KR20200053308A - Drug delivery system comprising enzyme-responsive amphiphilic peptide - Google Patents

Abstract

The present invention relates to a drug delivery system including an enzyme-responsive amphiphilic peptide. Particularly, the enzyme-responsive amphiphilic peptide is a peptide including a sequence of arginine (R)- histidine (H)- [glycine (G)- leucine (L)- phenylalanine (F)- glycine (G)]_n. The peptide forms nanoparticles through self-assembly, and is characterized in that the hydrophilic sequence RH faces outwards and the hydrophobic sequence GLFG is positioned inwards, and can move into cells while including a hydrophobic drug, such as an anticancer agent, therein. In addition, GLFC is characteristically cleaved by cathepsin-B enzyme. Thus, when the nanoparticles permeate a cell membrane and come into cells, they are cleaved by cathepsin-B to release the drug.

Description

Drug delivery system comprising enzyme-responsive amphiphilic peptide

The present invention relates to a drug delivery system comprising an enzyme reaction amphiphilic peptide.

Peptides are not only being studied as drug delivery systems due to their excellent biocompatibility and biodegradability, but peptides can also be provided as strategic and efficient peptide-based systems by engaging in important biological mechanisms. For example, cell-penetrating peptides (CPP) can be applied to enhance cell influx. Lysine and arginine can induce interactions with cell membranes and deliver substances such as nanoparticles, small molecules and genes.

Since gene delivery systems require penetration of the nuclear membrane for effective gene expression, the nuclear localization sequence (NLS) can be used to introduce proteins into the cell nucleus by nuclear transfer. Therefore, NLS can be useful for gene delivery. On the other hand, it has been reported that mitochondrial target peptides can also be used as a useful tool in delivery systems, and has been studied as a new delivery system.

The self-assembled nanostructure of the amphipathic peptide (AP) has attracted much attention as a drug delivery system due to the self-assembly ability induced by amino acid design. A recent study showed that APs based on coiled-coil helix bundles form a hierarchical assembly. Research into the application of a polypeptide derived from a calcium channel as a drug delivery system has been conducted, and APs containing arginine and valine have been reported in siRNA delivery systems, and nanostructures based on AP are self-assisted ( self-adjuvant). Branched amphipathic peptide capsules have been studied for cell uptake and retention of encapsulated solutes.

Drug delivery systems (DDS) using amphipathic peptides have the potential as biocompatibility and efficient delivery carriers. Most therapeutic agents are hydrophobic and have low solubility in aqueous solutions, resulting in low therapeutic efficacy. Therefore, it is necessary to improve the solubility of the drug through encapsulation. The AP containing the hydrophilic head and the hydrophobic core can encapsulate the hydrophobic drug, thereby increasing therapeutic efficacy. The peptide is excellent in biocompatibility and biodegradability and is advantageous in drug delivery systems. Indeed, polymer-based delivery systems have been reported to have significant cytotoxicity due to low degradability and excessive intracellular accumulation.

However, the drug delivery system using the AP has not yet achieved satisfactory results compared to the polymer-based drug delivery system. Therefore, to improve the efficiency of the delivery system, the stability and control of drug release should be improved. .

On the other hand, as a technology related to the present invention, Korean Patent No. 1647178 discloses a technique related to stabilized plasmid-lipid particles using an enzyme cleavable linker or a polyethylene glycol-lipid containing oligolysine, and Korean Patent No. 0857389 Although the description of AP-GRR peptides or peptide chains comprising AP-GRR peptides and drug delivery carriers comprising the same is disclosed, no drug delivery systems comprising the enzymatically reactive amphipathic peptides of the present invention have been disclosed. .

The present invention has been derived by the above-described needs, the present invention provides a drug delivery system comprising a peptide consisting of the amino acid sequence represented by Formula 1, and the peptide nanoparticles of the present invention are self-assembled to seal the drug. The present invention was completed by confirming that there is no cytotoxicity and that the peptide nanoparticles encapsulating the target drug can deliver the drug into the cell and exhibit excellent anticancer effects.

In order to achieve the above object, the present invention provides a drug delivery system comprising a peptide consisting of an amino acid sequence represented by the following general formula (1).

