Cell designer molecule for imparting drug release properties to cell

Abstract

Provided are, for example: a compound that imparts drug release properties and cancer cell-targeting properties to a cell; a method for imparting drug release properties and cancer cell-targeting properties to a cell by using the compound; and a cell having cancer cell-targeting properties, the cell having the compound adsorbed thereto.

Description

Cellular designer molecules that confer drug-releasing properties to cells

[0001] The present invention relates to the fields of cell engineering, tissue engineering, medical materials, medicine, etc. In particular, the present invention relates to a compound that imparts drug-releasing properties to cells and uses thereof.

[0002] There are various types of cancer treatments. Among them, there is a treatment that directly delivers anticancer drugs such as antibodies to cancer cells (see Non-Patent Document 1, etc.). However, there is a problem that the delivery rate of the anticancer drugs to solid tumors is low, and sufficient therapeutic effect cannot be obtained. Furthermore, if the anticancer drugs can be administered to solid tumors over a long period of time, the therapeutic effect will be even greater.

[0003] Guidance for Cancer Immunotherapy Development 2015: Concept of Early Clinical Trials - Aiming for Safe and Effective Development - Report of the Review Committee for the Development of Guidance for the Ministry of Health, Labor and Welfare's Subsidy Program for Accelerated Drug Review, Innovative Drugs, Medical Devices and Regenerative Medicine Products Commercialization Promotion Project, January 30, 2015

[0004] It is necessary to improve the delivery rate of drugs to cancer and to find new systems that can release drugs in the vicinity of cancer.

[0005] The present inventors conducted extensive research to solve the above problems, and synthesized cellular designer molecules that impart drug-releasing properties to platelets. They also found that platelets adsorbing the designer molecules have drug-releasing properties and cancer-targeting properties and can be used as carriers for anticancer drugs, leading to the completion of the present invention.

[0006] That is, the present invention provides the following: (1) A compound comprising a hydrophilic polymer or a derivative thereof, a bile acid or a derivative thereof, and a cancer-targeting substance. (2) The compound according to (1), wherein the hydrophilic polymer is selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polyglutamic acid, polyaspartic acid, polylysine, and polyhistidine. (3) The compound according to (2), wherein the hydrophilic polymer is polyethylene glycol or polyvinyl alcohol. (4) The compound according to any one of (1) to (3), wherein the bile acid is selected from the group consisting of cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, ursodeoxycholic acid, hyodeoxycholic acid, glycocholic acid, and taurocholic acid. (5) The compound according to (4), wherein the bile acid is deoxycholic acid or cholic acid. (6) The compound according to any one of (1) to (5), wherein the carboxyl group of the bile acid or a derivative thereof is not used for binding. (7) A method for producing cells having drug-releasing properties and cancer cell tropism, comprising adsorbing the compound according to any one of (1) to (6) onto cells. (8) The method according to (7), wherein the cells are platelets. (9) An agent or kit for imparting drug-releasing properties and cancer cell tropism to cells, comprising the compound according to any one of (1) to (6). (10) The agent or kit according to (9), wherein the cells are platelets. (11) Cells to which the compound according to any one of (1) to (6) has been adsorbed. (12) The cells according to (11), which are platelets. (13) The cells according to (11) or (12), which carry, contain, express or produce a drug. (14) A composition for delivering a drug to cancer, comprising the cells according to (13). (15) Platelets to which a compound comprising a hydrophilic polymer or a derivative thereof, and a bile acid or a derivative thereof has been adsorbed. (16) The platelets according to (15), wherein the hydrophilic polymer in the compound is selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polyglutamic acid, polyaspartic acid, polylysine, and polyhistidine. (17) The platelets according to (16), wherein the hydrophilic polymer in the compound is polyethylene glycol or polyvinyl alcohol.(18) The platelets according to any one of (15) to (17), wherein the bile acid in the compound is selected from the group consisting of cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, ursodeoxycholic acid, hyodeoxycholic acid, glycocholic acid, and taurocholic acid. (19) The platelets according to (18), wherein the bile acid in the compound is deoxycholic acid or cholic acid. (20) The platelets according to any one of (15) to (19), wherein the carboxyl group of the bile acid or a derivative thereof in the compound is not used for binding. (21) The platelets according to any one of (15) to (20), which carry, contain, express, or produce a drug. (22) A composition for delivering a drug to cancer, comprising the platelets according to (20) or (21).

[0007] According to the present invention, by using the compound of the present invention, cells, particularly platelets, can be used as carriers for drugs such as antibodies. The delivery rate of drugs to solid tumors can be increased by using cells adsorbed with the compound of the present invention. Furthermore, drugs can be released from cells adsorbed with the compound of the present invention in response to a weakly acidic environment, such as the weakly acidic environment of tumors.

