CN118649243A - Preparation and application of a nucleic acid framework nanodrug that induces copper death and blocks PD-L1 - Google Patents

Abstract

The present invention belongs to the technical field of tumor treatment, and specifically discloses the preparation and application of a nucleic acid framework nano drug that can induce copper cell death and block PD-L1. Elesclomol-CuCl ₂ is carried on tFNAs to construct a tFNAs drug delivery system that can induce copper cell death. At the same time, the immune checkpoint inhibitor aPD-L1 is connected to tFNAs to block the interaction between PD-L1 and its ligand, thereby enhancing the immune response. Elesclomol-Cu and aPD-L1 are carried on tFNAs to solve the hydrophobicity of Elesclomol and the drug resistance of Elesclomol and aPD-L1, effectively induce copper cell death of tumor cells and block PD-L1, thereby enhancing the therapeutic effect of tumors.

Description

Preparation and application of a nucleic acid framework nanodrug that induces copper death and blocks PD-L1

Technical Field

The present invention relates to the technical field of tumor treatment, and in particular to the preparation and application of a nucleic acid framework nano drug capable of inducing copper death and blocking PD-L1.

Background Art

Osteosarcoma is the most common primary malignant bone tumor, and 80% of patients are adolescents aged 10-20 years old. Osteosarcoma is highly malignant and has a very poor prognosis, with extremely high amputation and mortality rates, which have a huge impact on families and society. Under the current standard treatment of adjuvant chemotherapy and radiotherapy combined with surgical treatment, the 5-year survival rate of osteosarcoma patients is still only 60-65%. One-third of patients experience recurrence or distant metastasis after treatment, and the average survival time of patients after metastasis is less than 1 year.

With the development of nanotechnology, a variety of nanomedicines such as liposomes, mesoporous silica, and metal nanomaterials have been used in the study of tumor treatment. Among them, DNA tetrahedrons (tFNAs) based on nucleic acids have been shown to have many advantages, including natural biocompatibility and biodegradability, structural stability, programmability, functional diversity, and easy cellular internalization. Compared with single-stranded DNA and other spatial nanomaterials, the most prominent feature of tFNAs is its significantly enhanced endocytosis. In recent years, many tumor studies have focused on the use of tFNAs nanostructures functionalized at the cellular level for tumor treatment. Although many chemotherapeutic drugs such as doxorubicin (DOX), fluoropyrimidines, and Elesclomol are first-line drugs, the main obstacles to their clinical application are low cellular uptake, lack of target specificity, hydrophobic form, and multidrug resistance (MDR). Based on the main mechanisms of MDR: reduced drug uptake, increased drug efflux, DNA repair, and correction of drug-induced apoptosis through cell cycle regulation and activation of detoxification systems. Kim et al. constructed self-assembled DNA tetrahedral nanoparticles to deliver DOX into drug-resistant breast cancer cells through physical coupling. Compared with free DOX, tFNA-loaded drugs were not excreted, which may be due to the protection of endosomes during the transmembrane process.

Copper death is a newly discovered controlled cell death mode with a mechanism that is significantly different from known apoptosis, pyroptosis, necroptosis and ferroptosis. The main process of copper death depends on the accumulation of intracellular copper ions. Copper ions directly bind to the acylated components in the tricarboxylic acid cycle (TCA), causing these proteins to aggregate and dysregulate, blocking the tricarboxylic acid cycle (TCA), triggering protein toxic stress, and inducing cell death. Although existing copper carriers, such as copper ion carrier drugs such as Elesclomol (ES), disulfiram (Disulfiram) and NSC319726, can cause cell death, their tumor cell endocytosis needs to be enhanced. At the same time, studies have also shown that copper death may cause the activation of immune checkpoint signaling axes (such as PD-1/PD-L1), allowing tumor cells to escape immune surveillance and inhibit the effect of tumor treatment.

Therefore, simultaneously inducing copper cell death in tumor cells and inhibiting the activation of immune checkpoint signaling pathways may become a new and effective tumor treatment method.

Summary of the invention

In order to solve the above technical problems, the present invention provides a preparation method and application of a nucleic acid framework nanodrug that induces copper death and blocks PD-L1.

