Dipeptide modified polymer and application thereof in intracellular delivery of protein

Abstract

The invention discloses a dipeptide modified polymer and application thereof in intracellular delivery of protein. The screened polymer and protein form a compound and are subjected to phase transformation, solute in a liquid phase is transformed into solid-phase particles insoluble in water, early release of the target protein is reduced, and delivery of the protein in the presence of serum is achieved. The protein intracellular delivery method provided by the invention has relatively high intracellular delivery efficiency in the presence of serum-free protein and serum, and the delivery efficiency is higher than that of a commercial protein transfection reagent PULSin; after beta-Gal and other enzyme proteins are delivered into cells, the activity can be recovered, a catalytic substrate is converted into a monitorable product, and the amount of the delivered and active recovered protein is less affected by serum protein in incubation conditions; in addition, the material has low cytotoxicity, the cell activity is higher than 90% under the experimental condition of protein delivery, and the material has good biocompatibility.

Description

Technical Field

[0001] The present invention belongs to the technical field of polymer materials, and in particular relates to a dipeptide-modified polymer and its application in protein intracellular delivery. Background Art

[0002] Protein delivery technology is a key technology in modern biomedical research and is of great significance for the treatment of genetic diseases, cancer, and many other diseases. Currently, protein delivery is mainly achieved through covalent modification and non-covalent carriers.

[0003] Covalent modification is a method of adding specific chemical groups to amino acid residues of proteins through chemical or enzymatic reactions, thereby altering their structure and function. This method enables precise targeting and delivery of proteins. A common covalent modification method is genetic engineering, whereby fragments such as transduction peptides are fused to native proteins to impart specific cellular entry pathways. Another method is chemical modification, using polymers or small molecule ligands to alter the protein's cellular entry pathway.

[0004] However, covalent protein modification also has limitations. First, delivering different proteins requires re-modification, which increases operational complexity and cost. Second, the modification process can disrupt the protein structure, leading to irreversible loss of activity. This loss of activity can affect protein function, thereby reducing therapeutic efficacy.

[0005] Compared to covalent modification, the non-covalent carrier method forms a complex by aggregating proteins and carriers, helping proteins overcome the hydrophobic, negatively charged cell membrane barrier. This method has greater flexibility and applicability and can be applied to different types of protein delivery. Non-covalent carriers generally include liposomes, polymers, metal-organic frameworks, etc. (for example, patent CN116377011A, document: Yang Xiaoti. Research on protein delivery and functional analysis based on metal-organic framework materials [D]. University of Chinese Academy of Sciences.). These carriers can form stable complexes with proteins and protect proteins from enzymatic degradation in the biological environment.

[0006] However, the non-covalent carrier approach also faces some challenges. Due to the large molecular weight and different charge distribution of proteins, they cannot directly form stable complexes with cationic carriers. To solve this problem, researchers need to modify functional groups on cationic carriers to improve the stability of the complex. Although most non-covalent carriers have overcome the stability problem of the complex in salt ion solutions, their tolerance to serum is very low. Proteins in serum will compete with the carrier for binding, causing the protein to be released before reaching the target cells. This serum instability is a bottleneck problem in protein delivery in biological applications. Summary of the Invention

[0007] The present invention aims to remedy the deficiencies in the prior art and proposes a strategy for delivering proteins of different isoelectric points and molecular weights in the presence of serum. A carrier library is constructed by subjecting polymers to different surface chemical modifications. The screened polymers help proteins overcome cell membrane barriers by non-covalently aggregating with proteins to form complexes, and proteins can restore enzymatic activity. In addition, proteins in biological fluids inevitably compete with target proteins, causing the release of target proteins. Therefore, commercially available protein delivery reagents such as PULSin require serum-free incubation conditions. The screened polymers form complexes with proteins and undergo phase transitions, transforming solutes in the liquid phase into solid-phase particles insoluble in water, reducing the premature release of target proteins and enabling the delivery of proteins in the presence of serum.

