KR20140041304A - Liposome comprising elastin-like polypeptide conjugated to a moiety containing a hydrophobic group, chemosensitizer and anticancer agent and use thereof - Google Patents

Abstract

Liposomes comprising a elastin-like polypeptide conjugated with a hydrophobic group, a chemical sensitizer and an anticancer agent, a pharmaceutical composition comprising the same, and a method for delivering a chemical sensitizer and an anticancer agent to a target site of an individual using the same Is provided.

Description

Liposome containing elastin-like polypeptide conjugated to a moiety containing a hydrophobic group, chemosensitizer and anticancer agent and use according to hydrophobic conjugated elastin-like polypeptide, chemical sensitizer and anticancer agent

Liposomal comprising an elastin-like polypeptide conjugated with a hydrophobic group, a chemical sensitizer and an anticancer agent, a pharmaceutical composition comprising the same, and a method for delivering a chemical sensitizer and an anticancer agent to a target site of an individual using the same. It is about.

Liposomes consist of one or more lipid bilayer membranes surrounding an aqueous inner compartment. Liposomes can be characterized by membrane type and size. Small Unilamellar vesicles (SUVs) have a single membrane and may have a diameter of 20 nm to 50 nm. Large Unilamellar vesicles (LUVs) can have diameters of 50 nm or more. Oligolamella large vesicles and multilamellar large vesicles have multiple, generally concentric, membrane layers and may be 100 nm or more in diameter. Liposomes with several non-concentric membranes, the several small vesicles contained in larger vesicles, are called multivesicular vesicles.

Liposomes can be formulated to have therapeutic agents, drugs or other active agents distributed in the aqueous interior space (water soluble active ingredient) or in the lipid bilayer (water insoluble active ingredient). In addition, hydrophobic substances such as cholesterol may be included in micelles, which may be included in the internal space of liposomes.

Active ingredients with short half-lives in the blood stream are particularly suitable for delivery through liposomes. For example, many anti-neoplastic agents have a short half-life in the blood stream, making parenteral administration unsuitable. However, the use of liposomes for site-specific delivery of the active ingredient through the blood stream can be limited by the rapid removal of liposomes from the blood by the cells of the reticuloendothelial system (RES).

Liposomes are not normally leaky, but can become liable if holes are formed in the liposome membrane, the membrane is degraded or dissolved, or the membrane temperature is increased to the phase transition temperature. An increase in temperature at the target site in the individual (hyperthermia) raises the liposome temperature above the phase transition temperature, thus causing release of the liposome contents. It can be used for the selective delivery of therapeutic agents. However, if the phase transition temperature of liposomes is significantly higher than the normal tissue temperature, this technique may be limited in use.

Thus, there is still a need for liposomes that can be used to efficiently deliver the active ingredient.

One aspect is to provide liposomes comprising an elastin-like polypeptide conjugated with a moiety comprising a hydrophobic group, a chemical sensitizer and an anticancer agent.

Another aspect is to provide a pharmaceutical composition comprising the liposome.

Another aspect is to provide a method for delivering a chemical sensitizer and an anticancer agent to a target site of an individual using the liposomes.

One aspect includes a lipid bilayer, an elastin-like polypeptide (ELP) conjugated with a hydrophobic moiety, a chemical sensitizer and an anticancer agent, and the elastin-like polypeptide conjugated to a moiety comprising the hydrophobic group is Provided is a liposome that is filled in a lipid bilayer.

The term “lipid bilayer” refers to a membrane consisting of two layers of lipid molecules. The lipid bilayer can be one that has a thickness similar to naturally occurring membranes such as cell membranes, nuclear membranes, and viral envelopes. For example, the thickness of the lipid bilayer may be 10 nm or less, for example, having a thickness of 1 nm to 9 nm, 2 nm to 8 nm, 2 nm to 6 nm, 2 nm to 4 nm, or 2.5 nm to 3.5 nm. Lipid bilayers are barriers that prevent ions, proteins and other molecules from spreading to where they do not need to be kept where they should be. Natural lipid bilayers are usually composed mostly of phospholipids. Phospholipids have a hydrophilic head and two hydrophobic tails. When phospholipids are exposed to water, they themselves are arranged in a two-layer sheet (bilayer) so that all the tails are towards the center of the sheet. The center of this bilayer also excludes molecules such as sugars or salts that contain little water and are soluble in water but not soluble in oil. Phospholipids with specific head groups can determine the surface chemistry of the bilayer. In addition, lipid tails can affect membrane properties, for example by determining the phase of the bilayer. The lipid bilayer takes a solid gel phase at low temperatures but can transition to a fluid state at higher temperatures. Packing of lipids in a lipid bilayer can also affect its mechanical properties, including resistance to stretching and bending. Biological membranes can usually contain several other lipid types than phospholipids. An especially important example in animal cells is cholesterol. Cholesterol can help strengthen lipid bilayers and lower permeability.

The "lipid molecule" that constitutes the lipid bilayer may be a molecule having a hydrophilic head and a hydrophobic tail. The lipid molecule may be one having a carbon atom of C14 to C50. The lipid molecule may be a phospholipid. The phospholipid may be one having a carbon atom of C16 to C24. The phospholipid is at least one selected from the group consisting of phosphatidyl choline, phosphatidyl glycerol, phosphatidyl inositol, and phosphatidyl ethanolamine, and the at least one phospholipid may have two acyl groups. In addition, the phospholipid may be one having a phase transition temperature of about 10 ℃ to about 70 ℃, for example, about 39 ℃ to about 45 ℃. The phospholipid may be an acyl group saturated or unsaturated. The phospholipid may be a mixture of two or more kinds of phospholipid molecules. By mixing two or more kinds of phospholipid molecules, lipid bilayers having various phase transition temperatures can be produced.

Phospholipid molecules include, for example, two acyl groups, C12 saturated chain phospholipids (Tc = 10 ° C), C14 saturated chain phospholipids (Tc = 24 ° C), C16 saturated chain phospholipids (Tc = 41 ° C), One or more selected from the group consisting of C18 saturated chain phospholipids (Tc = 55 ° C.), C20 saturated chain phospholipids (Tc = 65 ° C.), C22 saturated chain phospholipids (Tc = 70 ° C.), and combinations thereof. Similar to the phase transition temperatures in which phosphatidylcholine changes in a similar manner along the length of their acyl chains, phosphatidylglycerol, phosphatidyl inositol, phosphatidyl ethanolamine, sphingomyelin and gangliosides can also be used.

An example of a C16 saturated chain phospholipid may be dipalmitoyl phosphatidylcholine (DPPC). DPPC is a saturated chain (C 16) phospholipid having a phase transition temperature of about 41.5 ° C. An example of a C18 saturated chain phospholipid may be 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). DSPC is a saturated chain (C 18) phospholipid having a phase transition temperature of about 55.10 ° C.

