Organic molecule cytoplasm delivery carrier as well as preparation method and application thereof

Abstract

The invention provides an organic molecule cytoplasm delivery carrier as well as a preparation method and application thereof, the compound for cytoplasm delivery has the characteristic of fat-soluble aggregation, and can form electrostatic adsorption with small molecule protein goods such as RNase A (13.7 KDa), Trypsin (24KDa) and Saporin (32.8 KDa), medium molecule protein goods such as HRP (40KDa) and macromolecular protein goods such as beta-Gal (430KDa), so that the compound for cytoplasm delivery can be used for preparing a lipid-soluble protein carrier. And furthermore, transmembrane transportation is formed through the strong fat solubility property, so that the protection and intracellular delivery of carried protein molecules are realized. According to the delivery mechanism, cargo molecules can quickly penetrate through cell membranes, and carried protein molecules can enter cytoplasm to exert functions without release operation. Development of the organic molecule cytoplasm delivery carrier is expected to assist potential protein therapies with low bioavailability and off-target toxicity to complete clinical transformation, which is crucial to tumor therapy, gene therapy and the like requiring precise local administration.

Description

Technical Field

[0001] The present invention relates to the technical field of biological preparations, and in particular to an organic molecule cytoplasmic delivery carrier and a preparation method and application thereof. Background Art

[0002] In the fields of biotechnology and medicine, the delivery of proteins to the cytoplasm is of great significance, which provides a new strategy for manipulating cell functions and treating various diseases. However, due to the selective permeability of the cell membrane and the characteristics of the protein itself, achieving efficient and lossless cytoplasmic protein delivery has always been a difficult problem that needs to be overcome. Proteins are extremely important therapeutic molecules in biotechnology. About 20% of the drugs on the market are protein drugs, such as monoclonal antibodies, cytokines, etc., but most of the protein therapeutic drugs currently available in the clinic are only targeted at extracellular targets, because most intracellular proteins have poor membrane permeability and lack effective intracellular delivery methods. At present, although a variety of delivery technologies have been proposed, such as electroporation, cell-penetrating peptides, synthetic virus-like nanoparticles, etc., these methods have disadvantages such as complex operation, high cost, and possible effects on protein structure and activity.

[0003] In recent years, polymers have become popular candidates for cytoplasmic protein delivery due to their ease of synthesis, functionalization, and high immune evasion efficiency. However, existing methods lack universality for delivering proteins of varying isoelectric points and molecular weights, limiting the range of protein therapeutic candidates. Therefore, developing a carrier compound that can efficiently deliver most proteins without releasing the cargo molecule remains an urgent challenge.

[0004] In view of the above application requirements, the present invention is proposed. Summary of the Invention

[0005] The problem solved by the present invention is to provide an organic compound molecule for cytoplasmic delivery to make up for the shortcomings of existing protein delivery methods such as complex operation, high cost, and low activity of target protein.

[0006] To solve the above problems, the present invention provides an organic molecule cytoplasmic delivery carrier, wherein the carrier is the compound MTPABP, whose chemical structure can form electrostatic adsorption with protein molecules through the lipid-soluble aggregation effect, and mediate transmembrane transport to deliver protein molecules into the cytoplasm, and the protein molecules can function in the cell without the need for release operation.

[0007] Preferably, the chemical structure of the carrier MTPABP is as follows:

[0008]

[0009] The present invention also provides a method for preparing an organic molecule cytoplasmic delivery vector, wherein the method for preparing the compound MTPABP comprises the following steps:

[0010] 4-(7-bromobenzo[C][1,2,5]thiadiazol-4-yl)-N,N-bis(4-methoxyphenyl)aniline, (4-(6-chlorohexyl)oxy)phenyl)boric acid, Pd(PPh3)4 and K2CO3 were added to a flask, the flask was evacuated and purged, and then tetrahydrofuran, ethanol and water were added, and the mixture was heated under reflux with stirring for 24 h;

[0011] The reaction mixture was cooled to room temperature, the organic phase was separated and extracted again with an organic solvent, the organic phases were combined and dried, filtered, and concentrated in vacuo to obtain a crude product;

[0012] MTPABP was purified by column chromatography.

[0013] Preferably, the preparation method of the compound 4-((6-chlorohexyl)oxyphenyl)boric acid is:

[0014] 4-Hydroxyphenyl, boric acid and K2CO3 were added to the flask, the flask was evacuated and purged, acetonitrile and 1-bromo-6-chlorohexane were injected respectively under nitrogen atmosphere, and the mixture was heated under reflux and stirred for 12 h;

[0015] Add ice water, separate the organic phase and extract again with an organic solvent, combine the organic phases, dry them, filter them, and concentrate them in vacuo to obtain a crude product;

[0016] Purification by column chromatography gave 4-((6-chlorohexyl)oxyphenyl)boronic acid.

