KR20110073731A - Water-soluble nanostructure for photodynamic therapy - Google Patents

Abstract

PURPOSE: A soluble nanostructure containing soluble porphyrin derivatives is provided to ensure high photodynamic therapeutic agent and to prevent side effects. CONSTITUTION: A soluble nanostructure for photodynamic therapy contains soluble porphyrin derivative of chemical formula 1. The porphyrin derivative is prepared by amidation of protoporphyrin IX(chemical formula 2) with polyethylene glycol derivatives(chemical formulas 3 and 4) of amino terminal. A coupling agent for amidation is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC), N,N'-dicyclohexylcarbodiimide(DCC), or N,N'-diisopropylcarbodiimide(DIC).

Description

Water-soluble nanostructure for photodynamic therapy

The technology disclosed herein relates to a water-soluble nanostructure for treating photodynamics, and more particularly, has excellent solubility in water and improves stability in the body, thereby improving efficiency as a photosensitizer and reducing side effects due to residual photosensitizer. An improved water soluble porphyrin derivative and a water soluble nanostructure comprising the same.

Photodynamic therapy (PDT) is a technique that can treat incurable diseases such as cancer without surgery by using photosensitizer, which is selective and sensitive to various tumors, and has side effects such as chemotherapeutic agents. This is a kind of curiosity without. For example, by administering the photosensitive material to the subject by intravenous injection and irradiating with appropriate light, the excited photosensitive material activates an oxygen molecule to convert oxygen into a singlet state or to convert new radicals. It selectively attacks and destroys only cancer cells or various tumor tissues. Such photodynamic therapy can easily remove only the diseased cells while preserving normal cells, and in most cases, can eliminate the risk of general anesthesia, and can be easily operated by simple local anesthesia. Therefore, there is no need to extract the damaged organs, and the recovery after the minimally invasive procedure is quick and the hospitalization period is shortened, thereby improving the welfare of the patient.

Such photosensitive materials are typical of porphyrin-type compounds. Porphyrin-based compounds extracted from jambun, mulberry leaves, and green algae have spectroscopic properties suitable for use as photosensitive materials, and the most important property is relatively cellular. The red light (700-900 nm) having a large transmittance can efficiently generate an electron transition and an excited state accordingly.

Porphyrin derivatives as photosensitizers selectively penetrate and accumulate in cancer cells or tumor tissues, and are also used as early diagnosis of tumors because they exhibit fluorescence or phosphorescence due to the characteristics of the compounds. Porphyrin's cancer cell selectivity is explained by the fact that the receptor for low density lipoprotein (LDL), which is responsible for the transport of porphyrin in the body, is expressed in cancer cells more than normal cells. However, most photosensitizers are poorly soluble and have a long photoresist and are highly phototoxic. In addition, photo-sensitizers are expensive and the metabolism is slow in the human body, side effects of phototoxicity have been found.

It is preferable that the excellent photosensitizer satisfies the following conditions.

1. have high photoreaction efficiency from triplet oxygen to singlet oxygen,

2. To be distributed selectively to cancerous tissue and surrounding tissues,

3. Easy to remove from human body after administration,

4. have minimal side effects and toxicity,

5. Relatively low manufacturing cost, high productivity and high purity manufacturing

According to one embodiment, a water-soluble porphyrin derivative represented by the following Chemical Formula 1 is provided to provide a photosensitizer satisfying the above conditions.

<Formula 1>

In the above formula,

R ₁ and R ₂ are each independently -H or -CH ₃ ,

R ₃ and R ₄ are each independently -OH, -OCH ₃ , -NH ₂ , -NHCH ₃ , -NH (CH ₃ ) ₂ , -NHCOCH ₃ , -N (COCH ₃ ) ₂ , -COR ₅ , or- CH ₂ COR ₅ , wherein R ₅ = -OH, -OCH ₃ , -NH ₂ , -NHCH ₃ , or -NH (CH ₃ ) ₂ ,

m and n are each independently an integer from 21 to 226.

The water-soluble porphyrin derivative has a form in which water-soluble polyethylene glycol derivatives are bound to protoporphyrin IX (hereinafter referred to as PPIX), and the introduction of non-ionic polymer chains such as polyethylene glycol derivatives not only increases water solubility but also inhibits intermolecular association. Thus, the efficiency as the photosensitizer can be increased. It can be used in photodynamic therapy to treat various diseases by generating reactive oxygen species in response to long wavelength laser.