[0008] Figure 1 is a photograph showing the results of a fluorescence microscope study of the adsorption of 8-PEG-DCA-(81)-FGA to platelets. 8-PEG-DCA-FGA represents the fluorescence of 8-PEG-DCA-FGA, Calcein Red AM represents the fluorescence of calcein red, Ph represents a phase-contrast microscope image, and Merge represents a superimposed image. Figure 2 shows a fluorescence image showing the adsorption of 8-PEG40k-DCA-FGA to platelets and line scan results showing the fluorescence intensity. In the figure, "8-PEG40k-DCA-OH-FGA" represents the fluorescence of FGA derived from 8-PEG40k-DCA-OH-FGA, "Calcein Red" represents the fluorescence of calcein red derived from calcein red-encapsulated platelets, and "Merge" represents a superimposed image. The line scan shows the results of observing the fluorescence intensity along the white line on the upper right merged image. Distance indicates the distance from the starting point (left) of the white line. Figure 3 shows fluorescence images demonstrating the adsorption of 4-PEG20k-DCA-Cy3 to platelets. In the figure, "Calcein" indicates the fluorescence of calcein derived from calcein-encapsulated platelets, "4-PEG20k-DCA75-Cy3" indicates the fluorescence of Cy3 derived from 4-PEG20k-DCA75-Cy3, Ph indicates a phase-contrast microscope image, and "Merge (4-PEG20k-DCA75-Cy3 + Ph)" indicates an overlaid image. Figure 4 shows the calcein leakage rate from platelets after modification with the cell designer molecule 4-PEG20k-trz-DCA-COOH or 4-PEG2k-trz-DCA-COOH. In the figure, "Platelet" indicates unmodified platelets. Figure 5 shows the calcein leakage rate from platelets modified with the cell designer molecule 4-PEG20k-trz-DCA-COOH or 4-PEG2k-trz-DCA-COOH at pH 7.4 or pH 6.5. In the figure, "Platelet" indicates unmodified platelets. Figure 6 shows the results of a test of NB-1 cells (ALK-overexpressing cells) recognition by LMWH-FITC, PEG-LMWH-Cy3, or PEG-LMWH-Cy3 in competition with LMWH-FITC. In the figure, the vertical axis represents the number of cells counted, and the horizontal axis represents fluorescence intensity. "Non" indicates the control (NB-1 cells in the absence of fluorescently labeled molecules).FIG. 7 shows fluorescence images showing the adsorption of platelets modified with 4-PEG20k-DCA-LMWH to target cancer cells.

[0009] In one aspect, the present invention provides a compound comprising a hydrophilic polymer or a derivative thereof, a bile acid or a derivative thereof, and a cancer-targeting substance. Herein, the compound may be referred to as a "compound of the present invention" or a "cellular designer molecule of the present invention." The compound of the present invention can confer drug-releasing properties and cancer cell-targeting properties to cells.

[0010] In a preferred embodiment, the compound of the present invention can respond to the weakly acidic environment of a tumor by inducing cell membrane disruption of the cells to which the compound of the present invention is adsorbed, thereby releasing the drug from the cells.

[0011] A hydrophilic polymer is a polymer that has a charged functional group or a polar functional group and is capable of dissolving in or interacting with water or other polar substances. The hydrophilic polymer contained in the compound of the present invention carries a bile acid or its derivative, and a cancer-targeting substance. The hydrophilic polymer contained in the compound of the present invention may be of any type. There is no particular limitation on the molecular weight of the hydrophilic polymer, and it may be, for example, several thousand to several hundred thousand. There is no particular limitation on the structure of the hydrophilic polymer, and it may be linear, branched, cyclic, or the like. Preferably, the hydrophilic polymer contained in the compound of the present invention is highly biocompatible and has low or no cytotoxicity.

[0012] Examples of hydrophilic polymers include polyethylene glycol, polyvinyl alcohol, poly(vinylpyrrolidone), polyvinylamide, polyacrylic acid, polyacrylamide, polyamino acids, polysaccharides, proteins, etc. Preferred examples of hydrophilic polymers include polyethylene glycol (PEG), polyvinyl alcohol, polyglutamic acid, polyaspartic acid, polylysine, polyhistidine, etc. More preferred examples of hydrophilic polymers include PEG or polyvinyl alcohol. An even more preferred example of a hydrophilic polymer is PEG. Needless to say, the hydrophilic polymers contained in the compound of the present invention are not limited to the above examples.

[0013] When the hydrophilic polymer is PEG, any type of PEG may be used. The shape of the PEG is not particularly limited, and linear, Y-shaped, branched, and other types can be used. Preferably, branched PEG is used in the compounds of the present invention. PEG with many branches is preferred. 4-arm and 8-arm PEGs are commercially available. The molecular weight of the PEG is also not particularly limited, and it may be PEG with a molecular weight of several thousand to several tens of thousands, for example, about 2,000 to about 40,000, about 10,000 to about 40,000, or about 20,000 to about 40,000. For example, when the compound of the present invention is to be adsorbed to platelets, PEG with a molecular weight of several thousand to several tens of thousands is preferred, including, but not limited to, PEG with a molecular weight of about 2,000 to about 40,000.

[0014] Derivatives of hydrophilic polymers can be prepared appropriately by those skilled in the art and are also commercially available. Functional groups in the hydrophilic polymer can be amidated, esterified, alkylated, alkenylated, alkynylated, halogenated, or the like by known methods. For example, an amino group, a maleimide group, an N-hydroxysuccinimide group, a carboxyl group, or the like can be bound to the end of polyethylene glycol by known methods. The derivative of the hydrophilic polymer can be selected depending on the type and structure of the bile acid or its derivative to be bound to the hydrophilic polymer, and the cancer-targeting substance.

[0015] Bile acids are steroid compounds biosynthesized from cholesterol in the mammalian liver. Bile acids can disrupt cell membranes and contribute to the adsorption of the compounds of the present invention to cells and the imparting of drug release to cells. Various bile acids are known, and an appropriate one can be selected and used in the compounds of the present invention. Bile acids include primary bile acids such as cholic acid and chenodeoxycholic acid, secondary bile acids such as deoxycholic acid, lithocholic acid, ursodeoxycholic acid, and hyodeoxycholic acid, and conjugated bile acids such as glycocholic acid and taurocholic acid. Any of these may be used in the compounds of the present invention. Deoxycholic acid or cholic acid is preferred. Deoxycholic acid (DCA) is more preferred.

[0016] Bile acid derivatives are known, can be appropriately synthesized, and are commercially available. For example, any hydroxyl group of a bile acid may be attached or detached, or may be alkylated, azide, or the like. Furthermore, for example, a carboxyl group of a bile acid may be esterified or amidated. The bile acid derivatives are not limited to those described above.

