CN116115769A - Nucleoside phosphate protein intracellular delivery system and preparation method and application thereof - Google Patents

Nucleoside phosphate protein intracellular delivery system and preparation method and application thereof Download PDF

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CN116115769A
CN116115769A CN202310072809.4A CN202310072809A CN116115769A CN 116115769 A CN116115769 A CN 116115769A CN 202310072809 A CN202310072809 A CN 202310072809A CN 116115769 A CN116115769 A CN 116115769A
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protein
monomer
delivery system
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aba
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殷黎晨
李伟
刘寻
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Suzhou University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes

Abstract

The invention discloses a nucleoside phosphate protein intracellular delivery system, a preparation method and application thereof. The delivery system of the invention can deliver proteins with different charges and molecular weights into cells, effectively escape lysosome capture and maintain protein activity. The first example is a nucleic acid-like protein prodrug modified by a small molecular compound, the surface charge density and the binding site of the protein are effectively regulated and controlled, the combination of the protein and a cationic polymer carrier is enhanced, ABA and nucleotide can fall off from the protein in an acidic environment, and the nanocomposite is dissociated, so that the efficient intracellular delivery and intracellular traceless release of the protein are realized. The simple and efficient technology provides a new strategy for the modification and application of protein drugs and the functional modification of cationic polymers.

Description

Nucleoside phosphate protein intracellular delivery system and preparation method and application thereof
Technical Field
The invention relates to a nucleoside phosphate protein intracellular delivery system, a preparation method and application thereof, and belongs to the technical field of biological medicines.
Background
Proteins have great potential in the treatment of diseases due to their high degree of biological activity and specificity. The problems of complex molecular weight, size and structural composition of proteins, etc., lead to the failure of the proteins to cross cell membranes to participate in intracellular biochemical reactions, so far most protein drugs on the market are developed aiming at extracellular targets. However, proteins that act on intracellular targets can bring about more efficient therapeutic effects, and there is an urgent need to develop effective and versatile intracellular delivery strategies for proteins in order to accelerate the application of intracellular protein therapies.
The most critical issue for intracellular protein delivery is how to stably encapsulate the protein into a carrier. Over the last decade researchers have developed a number of intracellular delivery platforms of proteins such as gold nanoparticles, silica nanoparticles, metal organic frameworks, liposomes, nanovesicles, carbon nanotubes, nanogels and polymers. Among them, the polymer is one of the most advantageous delivery vehicles due to the features of simple synthesis and easy modification. However, proteins have low charge density and few surface binding sites, and the polymer/protein cannot form stable complexes. Based on this, researchers have developed a variety of functional polymers such as amphiphilic polymers, fluorinated polymers, boric acid-rich polymers, guanidine-rich polymers, and coordination polymers, which enhance binding to proteins by a variety of non-covalent interactions. On the other hand, researchers have also attempted to modify proteins such as sugar molecules, nucleic acids and other functional small molecules to increase binding sites and improve interactions with polymers. These strategies, while enhancing the binding between the protein and the polymeric carrier, achieve a highly effective intracellular delivery effect. However, the modification mode of covalent coupling with protein is complex, has low efficiency and is easy to cause protein inactivation. In particular, the modification of nucleic acid on protein further increases the difficulty of protein modification due to the large molecular weight and easy degradation of nucleic acid. Therefore, there is a need to develop a novel delivery method that can overcome the above-mentioned obstacles and deliver proteins into cells safely and efficiently.
Disclosure of Invention
In order to solve the technical problems, the invention designs an intracellular delivery system of the nucleic acid-like protein prodrug mediated by the functionalized cationic polymer, which improves the combination stability of the protein and a carrier, can directly deliver the protein into cytoplasm, avoids capturing and degrading of endosomes/lysosomes, and keeps the activity of the functional protein. Solves the problem that protein medicines used in the prior art all play a role by targeting cell surface receptors or extracellular specific structures.
The method comprises the steps of reacting monomer ABA (2-acetylphenylboronic acid) with primary amino groups of protein lysine, and covalently binding the ABA with the protein to obtain ABA-protein; then, the ABA-protein reacts with the monomer N to bond N to the protein, so as to obtain N-protein; and combining the N-protein with a functionalized cationic polymer to obtain a protein delivery system. In the protein delivery system, ABA modification groups are removed in an intracellular acidic environment, so that the protein activity is promoted to be recovered, and gene knockout of PLK1 and EGFP is realized in HeLa cells and 293T-EGFP cell models. In addition, the intracellular protein delivery system can deliver the protein to different cell lines, and has good cell universality delivery capability.