In addition to the phospholipid, other membrane-forming materials may be used for the lipid bilayer. Exemplary materials that form solid membranes can be boll lipids or bacterial lipids. In addition, block copolymers comprising water soluble polymers (eg polyethylene glycol) and water insoluble polymers (eg polypropylene oxide and polyethylethylene) can be used.

The term "primary lipid" refers to the main lipid component of the liposome bilayer material in the liposome bilayer. Thus, for example, in a liposome bilayer consisting of 70% phospholipid and 30% cholesterol, the phospholipid is the major lipid.

Lipid bilayers may have different phase behaviors depending on the temperature. At a given temperature, the lipid bilayer may be present in either liquid or gel (solid). Lipid molecules have a characteristic temperature that transitions from a gel phase to a liquid phase. Lipid molecules in both phases are inhibited from flip-flopping across the bilayer, but a given lipid molecule in the liquid bilayer will exchange positions with neighboring molecules. This random walk exchange allows lipid molecules to diffuse and travel across the membrane surface. Unlike the liquid phase bilayer, the lipid in the gel phase bilayer is fixed in position.

The phase behavior of the lipid bilayer can be determined by the strength of the attractive van der Waals interactions between neighboring lipid molecules. Lipid molecules with longer tails have a larger area of interaction, which increases the intensity of this interaction and consequently decreases lipid molecule motility. Thus, at a given temperature, lipid molecules with short tails may be more fluid than lipid molecules with other equal and longer tails. The transition temperature can also be affected by the degree of unsaturation of the lipid molecule tail. Unsaturated double bonds can create kinks in the alkane chain, which can destroy lipid packing. This breakdown can create additional free space in the bilayer that can give additional flexibility in neighboring chains.

Most natural membranes are complex mixtures of other lipid molecules. When some of the components of the complex mixture are liquid at a given temperature and the other component is a gel phase, the two phases coexist in a spatially separated region, such as an iceberg floating in the ocean.

The term "phase transition temperature" refers to the temperature at which a phase changes from a solid phase to a liquid phase (also called a melting temperature) or from a liquid phase to a solid phase. The substance comprises a lipid bilayer or liposome comprising a lipid molecule, a lipid bilayer or liposome without a elastin-like polypeptide conjugated to a moiety comprising a hydrophobic group, or an elastin-like polypeptide conjugated to a moiety comprising a hydrophobic group. do.

The liposome may comprise an elastin-like polypeptide conjugated to a moiety including a hydrophobic group, and the moiety including the hydrophobic group may be filled in the lipid bilayer.

The moiety comprising the hydrophobic group may be a molecule having a property capable of fixing an elastin-like polypeptide conjugated thereto to the lipid bilayer by filling the lipid bilayer, for example, a hydrophobic property. have. The moiety including the hydrophobic group may be the same or different lipid molecule as the lipid molecule constituting the lipid bilayer.

The moiety including the hydrophobic group may include a molecule composed only of a hydrophobic portion or an amphiphilic molecule including a hydrophobic portion and a hydrophilic portion. The amphiphilic molecule comprising the hydrophobic portion and the hydrophilic portion may be one in which the hydrophobic portion is disposed in the inner direction of the lipid bilayer and the hydrophilic portion is disposed in the outer direction of the lipid bilayer and bound to an elastin-like polypeptide. Here, "outer direction" refers to an outward direction from the center of the lipid bilayer, and indicates an inner direction of the liposome or an outward direction of the liposome.

The moiety including the hydrophobic group may be a lipid molecule that exists naturally in the biological membrane or a lipid molecule that does not exist naturally in the biological membrane but may constitute a lipid bilayer.

The lipid molecules naturally present in the biological membrane may be selected from the group consisting of phospholipids or derivatives thereof, sterols or derivatives thereof, sphingolipids or derivatives thereof, and combinations thereof. The phospholipid or a derivative thereof may be at least one selected from the group consisting of phosphatidyl choline, phosphatidyl glycerol, phosphatidyl inositol, phosphatidyl ethanolamine, and combinations thereof. The sterol or derivatives thereof may be cholesterol or derivatives thereof, or squalene or derivatives thereof. The sphingolipids may be sphingomyelin or gangliosides or derivatives thereof. The moiety including the hydrophobic group includes the intermediate or precursor of the phospholipid, sterol or sphingolipid produced during in vivo synthesis. For example, phosphoglycerides, sphingosine, ceramides or cerebrosides.

The moiety comprising the hydrophobic group may be a saturated or unsaturated hydrocarbon, a saturated or unsaturated acyl molecule or a saturated or unsaturated alkoxy molecule.

Conjugation of an elastin-like polypeptide with a moiety comprising a hydrophobic group may be one that is not cleavable under physiological or pathological conditions, or is made by cleavable linkage. Examples of cleavable linkages may be linkages made via a pH cleavable linker, a heat transferable linker, a radiation drying cleavable linker, a linker delivered in an aqueous solution.

The moiety including the hydrophobic group may be conjugated by binding to the N or C terminal carbonyl (-C (O)-) group of the elastin-like polypeptide N terminus. The moiety comprising the hydrophobic group is formed by a bond formed by reaction with a functional group selected from the group consisting of functional groups of side chains of the elastin-like polypeptide, for example, amino, carbonyl, hydroxy, thiol, and combinations thereof. It may be connected. The moiety including the hydrophobic group may be conjugated through an amine or amide bond with the N atom of the elastin-like polypeptide. The moiety including the hydrophobic group may be conjugated to the carbonyl group of the C terminal of the elastin-like polypeptide through an amide or ester bond.

The moiety including the hydrophobic group may be a hydrophobic group moiety having a carbon number of C4-C30, for example, C14-C24, or a carbon number of C16-C24. The moiety comprising the hydrophobic group is, for example, myristoyl (C14), palmitoyl (C16), stearoyl (C18), arachidonyl (C20), behenonyl (C22) or lignoseroyl (C24). The moiety comprising the hydrophobic group may be filled in the lipid bilayer by a hydrophobic effect, such that an elastin-like polypeptide conjugated to the moiety comprising the hydrophobic group may be immobilized on the liposome.

The term "elastin-like polypeptide" refers to a class of amino acid polymers that undergo conformational changes with temperature. In one embodiment, the elastin-like polypeptide may be a polymer having "inverse phase transitioning behavior". The inverse phase transition behavior is insoluble in an aqueous solution at a temperature below the "inverse phase transition temperature (T _t )", and becomes insoluble when the temperature rises to a temperature higher than the inverse phase transition temperature. Elastin-like polypeptides can transition from highly soluble elongated chains to tightly folded aggregates with greatly reduced solubility as the temperature rises. This reverse phase transition may be induced by having more β-turn and distorted β-structure of the elastin-like polypeptide as the temperature increases. In some cases, the elastin-like polypeptide can be defined based on the temperature range at which phase transition occurs. For example, in some cases, the phase transition can occur in a temperature range of about 10 ° C to about 70 ° C.

