Patent Application
20240123092
The Sequence Listing submitted as a XML file named “Q291846 sequence listing as filed,” created on Sep. 15, 2023, and having a size of 1.81 KB is hereby incorporated by reference.
The present invention relates to a conjugate for photodynamic diagnosis or treatment in which a peptide is conjugated with a photosensitizer via an intracellularly degradable linkage, and to a composition for photodynamic diagnosis or treatment including the same.
Photodynamic therapy (PDT) is a technology of treating malignant diseases such as cancers or treating diseases such as acne without performing a surgical operation by using a photosensitive material (hereinafter, referred to as “photosensitizer”). Such PDT has been actively studied from the early 21st century, and currently, is being used in order to increase immunity in cancer diagnosis and therapy, autologous bone marrow transplantation, antibiotics, AIDS therapy, skin graft, or arthritis therapy, and thus the application range thereof has gradually widened.
In particular, as for PDT used in cancer therapy, when a photosensitizer, which is a material sensitive to light, is administered into the body and external light is irradiated, the photosensitizer chemically reacts with fluent oxygen in the external light, to generate singlet oxygens or free radicals, and these singlet oxygens or free radicals induce apoptosis in various lesions and cancer cells to destruct them.
Currently, porphyrin derivatives, chlorine, bacteriochlorin, phthalocyanine, 5-aminolevulinic acid derivatives, and the like have been known as the photosensitizer used in PDT. Cyclic tetrapyrrole derivatives as the photosensitizer are characterized by being selectively accumulated in cancer cells and exhibiting fluorescence or phosphorescence due to compound property, and thus may be utilized as a reagent for early diagnosis. In addition, since metalloporphyrin in which metal is bound inside the cyclic tetrapyrrole exhibits several characteristics depending on the kind of metal bound thereto, metalloporphyrin is used as a contrasting agent at the time of magnetic resonance imaging (MRI) and thus is applied during the early diagnosis of tumor cells such as cancer cells. Also, 5-aminolevulinic acid derivatives, which are the most widely known photosensitizers, are simply used and have a small molecular weight, which thus comparatively facilitate skin permeation, and have few side effects and thus are stable.
However, the photosensitizer for photodynamic diagnosis or therapy used in the related art has low accumulation rate and target specificity to cancer cells and does not induce fluorescence interference, and thus exhibits cytotoxicity in general cells, that is, normal cells in vivo when the light is irradiated, resulting in reduced therapeutic outcomes and causing several side effects. That is, conventionally, the photosensitizers used in photodynamic therapy have a hydrophobic characteristic, so that after intravenous injection, it accumulates nonspecifically in normal tissues including skin as well as cancer tissues. This not only makes the tumor-to-background ratio worse and thus makes diagnostic imaging of the tumor site difficult, but also has the risk of damaging the important surrounding normal tissues during photodynamic therapy. Moreover, when a patient receiving photodynamic therapy is exposed to bright light such as sunlight, reactive oxygen is generated again from the photosensitizer that has accumulated in the skin, resulting in side effects of skin photosensitivity. For this reason, after photodynamic therapy, the patient is recommended to remain in the dark room for at least 6 weeks until the photosensitizer accumulated in the normal tissues of the skin, etc., disappears, which causes discomfort to the patient. There is an attempt to solve the skin photosensitivity problem by increasing the hydrophilicity of the photosensitizer, but in this case, since a large amount of the intravenously administered photosensitizer is excreted through urine, in order for a sufficient amount of photosensitizer for therapy to be accumulated in the tumor tissue, a higher dose of photosensitizer should be administered.
As a means for solving these problems and enhancing tumor selectivity, by developing a peptide-photosensitizer conjugate in which a peptide capable of specifically binding to a receptor or the like over-expressed on the surface of cancer cells is linked to a photosensitizer, an attempt was made so that the photosensitizer can be specifically introduced only into the target cancer cells. However, the peptide-photosensitizer conjugates that have been developed previously still have a disadvantage in that they generate fluorescence signals when they are outside the target cells or are introduced into the cells and exhibit phototoxicity, and even in the case of nonspecific adsorption to normal cells, it is phototoxic to normal cells when exposed to light. Accordingly, there was a limit in that one had to wait until the peptides-fluorescent dyes or peptide-photosensitizer conjugates that are circulating in blood or accumulated nonspecifically in normal cells are removed via urinary excretion or metabolism, and thereafter obtain fluorescence images or perform photodynamic therapy. However, since the pharmacokinetic characteristics are different depending on patients, there is a problem that it is difficult to know the optimal time for imaging or photodynamic therapy, and there is a limit to obtaining the tumor selective photodynamic therapy effect.
Accordingly, the present invention is designed to solve the problems that conventional fluorescent dyes and photosensitizers generally rely on cell permeability, resulting in low accumulation rate and target rate of cancer cells, low tumor-to-background signal ratio due to autofluorescence, significantly different in residence time affected by different pharmacokinetic characteristics among subjects, and generating noise in the fluorescence activated image. To this end, an attempt has been made to develop a photosensitizer-peptide conjugate with linker that does not produce fluorescence signals in its native state but selectively produces a strong fluorescence signal only when it enters the target cell (for example, cancer cells), so as to enable image detection of target cells with high tumor-to-background signal ratio without time limitation.
In addition, in the case of the degradable photosensitizer-peptide conjugate of the present invention, reactive oxygen is not produced when it is present outside the target cell, and reactive oxygen generation is activated only when it is ingested and entered into the target cell. By providing a target cell-specific photodynamic therapy effect, the present invention aims at inhibiting non-specific damage to normal tissues.
In order to accomplish the above object,
The photosensitizer and the cleavable linker may be linked by a covalent bond, but preferably linked by an amide bond, but may be linked by various types of bonds as long as the cleavage of the linker is not affected at a target site, and amino acids or chemicals may be additionally inserted between the photosensitizer and the linker. The amino acids or chemicals may increase the hydrophobicity or hydrophilicity of the conjugate, or improve the accessibility of a cleavage enzyme to the linker.
The cleavable linker and the peptide may be linked by a covalent bond, but preferably linked by an amide bond, but may be linked by various types of bonds as long as the cleavage of the linker is not affected at a target site, and amino acids or chemicals may be additionally inserted between the linker and the peptide. The amino acids or chemicals may increase the hydrophobicity or hydrophilicity of the conjugate, or improve the accessibility of a cleavage enzyme to the linker.
In the peptide consisting of 1 to 50 amino acids and containing at least one tryptophan, (1) tryptophans may be included in a peptide sequence having a target ability, (2) 1 to 3 tryptophans may be linked continuously, (3) amino acids may be additionally linked to improve hydrophilicity, or (4) phenylalanine or lipid may be additionally linked by a covalent bond in order to prepare nanoparticles, micelles, or liposomes using the conjugate.
The conjugate according to the present invention may be used as it is or prepared in the form of nanoparticles, micelles, or liposomes to obtain a passive targeting effect (corresponding to Formulas 3 to 15).
In addition, the conjugate according to the present invention may also obtain an active targeting effect by additionally introducing a targeting ligand (corresponding to Formulas 1 and 2).
In the conjugate according to the present invention, the distance between the photosensitizer and the tryptophan may be within 2 nm. When the tryptophan is located close to the photosensitizer within 2 nm, a photoinduced electron transfer phenomenon may occur between the tryptophan and the photosensitizer, and at this time, the photosensitizer may be quenched to be induced so as not to generate a fluorescence signal and not to generate reactive oxygen. In the present invention, the main function of tryptophan acts as a quenching agent when a three-dimensional distance with the bound photosensitizer is within 2 nm, thereby not only preventing fluorescence signals from being generated from the conjugate, but also suppressing the generation of reactive oxygen for treatment. After the conjugate is taken up into the target cell, while the linker in the conjugate is cleaved within the cell, the distance between the tryptophan and the photosensitizer may become far away to 2 nm or more, and the quenching action by tryptophan may disappear. From this time, a strong fluorescence signal may be generated to enable target cell-specific fluorescence imaging diagnosis, and the generation of reactive oxygen may also actively occur, thereby enabling selective photodynamic therapy.
For the purpose of improving the solubility of the conjugate according to the present invention in an aqueous solution, polyethyleneglycol (PEG) may be further introduced between the photosensitizer and the cleavable linker (corresponding to Formulas 1 and 2), and for the purpose of facilitating the accessibility of the enzyme to the linker or for the purpose of promoting aggregation between conjugates, 1 to 5 amino acids may be additionally introduced (corresponding to Formulas 9 to 15). At this time, tryptophan may not be included in the 1 to 5 amino acids. If tryptophan is included in the 1 to 5 amino acids, even if the conjugate reaches the cancer cells and the cleavable linker is cleaved, tryptophan continues to remain next to the photosensitizer, so that there is a problem that the quenching state may be continuously maintained. The number of ethylene glycol repeating units in the PEG may be 1 to 10, but is not limited thereto.
