Preparation and application of artificial peptide transporter for promoting transmembrane delivery of peptide drugs

Abstract

The invention provides preparation and application of an artificial peptide transporter for promoting transmembrane delivery of peptide drugs, the structural general formula of the artificial peptide transporter is as follows: # imgabs0 #. The compound involved in the invention is composed of three parts: (1) a bisamide structure as an anion binding unit which is bound with carboxyl anions of peptide molecules; (2) crown ether is used as a cation binding unit and is bound with amino positive ions of the peptide molecules; and (3) hydrophobic side chain: connecting the anion binding unit with the cation binding unit, so that the whole molecule can be used as molecular tweezers to form a stable compound with peptide drugs, and the stable compound is coated in a hydrophobic environment to promote transmembrane transportation of the peptide drugs. The obtained compound is cheap in raw material and easy to synthesize, the bioavailability of polypeptide drugs is remarkably improved, and the compound shows remarkable synergistic anti-cancer activity in in-vitro and in-vivo experiments, so that the compound has great potential as a pharmaceutical adjuvant.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the design and synthesis of an artificial peptide transporter and its application in promoting transmembrane delivery of peptide drugs, especially in anti-cancer treatment. Background Art

[0002] Peptide drugs have shown great application potential in the fields of anticancer, anti-infection and metabolic regulation due to their good biological activity, strong specificity and low toxicity. However, the delivery of peptide drugs faces many challenges in drug development, mainly due to their unique chemical properties.

[0003] First, peptide molecules usually have large volumes and polar functional groups, which result in extremely low permeability on non-polar barriers such as cell membranes. Secondly, most peptide drugs exist in the form of zwitterions under physiological conditions and are prone to aggregation in aqueous solutions, further reducing their ability to penetrate membranes. Even for shorter-chain neutral peptides, the absorption rate and bioavailability in the body are still greatly limited due to the lack of a natural transport mechanism. In addition, during the delivery process, peptide drugs are susceptible to rapid degradation by gastrointestinal enzymes, nonspecific binding of plasma proteins, and rapid clearance by the kidneys, which significantly shortens their half-life in the body. These factors together limit the clinical application of peptide drugs, which often require higher doses to achieve the desired therapeutic effect, while increasing toxic side effects and production costs.

[0004] In response to the above problems, the research focus in recent years has gradually shifted to the development of artificial transport systems that can effectively deliver peptide drugs. By simulating the functions of natural transporters, artificial peptide transporters can effectively protect peptide drugs from degradation, while significantly improving their transmembrane transport capacity. However, the current types of artificial peptide transport systems are extremely limited, and the delivery of peptides to electrical centers still faces problems such as low efficiency, poor biocompatibility, and complex preparation. New molecular design strategies are urgently needed to address these challenges.

[0005] In response to the above-mentioned difficulties, the present invention proposes a new artificial peptide transporter, which integrates the transport functions of anions and cations. By assembling with peptide drugs to form an efficient transmembrane transport system, it can significantly improve the transmembrane transport efficiency and bioavailability of peptide drugs, and provide a new strategy for solving the problem of peptide drug delivery and absorption. Summary of the invention

[0006] The present invention provides an artificial peptide transporter for promoting the transmembrane transport of neutral peptide drugs. The transporter integrates anion binding function, cation binding function and hydrophobic effect through molecular design, which can significantly improve the transmembrane transport efficiency of neutral peptide drugs, enhance their bioavailability and therapeutic effect, and especially shows significant advantages in the field of anti-cancer.

[0007] The present invention aims to develop an efficient artificial peptide transporter to meet the following challenges: first, the cell membrane penetration problem of peptide compounds, by coating the polar part of the peptide molecule in a hydrophobic structure to overcome its permeability problem on the cell membrane; second, to improve the bioavailability of peptide drugs by enhancing their stability and absorption efficiency in the body.

[0008] The technical solution adopted by the present invention is mainly divided into three parts: one is to prepare artificial peptide transporters, the second is to detect the transmembrane transport activity of artificial peptide transporters for peptides, and the third is to evaluate the synergistic anti-cancer activity of the series of compounds and peptide drugs such as tyroserleutin.

