WO2023284554A1 - Carrier-free intracellular protein delivery prodrug, and preparation method therefor and application thereof - Google Patents

Abstract

The present invention provides a carrier-free intracellular protein delivery prodrug, and a preparation method therefor and an application thereof, relating to a carrier-free intracellular protein delivery system for transport by means of L-type amino acid transporter 1 (LAT1). The delivery system in the present invention can directly deliver proteins having different charges/molecular weights into cells, avoid endosomal/lysosomal entrapment, maintain protein activities, have excellent tumor targeting performance, and can reduce toxic side effects. This is the first case of trans-membrane transporter-mediated carrier-free intracellular protein transport by a prodrug, and the technology provides a new strategy for potential clinical applications of anti-tumor protein drugs.

Description

A carrier-free protein intracellular delivery prodrug and its preparation method and application technical field

The invention relates to protein modification and transport technology, more specifically to a protein/polypeptide delivery system, specifically a carrier-free protein intracellular delivery prodrug and its preparation method and application.

Background technique

Proteins are an important class of biomaterials with good biocompatibility, and many proteins (such as enzymes) also have special biological functions, so they are expected to be used in a variety of applications such as: cancer therapy, immunotherapy, and biological imaging, etc. . The vast majority of biological reactions are carried out in cells, and protein preparations acting in cells have broad application prospects. However, the protein has a large molecular weight and cannot pass through the biomembrane barrier. The current protein preparations mostly target extracellular receptors or domains, which largely limits the application of protein preparations. It is of great significance to develop an efficient and safe protein intracellular delivery system to deliver functional proteins into dysfunctional cells to achieve cell death or functional recovery while reducing the toxic side effects of normal cells.

At present, most intracellular protein delivery methods are based on carrier-assisted delivery. This delivery method has the following problems: 1) In order to achieve a better delivery effect, cationic polymers are mostly used as protein carriers, and cationic polymers are unstable in serum. its application in vivo. 2) The carrier-assisted delivery method enters cells through the endocytic pathway. Most of the proteins are captured by endosomes/lysosomes and degraded in lysosomes. Only a few proteins enter the cytoplasm to function. 3) Lack of targeting function, can enter normal cells and produce toxic side effects. 4) The interaction between the carrier and the protein may destroy the three-dimensional structure of the protein and cause the loss of activity. 5) The carrier needs a long time to degrade in the body, and there are potential safety problems.

Therefore, there is an urgent need to develop a new delivery method that can overcome the above obstacles and deliver proteins safely and efficiently into the lesion cells.

technical problem

The present invention designs a carrier-free protein delivery system, which can be recognized and transported by tumor cells, can directly deliver proteins into the cytoplasm, avoid endosome/lysosome encapsulation, preserve functional protein activity, especially, reduce Toxic side effects of protein delivery systems in normal cells. It solves the problem that protein drugs used in the prior art all function by targeting cell surface receptors or extracellular specific structures.

technical solution

In the present invention, monomer N reacts with the primary amino group of protein lysine residue in sodium bicarbonate solution, and N is covalently combined with protein to obtain N-protein; then N-protein is reacted with monomer P Binding of P to the protein yields PN-protein. PN-protein has ROS responsiveness and can be broken under the condition of high concentration of ROS in tumor cells to restore the original protein structure and activity. Relevant experiments have shown that this carrier-free delivery method is universal, can deliver proteins of various charges/molecular weights, and avoid endosome/lysosome wrapping to prevent protein degradation. In addition, the protein delivery system of the present invention can accurately identify tumor cells, reduce toxic and side effects in normal cells, and remove N-modified groups under high levels of ROS in tumor cells, promote the recovery of protein activity, and achieve tumor cell-specific killing . In the 4T1 tumor-bearing mouse model, the toxin protein saporin prodrug (PN-saporin) was injected into mice by tail vein injection, successfully inhibiting tumor growth and significantly prolonging the survival of mice.

The present invention adopts the following technical scheme: a carrier-free protein intracellular delivery prodrug, its structure is D-N-P, wherein structure D is a protein, structure N is a monomer structure covalently linked with the protein, and structure P is a covalent The linked monomer structure is the LAT1 substrate molecule. Preferably, the structure N can drop from the structure D in the tumor cell environment.

The preparation method of the above carrier-free protein intracellular delivery prodrug is as follows: react monomer N with protein to obtain N-protein; then react N-protein with monomer P to obtain carrier-free protein intracellular delivery prodrug; wherein The monomeric N structure is located between the protein D and monomeric P structures.