For the purpose of encapsulating the conjugate according to the present invention in nanoparticles, micelles or liposomes, polyethyleneglycol (PEG), phenylalanine, or lipid may be further introduced into 1 to 5 amino acids introduced between the photosensitizer and the cleavable linker, and polyethyleneglycol (PEG), phenylalanine, or lipid may be further introduced into the peptide consisting of 1 to 50 amino acids and containing at least one tryptophan. The number of ethylene glycol repeating units in the PEG may be 1 to 10, but is not limited thereto. The lipid may use fatty acid having 6 to 25 carbon atoms, but is not limited thereto.
For the purpose of improving in vivo stability of the conjugate according to the present invention, a carboxyl group at an amino acid terminal may be substituted with an amide group (corresponding to Formulas 8 to 10).
The conjugate according to the present invention may be used for photodynamic diagnosis or photodynamic therapy for cancer. A cancer target delivery method of the conjugate may use both conventional active targeting and passive targeting.
In the case of the active targeting, a cancer cell-specific ligand may be introduced into the conjugate, and the cancer cell-specific ligand may use an antibody, an aptamer, a peptide, and the like that specifically bind to cancer cells, thereby further improving a specific uptake ability to targeting cancer cells. When the peptide itself capable of specifically binding to specific cells includes tryptophan, such a peptide and the photosensitizer may be linked with a cleavable linker to induce the distance between the tryptophan and the photosensitizer to be within 2 nm, and even at this time, a quenching phenomenon of the photosensitizer may be obtained. Examples of the peptide that specifically binds to the cancer cells may be those that specifically bind to at least one of epidermal growth factor receptor 1, epidermal growth factor receptor 2, epidermal growth factor receptor 3, and epidermal growth factor receptor 4.
In the case of the passive targeting, since a cancer tissue has a loose vascular structure and no lymph nodes, a characteristic in which a nano-sized drug is well absorbed may be used, so that the conjugate may be used as it is without the introduction of a cancer cell-specific ligand, or the conjugate may be encapsulated and used in nanoparticles, micelles or liposomes having sizes of tens to hundreds of nm.
In the conjugate according to the present invention, the cleavable linker may use:
Chemical structures of the conjugate according to an embodiment of the present invention may be represented by Formulas 1 to 15 below.
Further, the present invention provides a liposome including the conjugate. In order to smoothly encapsulate the liposome and improve the in vivo stability of the conjugate, when a carboxyl group remains in the amino acid in the conjugate according to the present invention, the carboxyl group may be substituted and used with an amide group. This may correspond to Formulas 8 to 10.
Furthermore, the present invention provides a micelle including the conjugate. To prepare the micelle, polyethyleneglycol (PEG) or lipid in the form of side chains may be introduced into the conjugate according to the present invention. This may correspond to Formulas 11 to 15.
In addition, the present invention provides a composition for photodynamic diagnosis or photodynamic therapy including the conjugate, liposome or micelle.
Furthermore, the present invention provides a method for treating a cancer patient including administering a composition including the conjugate, liposome or micelle to a cancer patient.
In the photosensitizer-peptide conjugate of the present invention, when the distance between the photosensitizer and the tryptophan is maintained within 2 nm through the degradable linkage, the conjugate generates no fluorescent signal and reactive oxygen species in normal tissues or during the circulation in the blood by quenching a fluorescent signal and reactive oxygen generation ability of the photosensitizer. After the conjugate of the present invention is selectively absorbed into cells by interaction with an overexpressed antigen on the surface of the target cells, a linker is degraded in the cells to increase the distance between tryptophan included in the peptide and the photosensitizer, which becomes farther than 2 nm, and the quenching action is terminated to generate a strong fluorescence signal and induce active generation of reactive oxygen species. Therefore, the conjugate for photodynamic diagnosis or treatment including a peptide according to the present invention has high tissue permeability, shows a high photodynamic therapeutic effect only in target cells (for example, cancer cells) while being safe in normal cells, and can obtain a good diagnostic image having a high ratio of target-to-background signal ratio.
In order to achieve the above object, the present invention provides a conjugate for photodynamic diagnosis or treatment in which a tryptophan or a peptide including the tryptophan is conjugated with a photosensitizer via an intracellularly degradable linkage, and a composition for photodynamic diagnosis or treatment including the same.
Unless otherwise defined, the term “treatment” indicates the action that can reverse or relieve the disease itself or one or more symptoms of the disease targeted by the term, or that can inhibit the progress or prevent the target disease. As used herein, the term “treatment” refers to an act of treating when “treatment” is defined as above.
The main function of the tryptophan in the present invention is to prevent the fluorescence signal from being generated from the conjugate by acting as a quenching agent when the distance on the three-dimension with the bound photosensitizer is within 2 nm, and to inhibit the generation of therapeutic reactive oxygen species. After the conjugate is entered into target cells, a linker in the conjugate is degraded inside the cells to increase the distance between tryptophan and the photosensitizer to 2 nm or more, and the quenching action by tryptophan is terminated. From this time on, strong fluorescence signals are generated to enable fluorescence imaging diagnosis specific to target cells, and active generation of reactive oxygen species is also induced, thereby enabling selective photodynamic therapy. In particular, when the tryptophan and the photosensitizer conjugate bound via a degradable linker is further bound to an antibody, RNA or DNA aptamer, a peptide ligand, a nanoparticle, or the like capable of binding to a specific cell, it further improves the specificity to target cells.
When the peptide itself capable of specific binding to a specific cell includes tryptophan, the distance between the tryptophan and the photosensitizer may be induced to be within 2 nm by conjugating the peptide and the photosensitizer via a degradable linker. Also at this time, a quenching phenomenon of the photosensitizer may be obtained. The thus-quenched fluorophore (or photosensitizer) and the peptide conjugate is absorbed only into target cells of the peptide-photosensitizer conjugate by specifically bound to the surface of target cancer cells and inflammatory cells, and be entered into the target cells, the linker is degraded and the distance between the tryptophan and the fluorophore (or photosensitizer) is increased to 2 nm or more, and the quenching action by tryptophan is terminated. From this time on, strong fluorescence signals are generated to enable fluorescence imaging diagnosis specific to target cells, and active generation of reactive oxygen species is also induced, thereby enabling selective photodynamic therapy.
The intracellularly degradable linkage means that it can be degraded by an intracellular environment such as an enzyme, a signaling factor, a reducing agent, and a pH, and more specifically, it may be degraded in lysosome. Therefore, the intracellular degradable linkage may be any one or more selected from linkages that are degraded by intracellular enzymes, linkages that are degraded by intracellular reducing agents, or linkages that are degraded by intracellular low pH.
The enzyme that degrades a linker included in a conjugate for photodynamic diagnosis or treatment of the present invention may be any one selected from cathepsins and esterases or a combination thereof, and the cathepsins may be any one or more enzymes selected from the group consisting of cathepsin B, cathepsin L and cathepsin S. For example, it may be a bond consisting of arginine or lysine which is degraded by cathepsin B present in lysosome, an ester (—COO—) or a thioester (—COS—) bond which is degraded by esterase.
The intracellular reducing agent capable of degrading a linker included in a conjugate for photodynamic diagnosis or treatment of the present invention may be at least one selected from the group consisting of glutathione, sulfhydryl and nicotinamide adenine dinucleotide phosphate (NADPH), and may be, for example, a disulfide bond that may be degraded by glutathione in the lysosome. Glutathione is known to be present at a concentration of about 2 μM in the extracellular region including blood and at a high concentration of 10 mM in cancer cells.
A schematic diagram of the action of the present invention is illustrated in
More preferably, the distance on the three-dimension may be within 2 nm. When the intracellular degradable linker is cleaved in target cells and the distance between the tryptophan and the photosensitizer is further increased to 2 nm or more, the quenching effect by tryptophan is terminated and strong fluorescence signals are generated from the photosensitizer, thereby producing a photodynamic treatment effect (
As the photosensitizer used for photodynamic diagnosis or therapeutic conjugation according to the present invention, a photosensitizer that may be applied by a person skilled in the pertinent field may be applied. For example, it may be used from the group consisting of the porphyrin-based compound selected from the group consisting of porphyrins, chlorins, pheophorbides, bacteriochlorins, porphycenes, and phthalocyanines, or the non-porphyrin-based compound selected from the group consisting of hypericin, rhodamine, rose bengal, psoralen, phenothiazinum-based dyes and merocyanine, but is not limited thereto. More specifically, it may use those, alone or in combination, selected from the group consisting of the porphyrin-based compound selected from the group consisting of hematoporphyrins, porphycenes, pheophorbides, purpurins, chlorins, protoporphyrins, phthalocyanines in the form of free bases or metal complexes; or the non-porphyrin-based compound selected from the group consisting of hypericin, rhodamine, ATTO, rose Bengal, psoralen, phenothiazinium-based dyes and merocyanine.