A first object of the present invention is to provide a phosphonucleoside protein intracellular delivery system comprising a nucleic acid-like protein prodrug, and a cationic polymer carrier conjugated to the nucleic acid-like protein prodrug;
the nucleic acid protein prodrug comprises a protein, a monomer ABA which is covalently connected with the protein and a monomer N which is covalently connected with the monomer ABA, wherein the monomer ABA is a phenylboronic acid compound or a phenylboronic acid pinacol ester compound, and the general formula is as follows:
Figure BDA0004065198760000021
wherein R is hydrogen or a C1-C6 alkane chain;
the monomer N is adenosine phosphate (ATP, ADP and AMP), cytidine phosphate (CTP, CDP and CMP), guanosine phosphate (GTP, GDP and GMP), uridine phosphate (UTP, UDP and UMP), thymidine phosphate (TTP, TDP and TMP), adenosine, cytidine, guanosine, uridine or thymidine.
Further, the cationic polymer is:
Figure BDA0004065198760000031
wherein n=5-500;
m＝0-10；
r1 is a urea phosphate, a thymidine phosphate, an adenosine phosphate, a cytidine phosphate, a guanosine phosphate, an adenosine, a cytidine, a guanosine, a uridine, or a thymidine having base pairing with the monomer N.
Further, the mass ratio of the nucleic acid-like protein prodrug to the cationic polymer carrier is 1 (0.5-10).
Further, the structure of the monomer ABA is as follows:
Figure BDA0004065198760000032
further, the structure of the monomer N is as follows:
Figure BDA0004065198760000033
Figure BDA0004065198760000041
where n=0, 1, 2 or 3.
Further, the protein is a toxic protein, a non-toxic protein, an antibody or a gene editing tool enzyme.
A second object of the present invention is to provide a method for preparing the said nucleoside phosphate protein intracellular delivery system, comprising the steps of:
s1, reacting monomer ABA with protein to obtain ABA-protein;
s2, reacting ABA-protein with a monomer N to obtain the nucleic acid-like protein prodrug;
s3, mixing and reacting the nucleic acid-like protein prodrug with a cationic polymer to obtain the nucleoside phosphate protein intracellular delivery system.
Further, the molar ratio of the monomer ABA to the primary amino groups in the protein is (1-10) to 1.
Further, the molar ratio of the monomer N to the primary amino groups in the protein is (1-10) to 1.
Further, in the step S1, the reaction condition is that in an aqueous solution, the reaction is carried out for 10 to 30 minutes at the temperature of 10 to 40 ℃.
Further, in the step S2, the reaction condition is that in an aqueous solution, the reaction is carried out for 1 to 4 hours at a temperature of between 10 and 40 ℃.
Further, the aqueous solution in the present invention is sodium bicarbonate solution or pure water solution.
A third object of the present invention is to provide the use of said phosphonucleotidylated protein intracellular delivery system for the preparation of protein nanomedicines or gene editing.
The beneficial effects of the invention are as follows:
the invention has the advantages that the negative charge density of the pro-drug delivered in the cell of the nucleic acid-like protein and the binding site on the surface of the protein are increased, the combination with the nano-carrier is more stable, and meanwhile, the nucleic acid-like modification is carried out on the protein by using simple small molecules, so that the modification mode is rapid, efficient and simple, and the biological activity of the protein is not influenced. In addition, the invention can accurately deliver the protein into cells, and the cationic carrier can shield the activity of the protein prodrug outside the cells, and under the intracellular environment, the structural ABA can drop from the structural protein, for example, the structural ABA can drop in the acidic environment of an endosome to restore the activity of the protein, so that the protein can play a role in the cells.
Drawings
FIG. 1 is a diagram showing the binding of an N-modified protein prodrug to a functionalized cationic polymer LPP;
FIG. 2 shows the binding of N-modified protein prodrugs to the functionalized cationic polymer PEI-AZT;
FIG. 3 is a graph of the average fluorescence intensity of functionalized cationic polymer LPP-mediated delivery of a nucleic acid-like protein prodrug delivery system for modified BSA-FITC uptake in HeLa cells;
FIG. 4 is a laser confocal plot of functionalized cationic polymer LPP-mediated delivery of modified BSA-FITC by HeLa cells by a nucleophile-like protein prodrug delivery system;
FIG. 5 is a generic view of the protein delivery of functionalized cationic polymer PEI-AZT mediated nuclease-like protein prodrug delivery system;
FIG. 6 is a graph of the co-localization of functionalized cationic polymer LPP-mediated, nuclease-like protein prodrug delivery system with lysosomes at various time points in HeLa cells;
FIG. 7 is a graph showing the co-localization of functionalized cationic polymer PEI-AZT mediated nuclease-like protein prodrug delivery system with lysosomes at different time points in HeLa cells;
FIG. 8 is a laser confocal map of distribution of functionalized cationic polymer LPP-mediated, different molecular weight and isoelectric point, nucleic acid-like protein prodrug delivery systems in HeLa cells;
FIG. 9 is an in situ staining and quantitative analysis of functionalized cationic polymer LPP-mediated, nucleic acid-like protein prodrug delivery system delivering beta-galactosidase (beta-gal) in HeLa cells;
FIG. 10 is a staining and quantitation diagram of functionalized cationic polymer LPP-mediated delivery of horseradish peroxidase (HRP) by a nucleic acid-like protein prodrug delivery system in HeLa cells.
