KR20120045155A - Polymer conjugated photosensitizer for photodynamic therapy and preparation method thereof - Google Patents

Abstract

PURPOSE: A polymer-photosensitizer conjugate for photodynamic therapy is provided to ensure biodegration and biocompatibility in vivo. CONSTITUTION: A polymer-photosensitizer conjugate for photodynamic therapy contains 3-diethylaminopropyl(DEAP), photosensitizer, and polyethylene glycol-grafted biocompatible polymers. The biocompatible polymers include glyol chitosan, hyaluronic acid, pullulan, curdlan, pectin, starch, dextran, chitosan, poly-L-lysine, or polyaspartic acid. The photosensitizer is chlorine e6, protoporphyrin 9, benzoporphyrin, monoasplchlorine, or mezotetrahydroxyphenyl chlorine.

Description

Polymer-conjugated photosensitizer for photodynamic therapy and preparation method

The present invention provides a polymer-photoresponder conjugate for photodynamic therapy that can selectively react in a disease site having weakly acidic conditions to increase the efficiency of generating active oxygen (monooxygen, radical oxygen) and enhance the therapeutic effect, and a method for preparing the same. It is about.

Although the incidence of cancer in modern people is increasing exponentially and accounts for a large part of the causes of death, chemotherapy, which is the most widely used treatment for cancer, has the advantage of directly attacking and removing cancer cells. This may occur, and there are various problems such as side effects appearing as well as cancer cells as well as the surrounding normal tissues, it is difficult to effectively treat the disease.

On the other hand, photodynamic therapy (PDT) is one of the recent cancer treatment methods.

The photoensitizer used in photodynamic therapy has little cytotoxicity when it is not exposed to light, but when light of a specific wavelength is irradiated, the light energy generated by the photoresponder is excited by the oxygen in the tumor tissue. The oxygen in the ground state generates reactive oxygen species (singlet oxygen, oxygen radicals, superoxide and peroxide) that are highly chemically reactive. These reactive oxygen species start chemically destroying the surrounding cellular components and vascular tissues and proceed to apoptosis and necrosis. In addition, such photoresponders have a property of specifically accumulating in cancer tissues after intravenous administration, and when a certain wavelength of light is irradiated after a certain time, only cancer tissues may be necrotic and normal tissues may be preserved. Therefore, photodynamic therapy has a great advantage in alleviating side effects compared with general chemotherapy. As such, photodynamic therapy has the advantage of selectively removing only cancer cells while preserving normal cells, and most of them can eliminate the risk of general anesthesia and can be easily treated with simple local anesthesia. have. Photodynamic therapy with this advantage has been studied in earnest since the 1980s, and in the 1990s, the US FDA approved the treatment of esophageal cancer in January 1996 since the clinical procedure was approved in Canada, Germany and Japan. It is widely used worldwide in September, with approvals for early lung cancer treatment.

However, photodynamic therapy currently in use is not used for bulky tumors due to the restriction of light transmission, and in particular, low concentrations of photoresponders in tumors do not show an effective therapeutic effect. One of the important factors defining the efficiency of photodynamic therapy is target or selectivity, which indicates the extent of selective accumulation of photoresponders only in tumor tissues in tumor tissues and normal tissues. However, existing photoresponders have low selectivity and accumulation to tumors, and in a few cases, they also act on normal sites and cause side effects. Therefore, various efforts are being made to effectively deliver photoresponders.

Therefore, there is a demand for the development of a new photoresponder for reducing the side effects by increasing tumor selectivity and efficient treatment by laser.

In order to overcome the problems of most photoresponders having low selectivity and to effectively deliver the photoresponder to the disease site, the present inventors have introduced a photoresponder and a pH sensitive material into an amine group of a biocompatible and biodegradable polymer. The conjugate reacts even at small pH changes and decomposes in weakly acidic conditions, thereby reducing the weakly acidic environment. Eggplant has been found to be able to increase the efficiency of the generation of mono-antioxidants while high selectivity and accumulation rate for the disease and completed the present invention.

It is therefore an object of the present invention to provide a polymer-photoresponder conjugate for photodynamic therapy in which biocompatible and biodegradable chitosan derivatives, photoresponders, pH sensitive substances and polyethylene glycol are organically bound.

Another object of the present invention is to provide a method for preparing a polymer-photoresponder conjugate for photodynamic therapy.

In order to achieve the above object, the present invention is a polymer for photodynamic therapy comprising a biocompatible polymer grafted with 3-diethylaminopropyl (DEAP), a photoresponder and polyethylene glycol (grafting) Provide a photoresponder conjugate.

In one embodiment of the present invention, the biocompatible polymer is glycol chitosan, hyaluronic acid, pullulan, curdlan, pectin, starch, dex It may be selected from the group consisting of dextran, chitosan, poly-L-lysine and polyaspartic acid.

In one embodiment of the present invention, the photoresponsive agent may be selected from the group consisting of chlorine e6, protopophyrin 9, bonerine, benzoporphyrin, mono aspartyl chlorine and meso tetrahydroxyphenyl chlorine.

In one embodiment of the present invention, the biocompatibility The pH-sensitive substance may be substituted with 0.1 to 0.9 per primary amine of the polymer, the photoresponder may be substituted with 0.05 to 0.5, and the polyethylene glycol may be substituted with 0.01 to 0.5.

In one embodiment of the present invention, the polymer-photoresponder conjugate is selectively accumulated in the weakly acidic disease sites in the range of pH 6.0 ~ 7.0, it can generate free radicals by light irradiation.

In one embodiment of the present invention, the weakly acidic disease is any one selected from myopathy such as myasthenia gravis, Lou Gehrig's disease and muscular dystrophy, chronic obstructive pulmonary disease, rheumatoid arthritis and solid cancer Can be.

In addition, the present invention comprises the steps of dissolving the biocompatible polymer in a solvent; Adding 3-diethylaminopropyl to the solution in which the biocompatible polymer is dissolved to bind the amine group of the biocompatible polymer to the carboxyl group of the 3-diethylaminopropyl; Substituting the carboxyl group of the photoresponder and polyethylene glycol with succinimide and adding the solution to the amine group of the biocompatible polymer; And it provides a method for producing a polymer-photoresponder conjugate for photodynamic therapy comprising the step of dialysis and freeze-drying the combined conjugated conjugate.

In one embodiment of the present invention, 1 to 3 mol of 3-diethylaminopropyl, 0.5 to 1.5 mol of photoresponder, and 0.5 to 2 mol of polyethylene glycol may be added per 1 mol of each repeating chain of the biocompatible polymer.

In one embodiment of the present invention, the biocompatible polymer is glycol chitosan, hyaluronic acid, pullulan, curdlan, pectin, starch, dex Consisting of dextran, chitosan, poly-L-lysine and polyaspartic acid Can be selected from the group.

In one embodiment of the present invention, the photoresponsive agent may be selected from the group consisting of chlorine e6, protopophyrin 9, bonerine, benzoporphyrin, mono aspartyl chlorine and meso tetrahydroxyphenyl chlorine.