In the present invention, when the conjugate of the present invention is selectively accumulated in cancer tissue, fluorescent interference is released by the action of a reducing agent or an enzyme in cancer cells, and since the photosensitizer can exhibit fluorescence only in cancer cells by laser irradiation, this fluorescence can diagnose cancer. In addition, reactive oxygen generation that has been suppressed is activated again, so that a target cell-specific photodynamic therapy effect can be obtained.
The present invention provides a composition for photodynamic diagnosis or treatment including the conjugate for photodynamic diagnosis or treatment.
The composition for photodynamic therapy according to the present invention may include a pharmaceutically effective amount of the conjugate for photodynamic diagnosis or therapy of the present invention alone, or may further include at least one pharmaceutically acceptable carrier, excipient, or diluent. The term pharmaceutically effective amount means an amount sufficient to prevent, improve, and treat symptoms of diseases.
The pharmaceutically effective amount of the conjugate for photodynamic diagnosis or therapy according to the present invention is 0.5˜100 mg/day/kg body mass, and preferably 0.5˜5 mg/day/kg body mass. However, the pharmaceutically effective amount may be appropriately varied depending on the severity of the symptom, age, weight, health status, and gender of the patient, route of administration, duration of treatment, and the like.
Herein, the term pharmaceutically acceptable carrier, excipient or diluent means a composition that is physiologically acceptable and does not generally cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions when being administered to a human. Examples of the carrier, excipient, and diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil. In addition, they may further include filler, anti-coagulant, lubricant, wetting agent, flavoring, emulsifier, and preservative.
In addition, the composition of the present invention may be formulated by using the method known in the art so that an active component is provided in a manner of fast release, sustained release, or delayed release after the composition is administered to a mammal. The dosage form may be powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injectable solution, or sterile powder.
The composition for photodynamic therapy according to the present invention may be administered through several routes including oral, transdermal, subcutaneous, intravenous, and muscular routes, and the dosage of the active component may be appropriately selected depending on several factors such as route of administration, age, gender, weight, and severity of symptom of the patient.
In addition, in the case where the conjugate according to the present invention or the composition including the conjugate is used to conduct therapy or diagnosis of diseases, a light source usable in the present invention may be, but is not limited thereto, at least one selected from the group consisting of light sources for in vitro light irradiation selected from the group consisting of an ultrasonic irradiation emitter, a light emitting diode, a laser diode, a dye laser, a halogenated metal lamp, a flash lamp, a mechanically filtered fluorescent light source, a mechanically filtered incandescence, a filament light source, and the like; and light sources for in vivo light irradiation including a laser fiber for photodynamic therapy and the like. In the present invention, the photosensitizer may exhibit activity in the near infrared ray in the range of 600 nm to 900 nm.
Hereinafter, the present invention will be described in more detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.
Experiments were conducted to confirm the quenching effect of tryptophan on the photosensitizer.
More specifically, in order to confirm that the quenching effect occurs as the distance between the photosensitizer and the tryptophan approaches, Chlorin e4 (Ce4) and AIPcS4A1 (III) (phthalocyanine chloride tetrasulfonic acid), which are representative chlorine and cyanine photosensitizers, were used for the analysis. The concentration of Ce4 and AlPcS4 was fixed at a concentration of 5 μM and the ratio of the fluorescence signal (F) when tryptophan was added to the fluorescence signal (F0) when tryptophan was not added while increasing the concentration of tryptophan was obtained. (Ce4: λEx 400 nm, λEm 665 nm; AlPcS4: λEx 660 nm, λEm 690 nm). The results are illustrated in
According to
In Example 2, an experiment was conducted to analyze whether the quenching effect of the photosensitizer can be controlled by controlling the distance between the tryptophan included in the peptide ligand and the photosensitizer.
As an example of peptide ligands including tryptophan, GE11 peptide (YHWYGYTPQNVI; SEQ ID NO: 1), which is known to bind specifically to the epidermal growth factor receptor (EGFR) overexpressed on the surface of cancer cells was selected, and chlorin e4 (Ce4) was used as a photosensitizer. As a degradable linker, it was selected that a disulfide bond capable of being degraded by a reducing agent present in a high concentration in a cell was inserted in a cyclic form.
2.1. Synthesis of Photosensitizer-Peptide Conjugate (C-EGFR) Including a Cyclic Disulfide Bond Linker
For the synthesis of C-EGFR, peptides were prepared by Fmoc solid phase peptide synthesis (SPPS) using ASP48S (Peptron Inc, USA). The preparation process is provided as follows.
Eight equivalents of the amino acid with the protecting group (Fmoc) bound to H-Ile-2-chloro-Trityl resin (Anaspec, USA) and eight equivalents of 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/eight equivalents of N-Hydroxybenzotriazole (HOBt)/16 equivalents of 4-Methylmorpholine (NMM) were dissolved in Dimethylformaminde (DMF) and added, and reacted at room temperature for 2 hours, followed by washing with DMF, methanol and DMF in that orderly manner.
In order to isolate the protecting group Fmoc attached to the amino acid, DMF including 20% piperidine was added to the reaction solution and reacted twice at room temperature for 5 minutes, followed by washing with DMF, methanol and DMF in that orderly manner. This process was repeated to synthesize a resin-bound structure on the peptide scaffold
To the resin-peptide scaffold conjugate, four equivalents of Ce4 and four equivalents of HBTU/four equivalents of HOBt/eight equivalents of NMM were dissolved in dimethylsulfoxide (DMSO) and added, and then the mixture was reacted for 12 hours, suctioned, and then washed with DMF, methanol and DMF in that orderly manner.
When the peptide scaffold to be synthesized is formed through this process, trifluoroacetic acid (TFA)/1,2-ethanedithiol (EDT)/thioanisole/triisopropylsilane (TIS)/water were treated with solution diluted to 90/2.5/2.5/2.5/2.5 to remove the protecting group of the peptide residue prepared above and the peptide was separated from the resin to prepare [Ce4-miniPEG2-Cys(Trt)-Cys(Trt)-Tyr(t-Bu)-His(Trt)-Trp(Boc)-Tyr(t-Bu)-Gly-Tyr(t-Bu)-Pro-Gln(Trt)-Asn(Trt)-Val-Ile].
Thereafter, 10 times of cold diethyl ether was added to the reaction solution to precipitate the peptide, and centrifugation was carried out at 3000 rpm for 10 minutes. The filtrate was discarded and repeated 2 times. The conjugate thus obtained was purified by reversed phase high performance liquid chromatography (HPLC).
Peptides purified for cyclic disulfide bonds in the peptide were dissolved in 0.1% ammonium acetate solution and stirred strongly for 24 hours. The progress of the reaction was confirmed by HPLC and LC/MS. After confirming that the reaction was completed, the reaction mixture was purified by reversed phase HPLC using a Vydac C4, 5u, 300 A column (4.6×50 mm). Elution was performed using a water-acetonitrile linear gradient using 30 to 60% (v/v) acetonitrile solution including TFA 0.1%.
The chemical structure of the C-EGFR conjugate thus prepared is shown in the following Formula 1. That is, the photosensitizer and the EGFR target peptide, GE11, were bound via a cyclic disulfide bond. Due to the cyclic structure, Ce4, which is a photosensitizer, was located close to the tryptophan, and moreover, due to the limitation of mobility via a cyclic bond, Ce4 was induced to remain in close proximity to the tryptophan.
2.2. Characteristics of Cyclic C-EGFR Conjugate
1) Molecular Weight Measurement and Fluorescence Emission Characteristic Analysis
The molecular weight of the synthesized and purified C-EGFR was measured using LC/MS (Agilent Hewlett Packard 1100 series, USA) and the absorbance was measured using a UV/Vis absorption spectrometer (DU370, USA).
As a result of the evaluation, the purified conjugate of the present invention thus obtained, i.e., the C-EGFR conjugate, had a molecular weight of 2,423 g/mol (
2) Analysis of Fluorescence Signal and Reactive Oxygen Generation Recovery by Reducing Agent Treatment
The increase of fluorescence and reactive oxygen generation by reducing agent treatment was analyzed. The C-EGFR conjugate thus obtained was dissolved in phosphate-buffered saline (PBS, 6.7 mM, pH 7.4, 154 mM NaCl) to be 2 and then transferred to a multifunctional microplate reader (Safire 2, Tecan, Switzerland) to obtain fluorescence spectrum (λEx. 400 nm, λEm. 650˜850 nm) results.
In the dithiothreitol (DTT) treatment sample, which is a reducing agent, 2 μM of a conjugate was mixed with 2 μM and 5 mM DTT, and the same amount of phosphate-buffered saline (PBS) was added to the buffer-treated group. The fluorescent intensity of the mixed solution (200 μl) was measured at intervals of 30 minutes for 4 hours (λEx 400 nm, λEm 665 nm).
