An ido1 aptamer, nanoparticle and preparation method and application thereof

Abstract

The present application belongs to the technical field of molecular biology, and particularly relates to an IDO1 aptamer, nanoparticles, and a preparation method and application thereof. The present application utilizes protein-based SELEX technology to screen eight nucleic acid aptamers with small molecular weight, good IDO1 binding capacity, and high efficiency in inhibiting IDO1, and the specific nucleotide sequence is shown in any one of SEQ ID NO. 1-8. The eight nucleic acid aptamers are verified to be capable of efficiently binding to IDO1, and the eight nucleic acid aptamers and IDO1 protein are mixed and incubated to find that the concentration of kynurenine is significantly reduced, and the activity of IDO1 protein is inhibited, which provides a new idea and new tool for tumor diagnosis and treatment, and has important clinical value.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to an IDO1 nucleic acid aptamer, nanoparticles, their preparation methods, and applications. Background Technology

[0002] Aptamers are synthetically produced short single-stranded DNA or RNA molecules that bind to targets in a manner similar to an antigen-antibody interaction. Aptamers bind to targets with high affinity and specificity, and are smaller in molecular weight than traditional biological antibodies. They also offer advantages such as ease of synthesis, high stability, low immunogenicity, chemical modifiability, and no batch-to-batch effects. Currently, aptamers have wide applications in fields including diagnostics, therapy, bioimaging, and targeted drug delivery. With ongoing research into aptamers, an increasing number of functional aptamers are being developed as therapeutic agents. Therapeutic aptamers typically antagonize protein targets, exerting an inhibitory function.

[0003] Indoleamine 2,3-dioxygenase (IDO1) is an immune checkpoint molecule found in the cytoplasm. It is induced to express in many tumors, helping tumor cells achieve immune evasion. Currently, IDO1 is believed to exert its immunosuppressive effect by regulating tryptophan metabolism. The mechanism includes: in the tumor microenvironment, high levels of IDO1 expression consume tryptophan (Trp), suppressing effector T cells (T cells). eff ) function, promote regulatory T cells (T cells) reg IDO1 differentiates itself; on the other hand, its catalytic product, kynurenine (Kyn), is a natural ligand for the aryl hydrocarbon receptor (AHR), and activation of the AHR pathway further enhances immunosuppression. It is precisely because of IDO1's crucial role in maintaining the immunosuppressive tumor microenvironment that it has become an important target in anti-tumor immunotherapy. In recent years, numerous small-molecule inhibitors targeting IDO1 have been developed, and although many have entered clinical trials, there are no records of IDO1 inhibitors successfully passing clinical trials. Further research is needed on IDO1-targeting inhibitors. Functional aptamers have the potential to be therapeutic agents, and there are currently no reports on the nucleic acid aptamer sequences of IDO1. Therefore, functional aptamers targeting IDO1 hold promise as a new therapeutic approach. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an IDO1 nucleic acid aptamer that efficiently binds to and inhibits IDO1, constructs nanoparticles, improves the stability of the IDO1 nucleic acid aptamer, and targets the IDO1 protein in the cytoplasm.

[0005] The present invention provides an IDO1 nucleic acid aptamer, wherein the nucleotide sequence of the IDO1 nucleic acid aptamer is shown in any one of SEQ ID NO. 1 to 8.

[0006] The present invention also provides the application of the IDO1 nucleic acid aptamer described in the above technical solution in any one or more of the following (a) to (d);

[0007] (a) Preparation of a formulation that inhibits the activity of IDO1 enzyme;

[0008] (b) Preparations for treating tumors;

[0009] (c) Diagnostic agents for tumors;

[0010] (d) Preparations for detecting tumors.

[0011] Preferably, the formulation comprises nanoparticles.

[0012] The present invention also provides a nanoparticle based on the IDO1 nucleic acid aptamer described above, comprising a core and a shell;

[0013] The core comprises a complex of an IDO1 nucleic acid aptamer and a charge neutralizer; the shell comprises precipitates of cations and anions.

[0014] Preferably, the mass ratio of the IDO1 nucleic acid aptamer to the charge neutralizer is 4 to 8:1.

[0015] Preferably, the charge neutralizing agent comprises protamine sulfate;

[0016] The cation includes Ca. 2+ Fe 3+ Mg 2+ Mn 2+ Zn 2+ and Ni 2+ One or more of the following;

[0017] The anions include one or more of phosphate ions, hydrogen phosphate ions, citrate ions, oxalate ions, and carbonate ions.

[0018] The present invention also provides a method for preparing the nanoparticles described in the above technical solution, comprising the following steps:

[0019] The IDO1 nucleic acid aptamer, charge neutralizer, and solution containing anions are mixed to obtain the first mixture.

[0020] The first mixture was mixed with a solution containing cations, allowed to stand at 37°C for 20–60 min, centrifuged, and the precipitate was collected to obtain the nanoparticles.

[0021] The present invention also provides the application of the nanoparticles described in the above technical solution in the preparation of reagents for treating tumors.

[0022] The present invention also provides the application of the nanoparticles described above in the preparation of delivery reagents for nucleic acid aptamers targeting intracellular proteins.

[0023] Beneficial effects:

[0024] This invention expresses and purifies recombinant His-labeled mouse IDO1 protein using a prokaryotic system. Utilizing protein SELEX technology, after 13 rounds of DNA aptamer selection, eight nucleic acid aptamers with small molecular weights, good IDO1 binding ability, and high efficiency in inhibiting IDO1 were obtained. The specific nucleotide sequences are shown in any one of SEQ ID NO. 1–8. Examples verify that the binding strength of the eight nucleic acid aptamers to IDO1 ranges from 2929.7 to 27963.3 (mean fluorescence intensity, MFI). Furthermore, incubation with a mixture of the above eight nucleic acid aptamers and IDO1 protein significantly reduces the concentration of kynurenine, specifically to 22.0–32.0 μM.

[0025] Furthermore, this invention utilizes the IDO1 nucleic acid aptamer obtained from the above screening to combine with a charge neutralizing agent to prepare nanoparticles, which can further improve the stability of the IDO1 nucleic acid aptamer, enabling the aptamer to successfully target the IDO1 protein in the cytoplasm for tumor treatment. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0027] Figure 1 Flowchart for the selection of IDO1 aptamers;

[0028] Figure 2 The results show the binding affinity analysis between the IDO1 candidate aptamer and the target protein.

[0029] Figure 3 The results of the detection of the inhibition of IDO1 enzyme activity by the candidate aptamers of IDO1;

[0030] Figure 4 Predicted secondary structure diagram of the IDO1 aptamer;

[0031] Figure 5 A simulation diagram of the docking between the IDO1 aptamer and the IDO1 protein;

[0032] Figure 6 The results of affinity fitting analysis between the IDO1 aptamer and the IDO1 protein;

[0033] Figure 7The figure shows the results of gel migration retardation analysis of the binding between the IDO1 aptamer and the IDO1 protein.