[0017] Preferably, the preparation method of the compound 4-(7-bromobenzo[c][1,2,5]thiadiazol-4-yl)-N,N-bis(4-methoxyphenyl)aniline is:

[0018] 4-(bis(4-methoxyphenyl)amino)phenyl)boronic acid, 4,7-dibromobenzo[1,2,5]thiadiazole, Pd(PPh3)4 and K2CO3 were added to a flask, the flask was evacuated and purged, and then tetrahydrofuran, ethanol and water were added, and the mixture was heated under reflux with stirring for 24 h;

[0019] The reaction mixture was cooled to room temperature, the organic phase was separated and extracted again with an organic solvent, the organic phases were combined and dried, filtered, and concentrated in vacuo to obtain a crude product;

[0020] The residue was purified by column chromatography to obtain 4-(7-bromobenzo[c][1,2,5]thiadiazol-4-yl)-N,N-bis(4-methoxyphenyl)aniline.

[0021] The present invention also provides an application of an organic molecule cytoplasmic delivery carrier, wherein the organic molecule cytoplasmic delivery carrier MTPABP forms a complex with a protein through electrostatic adsorption to achieve transmembrane delivery; the protein is selected from a small molecule protein cargo, a medium molecule protein cargo or a large molecule protein cargo.

[0022] Preferably, the small molecule protein cargo includes RNaseA, Trypsin or Saporin, the medium molecule protein cargo includes HRP, and the large molecule protein cargo includes β-Gal.

[0023] Preferably, the delivery process satisfies:

[0024] The incubation mass ratio of the carrier MTPABP to the protein is 1:2.5 to 1:3;

[0025] Once the complex enters the cytoplasm, it can activate protein function without requiring a release procedure.

[0026] The present invention comprises lipophilic organic molecules comprising 4-((6-chlorohexyl)oxyphenyl)boronic acid and 4-(7-bromobenzo[C][1,2,5]thiadiazol-4-yl)-N,N-bis(4-methoxyphenyl)aniline. 4-(7-bromobenzo[C][1,2,5]thiadiazol-4-yl)-N,N-bis(4-methoxyphenyl)aniline is a widely used aggregation-induced emission molecule with low toxicity, strong lipophilicity, and the ability to easily penetrate cell membranes and enter the cytoplasm. Furthermore, the 4-(7-bromobenzo[C][1,2,5]thiadiazol-4-yl)-N,N-bis(4-methoxyphenyl)aniline, by linking to 4-((6-chlorohexyl)oxyphenyl)boronic acid, enables rapid and specific covalent binding of MTPABP to the self-labeled HaloTag fusion protein. After incubating MTPABP with HaloTag fusion protein or other cargo molecules in a certain ratio, unbound MTPABP is removed by ultrafiltration, and then the incubation complex is incubated with cells to deliver the cargo molecules to the cytoplasm. In this process, MTPABP forms electrostatic adsorption with the cargo molecules in the solution, thereby exposing the cargo molecules in the cytoplasm without the need for a release step, greatly improving the release of the cargo molecules and promoting their function in the cell.

[0027] Compared with the prior art, the organic compound carrier for cytoplasmic delivery and the preparation method thereof of the present invention have the following beneficial effects:

[0028] 1) The compound can form electrostatic adsorption with cargo proteins in solution through its significant aggregation effect, effectively achieving intracellular delivery of protein molecules, releasing the contained proteins without decomposition, and has a wide range of applications;

[0029] 2) By forming electrostatic adsorption after incubation with cargo proteins in an in vitro solution, the cargo protein molecules are anchored, which has the advantages of strong controllability, good uniformity, and easy operation;

[0030] 3) It is expected to help potential protein therapies with low bioavailability and off-target toxicity to complete clinical transformation, which is crucial for tumor treatment and gene therapy that require precise local delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the synthesis of the compound for cytoplasmic delivery described in Example 1 of the present invention;

[0032] Figure 2 The results of the study on the uptake mechanism of HaloTag-EGFP in HEK 293T cells in Example 2 of the present invention are shown in the scale bar 10.

[0033] Figure 3 The results of the study on the uptake mechanism of HaloTag-EGFP in HeLa cells in Example 2 of the present invention are shown. The scale bar is 10 μm.

[0034] Figure 4 The results of the study on the uptake mechanism of HaloTag-EGFP in HT22 cells in Example 2 of the present invention are shown. Scale bar: 10 μm.

