CN117946219A - Ring-shaped human leukemia cell targeting membrane-penetrating peptide and preparation method and application thereof - Google Patents

Abstract

The invention discloses a targeting membrane-penetrating peptide of a ring-shaped human leukemia cell, a preparation method and application thereof, and belongs to the technical field of tumor targeting treatment. The targeting membrane penetrating peptide is obtained by modifying a non-key amino acid site (CGFYWLRSC) of C9C, and specifically comprises the following steps: and cyclizing the non-key amino acid locus of the C9C after single-point mutation, multi-point mutation or D-type amino acid substitution to obtain the targeted membrane penetrating peptide of the annular human leukemia cells. The targeting membrane penetrating peptide has the capability of leukemia cell targeting and membrane penetration, basically does not influence the cell viability of K562 cells, KU812 cells, CCRF-CEM cells and THP-1 cells, can be used for preparing antitumor drugs (in the aspects of targeting delivery and membrane penetration), has good application prospect in preparing drugs for targeting human chronic myelogenous leukemia cells, human peripheral blood basophilic leukemia cells and human acute T lymphocyte leukemia cells, and can be used as another important field of C9C series polypeptide development.

Description

Ring-shaped human leukemia cell targeting membrane-penetrating peptide and preparation method and application thereof

Technical Field

The invention belongs to the technical field of tumor targeted therapy, and particularly relates to a targeting membrane-penetrating peptide for a ring-shaped human leukemia cell, and a preparation method and application thereof.

Background

Leukemia is a malignant clonal disease of hematopoietic stem cells, and is one of the major malignant tumors that endanger humans. Clonal leukemia cells accumulate in bone marrow and other hematopoietic tissues due to proliferation control, differentiation disorders, apoptosis resistance, and other mechanisms, and infiltrate other non-hematopoietic tissues and organs while inhibiting normal hematopoietic function. Different degrees of anemia, hemorrhage, infectious fever, hepatopathy, splenomegaly, lymphadenopathy and bone pain are seen clinically.

The leukemia is treated by adopting a drug targeting treatment strategy, so that the efficiency of leukemia cell targeting treatment can be improved. Karjalainen and the like, and a nine peptide CGFYWLRSC (C9C for short) is obtained by screening a combined phage polypeptide library, and the C9C can be used as a research direction (Karjalainen K,Jaalouk DE,Bueso-Ramos CE,et al.Targeting neuropilin-I in human leukemia and lym-phoma[J].Blood,2011,117(3):920-927.doi:10.1182/blood-2010-05-282921). of leukemia targeted therapeutic ligand because the nine peptide CGFYWLRSC can be combined with different leukemia cell lines and bone marrow samples from patients, but the problem of low membrane permeability of the C9C in the leukemia targeted therapeutic ligand is solved, so that the leukemia therapeutic efficiency of the C9C is seriously influenced. At present, few reports about modification of C9C are provided, and reports about improvement of C9C targeting membrane permeability are not provided.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a cyclic human leukemia cell targeting membrane penetrating peptide, a preparation method and application thereof, and solve the problem of low membrane permeability in the C9C targeting leukemia cell process.

In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:

The invention discloses a targeting membrane-penetrating peptide of a ring-shaped human leukemia cell, which is obtained by mutating and cyclizing amino acid of a C9C non-key site; the sequence of the C9C is shown as SEQ ID NO.1, and the amino acid of the non-key site is L-cysteine at the first position, glycine at the second position, tyrosine at the fourth position, tryptophan at the fifth position and L-cysteine at the ninth position of the C9C; the cyclization is an end-to-end cyclization.

Preferably, the mutation is a single point mutation, a multiple point mutation or a D-form amino acid substitution.

Further preferably, the mutation introduces an amino acid that is one or more of arginine, phenylalanine, and benzothiophene alanine.

Preferably, the sequence of the targeting membrane penetrating peptide of the annular human leukemia cells is shown as SEQ ID NO. 2-SEQ ID NO. 15.

