Patent Application
20230000984
The present invention relates to a biocompatible photo thermal composition that can be used in various fields including the treatment of cancer and skin diseases.
Cancer, which is caused by various reasons including stress and pollution, is the leading cause of death of modern people. Cancer is caused by gene mutation in normal cells. Cancer indicates a malignant tumor among tumors which do not follow normal path of cell differentiation, cell growth and cell apoptosis. Methods of treating cancer include surgical operation, chemotherapy, and radiotherapy.
In the case of chemotherapy, a drug is administered systemically, and the drug not only kills cancer cells but also spreads to normal tissues to cause toxicity in normal cells as well. Accordingly, serious side effects such as gastrointestinal side effects, thrombocytopenia and hair loss are caused.
Chemotherapy based anticancer treatment is limited in the case of tumors resistant to chemotherapy by expressing p-glycoproteins.
In particular, a solid tumor has heterogeneity in tumor tissue, indicating that both the tumor cells having sensibility to chemotherapeutic agents and the other tumor cells showing resistance to chemotherapeutic agents exist together in the tissue so that the elimination of the resistant tumor cells alone is very difficult even after the administration of chemotherapeutic agents.
To solve the problem above, it is necessary to develop a novel anticancer agent based on a novel anticancer mechanism which can be applied locally to tumor tissues and overcome different sensibility among tumor cells. Thus, studies reflecting such necessity have been actively going on.
In particular, photothermal therapy is one of the most popular anticancer treatment methods. This method uses the weakness of cancer cells on heat, compared with normal cells, so that a photoresponsive material is located in a local area where cancer cells are located and then heat is generated by a stimulus given from outside to kill cancer cells selectively.
For example, methods using gold nanoparticles, nanoporous silica or carbon nanotubes as a photoresponsive material or using organic polymer nanoparticles have been developed (Patent Reference 1, Korean Patent Publication No. 10-2012-0107686).
The present inventors have been tried to develop an anticancer agent that can overcome side effects according to systemic administration and difficulty in eliminating tumor cells having resistance to chemotherapeutic agents. In the course of our study, the present inventors developed a biocompatible photothermal composition capable of acting selectively on a local site, and confirmed its photothermal effect and photothermal therapeutic effect on cancer cells, leading to the completion of the present invention.
The present inventors also confirmed the antimicrobial effect of the said photothermal composition and thereafter applied the composition to the treatment of skin disease and extended the use of the composition for accelerating absorption of functional materials for cosmetics.
It is an object of the present invention to provide a photothermal composition comprising a metal salt and a benzene ring compound derivative containing two or more hydroxy groups.
To achieve the above object, the present invention provides a photothermal composition comprising a metal salt; and a catechol derivative.
The composition of the present invention displays a remarkable effect on photothermal therapy since the temperature of the applied area can be raised at least 50° C. by near infrared ray irradiation, after the injection. The composition can be combined with a biocompatible material to have biocompatibility and can act selectively on a local site to minimize side effects. The composition also has an effect of continuous photothermal treatment because it is present in the administration site for a few days after injection. Therefore, the composition of the present invention can be used for anticancer treatment. In addition, the composition of the present invention exhibits an antibacterial effect, so that it can be used for treating skin disease and for increasing skin absorption of functional materials for cosmetics through photothermal effect.
Hereinafter, the present invention is described in detail.
The present invention provides a photothermal composition comprising a metal salt; and a catechol derivative.
Herein, the photothermal composition is characterized by comprising tannic acid or the compound represented by formula 1 below as the catechol derivative.