The polymer-photoresponder conjugate according to the present invention can ensure biodegradability and biocompatibility in vivo based on a biocompatible polymer, and selectively act in weakly acidic conditions due to the use of a pH-sensitive substance to the disease site. The photoresponder can be delivered quickly to increase selectivity and accumulator, and can increase the efficiency of photodynamic therapy by increasing the generation efficiency of active oxygen (single oxygen, radical oxygen) generated during laser irradiation.

Figure 1 shows the synthesis of GCS- g -DEAP- g -Ce6- g -PEG in accordance with an embodiment of the present invention.
2 is a photodynamic therapy using a GCS- g -DEAP- g -Ce6- g -PEG conjugate according to an embodiment of the present invention The principle is shown.
Figure 3 shows the ¹ H-NMR peak of the GCS- g -DEAP- g -Ce6- g -PEG conjugate according to an embodiment of the present invention.
Figure 4 is a graph showing the particle size distribution of the GCS- g -DEAP- g -Ce6- g -PEG conjugate according to an embodiment of the present invention. (a) is pH 7.4, (b) is pH 6.8.
5 is a FE-SEM image showing the shape of the GCS- g -DEAP- g -Ce6- g -PEG conjugate according to an embodiment of the present invention. (a) is pH 7.4, (b) is pH 6.8.
Figure 6 is a graph showing the change in zeta potential according to the pH of the GCS- g -DEAP- g -Ce6- g -PEG conjugate according to an embodiment of the present invention.
FIG. 7 is a fluorescence image for examining changes in pH of a GCS- g -DEAP- g -Ce6- g -PEG conjugate and a GCS- g -Ce6- g -PEG conjugate (control) according to an embodiment of the present invention. to be.
Figure 8 is a mono-oxygen of (a) GCS- g -DEAP- g -Ce6- g -PEG conjugate and (b) GCS- g -Ce6- g -PEG conjugate according to the pH change according to an embodiment of the present invention The generation amount is measured by changing the fluorescence intensity of 9,10-dimethylanthracene (DMA). pH 7.4 (●, black), pH 6.8 (■, red), pH 6.4 (▲, blue).
9 shows GCS- g- DEAP- g- Ce6- g- PEG conjugate (shown in black) and free Ce6 (control, shown in white) in human epithelial ovarian cancer HeLa cells according to one embodiment of the present invention. It is a graph showing the change of phototoxicity according to the pH change in each treatment.
10 was treated with GCS- g- DEAP- g- Ce6- g- PEG conjugate (1 to 400 μg / ml) for 24 hours without light irradiation on human epithelial ovarian cancer HeLa cells according to one embodiment of the present invention. It is a graph showing the cytotoxicity when.
FIG. 11 shows apoptosis according to pH change when human epidermal ovarian cancer HeLa cells were treated with GCS- g -DEAP- g -Ce6- g- PEG conjugate and free Ce6 (control), respectively, according to an embodiment of the present invention. (fluorescence image showing the level of (apoptosis).
FIG. 12 shows GCS- g -DEAP- g -Ce6- g- PEG conjugate and free Ce6 (control) at pH 7.4, pH 6.8 and 6.4, respectively, in human epithelial ovarian cancer HeLa cells according to one embodiment of the present invention. When is an optical micrograph.
13 is in accordance with one embodiment of the present invention To confirm the effect in vivo , (a) GCS- g -DEAP- g -Ce6- g -PEG (right), GCS- g -Ce6- g -PEG (middle) and free Ce6 (left) were respectively applied to HeLa. Fluorescence images were obtained 30 minutes after intravenous administration to tumor nude mice. In addition, (b) GCS- g -DEAP- g -Ce6- g -PEG and (c) GCS- g -Ce6- g -PEG is a fluorescence image obtained every hour to see the change over time after administration.
Figure 14 is an optical micrograph measured to confirm the tumor size of HeLa tumor nude mouse when treated with GCS- g -DEAP- g -Ce6- g -PEG according to an embodiment of the present invention.
FIG. 15 is a graph showing tumor volume changes of HeLa tumor nude mice when treated with GCS- g -DEAP- g -Ce6- g- PEG and free Ce6 (control) according to an embodiment of the present invention.

The present invention effectively delivers photoresponders to disease sites showing weak acidity, thereby increasing the selectivity and accumulation of photoresponders, and polymer-photoresponse for photodynamic therapy that can selectively act only on disease sites without acting on other sites. It relates to a first conjugate.

In most solid cancers, most of the cancer cells are hypoxic, so they look for anaerobic glycolysis and release acidic substances, such as lactic acid, out of the cells, so that the external microstructure of the cancer cells is different from the normal tissue pH of 7.4. The environment differs slightly in acidity (pH 6.5 to 7.0) (Mol Med Today. 2000, 6, 15-19).

Therefore, the development of polymer conjugated smart photoresponders for photodynamic therapy acting according to pH changes not only reduces side effects on normal tissues, but also provides a new route to effectively deliver photoresponders to tumor tissues.

Recent studies have shown that nanotransmitters containing photoresponders are sensitive below pH 6.0 to effectively deliver photoresponders (CL Peng, LY Yang, TY Luo, PS Lai, SJ Yang, WJ Lin). , MJ Shieh, Nanotechnology. 2010, 21, 155103-15513), a technique was developed in which the photoresponder was physically incorporated into the polymer micelle so that the photoresponder and the polymer micelle were completely separated in an environment of pH 6.5 or lower. (SJ Lee, K. Park, Y, K. Oh, SH Kwon, S. Her, IS Kim, K. Choi, SJ Lee, H. Kim, SG Lee, K. Kim, IC Kwon, Biomaterials. 2009, 30 , 2929-2939).

Accordingly, the present inventors have prepared a new polymer-photoresponder conjugate by chemical bonding between the photoresponder and the polymer material. These polymer conjugated smart photoresponders can be injected into the body without loss of photoresponders caused by the physical incorporation of photoresponders into nanocarriers and weakly acidic (pH 6.8) It is formulated to be sensitive to conditions so that cancer cells can be treated more effectively.

In the present invention, in order to prepare a polymer conjugated smart photoresponder that can be selectively treated in a weak acid, a micro-environment outside the cancer cell, a biochemically and biodegradable polymer and an organic chemical combination of a photoresponder, a pH-sensitive substance and polyethylene glycol are newly synthesized. Polymer-photoresponder conjugates were synthesized. In particular, a pH-sensitive functional group capable of recognizing the surrounding environment of solid cancer cells is characterized in that it is prepared to act in a weakly acidic environment by bonding to a biocompatible and biodegradable polymer.

More specifically, the present invention relates to a polymer-photoresponder for photodynamic therapy including 3-diethylaminopropyl (DEAP), a photoresponder and a biocompatible polymer grafted with polyethylene glycol. Provide the conjugate.