The invention also discloses a preparation method of the cyclic human leukemia cell targeting membrane-penetrating peptide, which is prepared by an artificial solid phase synthesis method based on FMOC amino acid.

The invention also discloses application of the annular human leukemia cell targeting membrane penetrating peptide in preparation of an anti-tumor pharmaceutical preparation.

Preferably, the antitumor drug is an antitumor drug.

Further preferably, the anti-leukemia drug is a drug targeting human chronic myelogenous leukemia cells, human peripheral blood basophilic leukemia cells, or human acute T-lymphoblastic leukemia cells.

Preferably, the concentration of the cyclic human leukemia cell targeting transmembrane peptide is 1.0X10- ^-2 to 20.0. Mu. Mol/L.

The invention also discloses an anti-leukemia composition, which comprises other active ingredients with anti-leukemia effect, the cyclic human leukemia cell targeting membrane-penetrating peptide and one or more pharmaceutically acceptable carriers or excipients.

Compared with the prior art, the invention has the following beneficial effects:

The invention provides a targeting membrane-penetrating peptide of a cyclic human leukemia cell, which is obtained by mutating and cyclizing a non-key amino acid site of C9C. The cyclic human leukemia cell targeting transmembrane peptide can specifically target tumor cells, obviously increase the transmembrane property of K562 cells, KU812 cells and CCRF-CEM cells, has high action efficiency, can improve the stability of the transmembrane peptide, and reduces the dosage of the transmembrane peptide. Particularly, the cyclic-C9C-R in the cyclic human leukemia cell targeting transmembrane peptide can obviously improve the membrane permeability of C9C, and meanwhile, the targeting of C9C is maintained, and the uptake in K562 cells and KU812 cells is obviously higher than that in THP1 cells; secondly, the permeable membranes in K562 cells and KU812 cells show concentration dependence and time dependence, and the higher the concentration is, the higher the uptake in the cells is; uptake in cells varies with time; thirdly, the cell line has no obvious cytotoxicity on K562 cells, KU812 cells, CCRF-CEM cells and THP1 cells. Therefore, the human leukemia cell targeting membrane penetrating peptide can be used for preparing antitumor drugs (in the aspects of targeting delivery and membrane penetration), has good application prospect in preparing targeted human chronic myelogenous leukemia cells, human peripheral blood basophilic leukemia cells and human acute T lymphocyte leukemia cells, and can be used as another important field of C9C series polypeptide development.

The preparation method of the annular human leukemia cell targeting membrane-penetrating peptide provided by the invention is prepared through chemical synthesis, has mature technical line, is easy for mass preparation, has good stability, and has potential application prospect in research and development of less human leukemia cell targeting membrane-penetrating peptides at present.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be considered as limiting the scope.

FIG. 1 is a flow chart of a method for synthesizing linear human leukemia cell-targeted transmembrane peptide;

FIG. 2 is a graph showing the selective uptake results of the cyclic human leukemia cell-targeted transmembrane peptide on K562 cells;

FIG. 3 is a graph showing the selective uptake results of cyclic human leukemia cell-targeted transmembrane peptide on KU812 cells;

FIG. 4 is a graph showing the selective uptake of cyclic human leukemia cell-targeted transmembrane peptide into CCRF-CEM cells;

FIG. 5 is a graph of the concentration dependence and time dependent uptake results of Cyclo-C9C-R on K562 cells; wherein: (a) is a concentration-dependent flow chart, (b) is a concentration-dependent quantitative chart, (c) is a time-dependent flow chart, and (d) is a time-dependent quantitative chart;

FIG. 6 is a graph of the concentration dependence and time dependent uptake of Cyclo-C9C-R on KU812 cells; wherein: (a) is a concentration-dependent flow chart, (b) is a concentration-dependent quantitative chart, (c) is a time-dependent flow chart, and (d) is a time-dependent quantitative chart;