[0008] The present invention provides a dipeptide-modified polymer (DMP), the general chemical structure of which is Formula 1 or Formula 2, wherein:

[0009]

[0010] In formula 1-2, R1 is a cationic polymer,

[0011] R2 is a hydrophobic functional group, including at least one of methyl (A), isopropyl (V), sec-butyl (I), isobutyl (L), methylthio (M), phenyl (F), phenol (Y), and indolyl (W).

[0012]

[0013] R3 is a cationic functional group, including at least one of a guanidine group (R) and an amino group (K).

[0014]

[0015] X is the number of peptide connections, and X is an integer between 1 and 256. Preferably, the cationic polymer is at least one of polylysine, polyamidoamine dendrimer, branched polyethyleneimine, and linear polyethyleneimine.

[0016] Preferably, R1 is polylysine, R2 is phenyl, phenol or indole, R3 is guanidine, and X is 64.

[0017] More preferably, the chemical structure of the dipeptide-modified polymer is Formula 3 (named FR) or Formula 4 (named RW):

[0018]

[0019] The present invention also provides a method for preparing the dipeptide-modified polymer. The general formula of the dipeptide-modified polymer is Formula 1. The preparation method comprises the following steps:

[0020] (1) A first amino acid with an amino protecting group undergoes a coupling reaction with a cationic polymer having a Cy5-NH2 core and an amino terminal to obtain product 1;

[0021] (2) removing the amino protecting group of the first amino acid from the obtained product 1 to obtain the product 1 from which the amino protecting group has been removed;

[0022] (3) subjecting a second amino acid having an amino protecting group to an amidation reaction with the amino group of the product 1 from which the amino protecting group has been removed to obtain product 2;

[0023] (4) removing the amino protecting group of the second amino acid from the obtained product 2 to obtain the dipeptide-modified polymer.

[0024] Specifically, in step (1), Fmoc-first amino acid-OPFP, DIPEA, and polylysine with Cy5-NH2 as the core and amino terminal were added to N,N-dimethylformamide in a molar ratio of 128:256:1 and reacted at room temperature for 24 hours to obtain product 1;

[0025] In step (3), Boc-second amino acid (Pbf)-OH, EDCl, and DMAP were activated in dimethyl sulfoxide at a molar ratio of 140:128:128 for 5 minutes, and then the product 1 with the amino protecting group removed was added at a molar ratio of 1, and the reaction was carried out at room temperature for 24 hours.

[0026] The present invention also provides the use of the dipeptide-modified polymer in the intracellular delivery of proteins. The dipeptide-modified polymer is used as a protein intracellular delivery carrier to achieve intracellular release of proteins and restore their activity.

[0027] Preferably, the protein is at least one of bovine serum albumin (BSA), ovalbumin (OVA), immunoglobulin G (IgG), lysozyme, phycoerythrin (R-PE), β-galactosidase (β-Gal) and ribonuclease A (RNaseA).

[0028] Beneficial effects of the present invention:

[0029] The protein intracellular delivery method proposed in the present invention has a high intracellular delivery efficiency both in the absence of serum protein and in the presence of serum, and the delivery efficiency is higher than that of the commercial protein transfection reagent PULSin; the present invention can restore the activity of enzyme proteins such as β-Gal after delivery into the cell, catalyzing the conversion of substrates into monitorable products, and the amount of delivered and restored protein activity is less affected by serum proteins in the incubation conditions; the present invention has obvious cytotoxicity after delivering the toxic protein RNase A to HeLa cells in the presence of serum; in addition, the material has low cytotoxicity, and the cell activity is higher than 90% under the experimental conditions of protein delivery, and has good biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Figure 5 is the average fluorescence intensity of FITC in HeLa cells after BSA FITC was delivered by different dipeptide-modified polymers.

[0031] Figure 2 Cell viability after incubation of HeLa cells with different concentrations of dipeptide-modified polymers FR and RW for 24 hours.