The reverse phase transition behavior is due to the contraction and self-assembly of elastin-like polypeptides as the temperature rises from a lower temperature than the T _t of the elastin-like polypeptide when the elastin-like polypeptide is linked to a component of the lipid bilayer. Thereby destroying the lipid bilayer. Failure of the lipid bilayer may increase the permeability of the lipid bilayer. Thus, the active ingredient included in liposomes comprising a lipid bilayer can be released outward from the liposomes with higher permeability. However, one or more embodiments are not limited to specific mechanisms of action.

The destruction of the lipid bilayer constituting the liposomes by reverse phase transition behavior of the elastin-like polypeptide may depend on the lipid molecules constituting the lipid bilayer, ie the phase transition temperature of the lipid bilayer. The lipid bilayer exists as a gel phase below its phase transition temperature, and as a liquid (crystalline) phase above it. When the lipid bilayer is present in the gel, even if the elastin-like polypeptide is changed to a structure having a β-turn structure by reverse phase transition behavior, the destruction of the lipid biworm may not occur or may be limited. On the other hand, when the lipid bilayer is in the liquid phase, as the elastin-like polypeptide is changed to a structure having a β-turn structure by reverse phase transition behavior, destruction of the lipid biworm may be induced. That is, when the lipid bilayer is in the liquid phase as compared to the gel phase, the reverse phase transition of the elastin-like polypeptide leads to more efficient destruction of the lipid bilayer. Thus, the release temperature of the active ingredient included in liposomes can be controlled by controlling the phase transition temperature of the lipid bilayer of liposomes and the reverse phase temperature of elastin-like polypeptides. For example, the phase transition temperature of a lipid bilayer or liposome, including elastin-like polypeptides, may be about 10 ° C. to 70 ° C., for example 39 ° C. to 45 ° C.

The elastin-like polypeptide to which the moiety including the hydrophobic group is conjugated may be a moiety including the hydrophobic group to a terminal which is not a side chain of the elastin-like polypeptide. In addition, the elastin-like polypeptide to which the moiety including the hydrophobic group is conjugated is a moiety including a hydrophobic group having one chain, in which the moiety including the hydrophobic group is conjugated to the terminal, not the side chain of the elastin-like polypeptide. The tee may be bonded. For example, a moiety containing the hydrophobic group is conjugated by binding to an N or C terminal carbonyl (-C (O)-) group at the N-terminus of the elastin-like polypeptide, and a moiety including the hydrophobic group. The tee may be conjugated through an amine or amide bond with the N atom of the elastin-like polypeptide or may be conjugated through an amide or ester bond with a carbonyl group at the C-terminus of the elastin-like polypeptide. Here, the moiety including the hydrophobic group may be a hydrocarbon having a carbon number of C4-C30, for example, C14-C24, or a carbon number of C16-C24. In an elastin-like polypeptide conjugated with a moiety comprising a hydrophobic group, the ELP may be conjugated to a moiety comprising a hydrophobic group having a single chain.

In accordance with one or more embodiments, liposomes comprising an elastin-like polypeptide can be used to efficiently release the active ingredient included in liposomes, compared to liposomes that do not contain elastin-like polypeptides and only comprise a lipid bilayer. This is because the release of the active ingredient contained in the liposome to the outside of the liposome is simply induced by the diffusion of the lipid molecule in the case of using the phase transition of the lipid molecule of the lipid bilayer, whereas in the case of liposomes containing an elastin-like polypeptide, In addition to being induced by diffusion, it may be because the reverse phase inversion behavior of the elastin-like polypeptide, i.e., the release of the active ingredient is further induced by lipid bilayer destruction by contraction and assembly. Here, the active ingredient may be included in the interior space of the liposomes or inside or both of the lipid bilayer.

In one embodiment, the elastin-like polypeptide is partially or entirely of VPGXG (SEQ ID NO: 1), PGXGV (SEQ ID NO: 2), GXGVP (SEQ ID NO: 3), XGVPG (SEQ ID NO: 4), GVPGX (SEQ ID NO: 5) and One or more repeating units selected from the group consisting of combinations thereof, wherein V may be valine, P is proline, G is glycine, and X is a natural or non-natural amino acid that is not proline. Here, X of each repeating unit may be the same or different amino acids. The repeating units may be separated from one another by one or more amino acids or other linker moieties that do not eliminate the phase transition properties of the resulting elastin-like polypeptide, or the ends may be part of the one or more amino acids or other linker moieties. The ratio of the repeating unit to another amino acid or linker moiety may be about 0.1% to about 99.9% of the repeating unit based on the amino acid different from the repeating unit. The selected repeating unit may be repeated two or more times, for example, 2 to 200 times.

In one embodiment, the elastin-like polypeptide is VPGXG, PGXGV, GXGVP, XGVPG, GVPGX or a combination of one or more tandemly-repeated blocks thereof or VPGXG, PGXGV, GXGVP, XGVPG, GVPGX or Combinations of these may include blocks repeated in series one or more times. The elastin-like polypeptide may be one having a structure of (VPGXG, PGXGV, GXGVP, XGVPG, GVPGX, or a combination thereof). An elastin-like polypeptide conjugated to a moiety comprising a hydrophobic group may be one having a structure of C8 to C24 fatty acid acyl- (VPGXG, PGXGV, GXGVP, XGVPG, GVPGX or a combination thereof) n. n is an integer of one or more as the number of repetitions, for example, 1-200, 2 to 200, 2 to 100, 2 to 80, 2 to 60, 2 to 40, 2 to 10, 2 to 12, 2 to 8, 2 To 6, 4 to 100, 8 to 80, 10 to 60, 12 to 40, 20 to 40, 4 to 10, 4 to 8, or an integer of 4 to 6. For example, stearoyl-VPGVG VPGVG VPGVG VPGVG VPGVG VPGVG-NH ₂ (SEQ ID NO: 6: also referred to as “SA-V6-NH ₂ ”).

The elastin-like polypeptide is not only composed of VPGXG, PGXGV, GXGVP, XGVPG, GVPGX, or a combination thereof, as long as reverse phase transition behavior is maintained, but also other portions of the molecule, such as a linker. And breakers. The elastin-like polypeptide may be one in which the N- or C-terminal is bonded to a moiety including a hydrophobic group. In addition, moieties comprising hydrophobic groups can be conjugated by binding to reactive groups in the side chain of the amino acid residue of the elastin-like polypeptide. The reactive group may be an amino group, hydroxyl group, thiol group or carboxyl group. Opposite ends that are not bound to a moiety comprising a hydrophobic group may or may not be blocked. For example, when a moiety comprising a hydrophobic group and an elastin-like polypeptide are bound through the N terminus of an elastin-like polypeptide, the carboxyl group at the C terminus of the elastin-like polypeptide may be blocked or unblocked. The blocking may be by binding to or reacting with a material that is biocompatible, non-immunogenic, aids in specific delivery, or may be avoided from a biodegradation system. For example, the blocking can be accomplished by forming an amide bond by bonding an amino group with a carboxyl group at the C terminus of an elastin-like polypeptide. The amino group may be an ammonia molecule, a primary amine, a secondary amine or a tertiary amine. The primary amine, secondary amine or tertiary amine may be each having a carbon number of C1-C10, for example, C1-C6. X may be valine or alanine.