According to one embodiment, the water-soluble porphyrin derivative may be prepared by amidation of protoporphyrin IX (Formula 2) with polyethylene glycol derivatives (Formula 3 and Formula 4) at the amine end. The mass average molecular weight (Mw) of the polyethylene glycol derivatives at the amine end may be 1,000 or more and 10,000 or less. Coupling agents can be used for the amidation. Examples of the coupling agent include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N, N'-dicyclhexylcarbodiimide (DCC), N, N'-diisopropyl One or more selected from the group consisting of carbodiimide (DIC) can be used.

<Formula 2>

<Formula 3>

(R ₁ is -H or -CH ₃ , R ₃ is -OH, -OCH ₃ , -NH ₂ , -NHCH ₃ , -NH (CH ₃ ) ₂ , -NHCOCH ₃ , -N (COCH ₃ ) ₂ , -COR ₅ , or -CH ₂ COR ₅ (where R ₅ = -OH, -OCH ₃ , -NH ₂ , -NHCH ₃ , or -NH (CH ₃ ) ₂ ) and m is an integer from 21 to 226 .)

<Formula 4>

(R ₂ is -H or -CH ₃ , R ₄ is -OH, -OCH ₃ , -NH ₂ , -NHCH ₃ , -NH (CH ₃ ) ₂ , -NHCOCH ₃ , -N (COCH ₃ ) ₂ , -COR ₅ , or -CH ₂ COR ₅ (where R ₅ = -OH, -OCH ₃ , -NH ₂ , -NHCH ₃ , or -NH (CH ₃ ) ₂ ) and n is an integer from 21 to 226 .)

The scheme below shows one embodiment of a method of preparing a water soluble porphyrin derivative.

According to the above reaction scheme, PPIX and methoxy polyethyleneglycol amine were converted into 1-hydroxybenzotriazole (HOBT) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Water-soluble porphyrin derivative PEGylated-PPIX can be prepared by amidation in a N, N'-dimethylformamide (DMF) solvent in the presence.

According to one embodiment, there is provided a water-soluble nanostructure for photodynamic therapy comprising the water-soluble porphyrin derivative of Formula 1. The water-soluble porphyrin derivative of Formula 1 may be improved in poor solubility of the conventional photosensitizer by having a hydrophobic PPIX moiety and a hydrophilic amine-terminated polyethylene glycol derivative. That is, it is possible to form a stable polymer micelles of several hundred nanometers in size. For example, the water-soluble nanostructure may have a form of nanospheres having a diameter of 100 to 300 nm, and may be stably maintained in the aqueous solution with little change in particle size over time. Therefore, when the water-soluble nanostructure is added to the body it can prevent the outflow of the photosensitizer and to reach the target site.

The water-soluble porphyrin derivative represented by Formula 1 above and the water-soluble nanostructure for photodynamic therapy using the same as an active ingredient have high stability in the body and thus have high efficiency as a photodynamic therapeutic agent and prevent side effects in which the drug is deposited in the organ by reducing the residence time in the body. can do. From the results shown in the following examples it can be seen that the excellent results in terms of singlet oxygen production rate and cytotoxicity can be used suitably for photodynamic therapy.

Hereinafter, the technology disclosed herein will be described in more detail with reference to examples, but the following examples are merely examples for demonstrating the construction and effects of the disclosed technology, and the spirit of the disclosed technology is not limited to the following examples. .

[Example]

Preparation Example: PEGylated-PPIX Synthesis

Protoporphyrin IX 100 mg (0.178 mmol), methoxy polyethylene glycol amine (Mw 2000) 593 mg (0.296 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (1-ethyl -3- (3-dimethylaminopropyl) carbodiimide hydrochloride) 101mg (0.527mmol) and 1-hydroxybenzotriazole 90mg (0.666mmol) N, N'-dimethylformamide (N, N'-dimethylformamide ) Into 20ml and stirred at 70 ° C or less. After completion of the reaction, N, N'-dimethylformamide was removed by distillation under reduced pressure, dissolved in dichloromethane and washed with distilled water, Na ₂ CO ₃ aqueous solution and 1N-hydrogen chloride aqueous solution, respectively. Dichloromethane was removed by distillation under reduced pressure and purified by column chromatography using a developing solution (volume ratio of dichloromethane: methanol = 90: 10).

¹ H-NMR analysis of PEGylated-PPIX obtained by the above method is shown in FIG. 1.

2. Characterization and Photochemical Characterization of PEGylated-PPIX

The effect of PEGylated-PPIX synthesized in the above preparation and the change of the average particle size over time, the measurement of singlet oxygen generation rate, the phototoxicity evaluation of the cells was confirmed excellent effect.