[0017] A cancer-targeting substance is a substance that has high affinity or specific binding ability for cancer cells or cancer tissues. Cancer-targeting substances contribute to conferring tropism to cells for cancer cells or cancer tissues. Substances with high affinity or specific binding ability vary depending on the type of cancer. Those skilled in the art can select a cancer-targeting substance depending on the type of cancer. For example, heparin, low-molecular-weight heparin, or a derivative thereof may be selected as a targeting substance for glioblastoma. Folic acid or a derivative thereof may also be selected as a targeting substance for ovarian cancer or endometrial cancer. An ALK receptor aptamer may also be selected as a targeting substance for lung cancer and blood cancer.

[0018] The cancer-targeting substance may be of any type, such as a peptide, sugar, lipid, or nucleic acid. The cancer-targeting substance may be naturally occurring or artificially obtained. Those skilled in the art can synthesize a cancer-targeting substance using known methods and materials. The cancer-targeting substance may also be a commercially available compound.

[0019] Those skilled in the art can synthesize the compound of the present invention by appropriately selecting a hydrophilic polymer or a derivative thereof, a bile acid or a derivative thereof, and a cancer-targeting substance depending on the type and amount of cells to which the compound of the present invention is applied, the type of cancer to which the cells are targeted, etc.

[0020] The compounds of the present invention can be synthesized by conjugating a hydrophilic polymer or its derivative, a bile acid or its derivative, and a cancer-targeting substance. The compounds of the present invention can be synthesized using known methods. Typically, the compounds of the present invention are synthesized by covalently binding a bile acid or its derivative and a cancer-targeting substance to a hydrophilic polymer or its derivative. Functional groups in the hydrophilic polymer or its derivative, functional groups in the bile acid or its derivative, and functional groups in the cancer-targeting substance can be used for conjugation. Those skilled in the art can appropriately select the conjugation mode between the hydrophilic polymer or its derivative, the bile acid or its derivative, and the cancer-targeting substance in the compounds of the present invention depending on the type of functional group. The bile acid or its derivative, and the cancer-targeting substance may be conjugated to any position on the hydrophilic polymer or its derivative, but are preferably conjugated near or at the end. These conjugation modes can be appropriately selected by those skilled in the art. For example, the bile acid or its derivative and the cancer-targeting substance can be conjugated to the hydrophilic polymer using known methods such as amide formation, ester formation, nucleophilic substitution reaction (e.g., SN2 reaction using a tosyl group), and click reaction. For example, PEG and deoxycholic acid may be conjugated by using PEG having an amino group at its terminus and condensing the amino group with the carboxyl group of deoxycholic acid using a condensing agent such as DMT-MM to form an amide (peptide bond). Alternatively, PEG and a peptidic cancer-targeting substance may be conjugated by forming a peptide bond in the same manner as described above using PEG having an amino or carboxyl group at its terminus and the peptidic cancer-targeting substance. Alternatively, PEG and a cancer-targeting substance and / or bile acid may be conjugated by forming an ester bond between the carboxyl group at its terminus and the hydroxyl group of the cancer-targeting substance and / or the hydroxyl group of a bile acid or its derivative. PEGs with functional groups attached to their termini can be synthesized using known methods and materials. PEGs with functional groups attached to their termini are commercially available, and these may also be used.The proportions of the hydrophilic polymer or its derivative, the bile acid or its derivative, and the cancer-targeting substance contained in the compound of the present invention can also be determined appropriately.

[0021] In the compounds of the present invention, it is preferable that the carboxyl group of a bile acid or its derivative is not used in binding (or is free). The term "the carboxyl group of a bile acid or its derivative is not used in binding (or is free)" typically refers to the fact that the carboxyl group is not covalently bound to a compound, a compound moiety, or a group (e.g., an alkyl group, a phenyl group, etc.). For example, when deoxycholic acid (DCA) is conjugated to PEG, an ester bond may be formed between the hydroxyl group of DCA and the carboxyl group located at the PEG terminal, leaving the carboxyl group of DCA. Because the isoelectric point of the carboxyl group of a bile acid or its derivative is on the weakly acidic side, it is protonated under weakly acidic conditions, resulting in hydrophobicity. Because cell membranes are lipid bilayers, hydrophobic bile acids or their derivatives are easily taken up, making the cell membrane more susceptible to destruction. Because tumors are weakly acidic, it is possible to selectively destroy cell membranes (e.g., platelets) in the tumor environment.

[0022] The compound of the present invention may contain one or more types of hydrophilic polymers. The compound of the present invention may contain one or more types of bile acids. The compound of the present invention may contain one or more types of cancer-targeting substances. The compound of the present invention may further contain functional molecules such as molecules that promote cancer tissue-specific binding, therapeutic agents, and labels.

[0023] In another aspect, the present invention provides a method for producing cells having drug-releasing properties and cancer cell-tropism, which comprises adsorbing the compound of the present invention onto cells.

[0024] The cells to which the compound of the present invention is adsorbed may be of any type and origin, and can be appropriately selected depending on the purpose. The cells may carry, contain, express, or produce the desired drug. An example of a cell is platelet. Platelets have cancer tropism and have the property of reaching the vicinity of cancer, covering the cancer, or agglutinating to the cancer. The cell may be a platelet or not. The cell may be a cell isolated directly from a living body or an established cell line. The cell may be derived from a stem cell such as an iPS cell or an ES cell. The cells to which the compound of the present invention is adsorbed are not limited to those described above. A preferred example of a cell is platelet from a cancer patient. Using the patient's own platelets can suppress rejection reactions and enable long-term administration.