Singlet oxygen generation (SOG) was measured by the following method. The C-EGFR conjugate thus obtained was dissolved in PBS to be 2 and then DTT-treated samples were mixed with 2 μM and 5 mM DTT, respectively, and the same amount of PBS was mixed in the buffer-treated group. The mixed solution (200 μl) was cultured at 37° C. for 4 hours. Subsequently, oxygen-saturated PBS including concentrated singlet oxygen sensor green (SOSG) was mixed and the final SOSG concentration was adjusted to 5 μM. Fluorescence intensity change of the SOSG was periodically measured using a 670 nm continuous wavelength laser device during the light irradiation (light irradiation dose: 50 mW/cm 2) of the sample.
According to
3) Cell Culture
The human breast cancer cell line HCC70 overexpressing epithelial growth factor receptor (EGFR) and the human normal cell line PCA-SMC (primary coronary artery smooth muscle cells) not expressing EGFR were all obtained from the American Type Culture Collection (ATCC, USA).
The HCC70 cells were cultured in RPMI (Roswell Park Memorial Institute) 1640 medium including 10% fetal bovine serum and 1% penicillin/streptomycin (Life Technologies) at 37° C. under 5% carbon dioxide and standard humidity conditions. PCA-SMC cells were cultured in a vascular cell basal medium including recombinant human insulin, ascorbic acid, glutamine, recombinant human epithelial growth factor, and fetal bovine serum 10% at 37° C. under 5% carbon dioxide and standard humidity conditions.
4) Measurement of EGFR Expression Level Using Flow Cytometer
Expression level of EGFR, a target protein, in each cell was measured by flow cytometer. PCA-SMC and HCC70 cells at 3×105 each were immobilized in a phosphate-buffered solution including 4% paraformaldehyde and washed three times.
The immobilized cells were reacted with EGFR antibody (4 μg/1 mL in 1% BSA/0.25% Tween20/PBS) for 30 minutes and washed with cold phosphate-buffered solution. Thereafter, the cells were reacted with a secondary antibody (1:100 dilution) bound with a fluorescent dye, FITC, for 30 minutes, washed once again with cold phosphate-buffered solution, and analyzed with a flow cytometer to verify the degree of EGFR expressed on the cell surface (
5) Real-Time Observation of Fluorescence Quenching and Fluorescence Recovery
An experiment was conducted to verify whether a C-EGFR conjugate of the present invention in which fluorescence signal generation has been quenched from outside the target cell strongly generates fluorescence after the absorption of the C-EGFR conjugate into target cells. EGFR-overexpressing HCC70 cells were treated with the C-EGFR conjugate and free photosensitizer (Ce4), and then fluorescence images were obtained at regular time intervals without washing. The detailed method is provided as follows.
Each 1×106 of HCC70 cells per well were put in a 12-well plate (BD Biosciences, USA), and the cells were cultured for 24 hours to allow the cells to be attached well, and then C-EGFR conjugates and free photosensitizer were mixed and diluted. The existing medium was then replaced with 0.5 ml of fresh medium including the conjugate and the free photosensitizer. Thereafter, each of the near infrared light fluorescence images (λEx 640±15 nm, λEm. 690±25 nm) were obtained at intervals of 15 minutes for 4 hours using the Live Cell Imaging System (Axio Observer Z1, Carl Zeiss, Germany) without washing the cells.
According to
6) Specific Cell Absorption and Fluorescence Activity of the Conjugate Accordingly
PCA-SMC cells and HCC70 cells per well were put in an 8-well Lab-Tek chamber, PCA-SMC cells were put at a cell number of 5×103 in each well, and HCC70 cells were put at a cell number of 5×104 in each well, and were cultured for 24 hours to allow the cells to be attached well. Thereafter, the existing medium was replaced with 200 μl of fresh medium in which the C-EGFR conjugate (2 μM) was dissolved, and then the cells were cultured for 4 hours.
Finally, the cells were washed three times with the culture medium, and then a near-infrared fluorescence image (λEx 405 nm, λEm 625˜754 nm) was obtained using a confocal laser microscope (CLSM, ZEISS LSM 510 META, Germany).
Experiments were performed to determine the intracellular location of the C-EGFR conjugate of the present invention. In the 8-well Lab-Tek chamber, 5×103 PCA-SMC cells and 5×104 HCC70 cells were put in each well. The cells were cultured for 24 hours to allow the cells to be attached well, and then 2 μM C-EGFR was added and cultured for 4 hours, followed by washing three times. Thereafter, after replacing the existing medium with fresh medium including 100 nM LysoTracker Blue DND-22 dye, the cells were cultured for 45 minutes. Fluorescent images of the C-EGFR conjugate (λEx 405 nm, λEm 625˜754 nm) and LysoTracker (λEx 405 nm, λEm 411˜497 nm) were obtained for these samples using confocal microscopy.
According to
7) Cytotoxicity and Photodynamic Therapy Efficacy Experiment
In the dark state without light, the C-EGFR conjugate of the present invention was treated with 0˜10 μM concentration in HCC70 cells and cytotoxicity was evaluated. As a result, as illustrated in
In order to evaluate the effect of target photodynamic therapy, PCA-SMC cells were put in a 1×103/well and HCC70 cells were put in a 1×104/well in a 96-well plate, and were cultured for 24 hours to allow the cells to be adhered well. C-EGFR conjugates were prepared at concentrations of 0.1 μM, 0.5 μM, 1 μM, 2 μM, 5 μM and 10 μM, respectively, treated with cells, and then cultured for 4 hours. Thereafter, the C-EGFR conjugate that had not been introduced into the cells was washed and removed, and then irradiated with a 670 nm laser at an irradiation dose density of 50 mW/cm 2 for 6 minutes and 40 seconds. Thereafter, after culturing in an incubator overnight for stabilization, the cytotoxicity was assessed by CCK-8 assay.
As a result, according to
In Example 3, the linker between the photosensitizer and the tryptophan was linear, and it was verified that the fluorescence and reactive oxygen generation, which were quenched when the linear linker degraded in the cells, was activated. To this end, Ce4 was selected as a photosensitizer, and conjugates of GE11 peptide including tryptophan (YHWYGYTPQNVI) and a photosensitizer linked via a linear disulfide bond were synthesized and analyzed for their efficacy.
3.1. Synthesis of a Photosensitizer-Peptide Conjugate (L-EGFR) Including Linear Disulfide Bond Linkers
The peptides were prepared by Fmoc solid phase peptide synthesis method using ASP48S, and the preparation process was provided as follows.
Eight equivalents of Fmoc-miniPEG2-OH and eight equivalents of HBTU/eight equivalents of HOBt/16 equivalents of NMM were dissolved in DMF and were added to H-Cys(Trt)-2-chloro-Trityl resin (Anaspec, USA) and reacted at room temperature for 2 hours, followed by washing with DMF, methanol and DMF in that orderly manner.
In order to isolate the protecting group Fmoc attached to the amino acid, DMF including 20% piperidine was added to the reaction solution and reacted twice at room temperature for 5 minutes, followed by washing with DMF, methanol and DMF in that orderly manner.
Four equivalents of chlorin e4(Ce4) and four equivalents of HBTU/four equivalents of HOBt/eight equivalents of NMM were dissolved in DMSO and added to the peptide resin for reaction for 12 hours and suction. The resulting Ce4-miniPEG2-Cys(Trt)-2-chloro-Trityl resin was washed with methanol and DMF in that orderly manner.
The resulting peptide resin was treated with a solution of TFA/EDT/thioanisole/TIS/water diluted to 90/2.5/2.5/2.5/2.5 to remove the protecting group of the peptide residue and isolate the peptide from the resin to obtain Ce4-miniPEG2-Cys.
Thereafter, 10 times of cold diethyl ether was added to the reaction solution to precipitate the peptide, and centrifugation was carried out at 3000 rpm for 10 minutes. The filtrate was discarded and repeated twice. The conjugate thus obtained was purified by reverse phase HPLC.
The Ce4-miniPEG2-Cys and Aldrithiol-2 thus obtained were dissolved in acetic acid and reacted at room temperature for 4 hours. The progress of the reaction was confirmed by HPLC and LC/MS. When the reaction was completed, the reaction mixture was centrifuged and purified to obtain lyophilized Ce4-miniPEG-Cys(Pys).
On the other hand, for the peptide scaffold synthesis, eight equivalents of Fmoc-amino acid and eight equivalents of HBTU/eight equivalents of HOBt/16 equivalents of NMM were dissolved in DMF and were added to H-Ile-2-chloro-Trityl resin (Anaspec, USA) and reacted at room temperature for 2 hours, followed by washing with DMF, methanol and DMF in that orderly manner.
In order to isolate the protecting group Fmoc attached to the amino acid, DMF including 20% piperidine was added to the reaction solution and reacted twice at room temperature for 5 minutes, followed by washing with DMF, methanol and DMF in that orderly manner.
