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

Abstract

This invention provides the preparation and application of a class of artificial peptide transporters for promoting transmembrane delivery of peptide drugs. The general structural formula is as follows: The compound involved in this invention consists of three parts: (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: connecting the anion-binding unit and the cation-binding unit, so that the whole molecule can act as molecular tweezers to form a stable complex with the peptide drug and encapsulate it in a hydrophobic environment to promote its transmembrane transport. The compound obtained in this application has inexpensive raw materials, is easy to synthesize, and significantly improves the bioavailability of peptide drugs. It has shown significant synergistic anticancer activity in both in vitro and in vivo experiments, and therefore has great potential as a pharmaceutical excipient.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the design, synthesis, and application of a class of artificial peptide transporters in promoting transmembrane delivery of peptide drugs, particularly in anticancer treatment. Background Technology

[0002] Peptide drugs, due to their excellent biological activity, high specificity, and low toxicity, have shown great potential in the treatment of cancer, infection, and metabolic regulation. However, the delivery of peptide drugs faces many challenges in drug development, mainly due to their unique chemical properties.

[0003] First, peptide molecules typically possess large volumes and polar functional groups, resulting in extremely low permeability to nonpolar barriers like cell membranes. Second, most peptide drugs exist as zwitterions under physiological conditions, readily aggregating in aqueous solutions, further reducing their membrane permeability. Even shorter-chain neutral peptides suffer from significantly limited absorption and bioavailability in vivo due to the lack of natural transport mechanisms. Furthermore, during delivery, peptide drugs are susceptible to rapid degradation by gastrointestinal enzymes, nonspecific binding to plasma proteins, and rapid renal clearance, significantly shortening their in vivo half-life. These factors collectively limit the clinical application of peptide drugs, often requiring higher doses to achieve the desired therapeutic effect, while also increasing toxicity, side effects, and production costs.

[0004] To address the aforementioned issues, recent research has increasingly focused on developing artificial transport systems capable of efficiently delivering peptide drugs. By mimicking the function of natural transporters, artificial peptide transporters can effectively protect peptide drugs from degradation while significantly enhancing their transmembrane transport capabilities. However, the current variety of artificial peptide transport systems is extremely limited, and the delivery of peptides to electrochemical centers still faces problems such as low efficiency, poor biocompatibility, and complex preparation. New molecular design strategies are urgently needed to overcome these challenges.

[0005] To address the aforementioned challenges, this invention proposes a novel artificial peptide transporter that integrates the transport functions of anions and cations. By assembling it with peptide drugs to form a highly efficient transmembrane transport system, it can significantly improve the transmembrane transport efficiency and bioavailability of peptide drugs, providing a new strategy for solving the problems of peptide drug delivery and absorption. Summary of the Invention

[0006] This invention provides an artificial peptide transporter for promoting the transmembrane transport of neutral peptide drugs. This transporter integrates anion binding, cation binding and hydrophobic interactions through molecular design, which can significantly improve the transmembrane transport efficiency of neutral peptide drugs, enhance their bioavailability and therapeutic effect, and show significant advantages, especially in the field of anticancer.

[0007] This invention aims to develop a highly efficient artificial peptide transporter to address the following challenges: first, the cell membrane penetration problem of peptide compounds, by encapsulating 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 vivo.

[0008] The technical solution adopted in this invention mainly consists of three parts: first, preparing artificial peptide transporters; second, detecting the transmembrane transport activity of artificial peptide transporters for peptides; and third, evaluating the synergistic anticancer activity of this series of compounds and peptide drugs such as tyrosine leucine.

[0009] The general structural formula of the artificial peptide transporter with peptide transport activity described in this invention is as follows:

[0010]

[0011] The compound involved in this invention consists of three parts: (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: connecting the anion binding unit and the cation binding unit, so that the whole molecule can act 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 for the target compound is as follows:

[0013]

[0014] The specific synthesis process is as follows: (1) Preparation of anion-binding unit: the target anion-binding unit is prepared by condensation reaction of fluorinated anhydride and amine; (2) Preparation of cation-binding unit: crown ether modified unit is synthesized by alkylation reaction; (3) Assembly of transporter: anion and cation-binding units are covalently connected to hydrophobic chain by chemical bond to form complete molecule.