[0035] Figure 5a and Figure 5b This is the result of intracellular delivery and uptake of β-Gal in HeLa cells in Example 3 of the present invention;

[0036] Figure 6a and Figure 6b The results of intracellular toxicity and activity of β-Gal in HeLa cells in Example 3 of the present invention are as follows;

[0037] Figures 7a-7f This is the DLS and Zeta results of the intracellular delivery of β-Gal in HeLa cells in Example 3 of the present invention;

[0038] Figures 8a-8c This is the result of intracellular delivery of HRP in HeLa cells in Example 4 of the present invention;

[0039] Figure 9 The results of intracellular toxicity of HRP delivered to HeLa cells and the activity of the delivered HRP in Example 4 of the present invention are as follows;

[0040] Figures 10a-10f This is the DLS, Zeta result of the intracellular delivery of HRP in HeLa cells in Example 4 of the present invention;

[0041] Figures 11a-11cThe intracellular delivery activity and DLS and Zeta results of Trypsin in Example 5 of the present invention are shown;

[0042] Figures 12a-12c The intracellular delivery activity and DLS, Zeta results of RNaseA in Example 6 of the present invention;

[0043] Figures 13a-13c The intracellular delivery activity and DLS and Zeta results of Saporin in Example 7 of the present invention are shown. DETAILED DESCRIPTION

[0044] To make the above-mentioned objects, features and advantages of the present invention more clearly understood, the following describes in detail the specific embodiments of the present invention in conjunction with the accompanying drawings. It should be noted that the technical features in the embodiments of the present invention can be combined with each other.

[0045] The organic molecule cytoplasmic delivery carrier of the present invention is the compound MTPABP, whose chemical structure can form electrostatic adsorption with protein molecules through the lipid-soluble aggregation effect, and mediate transmembrane transport to deliver protein molecules into the cytoplasm, and can enable protein molecules to function in the cell without the need for release operation.

[0046] The chemical structure of the compound MTPABP is as follows:

[0047]

[0048] Example 1 Synthesis of MTPABP

[0049] The overall synthetic route is as follows:

[0050]

[0051] Reaction conditions: (1) K2CO3, MecN, N2, 12h, 85.5%; (2) EtOH / THF / H2O, Pd(PPh3)4, K2CO3, N2, 12h, 88.2%; (3) EtOH / THF / H2O, Pd(PPh3)4, K2CO3, N2, 12h, 72.13%

[0052] 1) Synthesis of Compound 1: 4-((6-chlorohexyl)oxyphenyl)boronic acid

[0053] (4-Hydroxyphenyl)boronic acid (5 mmol) and K2CO3 (7.5 mmol) were added to a 100 ml two-necked round-bottom flask. The flask was evacuated and purged three times with nitrogen. Acetonitrile (40 mL) and 1-bromo-6-chlorohexane (7.5 mmol) were added under a nitrogen atmosphere, respectively, and the mixture was heated under reflux with stirring for 12 h. Ice water was then added, and the organic phase was separated and extracted three times with dichloromethane. The organic phase was combined, dried over Na2SO4, and the solvent was evaporated under reduced pressure. Filtered and concentrated in vacuo to obtain the crude product, which was purified by column chromatography on silica gel (eluent: dichloromethane / hexane 1 / 2) to yield (4-((6-chlorohexyl)oxy)phenyl)boronic acid as a white solid (85.5%, 1.095 g).

[0054] 1H NMR(400MHz,chloroformd)δ8.15(d,J=8.0Hz,2H),7.00(d,J=8.0Hz,2H),4.05( t, J=6.4Hz, 2H), 3.57 (t, J=6.6Hz, 2H), 1.83 (p, J=6.5Hz, 4H), 1.58-1.50 (m, 4H).

[0055] 2) Synthesis of Compound 2: 4-(7-bromobenzo[c][1,2,5]thiadiazol-4-yl)-N,N-bis(4-methoxyphenyl)aniline

[0056] 4-(Bis(4-methoxyphenyl)amino)phenyl)boronic acid (1 mmol), 4,7-dibromobenzo[C][1,2,5]thiadiazole (1 mmol), Pd(PPh3)4 (0.01 mmol), and K2CO3 (3 mmol) were added to a 100 mL two-necked round-bottom flask. The flask was evacuated and purged with nitrogen three times. Tetrahydrofuran (20 mL), ethanol (20 mL), and water (5 mL) were then added and heated under reflux with stirring for 24 hours. The reaction was then cooled to room temperature, the organic phase was separated, and the mixture was extracted again with dichloromethane three times. The organic phases were combined and dried over Na2SO4, and the solvent was evaporated under reduced pressure. The reaction mixture was filtered and concentrated in vacuo to give a crude product, which was purified by column chromatography on silica gel (eluent: dichloromethane / hexane 1 / 4) to give 4-(7-bromobenzo[C][1,2,5]thiadiazol-4-yl)-N,N-bis(4-methoxyphenyl)aniline as a red solid (88.2%, 457.3 mg).