[0009] The general structure of the artificial peptide transporter with polypeptide transport activity described in the present invention is as follows:

[0010]

[0011] The compound involved in the present invention is composed of three parts, namely: (1) a diamide structure as an anion binding unit, which binds to the carboxyl anion of the peptide molecule; (2) a crown ether as a cation binding unit, which binds to the amino cation of the peptide molecule; and (3) a hydrophobic side chain: the anion binding unit is connected to the cation binding unit, so that the entire molecule can serve as a molecular tweezer to form a stable complex with the peptide drug, and encapsulate it in a hydrophobic environment to promote its transmembrane transport.

[0012] The synthetic route of the target compound is as follows:

[0013]

[0014] The specific synthesis process is as follows: (1) Preparation of anion binding unit: prepare the target anion binding unit through the condensation reaction of fluorinated anhydride and amine; (2) Preparation of cation binding unit: synthesize the crown ether modified unit through alkylation reaction; (3) Assembly of transporter: covalently connect the anion and cation binding units to the hydrophobic chain through chemical bonds to form a complete molecule.

[0015] Furthermore, the compound obtained by the above preparation method is used in the anti-cancer treatment of the polypeptide drug tyroserleutide.

[0016] The beneficial effects of this application are:

[0017] 1. Efficient transport: The transporter of the present invention effectively improves the transmembrane permeability of neutral peptide drugs through molecular design, thereby greatly improving their bioavailability.

[0018] 2. Enhanced drug efficacy: Significantly improve the bioavailability of peptide drugs, and show significant synergistic anti-cancer activity in both in vitro and in vivo experiments.

[0019] 3. Good biocompatibility: It adopts a simple and green synthesis route with excellent biocompatibility, which facilitates structural optimization and clinical transformation.

[0020] 4. Wide applicability: It is not only suitable for the delivery of tyroserleutide (YSL), but can also be extended to other peptide drugs, with great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The fluorescence intensity changes of compound 1 and compound 4 at different concentrations of calcein, and the corresponding EC values ​​of compound 1 and compound 4 to these changes 50 value.

[0022] Figure 2 For Cu 2+ -Calculated observed influx rate constant (K) of YSL at different concentrations of compound 1 in the calcein liposome fluorescence assay obs );

[0023] Figure 3 Confocal images of MCF-7 cells treated with YSL-py in the presence of different concentrations of compound 1 followed by staining with rhodamine phalloidin;

[0024] Figure 4 Quantitative analysis of the enhanced cellular uptake of YSL-py after adding compound112 h using flow cytometry;

[0025] Figure 5 is the IC of YSL against MCF-7 cells in the presence of different concentrations of compound 1 50 value;

[0026] Figure 6 The measurement of MCF-7 tumor volume in nude mice with different treatments (n=5 in each group);

[0027] Figure 7 To perform histological analysis of tumors after different treatments by H&E, Ki-67, and TUNEL staining;

[0028] Figure 8 Figure 3 is a graph showing the body weight changes of mice treated with different methods. DETAILED DESCRIPTION

[0029] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. The experimental methods in the following embodiments, unless otherwise specified, are conventional methods, and the materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0030] Embodiment one:

[0031] Preparation of compounds:

[0032]

[0033]

[0034] 1) Preparation of intermediate 1a:

[0035] 1 H NMR (600 MHz, CDCl 3 )δ7.60(s,2H),7.15(s,1H),2.35(s,6H),1.59(s,9H). 13 C NMR (151 MHz, CDCl 3 )δ166.2,137.8,134.1,131.9,127.2,80.8,28.2,21.2.MS-ESI:calculatedfor[M+H] + (C 13 H 19 O 2 ): m / z 207.1385, found: m / z 207.1377.

[0036] 2) Preparation of intermediate 1b:

[0037] NMR(600MHz, CDCl3)δ7.93(d,J=1.7Hz,2H),7.59(t,J=1.8Hz,1H),4.50(s,4H),1.60(s,9H).13C NMR (151MHz, CDCl3) δ 138.8, 133.5, 131.2, 129.9, 81.8, 32.2, 28.2.MS-ESI: calculated for [M+H]+ (C13H17Br2O2): m / z 364.9570, found: m / z 364.9536.