FIG. 11 is a graph of toxicity assays of functionalized cationic polymer LPP-mediated delivery of ribonuclease A prodrug (A-RNase A) and saporin prodrug (A-saporin) in HeLa cells by a nucleoprotein-like prodrug delivery system;
FIG. 12 is a laser confocal plot of functionalized cationic polymer LPP-mediated, nucleic acid-like protein prodrug delivery system delivering modified IgG-RBITC for uptake in HeLa cells;
FIG. 13 is a functionalized cationic polymer LPP-mediated delivery of A-RNP by a nucleic acid-like protein prodrug delivery system EGFP Mean fluorescence intensity profile and laser confocal profile of gene editing on 293T-EGFP cells;
FIG. 14 is a functionalized cationic polymer LPP-mediated delivery of A-RNP by a nucleic acid-like protein prodrug delivery system PLK1 DNA cleavage effect map of gene editing on HeLa cells;
FIG. 15 is a laser confocal plot of functionalized cationic polymer PEI-AZT mediated nuclease-like protein prodrug delivery system in tumor cell line A549 and normal cell lines H9C2 and 3T 3.
Detailed Description
The present invention will be further described with reference to specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the present invention and practice it.
As a specific example, the method of the present invention for preparing ABA and N modified nuclease-like protein prodrugs is illustrated below:
Figure BDA0004065198760000061
protein is a Protein.
The specific preparation method is as follows:
dissolving monomer ABA in NaHCO 3 Obtaining a monomer ABA solution in the solution; dissolving the protein in NaHCO 3 Obtaining protein solution in the solution; mixing the monomer ABA solution and the protein solution according to a certain molar ratio of the monomer ABA to the protein amino, and stirring for 20 minutes at room temperature; dissolving monomer N in NaHCO 3 In solution (10 mg/mL); mixing ABA-protein and monomer according to a certain molar ratio, stirring for 3 hours at room temperature, transferring the reaction solution into a ultrafiltration tube (MWCO=3 kDa), and washing with ultrapure water for 5 times to obtain N-protein, which is a nucleic acid-like protein prodrug.
For a further understanding of the present invention, preferred embodiments of the invention are described below in conjunction with the examples, which are provided to further illustrate the features and advantages of the invention, and not to limit the claims of the invention. The raw materials involved in the invention are all conventional products, and can be purchased commercially or prepared conventionally according to the prior art; the specific methods of operation involved, such as stirring, lyophilization, are all conventional methods, and specific tests are also conventional in the art.
Synthesis example
Monomer ABA is a structure shown in the following formula:
Figure BDA0004065198760000062
monomer N has the structure shown in the following formula:
Figure BDA0004065198760000063
monomer ABA, monomer N were used in the following examples.
Example 1:
LPP was dissolved in phosphate buffer (phosphate buffer,10mM, pH 5.0) at a concentration of 1 mg/mL. LPP and A-BSA were mixed in different mass ratios (LPP/A-BSA=1, 2.5, 5, 10 and 15, w/w), and after shaking, they were allowed to stand at room temperature for 30 minutes to give LPP/A-BSA NCs of different mass ratios (final concentration 50. Mu.g BSA/mL). NCs were centrifuged (12000 rpm,4 ℃ C., 20 minutes) and the supernatant was collected by fluorescence spectrophotometry (excitation light lambda) ex Emission light λ=488 nm em =525 nm) and the fluorescence intensity of the protein in the supernatant was measured, thereby determining the concentration of the protein in the supernatant. Referring specifically to fig. 1, for LPP/a-BSA NCs, the binding efficiency of a-BSA to LPP gradually increased from 72% (LPP/a-bsa=1) to 99% (LPP/a-bsa=15) with increasing mass ratio. Whereas for LPP/BSA NCs, the binding efficiency of BSA to LPP showed a tendency to increase followed by decrease with increasing mass ratio, which was highest when LPP/bsa=5, up to 54%. Therefore, the binding efficiency of LPP/A-BSA NCs is higher than that of LPP/BSA NCs.