In the present invention, the molar ratio of the monomer N to the primary amino group of the protein is (1-20): 1; the molar ratio of the monomer P to the primary amino group of the protein is 1: (0.5-10); preferably, the single The molar ratio of the monomer N to the primary amino group of the protein is (2-15):1, and the molar ratio of the monomer P to the primary amino group of the protein is 1:(1-5).

In the present invention, the N-terminal of the monomer is a group that can react with the primary amino group of the protein, such as a nitro group, an alkynyl group, a carboxyl group, a succinyl group, an aldehyde group, an epoxy group, etc.; It is a group that can react with monomer P; the reaction between monomer N and protein is carried out in solution at room temperature to obtain N-protein; when N-protein reacts with monomer P, the reaction site can be the original monomer N The end group of the monomer N can also be the group converted from the original end group of the monomer N after the reaction between the monomer N and the protein. The P-terminus of the monomer is a group capable of reacting with the N-terminus of the N-protein. Preferably, the reaction between N-protein and monomer P is carried out in solution at room temperature, so the end where monomer N and monomer P can react is not particularly limited, as long as they can react in water at room temperature. Preferably, the LAT1 substrate molecule is L-leucine, L-methionine, L-phenylalanine, L-pyridine, L-tryptophan, L-tyrosine, L-isoleucine amino acid, L-histidine, etc.

As a specific example, the monomer N is one of the structures shown in the following formula:

.

Monomer P is one of the structures shown in the following formula:

.

In the present invention, the reaction between the monomer N and the protein is carried out in a solution at room temperature. Specifically, the monomer N solution and the protein solution are mixed and reacted for 5 to 20 hours. Preferably, the reaction condition is to react at room temperature for 8 to 20 hours. 15 hours; after the reaction was completed, dialyzed to obtain N-protein. In the protein solution, the solvent is lye, and the alkaline solution can be prepared with a base such as an inorganic base such as sodium bicarbonate.

In the present invention, the reaction condition of N-protein and monomer P is room temperature for 5 to 60 minutes, preferably, the reaction condition of N-protein and monomer P is room temperature for 15 to 30 minutes; after the reaction, ultrafiltration, The carrier-free intracellular delivery prodrug, namely PN-protein, was obtained. As an advantage, the reaction of the N-protein of the present invention with the monomer P is carried out in water without the participation of other substances, thus ensuring the purity of the protein prodrug.

The invention further discloses the application of the carrier-free protein intracellular delivery prodrug in the preparation of protein medicine or antitumor medicine.

In the present invention, the protein is a toxic protein, a non-toxic protein or an enzyme, all of which are aimed at tumor cells.

Beneficial effect

The advantage of the present invention is that the intracellular delivery of prodrug without carrier protein does not require an endocytic mechanism, directly delivers the protein into the cytoplasm, avoids the link of endosome/lysosome escape, and greatly retains the activity of the protein. At the same time, due to the use of simple small molecules to modify the protein, its stability in serum is much higher than that of carrier-based delivery methods. In addition, the present invention can accurately deliver proteins into tumor cells, and outside the cells, small molecules (structure N, structure P) can shield the activity of proteins, and in the intracellular environment, structure N can drop from structure D, For example, under the condition of a high level of reactive oxygen species (ROS) in tumor cells, the protein activity is dropped and restored, and the existence of double insurance greatly reduces the toxic and side effects on normal cells.

Description of drawings

Figure 1 depicts matrix-assisted laser desorption ionization time mass spectrometry (MALDI-TOF) of protein molecular weight after N modification and H ₂ O ₂ deprotection.

Figure 2 depicts the mean fluorescence intensity graph of the uptake of BSA-FITC in HeLa cells with different modifications delivered by the LAT1-mediated carrier-free delivery system and H ₂ O ₂ pretreated BSA-FITC.

Figure 3 depicts the confocal images of HeLa cells delivered by LAT1-mediated carrier-free delivery system with different modifications and H ₂ O ₂ pretreated BSA-FITC.

Figure 4 depicts the uptake levels of PN-BSA-FITC treated with different endocytosis inhibitors in HeLa cells delivered by LAT1-mediated carrier-free delivery system.

Figure 5 depicts the co-localization of PN-BSA-FITC delivered by LAT1-mediated carrier-free delivery system with lysosomes at different time points in HeLa cells.

Figure 6 depicts the laser confocal images of the distribution of PN-BSA-FITC delivered in tumor cells and normal cells by the LAT1-mediated carrier-free delivery system.

