water phase surround the lipid microspheres to prevent reagglomeration, resulting in an o / w emulsion. The ultrasonic machine generates heat during operation, which helps to remove volatile organic solvent by volatilization. If the ultrasonic machine is operated for too long, the surfactant may be broken and the surfactant may aggregate with each other. Therefore, it is desirable to use it for an appropriate intensity and time.

After the o / w emulsion is produced, a process of removing a photosensitizer or a foreign substance that is not incorporated by filtration is performed. The filtration may use a filtration device that can filter particles of 800 nm or less.

The filtered emulsion may be cooled to below the melting point of the saturated lipids to solidify the lipid particles of the emulsion. Solidifying the lipid particles is useful for maintaining dispersion, so it is desirable to store or freeze at low temperatures within a short time after the emulsion is produced. Unsaturated lipids, which existed together during the solidification of lipid particles, serve to form gaps in the solidified lipid particles, thereby increasing the incorporation efficiency in the lipid nanoparticles of the photosensitizer. Thus, the prepared lipid nanoparticles can be easily recovered using lyophilization, and can be rapidly lyophilized using liquid nitrogen during lyophilization. Freeze-drying may be carried out by completely freezing in liquid nitrogen in a short time and then subliming and blowing off the moisture using a vacuum dryer.

In still another aspect of the present invention, the present invention provides a tumor therapeutic agent by photodynamic therapy and a tumor diagnostic agent by photodynamic diagnosis comprising the lipid nanoparticles according to the present invention. The photodynamic therapy and diagnosis can be carried out in the same manner as conventional photodynamic therapy, and the dosage of the lipid nanoparticles can be appropriately determined in consideration of the condition of the patient under the judgment of the physician performing the clinic. The lipid nanoparticles can be administered by injection, preferably by intravenous injection. Lipid nanoparticulates according to the present invention can make it possible to efficiently diagnose or treat a photosensitizer at a target site without giving adverse effects to fluorescence in normal cells.

In the following example, after preparing a lipid nano-microspheres containing the photosensitizer according to an embodiment of the present invention, fluorescence interference caused by the cell mechanism when fluorescence interference occurs during blood circulation into the cancer cells by the target material The pulleys were identified. In addition, confocal microscopy confirmed that the photosensitizer was introduced into the nucleus in the cell, and the location and amount of the stay were also confirmed. In addition, the analysis using an imaging machine called an image station confirmed that it can be used for imaging of a photosensitizer material. Through this, it could be expected that the lipid nanoparticles according to the present invention may not only diagnose cancer cells but also treat them.

As described above, the lipid nanoparticles according to the present invention do not generate fluorescence by fluorescence interference before reaching the target site, thereby preventing the occurrence of side effects caused by fluorescence, and when they reach the target site, they are incorporated into cells. It is degraded to enable diagnosis and treatment by photosensitisers. Therefore, the lipid nanoparticles according to the present invention can enable the diagnosis or treatment by a photosensitizer efficiently at a target site without giving side effects due to the generation of fluorescence in normal cells. In addition, the lipid nanoparticles according to the present invention has a high incorporation efficiency of the photosensitizer to enable the treatment and diagnosis by the photosensitizer efficiently.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for the understanding of the present invention, and the scope of the present invention is not limited by them in any sense.

Example 1-3 Preparation of Lipid Nanoparticles Impregnated with Photosensitizer

Purified pullulan (1 g, 6.173 mmol per glucose unit) was dissolved in 30 ml of dehydrated DMSO solvent at 60 ° C. for 20 minutes, and then an appropriate amount of folic acid and 4-dimethylaminopyridine (DMAP) were added thereto. Stir at room temperature overnight for folic acid to dissolve cleanly. After that, a little amount of 1,3-dicyclohexylcarbodiimide (DCC) was added, and then reacted at 25 ° C. for 2 days. Upon completion of the reaction, the solution filtered through a 800 nm filtration membrane was immediately dialyzed using a dialysis membrane. The dialyzed solution was frozen and dried in vacuo to form a powder. The material was kept out of light and stored at -20 ° C (yield 89-93%). The results of NMR analysis of the prepared pullan-folic acid surfactant are shown in FIG. 2.