PEI-AZT (or PEI) was dissolved in ultrapure water at a concentration of 2 mg/mL. Mixing PEI-AZT with A-BSA (G-BSA or G-BSA), shaking, and standing at room temperature for 20 min to obtain PEI/A-BSA NCs, PEI-AZT/G-BSA NCs andPEI-AZT/C-BSA NCs (final concentration 20. Mu.g protein/mL). NCs were centrifuged (12000 rpm,4 ℃ C., 20 minutes), and the supernatant was collected by fluorescence spectrophotometry (excitation light) λex =488 nm, emitted light λem =525 nm) and the fluorescence intensity of the protein in the supernatant was measured, thereby determining the concentration of the protein in the supernatant. Referring specifically to FIG. 2, the binding efficiency of PEI-AZT/A-BSA-FITC NCs was 97.5%, which is significantly higher than that of the control PEI-AZT/G-BSA-FITC NCs (77.9%) and PEI-AZT/C-BSA-FITC NCs (89.7%), and the incorporation of base complementary pairing hydrogen bonding in the protein prodrug A-BSA and the thymidylated cationic polymer PEI-AZT significantly enhanced the binding of both. The unmodified cationic polymer PEI binds to the protein prodrug A-BSA-FITC only by electrostatic interactions with a binding efficiency of 91.2% which is significantly lower than that of PEI-AZT/A-BSA-FITC NCs, indicating that the multiple interactions introduced between the protein and the cationic polymer can increase the binding efficiency of NCs.
Example 2:
BSA was labeled with Fluorescein Isothiocyanate (FITC) at 20mM NaHCO 3 In the buffer solution, according to the protein: fitc=1:3 (mass ratio) in the dark, reacted overnight, then dialyzed (mwco=3.5 kDa) against ultrapure water for two days, unreacted FITC molecules were removed to obtain FITC-labeled proteins, and the proteins were modified to obtain a-BSA-FITC using the method of example one. Human cervical cancer cells (HeLa) were then plated at 1X 10 per well 5 Is inoculated into 24-well plates and cultured in DMEM medium containing 10% FBS for 24 hours. After complete cell attachment, fresh serum-free medium was used instead, and different protein samples were added to the wells at a concentration of 2. Mu.g/mL at 37℃and 5% CO 2 Is incubated for 4 hours. After three washes with PBS, treatment with trypan blue solution for 10 min was continued for three washes with PBS, and finally the uptake of cells was analyzed by flow cytometry. Referring specifically to FIG. 3, experimental results show that after the ABA and A modified proteins are complexed with LPP, the cellular uptake is highest compared with unmodified proteins and the helicated cationic polypeptide DL LPP with 73-fold and 22-fold increase in fluorescence intensity, respectively.
Further studies on the internalization of LPP-mediated nuclease-like protein prodrugs using laser confocal experiments, heLa cells were first 1X 10 per well 4 Is inoculated into a confocal dish. Culturing in DMEM medium containing 10% FBS for 24 hr to allow cells to adhere completely, replacing with fresh medium without serum, adding different protein samples into the wells at concentration of 2 μg/mL, and heating at 37deg.C and 5% CO 2 For 4 hours, see in particular figure 4. The confocal experimental result is consistent with the flow cytometry analysis result, and the results together indicate that the nucleic acid-like protein prodrug can be effectively combined with a cationic carrier, and high-efficiency intracellular delivery is realized through the nucleic acid-like protein prodrug.
