KR20170040748A - Multi-block Polypeptides and Their Self-assembled Nanostructures for Enhanced Intracellular Delivery of Multiple Drugs - Google Patents

Abstract

Provided is a drug delivery containing multiblock copolypeptide which comprises: a cell-permeable polypeptide (CPP) block; a cysteine (Cys) block connected to one terminal end of the CPP block; a charged and hydrophilic elastin-based polypeptide (EBP) block connected to the Cys block; and a hydrophobic EBP block connected to the hydrophilic EBP.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to drug delivery systems and self-assembled nanostructures including multiblock polypeptides for efficient intracellular delivery of multiple drugs,

The present invention relates to drug delivery vehicles and self-assembled nanostructures comprising multiblock polypeptides for efficient intracellular delivery of multiple drugs. More specifically, the invention relates to a cell permeable polypeptide block; Cysteine block; A charged hydrophilic elastin-based polypeptide block; And a multimolecular polypeptide comprising a hydrophobic elastin-based polypeptide block, a drug delivery vehicle comprising the same, and a self assembly thereof.

Nanoparticles, such as micelles or nanostructures, play an important role as drug carriers, stabilizing drugs in vivo and allowing drugs to be delivered only to targets.

Molecular drugs used for treatment, such as chemotherapy, have the following impairments: insoluble in aqueous conditions, low biostability to access target cells, and damage to normal tissue.

Various carriers regulate drug release by increasing the half-life of the drug in the blood circulation until the drug reaches the target, reducing the damage of normal cells by overcoming the obstacles, and controlling the characteristics of the carriers according to the target environment. Polymers are valuable as polymer carriers to form nanoparticles, such as nanocapsules, liposomes or micelles, that effectively deliver drugs. The structure of the polymer is composed of amphiphilic block copolymers wherein the copolymer consists of a diblock and is controlled in length ratio between the two polymers, B-poly (caprolactone) (MePEG-b-PCL)} and MePEG-b-poly (lactic-co-glycolic acid) (hereinafter referred to as " MePEG- MePEG-b-PLGA) {MePEG-b-poly (lactic-co-glycolic acid) (MePEG-b-PLGA)} can be formed into nanocapsules, liposomes or micelles depending on the length of each block. Compared to micelles, polymers made of nanocapsules or liposomes are located on the surface of a structure that contains a larger amount of drug with a larger core (core). However, nanocapsules or liposomes are less stable than micelles due to their size and location of polymers, while micelles penetrate into cells more effectively due to their small size.

Polypeptides have the advantage of including biocompatibility, biodegradability and specific functionality. Can be precisely controlled on a monomer scale such as the exact number of monomers in one polypeptide. And can be finely controlled with specific functional groups in each monomer. In addition, the polypeptides may be used as drug delivery vehicles. For example, gelatin has been used as microspheres and nanoparticles for colloidal drug delivery systems. Also, silk fibroin (SF) was used as a nanoscale drug delivery vehicle. Polypeptides are assembled by the interaction between repetitive peptides having a secondary structure to form nanoparticles. Both amphiphilic block copolymers based on polypeptides form micelle structures. Hydrophilic blocks of both hydrophilic blocks are conjugated with functional peptides such as ligands or functional peptides such as targeting moieties or antigens for therapeutic effect.

The hydrophobic blocks of both hydrophilic block copolypeptides are very important for micelle formation and micelle disintegration for drug release. Hydrophobic blocks with stimuli-responsiveness control their hydrophobicity and micelle formation. Polypeptides having mainly glutamine acid or lysine have pH sensitivity, and their proton additive action and deprotonation effect depending on pH affect the hydrophobicity of polypeptides. Thermal-responsive polypeptides having a low critical solution temperature (LCST) or an upper critical solution temperature (UCST) are hydrophobic at certain temperature ranges. The stimulus-responsiveness of the polypeptides is tailored to the environment of the target cells and organs for the delivery and release of encapsulated drugs and molecules.

Elastin-based polypeptides (EBPs) are heat-reactive biopolymers derived from the elastomeric domain. Elastin is a major protein component in the extracellular matrix (ECM). EBPs have been modified to have thermal sensitivity based on elastomeric domains and have been found to contain Val-Pro- (Gly or Ala) -X, a repeat unit of the pentapeptide, a peptide consisting of five amino acids _aa- Gly. EBPs are heat-sensitive polypeptides, and their transition temperature is readily controlled to form drug delivery nanostructures.

X _aa is a guest residue and may be any amino acid except proline. According to the sequence of the repeating unit, two kinds of EBPs can be distinguished, one being an elastin-based polypeptide with elasticity of the sequence Val-Pro-Gly-X _aa -Gly (EBPE ) and the other is elastin having a plasticity of sequences _{Val-Pro-Ala-X aa} -Gly - is based polypeptides (elastin-based polypeptide with plasticity, EBPP).

EBPs have lower critical solution temperature (LCST) behavior with reversible phase transition with temperature. The LCST has the advantage of using an easy purification method such as inverse transition cycling (ITC) And also provides the advantage of being thermally triggered to self-assemble into particles, gels, fibers and other structures. Block copolypeptides composed of EBPs blocks with different sequences are used to form self assembled structures. EBPs Diblock copolymers consist of two EBPs, which have different sequences and transition temperatures (T _t ) to form self-assembled micelle structures. When the temperature of the EBPs diblock copolymer solution is increased above the lower limit T _t , EBPs with low T _t become insoluble while EBPs with high T _t become soluble and both amphiphilic diblock EBPs Self-assembled into a micelle structure. EBPs diblock copolymers can be fused with other functional peptides to have functional multivalency as micellar constructs, for example, they can be fused with cell permeable peptides having cell permeability.

Cell Penetrating Peptides (CPPs) can carry cargo that is much larger than their size. These cell permeable peptides are used for cell membrane permeation along with cargo, where sequences that play a very important role in cell permeability are isolated from the original protein and transformed into short peptides (10-30 amino acids). Although the exact mechanism of cell uptake is not known, the mechanism is independent of the receptor, and CPPs interact with the cell membrane with an appropriate affinity to penetrate the cell membrane without interfering with it. Since CPPs have positive charge and proper hydrophobicity, they can approach and penetrate mainly cell membranes composed of lipids with negative charge. If the hydrophobicity is too high, the peptides will be trapped in cell membrane lipids. In addition, these secondary structures are suitable for interacting with lipids.

The present inventors have continued the study on multimeric drug delivery systems using cell permeable peptides and elastin proteins. As a result, a new drug delivery system with a cell-permeable peptide and an elastin-based polypeptide was constructed.

Journal of Controlled Release, 2005, 108 (2-3), 396-408

The present invention is to provide a drug delivery vehicle comprising a novel multiblock copolypeptide.

Another object of the present invention is to provide a self assembled nanostructure of a multiblock copolypeptide.

It is another object of the present invention to provide a drug delivery system capable of sequential release of multiple drugs.

The present invention provides a drug delivery vehicle comprising:

A polypeptide block (CPP block); And

A cysteine block (Cys) connected to one end of the cell permeable polypeptide block; And

An elastin-based polypeptide block (hydrophilic EBP block) that is charged and hydrophilic and connected to the cysteine block; And

A hydrophobic elastin-based polypeptide block (hydrophobic EBP block) linked to the hydrophilic EBP;

≪ / RTI >

The hydrophilic elastin-based polypeptides (EBP) block, a _{Val-Pro-Gly-X aa} -Gly [ SEQ ID NO: 1] or _{Val-Pro-Ala-X aa} -Gly -penta-peptide consisting of [SEQ ID NO: 2] 6n times And is composed of an amino acid sequence represented by the following Formula 1 or Formula 2:

[Formula 1]

[Val-Pro-Gly-X _aa- Gly] _6n ; or

[Formula 2]

[Val-Pro-Ala-X _aa- Gly] _6n ,

In the above formula (1) or (2)

N is an integer of 1 or more,

Wherein X _aa is an amino acid other than proline, and is selected from any natural or artificial amino acid when the pentapeptide is repeated, and at least one of the X _aa is a charged amino acid and a hydrophilic amino acid.

The hydrophobic elastin-based polypeptide (EBP)

_{Val-Pro-Gly-X aa} -Gly [ SEQ ID NO: 1] or _{Val-Pro-Ala-X aa} -Gly repeats [SEQ ID NO: 2] penta-peptide consisting of 6n times, represented by the following formula 1 or formula 2 Lt; RTI ID = 0.0 > amino acid < / RTI >

[Formula 1]

[Val-Pro-Gly-X _aa- Gly] _6n ; or

[Formula 2]

[Val-Pro-Ala-X _aa- Gly] _6n ,

In the above formula (1) or (2)

N is an integer of 1 or more,

X _aa is an amino acid other than proline, which is selected from any natural or artificial amino acid when the pentapeptide is repeated, and at least one of X _aa is a hydrophobic or aliphatic amino acid.

The cell permeable peptide (CPP) block may be selected from the group consisting of Penetratin, SynBl, SynBl-NLS, poly-arginine, VP22, MAP, hCT-derived CPP, MPG, Buforin 2, PEP- But are not necessarily limited thereto. The CPP sequence information is well known in the art.

The cysteine (Cys) block refers to a peptide sequence containing cysteine. Specifically, the amino acid sequence of the cysteine block may be (Gly-Ala-Cys) repeated one or more times. More specifically, a peptide sequence consisting of (Gly-Ala-Cys) n [n represents a repetition number] can be used as a cysteine block, but is not necessarily limited thereto.

A cysteine block of (Gly-Ala-Cys-Gly-Ala-Cys-Gly-Ala-Cys-Gly-Ala-Cys) [SEQ ID NO: 43] was used in one embodiment of the present invention.

In the hydrophilic EBP block, n is 1 in the formula 1, X _aa of each of the repeated pentapeptides is 1,

A (Ala), G (Gly) and I (Ile) in a ratio of 1: 4: 1 [SEQ ID NO: 20];

K (Lys), G (Gly) and I (Ile) in a ratio of 1: 4: 1 [SEQ ID NO: 22];

D (Asp), G (Gly) and I (Ile) in a ratio of 1: 4: 1 [SEQ ID NO: 24]; or

E (Glu), G (Gly) and I (Ile) in a ratio of 1: 4: 1 [SEQ ID NO: 26]; Or,

or

Wherein n is 1 in the formula 2, X _aa of each of the repeated pentapeptides is

A (Ala), G (Gly) and I (Ile) in a ratio of 1: 4: 1 [SEQ ID NO: 21];

K (Lys), G (Gly) and I (Ile) in a ratio of 1: 4: 1 [SEQ ID NO: 23];

D (Asp), G (Gly) and I (Ile) in a ratio of 1: 4: 1 [SEQ ID NO: 25]; or

(SEQ ID NO: 27) in which E (Glu), G (Gly) and I (Ile) have a ratio of 1: 4: 1.

The hydrophobic EBP block comprises

Wherein n is 1 in the formula 1, X _aa of each of the repeated pentapeptides,

(SEQ ID NO: 28) wherein G (Gly), A (Ala) and F (Phe) are in a ratio of 1: 3: 2

Wherein n is 1 in the formula 2, X _aa of each of the repeated pentapeptides is

G (Gly), A (Ala) and F (Phe) in a ratio of 1: 3: 2 [SEQ ID NO: 29];

K (Lys), A (Ala) and F (Phe) in a ratio of 1: 3: 2 [SEQ ID NO: 30];

D (Asp), A (Ala) and F (Phe) in a ratio of 1: 3: 2 [SEQ ID NO: 31];

K (Lys) and F (Phe) in the ratio of 3: 3 [SEQ ID NO: 32];

D (Asp) and F (Phe) in a ratio of 3: 3 (SEQ ID NO: 33);

H (His), A (Ala) and I (Ile) in a ratio of 3: 2: 1 [SEQ ID NO: 34];

H (His), G (Gly) in a ratio of 5: 1 [SEQ ID NO: 35]; or

(SEQ ID NO: 36) in which G (Gly), C (Cys) and F (Phe) are in a ratio of 1: 3: 2.

The hydrophilic EBP block may comprise,

In Formula 2, n is 2, and X _aa of each of the repeated pentapeptides is represented by Formula

(SEQ ID NO: 44) in which E (Glu), G (Gly) and I (Ile) have a ratio of 1: 4: 1.

The hydrophobic EBP block

In Formula 2, n is 2, and X _aa of each of the repeated pentapeptides is represented by Formula

G (Gly), A (Ala) and F (Phe) in a ratio of 1: 3: 2 [SEQ ID NO: 45] or

In the above formula 2, n is 4, and X _aa of each of the repeated pentapeptides is

(SEQ ID NO: 46) in which G (Gly), A (Ala) and F (Phe) are present in a ratio of 1: 3: 2.

The drug delivery system comprises:

CPP block-cysteine blocks (Cys) ₄ - hydrophilic EBP block _{EBPP [E 1 G 4 I 1} ] 12 [ SEQ ID NO: 44; - hydrophobic EBP block EBPP composed of _{[G 1 A 3 F 2]} 12 [ SEQ ID NO: 45; or ; or

CPP block Cysteine block (Cys) ₄ hydrophilic EBP block EBPP [E ₁ G ₄ I ₁ ] ₁₂ [SEQ ID NO 44] hydrophobic EBP block EBPP [G ₁ A ₃ F ₂ ] ₂₄ [SEQ ID NO: 46] Lt; / RTI >

The CPP block may be Phenetatin (amino acid sequence SEQ ID NO: 38) or SynB1 (amino acid sequence SEQ ID NO: 40), and the drug delivery vehicle may be one selected from SEQ ID NO: 51, 52, 53 and 54.

The multiblock copolypeptide may comprise,

A self-assembled nanostructure of the core-shell structure can be formed while the hydrophobic EBP block forms a core structure and the CPP block-cysteine block-hydrophilic EBP block forms a shell structure by temperature stimulation.

The self-assembled nanostructure can be stabilized by the oxidation reaction of the cysteine block.

The hydrophilic EBP block and the acrylate are chemically bonded to each other by introducing an acrylate into the carboxyl group of the hydrophilic EBP block in the shell portion and irradiating ultraviolet rays to stabilize the self assembled nanostructure.

The acrylate may be glycidyl methacrylate (GMA).

The hydrophobic drug is captured in the core portion of the self-assembled nanostructure, and the chargeable drug is captured in the shell portion to control the release of the hydrophobic drug and the chargeable drug to release multiple drugs.

The hydrophobic drug is preferably a cell membrane dye, a photosensitiser, an anti-cancer agent, and antibiotics, but is not limited thereto.