In formula 1 above,
R1 and R2 are —OH;
R3 is —H, —OH, —CN, —NO2, halogen, —COOM, amine C1-5 straight or branched alkyl, C1-5 straight or branched alkyl, C1-5 straight or branched alkoxy, unsubstituted or substituted C6-10 aryl, unsubstituted or substituted C3-10 cycloalkyl, unsubstituted or substituted 5-10 membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O and S, or unsubstituted or substituted 5-10 membered heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O and S, and R3 is linked together with R4 to form unsubstituted or substituted C6-10 aryl,
wherein, M is —H, C1-5 straight or branched alkyl or epigallocatechinyl,
the substituted C6-10 aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl and 5-10 membered heterocycloalkyl are independently C6-10 aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl and 5-10 membered heterocycloalkyl substituted with one or more substituents selected from the group consisting of —OH, C1-5 straight or branched alkyl and C1-5 straight or branched alkoxy;
R4 is —H, —OH, —CN, —NO2, halogen, —COOM, —CH(OH)—CH2—NHA1, amine C1-5 straight or branched alkyl, C1-5 straight or branched alkyl, C1-5 straight or branched alkoxy, unsubstituted or substituted C6-10 aryl, unsubstituted or substituted C3-10 cycloalkyl, unsubstituted or substituted 5-10 membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O and S, or unsubstituted or substituted 5-10 membered heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O and S, and R4 is linked together with R5 to form unsubstituted or substituted C6-10 aryl,
wherein, M is —H, C1-5 straight or branched alkyl or epigallocatechinyl,
A1 is —H or C1-5 straight or branched alkyl,
the substituted C6-10 aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl and 5-10 membered heterocycloalkyl are independently C6-10 aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl and 5-10 membered heterocycloalkyl substituted with one or more substituents selected from the group consisting of —OH, C1-5 straight or branched alkyl and C1-5 straight or branched alkoxy;
R5 is —H, —OH, —CN, —NO2, halogen, —COOM, amine C1-5 straight or branched alkyl, C1-5 straight or branched alkyl, C1-5 straight or branched alkoxy, unsubstituted or substituted C6-10 aryl, unsubstituted or substituted C3-10 cycloalkyl, unsubstituted or substituted 5-10 membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O and S, or unsubstituted or substituted 5-10 membered heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O and S, and R5 is linked together with R6 to form unsubstituted or substituted C6-10 aryl,
wherein, M is —H, C1-5 straight or branched alkyl or epigallocatechinyl,
the substituted C6-10 aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl and 5-10 membered heterocycloalkyl are independently C6-10 aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl and 5-10 membered heterocycloalkyl substituted with one or more substituents selected from the group consisting of —OH, C1-5 straight or branched alkyl and C1-5 straight or branched alkoxy; and
R6 is —H, —OH, —CN, —NO2, halogen, —COOM, amine C1-5 straight or branched alkyl, C1-5 straight or branched alkyl, C1-5 straight or branched alkoxy, unsubstituted or substituted C6-10 aryl, unsubstituted or substituted C3-10 cycloalkyl, unsubstituted or substituted 5-10 membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O and S, or unsubstituted or substituted 5-10 membered heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O and S,
wherein, M is —H, C1-5 straight or branched alkyl or epigallocatechinyl,
the substituted C6-10 aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl and 5-10 membered heterocycloalkyl are independently C6-10 aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl and 5-10 membered heterocycloalkyl substituted with one or more substituents selected from the group consisting of —OH, C1-5 straight or branched alkyl and C1-5 straight or branched alkoxy.