[0057] 1H NMR (400MHz, DMSO-d6) δ7.95 (d J=8.7Hz, 2H), 7.89-7.81 (m, 4H), 7.10 (dt, J=8.6, 4.3Hz, 6H), 6.98-6.92 (m, 4H), 6.88 (d J=8.8Hz,2H),4.04(t,J=6.5Hz,2H),3.76(s,6H),3.65(t,J=6.6Hz,2H),1.76(s,4H),1.51-1.42(m,4H). 13C NMR (101MHz, DMSO) δ159.24, 156.50, 153.97, 148.99, 140.22, 131.77, 131.34, 130.72, 130.30, 129.6 2. 128.57, 127.97, 127.54, 119.01, 115.52, 114.98, 67.90, 55.73, 45.85, 32.47, 29.01, 26.52, 25.29.

[0058] 3) Synthesis of compound MTPABP

[0059] 4-(7-Bromobenzo[C][1,2,5]thiadiazol-4-yl)-N,N-bis(4-methoxyphenyl)aniline (0.5 mmol), (4-(6-chlorohexyl)oxy)phenyl)boronic acid (0.75 mmol), Pd(PPh3)4 (0.005 mmol), and K2CO3 (1.5 mmol) were added to a 50 mL two-necked round-bottom flask. The flask was evacuated and purged with nitrogen three times. Tetrahydrofuran (10 mL), ethanol (10 mL), and water (2 mL) were then added and heated under reflux with stirring for 24 hours. The reaction was then cooled to room temperature, the organic phase was separated, and the mixture was extracted three times with dichloromethane. The organic phase was combined, dried over Na2SO4, and the solvent was evaporated under reduced pressure. The residue was filtered and concentrated in vacuo to give a crude product, which was purified by column chromatography on silica gel (eluent: dichloromethane / hexane 1 / 2) to give 4-(7-bromobenzo[C][1,2,5]thiadiazol-4-yl)-N,N-bis(4-methoxyphenyl)aniline as a red solid (72.13%, 234.5 mg).

[0060] 1H NMR(400MHz,DMSO-d6)δ7.95(d,J=8.7Hz,2H),7.89-7.81(m,4H),7.10(dt,J=8.6,4.3Hz,6H),6.98-6.92(m,4H),6 .88(d,J=8.8Hz,2H),4.04(t,J=6.5Hz,2H),3.76(s,6H),3.65(t,J=6.6Hz,2H),1.76(s,4H),1.51-1.42(m,4H).13C NMR(101MHz,DMSO)δ159.24,156.50,153.97,148.99,140.22,131.77,131.34,130.72,130.30,129.62,128.5 7,127.97,127.54,119.01,115.52,114.98,67.90,55.73,45.85,32.47,29.01,26.52,25.29.m / z:calcd.for C38H36ClN3O3S:649.2166; found:649.2167.

[0061] The organic molecule cytoplasmic delivery carrier MTPABP forms a complex with the protein through electrostatic adsorption to achieve transmembrane delivery; the incubation mass ratio of the carrier MTPABP to the protein is 1:2.5 to 1:3; after the complex enters the cytoplasm, it can activate the protein function without the need for release operation.

[0062] The protein molecule can be a small molecular weight protein cargo (molecular weight ≤ 35 kDa) such as RNaseA, Trypsin, Saporin, a medium molecular weight protein cargo (35 kDa < molecular weight ≤ 100 kDa) such as HRP, or a large molecular weight protein cargo (molecular weight > 100 kDa) such as β-Gal.

[0063] Example 2 MTPABP delivers HaloTag-EGFP into the cytoplasm of HEK 293T, HeLa, and HT22 cells

[0064] HEK 293T, HeLa, and HT22 cells (1×10 4) were seeded into 3-well confocal culture dishes and allowed to attach for 24 hours. The culture conditions were: DMEM (DMEM) culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO2. Subsequently, enhanced green fluorescent protein HaloTag-EGFP (5 μg) and MTPABP (1 μg) dissolved in phosphate buffer (pH 7.4) were incubated overnight at room temperature in the dark. The product was added to each well containing 200 μL of DMEM culture medium (without FBS) and incubated at 37°C and 5% CO2 for 1 hour. After incubation, the cells were stained with 1× Cell Mask Deep Red at 37°C for 10 minutes and imaged using a Nikon inverted confocal microscope (LSCM) equipped with a Ti2 living cell workstation.