Example 3:
HeLa cells were grown at 1X 10 4 Individual cells/wells were seeded into 24-well plates and incubated at 37 ℃ for 24 hours. The cell culture medium was replaced with serum-free DMEM, followed by addition of BSA-FITC, PEI/A-BSA-FITC NCs, PULSin/BSA-FITC NCs, PEI-AZT/C-BSA-FITC NCs, PEI-AZT/G-BSA-FITC NCs and PEI-AZT/A-BSA-FITC NCs (final concentration of 5. Mu.g BSA-FITC/mL) and incubation for 4 hours. The medium was removed and washed three times with heparin sodium in PBS. 0.4% trypan blue solution was treated for 10 min and washed three times with PBS. 4% paraformaldehyde was fixed, hoechst 33258 (5. Mu.g/mL) stained for 15 min, washed three times with PBS, and cells were observed by CLSM. The cellular uptake of PEI-AZT/A-RNase A NCs, PEI-AZT/A-Cyt C NCs and PEI-AZT/A-IgG NCs was investigated by the same method. Referring specifically to FIG. 5, after incubation of PEI-AZT/A-protein NCs with HeLa cells, significant and evenly distributed fluorescence appears in the cytoplasm, indicating efficient transport of the protein into HeLa cells. Using the cationic polymer PEI and the commercial agent PLUSin as controls, no apparent fluorescence of the protein appears in the cytoplasm, indicating that its delivery efficiency is lower than that of the thymidylated cationic polymer PEI-AZT mediated intracellular delivery of the protein. On the other hand, by using protein prodrugs (C-protein and G-protein) modified with different base groups as a control, no obvious protein fluorescence appears in cells after NCs are incubated with HeLa cells, and the results show that the PEI-AZT-mediated intracellular delivery of protein is a thymidylated cationic polymerThe delivery system is universal to different proteins.
Example 4:
co-localization of the nanocomplex (LPP/A-BSA-FITC) and lysosomes/endosomes was observed using the laser confocal method. HeLa cells were first plated at 1X 10 per well 5 Is inoculated into a confocal dish. Cells were completely adherent by culturing in DMEM medium containing 10% FBS for 24 hours, replacing with fresh medium without serum, and then adding protein samples to the wells at a concentration of 2. Mu.g/mL at 37℃and 5% CO 2 Respectively 1, 2 and 4 hours. After three washes with PBS, incubation with trypan blue solution for 10 min, washing with PBS was continued three times, staining with Hoechst33342 (5. Mu.g/mL) for 20 min, labelling lysosomes/inclusion bodies with Lysotracker deep red (200 nM) for 1h, washing with PBS three times and observing co-localization of cells under confocal laser scanning microscopy. See in particular fig. 6. The experimental results show that after 1-2 hours of incubation, LPP/A-BSA-FITC NCs (green fluorescence) are mainly distributed on the cell membrane, and the intracellular distribution is less and mainly distributed on the cell membrane, and the intracellular distribution is less. When incubating. After 4 hours of incubation, green fluorescence in the cytoplasmic cytoplasm gradually increased and overlapped less with red fluorescence (endosome/lysosome). These results indicate that LPP/a-BSA-FITC can successfully escape endosomes, since LPP has a "proton sponge" effect, helping to reduce the risk of protein degradation in lysosomes.
To investigate the endosome/lysosomal escape of PEI-AZT/A-protein NCs. HeLa cells were seeded at 1X 104 cells/well into confocal dishes and incubated at 37℃for 24 hours. The cell culture medium was replaced with serum-free DMEM, followed by PEI-AZT/A-BSA-FITC NCs (final concentration 5. Mu.g BSA-FITC/mL). Different time points (1, 2 and 4 hours) were incubated at 37 ℃. The medium was removed and washed three times with heparin sodium in PBS. 0.4% trypan blue solution was treated for 10 min and washed three times with PBS. lysotracker deep red (LDR, 200 nM) for 2 hours, hoechst 33258 (5. Mu.g/mL) for 10 minutes, and CLSM observed cells. Referring specifically to FIG. 7, after 1 hour incubation, the green fluorescence (BSA-FITC) was less distributed in the cytoplasm, increased in the cytoplasm by hour 2, and gradually separated from the red fluorescence (endosome/lysosome), and uptake of PEI-AZT/A-BSA-FITC NCs by cells increased. When the incubation time was prolonged to 4 hours, the green fluorescence in the cytoplasm increased significantly, and the overlap with the red fluorescence was less, and the fluorescence was distributed in the cytoplasm in large amounts. These results indicate that PEI-AZT/A-BSA-FITC NCs can successfully escape the lysosomes, reducing the risk of degradation of the protein in the lysosomes.
Example 5:
the LPP-mediated, nucleic acid-like protein prodrug delivery system of the present invention can be used to deliver proteins of different molecular weights/charges. Cytochrome C (Cyt C-FITC), ribonuclease A (RNase A-FITC), alpha-trypsin (alpha-Chut-FITC) and ovalbumin (OVA-FITC) were labeled with FITC, and monomeric A-modified protein prodrugs were prepared as described in example 1, respectively; in contrast to the delivery effect of unmodified proteins.