The polymer-photoresponder conjugate enhances the efficacy of cancer treatment through photodynamic therapy by specifically accumulating the photoresponder by reacting specifically in the weak acid disease site, which is an external microenvironment of cancer cells.

In addition, the present invention uses a biodegradable biodegradable and biocompatible polymer to prepare a polymer-photoresponder conjugate.

"Biodegradable" means that the block copolymer can be chemically degraded in the body to form non-toxic compounds. The rate of degradation is the same or different than the rate of drug release. And "biocompatible" refers to the nature of interacting with the body without undesirable subsequent effects.

The biodegradable polymers used in the present invention include, but are not limited to, glycol chitosan, hyaluronic acid, pullulan, curdlan, pectin, starch, starch. , Biodegradable polymers and derivatives thereof selected from the group consisting of dextran, chitosan, poly-L-lysine and polyaspartic acid, preferably chitosan derivatives, and more Preferably, glycol chitosan, which is a chitosan derivative of the following structural formula, may be used.

Chitosan is a generic term for compounds obtained by deacetylating chitin, a natural polymer material rich in cellulose after nature. It is 2-amino-2-dioxy-β-D-glucopyrano It is a polysaccharide composed of 2-amino-2-deoxy-β-D-glucopyranose. Unlike other polysaccharides present in nature, chitosan contains primary amines in its main chain, and because of this, it has very unique properties and is applied in various fields such as environment, agriculture, and medicine. Especially, it has excellent biocompatibility and biodegradability. As a component of genes and drug carriers, scaffolds for tissue engineering, injectable hydrogels, etc., they have been the subject of intensive research in medicine.

The chitosan exhibits a sol-gel transition characteristic at a specific pH, and gelation occurs at pH 7.0 to 7.4, which is similar to that in the body, and solvates below the range, thereby preventing clogging of the injection needles seen in conventional hydrogels. Gels can be formed safely in the body without occurrence.

In the present invention, the photoresponder may be selected from the group consisting of chlorine e6, protopophyrin 9, bonerine, benzoporphyrin, monoaspartylchlorine and meso tetrahydroxyphenyl chlorine, and is preferably used for light of a specific wavelength. Chlorin e6 with high photochemical activity can be used.

Meanwhile, in the present invention, 3-diethylaminopropyl (DEAP) having a structure of Chemical Formula 1 may be used as a pH sensitive material.

The DEAP block has a tertiary amine and thus may exhibit sensitivity to pH in the body, particularly at weak acidity (pH 6.8). The tertiary amine group has ionization characteristics in which the solubility in water varies depending on pH, thereby forming a gel or maintaining a sol state according to pH change. The tertiary amine groups are chemically unreactive and typically exhibit reduced toxicity compared to secondary or primary amine groups in normal tissues. In addition, unlike primary or secondary amine groups, tertiary amines are not converted to acyl derivatives bound to carboxylic acids of biological proteins.

In the present invention, the organic combination of the biocompatible polymer and the pH sensitive material, the photoresponsive agent and the polyethylene glycol is characterized by the reaction of the free amine group of the highly reactive biodegradable polymer by reacting with the carboxyl group of the pH sensitive material, the photoresponsive agent and the polyethylene glycol. Responder conjugates can be synthesized.

In particular, by coupling the 3-diethylaminopropyl component having pH sensitivity, a conjugate capable of biodegradation in the human body and complete discharge into the body can be produced.

In one embodiment of the present invention, by grafting 3-diethylaminopropyl (DEAP) as a pH-sensitive material to glycol chitosan (GCS), chlorine e6 (Ce6) and polyethylene glycol as a photoresponder (grafting) GCS- g- DEAP - g - Ce6 - g - PEG, a polymer-photoresponder conjugate that selectively reacts to disease sites under conditions, was prepared.

In the polymer-photoresponder conjugate according to the present invention, the substitution degree of pH sensitive material per primary amine of the biocompatible polymer is 0.1 to 0.9, the substitution degree of the photoresponder is 0.05 to 0.5, and the degree of substitution of polyethylene glycol is 0.01. It is preferably in the range of ˜0.5, and most preferred is a case where the degree of substitution of the pH sensitive material is 0.40, the degree of substitution of the photoresponder is 0.20, and the degree of substitution of polyethylene glycol is 0.38. This is because an optimal conjugate capable of successfully delivering a photoresponder in the range of the substitution degree can be formed.

The polymer-photoresponder conjugate of the present invention is self-organized in an aqueous solution, spherical magnetic in water due to amphiphilicity by hydrophobic groups of the pH-sensitive DEAP and Ce6 group and the hydrophilic group of the biocompatible polymer and polyethylene glycol group The assembly can be formed.

In general, self-assembling forms spherical aggregates in which amphiphilic molecules having both hydrophilicity and hydrophobicity associate in an aqueous solution. Hydrophilic groups are collected outside the spherical aggregate and hydrophobic groups are collected inside. In the present invention, the GCS and PEG blocks are located in the hydrophilic outer shell and DEAP and Ce6 are located in the hydrophobic inner core, in particular, the hydrophobicity of the DEAP block is the ionization of the DEAP block under acidic conditions (~ pK _b ). It will depend on the pH of the solution in that it will increase. When such polymer-photoresponder conjugates face the tumor environment, the GCS- g- DEAP - g - Ce6 - g - PEG is changed to a partially loosened structure.

In addition, the polymer-photoresponder conjugate according to the present invention may be destabilized in a weakly acidic environment of pH 7.0 or less, that is, the structure of the polymer-photoresponder conjugate may be changed so that the photoresponder encapsulated inside the conjugate may be released to the outside. .

According to one embodiment of the present invention, the particle size of the GCS- g -DEAP - g - Ce6 - g - PEG conjugate is significantly reduced as the pH is changed from pH 7.4 to pH 6.8, which is due to the loosening of the structure of the conjugate in a weakly acidic environment. Because.

Therefore, the GCS- g- DEAP - g - Ce6 - g - PEG conjugate is characterized by the ability to specifically react to the disease site showing a weak acid pH of less than 7.0, preferably a site showing a weak acidity of pH 5.0 to 7.0 Can react specifically to Most preferably, it can react in a weakly acidic environment of pH 6.0-7.0.

Therefore, the polymer-photoresponder conjugate of the present invention selectively reacts at about pH 6.0 to 7.0, which is a weakly acidic condition formed in a disease site such as cancer. That is, at neutral pH, the DEAP block can serve as a reservoir for hydrophobic photoresponders, whereas in an acidic pH environment, the DEAP block causes degradation of the conjugate due to protonation of the inner core, As a result, the photoresist is released out of the conjugate.

Therefore, the polymer-photoresponder conjugate containing the pH-sensitive DEAP group of the present invention has a high selectivity for cancer tissues, and thus, a larger amount can be selectively accumulated in cancer tissues to exert an effective photodynamic therapeutic effect.