To achieve the above object, the present invention is implemented according to the following technical solutions:

One of the purposes of the present invention is to provide a method for preparing a nucleic acid framework nanodrug that induces copper death and blocks PD-L1, comprising the following steps:

S1. Take four 1uM single-stranded DNAs of equal amount and mix them in 100uL 1x TM buffer, react at 95°C for 15min, and then anneal at 4°C to obtain tetrahedral framework nucleic acids tFNAs;

S2, mixing equal amounts of 300 nM Elesclomol and _CuCl2 , adding the mixture to the tetrahedral framework nucleic acid tFNAs, mixing overnight at 4°C, then centrifuging at 14,000 g for 10 minutes using a 30 kDa ultrafiltration tube, collecting the supernatant to obtain the tFNAs complex loaded with Elesclomol-Cu, i.e., tFNAs@ESCu;

S3. The immune checkpoint inhibitor aPD-L1 was mixed with 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester SPDP at a molar ratio of 1:2.5, and the mixture was reacted at 20°C for 2 hours, and then the excess 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester SPDP was removed using a 10kDa ultrafiltration tube, and then 100uL 1xTM buffer was added to collect aPD-L1-SPDP; aPD-L1-SPDP was added to the above-mentioned tFNAs@ESCu, and the mixture was reacted at 20°C for 2 hours. The unreacted aPD-L1-SPDP was removed using a 100kDa ultrafiltration tube and centrifuged at 14000g for 15 minutes to obtain a nucleic acid framework nanodrug that induces copper death and blocks PD-L1, namely tFNAs@ESCu/aPD-L1.

The second purpose of the present invention is to provide a nucleic acid framework nanodrug that induces copper death and blocks PD-L1 prepared by the above method.

The third purpose of the present invention is to provide a nucleic acid framework nano drug that induces copper death and blocks PD-L1 for use in the preparation of drugs for treating osteosarcoma.

Compared with the prior art, the present invention constructs a tFNAs drug delivery system that can induce copper cell death by carrying Elesclomol-CuCl ₂ on tFNAs; at the same time, the immune checkpoint inhibitor aPD-L1 is connected to tFNAs to block the interaction between PD-L1 and its ligand, thereby enhancing the immune response; tFNAs are loaded with Elesclomol-Cu and aPD-L1, which can solve the hydrophobicity of Elesclomol and the drug resistance of Elesclomol and aPD-L1, effectively induce copper cell death of tumor cells and block PD-L1, thereby enhancing the therapeutic effect of tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows transmission electron microscopy images of tFNAs and tFNA/aPD-L1.

FIG2 shows atomic force microscopy images of tFNAs and tFNA/aPD-L1.

Figure 3 shows the ICP-MS results of tFNAs and tFNAs@ESCu.

FIG. 4 shows the BCA detection results of tFNAs and tFNA/aPD-L1.

FIG. 5 shows the PAGE gel detection results of tFNAs and tFNA/aPD-L1.

Figure 6 shows the cell viability of h-BMSCs, 143b, U2OS, and sjsa1 cells after adding different concentrations of tFNAs@ESCu/aPD-L1 for 24 hours.

Figure 7 shows the killing effect of adding FNAs, tFNAs@ESCu, and tFNAs@ESCu/aPD-L1 on 143b cells.

Figure 8 shows the expression results of the key copper death proteins DLAT, FDX1, and LIAS in 143b cells after adding FNAs, tFNAs@ESCu, and tFNAs@ESCu/aPD-L1.

Figure 9 shows the effect of adding FNAs, tFNAs@ESCu, and tFNAs@ESCu/aPD-L1 on the expression of PD-L1 in 143b cells.

Figure 10 shows the tumor growth inhibition effects of FNAs, FNAs@ESCu and tFNAs@ESCu/aPD-L1.

DETAILED DESCRIPTION

In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

Unless otherwise specified, the experimental materials and equipment used in the following examples were all commercially available; among them, the experimental cells include:

143b cells and 143b-luc were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.;

U2Os and sjsa1 were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.

h-BMSCs cells were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.