This process was repeated to synthesize a resin-bound structure on the peptide basic scaffold [H-Cys(Trt)-Tyr(t-Bu)-His(Trt)-Trp(Boc)-Tyr(t-Bu)-Gly-Tyr(t-Bu)-Thr(t-Bu)-Pro-Gln(Trt)-Asn(Trt)-Val-Ile-2-chloro-Trityl Resin].
The resulting peptide resin was treated with a solution of TFA/EDT/thioanisole/TIS/water diluted to 90/2.5/2.5/2.5/2.5 to remove the protecting group of the peptide residue and isolate the peptide from the resin.
Thereafter, 10 times of cold diethyl ether was added to the reaction solution to precipitate the peptide, and centrifugation was carried out at 3000 rpm for 10 minutes. The filtrate was discarded and repeated 2 times. The conjugate thus obtained was purified by reversed phase HPLC.
The purified peptide scaffold and Ce4-miniPEG2-Cys(Pys) were dissolved in a 1:1 solution of water and acetonitrile and reacted at room temperature for 12 hours. The progress of the reaction was confirmed by HPLC and LC/MS. After confirming the completion of the reaction, the reaction mixture was purified by reversed phase HPLC. Elution was performed using a water-acetonitrile linear gradient using a 40 to 70% (v/v) acetonitrile solution including TFA 0.1%.
The chemical structure of the L-EGFR conjugate thus prepared is shown in the following Formula 2. That is, the photosensitizer and the EGFR target peptide were bound via a linear disulfide bond.
3.2. Characteristics of a Linear L-EGFR Conjugate
1) Molecular Weight and Absorption Spectrum
The molecular weight was measured using LC/MSD and the absorbance was measured using a UV/Vis spectrometer.
As a result of the evaluation, the molecular weight of Formula 2 of the purified conjugate of the present invention thus obtained was 2,441 g/mol (
2) Analysis of Fluorescence and Reactive Oxygen Generation Recovery by Reducing Agent Action
Fluorescence recovery of the conjugate thus obtained by the reaction in response to a reducing agent present in a high concentration in cancer cells was performed and analyzed in the same manner as in Example 2. On the other hand, a sample prepared by dilution was put into a multifunctional microplate reader to obtain fluorescence spectrum (λEx 400 nm, λEm 620˜850 nm) results.
In addition, in order to confirm the generation of singlet oxygen (SOG), the same method as in Example 2 was performed and analyzed.
According to
In addition, in the quenching state, the generation of singlet oxygen was suppressed, and in the case of 5 mM DTT treatment, the quenching state was terminated and the generation of singlet oxygen was increased 2 times. Therefore, it can be understood that L-EGFR of Example 3 of the present invention activates fluorescence and singlet oxygen generation as the disulfide bond breaks upon absorption into target cells.
3) Serum Stability
The L-EGFR conjugate of the present invention was mixed with PBS including 10% of PBS, fetal bovine serum (FBS, Gibco, USA), respectively, and cultured, and the stability in the serum was evaluated. The final concentration of the conjugate in solution was adjusted to 2 μM. The solution was allowed to react at room temperature for 4 hours and then put in a multifunctional microplate reader (Safire 2, Tecan, Switzerland) to obtain a fluorescence spectrum (λEx. 400 nm) result.
According to
4) Cell Culturing
The human breast cancer cell line MDA-MB-468 overexpressing epithelial growth factor receptor (EGFR), the moderately expressing MDA-MB-231 and the human normal cell line PCA-SMC not expressing EGFR were all obtained from the American Type Culture Collection (ATCC, USA).
The MDA-MB-231 cells were cultured in RPMI (Roswell Park Memorial Institute) 1640 medium including 10% fetal bovine serum and 1% penicillin/streptomycin (Life Technologies) at 37° C. under 5% carbon dioxide and standard humidity conditions. The MDA-MB-468 cells were cultured in DMEM (Dulbecco's modified Eagle's medium) including 10% FBS and 1% penicillin/streptomycin (Life Technologies) at 37° C. under 5% carbon dioxide and standard humidity conditions. PCA-SMC cells were cultured in vascular cell basal medium including recombinant human insulin, ascorbic acid, glutamine, recombinant human epithelial growth factor, and FBS 10% at 37° C. under 5% carbon dioxide and standard humidity conditions.
5) Immunocytochemical Analysis
The expression level of EGFR, a target protein, in each cell was measured by immunocytochemistry. PCA-SMC, MDA-MB-231 and MDA-MB-468 cells were attached to the cover glass and immobilized with PBS including 4% paraformaldehyde for 15 minutes at room temperature and washed twice. Immobilized cells were reacted with 0.25% triton X-100 for 10 minutes and washed three times with PBS for 5 minutes. Thereafter, PBST buffer solution including 1% BSA (Bovine serum albumin) was treated in cells for 30 minutes, and then reacted with EGFR antibody (1:500 dilution) for 1 hour, and then washed three times with PBS for 5 minutes. Thereafter, the cells were reacted with secondary antibody (1:500 dilution) in the dark for 1 hour and then washed three times with PBS for 5 minutes. A cell nucleus was stained with DAPI for 1 minute, washed with PBS and mounted.
According to
6) Intracellular Absorption of a Conjugate and the Resulting Fluorescence Activity
PCA-SMC, MDA-MB-231 and MDA-MB-468 cells were put in each well of an 8-well Lab-Tek chamber, PCA-SMC cells were put, in each well, at a cell number of 5×103, and MDA-MB-231, MDA-MB-468 cells were put, in each well, at a cell number of 5×104, and were cultured for 24 hours to allow the cells to be adhered well. Thereafter, the existing medium was replaced with 200 μl of a fresh medium in which 2 μM the conjugate was dissolved, and the cells were cultured for 30 minutes, 1 hour, and 4 hours, respectively.
Finally, the cells were washed three times with fresh culture medium to remove the conjugate present outside the cells, and a near-infrared fluorescence image (λEx 405 nm, λEm 625˜754 nm) was obtained using a confocal laser microscope.
Experiments were performed to determine the intracellular location of the L-EGFR conjugate of the present invention. 1×105 cells per well were put in an 8-well Lab-Tek chamber and cultured for 24 hours to allow the cells to be attached well. L-EGFR conjugate was treated at a concentration of 2 μM and was cultured for 4 hours, followed by washing three times. To stain the lysosomes, the existing medium was replaced with fresh medium including 100 nM LysoTracker Blue DND-22 dye, and the cells were cultured for 45 minutes. As to the sample, fluorescence images were obtained for the conjugate (λEx 405 nm, λEm 625˜754 nm) and LysoTracker (λEx 405 nm, λEm 411˜497 nm) using a confocal microscope.
According to
According to
7) Cytotoxicity and Photodynamic Therapy Efficacy Experiment
In the dark state without light, the L-EGFR conjugate of the present invention was added to MDA-MB-468 cells at a concentration of 0˜10 μM and cytotoxicity was evaluated. As a result, as illustrated in
In order to evaluate the efficacy of photodynamic therapy, PCA-SMC cells were put in a 1×103/well and MDA-MB-231 cells and MDA-MB-468 cells were put as much as 1×104/well in a 96-well plate, respectively, and were cultured for 24 hours to allow the cells to be adhered well. L-EGFR conjugates were prepared at concentrations of 0.1 μM, 0.5 μM, 1 μM, 2 μM, 5 μM and 10 μM, respectively, treated with cells, and then cultured for 4 hours. Thereafter, the conjugate that had not been absorbed into cells was washed and removed, and then irradiated with a 670 nm laser at an irradiation dose density of 50 mW/cm 2 for 6 minutes and 40 seconds. Thereafter, after further culturing in an incubator until the next day, the cytotoxicity was assessed by CCK-8 assay.
As a result, according to
4.1. Preparation of a Conjugate Including a Photosensitizer, Arginine and Tryptophan
The peptides were prepared by Fmoc solid phase peptide synthesis (SPPS) using ASP48S. The preparation process is provided as follows.
The amino acid with the protecting group (Fmoc) bound to Trt-Cl resin (GL Biochem, China) was strongly mixed with DMF including 20% piperidine and reacted twice at room temperature for 10 minutes, followed by washing with DMF and methanol twice. Thereafter, eight equivalents of amino acid and eight equivalents of HBTU/eight equivalents of HOBt/16 equivalents of N,N-Diisopropylethylamine (DIPEA) were dissolved in DMF and added, and reacted at room temperature for 2 hours, followed by washing with DMF and methanol twice. This process was repeated to make RRW-resin, RRWW-resin, RRWWW-resin and RWR-resin, respectively.
The photosensitizer Ce4 and HBTU/HOBt/DIPEA were dissolved in DMF and added to the resin-peptide basic scaffold conjugate, and then the mixed solution was reacted for 12 hours and suctioned, followed by washing with DMF, methanol and DMF in that orderly manner.