Figure 7 depicts the in situ staining and quantitative analysis of the delivery of β-galactosidase (β-gal) in HeLa cells by the LAT1-mediated carrier-free delivery system.

Figure 8 depicts the LAT1-mediated carrier-free delivery system and the confocal images of different proteins delivered by the commercial reagent PULSin/protein complex in HeLa cells.

Figure 9 depicts the staining of horseradish peroxidase (HRP) delivered by the LAT1-mediated carrier-free delivery system in HeLa cells.

Fig. 10 has described LAT-mediated carrier-free delivery system delivery ribonuclease A prodrug (PN-RNase A) Toxicity test graph in HeLa, NIH-3T3 and 293T cells.

Figure 11 depicts the toxicity test graphs of saporin prodrug (PN-RNase A) delivered by LAT-mediated carrier-free delivery system in 4T1, NIH-3T3 and 293T cells.

Figure 12 depicts the tissue distribution of the toxic protein saporin prodrug (PN-saporin) in the mouse 4T1 xenograft tumor model 6 hours after the LAT1-mediated carrier-free delivery system.

Fig. 13 depicts the graph of inhibiting tumor growth and the survival cycle of PN-saporin delivered by the carrier-free delivery system mediated by LAT1 on the mouse 4T1 xenograft tumor model.

Figure 14 depicts the H&E staining images of the main organs and tumor sections in the mouse 4T1 xenograft tumor model delivered by the LAT1-mediated carrier-free delivery system to deliver PN-saporin.

Figure 15 depicts the graph of hemolysis rate after co-incubation of saporin prodrug (PN-saporin) with erythrocytes.

Embodiments of the present invention

As a specific example, the method for preparing N- and P-modified protein preparations (carrier protein-free intracellular delivery of prodrugs) in the present invention is schematically shown as follows:

.

Protein is protein.

The specific preparation method is exemplified as follows: (1) Dissolving 4-(hydroxymethyl)phenylboronic acid pinacol ester in anhydrous THF. Add triethylamine, then add 4-nitrophenyl chloroformate, and stir the reaction at room temperature. The reaction mixture was diluted with ethyl acetate, washed with HCl followed by saturated NaHCO ₃ . The organic layer was dried over MgSO ₄ , filtered and concentrated. It was then purified on a silica gel column. Elute with hexane solution containing 5% ethyl acetate to obtain white solid as monomer N; (2) Dissolve monomer N in dimethyl sulfoxide solution to obtain monomer N solution; dissolve protein in NaHCO ₃ solution to obtain a protein solution; then mix the monomer N solution and the protein solution according to a certain molar ratio of monomer N and protein amino groups, and after stirring at room temperature, transfer the reaction solution to a dialysis bag for ultrapure water dialysis to obtain N- Protein; (3) Dissolve monomer P in water (1 mg/mL); mix N-protein with monomer according to a certain molar ratio, stir at room temperature, transfer the reaction solution to an ultrafiltration tube, and ultrapure water After washing, PN-protein is obtained, which is a prodrug for intracellular delivery without carrier protein.

In order to further understand the present invention, the preferred embodiments of the present invention are described below in conjunction with the examples, and these descriptions are only to further illustrate the features and advantages of the present invention, rather than to limit the claims of the present invention. The raw materials involved in the present invention are all conventional products, which are commercially available and can also be conventionally prepared according to the prior art; the specific operation methods involved, such as stirring and freeze-drying, are conventional methods, and the specific tests are also conventional methods in the art.

Synthesis example: (1) Dissolve 4-(hydroxymethyl)phenylboronic acid pinacol ester (0.5 g, 2.1 mmol) in 20 mL of anhydrous THF. Triethylamine (0.6 mL, 4.5 mmol) was added, followed by 4-nitrophenyl chloroformate (0.47 g, 2.3 mmol), followed by stirring at room temperature for 1 hour. The reaction mixture was diluted with ethyl acetate and washed with 1.0 M HCl followed by saturated aqueous NaHCO ₃ . The organic layer was dried _over MgSO, filtered and concentrated, then purified on a silica gel column eluting with 5% ethyl acetate in hexanes to give a white solid as monomer N:

.

Characterized by ¹ H NMR (400 MHz, CDCl3), δ = 8.25 (d, J = 9.2 Hz, 2H), 7.85 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 9.2 Hz, 2H), 5.31 (s, 2H), 1.35 (s, 12H); monomer P is the structure shown in the following formula:

Monomer N and monomer P are used in the following examples.