[0025] The cells may carry, contain, express, or produce a drug. For example, a vector prepared to express or produce a drug may be introduced into the cells. Alternatively, the drug may be bound to the cells by, for example, chemical bonding. The drug contained in or produced within the cells may be released from the cells by the action of the compound of the present invention. In one embodiment, the drug is slowly released from the cells. The drug may be any drug and may be selected depending on the purpose. An example of the drug is an anticancer drug, such as an antibody used in cancer immunotherapy.

[0026] The compound of the present invention can be adsorbed to cells by contacting the compound of the present invention with cells. For example, the compound of the present invention can be adsorbed to cells by incubating the compound of the present invention with cells in a medium. Preferably, the medium is an aqueous medium, and may be a cell culture medium. Cell culture media are known and can be selected appropriately depending on the type of cells. Conditions such as incubation temperature and time can also be determined appropriately by those skilled in the art. The ratio of the compound of the present invention to cells to be contacted can also be determined appropriately by those skilled in the art. For example, 10 ４ ~10 ７ The compound of the present invention may be incubated at a concentration of several μM to several tens of μM per cell / mL.

[0027] In a further aspect, the present invention provides an agent for imparting drug-releasing properties and cancer cell tropism to cells, comprising a compound of the present invention. The dosage form of the agent can be selected appropriately, and may be solid (powder, granules, etc.), liquid (solution, suspension, etc.), or semi-solid (paste, etc.). The above explanation of the method for producing cells with drug-releasing properties and cancer cell tropism can be applied to the usage and dosage of the agent.

[0028] In a further aspect, the present invention provides a kit for imparting drug release properties and cancer cell tropism to cells, comprising the compound of the present invention. The kit includes the compound of the present invention as a component. For example, the kit may include the agent in its packaging. Usually, the kit is accompanied by an instruction manual.

[0029] In a further aspect, the present invention provides cells to which the compounds of the present invention are adsorbed. Examples of cells include, but are not limited to, platelets. Cells to which the compounds of the present invention are adsorbed have drug-releasing and cancer-specific properties, allowing them to target cancer and are expected to provide effective treatment. When administering cells to which the compounds of the present invention are adsorbed to a living organism, any administration route commonly used for administering cells to a living organism can be used. Examples of administration routes include, but are not limited to, intravenous injection, local injection, and catheter administration. The term "living organism" refers to a living animal, preferably a mammal such as a human, monkey, dog, cat, horse, cow, or pig, and more preferably a human.

[0030] The type of cancer to be treated using the cells to which the compound of the present invention is adsorbed is not particularly limited, but is typically a solid tumor. Examples of solid tumors include, but are not limited to, gastric cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer, brain tumor, etc.

[0031] Therefore, in further aspects, the present invention provides the following: a method for treating cancer, comprising administering to a living body cells to which a compound of the present invention is adsorbed and which carry, contain, express, or produce a drug; cells to which a compound of the present invention is adsorbed and which carry, contain, express, or produce a drug for use in cancer treatment; use of cells to which a compound of the present invention is adsorbed and which carry, contain, express, or produce a drug in the manufacture of a medicament for cancer treatment; and a pharmaceutical composition for cancer treatment, comprising cells to which a compound of the present invention is adsorbed and which carry, contain, express, or produce a drug. The pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient. Methods for producing the pharmaceutical composition are known to those skilled in the art, and can be prepared in any desired dosage form. The dosage of the pharmaceutical composition can also be determined appropriately by a physician.

[0032] In further aspects, the present invention provides the following: a method for delivering a drug to cancer, comprising administering to a living body cells to which a compound of the present invention has been adsorbed and which carry, contain, express, or produce a drug; cells to which a compound of the present invention has been adsorbed and which carry, contain, express, or produce a drug for delivering a drug to cancer; use of cells to which a compound of the present invention has been adsorbed and which carry, contain, express, or produce a drug in the manufacture of a composition for delivering a drug to cancer; and a composition for delivering a drug to cancer, comprising cells to which a compound of the present invention has been adsorbed and which carry, contain, express, or produce a drug. In the above-mentioned embodiments, the composition may be a pharmaceutical composition. The delivery route may be any route.

[0033] Platelets have the property of being cancer-targeting, reaching the vicinity of cancer, covering cancer, or agglutinating to cancer. Therefore, platelets adsorbed with a compound of the present invention that does not contain a cancer-targeting substance can reach the vicinity of cancer or agglutinate to cancer. Therefore, when the cells to which the compound of the present invention is adsorbed are platelets, the compound of the present invention may or may not contain a cancer-targeting substance. Therefore, when the cells to which the compound of the present invention is adsorbed are platelets, the compound of the present invention can be said to be a compound containing a hydrophilic polymer or a derivative thereof, and a bile acid or a derivative thereof. In this specification, such a compound may be referred to as "compound A." One specific example of compound A is a compound that contains a hydrophilic polymer or a derivative thereof, and a bile acid or a derivative thereof, but does not contain a cancer-targeting substance. Another specific example of compound A is a compound that contains a hydrophilic polymer or a derivative thereof, a bile acid or a derivative thereof, and a cancer-targeting substance.

[0034] The above explanation of the compound of the present invention can be applied to Compound A and its uses.

[0035] In a further aspect, the present invention provides platelets having compound A adsorbed thereon. Such platelets may carry, contain, express, or produce a drug. Platelets carrying, containing, expressing, or producing a drug can be used to deliver a drug to cancer. For example, a vector prepared to express or produce a drug may be introduced into the platelets. Alternatively, the drug may be bound to the platelets by, for example, chemical bonding. In one specific example, a drug contained in platelets having compound A adsorbed thereon or produced intracellularly can be released from the platelets by the action of compound A. In one specific example, the drug is sustainedly released from the cells. The drug may be any drug and can be selected depending on the purpose. An example of the drug is an anticancer drug, and may be, for example, an antibody used in cancer immunotherapy.