[0034] Figure 8 Results of CT26 cell induction of IDO1 expression (A) and IDO1 aptamer-mediated pull-down (B);

[0035] Figure 9 Results of aptamer-protamine complex assembly efficiency assay;

[0036] Figure 10 Transmission electron microscopy image of protamine-IDO1 aptamer nanoparticles (NP-IDO-APT), scale bar: 100 nm;

[0037] Figure 11 The results of confocal microscopy examination in Example 11 are shown. Scale bar: 10 μm.

[0038] Figure 12 The results show the stability of protamine-IDO1 aptamer nanoparticles.

[0039] Figure 13 The effect of protamine-IDO1 aptamer nanoparticles on cell proliferation;

[0040] Figure 14 The effect of protamine-IDO1 aptamer nanoparticles on cell apoptosis;

[0041] Figure 15 For protamine-IDO1 aptamer nanoparticles to T reg Effects on cell differentiation;

[0042] Figure 16 For protamine-IDO1 aptamer nanoparticles to target CD8 + The effect of TNF-α expression on T cells;

[0043] Figure 17 For protamine-IDO1 aptamer nanoparticles to target CD8 + The effect of IFN-γ expression on T cells;

[0044] Figure 18 To analyze the effects of protamine-IDO1 aptamer nanoparticles on lymphocytes using RNA-seq.

[0045] Figure 19 The effect of protamine-IDO1 aptamer nanoparticles on tumor growth in a CT26 mouse model;

[0046] Figure 20 The image shows the actual tumor structure and weight of a CT26 mouse model after treatment with protamine-IDO1 aptamer nanoparticles.

[0047] Figure 21 The effect of different doses of protamine-IDO1 aptamer nanoparticles on the growth of CT26 tumors; where A is the tumor growth curve; and B is the tumor volume at the monitoring endpoint.

[0048] Figure 22 The effect of protamine-IDO1 aptamer nanoparticles on the survival rate of CT26 tumor-bearing mice;

[0049] Figure 23 The effect of protamine-IDO1 aptamer nanoparticles on tumor growth in NOD / SCID mice;

[0050] Figure 24 Changes in the kynurenine / tryptophan ratio in plasma and tumor of CT26 tumor-bearing mice after treatment with protamine-IDO1 aptamer nanoparticles.

[0051] Figure 25 Lymphocytes (A) and CD8 infiltrating CT26 tumors after protamine-IDO1 aptamer nanoparticle therapy + Changes in the number of T cells (B);

[0052] Figure 26 For T26 tumors infiltrating after protamine-IDO1 aptamer nanoparticle therapy reg Changes in cell number;

[0053] Figure 27 CD86 tumor infiltrating CT26 cells after protamine-IDO1 aptamer nanoparticle therapy + T cell effector cytokine secretion; where A is CD8. + TNF-α + Cell proportion, B is CD8 + IFN-γ + Cell ratio;

[0054] Figure 28 Changes in the number of tumor-associated macrophages (A) and G-MDSCs (B) infiltrating CT26 tumors after protamine-IDO1 aptamer nanoparticle therapy;

[0055] Figure 29 The changes in body weight (A) and blood biochemical parameters (B) of CT26 tumor-bearing mice after treatment with protamine-IDO1 aptamer nanoparticles. Detailed Implementation

[0056] The present invention provides an IDO1 nucleic acid aptamer, the nucleotide sequence of which is shown in any one of SEQ ID NO. 1 to 8, and the specific sequence information is shown in Table 1.

[0057] This invention utilizes protein-based SELEX technology and, after 13 rounds of screening, obtained 8 small molecular weight nucleotide sequences, any one of which has good IDO1 binding ability and is a nucleic acid aptamer that efficiently inhibits IDO1.

[0058] In view of the functions of the IDO1 nucleic acid aptamer provided by this invention, the IDO1 nucleic acid aptamer described in the above technical solution is protected within the scope of this invention in one or more of the following applications: preparation of agents to inhibit IDO1 enzyme activity, preparation of agents to treat tumors, preparation of agents to diagnose tumors, and preparation of agents to detect tumors. The IDO1 nucleic acid aptamer provided by this invention offers new ideas and tools for targeted diagnosis and treatment of tumors, and has significant clinical value.

[0059] In this invention, the formulation comprises nanoparticles.

[0060] The present invention also provides a nanoparticle based on the IDO1 nucleic acid aptamer described above, comprising a core and a shell;

[0061] The core comprises a complex of an IDO1 nucleic acid aptamer and a charge neutralizer; the shell comprises precipitates of cations and anions.

[0062] In this invention, the mass ratio of the IDO1 nucleic acid aptamer to the charge neutralizing agent is preferably 4 to 8:1, more preferably 4:1. The charge neutralizing agent of this invention preferably includes protamine sulfate.

[0063] In this invention, the cation preferably includes Ca. 2+ Fe 3+ Mg 2+ Mn 2+ Zn 2+ and Ni 2+ One or more of the following;

[0064] The anion preferably includes one or more of phosphate ions, hydrogen phosphate ions, citrate ions, oxalate ions, and carbonate ions.

[0065] The present invention also provides a method for preparing the nanoparticles described in the above technical solution, comprising the following steps:

[0066] The IDO1 nucleic acid aptamer, charge neutralizer, and solution containing anions are mixed to obtain the first mixture.

[0067] The first mixture was mixed with a solution containing cations, allowed to stand at 37°C for 20–60 min, centrifuged, and the precipitate was collected to obtain the nanoparticles.

[0068] This invention mixes the IDO1 nucleic acid aptamer, a charge neutralizing agent, and a solution containing anions to obtain a first mixture. The anions preferred in this invention include one or more of phosphate ions, hydrogen phosphate ions, citrate ions, oxalate ions, and carbonate ions. This invention does not have strict requirements on the ratio of the mass of the IDO1 nucleic acid aptamer to the concentration of anions in the solution containing anions; conventional selection is acceptable. In this invention, the IDO1 nucleic acid aptamer, charge neutralizing agent, and anion solution are mixed, and the IDO1 nucleic acid aptamer and charge neutralizing agent condense to obtain a complex of the IDO1 nucleic acid aptamer and charge neutralizing agent; the first mixture is a mixed solution of the complex of the IDO1 nucleic acid aptamer and charge neutralizing agent and anions. The mixing time is preferably 20–60 min, more preferably 20–40 min, more preferably 20–30 min, and most preferably 30 min.