[0032] Figure 3 Figure 3 (a) Mean fluorescence intensity of FITC in HeLa cells after BSA FITC was delivered by dipeptide-modified polymers FR and RW in the presence of different serum concentrations, and confocal microscopy images (b). The commercial delivery reagent PULSin was used as a control.

[0033] Figure 4 Cellular distribution of BSAFITC delivered by dipeptide-modified polymers FR (a) and RW (b) at different time points.

[0034] Figure 5 Laser confocal microscopy images of dipeptide-modified polymers FR and RW delivering fluorescently labeled proteins with different isoelectric points and molecular weights.

[0035] Figure 6 Figure 3 shows the enzyme activity staining after β-gal delivery using dipeptide-modified polymers FR and RW. The commercial delivery reagent PULSin was used as a control.

[0036] Figure 7 Cell survival rate after RNase A delivery to HeLa cells using dipeptide-modified polymers. DETAILED DESCRIPTION

[0037] Example 1: Synthesis of dipeptide-modified polymers

[0038] The preparation method of the dipeptide-modified polymer of the present invention is:

[0039] Fmoc-amino acid (side chain protecting group)-OPFP (where Fmoc represents 9-fluorenylmethyloxycarbonyl, an amino protecting group; amino acid side chains include A, V, I, L, M, F, Y, and W, with A, V, I, L, M, and F lacking side chain protecting groups, Y having a side chain protecting group of tert-butyl O-tBu, and W having a side chain protecting group of tert-butyloxycarbonyl N-Boc), N,N-diisopropylethylamine (DIPEA), and a fifth-generation polylysine dendrimer with a Cy5-NH2 core and amino termini at a molar ratio of 128:256:1 were added to N,N-dimethylformamide (DMF) and reacted at room temperature for 24 hours. The reaction was followed by ether precipitation and vacuum drying. Deprotection was then carried out by adding 5% diethylamine (DIPEA:DMF = 5:95) for 1 hour, followed by ether precipitation and vacuum drying to obtain the intermediate product.

[0040] Boc-lysine-(Boc)-OPFP, DIPEA, and the aforementioned intermediate were added to DMF at a molar ratio of 128:256:1 and reacted at room temperature for 24 hours. The mixture was precipitated with diethyl ether and dried in a vacuum dryer. Deprotection was carried out in a solution of 50% trifluoroacetic acid (TFA) in dichloromethane (DCM) (TFA:DCM = 50:50) for 5 hours. The mixture was then precipitated with diethyl ether and dried in a vacuum dryer. The resulting products were designated AK, VK, IK, LK, MK, FK, YK, and WK. GPC analysis confirmed that the dendrimer surface was fully modified with amino acids, with 64 dipeptide-modified linkages.

[0041] Example 2 Synthesis of dipeptide-modified polymers

[0042] The preparation method of the dipeptide-modified polymer of the present invention is:

[0043] Fmoc-amino acid (side chain protecting group)-OPFP (where Fmoc represents 9-fluorenylmethoxycarbonyl, an amino protecting group, and the amino acid side chains include A, V, I, L, M, F, Y, and W, with A, V, I, L, M, and F lacking side chain protecting groups, Y having a tert-butyl O-tbu side chain protecting group, and W having an N-Boc side chain protecting group), DIPEA, and a fifth-generation polylysine dendrimer with a Cy5-NH2 core and amino termini at a molar ratio of 128:256:1 were added to DMF and reacted at room temperature for 24 hours. The reaction was followed by ether precipitation and vacuum drying. Deprotection was then carried out by adding 5% diethylamine (diethylamine:DMF = 5:95) for 1 hour, followed by ether precipitation and vacuum drying to obtain an intermediate product.