When the peptide scaffold to be synthesized is formed through this process, a solution in which TFA/EDT/thioanisole/TIS/water was diluted with 90/2.5/2.5/2.5/2.5 was treated for 2 hours. The protecting group of the peptide residue thus prepared was removed and the peptide was isolated from the resin. Thereafter, cold diethyl ether was added to the reaction solution to precipitate the peptide, centrifugation was carried out, and the obtained product was dried.
The finally obtained product was dissolved in DMSO and purified by reverse phase HPLC using a Vydac Everest C18 column (250 mm×22 mm×10 ium, USA). Elution was performed using a water-acetonitrile linear gradient using 10 to 75% (v/v) acetonitrile solution including TFA 0.1%, and the purified product was lyophilized.
The chemical structure of the prepared conjugate (Example 4) prepared as above is as shown in the following Formulas 3 to 6, and the photosensitizer and tryptophan are linked via arginine.
4.2. Characteristics of a Conjugate Including a Photosensitizer, Arginine, Tryptophan
1) Molecular Weight and Fluorescence Emission
The molecular weight was measured using LC/MSD and the absorbance was measured using a UV/Vis spectrometer.
As a result of the evaluation, the molecular weight of the purified conjugate of the present invention thus obtained was measured to be 1,051 g/mol for Ce4-RRW, 1,237 g/mol for Ce4-RRWW, 1,423 g/mol for Ce4-RRWWW, 1,051 g/mol for Ce4-RWR, respectively (see
2) Increase in fluorescence signal due to enzyme action 1 nmol of each of Ce4-RRWW and Ce4-RRWWW thus obtained was prepared and dissolved in a PBS solution. The trypsin enzyme was mixed with each 50 nmol of each conjugate and fluorescence changes were observed over time at 37° C.
According to
The Ce4-RRW thus obtained was diluted in 93.5 μl of sodium acetate buffer (20 mM sodium acetate buffer, 1 mM EDTA, pH 5.0), and then 97 pmol of cathepsin B (Calbiochem, USA) was mixed in the enzyme-treated sample. The same amount of sodium acetate buffer solution was mixed in the buffer solution-treated group. The mixed solution (100 μl) was cultured at 37° C. for 2 hours.
3) Serum Stability
The Ce4-RRW conjugate of the present invention was mixed with PBS including 10% of FBS and cultured to evaluate the stability in serum. The final concentration of the conjugate in solution was adjusted to 2 μM. The solution was allowed to react at room temperature for 4 hours and then put in a multifunctional microplate reader to obtain a fluorescence spectrum (λEx 610 nm, λEm. 620˜850 nm) result.
According to
4) Cell Culturing
Human cancer KB cell line was obtained from ATCC. The KB cells were cultured in MEM (Minimum Essential Media) medium (Gibco) including 10% FBS and 1% penicillin/streptomycin at 37° C. under 5% carbon dioxide condition.
5) Specific Cell Absorption and Fluorescence Activity of the Conjugate Accordingly
KB cells per well were put in a 4-well Lab-Tek chamber per 2×105, and were cultured for 24 hours to allow the cells to be attached well. Thereafter, the existing medium was replaced with 500 μl of fresh medium in which the Ce4-RRW conjugate (2 μM) was dissolved, and then the cells were cultured for 4 hours.
Finally, the cells were washed three times with the culture medium, and then a near-infrared fluorescence image (λEx 405 nm, λEm 625˜754 nm) was obtained using a confocal laser microscope.
Experiments were performed to determine the intracellular location of the Ce4-RRW of the present invention. In the 8-well Lab-Tek chamber, 5×104 KB cells were put in each well. The cells were cultured for 24 hours to allow the cells to be attached well, and then 2 μM Ce4-RRW was added and cultured for 4 hours, followed by washing three times. Thereafter, after replacing the existing medium with fresh medium including 100 nM LysoTracker Blue DND-22 dye, the cells were cultured for 45 minutes. Fluorescent images of the Ce4-RRW (λEx 405 nm, λEm. 625˜754 nm) and LysoTracker (λEx 405 nm, λEm 411˜497 nm) were obtained for these samples using confocal microscopy.
According to
According to
Even when the fluorescence images were superimposed on each other, the position looked superimposed exactly. It was confirmed that Ce4-RRW was located in the lysosome after absorption into cells and was activated by the enzyme.
In Example 5, when the photosensitizer, which had been quenched by tryptophan, was degraded by a complex action of an intracellular reducing agent and an enzyme, a strong fluorescence signal was generated while the quenching effect was terminated, and at the same time, a conjugate was prepared in which the photodynamic therapy efficacy was also recovered.
5.1. Synthesis of a Reducing Agent/Enzyme Complex Reaction Type Conjugate
The conjugate is a model of a conjugate having both a disulfide bond degraded by a reducing agent and a bond including arginine degraded by an enzyme, and Ce4-CRRCW conjugate was synthesized.
Peptides were prepared by Fmoc solid phase peptide synthesis (SPPS) using ASP48S. The preparation process is provided as follows.
The amino acid with the protecting group (Fmoc) bound to Trt-Cl resin (GL Biochem, China) was strongly mixed with DMF including 20% piperidine and reacted twice at room temperature for 10 minutes, followed by washing with DMF and methanol twice. Thereafter, eight equivalents of amino acid and eight equivalents of HBTU/eight equivalents of HOBt/16 equivalents of N,N-Diisopropylethylamine (DIPEA) were dissolved in DMF and added, and reacted at room temperature for 2 hours, followed by washing with DMF and methanol twice. This process was repeated to make CRRCW-resin.
The photosensitizer Ce4 and HBTU/HOBt/DIPEA were dissolved in DMF and added to the resin-peptide basic scaffold conjugate, and then the mixed solution was reacted for 12 hours and suctioned, followed by washing with DMF, methanol and DMF in that orderly manner.
When the peptide scaffold to be synthesized was formed through this process, a solution in which TFA/EDT/thioanisole/TIS/water was diluted with 90/2.5/2.5/2.5/2.5 was treated for 2 hours. The protecting group of the peptide residue thus prepared was removed and the peptide was isolated from the resin. Thereafter, cold diethyl ether was added to the reaction solution to precipitate the peptide, centrifugation was carried out, and the obtained product was dried.
The product obtained for cyclic disulfide bond generation was dissolved in a mixed solution of water/acetonitrile (9:1, v/v) at a concentration of 0.5 mg/mL, and ammonium acetate solution was added thereto and stirred (peptide solution: ammonium acetate=9:1, v/v). The progress of the reaction was confirmed by Elman reagent.
The finally obtained product was dissolved in DMSO and purified by reverse phase HPLC using a Vydac Everest C18 column (250 mm×22 mm×10 μm, USA). Elution was performed using a water-acetonitrile linear gradient using 10 to 75% (v/v) acetonitrile solution including TFA 0.1%, and the purified product was lyophilized.
The chemical structure of the conjugate Ce4-CRRCW prepared as above is as shown in the following Formula 7.
5.2. Characteristic Analysis of a Reducing Agent/Enzyme Complex Reaction Type Conjugate
1) Molecular Weight and Fluorescence Emission
The molecular weight of the synthesized Ce4-CRRCW was measured using LC/MSD and the absorbance was measured using a UV/Vis spectrometer. The maximum absorption peaks were measured at 402, 508 and 665 nm.
As a result of the evaluation, the purified conjugate of the present invention thus obtained had a molecular weight of 1,255 g/mol (
2) Increase in Fluorescence Signal Due to a Reducing Agent and Enzyme Action
Meanwhile, in order to analyze the reactivity of the reducing agent/enzyme complex reaction type Ce4-CRRCW conjugate to the reducing agent, the conjugate was treated with reducing agents DTT 2 μM and 5 mM for 6 hours and put into a multifunctional microplate reader to obtain the fluorescence spectrum (λEx 400 nm, λEm 620˜850 nm) results. In order to analyze the reactivity to the enzyme, the conjugate was treated with cathepsins B, S, L, and S, or cathepsin B pretreated with cathepsin inhibitor E64 for 6 hours and put into a multifunctional microplate reader to obtain the fluorescence spectrum (λEx 400 nm, λEm 620˜850 nm) results. In order to analyze the reducing agent/enzyme complex reaction, 5 mM DTT and cathepsin B enzyme were treated together for 6 hours and put into a multifunctional microplate reader to obtain the fluorescence spectrum (λEx 400 nm, λEm 620˜850 nm) results.
According to
3) Cell Culturing
SCC7 cancer cell lines were cultured in DMEM (Dulbecco's modified Eagle's medium) medium (Gibco) including 10% FBS and 1% penicillin/streptomycin at 37° C. under 5% carbon dioxide condition.
4) Absorption of the Conjugate in Cancer Cells and Fluorescence Activity Analysis Accordingly
SCC7 cancer cells per well were put in a 4-well Lab-Tek chamber per 2×105, and were cultured for 24 hours to allow the cells to be attached well. Thereafter, the existing medium was replaced with 500 μl of fresh medium in which the Ce4-RRW conjugate 2 μM was included, and then the cells were cultured for 2 to 6 hours. Finally, the cells were washed three times with the culture medium, and a near-infrared fluorescence image (λEx 633 nm, λEm. 646˜753 nm) was obtained using a confocal laser microscope.