[0065] The experiment found that after MTPABP specifically covalently binds to HaloTag fusion protein, it can deliver EGFP protein fused to HaloTag into cells. This experimental phenomenon has been verified in three cell lines: HEK 293T, HeLa and HT22. The co-localization of MTPABP and cytoplasm-delivered EGFP fluorescence is shown. Figure 2-Figure 4 .

[0066] Example 3 MTPABP delivery of β-Gal

[0067] 3.1 MTPABP delivers β-Gal into the HeLa cytoplasm

[0068] HeLa cells (1×10 4 ) were seeded in 24-well cell culture plates and attached for 24 hours. The culture conditions were: 37°C, 5% CO2 in MEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. β-Gal (5 μg) and MTPABP (2 μg) dissolved in phosphate buffer (pH 7.4) were then incubated overnight in the dark at room temperature. The product was added to each well containing MEM medium (without FBS) and incubated at 37°C, 5% CO2 for 6 hours. The cells were then washed three times with PBS buffer and stained using a β-galactosidase in situ staining kit (Biyuntian). The β-Gal delivered into the cells was imaged by optical microscopy.

[0069] As shown in the figure, MTPABP can deliver β-Gal into cells. Compared with commercial protein delivery vectors Lipo8000 and TranEx, MTPABP has the highest efficiency in delivering β-Gal into the cytoplasm. Figure 5a and 5b .

[0070] 3.2 Cytotoxicity of β-Gal delivered by MTPABP

[0071] In order to determine the cytotoxicity of the organic molecule cytoplasmic delivery carrier MTPABP after encapsulating the cargo molecules, we used the CCK8 kit (Synthemi) to detect the toxicity of the incubation mixture on cells.

[0072] HeLa cell suspension (1×10 4 ) and pre-incubate the culture plates in an incubator for 24 hours. Add a mixture of β-Gal (5 μg) and MTPABP (2 μg), β-Gal (5 μg) and Lipo8000 (2 μg), and β-Gal (5 μg) and TranEX (2 μg) to the 96-well plate and incubate the plates at 37°C, 5% CO2 for 24 hours. Add 10 μL of CCK-8 solution to each well, incubate the plates at 37°C, 5% CO2 for 1 hour, and measure absorbance at 450 nm using a microplate reader.

[0073] The results showed that MTPABP can deliver β-Gal into cells. Compared with commercial protein delivery vectors Lipo8000 and TranEx, MTPABP and β-Gal have the lowest intracellular toxicity. Figure 6b .

[0074] 3.3 β-Gal activity delivered into cells by MTPABP

[0075] For consistency comparison, based on the successful cytoplasmic delivery, the activity of the β-Gal enzyme delivered into the cells was tested according to the protocol reported in the previous literature. The specific operation is as follows:

[0076] Resuspend the cell pellet in 100 μL of pre-chilled Solution A (extraction solution) and quickly transfer to a 1.5 mL centrifuge tube. Add 0.05 g of glass beads and vortex vigorously for 5-10 minutes (intermittently cooling in an ice bath). Recover the extract with a pipette. Add another 100 μL of pre-chilled Solution A (extraction solution) to the remaining glass beads, vortex briefly, and recover the extract again. Combine the two extracts to form the total protein sample. Centrifuge at 12,000 rpm at 4°C for 15 minutes and collect the supernatant. The extracted total protein sample was used directly in subsequent activity assays. Protein concentration was determined using a Bradford protein assay kit (Yamei). According to the instructions for the β-galactosidase (β-Gal) Assay Kit (ONPG Method) (Coolaber), add total protein, ONPG, deionized water, Solution B, and Solution C in order, mix thoroughly, and incubate at 37°C for 30 minutes. Preheat the microplate reader / spectrophotometer for more than 30 minutes, set the temperature to 37°C, adjust the wavelength to 420 nm, and set the blank group to zero.

[0077] The results showed that MTPABP can deliver β-Gal into cells. Compared with commercial protein delivery vectors Lipo8000 and TranEx, MTPABP has the highest β-Gal activity in delivering β-Gal into cells. Figure 6a .

[0078] 3.4 Particle size (DLS) and surface potential (Zeta) of MTPABP-delivered β-Gal

[0079] Dynamic light scattering (DLS) and Zeta detection methods involve dissolving β-Gal and its corresponding β-Gal-MTPABP complex in distilled water at appropriate concentrations and directly detecting them using a DLS and Zeta method module (Zetasizer NanoZS 90, Malvern). DLS was used to characterize the particle size of MTPABP and the β-Gal-MTPABP complex at 25°C.