[0038] 3) Preparation of intermediate 1c:

[0039] MHz,CDCl 3 )δ7.90(s,2H),7.46(s,1H),4.43(s,4H),1.61(s,9H). 13 C NMR (151 MHz, CDCl 3 )δ164.8,136.5,133.3,131.5,128.9,81.8,54.2,28.2.MS-ESI:calculated for[M+Na] + (C 13 H 16 N 6 O 2 Na): m / z 311.1232, found: m / z 311.1224.

[0040] 4) Preparation of intermediate 1d:

[0041]

[0042] 5) Preparation of target compound 3F:

[0043] 4H),1.58(s,9H). 13 C NMR (151 MHz, CDCl 3 )δ164.8,157.9,137.2,133.4,130.8,128.3,82.1,43.4,28.1.MS-ESI:calculated for[M+Na] + (C 17 H 18 F 6 N 2 O 4 Na): m / z 451.1068, found: m / z451.1056.

[0044] 6) Preparation of target compound 5F:

[0045] m / z 551.0992.

[0046] 7) Preparation of target compound 7F:

[0047]

[0048] 8) Preparation of intermediate 5F-COOH:

[0049] 6.0Hz,4H). 13 C NMR (151 MHz, DMSO-d 6 )δ167.3,157.6,157.4,157.3,138.9,131.7,130.8,127.7,42.8.MS-ESI:calculated for[M+Na] + (C 15 H 10 F 10 N 2 O 4 Na): m / z 495.0374, found: m / z 495.0366.

[0050] 9) Preparation of intermediate 7F-COOH:

[0051]

[0052] 10) Preparation of target compound C8:

[0053] The product was purified by flash column chromatography to obtain pure product C8. Yield: 88 mg, 35%. 1 H NMR (600 MHz, CDCl 3 )δ7.65(d,J=8.4Hz,1H),7.53(s,1H),6.86(d,J=8.4Hz,1H),4.27(t,J=6 .7Hz,2H),4.21(dt,J=7.1,2.5Hz,4H),3.94(dtd,J=10.5,4.4,1.8Hz,4H ),3.77(dq,J=4.4,2.5,2.0Hz,4H),3.75–3.67(m,8H),1.75(p,J=6.9Hz, 2H),1.42(q,J=7.6Hz,2H),1.34–1.27(m,8H),0.88(t,J=6.9,1.6Hz,3H). 13 C NMR (151 MHz, CDCl 3 )δ166.5,152.8,148.2,123.7,123.1,114.1,111.9,70.9,70.8,70.7,70.6,69.4,6 9.3,68.9,68.7,65.0,31.8,29.3,29.2,28.8,26.9,22.7,14.1.MS-ESI:calculated for[M+Na] + (C 25 H 40 O 8Na):m / z 491.2621,found:m / z491.2609.

[0054] 11) Preparation of target compound C10:

[0055] 2.4Hz,4H),3.78(tq,J=4.4,2.5Hz,4H),3.74–3.67(m,8H),1.81–1.68(m,2H),1.42 (dd,J=11.4,4.8Hz,2H),1.28(dq,J=12.8,6.4Hz,12H),0.88(t,J=7.0,2.1Hz,3H). 13 C NMR (151 MHz, CDCl 3 )δ166.5,152.8,148.2,123.7,123.2,114.2,111.9,70.9,70.8,70.7,70.6,69.4,69.3 ,69.0,68.8,65.0,31.9,29.6,29.4,29.3,28.8,26.1,22.7,14.2.MS-ESI:calculated for[M+Na] + (C 27 H 44 O 8 Na): m / z 519.2934, found: m / z 519.2920.

