Aptamers, conjugates and compositions and methods of making and using

Abstract

The present disclosure provides an aptamer. The aptamer comprises a continuous nucleotide sequence, and the continuous nucleotide sequence has a sequence shown in formula (1). The present disclosure further provides a complex. The complex comprises a delivery group and a functional group formed by the aptamer of the present disclosure. The aptamer of the present disclosure can specifically target and be internalized in tumor cells, and the complex of the present disclosure can be efficiently targeted and delivered to tumor tissues, thereby effectively diagnosing and / or treating tumors and tumor-related diseases.

Description

[Technical field]

[0001] The present disclosure relates to conjugates and pharmaceutical compositions that include aptamers and delivery groups based on the aptamers. The present disclosure also relates to methods of making and using the aptamers, conjugates and pharmaceutical compositions. [Background technology]

[0002] A tumor refers to a neoplasm formed by the proliferation of local tissue cells under the action of various tumorigenic factors in a living body. A state in which tumor cells metastasize and invade surrounding tissues is called a malignant tumor. When classified according to the tissue cells from which the tumor is generated, it is generally classified into malignant tumors (carcinomas) arising from epithelial cells, malignant tumors (sarcomas) arising from mesenchymal tissue cells, malignant tumors (leukemia, etc.) arising from blood stem cells, and malignant tumors (gliomas) arising from neuroglial cells. Gliomas are the most common intracranial primary malignant tumors, accounting for approximately 40% to 50% of brain tumors, with an annual incidence rate of 3 to 8 cases per 100,000 people worldwide. According to the WHO pathological classification criteria, gliomas belong to neuroepithelial tumors and include multiple pathological types, including but not limited to pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglioma, and anaplastic oligodendroglioma.

[0003] Currently, one of the key problems in the field of diagnosis and treatment of tumors, especially gliomas, is how to specifically deliver diagnostic and / or therapeutic agents into tumor tissues and cells so that these diagnostic and / or therapeutic agents produce their corresponding diagnostic and / or therapeutic effects at the right time and in the right manner.

[0004] Aptamers or nucleic acid aptamers are oligonucleotide molecules that can bind to various target molecules, such as small molecular compounds, proteins, nucleic acids, and even cells, tissues, and organs. Aptamers can provide the important property of "recognizing specific molecules", and are therefore often used in biological technology and treatment, just like antibodies. Aptamers have the excellent properties of being designed in a test tube, being quickly synthesized by chemical methods, being easy to store, and being low or non-immunogenic, and therefore have attracted the attention of researchers in this field in recent years. However, aptamers suitable for tumor targeting delivery are still in need of further development and use in this field. Summary of the Invention [Problem to be solved by the invention]

[0005] The inventors of the present disclosure have unexpectedly discovered an aptamer that can specifically target tumor cells, particularly glioma cells. The aptamer exhibits high specificity for tumor cells, particularly glioma cells, and can thereby be effectively concentrated in tumor cells, particularly glioma cells. Furthermore, by covalently binding the aptamer to various functional groups to form a complex, the resulting complex can effectively deliver diagnostic and / or therapeutic agents suitable for various tumors to tumor cells, particularly glioma tissues and / or cells, and can exhibit excellent diagnostic and / or therapeutic effects. Therefore, the inventors have made the following invention. [Means for solving the problem]

[0006] In one aspect, the disclosure provides an aptamer, comprising a contiguous nucleotide sequence, wherein the groups connecting two adjacent nucleotides are independently a phosphate group or a phosphate group having a modified group, and each nucleotide is selected from one of modified or unmodified A, U, C, or G, and the contiguous nucleotide sequence has a sequence as shown in formula (1): 5'-T1-S1-N a -S2-N b-S3-N c -S4-T2-3' Formula (1) where T1 is a motif consisting of 1 to 3 nucleotides, T2 is a motif consisting of 0 to 15 nucleotides, and T2 does not contain a motif that is completely reverse complementary to T1; S1 and S4 are motifs each consisting of 3 to 7 nucleotides, S1 is the same length as S4 and is completely reverse complementary; N a and N c are motifs consisting of 1 to 4 nucleotides, respectively, and N a Each nucleotide in c is not complementary to any of the nucleotides in a and N c The total number of U's in a and N c accounts for more than 50% of the total number of all nucleotides in S2 and S3 are motifs each consisting of 1 to 4 nucleotides, S2 is the same length as S3 and is a perfect reverse complement; N b is a motif consisting of 3 to 6 nucleotides and N b The nucleotides on either end of the sequence do not form an AU or GC complement.

[0007] In another aspect, the present disclosure further provides a conjugate comprising one or more delivery groups and one or more functional groups, wherein the delivery groups are formed by removing a hydrogen atom or functional group from an aptamer of the present disclosure, and each of the delivery groups is independently attached to the functional group via a covalent bond or via a linking group, and each of the functional groups is independently one of a diagnostic agent group, a small molecule therapeutic agent group having a therapeutic effect against tumors, particularly gliomas, a functional oligonucleotide group having a therapeutic effect against tumors, particularly gliomas, and a delivery aid group.

[0008] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a conjugate according to the present disclosure and a pharma- ceutically acceptable carrier.

[0009] In yet another aspect, the present disclosure further provides the use of the aptamer and / or conjugate and / or pharmaceutical composition according to the present disclosure in the manufacture of a medicament for diagnosing and / or treating tumors and tumor-related diseases or conditions.

[0010] In yet another aspect, the present disclosure further provides a method for diagnosing and / or treating tumors and tumor-related diseases or conditions, comprising administering to a subject in need thereof an effective amount of a conjugate and / or pharmaceutical composition according to the present disclosure.

[0011] In yet another aspect, the present disclosure further provides kits comprising the conjugates and / or pharmaceutical compositions of the present disclosure.

[0012] INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Effect of the Invention

[0013] The aptamers of the present disclosure can be used to specifically deliver functional groups to various tumor cells, particularly glioma tissues and cells.

[0014] The conjugates and pharmaceutical compositions of the present disclosure have an excellent ability to target tumors, particularly glioma tissues and cells, and can significantly improve the accuracy of diagnosis of tumor-related diseases and / or symptoms, and / or significantly treat or alleviate tumor-related diseases and / or symptoms.

[0015] In one embodiment, the complex according to the present disclosure can efficiently and specifically enter tumor cells in vitro. For example, compared with the comparative complex of random sequence, the complex according to the present disclosure exhibits a significantly higher mean fluorescence intensity in U118MG glioma cells, and the R value is basically maintained at 2 or more, which indicates that the complex according to the present disclosure has an excellent ability to enter U118MG glioma cells. In addition, for example, the complex according to the present disclosure has a very strong ability in various cancer cells such as U118MG glioma cells, U251 human glioma cells, A549 human non-small cell lung cancer cells, and MCF-7 human breast cancer cells, and does not basically enter normal cells such as SVGp12 normal astrocytes and 293T human renal epithelial cells. In addition, for example, the complex according to the present disclosure has a very high ability to enter the inside of U118MG and A549 tumor spheres compared with the comparative AP10. In addition, for example, the complex according to the present disclosure of different lengths and sequences can all effectively target U118MG glioma tissues.

[0016] In another embodiment, the aptamer according to the present disclosure can deliver various diagnostic groups, such as different fluorescent groups, to tumor tissue. For example, different complexes containing the fluorescent group according to the present disclosure can all show strong fluorescent signals at the tumor inoculation site in the mouse body, thereby indicating that the aptamer according to the present disclosure can specifically target U118MG glioma, and further, the complex according to the present disclosure still shows strong fluorescent signals 24h to 48h after administration, indicating that it can stably target glioma tissue for a long period of time. In addition, for example, the complex according to the present disclosure can still reach and target brain glioma when administered via the tail vein, suggesting that the aptamer according to the present disclosure has an excellent ability to penetrate the blood-brain barrier (Blood-Brain-Barrier, BBB) and deliver diagnostic groups to brain glioma across the blood-brain barrier. In addition, for example, the complexes according to the present disclosure having various different modifications can stably target U118MG glioma, A549 human non-small cell lung cancer tumor, and PAN02 pancreatic cancer tumor cells, showing a wide range of specific tumor targeting capabilities. In addition, for example, by forming a complex, the aptamer according to the present disclosure can efficiently and specifically deliver different diagnostic groups (e.g., different fluorescent groups Cy3 or Cy5) to U118MG glioma and can stably retain them for a long period of time. In addition, for example, as is clear from the results of observation using a laser confocal microscope, the complexes according to the present disclosure can efficiently and specifically deliver diagnostic groups to the inside of tumor cells of U118MG glioma, showing excellent targeting effects and potential diagnostic capabilities.

[0017] In another aspect, the conjugate of the present disclosure can specifically deliver various small molecule drug groups, such as small molecule toxin groups, to tumor tissues, and can show excellent tumor inhibition effects.For example, the conjugate of the present disclosure can effectively deliver MMAE to tumor tissues, showing tumor targeting ability and reducing the toxicity risk caused by the distribution of MMAE molecules to other tissues, and various administration methods can effectively inhibit the rate of increase in tumor volume and tumor weight, thereby showing that the conjugate of the present disclosure can effectively inhibit tumor growth.In addition, when the dosage of the conjugate is further increased, the tumor volume hardly increases during the test period, showing a better antitumor effect.

[0018] In another embodiment, the complex according to the present disclosure can specifically deliver various functional oligonucleotide groups such as siRNA groups to tumor tissues and can exhibit excellent tumor cell viability suppression effects. For example, the aptamer according to the present disclosure can efficiently target tumor cells and suppress the content of target mRNA after complexing with siRNA, and also has excellent target delivery efficiency and high safety without significantly affecting the corresponding mRNA level in normal cells, and has excellent target delivery efficiency and high safety. Also, for example, various complexes containing siRNA groups of the present disclosure can effectively target and concentrate in U118MG tumor tissues at different time points after administration. Also, for example, the complex of the present disclosure can be freely taken up into PANC1 human pancreatic cancer cells in in vitro experiments and can exhibit a significant effect of inhibiting hSTAT3 mRNA. Also, for example, as is clear from the imaging results in laser confocal imaging and high content imaging systems, the complex containing siRNA groups of the present disclosure can effectively enter U118MG tumor cells, thereby helping to efficiently generate RNAi effects in tumor cells.

[0019] Furthermore, the inventors of the present disclosure have unexpectedly discovered that the conjugates and / or pharmaceutical compositions of the present disclosure can efficiently cross the blood-brain barrier and target gliomas in the brain when administered systemically, thereby further improving the delivery efficiency of functional groups, saving costs, and reducing undesirable side effects. For example, the conjugates of the present disclosure have unexpectedly discovered that when administered subcutaneously, they can cross the blood-brain barrier, effectively target and enter in-situ U118MG gliomas, and significantly inhibit the increase in tumor volume, thereby reducing the volume to 1 / 10 or less of the initial volume, and even to 1 / 100 or less compared to the control group. This shows that the conjugates of the present disclosure can effectively penetrate the blood-brain barrier, efficiently target and enter gliomas, have excellent effects of inhibiting tumor growth, and exhibit good treatment compliance and the ability to efficiently inhibit tumors.

[0020] It can be seen that the aptamer of the present disclosure has excellent ability to target tumors, especially glioma tissues and cells, the conjugate comprising the aptamer of the present disclosure and a diagnostic group (e.g., a fluorescent developer group) can significantly improve the success rate of tumor diagnosis, and the conjugate comprising the aptamer of the present disclosure and a therapeutic group can significantly and effectively inhibit tumor growth or reduce the expression level of cancer-related genes in tumor cells, and has good application prospects. [Brief description of the drawings]

[0021] [Figure 1A-1B] Photographs of high content imaging showing that AP1 and comparative AP10 enter U118MG glioma cells and SVGp12 normal astrocytes, respectively. [Fig. 1C-1G] Photographs of high content imaging showing that AP1 and comparative AP10 enter U251 human glioma cells, A549 human non-small cell lung cancer cells, MCF-7 human breast cancer cells, and 293T human kidney epithelial cells, respectively. [Figure 2A-2B]Photographs of high content imaging showing that AP1 and comparative AP10 enter U118MG glioma tumor spheres and A549 human non-small cell lung carcinoma tumor spheres, respectively. [Figure 3A-3D] The following are photographs of intravital imaging and tumor tissue imaging of U118MG subcutaneous tumor model mice 1 h, 4 h, 24 h, and 48 h after administration of different complexes, respectively. [Figure 4A-4B] These are photographs of fluorescent imaging of mouse brain tissues in which an in-situ tumor model of U118MG was constructed, 24 h and 48 h after administration of different complexes, respectively. [Figure 5A-5E] 13 shows photographs of fluorescence imaging in mice at different time points after administration of a complex according to the present disclosure and a comparative complex having different sequences. [Fig. 5F-5H] Photographs of tumor tissue imaging at different time points. [Figure 6] Photographs of fluorescent images of organs from one mouse in each group 1 h after administration of the complex. [Figure 7] 13 shows photographs of fluorescent imaging of various organ tissues in a mouse in which a PAN02 subcutaneous tumor model was constructed after administration of a conjugate according to the present disclosure. [Figure 8A-8E] 13 shows photographs of organ fluorescence images of mice 48 h or 96 h after administration of a conjugate according to the present disclosure. [Figure 9] 13 shows photographs of tumor fluorescence images of mice in each group on D9 after administration of the complex. [Figure 10] 1 is a line graph showing the change in tumor volume over time in each group of mice after administration of a conjugate according to the present disclosure or a control compound. [Figure 11A-11B] FIG. 2 shows the effect of different concentrations of complex AP1 and complex 19, respectively, on cell viability of U118-MG glioma cells and SVGp12 human astrocytes. [Figure 12] 13 is a photograph showing the results of fluorescence imaging in a mouse body after administration of a conjugate according to the present disclosure having different binding groups. [Figures 13A-13C]13 is a set of photographs showing the results of fluorescent imaging in mice 1 h, 24 h, and 48 h after administration of different complexes. [Figure 13D] Photographs of fluorescent signal imaging of tumor tissues and kidneys of mice in each group after the mice were sacrificed on D5. [Figures 14A-14C] 13 is a set of photographs showing the results of fluorescent imaging in mice 1 h, 24 h, and 48 h after administration of different complexes. [Figure 14D] Photographs of fluorescent signal imaging of tumor tissues and kidneys of mice in each group after the mice were sacrificed on D6. [Figure 15] 13 is a photograph showing the results of laser confocal imaging within U118MG glioma 24 hours after administration of different complexes. [Figure 16A-16B] 13 is a set of photographs showing the results of fluorescent imaging in mice after administration of different complexes. [Figure 17] 1 is a histogram showing the relative inhibition rate of hSTAT3 mRNA in PANC-1 human pancreatic cancer cells by conjugates of the present disclosure. [Figure 18] 1 is a photograph showing the results of laser confocal imaging of the complex of the present disclosure in U118MG glioma cells. [Figure 19] 1 is a histogram showing relative fluorescence intensity results of conjugates of the present disclosure in U118MG glioma. [Figure 20] 1 is a line graph showing the change in tumor fluorescence intensity over time in a U118MG in situ tumor model mouse after administration of a conjugate according to the present disclosure or a control compound. [Figure 21] 1 is a line graph showing the change in tumor volume over time in U118MG subcutaneous tumor model mice after administration of a conjugate according to the present disclosure or a control compound. [Figure 22] 1 is a line graph showing the change in tumor volume over time in U118MG subcutaneous tumor model mice after administration of different concentrations of a conjugate according to the present disclosure or a control compound. [Figure 23] 1 is a line graph showing the change in tumor volume over time in U118MG subcutaneous tumor model mice after administration of different concentrations of a conjugate according to the present disclosure or a control compound. [Figure 24] 1 is a line graph showing the change in tumor volume over time in A549 subcutaneous tumor model mice after administration of different concentrations of a conjugate according to the present disclosure or a control compound. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Hereinafter, specific embodiments of the present disclosure will be described in detail. It should be understood that the specific embodiments described herein are merely for the purpose of explaining and interpreting the present disclosure, and are not intended to limit the present disclosure.

[0023] definition In this disclosure, unless otherwise stated, A, U, C, G and T refer to adenine nucleotide, uracil nucleotide, cytosine nucleotide, guanine nucleotide and thymine nucleotide, respectively, and 2-methylcytosine nucleotide refers to a nucleotide in which the hydrogen at the 2' position on the cytosine base in the cytosine nucleotide is replaced with a methyl group. The structure of these nucleotides is well known to those skilled in the art. As used in this disclosure, a "nucleic acid motif" or "motif" is a nucleic acid sequence fragment in an aptamer, and is composed of one or more nucleotides. In some embodiments, the motif is a nucleic acid sequence fragment having a biological function.

[0024] As used in this disclosure, "alkyl group" refers to straight and branched chain saturated hydrocarbon groups having a specified number of carbon atoms, generally 1 to 20 carbon atoms, e.g., 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms. For example, C1-C6 alkyl group refers to straight and branched chain alkyl groups containing 1 to 6 carbon atoms. When referring to an alkyl residue having a specific number of carbons, it is intended to include all branched and straight chain forms having that number of carbons. Thus, for example, "butyl group" is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl groups, and "propyl" includes n-propyl and isopropyl groups. Alkylene groups are a subset of alkyl groups and refer to residues similar to alkyl groups but having two points of attachment.

[0025] As used herein, an "alkenyl group" refers to an unsaturated branched or straight chain alkyl group having one or more carbon-carbon double bonds obtained by removing one hydrogen from adjacent carbon atoms of a parent alkyl group. The group may be in the cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to, vinyl groups, propenyl groups such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl group), prop-2-en-2-yl, and propenyl groups such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, and buta-1,3-dien-2-yl. In some embodiments, alkenyl groups have from 2 to 20 carbon atoms, and in other embodiments, from 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenylene is a subset of the alkenyl group, referring to residues similar to the alkenyl group, but that have two points of attachment.

[0026] As used herein, an "alkynyl group" refers to an unsaturated branched or straight chain alkyl group having one or more carbon-carbon triple bonds obtained by removing two hydrogen atoms from adjacent carbon atoms of a parent alkyl group. Typical alkynyl groups include, but are not limited to, ethynyl groups, propynyl groups such as prop-1-yn-1-yl, prop-2-yn-1-yl, and butynyl groups such as but-1-yn-1-yl, but-1-yn-3-yl, and but-3-yn-1-yl. In some embodiments, alkynyl groups have 2-20 carbon atoms, while in other embodiments, 2-10, 2-8, or 2-6 carbon atoms. An alkynylene group is a subset of the alkynyl group and refers to a residue that is the same as the alkynyl group but has two points of attachment.

[0027] As used herein, a "heterocyclic group" refers to a stable 3- to 18-membered non-aromatic cyclic group containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise stated in the specification, a heterocyclic group is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, and may include fused or bridged ring systems. The heteroatoms in a heterocyclic group may be oxidized heteroatoms. One or more nitrogen atoms, if present, may be quaternized nitrogen atoms. A heterocyclic group is partially saturated or fully saturated. A heterocyclic group may be attached to the remainder of the molecule through any ring atom. Examples of such heterocyclic groups include dioxanyl, thiophenyl[1,3]disulfonyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindole, octahydroisoindole, 2-oxapiperazinyl, 2-oxapiperidyl, 2-oxapyrrolidinyl, oxazolidinyl, piperidyl, piperazinyl, and 4-piperidonyl. Examples of heterocyclic groups include, but are not limited to, pyrrolidinyl, pyrazolidyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Heterocycle is a subset of heterocyclic and refers to the same residues but with two points of attachment.

[0028] As used herein, an "aryl group" refers to a group formed by removing a hydrogen atom from a ring carbon atom derived from an aromatic monocyclic or polycyclic hydrocarbon ring system. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon from 6 to 18 carbon atoms, and one or more rings in the ring system are fully unsaturated, i.e., contain a cyclic, delocalized (4n+2) π-electron system according to the Hückel theory. Aryl groups include, but are not limited to, groups such as phenyl, fluorenyl, and naphthyl. An arylene group is a subset of the aryl group and refers to a residue that is the same as the aryl group but has two points of attachment.

[0029] "Heteroaryl group" refers to a group derived from a 3-18 membered aromatic ring radical containing 2-17 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen and sulfur. As used in this disclosure, a heteroaryl group may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system in which one or more rings are fully unsaturated, i.e., contain a cyclic, delocalized (4n+2) π-electron system according to the Hückel theory. Heteroaryl groups include fused or bridged ring systems. Heteroatoms in a heteroaryl group may be oxidized heteroatoms. One or more nitrogen atoms (if present) may be quaternized nitrogen atoms. A heteroaryl group is bonded to the remainder of the molecule through any ring atom. Examples of heteroaryl groups include azepinyl, acridinyl, benzimidazolyl, benzoindole, 1,3-benzodioxazolyl, benzofuryl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyronyl, benzofuryl, and benzofuranonyl. group, benzothiophenyl group, benzothieno[3,2-d]pyrimidinyl group, benzotriazolyl group, benzo[4,6]imidazo[1,2-a]pyridyl group, carbazolyl group, cinnolinyl group, cyclopenta[d]pyrimidinyl group, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl group, 5,6-dihydrobenzo[h]quinazolinyl group, 5,6-dihydrobenzo[h]cinnolinyl group, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl group, dibenzofuryl group, dibenzothiophenyl group, furyl group, furanonyl group, furo[3,2-c]pyridyl group, 5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl group, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl group, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridyl group, isothiazolyl group, imidazolyl group, indazolyl group, indole group, isoindole group, indolinyl group, isoindolinyl group, isoquinolyl group, indolizinyl group, isoxazolyl group, 5,8-methano-5,6 ,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[H]quinazolinyl, 1-phenyl-1H-pi aryl group, phenazinyl group, phenothiazinyl group, phenoxazinyl group, phthalazinyl group, pteridinyl group, purinyl group, pyrrolyl group, pyrazolyl group, pyrazolo[3,4-d]pyrimidinyl group, pyridyl group, pyrido[3,2-d]pyrimidinyl group, pyrido[3,4-d]pyrimidinyl group, pyrazinyl group, pyrimidinyl group, pyridazinyl group, pyrrolyl group, quinazolinyl group, quinoxalinyl group, quinolyl group, tetrahydroquinolyl group, 5,6,7 ,8-tetrahydroquinazolinyl group, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl group, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl group, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl group, thiazolyl group, thiadiazolyl group, triazolyl group, tetrazolyl group, triazinyl group, thieno[2,3-d]pyrimidinyl group, thieno[3,2-d]pyrimidinyl group, thieno[2,3-c]pyridinyl group (thieno[2,Heteroarylene is a subset of the heteroaryl group and refers to residues similar to the heteroaryl group but that have two points of attachment.

[0030] Aptamers according to the present disclosure In one aspect, the disclosure provides an aptamer, comprising a contiguous nucleotide sequence, wherein the groups connecting two adjacent nucleotides are independently a phosphate group or a phosphate group having a modified group, and each nucleotide is selected from one of modified or unmodified A, U, C, or G, and the contiguous nucleotide sequence has a sequence as shown in formula (1): 5'-T1-S1-N a -S2-N b -S3-N c -S4-T2-3' Formula (1) Here, T1 is a motif consisting of 1-3 nucleotides. The present inventors have found that the presence of T1 helps the aptamer according to the present disclosure to exhibit high tumor targeting effect. In some embodiments, T1 consists of two nucleotides, in which case the aptamer according to the present disclosure has better tumor targeting ability. In some embodiments, T1 consists of two nucleotides and includes at least one C. In some embodiments, from 5' to 3', T1 is CU, UC, or AC.

[0031] T2 is a motif consisting of 0-15 nucleotides. The present inventors have found that T2 having these numbers of nucleotides and various nucleotide sequences does not significantly affect the tumor targeting ability of the aptamer. In some embodiments, T2 consists of 0-10 nucleotides. In some embodiments, T2 consists of 1-9 nucleotides starting with U from 5' to 3', in which case the aptamer may have better stability.

[0032] T2 does not contain a motif that is completely reverse complementary to T1. In the context of this disclosure, "reverse complementary" refers to a pair of nucleotide sequences or motifs that can form hydrogen bonds according to the rules of nucleic acid base pairing, and each nucleotide from 5' to 3' of one nucleotide sequence or motif can form base pairs with each nucleotide from 3' to 5' of the other nucleotide sequence or motif. In some embodiments, "reverse complementary" includes one or more of the complements of AU, GC, and UG.

[0033] In the aptamer according to the present disclosure, S1 and S4 are motifs consisting of 3 to 7 nucleotides, respectively, and S1 is the same length as S4, and is completely reverse-complementary. The aptamer having the above S1 and S4 motifs has excellent stability and can target tumor tissues and cells for a long time. In some embodiments, S1 and S4 are each consisting of 3 to 5 nucleotides, respectively, and are the same length. In some embodiments, in the reverse complement formed by S1 and S4, GC complementation accounts for 40% or more of all complementation numbers, in which case the aptamer according to the present disclosure has even better stability and tumor targeting ability. In some embodiments, from 5' to 3', S1 is GCU and S4 is AGC, or S1 is GAGU and S4 is GCUC, or S1 is GGAGU and S4 is GCUCU, or S1 is UAUGG and S4 is CCAUG.

[0034] In the aptamer according to the present disclosure, N a and N c are motifs consisting of 1 to 4 nucleotides, respectively, and N a Each nucleotide in c is not complementary to any of the nucleotides in a and N c The total number of U's in a and N c It accounts for more than 50% of the total number of all nucleotides in the a and Nc Aptamers bearing the N motif have shown excellent tumor tissue targeting ability. a and / or N c If the motif is missing or N a and N c In some embodiments, when the total number of U in the N a and N c The sum of the numbers of nucleotides in N a and N c The sum of the number of nucleotides in N is 3 or 4, and a and N c The sum of the number of U's in is 2 or 3. a and / or N c is U, UU, UC, or CU.

[0035] In the aptamer according to the present disclosure, S2 and S3 are motifs consisting of 1-4 nucleotides, respectively, S2 is the same length as S3, and is completely reverse-complementary. By including S2 and S3 motifs, the aptamer according to the present disclosure exhibits good stability and excellent tumor targeting ability. In some embodiments, S2 and S3 are each consisting of 2-3 nucleotides, respectively, and are the same length. In some embodiments, the reverse complement formed by S2 and S3 includes at least one GC complement, in which case the reverse complement has better stability. In some embodiments, from 5' to 3', S2 is CA and S3 is UG, or S2 is AC and S3 is GU, or S2 is GCC and S3 is GGU.

[0036] N b is a motif consisting of 3 to 6 nucleotides, N b The nucleotides between the two ends of the N are not AU or GC complementary. bAptamers having the motif can retain a specific spatial arrangement, which allows the aptamers of the present disclosure to stably and efficiently target tumor tissues and cells. b In some embodiments, from 5' to 3', N b is GACG, GACGU, GACCG, UACU, GUUG, or GAUCU.

[0037] The inventors of the present disclosure have unexpectedly discovered that the aptamer according to the present disclosure having the sequence shown in the above formula (1) can effectively target tumors, particularly glioma tissues, and thus the aptamer according to the present disclosure can be used as a delivery carrier to deliver functional groups having diagnostic / therapeutic effects on tumors to tumors. Furthermore, the aptamer according to the present disclosure can specifically enter tumor cells, thereby enabling more effective delivery of diagnostic / therapeutic groups from the cellular level, and even the genetic level.

[0038] In some embodiments, in the aptamer according to the present disclosure, the length of the contiguous nucleotide sequence is 18 to 50 nucleotides, or 20 to 40 nucleotides, or 21 to 36 nucleotides, or 24 to 32 nucleotides. Aptamers having these contiguous nucleotide sequence lengths can more easily target tumors, and have a good balance between synthesis cost and targeting effect.

[0039] In some embodiments, in the aptamer of the present disclosure, the contiguous nucleotide sequence has the sequence shown in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3: 5'-CUGCUUCAGACGUGUUAGCUU-3' (SEQ ID NO: 1) where, from 5' to 3', T1 is CU, S1 is GCU, and N a is U, S2 is CA, and N b is GACG, S3 is UG, and N cis UU, S4 is AGC, T2 is UU, 5'-CUGAGUUCAGACGUGUUGCUCU-3' (SEQ ID NO: 2) where, from 5' to 3', T1 is CU, S1 is GAGU, and N a is U, S2 is CA, and N b is GACG, S3 is UG, and N c is UU, S4 is GCUC, T2 is U, 5'-UCUAUGGCUGCCGAUCUGGUCUCCAUGUACGU-3' (SEQ ID NO: 3) where, from 5' to 3', T1 is CU, S1 is GAGU, and N a is U, S2 is CA, and N b is GACG, S3 is UG, and N c is UU, S4 is GCUC, and T2 is U.