The repeating unit may be included by repeating one or more integer times independently of each other. The repetition frequency is independently 2 to 200, 2 to 100, 2 to 80, 2 to 60, 2 to 40, 2 to 10, 2 to 12, 2 to 8, 2 to 6, 4 to 100, 8 to 80 , 10 to 60, 12 to 40, 20 to 40, 4 to 10, 4 to 8, or may be an integer of 4 to 6.

In the liposomes, the molar ratio of the major lipid molecule of the lipid bilayer to the elastin-like polypeptide conjugated to the moiety comprising the hydrophobic group is the property of the lipid bilayer selected and the property of the elastin-like polypeptide conjugated to the moiety comprising the hydrophobic group. It may be appropriately selected according to. For example, the molar ratio of major lipid molecule: elastin-like polypeptide conjugated to a moiety comprising a hydrophobic group may be a molar ratio of 50 to 99.9: 0.1 to 50. For example, a major lipid molecule (DPPC or a mixture of DPPC and DSPC): an elastin-like polypeptide conjugated with a moiety containing a hydrophobic group (palmitoyl (VPGXG) n or stearoyl (VPGXG) n, n is 2 to 12) Molar ratio) may be 50 to 99.0: 0.1 to 50.

"Chemosensitizers" include drugs that make tumor cells more sensitive to the effects of chemotherapy. The chemosensitizer may be an inhibitor of a protein that confers drug resistance. The chemical sensitizer may be an MDR1 protein (multidrug resistance protein-1) inhibitor, an MDR-2 protein inhibitor, an MRP-1 protein (multidrug resistance related protein-1) inhibitor, a BCRP protein (breast cancer resistance protein) inhibitor, or a combination thereof It may be selected from the group consisting of. The chemical sensitizer may be selected from the group consisting of cyclosporin A, verapamil, bricodar, leberic acid, and combinations thereof.

The chemical sensitizer may be one having lipid bilayer stabilizer activity. For example, phenethylamine or a derivative thereof may be included. Phenethylamine or its derivatives are, for example, verapamil [( RS ) -2- (3,4-dimethoxyphenyl) -5-{[2- (3,4-dimethoxyphenyl) ethyl]-(methyl) amino} -2 -prop-2-ylpentanenitrile) or a derivative thereof. The liposomes may be free of lipid bilayer stabilizers other than chemical sensitizers. For example, it may not include steroids or derivatives thereof including cholesterol.

The anticancer agent may be an anthracycline anticancer agent. The anthracycline anticancer agent may be doxorubicin, daunorubicin, epirubicin, idarubicin, valerubicin, mitoxantrone, or a combination thereof.

The chemical sensitizer and anticancer agent may be located in the interior space of the liposome, the lipid bilayer or both the internal space and the lipid bilayer. In addition, the chemical sensitizer and the anticancer agent is located in the lipid bilayer itself has a lipid bilayer stabilizing activity may be included in place of or together with the lipid bilayer stabilizer. The liposome may be one having a phase transition temperature of 39 ℃ to 45 ℃. The liposome may be in gel form at room temperature.

The liposomes may further comprise a lipid stabilizer. The stabilizer may be a lipid having a phase transition temperature of at least the phase transition temperature of the lipid bilayer. The lipid bilayer stabilizer may be selected from the group consisting of steroids, sphingolipids or derivatives thereof and combinations thereof. The lipid bilayer stabilizer may be a steroid, or a derivative thereof, or a combination thereof that has the property of being incorporated into the lipid bilayer. The term "steroid" includes the form of four fused cycloalkane rings, namely three cyclohexane rings and one cyclopentane ring (D ring), designated A, B and C rings from left to right. Or an organic compound comprising a gonane or a skeleton derived therefrom. As used herein, the "skeleton derived therefrom" includes the introduction of an unsaturated bond into the scaffold. The steroid may vary depending on the functional groups attached to the four rings and the oxidation state of the ring. For example, the steroid may be to include a hydrophilic functional group in the ring. For example, the steroid may be one having a hydroxyl group in the ring. The steroid may be sterol. The term "sterol" refers to a form of steroid, having a hydroxyl group at position 3 and having a skeleton derived from cholestane. As used herein, the term "derived backbone" includes the introduction of unsaturated bonds in the cholestan backbone. Such steroids include steroids that are found in plants, animals and fungi. For example, it may be made from cycloartenol as in lanosterol or in plants as in animals or fungi. The sterols include cholesterol or derivatives thereof. As used herein, "derivative" means a derivative of cholesterol that retains its property of being incorporated into a lipid bilayer. The stabilizer may be selected from the group consisting of cholesterol, cystosterol, ergosterol, stigmasterol, 4,22-stigmasteradien-3-one, stigmasterol acetate, lanosterol, cycloarthenol, and combinations thereof. have. The lipid bilayer stabilizer may be included in an amount effective to cause the liposomes to remain stable at 37 ° C., but at a temperature above a certain temperature, for example, at temperatures above 39 ° C. For example, the lipid bilayer stabilizer may be included in an amount effective to maintain liposomes at 37 ° C. for at least 30 minutes, but at a temperature above 39 ° C. to break 50% or more of the liposomes within 30 minutes.

Main lipid molecule: The stabilizer, for example cholesterol may be a molar ratio of 50 to 99.9: 0.1 to 50. Main lipid molecule: The stabilizer, for example cholesterol, is 50 to 99.9: 0.1 to 50, for example 50 to 99.9: 1 to 50, 50 to 99.9: 3 to 50, 50 to 99.9: 5 to 50, 50 To 99.9: 7 to 50, 50 to 99.9: 9 to 50, 50 to 99.9: 11 to 50, 50 to 99.9: 15 to 50, 50 to 99.9: 20 to 50, 50 to 99.9: 20 to 35, 50 to 99.9 : 20-30, 50-99.9: 25-30, 50-99.9: 25-50, 50-99.9: 30-50, 50-99.9: 35-50, 50-99.9: 1-35, 50-99.9: 3 To 30, 50 to 99.9: 5 to 25, 50 to 99.9: 7 to 20, or 50 to 99.9: 9 to 15.