[0080] As can be seen from the figure, MTPABP carries a negative charge. After forming electrostatic adsorption with β-Gal, the potential decreases and the particle size increases slightly. Compared with commercial protein delivery carriers Lipo8000 and TranEx, MTPABP carries β-Gal, and DLS and Zeta are more stable and can be better delivered into cells. The results are shown in Figures 7a-7f .

[0081] Example 4: Delivery of HRP by MTPABP

[0082] 4.1 MTPABP delivers HRP into the HeLa cytoplasm

[0083] HeLa cells (1×10 4 ) were seeded into 24-well cell culture plates and allowed to attach for 24 hours at 37°C, 5% CO2 in MEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. HRP (6 μg) and MTPABP (2 μg) dissolved in phosphate buffer (pH 7.4) were then added to each well containing MEM medium (without FBS) and incubated overnight in the dark at room temperature. The cells were then incubated at 37°C, 5% CO2 for 6 hours. The cells were then washed three times with PBS buffer, and TMB substrate (50 mg / ml) dissolved in acetate buffer (pH 5.0) containing 3 mM hydrogen peroxide was added to each well. After incubation at room temperature for 10 minutes, the mixture in each well was imaged using a light microscope.

[0084] As shown in the figure, MTPABP can deliver HRP into cells. Compared with commercial protein delivery vectors Lipo8000 and TranEx, MTPABP can better deliver HRP into cells, so that under the oxidation of hydrogen peroxide, the blue color of TMB cells deepens compared with other groups. The results are shown in Figure 8c .

[0085] HeLa cells (1×10 4 ) were seeded in 24-well cell culture plates and attached for 24 hours. The culture conditions were: 37°C, 5% CO2 in MEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. HRP (6 μg) and MTPABP (2 μg) dissolved in phosphate buffer (pH 7.4) were then added to each well containing MEM medium (without FBS) and incubated in the dark overnight at room temperature and incubated for 6 hours at 37°C, 5% CO2. The cells were then washed three times with PBS buffer, and then washed three times with PBS and incubated in PBS containing Amplex Red (50 mM) and hydrogen peroxide (500 mM). After incubation at room temperature for 30 minutes, the treated cells were washed three times and imaged using a Nikon inverted confocal microscope (LSCM) equipped with a Ni2-type live cell workstation.

[0086] The results showed that MTPABP can deliver HRP into cells. Compared with commercial protein delivery vectors Lipo8000 and TranEx, MTPABP can better deliver HRP into cells, so that under the oxidation of hydrogen peroxide, the red fluorescence of AmplexRed is the brightest compared with other groups. The results are shown in Figure 8b .

[0087] 4.2 Cytotoxicity of HRP delivered by MTPABP

[0088] In order to determine the cytotoxicity of the organic molecule cytoplasmic delivery carrier MTPABP after encapsulating the cargo molecules, we used the CCK8 kit (Synthemi) to detect the toxicity of the incubation mixture on cells.

[0089] HeLa cell suspension (1×10 4 ) and pre-incubate the culture plates in an incubator for 24 hours. Add a mixture of HRP (6 μg) and MTPABP (2 μg), HRP (6 μg) and Lipo8000 (2 μg), and HRP (6 μg) and TranEX (2 μg) to the 96-well plate and incubate the plates at 37°C, 5% CO₂ for 24 hours. Add 10 μL of CCK-8 solution to each well, incubate the plates at 37°C, 5% CO₂ for 1 hour, and measure absorbance at 450 nm using a microplate reader.

[0090] The results showed that MTPABP could deliver HRP into cells. Compared with commercial protein delivery vectors Lipo8000 and TranEx, MTPABP and HRP had the lowest intracellular toxicity. Figure 9 .

[0091] 4.3 Particle Size (DLS) and Surface Potential (Zeta) of MTPABP-Delivered β-Gal

[0092] The DLS and Zeta assay methods involved dissolving HRP and its corresponding HRP-MTPABP complex in distilled water and directly detecting them at appropriate concentrations using a DLS and Zeta assay module (Zetasizer NanoZS 90, Malvern). The particle size of MTPABP and the HRP-MTPABP complex was determined using DLS at 25°C.

[0093] As can be seen from the figure, MTPABP carries a negative charge. After forming electrostatic adsorption with HRP, the potential decreases and the particle size increases slightly. Compared with commercial protein delivery carriers Lipo8000 and TranEx, MTPABP carries β-Gal, and DLS and Zeta are more stable and can be better delivered into cells. The results are shown in Figures 10a-10f .

[0094] Example 5: Delivery of Trypsin by MTPABP

[0095] 5.1 Intracellular Activity of Trypsin Delivered by MTPABP

[0096] In order to determine the cytotoxicity of the organic molecule cytoplasmic delivery carrier MTPABP after encapsulating the cargo molecules, we used the CCK8 kit (Synthemi) to detect the toxicity of the incubation mixture on cells.