[0015] Furthermore, the compounds obtained by the above preparation method can be used in the anticancer treatment of the polypeptide drug tyrosine leucine.

[0016] The beneficial effects of this application are:

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

[0018] 2. Enhanced efficacy: Significantly improves the bioavailability of peptide drugs, exhibiting significant synergistic anticancer activity in both in vitro and in vivo experiments.

[0019] 3. Good biocompatibility: It adopts a simple and green synthetic route, has excellent biocompatibility, and is easy to optimize structure and translate into clinical applications.

[0020] 4. Wide applicability: It is not only applicable to the delivery of tyrosine leucine peptide (YSL), but can also be extended to other peptide drugs, with huge application potential. Attached Figure Description

[0021] Figure 1 The fluorescence intensity changes of calcein at different concentrations of compound 1 and compound 4, and the corresponding EC50 values ​​of compound 1 and compound 4 for these changes are shown. 50 value.

[0022] Figure 2 For in Cu 2+ - In the fluorescence assay of calcein liposomes, the observed influx rate constant (KL) of YSL was calculated at different concentrations of compound 1. obs );

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

[0024] Figure 4 To perform quantitative analysis of enhanced uptake in YSL-py cells after 12 h of compound 1 addition using flow cytometry;

[0025] Figure 5 To determine the IC50 of YSL on MCF-7 cells in the presence of different concentrations of compound 1. 50 value;

[0026] Figure 6 To measure the MCF-7 tumor volume in nude mice (n=5 per group) under different treatments;

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

[0028] Figure 8 A graph showing the trend of weight change in mice under different treatments. Detailed Implementation

[0029] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further described below with reference to specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0030] Example 1:

[0031] Preparation of compounds:

[0032]

[0033]

[0034] 1) Preparation of intermediate 1a:

[0035] 1 H NMR (600MHz, CDCl3) δ7.60(s,2H),7.15(s,1H),2.35(s,6H),1.59(s,9H). 13 C NMR(151MHz, CDCl3)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 O2): 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, CDCl3)δ7.90(s,2H),7.46(s,1H),4.43(s,4H),1.61(s,9H). 13 C NMR(151MHz, CDCl3)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 N6O2Na): 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(151MHz, CDCl3)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 F6N2O4Na): 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). 13C NMR(151MHz,DMSO-d6)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 N2O4Na):m / z 495.0374, found:m / z495.0366.

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

[0051]

[0052] 10) Preparation of target compound C8:

[0053] The product was purified by rapid column chromatography to obtain pure product C8. Yield: 88 mg, 35%. 1 H NMR (600MHz, CDCl3) 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 (151MHz, CDCl3) δ166.5,152.8,148.2,123.7,123.1,114.1,111.9,70.9,70.8,70.7,70. 6,69.4,69.3,68.9,68.7,65.0,31.8,29.3,29.2,28.8,26.9,22.7,14.1.MS-ESI:calculated for[M+Na] + (C 25 H 40 O8Na): 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 (151MHz, CDCl3) δ166.5,152.8,148.2,123.7,123.2,114.2,111.9,70.9,70.8,70.7,70.6,6 9.4,69.3,69.0,68.8,65.0,31.9,29.6,29.4,29.3,28.8,26.1,22.7,14.2.MS-ESI:calculated for[M+Na] + (C 27 H 44 O8Na): 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 (151MHz, CDCl3) δ166.4,152.9,148.3,123.8,123.2,114.6,112.2,71.0,70.8,70.7,69.5,6 9.4,69.2,68.9,65.0,32.0,29.7,29.6,29.4,29.3,28.8,26.1,22.7,14.2.MS-ESI:calculated for[M+Na] + (C 29 H 48 O8Na): 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 (151MHz, CDCl3) δ166.5,152.8,148.2,123.8,123.2,114.2,111.9,70.9,70.8,70.7,70.6,6 9.4,69.3,69.0,68.8,65.0,32.0,29.7,29.6,29.4,28.8,26.1,22.7,14.2.MS-ESI:calculated for[M+Na] + (C 31 H 52 O8Na): m / z 575.3560, found: m / z 575.3548.