Liposomes are relatively short in blood half-life due to rapid ingestion by the liver and interest of the endothelial system or macrophages of organs of the RES, and thus may not accumulate in Ricky tumor tissue. Liposomes can be designed to increase blood circulation time by avoiding fast RES uptake. The lipid bilayer may be, for example, a lipid derivative derivatized with a hydrophilic polymer, for example, a phospholipid derivative. The hydrophilic polymer may be selected from the group consisting of polyethylene glycol, polylactic acid, polyglycolic acid, polylactic acid and polyglycolic acid copolymers, polyvinyl alcohol, polyvinylpyrrolidone, oligosaccharides, and mixtures thereof. The derivative may be a PEG conjugated to a phospholipid of C4-C30, for example C16-C24. The derivative may be DPPC-PEG, or DSPE-PEG. The PEG may have a weight average molecular weight of 180 to 50,000 Da.

The liposomes may be unilamellar vesicles (SUVs) or multivesicular vesicles. The liposome may have a diameter of 50 nm to 500 nm, for example, 50 nm to 400 nm, 50 nm to 300 nm, 50 nm to 200 nm, 100 nm to 500 nm, 100 nm to 400 nm, 100 nm to 300 nm, or 100 nm to 200 nm.

One embodiment comprises a phospholipid bilayer, an elastin-like polypeptide conjugated with a moiety comprising a hydrophobic group, a chemical sensitizer and an anticancer agent, wherein the elastin-like polypeptide conjugated to the moiety comprising the hydrophobic group is filled in the phospholipid bilayer It may be. Phospholipid bilayer refers to the inclusion of phospholipids as major lipid molecules. The phospholipids are dipalmitoylphosphatidylcholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-distearoyl-sn-glycero-3-phosphocholine: DSPC), or these It can be a combination of. The phospholipid bilayer may comprise a conjugate of a phospholipid derivative derivatized with a hydrophilic polymer, eg, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and PEG. The conjugate (DSPE-PEG) is 1,2-dstearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (ammonium salt) [1,2- distearoyl- sn -glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (ammonium salt)]. The phospholipid bilayer may comprise cholesterol.

One embodiment of the liposomes comprises a phospholipid bilayer, stearoyl-VPGVG VPGVG VPGVG-NH ₂ , verapamil and doxorubicin of DPPC, DSPC or a combination thereof, wherein the stearoyl-VPGVG VPGVG VPGVG-NH ₂ is The double layer may be filled. The phospholipid may have a DPPC: DSPC molar ratio of 1: 0 to 0.5, for example, 1: 0.1 to 0.5. The phospholipid bilayer may comprise a conjugate of a phospholipid derivative derivatized with a hydrophilic polymer, eg, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and PEG. The conjugate (DSPE-PEG) is 1,2-dstearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (ammonium salt) [1,2- distearoyl- sn -glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (ammonium salt)]. The phospholipid bilayer may comprise cholesterol.

The liposome has a phase transition temperature of about 10 ° C. to about 70 ° C., for example, 10 ° C. to 60 ° C., 10 ° C. to 55 ° C., 10 ° C. to 45 ° C., 20 ° C. to 60 ° C., 20 ° C. to 55 ° C., and 25 ° C. To 45 ° C., 30 ° C. to 45 ° C., 35 ° C. to 45 ° C., or 39 ° C. to 45 ° C. The phase transition temperature can be controlled by the carbon chain length of the main lipid molecule, the number of unsaturated bonds, the mixture of lipid molecules, and combinations thereof. For example, when DSPC having a lower phase transition temperature is mixed with DSPC higher than DPPC, a liposome composed of a mixture of DPPC and DSPC may have a higher phase transition temperature than a liposome composed of only DPPC. The liposome may be in gel form at room temperature.

Another aspect is a pharmaceutical composition for delivering a chemical sensitizer and an anticancer agent to a target site of an individual comprising a liposome, and a pharmaceutically acceptable carrier or diluent, wherein the liposome is a lipid bilayer, a moiety comprising a hydrophobic group And a elastin-like polypeptide conjugated to a moiety comprising the hydrophobic group, wherein the elastin-like polypeptide is conjugated to the lipid bilayer.

Such pharmaceutically acceptable carriers or diluents may be those known in the art. The carrier or diluent may be selected from the group consisting of water, for example saline and sterile water, Ringer's solution, buffers, dextrose solution, maltodextrin solution, glycerol, ethanol and combinations thereof.

The liposomes are as described above.

The liposomes may be dispersed in an aqueous medium. The aqueous medium may be one containing saline or PBS.

Another aspect is a method for delivering a chemical sensitizer and an anticancer agent to a target site of an individual, comprising a lipid bilayer, an elastin-like polypeptide conjugated with a moiety comprising a hydrophobic group, a chemical sensitizer and an anticancer agent, including the hydrophobic group Administering to the subject a liposome wherein the elastin-like polypeptide conjugated to the moiety is filled in the lipid bilayer; And heating the target site of the subject to release the chemical sensitizer and anticancer agent from the liposomes at the target site.

The method comprises a lipid bilayer, an elastin-like polypeptide conjugated with a moiety comprising a hydrophobic group, a chemical sensitizer and an anticancer agent, wherein the elastin-like polypeptide conjugated to a moiety comprising the hydrophobic group is filled in the lipid bilayer. Administering to the subject a liposome that is present. The liposomes are as described above.

The administration can be parenteral administration. The parenteral administration can be administered, for example, by intravenous administration, subcutaneous, intramuscular, intrathecal (abdominal cavity, joint, or facial), or by direct injection. Direct injection may be direct injection at the site of the condition, eg, at the tumor site. The liposomes can be administered in blood, such as veins, and delivered to a target site, such as a tumor site, by the bloodstream. The target site may have leaky properties.

The method includes heating the target site of the subject to release the chemical sensitizer and anticancer agent from the liposome at the target site. The heating may be heated by a clinical process or may be associated with an inherently higher target site compared to other sites in the body such as inflammation. Heating by the clinical process may involve direct heat transfer, eg contacting the body with a bath of hot liquid medium, for example water, ultrasound at a target site, for example high intensity focused ultrasound. ) Or by applying a magnetic field, such as an amplified magnetic field (AMF), ultrasound and / or radiofrequency. The target site may be a location where a pathological condition exists, such as a tumor site (eg, a solid tumor), or a site where inflammation is present. The heating may be to heat to 39 ℃ to 45 ℃.

According to a liposome according to one aspect, permeability may be controlled by contraction or self-assembly of an elastin-like polypeptide conjugated with a moiety including a hydrophobic group according to temperature. Thus, the liposomes can be used as vehicles to efficiently deliver chemical sensitizers and anticancer agents to the target site of the individual.

According to another aspect of the present invention, a pharmaceutical composition for delivering a chemical sensitizer and an anticancer agent to a target site of the individual can be efficiently delivered to the target site of the individual.

According to a method for delivering a chemical sensitizer and an anticancer agent to a target site of an individual according to another aspect, the chemical sensitizer and the anticancer agent can be efficiently delivered to a target site in the body of the individual.