In one embodiment of the present invention, a change in zeta potential at different pH conditions of the GCS - g -DEAP - g - Ce6 - g - PEG conjugate was observed. As a unit of attraction size, it is used as an important factor in controlling static dispersion. The zeta potential of the GCS- g -DEAP - g - Ce6 - g - PEG conjugate of the present invention was measured at about -8.0 mV at pH 7.4, and as the pH was lowered, the zeta potential increased to +1.3 mV at pH 6.8. Was measured. These results indicate that the GCS- g -DEAP - g - Ce6 - g - PEG conjugate was destabilized under weakly acidic conditions, and the exposure of the cation increased as the cation increased in the conjugate due to decomposition of the assembly. In other words, this means that the negative zeta potential generated from GCS and PEG at pH 7.4 is offset by the protonation of the DEAP block at pH 6.8.

In addition, in one embodiment of the present invention, the morphological images obtained from FE-SEM for the GCS - g -DEAP - g - Ce6 - g - PEG conjugate are also almost spherical GCS- g -DEAP - g- Ce6 at pH 7.4. g- PEG conjugates become unstable and partially disappear at pH 6.8. That is, the structure of the GCS - g- DEAP - g- Ce6 - g- PEG conjugate remains stable at neutral conditions of pH 7.4, but the structure of the conjugate is degraded at weakly acidic conditions of pH lower than physiological pH.

The polymer-photoresponder conjugate according to the present invention secures biocompatibility and biodegradability by using biocompatible and biodegradable polymer derivatives, and the photoresponder and pH sensitive functional group are organic chemically bonded to the site to express weak acidity. Can only target tumor tissue effectively. In addition, the polymer-photoresponder conjugate of the present invention acts sensitively under weakly acidic conditions due to the use of a pH sensitive material so that the structure of the conjugate collapses (i.e., 3-diethylaminopropyl, a pH sensitive material, is affected by a change in pH). Chemically modified to eventually expose the amine group of glycol chitosan and ultimately modify the structure of the conjugate as the ionic bonds dissociate), thereby delivering a photoresponder present within the nanocomposite to the target site in a short time. And 670 nm, a specific wavelength at which the photoresponsive chlorine e6 can exhibit photoactivity. Irradiation of the laser can increase the efficiency of the generation of active oxygen (single oxygen, radical oxygen) through the photoresponder to enhance the photodynamic therapy effect on the disease site.

Therefore, the polymer-photoresponder conjugate of the present invention can increase the therapeutic effect by selectively and efficiently delivering a drug to a specific disease or a disease such as acidosis having an environmental change of a weakly acidic condition, and in particular, changes the environmental change of the weakly acidic condition. Eggplants can be used to treat certain diseases, such as myotitis, Lou Gehrig's disease and muscular dystrophy, such as neuromuscular diseases, chronic obstructive pulmonary diseases, rheumatoid arthritis and solid cancer.

In the present invention, "solid cancer" refers to a cancer composed of all clumps except blood cancers, and cancer is defined as a disease characterized by fast and uncontrolled growth of abnormal cells, and cancer cells are spread locally or in the bloodstream and It can spread through the lymphatic system to other parts of the body. Types of solid cancer include, but are not limited to, stomach cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, fallopian tube carcinoma, vaginal carcinoma, vulvar carcinoma, pancreatic cancer, kidney cancer, bladder cancer , Urethral wall, prostate cancer, testicular cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colon cancer, anal cancer, colon cancer, rectal cancer, brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, brain stem tumor, Cerebrospinal tumors, head and neck cancers, bone cancer, thymoma, mesothelioma, germ cell tumor, multiple myeloma, sarcoma, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, renal pelvic carcinoma, glioma cancer, central nervous system (CNS) tumor, hematopoietic tumor , Fibrosarcoma, neuroblastoma, astrocytoma, lymphoma, malignant melanoma, oral cancer, nasopharyngeal cancer, nasal / sinus cancer, skin cancer and the like.

The present invention further provides a method for preparing a polymer-photoresponder conjugate for photodynamic therapy according to the present invention.

The polymer-photoresponder conjugate for photodynamic therapy according to the present invention can be prepared by the organic synthesis of chitosan derivatives, photoresponders, pH sensitive materials, polyethylene glycol.

The polymer-photoresponder conjugate for photodynamic therapy of the present invention may be prepared using a method apparent to those skilled in the art, but preferably (a) dissolving the biocompatible polymer in a solvent; (b) adding 3-diethylaminopropyl to the solution in which the biocompatible polymer is dissolved to bind the amine group of the biocompatible polymer to the carboxyl group of the 3-diethylaminopropyl; (c) substituting the photoresponder and the carboxyl group of polyethylene glycol with succinimide and adding the solution to the amine group of the biocompatible polymer; And (d) dialysis and lyophilizing the conjugated and synthesized conjugate.

In the manufacturing method of the polymer-photoresponder conjugate according to the present invention, any solvent capable of dissolving the biocompatible polymer may be used as long as it is a solvent capable of dissolving the biocompatible polymer, preferably dimethyl sulfoxide Side (DMSO) can be used.

In addition, 3-diethylaminopropyl (DEAP) added to the solution in which the biocompatible polymer is dissolved may be added in an amount of 1 to 3 mol per mole of the biocompatible polymer, and triethylamine, pyridine, etc., to the reaction solution. May be added to bond the amine group of the biocompatible polymer with the carboxyl group of 3-diethylaminopropyl.

Next, after replacing the carboxyl group, which is a reactive group between the photoresponder and polyethylene glycol, with succinimide, the photoresponder in the reaction solution is 0.5 to 1.5 mol per mol of the biocompatible polymer, and polyethylene glycol per mol of the biocompatible polymer. It is added in an amount of 0.5 to 2 mol to combine with the amine group of the biocompatible polymer. The reaction is then dialyzed and lyophilized to obtain the polymer-photoresponder conjugate.

The synthesized material spontaneously forms hydrophobic centers and hydrophilic surfaces at physiological pH, so that biocompatible polymers and polyethylene glycols are concentrated on the hydrophilic surfaces, and photoresponders and 3-diethylaminopropyl are concentrated on the hydrophilic surfaces. The ionization of diethylaminopropyl releases entanglement between the polymer chains and releases denseness between the photoresponders. As a result, when the polymer conjugated photoresponder, which is concentrated in the hydrophobic center and interacts with the photoresponders, contacts the weakly acidic environment when the polymer conjugated photoresponder, which has suppressed the photoresponse efficiency, disappears, the interaction between the photoresponders decreases. The optical response efficiency will be enhanced. In particular, when chlorine e6 used as a photoresist in one embodiment is irradiated with a 670nm laser having a photoactivity to a specific cancer site, it is possible to selectively generate a single oxygen only in the cancer site to enhance the therapeutic effect.

In an embodiment of the present invention, glycol chitosan was used as a biocompatible polymer, and 3-diethylaminopropyl (DEAP) was used as a pH-sensitive substance. First, glycol chitosan and 3-diethylaminopropyl (DEAP) were used. GCS- g -DEAP was synthesized. Subsequently, the carboxyl group of chlorine e6 and polyethylene glycol, a photoresponder, was substituted with succinimide, and then reacted with glycol chitosan to synthesize a GCS - g -DEAP - g - Ce6 - g - PEG conjugate.