[0069] After obtaining the first mixture, the present invention mixes the first mixture with a solution containing cations and allows it to stand at 37°C for 20–60 minutes. The cations in the present invention preferably include Ca. 2+ Fe 3+ Mg 2+ Mn 2+ Zn 2+ and Ni 2+ One or more of the following. In this invention, there are no strict requirements on the ratio of the mass of the IDO1 nucleic acid aptamer to the concentration of cations in the cation-containing solution; conventional selection is acceptable. During the settling process, cations spontaneously form microprecipitates around the complex of the IDO1 nucleic acid aptamer and the charge neutralizer, encapsulating the complex and forming nanoparticles.

[0070] After the settling period, the product obtained by settling is centrifuged, and the precipitate is collected; the precipitate is the nanoparticle. The centrifugation method is not strictly required; conventional methods in the art can be used.

[0071] The IDO1 nucleic acid aptamer described in this invention carries a negative charge and repels the cell membrane, making it difficult to enter the cell. Using a charge neutralizing agent, it can condense with the negatively charged IDO1 nucleic acid aptamer to obtain a complex. This complex serves as the core of nanoparticles, which are then prepared into mineralized nanoparticles using a biomimetic biomineralization strategy. This promotes the efficient entry of the IDO1 nucleic acid aptamer into tumor cells, thereby exerting an anti-tumor therapeutic effect. Furthermore, the protamine sulfate described is listed in the 2012 edition of the National Essential Medicines List of China and can be applied to medical research.

[0072] Given the effects of the nanoparticles provided by this invention, the application of the nanoparticles described in the above-mentioned technical solutions in the preparation of reagents for treating tumors and / or reagents for delivering drugs for treating tumors falls within the protection scope of this invention. The nanoparticles provided by this invention exhibit good delivery capability to the IDO1 nucleic acid aptamer. Example results show that the nanoparticles can promote CD8 in vitro. + Lymphocyte proliferation, inhibition of T cells reg Cell differentiation. Intratumoral injection of the nanoparticles inhibited the growth of CT26 tumors.

[0073] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides an IDO1 nucleic acid aptamer, nanoparticles, their preparation method, and applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0074] Example 1

[0075] Nucleic acid aptamer screening and cloning of IDO1 protein

[0076] 1. Expression and purification of IDO1 protein

[0077] The CDS region of IDO1 mRNA (NM_008324.2) was inserted into a pET28a(+) vector containing an N-terminal histidine (His6) tag. This vector was transformed into *E. coli* BL21(DE3) and cultured in LB medium. When OD... 600 When the pH reaches approximately 0.6, add 1 mM IPTG for induction for 4 hours. Collect the bacterial culture and centrifuge, then resuspend the bacterial pellet in TBS solution and perform sonication lysis. After centrifuging the lysis buffer, collect the supernatant and add it to a Ni-NTA column. Then, purify the protein sequentially with 50 mL of wash buffer 1 (composed of 20 mM Tris, 500 mM NaCl, and the remainder ddH2O, pH 8.0), 50 mL of wash buffer 2 (composed of 20 mM Tris, 500 mM NaCl, 20 mM imidazole, and the remainder ddH2O, pH 8.0), 50 mL of wash buffer 3 (composed of 20 mM Tris, 500 mM NaCl, 40 mM imidazole, and the remainder ddH2O, pH 8.0), and 25 mL of elution buffer (composed of 10 mM Tris, 500 mM NaCl, 60 mM imidazole, and the remainder ddH2O, pH 8.0). The eluted protein was concentrated by ultrafiltration and then further purified by molecular sieve (GE Healthcare Life Sciences) to obtain IDO1 protein.

[0078] 2. ssDNA library and amplification primers

[0079] The ssDNA library contains 40 random nucleotides (random nucleotides are one of A, T, C, G), and two primer binding sequences on both sides. The specific sequence is: 5'-CAGCACCGTCAACTGAATNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN GTGATGCGATGGAGATGT-3' (SEQ ID NO. 9), where N is one of A, T, C, G.

[0080] The upstream primer sequence for amplifying the aptamer is 5'-CAGCACCGTCAACTGAAT-3' (SEQ ID NO.10), and the downstream primer sequence is 5'-ACATCTCCATCGCATCAC-3' (SEQ ID NO.11).

[0081] The ssDNA library and amplification primers mentioned above were synthesized by Ribo Biotechnology Co., Ltd. (Beijing, China).

[0082] 3. Preparation of screening targets

[0083] At the start of the screening process, the IDO1 protein obtained in step 1 was immobilized on Ni-NTA beads to obtain Ni-NTA beads bound to the target protein, denoted as IDO1-beads. During screening, IDO1-beads were used as positive screening targets, and naked Ni-NTA beads (Ni-beads) were used as negative screening targets.

[0084] 4. Protein-based aptamer screening

[0085] IDO1 aptamers were screened using protein SELEX technology, according to... Figure 1 The steps shown involve incubating the ssDNA library at 95°C for 3 minutes, then cooling it on ice for 5 minutes. Following this, 13 rounds of screening are performed, with the first 3 rounds consisting only of positive screening and the remaining 10 rounds involving a combination of reverse and positive screening (one round of screening consists of one reverse and one positive screening performed sequentially). The specific steps for the entire nucleic acid aptamer screening process are as follows:

[0086] 4.1 First 3 rounds of positive screening:

[0087] 4.1.1 Mix the IDO1-beads obtained in step 3 with 5 nmol ssDNA library, add 1 mL of binding buffer (composed of 10 mM Tris, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2 and the balance ddH2O, pH 7.5), add 1 mg / mL BSA and 100 μg / L tRNA, incubate at 4 °C for 1 hour, centrifuge at 12000 rpm at 4 °C for 5 min, discard the supernatant to leave about 50 μL of liquid. The Ni-NTA beads (IDO1-beads) bound to the target protein were washed with 500 μL of washing buffer (composed of 10 mM Tris, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.05% (v / v) Tween 20 and the balance ddH2O, pH 7.5). The beads were centrifuged at 12,000 rpm for 5 min at 4 °C, the supernatant was discarded, and the IDO1-beads were resuspended in 200 μL of ddH2O. The IDO1-beads were then treated at 95 °C for 10 min, centrifuged at 12,000 rpm for 5 min at 4 °C, and the supernatant was collected.

[0088] Using the supernatant as a template, the first PCR amplification was performed according to the PCR amplification system (total volume 250 μL) and conditions in Table 1 to obtain the first PCR amplification product.

[0089] Table 1. Reaction system and conditions for the first PCR amplification.

[0090]

[0091] Using the first PCR amplification product as a template, a second PCR amplification was performed according to the PCR amplification system (total volume 2000 μL) and conditions in Table 2 to obtain the second PCR amplification product.

[0092] Table 2. Reaction system for the second PCR amplification

[0093]

[0094] The 10× buffer, Taq enzyme, and dNTPs used in the PCR experiments were purchased from TAKARA.