[0040] In some embodiments, the contiguous nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO:4, 5'-N6GGAGUUCAN1N2N3N4UGN5GCUCN7-3' (SEQ ID NO: 4) Here, N1, N2 and N3 are each independently one of A, U, C and G, N4 is U, C or G, or a motif consisting of two of U, C or G, N5 is U, CU or UU, N6 is CU, UC or AC, N7 is U, UU or UUN8, and N8 is a motif consisting of 1 to 15 nucleotides.

[0041] In the above nucleotide sequence, from 5' to 3', T1 is a motif represented by N6, S1 is a motif represented by GGAGU, and N a is U, S2 is CA, and N b is the motif N1N2N3N4 consisting of N1, N2, N3 and N4, S3 is UG, and N cis the motif represented by N5, S4 is a motif consisting of GCUC and the first nucleotide in N7, and T2 is a motif consisting of the other nucleotides in N7.

[0042] An aptamer comprising the nucleotide sequence shown in SEQ ID NO:4 can more effectively target tumors, particularly gliomas, and be concentrated in tumor tissue.

[0043] Experimental verification shows that in the nucleotide sequence shown in SEQ ID NO: 4, the above selection of N1, N2, N3 and N4 does not significantly affect the tumor targeting ability of the aptamer of the present disclosure. In some embodiments, the motif N1N2N3N4 consisting of N1, N2, N3 and N4 is one of GACG, GACGU, GACCG, UACU, GUUG or GAUCU, and the aptamer containing these motifs has a higher tumor-specific targeting effect.

[0044] In some embodiments, in the nucleotide sequence shown in SEQ ID NO: 4, N5 is U or UU. In this case, the aptamer according to the present disclosure has excellent targeting effect on tumors.

[0045] In some embodiments, the contiguous nucleotide sequence has a nucleotide sequence shown in any one of SEQ ID NOs:5-11. 5'-CUGGAGUUCAGACGUGUUGCUCUU-3' (SEQ ID NO:5) 5'-CUGGAGUUCAGACGUUGUGCUCUU-3' (SEQ ID NO:6) 5'-CUGGAGUUCAGACCGUGUGCUCUU-3' (SEQ ID NO:7) 5'-CUGGAGUUCAGACGUGUUGCUCU-3' (SEQ ID NO:8) 5'-ACGGAGUUCAGACGUGUUGCUCUU-3' (SEQ ID NO: 9) 5'-CUGGAGUUCACUACUGUUGCUCUU-3' (SEQ ID NO: 10) 5'-CUGGAGUUCAGUUGUGUUGCUCUU-3' (SEQ ID NO:11)

[0046] The aptamer according to the present disclosure having the above nucleotide sequence exhibits high targeting effect against tumors.

[0047] In some embodiments, motif N8 consists of 1 to 15 nucleotides. In some embodiments, N8 consists of 1 to 8 nucleotides.

[0048] In some embodiments, the presence of motif N8 makes the aptamer of the present disclosure more stable against exonucleases in the body, thereby enabling it to exert a tumor targeting effect in the body for a longer period of time. In some embodiments, N8 can increase or maintain the tumor targeting effect of the aptamer of the present disclosure. Considering the balance between stability, targeting, and synthesis efficiency, in some embodiments, motif N8 consists of 8 nucleotides. In some embodiments, the nucleotide sequence of motif N8 from 5' to 3' is CCGAUCUC. In some embodiments, the continuous nucleotide sequence has a nucleotide sequence shown in any one of SEQ ID NOs: 12 to 14. 5'-CUGGAGUUCAGACGUGUUGCUCUUCCGAUCUC-3' (SEQ ID NO: 12) 5'-CUGGAGUUCAGACGUUGUGCUCUUCCGAUCUC-3' (SEQ ID NO: 13) 5'-CUGGAGUUCAGACCGUGUGCUCUUCCGAUCUC-3' (SEQ ID NO: 14)

[0049] In the aptamers of the present disclosure, the terminal groups of the ribose 5'-end of the 5'-terminal nucleotide and the ribose 3'-end of the 3'-terminal nucleotide are independently a hydroxyl group or a phosphate group, and the selection of these terminal groups does not change the targeting ability of the aptamers of the present disclosure. In some embodiments, in the aptamers of the present disclosure, the terminal groups of the ribose 5'-end of the 5'-terminal nucleotide and the ribose 3'-end of the 3'-terminal nucleotide are both hydroxyl groups.

[0050] In the aptamer of the present disclosure, each nucleotide may be modified or unmodified. In general, modification of nucleotide may change the stability and / or tumor targeting ability of the aptamer of the present disclosure. In some embodiments, at least one nucleotide in the aptamer of the present disclosure is a modified nucleotide. In some embodiments, at least one group that connects two adjacent nucleotides in the aptamer of the present disclosure is a phosphate group that has a modified group.

[0051] Modifications of the nucleotide include, but are not limited to, modifications to the sugar, modifications to the base, and / or substitution of the nucleotide with a nucleotide analog. In some embodiments, in the aptamers of the present disclosure, each of the modified nucleotides is independently one of a 2'-halogen modified nucleotide, a 2'-alkoxy modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-substituted alkyl modified nucleotide, a 2'-amino modified nucleotide, a 2'-substituted amino modified nucleotide, a 2'-deoxyribonucleotide, a base modified nucleotide, and a nucleotide analog.

[0052] In the context of this disclosure, a "fluoro-modified nucleotide" refers to a nucleotide having the structure shown in formula (7) below, in which the hydroxyl at the 2' position of the ribose group of the nucleotide is replaced with fluorine. A "non-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl at the 2' position of the ribose group of the nucleotide is replaced with a non-fluorine group. In some embodiments, each non-fluoro-modified nucleotide is an independently selected one from among nucleotides or nucleotide analogs in which the hydroxyl at the 2' position of the ribose group of the nucleotide is replaced with a non-fluorine group.

[0053] These nucleotides in which the 2'-hydroxyl of ribose is substituted with a non-fluorine group are well known to those skilled in the art, and these nucleotides may be one selected from 2'-alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, and 2'-deoxynucleotides.

[0054] In some embodiments, the 2'-alkoxy modified nucleotide is a methoxy modified nucleotide (2'-OMe) as shown in formula (8). In some embodiments, the 2'-amino modified nucleotide (2'-NH2) is as shown in formula (9). In some embodiments, the 2'-deoxyribonucleotide (DNA) is as shown in formula (10).

[0055] [ka]

[0056] Those skilled in the art are aware of various ways to modify the base of a nucleotide. In some embodiments, the base modification includes, but is not limited to, adding one or more methyl groups to the base. In some embodiments, thymine (T) is considered a type of base modification uracil (U). In some embodiments, 2-methylcytosine is considered a type of base modification cytosine (C).

[0057] A nucleotide analog refers to a group that can substitute for a nucleotide in a nucleic acid, but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. In some embodiments, a nucleotide analog can be an isonucleotide, a bridged nucleotide (abbreviated as BNA), or an acyclic nucleotide.

[0058] BNA refers to a constrained or inaccessible nucleotide. BNAs may include five-, six-, or seven-membered bridged structures with a "fixed" C3'-endo sugar puckering. Typically, the bridge is introduced at the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide. In some embodiments, the BNA may be an LNA as shown in formula (12), an ENA as shown in formula (13), a cET BNA as shown in formula (14), or the like.

[0059] [ka]

[0060] An acyclic nucleotide is a nucleotide in which the sugar ring of the nucleotide is opened. In some embodiments, the acyclic nucleotide may be an unlocked nucleic acid (UNA) as shown in formula (15), a glycerol nucleic acid (GNA) as shown in formula (16), or a peptide nucleic acid (PNA).

[0061] [ka]

[0062] In the above formula (15) and formula (16), R is selected from H, OH, or alkoxy (O-alkyl group).

[0063] Peptide nucleic acids are nucleotide analogs formed from a polypeptide backbone substituted glycoside-phosphate backbone. In some embodiments, peptide nucleic acids may be nucleotide analogs formed from, for example, 2-aminoethylglycine-linked substituted glycoside-phosphate units.

[0064] An isonucleotide refers to a compound in which the position of the base in the ribose ring in a nucleotide is changed. In some embodiments, an isonucleotide may be a compound in which the base is shifted from the 1'-position to the 2'-position or 3'-position of the ribose ring, as shown in formula (17) or (18).

[0065] In the compounds of formula (17) and formula (18) above, Base represents a nucleic acid base such as A, U, G, C or T, and R is selected from H, OH, F or the non-fluorine groups described above.

[0066] [ka]

[0067] In some embodiments, the nucleotide analog is one selected from an isonucleotide, an LNA, an ENA, a cET, an UNA, and a GNA.

[0068] In some embodiments, each non-fluoro modified nucleotide is a methoxy modified nucleotide, which in context refers to a nucleotide in which the 2'-hydroxy group of the ribose is replaced with methoxy.

[0069] In this context, "fluoro-modified nucleotide", "2'-fluoro-modified nucleotide", "nucleotide in which the 2'-hydroxy group of the ribose group is replaced with fluorine" and "nucleotide having 2'-fluoro ribose" have the same meaning and all refer to a compound having the structure shown in formula (7) in which the 2'-hydroxy group of the nucleotide is replaced with fluorine, and "methoxy-modified nucleotide", "2'-methoxy-modified nucleotide", "nucleotide in which the 2'-hydroxy group of the ribose group is replaced with methoxy" and "nucleotide having 2'-methoxy ribose" have the same meaning and all refer to a compound having the structure shown in formula (8) in which the 2'-hydroxy group of the ribose group of the nucleotide is replaced with methoxy.

[0070] In some embodiments, each cytosine nucleotide in the contiguous nucleotide sequence of the aptamer of the present disclosure is a fluoro-modified cytosine nucleotide and / or each uracil nucleotide in the contiguous nucleotide sequence is a fluoro-modified uracil nucleotide. In some embodiments, each nucleotide in the contiguous nucleotide sequence of the aptamer of the present disclosure is a 2'-methoxy modified nucleotide. In some embodiments, one or more uracil nucleotides in the aptamer of the present disclosure have a modified base.

[0071] The group that connects two adjacent nucleotides may be a phosphate group or a modified phosphate group. The modification of the phosphate group may, for example, replace at least one non-bridging oxygen atom in the phosphate group with a sulfur atom to form a thiophosphate group or a dithiophosphate group. In some embodiments, at least one group that connects two adjacent nucleotides in the aptamer according to the present disclosure is a thiophosphate group. In some embodiments, at least one of the three groups that connect two adjacent nucleotides between the first four nucleotides at the 5' end in the aptamer according to the present disclosure is a thiophosphate group. In some embodiments, at least two of the three groups that connect two adjacent nucleotides between the first four nucleotides at the 5' end in the aptamer according to the present disclosure are thiophosphate groups. In some embodiments, at least one of the groups that connect two adjacent nucleotides between the first four nucleotides at the 3' end in the aptamer according to the present disclosure is a thiophosphate group. In some embodiments, at least two of the three groups linking two adjacent nucleotides between the first four nucleotides at the 5' end of the aptamer disclosed herein are phosphorothioate groups. In some embodiments, each group linking two adjacent nucleotides of the aptamer disclosed herein is a phosphorothioate group.

[0072] The aptamer with the above modification is not only low in cost, but also makes the aptamer less susceptible to cleavage by ribonucleases in the body, thereby increasing the stability of the aptamer and making it more resistant to nuclease hydrolysis.The modified aptamer also has high activity of targeting tumor tissues and / or cells.

[0073] In some embodiments, the contiguous nucleotide sequence has a nucleotide sequence set forth in one of SEQ ID NOs: 15-39. 5'-CfUfGGAGUfUfCfAGACfGUfGUfUfGCfUfCfUfUfCfCfGAUfCfUfCf-3' (SEQ ID NO: 15) 5'-CfUfGGAGUfUfCfAGACfGUfGUfUfGCfUfCfUfUf-3' (SEQ ID NO: 16) 5'-CfUfGGAGUfUfCfAGACfGUfUfGUfGCfUfCfUfUfCfCfGAUfCfUfCf-3' (SEQ ID NO: 17) 5'-CfUfGGAGUfUfCfAGACfGUfUfGUfGCfUfCfUfUf-3' (SEQ ID NO: 18) 5'-CfUfGGAGUfUfCfAGACfCfGUfGUfGCfUfCfUfUfCfCfGAUfCfUfCf-3' (SEQ ID NO: 19) 5'-CfUfGGAGUfUfCfAGACfCfGUfGUfGCfUfCfUfUf-3' (SEQ ID NO: 20) 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmUmCmCmGmAmUmCmUmCm-3' (SEQ ID NO: 21) 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUmUm-3' (SEQ ID NO: 22) 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmUmGmUmCmUmCmUmCmGmAmUmCmUmCm-3' (SEQ ID NO: 23) 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmUmGmUmGmCmUmCmUmUm-3' (SEQ ID NO: 24) 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmCmGmUmGmUmCmUmCmUmCmGmAmUmCmUmCm-3' (SEQ ID NO: 25) 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmCmGmUmGmUmGmCmUmCmUmUm-3' (SEQ ID NO: 26) 5'-CmUmGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUm-3' (SEQ ID NO: 27) 5'-CmUmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUm-3' (SEQ ID NO: 28) 5'-CmUmGmCmUmUmCmAmGmAmCmGmUmGmUmUmAmGmCmUmUm-3' (SEQ ID NO: 29) 5'-AmCmGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUmUm-3' (SEQ ID NO: 30) 5'-CmUmGmGmAmGmUmUmCmAmCmUmAmCmUmGmUmUmGmCmUmCmUmUm-3' (SEQ ID NO: 31) 5'-CmUmGmGmAmGmUmUmCmAmGmUmUmGmUmUmGmUmUmGmCmUmCmUmUm-3' (SEQ ID NO: 32) 5'-UmCmUmAmUmGmGmCmUmGmCmCmGmAmUmCmUmGmGmUmCmCmCmAmUmGmUmAmCmGmUm-3' (SEQ ID NO: 33) 5'-CmsUmsGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmUmCmCmGmAmUmCmsUmsCm-3' (SEQ ID NO: 34) 5'-CmsUmsGmGmAmGmUmUmCmAmGmAmCmGmUmGmUmUmGmCmUmCmsUmsUm-3' (SEQ ID NO: 35) 5'-CmsUmsGmGmAmGmUmUmCmAmGmAmCmGmUmUmGmUmCmUmCmUmCmGmAmUmCmsUmsCm-3' (SEQ ID NO: 36) 5'-CmsUmsGmGmAmGmUmUmCmAmGmAmCmGmUmUmGmUmGmCmUmCmsUmsUm-3' (SEQ ID NO: 37) 5'-CmsUmsGmGmAmGmUmUmCmAmGmAmCmCmGmUmGmUmCmUmCmUmCmCmGmAmUmCmsUmsCm-3' (SEQ ID NO: 38) 5'-CmsUmsGmGmAmGmUmUmCmAmGmAmCmCmGmUmGmUmGmCmUmCmsUmsUm-3' (SEQ ID NO: 39) Here, the capital letters C, G, U, and A represent the base composition of the nucleotide, the lower case letter m represents that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide, the lower case letter f represents that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide, and the lower case letter s represents that the two nucleotides on the left and right of the letter s are bonded by a thiophosphate group.

[0074] The aptamer according to the present disclosure can be obtained by a method for producing oligonucleotides (e.g., solid-phase synthesis and liquid-phase synthesis) that is common in the art. Solid-phase synthesis is already available as a commercial customization service. Modified nucleotides can be introduced into the aptamer according to the present disclosure by using nucleoside monomers having corresponding modifications, and the methods for producing nucleoside monomers having corresponding modifications and the methods for introducing modified nucleotides into aptamers are also well known to those skilled in the art. All modified nucleoside monomers may be purchased commercially or may be produced by known methods.

[0075] Complex In another aspect, the present disclosure further provides a conjugate, the conjugate comprising one or more delivery groups and one or more functional groups, the delivery groups being formed by removing one hydrogen atom or functional group from the aptamer of the present disclosure, each of the delivery groups being independently linked to the functional group via a covalent bond or via a linking group, each of the functional groups being independently one of a diagnostic agent group, a small molecule therapeutic agent group having a therapeutic effect on tumors, a functional oligonucleotide group having a therapeutic effect on tumors, and a delivery aid group. By linking the functional groups via a covalent bond or a linking group to form a conjugate, the aptamer of the present disclosure can deliver the functional group to the tumor.

[0076] The delivery group is formed by removing one or more hydrogen atoms or functional groups from an aptamer according to the present disclosure. In some embodiments, the ribose 5' group of the 5'-terminal nucleotide and the ribose 3' group of the 3'-terminal nucleotide of an aptamer according to the present disclosure are both hydroxyl groups, and the delivery group is formed by removing one hydrogen atom from the 5'-hydroxyl group of the 5'-terminal nucleotide of an aptamer according to the present disclosure. In some embodiments, the delivery group is formed by removing one hydrogen atom from the 3'-hydroxyl group of the 3'-terminal nucleotide of an aptamer according to the present disclosure. In some embodiments, the delivery group is formed by removing the 5'-hydroxyl group from the 5'-terminal nucleotide of an aptamer according to the present disclosure. In some embodiments, the delivery group is formed by removing the 3'-hydroxyl group from the 3'-terminal nucleotide of an aptamer according to the present disclosure. In some embodiments, the delivery group is formed by removing the ribose 2'-hydroxyl group from a nucleotide included in an aptamer according to the present disclosure.

[0077] In some embodiments, the complex of the present disclosure has a structure shown in formula (101):

[0078] [ka] (101) In the formula, each R AP The groups are independently a group having the structure shown in formula (102).

[0079] [ka] (102) wherein each AP group is the same or different and independently represents one of the delivery groups; each A0 group is the same or different and independently represents one of the functional groups; R j , each R k Or each R i are the same or different and each independently represent a covalent bond or a linking group, and R i and Rk are not both a covalent bond at the same time; m0 is an integer of 1 to 6; n0 is an integer of 1 to 6; and each n1 independently represents an integer of 0 to 4; [ka] represents the site at which the group is covalently attached.

[0080] In some embodiments, m0 is an integer of 1 to 6, i.e., the conjugate represented by formula (101) contains 1 to 6 functional groups A0. In some embodiments, m0 is an integer of 1 to 4, i.e., the conjugate represented by formula (101) contains 1 to 4 functional groups A0, from the viewpoint of delivery efficiency and cost. In some embodiments, m0 is 1, i.e., the conjugate represented by formula (101) contains one functional group A0.

[0081] In some embodiments, n0 is an integer from 1 to 6, i.e., the conjugate of formula (101) comprises 1 to 6 R AP From the viewpoint of delivery efficiency and cost, in some embodiments, n0 is an integer of 1 to 3, i.e., the conjugate represented by formula (101) contains 1 to 3 R AP In some embodiments, n0 is 1, i.e., the conjugate of formula (101) contains one R AP Contains a group.

[0082] In some embodiments, each n1 independently represents an integer from 0 to 4, and R i and R k are not both covalent bonds at the same time, so that each R AP In some embodiments, each n1 independently represents an integer from 0 to 1, whereby each R APThe group contains 1 to 2 delivery groups AP. In some embodiments, n0 is 1 and n1 is 0, in which case the conjugate shown in formula (101) contains one delivery group AP.

[0083] R AP In the group, R k and R i The role of the delivery group AP is to j Covalently bonded to the R j The purpose of the present invention is to bind the functional group A0 via a group. Therefore, any R group that can achieve the above-mentioned binding and does not adversely affect the effectiveness of the delivery group AP and the functional group A0 can be used. k or R i In some embodiments, any of the R k or each of the above R i are independently a straight chain alkylene group having a length of 1 to 70 carbon atoms, or one or more of the carbon atoms in the straight chain alkylene group is selected from the group consisting of C(O), NH, O, S, CH=N, S(O)2, OP(O)2, OP(O)(S), a C5-C8 glycosidic group, a C2-C 10 Alkenylene group, C2-C 10 Alkynylene group, C6-C 10 Arylene group, C3-C 18 Heterocyclylene groups and C5-C 10 heteroarylene groups, and the linear alkylene group is substituted with one or more selected from the group consisting of C1 to C 10 Alkyl groups, C6-C 10 Aryl groups, C5-C 10 Heteroaryl groups, C1-C 10 Halogenated alkyl groups, -OC1~C 10 Alkyl group, -OC1~C 10 Alkylphenyl group, -C1~C 10 Alkyl-OH, -OC1~C 10 Halogenated alkyl groups, -SC1~C 10 Alkyl groups, -SC1~C 10 Alkylphenyl group, -C1~C 10 Alkyl-SH, -SC1~C 10Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH2, -C1~C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl group), -NH(C1-C 10 Alkyl group), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl group), -NH(C1-C 10 alkylphenyl group), cyano group, nitro group, -CO2H, -C(O)O(C1-C 10 Alkyl group), -CON(C1-C 10 Alkyl)(C1-C 10 Alkyl group), -CONH(C1-C 10 Alkyl group), -CONH2, -NHC(O)(C1-C 10 alkyl group), -NHC(O)(phenyl group), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl group), -N(C1-C 10 Alkyl)C(O)(phenyl group), -C(O)C1~C 10 Alkyl group, -C(O)C1~C 10 Alkylphenyl group, -C(O)C1~C 10 Halogenated alkyl groups, -OC(O)C1~C 10 Alkyl group, -SO2(C1-C 10 Alkyl group), -SO2(phenyl group), -SO2(C1-C 10 Halogenated alkyl group), -SO2NH2, -SO2NH(C1-C 10 alkyl group), -SO2NH (phenyl group), -NHSO2 (C1-C 10 alkyl group), -NHSO2 (phenyl group) and -NHSO2 (C1-C 10 In consideration of the synthesis cost, difficulty, and tumor targeting effect of the complex, in some embodiments, each n1 is 0, and each R i are independently a covalent bond or a C1-C 20The bond may be one or a combination of more than one of an alkylene group, a phosphate ester bond, a thiophosphate ester bond, an amide bond, an ester bond, an ether bond, a thioether bond, a disulfide bond, a 1,2,3-triazole subunit, a polyethylene glycol subunit, a pyrrolidine subunit, a 2-oxopyrrolidine subunit, a phenylene group, a cyclohexylene group, a 2-succinimide subunit, a 2-thiosuccinimide subunit, an amino acid subunit, and a nucleotide subunit.

[0084] In some embodiments, the linking group R j includes any linking group that can be used for antibody drug conjugates known to those skilled in the art. j may be degradable or non-degradable. In some embodiments, the linking group R j In this context, "degradable" refers to the fact that the linking group R can be degraded after the conjugate of the present disclosure has been targeted to the tumor. j This refers to the fact that the linking group R j comprises one or more of an activated enzyme linking group, a sulfatase-cleavable linking group, a galactose-cleavable linking group, a lysosomal protease-sensitive linking group, a peptide linking group, a glucuronide linking group, an acid-sensitive cleavable linking group, or a glutathione-sensitive disulfide linking group. In some embodiments, the linking group R j comprises a peptide linking group. In some embodiments, the peptide linking group is one or more selected from the following: a valine-citrulline dipeptide linker (Val-Cit), an alanine-alanine dipeptide linker (Ala-Ala), a valine-alanine dipeptide linker (Val-Ala), a glycine-glycine-phenylalanine-glycine tetrapeptide linker (Gly-Gly-Phc-Gly). In some embodiments, the linking group R jis one selected from N-succinylidene 4-(2-dithiopyridine)butyrate (SPDB), N-succinimido-4-(2-thiopyridylidene)valerate (SPP), (S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanoic acid (Val-Cit-PAB-OH), N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or 2-(phosphate-(CH2)6-S-)-maleimidocaproyl-valine-citrulline-p-aminobenzyl subunit. In some embodiments, the linking group R j includes the linking groups listed in Mckertish CM, Kayser V. Advances and Limitations of Antibody Drug Conjugates for Cancer. Biomedicines. 2021 Jul 23;9(8):872., the contents of which are incorporated by reference in their entirety into this specification.

[0085] In some embodiments, the linking group R j comprises one or more of a valine-citrulline dipeptide linker (Val-Cit), a polyethylene glycol subunit, an iminohexyl subunit, an N-succinimidyl group, or a GAU trinucleotide linking group. i is independently a combination of one or two of a covalent bond, a disulfide bond, a dodecylene group, a valine-citrulline dipeptide linker (Val-Cit), a polyethylene glycol subunit, an iminohexyl subunit, an N-succinimidyl group, or a GAU trinucleotide subunit.

[0086] R j The role of the group is R AP By attaching a group to the functional group A0, R APThe objective of the present invention is to specifically deliver the functional group A0 to tumor tissues and / or cells through the tumor targeting effect of the delivery group AP in the group. Therefore, any R that can achieve the above-mentioned binding and does not affect the tumor targeting effect of the delivery group AP and the effect of the functional group A0 can be used. j Any group can achieve the object of the present invention and solve the technical problem that the present invention is intended to solve. In some embodiments, after the complex of formula (101) reaches the tumor tissue and / or enters the tumor cell, the R j is decomposed to release a pharmacoactive molecule corresponding to the single functional group A0. In some embodiments, the R j is not broken down in the body, and in this case, R in the complex j Groups and R AP The presence of the group does not affect the functional group A0 from exerting its diagnostic and / or therapeutic effect.

[0087] In some embodiments, R j is a covalent bond, and m is 1, in which case the complex shown in formula (101) has one functional group A and one R AP groups, each R AP A group is directly bonded to the functional group A. In some embodiments, each R AP groups are attached to the same atom of functional group A. In some embodiments, each R AP The groups are attached to different atoms of the functional group A0.

[0088] In some embodiments, R j is a bonding group, and the bonding group R j comprises a main chain portion, a side chain portion and a complex linkage portion.

[0089] The backbone moiety is attached to the complex linkage and the side chain moiety, respectively. In some embodiments, the backbone moiety is a straight chain alkylene group having a length of 1 to 70 carbon atoms, or one or more of the carbon atoms in the straight chain alkylene group is selected from the group consisting of C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycosidic groups, C2-C 10 Alkenylene group, C2-C10 Alkynylene group, C6-C 10 Arylene group, C3-C 18 Heterocyclylene groups and C5-C 10 heteroarylene groups, and the linear alkylene group is substituted with one or more selected from the group consisting of C1 to C 10 Alkyl groups, C6-C 10 Aryl groups, C5-C 10 Heteroaryl groups, C1-C 10 Halogenated alkyl groups, -OC1~C 10 Alkyl group, -OC1~C 10 Alkylphenyl group, -C1~C 10 Alkyl-OH, -OC1~C 10 Halogenated alkyl groups, -SC1~C 10 Alkyl groups, -SC1~C 10 Alkylphenyl group, -C1~C 10 Alkyl-SH, -SC1~C 10 Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH2, -C1~C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl group), -NH(C1-C 10 Alkyl group), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl group), -NH(C1-C 10 alkylphenyl group), cyano group, nitro group, -CO2H, -C(O)O(C1-C 10 Alkyl group), -CON(C1-C 10 Alkyl)(C1-C 10 Alkyl group), -CONH(C1-C 10 Alkyl group), -CONH2, -NHC(O)(C1-C 10 alkyl group), -NHC(O)(phenyl group), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl group), -N(C1-C 10 Alkyl)C(O)(phenyl group), -C(O)C1~C 10 Alkyl group, -C(O)C1~C 10Alkylphenyl group, -C(O)C1~C 10 Halogenated alkyl groups, -OC(O)C1~C 10 Alkyl group, -SO2(C1-C 10 Alkyl group), -SO2(phenyl group), -SO2(C1-C 10 Halogenated alkyl group), -SO2NH2, -SO2NH(C1-C 10 alkyl group), -SO2NH (phenyl group), -NHSO2 (C1-C 10 alkyl group), -NHSO2 (phenyl group) and -NHSO2 (C1-C 10 halogenated alkyl groups).