[Formula 1]

Arginine (R) -histidine (H)-[glycine (G) -leucine (L) -phenylalanine (F) -glycine (G)] _n

In the general formula 1, n is an integer from 1 to 10.

The present invention relates to a drug delivery system comprising an enzymatically reactive amphipathic peptide, wherein the enzymatically reactive amphipathic peptide is specifically arginine (R) -histidine (H)-[glycine (G) -leucine (L) -phenylalanine ( F) -glycine (G)] A peptide consisting of the sequence of _n , wherein the peptide forms nanoparticles by self-assembly, the hydrophilic sequence RH is directed outward, and the hydrophobic sequence GLFG is inward. It has the characteristic of being located, and it has the characteristic of being able to move into the cell while holding a hydrophobic drug such as an anti-cancer agent inside. In addition, GLFG is characterized by being cut by the cathepsin-B enzyme, so that when the nanoparticles according to the present invention penetrate the cell membrane and enter the cell, the drug is cleaved by cathepsin-B to release the drug. Therefore, the drug delivery system of the present invention can be usefully used as a delivery agent for anti-cancer drugs, which are hydrophobic drugs.

1 is an example of a drug delivery system, which is a schematic diagram of RH- (GFLG) ₃ nanoparticles.
2 is a result confirming the physical properties of the RH- (GFLG) ₃ nanoparticles. (A) is a histogram showing the size distribution of LL nanoparticles, (B) is an image measured by field emission scanning electron microscopy of LL peptide nanoparticles, and (C) is the size according to the storage period of LL and DD nanoparticles A graph showing the change, (D) is a CD (Circular dichroism) spectrum of LL and DD nanoparticles, (E) is a result of confirming the minimum micelle concentration (CMC) of the LL peptide nanoparticles.
3 is a result confirming the cytotoxicity of the RH- (GFLG) ₃ nanoparticles. (A) is the MTT assay result, (B) is the LDH assay result, (C) is the hemolysis assay result, and (D) the zebra fish embryo development test result. LL is ^L RH- ^L (GFLG) ₃ , LD is ^L RH- ^D (GFLG) ₃ , DD is ^D RH- ^D (GFLG) ₃ , DL is ^D RH- ^L (GFLG) ₃ , PEI 25KD Is a cationic polymer as a positive control.
4 is a result of the cell influx assay of NH red bound RH- (GFLG) ₃ nanoparticles. (A) is an image confirmed by confocal microscopy, and (B) is the result of FACS (Fluorescence-activated cell sorting) analysis.
Figure 5 shows the results of an enzyme sensitivity assay using MALDI-TOF mass spectrometry. (A) LL peptide (control), (B) LL peptide treated with cathepsin B; (C) LD peptide (control) (D) LD peptide treated with cathepsin B; (E) DL peptide (control) (F) DL peptide treated with cathepsin B; (G) DD peptide (control) (H) DD peptide treated with cathepsin B.
6 is a result showing the cumulative release amount of the drug (doxorubicin) released from LL-Dox and DD-Dox by enzyme treatment.
7 is a result of a cell influx assay of RH- (GFLG) ₃ nanoparticles in which Doxorubicin (Dox) is sealed in HeLa cells.
8 is a result confirming the anticancer activity of Dox-RH- (GFLG) ₃ nanoparticles in HeLa and SW480 cells. (A) is the cell viability after 48 hours incubation in SW480 cells, and (B) is the cell survival rate after 72 hours incubation in HeLa cells. ** and *** are statistically significantly reduced cell viability of Dox-RH- (GFLG) ₃ nanoparticles of the present invention compared to treatment with doxorubicin alone, ** is p <0.01, * ** is p <0.001.
9 is a result confirming the anticancer activity of Dox-RH- (GFLG) ₃ nanoparticles.

The present invention relates to a drug delivery system comprising a peptide consisting of the amino acid sequence represented by the following general formula (1).

[Formula 1]

Arginine (R) -histidine (H)-[glycine (G) -leucine (L) -phenylalanine (F) -glycine (G)] _n

In the general formula 1, n is an integer from 1 to 10.

The peptide is self-assembled and the amino acid sequence of hydrophilic arginine (R) -histidine (H) is located outward, and glycine (G) -leucine (L) -phenylalanine (F)-of hydrophobic residues is located inward. It is characterized by forming micelle-shaped nanoparticles in which the amino acid sequence of glycine (G) is located.