Preferably,
R1 and R2 are —OH;
R3 is —H, —OH, —CN, —NO2, halogen, —COOM, amine C1-3 straight or branched alkyl, C1-3 straight or branched alkyl, C1-3 straight or branched alkoxy, unsubstituted or substituted C6-10 aryl, unsubstituted or substituted C3-10 cycloalkyl, unsubstituted or substituted 5-10 membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O and S, or unsubstituted or substituted 5-10 membered heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O and S, and R3 is linked together with R4 to form unsubstituted or substituted C6-10 aryl,
wherein, M is —H, C1-5 straight or branched alkyl or epigallocatechinyl,
the substituted C6-10 aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl and 5-10 membered heterocycloalkyl are independently C6-10 aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl and 5-10 membered heterocycloalkyl substituted with one or more substituents selected from the group consisting of —OH, C1-3 straight or branched alkyl and C1-3 straight or branched alkoxy;
R4 is —H, —OH, —CN, —NO2, halogen, —COOM, —CH(OH)—CH2—NHA1, amine C1-3 straight or branched alkyl, C1-3 straight or branched alkyl, C1-3 straight or branched alkoxy, unsubstituted or substituted C6-10 aryl, unsubstituted or substituted C3-10 cycloalkyl, unsubstituted or substituted 5-10 membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O and S, or unsubstituted or substituted 5-10 membered heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O and S, and R4 is linked together with R5 to form unsubstituted or substituted C6-10 aryl,
wherein, M is —H, C1-5 straight or branched alkyl or epigallocatechinyl,
A1 is —H or C1-5 straight or branched alkyl,
the substituted C6-10 aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl and 5-10 membered heterocycloalkyl are independently C6-10 aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl and 5-10 membered heterocycloalkyl substituted with one or more substituents selected from the group consisting of —OH, C1-3 straight or branched alkyl and C1-3 straight or branched alkoxy;
R5 is —H, —OH, —CN, —NO2, halogen, —COOM, amine C1-3 straight or branched alkyl, C1-3 straight or branched alkyl, C1-3 straight or branched alkoxy, unsubstituted or substituted C6-10 aryl, unsubstituted or substituted C3-10 cycloalkyl, unsubstituted or substituted 5-10 membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O and S, or unsubstituted or substituted 5-10 membered heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O and S. and R5 is linked together with R6 to form unsubstituted or substituted C6-10 aryl,
wherein, M is —H, C1-5 straight or branched alkyl or epigallocatechinyl,
the substituted C6-10 aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl and 5-10 membered heterocycloalkyl are independently C6-10 aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl and 5-10 membered heterocycloalkyl substituted with one or more substituents selected from the group consisting of —OH, C1-3 straight or branched alkyl and C1-3 straight or branched alkoxy; and
R6 is —H, —OH, —CN, —NO2, halogen, —COOM, amine C1-3 straight or branched alkyl, C1-3 straight or branched alkyl, C1-3 straight or branched alkoxy, unsubstituted or substituted C6-10 aryl, unsubstituted or substituted C3-10 cycloalkyl, unsubstituted or substituted 5-10 membered heteroaryl containing one or more hetero atoms selected from the group consisting of N, O and S, or unsubstituted or substituted 5-10 membered heterocycloalkyl containing one or more hetero atoms selected from the group consisting of N, O and S,
wherein, M is —H, C1-5 straight or branched alkyl or epigallocatechinyl,
the substituted C6-10 aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl and 5-10 membered heterocycloalkyl are independently C6-10 aryl, C3-10 cycloalkyl, 5-10 membered heteroaryl and 5-10 membered heterocycloalkyl substituted with one or more substituents selected from the group consisting of —OH, C1-3 straight or branched alkyl and C1-3 straight or branched alkoxy.
In addition, the metal salt is a lanthanide metal salt or a transition metal salt. Examples of the metal of the lanthanide metal salt are cerium (Ce), europium (Eu), gadolinium (Gd) and terbium (Tb). Examples of the metal of the transition metal salt are aluminum (Al), vanadium (V), manganese (Mn), iron (Fe), zinc (Zn), zirconium (Zr), molybdenum (Mo), ruthenium (Ru) and rhodium (Rh).
Further, the metal ion of the metal salt characteristically forms a complex with the catechol derivative above.
The catechol derivative is characteristically linked to a biocompatible substance through covalent bond.
Herein, the biocompatible substance is exemplified by hyaluronic acid, methylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, alginate, chitosan, gelatin and collagen.
For example, the compound represented by formula 1 can be fixed with hyaluronic acid which is one of biocompatible substances, as shown in formula 2 below.