[0097] HeLa cell suspension (1×10 4 ) and pre-incubate the culture plates in an incubator for 24 hours. Add trypsin (20 μg / mL) and MTPABP (2 μg) to the plates, and then incubate the mixture of trypsin (20 μg / mL) and MTPABP (2 μg) in a 96-well plate. Incubate the plates at 37°C, 5% CO2 for 24 hours. Add 10 μL of CCK-8 solution to each well, incubate the plates at 37°C, 5% CO2 for 1 hour, and measure the absorbance at 450 nm using a microplate reader.

[0098] The results showed that MTPABP can deliver Trypsin into cells. Compared with the delivery of Trypsin alone, the Trypsin carried by MTPABP entered the cells, causing cell apoptosis and detecting higher cytotoxicity. The results are shown in Figure 11a .

[0099] 5.2 Particle Size (DLS) and Surface Potential (Zeta) of Trypsin Delivered by MTPABP

[0100] The DLS and Zeta assay methods involved dissolving trypsin and its corresponding trypsin-MTPABP complex in distilled water at appropriate concentrations and directly detecting them using a DLS and Zeta assay module (Zetasizer NanoZS 90, Malvern). DLS was used at 25°C to characterize the particle size of MTPABP and the trypsin-MTPABP complex.

[0101] As can be seen from the figure, MTPABP has a negative charge. After forming electrostatic adsorption with Trypsin, the potential is reduced and the particle size is slightly increased, which enables better delivery into cells. The results are shown in Figure 11b 、 11c .

[0102] Example 6 MTPABP delivery of RNaseA

[0103] 6.1MTPABP delivers RNaseA intracellular activity

[0104] In order to determine the cytotoxicity of the organic molecule cytoplasmic delivery carrier MTPABP after encapsulating the cargo molecules, we used the CCK8 kit (Synthemi) to detect the toxicity of the incubation mixture on cells.

[0105] HeLa cell suspension (1×10 4 ) and pre-incubate the culture plates in an incubator for 24 hours. Add RNaseA (50 μg / mL) and MTPABP (2 μg) to the culture plates, and then incubate the mixtures of RNaseA (50 μg / mL) and MTPABP (2 μg) in a 96-well plate. Incubate the plates at 37°C, 5% CO2 for 24 hours. Add 10 μL of CCK-8 solution to each well, incubate the plates at 37°C, 5% CO2 for 1 hour, and measure the absorbance at 450 nm using a microplate reader.

[0106] The results showed that MTPABP can deliver RNaseA into cells. Compared with only delivering RNaseA, the RNaseA carried by MTPABP entered the cells, causing cell apoptosis and detecting higher cytotoxicity. The results are shown in Figure 12a .

[0107] 6.2 Particle Size (DLS) and Surface Potential (Zeta) of MTPABP-Delivered RNaseA

[0108] The DLS and Zeta assay methods involved dissolving RNaseA and its corresponding RNaseA-MTPABP complex in distilled water and directly detecting them at appropriate concentrations using a DLS and Zeta assay module (Zetasizer NanoZS 90, Malvern). DLS was used at 25°C to characterize the particle size of MTPABP and the RNaseA-MTPABP complex.

[0109] As can be seen from the figure, MTPABP has a negative charge. After forming electrostatic adsorption with RNaseA, the potential decreases and the particle size increases slightly, which enables better delivery into cells. The results are shown in Figure 12b 、 12c .

[0110] Example 7: MTPABP delivery of Saporin

[0111] 7.1 Intracellular Activity of Saporin Delivered by MTPABP

[0112] In order to determine the cytotoxicity of the organic molecule cytoplasmic delivery carrier MTPABP after encapsulating the cargo molecules, we used the CCK8 kit (Synthemi) to detect the toxicity of the incubation mixture on cells.

[0113] HeLa cell suspension (1×10 4 ) and pre-incubate the culture plates in an incubator for 24 hours. Add saporin (5 μg / mL) and MTPABP (2 μg), and then incubate the mixture of saporin (5 μg / mL) and MTPABP (2 μg) to the 96-well plate. Incubate the plates at 37°C, 5% CO2 for 24 hours. Add 10 μL of CCK-8 solution to each well, incubate the plates at 37°C, 5% CO2 for 1 hour, and measure the absorbance at 450 nm using a microplate reader.

[0114] The results showed that MTPABP can deliver Saporin into cells. Compared with delivering Saporin alone, RNaseA carried by MTPABP enters cells, causing cell apoptosis and detecting higher cytotoxicity. Figure 13a .