The anticancer agent may be selected from the group consisting of Calcein, gemcitabine, Busulfan, Chlorambucil, Cyclophosphamide, Melphalan, Cisplatin, (6-fluorouracil (5-FU), methotrexate (MTX), daunorubicin, adriamycin, vinblastine, vincristine, ifosfamide, cytarabine, From the group consisting of Vincristine, Vindesine, Procarbazine, Tamoxifen, Megesterol acetate, Flutamide and Gosereline acetate (Zoladex) But it is not limited thereto.

The antibiotic may be selected from the group consisting of penicillin antibiotics, cephalosporine antibiotics, macrolide antibiotics, tetracycline antibiotics, quinolone antibiotics, antihistamines, which is any one selected from the group consisting of clindamycin, metronidazole, chloramphenicol, Actinomycin-D, Bleomycin and mitomycin-C But is not limited thereto.

The chargeable drug may be a negative charge drug or a positively charged drug. The chargeable drug may be a charge (negatively charged or positively charged) dye, a chargeable protein drug, and the like.

The chargeable drug exhibits hydrophilicity.

The positively charged dye is selected from the group consisting of pararosaniline, methylene blue, auramine o, chrysoidine G, malachite green, rhodamine B ), And rhodamine 6G (Rhodamine 6G), but the present invention is not limited thereto.

Wherein the negatively charged dye is selected from the group consisting of Eosin, Metanil yellow, Methyl orange, xylene cyanol, and bromophenol blue. But is not limited thereto.

Wherein said positively charged drug is selected from the group consisting of propafenone, procainamide, metformin, phentermine, atenolol, propranolol, prazosin, and la But is not limited to, any one selected from the group consisting of labetalol.

The negative charge drug is preferably one selected from the group consisting of naproxen, ibuprofen, dexamethasone sodium phosphate, but is not limited thereto.

The drug delivery system is capable of releasing multiple drugs by controlling the release of the hydrophobic drug and the chargeable drug or the hydrophilic drug by the oxidation-reduction reaction of the cysteine block.

A hydrophobic drug is captured in a core portion of the self-assembled nanostructure, and a charge drug having acrylate binding to a thiol group in a cysteine block of the shell portion is bonded to release the hydrophobic drug and the hydrophilic drug Multiple drug release is possible.

The chargeable drug to which the acrylate is conjugated may include the above-mentioned chargeable drug, and may include a drug having hydrophilicity.

The self-assembled nanospheres release the entrapped drug (hydrophilic drug) trapped in the shell, followed by release of the hydrophobic drug entrapped in the core; Or the hydrophobic drug entrapped in the core is released, and then the chargeable drug (hydrophilic drug) trapped in the shell is released, so that sequential drug release is possible.

Figures 5 and 6 schematically illustrate the concept of the present invention. 5 (b), the self-assembled nanostructure (micelle) is stabilized by the oxidation reaction of the cysteine block. At the same time, in FIGS. 5 (c) and 5 (d), the rate of multiple drug release of micelles can be controlled by the oxidation-reduction reaction of cysteine blocks. Example 12 and FIG. 12 (a) show the results of releasing multiple drugs after micelles were stabilized by the oxidation reaction of cysteine blocks.

6 (a) and 6 (b), methacrylate (MA) was introduced into the charge (hydrophilic) drug. Methacrylate is an acrylate capable of reacting with a thiol group of a cysteine block. Thus, since the MA-conjugated drug can chemically bind to the thiol group of the cysteine block, the drug can be captured in the shell portion and control drug release. Example 13 and Fig. 12 (b) show the result that an acrylate-bound hydrophilic drug is bound to a cysteine block and released. 6 (c), an acrylic moiety is introduced by conjugating a carboxyl group of a hydrophilic EBP block with glycidyl methacrylate (GMA) to form an ultraviolet The micelle structure was chemically stabilized through chemical bonding of acrylic groups by ultraviolet irradiation.

The term "amino acid" as used herein means a natural amino acid or an artificial amino acid, preferably a natural amino acid. For example, the amino acid means glycine, alanine, serine, valine, leucine, isoleucine, methionine, glutamine, asparagine, cysteine, histidine, phenylalanine, arginine, tyrosine or tryptophan.

The properties of the amino acids are well known in the art. Specifically, they exhibit hydrophilicity (negative charge or positive charge), exhibit hydrophobicity, and exhibit aliphatic or aromatic properties.

As used herein, the abbreviations Gly (G), Ala (A) and the like are amino acid abbreviations. Gly is an acronym for glycine, and Ala is an acronym for alanine. Glycine is represented by G and alanine by A. The abbreviations are expressions commonly used in the art.

In the present invention, the term "hydrophilic amino acid with charge" means an amino acid having a positive charge or a negative charge in the hydrophilic amino acid. For example, the positively charged hydrophilic amino acids include lysine, arginine, and the like.

The term "hydrophobic amino acid" is an amino acid showing hydrophobic properties, such as glycine and leucine.

The term "polypeptide" as used herein refers to any polymeric chain of amino acids. The terms "peptide" and "protein" may be used interchangeably with the term polypeptide, which also refers to a polymer chain of amino acids. The term "polypeptide" includes polypeptide analogs of natural or synthetic proteins, protein fragments and protein sequences. The polypeptide may be a monomer or a polymer.

"Copolipeptide" is a copolymer formed by linking two or more polypeptides. In the present invention, multiblock copolypeptides were prepared by linking four types of polypeptides, namely, a cell permeable peptide, cysteine, hydrophilic EBP and hydrophobic EBP (see FIGS. 5A and 6A).

The term " phase transition " means that the state of a material changes, such as when water turns into water vapor or ice turns into water.

The polypeptides according to the present invention are basically elastin-based polypeptides (EBPs) having stimulus-responsive properties. The "elastin-based polypeptide" is also referred to as "elastin-like polypepties (ELP) ". Is a term widely used in the technical field of the present invention.

In the present specification, _Xaa is referred to as "guest residue ". It is possible to manufacture a wide variety of EBP according to the present invention with a variety of introducing said X _aa.

The EBP undergoes a reversible phase transition at a lower critical solution temperature (LCST), also referred to as a transition temperature (T _t ). These are T _t , It becomes insoluble when the temperature exceeds T &_lt; _{t &} gt ;.

In the present invention, the physical and chemical properties of EBP are mainly controlled by a combination of Val-Pro- (Gly or Ala) penta peptide repeating units -X _aa -Gly. Specifically, the third amino acid of the repeat unit determines the relative mechanical properties. For example, in the present invention, the third amino acid Gly determines the elasticity, or Ala determines the plasticity. The elasticity or plasticity is a property that appears after the transition.

On the other hand, both the hydrophobicity of the guest residue X _aa , which is the fourth amino acid, and the multimerization of the pentapeptide repeat unit all have an effect on T _t .

The EBP according to the present invention can be a polypeptide in which the pentapeptide is repeated, and the repeated polypeptide can form a polypeptide block (EBP block). Specifically, a hydrophilic EBP block or a hydrophobic EBP block can be formed. The hydrophilic or hydrophobic properties of the EBP block of the present invention are deeply related to the transition temperature of EBP.

The transition temperature of EBP also depends on the amino acid sequence and its molecular weight. Urry et al. (2003) investigated the relationship between EBP sequence and T _t (Urry DW, Luan C.-H., Parker TM, Gowda DC, Parasad KU, Reid MC, and Safavy A. 1991. Temperature of polypeptide inverse temperature transition depends on the mean residue hydrophobicity. J. Am. Chem. Soc. 113: 4346-4348.). Urry et al. In the pentapeptide of Val-Pro-Gly-Val-Gly, when the fourth amino acid, "guest residue," is replaced with a residue that is more hydrophilic than Val, the T _t increases compared to the original sequence, Substitution of the guest residue with the residue results in a lower T _t than the original sequence. That is, hydrophilic EBPs have high T _t and hydrophobic EBPs have relatively low T _t . This finding led to the determination of which amino acid to use as the guest residue of the EBP sequence and to the change in the composition of the guest residues, thus making it possible to produce EBP having a specific T _t (Protein-Protein Interactions: A Molecular Cloning Manual , 2002, Cold Spring Harbor Laboratory Press, Chapter 18. pp. 329-343).

As described above, the high _t T represents the hydrophilic represents a hydrophobic if T _t is low. EBP blocks according to the present invention can also increase or decrease T _t by changing the amino acid sequence including the guest residue and the molecular weight. Thus, it is possible to prepare a hydrophilic EBP block or a hydrophobic EBP block.

For reference, EBP has a lower temperature than T _t may be used as the hydrophobic block, EBP has a higher T than the temperature _t may be used as the hydrophilic block. Due to this nature of EBP, hydrophilic and hydrophobic properties of EBP can be relatively defined when applied to biotechnology.

EBP example the sequence of the invention for example, Val-Pro-Ala-X aa -Gly a plastic penta peptide repeat polypeptide plastic block and _{Val-Pro-Gly-X aa} -Gly acoustic penta-peptide repeat elastic polypeptide which When the blocks are compared, Gly, the third amino acid, shows higher hydrophilicity than Ala. Therefore, the plastic block polypeptide when low is:: (EBPE Elastin-based polypeptide with elasticity) than T _t (Elastin-based polypeptide with plasticity EBPP) a polypeptide elastic block.

The EBPs according to the present invention can exhibit hydrophilic or hydrophobic properties by adjusting T _t as described above. And chargeable amino acids can be used to impart chargeability

The multiblock copolypeptides according to the invention are shown schematically in Figures 5A and 6A. The present invention utilizes a cell permeable peptide (CPP). Therefore, the multiblock copolypeptide of the present invention can easily enter into cells.

&Quot; Multiblock copolypeptide "of the present invention refers to a quadruple block copolypeptide composed of a CPP block-Cys block-hydrophilic EBP block-hydrophobic EBP block, EBPPs. " In the present specification, the copolypeptide to which two blocks are linked is also referred to as a "diblock copolypeptide ". In the case of a co-polypeptide in which three blocks are linked, it is also referred to as a "triblock copolypeptide ". In particular, "hydrophilic EBP block-hydrophobic EBP block" is also referred to as "die block EBP" or "die block EBPs"

In the examples of the present invention, two types of CPPs were used: Penetratin and SynB1. Phenetratin is part of a peptide derived from the homeodomain of Antennapedia (Drosophila homeoprotein) and has the amino acid sequence of RQIKIWFQNRRMKWKK. SynB1 is one of the SynB family members of the vectors derived from the antimicrobial peptide proteogrin-1 (PG-1) in porcine white blood cells and has the amino acid sequence of RGGRLSYSRRRFSTSTGR.

Penetrathin has a high penetration ability into cells, and SynB1 has low toxicity. CPPs can be conjugated to molecules such as proteins, polymers or dyes or drugs.

EBPs fused with CPPs are used as therapeutic polypeptides to target hyperthermia. The CPP-EBPs fusion polypeptides have T _t Soluble and long EBP chains, it is difficult to permeate into cell membranes. EBPs have been aggregated in the above T _t, wherein the fusion polypeptide are easily accumulated in the transmission to the tumor. Various CPPs can be fused with EBPs for a variety of applications.

Also, CPP-EBPs fusion polypeptides are used with other peptides or drugs. Fusion proteins, including CPP and mono block EBPs, can serve as therapeutic polypeptides that target the solid temperature. However, diblock EBPs can more effectively contain drugs and control their penetration into target cells and non-target cells.

In the present invention, self-assembled micelle structures as drug delivery vehicles capable of penetrating into cells were prepared. The micelles consisted of an EBP diblock (hydrophilic EBP-hydrophobic EBP), a cysteine (Cysteine or Cys) block and CPPs (located at the N-terminus of the polypeptide). The EBPs diblock copolymer was introduced and self-assembled into a heat-stimulated micellar structure to capture the drug. The transition temperature of the hydrophobic EBP having Gly, Ala, and Phe (ratio of 1: 3: 2) as guest residues was found to be the hydrophilic EBP having Glu, Gly, and Ile (ratio of 1: 4: 1) Transition temperature. (Gly-Ala-Cys) was repeated four times and cysteine blocks were located between CPPs and hydrophilic EBPPs, and CPPs were introduced to increase penetration efficiency on the surface of the micelle structure.

Two strategies have been applied to the self-assembled micelle structure to incorporate the drug into the micelle and stabilize the structure. The first involves physically incorporating two classes of drugs into each hydrophilic and hydrophobic EBP block and stabilizing the structure using cysteine blocks. As the micelles were formed, the drugs charged with positive (negative) charge physically interacted with the hydrophilic EBP blocks charged with negative (positive) charges in the shell, while the hydrophobic drugs interacted with the core ). After self assembly of the micelle structure with two other drugs, the CPP was located on the micelle surface and the cysteine block was located below the CPP block. Thiol groups in cysteine blocks stabilize the structure by forming disulfide bonds. 5 (b), the self-assembled nanostructure (micelle) is stabilized by the oxidation reaction of the cysteine block. Referring to FIGS. 5 (c) and 5 (d), the rate of multiple drug release of micelles can be controlled by the oxidation-reduction reaction of cysteine blocks. Example 12 and FIG. 12 (a) show the results of releasing multiple drugs after micelles were stabilized by the oxidation reaction of cysteine blocks.

Another strategy is to use a hydrophilic EBP block to chemically bond the drug to the cysteine block and stabilize the structure by using a photo-crosslinking linker. 6 (a) and 6 (b), methacrylate (MA) was introduced into the charge (hydrophilic) drug. Methacrylate is an acrylate capable of reacting with a thiol group of a cysteine block. Thus, since the MA-conjugated drug can chemically bind to the thiol group of the cysteine block, the drug can be captured in the shell portion and control drug release. Example 13 and Fig. 12 (b) show the result that an acrylate-bound hydrophilic drug is bound to a cysteine block and released. 6 (c), an acrylic moiety is introduced by conjugating a carboxyl group of a hydrophilic EBP block with glycidyl methacrylate (GMA) to form an ultraviolet The micelle structure was chemically stabilized through chemical bonding of acrylic groups by ultraviolet irradiation.

As the micelles were formed, the thiol groups in the cysteine blocks were conjugated with drugs, and the hydrophobic core contained other hydrophobic drugs. After the formation of the micelle, the carboxyl group of the hydrophilic EBP block was modified by connecting it with the photo-crosslinking linker, and the structure was stabilized by crosslinking the hydrophilic EBP block with UV. For the dual dye release test as a drug model, the three fluorescent dyes were chemically conjugated or physically interacted with the multiblock copolymers. The present invention provides stabilized nanocarriers with high penetration ability and thermal reactivity for advanced drug delivery systems.

The drug delivery vehicle comprising the multiblock copolypeptide according to the present invention is a stabilized nanocarrier which has high intracellular penetration, is thermally responsive, and provides sequential delivery of multiple drugs.

