CN114848837A

CN114848837A - Polypeptide drug conjugate and nucleic acid drug self-assembly nanoparticle and application thereof

Info

Publication number: CN114848837A
Application number: CN202210382840.3A
Authority: CN
Inventors: 王冬园; 张玉; 曾芳; 黄怡菲; 胡宽; 杜晶晶
Original assignee: Tongji Medical College of Huazhong University of Science and Technology
Current assignee: Tongji Medical College of Huazhong University of Science and Technology
Priority date: 2022-04-12
Filing date: 2022-04-12
Publication date: 2022-08-05
Anticipated expiration: 2042-04-12
Also published as: CN114848837B

Abstract

The present invention relates to a novel Polypeptide Drug Conjugate (PDC). The invention also provides a PDC-DNA aptamer induced self-assembly body which is formed by self-assembly of the designed PDC and the DAC aptamer AS 1411. The invention also provides that the PDC and the DNA aptamer can be mixed and incubated at normal temperature to be self-assembled into microspheres with the diameter not more than 100nm, and the nano microspheres are positively charged. The invention also provides a method for effectively carrying the nucleic acid medicament into the tumor cell nucleus by using the PDC-DNA aptamer self-assembly body. The invention also provides application of the PDC-DNA aptamer in treating malignant ovarian cancer, and the PDC-DNA aptamer can achieve the aim of resisting tumors from various signal paths. The invention also provides the self-assembly body of the polypeptide-nucleic acid, which has better biocompatibility.

Description

Polypeptide drug conjugate and nucleic acid drug self-assembly nanoparticle and application thereof

Technical Field

The invention belongs to the field of bioengineering, and relates to a polypeptide drug conjugate, in particular to a polypeptide drug conjugate-DNA aptamer induced co-assembly and application thereof.

Background

In recent decades, nucleic acid drugs have attracted global attention because of their superior efficacy in the treatment of a variety of diseases, which are often considered to be either untreatable or lack of effective treatments. Nucleic acid drugs are inherently structurally unstable and do not readily penetrate cell membranes, and therefore various strategies have been developed to encapsulate or stabilize nucleic acids for successful delivery into cells. DNA aptamer AS1411 is a G-quadruplex of 26 oligonucleotides rich in guanine bases, and has been shown to interact with nucleolin in various cancer cells to inhibit malignant proliferation of tumors. AS1411 is the nucleic acid drug historically first entered clinical trials for the treatment of a variety of malignancies. AS1411 therefore has a very promising prospect in the imaging of tumour cells or cancer therapy. However, one significant technical hurdle faced by AS1411 is the inefficient uptake of cells due to electrostatic repulsion of the cell membrane. Therefore, the most challenging problem at present is how to develop an efficient, secure delivery system for the intranuclear delivery of AS 1411.

Attempts have been made to develop various nucleic acid delivery methods, such as liposomes, silica nanoparticles, metal nanoparticles, inorganic nanoparticles, and the like. However, due to their effective delivery efficiency, tissue damaging or non-biodegradable properties, these drugs have unsatisfactory clinical therapeutic effects. Cell Penetrating Peptides (CPPs) have been the nucleic acid delivery tool of interest, which has several potential advantages over other approaches, including better biocompatibility, nucleic acid delivery potential, biodegradability, low toxicity, and ease of modification, among others. However, the CPP alone as a carrier often has problems of low drug-loading efficiency or poor therapeutic effect. Polypeptide Drug Conjugates (PDCs) are of interest because they have good target specificity and selective toxicity. Because the cell-penetrating mechanisms of the PDC medicament and the chemotherapeutic medicament are different, the PDC can effectively avoid the interference of a medicament discharge system and successfully enter cells to play an anti-tumor effect, and the clinical tumor drug resistance phenomenon can be expected to be overcome. Meanwhile, some PDCs can be designed to be positively charged, thus becoming an effective tool for nucleic acid drug delivery.

Histone Deacetylase (HDAC) is a common anti-cancer target for pharmaceutical chemists, and 5 HDAC inhibitors have been approved for cancer therapy in combination with other chemotherapeutic or targeted drugs, showing its potential in cancer therapy. 5-fluorouracil (5-FU) is a first-line anticancer drug for many cancer patients such as colon cancer, and has been widely used in combination therapy with other drugs or nano-drugs. Many studies have shown that HDAC inhibitors can enhance the anticancer effect of 5-FU in various cancer types, suggesting that this combination may be an alternative choice for cancer patients. Therefore, the HDAC inhibitor and the chemotherapeutic drug 5-FU are coupled to the cell-penetrating peptide together to form the multifunctional PDC, the electropositive characteristic of the multifunctional PDC is beneficial to self-assembly of the multifunctional PDC and the nucleic acid drug into nanoparticles, intracellular delivery of the nucleic acid drug is realized, and the aim of combined treatment of tumors from multiple paths is fulfilled. The invention is expected to provide a new combined treatment scheme for clinically treating malignant ovarian cancer and provide a new idea for clinically designing safe and effective polypeptide nano-drugs.