[0115] 7.2 Particle Size (DLS) and Surface Potential (Zeta) of Saporin Delivered by MTPABP

[0116] The DLS and Zeta assay methods involved dissolving saporin and its corresponding saporin-MTPABP complex in distilled water and directly detecting them at appropriate concentrations using a DLS and Zeta assay module (Zetasizer NanoZS 90, Malvern). DLS was used to characterize the particle size of MTPABP and the saporin-MTPABP complex at 25°C.

[0117] As can be seen from the figure, MTPABP has a negative charge. After forming electrostatic adsorption with Saporin, the potential decreases and the particle size increases slightly, which enables better delivery into cells. The results are shown in Figure 13b 、 13c .

Claims

1. An organic molecule cytoplasmic delivery vector, characterized in that: The carrier is the compound MTPABP, whose chemical structure can form electrostatic adsorption with protein molecules through the lipid-soluble aggregation effect, and mediate transmembrane transport to deliver protein molecules into the cytoplasm, and enable the protein molecules to function in the cell without the need for release operations.

2. The organic molecule cytoplasmic delivery vector according to claim 1, characterized in that The chemical structural formula of the carrier MTPABP is as follows: 。 3. A method for preparing an organic molecule cytoplasmic delivery vector, for preparing the organic molecule cytoplasmic delivery vector according to claim 1, characterized in that: The preparation method of the compound MTPABP comprises the following steps: 4-(7-bromobenzo[C][1,2,5]thiadiazol-4-yl)-N,N-bis(4-methoxyphenyl)aniline, (4-(6-chlorohexyl)oxy)phenyl)boric acid, Pd(PPh3)4 and K2CO3 were added to a flask, the flask was evacuated and purged, and then tetrahydrofuran, ethanol and water were added, and the mixture was heated under reflux with stirring for 24 h; The reaction mixture was cooled to room temperature, the organic phase was separated and extracted again with an organic solvent, the organic phases were combined and dried, filtered, and concentrated in vacuo to obtain a crude product; MTPABP was purified by column chromatography.

4. The method for preparing the organic molecule cytoplasmic delivery vector according to claim 3, characterized in that: The preparation method of the compound 4-((6-chlorohexyl)oxyphenyl)boric acid is as follows: 4-Hydroxyphenyl, boric acid and K2CO3 were added to the flask, the flask was evacuated and purged, acetonitrile and 1-bromo-6-chlorohexane were injected respectively under nitrogen atmosphere, and the mixture was heated under reflux and stirred for 12 h; Add ice water, separate the organic phase and extract again with an organic solvent, combine the organic phases, dry them, filter them, and concentrate them in vacuo to obtain a crude product; Purification by column chromatography gave 4-((6-chlorohexyl)oxyphenyl)boronic acid.

5. The method for preparing the organic molecule cytoplasmic delivery vector according to claim 3, characterized in that: The preparation method of the compound 4-(7-bromobenzo[c][1,2,5]thiadiazol-4-yl)-N,N-bis(4-methoxyphenyl)aniline is as follows: 4-(bis(4-methoxyphenyl)amino)phenyl)boronic acid, 4,7-dibromobenzo[1,2,5]thiadiazole, Pd(PPh3)4 and K2CO3 were added to a flask, the flask was evacuated and purged, and then tetrahydrofuran, ethanol and water were added, and the mixture was heated under reflux with stirring for 24 h; The reaction mixture was cooled to room temperature, the organic phase was separated and extracted again with an organic solvent, the organic phases were combined and dried, filtered, and concentrated in vacuo to obtain a crude product; The residue was purified by column chromatography to obtain 4-(7-bromobenzo[c][1,2,5]thiadiazol-4-yl)-N,N-bis(4-methoxyphenyl)aniline.

6. Use of the organic molecule cytoplasmic delivery vector according to claim 1 or 2, characterized in that: The organic molecule cytoplasmic delivery carrier MTPABP forms a complex with a protein through electrostatic adsorption to achieve transmembrane delivery; the protein is selected from a small molecule protein cargo, a medium molecule protein cargo or a large molecule protein cargo.

7. Use of the organic molecule cytoplasmic delivery vector according to claim 6, characterized in that: The small molecule protein cargo includes RNaseA, Trypsin or Saporin, the medium molecule protein cargo includes HRP, and the large molecule protein cargo includes β-Gal.

8. Use of the organic molecule cytoplasmic delivery vector according to claim 6, characterized in that: The delivery process satisfies: The incubation mass ratio of the carrier MTPABP to the protein is 1:2.5 to 1:3; Once the complex enters the cytoplasm, it can activate protein function without requiring a release procedure.

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