[0044] Boc-arginine-(Pbf)-OH (where Pbf is 2,2,4,6,7-pentamethyldihydrobenzofuran-3-sulfonyl, a side chain protecting group), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl), and 4-dimethylaminopyridine (DMAP) were activated in dimethyl sulfoxide at a molar ratio of 140:128:128 for 5 minutes. The intermediate product was then added at a molar ratio of 1 and allowed to react at room temperature for 24 hours. The product was then dialyzed against dimethyl sulfoxide and lyophilized using a freeze dryer. Deprotection was then performed in 95% TFA (TFA:DCM = 95:5) for 5 hours, followed by precipitation with diethyl ether and drying in a vacuum dryer. The resulting products were designated AR, VR, IR, LR, MR, FR, YR, and WR. GPC analysis confirmed that the surface of the dendrimer was fully modified with amino acids, with 64 dipeptide-modified linkages.

[0045] The structural formula of FR is shown in Formula 3:

[0046]

[0047] Example 3 Synthesis of dipeptide-modified polymers

[0048] The preparation method of the dipeptide-modified polymer of the present invention is:

[0049] Fmoc-Lys-(Boc)-OPFP, DIPEA, and a fifth-generation polylysine dendrimer with a Cy5-NH2 core and amino termini were added to DMF at a molar ratio of 128:256:1 and reacted at room temperature for 24 hours. The mixture was then precipitated with diethyl ether and dried in a vacuum dryer. Deprotection was then performed by adding 5% diethylamine (diethylamine:DMF = 5:95) for 1 hour, followed by precipitation with diethyl ether and drying in a vacuum dryer.

[0050] Boc-amino acid (side chain protecting group)-OPFP (amino acid side chains include A, V, I, L, M, F, Y, and W, where A, V, I, L, M, and F have no side chain protecting groups, Y has a side chain protecting group of tert-butyl O-tbu, and W has a side chain protecting group of N-Boc), DIPEA, and the above dendrimer (1 molar ratio of 1) were added to DMF at a molar ratio of 128:256:1 and reacted at room temperature for 24 hours. The reaction was followed by precipitation with diethyl ether and drying in a vacuum dryer. Deprotection was then carried out in 50% TFA (TFA:DCM = 50:50) for 5 hours, followed by precipitation with diethyl ether and drying in a vacuum dryer. The resulting products were designated KA, KV, KI, KL, KM, KF, KY, and KW. GPC analysis confirmed that the dendrimer surface was fully modified with amino acids, with 64 dipeptide-modified linkages.

[0051] Example 4 Synthesis of dipeptide-modified polymers

[0052] After activation in dimethyl sulfoxide (DMSO) at a molar ratio of 140:128:128, Fmoc-arginine-(Pbf)-OH, EDCl, and DMAP were added at a molar ratio of 1 for 5 minutes. A fifth-generation polylysine dendrimer with a Cy5-NH2 core and amino termini was then added at a molar ratio of 1. The reaction was allowed to proceed at room temperature for 24 hours. The product was then dialyzed against dimethyl sulfoxide and lyophilized using a freeze dryer. Deprotection was then performed in 5% diethylamine (DMF = 5:95) for 1 hour, followed by precipitation with diethyl ether and drying in a vacuum dryer.

[0053] Boc-amino acid (side chain protecting group)-OPFP (amino acid side chains include A, V, I, L, M, F, Y, and W, where A, V, I, L, M, and F have no side chain protecting groups, Y has a side chain protecting group of tert-butyl O-tbu, and W has a side chain protecting group of N-Boc), DIPEA, and the above dendrimer (1 molar ratio of 1) were added to DMF at a molar ratio of 128:256:1 and reacted at room temperature for 24 hours. The reaction was followed by precipitation with diethyl ether and drying in a vacuum dryer. Deprotection was then carried out in 95% TFA (TFA:DCM = 95:5) for 5 hours, followed by precipitation with diethyl ether and drying in a vacuum dryer. The resulting products were designated RA, RV, RI, RL, RM, RF, RY, and RW. GPC analysis confirmed that the dendrimer surface was fully modified with amino acids, with 64 dipeptide-modified linkages.