[0056] 12) Preparation of target compound C12:

[0057] 3.77(dq,J=4.6,2.0Hz,4H),3.75–3.67(m,8H),1.75(p,J=6.9Hz,2H),1.45–1.38( m,2H),1.35(dd,J=10.4,4.4Hz,2H),1.27(d,J=8.1Hz,14H),0.88(t,J=6.9Hz,3H). 13 C NMR (151 MHz, CDCl 3 )δ166.4,152.9,148.3,123.8,123.2,114.6,112.2,71.0,70.8,70.7,69.5,69.4,69.2 ,68.9,65.0,32.0,29.7,29.6,29.4,29.3,28.8,26.1,22.7,14.2.MS-ESI:calculated for[M+Na] + (C 29 H48 O 8 Na): m / z 547.3247, found: m / z 547.3232.

[0058] 13) Preparation of target compound C14:

[0059] 3.94(dt,J=11.5,4.4Hz,4H),3.78(dq,J=5.1,2.2Hz,4H),3.73–3.68(m,8H),1.74( p,J=6.9Hz,2H),1.45–1.39(m,2H),1.27(d,J=13.7Hz,20H),0.88(t,J=6.9Hz,3H). 13 C NMR (151 MHz, CDCl 3 )δ166.5,152.8,148.2,123.8,123.2,114.2,111.9,70.9,70.8,70.7,70.6,69.4,69.3 ,69.0,68.8,65.0,32.0,29.7,29.6,29.4,28.8,26.1,22.7,14.2.MS-ESI:calculated for[M+Na] + (C 31 H 52 O 8 Na): m / z 575.3560, found: m / z 575.3548.

[0060] 14) Preparation of target compound BrC12:

[0061] 3.77(t,J=4.9Hz,4H),3.74–3.66(m,8H),3.42(s,2H),1.85(d,J=21.8Hz ,2H),1.73(d,J=14.4Hz,2H),1.35(d,J=6.7Hz,2H),1.33–1.18(m,12H). 13 C NMR (151 MHz, CDCl 3 )δ166.5,152.8,148.2,123.7,111.9,69.4,69.3,68.9,68.7,65.0,32.9,28.8,28.2,26.1.MS-ESI:calculated for[M+Na] + (C 29 H 47 B O 8Na): m / z 625.2347, found: m / z 625.2336。

[0062] 15) Preparation of the target compound BrC14:

[0063]

[0064] 1H), 6.86 (d, J = 8.4 Hz, 1H), 4.27 (t, J = 6.7 Hz, 2H), 4.22–4.19 (m, 4H), 3.94 (ddd, J = 9.3, 5.0, 3.3 Hz, 4H), 3.77 (q, J = 3.3, 1.9 Hz, 4H), 3.71 (dt, J = 6.6, 3.3 Hz, 4H), 3.69 (s, 4H), 3.41 (t, J = 6.8 Hz, 2H), 1.87–1.82 (m, 2H), 1.74 (q, J = 7.1 Hz, 2H), 1.41 (d, J = 7.6 Hz, 2H), 1.36–1.24 (m, 18H). 13 C NMR (151 MHz, CDCl 3 ) δ 166.5, 152.8, 148.2, 123.8, 123.2, 114.2, 111.9, 71.0, 70.9, 70.8, 70.7, 70.6, 69.4, 69.3, 69.0, 68.8, 65.0, 34.1, 32.9, 29.6, 29.5, 29.3, 28.8, 28.2, 26.1. MS-ESI: calculated for [M+Na] + (C 31 H 51 BrO 8 Na): m / z653.2600, found: m / z 653.2634。

[0065] 16) Preparation of the target compound compound1:

[0066] The reaction mixture was stirred at 25 °C for 24 h. The solvent was removed under vacuum, the crude product was dissolved in dichloromethane and washed with water, and the crude product was purified by flash column chromatography to give the pure product compound1. Yield 190 mg, 50%. 1 H NMR (600 MHz, CDCl 3)δ7.81(d,J=1.7Hz,2H),7.61(dd,J=8.4,2.0Hz,1H),7.56(d,J=6.2Hz,2H),7.45(d,J=2 .0Hz,1H),7.33(d,J=1.9Hz,1H),6.82(d,J=8.5Hz,1H),4.54(d,J=6.0Hz,4H),4.27(dt, J=13.3,6.7Hz,4H),4.19–4.13(m,4H),3.93–3.87(m,4H),3.74(td,J=4.8,2.6Hz,4H),3 .71–3.65(m,8H),1.73(q,J=7.1Hz,4H),1.41(q,J=7.6Hz,4H),1.27(d,J=18.8Hz,12H). 13 C NMR (151 MHz, CDCl 3 )δ166.6,165.8,157.9,152.7,148.1,137.6,131.8,131.0,128.2,123.7,123.1,113.9,111.7,70.7,70. 6,70.5,70.4,69.3,69.2,68.6,65.6,65.1,43.3,29.4,29.3,29.2,28.7,26.0,25.9.MS-ESI:calculated for[M+Na] + (C 44 H 56 F 10 N 2 O 12 Na): m / z1017.3566, found: m / z 1017.3537.