1 is a diagram showing the survival rate of NCI / ADR-RES cells in culture in the presence of doxorubicin.
2 shows the expression of drug resistance genes in cancer cells.
FIG. 3 shows doxorubicin release profiles with temperature of doxorubicin-containing liposomes. FIG.
4 shows drug release with storage time at 37 ° C. storage temperature.
5 shows drug release with storage time at 42 ° C. storage temperature.
FIG. 6 shows cytotoxicity of free doxorubicin and doxorubicin and verapamil-induced liposomes at 37 ° C. against NCI / ADR-RES cells. FIG.
Figure 7 confirmed the cytotoxicity to the doxorubicin (A) and doxorubicin and verapamil (B) free liposomes collected at 45 ℃.
8 is a diagram showing the effect of the type of drug on the cell viability.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One: Liposomal  Preparation and introduction of drugs

Liposomes were prepared using the components and component ratios shown in Table 1 below.

number Ingredient and ingredient ratio Phospholipids (moles) DSPE-PEG ^* (Mall) Cholesterol (mol) ELP ^** (Mall) One 55 (DPPC) 2 15 0.41 2 55 (DPPC) 2 15 0.28 3 55 (DPPC) 2 20 0.41 4 55 (DPPC) 2 0 0.41 5 55 (DPPC: DSPC = 1: 3) 2 0 0.41 6 55 (DPPC: DSPC = 2: 2) 2 0 0.41 7 55 (DPPC: DSPC = 3: 1) 2 0 0.41 8 55 (DPPC) 2 10 0.41 9 55 (DPPC) 2 5 0.41 10 55 (DPPC) 2 15 0.83 11 55 (DPPC: DSPC = 9: 1) 2 0 0.41 12 55 (DPPC: DSPC = 8.5: 1.5) 2 0 0.41 13 55 (DPPC: DSPC = 8: 2) 2 0 0.41

* 1,2-dstearoyl- sn -glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (ammonium salt) [1,2-distearoyl- sn -glycero-3 -phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (ammonium salt)]

** Stearoyl-VPGVG VPGVG VPGVG-NH ₂ (SEQ ID NO: 7: SA-V3-NH ₂ below)

Specifically, SA-V3-NH ₂ was dissolved in ethanol and phospholipids (DPPC or DPPC / DSPC), DSPE-PEG and cholesterol were dissolved in chloroform. The ethanol solution and the chloroform solution were mixed in a round bottom flask and the solvent was evaporated under reduced pressure at room temperature using a rotary evaporator to form a thin lipid film on the inner wall of the vessel.

A 150 mM ammonium sulphate solution was added to the vessel at room temperature to hydrate the lipid thin film. The hydrated solution was vortexed and sonicated. The solution was extruded by using the ^{Avanti ® Mini-Extruder (Avanti Polar} Lipids, Inc.) containing polycarbonate membranes of pore size of 100nm was prepared in the uni-lamellar vesicles of the liposome type. The solvent of the prepared liposome solution was passed through PBS flowing through PD-10 (GE Healthcare) desalting column, and exchanged with PBS.

Liposomes were loaded with two types of drugs, doxorubicin and verapamil, as needed. Drug loading can be determined using the ammonium sulfate gradient method ( J. Control . Release 2009 , 139, 73-80) or the pH-gradient method ( Biochimica meat Biophysica Acta 1985 , 816, 294-302). Drug loading by ammonium sulphate gradient method was added to the liposome solution with the ammonium sulphate solvent (concentration 250 mM or 150 mM) inside the liposomes and Tris-HCl buffer (25 mM) outside. Drug loading was performed at 37 ° C. for 60 minutes. Drug loading by pH-gradient was added to the liposome solution with 300 mM citrate buffer (pH 4.0) inside the liposome and 20 mM HEPES buffer (150 mM NaCl, pH 7.4) outside. Drug loading was performed at 37 ° C. for 60 minutes.

The prepared liposome solution was passed through the PD-10 (GE Healthcare) desalting column with physiological saline to remove uncaptured drug. As a result, a liposome was prepared in which the drug was trapped in the aqueous inner or lipid bilayer of the liposome.

The prepared liposomes were measured for the size of liposomes prepared by a zeta-sizer (Malvern inst.) Instrument. Sized size had an average diameter of 100 nm to 1,000 nm. In addition, it was possible to control the size by adjusting the proportion of the components constituting the liposome. For example, when the content of ELP decreased, the size became smaller, and when the cholesterol content increased, the particle size became smaller.

Doxorubicin and verapamil collected in liposomes were analyzed by HPLC. Liposomes encapsulated with a certain amount of doxorubicin and verapamil were diluted and dissolved in DMSO (diemthyl sulfoxide) and injected into HPLC. HPLC was confirmed by eluting the drug with KH ₂ PO ₄ / MeCN as eluent and measuring the absorbance at 280 nm. As a result, inherent peaks of doxorubicin and verapamil were observed, confirming that these drugs were collected in liposomes.

In addition, the amount of drug collected according to the method of loading the drug was confirmed. At drug loading, the initial drug concentration was 500 μg of doxorubicin and 250 μg of verapamil per mL of liposome solution. The amounts of drugs captured by the ammonium sulfate method and the pH-gradient method are shown in Table 2.

Loading way Ammonium Sulfate Gradient Method pH-gradient method 250 mM ammonium sulfate (internal), 25 mM Tris HCl buffer (external) 150 mM ammonium sulfate (internal), 25 mM Tris HCl buffer (external) 300 mM citrate buffer (pH 4.0) (internal),
20 mM HEPES buffer (150 mM NaCl, pH 7.4) (external) Doxorubicin 404 μg / mL 36.5 ug / mL 42 ug / mL Doxorubicin / Verapamil 81.7 μg / mL / unidentified 40 ug / mL, and 38.8 ug / mL Not verified Verapamil Not verified 1.4 ug / mL Not verified

As shown in Table 2, when using the ammonium sulphate method (150 mM), two drugs, doxorubicin and verapamil, could be introduced into liposomes simultaneously.

In addition, the size and polydispersity of liposome particles before and after drug loading were confirmed. The prepared liposomes were loaded with two drugs doxorubicin and verapamil. Loading was loaded by ammonium sulphate method (150mM). Doxorubicin was added to liposome solution at a mass ratio of 1: 0.1 relative to the lipid component and verapamil was added to the liposome solution at the same time, and incubated at 37 ° C for 1 hour.

Liposome number
Before drug loading After drug loading Average particle size (nm) Polydispersity (pdi) Average particle size (nm) Polydispersity (pdi) 8 135 0.090 142.6 0.005 8 133.4 0.077 139.6 0.029 9 126.2 0.094 124.8 0.083 9 122 0.093 123.0 0.127

As shown in Table 3, even if two drugs were collected in one liposome, the particle size and polydispersity did not change significantly. That is, no aggregation was observed even when a hydrophobic drug was introduced.