Experiments were also conducted to analyze whether the conjugate migrates to a lysosome with a high concentration of intracellular reducing agent. In the 8-well Lab-Tek chamber, SCC7 cells were put at a cell number of 5×104 in each well. The cells were cultured for 24 hours to allow the cells to be attached well, and then 2 μM the conjugate was added and cultured for 6 hours, followed by washing three times. Thereafter, after replacing the existing medium with fresh medium including 100 nM LysoTracker Blue DND-22 dye, the cells were cultured for 45 minutes. Fluorescent images of the conjugate (λEx 405 nm, λEm 625˜754 nm) and LysoTracker (λEx 405 nm, λEm 411˜497 nm) were obtained for these samples using confocal microscopy.
According to
According to
6.1. Synthesis of Phenylalanine-Added Photosensitizer-Peptide Conjugate
The peptides were prepared by Fmoc solid phase peptide synthesis (SPPS) using ASP48S, and the preparation process was as follows.
An amino acid with a protecting group (Fmoc) bound to Trt-Cl resin (GL Biochem, China) was strongly mixed in DMF containing 20% piperidine, reacted twice for 10 minutes at room temperature, and then washed with DMF and methanol twice. Thereafter, 8 equivalents of amino acid and 8 equivalents of HBTU/8 equivalents of HOBt/16 equivalents of N,N-diisopropylethylamine (DIPEA) were dissolved in DMF and added, reacted at room temperature for 2 hours, and then washed with DMF and methanol twice. This process was repeated to make RRWGF-resin, WRRGKF-resin, and FWRRGK-resin, respectively.
The photosensitizer Ce4 and HBTU/HOBt/DIPEA were dissolved in DMF and added to the resin-peptide basic scaffold conjugate, and then the mixture was reacted for 12 hours and suctioned, and then washed with DMF, methanol and DMF in that orderly manner.
When the peptide basic scaffold to be synthesized was formed through this process, the peptide basic scaffold was treated with a solution of TFA/EDT/thioanisole/TIS/water diluted to 90/2.5/2.5/2.5/2.5 for 2 hours to remove a protecting group of the peptide residue generated above and to isolate the peptide from the resin. Thereafter, the peptide was precipitated and centrifuged by adding cold diethyl ether to a reaction solution, and the obtained product was dried.
The finally obtained product was dissolved in DMSO and purified by performing reverse-phase HPLC using a Vydac Everest C18 column (250 mm×22 mm×10 ium, USA). The product was eluted using a water-acetonitrile linear gradient using a 10 to 75%(v/v) acetonitrile solution containing 0.1% TFA, and the purified product was lyophilized.
Chemical structures of the conjugate (Example 6) prepared as described above were shown in Formulas 8 to 10 below.
6.2. Characteristic Analysis of Phenylalanine-Added Conjugates
1) Molecular Weight and Fluorescence Emission
The molecular weights of the synthesized conjugates were measured using LC/MSD. The absorbance of the conjugates was measured using a UV/Vis spectrometer. In order to analyze the fluorescence of the conjugates, the conjugates were dissolved in phosphate buffered saline (PBS) and dimethyl sulfoxide (DMSO) at a 2 μM Ce4 equivalent concentration, respectively and then the fluorescence spectra (λEx. 400 nm, λEm. 410 to 800 nm) were obtained on a microplate reader.
As a result of the evaluation, the molecular weights of the purified conjugates of the present invention obtained above were 1254.49 g/mol for Ce4-RRWGF-NH2, 1382.66 g/mol for WRRGK(Ce4)F-NH2, and 1382.66 g/mol for FWRRGK(Ce4)-NH2 (see
2) Preparation of Liposome Formulation Loaded with Conjugate
The peptide conjugates Ce4-RRWGF-NH2, WRRGK(Ce4)F-NH2, and FWRRGK(Ce4)-NH2 formed nanoparticles by self-assembly in an aqueous solution to obtain a fluorescence quenching effect, but due to the large particle size, a liposome formulation was prepared to obtain small-sized nanoparticles. Dipalmitoylphosphatidylcholine (DOPC) dissolved in chloroform, cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG2000) were added and mixed in a round flask in a molar ratio of 50:45:5 and then chloroform was volatilized using argon gas. Thereafter, a solvent was completely removed through a freeze dryer for 3 hours to prepare a lipid in the form of a thin film. Thereafter, a 350 mM ammonium sulfate solution was added thereto for hydration. To make the liposome size to 100 nm or less, the hydrated lipid solution was sonicated and then dialyzed with a 100 mM sodium acetate (pH 5.2) solution for 16 hours. After dialysis, each peptide conjugate was dissolved in dimethyl sulfoxide (DMSO), mixed with liposomes so that the volume of dimethyl sulfoxide was 5 v/v %, and then reacted for 1 hour. Thereafter, unreacted liposomes and peptides were removed using a PD-10 column, and liposomes were concentrated using an Amicon Ultra-0.5 centrifugal filter (molecular weight cut off: 100 kDa). In order to analyze the liposomes loaded with the peptide conjugates, some of the liposomes were dissolved in dimethyl sulfoxide, subjected to quantitative analysis, and then used in experiments.
3) Analysis of Size and Quenching State of Prepared Liposome
Liposomes loaded with conjugates, Lipo-Ce4F (Ce4-RRWGF-NH2@liposome), Lipo-WCe4F (WRRGK(Ce4)F-NH2@liposome), and Lipo-FCe4 (FWRRGK(Ce4)-NH2@liposome) were diluted with distilled water, respectively, and then when the sizes were measured with a particle size analyzer (dynamic light scattering (DLS)), the sizes were confirmed as 119.2±0.46 nm, 111.9±1.28 nm, and 124.6±0.72 nm (see
4) Cell Culturing
A human colon cancer HCT-116 cell line was obtained from ATCC. HCT-116 cells were cultured in a McCoy's medium (Gibco) containing 10% FBS and 1% antibiotic/antimycotic under conditions of 37° C. and 5% carbon dioxide.
5) Uptake of Liposomal Nanoparticles into Cancer Cells and Analysis of Fluorescence Activity Accordingly
After the quenched liposomal nanoparticles were taken up into cancer cells, the re-activation of fluorescence in the cancer cells was analyzed. HCT-116 cells were added in a 4-well Lab-Tek chamber at 5×104 per well and cultured for 24 hours to allow the cells to be attached well. The existing cell culture medium was replaced with a culture medium (500 iLd) containing liposomal nanoparticles at a 5 μM Ce4 equivalent concentration. Every each hour, the liposomal nanoparticle-treated cancer cells were washed three times with a fresh cell culture medium, and then near-infrared fluorescence images (λEx. 633 nm, λEm. 638 to 759 nm) were obtained using a confocal laser microscope. As a control, cancer cells treated with only a cell medium were used.
According to
6) Analysis of Cytotoxicity and Photodynamic Therapy Effect of Liposomal Nanoparticles
HCT-116 cells were added in a 96-well plate at 1×104 per well and cultured for 24 hours to allow the cells to be attached well. After the existing medium was replaced with 100 μl of a cell culture medium in which a free photosensitizer Ce4 and liposomal nanoparticles were diluted to a 2 μM Ce4 equivalent concentration, the cancer cells were treated for 4 hours. First, in order to confirm the dark toxicity of the liposomal nanoparticles, Ce4, Lipo-WCe4F, Lipo-FCe4, and Lipo-Ce4F were treated for 4 hours, respectively, and then the cells were washed three times with a fresh culture medium and after 24 hours, the cytotoxicity was observed (see
7.1. Synthesis and Analysis of PEG or Lipid-Added Photosensitizer-Peptide Conjugate
1) Synthesis of PEG or Lipid-Added Photosensitizer-Peptide Conjugate
The peptides were prepared by Fmoc solid phase peptide synthesis (SPPS) using ASP48S, and the preparation process was as follows.
An amino acid with a protecting group (Fmoc) bound to K(Dde) resin (GL Biochem, China) was strongly mixed in DMF containing 20% piperidine, reacted twice for 10 minutes at room temperature, and then washed with DMF and methanol twice. Thereafter, 8 equivalents of miniPEG2 or palmitoyl/8 equivalents of HBTU/8 equivalents of HOBt/16 equivalents of N,N-diisopropylethylamine (DIPEA) were dissolved in DMF and added, respectively, reacted at room temperature for 2 hours, and then washed with DMF and methanol twice.
To bind a photosensitizer chlorin e4 (Ce4) or pyropheophorbide a (PPA) to a side chain of lysine (K) of the resin-peptide basic scaffold conjugate, the protecting group of lysine was washed and removed twice for 10 minutes each in a hydrazine solution dissolved in DMF. In order to bind the photosensitizer to lysine from which the protecting group has been removed, 4 equivalents of Ce4 or pyropheophorbide a, DIC, and DIPEA were dissolved and added in DMF, respectively, and the mixture reacted for 12 hours, suctioned, and then washed with DMF, methanol and DMF in that order.