Disclosure of Invention

Aiming at the technical problems of nucleic acid drug carriers in the prior art, a novel polypeptide drug conjugate, a polypeptide drug conjugate-aptamer self-assembly and application thereof are provided. The polypeptide drug conjugate and the polypeptide drug conjugate-aptamer assembly aim to solve the technical problems of complicated preparation and purification, high toxicity, biocompatibility, poor effect and the like of a nucleic acid drug carrier in the prior art.

Regarding the polypeptide drug conjugate designed by the invention, the polypeptide is selected from a positively charged cell penetrating polypeptide RW9, the carbon end of which is coupled with HDAC inhibitor, and the nitrogen end of which is coupled with chemotherapeutic drug 5-fluorouracil. The polypeptide drug conjugate can be self-assembled into nano microspheres with nucleic acid drug AS1411 due to strong electropositivity, and the nucleic acid drug can be successfully delivered into cells, so that the anti-tumor effect can be exerted from multiple layers.

The sequence of the polypeptide drug couplet is as follows: 5-FU-caproic acid-arginine-tryptophan-arginine-caproic acid, has the following specific structure,

the invention has the beneficial effects that: in the invention, the multifunctional polypeptide drug conjugate is designed based on the reported sequence of the cell-penetrating peptide RW9 and is simultaneously coupled with an HDAC inhibitor and a chemotherapeutic drug 5-FU, so that the synthesis is simple and efficient, the yield is high, and the preparation method is suitable for mass production. On one hand, the invention can combine the dual effects of a targeted drug and a chemotherapeutic drug to improve the antitumor activity of the polypeptide, and on the other hand, the polypeptide drug conjugate is rich in positive charges and can be self-assembled into nanoparticles with negatively charged nucleic acid drugs to complete the intracellular delivery of the nucleic acid drugs so as to achieve the antitumor purpose from multiple layers. The assembly can be completed within 30 minutes of standing at normal temperature, and is very simple and efficient.

According to the invention, through a projection electron microscope, particle size potential and particle size test, the self-assembly body can form regular nanospheres with the diameter of about 100 nm.

According to the invention, a cytotoxicity experiment shows that the PDC-nucleic acid self-assembly nanoparticle has a remarkable inhibition effect on malignant ovarian cancer cell PA-1, and almost has no toxicity on normal cells under the same concentration.

The PDC medicament can effectively deliver the nucleic acid medicament AS1411 into cell nuclei through a flow cytometer and a laser confocal microscope. Through a flow cytometer, we find that the polypeptide nano-drug can effectively induce malignant ovarian cancer cell PA-1 to undergo apoptosis, and block the cell cycle at the G1 stage, thereby fully demonstrating the anti-tumor activity of the polypeptide nano-drug. The invention further discovers, through a protein co-immunoprecipitation experiment, that the polypeptide nano-drug not only can inhibit the function of HDAC in cells to cause acetylation of HDAC specific substrate histone H3, but also can inhibit expression of dry gene sox2 and WNT5A1 in a WNT channel, so as to improve the protein expression level of P21, and fully proves that the polypeptide nano-drug can synergistically achieve the effect of inhibiting PA-1 of malignant ovarian cancer cells from multiple channels.

Compared with the prior art, the invention has remarkable technical progress. The polypeptide drug couplet designed by the invention successfully completes the synthesis of the polypeptide drug couplet mainly through a solid-phase polypeptide synthesis strategy, does not relate to the introduction and complex synthesis of unnatural amino acid, simply and efficiently takes products, and is beneficial to mass production. The PDC medicament designed by the invention has better biocompatibility and cell selective toxicity, and has better selective toxicity on malignant ovarian cancer cell PA-1. Meanwhile, the polypeptide drug couplet is rich in arginine, has positive charge at physiological pH, can perform self-assembly with nucleic acid drugs with negative charge through electrostatic interaction to form nanoparticles, and successfully and efficiently transports the nucleic acid drugs into cell nuclei. The nano system can realize polypeptide-nucleic acid self-assembly by standing for 30 minutes at normal temperature, and is simple and efficient to operate. Meanwhile, the PDC-nucleic acid drug self-assembly still has better biocompatibility and tumor cell selective toxicity, has stronger selective toxicity on PA-1 cells and has almost no toxicity on normal cells under the same concentration. Meanwhile, the invention also discovers that the polypeptide nano-drug can achieve the anti-tumor purpose by influencing multiple cell pathways, such as sternness genes sox2, WNT5A1, P21, HDAC and the like, thereby achieving the purpose of multi-pathway synergistic anti-tumor. The invention provides a simple, efficient and safe tool for constructing a nucleic acid drug delivery system.