Figure 1 is a schematic diagram of the construction of plasmids encoding EBP gene libraries with different DNA sizes and their block polypeptides. The modified vector was designed to contain the recognition sites of three different restriction enzymes (RE), including XbaI (RE1), AcuI (RE2), and BseRI (RE3). For example, EBPP [G ₁ A ₃ F ₂ ] ₁₂ Was constructed by a linkage reaction between the plasmid-based gene vector for EBPP [G ₁ A ₃ F ₂ ] ₆ and the insert: a plasmid-borne gene for EBPP [G ₁ A ₃ F ₂ ] ₆ ) Were prepared by double digestion with XbaI and AcuI. On the other hand, the plasmid-based gene vector for EBPP [G ₁ A ₃ F ₂ ] ₆ was prepared by double digestion with XbaI and BseRI and dephosphorylation with alkaline phosphatase.
2 is an agarose gel electrophoresis image of the EBP gene library used in the present invention. _{(A) EBPE [A 1 G} 4 I 1], (B) EBPP [A 1 G 4 I 1], (C) EBPE [K 1 G 4 I 1], (D) EBPP [K 1 G 4 I 1 ], (E) EBPE [D 1 G 4 I 1], (F) EBPP [D 1 G 4 I 1], (G) EBPE [E 1 G 4 I 1], (H) EBPP [E 1 G 4 I ₁ ], EBPP [G ₁ A ₃ F ₂ ], (J) EBPP [K ₁ A ₃ F ₂ ], (K) EBPP [D ₁ A ₃ F ₂ ], and ₃ A ₂ I ₁ ]. The number of EBP repeating units was indicated below each DNA band. The bilateral lanes on all agarose gels represent different DNA size markers (from bottom to top 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 1.0, 1.5, 2.0, and 3.0 kbp).
Figure 3 is a copper stained SDS-PAGE gel (4-20% gradient) of EBP used in the present invention. _{(A) EBPE [A 1 G} 4 I 1], (B) EBPP [A 1 G 4 I 1], (C) EBPE [K 1 G 4 I 1], (D) EBPP [K 1 G 4 I 1 ], (E) EBPE [D ₁ G ₄ I ₁ ] and (F) EBPP [D ₁ G ₄ I ₁ ]. Two-sided lanes on SDS-PAGE gels contain standard protein size markers (7, 15, 24, 35, 40, 50, 65, 90, 110, 150 kDa from top to bottom).
4 is a thermal profile of the EBPs used in the present invention. _{(A) EBPE [A 1 G} 4 I 1] n, (B) EBPP [A 1 G 4 I 1] n, (C) EBPE [K 1 G 4 I 1] n, (D) EBPP [K 1 G ₄ I ₁ ] _n , (E) EBPP [D ₁ G ₄ I ₁ ] _n And (F) EBPP [G ₁ A ₃ F ₂ ] _n . The optical absorbance at 350 nm of the thermal profile was obtained by heating 25 [mu] M EBP solution in PBS buffer supplemented with 1-3 M NaCl at a heating rate of 1 [deg.] C / min.
Figure 5 is a schematic diagram of the molecular structure, design, and function of multiblock copolypeptides, according to one embodiment of the present invention. (a) Fusion proteins consist of a hydrophobic EBP block, a hydrophilic EBPP block, a cysteine block and a CPP. (b) Thiol groups in cysteine blocks form disulfide bonds, which chemically stabilize the structure when the micelles are self-assembled by thermal stimulation. (c) multiblock copolypeptides can capture hydrophobic drugs in the core, capture charge drugs in the shell, and cysteine blocks form disulfide bonds to stabilize the micelle structure. (d) After the disulfide bond is cleaved under reducing conditions, the double drug in the micelle is released.
Figure 6 is a schematic diagram of the molecular structure, design, and function of multiblock copolypeptides, according to another embodiment of the present invention. (a) Fusion proteins consist of a hydrophobic EBP block, a hydrophilic EBP block, a cysteine block and a CPP. (B) Thiol groups present in cysteine blocks can be conjugated to methacrylic acid-based drugs, and hydrophobic cores can capture hydrophobic drugs. (c) (i) The carboxyl groups of the hydrophilic EBP block are conjugated to glycidyl methacrylate, which helps stabilize the micelles by UV crosslinking in hydrophilic EBP blocks. (ii) Once the micelles are self-assembled by thermal stimulation, the methacrylate in the hydrophilic EBP block is crosslinked via UV exposure.
Figure 7 is a graph showing the results of a comparison of (a) pennnetatin- (GAC) 4-EBPP [E ₁ G ₄ I ₁ ] ₁₂ -EBPP [G ₁ A ₃ F ₂ ] ₁₂ , _{[E 1 G 4 I 1]} 12 -EBPP [G 1 A 3 F 2] 24, (c) SynB1- (GAC) 4-EBPP [E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F 2 ] _12, and (d) SynB1- (GAC) 4 -EBPP [E 1 G 4 I 1] 12 -EBPP [G 1 a 3 F 2] agarose gel (1%) in (a) of the agar and ₂₄ ( B) SDS-PAGE image (4-20% gradient gel). (A) The modified pET21-a (+) plasmid encoding the multiblock copolypeptide was cleaved with Xba I and BamH I. The length of the gene encoding the fusion proteins was indicated at the top of the gene fragment. The size of each DNA fragment differs from that in Table 4 because there are restriction enzyme cleavage sites outside the gene encoding the fusion proteins. (B) Fusion proteins were expressed in E. coli and purified by ITC. The 4-20% gradient gel was visualized by copper staining. The expected molecular weight is indicated at the top of the band.
Figure 8 is a turbidity profile of a multiblock copolypeptide of the invention. (A) Turbidity profile for the following blocks (25 uM each); _{(a) EBPP [E 1 G} 4 I 1] 12 -EBPP [G 1 A 3 F 2] 12, (b) (GAC) 4-EBPP [E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F _{2] 12,} (c) Fe net Latin - (GAC) 4-EBPP [ E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F 2] 12, and (d) SynB1- (GAC) 4- EBPP [E ₁ G ₄ I ₁ ] ₁₂ -EBPP [G ₁ A ₃ F ₂ ] ₁₂ . (B) turbidity profiles for the following blocks (25 uM each); _{(a) EBPP [E 1 G} 4 I 1] 12 -EBPP [G 1 A 3 F 2] 24, (b) (GAC) 4-EBPP [E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F _{2] 24,} (c) Fe net Latin - (GAC) 4-EBPP [ E 1 G 4 I 1] 24 -EBPP [G 1 A 3 F 2] 24, and (d) SynB1- (GAC) 4- EBPP [E ₁ G ₄ I ₁ ] ₁₂ -EBPP [G ₁ A ₃ F ₂ ] ₂₄ . The absorbance was measured at 350 nm while heating the sample at a heating rate of 1 / min. The phase transition occurred twice. The primary phase transition occurs due to the aggregation of the hydrophobic block, and the secondary phase transition is generated under the influence of the polarity block.
Figure 9 (a) on page net Latin - (GAC) 4-EBPP [ E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F 2] 12, (b) Fe net Latin - (GAC) 4-EBPP _{[E 1 G 4 I 1]} 12 -EBPP [G 1 A 3 F 2] 24, (c) SynB1- (GAC) 4-EBPP [E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F 2 ] _12, and (d) SynB1- (GAC) 4 -EBPP [E 1 G 4 I 1] 12 -EBPP [G 1 a 3 F 2] hydrodynamic radius (hydrodynamic radius, Rh) of ₂₄ is measured by a DLS mechanism . The hydrodynamic radius of the multiblock copolypeptide was measured at a concentration of 25 uM in 10 mM PBS. The hydrodynamic radius of multiblock copolypeptides before primary phase transfer was about 10 to 20 nm.
Figure 10 shows the hydrodynamic radius of pennantatin- (GAC) 4-EBPP [E ₁ G ₄ I ₁ ] ₁₂ -EBPP [G ₁ A ₃ F ₂ ] ₂₄ after GMA joining and UV crosslinking at 4 and 37 ° C radius, Rh). Once the multiblock copolypeptide forms a micelle structure, the hydrophilic block is conjugated to GMA and crosslinked by UV exposure. Radius was measured at a concentration of 25 uM in 10 mM PBS.
Fig. 11 shows the results of SynB1- (GAC) 4-EBPP [E ₁ G _4] at 4 ° C and 37 ° C before and after TCEP treatment (B), after syringe filtration (C), and after hydrogen peroxide treatment (D) I ₁ ] ₁₂ -EBPP [G ₁ A ₃ F ₂ ] ₂₄ (hydrodynamic radius, Rh). SynB1- (GAC) 4-EBPP [ E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F 2] 24 has been treated with TCEP in order to reduce the thiol group, a hydrogen peroxide treatment for the disulfide bond in cysteine block It was filtered with a syringe before. Radius was measured at a concentration of 25 uM in 10 mM PBS.
12 is a graph showing the results of (A) SynB1- (GAC) 4-EBPP [E ₁ G ₄ I ₁ ] ₁₂ -EBPP [G ₁ A ₃ F ₂ ] ₂₄ micelles stabilized through disulfide bonds by oxidation of cysteine blocks (GAC) 4-EBPP [E ₁ < / _RTI > (EAC) of the fluorinated sodium salt embedded in the hydrophobic EBP block and the acrylated rhodamine B chemically bonded to the cysteine block. G ₄ I ₁ ] ₁₂ -EBPP [G ₁ A ₃ F ₂ ] ₂₄ micelles. In micelles, the double dyes were released in PBS (pH 7.4). Partial samples were taken from dialysis buffer at 20 minutes, 40 minutes, 1 hour, 1 hour 20 minutes, 1 hour 40 minutes, 2 hours, 6 hours, 10 hours, and 12 hours after the start of release.

Example  1: Material

The pET-21a (+) vector and BL21 (DE3) E. coli cells were obtained from Novagen Inc. (Madison, Wis., USA) and Top10 cells were purchased from Invitrogen (Carlsbad, CA, USA). All custom oligonucleotides were synthesized at Cosmo Gene Tech (Seoul, Korea). Calf intestinal alkaline phosphatase (CIP), BamHI and XbaI were obtained from Fermentas (Ontario, Canada). AcuI and BseRI were purchased from New England Biolabs (Ipswich, Mass., USA). T4 DNA ligase was obtained from Elpis Bio-tech (Daejeon, Korea). DNA miniprep, gel extraction, and PCR purification kits were obtained from Geneall Biotechnology (Seoul, Korea). 'Dyne Agarose High' was obtained from DYNE BIO, Inc. (Seongnam, Korea). All Top 10 cell cultures were performed in 'TB DRY' culture medium from MO BIO Laboratories, Inc. (Carlsbad, CA, USA). All BL21 (DE3) cell cultures were performed in 'Circle Grow' culture medium obtained from MP Biomedicals (Solon, Ohio, USA). 'Ready Gels, Tris-HCl 2 - 20% precast gels' were purchased from Bio-Rad (Erkulles, California, USA). Phosphate buffered saline (PBS, pH 7.4), ampicillin, polyethyleneamine (PEI), rhodamine 6G, rhodamine B, fluorescein sodium salt, Sodium iodide, glycidyl methacrylate (GMA), and hydrogen peroxide were obtained from Sigma-Aldrich (St. Louis, Missouri, USA).

Example  2: Different EBP Blocks and  These block To the polypeptide  Notation for

Different EBP having the penta-peptide repeating units Val-Pro- (Gly or Ala) -Gly _aa -X are named as follows: _Xaa may be any amino acid except Pro. First, Val-Pro-Ala-X aa repeat of the penta-peptide -Gly with plasticity elastin-based polyester that has plasticity peptides: is defined as (elastin-based polypeptide with plasticity EBPP ). The Val-Pro-Gly-X _aa repeat of the penta-peptide are -Gly-based elastin polypeptides that elasticity: referred to as (the elastin-based polypeptide with elasticity EBPE). Second, in [X _i Y _j Z _k ] _n , the upper case letters in the parentheses are the amino acid code of the single letter of the guest residue, ie, the amino acid at the 4th position (X _aa ) of the EBP pentapeptide, And the ratio of the guest residue of the EBP monomer gene as a repeating unit. The subscript _n of [X _i Y _j Z _k ] _n represents the total number of repetitions of EBP. For example, EBPP [G ₁ A ₃ F ₂ ] ₁₂ is an EBPP block consisting of 12 Val-Pro-Ala-X _aa- Gly pentapeptide units repeatedly, wherein the fourth guest residue position (X _aa ) The ratio of Gly, Ala, and Phe is 1: 3: 2. Finally, multiblock polypeptides, such as CPP block-cysteine block-hydrophilic EBP block-hydrophobic EBP block, are named by the composition of each block of angle brackets, with a hyphen between the blocks.

Example  3: Seamless gene Cloning  Modified for pET Preparation of -21a vector

4 μg of pET-21a vector was digested and dephosphorylated with 50 U of XbaI, 50 U of BamHI, 10 U of thermosensitive alkaline phosphatase in FastDigest buffer for 20 minutes at 37 ° C. Restricted plasmid DNA was purified using a PCR purification kit and then eluted in 40 μL of distilled deionized water. (SEQ ID NO: 41) (5'-ctagaaataattttgtttaactttaagaaggaggagtacatatgggctactgataatgatcttcag-3 ') and SEQ ID NO: 42 (5'-gatcctgaagatcattatcagtagcccatatgtactcctccttcttaaagttaaacaaaattattt-3') having an adhesive end compatible with XbaI and BamHI were designed. Two oligonucleotides were annealed by heating 50 μL of 2 μM oligonucleotide concentration in T4 DNA ligase buffer at 95 ° C. for 2 minutes and the solution was slowly cooled to room temperature over 3 hours. By incubating 20 pmol of annealed dsDNA and 0.1 pmol of the linearized vector at 37 DEG C for 30 minutes in T4 DNA ligase buffer with 1 U of T4 DNA ligase, the DNA inserts to multiple replication sites in the pET-21a vector The ligation was performed. Modified pET-21a (mpET-21a) vectors for seamless gene cloning and expression were transformed into recipient cells of Top 10 and were then transformed into super-optimal broth (SOC) supplemented with 50 μg / ml of ampicillin with catabolite repression. The DNA sequence of the mpET-21a vector was then verified by fluorescent dye terminator DNA sequencing (Applied Biosystems Automatic DNA Sequencer ABI 3730).

Example  4: EBP  Synthesis of gene units and their objections Oligomerization

Fourth residues were each other to optimize the T _t of Val-Pro- (Gly or Ala) penta peptide repeating units being variable for EBP having the sequence -X _aa -Gly designed at the DNA level to be less than the physiological temperature a different molar ratio. For 17 EBP libraries, the DNA and amino acid sequences of EBPs with various pentapeptide repeat units are shown in Tables 1 and 2, respectively.