Example 1. Preparation and characterization of nucleic acid framework nanodrug tFNAs@ESCu/aPD-L1 that induces copper death and blocks PD-L1

S1. Take four 1uM single-stranded DNAs of equal amount and mix them in 100uL 1x TM buffer, react at 95°C for 15min, and then anneal at 4°C to obtain tetrahedral framework nucleic acids tFNAs;

S2, mixing equal amounts of 300 nM Elesclomol and _CuCl2 , adding the mixture to the tetrahedral framework nucleic acid tFNAs, mixing overnight at 4°C, then centrifuging at 14,000 g for 10 minutes using a 30 kDa ultrafiltration tube, collecting the supernatant to obtain the tFNAs complex loaded with Elesclomol-Cu, i.e., tFNAs@ESCu;

S3. Mix 60 uL of 10 mg/mL immune checkpoint inhibitor aPD-L1 with 940 uL 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester SPDP, react at 20°C for 2 hours, remove excess 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester SPDP with a 10kDa ultrafiltration tube, and then add 100uL 1xTM buffer to collect aPD-L1-SPDP; add aPD-L1-SPDP to the above tFNAs@ESCu, react at 20°C for 2 hours, use a 100kDa ultrafiltration tube, and centrifuge at 14000g for 15 minutes to obtain the nucleic acid framework nanodrug tFNAs@ESCu/aPD-L1 that induces copper death and blocks PD-L1.

The tFNAs, tFNAs@ESCu, and tFNAs@ESCu/aPD-L1 prepared in this example were subjected to transmission electron microscopy and atomic force microscopy, respectively; the supernatants before and after the reaction of tFNAs and Elesclomol-Cu complex were sent for ICP-MS detection; the supernatants before and after the reaction of tFNAs@ESCu and aPD-L1-SPDP were subjected to BCA detection.

In addition, tFNAs and tFNA/aPD-L1 (without Elesclomol-Cu complex, i.e., prepared without adding Elesclomol and CuCl ₂ in the above step S2) were subjected to PAGE gel detection.

The test results are shown in Figures 1 to 5. As shown in Figure 1, tFNAs present a triangular spatial conformation under a transmission electron microscope, with a size of about 15 nm, while the size of tFNAs carrying aPD-L1 increases; as shown in the atomic force microscopy results of Figure 2, the thickness of tFNAs carrying aPD-L1 increases to about 20 nm; as shown in the ICP-MS results of Figure 3, tFNAs can successfully carry Elesclomol-Cu complexes; as shown in the BCA test results of Figure 4 and the PAGE results of Figure 5, tFNAs can successfully carry aPD-L1.

Furthermore, in order to verify the biosafety and tumor killing effect of tFNAs@ESCu/aPD-L1 prepared in the above example, h-BMSCs, 143b, U2OS, and sjsa1 were inoculated in a 96-well plate at 4000 cells/well, and 100uL of tFNAs@ESCu/aPD-L1 with different concentrations (0nM, 40nM, 100nM, 200nM, 300nM) was added to the wells. After co-culture for 24h, the cell viability was detected by the standard CCK8 method.

The test results are shown in Figure 6. As shown in Figure 6, with the increase of concentration, the activity of h-BMSCs is not greatly affected, while the activity of tumor cells 143b, U2OS, and sjsa1 is greatly reduced with the increase of concentration, which proves that tFNAs@ESCu/aPD-L1 has good in vivo biosafety and good tumor killing effect.

Blank group, tFNAs group, tFNAs@ESCu group and tFNAs@ESCu/aPD-L1 group were set up. 143b cells were seeded in a 96-well low-adhesion plate at 10,000 cells/well and centrifuged at 300 g for 5 minutes. The cells grew in clusters and were induced into tumor spheres after 3 days. Live and dead staining was performed 12 hours after the addition of drugs (300 nM) in each group, and the tumor killing effect was observed under a confocal microscope.

The detection results are shown in FIG7 . As can be seen from FIG7 , the live-dead staining results show that the tFNAs@ESCu/aPD-L1 group has the best tumor killing effect.