Drawings

FIG. 1 is a schematic representation of the design of novel polypeptide drug conjugates and their delivery of nucleic acid drug AS1411 into the nucleus;

FIG. 2 is a graph showing the anti-tumor results of several polypeptides involved in the present invention;

FIG. 3 is a scheme showing the synthesis of the polypeptide drug conjugate of the present invention;

FIG. 4 shows the in vitro inhibition effect of HDAC enzyme activity of the polypeptide drug conjugate of the present invention;

FIG. 5 shows the encapsulation ability of the polypeptide drug conjugate of the present invention for nucleic acid drug AS 1411;

FIG. 6 is a diagram showing the flow cytometric analysis of the ability of the polypeptide drug conjugate of the present invention to deliver a nucleic acid drug intranuclear;

FIG. 7 is a diagram showing intracellular distribution of the polypeptide drug conjugates/nucleic acid nanoparticles of the present invention;

FIG. 8 is a graph showing the toxicity of the polypeptide drug conjugates/nucleic acid nanoparticles of the present invention in different tumor cells;

FIG. 9 is a graph showing the results of inducing PA-1 apoptosis by the polypeptide drug conjugate/nucleic acid nanoparticle of the present invention;

FIG. 10 is a graph showing the results of PA-1 cell cycle arrest induced by the polypeptide drug conjugates/nucleic acid nanoparticles of the present invention;

FIG. 11 is a diagram showing the result that PA-1 cell proliferation is influenced by the polypeptide drug conjugate/nucleic acid nanoparticle of the present invention through multiple pathways.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

First, design of polypeptide drug couplet in the invention

The invention adopts a cell-penetrating peptide consisting of 9 amino acids, an HDAC inhibitor is coupled at the carbon end of a polypeptide, a chemotherapeutic drug 5-FU is coupled at the nitrogen end, the name FU-NHOH is shown in figure 1, and the sequence of the polypeptide is as follows: 5-FU-alanine-arginine-tryptophan-arginine-hexanoic acid-hydroximic acid. The polypeptide drug conjugate body is positively charged, so that the nucleic acid drug AS1411 can be effectively encapsulated and delivered into cell nuclei, and the anti-tumor effect can be expected to be cooperatively achieved from multiple channels.

To further illustrate the anti-tumor effect of the polypeptide drug conjugate, we designed three additional negative control polypeptides, namely, cell-penetrating peptide RW9 (with the sequence arginine-tryptophan-arginine), polypeptide RW9-NHOH (with the sequence arginine-tryptophan-arginine-histidine-hydroxamic acid) which is singly coupled with HDAC inhibitor, and polypeptide FU-RW9 (with the sequence: 5-FU-caproic acid-arginine-tryptophan-arginine-5-FU), which is singly coupled with HDAC inhibitor. As can be seen from the antitumor effects of the negative control polypeptides on PA-1 cells, Huh7 cells and HT29, the polypeptide conjugate FU-NHOH coupled with HDAC inhibitor and 5-FU has more significant antitumor activity, which suggests that the coupling of the 2 small-molecule drugs can achieve better antitumor effect synergistically, as shown in FIG. 2.

Synthesis and preparation of polypeptide drug couplet FU-NHOH

(1) Resin swelling and deprotection:

the specific procedure for solid phase polypeptide synthesis is shown in scheme 3: the peptide solid phase synthesis with hydroximic acid uses special 2-Chlorotityl-N-Fmoc-hydroxamine resin (loading degree: 0.54 mmol/g). The operation is as follows: the 2-Chlorotityl-N-Fmoc-hydroxamine resin was swollen with DCM for 15 min. The Fmoc protecting group was removed twice each for 30min with 50% morphine (in DMF) and then washed 3 times with DMF/DCM, respectively.