[Table 1]

The gene sequence of the EBP library. Plastic EBP (EBPP) with pentapeptide repeats of Val-Pro-Ala-X _aa- Gly; (EBPE) with pentapeptide repeats of Val-Pro-Gly-X _aa -Gly were all cloned to have the same guest residue composition and ratio

[Table 2]

The amino acid sequence of the EBP library

In Table 1, SEQ ID NOS: 3-10 can be classified into a gene sequence for a hydrophilic EBP block, and SEQ ID NOS: 11-19 can be classified into a gene sequence for an EBP block containing Phe, His. In Table 2, amino acid sequence numbers 20-27 may be classified as hydrophilic, and amino acid sequence numbers 28-36 in which Phe and His are included may be classified as hydrophobic EBP blocks. In particular, in Table 2, SEQ ID NOS: 22 and 23 are classified into positively charged hydrophilic EBP blocks, and SEQ ID NOS: 24 to 27 are classified into negatively charged hydrophilic EBP blocks. That is, as described above, when the LCST of EBP is lower than the body temperature, it is hydrophobic. When the LCST of EBP is higher than the body temperature, it is hydrophilic. Due to this nature of EBP, the hydrophilic and hydrophobic properties of EBP can be relatively defined when applied to biotechnology.

Temperature and pH to have a unique stimulus reactive pentapeptide repeating unit, including Val-Pro- (Gly or Ala) -Gly _aa -X different EBP having [wherein X _aa is which may be any amino acid except Pro] Were designed at the DNA level. _{Val-Pro-Ala-X aa} -Gly the penta EBPP with a plastic having a peptide repeat (EBP with plasticity: EBPP) and, in the elastic EBPE having _{Val-Pro-Gly-X aa} -Gly the pentapeptide repeating ( EBP with elasticity: EBPE) were duplicated with the same guest residue composition and ratio. Tables 1 and 2 show the gene and amino acid sequence of different EBPs each having a pentapeptide repeating unit. For example, EBPE [G ₁ A ₃ F ₂ ] ₁₂ and EBPP [G ₁ A ₃ F ₂ ] ₁₂ have substantially the same molar mass, as well as the same combination of the fourth residues of the EBP pentapeptide repeating unit . And different third amino acid residues (Ala or Gly) of the pentapeptide repeating unit. EBPs with positive and negative charges were prepared by introducing charged amino acids such as Lys, Asp, Glu, and His into guest residues.

For each pair of oligonucleotides encoding various EBPs, 50 μL of each of the two oligonucleotides (tailored to a concentration of 2 μM each) was heated in T4 DNA ligase buffer at 95 ° C. for 2 minutes, Next, the reaction solution was annealed by slowly cooling at room temperature for 3 hours. A total of 4 μg of the modified mpET-21a replication vector was digested and dephosphorylated with 15 U of BseRI and 10 U of FastAP Alkaline Phosphatase for 30 min at 37 ° C. Restriction plasmid DNA was purified using a PCR purification kit and then eluted in 40 μL of distilled deionized water. By incubating 90 pmol of annealed dsDNA and 30 pmol of the linearized mpET-21a replication vector at 16 DEG C for 30 min in T4 DNA ligase buffer with 1 U of T4 DNA ligase, The DNA insert was ligated. The ligated plasmids were transformed into Top10 chemically accommodating cells, which were then applied onto SOC plates supplemented with 50 μg / ml of ampicillin. The DNA sequence was then confirmed by DNA sequencing. After constructing all EBP monomeric genes, each EBP gene was synthesized by ligating the same 36 repeat inserts into the corresponding vector containing 36 repetitive genes as follows. The replication procedure for EBP libraries and their convergence are schematically illustrated in FIG. A vector containing a copy of the gene for the monomeric EBP was digested and dephosphorylated with 10 U of XbaI, 15 U of BseRI, and 10 U of FastAP thermophilic alkaline phosphatase in Cutsmart buffer at 37 C for 30 minutes. Restricted plasmid DNA was purified using a PCR purification kit and then eluted in 40 μL of distilled deionized water. A total of 4 μg of EBP monomer genes were digested with 10 U of XbaI and 15 U of AcuI in Cutsmart buffer at 37 ° C. for 30 minutes to produce the insert part. After digestion, the reaction products were separated by electrophoresis on agarose gels and the inserts were purified using a gel extraction kit. The ligation reaction was performed by incubating 90 pmol of the purified insert with 30 pmol of the linearized vector with 1 U of T4 DNA ligase at 16 DEG C for 30 minutes in T4 DNA ligase buffer. The products were transformed into Top 10 chemically accommodating cells, which were then plated on SOC plates supplemented with 50 μg / ml of ampicillin. The transformants were initially selected on a agarose gel by a diagnostic restriction digest and further confirmed by DNA sequencing as described above.

As previously described, EBP gene libraries of different DNA sizes were synthesized using three different restriction endonucleases with the designed plasmid vectors. Figure 1 illustrates a recursive directional ligation (RDL) method in which EBP monomer genes are ligated to form oligomerized EBP genes. For example, EBPP [G ₁ A ₃ F ₂ ] ₁₂ Was constructed by a linkage reaction between the plasmid-based gene vector for EBPP [G ₁ A ₃ F ₂ ] ₆ and the insert: a plasmid-borne gene for EBPP [G ₁ A ₃ F ₂ ] ₆ ) Were prepared by double digestion with XbaI and AcuI. On the other hand, the plasmid-based gene vector for EBPP [G ₁ A ₃ F ₂ ] ₆ was prepared by double digestion with XbaI and BseRI and dephosphorylation with alkaline phosphatase. This RDL method using two different double restriction enzymes has several advantages. First, due to the different shapes of the protrusions of both the insert and the vector, the self-ligating reaction of the restriction vector did not occur and they were efficiently linked to the head-tail orientation. Second, it has no additional DNA sequence to encode the linker between each block due to the mechanism of type III restriction endonuclease. Each EBP gene was oligomerized to generate 36, 72, 108, 144, 180, and 216 EBP pentapeptide repeats. Using two restriction endonucleases Xba I and BamH I, oligomerized gene sizes with 540, 1080, 1620, 2160, 2700, and 3240 base pairs (bps) were determined. As characterized by agarose gel electrophoresis, Figure 2 shows the limited DNA bands of EBP libraries with DNA size markers on both terminal lanes. For example, EBPE [A ₁ G ₄ I ₁ ] of FIG. 2 (A) is a restriction DNA encoding an oligomerized pentapeptide sequence containing 1: 4: 1 ratio with Ala, Gly, Ile as guest residues It clearly shows the band. All of the restriction DNA bands are shown in their corresponding lengths as compared to the molecular size markers.

EBP genes and their block copolypeptides were cloned into E. coli strains carrying the T7 promoter And was purified by multiple cycles of ITC (inverse transition cycling). Figure 3 shows a copper stained SDS-PAGE gel image of purified EBP. EBPs migrated at least 20% greater than the theoretically calculated molecular weight. Two-sided lanes on SDS-PAGE gels contain standard protein size markers (7, 15, 24, 35, 40, 50, 65, 90, 110, and 150 kDa from bottom to top). EBPE [A ₁ G ₄ I ₁ ] and EBPP [A ₁ G ₄ I ₁ ] in Figures 3 (A) and 3 (B) represent a series of corresponding proteins with a molecular weight greater than the theoretical molecular weight (EBPE [A ₁ G ₄ I ₁ ] from left to right 14.0, 27.7, 41.3, 55.0, 68.6 kDa). In general, Figure 3 EBPE of (C) and _{3 (D) [K 1 G} 4 I 1] and _{EBPP [K 1 G 4 I 1} ] The EBP libraries positively charged including a can, EBPE [A ₁ G ₄ I ₁ ] and EBPP [A ₁ G ₄ I ₁ ]. In addition, negatively charged EBP libraries of EBPE [D ₁ G ₄ I ₁ ] and EBPP [D ₁ G ₄ I ₁ ] of Figures 3 (E) and 3 (F) Gt; EBP < / RTI > libraries.

The characteristics of the EBP library were analyzed. Figure 4 shows the thermal transfer behavior of EBPs by measuring the optical absorbance at ₃₅₀ nm (Absorbance ₃₅₀ ) at a heating rate of 1 캜 / min. The inverse transition temperature (T _t ) is defined as the temperature at which the first derivative ( d (OD ₃₅₀ ) / dT ) of the turbidity, which is a function of temperature, was the maximum. Depending on environmental conditions such as salt concentration and pH and different third and fourth amino acids of EBP pentapeptide repeating units, the T _t of EBP was finely controlled in PBS and PBS supplemented with 1-3 M NaCl. For example, EBPE [A ₁ G ₄ I ₁ ] ₁₂ (FIG. 4 (A)) with Gly at the third amino acid of the EBP pentapeptide repeat has a higher affinity for Gly than Ala at the third amino acid of the EBP pentapeptide repeat Which is higher than EBPP [A ₁ G ₄ I ₁ ] ₁₂ (FIG. 4 (B)) with Ala at the third amino acid of the EBP pentapeptide repeat in PBS with 1 M NaCl because of its high hydrophilicity. Generally, charged EBP libraries appear to have a higher T _t than nonpolar EBP libraries because they introduce charged residues into the 4 th amino acid of the EBP pentapeptide repeat. The negatively charged EBP libraries of EBPP [D ₁ G ₄ I ₁ (Figure 4 (E)) are characterized by different pKa values of Asp and Lys at the 4th amino acid of the EBP pentapeptide repeat, EBPE [K ₁ G Has higher T _t than the charged EBP libraries in the amount of ₄ I ₁ (Figure 4 (C)) and EBPP [K ₁ G ₄ I ₁ ] (Figure 4 (D)). For reference, in FIG. 4 (A), (B) , (C), (D) and (E) is a (F) indicates a hydrophilic _{EBPP [G 1 A 3 F 2} ] 12 and EBPP [G ₁ A ₃ F ₂ ] ₂₄ represents hydrophobicity.

Example  5: cell permeability Peptides ( CPP ) Gene production and cysteine Cys ) include block  making

The amino acid sequences of the pennetalin were derived from the homeodomain of Antennapedia (Drosophila homeoprotein) and SynB1 was derived from the porcine leukocytes with the antimicrobial peptide proteogrin-1 (PG -1) in the SynB family.

The amino acid sequence of the cysteine (Cys) block contained alanine (Alanine, Ala) and glycine (Gly) between repeated cysteines (Cys) and the amino acids were repeated four times, that is, (Gly- Cys) ₄ . Alanine and glycine were introduced to create a reversible intermolecular crosslinking of thiol groups in space and cysteine for drug conjugation. Genes encoding the cysteine-containing blocks, pennelatin, and the amino acid sequences of SynB1 are codons that contain two restriction enzyme recognition sites (AcuI and BseRI) at each end of the genes for seamless ligation (codon) optimization. Pairs of oligonucleotides of phenetratin, SynB1, and cysteine-containing blocks were chemically synthesized in Cosmo Genetech (Seoul, Korea) and bound to double stranded DNA (dsDNA) fragments cleaved with AcuI and BseRI.

Example  6: CPP block  And Cys Block  Include multiple block Copolypeptide  design

Block polypep- tides based on diblock EBPs (hydrophilic EBP-hydrophobic EBP) that are self-assembled into micelles along with cysteine (Cys) blocks and CPP blocks were designed. At this time, CPP was introduced to permeate the cell membrane. Table 3 below shows the DNA and amino acid sequences of penetratin and SynB1 used as CPP blocks.

[Table 3] Genes and amino acid sequences of CPPs

 Table 4 shows the sequence and amino acid sequence of the multi-block EBPs. Micelle structures, including hydrophilic EBP blocks and cysteine blocks, can deliver dual drugs and can be stabilized. Multivalent CPPs located on the micelle surface can increase the internalization of the construct into cells. The self-assembled micelle structure provides drug delivery and structural stabilization of the hydrophilic EBP block on the shell and cysteine block.

[Table 4] Sequence, gene length and molecular weight of multi-block EBPs

(CPP) block-cysteine (Cys) block-charged hydrophilic EBP block-hydrophobic EBP block. At this time, CPP was introduced to permeate the cell membrane. Table 4 shows the sequence and amino acid sequence of the four multiblock copolypeptides. Micelle structures containing hydrophilic EBP blocks and cysteine blocks can deliver and stabilize dual drugs and multivalent CPP located on the micelle surface allows the structure to penetrate well into the cell. The self-assembled micelle structure is provided with drug delivery and structural stabilization through a hydrophilic EBP block on the shell and cysteine block.

 Figure 5 illustrates the first strategy of a micelle structure for dual drug delivery and structural stabilization. Hydrophilic EBP positively charged with carboxyl groups is physically bound to positively charged drugs. As multiblock copolypeptides form self-assembled micelle structures, other hydrophobic drugs are captured as micelle cores. After the two drugs are incorporated as micelles, thiol groups on cysteine blocks form disulfide bonds to stabilize micelles. The two drugs are then bound to each block in the micelle through different physical interactions (electrostatic interactions and hydrophobic interactions). The chargeable drug trapped in the shell portion and the hydrophobic drug trapped in the core portion may be released at different rates. That is, the release pattern of the two drugs may be different.

Figure 6 shows another strategy of the micelle structure. Micelles are stabilized by using a photocrosslinker that is conjugated to the carboxyl groups of the hydrophilic EBP. The thiol groups in the cysteine block are chemically conjugated to the drugs. Similar to the first strategy of FIG. 5 above, while the multiblock copolypeptide forms a self-assembled micelle structure, other hydrophobic drugs are captured into the micelle core. After both drugs are trapped in the micelle, the micelle structure is stabilized by the formation of photo-crosslinking. Both drugs bind to each block in the micelle through chemical bonding and physical interaction. The chargeable drug conjugated to the shell portion and the hydrophobic drug entrapped in the core portion may be released at different rates. That is, the release pattern of the two drugs may be different.