HeLa cells were allowed to adhere completely by culturing in DMEM medium containing 10% FBS for 24 hours, replacing fresh medium with serum-free medium, and then adding protein complex samples of different molecular weights/charges to the wells at a final concentration of 2. Mu.g/mL at 37℃and 5% CO 2 Is incubated for 4 hours. After three washes with cold PBS, incubation with trypan blue solution for 10 min was continued for three washes with PBS, staining with Hoechst33342 (5. Mu.g/mL) for 20 min, and intracellular fluorescence distribution was observed under a laser confocal scanning microscope. See in particular fig. 8. Experimental results show that after modification with the monomeric A compound, all protein prodrugs are significantly and uniformly distributed in cells after complexing with LPP, and furthermore, compared with the unmodified protein, the internalization amount of the protein prodrugs is significantly increased. The experimental results show that the protein prodrug prepared by the small molecule modified protein has good universality.
Example 6:
to investigate the restoration of intracellular activity of the monomeric a-modified biologically active protein, a-modified β -galactosidase (β -gal) prodrugs were prepared. The preparation was similar to that in example 1.
HeLa cells per well1×10 4 Each was inoculated into 96-well plates and cultured in DMEM medium containing 10% FBS for 24 hours. Different modified and treated β -gal protein samples were added to the wells at a concentration of 2. Mu.g/mL at 37℃and 5% CO, with fresh serum-free medium replaced 2 Is incubated for 4 hours. Washed three times with PBS, and then cell-fixing solution was added thereto to fix the cells at room temperature for 10 minutes. The fixative was removed, washed three times with PBS, and substrate staining solution containing X-gal (0.1 mg/mL) was added. Placing the cell plates in the absence of CO 2 Is placed in an incubator at 37℃overnight. After that, the staining solution was removed and washed three times with PBS. Staining of cells was observed with an optical microscope. Further quantitative analysis of the activity of the enzyme using o-nitro- β -D-galactopyranoside (ONPG). After the intracellular delivery of β -gal was tested, PBS was used for three washes, 200. Mu.L of lysate was added to lyse cells, 50. Mu.L of lysate was added to 50. Mu.L of an enzyme activity detection solution containing ONPG, and the mixture was left at 37℃for 1 hour, followed by 150. Mu.L of NaHCO 3 (1M) terminate the reaction, transfer the solution to a 96-well plate and detect absorbance at 420 nm. The absorbance was defined as 100% with the enzyme activity of untreated β -gal at equal concentrations as a positive control. See in particular fig. 9. Experimental results indicate that LPP/A- β -gal shows the most blue deposition, indicating a large amount of β -gal internalization and performing its biological function. Quantitative experiments showed that the same results, LPP/a- β -gal, was almost completely restored in cells, demonstrating that LPP complex a modified protein prodrugs can effectively achieve protein internalization and restore their activity in cells.
Example 7:
intracellular delivery of horseradish peroxidase (HRP). The preparation was carried out in a similar manner to example 1 to give A-HRP.
HeLa cells were plated at 4X 10 cells per well 4 Each was inoculated into a 24-well plate and cultured in DMEM medium containing 10% FBS for 24 hours. Serum-free fresh medium was exchanged and different modified and treated HRP protein samples were then added to the wells at a concentration of 2 μg/mL and incubated for 4 hours at 37 ℃ and 5% CO 2. PBS was used for 5 times, tetramethylbenzidine (TMB, 10. Mu.g/mL) solution and hydrogen peroxide (3 mM) solution were added, incubated at room temperature for 10 minutes, and staining of each well was observed. See in particular fig. 10.The experimental results showed that the blue change was most pronounced in LPP/A-HRP and that the unmodified group had little change in color, indicating that LPP/A-HRP was able to internalize efficiently into cells and perform its biological function.
Example 8:
intracellular delivery of toxic proteins. Ribonuclease A (RNase A) and saporin (saporin) were selected as model proteins to examine intracellular delivery efficiency and biological function. The preparation was carried out in a similar manner to that in example 1, to give A-RNase A and A-saporin, respectively.
HeLa cells were plated at 6X 10 cells per well 3 Each was inoculated into 96-well plates and cultured in DMEM medium containing 10% FBS for 24 hours. The fresh serum-free medium was replaced, and the A-modified ribonuclease A was added to the wells at protein concentrations of 20. Mu.g/mL, 10. Mu.g/mL, 5. Mu.g/mL, 2.5. Mu.g/mL, 1.25. Mu.g/mL, 0.625. Mu.g/mL, and 0.3125. Mu.g/mL per well for further cultivation for 48 hours. In addition, A-modified saporin was added to the wells at protein concentrations of 1. Mu.g/mL, 0.75. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL per well, and incubated for 48h. Cell viability was determined using the MTT assay, with the cells without any treatment as reference, and the results were expressed as a percentage of control cells. See in particular fig. 11. Experimental results show that relative cell viability of HeLa cells treated with inverse LPP/A-RNase A gradually decreased with increasing NCs concentration, and relative cell viability decreased to 31% when the treatment concentration was 20. Mu.g/mL. LPP/A-RNase A NCs exhibit concentration-dependent cell killing ability, IC thereof 50 2.69. Mu.g/mL. LPP/A-saporin also showed excellent concentration-dependent cell killing ability, relative cell viability was 25% at a concentration of 1. Mu.g/mL, and IC thereof 50 0.45 μg/mL.