[0090] The side chain part is the main chain part and R AP In some embodiments, each side chain moiety is independently a covalent bond or a straight chain alkylene group that is 1-70 carbon atoms in length, or one or more of the carbon atoms in the straight chain alkylene group is selected from C(O), NH, O, S, CH=N, S(O)2, OP(O)2, a C5-C8 glycosidic group, a C2-C 10 Alkenylene group, C2-C 10 Alkynylene group, C6-C 10 Arylene group, C3-C 18 Heterocyclylene groups and C5-C 10 heteroarylene groups, and the linear alkylene group is substituted with one or more selected from the group consisting of C1 to C 10 Alkyl groups, C6-C 10 Aryl groups, C5-C 10 Heteroaryl groups, C1-C 10 Halogenated alkyl groups, -OC1~C 10 Alkyl group, -OC1~C 10 Alkylphenyl group, -C1~C 10 Alkyl-OH, -OC1~C 10 Halogenated alkyl groups, -SC1~C 10 Alkyl groups, -SC1~C 10 Alkylphenyl group, -C1~C 10 Alkyl-SH, -SC1~C 10Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH2, -C1~C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl group), -NH(C1-C 10 Alkyl group), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl group), -NH(C1-C 10 alkylphenyl group), cyano group, nitro group, -CO2H, -C(O)O(C1-C 10 Alkyl group), -CON(C1-C 10 Alkyl group) (C1-C 10 Alkyl group), -CONH(C1-C 10 Alkyl group), -CONH2, -NHC(O)(C1-C 10 alkyl group), -NHC(O)(phenyl group), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl group), -N(C1-C 10 Alkyl)C(O)(phenyl group), -C(O)C1~C 10 Alkyl group, -C(O)C1~C 10 Alkylphenyl group, -C(O)C1~C 10 Halogenated alkyl groups, -OC(O)C1~C 10 Alkyl group, -SO2(C1-C 10 Alkyl group), -SO2(phenyl group), -SO2(C1-C 10 Halogenated alkyl group), -SO2NH2, -SO2NH(C1-C 10 alkyl group), -SO2NH (phenyl group), -NHSO2 (C1-C 10 alkyl group), -NHSO2 (phenyl group) and -NHSO2 (C1-C 10 halogenated alkyl groups), The composite linkages are attached to the main chain portion and the functional group A0, respectively. In some embodiments, each composite linkage is independently a covalent bond or a C1-C 10The bond may be one or a combination of more than one of an alkylene group, a phosphate ester bond, a thiophosphate ester bond, an amide bond, an ester bond, an ether bond, a thioether bond, a disulfide bond, a 1,2,3-triazole subunit, a polyethylene glycol subunit, a pyrrolidine subunit, a 2-oxopyrrolidine subunit, a phenylene group, a cyclohexylene group, a 2-succinimide subunit, a 2-thiosuccinimide subunit, an amino acid subunit, and a nucleotide subunit.

[0091] In some embodiments, the linking group R j Each of the composite bonds in each of the functional groups A0 is bonded to the main chain portion, and the number of the side chain portions is n0. Each of the side chain portions is bonded to the main chain portion and one of the R AP Thus, each functional group A and R AP The groups each independently represent a bonding group R j In some embodiments, all of the side chain moieties are attached to the same atom in the main chain moiety, or alternatively, each side chain moiety is attached to a different atom in the main chain moiety.

[0092] In some embodiments, m0 is 1 and the linking group R j includes the structure shown in formula (301).

[0093] [ka] Formula (301) In the formula, k is an integer of 1 to 3, and L C is the main chain portion, and L A is the side chain moiety, and L B is the composite joint, [ka] represents the site at which the group is covalently attached.

[0094] The main chain portion LC is a covalent bond or a divalent to tetravalent, linear or branched C1 to C 25 or one or more carbon atoms in the saturated hydrocarbon group are selected from the group consisting of C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycoside group, C2-C5 alkenylene group, C2-C5 alkynylene group, C6-C 10 Arylene groups, C3-C8 heterocyclylene groups and C5-C 10 heteroarylene groups, and the saturated hydrocarbon group is substituted with one or more selected from the group consisting of C1 to C5 alkyl groups, C6 to C 10 Aryl groups, C5-C 10 Heteroaryl group, -O-C1-C5 alkyl group, -OC1-C5 alkylphenyl group, -C1-C5 alkyl-OH, -SC1-C5 alkyl group, nitro, -C(O)O(C1-C5 alkyl group), -CON(C1-C5 alkyl)(C1-C5 alkyl group), -CONH(C1-C5 alkyl group), -CONH2, -NHC(O)(C1-C5 alkyl group), -NHC(O)(phenyl group), -N(C1-C5 alkyl)C(O)(C1-C5 alkyl group), - It may have one or more substituents selected from the group consisting of N(C1-C5 alkyl)C(O) (phenyl group), -C(O)C1-C5 alkyl group, -C(O)C1-C5 alkylphenyl group, -OC(O)C1-C5 alkyl group, -SO2(C1-C5 alkyl group), -SO2(phenyl group), -SO2NH2, -SON2NH(C1-C5 alkyl group), -SON2NH(phenyl group), -NHSO2(C1-C5 alkyl group) and -NHSO2(phenyl group). In some embodiments, L C is divalent to tetravalent C5 to C 20 or one or more carbon atoms in the saturated hydrocarbon group are selected from the group consisting of C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 glycoside group, C2-C5 alkenylene group, C2-C5 alkynylene group, C6-C 10 Arylene groups, C3-C8 heterocyclylene groups and C5-C 10heteroarylene groups, and the saturated hydrocarbon group is substituted with one or more selected from the group consisting of C1 to C5 alkyl groups, C6 to C 10 Aryl groups, C5-C 10 In some embodiments, L may have one or more substituents selected from the group consisting of a heteroaryl group, an -O-C1 to C5 alkyl group, an -O-C1 to C5 alkylphenyl group, a -C1 to C5 alkyl-OH group, an -SC1 to C5 alkyl group, a nitro group, and -CONH2. C The length of L is 5 to 30 atoms. C The length of is L C L in A From an atom directly bonded to L B The number of chain atoms in the longest chain of atoms formed up to the atom directly bonded to L. C The length ranges from 8 to 25 atoms.

[0095] The side chain moiety L A is a covalent bond or C1-C 20 or one or more carbon atoms in the alkylene group are C(O), NH, O, S, CH=N, S(O)2, OP(O)2, a C5-C8 glycoside group, a C2-C5 alkenylene group, a C2-C5 alkynylene group, a C6-C 10 Arylene groups, C3-C8 heterocyclylene groups and C5-C 10 heteroarylene groups, and the alkylene group is substituted with one or more selected from the group consisting of C1 to C5 alkyl groups, C6 to C 10 Aryl groups, C5-C 10Heteroaryl group, -O-C1-C5 alkyl group, -OC1-C5 alkylphenyl group, -C1-C5 alkyl-OH-SC1-C5 alkyl group, -SC1-C5 alkylphenyl group, -C1-C5 alkyl group, -SH, -OH, -SH, -NH2, -C1-C5 alkyl group, -NH2, -N(C1-C5 alkyl)(C1-C5 alkyl group), -NH(C1-C5 alkyl group), -N(C1-C5 alkyl)(C1-C5 alkylphenyl group), -NH(C1-C5 alkylphenyl group), nitro group, -C(O)O(C1-C5 alkyl group), -CON(C1-C5 alkyl)(C1-C5 alkyl group), -CONH(C1-C5 alkyl may have one or more substituents selected from the group consisting of -CONH2, -NHC(O)(C1-C5 alkyl group), -NHC(O)(phenyl group), -N(C1-C5 alkyl)C(O)(C1-C5 alkyl group), -N(C1-C5 alkyl)C(O)(phenyl group), -C(O)C1-C5 alkyl group, -C(O)C1-C5 alkylphenyl group, -OC(O)C1-C5 alkyl group, -SO2(C1-C5 alkyl group), -SO2(phenyl group), -SO2NH2, -SON2NH(C1-C5 alkyl group), -SON2NH(phenyl group), -NHSO2(C1-C5 alkyl group) and -NHSO2(phenyl group).

[0096] The composite joint L B is one or a combination of one to five bonds selected from the group consisting of a phosphate bond, a thiophosphate bond, an amide bond, an ester bond, an ether bond, and a disulfide bond.

[0097] In some embodiments, k is an integer from 1 to 3, and L C contains any one of the groups represented by formulae (L1) to (L3), and is connected to L via an ether bond in the group represented by formulae (L1) to (L3). A is bonded to the part.

[0098] [ka] [ka] represents the site at which the group is attached to the remainder of the molecule. In some embodiments, k=1 and L C contains a group represented by formula (L1), and the O atom in the group (L1) is L A In some embodiments, k=2 and L C contains a group represented by the formula (L2), and each of the two O atoms in the group (L1) is one L A In some embodiments, k=4 and L C contains a group represented by the formula (L3), and each of the three O atoms in the group (L3) is one L A is directly bonded to

[0099] L B is a phosphate bond or a disulfide bond, Each L A is a covalent bond, or each L A is selected from the group consisting of groups (L4) to (L23) and combinations thereof.

[0100] [ka] In the formula, each j1 is an integer from 1 to 10, Each R' is C1 to C 10 is an alkyl group, Each Ra is a hydrogen atom, C1 to C 10 An alkyl group or a group selected from the group consisting of the groups (L24) to (L37).

[0101] [ka]

[0102] In some embodiments, L A The length of L is 3 to 35 atoms. A The length of is L A L inC From an atom directly bonded to L A R in AP In some embodiments, each L A is a combination of at least two bonds among groups (L4) to (L9), (L13), (L14), and (L18). A is a combination of at least two bonds among groups (L4), (L5), (L7), (L9), (L13), (L14), and (L18).

[0103] In some embodiments, L A has a structure containing an amide bond as shown in formula (302), and L B has a structure containing an N-acylpyrrolidine represented by formula (303), contains a carbonyl group and an oxygen atom, and L C is a linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trihydroxymethylaminomethane.

[0104] [ka] Formula (302) [ka] Formula (303) In the formula, n 302 , q 302 and p 302 are each independently an integer of 2 to 6, and preferably, 302 , q 302 and p 302 are each independently 2 or 3; n 303 is an integer from 4 to 16, preferably, 303 is an integer between 8 and 12, [ka] represents the site at which the group is covalently attached.

[0105] In some embodiments, each said side chain moiety L A Each is an R AP group via a phosphate ester bond, an ether bond or an ester bond, and C and the oxygen atom of the hydroxyl group in the main chain portion L C and are bonded by forming an ether bond, B represents the carbonyl group in formula (303) and the main chain portion L C and the nitrogen atom of the amino group in formula (303) is bonded to the functional group A0 by forming an amide bond, and the oxygen atom in formula (303) is bonded to the functional group A0 by forming a phosphate ester bond, an ether bond, or an ester bond. C is a linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trishydroxymethylaminomethane, and the main chain moiety L C The oxygen atom of the hydroxyl group is A and the nitrogen atom of the amino group is bonded to the complex bond L via an ether bond. B is bonded to the linking group R via an amide bond. j is a compound having 1 to 3 side chains bonded to the carbon atom of the same aminomethyl group and a complex bond L B via R containing the delivery group AP is bonded to the group.

[0106] In some embodiments, the complex has a structure shown in formula (305):

[0107] [ka] Formula (305)

[0108] In some embodiments, the linking group R j includes the structure shown in formula (306).

[0109] [ka] Formula (306) In the formula, n 306 is an integer from 0 to 3, and each p 306 are independently an integer from 1 to 6, [ka] represents the site at which the group is covalently attached, the combination of bonds formed by all of the pyrrolidine subunits and any possible phosphodiester groups constitutes the main chain moiety, and the chain of atoms between the carbonyl group attached to the nitrogen atom in the pyrrolidine subunit and the oxygen atom marked with an * constitutes each side chain moiety, the oxygen atom marked with an * being R AP At least one of the oxygen atoms marked with # is a complex bond and forms an ether bond, ester bond or phosphate bond with the functional group A0, and the remaining oxygen atoms marked with # are bonded to a hydrogen atom to form a hydroxyl group or to a C1-C3 alkyl group to form a C1-C3 alkoxy group. Therefore, the bonded group R j The 1 to 3 side chain moieties in the AP is bonded to the group.

[0110] In some embodiments, a complex according to the present disclosure has a structure shown in formula (307a), (307b), or (307c).

[0111] [ka]

[0112] In some embodiments, the complex of the present disclosure has the structure shown in formula (308):

[0113] [ka] Formula (308) In the formula, n 308 may be an integer of 1 to 10. In some embodiments, n is an integer of 1 to 10, taking into consideration various aspects such as ease of synthesis, structure / process cost, and tumor cell specificity. 308 is an integer from 2 to 6. In some embodiments, n 308 is 3 or 4.

[0114] Each R3 is independently a functional group A0, or an R AP In some embodiments, at least one R is a functional group A and at least one R is a R AP In some embodiments, one R is a functional group A and the remaining R is R AP It is based on

[0115] In some embodiments, each m 308 is independently selected from an integer of 2 to 10, it is believed that the spatial position between the multiple delivery groups AP in the complex can be made more suitable for interaction with the corresponding receptors on the surface of the tumor cell, and in order to make the compound represented by formula (308) simpler, easier to synthesize, and / or less costly, according to some embodiments of the present disclosure, each m 308 is independently an integer from 2 to 5, and in some embodiments, each m 308 are all equal.

[0116] Those skilled in the art will recognize that each R 308 H, C1~C 10 Alkyl groups, C1-C 10 Halogenated alkyl groups and C1-C 10 It will be appreciated that when independently selected from alkoxy groups, any of them can achieve the objectives of the present disclosure without changing the properties of the conjugate shown in formula (308). 308is independently selected from H, a methyl group, or an ethyl group. 308 are both H.

[0117] Each L1 bonded to the functional group A0 represents the composite bond, and the R AP Each L1 bonded to represents the side chain moiety. In some embodiments, one R3 is the functional group A0 and the remaining R3 is the R AP In some embodiments, one or more L1 is a group as the side chain moiety, R AP The nitrogen-containing backbone is provided with a linking group R j In the context of the present disclosure, a "nitrogen-containing backbone" refers to the chain structure in the structure shown in formula (308), and R 308 The carbon atom to which is bonded and the N atom are bonded to each other.

[0118] Considering the delivery efficiency and synthesis cost, in some embodiments, the length of each L1 is independently 3 to 25 atoms. In some embodiments, the length of each L1 is independently 4 to 15 atoms. For convenience, L1 is defined as a straight chain alkylene group, but it will be understood by those of skill in the art that the amino and alkenyl groups resulting from the above-mentioned replacement and / or substitution may not be straight chain groups or may have different names. For purposes of this disclosure, the length of L1 is the number of atoms in the chain connecting the two attachment points. For this purpose, for a ring (e.g., a heterocyclylene or heteroarylene group) obtained by replacing carbon atoms of the straight chain alkylene group, the length of the portion of the chain corresponding to the ring is calculated based on the minimum number of atoms between the attachment points on the ring.

[0119] In some embodiments, L1 is selected from the group consisting of the groups shown in formulas L4-L23 above and any bond combinations thereof. In some embodiments, each L1 is independently selected from the group consisting of a combination of at least two bonds among groups L4-L9, L13, L14, and L18. In some embodiments, each L1 is independently a combination of at least two bonds among groups L4, L5, L7, L9, L13, L14, and L18.

[0120] In some embodiments, in the conjugate of formula (308), each L1 has a binding site bonded to an N atom on the nitrogen-containing backbone and a functional group A0 or AP A bond site that is bonded to a group is included together with the site that is bonded to an N atom on the nitrogen-containing backbone to form an amide bond with the N atom. In some embodiments, one or more L1 is selected from B5, B6, B5', or B6'.

[0121] [ka] During the ceremony, [ka] represents the site at which the group is covalently attached, and q2 is an integer from 1 to 10. In some embodiments, q2 is an integer from 1 to 5.

[0122] In some embodiments, each R AP The group comprises one or more delivery groups. In some embodiments, the compound of formula (308) comprises multiple functional groups. In some embodiments, each functional group in the compound of formula (308) is the same functional group. In some embodiments, each functional group in the compound of formula (308) is a functional group for the same purpose and function. In some embodiments, the compound of formula (308) comprises different types of functional groups for different purposes and functions.

[0123] In some embodiments, the compound of formula (308) has a structure of formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), (422), (423), (424), (425), (426) or (427).

[0124] [ka] Expression (403) [ka] Expression (404) [ka] Formula (405) [ka] Formula (406) [ka] Expression (407) [ka] Expression (408) [ka] Expression (409) [ka] Formula (410)

change

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[0125] In some embodiments, the linking group R jcomprises a nucleotide sequence I and a nucleotide sequence II, each of which comprises 5-25 modified or unmodified nucleotides, the nucleotide sequence I and the nucleotide sequence II being at least partially reverse complementary, the delivery group being attached to the nucleotide sequence I, the functional group being attached to the nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II not eliciting an immune or toxic reaction in a subject. In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary or completely reverse complementary, or the nucleotide sequence I and the nucleotide sequence II are equal in length and each of which comprises 10-20 modified or unmodified nucleotides, or the nucleotide sequence I and the nucleotide sequence II each consist of 17 nucleotides and are completely reverse complementary. In some embodiments, the 3' terminus of the delivery group is linked via a phosphate bond to the 5' ribose position of the 5' terminal nucleotide of nucleotide sequence I and the functional group is linked via a phosphate bond to the 5' ribose position of the 5' terminal nucleotide of nucleotide sequence II, or the functional group comprises a nucleotide sequence, the 3' terminus of the nucleotide sequence is linked via a phosphate bond to the 5' ribose position of the 5' terminal nucleotide of nucleotide sequence I. In some embodiments, the nucleotide sequence I and the nucleotide sequence II have the sequences set forth in SEQ ID NO:40 and SEQ ID NO:41, respectively. 5'-GUACAUUCUAGAUAGCC-3' (SEQ ID NO:40) 5'-GGCUAUCUAGAAUGUAC-3' (SEQ ID NO:41)

[0126] In some embodiments, the nucleotide sequence I and the nucleotide sequence II have the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. 5'-GmUfAmCfAmUfUfCfUfAmGmAmUfAmGmCfCf-3' (SEQ ID NO: 42) 5'-GmGmCfUfAmUfCfUfAmGmAmAmUfGmUfAmCf-3' (sequence number 43).

[0127] The conjugate of the present disclosure may include one or more functional groups. In the context of the present disclosure, a functional group refers to a group containing a radical group formed by removing one or more atoms, functional groups, or substructure groups (e.g., removing one hydrogen atom) from an active agent molecule having a corresponding functionality (e.g., a diagnostic agent compound, a small molecule therapeutic agent compound having a therapeutic effect on tumors, particularly gliomas, and / or a functional oligonucleotide compound that controls the expression level of a cancer-related gene in tumor cells), and the functional group forms a covalent bond with another part of the molecule of the conjugate of the present disclosure through the radical group.

[0128] In some embodiments, at least one functional group in the conjugate of the present disclosure is a diagnostic group, and each of the diagnostic groups is independently selected from a contrast agent group or a fluorescent imaging group (fluorescent group). In some embodiments, all of the functional groups in the conjugate of the present disclosure are diagnostic groups. By including a diagnostic group, the conjugate of the present disclosure can deliver the diagnostic group to a tumor, thereby specifically, efficiently, and accurately diagnosing the presence of a tumor and associated disease and / or symptom information, such as disease progression status. In some embodiments, the diagnostic group can be used to diagnose tumor cells / tissues in a test sample in an in vitro experiment. In some embodiments, the diagnostic group can be used to diagnose the presence and / or characteristics of tumor cells / tissues in a subject's body.

[0129] In some embodiments, at least one functional group in the conjugate according to the present disclosure is a small molecule therapeutic group having a therapeutic effect on tumors, particularly gliomas, and each of the small molecule therapeutic groups is independently selected from a cytotoxin group, an antibiotic group, an angiogenesis inhibitor, or an antibody drug group. In some embodiments, all functional groups in the conjugate according to the present disclosure are the small molecule therapeutic group. By including the small molecule therapeutic group, the conjugate according to the present disclosure can specifically deliver the small molecule therapeutic group to tumors, thereby treating and / or alleviating the progression or symptoms of tumor disease through the effect of the small molecule therapeutic group, for example, the conjugate according to the present disclosure specifically delivers the cytotoxin group to tumors, thereby specifically eliminating cancer cells in the tumor, thereby reducing side effects due to the poor targeting of the cytotoxin itself and significantly reducing the number of cancer cells in the tumor, thereby treating the tumor.

[0130] In some embodiments, at least one functional group in the complex according to the present disclosure is a functional oligonucleotide group, and the functional oligonucleotide group can regulate the expression level of a cancer-related gene in a tumor cell. In some embodiments, all functional groups in the complex according to the present disclosure are the functional oligonucleotide groups. By including the functional oligonucleotide group, the complex according to the present disclosure can specifically deliver the functional oligonucleotide group to a tumor, thereby controlling the expression level of a cancer-related gene in a tumor cell through the effect of the functional oligonucleotide group, for example, the RNAi effect, and treating and / or alleviating the progression or symptoms of a tumor-related disease, for example, by inhibiting the expression of an oncogenic gene. In some embodiments, the functional oligonucleotide is an siRNA. In some embodiments, the functional oligonucleotide is an siRNA targeting STAT3 mRNA, RRM2 mRNA, or PLK1. In some embodiments, the functional oligonucleotide is one or more of an siRNA having a sense strand as shown in SEQ ID NO: 44 and an antisense strand as shown in SEQ ID NO: 45, or an siRNA having a sense strand as shown in SEQ ID NO: 67 and an antisense strand as shown in SEQ ID NO: 68, or an siRNA having a sense strand as shown in SEQ ID NO: 69 and an antisense strand as shown in SEQ ID NO: 70. 5'-CmsUmsAmGmAmAmAfAfCfUmGmGmAmUmAmAmCmGmUm-3' (SEQ ID NO:44) 5'-AmsCfsGmUmUmAfUmCmCmAmGmUmUmUfUmCfUmAmGmsCmsCm-3' (SEQ ID NO:45) 5'-CUUCUUAUUGACACUUACAdT-S-dT-3' (SEQ ID NO:67) 5'-UGUAAGUGUCAAUAAGAAGdT-S-dT-3' (SEQ ID NO:68) 5'-CAAGAAGAAUGAAUACAGUdT-S-dT-3' (SEQ ID NO:69) 5'-ACUGUAUUCAUUCUUCUUGdT-S-dT-3' (SEQ ID NO: 70)

[0131] The functional group may be included in the conjugate of the present disclosure in any suitable manner, for example, the functional group A0 and the main chain portion may be linked by the conjugate linkage described above.

[0132] In some embodiments, at least one of the functional groups in the conjugates of the present disclosure is a delivery aid group, the delivery aid group being 10 ~C 30 The delivery aid group is one or more selected from the group consisting of a hydrocarbon group, a cholesterol group, and a phospholipid group. By including such a delivery aid group, the complex according to the present disclosure can be more compatible with the internal environment in the central nervous system, have better bioavailability, and / or can be more efficiently delivered to the tumor.

[0133] One of skill in the art can prepare the conjugates of the present disclosure by any reasonable synthetic route.

[0134] In some embodiments, the method for synthesizing a complex according to the present disclosure includes contacting a protected complex with a deprotection reagent in a solvent under deprotection reaction conditions, and isolating the complex to obtain a complex according to the present disclosure. The protected complex is a compound formed by protecting any active functional group in the complex according to the present disclosure with a protecting group. In some embodiments, the active functional group includes, but is not limited to, a hydroxyl group, an amino group, and / or a phosphate group, and the protecting group is correspondingly a hydroxyl protecting group, an amino protecting group, and / or a hydroxyl phosphate protecting group (e.g., a cyanoethyl protecting group). Depending on the type of protecting group, the solvent, deprotection reaction conditions, and deprotection reagent to be used are selected and determined. In some embodiments, the deprotection reaction conditions, solvent, and deprotection reagent are the deprotection reaction conditions, solvent, and reagent used in solid-phase synthesis of nucleic acid. In some embodiments, the method includes adding the protected complex to a mixed solution of a methylamine aqueous solution and an ammonia solution, and the deprotection reaction conditions include reacting at room temperature and normal pressure for 1 to 5 h. In some embodiments, the methylamine aqueous solution and saturated concentrated ammonia water are mixed in equal volumes to obtain the mixed solution, and the amount of the solution relative to the protected complex is 0.1-10 ml / μmol. In some embodiments, the separation includes purification by column chromatography, collection of the product eluate, and removal of the solvent. The purification conditions may be, for example, using a preparative ion chromatography purification column and eluting with a gradient eluent of an aqueous sodium chloride solution and an aqueous sodium phosphate solution. In some embodiments, a solvent of 20 mM sodium phosphate (pH 8.1), water / acetonitrile = 9:1 (volume ratio) is used as eluent A, and a solvent of 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), water / acetonitrile = 9:1 (volume ratio) is used as eluent B, and gradient elution is performed with an elution gradient of eluent A:eluent B = 100:0-50:50.

[0135] In some embodiments, a conjugate according to the present disclosure has a structure according to formula (101), and a method for synthesizing the protected conjugate includes reacting an active group R x1and a compound containing a delivery group, the compound being treated with an active group R x2 and a functional group to obtain the protected complex, wherein the delivery group is formed by removing one or more hydrogen atoms or one or more functional groups from the aptamer of the present disclosure, each of the functional groups being one of a diagnostic agent group, a small molecule therapeutic agent group having a therapeutic effect against a tumor, and a functional oligonucleotide group having a therapeutic effect against a tumor, any active group of the delivery group and the functional group is protected by a protecting group, and the active group R x1 and the active group R x2 is converted into a covalent bond or a bonding group R j In some embodiments, the active group R x1 and an active group R x2 The molar ratio of the delivery group to the functional group to which it is attached is m0:n0. In some embodiments, the active groups in the delivery group and functional group include, but are not limited to, one or more of a hydroxyl group, an amino group, and a phosphate group.

[0136] Those skilled in the art can easily identify the active group R x1 and a delivery group. In some embodiments, the active group R x1 Compounds containing the active group R and the delivery group can be obtained by nucleic acid synthesis methods well known to those skilled in the art, such as phosphoramidite solid phase synthesis or phosphodiester / phosphotriester solution phase synthesis. x1 and a delivery group can be obtained by phosphoramidite solid phase synthesis, the method comprising the steps of sequentially linking nucleoside monomers under conditions of phosphoramidite solid phase synthesis according to the order of nucleotides in a single strand of oligonucleotide, at least one nucleoside monomer carrying an active group R x1 or after all the nucleoside monomers are linked, the activating group R x1and then removing the protecting group to leave an active group R x1 The phosphoramidite solid phase synthesis method is well known to those skilled in the art, and the process and conditions thereof are disclosed in detail in Methods in Molecular Biology, vol. 288: Oligonucleotide Synthesis: Methods and Applications, pp. 17-31, the entire contents of which are incorporated herein by reference.

[0137] In some embodiments, the coupling reaction conditions are condensation reaction conditions or mercapto-disulfide bond exchange reaction conditions.

[0138] In some embodiments, the coupling reaction conditions are condensation reaction conditions, the condensation reaction conditions being acylation condensation reaction conditions, dehydration condensation reaction conditions, or click chemistry reaction conditions, and the active group R x1 and active group R x2 is a group capable of the condensation reaction. In some embodiments, the condensation reaction conditions are acylation condensation reaction conditions, and the active group R x1 and R x2 generates an acylation condensation reaction to give R I In some embodiments, the condensation reaction conditions are dehydration condensation reaction conditions, and the active group R x1 and R x2 In some embodiments, the condensation reaction conditions are click chemistry conditions, and the active group R x1 and R x2 In some embodiments, the condensation reaction conditions are Michael addition reaction conditions, and the active group R x1 and R x2In some embodiments, the condensation reaction conditions are N-hydroxysuccinimide-carbodiimide (NHS-EDC)-assisted coupling reaction conditions, and the active group R x1 and R x2 One of them is a group containing an N-hydroxysuccinimide (NHS) group, and the other is a group containing a carbodiimide group (EDC).

[0139] In some embodiments, the active group R x1 and a compound containing a delivery group, which, under the conditions of the coupling reaction, converts to an active group R x0 The aptamer according to the present disclosure having the active group R X0 and the acylation group form a covalent bond via a coupling reaction, thereby attaching the click chemistry active group to the aptamer of the present disclosure.

[0140] In some embodiments, the active group R x1is an active group containing 1 to 3 click chemistry active groups at the terminal, and the click chemistry active group includes a terminal alkynyl group. In some embodiments, the acylation group is an active ester group, and may be, for example, one of an NHS ester group, an imino ester group, and a pentafluorophenyl group. Those skilled in the art can obtain the crosslinker in various ways, for example, when the acylation group is a pentafluorophenyl group and the click chemistry group includes a terminal alkynyl group, the crosslinker can be prepared according to the method described in Scheme 1a(A) in φstergaard, Michael E., et al. "Efficient synthesis and biological evaluation of 5'-GalNAc conjugated antisense oligonucleotides." Bioconjugate chemistry 26.8 (2015):1451-1455, the entire contents of which are incorporated herein by reference. In some embodiments, the active group R x0 is an amino group. In some embodiments, the coupling conditions are alkaline conditions. In some embodiments, the alkaline conditions are conditions in which a weak alkaline aqueous solution is present, for example, conditions in which a sodium bicarbonate aqueous solution is present.