[0060] 14) Preparation of the target compound BrCl2:

[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 (151MHz, CDCl3) 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 BrO8Na): m / z 625.2347, found: m / z 625.2336.

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

[0063]

[0064] 1H),6.86(d,J=8.4Hz,1H),4.27(t,J=6.7Hz,2H),4.22–4.19(m,4H),3.94(ddd,J=9.3,5.0,3.3Hz,4H),3.77(q,J=3.3,1.9Hz,4H),3.71(dt ,J=6.6,3.3Hz,4H),3.69(s,4H),3.41(t,J=6.8Hz,2H),1.87–1.82(m,2H),1.74(q,J=7.1Hz,2H),1.41(d,J=7.6Hz,2H),1.36–1.24(m,18H). 13 C NMR (151MHz, CDCl3) δ166.5,152.8,148.2,123.8,123.2,114.2,111.9,71.0,70.9,70.8,70.7,70. 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 BrO8Na): m / z653.2600, found: m / z 653.2634.

[0065] 16) Preparation of target compound 1:

[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 rapid column chromatography to give pure compound 1. Yield: 190 mg, 50%. 1H NMR (600MHz, CDCl3) 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,4 H),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 (151MHz, CDCl3) 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 N2O 12 Na): m / z1017.3566, found: m / z 1017.3537.

[0067] 17) Preparation of target 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 (151MHz, CDCl3) 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. 8,70.7,70.6,70.5,69.3,69.2,68.7,68.6,65.7,65.1,43.3,29.8,29.5,29.4,29.2,28.7,26.0.MS-ESI:calculated for[M+Na] + (C 46 H 60 F 10 N2O 12 Na): m / z1045.3879, found: m / z1045.3861.

[0069] 18) Preparation of target 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 (151MHz, CDCl3) 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 N2O 12 K): m / z 1133.3242, found: m / z1133.3219.

[0071] 19) Preparation of target compound 4:

[0072] (600MHz, CDCl3) 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 (151MHz, CDCl3) 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 N2O 12 ):m / z 1123.3996,found:m / z1123.3954.

[0073] Experimental Example 1

[0074] Detection of transmembrane transport activity of compounds

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

[0076] The preparation method of large monolayer liposomes (LUVs) of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) is 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 membrane was dried under vacuum overnight. Next, the lipid membrane was subjected to a process containing Cu... 2+A buffer solution of calcein (pH 7.4, 20 mM HEPES, 100 mM Na₂SO₄, 0.2 mM CuSO₄, 0.2 mM calcein) was mixed and vigorously stirred, then incubated on a rotary shaker at 300 rpm for 1 h to form a homogeneous suspension. This suspension was subjected to 10 freeze-thaw cycles, each cycle consisting of freezing in liquid nitrogen for 1 min followed by heating in a 55 °C water bath for 2 min. Afterwards, the suspension was extruded 25 times through a 200 nm polycarbonate membrane to obtain a uniformly dispersed suspension. Unencapsulated Cu was removed by gel filtration onto a Sephadex G-25 column using the buffer solution (pH 7.4, 20 mM HEPES, 100 mM Na₂SO₄) as the eluent. 2+ And calcein. The final result was a LUVs suspension with a diameter of approximately 200 nm and a concentration of 6.6 mM, which was stored overnight at 4 °C.

[0077] For each experiment, LUVs were mixed with Cu 2+ The external solution of tyrosine leucine peptide (YSL) (pH 7.4, 20 mM M HEPES, 100 mM Na₂SO₄, 0.2 mM CuSO₄, 30 mM YSL) was mixed to achieve a final lipid concentration of 0.2 mM, with a total volume of 2 mL. Then, 20 μL of transporter protein solution in DMSO was added to initiate the measurement of tyrosine leucine peptide transport. Changes in fluorescence emission (excitation wavelength λ) over 10 min were monitored using a fluorescence spectrophotometer. ex =495nm, emission wavelength λ em =515nm). The transport activity of transport proteins is quantitatively analyzed by calculating the change in fluorescence intensity, and the specific formula is as follows:

[0078] ΔI=I t -I0

[0079] Among them, I t It represents the fluorescence intensity at time t, and I0 represents the baseline fluorescence intensity.