Example 9:
antibody IgG was labeled with rhodamine B isothiocyanate (FITC) at 20mM NaHCO 3 In the buffer solution, according to the protein: RBITC=1:3 (mass ratio) was reacted overnight in the absence of light, then the unreacted RBITC molecules were removed by dialysis against ultra pure water (MWCO=3.5 kDa) for two days to give RBITC-labeled proteins, and then the proteins were modified to give A-IgG-RBITC using the method of example 1.
The internalization of LPP-mediated nuclease-like protein prodrugs was studied using laser confocal experiments by first 1X 10 HeLa cells per well 4 Is inoculated into a confocal dish. Culturing in DMEM medium containing 10% FBS for 24 hr to allow cells to adhere completely, replacing with fresh medium without serum, adding different protein samples into the wells at concentration of 2 μg/mL, and heating at 37deg.C and 5% CO 2 For 4 hours under the conditions of (a) and see in particular figure 12. The results showed a clear red fluorescence distribution in the cytoplasm, indicating efficient internalization of LPP/A-IgG-RBITC into the cell. The nucleophile-like protein prodrug is thus able to efficiently bind to the cationic carrier and achieve efficient intracellular delivery therethrough.
Example 10:
to investigate the ability of the monomer a modified CRISPR-Cas9 tool enzyme to perform gene editing. A modified CRISPR-Cas9 protein prodrugs are prepared. The preparation was similar to that in example 1, yielding a-Cas9.
293T-EGFP cells were plated at 1X 10 per well 4 The individual cells were inoculated into confocal dedicated dishes and incubated at 37℃for 24 hours. The cell culture medium was replaced with serum-free DMEM, and then different ribonucleoprotein complex samples were added to the wells at a concentration of 2 μg/mL Cas9 and 1 μg/mL sgEGFP, and incubated for 4 hours. PBS was washed three times and then incubated with DMEM containing 10% fbs at 37 ℃ for additional 44 hours. Cells were observed by CLSM after three washes with PBS. In addition, 293T-EGFP cells were plated at 1X 10 per well 5 Individual cells were seeded onto 24-well plates and incubated at 37 ℃ for 24 hours. Cells were treated as described above, and the cells were trypsinized and assayed for EGFP expression in 293T-EGFP cells using a flow cytometer. Commercial reagent (CMAX) was used to form CMAX/protein complexes for comparison according to the product instructions. Cell mean fluorescence quantitative data showed LPP/A-RNP EGFP The EGFP knockout efficiency of the kit reaches about 33 percent and is obviously higher than that of an unmodified protein sample group. Higher than the commercial reagent compounded protein complex sample group (12%). The same results were observed with laser confocal mapping, LPP/A-RNP EGFP The green fluorescence of the 293T-EGFP cells after treatment is obviously reduced.
Next, heLa cells were plated at 1X 10 per well 5 Individual cells were seeded in 6-well plates and incubated at 37 ℃ for 24 hours. The cell culture medium was replaced with serum-free DMEM, and then different ribonucleoprotein complex samples were added to the wells at a concentration of 2 μg/mL Cas9 and 1 μg/mL sgPLK1, and incubated for 4 hours. PBS was washed three times and then incubated with DMEM containing 10% FBS at 37 ℃ for additional 44 hours. Then, the whole genome DNA is extracted by using a cell/tissue genome DNA extraction kit, and after PCR amplification, the PCR product is dissolved in 1 XNEBuffer 2, and a mismatched double-stranded structure is formed through a denaturation/annealing process. The annealed PCR products were mixed with T7E1 enzyme, incubated at 37℃for 30 minutes, and then analyzed by 2% agarose gel electrophoresis.
Example 11:
to assess the ability of NCs to distinguish tumor cells from normal cells, the distribution of PEI-AZT/A-BSA-FITC NCs (5 μg BSA-FITC/mL) in other tumor cell lines (A549) as well as normal cell lines (H9C 2, NIH-3T 3) was observed using CLSM. All cells were seeded at 1×104 cells/well into dedicated confocal dishes and incubated at 37 ℃ for 24 hours. The cell culture medium was replaced with serum-free DMEM, followed by PEI-AZT/A-BSA-FITC NCs (final concentration 5. Mu.g/mL). Incubate for 4 hours at 37℃and wash three times with heparin sodium in PBS. 0.4% trypan blue solution was treated for 10 min and washed three times with PBS. Hoechst 33258 (5. Mu.g/mL) was stained for 15 min and the cells were observed by CLSM.