[0141] Those skilled in the art can easily convert the active group R x0 and in some embodiments, the active group R x0 Aptamers having the active group R are prepared by using phosphoramidite monomers containing active groups at the corresponding positions during synthesis of the aptamer. Those skilled in the art can obtain phosphoramidite monomers containing active groups in a variety of ways. In some embodiments, the active group R x0 is an amino group, and R x0 Phosphoramidite monomers containing R x0The phosphoramidite monomer containing the active group R may be 6-(trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite monomer, which is readily available commercially. x0 is an amino group, and the active group R x0 can be obtained by linking the phosphoramidite monomer to a single-stranded oligonucleotide by phosphoramidite solid-phase synthesis, followed by removal of the trifluoroacetyl protecting group by a deprotection reaction (e.g., aminolysis with concentrated aqueous ammonia) that can be easily accomplished by a person skilled in the art.

[0142] In some embodiments, the coupling reaction conditions are one of a mercapto-disulfide bond exchange reaction, and the active group R x1 and R x2 One of the active groups R is a group containing a mercapto group, and the other is a group containing a leaving group linked by a disulfide bond. x1 R in the phosphoramidite monomer containing x1 is a protected R x1 ', and the preparation method includes deprotecting the protected active group R x1 The compound prepared containing the ' and delivery group is contacted with a deprotection reagent to produce an active group R x1 In some embodiments, the compound further comprises obtaining a compound comprising the R x1 wherein R′ comprises a disulfide bond leaving group, the deprotection reaction conditions are mercapto-disulfide bond exchange reaction conditions, and the deprotection reagent is a disulfide bond activator. In some embodiments, the disulfide bond activator is dithiodipyridine. Those skilled in the art will appreciate that the activator R′ can be deprotected by a variety of methods. x1 or R x1 ', and in some embodiments, the active group R x1 or R x1Phosphoramidite monomers containing ' are commercially available, for example, the phosphoramidite monomer shown in formula (105) is commercially available.

[0143] [ka] Formula (105) In the formula, n 105 and m 105 are each independently an integer of 1 to 10.

[0144] Those skilled in the art can easily convert the active group R x2 and a functional group. In some embodiments, the coupling reaction conditions are mercapto-disulfide bond exchange reaction conditions, and the active group R x2 contains a mercapto group, and the active group R x2 Compounds containing functional groups R can be obtained in various ways known to those skilled in the art, for example, by phosphoramidite solid phase synthesis using phosphoramidite monomers containing mercapto groups, or are commercially available. In some embodiments, the functional group is a functional oligonucleotide group, the coupling reaction conditions are phosphoramidite solid phase synthesis reaction conditions, and the active group R x1 is a hydroxy group, and the active group R x2 is a phosphoramidite group, and the method comprises combining a delivery group and an active group R x1 and a solid support to which the functional group R is bound, in sequence to a nucleoside monomer in the manner of a phosphoramidite solid phase synthesis reaction according to the nucleic acid sequence of the functional oligonucleotide. In some embodiments, the functional group is a diagnostic agent group or a small molecule therapeutic agent group, the coupling reaction conditions are phosphoramidite solid phase synthesis reaction conditions, and the active group R x2 is a phosphoramidite group, and the active group R x2and the compound comprising the functional group may be, for example, a compound comprising a phosphoramidite group and a fluorescent group or a small molecule therapeutic group, which are readily available commercially. In some embodiments, the coupling reaction conditions are Michael addition reaction conditions, and the active group R x2 is an N-succinimide group, and the active group R x2 The compound comprising the functional group may be, for example, a compound comprising an N-succinimide group and a small molecule therapeutic agent group, which are readily available commercially. In some embodiments, the functional group is a functional oligonucleotide group, and the active group R x1 and active group R x2 are nucleotide sequence I and nucleotide sequence II, respectively, the nucleotide sequence I and the nucleotide sequence II each contain 5 to 25 modified or unmodified nucleotides, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary, the delivery group is bound to the nucleotide sequence I, the functional group is bound to the nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II do not induce an immune response in the body of a subject or a toxic response of the subject, and the coupling reaction conditions are reaction conditions that anneal to form a nucleic acid duplex. In some embodiments, the functional oligonucleotide group is an siRNA, the 5' end of the nucleotide sequence I and the delivery group are bound via a phosphodiester bond, and the 3' end of the nucleotide sequence II and the 5' end of the siRNA are bound via a phosphodiester bond. In some embodiments, the nucleotide sequence I and the nucleotide sequence II each consist of 17 nucleotides and are completely reverse complementary. In some embodiments, the nucleotide sequence I and the nucleotide sequence II have the sequences shown in SEQ ID NO: 40 and SEQ ID NO: 41, respectively. In some embodiments, the nucleotide sequence I and the nucleotide sequence II have the sequences shown in SEQ ID NO:42 and SEQ ID NO:43, respectively.

[0145] In some embodiments, the complex may be used in the present disclosure in the form of its pharma- ceutically acceptable salt or precursor compound. In the context of the present disclosure, "pharma-ceutically acceptable salt" refers to forming a corresponding salt of a drug, such as potassium salt, sodium salt, carboxylate, etc., that does not have any extra side effects on the human body, in order to increase the stability, solubility, and / or bioavailability of the drug. "Precursor compound" refers to a compound that is not completely the same in structure and function as the complex itself, but can react after entering the body or in a body fluid environment to form the complex according to the present disclosure, thereby exerting its effect and achieving the purpose according to the present disclosure. In some cases, these precursor compounds have the effect of increasing the stability of the drug, extending the sustained release time, increasing bioavailability, etc. In some embodiments, the precursor compound includes a precursor group that can react in the human body to form all the functional groups A0 in the complex. In some embodiments, the precursor compound includes a compound in which all the active hydroxyl groups in the complex are replaced with acetoxy groups. In some embodiments, the precursor compound includes a prodrug group, the prodrug being a residue formed from a precursor compound of a diagnostic agent, a therapeutic agent, and / or a functional oligonucleotide corresponding to the functional group in the complex. In some embodiments, the prodrug group may be, for example, a group in which an active hydrogen in a hydroxy group or an amino functional group in the functional group is replaced with an acyl group, an alkyl group, or a phosphoryl group. As will be appreciated by those skilled in the art, the use of these pharma- ceutical acceptable salts and precursor compounds is also within the scope of the present disclosure.

[0146] Pharmaceutical Compositions In one aspect, the present disclosure further provides a pharmaceutical composition comprising a conjugate according to the present disclosure and a pharma- ceutically acceptable carrier.

[0147] The pharma- ceutically acceptable carrier may be a carrier commonly used in the art, such as water, saline, magnetic nanoparticles (e.g., nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate ester ... Examples of suitable polymerizable compounds include, but are not limited to, one or more of poly(N,N-dimethylaminoethyl methacrylate, PPEEA) and poly(2-dimethylaminoethyl methacrylate, PDMAEMA) and derivatives thereof.

[0148] In some embodiments, the pharma- ceutically acceptable carrier comprises a physiologically acceptable compound, which, for example, serves to stabilize the pharmaceutical composition or to increase or decrease the absorption of the conjugate and / or pharmaceutical composition. The physiologically acceptable compound is one or more selected from carbohydrates such as glucose, sucrose, and / or dextran, antioxidants such as ascorbic acid and / or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of any co-administered substances, excipients, stabilizers, and buffers. Detergents can also be used to stabilize the composition or to increase or decrease the absorption of the pharmaceutical composition. The physiologically acceptable compound may further comprise one or more of a wetting agent, an emulsifying agent, a dispersing agent, or a preservative, which is particularly used to prevent the growth or action of microorganisms. The physiologically acceptable compounds are known to those skilled in the art and will not be described in detail in this disclosure. As one of ordinary skill in the art can readily appreciate, the choice of pharma- ceutically acceptable carrier and physiologically acceptable compound will depend, for example, on the route of administration and the particular physiochemical properties of any co-administered substances.

[0149] In some embodiments, the pharma- ceutically acceptable carrier is sterile and generally free of unnecessary substances.The pharmaceutical composition according to the present disclosure may further include pharma- ceutically acceptable auxiliary substances such as pH adjusting and buffering agents such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, toxicity adjusting agents, etc., as necessary to approximate physiological conditions, and the concentration of the complex according to the present disclosure in the pharmaceutical composition can vary within a wide range and is selected according to the specific method of administration, mainly based on the fluid volume, viscosity, body weight, etc.

[0150] In some embodiments, there is no special requirement for the content of the complex and the pharma- ceutically acceptable carrier in the pharmaceutical composition. In some embodiments, the weight ratio of the complex to the pharma- ceutically acceptable carrier may be 1:(1-500), and in some embodiments, the weight ratio is 1:(1-50).

[0151] In some embodiments, the pharmaceutical composition may include other pharma- ceutically acceptable additives, which may be one or more of various agents or compounds commonly used in the art. For example, the other pharma-ceutically acceptable additives may include at least one of a pH buffer, a protectant, and an osmolality regulator.

[0152] The pH buffer solution may be a trishydroxymethylaminomethane hydrochloride buffer solution having a pH of 7.5 to 8.5 and / or a phosphate buffer solution having a pH of 5.5 to 8.5, for example, a phosphate buffer solution having a pH of 5.5 to 8.5.

[0153] The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. The content of the protective agent may be 0.01 to 30% by weight based on the total weight of the pharmaceutical composition.

[0154] The osmotic pressure regulator may be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator is determined so that the osmotic pressure of the pharmaceutical composition is 200 to 700 milliosmoles per kilogram (mOsm / kg). Depending on the desired osmotic pressure, a person skilled in the art can easily determine the content of the osmotic pressure regulator. In some embodiments, the dosage during administration of the formulation prepared from the pharmaceutical composition is adjusted according to the administration method.

[0155] In some embodiments, the pharmaceutical composition may be a liquid formulation such as an injection solution, or a lyophilized powder injection that is mixed with a liquid additive at the time of administration to prepare a liquid formulation. The liquid formulation may be used for subcutaneous, intramuscular, or intravenous administration, but is not limited thereto, and the pharmaceutical composition may be delivered by a method such as, but not limited to, puncture injection, oral inhalation, or nasal administration. In some embodiments, the pharmaceutical composition is used for subcutaneous, intramuscular, intravenous, or intrathecal injection administration.

[0156] In some embodiments, the pharmaceutical composition may be in the form of a liposome formulation. In some embodiments, the pharma- ceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (hereinafter also referred to as organic amine), an auxiliary lipid, and / or a polyethylene glycolated lipid. The organic amine, auxiliary lipid, and polyethylene glycolated lipid may be one or more selected from the amine-containing transfection compound or its pharma- ceutically acceptable salt or derivative, auxiliary lipid, and polyethylene glycolated lipid described in Chinese patent application CN103380113A (incorporated herein by reference in its entirety).

[0157] In some embodiments, the organic amine may be a compound represented by formula (201) or a pharma- ceutically acceptable salt thereof, as described in Chinese Patent Application CN103380113A.

[0158] [ka] Formula (201) During the ceremony, X 101 and X 102 are each independently O, S, NA or CA, and A is hydrogen or C1-C 20 is a hydrocarbon chain, Y 101 and Z 101 are each independently C=O, C=S, S=O, CH-OH or SO2; R 101 , R 102 , R 103 , R 104 , R 105 , R 106 and R 107are each independently hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain heteroaliphatic group, a substituted or unsubstituted, branched or straight-chain acyl group, a substituted or unsubstituted, branched or straight-chain aryl group, or a substituted or unsubstituted, branched or straight-chain heteroaryl group; x is an integer from 1 to 10; n is an integer of 1 to 3, m is an integer of 0 to 20, and p is 0 or 1, where m=p=0, R 102 is hydrogen, When at least one of n and m is 2, R 103 and the nitrogen in formula (201) form a structure represented by formula (202) or formula (203).

[0159] [ka] Formula (202), [ka] Formula (203), In the formula, g, e, and f are each independently an integer of 1 to 6, "HCC" represents a hydrocarbon chain, and each * N represents a nitrogen atom in formula (201).

[0160] In some embodiments, R 103 is a polyamine. In another embodiment, R 103 In some embodiments, R in formula (201) is a ketal. 101 and R 102 are each independently an optionally substituted or unsubstituted, branched or straight chain alkyl or alkenyl group having 3 to about 20 carbon atoms, e.g., 8 to about 18 carbon atoms, and 0 to 4 double bonds, e.g., 0 to 2 double bonds.

[0161] In some embodiments, when each of n and m is independently 1 or 3, R 103 may be any one of the following formulas (204) to (213).

[0162] [ka] Formula (204), [ka] Formula (205), [ka] Formula (206), [ka] Formula (207), [ka] Formula (208), [ka] Formula (209), [ka] Formula (210), [ka] Formula (211), [ka] Equation (212) and [ka] Formula (213) In formulae (204) to (213), g, e, and f are each independently an integer of 1 to 6, each "HCC" represents a hydrocarbon chain, * is R 103 and the nitrogen atom in formula (201), and * Each H on position may be substituted to provide a bond with the nitrogen atom in formula (201).

[0163] Those skilled in the art can obtain the compound of formula (201) by any reasonable method. In some embodiments, the compound of formula (201) may be prepared according to the description in Chinese patent application CN103380113A.

[0164] In some embodiments, the organic amine is an organic amine represented by formula (214) and / or an organic amine represented by formula (215).

[0165] [ka] Formula (214), [ka] Formula (215) the co-lipid is cholesterol, a cholesterol analogue and / or a cholesterol derivative; The polyethylene glycolated lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.

[0166] In some embodiments, the molar ratio of the organic amine, the colipid, and the polyethylene glycolated lipid in the pharmaceutical composition is (19.7 to 80):(19.7 to 80):(0.3 to 50), and may be, for example, (50 to 70):(20 to 40):(3 to 20).

[0167] In some embodiments, the particles of the pharmaceutical composition formed by the conjugate of the present disclosure and the amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, and more typically, the average diameter of the liposome particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm, e.g., the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, or 160 nm.

[0168] In some embodiments, the weight ratio (weight / weight ratio) of the complex to total lipid (e.g., organic amine, auxiliary lipid, and / or PEGylated lipid) in the pharmaceutical composition formed by the complex of the present disclosure and the amine-containing transfection reagent is within the range of about 1:1 to about 1:50, about 1:1 to about 1:30, about 1:3 to about 1:20, about 1:4 to about 1:18, about 1:5 to about 1:17, about 1:5 to about 1:15, about 1:5 to about 1:12, about 1:6 to about 1:12, or about 1:6 to about 1:10, for example, the weight ratio of the complex of the present disclosure to total lipid is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, or 1:18.

[0169] In some embodiments, the pharmaceutical composition may be marketed with each component being independent, or may be in the form of a liquid formulation when used. In some embodiments, the pharmaceutical composition formed by the conjugate according to the present disclosure and the pharma- ceutical acceptable carrier may be produced according to various known methods, and the conjugate according to the present disclosure may be used instead of a conventional aptamer or conjugate. In some embodiments, the pharmaceutical composition may be produced according to the following method.

[0170] The organic amine, auxiliary lipid and polyethylene glycolated lipid are suspended in alcohol in the above molar ratio and mixed uniformly to obtain a lipid solution. The amount of alcohol is determined so that the total mass concentration of the obtained lipid solution is 2 to 25 mg / mL, for example, 8 to 18 mg / mL. The alcohol is one or more selected from pharma- ceutically acceptable alcohols, for example, alcohols that are liquid around room temperature, such as ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, and polyethylene glycol 400, and may be, for example, ethanol.

[0171] The complex according to the present disclosure is dissolved in a buffer salt solution to obtain an aqueous complex solution. The concentration of the buffer salt solution is 0.05 to 0.5 M, and may be, for example, 0.1 to 0.2 M. The pH of the buffer salt solution is adjusted to 4.0 to 5.5, and may be, for example, 5.0 to 5.2. The amount of the buffer salt solution is determined so that the concentration of the complex is 0.6 mg / mL or less, and may be, for example, 0.2 to 0.4 mg / mL. The buffer salt is one or more selected from soluble acetates and soluble citrates, and may be, for example, sodium acetate and / or potassium acetate.

[0172] After mixing the lipid solution and the aqueous complex solution, the resulting product is incubated at 40-60°C for at least 2 minutes, for example, 5-30 minutes, to obtain an incubated liposome formulation. The volume ratio of the lipid solution to the aqueous complex solution is 1:(2-5), and may be, for example, 1:4.

[0173] The incubated liposome preparation is concentrated or diluted, impurities are removed, and bacteria are removed to obtain the pharmaceutical composition according to the present disclosure. Its physicochemical parameters are pH 6.5-8, encapsulation efficiency not less than 80%, particle size 40-200 nm, polydispersity index not more than 0.30, and osmotic pressure 250-400 mOsm / kg. For example, the physicochemical parameters may be pH 7.2-7.6, encapsulation efficiency not less than 90%, particle size 60-100 nm, polydispersity index not more than 0.20, and osmotic pressure 300-400 mOsm / kg.

[0174] Here, the concentration or dilution may be performed before removing impurities, after removing impurities, or simultaneously. As a method for removing impurities, various conventional methods may be used, for example, ultrafiltration may be performed under conditions of 100K Da using a tangential flow system and a hollow fiber column, and the ultrafiltration exchange solution may be a phosphate buffer solution (PBS) of pH 7.4. As a method for sterilization, various conventional methods may be used, for example, sterilization may be performed by filtering through a 0.22 μm filter.

[0175] Uses of the aptamers and / or complexes according to the present disclosure In yet another aspect, the present disclosure further provides the use of the aptamer and / or conjugate and / or pharmaceutical composition according to the present disclosure in the manufacture of a medicament for diagnosing and / or treating tumors and tumor-related diseases or conditions.

[0176] In yet another aspect, the present disclosure further provides a method for diagnosing and / or treating tumors and tumor-related diseases or conditions, comprising administering to a subject in need thereof a conjugate and / or pharmaceutical composition according to the present disclosure.

[0177] By administering the conjugate and / or pharmaceutical composition of the present disclosure, the method of the present disclosure can effectively diagnose and / or treat tumors and tumor-related diseases or conditions, and the highly specific targeting effect of the conjugate of the present disclosure can reduce the distribution of the diagnostic and / or therapeutic agent in other organs / tissues of the body that are not intended, and reduce potential side effects. In particular, it has great significance and significant value for radiotherapy and / or chemotherapy drugs that are commonly used in the field of tumor treatment and are known to have serious side effects. In some embodiments, the drug can be used for diagnosis of tumor cells / tissues in a test sample in an in vitro experiment. In some embodiments, the drug can be used for diagnosis of tumor cells / tissues in a subject's body. In some embodiments, the drug can be used for treatment of tumors or tumor-related diseases and conditions in a subject's body.

[0178] The term "pharmaceutical administration / administration" as used in this disclosure refers to placing the complex and / or pharmaceutical composition into the body of a subject by a method or route that at least partially localizes the complex and / or pharmaceutical composition to a desired site to produce a desired effect. Routes of administration suitable for the methods of this disclosure include local administration and systemic administration. Generally, local administration delivers more of the complex and / or pharmaceutical composition to a particular site than to the entire body of the subject, while systemic administration delivers the complex and / or pharmaceutical composition to substantially the entire body of the subject.

[0179] Furthermore, the inventors of the present disclosure have unexpectedly discovered that the conjugates and / or pharmaceutical compositions of the present disclosure can efficiently cross the blood-brain barrier and target tumors in the brain when administered systemically, thereby further improving the delivery efficiency of functional groups, saving costs, and reducing undesirable side reactions.

[0180] Administration to a subject may be by any suitable route known in the art, including, but not limited to, oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, transdermal, intratracheal (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration. The frequency of administration may be once or more per day, week, biweekly, triweekly, monthly, or yearly.

[0181] The dose of the conjugate and / or pharmaceutical composition of the present disclosure may be a dose common in the art, which may be determined according to various parameters, in particular the age, weight and sex of the subject. Toxicity and therapeutic efficacy may be measured by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., LD 50 (the dose that kills 50% of the colony) and ED 50 (For a quantitative response, this refers to the dose which produces 50% of the maximum response strength; for a qualitative response, this refers to the dose at which a positive response occurs in 50% of experimental subjects) may be determined. A range of human dosages can be derived based on data obtained from cell culture assays and animal studies.

[0182] When administering the complex and / or pharmaceutical composition according to the present disclosure to, for example, a male or female, 6-12 week old, 18-25 g body weight C57BL / 6J or C3H / HeNCrlVr mouse, the dose of the complex formed by the complex and the pharma- ceutically acceptable conjugate molecule may be 0.001-100 mg / kg body weight, in some embodiments, 0.01-50 mg / kg body weight, in further embodiments, 0.05-20 mg / kg body weight, in still further embodiments, 0.1-15 mg / kg body weight, and in still further embodiments, 0.1-10 mg / kg body weight. When administering the complex and / or pharmaceutical composition according to the present disclosure, the above dose is preferred.

[0183] kit The present disclosure provides a kit, the kit comprising a conjugate and / or pharmaceutical composition according to the present disclosure.

[0184] In some embodiments, the kit according to the present disclosure may provide the conjugate and / or pharmaceutical composition in a container. In some embodiments, the kit according to the present disclosure may include a container providing a pharma- ceutically acceptable excipient. In some embodiments, the kit may include other components, such as a stabilizer or preservative. In some embodiments, the kit according to the present disclosure may include at least one other therapeutic agent in a container other than the container providing the conjugate and / or pharmaceutical composition according to the present disclosure. In some embodiments, the kit may include instructions for mixing the conjugate and / or pharmaceutical composition with a pharma- ceutically acceptable carrier and / or excipient or other components, if present.

[0185] In the kits of the present disclosure, the conjugate and pharma- ceutically acceptable carrier and / or additive, and the pharmaceutical composition, and / or pharma- ceutically acceptable additive, can be provided in any form, such as liquid, dry, or lyophilized. In some embodiments, the conjugate and pharma- ceutically acceptable carrier and / or additive, and the pharmaceutical composition and any pharma- ceutically acceptable additive, are essentially clean and / or sterile. In some embodiments, sterile water can be provided in the kits of the present disclosure.

[0186] The present disclosure will be further described below with reference to examples, but the present disclosure is not limited thereto in any way. EXAMPLES

[0187] Unless otherwise specified, all reagents and media used in the following examples are commercially available products, and all operations such as nucleic acid electrophoresis and real-time PCR are performed with reference to the methods described in Molecular Cloning (Cold Spring Harbor Borough Press (1989)).

[0188] Preparation Examples 1 to 9 and 12 Synthesis of the Complex According to the Present Disclosure The conjugates numbered AP1-AP8 and AP12 in Table 1A were synthesized by solid-phase synthesis, and all nucleoside monomers were linked in sequence from 3' to 5' according to the nucleotide sequences corresponding to AP1-AP8 and AP12 in Table 1A, respectively, and then Cy5 phosphoramidite monomer (purchased from Suzhou Jima Co., Ltd., lot number CY5P21H1B) was linked according to the method of linking nucleoside phosphoramidite monomers by solid-phase synthesis. Then, the nucleotide sequence was added to an equal volume mixture of methylamine aqueous solution and ammonia water, the amount of the solution for the conjugate was 0.5ml / μmol, and the reaction was carried out at 25°C for 2h, filtered to remove solids, and the supernatant was concentrated in vacuum and dried.

[0189] The purification of the produced complex was completed by gradient elution with NaCl aqueous solution using a production type ion chromatography purification column (Source 15Q). Specifically, 20 mM sodium phosphate (pH 8.1), water / acetonitrile = 9: 1 (volume ratio) solvent was used as eluent A, 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), water / acetonitrile = 9: 1 (volume ratio) solvent was used as eluent B, and gradient elution was performed with an elution gradient of eluent A: eluent B = 100: 0 to 50: 50. The product eluate was collected and then combined, and desalted using a reverse phase chromatography purification column. The specific conditions were desalted using a Sephadex column, the packing material was Sephadex G25, and eluted with deionized water. The resulting eluate was concentrated to remove the solvent and lyophilized to obtain complexes AP1 to AP8, each of which has the 5'-position of ribose in the 5'-terminal nucleotide bound to the fluorescent group Cy5 via a phosphate ester group linking group.

[0190] According to the same method, the conjugate AP9 was prepared in which the 5'-position of ribose in the 5'-terminal nucleotide was linked to the fluorescent group Cy3 via a phosphate linker group, with the only difference being that the nucleoside monomers were linked in order according to the nucleic acid sequence corresponding to AP9 in Table 1A, and Cy3 phosphoramidite monomers (purchased from Shanghai Jiaowei Co., Ltd., product number OP-038) were used instead of Cy5 phosphoramidite monomers.

[0191] After the synthesis of the above complexes AP1 to AP9 and AP12 was completed, the complexes obtained were washed with ultrapure water (Milli-Q ultrapure water system, resistivity 18.2 MΩ). * The mixture was diluted to a concentration of 0.2 mg / mL at 25°C (cm) and then the molecular weight was detected using a liquid chromatography mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model number: LCT Premier). As a result, the measured molecular weight value was consistent with the theoretical value, indicating that the desired complex was obtained.

[0192] Comparative Preparation Examples 10, 11, 13 and 14 Synthesis of Comparative Complexes According to the methods of Preparation Examples 1 to 9 and 12, the conjugates numbered Comparative AP10, Comparative AP11, Comparative AP13 and Comparative AP14 in Table 1A were synthesized, respectively. The only difference was that the nucleoside monomers were sequentially linked according to the sequences corresponding to Comparative AP10, Comparative AP11, Comparative AP13 and Comparative AP14 in Table 1A. Thus, Comparative conjugates Comparative AP10, Comparative AP11, Comparative AP13 and Comparative AP14 were obtained, respectively, in which the ribose 5' position of the 5'-terminal nucleotide is linked to the fluorescent group Cy5 via a phosphate ester group linking group. Comparative AP10 and Comparative AP11 are negative control conjugates having different sequences or modification schemes, respectively, and the aptamer sequences have a certain degree of randomness and have little sequence homology with the aptamers according to the present disclosure. Comparative AP13 and Comparative AP14 are comparative conjugates whose sequences have only a small amount of nucleotide difference from the conjugates AP2 and AP4, respectively. Specifically, comparative AP13 has two more nucleotides G and A at the 5' end, one less nucleotide C at the 3' end, and is missing one U nucleotide in the strand at the 17-18th nucleotide position from the 5' end corresponding to AP2, compared to AP2. Comparative AP14 has two more nucleotides G and A at the 5' end, one less nucleotide U at the 3' end, and is missing one U nucleotide in the strand at the 17-18th nucleotide position from the 5' end corresponding to AP2, compared to AP4.

[0193] Table 1A Nucleotide sequences of the complexes [Table 1]

[0194] In Table 1A, capital letters C, G, U, and A represent the base composition of nucleotides, lowercase letter m represents that one nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide, lowercase letter f represents that one nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide, lowercase letter s represents that the two nucleotides on the left and right of the letter s are linked by a thiophosphate group, and CY3 and CY5 represent the binding sites in the aptamer of the fluorescent dye groups Cy3 (Cyanine 3) and Cy5 (Cyanine 5), respectively.

[0195] Preparation Examples 15-16 Synthesis of Complexes 15-16 In this Production Example 15, Complex 15 was produced by the following steps.

[0196] (15-1) Preparation of Oligonucleotide C1 The oligonucleotide sequence in the complex 15 in Table 1B was synthesized by the methods of Preparation Examples 1 to 8 and 12. The only difference is that the sense strand sequence includes a sense strand of siRNA, and the 3' end of the sense strand of the siRNA is linked to a delivery group included in the complex AP2 via Spacer18-GfAfUf as a linking group, and Spacer18 refers to a hexapolyethylene glycol subunit linking group that includes six ethoxy subunits linked in sequence and has a length of 18 atoms. The linking group is linked in sequence from 3' to 5' with nucleoside monomers GfAfUf, and then linked with a hexapolyethylene glycol (HEG)-phosphoramidite monomer according to a solid phase synthesis method. The HEG-phosphoramidite monomer has a structure shown in formula (15-a).

[0197] [ka] Formula (15-a) During solid-phase synthesis, after the final nucleoside monomer at the 5'-end was attached, a phosphoramidite monomer containing an -NH-(CH2)6- group (purchased from Hongene Biotech) was further attached according to the method for attaching a nucleoside phosphoramidite monomer, the single-stranded oligonucleotide was cleaved from the solid support and concentrated by aminolysis, and the crude product was desalted by ultrafiltration, concentrated and dried to obtain the single-stranded oligonucleotide C1 (40.00 mg, 5.49 μmol) shown in formula (15-b).