[0080] Test results are as follows Figure 1 As shown, the addition of compound 1 caused a concentration-dependent increase in fluorescence intensity, reaching saturation at 20 μM. The maximum increase in fluorescence intensity (ΔI) obtained under these conditions was [not specified]. max Compound 1 and compound 4 are used as calibration references. The half-maximal effective concentration (EC50) required to achieve 50% transport is determined by compound 1 and compound 4. 50 The concentrations were 7.5 and 12.3 μM, respectively, indicating that compound 1 was more efficient than compound 4 in mediating YSL transport. Next, nonlinear fitting was performed on Cu... 2+- The time-process curves obtained by the calcein assay were used to derive the influx rate constant (Kobs) observed in YSL at different transporter protein concentrations. Figure 2 The study revealed a significant linear correlation between Kobs and transporter concentration, suggesting that compound1 operates in a single-molecule manner in mediating YSL transport.

[0081] Experimental Example 2

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

[0083] Cellular uptake of YSL based on laser confocal assay

[0084] MCF-7 cells were seeded in glass-bottomed culture dishes at a density of 1 × 10⁶ cells per dish. 6 Cells were incubated at 37°C and 5% CO2 for 24 h. Then, 0.2 μM Compound 1 and 0.2 mM YSL-py solution were added, and incubation continued for 12 h. The control group received only 0.2 mM YSL-py. Subsequently, the culture medium was removed, and the cells were washed once with 1×PBS buffer. 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% CO2 for 30 min. The supernatant was then aspirated, and the cells were washed 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, the fluorescence signal in the cytoplasm was almost negligible when YSL-py was treated at a concentration of 0.2 mM, which is attributed to the poor membrane permeability of peptides. In contrast, 0.2 μM of compound 1 significantly enhanced intracellular transport of peptides, as evidenced by the significant enhancement of cytoplasmic fluorescence.

[0086] Study on YSL uptake by cells based on flow cytometry

[0087] A total of 1×10⁶ cells were planted in each well of the 6-well plate. 6MCF-7 cells were incubated at 37°C and 5% CO2 for 24 h. After incubation, the cells were treated with 0.2 μM compound 1 and 0.2 mM YSL-py solution, respectively, and incubated for another 12 h. The control group was treated with only 0.2 mM YSL-py solution under the same conditions. After incubation, the cells were washed with 1×PBS buffer, digested with EDTA-free trypsin, and collected. The collected cells were centrifuged at 112×g for 2 min, washed twice with 1×PBS buffer, and finally resuspended in 1.0 mL DMEM medium. Flow cytometry was used for analysis, and the data were analyzed using FlowJo 10.6.2 software.

[0088] The results are as follows Figure 4 As shown: It was confirmed that the cellular uptake of YSL-py increased threefold in the presence of 0.2 μM compound1.

[0089] Study on the transport of tyrosine leucine by artificial peptide transporters based on MTT assay

[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 used to prepare DMEM culture solutions at different concentrations, which were then used to treat the cells for 24 hours. After treatment, 20 μL of MTT solution (5 mg / mL, dissolved in PBS) was added to each well, and the cells were cultured at 37°C for another 4 hours. The resulting mepirazine product was dissolved in 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 analytical tool proposed by Chou and Talalay for analyzing drug interactions in combination therapy. Its mathematical expression is:

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

[0094] Among them, D A D represents the experimental concentration of drug A. B The experimental concentration representing drug B; IC 50 A is the half-maximal inhibitory concentration (IC50) of drug A. 50B is the half-maximal inhibitory concentration (IC50) of drug B. The IC50 of YSL at different concentrations of compound 1 is... 50 The values ​​and corresponding CI values ​​for MCF-7 cells.