[0067] 17) Preparation of target compound compound 2:

[0068] =1.7Hz,2H),7.62(dd,J=8.4,2.0Hz,1H),7.47(d,J=2.0Hz,1H),7.36(d,J=4.7Hz,3 H),6.83(d,J=8.4Hz,1H),4.56(d,J=6.0Hz,4H),4.28(dt,J=18.8,6.7Hz,4H),4.19– 4.14(m,4H),3.91(dt,J=12.4,4.2Hz,4H),3.75(q,J=4.4Hz,4H),3.71–3.64(m,8H) ,1.74(ddt,J=10.7,7.0,2.9Hz,4H),1.43–1.40(m,4H),1.28(q,J=9.5,8.1Hz,16H). 13 C NMR (151 MHz, CDCl 3 )δ166.6,165.7,158.1,152.7,148.1,137.5,131.9,131.1,128.3,123.7,123.1,113.9,111.7,70.8,70.7,7 0.6,70.5,69.3,69.2,68.7,68.6,65.7,65.1,43.3,29.8,29.5,29.4,29.2,28.7,26.0.MS-ESI:calculated for[M+Na] + (C 46 H 60 F 10 N 2 O 12 Na): m / z1045.3879, found: m / z1045.3861.

[0069] 18) Preparation of target compound compound 3:

[0070] 7.63(dd,J=8.4,2.0Hz,1H),7.49(d,J=2.0Hz,1H),7.38(d,J=1.9Hz,1H),7.16(t,J=6.2Hz, 2H),6.84(d,J=8.4Hz,1H),4.58(d,J=6.0Hz,4H),4.29(t,J=6.8Hz,2H),4.26(t,J=6.7Hz,2 H),4.20–4.16(m,4H),3.94–3.90(m,4H),3.78–3.75(m,4H),3.72–3.69(m,4H),3.68(s,4H) ,1.74(ddd,J=10.1,7.1,3.5Hz,4H),1.41(ddd,J=9.2,4.6,2.4Hz,4H),1.30–1.25(m,12H). 13 C NMR (151 MHz, CDCl 3 )δ166.5,165.7,157.8,157.6,152.9,148.2,137.4,131.9,131.2,128.4,123.8,123.2,114.5,112.1,108.6,70.9,70.7,70 .6,69.5,69.3,69.0,68.8,65.6,65.0,43.4,29.7,29.4,29.3,29.2,28.7,28.6,26.0,25.9,22.7,14.1.MS-ESI:calculated for[M+K] + (C 46 H 56 F 14 N 2 O 12 K): m / z 1133.3242, found: m / z1133.3219.

[0071] 19) Preparation of target compound 4:

[0072] (600MHz,CDCl 3)δ7.83(d,J=1.7Hz,2H),7.61(dd,J=8.4,2.0Hz,1H),7.47(d,J=2.0Hz,1H),7.43(t,J=6 .2Hz,2H),7.36(d,J=1.9Hz,1H),6.82(d,J=8.4Hz,1H),4.55(d,J=6.0Hz,4H),4.27(dt, J=13.5,6.7Hz,4H),4.19–4.15(m,4H),3.93–3.88(m,4H),3.77–3.73(m,4H),3.71–3.65 (m,8H),1.73(q,J=7.1Hz,4H),1.40(dd,J=9.9,5.5Hz,4H),1.28(q,J=9.0,7.2Hz,18H). 13 C NMR (151 MHz, CDCl 3 )δ166.6,165.8,158.1,157.9,152.7,148.1,137.5,131.8,131.2,128.3,123.7,123.1,114.0,111.8,108.6,70.8,70.7, 70.6,70.5,69.4,69.2,68.8,68.6,65.7,65.1,43.4,29.7,29.5,29.4,29.2,28.7,28.6,26.0,25.9.MS-ESI:calculated for[M+H] + (C 48 H 61 F 14 N 2 O 12 ):m / z 1123.3996,found:m / z1123.3954.