[0095] The obtained second PCR amplification product was incubated with streptavidin agarose beads (GE Healthcare LifeSciences) at 37°C for 30 minutes. The double strands of the PCR product were separated using 200mM NaOH, and the collected single-stranded 5'FAM-labeled amplicon solution was neutralized with HCl to obtain the 5'FAM-labeled target strand, i.e., the secondary random nucleotide library, which was then added to the next SELEX cycle.

[0096] 4.1.2 Repeated screening: The secondary nucleotide library obtained in step 4.1.1 is repeated twice, that is, three rounds of positive screening are completed, and a third round of secondary nucleotide library is obtained.

[0097] 4.2 The last 10 rounds of reverse screening + forward screening:

[0098] 4.2.1 Starting from the fourth round of screening, a reverse screening operation is performed before each round of forward screening: The third round secondary nucleotide library is incubated at 95℃ for 3 min, cooled on ice for 5 min, and then 1 mg / mL BSA and 100 μg / L tRNA are added. The mixture is then incubated with naked Ni-NTA beads at 4℃ for 30 min. After centrifugation at 12000 rpm for 5 min at 4℃, the supernatant is collected and labeled as the reverse screening product for this round.

[0099] 4.2.2 Perform forward screening on the secondary reverse screening product obtained in step 4.1.1 to obtain the secondary random nucleotide library for this round.

[0100] 4.2.3 Repeated Screening: The secondary random nucleotide library obtained in step 4.2.2 is subjected to repeated operations of 4.2.1 and 4.2.2 nine times each, i.e., nine rounds of reverse screening + forward screening. In these nine rounds of reverse screening, the nucleotide library added in the next round of reverse screening is the secondary nucleotide library prepared in the previous round of forward screening; in these nine rounds of forward screening, the nucleotide library added in the next round of forward screening is the secondary nucleotide library prepared in the previous round of reverse screening. After repeated screening, the 13th round of enriched random nucleotide library is obtained.

[0101] In this embodiment, flow cytometry was used to monitor the enrichment of target sequences in the libraries acquired in each round. The enriched DNA pool from the 13th round was cloned into the TA vector and sequenced. Based on the sequencing results, the top 8 most frequent sequences were chemically synthesized as candidate aptamers and named IA1 to IA8, as shown in Table 3.

[0102] Table 3. IDO1 Nucleic Acid Aptamer Names and Sequence Information

[0103]

[0104] Example 2

[0105] Binding ability of IDO1 candidate aptamers to target proteins

[0106] To evaluate the binding ability of IDO1 nucleic acid aptamers to target proteins, the 5' ends of eight aptamers obtained in Example 1 were labeled with FAM and incubated with Ni-NTA magnetic beads (i.e., IDO1-beads) bound to IDO1 obtained in step 3 of Example 1. Fluorescence intensity was analyzed by flow cytometry, and the results are shown in Table 4. Figure 2 .

[0107] Table 4. Binding ability of nucleic acid aptamers to IDO1 protein

[0108]

[0109]

[0110] According to Table 4 and Figure 2 It can be seen that the nucleic acid aptamers numbered #1 (IA3), #3 (IA2), and #4 (IA1) among the eight nucleic acid aptamers showed the strongest IDO1 binding ability.

[0111] Example 3

[0112] IDO1 enzyme activity assay

[0113] Since the function of IDO1 is largely dependent on its enzymatic activity, recombinant mouse IDO1 protein was used for in vitro enzyme activity assays to examine the inhibitory effect of the candidate aptamer obtained in Example 1. 2 μg of recombinant His-labeled mouse IDO1 protein (prepared as in step 1 of Example 1) and 500 nM of the candidate aptamer were mixed in 100 μL of 50 mM potassium phosphate buffer (pH 7.5) and incubated at 4 °C for 30 min. Subsequently, 20 mM L-ascorbic acid (Sigma-Aldrich), 10 μM methylene blue (Macklin), and 40 μM L-tryptophan (Sigma-Aldrich) were added to the mixture, and it was incubated at 37 °C for 30 min. Then, 50 μL of 30% (w / v) trichloroacetic acid was added to the reaction solution, and the mixture was incubated at 65 °C for 15 min. After centrifugation, the supernatant was collected and mixed with an equal volume of 2% (w / v) 4-(dimethylamino)benzaldehyde (Sigma-Aldrich) and added to a 96-well plate. The absorbance was read at 490 nm using a microplate reader. The absorbance was determined according to the kynurenine standard curve (OD). 490 The concentration of kynurenine in the above product was determined by (kynurenine concentration × 0.0051 + 0.084), and the results are shown in Table 5. Figure 3 As shown.

[0114] Table 5. Inhibitory effect of nucleic acid aptamers on IDO1 enzyme activity

[0115]

[0116] According to Table 5 and Figure 3 It can be seen that the addition of candidate aptamer #4 (IA1) resulted in the lowest concentration of kynurenine (Kyn), the catalytic product of IDO1, indicating that among the eight candidate aptamers, candidate aptamer #4 (IA1) had the strongest inhibitory effect on IDO1.

[0117] Example 4

[0118] Aptamer structure prediction and aptamer protein docking simulation

[0119] Based on the results of Examples 2 and 3, candidate aptamer #4 (IA1, hereinafter referred to as IDO-APT) was further characterized: The secondary structure of IDO-APT was predicted using the UNAFold Web server (http: / / www.unafold.org) with the aid of nucleic acid structure analysis tools, and the results are as follows: Figure 4 As shown in the figure. The docking simulation of IDO1 protein and IDO-APT was performed using the HDOCK SERVER tool (http: / / hdock.phys.hust.edu.cn), and the results are as follows. Figure 5 As shown.

[0120] according to Figures 4-5 It can be seen that IDO-APT contains two stem-loop structures. Nucleotides at positions 42-44, 51-55, and 61-65 in IDO-APT may bind to amino acid residues at positions 119-126, 259-263, and 301-309 of the IDO1 protein.

[0121] Example 5

[0122] aptamer binding test

[0123] Experimental group: FAM-labeled IDO-APT of different concentrations and Ni-NTA magnetic beads (i.e. IDO1-beads) bound with IDO1 obtained in step 3 of Example 1 were incubated in binding buffer at 4°C for 30 minutes.

[0124] Control group: The nucleotide sequence of IDO-APT was scrambled to obtain a new nucleotide sequence, denoted as Scrambled APT, specifically: 5'-CAGCACCGTCAACTGATTTTTAACGGCATATCCGGACTTTTTTGAACCTAGGGCCTTTGTGATGCGATGGAGATGT-3' (SEQ ID NO.12); Scrambled APT with different concentrations of FAM labeling and Ni-NTA magnetic beads (i.e., IDO1-beads) bound to IDO1 obtained in step 3 of Example 1 were incubated in binding buffer at 4°C for 30 minutes;

[0125] After incubation in both the experimental and control groups, fluorescence intensity was analyzed by flow cytometry. Based on the average fluorescence intensity, a curve was fitted between IDO-APT and IDO1 proteins, Y = B. max *X / (Kd+X), calculate the dissociation constant Kd, where Y is the amount of aptamer capable of binding IDO1 protein, X is the aptamer concentration, and B... max The maximum aptamer amount that can bind to the IDO1 protein. Results are shown in Table 6 and... Figure 6 As shown.