Example 1: Monomer N was dissolved in dimethyl sulfoxide solution with a final concentration of (32 mg/mL) to obtain a monomer N solution; bovine serum protein (BSA) was dissolved in 0.1 M NaHCO ₃ aqueous solution (6 mg/mL) to obtain bovine serum protein solution; then mix the two solutions according to the molar ratio of monomer N and amino group of bovine serum protein as 2:1, stir at room temperature for 10 hours, and transfer the reaction solution into a dialysis bag (MWCO = 1 kDa), dialyzed with ultrapure water for 3 days, and freeze-dried to obtain N-modified protein monomer (N-BSA).

Dissolve monomer P and N-BSA in water respectively to obtain monomer P solution (1 mg/mL) and N-BSA solution (6 mg/mL). Mix the monomer P solution and the N-BSA solution in a molar ratio of 2:3 according to the molar ratio of the monomer P and the primary amino group of the protein BSA, stir at room temperature for 30 minutes, and then transfer the reaction solution to an ultrafiltration tube ( MWCO = 3 kDa), washed 5 times with ultrapure water, and freeze-dried to obtain PN-protein (PN-BSA), which is a prodrug for intracellular delivery without carrier protein.

Mix water and acetonitrile at a ratio of 1:1 to obtain a mixed solution, add erucic acid (final concentration: 10 mg/mL) to the mixed solution, and use the mixed solution as the matrix solution. The protein concentration was 1 mg/mL (in water, pH = 7) and mixed in equal volumes with the matrix solution. The obtained protein samples were characterized by matrix-assisted laser desorption ionization time mass spectrometry (MALDI-TOFMS), see Figure 1 for details. The results of MALDI-TOF experiments show that about 24 N molecules can be modified on each BSA, and the modification can be removed without trace in the presence of H ₂ O ₂ after modification, and the original molecular weight of BSA can be restored.

Example 2: Use fluorescein isothiocyanate (FITC) to label BSA, react overnight in 0.1 M NaHCO ₃ buffer solution according to protein: FITC = 1:4 (mass ratio) and avoid light, and then use ultra Dialyze with pure water (MWCO = 3.5 kDa) for two days to remove unreacted FITC molecules to obtain FITC-labeled protein, and then use the method in Example 1 to modify the protein to obtain PN-BSA-FITC. Then, human cervical cancer cells (HeLa) were inoculated into 24-well plates at an amount of 5×10 ⁴ per well, and cultured in DMEM medium containing 10% FBS for 24 hours. After the cells were completely adhered to the wall, protein samples with different modifications and a final concentration of 100 μM H ₂ O ₂ were added to the wells at a concentration of ₄ μg/mL for 12 hours. Incubate for 12 hours. After washing twice with PBS, incubate with trypan blue solution for 3 minutes, continue to wash three times with PBS, and finally analyze the uptake of cells by flow cytometry. See Figure 2 for details. The experimental results show that the protein modified by PN has the highest cellular uptake. Compared with the protein modified only by N, its fluorescence intensity increases by 35 times, but after being treated with H ₂ O ₂ , the modification is removed. The internalization ability is lost, so the fluorescence intensity is similar to that of the unmodified group.

The internalization of protein prodrugs with different modifications and H ₂ O ₂ treatment was further studied by confocal laser experiments. First, HeLa cells were seeded into special confocal dishes at a quantity of 10×10 ⁵ per well. Culture in DMEM medium containing 10% FBS for 24 hours to make the cells adhere completely, and then add protein samples with different modifications and a final concentration of 100 μM H ₂ O ₂ for 12 hours at a concentration of 4 μg/mL to the wells Incubate for 12 hours at 37°C and 5% CO ₂ , see Figure 3 for details. The results of confocal experiments are consistent with the results of flow cytometry analysis. The above results together indicate that the prodrug delivered intracellularly without carrier protein can be recognized by LAT1 and effectively internalized through its translocation.