Herein, L is C1-10 straight or branched alkylene or
m is an integer selected from 1-5; and
n is an integer selected from 1-1000.
R1, R2, R3, R4 and R6 are independently as defined in formula 1 above.
When hyaluronic acid is used as a biocompatible substance to combine with a metal salt and a catechol derivative, the resulting complex is preferably in the form of hydrogel.
In general, hydrogel is hard to be injected in the form of injections. However, when a catechol derivative in which a metal salt and hyaluronic acid are linked together is injected hypodermically using a dual syringe, it can form hydrogel under the subcutis, suggesting that it can be applied locally for selective photothermal therapy.
Further, a complex in the form of hydrogel can be formulated as a patch, a depot or an external preparation. When a drug is incorporated in the preparation of hydrogel, the prepared sustained-release hydrogel containing the drug can be used for prolonged release of a therapeutic drug.
The photothermal composition of the present invention demonstrated sustainability of photothermal effect at 50° C. or higher for at least 6 days by infrared ray irradiation after the injection of the composition. Thus, long term photothermal therapy can be achieved by a single administration of the composition.
The photothermal composition of the present invention can be administered by a pathway selected from the group consisting of intravenous injection, intraperitoneal injection, intramuscular injection, intracranial injection, intratumoral injection, intraepithelial injection, transdermal delivery, esophageal administration, abdominal administration, intraarterial injection, intraarticular injection and intraoral administration.
In addition, the photothermal composition of the present invention is characteristically used for treating cancer, and at this time the cancer can be a solid tumor or a blood cancer.
The solid tumor above is specifically exemplified by brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial carcinoma, ependymoma, brain stem tumor, head and neck cancer, laryngeal cancer, oropharyngeal cancer, nasal cavity cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, thoracic tumor, small cell lung cancer, non-small cell lung cancer, thymic carcinoma, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, small bowel cancer, colon cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, prostate cancer, female genital tumors, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female gonadal cancer, female urethral cancer or skin cancer. The blood cancer above is specifically exemplified by leukemia, malignant lymphoma, multiple myeloma or aplastic anemia.
Further, the photothermal composition of the present invention is characteristically used for the treatment of skin disease due to its antibacterial activity mediated by photothermal action. The skin disease above is specifically exemplified by acne, warts, atopy, eczema, lipomas, sebaceous cysts, epidermal cysts, epithelial cysts, subcutaneous cysts or skin fibrosis.
In addition, the photothermal composition of the present invention can be used for the improvement of skin absorption of a functional material for cosmetics. The functional material for cosmetics can be any liquid or solid substance having moisture containing, ultraviolet blocking, whitening, wrinkle reducing or irritation preventing functions.
The functional material above is exemplified by such extracts as avocado extract, fumitoli extract, carrot extract, Moutan cortex extract, Pueraria lobata root extract, Stone root extract, Lady's horsetail extract, lady mantil extract, horsetail extract, soybean embryo extract, wheat germ extract, radish extract, Laminaria japonica extract, Sanguisorba officinalis L. extract, Cinnamomum cassia bark extract, ginger extract, Ephedra distachya extract, herb extract, vitamin F and apple seed extract. The functional material can also be a substance containing components such as arbutin, ethylascorbyl ether, retinol, retinylpalmitate, adenosine, polyethoxylated retinamide, or a commercial product or a cosmetic ingredient for medicines containing them.
For example, the hyaluronic acid-gallic acid conjugate can be used for the purpose of promoting skin absorption of a functional substance by mixing with paste, gel, cream, lotion, powder, solid soap, wax, shampoo, rinse, solution, suspension, emulsion, mineral cosmetic, oil, emulsion foundation, soft lotion, nutritional lotion, nutritional cream, massage cream, essence, cleansing cream, cleansing foam, pack, pack base, eye cream, perfume, ointment, cleansing water, powder and spray, etc.
Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.