[0126] Table 6. Binding affinity of IDO-APT aptamer to IDO1 protein (MFI%)

[0127] FAM concentration (μM) 0 20 40 80 160 320 IDO-APT 1.4 12.1 23.1 59.1 95.4 100.0 ScrambledAPT 1.0 1.5 1.5 1.3 2.2 2.0

[0128] According to Table 6 and Figure 6 It can be seen that IDO-APT has a strong binding affinity for IDO1 protein, and the dissociation constant between IDO-APT and IDO1 protein is 127.39 nmol / L.

[0129] Example 6

[0130] Electrophoretic mobility transfer assay (EMSA)

[0131] The targeting ability of IDO1-APT was confirmed using electrophoretic mobility transfer assay (EMSA). Specific steps:

[0132] Experiment 1: 10 μL of 100 nM 5'FAM-labeled Scrambled-APT was incubated at 4°C for 1 hour; Experiment 2: 10 μL of a mixture containing 100 nM 5'FAM-labeled Scrambled-APT and 1 mg / mL BSA protein was incubated at 4°C for 1 hour; Experiment 3: 10 μL of a mixture containing 100 nM 5'FAM-labeled Scrambled-APT and 1 mg / mL mouse IDO1 protein (prepared as in step 1 of Example 1) was incubated at 4°C for 1 hour; Experiment 4: 10 μL of a mixture containing 100 nM 5'FAM-labeled Scrambled-APT and 1 mg / mL human IDO1 protein (prepared as in step 1 of Example 1) was incubated at 4°C for 1 hour.

[0133] Experiment 5: 10 μL of 100 nM 5'FAM-labeled IDO-APT was incubated at 4 °C for 1 hour; Experiment 6: 10 μL of a mixture containing 100 nM 5'FAM-labeled IDO-APT and 1 mg / mL BSA protein was incubated at 4 °C for 1 hour; Experiment 7: 10 μL of a mixture containing 100 nM 5'FAM-labeled IDO-APT and 1 mg / mL mouse IDO1 protein was incubated at 4 °C for 1 hour; Experiment 8: 10 μL of a mixture containing 100 nM 5'FAM-labeled IDO-APT and 1 mg / mL human IDO1 protein was incubated at 4 °C for 1 hour.

[0134] Using 0.5×TBE as the electrophoresis buffer, the incubated samples were electrophoresed in 8% natural PAGE gel at 70V for 10 minutes and then at 150V for 40 minutes. The migration of aptamer bands was observed using a fluorescence imager, and the results are as follows: Figure 7 As shown.

[0135] according to Figure 7 It can be seen that electrophoretic migration arrest can only be observed in the lanes where IDO-APT is incubated with mouse IDO1 protein, while the control aptamer does not exhibit this phenomenon, indicating that IDO-APT can specifically bind to IDO1 protein.

[0136] Example 7

[0137] Aptamer-mediated pull-down test

[0138] Aptamer-mediated pull-down assays were performed using an endogenous expression system based on the CT26 cell line.

[0139] (1) Detect the expression of IDO1 in CT26 cells treated with different doses of IFN-γ (PeproTech).

[0140] CT26 cell culture medium was supplemented with 50, 100, and 200 ng / mL IFN-γ, respectively, and cultured for 48 hours. CT26 cells were then harvested and lysed at 4°C for 30 min using Co-IP lysis buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl pH 8.0, 10% (v / v) glycerol, 0.5% NP40, 100× PMSF, and 100× protease inhibitor cocktail (Sigma-Aldrich)). Cells were then centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant of the cell lysate was collected for Western blot analysis. The results are shown below. Figure 8 As shown in Figure A.

[0141] according to Figure 8As can be seen from Figure A, a concentration of 50 ng / mL of IFN-γ is sufficient to induce adequate IDO1 expression.

[0142] (2) The 5' end of IDO-APT was labeled with biotin and incubated with IFN-γ-treated CT26 cell lysate. The IDO-APT-IDO1 protein complex was then captured using streptavidin agarose beads (GE Healthcare Life Sciences) and detected by Western blot. Specific procedures are as follows:

[0143] CT26 cells were administered at a rate of 1×10⁻⁶. 6 Cells were seeded at a count of 1 / mL in 60 mm culture dishes and allowed to grow overnight. IDO1 expression was induced for 48 hours with 50 ng / mL recombinant mouse IFN-γ (PeproTech). Harvested CT26 cells were lysed at 4°C for 30 min with Co-IP lysis buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl pH 8.0, 10% (v / v) glycerol, 0.5% NP40, 100× PMSF, and 100× protease inhibitor cocktail (Sigma-Aldrich)). After centrifugation at 12,000 rpm for 10 min at 4°C, the cell lysate supernatant was collected and incubated with 5' biotin-labeled IDO-APT or Scrambled-APT at 4°C for 2 h. Then, streptavidin agarose beads were added to the cell lysate, and incubation was continued at 4°C for 2 h to capture the protein aptamer complex. After washing three times with washing buffer, the protein precipitated by heating was eluted in protein loading buffer. The sample was then subjected to SDS-PAGE electrophoresis, followed by Western blotting with anti-IDO antibody (Abcam). The results are shown in Figure 8B. Figure 8 In the text, "-" indicates that it has not been added, and "+" indicates that it has been added.

[0144] according to Figure 8 As shown in Figure B, IDO-APT can successfully precipitate IDO1 protein, which is then competitively precipitated by free IDO-APT (without biotin labeling) in a dose-dependent manner.

[0145] Example 8

[0146] Validation of assembly efficiency of aptamer-protamine nanoparticles

[0147] IDO-APT and protamine were mixed in solutions containing phosphate and carbonate at mass ratios of 8:1, 4:1, 2:1, and 1:1. After standing for 30 min, CaCl2 was added, and the mixture was incubated at 37°C for 30 min. The nanoparticle precipitate was resuspended and subjected to agarose gel electrophoresis. The results are as follows: Figure 9As shown.

[0148] according to Figure 9 It can be seen that the ratio of IDO-APT to protamine is 4:1, which shows the best assembly efficiency of the aptamer-protamine complex.

[0149] Example 9

[0150] The preparation of IDO-APT-protamine nanoparticles consists of the following steps:

[0151] IDO-APT and protamine were mixed in a solution containing phosphate and carbonate at a mass ratio of 4:1. After standing for 30 min, CaCl2 was added and incubated at 37 °C for 30 min. The precipitate was collected by centrifugation to obtain IDO-APT-protamine nanoparticles, denoted as NP-IDO-APT.