[0073] Experimental Example 1

[0074] Transmembrane transport activity assay of compounds

[0075] Study on transmembrane transport activity of tyroserleutin based on Cu(II)-calcein assay

[0076] Large unilamellar vesicles (LUVs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were prepared as follows: First, POPC powder (25.0 mg) was dissolved in 1 mL of anhydrous chloroform, and then the solvent was removed under reduced pressure at 35°C. The resulting lipid film was dried in vacuum overnight. Next, the lipid film was mixed with a Cu-containing 2+ and calcein buffer solution (pH 7.4, 20 mM HEPES, 100 mM Na2 SO 4 , 0.2 mM CuSO 4 , 0.2 mM calcein) were mixed and stirred vigorously, and then incubated on a rotary shaker at 300 r / min for 1 h to form a uniform suspension. The suspension was subjected to 10 freeze-thaw cycles, each cycle consisting of freezing in liquid nitrogen for 1 min and then heating in a 55°C water bath for 2 min. Afterwards, it was extruded 25 times through a 200 nm polycarbonate membrane to obtain a uniformly dispersed suspension. Unencapsulated Cu was removed by gel filtration on a Sephadex G-25 column using a buffer solution (pH 7.4, 20 mM HEPES, 100 mM Na2SO4) as the eluent. 2+ The final product was a LUVs suspension with a diameter of about 200 nm and a concentration of 6.6 mM, which was stored at 4°C overnight.

[0077] For each experiment, LUVs were mixed with 2+ and tyroserleutin (YSL) in an external solution (pH 7.4, 20 mM HEPES, 100 mM Na 2 SO 4 , 0.2 mM CuSO 4 , 30 mM YSL) to a final lipid concentration of 0.2 mM in a total volume of 2 mL. Then, 20 μL of the transporter solution in DMSO was added to initiate the measurement of tyroserleutin transport, and the change in fluorescence emission (excitation wavelength λ ex =495nm, emission wavelength λ em =515nm). The transport activity of the transport protein was quantitatively analyzed by calculating the change in fluorescence intensity. The specific formula is as follows:

[0078] ΔI=I t -I 0

[0079] Among them, I t represents the fluorescence intensity at time t, I 0 represents the baseline fluorescence intensity.

[0080] The test results are as follows Figure 1 The addition of compound1 caused a concentration-dependent increase in fluorescence intensity, which reached saturation at 20 μM. The maximum fluorescence intensity increase (ΔI max ) was used as a calibration reference. The half effective concentration (EC 50) were 7.5 and 12.3 μM, respectively, indicating that compound 1 is more effective than compound 4 in mediating YSL transport. Next, nonlinear fitting was performed from Cu 2+ -Time course curves obtained from the calcein assay yielding the entry rate constants (Kobs) observed for YSL at different transporter concentrations. Figure 2 A clear linear correlation between Kobs and transporter concentrations was revealed, suggesting that compound 1 operates in a unimolecular manner in mediating YSL transport.

[0081] Experimental Example 2

[0082] In vivo and in vitro anticancer activity studies of compounds

[0083] Study on cellular uptake of YSL based on laser confocal microscopy

[0084] MCF-7 cells were seeded in glass-bottom culture dishes at a density of 1 × 10 6 cells. At 37°C, 5% CO 2 After incubation in a humidified environment for 24 h, 0.2 μM compound 1 and 0.2 mM YSL-py solution were added and incubated for another 12 h. Only 0.2 mM YSL-py was added to the control group. Subsequently, the culture medium was removed, the cells were rinsed once with 1× PBS buffer, and then 1.0 mL of fresh culture medium and rhodamine phalloidin staining solution were added. After thorough mixing, the cells were incubated at 37°C and 5% CO 2 The cells were incubated in a humidified environment for 30 min. The supernatant was then removed and the cells were rinsed twice with 1× PBS buffer. Finally, 1.0 mL of serum-free DMEM medium was added and fluorescence images were captured using a laser confocal microscope (LSM5 EXCITER, Zeiss).