Example 3: In order to further explore the internalization mechanism of protein prodrugs, a laser confocal method was used for research. First, HeLa cells were inoculated into special confocal dishes according to the number of 10×10 ⁵ per well. Cultivate in DMEM medium containing 10% FBS for 24 hours to make the cells adhere completely. Wash 3 times with PBS, replace with fresh medium, add endocytosis inhibitor: chlorpromazine (CPZ, 10 μg/mL) to inhibit clathrin mediated endocytosis; genistein (GNT, 100 μg/mL) inhibits caveolin-mediated endocytosis; methyl-β-cyclodextrin (mβCD, 50 μM) reduces the amount of cholesterol on the membrane inhibition Lipid raft-mediated endocytosis and macropinocytosis inhibited by wortmannin (WTM, 50 nM), incubated at 37°C for 1 hour. Wash with PBS 3 times, replace with fresh medium, then add PBS solution containing PN-BSA-FITC (final concentration 4 μg/mL) and incubate for 12 hours, incubate at 37 °C for 12 hours, wash 3 times with PBS, trypan blue ( 0.2 mg/mL, 500 μL) for 2 minutes, washed 3 times with PBS, and RIPA lysate (100 μL, 30 min) to lyse the cells. Cellular uptake levels were measured by spectrofluorometer ( λ _ex = 488 nm, λ _em = 530 nm). The fluorescence intensity of HeLa cells treated with PN-BSA-FITC (final concentration: 4 μg/mL) without inhibitors for 12 hours (37 °C) was taken as 100%. See Figure 4 for details. As shown in the figure, after the potential endocytic pathway is inhibited by using an endocytosis inhibitor, the internalization amount of the protein prodrug is almost not reduced, indicating that the protein prodrug is mainly composed of non-caveolin, lipid raft protein, macrocytic Endocytosis into cells through drinking and other pathways, and enter cells in a non-endocytic way.

Example 4: It is visually shown that the carrier-free protein prodrug intracellular delivery system enters cells in a non-endocytic manner, and the confocal laser method is used for the co-integration of protein prodrug (PN-BSA-FITC) and lysosome/endosome Positioning was observed. First, HeLa cells were inoculated into special confocal dishes according to the number of 10×10 ⁵ per well. Culture in DMEM medium containing 10% FBS for 24 hours to make the cells adhere completely, then add protein samples to the wells at a concentration of 4 μg/mL, and incubate at 37 °C and 5% CO ₂ for 1 and 2 days respectively. , 4, 6, 8, 12 hours. After washing twice with PBS, incubate with trypan blue solution for 3 minutes, continue to wash three times with PBS, stain with Hoechst 33342 (5 μg/mL) for 20 minutes, and label lysosomes/endosomes with Lysotracker deep red (20 nM) 30 After three minutes of washing with PBS, intracellular colocalization was observed under a confocal scanning microscope. See Figure 5 for details. The experimental results show that at any moment, the protein prodrug (green fluorescence) hardly overlaps with the lysosome/endosome (red fluorescence), indicating that the protein prodrug is not encapsulated in the lysosome/endosome. The above experimental results show that protein prodrugs enter cells in a non-endocytic manner.

Example 5: The carrier-free protein prodrug intracellular delivery system of the present invention can target tumor cells, and reduce the toxic and side effects on normal cells when delivering toxic functional proteins. In order to verify this theory, five tumor cells, 4T1, MDA-MB-231, CT-26, U87, and U251, and three normal cells, NIH-3T3, HEK293T, and H9C2, were selected as the control group. Firstly, different kinds of cells were inoculated into special confocal dishes according to the quantity of 10×10 ⁵ per well. Cultured in DMEM/1640 complete medium containing 10% FBS for 24 hours to make the cells adhere completely, and then fluorescein isothiocyanate (FITC)-labeled protein samples (PN-BSA-FITC) were added at a concentration of 4 μg/mL ) into the wells and incubated for 12 hours at 37 °C and 5% CO ₂ . After washing twice with cold PBS, incubate with trypan blue solution for 3 minutes, continue to wash with PBS three times, stain with Hoechst 33342 (5 μg/mL) for 20 minutes, and observe the fluorescence distribution in the cells under a laser confocal scanning microscope. See Figure 6 for details. The experimental results showed that a wide range of fluorescence distributions were observed in the five tumor cells, indicating that the protein prodrugs were internalized into the cells in large quantities, while there was almost no fluorescence distribution in normal cells, indicating that the protein prodrugs could specifically recognize tumor cells , to avoid internalization in normal cells.

Example 6: In order to study the activity recovery of P and N modified bioactive proteins in cells, P and N modified β-galactosidase (β-gal) prodrugs were prepared. The preparation method is similar to that in Example 1, prepared according to the molar ratio of P:N:protein primary amino group=1:4:1, and purified by dialysis.