5-FU-alanine-arginine-tryptophan-arginine-hexanoic acid-hydroximic acid

(2) Synthesis of resin polypeptide:

the deprotected resin was prepared by mixing a prepared solution of Fmoc-6-amino-hexanoic acid (5eq,0.4M, DMF), a solution of 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) (5eq,0.4M, DMF), and N, N-Diisopropylethylamine (DIPEA) (10eq) uniformly, and then adding nitrogen into the resin for 1H. Pumping out the reaction solution, washing the resin according to the method, then connecting the next amino acid, namely pumping out the reaction solution, washing the resin according to the method, then carrying out the next operation, after removing the Fmoc protecting group from the resin with morpholine, (2) Fmoc-Arg (pdf) -OH or (3) Fmoc-Arg (pdf) -OH or (4) Fmoc-Trp (Boc) -OH or (5) Fmoc-Arg (pdf) -OH or (6) Fmoc-Arg (pdf) -OH or (7) Fmoc-Trp (Boc) -OH or (8) Fmoc-Trp (Boc) -OH or (9) Fmoc-Arg (pdf) -OH or (10) Fmoc-Arg (pdf) -OH or (11) Fmoc-6-amino-hexanoic acid or (12)5-FU, PyBOP and DIPEA solutions are mixed, and added to the resin for 2h under nitrogen bubbling; the reaction solution was withdrawn, and the resin was washed as described above and then subjected to the next step. The polypeptide was cleaved from the resin with trifluoroacetic acid (TFA), Triisopropylsilane (TIPS) and H2O (v: v: v ═ 9.5:0.25:0.25) cleavage solution, and the cleavage solution was removed. Polypeptide was dissolved using 50% acetonitrile/ddH 2O (v/v), the reaction was filtered, the clear filtrate was separated by HPLC, and molecular weight of the polypeptide was identified by ESI mass spectrometry.

Enzyme activity inhibition effect of HDAC-targeted polypeptide drug conjugate

The HDAC enzyme activity inhibition effect is identified by using a commercially available HDAC enzyme activity detection kit, the operation is repeated at least three times each time, the HDAC enzyme is from HeLa nuclear extracts reported in various documents, the enzyme activity result is executed according to the operation of the commercially available kit, and finally, the inhibition activity of the polypeptide drug conjugate on the HDAC protein is measured by using an enzyme-labeling instrument, as shown in figure 4.

Fourth, the wrapping capability of polypeptide drug conjugate FU-NHOH targeting HDAC on nucleic acid drug AS1411

Firstly, the wrapping capacity of polypeptides with different concentrations on AS1411 is researched by adopting an agarose gel electrophoresis experiment, N/P ratios (nitrogen/phosphorus ratios) of 0,2.25,4.5,9,18,36 and 72 are respectively selected for mixing, standing is carried out for 30min at normal temperature, and a sample is loaded into a prepared 1.5% agarose gel electrophoresis for electrophoretic separation. The samples were analyzed using a gel imager to determine the optimal polypeptide/nucleic acid ratios for subsequent experiments, as shown in FIG. 5A.

Characterization of nanoparticles of FU-NHOH/AS1411

We chose the optimal polypeptide/nucleic acid ratio of FIG. 5A and performed subsequent characterization experiments. First, we characterized the polypeptide/nucleic acid nanoparticles with an N/P value of 72 by TEM (transmission electron microscope), and as shown in fig. 5B, the polypeptide nanoparticles showed a regular nanoparticle structure, which is directly about 100 nm. Then, we detected the electric potential before and after the nucleic acid was encapsulated, as shown in fig. 5C, the polypeptide itself was positively charged, the nucleic acid itself was negatively charged, and the polypeptide encapsulated the nucleic acid was positively charged, but the positive charge was lower than the polypeptide itself, suggesting that the polypeptide can successfully encapsulate the nucleic acid and make the nanoparticle positively charged, which is beneficial to the cell-penetrating membrane of the nanoparticle. Finally, we examined the diameter of the polypeptide nanoparticles, which is close to 100nm (as shown in FIG. 5D), in agreement with the TEM results.

Sixthly, the capability of delivering the polypeptide FU-NHOH to nucleic acid drugs in nucleus

We first examined the ability of the polypeptide drug conjugate to deliver nucleic acid AS1411 using flow cytometry. As shown in FIG. 6, in PA-1 cells, the polypeptide FU-NHOH has a strong ability to carry AS1411 into cells, and the delivery efficiency is positively correlated with the concentration of the polypeptide. It is fully demonstrated that the polypeptide can effectively carry the nucleic acid drug AS1411 into cells.