The invention discloses a functionalized cationic polymer-mediated nucleic acid-like protein prodrug intracellular delivery strategy. Covalent modification of base molecules on protein lysine residues produces protein prodrugs that can be efficiently and stably bound to cationic polymeric carriers. The intracellular delivery mode mediated by the functionalized cationic polymer carrier can prevent the protein from being captured by an endosome/lysosome, and can greatly improve the utilization rate of protein medicines. The modification groups on the protein prodrug can be released in the acidic environment in the cell, and the protein restores activity and exerts pharmacological activity. The delivery strategy is universal and can be used to deliver protein drugs of different molecular weights, isoelectric points and functions, including enzymes, antibodies, toxin proteins and CRISPR-Cas9 nucleases. In particular, protein prodrug complexes of the toxin proteins RNase a and saporin exhibit excellent antitumor effects in vitro. And, CRISPR-Cas9 nucleases exert a remarkable gene editing effect after delivery to cells. Meanwhile, the delivery mode has good cell universality delivery capability. The protein prodrug effectively regulates and controls the surface charge density and binding sites of the protein, and realizes the efficient intracellular delivery and traceless release of the protein. This simple and efficient technique provides a new approach for functional modification of proteins and cationic polymers.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims (10)

1. A phosphorylated protein intracellular delivery system, comprising a nucleic acid-like protein prodrug, and a cationic polymer carrier conjugated to the nucleic acid-like protein prodrug;
the nucleic acid protein prodrug comprises a protein, a monomer ABA which is covalently connected with the protein and a monomer N which is covalently connected with the monomer ABA, wherein the monomer ABA is a phenylboronic acid compound or a phenylboronic acid pinacol ester compound, and the general formula is as follows:
Figure FDA0004065198740000011
wherein R is hydrogen or a C1-C6 alkane chain;
the monomer N is adenosine phosphate, cytidine phosphate, guanosine phosphate, uridine phosphate, thymidine phosphate, adenosine, cytidine, guanosine, uridine or thymidine.
2. The phosphorylated protein intracellular delivery system of claim 1, wherein the cationic polymer is:
Figure FDA0004065198740000012
wherein n=5-500;
m＝0-10；
r1 is a urea phosphate, a thymidine phosphate, an adenosine phosphate, a cytidine phosphate, a guanosine phosphate, an adenosine, a cytidine, a guanosine, a uridine, or a thymidine having base pairing with the monomer N.
3. The phosphonucleoside protein intracellular delivery system of claim 1, wherein the monomeric ABA has the structure:
Figure FDA0004065198740000021
4. the intracellular delivery system of phosphorylated protein of claim 1, wherein the monomer N has the structure:
Figure FDA0004065198740000022
where n=0, 1, 2 or 3.
5. The phosphorylated protein intracellular delivery system of claim 1, wherein the protein is a toxic protein, a non-toxic protein, an antibody, or a gene editing tool enzyme.
6. A method of preparing an intracellular delivery system of a phosphonucleoside protein according to any one of claims 1 to 5, comprising the steps of:
s1, reacting monomer ABA with protein to obtain ABA-protein;
s2, reacting ABA-protein with a monomer N to obtain the nucleic acid-like protein prodrug;
s3, mixing and reacting the nucleic acid-like protein prodrug with a cationic polymer to obtain the nucleoside phosphate protein intracellular delivery system.
7. The method according to claim 6, wherein the molar ratio of the monomer ABA to the primary amino groups in the protein is (1-10) to 1; the molar ratio of the monomer N to the primary amino groups in the protein is (1-10) to 1.
8. The process according to claim 6, wherein in the step S1, the reaction is carried out at 10 to 40℃for 10 to 30 minutes in an aqueous solution.
9. The process according to claim 6, wherein in the step S2, the reaction conditions are that the reaction is carried out in an aqueous solution at 10 to 40℃for 1 to 4 hours.
10. Use of the phosphonucleoside protein intracellular delivery system of any one of claims 1 to 5 in the preparation of protein nanomedicines or gene editing.
CN202310072809.4A 2023-02-07 2023-02-07 Nucleoside phosphate protein intracellular delivery system and preparation method and application thereof Pending CN116115769A (en)

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