Example  7: CPP - Cys - Hydrophilic EBP - hydrophobic Of EBP  multiple block Of the copolypeptide  Gene construction

CPP double-stranded DNA fragments, Cys block double-stranded DNA fragments, and hydrophilic EBPs and hydrophobic EBPs were synthesized using CPP-Cys-EBPP [E ₁ A ₄ I ₁ ] ₁₂ -EBPP [G ₁ A ₃ F ₂ ] _{n &lt}; / RTI &_gt; fusion polypeptide library. The plasmid vector encoding EBPP [G ₁ A ₃ F ₂ ] _n was digested with 10 U of XbaI, 15 U of BseRI and 10 U of FastAP (a type of temperature sensitive alkaline phosphatase) in CutSmart buffer at 37 ° C And cleaved and dephosphorylated for 30 minutes. The digested plasmid DNA was purified with a PCR purification kit and then extracted with 40 μL of distilled and deionized water. To prepare the EBPP [E ₁ A ₄ I ₁ ] ₁₂ gene as an insert, a total of 4 μg of EBPP [E ₁ A ₄ I ₁ ] ₁₂ gene was added to 10 U of XbaI and 15 U of AcuI in CutSmart buffer Gt; 37 C < / RTI > for 30 minutes. After cleavage, the reaction product was separated by agarose gel electrophoresis, and a gel extraction kit was used to purify the insert. The ligation was performed by incubating 90 pmol of the purified insert and 30 pmol of the linearized vector with 1 U of T4 DNA ligase in T4 DNA ligase buffer for 30 minutes at 16 ° C. The resulting product was introduced into Top10 recipient cells (made by chemical methods) and then plated onto SOC plate media (supplemented with 50 μg / ml of ampicillin). Transformants were first verified by identifying restriction enzyme cleaved fragments on agarose gels and then further validated by DNA sequencing as mentioned above.

Hydrophilic EBP- die block of hydrophobic _{EBP "EBPP [E 1 A 4} I 1] 12 -EBPP [G 1 A 3 F 2] n" is a plasmid vector coding for the 15 U and 10 U BseRI in CutSmart buffer FastAP ( Gt; 37 C < / RTI > for 30 min. The cleaved plasmid DNA was purified with a PCR purification kit and then extracted with 40 μL of distilled and deionized water. The ligation was performed by incubating the cysteine block double stranded DNA fragments and the linearized vector. Cloning was confirmed by DNA sequencing as mentioned above. The plasmid vector encoding the triblock "cysteine block-EBPP [E ₁ A ₄ I ₁ ] ₁₂ -EBPP [G ₁ A ₃ F ₂ ] _n " was prepared as a linearized vector, and the CPP double strand DNA fragments. In addition, the cloning was confirmed by DNA sequencing as mentioned above.

Example 8: Multiple block Of the copolypeptide  Gene expression and purification

g, 4℃, 10 분)로 모아진 후, 20 ml의 10 mM PBS로 재부유 되었고, 그 다음으로 초음파 처리(얼음에서, 20초 간격으로 10 초간 초음파를 부가하는 조건으로 하여, 전체 5 분간 초음파 처리함)를 통해서 용해되었다. 상기 세포 용해물에서 불용성 세포 잔해를 제거하기 위해 원심분리(16,000 rpm, 4℃, 15 분)를 실시했다. 핵산은 폴리에틸렌아민(polyethylenimine)(0.5% w/v) 침전 및 원심분리(16,000 rpm, 4℃, 15 분)로 제거되었다. 그 다음 EBP로 이루어진 다중 블럭 폴리펩타이드들는 ITC로 정제되었다. EBP는 염화나트륨(3-5 M)을 첨가하고 40℃로 가열함으로써 응집되었다. 상기 응집된 단백질은 원심분리(16,000 rpm, 4℃, 15 분)를 통해서 침전물로 가라앉혀졌다. 가용성 불순물을 포함한 상층액은 버렸고, 상기 단백질 침전물은 4℃에서 10 mM PBS로 완전히 재부유 되었다. 낮은 온도에서의 불용성 불순물은 저온 조건에서 원심분리(16,000 rpm, 4℃, 15 분)함으로써 제거되었다. EBPP를 기반으로 한 단백질은 아래의 과정을 반복함으로써 정제되었다; 37℃에서 염화나트륨과 함께 원심분리 후 새로운 PBS로 재부유 그리고 4℃ 원심분리. 37℃ 원심분리 후 매 PBS 첨가 단계에서, 단백질 농도를 증가시키기 위해서 더해진 새로운 PBS의 양을 줄였다.The multiblock EBPs in Table 4 were overexpressed in E. coli by IPTG induction and purified by inverse transition cycling (ITC). All plasmids containing multiblock EBP genes were addressed in Top 10 E. coli during cloning. For expression, plasmids were introduced into BL21 (DE3) Escherichia coli cells prepared by a chemical method with heat shock, and Escherichia coli was plated on SOC plate medium and incubated overnight at 37 ° C. One colony of the transformants was shaked at 100 rpm at 37 ° C overnight in CircleGrow culture medium (supplemented with 50 μg / ml ampicillin). 4 ml of the primary culture medium was added to 500 ml of CircleGrow culture medium (supplemented with 50 ug / ml ampicillin) contained in a 2 L flask and cultured at 37 ° C with shaking at 180 rpm until the value of OD ₆₀₀ reached 0.8 . Protein expression was induced by IPTG (isopropyl beta-thiogalactopyranoside) induction (final concentration of 1 mM). After induction of IPTG, cultures were shaken at 180 rpm overnight at 37 ° C, and the cultured cells were centrifuged (3000

g, 4 캜, 10 minutes), and then resuspended in 20 ml of 10 mM PBS. Then, ultrasonication was performed under the condition of adding ultrasonic waves for 10 seconds at intervals of 20 seconds on ice, Lt; / RTI > The cell lysate was centrifuged (16,000 rpm, 4 ° C, 15 minutes) to remove insoluble cell debris. Nucleic acid was removed by precipitation with polyethylenimine (0.5% w / v) and centrifugation (16,000 rpm, 4 ° C, 15 min). The multiblock polypeptides consisting of EBP were then purified by ITC. EBP was aggregated by adding sodium chloride (3-5 M) and heating to 40 占 폚. The aggregated protein was submerged in the precipitate through centrifugation (16,000 rpm, 4 캜, 15 min). The supernatant containing soluble impurities was discarded and the protein precipitate was completely resuspended in 10 mM PBS at 4 < 0 > C. Insoluble impurities at low temperature were removed by centrifugation (16,000 rpm, 4 캜, 15 min) at low temperature. EBPP-based proteins were purified by repeating the following steps; Centrifuge with sodium chloride at 37 ° C, resuspend in fresh PBS and centrifuge at 4 ° C. At the time of every PBS addition after centrifugation at 37 ° C, the amount of new PBS added to increase the protein concentration was reduced.

Example  9: Multiple block Of the copolypeptide  Characterization

The expression and impurity of multiblock copolypeptides were confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and their LCST was confirmed by measuring the turbidity with temperature. In addition, dynamic light scattering (DLS) was measured to confirm that multiblock copolypeptides self-assemble into micellar structures depending on temperature. The concentration of the polypeptide solution was adjusted by dilution of the stock solution wherein the concentration was adjusted to a Tyr (1280 M ^{- 1} cm ^-1 ) of pET-21a (+) modified at 280 nm and two Trp ^- using the molar extinction gyesueul (5690 M ^-1 cm ¹⁾ and ^{SynB1 Tyr (1280 M -1 cm -1} ) was measured with a spectrophotometric method. SDS-PAGE gels were 4-20% gradient gels and visualized with copper staining. LCST and DLS were measured as a function of temperature with a heating rate of 1 [deg.] C / min in 10 mM PBS. The OD ₃₅₀ of multiblock copolypeptide and DLS was measured. The transition temperature is the temperature corresponding to the inflection point on the OD ₃₅₀ graph.

The genes of the multiblock copolypeptides were prepared by molecular cloning, digested with Xba I and BamH I and confirmed by agarose gel electrophoresis. Figure 7 (A) is a multi-block co shows the gene fragment of the polypeptide, page net Latin - (GAC) 4-EBPP [ E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F 2] 12, page net Latin - (GAC) 4-EBPP [ E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F 2] 24, SynB1- (GAC) 4-EBPP [E 1 G 4 I 1] 12 -EBPP [ _{G 1 a 3 F 2] 12} , and SynB1- (GAC) 4-EBPP [ E 1 G 4 I 1] 12 -EBPP [G 1 a 3 F 2] the length (2247, 3327, 2253 of the gene encoding the ₂₄ , And 3333 bp) are indicated on the gene fragment. The values indicated on the agarose gel are 66 bp larger than the values in Table 4 because restriction sites of the restriction enzyme are located on both sides of the genes encoding the multiblock copolypeptide. Figure 7 (B) shows a multiblock copolypeptide expressed in E. coli, purified by ITC, and characterized by SDS-PAGE. 7 (B), copper-stained SDS-PAGE (4-20% gradient) shows that the multiblock copolypeptide was completely purified by ITC. Fe net Latin - (GAC) 4-EBPP [ E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F 2] 12, page net Latin - (GAC) 4-EBPP [ E 1 G 4 I 1] 12 _{-EBPP [G 1 A 3 F 2} ] 24, SynB1- (GAC) 4-EBPP [E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F 2] 12, and SynB1- (GAC) 4-EBPP _{[E 1 G 4 I 1]} 12 -EBPP [G 1 a 3 F 2] expected molecular weight (62.7, 107.0, 62.6, and 106.85 kDa) of ₂₄ was displayed on the band. The multiblock copolypeptide bands appeared at a higher position compared to the expected molecular weight because the polypeptide-based EBP shifted about 20% from the theoretical molecular weight, which is in good agreement with previous studies.

The thermal behavior of multiblock copolypeptides was characterized by a turbidity profile. Turbidity profiles were obtained by measuring the absorbance at 350 nm while heating a 25 μM protein solution in 10 mM PBS at a heating rate of 1 ° C / min. Figure 8 shows LCST plots of diblock EBPs, diblock EBPs with cysteine blocks, and multiblock copolypeptides, and Table 5 shows the total ionization.

[Table 5] Transition temperature

The transition temperature (T _t ) in Table 5 is the inflection point of the LCST graphs and is calculated from the LCST graphs of FIG. The inflection point was calculated from the graph of absorbance and DLS at 350 nm. The primary T _t between UV and DLS is different because low levels of EBP aggregation do not show turbulence in solution, but DLS measures aggregation. Thus, the transition temperature from the DLS is lower than the transition temperature from the absorbance. Phase transitions occur twice. The first transition is the hydrophobic EBP block, EBPP [G ₁ A ₃ F ₂ ] ₁₂ and EBPP [G ₁ A ₃ F ₂ ] ₂₄ . (GAC) 4-EBPP [E ₁ G ₄ I ₁ ] ₁₂ -EBPP [G ₁ A ₃ F ₂ ] ₂₄ and SynB1- (GAC) 4-EBPP with long hydrophobic EBPP blocks EBPP [E ₁ G ₄ I ₁ ] ₁₂ -EBPP [G ₁ A ₃ F ₂ ] ₂₄ (31.02 and 31.37 ° C) is a multiblock copolypeptide with short hydrophobic EBPP blocks _{EBPP [E 1 G 4 I 1} ] 12 -EBPP [G 1 A 3 F 2] 12 and SynB1- (GAC) 4-EBPP [ E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F 2] 12 ) has a lower than T _t (38.57 and 40.61). EBPP hydrophilic _{block, EBPP [E 1 G 4 I} 1] Since the secondary transfer is caused by _12, aggregation of, Fe net Latin - (GAC) 4-EBPP [ E 1 G 4 I 1] 12 -EBPP [G 1 A ₃ F _{2] 12,} page net Latin - (GAC) 4-EBPP [ E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F 2] 24, SynB1- (GAC) 4-EBPP [E 1 G ₄ I ₁ ] ₁₂ -EBPP [G ₁ A ₃ F ₂ ] ₁₂ and SynB1- (GAC) 4-EBPP [E ₁ G ₄ I ₁ ] ₁₂ -EBPP [G ₁ A ₃ F ₂ ] ₂₄ (83.55, 82.97 , 52.57, and 84.37 kDa) have similar secondary transition temperatures. The primary transition temperature indicates that the cysteine blocks are hydrophilic and are multiblock EBPPs with hydrophilic cysteine blocks (GAC) 4-EBPP [E ₁ G ₄ I ₁ ] ₁₂ -EBPP [G ₁ A ₃ F ₂ ] ₁₂ and _{GAC) 4-EBPP [E 1} G 4 I 1] 12 -EBPP [G 1 a 3 F 2] the primary transition temperature of the die block _{24 EBPPs (EBPP [E 1 G} 4 I 1] 12 -EBPP [G 1 a ₃ F ₂ ] ₂₄ and EBPP [E ₁ G ₄ I ₁ ] ₁₂ -EBPP [G ₁ A ₃ F ₂ ] ₂₄ ) (ΔT _t is 2.25 and 3.25). (GAC) 4-EBPP [E ₁ G ₄ I ₁ ] ₁₂ -EBPP [G ₁ A ₃ F ₂ ] ₁₂ and penninalatin- (GAC) 4 -EBPP [E ₁ G ₄ I ₁ ] ₁₂ -EBPP [G ₁ A ₃ F ₂ ] ₂₄ ) was 2.7 and 1.35 lower than that of multiblock EBPPs with cysteine blocks. Also, SynB1 reduces the primary transition point of the multi-block EBPPs, multi-block co-polypeptide (SynB1- (GAC) having a _{SynB1 4-EBPP [E 1 G} 4 I 1] 12 -EBPP [G 1 A 3 F 2 ] ₁₂ and SynB1- (GAC) 4-EBPP [ E 1 G 4 it 1] 12 -EBPP [G 1 a 3 F 2] transition temperature of ₂₄₎ was lower than the multi-block EBPPs having a cysteine block (ΔT _t 0.66 And 1.00). Although phennetatin and SynB1 have positively charged amino acids, hydrophobicity for interaction with cell membranes has a greater effect. Because of this hydrophobicity, the secondary transition temperature of the multiblock copolypeptide with CPP was lower than that of the triple block copolypeptide with cysteine blocks.

The formation and size of the self-assembled micelles were confirmed by dynamic light scattering (DLS), in which samples were heated at a heating rate of 1 ° C / min from 20 ° C to 60 ° C, eleven times at each temperature (Fig. 9). The hydrodynamic radius (Rh) of the multi-block EBPPs was measured in 25 μM protein solution in 10 mM PBS. Table 5 above shows the absorbance at 350 nm and the transition temperatures of the multi-block EBPP calculated from DLS. According to the DLS data, the radius of the fusion protein prior to the primary transfer is about 10 nm, indicating that the protein is a soluble monomer. As the temperature is increased, multiple block EBPPs (Petinalatin- (GAC) 4-EBPP [E ₁ G ₄ I ₁ ] ₁₂ -EBPP [G ₁ A ₃ F ₂ ] ₁₂ , _{[E 1 G 4 I 1]} 12 -EBPP [G 1 A 3 F 2] 24, SynB1- (GAC) 4-EBPP [E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F 2] 12, and SynB1- (GAC) 4-EBPP [ E 1 G 4 I 1] 12 -EBPP [G 1 a 3 F 2] 24) hydrodynamic radius (hydrodynamic radius, Rh) is at 270 at around the temperature T _t of 600 nm (49.17, 46.77, 44.15, and 47.21 nm), respectively, after reaching a stable stage. This result means that the multiblock copolypeptide of the present invention formed a metastable nanostructure before forming stable micelles.