HeLa cells were seeded into 24-well plates at ⁴ ×104 per well and cultured in DMEM medium containing 10% FBS for 24 hours. In order to explore the effect of intracellular ROS on the recovery of PN-β-gal activity, the cells were pre-treated with vitamin C (VC, 2 h, to remove the original ROS in the cells), and the different modified and treated β-gal protein samples were mixed at 4 μg /mL concentration was added to the wells and incubated at 37°C and 5% CO ₂ for 12 hours. Wash three times with PBS, add cell fixative, and fix at room temperature for 10 minutes. Remove the fixative, wash with PBS three times, and add the substrate staining solution containing X-gal (0.1 mg/mL). Place the cell plate overnight in a 37°C incubator without CO ₂ . Then remove the staining solution and wash three times with PBS. Observe the staining of cells with a light microscope. Further, o-nitro-β-D-galactopyranoside (ONPG) was used to quantify the activity of the enzyme. After the treatment of β-gal intracellular delivery experiment, wash with PBS three times, add 200 μL lysis solution to lyse the cells, take 50 μL lysis solution and add 50 μL enzyme activity detection solution containing ONPG, leave at 37 ℃ for 1 h, then add 150 μL NaHCO ₃ (1 M) to terminate the reaction, transfer the solution to a 96-well plate and detect the absorbance at 420 nm. The enzyme activity of untreated β-gal at an equal concentration was used as a positive control, and the absorbance was defined as 100%. See Figure 7 for details. The experimental results showed that PN-β-gal showed the most blue deposition, indicating that a large amount of β-gal was internalized and performed its biological function, and after VC treatment, the blue deposition decreased, indicating that ROS reduction prevented PN- Restoration of β-gal activity. Quantitative experiments showed the same results, and PN-β-gal could almost completely restore its activity in cells, which proved that P and N modified protein prodrugs could effectively achieve protein internalization and restore its activity in tumor cells.

Example 7: The carrier-free delivery system of the present invention can be used to deliver proteins with different molecular weights/charges. Cytochrome C (Cyt C-FITC), ribonuclease A (RNase A-FITC), α-trypsin (α-Chyt-FITC), superoxide dismutase (SOD-FITC), egg white albumin were labeled with FITC (Egg W-FITC), immunoglobulin (IgG-FITC) and TRITC (5/6-carboxy-tetramethyl-rhodamine succinimidyl ester) labeled trypsin (TRP-TRITC), lysozyme (LYZ -TRITC) were prepared into P and N modified protein prodrugs according to the method in Example 1; according to the product instructions, a commercial reagent (PULSin) was used to form a PLUSin/protein complex for comparison.

HeLa cells were cultured in DMEM medium containing 10% FBS for 24 hours to completely adhere to the wall, and then protein prodrugs or PLUSin/protein complex samples with different molecular weights/charges were added to the wells at a final concentration of 4 μg/mL. Incubate for 12 hours at 37°C and 5% CO ₂ . After washing twice with cold PBS, incubate with trypan blue solution for 3 minutes, continue to wash with PBS three times, stain with Hoechst 33342 (5 μg/mL) for 20 minutes, and observe the fluorescence distribution in the cells under a laser confocal scanning microscope. See Figure 8 for details. The experimental results show that after modification by P and N compounds, all protein prodrugs are significantly and evenly distributed in the cells. In addition, compared with the commercial reagent PULSin, the internalization amount of the protein prodrugs of the present invention is significantly increased. The above experimental results collectively show that the preparation of protein prodrugs by modifying proteins with small molecules in the present invention has good universality, and its delivery ability is better than that of existing commercial reagents.

Example 8: Intracellular delivery of horseradish peroxidase (HRP). The preparation method was similar to that of Example 1, prepared according to the molar ratio of P:N:primary amino group=1.5:7:1, and purified by dialysis to obtain PN-HRP.

HeLa cells were seeded into 24-well plates at ⁴ ×104 per well and cultured in DMEM medium containing 10% FBS for 24 hours. Then, different modified and treated HRP protein samples were added to the wells at a concentration of 4 μg/mL and incubated at 37 °C and 5% CO ₂ for 12 hours. Wash 6 times with PBS, add tetramethylbenzidine (TMB, 10 μg/mL) solution and hydrogen peroxide (3 mM) solution, incubate at room temperature for 10 minutes, and observe the staining of each well. See Figure 9 for details. The experimental results showed that the blue color change was the most obvious in PN-HRP, and there was almost no color change in the unmodified group, indicating that PN-HRP could be effectively internalized into cells and perform its biological functions.