[0152] Comparative Example 1

[0153] The preparation of individual nanoparticles consists of the following steps:

[0154] Fish protamine was mixed in a solution containing phosphate and carbonate, and after standing for 30 min, CaCl2 was added and incubated at 37 °C for 30 min. The precipitate was collected by centrifugation to obtain blank nanoparticles, denoted as NPs.

[0155] Comparative Example 2

[0156] The preparation of the aptamer-protamine nanoparticles consists of the following steps:

[0157] ScrambledAPT and protamine were mixed in a solution containing phosphate and carbonate at a mass ratio of 4:1. After standing for 30 min, CaCl2 was added and incubated at 37 °C for 30 min. The precipitate was collected by centrifugation to obtain the control aptamer-protamine nanoparticles, denoted as NP-Scr-APT.

[0158] Example 10

[0159] Morphological observation of IDO-APT-protamine nanoparticles

[0160] The NP-IDO-APT obtained in Example 9 was dropped onto a copper grid of a carbon support film. The nanoparticles were then imaged using a transmission electron microscope (JEM-1400PLUS). First, 10 μL of the sample was dropped onto a copper grid of a standard carbon support film and allowed to stand for 1 min. The sample was then stained with 1% uranium acetate for 1 min. After the stain was absorbed, the sample was washed with distilled water, dried, and its particle size and morphology were observed using a transmission electron microscope. The results are as follows: Figure 10 As shown.

[0161] according to Figure 10As can be seen, the nanoparticles have a regular spherical structure and a particle size of about 50 nm.

[0162] Example 11

[0163] Aptamer cell uptake assay

[0164] 1×10 5 CT26 cells were seeded in 20 mm glass-bottom cell culture dishes (NEST) and cultured for 24 hours. They were divided into experimental and control groups. The experimental group was added to the cell culture medium with 500 nM 5'FAM-labeled NP-IDO-APT obtained in Example 9 and cultured at 37°C for 6 hours. The control group was added to the cell culture medium with 500 nM 5'FAM-labeled IDO-APT and cultured at 37°C for 6 hours.

[0165] After the experimental and control groups were cultured, the cells were treated with 30 μM Hoechst for 10 minutes, followed by washing three times with PBS. Images were taken using a Zeiss LSM 880 confocal laser scanning microscope. The results are as follows: Figure 11 As shown.

[0166] according to Figure 11 As can be seen, after incubating CT26 cells with 5'FAM-labeled NP-IDO-APT, a fluorescent signal of IDO-APT was observed in the intracellular space. No similar signal was observed after incubating IDO-APT alone with cells, demonstrating that nanoparticles facilitate the penetration of IDO-APT into the cell membrane.

[0167] Example 12

[0168] Serum stability test

[0169] Experimental group: 5'Cy5 labeled NP-IDO-APT was incubated with 10% (v / v) FBS at 37°C for 12 h, 24 h, 36 h, 48 h and 72 h.

[0170] Control group: 5'Cy5 labeled IDO-APT was incubated with 10% (v / v) FBS at 37°C for 12 h, 24 h, 36 h, 48 h and 72 h.

[0171] Samples from the experimental and control groups incubated for different time periods (1–72 hours) were electrophoresed on 2% agarose gels. After electrophoresis, the degradation degree of the aptamers was observed using an Amersham Imager 600 (GE Healthcare Life Sciences). The results are shown in Table 7. Figure 12 As shown.

[0172] Table 7 Stability of Nucleic Acid Aptamers

[0173] Time (h) 0 12 24 36 48 72 IDO-APT 1.0 0.277 0.080 0.025 0.017 0.007 NP-IDO-APT 1.000 0.711 0.630 0.597 0.515 0.433

[0174] Note: The table shows the relative ratio of grayscale values, specifically the ratio of grayscale values ​​relative to 0h.

[0175] According to Table 7 and Figure 12 It can be seen that NP-IDO-APT can exist stably in 10% FBS with a half-life of 48 hours, while the bands of IDO-APT decrease rapidly with a half-life of less than 12 hours. Therefore, the nanoparticles obtained in Example 9 can protect IDO-APT from degradation and prolong its half-life.

[0176] Example 13

[0177] Cell proliferation assay

[0178] CT26 cells were grown at a rate of 2 × 10⁶ cells per well. 3 Cells were seeded at a density of 1000 cells in 96-well plates and allowed to grow overnight. They were then randomly divided into four groups, designated as UT group, NPs group, NP-Scr-APT group, and NP-IDO1-APT group, respectively.

[0179] CT26 cells in the UT group grew normally without any treatment;

[0180] The NPs group was prepared by adding 100 nM of the NPs obtained in Comparative Example 1 to the cell culture medium;

[0181] In the NP-Scr-APT group, 100 nM of NP-Scr-APT obtained in Comparative Example 2 was added to the cell culture medium;

[0182] 100 nM of NP-IDO1-APT obtained in Example 9 was added to the cell culture medium for the NP-IDO1-APT group.

[0183] After culturing cells for 24, 48, and 72 hours, 10 μL CCK-8 reagent was added to each well, and the cells were incubated at 37°C for 3 hours. The absorbance at 450 nm was measured to detect the effect of aptamers or protamine on cell proliferation. The results are as follows: Figure 13 As shown.

[0184] according to Figure 13 It can be seen that the proliferation of CT26 cells was not affected after administration of NP-IDO-APT, NP-Scr-APT or NPs.

[0185] Example 14

[0186] Apoptosis assay

[0187] CT26 cells were added at a rate of 1 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 cells per well in 12-well plates and allowed to grow overnight. They were randomly divided into 4 groups, designated as UT group, NPs group, NP-Scr-APT group, and NP-IDO1-APT group, respectively.

[0188] CT26 cells in the UT group grew normally without any treatment;

[0189] The NPs group added 100 nM individual nanoparticles to the cell culture medium;

[0190] In the NP-Scr-APT group, 100 nM control aptamer-protamine nanoparticles (NP-Scr-APT) were added to the cell culture medium.

[0191] 100 nM of NP-IDO1-APT obtained in Example 9 was added to the cell culture medium for the NP-IDO1-APT group.

[0192] After culturing cells for 24, 48, and 72 hours in each treatment group, apoptosis of CT26 cells was detected using an Annexin V-mCherry and SYTOX Green (Beyotime) apoptosis detection kit: cells were digested and washed once with PBS, then stained for 15 minutes at room temperature with 200 μL binding buffer containing 5 μL Annexin V-mCherry and 1 μL SYTOX Green. The stained cells were immediately analyzed by flow cytometry, and the results are as follows: Figure 14 As shown.

[0193] according to Figure 14 It can be seen that NP-IDO-APT, NP-Scr-APT, or NPs have no significant effect on apoptosis in CT26 cells.