FIG. 7 is a graph of the concentration-dependent and time-dependent uptake of Cyclo-C9C-Rf (3 Bta) on CCRF-CEM cells; wherein: (a) is a concentration-dependent flow chart, (b) is a concentration-dependent quantitative chart, (c) is a time-dependent flow chart, and (d) is a time-dependent quantitative chart;

FIG. 8 is a graph showing the results of the targeted uptake of Cyclo-C9C-R into tumor cells (K562 cells) and control cells (THP 1 cells);

FIG. 9 is a graph showing the results of the targeted uptake of Cyclo-C9C-R into tumor cells (KU 812 cells) and control cells (THP 1 cells);

FIG. 10 is a graph showing the results of cytotoxicity analysis; wherein: (a) is a cytotoxicity map of Cyclo-C9C-R against K562 cells, (b) is a cytotoxicity map of Cyclo-C9C-R against KU812 cells, (C) is a cytotoxicity map of Cyclo-C9C-Rf (3 Bta) against CCRF-CEM cells, and (d) is a cytotoxicity map of Cyclo-C9C-Rf (3 Bta) against THP1 cells.

Detailed Description

The invention provides a targeting membrane-penetrating peptide of a ring-shaped human leukemia cell, which is obtained by mutating a non-key amino acid locus (CGFYWLRSC) of C9C (the sequence of which is shown as SEQ ID NO.1 in table 1) and then cyclizing the amino acid locus end to end, wherein the sequence of the targeting membrane-penetrating peptide of the ring-shaped human leukemia cell is shown as SEQ ID NO. 2-SEQ ID NO.15 in table 1.

Wherein the mutation is single-point mutation, multi-point mutation or D-type amino acid substitution, and the amino acid introduced by the mutation is one or more of arginine, phenylalanine and benzothiophene alanine.

Table 1 sequence listing

The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the teachings of the present application, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

The following examples use instrumentation conventional in the art. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The following examples used various starting materials, unless otherwise indicated, were conventional commercial products, the specifications of which are conventional in the art. In the description of the present invention and the following examples, "%" indicates weight percent unless otherwise specified.

1. Experimental materials

1. Experimental drugs and reagents: RINK AMIDE-MBHA resin (carbofuran), fmoc protected amino acid, piperidine (national medicine group), DMF (Kemeou chemical reagent), DCM (Kemeou chemical reagent), HATU (Pico medicine), HOBt (Pico medicine), DIPEA (Pico medicine), pyBop (Pico medicine), TFA (Aldine), TIS (Aldine), 1, 3-dimethoxybenzene (Aldine), DODT (Aldine), chromatographic acetonitrile (Aldine), PBS buffer (CORNING), RPMI 1640 (CORNING), penicillin-streptomycin solution (diabody) (MCE), fetal bovine serum (NEWZERUM), trypsin (CORNING), DMSO (MP Biomedicals), MTT (Solarbio).

2. Experimental cells: k562 (human chronic myelogenous leukemia cells), KU812 (human peripheral blood basophilic leukemia cells), CCRF-CEM (human acute T-lymphocytic leukemia cells), and THP-1 (human leukemia monocytic cell line).

2. Synthetic cyclic human leukemia cell targeting membrane-penetrating peptide

As shown in FIG. 1, cyclic human leukemia cell-targeted transmembrane peptides were synthesized using FMOC amino acid-based solid phase synthesis.