[0036] It is also preferable that the carboxyl group of bile acid or its derivative is not used for binding in Compound A. The reason and mechanism for this are as described above. That is, Compound A can selectively destroy the membrane of platelets adsorbed thereto in response to the weakly acidic environment of tumors, thereby enabling selective drug delivery to cancer.

[0037] Platelets to which Compound A is adsorbed can be used in the treatment of cancer, similar to the cells to which the compound of the present invention is adsorbed as described above.

[0038] Thus, in a further aspect, the present invention provides the following: a method for treating cancer, comprising administering to a living body platelets having compound A adsorbed thereon and carrying, containing, expressing, or producing a drug; platelets having compound A adsorbed thereon and carrying, containing, expressing, or producing a drug for use in cancer treatment; use of platelets having compound A adsorbed thereon and carrying, containing, expressing, or producing a drug in the manufacture of a medicament for cancer treatment; and a pharmaceutical composition for treating cancer, comprising platelets having compound A adsorbed thereon and carrying, containing, expressing, or producing a drug according to a further aspect of the invention. The pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient. Methods for producing the pharmaceutical composition are known to those skilled in the art, and can be prepared in any desired dosage form. The dosage of the pharmaceutical composition can also be determined appropriately by a physician. The route of administration of platelets having compound A adsorbed thereon to a living body may be any route and can be selected appropriately.

[0039] In a further aspect, the present invention provides the following: a method for delivering a drug to cancer, comprising administering to a living body platelets having compound A adsorbed thereon and carrying, containing, expressing, or producing a drug; platelets having compound A adsorbed thereon and carrying, containing, expressing, or producing a drug for delivering a drug to cancer; use of platelets having compound A adsorbed thereon and carrying, containing, expressing, or producing a drug in the manufacture of an agent for delivering a drug to cancer; and a composition for delivering a drug to cancer, comprising platelets having a compound of the invention adsorbed thereon and carrying, containing, expressing, or producing a drug. In the above, the composition may be a pharmaceutical composition. The delivery route may be any route and can be selected as appropriate.

[0040] The present invention will be explained in more detail and specifically below by showing examples, but the examples should not be construed as limiting the scope of the present invention.

[0041] (1) 8-PEG-DCA-(81)-FGA (a compound in which DCA (introduction rate 81%) and FGA (fluorescein glycinamide) (introduction rate 1%) were introduced into an 8-branched PEG) was synthesized by the method described below. Aminated DCA was synthesized by reacting DCA with a large excess of ethylenediamine and DCC / NHS. 8-PEG-DCA was synthesized by mixing this aminated DCA with 8-PEG-NHS and reacting. 8-PEG-DCA-FGA was synthesized by mixing a small amount of FGA with 8-PEG-DCA and reacting with DMT-MM. １ The introduction rates of DCA and FGA were calculated using H-NMR. The introduction rate of DCA in the synthesized compound (referred to as 8-PEG-DCA-(81)-FGA) was 81% (DCA was introduced into 6 to 7 of the 8 PEG chains), and the introduction rate of FGA was 1%. The structure of 8-PEG-DCA-(81)-FGA is shown below.

[0042] (2) It was confirmed that the 8-PEG-DCA-(81)-FGA obtained as described above was adsorbed onto platelets. ８500 μL of 8-PEG-DCA-(81)-FGA (12.5 μM) was added to the 500 μL of 8-PEG-DCA-(81)-FGA (12.5 μM) in 5% CO ２ The platelets were incubated in PBS at 37°C for 2 hours under a microscope, and the platelets were examined for fluorescence using a fluorescence microscope. The results are shown in Figure 1. Figure 1 shows the fluorescence of 8-PEG-DCA-FGA (8-PEG-DCA-FGA), calcein red (Calcein red AM), phase contrast microscopy (Ph), and merged images (Merge). The fluorescence of the calcein red-stained platelets and 8-PEG-DCA-FGA overlapped, confirming the insertion of 8-PEG-DCA-FGA into the platelet membrane.

[0043] Drug release from platelets in response to a weakly acidic environment was confirmed. (Experimental Method) DCA was introduced to the end of an 8-branched PEG (molecular weight: 40 kDa) via an amide bond. 8-PEG-DCA-COOH, which has a carboxylic acid moiety, was synthesized. The introduction rate of DCA-COOH was 44%. Calcein-containing (encapsulated) bovine whole blood-derived platelets (3.0 x 10 ８ 8-PEG-DCA-COOH (0.25 mg / mL) was added to the cells and incubated at 37°C in 5% CO ２ The platelets were allowed to stand for 1 hour under atmospheric pressure to allow 8-PEG-DCA-COOH to adsorb to the platelets. Adsorption was confirmed in the same manner as in (2) of Example 1. The pH of the suspension of platelets adsorbed with 8-PEG-DCA-COOH was adjusted to 7.4 or 6.5, and the fluorescence intensity of the supernatant was measured to evaluate the calcein leakage rate.

[0044] (Results and Discussion) The ratio of the fluorescence intensity of the supernatant at pH 7.4 to that at pH 6.5 was approximately 3:8, confirming that calcein leaked in response to a pH change (weakly acidic conditions). This result suggests that when a compound in which the carboxyl group of bile acid or its derivatives is not used for binding is used, the drug is released from platelets in response to the weakly acidic environment of tumors.

[0045] Example 3: Cellular designer molecules for platelets (3-1) Synthesis of cellular designer molecules that do not contain cancer-targeting substances Cellular designer molecules for adsorption to platelets were synthesized. Because platelets themselves have cancer-targeting properties, cellular designer molecules for platelets do not necessarily need to contain cancer-targeting substances. Therefore, cellular designer molecules that do not contain cancer-targeting substances were synthesized.