(1) Particle size and particle size distribution

Figure 2 compares the particle size distribution of free-PPIX, methoxy polyethylene glycol amine (mPEG), a mixture of PPIX and mPEG and the PEGylated-PPIX synthesized in the above preparation. PEGylated-PPIX synthesized as shown in Figure 2 shows a narrower particle size distribution compared to the mixture of free-PPIX, PPIX, mPEG, and has a mean average diameter of mPEG and free-PPIX.

Figure 3 is a photograph of visual observation of the solubility of free-PPIX, mPEG, a mixture of PPIX and mPEG and PEGylated-PPIX synthesized in the preparation with water. All four samples were dissolved in water at [4X10 ^-4 M], and the mixture of free-PPIX and mPEG and PPIX was opaque, while PEGylated-PPIX was transparent, indicating that PEGylated-PPIX had excellent solubility in water.

4 and 5 show the concentration of PEGylated-PPIX synthesized in Preparation Example and average particle size over time, respectively. It can be seen that the PEGylated-PPIX synthesized as shown in FIGS. 4 and 5 forms nanoparticles having an average diameter of 200 nm in an aqueous solution, and maintains stable particle size over time.

(2) singlet oxygen generation measurement

Experimental Example 1

Singlet oxygen generation efficiency of PEGylated-PPIX synthesized in the preparation was measured using N, N-dimethyl-4-nitroaniline (N, N-dimethyl-4-nitrosoaniline, RNO). 1.5 ml of PEGylated-PPIX diluted to [ ⁴ × 10 ⁻⁴ M], 50 μl of 250 μM RNO, 1 μl of 0.03 M L-histidine, 0.5 ml of PBS (Phosphate buffered saline) were mixed and irradiated with a 633 nm He-Ne laser for 60 minutes. Absorbance at 440 nm was measured at 5-10 minute intervals. Singlet oxygen generated after the laser irradiation can be discolored by the RNO reduce the absorbance at 440nm This referred to the decrease amount (C / C _0), the amount of generation of singlet oxygen may account for changes in RNO concentration. As a result of the measurement of singlet oxygen generation, it was found that the singlet oxygen generation efficiency was very good (about 75%) until the first 20 minutes, and after 40 minutes, the absorbance value was close to zero.

The singlet oxygen generation rate of PEGylated-PPIX measured by the above method is shown in FIG. 6.

Comparative Example 1

RNO was used to measure singlet oxygen generation efficiency of free-PPIX. Free-PPIX was diluted in 0.1M SDS (Sodium dodecyl sulfate) / H ₂ O to a concentration of [4X10 ^-4 M], and then experimented in the same manner as in Experiment 1.

As shown in FIG. 6, the singlet oxygen generation efficiency of free-PPIX was about 15%, which is significantly lower than that of PEGylated-PPIX.

(3) Cytotoxicity Assessment

Experimental Example 2

Fibroblast cells (1 × 10 ⁵ ) were seeded in 96 well plates and then cultured for one day. Cells were treated for 12 hours at different concentrations of PEGylated-PPIX. After 12 hours, washed twice with PBS and MTT solution was added. The MTT assay is a test that uses the ability of mitochondria to reduce yellow water-soluble substrate MTT tetrazolium by dehydrogenase action to water-soluble MTT formazan, which has a blue purple color. It means to be alive. To the treated cells 3- (T 5-dimethyl-2-thiazolyl-2 5-diphenyl-tetrazolium bromide) (3- (T 5-dimethyl-2-thiazolyl-2 5-diphenyl-tetrazolium bromide)) The reagent was added and incubated for 3 hours to form formazan, and then the amount of formazan was measured for absorbance at 570 nm using an ELISA reader.

As shown in FIG. 7, PEGylated-PPIX did not show toxicity to cells even at a concentration of 200 μg / ml.

Comparative Example 2

Cytotoxicity of free-PPIX was evaluated in the same manner as in Experimental Example 2 above. In the case of free-PPIX concentration of 5μg / ml did not show the toxicity to cells, but it can be seen that the cytotoxicity increases as the concentration increases.

(4) light cytotoxicity assessment

Experimental Example 3

Fibroblast cells (1 × 10 ⁵ ) were seeded in 96 well plates and then cultured for one day. After treatment with 200 μg / ml of the synthesized PEGylated-PPIX based on the PPIX concentration to the cells were cultured for 1 hour to be absorbed into the cells. After washing the cells with the culture, the cells were irradiated with a 633 nm He-Ne laser for 5 to 20 minutes, and cultured for 12 hours, followed by MTT analysis.

As shown in FIG. 8, PEGylated-PPIX increased photocytotoxicity with increasing laser irradiation time and showed about 40% toxicity after laser irradiation for 20 minutes.