RINK AMIDE-MBHA resin (50.0 mg,0.32 mmol.g ^-1, 0.016 mmol) was taken, 5mL DMF/DCM (1:1) was added and swollen for 45min, and the filtrate was removed by suction filtration under reduced pressure. Adding 20% piperidine/DMF to deprotect the group for 10min, filtering under reduced pressure to remove the filtrate, repeating the above operation twice, and completely removing the Fmoc protecting group at the N end of RINK AMIDE-MBHA resin. After suction filtration, the mixture was washed thoroughly with DCM/DMF. Fmoc-Glu-Oall (32.7 mg,0.08 mmol), HATU (30.3 mg,0.08 mmol), HOBt (12.2 mg,0.08 mmol) and DIPEA (27.84 mg,0.08 mmol) were added to the column, reacted for 45min, suction filtered and washed thoroughly with DCM/DMF. Amino acids are sequentially linked according to the sequence sequences shown in SEQ ID No.2 to SEQ ID No.15 in Table 1, respectively. Cyclization was performed with PyBop (41.6 mg,0.08 mmol), HOBt (12.2 mg,0.08 mmol), DIPEA (27.8 mg,0.08 mmol) twice for 3h and overnight, respectively. After the synthesis is completed, a cleavage reagent is added to cleave the product from the resin. And adding DCM into a synthesis tube, fully washing the product, collecting the liquid, volatilizing the solvent, adding glacial ethyl ether, centrifuging at 8000r/min for 20min, discarding the supernatant, adding the glacial ethyl ether again, slightly swirling for washing, centrifuging at 8000rpm for 5min, discarding the supernatant to obtain a crude product of the annular human leukemia cell targeted membrane penetrating peptide, and storing in a refrigerator at-80 ℃.

After the structure of the obtained crude product of the targeting membrane-penetrating peptide of the annular human leukemia cells is confirmed by MALDI-TOF (Hexin instrument CMI-1600), the crude product of the targeting membrane-penetrating peptide of the annular human leukemia cells is separated and purified by HPLC (Shimadzu LC-2030 plus), and the targeting membrane-penetrating peptide of the annular human leukemia cells is obtained.

3. Detection of membrane permeability of cyclic human leukemia cell targeting transmembrane peptide and leukemia cell targeting

And (3) carrying out activity screening on the membrane permeability, the membrane permeability time dependence and the membrane permeability concentration dependence of the prepared annular human leukemia cell targeting membrane penetrating peptide and the targeting of leukemia cells by adopting a flow cytometry. The specific method comprises the following steps:

1. cell inoculation: cells in good growth and logarithmic phase were collected, centrifuged at 1200rpm for 5min, the supernatant was discarded, and after resuspension of the cells with medium, the cells were inoculated in a 1.5X10 ⁵ cells/well volume into a 6-well cell culture plate at a volume of 1 mL/well. After inoculation, the cells were incubated in a constant temperature incubator at 37℃with 5% CO ₂ for 24h.

2. Preparing a sample solution to be tested: and (3) gradually diluting the prepared polypeptide mother solution with PBS to obtain 1 mu mol/L, 5 mu mol/L and 10 mu mol/L of sample solution to be detected.

3. Administration: 110. Mu.L of the sample solution to be tested was added to each well. After mixing by gentle shaking, the cells were placed in an incubator for incubation for 2h.

4. Cell collection: taking out the 6-hole plate, centrifuging at 1200rpm for 5min to collect cells, sucking off the culture medium, washing the cells twice with PBS (centrifuging at 1000rpm for 5min and at 800rpm for 5min in sequence), and discarding the supernatant to obtain a cell sample.

5. And (3) detection: 300. Mu.L of PBS was added to each tube, and the cells were dispersed by gentle vortexing. Cell uptake was detected by flow cytometry as soon as possible.

The membrane permeability, membrane permeation time dependence, membrane permeation concentration dependence and targeting of the cyclic human leukemia cell targeting membrane-penetrating peptide are shown in fig. 2 to 9.