[0054] Among them, the structural formula of RW is shown in Formula 4:

[0055]

[0056] Example 5: Screening of protein delivery efficiency of dipeptide-modified polymers

[0057] Using fluorescein isothiocyanate (FITC)-labeled BSA (BSA-FITC) as a model protein, the delivery efficiency of BSA by the dipeptide-modified polymers prepared in Examples 1 to 4 of the present invention was evaluated on HeLa cells.

[0058] The specific method is as follows: HeLa cells were seeded into 12-well plates (10 5 / well) and cultured for 24 hours, a protein delivery experiment was performed. BSA-FITC was mixed with the dipeptide-modified polymer prepared in Examples 1 to 4 in 100 μl of serum-free 1640 culture medium, incubated at room temperature for 15 minutes, and then 900 μl of 1640 culture medium containing 10% fetal bovine serum (FBS) was added. After washing the 12-well plate once with PBS, 500 μl of culture medium containing DMP / BSA-FITC complex was added to each well and incubated in a 37°C incubator for 4 hours. The culture medium was removed, and the cells were washed three times with PBS and digested with trypsin. The FITC fluorescence intensity in the cells was quantitatively analyzed using a flow cytometer. The concentration of BSA-FITC is 5 μg·ml -1 The dose of DMP was 5 μg ml -1 .

[0059] Experimental results: Figure 1 As shown, the protein delivery efficiency of the dipeptide-modified polymers with different structures varies greatly, among which the polymer FR prepared in Example 2 and the polymer RW prepared in Example 4 showed the highest protein delivery efficiency after pre-incubation with BSA-FITC.

[0060] Example 6: Cytotoxicity of dipeptide-modified polymers

[0061] The CCK8 method was used to detect the toxicity of the dipeptide-modified polymers prepared in Examples 2 and 4 of the present invention to cells under different material concentration conditions.

[0062] The specific method is as follows: HeLa cells were seeded into 96-well plates (5000 / well) and cultured for 24 hours before cytotoxicity experiments. The culture medium was removed and 200 μl of DMP containing different concentrations (2.5 μg ml -1 -160μg·ml -1 The cells were cultured in 1640 medium containing 10% FBS for 24 hours and then placed in a 37° C. incubator for 24 hours. The cell viability was determined using the standard CCK8 assay.

[0063] Experimental results: Figure 2 As shown, the dipeptide-modified polymers prepared in Examples 2 and 4 of the present invention were effective in delivering the experimental concentration (5 μg·ml -1 ) under the conditions of HeLa cell survival rate is higher than 90%, and at a concentration of 20μg.ml -1 The above results indicate that the dipeptide-modified polymer prepared by the present invention has low cytotoxicity and good biocompatibility.

[0064] Example 7: Intracellular delivery efficiency of proteins by dipeptide-modified polymers at different serum concentrations

[0065] Using BSA-FITC as a model protein, the intracellular delivery efficiency of the dipeptide-modified polymers prepared in Examples 2 and 4 of the present invention at different serum concentrations was evaluated on HeLa cells.

[0066] The specific method is as follows: HeLa cells were seeded into 12-well plates (10 5 / well) and cultured for 24 hours before protein delivery experiments. BSA-FITC and dipeptide-modified polymers were mixed in 100 μl of serum-free 1640 medium and incubated at room temperature for 15 minutes. Then, 900 μl of 1640 medium containing 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% FBS was added (named SFM, SM-10, SM-20, SM-30, SM-40, SM-50, SM-60, SM-70, SM-80, SM-90, and SM-100, respectively). After washing the 12-well plate once with PBS, 500 μl of medium containing DMP / BSA-FITC complex was added to each well and incubated in a 37°C incubator for 4 hours. The medium was removed, the cells were washed three times with PBS, and then trypsinized. The FITC fluorescence intensity in the cells was quantitatively analyzed using a flow cytometer.