However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
5 mg/ml of catechol dissolved in TDW (triple distilled water) was mixed with 5 mg/ml of iron chloride dissolved in TDW at the ratio of 1:1 (v/v) to form coordination bonds, leading to the preparation of a coordination complex.
5 mg/ml of catechol amine dissolved in TDW (triple distilled water) was mixed with 5 mg/ml of iron chloride dissolved in TDW at the ratio of 1:1 (v/v) to form coordination bonds, leading to the preparation of a coordination complex.
5 mg/ml of epigallocatechin gallate dissolved in TDW (triple distilled water) was mixed with 10 mg/ml of iron chloride dissolved in TDW at the ratio of 1:1 (v/v) to form coordination bonds, leading to the preparation of a coordination complex.
5 mg/ml of gallic acid dissolved in TDW (triple distilled water) was mixed with 5 mg/ml of iron chloride dissolved in TDW at the ratio of 1:1 (v/v) to form coordination bonds, leading to the preparation of a coordination complex.
5 mg/ml of tannic acid dissolved in TDW (triple distilled water) was mixed with 10 mg/ml of iron chloride dissolved in TDW at the ratio of 1:1 (v/v) to form coordination bonds, leading to the preparation of a coordination complex.
100 g of hyaluronic acid was dissolved in 0.3 M NaHCO3, to which 63 mg of EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 50 mg of NHS (N-hydroxysuccinimide) were added, followed by reaction for 3 hours.
50μ of N-Boc-2, 2-(ethylenedioxy) diethylamine was added to the mixture above, followed by reaction at room temperature for 12 hours (Compound 1,
A mixture of 5 ml of TFA (trifluoroacetic acid) and 5 ml of DCM (dichloromethane) at the ratio of 1:1 was added thereto, followed by reaction at 0° C. for 3 hours (Compound 2,
90 mg of gallic acid was dissolved in 0.3 M NaHCO3, to which 126 mg of EDC and 100 mg of NHS were added, followed by reaction at room temperature for 3 hours. DCM and TFA were eliminated using a defreezer. The mixture above was mixed with Compound 2, followed by reaction for 12 hours. Unreacted gallic acid, EDC and NHS were eliminated by using a dialysis bag (molecular weight cutoff: 2000).
Hyaluronic acid-gallic acid conjugate prepared in step 1 dissolved in PBS (phosphate buffered saline, 15 mg/ml) was mixed with iron chloride dissolved in PBS (phosphate buffered saline, 5 mg/ml) at the ratio of 1:1 (v/v). As soon as the two solutions were added, hydrogel was formed as they were mixed.
<1-1> Experiment Method
50μ of the catechol/iron ion coordination complex prepared in Example 1 was placed in an EP-tube. The tube was irradiated with 1.2 W 808 nm laser for 1 minute. Then, the temperature was measured using a thermal sensing camera.
<1-2> Experiment Result
The results are shown in
It was confirmed that the temperature of the catechol/iron ion coordination complex was raised to 60° C. or more.
<2-1> Experiment Method
50μ of the dopamine/iron ion coordination complex prepared in Example 2 was placed in an EP-tube. The tube was irradiated with 1.2 W 808 nm laser for 1 minute. Then, the temperature was measured using a thermal sensing camera.
<2-2> Experiment Result
The results are shown in
It was confirmed that the temperature of the catecholamine/iron ion coordination complex was raised to 60° C. or more.
<3-1> Experiment Method
50μ of the epigallocatechin gallate (EGCG)/iron ion coordination complex prepared in Example 3 was placed in an EP-tube. The tube was irradiated with 1.2 W 808 nm laser for 1 minute. Then, the temperature was measured using a thermal sensing camera.
<3-2> Experiment Result
The results are shown in
It was confirmed that the temperature of the epigallocatechin/iron ion coordination complex was raised to 60° C. or more.