Example  10: Multiple block To the copolypeptide Glycidyl Methacrylate ( glycidyl  methacrylate), and its bonding Light bridging ( photocrosslinking ) And characterization

Glycidyl methacrylate (GMA) was conjugated to the multiblock copolypeptide to introduce the double bond used to stabilize the micelle by UV crosslinking. To the multiblock copolypeptide present at 1 w / v% in pH 3.5 deionized water, 70 umol of GMA was added and incubated at 4 [deg.] C for 24 hours with agitation. After the reaction, the GMA modified polypeptides were purified with ITC to remove unreacted GMA and resuspended in PBS. 6.25 uM of polypeptides modified with GMA were incubated at 37 DEG C to form micelles with 0.1% initiator and then exposed to UV for 10 minutes to crosslink the micelle structure formed . The hydrodynamic radius (Rh) of the micelles was measured by DLS at 4 and the structure retention at 37 ° C was confirmed.

Fe net Latin - (GAC) 4-EBPP [ E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F 2] introducing a methacrylic acid salt moiety (methacrylate moiety) to the carboxyl groups in the hydrophilic block of ₂₄ EBPPs , Multi-block EBPPs were conjugated with glycidyl methacrylate (GMA), and then when the multi-block EBPPs were formed into a micellar structure through UV exposure, the conjugation was crosslinked to stabilize the micelles . After UV exposure, the hydrodynamic radius of pennantatin- (GAC) 4-EBPP [E ₁ G ₄ I ₁ ] ₁₂ -EBPP [G ₁ A ₃ F ₂ ] ₂₄ present at 25 uM in 10 mM PBS radius, Rh) were measured at 4 캜 and 37 캜 (Fig. 10). The hydrodynamic radius (Rh) of the micelle was 123.3 nm at 4 ° C because the hydrophobic block of the hydrophobic core becomes hydrophilic at 4 ° C at 37 ° C and the micelle maintains the cross- It is water-soluble and larger than non-crosslinked micelles (46.77 nm). At 37 [deg.] C, a narrow peak at 46.57 nm and a broad peak at 401 nm were observed. The micelles at 123.3 nm at 4 ° C were not completely restored to the previous micelle structure and assembled into a random structure (wide 401 nm structure).

Example  11: Lee Sang Hwa  Disulfide bond formation and characterization

Once the micelle is formed, the four thiol groups in the cysteine block form a disulfide bond in the presence of an oxidizing agent, thereby crosslinking the shell. To separate the disulfide bonds, 100 uM of multiblock EBPPs at 4 占 폚 were treated with 500 uM of TCEP (reducing agent), and until the TCEP lost its reducing ability by self-oxidization, i.e., 72 hours Lt; / RTI > To remove debris from the polypeptide solution, the polypeptide solution was filtered through a 0.2 nm syringe filter. The filtered 25 μM polypeptide solution was incubated at 37 ° C. to form micelles in the presence of sodium iodide (0.25 uM) acting as a catalyst, and 250 μM of hydrogen peroxide was added to form disulfide bonds. After each step, the hydrodynamic radius (Rh) of the structure in solution was measured by DLS at 37 ° C and the retention of the micelle structure at 4 ° C was confirmed.

SynB1- (GAC) 4-EBPP [ E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F 2] a thiol group in the cysteine in the block ₂₄ are sikyeotgo stabilize the micelles to form a disulfide bond, said bond formation is DLS. Prior to forming the disulfide bond, SynB1- (GAC) 4-EBPP [E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F 2] was treated a TCEP (reducing agent) to reduce the thiol group of _24, The debris was removed using a syringe filter. Hydrogen peroxide was then treated to form disulfide bonds. After each step, the hydrodynamic radius (Rh) of the structure in solution was measured at 4 [deg.] C and 37 [deg.] C in 10 mM PBS (Figure 11). TCEP prior to treatment, SynB1- (GAC) 4-EBPP [E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F 2] 24 solution of the monolithic type polypeptide (11.36 nm), and the nanostructure at 4 ℃ (174.8 nm), as shown in FIG. 11 (A). After TCEP treatment, random disulfide bonds were reduced. As shown in Fig. 11 (B), the hydrodynamic radius (Rh) of the polypeptides in the monolithic form (4.32 nm) and the nanostructure (75.43 nm) The strength of the polypeptides increased and the strength of the nanostructures decreased. This result implies that the multiblock copolypeptide of the present invention comprises a multimeric polypeptide linked by a disulfide bond. Figure 11 (C) shows only one monolith (7.05 nm) free of impurities at 4 占 폚. After the hydrogen peroxide treatment, the shell in the micelle was crosslinked with a disulfide bond, and the micelle structure was maintained below the primary transition temperature. Fig. 11 (D) shows a stable micelle structure at 4 캜.

Example  12: Physically captured rhodamine 6G and fluorescein Sodium salt  In vitro release

The rhodamine 6G charged to a positive charge is at a final concentration of 25 uM 25 uM multi-block co-polypeptide (SynB1- (GAC) 4-EBPP [E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F 2] 24 ) And the solution was incubated for 30 min at 4 DEG C such that the positively charged rhodamine 6G interacted with the negative carboxyl groups in the multiblock copolypeptide. Fluorescein sodium salt was added to a mixture of rhodamine 6G and multiblock copolypeptide at a final concentration of 25 uM. The solution was heated to 37 DEG C to capture the fluorescein sodium salt in the micelle core (hydrophobic EBP portion). The multiblock copolypeptide, including rhodamine 6G and the fluorescein sodium salt, was incubated at 37 占 폚 with 0.25 uM sodium iodide and 250 uM hydrogen peroxide as a catalyst to form a disulfide bond. The mixture was transferred into the dialysis membrane and dialyzed while stirring at 37 rpm at 170 rpm in 50 ml of PBS at 37 < 0 > C. For measurements up to 12 hours, 1 ml of the aliquot was withdrawn and replaced with 1 ml of PBS. The fluorescence intensity of the partial sample at each time was measured at excitation wavelength of 524 nm and emission wavelength at 555 nm for Rhodamine 6G and excitation at 460 nm for fluorescein sodium salt, The wavelength and the emission wavelength at 513 nm were measured. For this measurement, a "Synergy Mx monochromator-based multi-mode microplate reader"

For the dual emission test, as described above, two fluorescent dyes (rhodamine 6G and fluorous sodium salt) were used as model drugs. The negatively charged block in the multiblock copolypeptide first undergoes a charge-charge interaction with positively charged rhodamine 6G at 4 DEG C and then the fluorosine sodium salt is reacted with the multiblock copoly Was added to the peptide. The resulting solution was heated to 37 占 폚. At this time, the fluorosine sodium salt was captured by the hydrophobic interaction.

Multiple block copolypeptides including rhodamine 6G and fluorousin sodium salt were treated with sodium iodide and hydrogen peroxide as catalysts at 37 ° C to form disulfide bonds and multiple drug release experiments were performed using them. The multiblock copolypeptide, including 6G and the fluorescein sodium salt, was dialyzed in PBS at pH 7.4. Partial samples were taken from the dialysis buffer at 20 minutes, 40 minutes, 1 hour, 1 hour 20 minutes, 1 hour 40 minutes, 2 hours, 6 hours, 10 hours, and 12 hours after the start of release (FIG. 12 (A) . Rhodamine 6G was not released for 20 minutes, and after 2 hours, 71.18% of rhodamine 6G was continuously released. 51.23% of the fluorescein sodium salt was released after the first 20 minutes, and also 80.51% of the sodium fluorescein salt was released after 2 hours. Fluorescein sodium salt was released faster and more rapidly than rhodamine 6G.

Example  13: chemically Bonded  In vitro release of rhodamine B and physically entrapped fluorescein sodium salt

The chemical bonding of rhodamine B was carried out as follows: The thiol groups in the cysteine block were modified by glycidyl methacrylate (GMA) to form a bond with rhodamine B incorporating methacrylate residues . 0.2 g of rhodamine B was prepared in 20 ml of deionized water at pH 3.5 and 70 ul of GMA was added to the dye solution (including rhodamine B) and reacted at 50 ° C for 24 hours. After the reaction was terminated, the solution was lyophilized and the dried dyes were reprecipitated in diethyl ether to remove unreacted GMA. Rhodamine B modified with methacrylate was lyophilized to bond with thiol groups.

0.04 g of the multi-block co-polypeptide (SynB1- (GAC) 4-EBPP [E 1 G 4 I 1] 12 -EBPP [G 1 A 3 F 2] 24) is phosphoric acid of 3 ml in a nitrogen atmosphere (nitrogen atmosphere) 0.0 > TCET < / RTI > in buffer (pH 10) for 24 hours at 4 ° C. Rhodamine B modified with 0.09 g of methacrylate contained in 0.8 ml of hexylamine contained in 1 ml of phosphate buffer solution (pH 10) and 1 ml of phosphate buffer solution (pH 10) ≪ / RTI > The reaction was carried out at room temperature for 4 hours. The multimeric block polypeptides conjugated with rhodamine B were purified by ITC and unreacted rhodamine B was removed by dialysis.

Physical uptake of sodium fluorescein sodium salt was performed as follows: 25 uM rhodamine B-conjugated multiblock copolypeptides were prepared in PBS and the fluorescein sodium salt was added to a final concentration of 25 uM. The mixed solution was heated to 37 캜 so as to capture the fluorescein sodium salt with the micelle core. The mixture was transferred into the dialysis membrane and dialyzed while stirring at 37 rpm at 170 rpm in 50 ml of PBS at 37 < 0 > C. For measurements up to 12 h, 1 ml aliquot was removed and replaced with 1 ml PBS. The fluorescence intensity of the partial sample at each time was measured at 530 nm excitation wavelength and 578 nm emission wavelength for rhodamine B and excitation at 460 nm for the fluorescein sodium salt, The wavelength and the emission wavelength at 513 nm were measured. For this measurement, a "Synergy Mx monochromator-based multi-mode microplate reader"

For the dual emission test, two fluorescent dyes (rhodamine B and sodium fluorescein salt) were used as model drugs. As noted above, rhodamine B was chemically conjugated to the hydrophilic EBP block of the multiblock copolypeptide. Fluorescein sodium salt was added to the rhodamine B-conjugated multiblock copolypeptide, and the resulting solution was heated to 37 ° C, where the hydrophobic interaction trapped the fluorosine sodium salt in the hydrophobic EBP portion. Multiblock copolypeptides with fluorsin sodium salt with rhodamine B conjugated and physically entrapped were dialyzed in PBS (pH 7.4). Partial samples were taken from dialysis buffer at 20 minutes, 40 minutes, 1 hour, 1 hour 20 minutes, 1 hour 40 minutes, 2 hours, 6 hours, 10 hours, and 12 hours after the start of release (FIG. 12B) . 24.69% of rhodamine B was released after the first 20 minutes, 83.83% of the sodium fluorescein salt was released relatively slowly for 6 hours, 47.68% of the sodium fluorescein salt was released for 20 minutes, and 82.42% Fluorescein sodium salt was released for 2 hours. Fluorescein sodium salt was released faster and more rapidly than rhodamine B. Dyes chemically conjugated in PBS were released slower than physically trapped dyes.

Transferring dual drugs into cells using stabilized micelles is important for advanced drug delivery systems. We have specifically fabricated self-assembled micelle structures as advanced drug delivery carriers. The micellar constructs were based on thermally responsive elastin-based polypeptides (EBPs) containing cell penetrating peptides (CPPs) necessary for cell membrane penetration. EBPs diblock copolymers self-assembled with a thermal-triggered micelle structure containing the drug were introduced. In addition, cysteine (Cys) blocks containing cysteine amino acids were introduced between CPPs and EBPs for micelle stabilization or drug conjugation, and these CPPs allowed micelles to penetrate cell membranes more efficiently with drugs. CPPs were located on the surface of the micelle structure, and when the EBPs diblock copolymer physically entrapped two different drugs and formed self-assembled micelle structures, the cysteine blocks formed disulfide bonds. Also, while the multiblock copolypeptide forms the self-assembled micelle structure, the cysteine block is used as the drug conjugation site, and when the thermo-stimulated micelles comprise the drug in the hydrophobic core, which was chemically stabilized by crosslinking of the shell. For release testing as a drug model, three fluorescent dyes (rhodamine 6G, rhodamine B, and fluorescein sodium salt) were chemically conjugated or physically interacted with multiblock copolypeptides. The present invention provides stabilized nano-carriers with high penetration ability based on thermally responsive fusion polypeptides for advanced drug delivery systems.