[0085] The results are as follows Figure 3 As shown: In the absence of compound 1, YSL-py treated at 0.2 mM concentration showed negligible fluorescence signal in the cytoplasm, which was attributed to the poor membrane permeability of the peptide. In contrast, 0.2 μM compound 1 significantly enhanced the intracellular delivery of the peptide, as evidenced by a significant increase in cytoplasmic fluorescence.

[0086] Study on the detection of cellular uptake of YSL based on flow cytometry

[0087] A total of 1 × 10 6 MCF-7 cells and cultured at 37°C with 5% CO 2environment for 24 hours. After the incubation, the cells were treated with 0.2μM compound1 and 0.2mM YSL-py solution, respectively, and incubated for another 12 hours. The control group was treated with only 0.2mM YSL-py solution under the same conditions. After the incubation, the cells were first rinsed with 1×PBS buffer, then digested with EDTA-free trypsin and collected. The collected cells were centrifuged at 112×g for 2 minutes, then rinsed twice with 1×PBS buffer, and finally resuspended in 1.0mL DMEM medium. Flow cytometer analysis was used, and the data were analyzed by FlowJo 10.6.2 software.

[0088] The results are as follows Figure 4 As shown: It was confirmed that in the presence of 0.2 μM compound 1, the cellular uptake of YSL-py increased by 3 times.

[0089] Study on the transport of tyroserleutin by artificial peptide transporter based on MTT experiment

[0090] Human breast cancer cells (MCF-7) were seeded in 96-well plates and cultured to a density of 60% for cell viability testing. Compound 1 and YSL were prepared into DMEM medium solutions at different concentrations and then used to treat cells for 24 hours. After the treatment, 20 μL of MTT solution (5 mg / mL in PBS) was added to each well, and the cells were cultured at 37°C for another 4 hours. The formed meperazine product was dissolved by DMSO, and its absorbance at 490 nm was measured using a microplate reader.

[0091] The collected data were compared with the logarithmic values ​​of compound concentrations, and the IC was calculated using nonlinear regression curve fitting in GraphPad Prism 8.0.1 software. 50 value.

[0092] The combination index (CI) is a powerful quantitative analysis tool proposed by Chou and Talalay for analyzing drug interactions in combined medication. Its mathematical expression is:

[0093] CI=D A / IC 50,A +D B / IC 50,B

[0094] Among them, D A represents the experimental concentration of drug A, D B represents the experimental concentration of drug B; IC 50 , A is the half-inhibitory concentration of drug A, IC 50, B is the half-inhibitory concentration of drug B. The IC of YSL in the presence of different concentrations of compound1 50 Values ​​and the corresponding CI values ​​for MCF-7 cells.

[0095]

[0096] The results in Table 1 show that due to its zwitterionic properties and easy aggregation at physiological pH, YSL has limited membrane permeability in cancer treatment and requires high doses to effectively fight tumors. Studies have shown that compound 1 can effectively improve the membrane permeability and anticancer effect of YSL. Through MTT experiments, it was found that YSL had limited anticancer activity when used alone, and IC 50 The value was 4.34mM. When used in combination with compound 1 (1-8μM, non-toxic to MCF-7 cells), the anti-cancer effect of YSL was significantly enhanced. Figure 5 It was shown that, especially at 8 μM compound 1, the IC 50 When the concentration of compound 1 was reduced to 0.19 mM, the anticancer activity increased by 22.8 times. The CI value calculated by the Chou-Talalay method was 0.42, indicating that compound 1 and YSL had a synergistic anticancer effect.