Then, each of 2 (Example 1), 5 (Example 2), and 10 mg (Example 3) of pheophorbide a was dissolved in 1 mL acetone, 130 mg of saturated cacaoter and oleic acid of unsaturated lipid, respectively. 60 mg was added and mixed up to the melting point (50 ° C.) of the cacao butter to prepare an oily solution.

0.1 g of the pullulan-folate surfactant prepared above was added dropwise to the aqueous solution dissolved in 20 ml of water. The emulsion solution thus prepared was added to an ultrasonic machine for 8 minutes (5 seconds operation, 5 seconds stop 400W), and then filtered using a microfiltration device using a pore size 0.8um syringe filter. Then cooled to near 0 ° C.

Comparative Example 1: Preparation of Lipid Nanoparticles Without Photosensitizer

A photosensitive agent-free lipid nanoparticles were prepared in the same manner as in Example 1, except that the oily solution was prepared without containing a photosensitizer.

Experimental Example 1 Particle Size Analysis of Lipid Nanoparticles

The particle size of the lipid nanoparticles prepared in Examples 1-3 and Comparative Example 1 was analyzed using zeta potential zeta sizer SZ (Malvern, UK). Lipid nanoparticles were measured by dispersing in deionized water at a concentration of 1 mg / mL.

The results are shown in Figure 3 below.

A: Comparative Example 1, B: Example 1 (photosensitizer 2 mg), C: Example 2 (photosensitizer 5 mg), C: Example 3 (photosensitizer 10 mg)

Experimental Example 2: presence or absence of fluorescence interference of the photosensitizer in the oil phase and the water phase

The photosensitive agent and the lipid nanoparticles containing the photosensitizer prepared in Examples 1-3 were used to calculate the concentration of the photosensitizer by using a fluorescence spectrometer to have the same concentration of the photosensitizer, and both materials were organic solvents or phosphoric acid. Diluted in buffer solution (PBS) and then analyzed by dilution factor using a fluorescence analyzer. Results according to the dilution factor are shown in FIG. 4.

Experimental Example 3: Presence of Fluorescence Interference between Photosolvent and Organic Solvent by PBS

The presence or absence of fluorescence interference can be represented as an image through a machine called an image station (Kodak, Japan). An image station machine is a device that shoots light at near-infrared wavelengths and photographs the image when a material that absorbs the wavelength fluoresces. Each of the lipid nanoparticles prepared in Examples 1-3 was diluted to a concentration of 1 mg / ml in DMSO or PBS and imaged using an image station machine. For comparison, the pheophorbide a was diluted at a concentration of 1 mg / ml in DMSO or PBS, and the same imaging was performed on each of DMSO and PBS. A photograph taken of an image station machine is shown in FIG. 5. According to FIG. 5, the lipid nanoparticles according to the present invention did not show fluorescence due to fluorescence interference in aqueous PBS, but did not show fluorescence interference in oily DMSO, and it was confirmed that fluorescence occurred.

Experimental Example 4: MTT Assay of Photosensitive Agent and Lipid Nanoparticles Impregnated with Photosensitive Agent

Only pheophorbide a was treated with HeLa cells at concentrations of 0.1 μg / ml, 0.5 μg / ml, 2.5 μg / ml and 12.5 μg / ml, respectively, and immediately after red light (42 kJ / m ² (light exposure time 10 min,) MTT quantification was performed for the exposure of the VA-II DPSS LASER DRIVER 010888 and the exposure of the red light (42 kJ / m ² (light exposure time 10 min,) VA-II DPSS LASER DRIVER 010888) after 6 hours. It was. The results are shown in Figure 6a.