The chirality of the amino acids forming the peptide may be L-type or D-type, but preferably the hydrophilic arginine and histidine amino acids may be L- or D-type, likewise hydrophobic glycine, leucine, phenylalanine and The glycine amino acid may be of L- or D-type, more preferably the chirality of the amino acids forming the peptide is ^L RH- ^L (GFLG) _n , ^L RH- ^D (GFLG) _n or ^D RH- ^L (GFLG) _n (DL), and most preferably ^L RH- ^L (GFLG) _n , but is not limited thereto.

A hydrophobic anticancer agent may be contained inside the micelle, and the hydrophobic anticancer agent is preferably at least one anticancer agent selected from doxorubicin, docetaxel, paclitaxel, irinotecan, camptothecin and actinomycin D, and more preferably doxorubicin. It is not limited.

The peptide is characterized by being cleaved by the cathepsin B enzyme.

Hereinafter, the present invention will be described in more detail using examples. It is obvious to those skilled in the art that these examples are only intended to illustrate the present invention more specifically and that the scope of the present invention is not limited by them.

Example  1. Peptide nanoparticles of  formation

RH-GFLG (SEQ ID NO: 1), RH- (GFLG) ₂ (SEQ ID NO: 2), ^L RH- ^L (GFLG) ₃ (LL) (SEQ ID NO: 3), ^L RH- ^D (GFLG) ₃ (LD) ( SEQ ID NO: 3), ^D RH- ^L (GFLG) ₃ (DL) (SEQ ID NO: 3), and ^D RH- ^D (GFLG) ₃ (DD) (SEQ ID NO: 3) were purchased from peptron and among the peptides, isomers LD, DL, DD peptide containing was used as a control.

Peptides were prepared by dissolving in water so that the final concentration was 1-10 mg / ml, and then sonicating for 15 minutes to form peptide nanoparticles and storing at 4 ° C for one day before use.

Peptide nanoparticles tagged with Nile Red were prepared for a cell influx assay, and Nile Red was prepared to have a concentration of 100 nM using methanol as a solvent, followed by solution of the peptide such that the Nile Red: peptide became a molar ratio of 1:10 ⁵ And nile were mixed and sonicated for 15 minutes. Nileed that was not tagged to the peptide was removed by spin column, and the nanoparticle of the nied-tagged peptide was stored at 4 ° C.

Example  2. Peptide nanoparticles of  Physical properties

The size of the peptide nanoparticles was analyzed using a phosphorescent ELS-Z2 dynamic light scatterometer at a concentration of 0.5 mg / ml. The surface charge value of the peptide nanoparticles was measured by Nano-ZS from Malvern. To confirm the stability of the nanoparticles, LL peptide nanoparticles were continuously analyzed with a dynamic light scattering particle sizer for 30 days. The form of the LL peptide nanoparticles was analyzed by Hitachi scanning electron microscope. For scanning electron microscope measurements, the peptide nanoparticles were dried on a silicon wafer and then coated with osmium. The prepared peptide nanoparticles were analyzed under a scanning electron microscope under a voltage condition of 5-10 kV. The structure of the peptide nanoparticles formed by self-assembly was measured using a Jasco CD analyzer. LL and DD peptide nanoparticle solutions were measured in the 190-260 nm wavelength range.

As a result, as shown in (A) and (B) of Figure 2, the size (diameter) of the LL peptide nanoparticles according to the present invention was found to be about 100 nm level; FIG. 2 (C) shows that LL and DD peptide nanoparticles retain their size (diameter) of about 200 nm even when stored at 4 ° C. for about 30 days; 2 (D), it was confirmed that 91.5% of LL peptide nanoparticles have a turn structure, 8.5% have a random structure, 29.2% of DD peptide nanoparticles have a beta-sheet structure, 15% have a turn structure, It was confirmed that 55.8% were random structures.