[0067] In another independent experiment, HeLa cells were seeded into confocal microplates (10 5 After culturing for 24 hours (per well), the confocal dish was washed once with PBS, and 1 ml of the above-mentioned culture medium containing the DMP / BSA-FITC complex was added to each dish. The dish was placed in a 37°C incubator and incubated for 4 hours. The culture medium was removed and the cells were washed three times with PBS. The fluorescence intensity and distribution within the cells were observed using a laser confocal microscope. The commercial delivery reagent PULSin was used as a control according to the instructions. The concentration of BSA-FITC was 5 μg ml -1 , the concentration of DMP was 10 μg ml -1 .

[0068] Experimental results: Figure 3 As shown, the dipeptide-modified polymers prepared in Examples 2 and 4 of the present invention exhibited a certain degree of serum tolerance. The delivery rate of the FR / BSA-FITC complex gradually increased with increasing serum concentration during culture, while the protein delivery efficiency of the RW / BSA-FITC complex was essentially unaffected at concentrations ranging from 0% to 100% FBS. These results demonstrate that loading the model protein BSA-FITC onto FR and RW can, to a certain extent, avoid nonspecific competition from serum proteins and retain the BSA-FITC loaded during pre-incubation. The stability of the complex in biological fluids is crucial for the in vivo application of protein delivery reagents.

[0069] Example 8: Cellular distribution of dipeptide-modified polymer and BSA-FITC complex

[0070] Using BSA-FITC as a model protein, the changes in cellular distribution over time and the endosomal escape process of the dipeptide-modified polymers prepared in Examples 2 and 4 of the present invention were evaluated on HeLa cells.

[0071] The specific method is as follows: HeLa cells were seeded into a confocal dish (10 5 / well) after culturing for 24 hours, the protein delivery experiment was performed. BSA-FITC and dipeptide-modified polymers were mixed in 100 μl of serum-free 1640 medium, incubated at room temperature for 15 minutes, and then 900 μl of serum-free medium was added. After washing the confocal dish once with PBS, 1 ml of culture medium containing DMP / BSA-FITC complex was added to each dish, and the dish was placed in a 37°C incubator for incubation for 0.5-4 hours. After removing the culture medium and washing three times with PBS, the fluorescence intensity and distribution in the cells were observed using a laser confocal microscope. The concentration of BSA-FITC was 5 μg ml -1 , the concentration of DMP is 10μg.ml -1 .

[0072] Experimental results: Figure 4 As shown in (a), the FR / BSA-FITC complex bypasses the lysosomal pathway and enters the cell rapidly within 0.5 hours, directly achieving cytoplasmic delivery of the protein. The fluorescence intensity reaches the highest at 2 hours, and the fluorescence of BSA-FITC is diffused in the cytoplasm. Figure 4 As shown in (b), the fluorescence intensity of the RW / BSA-FITC complex gradually increases over time from 0.5 to 4 hours. At 4 hours, the fluorescence of BSA-FITC overlaps minimally with lysosomes, achieving protein cytoplasmic delivery. These results demonstrate that both FR and RW can load the model protein BSA-FITC into the cytoplasm without being degraded by lysosomes, which is key to protein recovery and function in cells.

[0073] Example 9: Delivery of proteins with different isoelectric points (pI) and molecular weights (Mw) by dipeptide-modified polymers

[0074] FITC-labeled OVA, IgG, Lysozyme and natural fluorescent protein R-PE were used as model proteins to evaluate the ability of the dipeptide-modified polymers prepared in Examples 2 and 4 of the present invention to deliver proteins of different isoelectric points and molecular weights on HeLa cells.