As can be seen from FIGS. 2 and 3, the membrane permeability of Cyclo-C9C-R (sequence shown as SEQ ID NO.5 in Table 1) to K562 cells and KU812 cells was significantly increased to 9.24 and 7.99 times that of C9C, respectively, compared to C9C. As can be seen from FIG. 4, the membrane permeability of Cyclo-C9C-Rf (3 Bta) (sequence shown as SEQ ID NO.14 in Table 1) to CCRF-CEM cells was significantly increased to 4.10 times that of C9C compared to C9C. The membrane permeability modification of C9C is shown to be beneficial to improving the membrane permeability of C9C to K562 cells, KU812 cells and CCRF-CEM cells.

As can be seen from FIGS. 5 and 6, the permeabilization of Cyclo-C9C-R in K562 and KU812 cells shows a concentration dependency, the higher the concentration, the higher the uptake in the cells. The permeant of Cyclo-C9C-R in K562 cells and KU812 cells varied with time. In K562 cells, the higher the uptake in the cells, the less the uptake in the cells from 120 to 240min, at 5 to 120min, the longer the time. In KU812 cells, the higher the uptake in the cells, the less the uptake in the cells, at 5-30 min, the longer the time. As can be seen from FIG. 7, the permeabilization of Cyclo-C9C-Rf (3 Bta) in CCRF-CEM cells shows a concentration dependency, the higher the concentration, the higher the uptake in the cells. The permeabilization of Cyclo-C9C-Rf (3 Bta) in CCRF-CEM cells is time dependent. The uptake in the cells is substantially unchanged at 5-30 min, and is higher at 60-240 min with prolonged time.

As can be seen from fig. 8 and 9, the altered cyclic human leukemia cell-targeting transmembrane peptide remained targeted to leukemia cells. The uptake difference of Cyclo-C9C-R in leukemia cells (K562 and KU812 cells) and control cells (THP 1 cells) can reach 33.30 and 11.74 times, and compared with C9C, the uptake difference is improved to a certain extent (2.22 times and no obvious targeting). In addition, the Cyclo-C9C-RF and Cyclo-C9C-R (3 Bta) had a more pronounced targeting on K562 cells, and the Cyclo-C9C-RF and Cyclo-C9C-F (3 Bta) had a more pronounced targeting on KU812 cells.

4. Detection of cytotoxicity of cyclic human leukemia cell-targeted transmembrane peptide

And determining cytotoxicity of the targeting transmembrane peptide of the annular human leukemia cells on tumor cells and control cells by adopting an MTT method. The specific method comprises the following steps:

1. Cell inoculation: k562 cells, KU812 cells, CCRF-CEM cells and THP-1 cells in the growth exponential phase were diluted with RPMI 1640 medium to a cell solution on the order of 10 ⁴ cells/ml, inoculated in parallel into 96-well plates (2000-4000 cells/well) with a volume of 180. Mu.L per well, and cultured at 37℃under 5% CO ₂ for 12 hours.

2. Administration: each well was charged with either a different concentration of Cyclo-C9C-R20. Mu.L or Cyclo-C9C-Rf (3 Bta) 20. Mu.L to give the final concentration of compound in the well: 1.0X10 ^-2μmol/L,1.0×10^-1. Mu. Mol/L, 1.0. Mu. Mol/L, 5.0. Mu. Mol/L, 10.0. Mu. Mol/L, 20.0. Mu. Mol/L, 3 wells per concentration, 6 wells per negative control, cells per well without compound, and cultivation at 37℃under 5% CO ₂ was continued for 48h.

3. And (3) detection: adding 22 mu L of MTT (5 mg/mL) into each well to obtain the final concentration of MTT in each well as 0.5mg/mL, culturing at 37 ℃ under 5% CO ₂ for 4 hours, carefully sucking out the supernatant, adding 150 mu L of DMSO into each well, oscillating for 10 minutes, measuring the ultraviolet absorption value (OD value) at 490nm of each well by using an ELISA (enzyme-linked immunosorbent assay), and then calculating the cell inhibition rate;

The cell inhibition rate was calculated as:

Inhibition% = (control well mean OD value-drug group mean OD value)/control well mean OD value x 100%;