Synthesis of the GCS- g- DEAP - g - Ce6 - g - PEG conjugate is shown in Figure 1, the free amine group of glycol chitosan (GCS) isothiocyanate group of 3-diethylaminopropyl (DEAP) Reaction with (isothiocyanate) can be synthesized by introducing 3-diethylaminopropyl (DEAP) to the amine group, wherein the reaction can be reacted for 5 to 7 days at room temperature. Next, the carboxyl group, which is a reactive group of chlorine e6 and polyethylene glycol, is substituted with succinimide as a photoresponder, and then added and reacted at room temperature for 1 to 2 hours to bind a free amine group of glycol chitosan.

The synthesized polymer-photoresponder conjugate can be obtained in a powder state through dialysis and freeze-drying, and has a negative charge (-) when dissolved in an aqueous phase.

In general, the body's metabolic process must be done quickly to remove a large amount of acid produced in the body to maintain the normal concentration of hydrogen ions. However, this impairment in maintenance function causes acidosis when the hydrogen ion concentration is lowered below 7.35, and alkaline symptoms when raised above 7.45. In addition, the acidosis (acidosis) is one of the symptoms seen when the disease occurs, myopathy (neuromuscular diseases, such as myopathy, Lou Gehrig's disease and muscular dystrophy), chronic obstructive pulmonary disease, rheumatoid arthritis and solid cancer The same disease area is characterized by weak acidity, in particular the extracellular pH of solid cancer is 7.2 to 6.0.

However, the polymer-photoresponder conjugate according to the present invention has a pH of 7.0 There is a characteristic that can specifically react to the disease site showing the weak acidity. Preferably it may specifically react to the site showing a weak acidity of pH 5.0 to 7.0. More preferably, it can react specifically at pH 6.0-7.0, most preferably pH 6.8.

The photoresponder emission principle of the polymer-photoresponder conjugate of the present invention is neutral pH, that is, pH 7.4, as shown in Figure 2, the photoresponder material is spherical by ionic bonding inside the polymer synthesized in the present invention When the nanoparticles are formed and the pH is lowered to become a weakly acidic condition, ionic bonds are not balanced due to an increase in cations, and the photoresist existing inside is released to the outside as the ionic bonds dissociate.

As such, the polymer-photoresponder conjugate of the present invention is based on the principle that the conjugate can be decomposed to release the drug at about pH 6.8, which is a weakly acidic condition formed at the disease site.

Therefore, the polymer-photoresponder conjugate of the present invention can rapidly release the photoresponder present therein through structural modification of the complex under conditions of pH 7.0 which is weakly acidic.

In another aspect, the polymer-photoresponder conjugate according to the present invention may be used as a pharmaceutical composition for photodynamic therapy.

The term “treatment”, unless stated otherwise, means to reverse, alleviate, inhibit the progression of, or prevent the disease or condition to which the term applies, or one or more symptoms of the disease or condition. The term "treatment" as used herein refers to the act of treating when "treating" is defined as above. Thus, the “treatment” or “therapeutic regimen” of diseases with environmental changes in mildly acidic conditions in mammals may include one or more of the following:

(1) inhibits the growth of diseases with environmental changes in weakly acidic conditions, i.e. inhibits their development,

(2) preventing the spread of diseases with environmental changes in weakly acidic conditions, i.e., preventing metastasis,

(3) alleviate diseases with environmental changes in weakly acidic conditions.

(4) prevent recurrence of diseases with environmental changes in weakly acidic conditions, and

(5) alleviating the symptoms of diseases with environmental changes in weakly acidic conditions;

The pharmaceutical composition for treating photodynamics according to the present invention may include a pharmaceutically effective amount of a polymer-photoresponder conjugate or a salt thereof alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents. The pharmaceutically effective amount herein refers to an amount sufficient to prevent, ameliorate, and treat a symptom of a disease having environmental changes in weakly acidic conditions.

According to the invention Polymer-Photoresponders The pharmaceutically effective amount of the conjugate or salt thereof may be 0.01 to 100 mg / day / kg body weight. However, the pharmaceutically effective amount may be appropriately changed according to the degree of symptoms of immune disease, the age, weight, health condition, sex, route of administration and duration of treatment of the patient.

In addition, the pharmaceutically acceptable refers to a composition that is physiologically acceptable and does not cause an allergic reaction such as gastrointestinal disorders, dizziness or the like when administered to humans. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.

In addition, the compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

In addition, the composition according to the present invention can be administered orally or parenterally according to the desired method, the dosage is the weight, age, sex, health status, diet, time of administration, method of administration, excretion rate and disease of the patient. It may be appropriately changed depending on the severity and the like.

The compositions of the present invention can be delivered by injection or other means, such as implantation, celiac or possible space, tissue of the body, as appropriate for a person or other mammal with a therapeutically effective disease state or condition, depending on the desired method. By coating the surface or by coating the surface of the implantable device), in particular, the composition is preferably delivered parenterally. By parenteral means intramuscular, intraperitoneal, intraperitoneal, subcutaneous, intravenous and intraarterial. Therefore, compositions comprising the polymer-photoresponder conjugates of the present invention may typically be formulated into injectable formulations.

Injectable compositions of the invention may be injected or inserted into the body of a human or other mammal by any suitable method, preferably by injection through a hypodermic needle.

For example, by injection or in other ways, intraarterial, intravenous, genitourinary, subcutaneous, Intramuscular, subcutaneous, intracranial, intracardiac, pleural, or other body cavity or possible space may be administered. Alternatively, via a catheter or syringe, for example, during arthroscopy, intraarticularly, into the urogenital tract, into the vasculature, into the palate or into the pleura, or into the body It can be introduced during any surgical, surgical, diagnostic or interventional procedure, into any body cavity or space available.

In addition, the compositions of the present invention can be used alone or in combination with methods using surgery, hormonal therapy, drug treatment and biological response modifiers for the treatment of diseases with environmental changes in weakly acidic conditions.

Hereinafter, the present invention will be described in detail with reference to the following examples and accompanying drawings. The following examples are only intended to describe the present invention in more detail, and the protection scope of the present invention is not limited to the examples described in the following examples. In addition, not only the following examples but also matters that can be easily inferred by those skilled in the art are obviously included in the protection scope of the present invention.

Material Preparation

First, the following materials were prepared to prepare polymer-photoresponder conjugates for photodynamic therapy.

Glycol chitosan (GCS, Mw = 86kDa), triethylamine (TEA), dimethylsulfoxide (DMSO), 3-diethylaminopropyl isothiocyanate (DEAP), 9,10-dimethylanthracene (DMA), N-hydroxysuccinimide (NHS), and N, N'-dicyclohexylcarbodimide (DCC) are Sigma-Aldrich (USA). ) And chlorine e6 (Ce6) was purchased from Frontier Scientific Inc. (USA).