<110> Industry-University Cooperation Foundation Hanyang University ERICA Campus <120> Multi-block Polypeptides and Their Self-assembled Nanostructures          for Enhanced Intracellular Delivery of Multiple Drugs <130> P16U22D1659 <150> KR 15 / 139,205 <151> 2015-10-02 <160> 54 <170> Kopatentin 2.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Elastin-based peptide, Xaa can be any amino acid, natural or          non-natural <400> 1 Val Pro Gly Xaa Gly   1 5 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Elastin-based peptide, Xaa can be any amino acid, natural or          non-natural <400> 2 Val Pro Ala Xaa Gly   1 5 <210> 3 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> EBP <400> 3 gtcccaggtg gaggtgtacc cggcgcgggt gtcccaggtg gaggtgtacc tgggggtggg 60 gtccctggta ttggcgtacc tggaggcggc 90 <210> 4 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> EBP <400> 4 gttccagctg gcggtgtacc tgctgctgct gttccggccg gtggtgttcc ggcgggcggc 60 gtgcctgcaa taggagttcc cgctggtggc 90 <210> 5 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> EBP <400> 5 gttggggtg gtggtgttcc gggtaaaggt gttccgggtg gtggtgttcc gggtggtggt 60 ggtgttccgg gtatcggtgt tccgggtggc 90 <210> 6 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> EBP <400> 6 gttccggcgg gtggtgttcc ggcgaaaggt gttccggcgg gtggtgttcc ggcgggtggt 60 gttccggcga tcggtgttcc ggcgggtggc 90 <210> 7 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> EBP <400> 7 gttggggtg gtggtgttg gggtgatggt gttccgggtg gtggtgttcc gggtggtggt 60 ggtgttccgg gtatcggtgt tccgggtggc 90 <210> 8 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> EBP <400> 8 gttggggggg gtggtgttg ggcggatggt gttccggcgg gtggtgttcc ggcgggtggt 60 gttccggcga tcggtgttcc ggcgggtggc 90 <210> 9 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> EBP <400> 9 gttggggtg gtggtgttcc gggtgaaggt gttccgggtg gtggtgttcc gggtggtggt 60 ggtgttccgg gtatcggtgt tccgggtggc 90 <210> 10 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> EBP <400> 10 gttgggcgg gtggtgttcc ggcggaaggt gttccggcgg gtggtgttcc ggcgggtggt 60 gttccggcga tcggtgttcc ggcgggtggc 90 <210> 11 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> EBP <400> 11 gtcccgggtg cgggcgtgcc gggatttgga gttccgggtg cgggtgttcc aggcggtggt 60 gttccgggcg cgggcgtgcc gggctttggc 90 <210> 12 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> EBP <400> 12 gtgccggcgg cgggcgttcc agcctttggt gtgccagcgg cgggagttcc ggccggtggc 60 gtgccggcag cgggcgtgcc ggcttttggc 90 <210> 13 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> EBP <400> 13 gtgccggcgg cgggcgttcc agcctttggt gtgccagcgg cgggagttcc ggccaaaggc 60 gtgccggcag cgggcgtgcc ggcttttggc 90 <210> 14 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> EBP <400> 14 gtgccggcgg cgggcgttcc agcctttggt gtgccagcgg cgggagttcc ggccgatggc 60 gtgccggcag cgggcgtgcc ggcttttggc 90 <210> 15 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> EBP <400> 15 gttccagcgt ttggcgtgcc agcgaaaggt gttccggcgt ttggggttcc cgcgaaaggt 60 gtgccggcct ttggtgtgcc ggccaaaggc 90 <210> 16 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> EBP <400> 16 gttccagcgt ttggcgtgcc agcggatggt gttccggcgt ttggggttcc cgcggatggt 60 gtgccggcct ttggtgtgcc ggccgatggc 90 <210> 17 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> EBP <400> 17 gtgccggcgc atggagttcc tgccgccggt gttcctgcgc atggtgtacc ggcaattggc 60 gttccggcac atggtgtgcc ggccgccggc 90 <210> 18 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> EBP <400> 18 gttccggccg gaggtgtacc ggcgcatggt gttccggcac atggtgtgcc ggctcacggt 60 gtgcctgcgc atggcgttcc tgcgcatggc 90 <210> 19 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> EBP <400> 19 gtgccggcgt gcggcgttcc agcctttggt gtgccagcgt gcggagttcc ggccggtggc 60 gtgccggcat gcggcgtgcc ggcttttggc 90 <210> 20 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> EBP <400> 20 Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val   1 5 10 15 Pro Gly Gly Gly Val Pro Gly Ile Gly Val Pro Gly Gly Gly              20 25 30 <210> 21 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> EBP <400> 21 Val Pro Ala Gly Gly Val Pro Ala Ala Gly Val Ala Gly Gly Val   1 5 10 15 Pro Ala Gly Aly Gly Val Ala Gly Val Ala Gly Gly              20 25 30 <210> 22 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> EBP <400> 22 Val Pro Gly Gly Gly Val Pro Gly Lys Gly Val Pro Gly Gly Gly Val   1 5 10 15 Pro Gly Gly Gly Val Pro Gly Ile Gly Val Pro Gly Gly Gly              20 25 30 <210> 23 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> EBP <400> 23 Val Pro Ala Gly Gly Val Pro Ala Lys Gly Val Pro Ala Gly Gly Val   1 5 10 15 Pro Ala Gly Aly Gly Val Ala Gly Val Ala Gly Gly              20 25 30 <210> 24 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> EBP <400> 24 Val Pro Gly Gly Gly Val Pro Gly Asp Gly Val Pro Gly Gly Gly Val   1 5 10 15 Pro Gly Gly Gly Val Pro Gly Ile Gly Val Pro Gly Gly Gly              20 25 30 <210> 25 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> EBP <400> 25 Val Pro Ala Gly Gly Val Pro Ala Asp Gly Val Pro Ala Gly Gly Val   1 5 10 15 Pro Ala Gly Aly Gly Val Ala Gly Val Ala Gly Gly              20 25 30 <210> 26 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> EBP <400> 26 Val Pro Gly Gly Gly Val Pro Gly Gly Gly Val Pro Gly Gly Gly Val   1 5 10 15 Pro Gly Gly Gly Val Pro Gly Ile Gly Val Pro Gly Gly Gly              20 25 30 <210> 27 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> EBP <400> 27 Val Pro Ala Gly Gly Val Ala Glu Gly Val Ala Gly Gly Val   1 5 10 15 Pro Ala Gly Aly Gly Val Ala Gly Val Ala Gly Gly              20 25 30 <210> 28 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> EBP <400> 28 Val Pro Gly Ala Gly Val Pro Gly Phe Gly Val Pro Gly Ala Gly Val   1 5 10 15 Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Phe Gly              20 25 30 <210> 29 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> EBP <400> 29 Val Pro Ala Ala Gly Val Ala Phe Gly Val Ala Ala Gly Val   1 5 10 15 Pro Ala Gly Gly Val Pro Ala Ala Gly Val Ala Phe Gly              20 25 30 <210> 30 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> EBP <400> 30 Val Pro Ala Ala Gly Val Ala Phe Gly Val Ala Ala Gly Val   1 5 10 15 Pro Ala Lys Gly Val Ala Ala Gly Val Ala Phe Gly              20 25 30 <210> 31 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> EBP <400> 31 Val Pro Ala Ala Gly Val Ala Phe Gly Val Ala Ala Gly Val   1 5 10 15 Pro Ala Asp Gly Val Ala Ala Gly Val Ala Phe Gly              20 25 30 <210> 32 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> EBP <400> 32 Val Ala Phe Gly Val Ala Lys Gly Val Ala Phe Gly Val   1 5 10 15 Pro Ala Lys Gly Val Pro Ala Phe Gly Val Pro Ala Lys Gly              20 25 30 <210> 33 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> EBP <400> 33 Val Pro Ala Phe Gly Val Pro Ala Asp Gly Val Pro Ala Phe Gly Val   1 5 10 15 Pro Ala Asp Gly Val Pro Ala Phe Gly Val Pro Ala Asp Gly              20 25 30 <210> 34 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> EBP <400> 34 Val Ala His Gly Val Ala Ala Gly Val Ala His Gly Val   1 5 10 15 Pro Ala Ile Gly Val Pro Ala His Gly Val Pro Ala Ala Gly              20 25 30 <210> 35 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> EBP <400> 35 Val Ala Gly Gly Val Ala Gly Val Ala Gly Val   1 5 10 15 Pro Ala His Gly Val Ala His Gly Val Ala His Gly              20 25 30 <210> 36 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> EBP <400> 36 Val Pro Ala Cys Gly Val Pro Ala Phe Gly Val Pro Ala Cys Gly Val   1 5 10 15 Pro Ala Gly Gly Val Pro Ala Cys Gly Val Pro Ala Phe Gly              20 25 30 <210> 37 <211> 48 <212> DNA <213> Penetratin of cell penetrating pepatide <400> 37 cgccagatta agatctggtt ccagaatcgc cgcatgaaat ggaagaaa 48 <210> 38 <211> 16 <212> PRT <213> Penetratin of cell penetrating pepatide <400> 38 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys   1 5 10 15 <210> 39 <211> 54 <212> DNA <213> SynB1 of cell penetrating pepatide <400> 39 cgggggggcc gtctgtcata tagccgtcgt cgttttagca ccagcaccgg tcgt 54 <210> 40 <211> 18 <212> PRT <213> SynB1 of cell penetrating pepatide <400> 40 Arg Gly Gly Arg Leu Ser Tyr Ser Arg Arg Phe Ser Thr Ser Thr   1 5 10 15 Gly Arg         <210> 41 <211> 66 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 41 ctagaaataa ttttgtttaa ctttaagaag gaggagtaca tatgggctac tgataatgat 60 cttcag 66 <210> 42 <211> 66 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 42 gatcctgaag atcattatca gtagcccata tgtactcctc cttcttaaag ttaaacaaaa 60 ttattt 66 <210> 43 <211> 12 <212> PRT <213> Cys block <400> 43 Gly Ala Cys Gly Aly Cys Gly Aly Cys Gly Aly Cys   1 5 10 <210> 44 <211> 60 <212> PRT <213> hydrophilic EBP <400> 44 Val Pro Ala Gly Gly Val Ala Glu Gly Val Ala Gly Gly Val   1 5 10 15 Pro Ala Gly Gly Val Pro Ala Ile Gly Val Pro Ala Gly Gly Val Pro              20 25 30 Ala Gly Aly Gly Aly Gly Aly Gly Aly Gly          35 40 45 Gly Gly Val Pro Ala Ile Gly Val Pro Ala Gly Gly      50 55 60 <210> 45 <211> 60 <212> PRT <213> hydrophobic EBP <400> 45 Val Pro Ala Ala Gly Val Ala Phe Gly Val Ala Ala Gly Val   1 5 10 15 Pro Ala Gly Gly Val Pro Ala Ala Gly Val Pro Ala Phe Gly Val Pro              20 25 30 Ala Ala Gly Val Ala Phe Gly Val Ala Ala Gly Val Ala          35 40 45 Gly Gly Val Pro Ala Ala Gly Val Ala Phe Gly      50 55 60 <210> 46 <211> 120 <212> PRT <213> hydrophobic EBP <400> 46 Val Pro Ala Ala Gly Val Ala Phe Gly Val Ala Ala Gly Val   1 5 10 15 Pro Ala Gly Gly Val Pro Ala Ala Gly Val Pro Ala Phe Gly Val Pro              20 25 30 Ala Ala Gly Val Ala Phe Gly Val Ala Ala Gly Val Ala          35 40 45 Gly Gly Val Pro Ala Ala Gly Val Ala Phe Gly Val Ala Ala      50 55 60 Gly Val Pro Ala Phe Gly Val Pro Ala Ala Gly Val Pro Ala Gly Gly  65 70 75 80 Val Pro Ala Ala Gly Val Ala Phe Gly Val Ala Ala Gly Val                  85 90 95 Pro Ala Phe Gly Val Pro Ala Ala Gly Val Pro Ala Gly Gly Val Pro             100 105 110 Ala Ala Gly Val Ala Phe Gly         115 120 <210> 47 <211> 120 <212> PRT <213> hydrophilic EBP-hydrophobic EBP <400> 47 Val Pro Ala Gly Gly Val Ala Glu Gly Val Ala Gly Gly Val   1 5 10 15 Pro Ala Gly Gly Val Pro Ala Ile Gly Val Pro Ala Gly Gly Val Pro              20 25 30 Ala Gly Aly Gly Aly Gly Aly Gly Aly Gly          35 40 45 Gly Gly Val Pro Ala Ile Gly Val Pro Ala Gly Gly Val Pro Ala Ala      50 55 60 Gly Val Pro Ala Phe Gly Val Pro Ala Ala Gly Val Pro Ala Gly Gly  65 70 75 80 Val Pro Ala Ala Gly Val Ala Phe Gly Val Ala Ala Gly Val                  85 90 95 Pro Ala Phe Gly Val Pro Ala Ala Gly Val Pro Ala Gly Gly Val Pro             100 105 110 Ala Ala Gly Val Ala Phe Gly         115 120 <210> 48 <211> 180 <212> PRT <213> hydrophilic EBP-hydrophobic EBP <400> 48 Val Pro Ala Gly Gly Val Ala Glu Gly Val Ala Gly Gly Val   1 5 10 15 Pro Ala Gly Gly Val Pro Ala Ile Gly Val Pro Ala Gly Gly Val Pro              20 25 30 Ala Gly Aly Gly Aly Gly Aly Gly Aly Gly          35 40 45 Gly Gly Val Pro Ala Ile Gly Val Pro Ala Gly Gly Val Pro Ala Ala      50 55 60 Gly Val Pro Ala Phe Gly Val Pro Ala Ala Gly Val Pro Ala Gly Gly  65 70 75 80 Val Pro Ala Ala Gly Val Ala Phe Gly Val Ala Ala Gly Val                  85 90 95 Pro Ala Phe Gly Val Pro Ala Ala Gly Val Pro Ala Gly Gly Val Pro             100 105 110 Ala Ala Gly Val Ala Phe Gly Val Ala Ala Gly Val Ala         115 120 125 Phe Gly Val Pro Ala Ala Gly Val Ala Gly     130 135 140 Gly Val Pro Ala Phe Gly Val Ala Ala Gly Val Ala Phe Gly 145 150 155 160 Val Pro Ala Ala Gly Val Ala Gly Aly Ala Gly Val                 165 170 175 Pro Ala Phe Gly             180 <210> 49 <211> 132 <212> PRT <213> Cys-hydrophilic EBP-hydrophobic EBP <400> 49 Gly Ala Cys Gly Ala Cys Gly Ala Cys Gly Ala Cys Val Ala Gly   1 5 10 15 Gly Val Pro Ala Glu Gly Val Pro Ala Gly Gly Val Pro Ala Gly Gly              20 25 30 Val Pro Ala Ile Gly Val Pro Ala Gly Gly Val Pro Ala Gly Gly Val          35 40 45 Pro Ala Glu Aly Gly Aly Gly Aly Gly Gly Val Pro      50 55 60 Ala Ile Gly Val Ala Gly Aly Aly Gly Val Ala  65 70 75 80 Phe Gly Val Pro Ala Ala Gly Val Ala Gly                  85 90 95 Gly Val Pro Ala Phe Gly Val Ala Ala Gly Val Ala Phe Gly             100 105 110 Val Pro Ala Ala Gly Val Ala Gly Aly Ala Gly Val         115 120 125 Pro Ala Phe Gly     130 <210> 50 <211> 192 <212> PRT <213> Cys-hydrophilic EBP-hydrophobic EBP <400> 50 Gly Ala Cys Gly Ala Cys Gly Ala Cys Gly Ala Cys Val Ala Gly   1 5 10 15 Gly Val Pro Ala Glu Gly Val Pro Ala Gly Gly Val Pro Ala Gly Gly              20 25 30 Val Pro Ala Ile Gly Val Pro Ala Gly Gly Val Pro Ala Gly Gly Val          35 40 45 Pro Ala Glu Aly Gly Aly Gly Aly Gly Gly Val Pro      50 55 60 Ala Ile Gly Val Ala Gly Aly Aly Gly Val Ala  65 70 75 80 Phe Gly Val Pro Ala Ala Gly Val Ala Gly                  85 90 95 Gly Val Pro Ala Phe Gly Val Ala Ala Gly Val Ala Phe Gly             100 105 110 Val Pro Ala Ala Gly Val Ala Gly Aly Ala Gly Val         115 120 125 Pro Ala Phe Gly Val Pro Ala Ala Gly Val Pro Ala Phe Gly Val Pro     130 135 140 Ala Ala Gly Val Ala Gly Aly Ala Gly Val Ala 145 150 155 160 Phe Gly Val Pro Ala Ala Gly Val Ala Phe Gly Val Ala Ala                 165 170 175 Gly Val Pro Ala Gly Gly Val Pro Ala Ala Gly Val Ala Phe Gly             180 185 190 <210> 51 <211> 148 <212> PRT <213> Penetratin-Cys-hydrophilic EBP-hydrophobic EBP <400> 51 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys   1 5 10 15 Gly Ala Cys Gly Ala Cys Gly Ala Cys Gly Ala Cys Val Ala Gly              20 25 30 Gly Val Pro Ala Glu Gly Val Pro Ala Gly Gly Val Pro Ala Gly Gly          35 40 45 Val Pro Ala Ile Gly Val Pro Ala Gly Gly Val Pro Ala Gly Gly Val      50 55 60 Pro Ala Glu Aly Gly Aly Gly Aly Gly Gly Val Pro  65 70 75 80 Ala Ile Gly Val Ala Gly Aly Aly Gly Val Ala                  85 90 95 Phe Gly Val Pro Ala Ala Gly Val Ala Gly             100 105 110 Gly Val Pro Ala Phe Gly Val Ala Ala Gly Val Ala Phe Gly         115 120 125 Val Pro Ala Ala Gly Val Ala Gly Aly Ala Gly Val     130 135 140 Pro Ala Phe Gly 145 <210> 52 <211> 208 <212> PRT <213> Penetratin-Cys-hydrophilic EBP-hydrophobic EBP <400> 52 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys   1 5 10 15 Gly Ala Cys Gly Ala Cys Gly Ala Cys Gly Ala Cys Val Ala Gly              20 25 30 Gly Val Pro Ala Glu Gly Val Pro Ala Gly Gly Val Pro Ala Gly Gly          35 40 45 Val Pro Ala Ile Gly Val Pro Ala Gly Gly Val Pro Ala Gly Gly Val      50 55 60 Pro Ala Glu Aly Gly Aly Gly Aly Gly Gly Val Pro  65 70 75 80 Ala Ile Gly Val Ala Gly Aly Aly Gly Val Ala                  85 90 95 Phe Gly Val Pro Ala Ala Gly Val Ala Gly             100 105 110 Gly Val Pro Ala Phe Gly Val Ala Ala Gly Val Ala Phe Gly         115 120 125 Val Pro Ala Ala Gly Val Ala Gly Aly Ala Gly Val     130 135 140 Pro Ala Phe Gly Val Pro Ala Ala Gly Val Pro Ala Phe Gly Val Pro 145 150 155 160 Ala Ala Gly Val Ala Gly Aly Ala Gly Val Ala                 165 170 175 Phe Gly Val Pro Ala Ala Gly Val Ala Phe Gly Val Ala Ala             180 185 190 Gly Val Pro Ala Gly Gly Val Pro Ala Ala Gly Val Ala Phe Gly         195 200 205 <210> 53 <211> 150 <212> PRT <213> SynB1-Cys-hydrophilic EBP-hydrophobic EBP <400> 53 Arg Gly Gly Arg Leu Ser Tyr Ser Arg Arg Phe Ser Thr Ser Thr   1 5 10 15 Gly Arg Gly Ala Cys Gly Ala Cys Gly Ala Cys Gly Ala Cys Val Pro              20 25 30 Ala Gly Aly Gly Aly Gly Aly Gly Aly Gly          35 40 45 Gly Gly Val Pro Ala Ile Gly Val Pro Ala Gly Gly Val Pro Ala Gly      50 55 60 Gly Val Pro Ala Glu Gly Val Pro Ala Gly Gly Val Pro Ala Gly Gly  65 70 75 80 Val Pro Ala Ile Gly Val Pro Ala Gly Gly Val Pro Ala Ala Gly Val                  85 90 95 Pro Ala Phe Gly Val Pro Ala Ala Gly Val Pro Ala Gly Gly Val Pro             100 105 110 Ala Ala Gly Val Ala Phe Gly Val Ala Ala Gly Val Ala         115 120 125 Phe Gly Val Pro Ala Ala Gly Val Ala Gly     130 135 140 Gly Val Pro Ala Phe Gly 145 150 <210> 54 <211> 210 <212> PRT <213> SynB1-Cys-hydrophilic EBP-hydrophobic EBP <400> 54 Arg Gly Gly Arg Leu Ser Tyr Ser Arg Arg Phe Ser Thr Ser Thr   1 5 10 15 Gly Arg Gly Ala Cys Gly Ala Cys Gly Ala Cys Gly Ala Cys Val Pro              20 25 30 Ala Gly Aly Gly Aly Gly Aly Gly Aly Gly          35 40 45 Gly Gly Val Pro Ala Ile Gly Val Pro Ala Gly Gly Val Pro Ala Gly      50 55 60 Gly Val Pro Ala Glu Gly Val Pro Ala Gly Gly Val Pro Ala Gly Gly  65 70 75 80 Val Pro Ala Ile Gly Val Pro Ala Gly Gly Val Pro Ala Ala Gly Val                  85 90 95 Pro Ala Phe Gly Val Pro Ala Ala Gly Val Pro Ala Gly Gly Val Pro             100 105 110 Ala Ala Gly Val Ala Phe Gly Val Ala Ala Gly Val Ala         115 120 125 Phe Gly Val Pro Ala Ala Gly Val Ala Gly     130 135 140 Gly Val Pro Ala Phe Gly Val Ala Ala Gly Val Ala Phe Gly 145 150 155 160 Val Pro Ala Ala Gly Val Ala Gly Aly Ala Gly Val                 165 170 175 Pro Ala Phe Gly Val Pro Ala Ala Gly Val Pro Ala Phe Gly Val Pro             180 185 190 Ala Ala Gly Val Ala Gly Aly Ala Gly Val Ala         195 200 205 Phe Gly     210