When the peptide basic scaffold to be synthesized was formed through this process, the peptide basic scaffold was treated with a solution of TFA/EDT/thioanisole/TIS/water diluted to 90/2.5/2.5/2.5/2.5 for 2 hours to remove a protecting group of a peptide residue generated above and to isolate the peptide from the resin. Thereafter, the peptide was precipitated and centrifuged by adding cold diethyl ether to a reaction solution, and the obtained product was dried.
The finally obtained product was dissolved in DMSO and purified by performing reverse-phase HPLC using a Vydac Everest C18 column (250 mm×22 mm×10 ium, USA). The product was eluted using a water-acetonitrile linear gradient using a 10 to 75% (v/v) acetonitrile solution containing 0.1% TFA, and the purified product was lyophilized.
Chemical structures of the conjugates prepared as described above were shown in Formulas 11 to 15 below.
2) Analysis of Molecular Weights and Fluorescence Characteristics of the Synthesized Conjugates
The molecular weights of the synthesized conjugates were measured using LC/MSD, and the absorbance was measured using a UV/Vis spectrometer.
As a result of the evaluation, the molecular weights of the purified conjugates of the present invention obtained above were 1528.50 g/mol for miniPEG2-K(Ce4)RRWGF, 1621.65 g/mol for Palmitoyl-WRRGK(Ce4)F, and 1621.65 g/mol for Palmitoyl-FWRRGK(Ce4), 1447.04 g/mol for Palmitoyl-WERGK (Ce4), and 1429.04 g/mol for Palmitoyl-WERGK (PPA), respectively (see
7.2. Preparation and Efficacy Evaluation of Micellar Nanoparticles Using Lipid-Added Photosensitizer-Peptide Conjugate
In the case of mPEG2-Ce4F, since a difference in fluorescence intensity between the phosphate buffer and dimethyl sulfoxide was measured smaller than that of other conjugates, the mPEG2-Ce4F conjugate was excluded from preparing the micellar nanoparticles.
1) Preparation of Micellar Nanoparticles Using Conjugate
In order to prepare small-sized micellar nanoparticles, micellar nanoparticles were prepared by mixing a surfactant with the conjugate. The surfactant F68 or F127 was dissolved in distilled water at 10 wt/v %, and the peptide was dissolved in an ethanol solvent. Peptides and the surfactant were mixed in a mass ratio of 1:10, and ethanol was evaporated and removed while stirring for 3 hours. The prepared micellar nanoparticles were washed with distilled water and concentrated using an Amicon Ultra-0.5 centrifugal filter (molecular weight cut off: 100 kDa) three times.
2) Analysis of Size Distribution and Quenching Characteristics of Micellar Nanoparticles
The micellar nanoparticles PA-WCe4F/F68, PA-FCe4/F68, PA-FCe4/F127, PA-WERCe4/F127, and PA-WERPPA/F127 prepared above were diluted with distilled water, respectively, and the sizes were measured with a particle size analyzer (Dynamic light scattering, DLS). As a result, the sizes were confirmed as 169.8±2.33 nm, 157±0.50 nm, 193.5±10.14 nm, 243.6±5.31 nm, and 90.3±5.23 nm (see
In order to confirm that the fluorescence signal of the photosensitizer was quenched after micelle formation, the prepared micellar nanoparticles were diluted in a phosphate buffer and dimethyl sulfoxide to a 2 μM photosensitizer equivalent concentration, respectively, and each fluorescence spectrum was analyzed with a microplate reader. As a result, it was confirmed that the fluorescence in the phosphate buffer was 1399.7 times, 485.1 times, 20.6 times, 127.3 times, and 398.1 times lower than the fluorescence intensity of the micellar nanoparticles dissolved in dimethyl sulfoxide (λEx. 400 nm, see
3) Increase in Fluorescence Signal Due to Enzymatic Action
It was analyzed whether the fluorescence of the micellar nanoparticles prepared above was increased again by the enzyme treatment. For a sample in which micellar nanoparticles were dispersed in a phosphate buffer at a 2 μM Ce4 equivalent concentration and a sample treated with 2.5 unit cathepsin B enzyme, an increase in fluorescence intensity over time was measured (λEx. 360/35 nm, λEm. 665/8 nm). The fluorescence of micellar nanoparticles PA-WCe4F/F68 and PA-FCe4/F68 increased 5.26 times and 6.53 times, respectively, after 18 hours of enzymatic treatment (see
4) Cell Culturing
A human colon cancer HCT-116 cell line was obtained from ATCC. The HCT-116 cells were cultured in a McCoy's medium (Gibco) containing 10% FBS and 1% antibiotic/antimycotic under conditions of 37° C. and 5% carbon dioxide.
5) Uptake of Micellar Nanoparticles into Cancer Cells and Analysis of Fluorescence Activity Accordingly
HCT-116 cells were added in a 4-well Lab-Tek chamber at 5×104 per well and cultured for 24 hours to allow the cells to be attached well. The existing medium was replaced with 500 μl of a medium in which the micellar nanoparticles were diluted at a 2 μM Ce4 or PPA equivalent concentration, and cancer cells were treated for 4 hours. Finally, after the cancer cells were washed three times with a fresh culture medium, near-infrared fluorescence images (λEx. 633 nm, λEm. 638 to 759 nm) of the cancer cells were obtained using a confocal laser microscope.
According to
6) Analysis of Cytotoxic and Phototoxic Effects of Micellar Nanoparticles
HCT-116 colon cancer cells were added in a 96-well plate at 1×104 per well and cultured for 24 hours to allow the cells to be attached well. A free photosensitizer (free Ce4) and PA-FCe4/F68 micellar nanoparticles were treated at 0, 0.625, 1.25, 2.5, 5, and 10 μM Ce4 equivalent concentrations. In addition, the free photosensitizer (free Ce4) and the PA-FCe4/F127 micellar nanoparticles were treated at 0, 0.125, 0.25, 0.5, 1, and 2 μM Ce4 equivalent concentrations. The photosensitizer was treated for 4 hours. For dark toxicity analysis of micellar nanoparticles, after treatment with the photosensitizer for 4 hours, cancer cells were washed three times with a fresh culture medium and further cultured for 24 hours, and then cell viability was analyzed. In order to confirm the phototoxicity of micellar nanoparticles, after treatment with the photosensitizer for 4 hours, the cancer cells were washed three times with a fresh culture medium, and then irradiated with a 670 nm laser at a 10 J/cm 2 light dose. After further culturing the cancer cells for 24 hours, the cell viability was analyzed. As a result, when the laser was not irradiated, in the free photosensitizer and micellar nanoparticle-treated groups, the cytotoxicity was not exhibited even at high concentrations. On the other hand, when irradiated with the laser, the cancer cells treated with the micellar nanoparticles exhibited a very high cancer therapy effect compared to cancer cells treated with a free photosensitizer (see
7) Phototoxic Effect Using Micellar Nanoparticles in Mouse Colon Cancer Model
Tumor production was induced by injecting 1×107 cells/200 μL of HCT-116 cells subcutaneously in the right legs of 5-week-old Balb/c-nu mice. When the sizes of the tumor reached 100 to 200 mm2, the mice were divided into a control group, a free photosensitizer treated group, and a PA-FCe4/F127 micellar nanoparticle-treated group and used for the experiments. In the case of a near-infrared fluorescence imaging analysis group, a photosensitizer was injected intravenously into the tails of mice at doses of 2 mg Ce4 eq./kg and 5 mg Ce4 eq./kg, and fluorescent images were taken before injection and on 24 hours after injection. In the case of the photodynamic treated group, the photosensitizer was injected intravenously into the tails of the mice at doses of 2 mg Ce4 eq./kg and 5 mg Ce4 eq./kg, and after 24 hours, the tumor was irradiated with a 670 nm laser at a 50 J/cm 2 light dose. The sizes of the tumor and the body weights of the mice were measured periodically until day 10.
According to the result of analyzing the near-infrared fluorescent images (see
As discussed above, the specific portions of the contents of the present invention have been described in detail. Therefore, it is apparent to a person having ordinary skill in the pertinent art that such specific technology is merely a preferable embodiment, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention is defined by the appended claims and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2016-0088676 | Jul 2016 | KR | national |
This application is a Continuation-in-Part of U.S. application Ser. No. 16/316,555 filed Jan. 9, 2019, which is a National Stage Application of International Application No. PCT/KR2017/007068 filed Jul. 4, 2017, claiming priority based on Korean Patent Application No. 10-2016-0088676 filed Jul. 13, 2016, the respective disclosures of all of the above of which are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16316555 | Jan 2019 | US |
| Child | 18468674 | US |