[0194] Example 15

[0195] In vitro lymphocyte detection

[0196] High levels of IDO1 in the tumor microenvironment promote T reg It promotes cell differentiation and induces T cell exhaustion. Previous reports have shown that IDO1 inhibition can reverse DCs' promotion of T cell exhaustion. reg It plays a role in cell differentiation and increases the ability of DCs to activate T cells. Therefore, this embodiment examines whether IDO-APT treatment leads to T cell differentiation during in vitro culture. regCell suppression and T cell enhancement. Spleens were harvested from BALB / c mice, ground, and filtered through a 200-mesh sieve. Splenic cells were then resuspended in RPMI 1640 medium supplemented with 10% FBS, 1% L-glutamate, penicillin (100 U / mL), streptomycin (100 μg / mL), 2 μg / mL anti-CD3 (BioLegend), 1 μg / mL anti-CD28 (BioLegends), and 20 ng / mL IL-2 (PeproTech). The lymphocyte suspension was seeded into 12-well plates, with 1 × 10⁶ cells per well. 6 Cells were randomly divided into three groups, denoted as NPs group, NP-Scr-APT group and NP-IDO1-APT group respectively.

[0197] In the NPs group, 100 nM individual nanoparticles were added to the cell culture medium and cultured for 6 days.

[0198] In the NP-Scr-APT group, 100 nM control aptamer-protamine nanoparticles (NP-Scr-APT) were added to the cell culture medium and cultured for 6 days.

[0199] The NP-IDO1-APT group was cultured for 6 days with 100 nM of NP-IDO1-APT obtained in Example 9 added to the cell culture medium.

[0200] To assess T cell function, cells in each treatment group were stimulated for 5 hours before harvesting with a mixture of 100 ng / mL PMA, 500 ng / mL iomycin, and a protein transport inhibitor (eBioscience). Cells were then incubated with anti-CD45-PEcy7 and anti-CD8-FITC for surface staining, and subsequently incubated with IFN-γ-APC or TNF-α-PE after fixation and permeabilization. For T… reg Cellular analysis was performed by collecting cells and staining them with anti-CD4-PerCP, anti-CD25-PE, and anti-Foxp3-APC. The cells were then analyzed by flow cytometry, and the results are as follows: Figures 15-17 As shown.

[0201] according to Figures 15-17 It can be seen that NP-IDO1-APT inhibits T reg It also promotes T cell activation. Compared with NP-Scr-APT or NPs, NP-IDO-APT treatment reduced CD4+. + CD25 in T cells + FoxP3 + The proportion of cells. In addition, NP-IDO-APT also promotes CD8... + T cells expressed more IFN-γ and TNF-α, an effect not observed in the control group.

[0202] Example 16

[0203] RNA-seq analysis of the effects of aptamers on lymphocytes

[0204] To further elucidate the effect of IDO-APT on T cell function, RNA sequencing analysis was performed on lymphocytes treated with NP-IDO-APT or NP-Scr-APT in Example 15. The results are as follows: Figure 18 As shown.

[0205] according to Figure 18 It can be seen that cytokine activity-related pathways were significantly enriched in cells treated with NP-IDO-APT. Moreover, compared with the NP-Scr-APT group, the expression of T cell effector factors, such as Ifng, Gzmb, Tnf, and Il2, was significantly upregulated in the cells of the NP-IDO-APT group, indicating that the IDO-APT provided by this invention promotes the effector function of lymphocytes.

[0206] Example 17

[0207] IDO-APT inhibits tumor growth in a CT26 mouse model.

[0208] First, 6-8 week old female BALB / c mice were subcutaneously inoculated with CT26 cells and randomly divided into three groups: NPs group, NP-Scr-APT group, and NP-IDO1-APT group. The tumor volume of mice in each treatment group reached approximately 50 mm. 3 When this happens, proceed as follows:

[0209] The NPs group received intratumoral injection of NPs at a dose of 1.5 mg / kg;

[0210] The NP-Scr-APT group received intratumoral injection of 1.5 mg / kg of NP-Scr-APT;

[0211] The NP-IDO1-APT group received intratumoral injection of NP-IDO-APT at a dose of 1.5 mg / kg.

[0212] Each treatment group received injections every three days, for a total of three times. Tumor size was measured every two days until a significant difference between groups was observed, as shown in the following figures. Figure 19 As shown. According to Figure 19 It can be seen that there were significant differences between groups two days after the third injection.

[0213] After significant differences were observed between groups, the tumor was removed, photographed, and its quality was tested. The results are as follows: Figure 20 As shown.

[0214] according to Figure 20It can be seen that, compared with the NP-Scr-APT or NPs treatment group, NP-IDO-APT significantly inhibited the growth of CT26 tumors, reducing tumor weight by more than 50%.

[0215] Example 18

[0216] To further confirm the antitumor effect of NP-IDO-APT, 6-8 week old female BALB / c mice were subcutaneously inoculated with CT26 cells and randomly divided into 5 groups: NPs group, NP-Scr-APT group, low-dose NP-IDO1-APT group, medium-dose NP-IDO1-APT group, and high-dose NP-IDO1-APT group. In each treatment group, the tumor volume reached approximately 50 mm². 3 At that time, the NPs group received an intratumoral injection of NPs at a dose of 1.5 mg / kg;

[0217] The NP-Scr-APT group received intratumoral injection of 1.5 mg / kg of NP-Scr-APT;

[0218] The low-dose group received intratumoral injection of 0.5 mg / kg of NP-IDO-APT;

[0219] The NP-IDO1-APT medium-dose group received intratumoral injection of NP-IDO-APT at a dose of 1.5 mg / kg;

[0220] The high-dose group received intratumoral injection of NP-IDO-APT at a dose of 2.5 mg / kg;

[0221] Each treatment group received the injection once every three days, for a total of three times. Tumor growth and survival in the mice were monitored, and the results were as follows: Figures 21-22 As shown.

[0222] according to Figures 21-22 It can be seen that the tumor-suppressive effect of NP-IDO-APT is concentration-dependent. High-dose NP-IDO-APT (2.5 mg / kg) significantly inhibited tumor growth, while low-dose NP-IDO-APT (0.5 mg / kg) only showed a slight tumor control effect. In addition, high-dose NP-IDO-APT also significantly prolonged the survival time of tumor-bearing mice.

[0223] Example 19

[0224] The inhibitory effect of NP-IDO-APT on CT26 tumors depends on the presence of lymphocytes.

[0225] Given the regulatory role of IDO1 in the adaptive immune system within the tumor microenvironment, it is believed that the tumor-suppressive effect of NP-IDO-APT is mediated by lymphocytes, rather than its own toxicity to tumor cells. To verify this hypothesis, immunodeficient non-obese diabetic (NOD) / severe combined immunodeficiency (SCID) mice were subcutaneously inoculated with CT26 cells and intratumorally injected with the same conditions as in Example 17, using the same doses of NP-IDO-APT, NP-Scr-APT, or NPs. The results are as follows... Figure 23 As shown.