The lipid nanoparticles containing the photosensitizer prepared in Example 1-3 were treated to the same cells at the same concentration as above, and then lighted by the same method as described above, followed by MTT quantification. The results are shown in FIGS. 6B, 6C and 6D, respectively.

For reference, MTT quantification is called cytotoxicity investigation and is an experiment to determine how much compound (or substance) is toxic to cells. When MTT is applied to living cells, MTT is reduced by reductase in the mitochondria, forming a crystal called formazan. In dead cells, no formazan is formed because the mitochondria are already broken and no reductase is present. Therefore, if a compound is treated for a certain time by concentration to induce cell death sufficiently and then treated with MTT, formazan is formed at low concentrations without cytotoxicity and formazan is not formed at high concentrations. to be.

Experimental Example 5: Visualization of Influx Location and Amount and Mechanism by Fluorescence of Lipid Nanoparticles Impregnated with Photosensitizer by Cell

Hela cells were inoculated to about 6 000 plates in 6-well culture plates and then incubated for 24 hours for attachment to the culture plates (5% CO ₂ , 37 ° C.). Thereafter, the concentrations of the lipid nanoparticles containing the pheophorbide a prepared in Example 1 or 2 (labeled NLC2 and NLC5, respectively) and the photosensitizer pheophorbide a alone (labeled PPb) were 0.2 Dispense to a concentration of about ug / ml. Then, after 6 hours, 2 mL of PBS containing 5% paraformaldehyde solution was added to fix the cells for 10 minutes, followed by DAPI staining for 2 minutes for nuclear staining (1 μl; 3.63 mM). Then, each dish was covered with a glass cover and fluorescence was observed under a confocal microscope. Photographs of the results of fluorescence observation with a confocal microscope are shown in FIG. 7.

From the results of FIG. 7, it was confirmed that in the case of the lipid nanoparticles containing the photosensitizer, the photosensitizer was introduced into and stayed in the nucleus of the cancer cells. It can be seen that more photosensitizers incorporated into the nanoparticles entered the nucleus than the photosensitisers alone. From this, it can be seen that the lipid nanoparticles incorporating the photosensitizer according to the present invention act well by introducing the photosensitizer into the nucleus of cancer cells.

1 shows a reaction scheme of synthesizing a biocompatible surfactant using pullulan and folic acid according to an embodiment of the present invention.

Figure 2 is a graph showing the results of NMR analysis for the surfactant of the flulan-folic acid prepared according to an embodiment of the present invention.

Figure 3 is a graph of the size distribution of the particles using the zeta potential zeta sizer SZ (Malvern, UK) with the lipid nanoparticles prepared in accordance with an embodiment of the present invention together with the lipid nanoparticles containing no photosensitizer. .

Figure 4a is a photo of the result of diluting the lipid nanoparticles prepared by using a pheophorbide a 2 mg in DMSO or phosphate buffer (PBS) according to an embodiment and then analyzed by dilution factor using a fluorescence analyzer to be.

Figure 4b is a photo of the result of diluting the lipid nanoparticles prepared using pheophorbide a 5 mg according to an embodiment in DMSO or phosphate buffer (PBS) and then analyzed by dilution factor using a fluorescence spectrometer to be.

Figure 4c is a photo of the result of diluting the lipid nanoparticles prepared by using a pheophorbide a 10 mg in DMSO or phosphate buffer (PBS) according to an embodiment and then analyzed by dilution factor using a fluorescence spectrometer to be.

5 is a photograph taken of the lipid nanoparticles prepared according to an embodiment of the present invention diluted in a concentration of 1mg / ml in DMSO or PBS and imaged using an image station machine.

Figure 6a shows the results of MTT assay for pheophorbide a alone.

Figure 6b shows the results of MTT assay for lipid nanoparticles prepared using pheophorbide a 2 mg according to an embodiment of the present invention.

Figure 6c shows the results of the MTT assay for lipid nanoparticles prepared using pheophorbide a 5 mg according to an embodiment of the present invention.