[0046] Tetrahydrofuran (THF) 430 μL, t-butanol (t-BuOH) 285 μL, H ２ A mixed solution of 205 μL of DCA-N (final concentration: 1.5 / 1 / 1 (vol.)) was prepared. Next, 5 mg of DCA-N was added to a 10 mL flask. ３ (12 μmol), 50 mg of 4-PEG20k-alkyne (2.5 μmol), and 1 mg of CuSO ４ ・5H ２ 0 (4 μmol), the mixed solution was added, and the mixture was stirred under nitrogen gas. Subsequently, 80 μL of a 50 mg / mL sodium ascorbate solution in Milli-Q was added to bring the total volume to 1 mL. Nitrogen bubbling was performed for 5 minutes, and the mixture was then stirred at 60°C under a nitrogen atmosphere for 24 hours. Subsequently, 40 mg (12 μmol, 3 eq. / Cu) of 2,4,6-trimercaptotriazine silica gel (Si-TMT) was added to the reaction solution to remove copper. After stirring at room temperature for approximately 30 minutes, the Si-TMT was removed using a syringe filter with a pore size of 5.0 μm. The resulting solution was then placed in a regenerated cellulose dialysis membrane (3.5 kDa) and dialyzed against 2 L of methanol for two days. Before dialysis, the reaction solution was diluted approximately four times with methanol. Furthermore, dialysis was performed against 2 L of Milli-Q for one day. After dialysis, the resulting solution was transferred to a 50 mL centrifuge tube and lyophilized to purify the synthesized 4-PEG20k-DCA-COOH. The synthesis scheme is shown below. Note that this 4-PEG20k-DCA-COOH is also referred to as 4-PEG20k-trz-DCA-COOH because PEG and DCA are bonded via a triazole group. Hereinafter, the same expression will be used for various compounds containing PEG and DCA.

[0047]

[0048] PEG-DCA was synthesized using the same method as above, using PEGs with different molecular weights and branching numbers. The yields and grafting degrees of the various PEG-DCA-COOHs synthesized are shown in the table below.

[0049]

[0050] (3-2) Modification of platelets with cell designer molecules 2.0 × 10 ８ 1 μL of 1 mM calcein red-AM solution was added to platelets prepared at 1000 cells / mL, and the mixture was incubated at 37°C for 30 minutes. The mixture was then centrifuged at 850 × g for 8 minutes, the supernatant removed, and 1 mL of PBS was added and washed twice under the same conditions to prepare calcein red-encapsulated platelets. After washing, 8-PEG40k-DCA-OH-FGA solution was added to a final concentration of 0.25 mg / mL (12.5 μM), and the total volume was 0.5 mL, followed by incubation at 37°C for 2 hours. The mixture was then washed three times with 1 mL of PBS, and observed under a fluorescence microscope using a confocal laser scanning microscope (FV3000, Olympus Corporation). 8-PEG40k-DCA-OH-FGA was synthesized by mixing 8-PEG40k-DCA with a small amount of FGA and reacting it with DMT-MM, as described in Example 1. The results are shown in Figure 2. The fluorescence of FGA derived from 8-PEG40k-DCA-OH-FGA and the fluorescence of calcein red derived from calcein red-encapsulating platelets overlapped, confirming the insertion of 8-PEG40k-DCA into the platelet membrane, i.e., the modification of platelets with 8-PEG40k-DCA.

[0051] Next, 2 x 10 ８1 μL of 1 mM calcein-AM solution was added to platelets prepared at 1000 cells / mL, and the mixture was incubated at 37°C for 30 minutes. The mixture was then centrifuged at 850×g for 8 minutes, the supernatant was removed, and 1 mL of PBS was added and washed twice under the same conditions to prepare calcein-encapsulated platelets. After washing, a 4-PEG20k-DCA75-Cy3 (DCA incorporation rate: 75%) solution was added to 1x PBS (pH 7.4) to a final concentration of 0.25 mg / mL (12.5 μM), and the mixture was incubated in a total volume of 0.5 mL at 37°C for 2 hours. The platelets were then washed three times with 1 mL of PBS and observed under a fluorescence microscope using a confocal laser scanning microscope (FV3000, Olympus Corporation). The incorporation rates of DCA and Cy3 in 4-PEG20k-DCA were: １ The introduction rate of Cy3 was calculated by H-NMR. The introduction rate of Cy3 was 16.8%. 4-PEG20k-DCA75-Cy3 was prepared by mixing 4-PEG20k-DCA75 and azide-modified Cy3, and then dissolving the mixture in CuSO. ４ and sodium ascorbate. The results are shown in Figure 3. The fluorescence of Cy3 from 4-PEG20k-DCA75-Cy3 overlapped with the fluorescence of calcein from calcein-encapsulated platelets, confirming the insertion of 4-PEG20k-DCA into the platelet membrane, i.e., the modification of platelets with 4-PEG20k-DCA.

[0052] (3-3) Retention of encapsulated substances and leakage of encapsulated substances from cell designer molecule-modified platelets 4 × 10 ８ 2 μL of 1 mM calcein-AM solution was added to the platelets prepared at cells / mL, and the mixture was incubated at 37°C for 30 minutes. The mixture was then centrifuged at 850 × g for 8 minutes, the supernatant was removed, and the platelets were washed three times with 1 mL of PBS under the same conditions to prepare calcein-encapsulated platelets. The supernatant after the third wash was collected to measure the fluorescence intensity.