[0198] [ka] Formula (15-b) During the ceremony, [ka] represents the oligonucleotide sequence corresponding to complex 15.

[0199] (15-2) Synthesis of oligonucleotide C2: [ka] 346 mg of Cy5-NHS was dissolved in 5.4 ml of solvent DMF. The concentrated and dried oligonucleotide single-stranded C1 was taken and dissolved in 1.34 mL of 100 mM sodium bicarbonate-aqueous solution for use. The DMF solution of Cy5-NHS was added to the oligonucleotide single-stranded C1 solution, triethylamine was added, and the reaction was allowed to proceed overnight at room temperature in the dark. Methyl tert-butyl ether / ethanol solution (v:v=4:1) was added to the reaction solution. The amount added was three times the volume of the reaction solution. After vortexing at room temperature for 1 min and centrifuging at 3200 g for 15 min using a centrifuge, the supernatant was removed, and the precipitate was the desired oligonucleotide single-stranded C1 crude product. The nucleic acid sample was dissolved in pure water, purified by Agilent semi-preparative reverse phase column, and gradient eluted with the following mobile phase. Eluent A: 100 mM TEAA-water (pH = 7.2), eluent B: acetonitrile (ACN), gradient: 15-75% (Bv / v%). The solvent was removed by concentration to obtain 43.4 mg of oligonucleotide single-stranded C2 to which the Cy5 fluorescent group was bound via the -NH-(CH2)6- group and the phosphate ester group binding group.

[0200] The sense strand of complex 16 was synthesized according to the same method. The only difference is that the nucleotides were linked in order according to the nucleotide sequence of the sense strand of complex 16 in Table 1B. Unlike complex 3 in which the delivery group is linked to the 3' end of the siRNA sense strand, the 5' end of the siRNA sense strand of complex 4 is linked to the 3' end of the delivery group contained in complex AP2 via Spacer18-GfAfUf linkage as a linking group, and the fluorescent group Cy5 is linked to the ribose 5' position of the 5'-terminal nucleotide of the delivery group via a -NH-(CH2)6- group and a phosphate ester group linking group.

[0201] According to the nucleic acid solid phase synthesis method, the antisense strand sequences of the complexes numbered as complexes 15 and 16 in Table 1B were synthesized.

[0202] The sense strand sequence and the antisense strand sequence of each complex were dissolved in water for injection, respectively, to obtain a 40 mg / mL solution, mixed in an equimolar ratio, heated at 50 ° C for 15 min, cooled at room temperature, and then formed a double-stranded structure by hydrogen bonding. Thereafter, the obtained complexes were purified, desalted, solvent removed, and lyophilized according to the method described in Preparation Examples 1 to 2 to obtain complexes 15 and 16, respectively. After the synthesis was completed, the purity of the sense strand sequence and the antisense strand sequence of the obtained complexes 3 and 4 were detected by ion exchange chromatography (IEX-HPLC), respectively, and the molecular weight was analyzed by liquid chromatography (LC-MS). The theoretical values ​​of the sense strand MS of complexes 3 and 4 were both 18986.82, the actual values ​​of complex 3 were 18984.81, and the actual values ​​of complex 4 were 18984.84. The antisense strands of complex 3 and complex 4 are the same, the theoretical value is 6881.59, and the actual value of the MS molecular ion peak is 1719.2 (tetravalent anion), and the measured actual value and the theoretical value are consistent. Complex 15 and complex 16 are complexes containing a double-stranded siRNA and a delivery group contained in complex AP2, respectively, and the delivery group is bound to the 5'-end or 3'-end of the siRNA sense strand via a Spacer18-GfAfUf binding group, and a Cy5 fluorescent group is bound to the ribose 5'-position of the 5'-terminal nucleotide of the entire nucleotide sequence including the siRNA sense strand via an NH-(CH2)6- group and a phosphate ester group binding group.

[0203] Table 1B Nucleotide sequences in the complex [Table 2]

[0204] In Table 1B, capital letters C, G, U, A, and T represent the base composition of nucleotides, lower case letter m represents that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide, lower case letter d represents that one nucleotide adjacent to the right side of the letter d is a deoxynucleotide, lower case letter f represents that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide, and lower case letter s represents that the two nucleotides on the left and right of the letter s are linked by a thiophosphate group. CY3 and CY5 represent the binding sites in the aptamer of the fluorescent dye groups Cy3 (Cyanine 3) and Cy5 (Cyanine 5), respectively, and CY5-(NH-C6H12) represents the site in the siRNA that is linked to the fluorescent dye group Cy5 via an n-hexylamino linking group. The capital letter X represents a propylene-phosphate ester group, and the lowercase letter combination hd represents a 2'-O-hexadecyl modified nucleotide with one nucleotide adjacent to the left of the letter combination hd. Spacer18 represents the hexapolyethylene glycol subunit binding group, and MMAE represents the binding site in the aptamer for the small molecule drug group MMAE (monomethylauristatin E) group.

[0205] Preparation Examples 17 to 18: Synthesis of Complex 17 and Complex 18 According to the same method as in Preparation Examples 15-16, the complexes numbered as complex 17 and complex 18 in Table 1B were synthesized. The only difference is that the nucleoside monomers were sequentially linked according to the nucleic acid sequences corresponding to complex 17 and complex 18 in Table 1B, respectively, and when the sense strand was produced, a C3-spacer-phosphoramidite monomer (purchased from Shanghai Jiaowei Co., Ltd., product number OP-005) was linked instead of the Spacer18-GfAfUf linking group, and the linking was repeated four times, and then the nucleoside monomers were continued to be linked according to the nucleic acid sequence in Table 1B. That is, the obtained complex 17 is a complex containing a double-stranded siRNA and an aptamer according to the present disclosure, and the aptamer is linked to the 5' end of the siRNA sense strand via four propylene phosphate ester groups linked in sequence as linking groups, and a Cy5 fluorescent group is linked to the 5' end of the aptamer via an NH-(CH2)6- group and a phosphate ester group. Complex 18 is a complex in which the first two nucleotides at each end of the aptamer in complex 17 are both modified with 2'-O-hexadecyl groups.

[0206] Preparation Example 19 Synthesis of Complex 19 A complex numbered as complex 19 in Table 1B was synthesized according to the same method as in Preparation Examples 15-16. The only difference is that nucleoside monomers were sequentially linked according to the nucleic acid sequences corresponding to complex 19 in Table 1B, respectively, to obtain three nucleotide sequences, and when annealing, the obtained three nucleotide sequences were dissolved together in water for injection and mixed in an equimolar ratio. The obtained complex 19 is a complex containing a double-stranded siRNA and a complex AP2 according to the present disclosure, in which the aptamer is linked to the siRNA via a linking group, and the linking group is a double strand formed by complementary nucleotide sequences having the sequences shown in SEQ ID NO:28 and SEQ ID NO:29, respectively, the aptamer is linked to the 5' end of the sequence shown in SEQ ID NO:28, the siRNA is linked to the 3' end of the sequence shown in SEQ ID NO:29, and a Cy5 fluorescent group is linked to the 5' end of the aptamer via an NH-(CH2)6- group and a phosphate ester group. After the preparation was completed, a 12% polyacrylamide gel was prepared, and the prepared complex 19 was dissolved in a nucleic acid loading buffer and mixed uniformly, then loaded onto the polyacrylamide gel and subjected to vertical electrophoresis at a voltage of 80 V for 1 h. After the electrophoresis was completed, the gel was immersed in a 3x bubble staining solution by the Gelstain bubble staining method, and the gel was stained by slowly shaking at room temperature for 1 h, and imaged with a gel imager. As is clear from the imaging results, the prepared complex 19 showed a single band, indicating that a double-stranded complex in which a double-stranded complementary region was formed between each nucleotide sequence, i.e., complex 19, was successfully obtained.

[0207] Preparation Example 20: Synthesis of Complex 20 In this preparation example, conjugate 20 was prepared according to the following steps. Conjugate 20 includes an aptamer according to the present disclosure and a small molecule drug group MMAE, which is attached to the 5' end of the aptamer via a linking group, which is a 2-(phosphate-(CH2)6-S-)-maleimidohexanoyl-valine-citrulline-p-aminobenzyl subunit.

[0208] (20-1) (Production of aptamer S1) The aptamer sequence in complex 20 in Table 1C was synthesized by solid-phase synthesis. The only difference was that the nucleoside monomers were linked in order according to the aptamer sequence corresponding to complex 20 in Table 1C, and during solid-phase synthesis, the last nucleoside monomer at the 5' end was linked, and then a phosphoramidite monomer containing a HO-(CH2)6-SS-(CH2)6- group (purchased from Hongene Biotech) was linked according to the method of linking a nucleoside phosphoramidite monomer, and the aptamer single strand was cleaved from the solid-phase support to obtain the aptamer single strand S1 (70.00 mg, 6.42 μmol) shown in formula (20-a).

[0209] [ka] Formula (20-a) During the ceremony, [ka] represents the aptamer sequence corresponding to complex 20.

[0210] (20-2) Synthesis of aptamer S2: [ka] 70.0 mg of S1 (6.42 μmol) prepared in step (20-1) was dissolved in 10.0 ml of purified water, and then 105 mg of TCEP (tris(2-chloroethyl)phosphate, 0.37 mmol, purchased from BiDe Pharmaceutical, lot number: BD155793) was dissolved in 10.0 ml of purified water to obtain a TCEP aqueous solution. The mixture was mixed uniformly and reacted at room temperature for 2 hours. The reaction solution was diluted with 10 mL of purified water and filtered to obtain 28 mL of reaction solution. The reaction solution was transferred to a 3K standard ultrafiltration tube and centrifuged at 3900 rpm for 30 min. The ultrafiltration and centrifugation steps were repeated twice again to collect the product in the filtration membrane, and aptamer S2 (67.0 mg, yield: 95.7%) was obtained.

[0211] (20-3) Synthesis of aptamer 20: [ka] 24 mg of Vc MMAE (18.56 μmol, 5 eq, purchased from CSN, lot number CSN16143-005) was dissolved in 6.0 ml of DMF, and 60 μl of triethylamine was added to obtain a Vc MMAE solution. 40.0 mg of the aptamer S2 (3.71 μmol, 1 eq) produced in step (20-2) was dissolved in 6.0 ml of purified water, and the above Vc MMAE solution was added to the resulting solution. After reacting at room temperature for 2 hours, a crude product of complex 20 was obtained (represented as S3 in the process diagram).

[0212] The obtained crude product of complex 20 was diluted with 0.5 ml of purified water, filtered through a 0.45 μm filter membrane, and the filtrate was purified by an Agilent semi-preparative reversed-phase column. The column used was Kromasil 100-10-C18, 100 Å, 10 um, 21.2*250 mm. Gradient elution was performed using 100 mM triethylamine acetate buffer (TEAA, pH = 7.0-7.3):acetonitrile = 95:5-35:65 as the mobile phase. The peak eluent of the product was collected and the solvent was removed by evaporation to obtain complex 20 (55 mg, yield 56.7%). The molecular weight was measured by LC-MS, and the theoretical value was 12092.67, and the actual value was 12091.68, which agreed with the theoretical value. This shows that conjugate 20 has the structure shown in S3 and comprises the aptamer of the present disclosure and a small molecule drug group MMAE, the MMAE group being attached to the 5' end of the aptamer via a linking group that is 2-(phosphate-(CH2)6-S-)-maleimidohexanoyl-valine-citrulline-p-aminobenzyl subunit (2-(phosphate-(CH2)6-S-)-MC-Val-Cit-PAB).

[0213] Table 1C Nucleotide sequence of the single strand of the aptamer [Table 3]

[0214] In Table 1C, capital letters C, G, U, and A represent the base composition of a nucleotide, lower case letter m represents that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide, lower case letter f represents that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide, and MMAE represents the binding site in the aptamer of the small molecule drug group MMAE (monomethylauristatin E) group.

[0215] Production Examples 21 to 26 Synthesis of Complexes AP21 to AP26 according to the present disclosure According to the methods of Preparation Examples 1 to 8 and 12, conjugates numbered AP21 to AP26 in Table 2 were synthesized, respectively, and the molecular weights were detected to confirm the synthesized conjugates. The only difference was that nucleoside monomers were bound in order according to the sequences corresponding to AP21 to AP26 in Table 2, respectively. As a result, conjugates AP21 to AP26 in which the fluorescent group Cy5 is bound to the 5' end were obtained, respectively. AP21 has one less nucleotide at the 3' end compared to the AP4, AP22 has a structure in which one nucleotide is reduced from each of the S1 and S4 motifs in the sequence shown in formula (1) compared to the AP4, AP23 has a structure in which another nucleotide is reduced from the S1 motif in the sequence shown in formula (1) corresponding to AP22, AP24 is a conjugate in which the first two nucleotides at the 5' end of AP4 are changed, and AP25 and AP26 have N in the sequence shown in formula (1) compared to the AP4. b It is a complex with an altered motif.

[0216] After the synthesis of the above complexes AP11 to AP26 was completed, the complexes obtained were washed with ultrapure water (Milli-Q ultrapure water system, resistivity 18.2 MΩ *The mixture was diluted to a concentration of 0.2 mg / mL at 25°C (cm) and then the molecular weight was detected using a liquid chromatography mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model number: LCT Premier). As a result, the measured molecular weight value was consistent with the theoretical value, indicating that the desired complex was obtained.

[0217] The MS theoretical values ​​and MS measured values ​​of the complexes AP11 to AP26 are shown in the following Table 2-a.

[0218] [Table 4]

[0219] Comparative Preparation Examples 27-35 Synthesis of Reference Complexes Comparative AP27-Comparative AP35 According to the methods of Preparation Examples 1 to 9 and 12, conjugates numbered Comparative AP27 to Comparative AP35 in Table 2 were synthesized, respectively, and the molecular weights were detected to confirm the synthesized conjugates. The only difference was that nucleoside monomers were bound in order according to the sequences corresponding to Comparative AP27 to Comparative AP35 in Table 2, respectively. In this way, control aptamer conjugates Comparative AP27 to Comparative AP35 in which the fluorescent group Cy5 is bound to the 5' end were obtained, respectively. Comparative AP27 and Comparative AP28 are control conjugates having the same sequence base composition as AP4 but different nucleotide sequence orders, Comparative AP29 and Comparative AP30 are control conjugates having the same sequence base composition as AP2 but different nucleotide sequence orders, Comparative AP31 has two fewer nucleotides at the 5' end and one fewer nucleotide at the 3' end compared to Comparative AP4, and Comparative AP32 has N in the sequence shown in formula (1) compared to Comparative AP4. b The comparative AP33 has a deletion of the N motif in the sequence shown in formula (1) compared to the comparative AP4. a and N cThe motif was deleted, and comparative AP34 was a control complex in which multiple nucleotides were added to the S2 and S3 motif portions, respectively, and partial sequences were altered, compared to comparative AP35.

[0220] Table 2 Nucleotide sequences of the complexes [Table 5]

[0221] In Table 2, capital letters C, G, U, and A represent the base composition of nucleotides, lowercase letter m represents that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide, lowercase letter f represents that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide, and Cy5 represents the binding site in the aptamer of the fluorescent dye group Cy5 (Cyanine 5) group.

[0222] After the synthesis of Comparative AP27 to Comparative AP35 was completed, the composites obtained were dissolved in ultrapure water (Milli-Q ultrapure water system, resistivity 18.2 MΩ) and * The mixture was diluted to a concentration of 0.2 mg / mL at 25°C (cm) and then the molecular weight was detected using a liquid chromatography mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model number: LCT Premier). As a result, the measured molecular weight value was consistent with the theoretical value, indicating that the desired complex was obtained.

[0223] The MS theoretical values ​​and MS measured values ​​of Comparative AP27 to Comparative AP35 are as shown in Table 2-b.

[0224] Table 2-b: Mass spectrometry results of comparative complexes [Table 6]

[0225] Preparation Example 36: Synthesis of Complex 36 A conjugate numbered conjugate 36 in Table 1B was synthesized according to the same method as in Preparation Examples 1 to 8 and 12. The only difference is that when nucleoside monomers were bound in order according to the nucleic acid sequence corresponding to conjugate 36 in Table 1B and a sense strand was produced, the 3' end of the siRNA sense strand was bound to a delivery group contained in conjugate AP2 via six dT phosphate groups as binding groups, and a Cy5 fluorescent group was bound to the 5' position of the siRNA sense strand via a phosphate group binding group.

[0226] After the synthesis was completed, the purity of the sense strand sequence and the antisense strand sequence of the obtained complex 36 were detected by ion exchange chromatography (IEX-HPLC), and the molecular weight was analyzed by liquid chromatography (LC-MS). The sense strand of complex 36 had a theoretical value of 19660.11 and an actual value of 19658.23, which agreed with the actual value. The antisense strand of complex 36 had a theoretical value of 6881.59 and an actual value of 6880.83, which agreed with the actual value.

[0227] Preparation Examples 37 to 38: Synthesis of Complex 37 and Complex 38 Conjugates numbered Conjugate 37 and Conjugate 38 in Table 1B were synthesized by the same method as in Preparation Examples 1 to 8 and 12. The only difference was that all nucleoside monomers were sequentially linked from 3' to 5' according to the nucleotide sequences of the sense strand or antisense strand of Conjugate 37 and Conjugate 38 in Table 1B, respectively, to obtain the sense strand and antisense strand sequences of the conjugates.

[0228] For complex 37, the sense strand sequence includes the sense strand of an siRNA, the 3' end of the sense strand of the siRNA is linked to the 5' end of a delivery group contained in complex AP2 via Spacer18-GfAfUf as a linking group, and the fluorescent group Cy5 is linked to the 5' position of ribose in the 5'-terminal nucleotide of the antisense strand via a phosphate group linking group.

[0229] For complex 38, the sense strand sequence includes the sense strand of an siRNA, the 5' end of the sense strand of the siRNA is linked to the 3' end of a delivery group included in complex AP2 via four propylene phosphate ester groups linked in sequence, and the fluorescent group Cy5 is linked to the 5' position of ribose in the 5'-terminal nucleotide of the antisense strand via a phosphate ester group linking group.

[0230] After the synthesis was completed, the purity of the sense strand sequence and the antisense strand sequence of the obtained complex 37 and complex 38 were detected by ion exchange chromatography (IEX-HPLC), and the molecular weight was analyzed by liquid chromatography (LC-MS). The sense strand of complex 37 had a molecular weight of 18327.97, and the measured value was 18325.87, which was consistent with the theoretical value. The sense strand of complex 38 had a theoretical value of 19011.37, and the measured value was 19003.59, which was consistent with the theoretical value. The antisense strands of complex 37 and complex 38 were the same, with both theoretical values ​​of 7415.22 and both measured values ​​of 7413.32, which was consistent with the theoretical value. As is clear from the results, the obtained complexes 37 and 38 have the sequences and structures shown in Table 1B above.

[0231] Preparation Examples 39 to 44 Synthesis of Complexes 39 to 42 and Comparative Complexes 43 to 44 According to the same method as in Preparation Example 20, conjugate 39 was produced, and the molecular weight was measured by LC-MS. The theoretical value of conjugate 39 was 9473.03, and the actual value was 9472.06, which coincided with the theoretical value. This shows that the conjugate 39 coincides with the sequence structure in Table 1C. The molecular weights of conjugates 40, 41, 42, comparative conjugate 43, and comparative conjugate 44 were all detected by LC-MS, and the actual values ​​coincided with the theoretical values. According to the method in Preparation Example 20, conjugates 39, 40, 41, 42, comparative conjugate 43, and comparative conjugate 44, each of which has a number in Table 1C, were synthesized, and the molecular weights were detected to confirm the synthesized conjugates. The only difference is that the nucleoside monomers were linked in order according to the sequences corresponding to conjugates 39, 40, 41, 42, comparative conjugate 43, and comparative conjugate 44 in Table 1C, respectively.

[0232] The theoretical MS values ​​and actual MS values ​​of the complex are as shown in Table 3 below.

[0233] Table 3 Mass spectrometry results of the complex [Table 7]

[0234] Experimental Example 1: Comparison of the ability of the conjugate according to the present disclosure and a control conjugate to enter U118-MG glioma cells in vitro In this example, the ability of the complex AP1 and a control complex AP10 to enter U118-MG glioma cells in vitro was evaluated and compared.

[0235] U118MG glioma cells (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were cultured in DMEM medium (Thermo Fisher) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher) at 37°C in an incubator containing 5% CO2 / 95% air.

[0236] 5 × 10 U118MG cells 5The cells / well were seeded on a 96-well plate. The complex AP1 and yeast tRNA thus prepared were prepared into complex solutions 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, and 1I, respectively, using DMEM medium. The complex solutions 1A to 1I contained yeast tRNA at a final concentration of 100 μg / mL, and complex AP1 at final concentrations of 5 nM, 10 nM, 20 nM, 40 nM, 60 nM, 80 nM, 100 nM, 150 nM, 200 nM, and 500 nM, respectively.

[0237] Using DMEM medium, the prepared control complex Comparative AP10 and yeast tRNA were prepared into complex solutions 1A', 1B', 1C', 1D', 1E', 1F', 1G', 1H' and 1I', respectively. The complex solutions 1A' to 1I' contained yeast tRNA at a final concentration of 100 μg / mL, and control complex Comparative AP10 at final concentrations of 5 nM, 10 nM, 20 nM, 40 nM, 60 nM, 80 nM, 100 nM, 150 nM, 200 nM and 500 nM, respectively.

[0238] The above complex solutions 1A to 1I were added to the culture wells containing the U118MG cells, respectively, with the amount added being 200 μL / well, and recorded in order as test group 1A to test group 1I. The above complex solutions 1A' to 1I' were added to the culture wells containing the U118MG cells, respectively, with the amount added being 200 μL / well, and recorded in order as control group 1A' to test group 1I'. 200 μL of DMEM medium containing yeast tRNA at a final concentration of 100 μg / mL was added to another culture well containing U118MG cells, and recorded in order as blank control group 1J. The 96-well plate containing each of the above test groups and the blank control group was incubated at 37° C. for 1 h in the dark, and after the incubation was completed, the supernatant was sucked out from the reaction mixtures of each of the test groups and the blank control group, and DPBS buffer (purchased from Thermo Fisher) was added and washed three times with 100 μL per time. After removing the washing solution, 100 μL of DMEM medium was added to each culture well to obtain a cell suspension. After removing the supernatant by centrifugation, 500 μL of DPBS buffer was added to resuspend the cells, and flow cytometry was performed using a flow cytometer (BD Biosciences). 10,000 cells were analyzed in each test group or blank control group, and the fluorescence intensity distribution was measured to calculate the mean fluorescence intensity (MFI, au). Furthermore, the mean fluorescence intensity corresponding to the final concentration of each complex was compared as shown in Table 4 below, and the relative fluorescence difference R was calculated according to the following formula. R = (MFI AP1 -MFI CAP10 ) / MFI CAP10 MFI AP1 represents the mean fluorescence intensity in U118-MG glioma of complexed AP1, MFI CAP10represents the average fluorescence intensity of comparative AP10 in U118-MG glioma cells. The R value reflects the relative ability of the complex AP1 to enter U118-MG glioma, with R>2 indicating a high ability of the corresponding complex to enter cells (see Table 2 in Engineering of Targeted Nanoparticles for Cancer Therapy Using Internalizing Aptamers Isolated by Cell-Uptake Selection. ACS Nano. 2012 January 24; 6(1), the entire disclosure of which is incorporated herein by reference).

[0239] Table 4. Mean fluorescence intensity and R value of the complex [Table 8]

[0240] As can be seen from Table 4, the complex AP1 of the present disclosure exhibited significantly higher mean fluorescence intensity in U118MG glioma cells than the random sequence comparison AP10 at different concentrations, indicating that the complex of the present disclosure has a superior ability to enter U118MG glioma cells.

[0241] Experimental example 2: Observation of the complex entering different cells using a fluorescent imaging system In this experiment, a PerkinElmer high content imaging system (Operatta) was used to observe the entry of the complex AP1 of the present disclosure and the comparative complex AP10 into different cells.

[0242] U118MG glioma cells, SVGp12 normal astrocytes, T98G human brain glioma cells, U251 human glioma cells, A549 human non-small cell lung cancer cells, MCF-7 human breast cancer cells, and 293T human renal epithelial cells were all purchased from Shanghai Cell Bank, Chinese Academy of Sciences. Before the experiment, logarithmically grown cells were selected for each cell line, and 5000 cells per well were inoculated into a 96-well plate. Each cell line was inoculated into two culture wells and cultured at 37°C in an incubator containing 5% CO2 / 95% air for 24 h. The supernatant was aspirated, and DPBS buffer (purchased from Thermo Fisher) was added and washed twice with 100 μL each time.

[0243] The complex AP1 and yeast tRNA thus produced were each prepared into complex solution 2A using DMEM medium, which contained yeast tRNA at a final concentration of 100 μg / mL and complex AP1 at a final concentration of 100 nM.

[0244] Using DMEM medium, the prepared control complexes, Comparative AP10 and Yeast tRNA, were each prepared into Control Complex Solution 2B, which contained Yeast tRNA at a final concentration of 100 μg / mL and Comparative AP10 at a final concentration of 100 nM.

[0245] For each cell line, 200 μL of complex solution 2A was added to one culture well, incubated at 37° C. for 30 minutes in the dark, the supernatant was aspirated, and DPBS buffer was added to wash twice with 100 μL each time to obtain test group 2X.

[0246] For each cell line, 200 μL of control complex solution 2B was added to a separate culture well, incubated at 37° C. for 30 minutes in the dark, the supernatant was aspirated, and DPBS buffer was added to wash twice with 100 μL each time to obtain control group 2Y.

[0247] 100μL of 37℃ DMEM medium was added to each of the test group 2X and control group 2Y of each of the above cell lines, and imaged in a high content imaging system. The fluorescence intensity was normalized by the average fluorescence intensity of the control group 2Y, that is, the average fluorescence intensity of the control group 2Y was subtracted from the fluorescence intensity of the test group 2X before imaging. The results are shown in Figure 1A to Figure 1G, respectively.

[0248] 1A and 1B are high content imaging pictures showing AP1 and comparative AP10 entering U118MG glioma cells and SVGp12 normal astrocytes, respectively. As can be seen from Fig. 1A and 1B, only U118MG cells treated with AP1 showed strong Cy5 fluorescent signal, while SVGp12 cells had no fluorescent signal in either the test or control groups, indicating that the conjugate of the present disclosure has a higher ability to enter U118MG glioma cells and does not enter SVGp12 normal astrocytes compared to comparative AP10. As can be seen from the above results, the conjugate provided by the present invention can selectively target and deliver functional groups to U118MG glioma cells.

[0249] Furthermore, Figures 1C-1G are high content imaging photographs showing AP1 and comparative AP10 entering U251 human glioma cells, A549 human non-small cell lung cancer cells, MCF-7 human breast cancer cells, and 293T human renal epithelial cells, respectively. As can be seen from Figures 1C-1G, U251 human glioma cells, A549 human non-small cell lung cancer cells, and MCF-7 human breast cancer cells treated with AP1 all showed strong Cy5 fluorescent signals, but the tumor cells and 293T human renal epithelial cells treated with comparative AP10 in each group did not have any fluorescent signals in either the test group or the control group. This shows that, compared with comparative AP10, the conjugate according to the present disclosure has a high ability to enter various tumor cells, does not enter normal cells, and can selectively target and deliver functional groups to various tumor cells.

[0250] Experimental Example 3: Observation of the complex entering tumor spheres using a fluorescent imaging system In this experiment, a PerkinElmer high content imaging system (Operatta) was used to observe the entry of the complex AP1 of the present disclosure and the comparative complex Comparative AP10 into U118MG glioma tumor spheres and A549 human non-small cell lung carcinoma tumor spheres.

[0251] 80 μL of fresh, sterile, uncoagulated 2% agarose was added to the culture wells of a 96-well plate, and after complete coagulation, logarithmically grown cells were selected for each cell line and inoculated into a 96-well plate at 2000 cells per well. Each cell line was inoculated into two culture wells, 200 μL of cell suspension was added to each well, and the cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin double antibody, and cultured at 37°C in an incubator containing 5% CO2 / 95% air. Every 48 h, half of the supernatant was sucked out, applied to the wall of the culture well, and new DMEM medium was added. After 7 days of culture, observation under a microscope confirmed that complete cell spheres were obtained, indicating that the tumor sphere culture was successful.