Example  2: Doxorubicin Verapamil Loaded Liposome  And its effect

NCI / ADR-RES cells, which are cancer cells resistant to doxorubicin, were cultured in the presence of doxorubicin, and cell viability was examined. NCI / ADR-RES cells are cancer cells derived from OVCAR-8.

Medium containing different concentrations of doxorubicin (0.1, 0.5, 1.0, 2.5, 5.0, 10, 20 ug / mL) (Minimum Essential Media: MEM), 10% by volume FBS (Fetal Bovine Serum), 1% by weight PS (penicillin NCI / ADR-RES cells were incubated at 37 ° C. for 2 hours. The medium was then replaced with fresh medium and incubated at 37 ° C. for 2 days. Thereafter, cell viability was examined by water soluble tetrazolium (WST) assay. 1 is a diagram showing the survival rate of NCI / ADR-RES cells in culture in the presence of doxorubicin. As shown in FIG. 1, even when treated with 20 ug / mL of doxorubicin, 70% of the cells survived.

Next, the expression of drug resistance genes in NCI / ADR-RES cells and other cancer cells was confirmed. Drug resistance genes were multidrug resistance related protein (MRP1), multidrug resistance protein 1 (MDR1), and breast cancer resistance protein (BCRP).

2 shows the expression of drug resistance genes in cancer cells. As shown in Figure 2, it was confirmed that two or more drug resistance genes are expressed in cancer cells. The result of FIG. 2 shows the result of the electrophoresis of the product obtained by RT-PCR using the RNA sample derived from each cell as a template.

In addition, drug release profiles with temperature of drug-containing liposomes were investigated. FIG. 3 shows doxorubicin release profiles with temperature of doxorubicin-containing liposomes. FIG. The liposomes used in FIG. 3 have a composition of liposome number 8 in Table 1. As shown in FIG. 3, the liposomes were stable up to 37 ° C. without drug release, but began to release rapidly at about 39 ° C. FIG.

In addition, the temperature sensitivity of liposomes containing both doxorubicin and verapamil was investigated. In this case, verapamil contained 50% of the liposomes based on doxorubicin weight. 4 shows drug release with storage time at 37 ° C. storage temperature. As shown in FIG. 4, at 37 ° C., the drug was stably collected without a change in drug release. 5 shows drug release with storage time at 42 ° C. storage temperature. As shown in FIG. 5, at 42 ° C., the drug was released rapidly in a short time. Release of drug encapsulated in liposomes was measured using a plate reader. After incubation, the fluorescence intensity of the sample was measured at excitation wavelength (λex) = 485 nm and emission wavelength (λem) = 635 nm after appropriate dilution to determine the amount of doxorubicin released from the liposomes. Relative percentage fluorescence intensity with incubation at specific temperatures was calculated by comparing the total release of entrapped material obtained after destruction of the liposome sample by addition of 1% Triton X-100 (ethanol). The liposomes used in FIGS. 4 and 5 have the composition of liposome no. 8 in Table 1.

In addition, cytotoxicity against NCI / ADR-RES cells of liposomes into which doxorubicin and verapamil were introduced was confirmed. FIG. 6 shows cytotoxicity of free doxorubicin and doxorubicin and verapamil-induced liposomes at 37 ° C. against NCI / ADR-RES cells. FIG. In this case, verapamil contained 50% of the liposomes based on doxorubicin weight. As shown in FIG. 6, the survival rate was lower in liposomes into which doxorubicin and verapamil were introduced. The experiment was carried out using NCI / ADR-RES in medium containing doxorubicin, doxorubicin and verapamil-incorporated liposomes (Minimum Essential Media: MEM), 10% by volume FBS (Fetal Bovine Serum), and 1% by weight PS (penicillin-streptomycin). Cells were incubated at 37 ° C. for 48 hours. Specifically, 5.0 × 10 ⁴ NCI / ADR-RES cells were incubated for 48 hours in MEM medium containing 10% (v / v) FBS and 1% penicillin / streptomycin in 24 wells. Doxorubicin and verapamil containing liposomes were treated by concentration in cultured NCI / ADR-RES cells, and the cells were immediately transferred to a thermoshaker and incubated at 37 ° C. for 10 minutes. Next, the cells were incubated at 37 ° C. for 2 hours, and then changed to fresh media. Subsequently, the cells were cultured at 37 ° C. for 46 hours, and then cell viability was measured by water soluble tetrazolium (WST) assay using a CCK-8 assay kit (Dojindo). The liposomes used in FIG. 6 have a composition of liposome number 8 in Table 1.

Figure 7 confirmed the cytotoxicity for the doxorubicin (A) and doxorubicin and verapamil (B) that was released at 45 ℃. In this case, verapamil contained 50% of the liposomes based on doxorubicin weight. 5.0 × 10 ⁴ NCI / ADR-RES cells were incubated for 48 hours in MEM medium containing 10% (v / v) FBS and 1% penicillin / streptomycin in each well of a 24-well plate. Doxorubicin and verapamil containing liposomes were treated by concentration in cultured NCI / ADR-RES cells, and the cells were immediately transferred to a thermoshaker and incubated at 45 ° C. for 10 minutes. Next, the cells were incubated at 37 ° C. for 2 hours, and then changed to fresh medium. Subsequently, the cells were cultured at 37 ° C. for 46 hours, and then cell viability was measured by using a Water Soluble Tetrazolium (WST) assay using a CCK-8 assay kit (Dojindo). The liposomes used in FIG. 7 have a composition of liposome number 8 in Table 1. As shown in FIG. 7A, when free doxorubicin was treated at high concentration (20 ug / mL), cell viability was 50%. As shown in FIG. 7B, for doxorubicin and verapamil liposomes, cell viability at 10 ug / mL doxorubicin (+5 ug / mL verapamil) was about 30%. In contrast, as shown in FIG. 7A, the cell viability was about 70% for the same concentration (10 ug / mL) of free doxorubicin.

In addition, cytotoxicity was confirmed for cells treated with free doxorubicin or free doxorubicin and free verapamil simultaneously. 8 is a diagram showing the effect of the type of drug on the cell viability. As shown in FIG. 8, the cell survival rate of the group treated with doxorubicin and verapamil was lower than that of the group treated with doxorubicin alone. 5.0 × 10 ⁴ NCI / ADR-RES cells were incubated for 48 hours in MEM medium containing 10% (v / v) FBS and 1% penicillin / streptomycin in 24 wells. Doxorubicin and verapamil were treated by concentration in cultured NCI / ADR-RES cells, and the cells were immediately transferred to a thermoshaker and incubated at 37 or 45 ° C for 10 minutes. Next, the cells were incubated at 37 ° C. for 2 hours, and then changed to fresh medium. Subsequently, the cells were cultured at 37 ° C. for 46 hours, and then cell viability was measured by WST assay using a CCK-8 assay kit (Dojindo).