[0053] To 100 μL of calcein-encapsulated platelets resuspended in 1x PBS (pH 7.4), the cell designer molecule 4-PEG20k-trz-DCA-COOH or 4-PEG2k-trz-DCA-COOH synthesized in (3-1) was added to a concentration of 5 μM, 25 μM, 125 μM, or 250 μM. The resulting solution was incubated in a total volume of 500 μL at 37°C for 2 hours. The mixture was then centrifuged at 3600 rpm for 5 minutes, and 200 μL of the supernatant was added to a 96-well plate. Fluorescence intensity was measured using a plate reader. Based on the fluorescence intensity in the supernatant, the percentage of calcein leaked from the platelets upon modification with the cell designer molecule was calculated, with the amount of calcein encapsulated in the platelets defined as 100%. The amount of calcein encapsulated in the platelets was determined by preparing calcein-encapsulated platelets, dispersing the platelets, and quantifying the encapsulated calcein based on the fluorescence intensity of the resulting solution. The results are shown in Figure 4.

[0054] After removing the supernatant, the platelets were resuspended in 500 μL of 1x PBS (pH 7.4 or pH 6.5) and then incubated at 37°C for 4 hours. The platelets were then centrifuged at 3600 rpm for 5 minutes, and 200 μL of the supernatant was added to a 96-well plate. Fluorescence intensity was measured using a plate reader. Based on the fluorescence intensity in the supernatant, the percentage of calcein leaked from the cell designer molecule-modified platelets was calculated, with the amount of calcein encapsulated in the platelets defined as 100%. The results are shown in Figure 5.

[0055] As is clear from the results in Figure 4, the molecular weight and branching number of the PEG contained in the cellular designer molecule can improve the ability of platelets modified with the molecule to retain their encapsulated substance. In particular, when modifying platelets, a PEG with the same branching number was found to have a higher molecular weight, resulting in a better ability to retain the encapsulated substance. Furthermore, sustained release of the encapsulated substance (fluorescent substance) from cellular designer molecule-modified platelets was confirmed under low pH (weakly acidic) conditions (Figure 5). This demonstrates pH-responsive membrane disruption behavior by cellular designer molecules. Furthermore, pH-responsive membrane disruption behavior was observed at low concentrations even when cellular designer molecules containing low-molecular-weight (2k) PEG were used (Figure 5).

[0056] These results can also be applied to cellular designer molecules containing cancer-targeting substances. That is, these results demonstrate that even when a cellular designer molecule containing a cancer-targeting substance is adsorbed to a cell, the ability of the cell to retain the encapsulated substance can be improved by adjusting the molecular weight and number of branches of the PEG contained in the cellular designer molecule, and that the cell membrane is disrupted under low pH (weakly acidic) conditions, resulting in the sustained release of the encapsulated substance.

[0057] Example 4: Cellular designer molecules containing cancer-targeting substances Cellular designer molecules were synthesized using low molecular weight heparin (LMWH) as a cancer-targeting substance. LMWH is known to have the ability to recognize anaplastic lymphoma kinase (ALK), which is expressed in glioblastoma cells.

[0058] (4-1) ALK Recognition by LMWH 8-PEG40k was modified with LMWH by amide condensation and further fluorescently labeled with Cy3 to synthesize 8-PEG40k-LMWH-Cy3. LMWH was partially deacetylated and then fluorescently labeled with FITC (fluorescein isothiocyanate) to synthesize LMWH-FITC. 4.0 × 10 ５ NB-1 cells expressing full-length ALK or H3122 cells lacking full-length ALK cell surface expression were suspended in 200 μL of PBS containing 0.1% BSA and incubated on ice for 30 minutes with 8-PEG40k-LMWH-Cy3 or LMWH-FITC at 0.1 mg / mL or 0.01 mg / mL, or with 0.5 mg / mL LMWH-FITC and 0.1 mg / mL 8-PEG40k-LMWH-Cy3. After washing with PBS, the cells were analyzed by fluorescence-activated cell sorting (FACS). As a control, NB-1 cells not incubated with the above fluorescently labeled molecules were subjected to FACS analysis. The results are shown in Figure 6.

[0059] As a result, binding of the fluorescently labeled molecule to NB-1 cells was observed. On the other hand, no binding was observed to H3122 cells. Upon binding to NB-1 cells, a concentration-dependent increase in fluorescence intensity was observed for both LMWH and LMWH-modified PEG. This confirmed that LMWH recognizes target cells (ALK-expressing cells) even when conjugated with PEG. Furthermore, when LMWH and PEG-LMWH were coexisted (right graph in Figure 6), the fluorescence (Cy3 fluorescence) derived from PEG-LMWH was shifted to the left compared to the case of PEG-LMWH alone (center graph in Figure 6). This indicates that the coexistence of LMWH reduced the amount of PEG-LMWH bound to cells, i.e., LMWH and PEG-LMWH compete for binding to NB-1 cells.

[0060] (4-2) Synthesis of Cellular Designer Molecules Containing Cancer-Targeting Substances In a 10 mL flask, 50 mg of 4-PEG20k-DCA75 (2.5 μmol), 18.8 mg of LMWH-N, which were synthesized in the same manner as in Example 3, were placed. ３ (3.75 μmol), 750 μL of Milli-Q, and 100 μL of acetonitrile were added. ４ ・5H ２ 50 μL of 0 aqueous solution was added and stirred under a nitrogen atmosphere. Subsequently, 100 μL of a 10 mg / mL aqueous sodium ascorbate solution was added, and nitrogen bubbling was performed for 5 minutes, followed by stirring at 60°C under a nitrogen atmosphere for 24 hours. To remove copper, 10 mg of Si-TMT (3 μmol, 3 eq. / Cu) was added to the reaction solution, and the mixture was stirred at room temperature for approximately 30 minutes. The Si-TMT was then removed using a 5.0 μm pore-size syringe filter. The resulting solution was then placed in a regenerated cellulose dialysis membrane (12-14 kDa) and dialyzed against 2 L of milliQ for two days. After dialysis, the resulting solution was transferred to a 50 mL centrifuge tube and lyophilized to purify the synthesized 4-PEG20k-DCA-LMWH. The synthesis scheme is shown below. The yield of the synthesized PEG-DCA-LMWH and the incorporation rates of DCA and LMWH are shown in the table below.