[0075] The specific method is as follows: HeLa cells were seeded into a confocal dish (10 5 / well) and cultured for 24 hours, the protein delivery experiment was performed. FITC-labeled OVA, IgG and Lysozyme and the natural fluorescent protein R-PE were mixed with dipeptide-modified polymers FR and RW in 100 μl of serum-free 1640 culture medium, incubated at room temperature for 15 minutes, and then 900 μl of serum-free culture medium was added. After washing the confocal dish once with PBS, 1 ml of culture medium containing DMP / BSA-FITC complex was added to each dish, and the dish was placed in a 37°C incubator for incubation for 4 hours. After removing the culture medium and washing three times with PBS, the fluorescence intensity and distribution in the cells were observed using a laser confocal microscope. The concentrations of FITC-labeled OVA, IgG and Lysozyme were all 5 μg ml -1 , the concentration of R-PE was 1 μg ml -1 , the concentration of DMP was 10 μg ml -1 .

[0076] Experimental results: Figure 5 As shown, FR and RW can deliver FITC-labeled OVA, IgG, Lysozyme, and natural fluorescent protein R-PE into the cytoplasm. The above results show that the dipeptide-modified polymer prepared by the present invention can deliver proteins with different isoelectric points and molecular weights, and has broad application potential.

[0077] Example 10: Delivery of β-gal into HeLa cells using dipeptide-modified polymers

[0078] Using β-gal as a model protein, the ability of the dipeptide-modified polymers prepared in Examples 2 and 4 of the present invention to deliver proteins in the presence of serum was evaluated on HeLa cells, and the protein was able to recover activity.

[0079] The specific method is as follows: HeLa cells were seeded into 12-well plates (10 5 / well) and cultured for 24 hours, a protein delivery experiment was performed. β-gal and a dipeptide-modified polymer were mixed in 100 μl of serum-free 1640 culture medium, incubated at room temperature for 15 minutes, and then 900 μl of serum-free culture medium or culture medium containing 10% FBS was added. The 12-well plate was taken out, the culture medium was removed, and after washing once with PBS, 500 μl of culture medium containing DMP / β-gal complex was added to each well and incubated in a 37°C incubator for 4 hours. After removing the culture medium and washing three times with PBS, the cells were stained using a β-gal in situ staining kit to evaluate the delivery efficiency of β-gal. All steps were performed according to the kit manual. The staining of the cells was observed using an inverted microscope. The commercial delivery reagent PULSin was used as a control according to the instructions. The concentration of β-gal is 5 μg·ml -1 , the concentration of DMP is 10μg.ml-1 .

[0080] Experimental results: HeLa cells were stained using the β-gal in situ staining kit and observed. β-gal hydrolyzed the substrate 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal) to generate a visible blue precipitate.

[0081] like Figure 6 As shown, both FR / β-gal and RW / β-gal-treated HeLa cells had obvious blue precipitates in serum-free or 10% FBS-containing medium conditions, and the presence of serum slightly reduced the amount of delivery.

[0082] In contrast, the commercial delivery reagent PULSin produced almost no blue precipitate in the presence of serum, indicating that PULSin's protein delivery is significantly affected by serum proteins and is unsuitable for protein delivery in the presence of serum. Furthermore, no blue precipitate was observed in cells treated with fifth-generation polylysine dendrimers with unmodified amino termini, demonstrating the importance of surface-modified amphiphilic groups for protein delivery.

[0083] The above results indicate that the dipeptide-modified polymer prepared by the present invention can efficiently deliver β-gal into cells, can restore the activity of the enzyme after β-gal enters cells, and the delivery is less affected by serum.

[0084] Example 11: Delivery of RNase A into HeLa cells using dipeptide-modified polymers

[0085] Using RNase A as a model protein, the ability of the dipeptide-modified polymers prepared in Examples 2 and 4 of the present invention to deliver cytotoxic proteins in the presence of serum was evaluated on HeLa cells.

[0086] The specific method is as follows: HeLa cells were seeded into 96-well plates (10 5 / well) after culturing for 24 hours, a cytotoxicity experiment was performed. RNase A was diluted in 100 μl of serum-free medium in equal proportions, and the same concentration of dipeptide-modified polymer was added and mixed. After incubation at room temperature for 15 minutes, 900 μl of culture medium containing 10% FBS was added. The 96-well plate was taken out, the culture medium was removed, and after washing once with PBS, 200 μl of culture medium containing DMP / RNase A complex was added to each well and incubated in a 37°C incubator for 24 hours. The cell viability was detected according to the standard CCK8 method. The concentrations of RNase A were 0, 6.25, 12.5, 25, 50, 100, and 200 μg ml -1 , the concentration of DMP was 15 μg ml -1 .