Claims (19)

Cell permeable polypeptide block (CPP block); And
A cysteine block (Cys) connected to one end of the cell permeable polypeptide block; And
An elastin-based polypeptide block (hydrophilic EBP block) that is charged and hydrophilic and connected to the cysteine block; And
A hydrophobic elastin-based polypeptide block (hydrophobic EBP block) linked to the hydrophilic EBP;
&Lt; / RTI &gt; wherein said multiblock copolypeptide comprises multiple block copolypeptides.
The method according to claim 1,
The hydrophilic block EBP, Val-Pro-Gly-X aa -Gly [ SEQ ID NO: 1] or _{Val-Pro-Ala-X aa} -Gly [ SEQ ID NO: 2] is a penta-peptide consisting of the repeating 6n times, the formula 1 Or an amino acid sequence represented by Formula 2:
[Formula 1]
[Val-Pro-Gly-X _aa- Gly] _6n ; or
[Formula 2]
[Val-Pro-Ala-X _aa- Gly] _6n ,
In the above formula (1) or (2)
N is an integer of 1 or more,
Wherein X _aa is an amino acid other than proline, and is selected from any natural or artificial amino acid when the pentapeptide is repeated, and at least one of the X _aa is a charged amino acid and a hydrophilic amino acid.
The method according to claim 1,
The hydrophobic EBP block comprises
_{Val-Pro-Gly-X aa} -Gly [ SEQ ID NO: 1] or _{Val-Pro-Ala-X aa} -Gly repeats [SEQ ID NO: 2] penta-peptide consisting of 6n times, represented by the following formula 1 or formula 2 Wherein the drug delivery system comprises an amino acid sequence:
[Formula 1]
[Val-Pro-Gly-X _aa- Gly] _6n ; or
[Formula 2]
[Val-Pro-Ala-X _aa- Gly] _6n ,
In the above formula (1) or (2)
N is an integer of 1 or more,
X _aa is an amino acid other than proline, which is selected from any natural or artificial amino acid when the pentapeptide is repeated, and at least one of X _aa is a hydrophobic or aliphatic amino acid.
The method according to claim 1,
Wherein the CPP block is selected from the group consisting of Penetratin, SynBl, SynBl-NLS, poly-arginine, VP22, MAP, hCT-derived CPP, MPG, Buforin 2, PEP-1 and Magainin 2.
The method according to claim 1,
Wherein the amino acid sequence of the cysteine residue (Gly-Ala-Cys) is repeated one or more times.
6. The method of claim 5,
Wherein the amino acid sequence of the cysteine block is represented by (Gly-Ala-Cys-Gly-Ala-Cys-Gly-Ala-Cys-Gly-Ala-Cys) [SEQ ID NO: 43].

3. The method of claim 2,
The hydrophilic EBP block may comprise,
Wherein n is 1 in the formula 1, X _aa of each of the repeated pentapeptides,
A (Ala), G (Gly) and I (Ile) in a ratio of 1: 4: 1 [SEQ ID NO: 20];
K (Lys), G (Gly) and I (Ile) in a ratio of 1: 4: 1 [SEQ ID NO: 22];
D (Asp), G (Gly) and I (Ile) in a ratio of 1: 4: 1 [SEQ ID NO: 24]; or
E (Glu), G (Gly) and I (Ile) in a ratio of 1: 4: 1 [SEQ ID NO: 26]; Or,
or
Wherein n is 1 in the formula 2, X _aa of each of the repeated pentapeptides is
A (Ala), G (Gly) and I (Ile) in a ratio of 1: 4: 1 [SEQ ID NO: 21];
K (Lys), G (Gly) and I (Ile) in a ratio of 1: 4: 1 [SEQ ID NO: 23];
D (Asp), G (Gly) and I (Ile) in a ratio of 1: 4: 1 [SEQ ID NO: 25]; or
[SEQ ID NO: 27] wherein E (Glu), G (Gly) and I (Ile) are present in a ratio of 1: 4: 1;
That is. Drug delivery.
The method of claim 3,
The hydrophobic EBP block comprises
Wherein n is 1 in the formula 1, X _aa of each of the repeated pentapeptides,
(SEQ ID NO: 28) wherein G (Gly), A (Ala) and F (Phe) are in a ratio of 1: 3: 2
Wherein n is 1 in the formula 2, X _aa of each of the repeated pentapeptides is
G (Gly), A (Ala) and F (Phe) in a ratio of 1: 3: 2 [SEQ ID NO: 29];
K (Lys), A (Ala) and F (Phe) in a ratio of 1: 3: 2 [SEQ ID NO: 30];
D (Asp), A (Ala) and F (Phe) in a ratio of 1: 3: 2 [SEQ ID NO: 31];
K (Lys) and F (Phe) in the ratio of 3: 3 [SEQ ID NO: 32];
D (Asp) and F (Phe) in a ratio of 3: 3 (SEQ ID NO: 33);
H (His), A (Ala) and I (Ile) in a ratio of 3: 2: 1 [SEQ ID NO: 34];
H (His), G (Gly) in a ratio of 5: 1 [SEQ ID NO: 35]; or
(Gly), C (Cys), and F (Phe) in a ratio of 1: 3: 2.
3. The method of claim 2,
The hydrophilic EBP block may comprise,
In Formula 2, n is 2, and X _aa of each of the repeated pentapeptides is represented by Formula
SEQ ID NO: 44] wherein E (Glu), G (Gly) and I (Ile) are present in a ratio of 1: 4: 1.
The method of claim 3,
The hydrophobic EBP block
In Formula 2, n is 2, and X _aa of each of the repeated pentapeptides is represented by Formula
G (Gly), A (Ala) and F (Phe) in a ratio of 1: 3: 2 [SEQ ID NO: 45] or
In the above formula 2, n is 4, and X _aa of each of the repeated pentapeptides is
SEQ ID NO: 46] wherein G (Gly), A (Ala) and F (Phe) are in a ratio of 1: 3: 2.
2. The drug delivery device according to claim 1,
CPP block-cysteine blocks (Cys) ₄ - hydrophilic EBP block _{EBPP [E 1 G 4 I 1} ] 12 [ SEQ ID NO: 44; - hydrophobic EBP block EBPP composed of _{[G 1 A 3 F 2]} 12 [ SEQ ID NO: 45; or ; or
CPP block - Cysteine block (Cys) ₄ - hydrophilic EBP block EBPP [E ₁ G ₄ I ₁ ] ₁₂ [SEQ ID NO: 44] - hydrophobic EBP block EBPP [G ₁ A ₃ F ₂ ] ₂₄ [SEQ ID NO: 46] Lt; / RTI &gt;
12. The method of claim 11,
The CPP block is phenetlatin (amino acid SEQ ID NO: 38) or SynB1 (amino acid SEQ ID NO: 40)
Wherein the drug delivery vehicle is one selected from SEQ ID NOS: 51, 52, 53 and 54.
The method according to claim 1,
The multiblock copolypeptide may comprise,
Wherein the hydrophobic EBP block forms a core structure by thermal stimulation and the CPP block-cysteine block-hydrophilic EBP block forms a shell structure, thereby forming a self-assembled nanostructure of the core-shell structure.
14. The method of claim 13,
Wherein the self-assembled nanostructure is stabilized by an oxidation reaction of the cysteine block.
14. The method of claim 13,
Wherein the hydrophilic EBP block is chemically bonded with the hydrophilic EBP block by introducing an acrylate into the carboxyl group of the hydrophilic EBP block of the shell portion and irradiating ultraviolet rays to stabilize the self assembled nanostructure.
14. The method of claim 13,
The drug delivery system comprises:
Wherein the hydrophobic drug is captured in the core portion of the self-assembled nanostructure and the chargeable drug is captured in the shell portion to control the release of the hydrophobic drug and the chargeable drug to release multiple drugs.
14. The method of claim 13,
The drug delivery system comprises:
Wherein the release of the hydrophobic drug and the chargeable drug is controlled by oxidation-reduction reaction of the cysteine block so that multiple drug release is possible.
14. The method of claim 13,
The drug delivery system comprises:
A hydrophobic drug is trapped in a core portion of the self-assembled nanostructure, and a charge drug having acrylate binding to a thiol group in a cysteine block of the shell portion is bonded to release the hydrophobic drug and the charge- Lt; RTI ID = 0.0 &gt; drug delivery. &Lt; / RTI &gt;
17. The method of claim 16,
Said self-assembled nanospheres release the entrapped drug in the shell, followed by release of the hydrophobic drug entrapped in the core; or
Wherein the hydrophobic drug entrapped in the core is released and then the charge drug trapped in the shell is released so that sequential drug release is possible.

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