[0252] According to the method of Experimental Example 2, AP1 or comparative AP10 was added to each culture well and imaged with a high content imaging system. The only difference was that after culture, the wells were directly imaged without washing, and imaged according to the measured Cy5 fluorescence intensity. The results are shown in Figures 2A and 2B.

[0253] 2A and 2B are high content imaging photographs showing AP1 and Comparative AP10 entering U118MG glioma tumor spheres and A549 human non-small cell lung carcinoma tumor spheres, respectively. As can be seen from FIGS. 2A and 2B, U118MG tumor spheres and A549 tumor spheres treated with AP1 showed strong Cy5 fluorescent signals inside, while tumor spheres treated with Comparative AP10 showed clearly weak fluorescent signals only at the edge of the tumor sphere. This shows that the complex according to the present disclosure has a higher ability to enter the interior of U118MG and A549 tumor spheres than Comparative AP10. As is clear from the above results, the aptamer according to the present disclosure can effectively deliver functional groups into the tumor interior and has high drug discovery potential.

[0254] Experimental Example 4 Distribution of the complex in the body of U118MG cell subcutaneous tumor model mice U118MG human glioma cells (purchased from Guangzhou Genio Biotech Co., Ltd.) were cultured in DMEM complete medium (MACGENE, product number CM15019) supplemented with 10% fetal bovine serum (FBS, RMBIO) at 37°C in an incubator containing 5% CO2 / 95% air.

[0255] Logarithmically growing U118MG human glioma cells were taken and digested with (0.25% pancreatin), the cells were collected, centrifuged to remove the supernatant, and the cells were resuspended in DMEM medium supplemented with 10% FBS to a concentration of 1 × 10 8 A cell culture medium was prepared at 100 μg / mL.

[0256] The experimental animals were 10 12-week-old male NOD-SCID mice (purchased from Beijing Sibaifu Biotechnology Co., Ltd.). The above cell culture medium was inoculated into the right front leg subcutaneous site of the NOD-SCID mice, with an inoculation volume of 100 μL per mouse, i.e., 1 × 10 7 After injection, the mice were kept for 20 days.

[0257] AP1, AP2 and comparative AP11 were each dissolved in a solution with a concentration of 0.3 mg / mL (calculated as aptamer) using 1×DMEM (purchased from Zhongke Maichen (Beijing) Technology Co., Ltd., lot number K1902200) medium.

[0258] The mice inoculated with U118MG were randomly divided into three groups, with four mice in each group, and the administration volume of each of the four mice in each group was calculated as 10 μL / g body weight. AP1, AP2 and comparative AP11 were administered to different groups of mice by tail vein injection, and recorded as test group 4A1, test group 4A2 and control group 4A3, respectively. Thus, the dosage of each mouse was 3 mg / kg (calculated by aptamer). Another mouse inoculated with U118MG was injected with 1×DMEM medium via tail vein, and the administration volume was 10 μL / g body weight, and recorded as blank control group 4Y.

[0259] One hour after administration, the mice from each group were placed in the small animal bio-optical imaging system IVIS Lumina Series III (PerkinElmer) in turn, the mice were anesthetized with isoflurane gas, and the anesthetized mice were placed with their abdomens facing up in the small animal bio-optical imaging system for bio-imaging. One mouse from each group was sacrificed at 1 h, 4 h, 24 h, and 48 h after administration, respectively, and tumor tissue was taken for fluorescence imaging (no blank control group was recorded for the results at 24 h and 48 h), and the results are shown in Figures 3A, 3B, 3C, and 3D, respectively.

[0260] 3A-3D are photographs of bioimaging and tumor tissue imaging 1 h, 4 h, 24 h, and 48 h after administration, respectively, and blank represents the blank control group 4Y. As can be seen from FIG. 3A-3D, the blank control group 4Y and the control group 4A3 administered with the control AP11 having a random sequence showed little or only weak fluorescent signals in the mouse body, whereas the complexes AP1 and AP2 according to the present disclosure showed strong fluorescent signals when tumors were inoculated in the mouse body. This shows that the aptamer according to the present disclosure can specifically target and deliver fluorescent groups to U118MG glioma. Furthermore, the complex AP2 showed strong fluorescent signals even 24 h to 48 h after administration. This shows that the complex according to the present disclosure can stably target glioma tissue for a long period of time.

[0261] Experimental Example 5 Distribution of the complex in the body of U118MG cell in-situ tumor model mice Logarithmically growing U118MG human glioma cells cultured in Experimental Example 4 were taken, digested with 0.25 wt% pancreatin to harvest the cells, the supernatant was aspirated, and the cells were resuspended in DMEM medium supplemented with 10% FBS to a cell density of 4 × 10 7 A cell culture medium was prepared at 100 μg / mL.

[0262] The experimental animals were 16 male NOD-SCID mice aged 12 weeks (purchased from Beifong Biological Technology Co., Ltd.). The above cell culture fluid was inoculated into the NOD-SCID mice, and the cell culture fluid was injected into the right striatum of the mice by mouse lateral ventricle injection at AP (anteroposterior): 1 mm, ML (medial lateral): 1.5 mm, and DV (dorsal ventral): 3.5 mm, with an injection volume of 10 μL, i.e., 4 × 10 cells were injected into each mouse. 5 After injection, the mice were kept for 14 days.

[0263] AP1, AP2 and comparative AP11 were dissolved in a complex solution with a concentration of 2mg / mL (calculated by aptamer) using 1xDMEM (purchased from Zhongke Maichen (Beijing) Technology Co., Ltd., lot number K1902200) medium. Ten mice were taken and 50μL of AP1 and AP2 solutions were injected by intrathecal injection, and the dose per animal was 100μg according to the amount of aptamer. Three mice were administered in each group, and recorded as test groups 5A and 5B, respectively; two mice were injected with 50μL of control AP11 solution, and the dose per animal was 100μg according to the amount of aptamer, and recorded as control group 5C, and two mice were injected with 50μL of DMEM medium, and recorded as blank control group 5Y.

[0264] Using 1xDMEM medium, AP1, AP2 and comparative AP11 were dissolved in a complex solution of 0.3mg / mL concentration (calculated by aptamer).Another 6 mice were taken and injected with AP1, AP2 and comparative AP11 solutions by tail vein injection, and the dosage was calculated according to the body weight of each animal, the administration volume was 10μL / g, and the dosage per animal was 3mg / kg calculated by the amount of aptamer, and 2 mice were administered in each group, which were recorded as test group 5D, 5E and control group 5F, respectively.

[0265] 24 h after administration, one mouse from each group was sacrificed to obtain brain tissue, and 48 h after administration, the remaining mice were sacrificed to obtain brain tissue, and fluorescent imaging of mouse brain tissue was performed using an IVIS Lumina Series III. The results are shown in Figure 4A and Figure 4B, respectively.

[0266] 4A and 4B are photographs of fluorescent imaging of mouse brain tissues in which an in-situ tumor model of U118MG was constructed after 24 h and 48 h of administration of blank control group 5Y, test groups 5A, 5B, 5D, and 5E, and control groups 5C and 5F, respectively. As can be seen from the results of FIG. 4A and FIG. 4B, the blank control group and the control group of comparative AP11 did not show any significant fluorescent signal at the tumor location, indicating that there was no significant targeting effect on the in-situ tumor brain glioma. In contrast, test groups 5A and 5B administered with the complex according to the present disclosure showed strong fluorescent signals at the tumor inoculation location. This indicates that the tumor tissue can be effectively targeted, and also indicates that the aptamer according to the present disclosure can effectively deliver diagnostic groups such as fluorescent groups to tumor tissue. Furthermore, test group 5E showed significant fluorescent signals at both 24 h and 48 h. This demonstrates that the AP2 complex can still reach and target brain gliomas upon tail vein administration, and also demonstrates that the complexes of the present disclosure can penetrate the blood-brain-barrier (BBB) ​​and enter brain gliomas.

[0267] Experimental Example 6 Distribution of different modified conjugates in the body of U118MG subcutaneous tumor model mice According to the method described in Experimental Example 4, U118MG subcutaneous tumor model mice were prepared.

[0268] Using 1xDMEM medium, AP2, AP3 and AP12 were dissolved in a complex solution with a concentration of 0.3mg / mL (calculated by aptamer). Nine mice were divided into three groups, with three mice in each group, and the above AP2, AP3 and AP12 solutions were injected into each mouse in each group via the tail vein. The dosage was calculated according to the body weight of all animals, and the administration volume was 10μL / g for each animal. When calculated by the amount of aptamer, the dosage per animal was 3mg / kg, and the three groups of mice were recorded as test group 6A, test group 6B and test group 6C, respectively. The other two mice were administered DMEM medium, the administration volume was 10μL / g, and recorded as blank control group 6Y. 30 min, 1 h, 24 h, and 48 h after administration, one mouse from each group was placed in a small animal bio-optical imaging system IVIS Lumina Series III (PerkinElmer), the mouse was anesthetized with isoflurane gas, and the anesthetized mouse was placed in the small animal bio-optical imaging system with its abdomen facing up to perform bio-imaging. The results are shown in Figures 5A to 5D. Ten days after administration, bio-imaging was performed on one mouse from the blank control group 6Y and the test group 6A, and the results are shown in Figure 5E. 24 h and 48 h after administration, one mouse from each group was sacrificed, tumor tissue was taken, and fluorescent imaging was performed, and the results are shown in Figures 5F and 5G, respectively. Ten days after administration, one mouse from the blank control group 6Y and the test group 6A was sacrificed, tumor tissue was taken, and fluorescent imaging was performed, and the results are shown in Figure 5H. In Figures 5A to 5H, Blank represents the blank control group 6Y.

[0269] 5A to 5E are photographs of fluorescence imaging in mice at different time points after administration of the complex according to the present disclosure and the comparative complex of different sequences, respectively, and FIG. 5F to FIG. 5H are photographs of tumor tissue imaging at different time points. As is clear from the results of FIG. 5A to FIG. 5H, 30 min after administration, the subcutaneous tumors of each test group and the control group all showed fluorescent signals, and 4 h to 24 h after administration, the mice of test groups 6A, 6B, and 6C administered with AP2, AP3, and AP12 according to the present disclosure also showed high fluorescent intensity, and 48 h to 10 days after administration, the mice of test group 6A administered with AP2 also showed strong fluorescent signals. As is clear from the above results, compared to the control complex, the complex according to the present disclosure can stably target tumor tissue for a long period of time, and it has been shown that the aptamer according to the present disclosure can stably deliver the diagnostic agent group to the tumor for a long period of time.

[0270] Experimental Example 7 Distribution of the complex in the body of A549 subcutaneous tumor model mice A549 human non-small cell lung cancer cell subcutaneous tumor model mouse was prepared according to the method described in Experimental Example 4. The only difference was that the cell culture medium was prepared using A549 human non-small cell lung cancer cells instead of U118MG glioma cells.

[0271] Eleven 6-8 week-old male NOD-SCID mice (purchased from Beijing Sibaifu Biotechnology Co., Ltd.) were selected and randomly divided into groups, with two mice in the blank control group and three mice in each of the remaining groups. The above-mentioned medium containing A549 tumor cells was inoculated into the right forelimb subcutaneous site of each mouse. The inoculation volume of A549 tumor cells per mouse was 100 μL, and the calculated number of inoculated cells per mouse was 1 × 10 7 There were 100 pieces.

[0272] AP2, AP3 and AP12 were dissolved in a complex solution with a concentration of 0.3 mg / mL (calculated by aptamer) using 1×DMEM (purchased from Zhongke Maichen (Beijing) Technology Co., Ltd., lot number K1902200) medium. After inoculation of the A549 tumor cells for 21 days, 1×DMEM and the AP2, AP3 and AP12 complex solutions prepared above were injected into each mouse via the tail vein, and the mice injected with 1×DMEM, AP2, AP3 and AP12 complex solutions were named blank control group, AP2 group, AP3 group and AP12 group, respectively. The dosage of all animals was calculated according to body weight, and the administration volume of each mouse was 10 μL / g. That is, except for the blank control group, the dosage of each mouse in the remaining groups was 3 mg / kg (calculated by the amount of aptamer), and the blank control group was administered only 10 μL / g of DMEM medium.

[0273] One mouse from each group was sacrificed 1 h and 24 h after administration, and the tumor tissues of each sacrificed mouse were taken and subjected to fluorescence imaging using an IVIS Lumina Series III. The two organs of each mouse were placed laterally and photographed in the same field of view. The results are shown in Figure 6.

[0274] Figure 6 shows the organ fluorescence images of one mouse from each group 1 h after administration. As can be seen from Figure 7, except for the blank control group, strong fluorescent signal intensity was detected in the A549 tumors of the AP2, AP3 and AP12 groups, and the fluorescent signal intensity of the AP3 group was higher than that of the other groups.

[0275] As can be seen from the above results, all of the conjugates with different modifications of the present disclosure are able to specifically target A549 tumors.

[0276] Experimental Example 8 Ability of the Conjugates of the Present Disclosure to Enter PAN02 Tumor Cells In this example, a PAN02 tumor cell model was used to verify the ability of the conjugates of the present disclosure to enter PAN02 tumor cells.

[0277] Logarithmically grown PAN02 tumor cells (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were harvested and digested to obtain a cell density of 1 × 10 8 The cells were resuspended in DMEM medium supplemented with 10% fetal bovine serum until the number of cells / mL was reached.

[0278] One 6-week-old C57 / BL mouse (male) was selected, and the medium containing the above-mentioned PAN02 tumor cells was inoculated subcutaneously into the right forelimb of the mouse. The volume of the PAN02 tumor cells inoculated into the mouse was calculated as 100 μL, and the number of cells inoculated into the mouse was 1 × 10 7 There were 100 pieces.

[0279] AP2 was dissolved in a complex solution with a concentration of 0.3 mg / mL (calculated by aptamer) using 1×DMEM (purchased from Zhongke Maichen (Beijing) Technology Co., Ltd., lot number K1902200) medium. After inoculation of the above PAN02 tumor cells for 21 days, the AP2 complex solution was injected into the tail vein of the mouse. The dosage was calculated according to the mouse weight, and calculated by the amount of aptamer, the dosage volume was 10 μL / g and the dosage was 3 mg / kg.

[0280] 135 minutes after administration, the mice were sacrificed, and the heart, liver, spleen, lung and tumor tissues of the sacrificed mice were removed and subjected to fluorescent imaging using an IVIS Lumina Series III. Images were taken from the same field of view, and the results are shown in FIG.

[0281] Figure 7 shows the fluorescence imaging photographs of each organ tissue in a mouse in which a PAN02 subcutaneous tumor model was constructed after administration of the complex of the present disclosure. As can be seen from Figure 7, the fluorescence signal intensity in PAN02 tumor cells is obviously higher than that in other organs, which indicates that the complex of the present disclosure can specifically target PAN02 pancreatic cancer tumor cells.

[0282] Experimental Example 9 Ability of conjugates carrying different fluorescent groups to enter U118MG glioma cells In this example, a U118MG glioma model was used to examine the ability of conjugates comprising a CY5-fluorescent group and a CY3-fluorescent group according to the present disclosure to enter the U118MG glioma model.

[0283] Logarithmically grown U118MG glioma cells (purchased from Guangzhou Geniobiotech Co., Ltd.) were taken, digested, and resuspended in DMEM medium supplemented with 10% fetal bovine serum until the cell density of U118MG glioma cells reached 1× and then diluted with 10 8 The cells / mL.

[0284] Eleven 6-8 week-old male NOD-SCID mice (purchased from Beijing Sibaifu Biotechnology Co., Ltd.) were selected and randomly divided into groups: group 1 with 3 mice, group 2 with 2 mice, group 3 with 3 mice, and group 4 with 3 mice. The above U118MG glioma cells-containing medium was inoculated into the right forelimb subcutaneous site of each mouse. The cell inoculation volume per mouse was 100 μL, and the calculated cell inoculation number per mouse was 1 × 10 7 There were 100 pieces.

[0285] Using 1xDMEM (purchased from Zhongke Maichen (Beijing) Technology Co., Ltd., lot number K1902200) medium, AP2, AP9 and AP12 were dissolved in a complex solution with a concentration of 0.3mg / mL (calculated by aptamer). After inoculation of the above U118MG glioma for 21 days, 1xDMEM and the above-prepared AP2, AP9 and AP12 complex solutions were injected into each mouse via the tail vein. Three mice in the first group were injected with AP2 complex solution, two mice in the second group were injected with AP9 complex solution, three mice in the third group were injected with AP12 complex solution, and three mice in the fourth group were injected with 1xDMEM as a blank control group. The dosage was calculated according to the body weight for all animals, and the dosage volume for each mouse was 10μL / g. That is, the dosage for each mouse in the first to third groups was 3mg / kg (calculated by the amount of aptamer).

[0286] The day of administration was designated as D1, and 48 h, 96 h, and 11 days (D12) after administration, one mouse from each group was sacrificed, and the tumor, lung, liver, and kidney tissues of the sacrificed mice were removed, respectively, and fluorescent imaging was performed using an IVIS Lumina Series III. The four organs of each mouse were arranged vertically in order and photographed in the same field of view, and the results are shown in Figures 8A to 8E, respectively. Group 1 represents the test group administered with AP2, group 2 represents the test group administered with AP9, group 3 represents the test group administered with AP12, and group 4 represents the blank control group.

[0287] FIG. 8A shows the organ fluorescence images of one mouse in group 1, one mouse in group 3, and one mouse in group 4 after 48 h of administration. As can be seen from FIG. 8A, no fluorescent signal was detected in either group 3 or group 4. In addition, a significant fluorescent signal was detected in the tumor tissue of the mouse injected with AP2, but no fluorescent signal was detected in other tissues except the metabolic organ kidney. This shows that AP2 can be effectively targeted to tumor tissue and has no significant targeted delivery in other tissues compared to the comparative complex.

[0288] Figure 8B shows the organ fluorescence images of one mouse in group 1, one mouse in group 3, and one mouse in group 4 96 h after administration. As can be seen from Figure 8B, AP2 still showed significant fluorescence signals in the tumor tissues after 96 h.

[0289] Figure 8C shows the organ fluorescence images of one mouse in group 2 and one mouse in group 4 after 48 h administration. As can be seen from Figure 8C, significant fluorescent signals could be detected in the mouse tumor tissue injected with AP9 labeled with Cy3 compared to the control group. This shows that the aptamer according to the present disclosure can efficiently target and deliver different diagnostic groups (e.g., fluorescent groups) to tumor tissue, and the conjugates containing the diagnostic groups according to the present disclosure can stably target tumor tissue.

[0290] FIG. 8D shows the organ fluorescence images of one mouse in group 2 and one mouse in group 4 96 h after administration. As can be seen from FIG. 8D, significant fluorescent signals could be detected in the tumor tissue of the mouse injected with AP9 labeled with Cy3, but no fluorescent signals were detected in other tissues except the metabolic kidney. Thus, the aptamer according to the present disclosure can efficiently deliver different diagnostic groups (e.g., different fluorescent groups) to U118MG glioma and can be stably maintained for a long period of time.

[0291] FIG. 8E shows the organ fluorescence images of one mouse in group 1, one mouse in group 3, and one mouse in group 4 after 11 days of administration. As can be seen from FIG. 8E, after 11 days of administration, AP2 still had a significant fluorescent signal in tumor tissue, but no fluorescent signal was detected in other tissues except for the metabolic organ kidney. Thus, the complex of the present disclosure can still stably and highly specifically target U118MG glioma tissue for a long period of time.

[0292] As can be seen from the above results, the aptamer of the present disclosure can not only specifically target glioma tissue, but also deliver different diagnostic agent groups to glioma.Therefore, the aptamer of the present disclosure shows excellent delivery ability, and the complex containing the diagnostic agent group of the present disclosure can efficiently and stably target glioma, so that the presence of glioma can be diagnosed quickly and effectively, and the diagnostic effect can be stably maintained for a long period of time.

[0293] Experimental Example 10: Targeting effect of complexes with different sequences and lengths on U118MG glioma In this example, the U118MG glioma model was used to investigate the targeting effects of complexes with different sequences and lengths on U118MG glioma.

[0294] Logarithmically grown U118MG glioma cells (purchased from Guangzhou Genio Biotech Co., Ltd.) were taken and digested to obtain a cell density of U118MG glioma cells of 1 × 10 8 The cells were resuspended in DMEM medium supplemented with 10% fetal bovine serum until the number of cells / mL was reached.

[0295] Fourteen 6-8 week-old male NOD-SCID mice (purchased from Beijing Sibaifu Biotechnology Co., Ltd.) were selected and randomly divided into groups, with two mice per group. The above U118MG glioma cell-containing medium was inoculated into the right forelimb subcutaneous site of each mouse. The inoculation volume of U118MG glioma per mouse was 100 μL, and the calculated number of inoculated cells per mouse was 1 × 10 7 There were 100 pieces.

[0296] Using 1×DMEM (purchased from Zhongke Maichen (Beijing) Technology Co., Ltd., lot number K1902200), AP2, AP4, AP5, AP6, AP7, AP8 and AP12 were dissolved in a complex solution with a concentration of 0.3 mg / mL (calculated by aptamer). After inoculation of the above U118MG glioma for 21 days, AP2, AP4, AP5, AP6, AP7, AP8 and AP12 complex solutions were injected into each mouse via the tail vein. The mice in each group injected with AP2, AP4, AP5, AP6, AP7, AP8 and AP12 were named AP2 group, AP4 group, AP5 group, AP6 group, AP7 group, AP8 group and AP12 group, respectively. The dosage was calculated according to the body weight for all animals, and the dosage volume for each mouse was 10 μL / g when calculated by the amount of aptamer. That is, the dosage for each mouse was 3 mg / kg.

[0297] The day of administration was designated as D1, and after 1 h, 24 h (D2), 48 h (D3) and 72 h (D4), the mice in each group were placed in the small animal bio-optical imaging system IVIS Lumina Series III in turn, anesthetized with isoflurane gas, and placed in the small animal bio-optical imaging system with the abdomen of the anesthetized mouse facing up for bio-imaging, and Cy5 fluorescent signals were dynamically detected to track the distribution of different Cy5-labeled complexes in the animal body. The observation results were analyzed. After 1 h of administration, fluorescent signals were observed in all subcutaneous tumors in each mouse, and there was no significant difference in the fluorescent signal intensity between the mice in each group. After 24 h and 48 h of administration, fluorescent signals were still detectable in the mice in each group, but the fluorescent signals in the subcutaneous tumors in the mice injected with AP6 and AP8 were all stronger than the fluorescent signals in the subcutaneous tumors in the mice injected with AP2, AP4, AP5, AP7 and AP12.

[0298] On D9, one mouse from each group was sacrificed, and the tumor, lung, liver, and kidney tissues of each sacrificed mouse were taken out and subjected to fluorescence imaging using an IVIS Lumina Series III. The tumor tissues of each mouse were arranged vertically and photographed in the same field of view, and the results are shown in Figure 9.

[0299] Figure 9 is the tumor fluorescence image of each group of mice after administration D9. As can be seen from Figure 9, Cy5 fluorescent signal is detected in the tumor tissue of each group of mice, which shows that the complexes of different lengths and sequences of the present disclosure can stably and effectively target tumor tissue within a long time.

[0300] Experimental Example 11 Activity of the complex according to the present disclosure in mice In this experimental example, the antitumor activity of the prepared complex 20 in mice was investigated.

[0301] The mice used in this experiment were purchased from Siberia, the germ cell line was NOD-SCID, the grade was SPF, the sex was female, and the age was 6 to 8 weeks. U118MG glioma cells were purchased from Genio.

[0302] Logarithmically growing U118MG cells were digested and resuspended in DMEM medium (MACGENE, product number CM15019) containing 10% fetal bovine serum (FBS, GIBCO) to a cell density of 1 × 10 8 The U118MG cell-containing culture solution was then inoculated into the right forelimb subcutaneous site of each mouse, with an injection volume of 100 μL. Thus, each mouse received 1 × 10 7 U118MG glioma cells were inoculated.

[0303] The above-prepared complex AP2 was prepared into a 1.94 mg / mL solution using PBS. The complex 20 was prepared into 0.625 mg / mL, 1.25 mg / mL and 2.06 mg / mL solutions (all calculated based on the amount of aptamer) using PBS, and MMAE (purchased from Shanghai McKin Co., Ltd., lot number C12886583) was dissolved into a 0.038 mg / mL solution using a mixture of 10% DMSO + 90% PBS (volume ratio).

[0304] The day of cell inoculation was designated as D1, and administration was performed once each on D8, D12, D16, and D20.

[0305] Thirty-six mice were randomly divided into the following six groups, with six mice in each group:

[0306] For the blank control group 1, PBS was administered via tail vein injection, with a single administration volume of 10 μL / g.

[0307] For the control group 2, the above complex AP2 solution was administered by tail vein injection, with a single dose volume of 10 μL / g and a single dose amount of 15.5 mg / kg.

[0308] For control group 3, the above MMAE solution was administered via tail vein injection, with a single dose volume of 10 μL / g and a single dose of 0.3 mg / kg.

[0309] For test group 4, the above-mentioned concentration of 0.625 mg / mL of complex 20 solution was administered by tail vein injection, with a single-dose volume of 10 μL / g, a single-dose amount of 5 mg / kg (calculated by the mass of the aptamer), and a dose containing MMAE of 0.3 mg / kg.

[0310] For test group 5, the above-mentioned concentration of 2.06 mg / mL of complex 20 solution was administered by tail vein injection, with a single-dose volume of 10 μL / g, a single-dose amount of 16.5 mg / kg (calculated by the mass of the aptamer), and a dose containing MMAE of 1 mg / kg.

[0311] For test group 6, the above-mentioned concentration of 1.25 mg / mL of complex 20 solution was administered by subcutaneous injection, with a single-dose volume of 5 μL / g, a single-dose amount of 5 mg / kg (calculated by the mass of the aptamer), and a dose containing MMAE was equivalent to 0.3 mg / kg.

[0312] [3] Detection The long and short diameters of the tumor were measured by external measurement. The tumor volume was calculated using the formula 1 / 2 (long diameter × short diameter). 2 Before the first administration on D8, the tumor volume of each group was measured and the average tumor volume was recorded, and from D16, the tumor volume of each group was measured and recorded, and was measured twice a week.

[0313] FIG. 10 is a line graph showing the change in tumor volume over time in mice of each group. As can be seen from the results of FIG. 10, the tumor volume increased rapidly in blank control group 1 and control group 2, which were administered with only PBS and AP2, while the rate of increase in tumor volume slowed in control group 3, which was administered with only MMAE. This indicates that MMAE itself has an inhibitory effect on tumor growth. Furthermore, test groups 4 and 6, whose MMAE content corresponds to control group 3, both had significantly smaller tumor volumes during the test than control group 3, and showed better antitumor activity than control group 3, which was administered with MMAE alone. This indicates that the conjugate according to the present disclosure can effectively deliver MMAE to tumor tissue, demonstrating tumor targeting ability, while reducing the toxicity risk caused by the distribution of MMAE molecules to other tissues, and that various administration methods can effectively inhibit tumor growth. In addition, the tumor volume of test group 5, which was further increased in dosage, hardly increased during the test period, showing a better antitumor effect. This indicates that the conjugate containing the small molecule drug group according to the present disclosure can rapidly and stably produce an inhibitory effect on tumors.

[0314] As is evident from the above results, the conjugates according to the present disclosure can effectively target and deliver small molecule drug groups having inhibitory effects on tumors to tumor tissues, and the conjugates containing the small molecule drug groups according to the present disclosure exhibit excellent antitumor activity and dose-dependent effects.

[0315] Experimental Example 12: Effect of the complex according to the present disclosure on the viability of normal cells and tumor cells In this experiment, the effect of the prepared complex 19 on the viability of normal cells and tumor cells was investigated.

[0316] In this experiment, U118-MG glioma cells were purchased from Genio, SVGp12 human astrocytes were purchased from Shanghai Cell Bank, Chinese Academy of Sciences, and CCK8 kit was purchased from Dojindo Laboratories.

[0317] Logarithmically grown U118-MG cells and SVGp12 cells were selected, seeded at 5,000 cells per well into a 96-well plate of the CCK8 kit, and cultured at 37°C and 5% CO2 for 24 hours. When the cell fusion rate reached 80% under a microscope, the target U118-MG cells and target SVGp12 cells were obtained.