Example 9: Intracellular delivery of toxic proteins. Select ribonuclease A (RNase A) As a model protein to detect intracellular delivery efficiency and biological function. The preparation method is similar to that in Example 1, prepared according to the molar ratio of P:N:protein primary amino group=2:7:1, and purified by dialysis to obtain PN- RNase A.

HeLa, NIH-3T3 and 293T cells were inoculated into 96-well plates at 6×10 ³ per well, and cultured in DMEM medium containing 10% FBS for 24 hours. Add ribonuclease A modified by N monomer and monomer P to the Wells were cultured for 48 h. The viability of the cells was measured by CTL assay, compared to cells without any treatment, and the results were expressed as a percentage of control cells. See Figure 10 for details. The experimental results showed that PN-RNase A showed obvious toxicity in tumor cell HeLa, and its IC ₅₀ value was 1.707 μg/mL, but it showed almost no toxicity in normal cells NIH-3T3 and 293T, N-RNase A and RNase The effect of A is similar; the results show that PN-RNase A can be effectively recognized and taken up by tumor cells, and specifically kill tumor cells, see Figure 10 for details.

Example 10: Saporin protein (saporin) was selected as a model protein to detect intracellular delivery efficiency and biological function. The preparation method is similar to that in Example 1, prepared according to the molar ratio of P:N:protein primary amino group=1.5:4:1, and purified by dialysis. And in 0.1 M NaHCO ₃ buffer solution, Cy5-NHS was mixed with saporin or PN-saporin respectively, reacted overnight, and dialyzed with ultrapure water (MWCO = 3.5 kDa) to obtain fluorescently labeled protein prodrugs.

As determined by conventional CTL assay, PN-saporin has obvious toxicity to 4T1 cells, and its IC ₅₀ value is 0.050 μg/mL, and it has no obvious toxicity in normal cells (NIH-3T3, 293T), indicating that the protein The drug has no toxic and side effects on normal cells and has good safety, see Figure 11 for details.

Tissue distribution in vivo of the carrier-free protein delivery system of the invention. Inoculate well-growing breast cancer cells (4T1) into BALB/c (6-8 weeks) mice subcutaneously to establish a breast cancer xenograft tumor model. When the tumor volume reached about 200 mm ³ , Balb/c mice were injected with saporin-Cy5 and PN-saporin-Cy5 (0.5 mg saporin/kg) respectively in the tail vein, and at 6 hours, the mice were sacrificed and their The heart, liver, spleen, lung, kidney and tumor were excised, and the enrichment of protein in each part was observed under the small animal imager. At the same time, the organs and tumors were weighed and ground, lysed with tissue lysate, and their fluorescence intensity was measured with a microplate reader for quantitative analysis. See Figure 12 for details.

In vivo tumor suppressive effect of the carrier-free protein delivery system of the present invention. Inoculate well-growing breast cancer cells (4T1) into BALB/c (6-8 weeks) mice subcutaneously to establish a breast cancer xenograft tumor model. When the tumor volume reached about 50 mm ³ , the mice were randomly divided into three groups, 10 in each group, respectively (1) PBS group, (2) Saporin (Saporin) group, (4) PN-soap Herbaceous element (PN-sporin) group. Mice in each group were given 100 μL of PBS, saporin or PN-saporin through the tail vein, and the saporin protein dose was 0.5 mg/kg. They were administered on days 1, 3, 5, and 7, respectively. The tumor volume and body weight of the mice were measured every other day, and the survival status of the mice was detected at the same time. When the tumor volume reached 1000 ^mm3 , it was considered dead by default. As shown in the figure, it shows that the tumor growth of the mice in the PN-saproin administration group was significantly inhibited, and the survival period of the mice was extended to 40 days, indicating that PN-saproin has a good anti-tumor effect in vivo, see Figure 13 for details.

The above experiments showed that the tumor growth status of the Saporin administration group was similar to that of the PBS group, and grew rapidly during the 12-day observation period. In contrast, tumor growth was significantly inhibited in the PN-saporin group, with a tumor inhibition rate of 80% at 12 days. And the survival period of the mice was significantly prolonged. Within 30 days after administration, the survival rate of the mice in the PN-saporin group was 100%, while all the mice in the control group died within 24 days. On the 12th day, the tumor tissues of the mice in each group were collected, and the levels of tumor cell necrosis and apoptosis were detected. Among them, the tumor tissue treated with PBS and saporin showed tightly packed tumor cells and stroma, while the tumor tissue treated with PN-saporin showed obvious features of cell necrosis and apoptosis, such as nuclear condensation, cell shrinkage and vacuolation . During the 12-day observation period, the body weight of the mice did not decrease significantly after PN-saporin administration, and the H&E staining results of the main organ sections showed no abnormalities, indicating that there were no obvious side effects after PN-saporin system administration. See Figure 14 for details. . In the hemolysis experiment, the PN-saporin administration group had no obvious hemolysis phenomenon, indicating its good biological safety, see Figure 15 for details. These results suggest that PN-saporin is a highly efficient protein drug that can be used for in vivo tumor therapy without obvious toxic side effects.