Example 2: tFNAs@ESCu/aPD-L1 can induce copper cell death and block PD-L1 in vitro

Blank group, tFNAs group, tFNAs@ESCu group and tFNAs@ESCu/aPD-L1 group were set up. 143b cells were seeded in a 6-well plate and 2 mL of culture medium containing 300 nM tFNAs, tFNAs@ESCu and tFNAs@ESCu/aPD-L1 was added. Proteins were collected after 12 hours, and the expressions of key copper death proteins DLAT, FDX1 and LIAS were detected by western-blot.

The test results are shown in Figure 8. As shown in Figure 8, the content of the key protein DLAT of copper death increased, and the content of FDX1 and LIAS decreased, which met the criteria of copper death.

Blank group, tFNAs group, tFNAs@ESCu group and tFNAs@ESCu/aPD-L1 group were set up. 143b cells were seeded in a 6-well plate, and 2 mL of culture medium containing 300 nM tFNAs, tFNAs@ESCu and tFNAs@ESCu/aPD-L1 was added. After 12 hours, the cells were collected and flow cytometry antibodies ( ^1x106 cells/5uL) were added to detect PD-L1 by flow cytometry.

The detection results are shown in FIG9 . As can be seen from FIG9 , PD-L1 in the tFNAs@ESCu/aPD-L1 group was completely blocked.

Example 3: tFNAs@ESCu/aPD-L1 can effectively inhibit tumor growth in vivo

We set up blank group, tFNAs group, tFNAs@ESCu group and tFNAs@ESCu/aPD-L1 group, and established an orthotopic model of femoral osteosarcoma in mice (5x10 ⁵ 143b cells were injected into the right femoral bone marrow cavity). Five days later, 10uL PBS, 30uM tFNAs, 30uM tFNAs@ESCu, and 30uM tFNAs@ESCu/aPD-L1 were injected orthotopically. Samples were collected 21 days after treatment to observe the gross tumor photos.

The results of animal experiments are shown in Figure 10. As can be seen from Figure 10, there is no significant difference in tumor size between Blank and tFNAs. Both tFNAs@ESCu and tFNAs@ESCu/aPD-L1 significantly inhibited tumor growth, with the latter having the best effect.

In summary, the nucleic acid framework nanodrug tFNAs@ESCu/aPD-L1 prepared by the present invention that induces copper death and blocks PD-L1 can be used to prepare drugs for treating osteosarcoma to treat osteosarcoma.

The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims (3)

1. A method for preparing a nucleic acid framework nano-drug for inducing copper death and blocking PD-L1, which is characterized by comprising the following steps:

S1, mixing 1uM single-stranded DNA with the quantity of four substances in 100uL 1 xTM buffer solution, reacting for 15min at 95 ℃, and then annealing at 4 ℃ to obtain tetrahedral framework nucleic acid tFNAs;

S2, mixing the illitem Elesclomol with CuCl ₂ in the same amount of 300nM, adding the mixture into the tetrahedral framework nucleic acid tFNAs, mixing the mixture overnight at 4 ℃, then centrifuging the mixture for 10 minutes by using a 30kDa ultrafiltration tube with 14000g, and collecting the supernatant to obtain a tFNAs complex carrying Elesclomol-Cu, namely tFNas@ESCu;

S3, the molar ratio is 1:2.5 mixing the immune checkpoint inhibitor aPD-L1 with N-hydroxysuccinimide ester SPDP of 3- (2-pyridyldithio) propionic acid, reacting for 2 hours at 20 ℃, removing much of the N-hydroxysuccinimide ester SPDP of 3- (2-pyridyldithio) propionic acid by using a 10kDa ultrafiltration tube, and adding 100uL of 1xTM buffer to collect aPD-L1-SPDP; the aPD-L1-SPDP is added into the tFNAs@ESCu, reacted for 2 hours at 20 ℃, and the unreacted aPD-L1-SPDP is removed by centrifugation for 15 minutes by using 14000g of a 100kDa ultrafiltration tube, so that the nucleic acid framework nano-drug which induces copper death and blocks PD-L1, namely tFNAs @ ESCu/aPD-L1, is obtained.

2. A nucleic acid framework nano-drug that induces copper death and blocks PD-L1 produced by the method of claim 1.

3. Use of a nucleic acid framework nano-drug for inducing copper death and blocking PD-L1 according to claim 2 in the manufacture of a medicament for treating osteosarcoma.