<4-1> Experiment Method
50μ of the gallic acid/iron ion coordination complex prepared in Example 4 was placed in an EP-tube. The tube was irradiated with 1.2 W 808 nm laser for 1 minute. Then, the temperature was measured using a thermal sensing camera.
<4-2> Experiment Result
The results are shown in
It was confirmed that the temperature of the gallic acid/iron ion coordination complex was raised to 60° C. or more.
<5-1> Experiment Method
50μ of the tannic acid/iron ion coordination complex prepared in Example 5 was placed in an EP-tube. The tube was irradiated with 1.2 W 808 nm laser for 1 minute. Then, the temperature was measured using a thermal sensing camera.
<5-2> Experiment Result
The results are shown in
It was confirmed that the temperature of the tannic acid/iron ion coordination complex was raised to 60° C. or more.
<6-1> Experiment Method
Cancer cells (KB cells) were cultured in a 24 well plate, and the cultured cells were collected in an EP tube. Cell pellet was made using a centrifuge (1000 rpm, 3 minutes). 20μ of the supernatant was left, to which 40μ
of the catechol/iron ion coordination complex prepared in Example 1 dissolved in PBS was added. The mixture was irradiated with 1.2 W 808 nm laser for 5 minutes. After removing the supernatant, 400μ
of medium was added thereto. The cells were cultured again in a 24 well plate for a day. The effect on photothermal therapy was measured by MTT assay.
<6-2> Experiment Result
The results are shown in
As a result, when the coordination complex was irradiated with laser, the cell viability was reduced to 20% or less.
<7-1> Experiment Method
Cancer cells (KB cells) were cultured in a 24 well plate, and the cultured cells were collected in an EP tube. Cell pellet was made using a centrifuge (1000 rpm, 3 minutes). 20μ of the supernatant was left, to which 40μ
of the dopamine/iron ion coordination complex prepared in Example 2 dissolved in PBS was added. The mixture was irradiated with 1.2 W 808 nm laser for 5 minutes. After removing the supernatant, 400μ
of medium was added thereto. The cells were cultured again in a 24 well plate for a day. The effect on photothermal therapy was measured by MTT assay.
<7-2> Experiment Result
The results are shown in
As a result, when the coordination complex was irradiated with laser, the cell viability was reduced to 20% or less.
<8-1> Experiment Method
Cancer cells (KB cells) were cultured in a 24 well plate, and the cultured cells were collected in an EP tube. Cell pellet was made using a centrifuge (1000 rpm, 3 minutes). 20μ of the supernatant was left, to which 40μ
of the epigallocatechin gallate/iron ion coordination complex prepared in Example 3 dissolved in PBS was added. The mixture was irradiated with 1.2 W 808 nm laser for 5 minutes. After removing the supernatant, 400μ
of medium was added thereto. The cells were cultured again in a 24 well plate for a day. The effect on photothermal therapy was measured by MTT assay.
<8-2> Experiment Result
The results are shown in
As a result, when the coordination complex was irradiated with laser, the cell viability was reduced to 20% or less.
<9-1> Experiment Method
Cancer cells (KB cells) were cultured in a 24 well plate, and the cultured cells were collected in an EP tube. Cell pellet was made using a centrifuge (1000 rpm, 3 minutes). 20μ of the supernatant was left, to which 40μ
of the gallic acid/iron ion coordination complex prepared in Example 4 dissolved in PBS was added. The mixture was irradiated with 1.2 W 808 nm laser for 5 minutes. After removing the supernatant, 400μ
of medium was added thereto. The cells were cultured again in a 24 well plate for a day. The effect on photothermal therapy was measured by MTT assay.
<9-2> Experiment Result
The results are shown in
As a result, when the coordination complex was irradiated with laser, the cell viability was reduced to 20% or less.