Example  3. Critical micelle concentration analysis of peptide nanoparticles

The critical micelle concentration of the peptide nanoparticles was analyzed using hydrophobic Nile. Peptide nanoparticles were prepared in 1 ml increments at 0.001 to 0.3 mg / ml. The prepared nanoparticle solution and 5 μl of a 40 μM nile red solution were mixed. In order to remove the ethanol contained in the nile red solution, it was blown with nitrogen and reacted at room temperature for 1 hour. After the reaction was completed, excitation was performed at 515 nm using a fluorescent device to measure at 550 to 750 nm. The critical micelle concentration of the peptide nanoparticles was analyzed at 650 nm.

As a result, it was confirmed that the critical micelle concentration of the nanoparticles was 0.02 mg / ml, as shown in FIG. 2 (E).

Example  4. Peptide nanoparticles of  Cytotoxicity check

(1) Cell culture

HeLa cells were cultured at 37 ° C., 5% CO ₂ condition with 89% DMEM, 10% FBS and 0.1 mg / ml antibiotic-antimycotic reagent. Cytotoxicity of the peptide nanoparticles was analyzed using the cultured HeLa cells.

(2) WST -1 analysis

HeLa cells were incubated for 24 hours by dispensing 1.0 × 10 ⁴ cells per well into a 96-well microplate. PEI 25KD, LL, LD, DL and DD were prepared at concentrations of 0.025 to 0.2 μg / μl. After stabilization for 24 hours, 10 μl of WST-1 was treated and reacted for 2 hours. For WST-1 foam, absorbance was measured at 450 nm using a VERASmax micro reader.

As a result, as shown in Figure 3 (A), it was found that the LL, DD, DL and LD peptide nanoparticles have little cytotoxicity, and thus do not affect the cell viability (%).

(3) LDH  Active analysis

Cells treated with 20% Tween-20 were used as a control showing the maximum activity of LDH, and after 24 hours of processing each sample, 10 μl of the supernatant of each sample was taken, and this supernatant was 0.2 mM. The reaction solution was reacted with NADH and 2.5 mM sodium fibrate at 37 ° C for 45 minutes. The absorbance of the sample after the reaction was measured at 490 nm.

As a result, as shown in FIG. 3 (B), it was found that the LL, DD, DL and LD peptide nanoparticles did not damage the cell membrane and thus little LDH release occurred.

Example  5. Analysis of hemolysis of nanoparticles

Hemolysis analysis of LL, LD, DL, and DD peptide nanoparticles was performed using human red blood cells, and PEI 25KD was used as a positive control. Blood used in Example 6 was taken from healthy volunteers. After treating EDTA with blood to prevent coagulation, red blood cells were separated from plasma by centrifugation at 5000 rpm for 5 minutes. The isolated red blood cells were washed twice with PBS, and then prepared with 2.0 × 10 ⁸ cells per ml.

In order to compare and analyze the hemolytic action, the group treated with 0.2% Triton X-100 was set as the maximum hemolysis control, and 0.25 ml of red blood cells and 0.25 ml of the sample were mixed to give a final concentration of 0.1 or 0.2 mg / ml. Prepared as much as possible and left at 37 ° C for 2 hours. After completion of the reaction, the sample was centrifuged at 5000 rpm for 5 minutes, and 30 µl of the supernatant was taken. The supernatant was diluted 10-fold with PBS and analyzed by measuring with a VERSAmax microplate reader at a wavelength of 450 nm.

As a result, as shown in Fig. 3 (C), it was shown that LL, DD, DL and LD peptide nanoparticles do not lyse red blood cells.

Example  6. Cell influx Assay

The ability of the peptide nanoparticles to enter the cells was analyzed by a fluorescence microscope from Reika, a confocal microscope from Zeiss, and a flow cytometer from BD Biosciences. The nano-labeled LL, DL, LD, and DD peptide nanoparticles were treated to a final concentration of 0.2 mg / ml and left for 6 hours or 16 hours. Thereafter, HeLa cells were treated with 20 μM of Hoechst 33324 for 15 minutes to stain the nuclei and analyzed with a fluorescence microscope and confocal microscope. In the flow cytometry analysis, 1.0 × 10 ⁶ HeLa cells per well were dispensed and stabilized for 24 hours, then treated with a final concentration of 0.2 mg / ml and left for 16 hours. After that, HeLa cells were washed twice with DPBS and harvested using trypsin-EDTA. HeLa cells were harvested by centrifugation at 1500 rpm for 3 minutes, and then dispersed with 500 μl of PBS, and then the flow cytometry was used to check whether the cells were introduced.