Comparative Example 3

As a result of cytotoxicity evaluation of free-PPIX, it was confirmed that cytotoxicity was observed at a concentration of 50 μg / ml or more, and after treatment with 5 μg / ml of free-PPIX based on PPIX concentration to cells, free-PPIX in the same manner as in Experimental Example 3 above. The photocytotoxicity of was evaluated.

As shown in FIG. 8, the free-PPIX showed about 20% toxicity after laser irradiation for 20 minutes, and it can be confirmed that the PDT effect of the synthesized PEGylated-PPIX was superior to that of Experimental Example 3.

1 is a ¹ H-NMR spectrum of PEGylated-PPIX synthesized according to the preparation of the present disclosure.

2 is a photograph after dissolving free-PPIX, PEGylated-PPIX, methoxy polyethylene glycol amine (mPEG), and a mixture of PPIX and mPEG in water.

3 is a measurement result of particle size distribution of PEGylated-PPIX, free-PPIX, mPEG, and a mixture of PPIX and mPEG.

4 is a measurement result of the average particle size according to the concentration of PEGylated-PPIX.

5 is a result of measuring the average particle size of PEGylated-PPIX over time.

6 shows the results of measuring singlet oxygen generation efficiency of free-PPIX and PEGylated-PPIX.

7 and 8 are the results of analysis of the cytotoxicity after laser stability and laser irradiation in the cancer state of free-PPIX and PEGylated-PPIX by MTT assay.

Claims (6)

Water-soluble nanostructure for photodynamic therapy comprising a water-soluble porphyrin derivative represented by the following formula (1): <Formula 1>

In the above formula, R ₁ and R ₂ are each independently -H or -CH ₃ , R ₃ and R ₄ are each independently -OH, -OCH ₃ , -NH ₂ , -NHCH ₃ , -NH (CH ₃ ) ₂ , -NHCOCH ₃ , -N (COCH ₃ ) ₂ , -COR ₅ , or- CH ₂ COR ₅ , wherein R ₅ = -OH, -OCH ₃ , -NH ₂ , -NHCH ₃ , or -NH (CH ₃ ) ₂ , m and n are each independently an integer from 21 to 226. The water-soluble nanostructure for photodynamic therapy according to claim 1, having a form of nanospheres having a diameter of 100 to 300 nm in an aqueous system. A process for preparing a water-soluble porphyrin derivative prepared by amidation of protoporphyrin IX (Formula 2) with amine-terminated polyethylene glycol derivatives (Formula 3 and Formula 4). <Formula 2>

<Formula 3>

(R ₁ is -H or -CH ₃ , R ₃ is -OH, -OCH ₃ , -NH ₂ , -NHCH ₃ , -NH (CH ₃ ) ₂ , -NHCOCH ₃ , -N (COCH ₃ ) ₂ , -COR ₅ , or -CH ₂ COR ₅ (where R ₅ = -OH, -OCH ₃ , -NH ₂ , -NHCH ₃ , or -NH (CH ₃ ) ₂ ) and m is an integer from 21 to 226 .) <Formula 4>

(R ₂ is -H or -CH ₃ , R ₄ is -OH, -OCH ₃ , -NH ₂ , -NHCH ₃ , -NH (CH ₃ ) ₂ , -NHCOCH ₃ , -N (COCH ₃ ) ₂ , -COR ₅ , or -CH ₂ COR ₅ (where R ₅ = -OH, -OCH ₃ , -NH ₂ , -NHCH ₃ , or -NH (CH ₃ ) ₂ ) and n is an integer from 21 to 226 .) The method for producing a water-soluble porphyrin derivative according to claim 3, wherein the weight average molecular weight (Mw) of the polyethylene glycol derivative at the amine end is 1,000 or more and 10,000 or less. The method of claim 3, wherein the coupling agent used for the amidation is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N, N'-dicyclhexylcarbodiimide ( DCC), N, N'-diisopropylcarbodiimide (DIC) A method for producing a water-soluble porphyrin derivative is at least one member selected from the group consisting of. Water-soluble porphyrin derivatives represented by the following formula (1): <Formula 1> In the above formula, R ₁ and R ₂ are each independently -H or -CH ₃ , R ₃ and R ₄ are each independently -OH, -OCH ₃ , -NH ₂ , -NHCH ₃ , -NH (CH ₃ ) ₂ , -NHCOCH ₃ , -N (COCH ₃ ) ₂ , -COR ₅ , or- CH ₂ COR ₅ , wherein R ₅ = -OH, -OCH ₃ , -NH ₂ , -NHCH ₃ , or -NH (CH ₃ ) ₂ , m and n are each independently an integer from 21 to 226.

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