<10-1> Experiment Method
Cancer cells (KB cells) were cultured in a 24 well plate, and the cultured cells were collected in an EP tube. Cell pellet was made using a centrifuge (1000 rpm, 3 minutes). 20μ of the supernatant was left, to which 40μ
of the tannic acid/iron ion coordination complex prepared in Example 5 dissolved in PBS was added. The mixture was irradiated with 1.2 W 808 nm laser for 5 minutes. After removing the supernatant, 400μ
of medium was added thereto. The cells were cultured again in a 24 well plate for a day. The effect on photothermal therapy was measured by MTT assay.
<10-2> Experiment Result
The results are shown in
As a result, when the coordination complex was irradiated with laser, the cell viability was reduced to 20% or less.
The hyaluronic acid-gallic acid conjugate synthesized in step 1 of Example 6 was dissolved in 1 m of D2O at the concentration of 5 mg/m
.
To confirm the synthesis of the hyaluronic acid-gallic acid conjugate, H NMR was analyzed up to 0-10 ppm by using 600 MHz NMR. As a result, hyaluronic acid peak was confirmed at 1.8-2.0 ppm, hydrogen peak was confirmed at 3.0-4.0 ppm, and gallic acid hydrogen (conjugate) peak was confirmed at 7.5 ppm. In addition, diethylamine hydrogen (linker) peak was confirmed at 2.8-2.9 ppm. From the above results, it was confirmed that the hyaluronic acid-gallic acid conjugate was successfully synthesized.
<12-1> Experiment Method
All moisture of the hydrogel formed in Example 6 was removed using a freeze-dryer and the weight of the dried hydrogel was measured.
TDW was added to the hydrogel, and the weight was measured at each time point using a balance. The temperature was fixed at 37° C. Swelling of the hydrogel was measured using the following equation. Swelling=(weightswelled hydrogel-weightdried hydrogel)/weightdried hydrogel×100%.
<12-2> Experiment Result
The results are shown in
As a result, it was confirmed that swelling was increased over the time up to 1500%.
<13-1> Experiment Method
Viscosity and viscoelasticity of the hydrogel formed in Example 6 were measured using a rotational rheometer. Viscosity, loss modulus and storage modulus were measured in the range between 0.1 and 50 Hz.
<13-2> Experiment Result
The results are shown in
As a result, it was confirmed that the hydrogel formed by hyaluronic acid-gallic acid conjugate and iron ions had viscosity and viscoelasticity.
<14-1> Experiment Method
The hydrogel formed in Example 6 was irradiated with 1.2 W 808 nm near infrared ray. Then, time dependent temperature changes were measured using a thermal imaging camera.
<14-2> Experiment Result
The results are shown in
As a result, when the formed hydrogel was irradiated with infrared laser, the temperature of the hydrogel was raised to 55° C. or more.
<15-1> Experiment Method
Cancer cells (KB cells) were cultured in a 24 well plate, and the cultured cells were collected in an Eppendorf tube.
Cancer cell pellet was made using a centrifuge (1000 rpm, 3 minutes). 40μ of the supernatant was left, to which the hydrogel formed in Example 6 was added.
The mixture was irradiated with 1.2 W 808 nm laser for 5 minutes. After removing the supernatant, 400μ of medium was added thereto. The cells were cultured again in a 24 well plate for a day. The effect on photothermal therapy was measured by MTT assay and live cell assay.
<15-2> Experiment Result
The results are shown in
As a result, when the hydrogel was irradiated with laser, the cell viability was reduced to 20% or less.
<16-1> Experiment Method
The hyaluronic acid-gallic acid conjugate prepared in step 1 of Example 6 dissolved in phosphate buffer (15 mg/ml) and iron chloride dissolved in phosphate buffer (5 mg/ml) were filled in two sections of a dual syringe, respectively, which was injected into the upper part of the right hind leg of Balb/c mouse. The mouse was then euthanized and the formation of hydrogel was confirmed using anatomical tools.
<16-2> Experiment Result
The results are shown in
As a result, it was confirmed that hydrogel was formed well under the mouse subcutis.