Carboxylate Polyethyleneglycol (PEG-COOH, Mw = 2kDa) has been reported by our inventors (ES Lee, Z. Gao, D. Kim, K. Park, IC Kwon, YH Bae, J. Control.Release 2008, 129 , 228-236).

DMEM (Dulbecco's Modified Eagle's Medium), FBS (fetal bovine serum), penicillin (penicillin) and streptomycin were purchased from Welgene Inc. (Korea).

Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies Inc (Japan) and used, and Annexin V-FITC fluorescence microscopy kit was purchased from BD Pharmingen (TM).

< Example  1>

GCS - g - DEAP - g - Ce6 - g - PEG Synthesis of

First, glycol chitosan (GCS) (0.01 mM) was dissolved in 40 ml of DMSO, and then reacted for 1 week at room temperature using 2 ml of triethylamine (TEA) and 0.1 ml of pyridine, and grafted DEAP (2 mM) to GCS. g- DEAP was prepared.

The GCS- g- DEAP solution was pre-activated with DCC (1.2 mM) and NHS (1.2 mM) in 10 ml of DMSO and mixed with Ce6 (1.1 mM) substituted with succinimide for 2 days to react. Then, PEG-COOH (3 mM) in which carboxyl group was substituted with succinimide by preactivation with DCC (3.2 mM) and NHS (3.2 mM) in 5 ml of DMSO. After 3 days of reaction, the solution was transferred to a pre-swollen dialysis membrane tube (Spectra / Por MWCO 15K) and dialyzed against PBS pH 5.0 solution to remove unreacted DEAP, Ce6, PEG-COOH, and NHS. The resulting solution was then filtered to remove dicyclohexyurea, a byproduct of DCC, and then lyophilized under reduced pressure. Then, the degree of substitution (number of DEAP or Ce6 or PEG blocks substituted per primary amine of GCS) is given as δ 1.2 (-CH ₃ , DEAP block) or δ 7.6 (-CH-, Ce6 block). Or ¹ H-NMR (DMSO-d ₆ with TMS) peak using ratio of introduction of peaks derived from δ 3.54 (-CH ₂ , PEG block) and δ2.73 (-CH-, a repeating sugar unit of GCS) Measured by analyzing

As a result, as shown in FIG. 3, the degree of substitution analyzed by ¹ H-NMR was 0.40 for the DEAP block, 0.20 for the Ce6 block, and 0.38 for the PEG block. In addition, for GCS- g- Ce6- g- PEG (control) synthesized without DEAP as a control, the degree of substitution analyzed by ¹ H-NMR was 0.23 for Ce6 blocks and 0.43 for PEG blocks.

< Example  2>

In aqueous solution GCS - g - DEAP - g - Ce6 - g - PEG  Preparation of the conjugate

To prepare self-assembled GCS- g -DEAP- g -Ce6- g- PEG in aqueous solution, pre-swollen dialysis of GCS- g -DEAP- g -Ce6- g -PEG (50 mg) dissolved in 5 ml of DMSO Transferred to membrane tube (Spectra / Por MWCO 15K) and dialyzed against Na ₂ B ₄ O ₇ buffer solution for 24 hours. The outer phase was exchanged three times with fresh Na ₂ B ₄ O ₇ buffer solution (0.1 mM). The pH of Na ₂ B ₄ O ₇ buffer solution containing GCS- g -DEAP- g -Ce6- g -PEG was adjusted to pH 6.0-7.4 using PBS buffer (150 mM), 0.1M HCl or 0.1M NaOH. .

< Example  3>

In aqueous solution GCS - g - DEAP - g - Ce6 - g - PEG  Characteristics of the conjugate

In order to examine the physicochemical properties of the self-assembled GCS- g -DEAP- g -Ce6- g -PEG in the aqueous solution prepared in Example 2, the particle size distribution and shape were as follows. (morphology), zeta potential change was measured.

GCS- g- DEAP- g- Ce6- g- PEG is self-organized in aqueous solution, GCS and PEG blocks are located in the hydrophilic outer shell and DEAP and Ce6 are located in the hydrophobic inner core. In the face of the tumor environment, GCS- g -DEAP- g -Ce6- g- PEG is transformed into a partially unwrapped structure, which is characterized by the specific characteristics of GCS- g -DEAP- g -Ce6- g -PEG. It can be confirmed through to FIG.

<3-1> particle size distribution

The particle size distribution of GCS- g -DEAP- g -Ce6- g- PEG (0.1 mg / ml) at pH 7.4 or pH 6.8 in PBS (150 mM) solution was determined by Zetasizer 3000 (Malvern Instruments with He-Ne laser beam). , USA), at a wavelength of 633 nm and a fixed scattering angle of 90 °.

As a result, as shown in Figs. 4 (a) and 4 (b), the average particle size of GCS- g -DEAP- g -Ce6- g- PEG is 150 nm at pH 7.4 and 3.4 nm at pH 6.8. appear. As a result, as the pH was lowered from 7.4 to 6.8, it was found that the particle size of GCS- g -DEAP- g -Ce6- g -PEG decreased.

<3-2> Morphological observation

The shape of GCS- g -DEAP- g -Ce6- g -PEG was confirmed using a Field Emission Scanning Electron Microscope (FE-SEM, Hitachi s-4800, Japan). Briefly, a sample was prepared by casting a GCS- g- DEAP- g- Ce6- g- PEG solution (0.1 mg / ml) diluted in 150 mM PBS on a slide glass at pH 7.4 or 6.8, which was then vacuumed. Dried. The shape of each sample was imaged by FE-SEM.

As a result, as shown in Figs. 5 (a) and 5 (b), the shape of GCS- g -DEAP- g -Ce6- g -PEG showed the most spherical shape at pH 7.4, but at pH 6.8 GCS- g -DEAP- g -Ce6- g -PEG became unstable and unstructured and several aggregates were observed.

<3-3> zeta potential ( zeta potential ) Measure

Zeta potential changes of GCS- g -DEAP- g -Ce6- g- PEG solutions (0.1 mg / ml) at different pH values (pH 7.4-6.0, PBS 150 mM) were measured using Zetasizer 3000. Prior to testing, the GCS- g -DEAP- g -Ce6- g -PEG solution was stabilized for 2 hours at room temperature.

As a result, as shown in FIG. 6, the zeta potential of GCS- g -DEAP- g -Ce6- g -PEG changed from -8.0 mV to +1.3 mV as the pH of the solution decreased from 7.4 to pH 6.8. These results suggest that the negative zeta potential resulting from PEG at pH 7.4 is offset by protonation of DEAP (pK _b 6.8) blocks at pH 6.8.

<3-4> pH For Magnetic quenching ( self - quenching ) effect

The self-quenching effect of GCS- g -DEAP- g -Ce6- g -PEG solution (0.1 mg / ml, PBS 150 mM) on the pH of the solution (pH 7.4 to 6.4) was determined by the KODAK image at each given pH. analyzed by station.