4. Experimental results: as can be seen from FIG. 10, cytotoxicity analysis showed that the compound Cyclo-C9C-R had no significant cytotoxicity against K562 cells and KU812 cells in the concentration range of 1.0X10 ^-2. Mu. Mol/L to 20.0. Mu. Mol/L. When the concentration of Cyclo-C9C-R administered was 20. Mu. Mol/L or less (the concentration used for measurement of the permeabilities and targeting was 5. Mu. Mol/L), it had substantially no effect on the cell viability of K562 cells and KU812 cells. The compound Cyclo-C9C-Rf (3 Bta) had no significant cytotoxicity to CCRF-CEM cells and THP-1 cells. When the administration concentration of Cyclo-C9C-R is 20 mu mol/L or less (the concentration used for measuring the membrane permeability and targeting is 5 mu mol/L), the Cyclo-C9C-R does not basically influence the cell viability of CCRF-CEM cells and THP-1 cells, and the safety of the targeting membrane penetrating peptide of the ring-shaped human leukemia cells is proved.

From the above, it is known that the cyclic human leukemia cell targeting transmembrane peptide can be used for preparing antitumor drugs (in the aspects of targeting delivery and transmembrane), has certain leukemia cell targeting and transmembrane capability, and basically does not influence the cell viability of K562 cells, KU812 cells, CCRF-CEM cells and THP-1 cells. The membrane permeability modification can expand the structural diversity of the C9C series polypeptide, and an activity test shows that the introduction of arginine (R), phenylalanine (F), benzothiophene alanine (3 Bta) and cyclization plays an important role in the membrane permeability modification of C9C, can increase the application scene of the C9C series polypeptide, and can be used as another important field of the development of the C9C series polypeptide.

The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims (10)

1. The targeting membrane penetrating peptide of the ring-shaped human leukemia cells is characterized by being obtained by mutating and cyclizing amino acids of non-key sites of C9C; the sequence of the C9C is shown as SEQ ID NO.1, and the amino acid of the non-key site is L-cysteine at the first position, glycine at the second position, tyrosine at the fourth position, tryptophan at the fifth position and L-cysteine at the ninth position of the C9C; the cyclization is an end-to-end cyclization.

2. The cyclic human leukemia cell-targeted transmembrane peptide of claim 1, wherein the mutation is a single point mutation, a multiple point mutation or a D-amino acid substitution.

3. The cyclic human leukemia cell-targeted transmembrane peptide of claim 2, wherein the mutation introduces an amino acid that is one or more of arginine, phenylalanine and benzothiophene alanine.

4. The cyclic human leukemia cell targeting transmembrane peptide according to claim 1, wherein the sequence of the cyclic human leukemia cell targeting transmembrane peptide is shown in SEQ ID NO. 2-SEQ ID NO. 15.

5. The method for preparing the cyclic human leukemia cell targeting transmembrane peptide according to any one of claims 1 to 4, wherein the cyclic human leukemia cell targeting transmembrane peptide is prepared by an artificial solid phase synthesis method based on FMOC amino acid.

6. Use of the cyclic human leukemia cell-targeted transmembrane peptide of any one of claims 1-4 in the preparation of an anti-tumor pharmaceutical formulation.

7. The use according to claim 6, wherein the antineoplastic agent is an antineoplastic agent.

8. The use according to claim 7, wherein the anti-leukemia drug is a drug targeting human chronic myeloid leukemia cells, human peripheral blood basophilic leukemia cells or human acute T-lymphoblastic leukemia cells.

9. The use according to claim 6, wherein the concentration of the cyclic human leukemia cell-targeted transmembrane peptide is 1.0X10- ^-2 to 20.0. Mu. Mol/L.

10. An anti-leukemia composition comprising an additional active ingredient having anti-leukemia effect, a cyclic human leukemia cell-targeting transmembrane peptide of any one of claims 1-4, and one or more pharmaceutically acceptable carriers or excipients.