[0318] Using DMEM complete medium (MACGENE, product number CM15019) supplemented with 10% fetal bovine serum (FBS, GIBCO), AP1, complex 19 and yeast tRNA were each gradient-diluted to obtain complex solutions AP1 solution 1 (concentration 0 nM), AP1 solution 2 (concentration 50 nM), AP1 solution 3 (concentration 200 nM), AP1 solution 4 (concentration 800 nM), and complex solutions complex 19 solution 1 (concentration 0 nM), complex 19 solution 2 (concentration 25 nM), complex 19 solution 3 (concentration 50 nM), complex 19 solution 4 (concentration 100 nM), complex 19 solution 5 (concentration 200 nM), complex 19 solution 6 (concentration 400 nM), and complex 19 solution 7 (concentration 800 nM). Each of the above complex solutions contains yeast tRNA at a final concentration of 100 μg / ml.

[0319] AP1 solution 1, AP1 solution 2, AP1 solution 3 and AP1 solution 4 were added to the target U118-MG cells, and incubated at 37°C for 72 h in the dark. Each group was repeated five times and recorded as blank control group 12A1, test groups 12A2, 12A3 and 12A4, respectively.

[0320] AP1 solution 1, AP1 solution 2, AP1 solution 3 and AP1 solution 4 were added to the SVGp12 cells to be detected, and incubated at 37°C for 72 hours in the dark. Each group was repeated five times and recorded as blank control group 12B1, test groups 12B2, 12B3 and 12B4, respectively.

[0321] Complex 19 solution 1, complex 19 solution 2, complex 19 solution 3, complex 19 solution 4, complex 19 solution 5, complex 19 solution 6 and complex 19 solution 7 were added to the target U118-MG cells, and incubated at 37°C for 72h in the dark. The experiment for each group was repeated five times, and recorded as blank control group 12C1 and test groups 12C2 to 12C7, respectively.

[0322] Complex 19 solution 1, complex 19 solution 2, complex 19 solution 3, complex 19 solution 4, complex 19 solution 5, complex 19 solution 6 and complex 19 solution 7 were added to the SVGp12 cells to be detected, and incubated at 37°C for 72h in the dark. The experiment for each group was repeated five times, and recorded as blank control group 12D1 and test groups 12D2 to 12D7, respectively.

[0323] Using CC8 kit, the UV absorbance of each test group and the control group was detected at a detection wavelength of 450 nm in the UV absorption spectrum according to the method of the specification, and the average value was taken from repeated experiments for each group. The cell viability was calculated according to the following formula:

[0324] Cell viability% = (OD450 of test group - OD450 of blank group) / (OD450 of control group - OD450 of blank group) x 100% Test group OD450 refers to the absorbance value of each test group at 450 nm, control group OD450 is the absorbance value of the blank control group corresponding to each test group at 450 nm, and blank group OA450 is the absorbance value of the blank cell used for ultraviolet detection at 450 nm. The results are shown in Figure 11A and Figure 11B.

[0325] FIG. 11A shows the effect of different concentrations of complex AP1 on the cell viability of U118-MG glioma cells and SVGp12 human astrocytic tumor cells, and FIG. 11B shows the effect of different concentrations of complex 19 on the cell viability of U118-MG glioma cells and SVGp12 astrocytic tumor cells.

[0326] As can be seen from the results of FIG. 11A, the complex AP1 containing only the diagnostic group CY5 does not affect the cell viability of normal cells and tumor cells at different concentrations. As can be seen from the results of FIG. 11B, the complex 19 containing siRNA can significantly reduce the cell viability of tumor cells, and the inhibitory efficacy improves with increasing dose. In addition, for normal cells, the cell viability only decreases slightly and can be basically maintained stable. This shows that the aptamer according to the present disclosure can not only efficiently target tumor cells to suppress the content of target mRNA after complexing with siRNA, but also has no significant effect on the corresponding mRNA level in normal cells, and has excellent target delivery efficiency and high safety. In short, as is clear from the above results, the complex containing the functional oligonucleotide according to the present disclosure can effectively target and deliver functional oligonucleotides such as siRNA that have an inhibitory effect on tumors to tumor tissues, and can show excellent antitumor activity and dose-dependent effect.

[0327] Experimental Example 13: Targeting of the complex according to the present disclosure in the mouse body In this experimental example, the targeting properties of the prepared conjugates 15, 16, 17 and 18 in mice were investigated.

[0328] Conjugates 15 to 18 contain the same aptamer sequence. For conjugate 15, the sense strand sequence includes a sense strand of an siRNA, the 5' end of the sense strand of the siRNA is bound to a fluorescent group Cy5, and the 3' end is bound to an aptamer according to the present disclosure via a GAU trinucleotide subunit as a binding group.

[0329] For complex 16, the sense strand sequence includes a sense strand of an siRNA, the 5' end of the sense strand of the siRNA is bound to an aptamer of the present disclosure via a GAU trinucleotide subunit as a linking group, and the 5' end of the aptamer is bound to the fluorescent group Cy5.

[0330] Complex 17 is a complex comprising a double-stranded siRNA and an aptamer according to the present disclosure, in which the aptamer is bound to the 5' end of the siRNA sense strand via four propylene phosphate ester groups linked in sequence as linking groups, and a Cy5 fluorescent group is bound to the 5' end of the aptamer.

[0331] Conjugate 18 is structurally similar to conjugate 17, and the aptamer structure is slightly different from that in conjugate AP2, see Table 1B and Preparation Example 18.

[0332] According to the method of Experimental Example 4, U118MG cells were cultured and subcutaneously inoculated into mice.

[0333] Using PBS, each of the above-prepared conjugates 15, 16, 17 and 18 was prepared into a 0.3 mg / mL solution.

[0334] Administration began 30 days after inoculation of U118MG cells, and the day of administration was designated as D1. In the experiment, the mice were administered once a day via tail vein injection for a total of three times.

[0335] Twelve mice (each weighing approximately 25 g) were randomly divided into four groups, with three mice in each group.

[0336] For test group 1, complex 15 was used, the single dose volume was 250 μL, and the single dose amount was 3 mg / kg.

[0337] For test group 2, conjugate 16 was used, with a single dose volume of 250 μL and a single dose of 3 mg / kg.

[0338] For test group 3, conjugate 17 was used, with a single dose volume of 250 μL and a single dose of 3 mg / kg.

[0339] For test group 4, conjugate 18 was used, with a single dose volume of 250 μL and a single dose of 3 mg / kg.

[0340] [3] Detection Twenty-four days after the initial administration, bioimaging was performed on each mouse using the small animal biooptical imaging system IVIS Lumina Series III.

[0341] FIG. 12 is a photograph showing the results of fluorescence imaging in mice after administration of different conjugates. As can be seen from FIG. 12, each conjugate using different binding groups, different binding methods, and different aptamers was concentrated in tumor cells. As can be seen from the above results, although the aptamers contained therein are bound to siRNA in different ways or the aptamer sequences are changed, the various conjugates according to the present disclosure can all effectively target tumor tissues. Furthermore, compared with other conjugates, the fluorescent signal of conjugate 18 was stronger and showed a dispersive distribution in the body. This indicates that conjugate 18 may have a more sustained targeting effect because it is not rapidly metabolized and excreted from the body, but has a longer duration in the circulatory system.

[0342] Experimental Example 14: Targeting of the complex in mice In this experimental example, the targeting properties of the prepared complexes AP2, AP21 to AP26, and comparative AP27 to comparative AP30 in mice were investigated.

[0343] According to the method of Experimental Example 4, U118MG cells were cultured and subcutaneously inoculated into 24 mice (all female) to obtain mice bearing U118MG subcutaneous tumors.

[0344] Using DMEM medium, the complexes AP2, AP21 to AP26, and Comparative AP27 to Comparative AP30 produced above were each prepared into a 0.3 mg / mL solution.

[0345] Administration began 14 days after inoculation of U118MG cells, and the day of administration was designated as D1. In the experiment, the mice were administered once a day via tail vein injection for a total of three times.

[0346] Twenty-four mice inoculated with U118MG subcutaneous tumors were randomly divided into 12 groups with 2 mice in each group.

[0347] For 7 groups of mice, AP2, AP21, AP22, AP23, AP24, AP25, or AP26 was administered to each mouse in each group. The single-dose volume was 10 μL / g mouse body weight, and the calculated single-dose amount was 3 mg / kg. These were recorded as test groups 14A to 14G, respectively.

[0348] For the other four groups of mice, comparative AP27, comparative AP28, comparative AP29, or comparative AP30 was administered to each mouse in each group. The single-dose volume was 10 μL / g mouse body weight, and the calculated single-dose amount was 3 mg / kg, and these were recorded as control groups 14H to 14K, respectively.

[0349] For another group of 2 mice, each mouse was administered with DMEM medium, with an administration volume of 10 μL / g mouse body weight, and recorded as the blank control group 15Y.

[0350] 1 h, 24 h, and 48 h after the first administration, each mouse was subjected to bioimaging using a small animal biooptical imaging system IVIS Lumina Series III. On D5, the mice in each group were sacrificed, and tumor tissues and kidneys were taken for fluorescence imaging.

[0351] 13A-13C are photographs showing the results of fluorescence imaging in mice after administration of different complexes for 1 h, 24 h, and 48 h, respectively. The mouse on the left side of the three mice in each photograph is a mouse in the blank control group 15Y. As can be seen from FIG. 13A, the blank control group did not show any fluorescence signal, but in contrast, after administration for 1 h, the mice in each test group and the control group all showed fluorescence signals in the subcutaneous tumor. As can be seen from FIG. 13B and 13C, after administration for 24 h and 48 h, only the mice in the test groups 14A-14G showed strong fluorescence signals in the subcutaneous tumor, while the mice in the control groups 14H-14K showed little or only weak fluorescence signals. Furthermore, FIG. 13D is a photograph of the fluorescence signal imaging of the tumor tissue and kidney of the mice in each group after the mice were sacrificed on D5, and Blank represents the blank control group 14Y. As can be seen from Figure 13D, the tumor tissues of the mice in the blank control group 14Y and the control groups 14H-14K showed little or only very weak fluorescent signals, whereas the tumor tissues of the mice in the test groups 14A-14G administered with the conjugates according to the present disclosure all showed strong fluorescent signals, and only weak fluorescent signals were shown in the metabolic organ kidney. This shows that, compared to the control conjugates, the various aptamers according to the present disclosure can all stably and efficiently target and deliver fluorescent groups to tumor tissues, and the various conjugates containing the diagnostic agent groups according to the present disclosure can all stably and efficiently target tumor tissues, thereby contributing to the successful diagnosis and monitoring of the presence of tumors.

[0352] Experimental Example 15: Targeting of the complex in mice In this experimental example, the targeting properties of the prepared complexes AP2, AP12, and comparative AP31 to comparative AP35 in mice were investigated.

[0353] According to the method of Experimental Example 4, U118MG cells were cultured and subcutaneously inoculated into 16 mice (all male) to obtain mice bearing U118MG subcutaneous tumors.

[0354] Using DMEM medium, the complexes AP2, AP12 and Comparative AP31 to Comparative AP35 produced above were each prepared into a 0.3 mg / mL solution.

[0355] Administration began 21 days after inoculation of U118MG cells, and the day of administration was designated as D1. In the experiment, the mice were administered once a day via tail vein injection for a total of three times.

[0356] Sixteen mice inoculated with U118MG subcutaneous tumors were randomly divided into eight groups with two mice in each group.

[0357] For the two groups of mice, AP2 or AP12 was administered to each mouse in each group. The single-dose volume was 10 μL / g mouse body weight, and the calculated single-dose amount was 3 mg / kg. These were recorded as test groups 15A to 15B, respectively.

[0358] For the other five groups of mice, comparative AP31, comparative AP32, comparative AP33, comparative AP34, or comparative AP35 was administered to each mouse in each group. The single-dose volume was 10 μL / g mouse body weight, and the calculated single-dose amount was 3 mg / kg. These were recorded as control groups 15C to 15G, respectively.

[0359] For another group of 2 mice, each mouse was administered with DMEM medium, with an administration volume of 10 μL / g mouse body weight, and recorded as the blank control group 15Y.

[0360] 1 h, 24 h, and 48 h after the first administration, each mouse was subjected to bioimaging using a small animal biooptical imaging system IVIS Lumina Series III. On D6, the mice in each group were sacrificed, and tumor tissues and kidneys were taken for fluorescence imaging.

[0361] 14A-14C are photographs showing the results of fluorescence imaging in mice after administration of different complexes for 1 h, 24 h, and 48 h, respectively. The mouse on the left side of the three mice in each photograph is a mouse in the blank control group 15Y. As can be seen from FIG. 14A, the blank control group did not show any fluorescence signal, but in contrast, after administration for 1 h, the mice in each test group and the control group all showed fluorescence signals in the subcutaneous tumor. As can be seen from FIG. 14B and 14C, after administration for 24 h and 48 h, only the mice in the test groups 15A and 15B showed strong fluorescence signals in the subcutaneous tumor, while the mice in the blank control groups 15Y and the control groups 15C-15G did not show any fluorescence signals. Furthermore, FIG. 14D is a photograph of the fluorescence signal imaging of the tumor tissue and kidney of the mice in each group after the mice were sacrificed on D6, and Blank represents the blank control group 15Y. As can be seen from Figure 14D, the tumor tissues of the mice in the blank control group 15Y and the control groups 15C to 15G did not show any fluorescent signal, whereas the tumor tissues of the mice in the test groups 15A and 15B administered with the conjugate according to the present disclosure all showed strong fluorescent signals, and only a very weak fluorescent signal in the metabolic organ kidney. This shows that the conjugate according to the present disclosure can stably and efficiently target tumor tissues compared to the control conjugate, and that none of the various conjugates that do not have the sequence shown in formula (1) showed a targeting effect on tumor tissues.

[0362] Experimental Example 16 Distribution of the complex in U118MG human glioma cells U118MG human glioma cells (purchased from Guangzhou Geniobiotech Co., Ltd.) were cultured according to the method of Experimental Example 4. Logarithmically grown U118MG human glioma cells were taken and digested with (0.25% pancreatin), the cells were harvested, centrifuged to remove the supernatant, and the cells were resuspended in serum-free DMEM medium to a concentration of 1 × 10 8 A cell culture medium was prepared at 100 μg / mL.

[0363] The experimental animals were eight 12-week-old male NOD-SCID mice (purchased from Beijing Schiff Biotechnology Co., Ltd.). The above cell culture medium was inoculated into the subcutaneous site of the right dorsum of the NOD-SCID mice, with an inoculation volume of 100 μL per mouse, i.e., 1 × 10 7 After injection, the mice were kept for 21 days.

[0364] The complexes AP2, AP4 and comparative complex AP13 were dissolved in a complex solution at a concentration of 0.3 mg / mL (calculated based on the aptamer) using serum-free DMEM medium.

[0365] On the 21st day after U118MG cell inoculation, 8 mice were randomly divided into 4 groups, with 2 mice in each group. The mice in each group were administered the drug, and the day of administration was designated as D1. In the experiment, the drug was administered once via tail vein injection.

[0366] For three groups of mice, each mouse in each group was administered with conjugate AP2, AP4 or comparative conjugate AP13, with a single dose volume of 10 μL / g mouse body weight and a calculated single dose of 3 mg / kg, which were recorded as test groups 16a, 16b and 16c, respectively.

[0367] For the other group of mice, DMEM was administered to each group of mice respectively, with a single administration volume of 10 μL / g mouse body weight, and recorded as the blank control group 16Y.

[0368] 24 h after administration, the mice were euthanized, and tumor tissues were collected and fixed in 4% paraformaldehyde solution. The tumor tissues were placed in 15% sucrose for 24 h to dehydrate, then placed in 30% sucrose for another 24 h to dehydrate, and the tissues were infiltrated with OCT embedding medium, then rapidly cooled in liquid nitrogen. The rapidly cooled tissues were sliced ​​to a thickness of 10 μm using a freezing microtome (model number POLAR-D-JC, purchased from Sakura Seiki Co., Ltd.) to obtain tumor tissue slices.

[0369] The tumor tissue slices were left at room temperature for 5 min, fixed with 4% paraformaldehyde for 15 min, washed with PBS 3 times at 5 mL each time, stained with DAPI for 3 min in the dark, and then blocked by repeating the above washing process three times to obtain DAPI-stained slices.

[0370] Imaging analysis was performed on the DAPI stained slices obtained above using a laser confocal imager (model number LSM 900 Basic Operation, purchased from Carl Zeiss (Shanghai) Management Co., Ltd.), with the following parameters selected: Cy5 laser intensity 20% (em 650nm, ex 670nm), DAPI laser intensity 1.5% (em 360nm, ex 460nm), and Best signal mode. The results are shown in Figure 15.

[0371] 15 is a photograph showing the results of laser confocal imaging of U118MG glioma cells 24 h after administration of different conjugates to mice subcutaneously inoculated with U118MG glioma. As can be seen from the experimental results, compared with the blank control DMEM and the comparative conjugate AP13, all of the conjugates containing the delivery group of the present disclosure can efficiently and specifically deliver the diagnostic group into the interior of U118MG glioma, showing excellent targeting effect and potential diagnostic ability.

[0372] Experimental Example 17 Delivery effect of complex 15 and complex 36 to U118MG subcutaneous tumor U118MG human glioma cells (purchased from Guangzhou Geniobiotech Co., Ltd.) were cultured according to the method of Experimental Example 4. Logarithmically grown U118MG human glioma cells were taken and digested with (0.25% pancreatin), the cells were harvested, centrifuged to remove the supernatant, and the cells were resuspended in serum-free DMEM medium to a concentration of 1 × 10 8 A cell culture medium was prepared at 100 μg / mL.

[0373] The experimental animals were 9 male NOD-SCID mice aged 12 weeks (purchased from Beijing Sibaifu Biotechnology Co., Ltd.). The above cell culture medium was inoculated into the subcutaneous site of the right dorsum of the NOD-SCID mice, with an inoculation volume of 100 μL per mouse, i.e., 1 × 10 7 After injection, the mice were kept for 21 days.

[0374] Using serum-free DMEM medium, complex AP2, complex 15, and complex 36 were dissolved in a complex solution at a concentration of 0.5 mg / mL (calculated by aptamer). Administration was started 21 days after inoculation of U118MG cells, and the day of administration was designated as D1. In the experiment, a single administration was performed via tail vein injection.

[0375] For the control group 17a, each mouse was administered with the AP2 complex, with a single dose volume of 10 μL / g mouse body weight, and the calculated single dose was 5 mg / kg.

[0376] For the control group 17b, each mouse was administered with Complex 15, with a single dose volume of 10 μL / g mouse body weight, and the calculated single dose was 5 mg / kg.

[0377] For the control group 17c, each mouse was administered with Complex 36. The single dose volume was 10 μL / g mouse body weight, and the calculated single dose was 5 mg / kg.

[0378] 24 h (D2) and 48 h (D3) after administration, the mice were anesthetized with isoflurane (0.41 ml / min of isoflurane in 4 L / min fresh air flow), and intravital imaging was performed on each mouse using the small animal bio-optical imaging system IVIS Lumina Series III.

[0379] Figure 16A and Figure 16B show the fluorescence imaging results in mice after administration of different complexes.As can be seen from the results, at different times after administration, all of the various complexes that contain the siRNA group of the present disclosure and the delivery group formed by the aptamer of the present disclosure can effectively target and concentrate in tumor tissue, and have excellent drug discovery potential.

[0380] Experimental Example 18 Evaluation of the activity of complex 37 and complex 38 to enter PANC-1 (human pancreatic cancer cells) PANC-1 human pancreatic cancer cells (purchased from the China National Experimental Cell Resources Sharing Platform) were cultured in DMEM complete medium (MACGENE, part number CM15019) containing 10% FBS (Gibco, part number 10099-141) at 37°C in an incubator containing 5% CO2 / 95% air.

[0381] Logarithmically grown PANC-1 human pancreatic cancer cells were taken and digested with (0.25% pancreatin), the cells were collected, centrifuged to remove the supernatant, and the cells were resuspended in DMEM medium containing 10% FBS to a concentration of 1 × 10 5 The cell culture medium was prepared at 1 × 10 cells / mL, mixed thoroughly and evenly, and then added to a 24-well plate at 1 mL / well, i.e., 1 × 10 cells. 5 cells / well.

[0382] Using DMEM, complex 37, complex 38 and complex AP2 were each dissolved in a complex solution at a concentration of 20 μM.

[0383] After culturing for 24 h in different culture wells of a 24-well plate, the supernatant was aspirated, and 900 μL of DMEM medium containing 10% FBS was added to each well. 100 μL of blank control group DMEM (hereinafter referred to as control group), complex 37, complex 38 or complex AP2 was added to the corresponding medium, and allowed to be freely taken up at a concentration of 2 μM. After 7 days, RNA was extracted and recorded as the control group, complex 9 group, complex 10 group and complex AP2 group (the latter three groups are collectively referred to as test groups).

[0384] Then, total RNA was extracted from the cells in each well using a tissue RNA extraction kit based on magnetic beads (purchased from Wuhan Naji Biotechnology Co., Ltd., product number NMR0211-20) according to the operating instructions in the specification, and a solution containing total RNA was obtained.

[0385] A solution containing 1 μg of total RNA was taken from each well of the cells, and the total RNA of the cells in each well was reverse transcribed using a reverse transcription kit (purchased from Promega Corporation, product number A3500) with Oligo(dT)18 as a primer according to the reverse transcription operation procedure in the kit's instructions. After the reaction was completed, 80 μl of DEPC water was added to the reverse transcription reaction system to obtain 100 μL of a solution containing cDNA.

[0386] For each reverse transcription reaction system, 5 μl of the solution containing the above cDNA was used as a template. SYBR Select Master Mix kit (purchased from Thermo Fisher Scientific, product number 4472908) was used and SteponePlus was used according to the operating instructions in the instructions. TM The target gene hSTAT3 and the reference gene hGAPDH were amplified using a real-time fluorescent quantitative PCR device (purchased from Thermo Fisher Scientific). The amplification procedure consisted of 10 min of pre-denaturation at 95°C, 30 s of denaturation at 95°C, 30 s of annealing at 60°C, and 30 s of extension at 72°C, with 40 cycles of denaturation, annealing, and extension. The melting procedure was the default procedure for the device.

[0387] Table 5: Primer information [Table 9]

[0388] The above quantitative PCR detection was carried out twice for each of the test groups and the control group.

[0389] The above quantitative PCR detection was carried out twice for each of the test groups and the control group.

[0390] Relative Quantitation 2 -ΔΔCt The fluorescence quantitative data was analyzed by the method. The calculation method is as follows: ΔCt(test group)=Ct(target gene in test group)-Ct(endogenous reference gene in test group) ΔCt(control group)=Ct(gene of interest in control group)-Ct(endogenous reference gene in control group) ΔΔCt(test group)=ΔCt(test group)-ΔCt(average value of control group) ΔΔCt(control group) = ΔCt(control group) - ΔCt(mean value of the control group) Inhibition rate (%) = (control group 2 -ΔΔCt Mean value - Test group 2 -ΔΔCt ) / Control group 2 -ΔΔCt Average value x 100

[0391] The experimental results were visually represented as a histogram of Mean±SD values ​​using Graph Prism software. The results are shown in Figure 17, where blank represents the control group.

[0392] Figure 17 is a bar graph showing the inhibition rate of hSTAT3 mRNA in PANC-1 human pancreatic cancer cells by the complex 37, complex 38 and complex AP2 of the present disclosure, respectively. As can be seen from the results of Figure 17, after administration of complex 37, the inhibition rate of hSTAT3 mRNA in PANC1 human pancreatic cancer cells was 33%, and after administration of complex 38, the inhibition rate of hSTAT3 mRNA in PANC1 human pancreatic cancer cells reached 44%. As can be seen from the above results, the complexes of the present disclosure having different binding modes, different binding groups and different modified nucleotide sequences can all be freely taken up into tumor cells and exhibit the effect of inhibiting target mRNA.

[0393] Experimental Example 19: Evaluation of internalization of complexes 15-19 in U118MG cells According to the method of Experimental Example 4, U118MG human glioma cells (purchased from Guangzhou Genio Biotech Co., Ltd.) were cultured. U118MG cells were digested with (0.25% pancreatin) and diluted to 2 × 10 5The cells / well were cultured in a 12-well plate and incubated in a cell incubator at 37°C with 5% CO2 for 24 h.

[0394] Using serum-free DMEM medium, complex 15, complex 16, complex 17, complex 18 and complex 19 were dissolved in a complex solution with a concentration of 200 μM. 10 μL of the compound was added to a 990 μL cell culture well, i.e., the final concentration of the compound was 2 μM. After incubation at 37 °C for 2 h, the supernatant was aspirated and discarded, washed three times with PBS, fixed for 10 min by adding 100 μL of 4% paraformaldehyde solution, washed three times with PBS, added 100 μL of DAPI staining solution, stained for 10 min, washed three times with PBS, and then laser confocal imaging was performed.

[0395] Laser confocal imaging: The parameters were Cy5 laser intensity 2% (em 650nm, ex 670nm) and DAPI laser intensity 1.5% (em 360nm, ex 460nm). Images were taken in Best signal mode, and the results are shown in Figure 18.

[0396] FIG. 18 is a photograph showing the results of laser confocal imaging of the complex of the present disclosure in U118MG glioma.

[0397] As can be seen from the results in Figure 18, the conjugates of the present disclosure using different binding modes, different binding groups and different modified nucleotide sequences can all be freely taken up by tumor cells, and conjugate 18 with 2'-O-hexadecyl group modification has a better ability to enter tumor cells.

[0398] Experimental Example 20: Evaluation of internalization of complex 15 and complex 18 in U118MG cells In this example, the ability of complex 15, complex 18 and complex AP2 to be freely taken up by U118-MG glioma cells in vitro was evaluated and compared. Logarithmically grown U118MG human glioma cells cultured in Example 4 were taken, digested with 0.25 wt% pancreatin to harvest the cells, centrifuged to remove the supernatant, and the cells were resuspended in DMEM medium supplemented with 10% FBS to a concentration of 1 × 10 5 The cell culture medium was prepared at 1000 μL / well, i.e., 1 × 10 cells / mL, and mixed thoroughly and homogenously. Then, 1000 μL / well of a 24-well plate was added. 5 cells / well.

[0399] Using DMEM, complex 15, complex 18 and complex AP2 were dissolved in a complex solution having a concentration of 40 μM, and recorded as test group 20a, test group 20b and control group 20c.

[0400] After 24 h of incubation, the medium was sucked off, and 900 μL of serum-free DMEM was added to each well. 100 μL of blank control DMEM, complex 15, complex 18 and complex AP2 were added to the corresponding medium and allowed to be freely incorporated at a concentration of 4 μM, and the fluorescence value of the cells was measured after 48 h.

[0401] For the above test group 20a, test group 20b and control group 20c, 100 μL of DMEM medium was taken and imaged in a high content imaging system. The average fluorescence intensity of the blank control group DMEM was normalized, that is, the average fluorescence intensity of the blank control group was subtracted from the fluorescence intensity of the test group 20a, test group 20b and control group 20c, and then imaging was performed. The results are shown in FIG.

[0402] Figure 19 is a bar graph showing the results of the fluorescence intensity of the complex of the present disclosure in U118MG glioma, and DMEM is the blank control group. As can be seen from the results of Figure 6, the complex of the present disclosure can effectively deliver siRNA groups into tumor cells, thereby helping to efficiently produce RNAi effects in tumor cells.

[0403] Experimental Example 21: Activity of the complex in vivo in U118MG in-situ tumor model mice According to the method of Experimental Example 4, U118MG human glioma cells expressing the luciferase (Photinus pyralis) reporter gene were cultured, hereinafter referred to as U118MG-luc human glioma cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.). Logarithmically grown U118MG-luc human glioma cells were taken and digested with 0.25 wt% pancreatin to collect the cells. After centrifugation, the supernatant was sucked out and the cells were resuspended in serum-free DMEM medium until the cell density reached 4 × 10 7 A cell culture medium was prepared at 100 μg / mL.

[0404] The experimental animals were 24 12-week-old male Balb / C-nude mice (purchased from Beifong Biological Technology Co., Ltd.). The above cell culture solution was inoculated into the striatum of Balb / C-nude mice, and the cell culture solution was injected into the right striatum of the mice by mouse striatal injection at AP (anteroposterior): 1 mm, ML (medial lateral): 1.5 mm, and DV (dorsal ventral): 3.5 mm, with an injection volume of 10 μL, i.e., 4 × 10 5 After in-situ tumor inoculation, the mice were kept for 14 days.

[0405] Using 1x DMEM (purchased from Zhongke Minchen (Beijing) Technology Co., Ltd., lot number K1902200), complex 20 was dissolved in a complex solution with a concentration of 1 mg / mL (calculated based on aptamer). Complex 39 and comparative complex 43 were each dissolved in a complex solution with a concentration of 0.8 mg / mL (calculated based on aptamer).