As a result, by treating a tumor cell with a chemosensitizer together with an anticancer agent, the effect of the anticancer agent could be enhanced. When doxorubicin and verapamil are treated in vivo without encapsulating liposomes, the two drugs cannot act on cancer at the same time (the location where the drug is absorbed, the amount absorbed, and the half-life in the body). However, liposomes encapsulated with doxorubicin and verapamil may be simultaneously released from the cancer site by heat stimulation and accumulate in the cancer site, thereby increasing the effect of the anticancer agent. In simultaneously delivering an anticancer agent and a chemical sensitizer, liposomes according to one aspect may be used.

<110> Samsung Electronics Co., Ltd. <120> Liposome comprising elastin-like polypeptide conjugated to a          moiety containing a hydrophobic group, chemosensitizer and          anticancer agent and use <130> PN098881 <150> 12/108269 <151> 2012-09-27 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> elastin-like polypeptide unit sequence <220> <221> VARIANT <222> (4) <223> Xaa denotes amino acid other than proline <400> 1 Val Pro Gly Xaa Gly   1 5 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> elastin-like polypeptide unit sequence <220> <221> VARIANT <222> (3) <223> Xaa denotes amino acid other than proline <400> 2 Pro Gly Xaa Gly Val   1 5 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> elastin-like polypeptide unit sequence <220> <221> VARIANT <222> (2) <223> Xaa denotes amino acid other than proline <400> 3 Gly Xaa Gly Val Pro   1 5 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> elastin-like polypeptide unit sequence <220> <221> VARIANT <222> (1) <223> Xaa denotes amino acid other than proline <400> 4 Xaa Gly Val Pro Gly   1 5 <210> 5 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> elastin-like polypeptide unit sequence <220> <221> VARIANT <222> (5) <223> Xaa denotes amino acid other than proline <400> 5 Gly Val Pro Gly Xaa   1 5 <210> 6 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> elastin-like polypeptide unit sequence modified with          stearoylation and amidation <220> <221> VARIANT <222> (1) <223> Amino terminal nitrogen is stearoylated <220> <221> VARIANT <30> <223> Carboxy terminal carboxy group is amidated with -NH2 <400> 6 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val   1 5 10 15 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly              20 25 30 <210> 7 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> elastin-like polypeptide unit sequence modified with          stearoylation at amino terminal and amidation at carboxy terminal <220> <221> VARIANT <222> (1) <223> Amino terminal nitrogen is stearoylated <220> <221> VARIANT &Lt; 222 > (15) <223> Carboxy terminal carboxyl group is amidated with -NH2 <400> 7 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly   1 5 10 15

Claims (21)

A lipid bilayer, an elastin-like polypeptide (ELP) conjugated with a moiety comprising a hydrophobic group, a chemical sensitizer and an anticancer agent, and an elastin-like polypeptide conjugated to a moiety comprising the hydrophobic group is Liposomes are filled in the lipid bilayer. The method according to claim 1, wherein the chemical sensitizer MDR1 protein (multidrug resistance protein-1) inhibitors, MDR-2 protein inhibitors, MRP-1 protein (multidrug resistance related protein-1) inhibitors, BCRP protein (breast cancer resistance protein) inhibitors Liposomes selected from the group consisting of, and combinations thereof. The liposome of claim 1, wherein the chemical sensitizer is selected from the group consisting of cyclosporin A, verapamil, bricodar, leberic acid, and combinations thereof. The liposome according to claim 1, wherein the anticancer agent is an anthracycline anticancer agent. The liposome of claim 1, further comprising a lipid bilayer stabilizer. The liposome of claim 5, wherein the lipid bilayer stabilizer is a steroid or a derivative thereof. The elastin-like polypeptide of claim 1, wherein the elastin-like polypeptide comprises one or more repeat units selected from the group consisting of VPGXG, PGXGV, GXGVP, XGVPG, GVPGX, and combinations thereof, wherein V is valine, P is proline, G Is glycine, and X is an amino acid other than proline. The liposome of claim 7, wherein the selected repeating unit is repeated 2 to 200 times. A pharmaceutical composition for delivering a chemical sensitizer and an anticancer agent to a target site of an individual comprising a liposome and a pharmaceutically acceptable carrier or diluent, the liposome comprising an elastin-conjugated moiety comprising a lipid bilayer, a hydrophobic group. A pharmaceutical composition comprising an analogous polypeptide, a chemical sensitizer, and an anticancer agent, wherein the elastin-like polypeptide conjugated to a moiety comprising the hydrophobic group is filled in the lipid bilayer. The pharmaceutical composition of claim 9, wherein the chemical sensitizer is selected from the group consisting of MDR1 protein inhibitors, MDR-2 protein inhibitors, MRP-1 protein inhibitors, BCRP protein inhibitors, and combinations thereof. The pharmaceutical composition of claim 9, wherein the chemical sensitizer is selected from the group consisting of cyclosporin A, verapamil, bricodar, leberic acid, and combinations thereof. The pharmaceutical composition of claim 9, wherein the anticancer agent is an anthracycline anticancer agent. The pharmaceutical composition of claim 9, further comprising a lipid bilayer stabilizer. The pharmaceutical composition of claim 13, wherein the lipid bilayer stabilizer is a steroid or a derivative thereof. The method of claim 9, wherein the elastin-like polypeptide comprises one or more repeating units selected from the group consisting of VPGXG, PGXGV, GXGVP, XGVPG, GVPGX, and combinations thereof, wherein V is valine, P is proline, G Is glycine, X is an amino acid other than proline. The pharmaceutical composition of claim 15, wherein the selected repeating unit is repeated 2 to 200 times. A method for delivering a chemical sensitizer and an anticancer agent to a target site of an individual, comprising: an elastin-like polypeptide conjugated with a lipid bilayer, a moiety comprising a hydrophobic group, a chemical sensitizer, and an anticancer agent, the moiety comprising the hydrophobic group Administering the liposome to which the elastin-like polypeptide conjugated is filled in the lipid bilayer; And
Heating the target site of the subject to release the chemical sensitizer and anticancer agent from the liposome at the target site. The method of claim 17, wherein the chemical sensitizer is selected from the group consisting of MDR1 protein inhibitors, MDR-2 protein inhibitors, MRP-1 protein inhibitors, BCRP protein inhibitors, and combinations thereof. The method of claim 17, wherein the anticancer agent is an anthracycline anticancer agent. The method of claim 17, wherein the liposome is a lipid bilayer stabilizer, further comprising a steroid or a derivative thereof. The method of claim 17, wherein the elastin-like polypeptide comprises one or more repeating units selected from the group consisting of VPGXG, PGXGV, GXGVP, XGVPG, GVPGX, and combinations thereof, wherein V is valine, P is proline, G Is glycine and X is an amino acid other than proline.

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