<17-1> Experiment Method
The hyaluronic acid-gallic acid conjugate prepared in step 1 of Example 6 dissolved in phosphate buffer (15 mg/ml) and iron chloride dissolved in phosphate buffer (5 mg/ml) were filled in two sections of a dual syringe, respectively, which was injected into the upper part of the right hind leg of Balb/c mouse.
After the in vivo injection, the hyaluronic acid-gallic acid conjugate and iron chloride formed hydrogel therein. The hydrogel was irradiated with 1.2 W 808 nm near infrared ray for 1 minute, and then the temperature changes were measured using a thermal imaging camera.
As the control, the hyaluronic acid-gallic acid conjugate prepared in step 1 of Example 6 dissolved in phosphate buffer (15 mg/ml) was injected into the upper part of the right hind leg of Balb/c mouse, followed by measurement of the temperature changes by the same manner as described above.
<17-2> Experiment Result
The results are shown in
As a result, compared with the control mouse subcutaneously injected with the hyaluronic acid-gallic acid conjugate alone, the photothermal effect was constantly observed in the experimental group mouse in which the hydrogel was formed.
Particularly, when near infrared ray was irradiated once a day for 4 days, the temperature was raised to 50° C. or more due to the sustainability of the hydrogel, confirming the photothermal effect.
<18-1> Experiment Method
A tumor was generated in the size of 100 mm3−200 mm3 by injecting 3×106 cancer cells (KB cell) in the upper part of the right hind leg of Balb/c mouse.
The hyaluronic acid-gallic acid conjugate prepared in step 1 of Example 6 dissolved in phosphate buffer (15 mg/ml) and iron chloride dissolved in phosphate buffer (5 mg/ml) were filled in two sections of a dual syringe, respectively, followed by intra tumoral injection.
Then, the tumor was irradiated with 1.2 W 808 nm near infrared ray for 5 minutes and the tumor size was measured every 3 days.
<18-2> Experiment Result
The results are shown in
As a result, it was confirmed that the tumor size was suppressed by the effect of the hydrogel formed by hyaluronic acid-gallic acid conjugate and iron ions on photothermal therapy.
<19-1> Experiment Method
The gallic acid/iron coordination complex prepared in Example 4 was mixed with hydrogel at the ratio of 5:10 (w/w). The prepared hydrogel and gallic acid/iron coordination complex mixture was applied thinly, followed by irradiation with 808 nm near infrared ray at the intensity of 0.5-0.75 W from the distance of 1 cm, 2 cm and 4 cm, respectively.
<19-2> Experiment Result
The results are shown in
<20-1> Experiment Method
E. coli (gram negative) and S. aureus (gram positive) were cultured and then diluted to optical density (OD) of 0.3. 100 μl of the diluted cells was loaded in an EP tube. Pellet was made using a centrifuge and the supernatant was removed. The coordination complex prepared in Example <2-5> was added thereto by 50μ, followed by irradiation with 1.2 W 808 nm laser for 5 minutes. The mixture was plated on an agar plate, followed by incubation for 24 hours.
<20-2> Experiment Result
The results are shown in
The composition of the present invention displays a remarkable effect on photothermal therapy since the temperature of the applied area can be raised at least 50° C. by near infrared ray irradiation, after the injection. The composition can be combined with a biocompatible material to have biocompatibility and can act selectively on a local site to minimize side effects. The composition also has an effect of continuous photothermal treatment because it is present in the administration site for a few days after injection. Therefore, the composition of the present invention can be used for anticancer treatment.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2017-0001171 | Jan 2017 | KR | national |
This application is a continuation of U.S. patent application Ser. No. 16/475,959, filed Aug. 6, 2019, which is 371 of PCT/KR2018/000063, filed Jan. 2, 2018 which claims the benefit of Korean Patent Application No. 10-2017-0001171, filed Jan. 4, 2017, the contents of each of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16475959 | Aug 2019 | US |
| Child | 17821471 | US |