Fluorescence images of GCS- g- DEAP- g- Ce6- g- PEG and GCS- g- Ce6- g- PEG were analyzed, and as shown in FIG. 7, GCS- g -DEAP- g -Ce6- g- PEG exhibited fluorescent self-quenching at pH 7.4 and dequenching at pH 6.8.

< Example  4>

Monoacidic  Measure

The close geometry between the Ce6 molecules is grafted with GCS due to the hydrophobic interaction of the DEAP block at pH 7.4, which is due to the self-quenching effect. In addition, this self-quenching affects the mono-oxygen generation of GCS- g -DEAP- g -Ce6- g- PEG due to the photosensitivity of Ce6 blocks. Therefore, to determine how the occurrence of monoantioxidants of GCS- g -DEAP- g -Ce6- g- PEG changes as the pH is changed, we present a rapid chemical trap for monoantioxidants. It was confirmed using, 10-dimethylanthracene (DMA). DMA selectively reacts with monoantioxidants to form non-fluorescent endoperoxides.

First, DMA (20 mmol) was mixed with GCS- g -DEAP- g -Ce6- g -PEG solution (0.1 mg / ml, PBS 150 mM) at different pH conditions (pH 6.4-7.4), and then 670 nm light source Was irradiated with light of 5.2 mW / cm ² intensity for 10 minutes. When DMA fluorescence intensity (measured using a Shimadzu RF-5301PC spectrofluorometer at λ _ex 360 nm and λ _em 380-550 nm) is stabilized after 1 hour, the change in DMA fluorescence intensity is determined by the overall DMA fluorescence spectrum (DMA in PBS). The fluorescence spectrum of each sample is shown below.

As a result, as shown in Fig. 8 (a), in the case of GCS- g -DEAP- g -Ce6- g -PEG, the occurrence of mono-antioxidant was reduced at pH 7.4, which is GCS- g -Ce6 self-quenching of GCS- g -DEAP- g -Ce6- g -PEG compared to the result that the monoantigen generation of g -PEG (see Figure 8 (b)) or free Ce6 (data not shown) is pH independent. (self-quenching) is believed to be due to recovery at pH 6.8 or 6.4.

< Example  5>

Phototoxicity ( Phototoxicity )

<5-1> Cell Culture

Human epidermal ovarian cancer HeLa cells (Korea Cell Line Bank) were wetted with DMEM / PBS medium containing DMEM / PBS medium containing 0.5M PBS, 2mM L-glutamine, 1% penicillin-streptomycin, and 10% FBS. It was maintained under 37 ° C. and 5% CO ₂ air conditions. Prior to testing, the cells growing in a single layer (1 × 10 ⁵ cells / ml) were obtained by trypsinization with 0.25% (w / v) trypsin / 0.03% (w / v) EDTA solution. It was. Seeding of HeLa cells that are attached to the DMEM medium (200ml) to the well plate and in vitro (in In vitro ) cells were incubated for 24 hours before testing.

<5-2> Phototoxicity  Measure

Phototoxicity of the irradiated GCS- g -DEAP- g -Ce6- g- PEG was tested against HeLa cancer cells prepared in <5-1> above. GCS- g- DEAP- g- Ce6- g- PEG (equivalent Ce6 1 μg / ml) or GCS- g -DEAP- g -PEG (1 dissolved in DMEM / PBS medium at different pH conditions (pH 6.4 to 7.4) ug / ml) was administered to cells attached to 96-well plates. The cells were irradiated with light of 5.2 mW / cm ² intensity for 10 minutes using a 670 nm light source to the solution, followed by further incubation for 12 hours. Phototoxicity was measured using Cell Counting Kit-8 (CCK-8).

As a result, as shown in FIG. 9, the monoantioxidant generation of GCS- g -DEAP- g -Ce6- g- PEG is different depending on the pH, so that human epithelial ovarian cancer HeLa at pH 6.8 and 6.4 than at pH 7.4. Phototoxicity to cells was relatively higher.

<5-3> Cell Viability Test

GCS- -DEAP- g g g -Ce6- -PEG to measure the toxicity of the polymer, light GCS- g -DEAP- g -Ce6- g -PEG ( 1 ~ 400 μg / ml) of HeLa cells treated with no irradiation Cell viability was tested. Cells were treated with GCS- g -DEAP- g -Ce6- g -PEG and incubated for 24 hours, and then evaluated by CCK-8 analysis.

As a result, as shown in FIG. 10, it was confirmed that GCS- g- DEAP- g- Ce6- g- PEG did not show cytotoxicity for 24 hours before the cells were cultured.

<5-4> apoptosis ( apoptosis )

Apoptosis was visualized using the Annexin V-FITC fluorescence microscopy kit, and fluorescence images were obtained by treating Annexin V-FITC as apoptosis marker. First, the light irradiated cells were washed twice with PBS and stained with 1 ml of Annexin V-FITC (10% by weight) at room temperature for 15 minutes. After staining was completed, the cells were washed twice with PBS and confirmed using an optical microscope (λ _ex 570nm and λ _em 595nm, ESCOPE 1500F).

As a result, as shown in FIG. 11, it was found that GCS- g- DEAP- g- Ce6- g- PEG leads to relatively higher levels of apoptosis on HeLa cells at pH 6.8 and 6.4 than at pH 7.4. On the other hand, in the case of free Ce6 or GCS- g -Ce6- g- PEG (data not shown), no significant difference was observed in apoptosis with pH.

In addition, the cells were observed under an optical microscope, and as a result, as shown in FIG. 12, when treated with GCS- g -DEAP- g -Ce6- g -PEG compared to the cells without any treatment, Although the number of changes was not large, it was found that the pH of 6.8 and 6.4 significantly reduced the number of cells. However, treatment with free Ce6 significantly reduced cell numbers at pH 7.4, pH 6.8 and 6.4.

Through these results GCS- -DEAP- g g g -Ce6- -PEG is was found that the selective action only for cancer cells represents the weak acid without having an effect, the normal cells (pH 7.4), through which GCS- g It was found that -DEAP- g -Ce6- g -PEG can be effectively used for photodynamic therapy of cancer.

< Example  6>

in vivo in GCS - g - DEAP - g - Ce6 - g - PEG Effect

<6-1> laboratory animals ( Animal care )

in In vivo experiments consisted of 4-6 week-old female nude mice (BALB / c nu / nu mice, Institute of Medical Science, Tokyo), who used the Institutional Animal Care and Use Committee (IACUC) in Yonsei University, Korea. In accordance with the guidelines of approved protocols.

<6-2> in vivo  Fluorescence Image Analysis

We used a BALB / c nu / nu female mouse model infused with HeLa cancer cells to determine tumor specificity of GCS- g -DEAP- g -Ce6- g- PEG, GCS- g -Ce6- g- PEG and free Ce6. I looked at it.