[0087] Experimental results: Figure 7 As shown in Figure 2, the dipeptide-modified polymer delivered RNase A under serum-containing conditions and showed obvious cytotoxicity. This is because RNase A enters the cytoplasm and can cleave RNA, leading to cell apoptosis. At a concentration of RNase A of 25 μg ml -1 When the FR / RNase A complex was added, the cell survival rate was only 17%, compared with RNase A alone at 200 μg ml -1 The results indicate that the dipeptide-modified polymer prepared by the present invention can achieve protein delivery and activity recovery in the presence of serum, and has the potential for in vivo application.

Claims

1. A dipeptide-modified polymer, characterized in that: The general chemical structure is Formula 1 or Formula 2, wherein: In formula 1-2, R1 is a cationic polymer, R2 is a hydrophobic functional group, including at least one of methyl, isopropyl, sec-butyl, isobutyl, methylthio, phenyl, phenol, and indolyl. R3 is a cationic functional group, including at least one of a guanidine group and an amino group, X is the number of peptide connections, and X is an integer between 1 and 256.

2. The dipeptide-modified polymer according to claim 1, characterized in that The cationic polymer is at least one of polylysine, polyamide-amine dendrimer, branched polyethyleneimine, and linear polyethyleneimine.

3. The dipeptide-modified polymer according to claim 2, characterized in that The R1 is polylysine, R2 is phenyl, phenol or indole, R3 is guanidine, and X is 64.

4. The dipeptide-modified polymer according to claim 3, characterized in that The chemical structure of the dipeptide-modified polymer is Formula 3 or Formula 4:

5. The method for preparing a dipeptide-modified polymer according to any one of claims 1 to 4, characterized in that: The general formula of the dipeptide-modified polymer is Formula 1, and the preparation method comprises the following steps: (1) A first amino acid with an amino protecting group undergoes a coupling reaction with an amino-terminal cationic polymer to obtain product 1; (2) removing the amino protecting group of the first amino acid from the obtained product 1 to obtain the product 1 from which the amino protecting group has been removed; (3) subjecting a second amino acid having an amino protecting group to an amidation reaction with the amino group of the product 1 from which the amino protecting group has been removed to obtain product 2; (4) removing the amino protecting group of the second amino acid from the obtained product 2 to obtain the dipeptide-modified polymer.

6. The method for preparing a dipeptide-modified polymer according to claim 5, wherein: In step (1), Fmoc-first amino acid-OPFP, DIPEA and amino-terminal polylysine were added to N,N-dimethylformamide at a molar ratio of 128:256:1 and reacted at room temperature for 24 hours to obtain product 1; In step (3), Boc-second amino acid-OH, EDCl, and DMAP were activated in dimethyl sulfoxide at a molar ratio of 140:128:128 for 5 minutes, and then the product 1 with the amino protecting group removed was added at a molar ratio of 1, and the reaction was carried out at room temperature for 24 hours.

7. The method for preparing a dipeptide-modified polymer according to claim 5, wherein: The first amino acid is alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine or tryptophan, and the second amino acid is lysine or arginine.

8. The method for preparing a dipeptide-modified polymer according to claim 7, wherein: When the first amino acid is tyrosine or tryptophan, the side chain of the first amino acid further has a side chain protecting group.

9. Use of the dipeptide-modified polymer according to any one of claims 1 to 4 in intracellular protein delivery.

10. The use according to claim 9, characterized in that The protein is at least one of bovine serum albumin, ovalbumin, immunoglobulin G, lysozyme, phycoerythrin, β-galactosidase and ribonuclease A.

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