As illustrated in FIG. 4, the nanoparticles stained with nile red, LL, DL, LD, and DD peptides were introduced into the cytoplasm, and the degree of introduction was in the order of LL> DL, LD >> DD.

Example  7. Doxorubicin  Sutured RH- ( GFLG ) ₃ Preparation of nanoparticles

In order to evaluate the efficacy as a drug delivery agent by sealing doxorubicin to peptide nanoparticles, peptide nanoparticles sealed with doxorubicin were prepared.

In order to remove the salt of doxorubicin hydrochloride, treatment with trimethylamine 3 times was left for 16 hours. The doxorubicin from which the salt was removed was extracted with chloroform about 5 times and used. To optimize the sealing efficiency of doxorubicin in peptide nanoparticles, various molar ratios (about 0.1 to 2 times) were prepared. After the doxorubicin dissolved in chloroform was placed in a glass vial, chloroform was removed with nitrogen gas, and a peptide solution of 1 µg / µl was added. After performing the vortex so that the sample was well mixed, the suture was exposed to ultrasound for 15 minutes, and then kept refrigerated overnight. Dialysis was performed overnight using a 1000Da dialysis membrane to remove unsealed doxorubicin. The prepared doxorubicin-sealed peptide nanoparticles were refrigerated.

Example  8. Enzyme sensitivity analysis

 To analyze the enzyme sensitivity of LL, LD, DL, and DD peptide nanoparticles, they were analyzed using a MALDI-TOF mass spectrometer (matrix-assisted laser mass spectroscopy). LL, LD, DL, and DD peptides were prepared by treating 0.02 μM cathepsin B at 37 ° C. for 6 hours. The prepared peptides were mass analyzed using Voyager's MALDI-TOF MS using α-cyano-4-hydroxycinnamic acid (CHCA) as a matrix.

As shown in Figure 5, the sensitivity to cathepsin B enzyme was found to be the best LL peptide nanoparticles.

Example  9. Drug Release ability  analysis

A dialysis method was used to analyze the drug release capacity of the peptide nanoparticles containing doxorubicin. Briefly, 300 μl of doxorubicin-sealed peptide nanoparticles (LL-Dox and DD-Dox) were placed in a dialysis membrane of 1000 Da, placed in 2.7 ml of PBS, and the amount of doxorubicin released over time was analyzed while shaking at 37 ° C. .

In the same manner as above, 300 μl of doxorubicin-sealed peptide nanoparticles (LL-Dox and DD-Dox) and 0.02 μM cathepsin B were placed in a dialysis membrane of 1000 Da, placed in 2.7 mL of PBS, and 37 ° C. The amount of doxorubicin released over time was analyzed while shaking at.

At this time, the amount of emitted doxorubicin (Dox) was analyzed at a wavelength of 480 nm using a micro reader.

As a result, as shown in FIG. 6, it was confirmed that the amount of doxorubicin release of LL-Dox + cathepsin B is greater than the amount of doxorubicin release of the remaining LL-Dox, DD-Dox and DD-Dox + cathepsin B.

Example  10. Doxorubicin  Sutured RH- ( GFLG ) ₃ Peptide nanoparticle cells permeability  analysis

The cell permeability of the doxorubicin sealed peptide nanoparticles was analyzed by confocal microscopy in HeLa cells. The HeLa cells were divided into 1.0 × 10 ⁴ pieces, stabilized in an 8-well confocal microscope dish, and treated with LL and DD peptide nanoparticles sealed with doxorubicin and doxorubicin in HeLa cells. At this time, the cell nuclei were stained with 20 μM Hoechst 33324 for 15 minutes. After 6 and 16 hours, the samples prepared with a confocal microscope were analyzed.

In addition, similar to the HeLa cells, cell permeability was analyzed using a 3D spheroid model. The peptide nanoparticles used in this example were prepared such that the nile was tagged and appeared red when observed.

As a result, as shown in Figure 7, LL-Dox of the present invention was treated with doxorubicin alone, or DD peptide nanoparticles, cathepsin B-bound DD-Dox was superior to the cell.