First, in In vivo animal models, HeLa cancer cells were suspended in PBS pH 7.4 (ion strength: 0.15) medium and injected into female nude mice by subcutaneous injection of 1 × 10 ⁴ cells. Then, when the cancer volume is 100 mm ³ , GCS- g -DEAP- g -Ce6- g -PEG (equivalent Ce6 0.1 mg / kg body), GCS- g -Ce6- g -PEG (equivalent Ce6 0.1 mg / kg body) or free Ce6 (2.5 mg / kg body) was injected through the tail vein of HeLa tumor nude mice. Fluorescence images of nude mice were measured using a 12-bit CCD camera (Image Station 4000 MM; Kodak, New Haven, CT) equipped with a cmount lens and a long wavelength transmission filter (600-700 nm; Omega Optical, USA). Fluorescence images were observed for 24 hours.

As a result, FIG. 13 (a) ~ in of the various cases as shown in (c) vivo It was confirmed that there was a marked difference in fluorescence intensity.

Fluorescence images were obtained 30 minutes after intravenous administration of GCS- g- DEAP- g- Ce6- g- PEG (equivalent Ce6 0.1 mg / Kg body) to nude mice, as shown in FIG. 13 (a). Strong fluorescence Ce6 signals appeared at the tumor site compared to the results obtained with the administration of g- Ce6- g- PEG (equivalent Ce6 0.1 mg / Kg body) and free Ce6 (2.5 mg / Kg body), which indicates a GCS- It is believed that this is due to the high accumulation of g- DEAP- g- Ce6- g- PEG conjugates. In particular, in the case of administration of free Ce6, despite the high dose of 2.5 mg / Kg body, the fluorescent Ce6 signal was very weak at the tumor site, and for this reason, it was found that free Ce6 was not suitable as a tumor target.

In addition, as shown in Figure 13 (b) GCS- g -DEAP- g -Ce6- g -PEG showed a fluorescent Ce6 signal in the form of a pulse for 24 hours: the intensity was relatively high at 30 minutes, 30 Compared with minutes, the intensity was lower at 1 hour, the intensity was higher at 6 hours than at 1 hour, the intensity was lower at 12 hours than at 6 hours, and at 24 hours compared to 12 hours. Was high. This tendency differs from that most GCS nanoparticles show a constant fluorescence signal at the tumor site for 2-3 days. One possible hypothesis of this property is that dequenching of GCS- g -DEAP- g -Ce6- g- PEG at acidic tumor sites amplifies the fluorescence signal and also GCS- g -DEAP- g -Ce6 g- PEG helps to freely diffuse or clarify with decreasing fluorescence intensity. On the other hand, even with strong fluorescence intensity in the liver, local light irradiation can limit photodestruction by mono-oxygen at the site of the tumor and thus phototoxicity of GCS- g -DEAP- g -Ce6- g -PEG to the liver. Is so small that it can be ignored.

On the other hand, as shown in Fig. 13 (c), the fluorescence intensity of free Ce6 at the tumor site was shown to gradually decrease with time, which may possibly result in improved kidney clearance.

<6-3> Change in volume of tumor

24 hours after the injection of GCS- g- DEAP- g- Ce6- g- PEG, the cancerous region of nude mice was irradiated with light of 5.2 mW / cm ² intensity for 40 minutes using a 670 nm light source. Changes in the cancer volume were observed over time, and the cancer volume was calculated using the following formula.

tumor volume = length × (width) 2/2.

As a result, when the GCS- g -DEAP- g -Ce6- g -PEG treatment, the tumor volume change of HeLa tumor nude mouse, as shown in Figure 14, HeLa tumor showed significant degeneration over time .

In addition, as shown in FIG. 15, the treatment with GCS- g- DEAP- g- Ce6- g- PEG was more effective in tumor regression compared to the high dose administration of free Ce6 (2.5 mg / kg body). there was.

Finally, GCS- g -DEAP- g -Ce6- g -PEG is safe for normal cells and selectively accumulates only for cancer cells, resulting in an effective cancer cell killing effect. It can be seen that it can be used efficiently.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (10)

A polymer-photoresponder conjugate for photodynamic therapy comprising 3-diethylaminopropyl (DEAP), a photoresponder and a biocompatible polymer grafted with polyethylene glycol. The method of claim 1,
The biocompatible polymer is glycol chitosan, hyaluronic acid, pullulan, curdlan, pectin, starch, dextran, chitosan. Polymer-photoresponder conjugates for photodynamic therapy, characterized in that selected from the group consisting of poly-L-lysine and polyaspartic acid. The method of claim 1,
The photoresponder is a polymer-photoresponder conjugate for photodynamic therapy, characterized in that selected from the group consisting of chlorine e6, protopophyrin 9, bonerine, benzoporphyrin, mono aspartyl chlorine and meso tetrahydroxyphenyl chlorine . The method of claim 1,
The biocompatibility Polymer-photoresponder for photodynamic therapy, characterized in that the pH-sensitive substance is replaced with 0.1 to 0.9 per one primary amine of the polymer, the photoresponder is replaced with 0.05 to 0.5, and the polyethylene glycol is replaced with 0.01 to 0.5. Conjugate. The method of claim 1,
The polymer-photoresponder conjugate is selectively accumulated in a weakly acidic disease site in the range of pH 6.0 ~ 7.0, the polymer-photoresponder conjugate for photodynamic therapy, characterized in that to generate free radicals by light irradiation. The method of claim 5,
The weakly acidic disease is any one selected from myopathy, neuromuscular diseases, such as myasthenia gravis, muscular dystrophy, chronic obstructive pulmonary disease, rheumatoid arthritis, and solid cancer. Polymer-photoresponder Conjugates. Dissolving the biocompatible polymer in a solvent;
Adding 3-diethylaminopropyl to the solution in which the biocompatible polymer is dissolved to bind the amine group of the biocompatible polymer to the carboxyl group of the 3-diethylaminopropyl;
Substituting the carboxyl group of the photoresponder and polyethylene glycol with succinimide and adding the solution to the amine group of the biocompatible polymer; And
A method for preparing a polymer-photoresponder conjugate for photodynamic therapy comprising dialysis and freeze drying the conjugated conjugate. The method of claim 7, wherein
1 to 3 moles of 3-diethylaminopropyl, 0.5 to 1.5 moles of photoresponder, and 0.5 to 2 moles of polyethylene glycol per 1 mole of each repeating chain of the biocompatible polymer. Method for producing a responder conjugate. The method of claim 7, wherein
The biocompatible polymer is glycol chitosan, hyaluronic acid, pullulan, curdlan, pectin, starch, dextran, chitosan. , Consisting of poly-L-lysine and polyaspartic acid Method for producing a polymer-photoresponder conjugate for photodynamic therapy, characterized in that selected from the group. The method of claim 7, wherein
The photoresponder is a polymer-photoresponder conjugate for photodynamic therapy, characterized in that selected from the group consisting of chlorine e6, protopophyrin 9, bonerine, benzoporphyrin, mono aspartyl chlorine and meso tetrahydroxyphenyl chlorine Manufacturing method.

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