Figure 6d shows the results of MTT assay for lipid nanoparticles prepared using pheophorbide a 10 mg according to an embodiment of the present invention.

FIG. 7 shows the results of fluorescence observation using confocal microscopy after applying lipid nanoparticles prepared using feophorbide a 2 mg and 5 mg respectively to Hela cells and DAPI staining according to an embodiment of the present invention. The photograph was taken in comparison with the case where the carbide a was applied alone.

<Explanation of symbols in the drawings>

PPb: Pheophorbide a sole

PF: pheophorbide a

NLC 2: Lipid Nanoparticulates Prepared with Pheophorbide a 2 mg

NLC 5: Lipid Nanoparticles Prepared with Pheophorbide a 5 mg

NLC 10: Lipid Nanoparticulates Prepared with Pheophorbide a 10 mg

non-internalized: no photosensitizer enters the cell

internalized: A state where photosensitizers enter the cell

cell viability: cell viability

Claims (14)

A lipid nanoparticulate containing a hydrophobic photosensitizer, wherein the hydrophobic photosensitizer is formed by emulsification with a biocompatible surfactant together with a saturated lipid and an unsaturated lipid. The lipid nanoparticulate of claim 1, wherein the hydrophobic photosensitizer fluoresces in the near infrared. The method of claim 2, wherein the hydrophobic photosensitizer is a lipid nano, characterized in that the pheophorbide a, pyrofeophorbide a, tetrabenzoporphine, mesotetraphenylformin, or Fe (III) mesoporphyrin chloride Microspheres. The method of claim 1, wherein the saturated fat is cacao butter, butter, shortening, laard, palm oil, coconut oil, palm kernel oil, palm oil, or a combination thereof, the unsaturated fat is oleic acid, olive oil, camellia oil, macadamia nut oil, Lipid nanoparticles, characterized in that castor oil, lauric acid, myristic acid, palmitic acid, stearic acid, isostearic acid, almond oil, avocado oil, evening primrose oil, malt oil, jojoba oil, corn oil, rice bran oil, or a combination thereof . The lipid nanoparticulate of claim 1, wherein the biocompatible surfactant is a hydrophobic tumor target material bound to a biocompatible aqueous polymer. 6. The lipid nanoparticulate of claim 5, wherein the biocompatible aqueous polymer is flulan, dextran, chondroitin sulfate, heparin, pluronic, hyaluronic acid, chitosan, or fucoidan. 6. The lipid nanoparticulate of claim 5, wherein the hydrophobic tumor target is folic acid, antibody, cobalamin, vitamin A, vitamin C, or vitamin B12. Preparing a biocompatible surfactant by binding a hydrophobic tumor target material to the biocompatible aqueous polymer; Dissolving the biocompatible surfactant in water to prepare an aqueous phase; Dissolving saturated and unsaturated lipids in a volatile organic solvent with a photosensitizer to prepare a lipid phase; Mixing the aqueous phase and the lipid phase to produce an o / w emulsion; Filtering the prepared emulsion to remove unincorporated photosensitizers or foreign matter; And Cooling the filtered emulsion to a temperature below the melting point of the solid lipid to solidify the emulsion particles, A method for preparing lipid nanoparticles according to any one of claims 1 to 7. The method of claim 8, wherein the volatile organic solvent is acetone, methylene chloride, acetonitrile, toluene, ethanol , or a combination thereof. 9. The method of claim 8, wherein the lipid phase is added dropwise to the aqueous phase in preparing the o / w emulsion. A tumor therapeutic agent by photodynamic therapy comprising the lipid nanoparticulates according to any one of claims 1 to 7. A tumor diagnostic agent by photodynamic diagnosis comprising the lipid nanoparticulates according to any one of claims 1 to 7. The tumor therapeutic agent according to claim 11, which is an injection. 13. The tumor diagnostic agent according to claim 12, which is an injection.

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