The invention discloses a carrier-free protein prodrug intracellular delivery strategy. Covalent modification of LAT1 substrate molecules on protein lysine residues prepares protein prodrugs, which can be efficiently and selectively delivered to tumor cells through translocation, avoiding the uptake of normal cells. This intracellular delivery method does not need to go through the endocytic pathway and endosome/lysosome capture, which can greatly improve the utilization rate of protein drugs. Under the action of high concentration of H2O2 in tumor cells, the modification group on the protein prodrug can fall off, and the protein recovers its activity and exerts pharmacological activity. This delivery strategy is universal to tumor cells and can be used to deliver protein drugs with different molecular weights, isoelectric points, and functions, including enzymes, antibodies, toxin proteins, and CRISPR-Cas9 nucleases. In particular, PN-saporin, a prodrug of the toxin protein saporin, exhibited excellent antitumor effects in vitro and in vivo without toxic side effects on normal cells/tissues. This is the first case of transmembrane transporter-mediated intracellular transport of carrier-free proteins, which enables highly sensitive and selective regulation of protein activity in tumor cells. This simple and efficient technique provides a new strategy for the potential clinical application of anti-tumor protein drugs.

Claims (10)

1. A carrier-free protein intracellular delivery prodrug, characterized in that the structure of the carrier-free protein intracellular delivery prodrug is D-N-P, wherein structure D is a protein, and structure N is a monomer structure covalently linked to the protein, the structure P is a LAT1 substrate molecule.
2. According to claim 1, the carrier-free protein intracellular delivery prodrug is characterized in that the structure N can drop from the structure D in the tumor cell environment; the LAT1 substrate molecule includes L-leucine, L-formazine Thionine, L-phenylalanine, L-xyrxine, L-tryptophan, L-tyrosine, L-isoleucine, or L-histidine.
3. According to claim 1, the carrier-free protein intracellular delivery prodrug is characterized in that the preparation method of the carrier-free protein intracellular delivery prodrug is to react monomer N with protein to obtain N-protein; The N-protein was reacted with monomeric P to obtain a prodrug for intracellular delivery without a carrier protein.
4. According to claim 3, the carrier-free protein intracellular delivery prodrug is characterized in that the molar ratio of the monomer N to the primary amino group of the protein is (1-20): 1; the ratio of the monomer P to the primary amino group of the protein The molar ratio is 1: (0.5~10); the N-terminal of the monomer is a group that can react with the primary amino group of the protein; the N-terminal of the protein is a group that can react with the monomer P; the end of the monomer P is a group capable of reacting with the N-terminus of an N-protein.
5. According to claim 4, the carrier-free protein intracellular delivery prodrug is characterized in that the N-terminus of the monomer can react with the primary amino group of the protein, including an aldehyde group, an epoxy group, a nitro group, an alkynyl group, and a carboxyl group. Or succinamide.
6. According to the described carrier-free protein intracellular delivery prodrug of claim 3, it is characterized in that, the reaction of monomer N and protein is carried out in solution, at room temperature, obtains N-protein; The reaction of N-protein and monomer P is in solution Carry out at medium and room temperature.
7. The method for preparing a prodrug without a carrier protein intracellular delivery according to claim 1, characterized in that it comprises the following steps of mixing the monomer N solution with the protein solution and reacting for 5 to 20 hours to obtain N-protein; and then mixing N- The protein is reacted with the monomer P at room temperature for 5-60 minutes to obtain the prodrug for intracellular delivery without carrier protein.
8. The method for preparing a prodrug for intracellular delivery without a carrier protein according to claim 7, wherein the protein is a toxic protein, a non-toxic protein or an enzyme.
9. The application of the carrier-free protein intracellular delivery prodrug according to claim 1 in the preparation of protein drugs or anti-tumor drugs.
10. The use according to claim 9, characterized in that the protein drug or antitumor drug does not contain polymer materials.