Example  11. Doxorubicin  Sutured RH- ( GFLG ) ₃ Anticancer effect of peptide nanoparticles

The anticancer effect of the doxorubicin-sealed peptide nanoparticles was analyzed by MTT in HeLa and SW480 cells. The HeLa and sw480 cells were divided into 1.0 × 10 ⁴ cells and stabilized in a 96-well micro dish for 24 hours. Prepared HeLa and SW480 cells were treated with doxorubicin (Dox) and doxorubicin-sealed peptide nanoparticles (LL-Dox and DD-Dox) with 0.25 to 2 μM, and after 48 hours, MTT assay was performed on SW480 cells. After 72 hours, MTT assay was performed on HeLa cells.

As a result, as shown in Figure 8 (A), it can be seen that the survival rate (%) of sw480 cells was reduced to about 50% level, as shown in Figure 8 (B), the cell survival rate in HeLa cells It was confirmed that (%) decreased to about 25%. On the other hand, when treated with doxorubicin alone, it was found that the anticancer activity against cancer cells was minimal.

Example  12. Zebrafish  In the model Doxorubicin  Confirmation of anticancer effect of sealed peptide nanoparticles

The anticancer effect of the doxorubicin-sealed peptide nanoparticles was confirmed in the zebrafish model. The zebrafish eggs were incubated in a fish incubator for 2 days and the eggshell was removed. Meanwhile, SW480 cells were treated with tracker green CMFDA and stained through cell culture for 30 minutes, and then obtained using trypsin-EDTA, and washed twice with PBS containing 10% FBS. The thus prepared SW480 cells were micro-injected into zebrafish larvae. The zebrafish larvae were then dispensed into 96-well microplates and allowed to stabilize for 2 days. Then, doxorubicin and doxorubicin-sealed LL and DD peptide nanoparticles were injected and stabilized again for 2 days, followed by anesthetization of zebrafish larvae with 0.04% tricaine and analyzed by Reika's fluorescence microscope.

As a result, as shown in FIG. 9, green fluorescence was confirmed in zebrafish injected with the cancer cell sw480, and when injected with doxorubicin alone, the green fluorescent cancer cells remained and exhibited orange fluorescence. Cancer cells were almost disappeared when -Dox was injected, and green fluorescence was much removed in the case of DD-Dox compared to doxorubicin alone, but it was confirmed that it was weaker than LL-Dox.

<110> The Industry & Academic Cooperation in Chungnam National University <120> Drug delivery system comprising enzyme-responsive amphiphilic          peptide <130> PN18213 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> RH- [GFLG] 1 <400> 1 Arg His Gly Phe Leu Gly   1 5 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> RH- [GFLG] 2 <400> 2 Arg His Gly Phe Leu Gly Gly Phe Leu Gly   1 5 10 <210> 3 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> RH- [GFLG] 3 <400> 3 Arg His Gly Phe Leu Gly Gly Phe Leu Gly Gly Phe Leu Gly   1 5 10

Claims (6)

A drug delivery system comprising a peptide consisting of the amino acid sequence represented by Formula 1 below:
[Formula 1]
Arginine (R) -histidine (H)-[glycine (G) -leucine (L) -phenylalanine (F) -glycine (G)] _n
In the general formula 1, n is an integer from 1 to 10. According to claim 1, wherein the peptide is self-assembled (self-assembly) is located in the amino acid sequence of the hydrophilic arginine (R) -histidine (H) outward, glycine (G)-leucine (L) of the hydrophobic residue inwardly -Phenylalanine (F)-glycine (G) drug delivery system, characterized in that to form a nanoparticle in the form of micelles in which the amino acid sequence is located. The drug of claim 1, wherein the chirality of the amino acids forming the peptide is ^L RH- ^L (GFLG) _n , ^L RH- ^D (GFLG) _n or ^D RH- ^L (GFLG) _n . Carrier. The drug delivery system according to claim 2, wherein a hydrophobic anticancer agent is sealed inside the amino acid of the hydrophobic residue of the nanoparticle. 5. The drug delivery system according to claim 4, wherein the hydrophobic anti-cancer agent is doxorubicin. The drug delivery system of claim 1, wherein the peptide is cleaved by a cathepsin B enzyme.

Images