[0097] In vivo anticancer effect evaluation based on animal experiments

[0098] In this study, we aimed to evaluate the effect of the combination of the artificial peptide transporter compound1 and YSL on the growth of MCF-7 breast cancer tumors. The experiment followed the international ethical guidelines for experimental animals and was approved by the Experimental Animal Ethics Committee of Xiamen University (approval number: XMULAC20220041). Thirty SPF-grade female BALB / c nude mice were selected and a breast cancer model was established by subcutaneous injection of MCF-7 cells. When the tumor volume exceeded 50 mm 3 The mice were randomly divided into six groups, each containing 5 mice. Thirty SPF female BALB / c nude mice (6-7 weeks old, weighing 20±2g) were selected and subcutaneously injected with 1×10^7 MCF-7 cells per mouse to establish a breast cancer model. When the tumor volume exceeded 50mm 3 The mice were randomly divided into six groups, with 5 mice in each group: Group G0: control group, intratumoral injection of PBS once every two days for two weeks; Group G1: low-dose compound 1 group (0.8 mg / kg); Group G2: high-dose compound 1 group (1.6 mg / kg); Group G3: YSL treatment group (8 mg / kg); Group G4: YSL (8 mg / kg) + low-dose compound 1 (0.8 mg / kg); Group G5: YSL (8 mg / kg) + high-dose compound 1 (1.6 mg / kg).

[0099] During the treatment period, the body weight of the mice was monitored daily to assess the systemic toxicity of the drug, and the three dimensions of the tumor were measured using calipers to calculate the tumor volume. Figure 6 The results showed that the tumor volume of the control group increased rapidly, while compound 1 alone had limited inhibitory effect on tumor growth. The YSL treatment group showed moderate tumor growth inhibition, while the combined treatment group significantly enhanced the anti-tumor activity, especially the high-dose compound 1 combined with YSL treatment group, with a tumor growth inhibition rate of 81.1%.

[0100] The results of weight monitoring showed that Figure 8 As shown in the figure, there was no significant difference in the weight changes of mice in all treatment groups compared with the control group, indicating that the combined treatment of compound1 and YSL was well tolerated and did not cause obvious systemic toxicity. After the treatment, the mice were killed and the tumor tissues were removed for H&E staining, Ki-67 immunohistochemical staining, and TUNEL staining. Figure 7 As shown: H&E staining showed that a large number of necrosis and apoptosis areas appeared in the tumor tissues of the combined treatment group, and Ki-67 staining showed that the proliferation of tumor cells in the combined treatment group was significantly inhibited. TUNEL staining showed that the number of apoptotic cells in the combined treatment group increased significantly, further confirming the synergistic anti-cancer effect of the combined treatment of compound1 and YSL.

[0101] Data statistics and analysis showed that the experimental data were expressed as mean ± standard error, n = 5. GraphPad Prism 8.01 software was used for statistical analysis, using one-way analysis of variance and T test. P values ​​less than 0.0001 were considered statistically significant. The above results show that the combined treatment of the artificial peptide transporter compound1 and YSL has a significant synergistic anti-cancer effect on the MCF-7 breast cancer model in vivo and has good tolerability. This discovery has laid an important theoretical foundation and experimental support for the synergistic cancer treatment strategy of artificial peptide transporters, and has important clinical development potential.

Claims

1. An artificial peptide transporter for promoting transmembrane delivery of peptide drugs, characterized in that: It contains at least one anionic structural unit and at least one cationic structural unit, and forms a structure capable of forming a stable 1:1 complex with a peptide drug by combining with a carboxyl anion and an amino cation.

2. The artificial peptide transporter according to claim 1, characterized in that The general structure is as follows:

3. The method for preparing an artificial peptide transporter for promoting transmembrane delivery of peptide drugs according to claim 1 or 2, characterized in that: The following steps are involved: Synthesize anion recognition units and cation recognition units; covalently link the above structural units through chemical reactions to form an artificial peptide transporter.

4. The preparation method according to claim 3, characterized in that: The specific reaction process is as follows:

5. Use of the artificial peptide transporter as claimed in claim 1 or 2 in drug therapy, especially in improving the bioavailability and therapeutic effect of peptide drugs.

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