Next, we further investigated the distribution of the AS1411 carried into the cells in the cells using confocal laser microscopy. As shown in FIG. 7, in PA-1 cells, the polypeptide FU-NHOH successfully transported AS1311 to the nucleus, indicating a strong intranuclear delivery capacity of the polypeptide.

Evaluation of killing effect of polypeptide/nucleic acid nanoparticles on different cells

A CCK8 cytotoxicity detection experiment is adopted to evaluate the killing effect of the polypeptide nanoparticles on different tumor cells. As shown in FIG. 8, in the stem cell-like ovarian cancer cell PA-1, FU-NHOH/AS1411 showed the strongest antitumor effect compared to the pure polypeptide FU-NHOH or the polypeptide non-functional nucleic acid nanoparticle FN-NHOH/NC, even stronger than the nucleic acid transfection reagent oligofectamin, and similar toxicity differences were seen in the liver cancer cell Huh7, the colon cancer cell HT29 and the like, but the toxicity was weaker to the normal cell 293T. The result fully indicates that the polypeptide drug conjugate can achieve more remarkable anti-tumor activity under the synergistic effect after being combined with nucleic acid drugs.

Effect of FU-NHOH/AS1411 nanoparticles on PA-1 cell function

We evaluated the ability of polypeptide nanoparticles to induce tumor cell PA-1 apoptosis, AS shown in FIG. 9, PA-1 cells were only able to undergo apoptosis by 20% under the treatment of low concentration of 20 μ M FU-NHOH or FU-NHOH/NC nanoparticles, while AS1411 was coated under the same concentration, i.e., 20 μ M FU-NHOH/AS1411 was able to induce PA-1 apoptosis by about 60%, which is superior to the commercial transfection reagent Oligofectamin.

Next, we continued to evaluate the effect of the polypeptide nanoparticles on cell cycle arrest of tumor cells PA-1. As shown in FIG. 10, the treatment of pure 20 μ M FU-NHOH or FU-NHOH/NC nanoparticles could not cause cell cycle arrest of PA-1 cells, while the 20 μ M FU-NHOH/AS1411 nanoparticles could effectively arrest the cell cycle of PA-1 at G1, which is similar to the marketed HDAC inhibitor SAHA, suggesting that the nanoparticles could achieve the cell cycle arrest effect by inhibiting the activity of HDAC.

Influence of polypeptide/nucleic acid nanoparticles on PA-1 signal pathway of tumor cells

Firstly, we verified through western blot experiments that the polypeptide nanoparticles can effectively inhibit the activity of HDAC in PA-1 cells, resulting in the increase of acetylation level of histone H3 AS a specific substrate, the inhibition effect is similar to that of the positive control HDAC inhibitor SAHA, while the oligofectamine/AS 1411 nanoparticles wrapped by transfection reagent can not effectively inhibit the activity of HDAC in cells, AS shown in fig. 11. Next, we evaluated the expression effect of different nano-drugs on the sternness gene of the PA-1 stem cell-like tumor cell, AS shown in FIG. 8, like SAHA, FU-NHOH/AS1411 can significantly inhibit the expression of SOX2 protein, indicating that the nano-drugs can effectively reduce the sternness and malignancy of the PA-1 cell. Studies have shown that the HDAC pathway interacts with the WNT pathway. We detect the influence of the polypeptide nano-drug on the protein expression of WNT5A1, find that the polypeptide nano-drug can effectively inhibit the expression of WNT5A1, is significantly better than a positive control drug SAHA or oligofectamin/AS1411 nanoparticle, and prove that the polypeptide nano-drug has a stronger WNT5A1 inhibition effect. The expression of the P21 protein induced by the polypeptide nano-drug is also fully proved that the drug can effectively block PA-1 cells in the G1 stage, which is consistent with the cell cycle result of the invention.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A polypeptide drug couplet is characterized in that amphiphilic cell-penetrating peptide RW9 is utilized, chemotherapeutics 5-fluorouracil is coupled to the nitrogen end of the polypeptide, histone deacetylase HDAC inhibitor is coupled to the carbon end of the polypeptide, and a multifunctional polypeptide drug couplet is formed (the sequence of the cell-penetrating peptide RW9 is arginine-tryptophan-arginine-tryptophan-arginine), the structure of the polypeptide drug couplet is shown as follows,

2. the polypeptide drug conjugate of claim 1 and the DNA aptamer can self-assemble into a nanoparticle.

3. The use of the polypeptide drug conjugate-DNA aptamer self-assembly of claim 2 for delivery in a cell.

4. The use of the polypeptide drug conjugate-DNA aptamer self-assembly of claim 3 in the preparation of a medicament for treating malignant ovarian cancer.