[0061]

[0062]

[0063] (4-3) Evaluation of the ability of cell designer molecule-modified platelets to recognize target cancer cells. ５ NB-1 cells were seeded at 1000 cells / well and cultured for 1 day in RPMI1640 / EMEM medium. 2.15 μL of DMSO was added to 15 μg of Cell Tracker Deep Red to prepare a 10 mM Cell Tracker / DMSO solution, and 2 μL of this solution was added to 2 mL of phenol red- and FBS-free DMEM to prepare a 10 μM Cell Tracker / DMEM solution. The medium was aspirated, washed with 1 mL of PBS, and then 1 mL of Cell Tracker / DMEM solution was added to each well. The cells were incubated at 37°C in 5% CO ２ After washing, the cells were incubated in RPMI1640 / EMEM medium (containing 10% FBS and 1% antibiotics) at 37°C and 5% CO ２ The incubation was carried out under reduced pressure for 1 day.

[0064] 2 x 10 ８ 1 μL of 1 mM calcein-AM solution was added to the platelets prepared at 10 cells / mL, and the mixture was incubated at 37°C for 30 minutes. After that, the mixture was centrifuged at 850 × g for 8 minutes, the supernatant was removed, and 1 mL of PBS was added and the mixture was washed twice under the same conditions to prepare calcein-encapsulated platelets. After washing, 1 × 10 ８A 4-PEG20k-DCA-LMWH solution was added to the platelets at a final concentration of 0.05 mg / mL (2.5 μM) per cell / mL of platelets, and the resulting mixture was incubated in 1 mL of 1x PBS at 37°C for 2 hours to prepare calcein-encapsulated platelets modified with 4-PEG20k-DCA-LMWH (cell designer molecule-modified platelets). After washing twice, the platelets were suspended in 1 mL of RPMI1640 / EMEM medium to prepare a cell designer molecule-modified platelet suspension. After aspirating the medium from the 24-well plate seeded with the NB-1 cells, 500 μL of the cell designer molecule-modified platelet suspension was added to each well of the plate, and an additional 500 μL of medium was added to bring the total volume of each well to 1 mL (5.0 × 10 ７ cells / mL), and then incubated at 37°C, 5% CO ２ The plates were incubated under a 5% CO₂ atmosphere for 4 hours, during which time they were observed every hour using a fluorescent microscope using an FV3000. RPMI1640 / EMEM medium (containing 10% FBS and 1% antibiotics) was used as the culture medium. As a control, a similar experiment was performed except that an unmodified platelet suspension was added to the 24-well plate containing the NB-1 cells instead of the cell designer molecule-modified platelet suspension. The results are shown in Figure 7. As is clear from Figure 7, the cell designer molecule-modified platelets were found to adsorb to NB1 cells to a greater extent than the unmodified platelets.

[0065] The present invention can be used in the fields of cell engineering, tissue engineering, medical materials, medicine, etc.

Claims

1. Compounds containing hydrophilic polymers or their derivatives, bile acids or their derivatives, and cancer-targeting substances.

2. The compound of claim 1, wherein the hydrophilic polymer is selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polyglutamic acid, polyaspartic acid, polylysine and polyhistidine.

3. The compound according to claim 2, wherein the hydrophilic polymer is polyethylene glycol or polyvinyl alcohol.

4. The compound according to any one of claims 1 to 3, wherein the bile acid is selected from the group consisting of cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, ursodeoxycholic acid, hyodeoxycholic acid, glycocholic acid, and taurocholic acid.

5. The compound of claim 4, wherein the bile acid is deoxycholic acid or cholic acid.

6. The compound according to any one of claims 1 to 5, wherein the carboxyl group of the bile acid or its derivative is not used for binding.

7. A method for producing cells having drug-releasing properties and cancer cell-tropism, comprising adsorbing the compound according to any one of claims 1 to 6 onto cells.

8. The method of claim 7, wherein the cells are platelets.

9. An agent or kit for imparting drug-releasing properties and cancer cell-targeting properties to cells, comprising the compound according to any one of claims 1 to 6.

10. The agent or kit according to claim 9, wherein the cells are platelets.

11. Cells to which the compound according to any one of claims 1 to 6 has been adsorbed.

12. The cell of claim 11, which is a platelet.

13. A cell according to claim 11 or 12, which carries, contains, expresses or produces a drug.

14. A composition for delivering a drug to cancer, comprising the cells of claim 13.

15. Platelets adsorbed with compounds including hydrophilic polymers or their derivatives and bile acids or their derivatives.

16. The platelet of claim 15, wherein the hydrophilic polymer in said compound is selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polyglutamic acid, polyaspartic acid, polylysine and polyhistidine.

17. The platelet of claim 16, wherein the hydrophilic polymer in said compound is polyethylene glycol or polyvinyl alcohol.

18. The platelets of any one of claims 15 to 17, wherein the bile acid in the compound is selected from the group consisting of cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, ursodeoxycholic acid, hyodeoxycholic acid, glycocholic acid, and taurocholic acid.

19. The platelet of claim 18, wherein the bile acid in said compound is deoxycholic acid or cholic acid.

20. The platelet according to any one of claims 15 to 19, wherein the carboxyl group of the bile acid or its derivative in the compound is not used for binding.

21. The platelet according to any one of claims 15 to 20, which carries, contains, expresses or produces a drug.

22. A composition for delivering a drug to cancer, comprising the platelets of claim 20 or 21.

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