[0095]

[0096] Table 1 shows that YSL's zwitterionic properties and tendency to aggregate at physiological pH limit its membrane permeability in cancer treatment, requiring high doses for effective antitumor activity. Studies have shown that compound 1 can effectively improve the membrane permeability and anticancer effect of YSL. MTT assays revealed that YSL alone has limited anticancer activity, with an IC50 value of [missing value]. 50 The value was 4.34 mM. When used in combination with compound 1 (1-8 μM, non-toxic to MCF-7 cells), the anticancer effect of YSL was significantly enhanced, such as... Figure 5 The results show that, especially at 8 μM compound 1, YSL's IC... 50 When the concentration 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 compound1 and YSL have a synergistic anticancer effect.

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

[0098] This study aimed to evaluate the effect of combined treatment with the artificial peptide transporter compound1 and YSL on the growth of MCF-7 breast cancer tumors. The experiment followed international guidelines for laboratory animal ethics and was approved by the Laboratory Animal Ethics Committee of Xiamen University (Approval No.: 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 Mice were randomly divided into six groups of five mice each. Thirty SPF-grade female BALB / c nude mice (6-7 weeks old, weighing 20±2g) were selected and a breast cancer model was established by subcutaneous injection of 1×10^7 MCF-7 cells per mouse. When the tumor volume exceeded 50mm... 3 Mice were randomly divided into six groups of five each: G0 group: control group, intratumoral injection of PBS every two days for two weeks; G1 group: low-dose compound 1 (0.8 mg / kg); G2 group: high-dose compound 1 (1.6 mg / kg); G3 group: YSL treatment group (8 mg / kg); G4 group: YSL (8 mg / kg) + low-dose compound 1 (0.8 mg / kg); G5 group: YSL (8 mg / kg) + high-dose compound 1 (1.6 mg / kg).

[0099] During treatment, the mice's body weight was monitored daily to assess the systemic toxicity of the drug, and tumor volume was calculated by measuring three dimensions of the tumor using calipers. Results are as follows: Figure 6 The results showed that tumor volume increased rapidly in the control group, while compound1 alone had limited inhibitory effect on tumor growth. The YSL treatment group showed moderate tumor growth inhibition, while the combination therapy group significantly enhanced anti-tumor activity, especially the high-dose compound1 and YSL combination therapy group, which achieved a tumor growth inhibition rate of 81.1%.

[0100] Weight monitoring results show that, Figure 8 As shown, there was no significant difference in body weight change between the treatment groups and the control group, indicating that the combined treatment of compound1 and YSL was well tolerated and did not cause significant systemic toxicity. After treatment, the mice were euthanized and tumor tissue was removed for H&E staining, Ki-67 immunohistochemical staining, and TUNEL staining. Figure 7 As shown: H&E staining revealed numerous necrotic and apoptotic areas in the tumor tissue of the combined treatment group, and Ki-67 staining showed that tumor cell proliferation was significantly inhibited in the combined treatment group. TUNEL staining showed a significant increase in the number of apoptotic cells in the combined treatment group, further confirming the synergistic anticancer effect of compound1 and YSL combined treatment.

[0101] Statistical analysis showed that experimental data are expressed as mean ± standard error, n=5. Statistical analysis was performed using GraphPadPrism 8.01 software, employing one-way ANOVA and t-tests. A p-value less than 0.0001 was considered statistically significant. These results indicate that the combined treatment of the artificial peptide transporter compound1 and YSL has a significant synergistic anti-cancer effect in the MCF-7 breast cancer model in vivo, and is well-tolerated. This discovery lays an important theoretical foundation and experimental support for the synergistic cancer treatment strategy using artificial peptide transporters, and has significant potential for clinical development.

Claims

1. A class of artificial peptide transporters for promoting transmembrane delivery of peptide drugs, characterized in that, It comprises 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 peptide drugs by binding with carboxyl anions and amino cations. The general structural formula is as follows:

2. The method for preparing an artificial peptide transporter for promoting transmembrane delivery of peptide drugs according to claim 1, characterized in that, The specific reaction process is as follows:

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