[0226] according to Figure 23 It can be seen that NOD / SCID mice carrying CT26 tumors did not show significant differences in tumor growth among the three different treatment groups.

[0227] Example 20

[0228] The following tests were performed using the same intratumoral injection dose of NP-IDO-APT, NP-Scr-APT, or NPs as in Example 17:

[0229] (1) Measurement of tryptophan (Trp) and kynurenine (Kyn) levels in plasma and tumor tissue

[0230] In vivo IDO1 enzyme activity was indicated by measuring Trp consumption and Kyn production. Trp and Kyn levels in plasma and tumor tissues were determined using LC-MS / MS.

[0231] Forty-eight hours after the last treatment, blood from mice was collected in heparinized tubes and centrifuged at 3000 rpm for 30 minutes at 4°C. 50 μL of the supernatant plasma was mixed with an equal volume of 0.1% formic acid solution, followed by 400 μL of cold methanol. The mixture was then incubated at -20°C for 1 hour. The sample was centrifuged at 12000 rpm for 10 minutes at 4°C, and the supernatant was collected. The Kyn / Trp ratio was determined by mass spectrometry. The results are as follows: Figure 24 As shown.

[0232] 50 mg of tumor tissue homogenate was homogenized and mixed with an equal volume of acetonitrile. The mixture was centrifuged at 12000 rpm for 10 minutes at 4°C. The supernatant was collected, mixed with an equal volume of methanol, and centrifuged again. The supernatant was used for subsequent mass spectrometry detection of the Kyn / Trp ratio. The results are as follows: Figure 24 As shown.

[0233] according to Figure 24 It can be seen that after NP-IDO-APT treatment, the Kyn / Trp ratio in the blood and tumor decreased significantly.

[0234] (2) Flow cytometry analysis of tumor-infiltrating lymphocytes (TILs)

[0235] The lymphocyte population infiltrating tumor tissue was analyzed by flow cytometry. T lymphocytes, tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment were detected. The specific procedures were as follows: Mice were euthanized two days after the third treatment, and tumor tissue was collected. The tumor tissue was cut into small pieces and digested at 37°C with collagenase D and DNase I for 30 minutes. After grinding and filtering through a 200-mesh sieve, tumor-infiltrating lymphocytes were isolated from the tissue digest using Ficoll-Hypaque. For intracellular cytokine analysis, cells were treated with 100 ng / mL PMA, 500 ng / mL iomycin, and a protein transport inhibitor (eBioscience) for 5 hours. After staining with anti-CD45-PE-Cy7, anti-CD4-PerCP, and anti-CD8-FITC, cells were fixed and permeabilized, then stained with IFN-γ-APC or TNF-α-PE. For T lymphocytes... reg Cellular analysis involved staining cells with anti-CD4-PerCP, anti-CD25-PE, and anti-Foxp3-APC. For tumor-associated macrophage analysis, cells were stained with anti-CD45-PEcy7, anti-CD11b-FITC, and anti-F4 / 80-APC. For granulocyte or monocyte MDSCs analysis, cells were stained with anti-CD45-PEcy7, anti-CD11b-FITC, anti-Ly6C-PE, or anti-Ly6G-PerCP. The stained cells were then analyzed by flow cytometry (BD Biosciences), and the results are as follows: Figures 26-27 As shown.

[0236] according to Figures 26-27 It can be seen that tumors treated with NP-IDO-APT have more tumor-associated lymphoid tissue (TILs). NP-IDO-APT treatment also increases CD8+ in TILs. + The proportion of T cells. CD8+ in the tumor after NP-IDO-APT treatment. + T cells showed significantly increased expression of TNF-α and IFN-γ. Less CD4+ was observed in TILs. + CD25 + FoxP3 + Cells, indicating that T cells in tumors treated with NP-IDO-APT reg The proportion of cells decreased. In tumors treated with NP-IDO-APT, the number of TAMs (CD45) decreased. + CD11b + F4 / 80 + (This is a significant reduction.) In tumors treated with NP-IDO-APT, CD45... + CD11b + Ly6G+ The number of defined granulocyte MDSCs (G-MDSCs) was significantly reduced in all treatments. This was due to CD45... + CD11b + Ly6C + The number of defined monocyte MDSCs (M-MDSCs) did not differ significantly. NP-IDO-APT treatment induced a more immune-responsive tumor microenvironment, an effect that may depend on the inhibition of IDO1 activity, leading to tumor suppression and prolonged survival.

[0237] (3) Biosafety evaluation of NP-IDO-APT

[0238] Changes in mouse body weight and vital signs were monitored during the experiment. In addition, changes in blood biochemical indicators (liver and kidney function) were also measured. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured to assess liver function, and blood urea nitrogen (BUN) and creatinine (CREA) levels were measured to assess kidney function. Results are as follows: Figure 29 As shown.

[0239] according to Figure 29 As can be seen, no abnormal signs were observed during the experiment, and the mice were in good health. Their body weight remained stable throughout all administrations. Therefore, the mice tolerated these treatments well. Blood biochemical results showed no significant liver or kidney toxicity. The experimental results indicate that NP-IDO-APT has good biocompatibility.

[0240] As can be seen from the above, the IDO1 nucleic acid aptamer provided by the present invention has a high affinity for IDO1 and can effectively inhibit IDO1 activity, with good safety.

[0241] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An IDO1 nucleic acid aptamer, the nucleotide sequence of which is shown in SEQ ID NO.

4.

2. A nanoparticle based on the IDO1 nucleic acid aptamer of claim 1, comprising a core and a shell; The core comprises a complex of an IDO1 nucleic acid aptamer and a charge neutralizer; the shell comprises precipitates of cations and anions.

3. The nanoparticles according to claim 2, characterized in that, The mass ratio of the IDO1 nucleic acid aptamer to the charge neutralizer is 4~8:

1.

4. The nanoparticles according to claim 2, characterized in that, The charge neutralizer includes protamine sulfate; The cation includes Ca. 2+ Fe 3+ Mg 2+ Mn 2+ Zn 2+ and Ni 2+ One or more of the following; The anions include one or more of phosphate ions, hydrogen phosphate ions, citrate ions, oxalate ions, and carbonate ions.

5. A method for preparing the nanoparticles according to any one of claims 2 to 4, comprising the following steps: The IDO1 nucleic acid aptamer, charge neutralizer, and solution containing anions are mixed to obtain the first mixture. The first mixture was mixed with a solution containing cations, allowed to stand at 37°C for 20-60 minutes, centrifuged, and the precipitate was collected to obtain the nanoparticles.

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