[0406] On the 15th day after in-situ tumor inoculation, bioimaging was performed on each mouse using the small animal bio-optical imaging system IVIS Lumina Series III, and the mice were divided into groups based on brain fluorescence intensity, with 6 mice per group. The day of administration was designated as D1 (i.e., day 1 of the experiment; hereafter, D4, D8, etc. represent days 4 and 8 of the experiment, and will be inferred in this manner).

[0407] The bioimaging method is as follows. Each mouse was intraperitoneally injected with 15 mg / mL D-fluorescein potassium salt working solution (purchased from Yeasen Biotechnology (Shanghai) Co., Ltd.) at a concentration of 10 μL / g body weight, and bioimaging (IVIS® Lumina III small animal bioimaging system) was performed 10 min after injection. After imaging, the fluorescent region of interest (ROI) in the mouse brain was surrounded, and the fluorescence intensity (Radiance) was measured by software. Under this condition, the Luciferase (Photinus pyralis) reporter gene expressed in U118MG-luc human glioma cells can generate a fluorescent response, so the fluorescence intensity can reflect the proliferation number of glioma cells. The higher the fluorescence intensity, the higher the number of glioma cells.

[0408] In the experiment, the mice were administered subcutaneously on D1, D4, D8 and D12. The mice were weighed before administration and administered according to their body weight.

[0409] For test group 21a, each mouse was administered with conjugate 20. The single dose volume was 5 μL / g mouse body weight, and the calculated single dose was 5 mg / kg, which corresponds to a dose of 0.3 mg / kg containing MMAE.

[0410] For test group 21b, each mouse was administered with conjugate 39. The single dose volume was 5 μL / g mouse body weight, and the calculated single dose was 4 mg / kg, which corresponds to a dose of 0.3 mg / kg containing MMAE.

[0411] For test group 21c, each mouse was administered with comparative conjugate 43. The single dose volume was 5 μL / g mouse body weight, and the calculated single dose was 4 mg / kg, which corresponds to a dose of 0.3 mg / kg containing MMAE.

[0412] For the blank control group, each mouse was administered with DMEM medium respectively, with a single administration volume of 5 μL / g mouse body weight.

[0413] According to the above method, the mice of each group were subjected to bioimaging analysis on D1, D22, D31 and D39, respectively, to measure the fluorescence intensity. The results are shown in Figure 20.

[0414] FIG. 20 is a line graph showing the change in tumor fluorescence intensity over time in U118MG in situ tumor model mice after administration of a conjugate according to the present disclosure or a control compound.

[0415] As can be seen from the results of FIG. 20, the tumor fluorescence intensity (Radiance) of the blank control group and the control group increased obviously with the extension of the observation time, compared with D1 after administration. This indicates that the number of U118MG human glioma cells increased obviously, and in contrast, the tumor fluorescence intensity of the test groups 21a and 21b administered with the complex according to the present disclosure decreased obviously, with the decrease reaching a maximum of one digit, and reaching more than two digits compared with the control group. This indicates that the number of U118MG human glioma cells decreased obviously, decreasing to 1 / 10 of the start of the experiment compared with the start of the experiment, and may even decrease to 1% or less of the control group. From the above, even by subcutaneous administration alone, the complex including the tumor therapeutic agent group and the delivery group formed by the target aptamer according to the present disclosure can effectively penetrate the blood-brain barrier and efficiently target and enter brain glioma, has an excellent effect of inhibiting tumor growth, and shows good treatment compliance and high drug discovery potential for efficiently inhibiting tumors.

[0416] Experimental Example 22: Inhibitory activity of the complex against U118MG subcutaneous tumor in mice U118MG human glioma cells (purchased from Guangzhou Geniobiotech Co., Ltd.) were cultured according to the method of Experimental Example 4. Logarithmically grown U118MG human glioma cells were taken and digested with (0.25% pancreatin), the cells were harvested, centrifuged to remove the supernatant, and the cells were resuspended in serum-free DMEM medium to a concentration of 1 × 10 8 A cell culture medium was prepared at 100 μg / mL.

[0417] The experimental animals were 36 12-week-old male NOD-SCID mice (purchased from Beijing Sibaifu Biotechnology Co., Ltd.). The above cell culture medium was inoculated into the subcutaneous site of the right dorsum of the NOD-SCID mice, with an inoculation volume of 100 μL per mouse, i.e., 1 × 10 7 After injection, the mice were kept for 7 days.

[0418] Using serum-free DMEM medium, complex 20 was dissolved in a complex solution with a concentration of 1 mg / mL (calculated by aptamer). Complex 39, complex 40, and complex 41 were each dissolved in a complex solution with a concentration of 0.8 mg / mL (calculated by aptamer). MMAE was dissolved in a solution with a concentration of 0.06 mg / mL using 10% DMSO + 90% serum-free DMEM medium (volume ratio).

[0419] The administration was started 7 days after the inoculation of U118MG cells, and the day of administration was designated as D8. In the experiment, the mice were administered subcutaneously in the abdomen once on D8, D12, D16, and D20, for a total of four administrations.

[0420] For the blank control group 22a, mice in each group were administered DMEM, with a single administration volume of 5 μL / g mouse body weight.

[0421] For the control group 22b, MMAE was administered to the mice in each group, with the single-dose volume set at 5 μL / g mouse body weight, and the calculated single-dose dose was 0.3 mg / kg.

[0422] For test group 22c, each mouse was administered with conjugate 20. The single dose volume was 5 μL / g mouse body weight, and the calculated single dose was 5 mg / kg, which corresponds to a dose of 0.3 mg / kg containing MMAE.

[0423] Mice were divided into three groups and administered with conjugate 39, conjugate 40 or conjugate 41. The single dose volume was 5 μL / g mouse body weight, and the calculated single dose was 4 mg / kg, which was equivalent to a dose of 0.3 mg / kg containing MMAE. These groups were recorded as test groups 22d, 22e and 22f, respectively.

[0424] Tumor volumes were measured on D16, D20, D25, D29, D33, D36, D41, D48, and D60. The long and short diameters of the tumor were measured by external measurement. Tumor volumes were calculated using the formula 1 / 2 (long diameter × short diameter) 2 ) was calculated. After the experiment was completed, tumor tissue was taken and weighed on D60. FIG. 21 is a line graph showing the time course of mouse tumor volume on different days after administration of different complexes. As can be seen from FIG. 21, the tumor volume and tumor weight of mice in test groups 22c to 22f administered with the complex of the present disclosure were all obviously reduced compared to control group 22b or blank control group. As is clear from the above results, the complexes containing delivery groups formed by these aptamers can effectively reach tumor tissues and exhibit excellent antitumor activity.

[0425] Experimental Example 23: In vivo activity of U118MG subcutaneous tumor model mice by long-term administration of the complex According to the method of Experimental Example 4, 42 mice inoculated with U118MG subcutaneous tumors were obtained, and the mice were kept after injection.

[0426] Using 10% DMSO + 90% serum-free DMEM medium (volume ratio), MMAE was dissolved in solutions of 0.03 mg / mL and 0.01 mg / mL concentrations, complex 39 was dissolved in solutions of 0.5 mg / mL and 0.165 mg / mL concentrations (calculated based on aptamer), and comparative complex 44 was dissolved in solutions of 0.5 mg / mL and 0.165 mg / mL concentrations (calculated based on aptamer).

[0427] Seven days after inoculation with U118MG cells, all mice were randomly divided into 7 groups, with 6 mice in each group, and the mice in each group were administered the drug on the day of administration, which was designated as D8. In the experiment, the mice were administered the drug subcutaneously in the abdomen once on D8, D11, D15, D29, D32 and D36, for a total of 6 doses. Before administration, the mice were weighed, and the administration volume was calculated according to their body weight.

[0428] For the blank control group, each mouse was administered with DMEM medium respectively, with a single administration volume of 10 μL / g mouse body weight.

[0429] For test group 23a, each mouse was administered MMAE at a concentration of 0.01 mg / mL. The single dose volume was 10 μL / g mouse body weight, and the calculated single dose was 0.1 mg / kg.

[0430] For test group 23b, each mouse was administered 0.165 mg / mL of conjugate 39. The single dose volume was 10 μL / g mouse body weight, and the calculated single dose was 1.65 mg / kg (corresponding MMAE dose was 0.1 mg / kg).

[0431] For the control group 23c, each mouse was administered a 0.165 mg / mL concentration of comparative conjugate 44. The single dose volume was 10 μL / g mouse body weight, and the calculated single dose was 1.65 mg / kg (corresponding MMAE dose was 0.1 mg / kg).

[0432] For test group 23d, each mouse was administered MMAE at a concentration of 0.03 mg / mL. The single dose volume was 10 μL / g mouse body weight, and the calculated single dose was 0.3 mg / kg.

[0433] For test group 23e, each mouse was administered 0.5 mg / mL of conjugate 39. The single dose volume was 10 μL / g mouse body weight, and the calculated single dose was 5 mg / kg (corresponding MMAE dose was 0.3 mg / kg).

[0434] For the control group 23f, each mouse was administered a 0.5 mg / mL concentration of comparative conjugate 44. The single dose volume was 10 μL / g mouse body weight, and the calculated single dose was 5 mg / kg (corresponding MMAE dose was 0.3 mg / kg).

[0435] Tumor volumes were measured on D1, D9, D16, D19, D21, D24, D26, D29, D32, D36, D39, D43, D47, D53, D57, D60, D64, D67, D71, D74, D78, D81, D84, D88, D92, D95 and D99. The blank control group was measured on D53, and the test groups 7a, 7b and control group 7c were measured on D60, after which the experiment was terminated.

[0436] The long and short diameters of the tumor were measured by external measurement. The tumor volume was calculated using the formula 1 / 2 (long diameter × short diameter). 2 After the experiment was stopped, the tumor tissues were taken and weighed, and the average values ​​were calculated. The results are shown in FIG.

[0437] FIG. 22 is a line graph showing the change in tumor volume over time in U118MG subcutaneous tumor model mice after administration of different concentrations of a conjugate according to the present disclosure or a control compound.

[0438] As can be seen from the results in Figure 22, the tumor volume increased rapidly in the blank control group, and the tumor volume increase rate slowed down in the test group administered with MMAE alone, indicating that MMAE itself has an inhibitory effect on tumor growth.

[0439] Furthermore, the tumor volume of test group 23b was significantly smaller than that of test group 23a and control group 23c, both of which correspond to the MMAE content, during the test period, and the tumor volume of test group 23e was significantly smaller than that of test group 23d and control group 23f, both of which correspond to the MMAE content, during the test period, showing superior antitumor activity to that of MMAE test groups 23a and 23d alone and that of comparative complex 44. After the experiment was stopped, the tumor weight in the mice administered the complex of the present disclosure was also significantly lower than that of the MMAE group and the control group. As is clear from the above results, the complex of the present disclosure can effectively deliver MMAE to tumor tissue, exhibiting tumor targeting ability, while reducing the toxicity risk caused by the distribution of MMAE molecules to other tissues, and exhibiting dose-related and excellent antitumor effects.

[0440] As is clear from the above results, the conjugate of the present disclosure can effectively target and deliver a small molecule drug group having an inhibitory effect on tumors to tumor tissue, and can exhibit excellent antitumor activity and dose-dependent effects.

[0441] Experimental Example 24: In vivo activity of the complex in U118MG subcutaneous tumor model mice According to the method of Experimental Example 4, 42 mice inoculated with U118MG subcutaneous tumors were obtained, and the mice were kept after injection.

[0442] MMAE was dissolved in a solution of 0.02 mg / mL concentration in 10% DMSO + 90% serum-free DMEM medium (volume ratio), complex 20 and comparative complex 44 were dissolved in a solution of 0.33 mg / mL concentration (calculated with aptamer), complex 39 and complex 41 were dissolved in a solution of 0.26 mg / mL concentration (calculated with aptamer), and complex 42 was dissolved in a solution of 0.23 mg / mL concentration (calculated with aptamer).

[0443] Seven days after inoculation of U118MG cells, the mice were divided into groups, with six mice in each group, and the mice were administered the drug on the day of administration, designated as D8. Before administration, the mice were weighed, and the administration volume was calculated assuming that the average weight of each mouse was 20 g.

[0444] In the experiment, the mice were administered subcutaneously in the abdomen once on D8, D12, D15 and D19, respectively, for a total of four times.

[0445] For the blank control group, each mouse was administered with DMEM medium respectively, with a single administration volume of 100 μL / g.

[0446] For test group 24a, each mouse was administered MMAE, with a single dose volume of 100 μL and a calculated single dose of 0.1 mg / kg.

[0447] For test group 24b, each mouse was administered with Conjugate 20. The single dose volume was 100 μL, and the calculated single dose was 1.65 mg / kg.

[0448] For the control group 24c, each mouse was administered with comparative complex 44. The single dose volume was 100 μL, and the calculated single dose was 1.65 mg / kg.

[0449] For test group 24d, each mouse was administered with Conjugate 39. The single dose volume was 100 μL, and the calculated single dose was 1.32 mg / kg.

[0450] For test group 24e, each mouse was administered with conjugate 41. The single dose volume was 100 μL, and the calculated single dose was 1.32 mg / kg.

[0451] For test group 24f, each mouse was administered with conjugate 42. The single dose volume was 100 μL, and the calculated single dose was 1.17 mg / kg.

[0452] In each of the above test groups and control groups 24a to 24f, the MMAE dose corresponding to a single administration was 0.1 mg / kg.

[0453] Tumor volumes were measured on D1, D9, D16, D19, D22, D26, D30, D36, D40, D43, D47, D50, D54, D57, D61, D64, D68, and D71. The blank control group was measured on D54, and group 8a (administered only MMAE) was measured on D64, after which the experiment was terminated.

[0454] The long and short diameters of the tumor were measured by external measurement. The tumor volume was calculated using the formula 1 / 2 (long diameter × short diameter). 2 After the experiment was terminated, tumor tissues were taken from each group, weighed, and the average value was calculated. The results are shown in FIG.

[0455] Figure 23 shows a line graph of the change in tumor volume over time in mice of each group and the tumor weight at D72. As can be seen from the results of Figure 23, the tumor volume of the blank control group increased rapidly, and the tumor volume increase rate of each of the other groups decreased. However, compared with test group 24a and control group 24c, which were administered with only MMAE, the tumor volume increase rate of each of the test groups administered with the conjugate of the present disclosure was significantly further decreased when the single dose of MMAE was equivalent to 0.1 mg / kg. In addition, conjugate 20, conjugate 39, conjugate 41 and conjugate 42 showed a better antitumor effect at the end of the experiment D72, with a tumor weight reduction of at least 58% compared with test group 24a.

[0456] Experimental Example 25: Activity of the complex in vivo in A549 subcutaneous tumor model mice A549 human lung adenocarcinoma cells (purchased from Guangzhou Genio Biotech Co., Ltd.) were cultured in DMEM complete medium (MACGENE, product number CM15019) containing 10% FBS (Gibco, product number 10099-141) at 37°C in a 5% CO2 / 95% air incubator. Cells were harvested by digestion with 0.25 wt% pancreatin, the supernatant was aspirated, and the cells were resuspended in serum-free DMEM medium to a cell density of 1 × 10 8 A cell culture medium was prepared at 100 μg / mL.

[0457] According to the method of Experimental Example 4, 30 mice inoculated with A549 subcutaneous tumor were obtained, and the mice were kept after injection.

[0458] Using serum-free DMEM medium, complex 20 and complex 39 were dissolved in a complex solution with a concentration of 1 mg / mL (calculated by aptamer). Comparative complex 43 was dissolved in a complex solution with a concentration of 0.8 mg / mL (calculated by aptamer). MMAE was dissolved in a solution with a concentration of 0.06 mg / mL using 10% DMSO + 90% serum-free DMEM medium (volume ratio).

[0459] Seven days after inoculation of A549 human lung cancer cells, all mice were divided into groups, with 6 mice in each group, and each mouse was administered the drug on the day of administration, designated as D8. The mice were weighed before administration, and the administration volume was calculated according to their weight.

[0460] Mice in each group were administered once on D8, D12, D15 and D19, totaling four doses. For the blank control group 25a, mice in each group were administered DMEM, respectively, with a single dose volume of 5 μL / g mouse body weight.

[0461] For the control group 25b, MMAE was administered to each group of mice, with the single-dose volume of 5 μL / g mouse body weight, and the calculated single-dose amount was 0.3 mg / kg.

[0462] For test group 25c, each mouse was administered with conjugate 20. The single dose volume was 5 μL / g mouse body weight, and the calculated single dose was 5 mg / kg, which corresponds to a dose of 0.3 mg / kg containing MMAE.

[0463] For test group 25d, each mouse was administered with conjugate 39. The single dose volume was 5 μL / g mouse body weight, and the calculated single dose was 5 mg / kg, which corresponds to a dose of 0.3 mg / kg containing MMAE.

[0464] For test group 25e, each mouse was administered with comparative conjugate 43. The single dose volume was 5 μL / g mouse body weight, and the calculated single dose was 5 mg / kg, which corresponds to a dose of 0.3 mg / kg containing MMAE.

[0465] Mice in each group were administered once on D46, D50, and D54, respectively. The single-dose volume was 10 μL / g mouse body weight, and the calculated dose of MMAE contained in the single dose was equivalent to 0.6 mg / kg.

[0466] Tumor volumes were measured on D1, D9, D16, D19, D22, D26, D30, D36, D40, D43, D47, D50, D54 and D57, and the blank control group was measured on D50, after which the experiment was terminated.

[0467] The long and short diameters of the tumor were measured by external measurement. The tumor volume was calculated using the formula 1 / 2 (long diameter × short diameter). 2 The results are shown in Figure 24.

[0468] Figure 24 is a line graph showing the time course of tumor volume in A549 subcutaneous tumor model mice after administration of different concentrations of the conjugate of the present disclosure or the control compound. As can be seen from the results of Figure 24, the tumor volume of the mice in the blank control group increased rapidly, the tumor volume increase rate of each of the remaining groups all decreased, and the tumor volumes of the mice administered with conjugate 20 and conjugate 39 at each time point were all smaller than those of the control groups 25b and 25e. As is clear from the above results, the conjugate containing the delivery group formed by the aptamer of the present disclosure can effectively target tumor tissue and exhibit excellent antitumor activity.

[0469] Although several embodiments of the present disclosure have been described in detail above, the present disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical idea of ​​the present disclosure, several simple modifications can be made to the technical solutions of the present disclosure, and all of these simple modifications fall within the protection scope of the present disclosure.

[0470] It should be noted that the specific technical features described in the above embodiments can be combined in any suitable manner unless they are contradictory, and in order to avoid unnecessary duplication, the present disclosure does not separately describe various possible combination manners.

[0471] Furthermore, various different embodiments of the present disclosure can be arbitrarily combined, and such combinations should also be considered as being disclosed in the present disclosure, unless they deviate from the spirit of the present disclosure.

Claims

1. The sequence comprises a continuous nucleotide sequence, and the group connecting two adjacent nucleotides is independently either a phosphate ester group or a phosphate ester group with a modifying group, each nucleotide is selected from one of modified or unmodified A, U, C, or G, and the continuous nucleotide sequence has the sequence shown in formula (1). 5'-T １ -S １ -N ａ -S ２ -N ｂ -S ３ -N ｃ -S ４ -T ２ -3' Formula (1) Here, T １ This is a motif consisting of 1 to 3 nucleotides, T ２ It is a motif consisting of 0 to 15 nucleotides, and T ２ T １ It does not contain any motifs that are completely inversely complementary to it. S １ and S ４ Each of these is a motif consisting of 3 to 7 nucleotides, S １ is, S ４ They have the same length and are completely inversely complementary. N ａ and N ｃ Each of these is a motif consisting of 1 to 4 nucleotides, N ａ Each nucleotide inside is N ｃ None of the nucleotides inside are complementary to N ａ and N ｃ The total number of U's inside is N ａ and N ｃ S accounts for more than 50% of the total number of nucleotides in the body. ２ and S ３ Each of these is a motif consisting of 1 to 4 nucleotides, S ２ is, S ３ They have the same length and are completely inversely complementary. N ｂ This is a motif consisting of 3 to 6 nucleotides, and N ｂ An aptamer in which the nucleotides at both ends do not form AU or GC complementarily.

2. The aptamer according to claim 1, wherein the length of the continuous nucleotide sequence is 18 to 50 nucleotides, or 20 to 40 nucleotides, or 21 to 36 nucleotides, or 24 to 32 nucleotides.

3. T １ It consists of two nucleotides, or T １ It consists of two nucleotides and contains at least one C, or from 5' to 3', T １ is CU, UC, or AC, or T ２ It consists of 0 to 10 nucleotides, or from 5' to 3', T ２ It consists of 1 to 9 nucleotides starting with U, or S １ and S ４ Each consists of 3 to 5 nucleotides and has the same length, or S １ and S ４ In the inverse complement formed by , GC complement accounts for at least 40% of all complements, or from 5' to 3', S １ is GCU and S ４ It is AGC, or S １ It is GAGU and S ４ GCUC, or S １ It is GGAGU and S ４ It is GCUCU, or S １ is UAUGG and S ４ is CCAUG, or The aforementioned N ａ and N ｃ The sum of the number of nucleotides inside is an integer between 2 and 4, or the aforementioned N ａ and N ｃ The sum of the number of nucleotides in the N is 3 or 4, and the N ａ and N ｃ The sum of the numbers of U inside is 2 or 3, or N from 5' to 3'. ａ or N ｃ These are independently U, UU, UC, or CU, or S ２ and S ３ Each consists of 2 to 3 nucleotides and is of the same length, or S ２ and S ３ The inverse complement formed by includes at least one GC complement, or S from 5' to 3'. ２ is CA and S ３ is UG, or S ２ AC and S ３ It is GU, or S ２ It is GCC and S ３ It is GGU, N ｂ It consists of four or five nucleotides, or from 5' to 3', N ｂ The aptamer according to claim 1, wherein is GACG, GACGU, GACCG, UACU, GUUG, or GAUCU.

4. The aforementioned continuous nucleotide sequence has the sequence shown in any of SEQ ID NOs: 1-3 and SEQ ID NOs: 5-11, or The aforementioned continuous nucleotide sequence has the nucleotide sequence shown in Sequence ID No. 4, 5'-N ６ GGAGUUCAN １ N ２ N ３ N ４ UGN ５ GCUCN ７ -3' (Sequence ID 4) Here, N １ , N ２ and N ３ Each of these is independently one of A, U, C, and G, and N ４ is a motif consisting of U, C, or G, or two of U, C, or G, N ５ is U, CU, or UU, N ６ is CU, UC, or AC, and N ７ is U, UU, or UUN ８ And N ８ This is a motif consisting of 1 to 15 nucleotides, or N １ 、N ２ 、N ３ and N ４ motif N consisting of １ N ２ N ３ N ４ is one of GACG, GACGU, GACCG, UACU, GUUG or GAUCU, or N ８ This is a motif consisting of 1 to 8 nucleotides, or, from 5' to 3', N ８ The aptamer according to claim 1, wherein the nucleotide sequence is CCGAUCUC, or the continuous nucleotide sequence has the sequence shown in one of sequence numbers 12 to 14.

5. Each cytosine nucleotide in the continuous nucleotide sequence is a fluoromodified cytosine nucleotide, and / or each uracil nucleotide in the continuous nucleotide sequence is a fluoromodified uracil nucleotide, or each nucleotide in the continuous nucleotide sequence is a 2'-methoxymodified nucleotide, or N in the continuous nucleotide sequence ｂ and S ３ One or more uracil nucleotides in the motif have a modified base, or The aptamer according to claim 1, wherein the continuous nucleotide sequence has the nucleotide sequence shown in one of sequence numbers 15 to 33.

6. In the aptamer, at least one group that binds two adjacent nucleotides is a thiophosphate ester group, or each of the groups that bind two adjacent nucleotides is a thiophosphate ester group, The aptamer according to claim 16, wherein the continuous nucleotide sequence has the nucleotide sequence shown in one of sequence numbers 34 to 39.

7. A complex comprising one or more delivery groups and one or more functional groups, wherein the delivery groups are formed by removing one or more hydrogen atoms or one or more functional groups from an aptamer according to any one of claims 1 to 6, each delivery group is independently bonded to the functional group via a covalent bond or via a binding group, and each functional group is independently one of a diagnostic group, a low molecular weight therapeutic group having a therapeutic effect on tumors, a functional oligonucleotide group having a therapeutic effect on tumors, and a delivery aid group.

8. Having the structure shown in formula (101), 【Chemistry 1】 （１０１） In the formula, each R ＡＰ group is independently a group having the structure represented by the formula (102), 【Chemistry 2】 （１０２） In the formula, each AP group is the same or different, independently representing one of the aforementioned serving groups, R ｊ , each R ｋ or each R ｉ These are the same or different, and each independently represents a covalent bond or bonding group, and R ｉ and R ｋ It is not possible for both to be covalently bonded at the same time, and each n １ Each of these independently represents an integer from 0 to 4. Each A ０ The groups, whether the same or different, independently represent one of the aforementioned functional groups, m ０ n is an integer from 1 to 6. ０ These are integers from 1 to 6, 【Transformation 3】 represents the site where the group is covalently bonded, or I understand ０ is an integer from 1 to 4, and / or n ０ is an integer from 1 to 3, and / or each n １ These are independent integers between 0 and 1. Or, m ０ is 1 and / or n ０ is 1 and / or at least one n １ or each n １ is 0, or Each of the R ｋ or each of the R ｉ These are independently covalent or linear alkylene groups having a length of 1 to 70 carbon atoms, or one or more carbon atoms in the linear alkylene group are C(O), NH, O, S, CH=N, S(O) ２ , OP(O) ２ ,OP(O)(S),C ５ ~C ８ Glycoside group, C ２ ~C １０ Alkenylene group, C ２ ~C １０ Alkynylene group, C ６ ~C １０ Arylene group, C ３ ~C １８ Heterocyclylene group and C ５ ~C １０ The linear alkylene group is substituted with one or more selected from the group consisting of heteroarylene groups, and the linear alkylene group is C １ ~C １０ alkyl group, C ６ ~C １０ Aryl group, C ５ ~C １０ heteroaryl group, C １ ~C １０ Alkyl halogenated groups, -OC １ ~C １０ Alkyl alkyl group, -OC １ ~C １０ Alkylphenyl group, -C １ ~C １０ Alkyl-OH, -OC １ ~C １０ Alkyl halogenates, -SC １ ~C １０ Alkyl alkyl, -SC １ ~C １０ Alkylphenyl group, -C １ ~C １０ Alkyl-SH, -SC １ ~C １０ Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH ２ , -C １ ~C １０ Alkyl-NH ２ , -N(C １ ~C １０ (Alkyl) (C １ ~C １０ Alkyl alkyl group), -NH(C １ ~C １０ Alkyl alkyl group), -N(C １ ~C １０ (Alkyl) (C １ ~C １０ Alkylphenyl group), -NH(C １ ~C １０ Alkylphenyl group, cyano group, nitro group, -CO ２ H, -C(O)O(C １ ~C １０ Alkyl(alkyl group), -CON(C １ ~C １０ (Alkyl) (C １ ~C １０ Alkyl(alkyl group), -CONH(C １ ~C １０ Alkyl(alkyl group), -CONH ２ , -NHC(O)(C １ ~C １０ Alkyl alkyl group), -NHC(O)(phenyl group), -N(C １ ~C １０ Alkyl)C(O)(C １ ~C １０ Alkyl alkyl group), -N(C １ ~C １０ Alkyl)C(O)(phenyl group), -C(O)C １ ~C １０ Alkyl alkyl group, -C(O)C １ ~C １０ Alkylphenyl group, -C(O)C １ ~C １０ Alkyl halogenated groups, -OC(O)C １ ~C １０ Alkyl, -SO ２ (C １ ~C １０ Alkyl(alkyl group), -SO ２ (phenyl group), -SO ２ (C １ ~C １０ (Halogenated alkyl group), -SO ２ NH ２ , -SO ２ NH(C) １ ~C １０ Alkyl(alkyl group), -SO ２ NH (phenyl group), -NHSO ２ (C １ ~C １０ (Alkyl group), -NHSO ２ (phenyl group) and -NHSO ２ (C １ ~C １０ It may have one or more substituents selected from the group consisting of halogenated alkyl groups, or each n １ All of these are 0, and each R ｉ These are, independently, covalently bonded or bonded to a C group. １ ~C ２０ A combination of one or more bonds from alkylene groups, phosphate ester bonds, thiophosphate ester bonds, amide bonds, ester bonds, ether bonds, thioether bonds, disulfide bonds, 1,2,3-triazole subunits, polyethylene glycol subunits, pyrrolidine subunits, 2-oxopyrrolidine subunits, phenylene groups, cyclohexylene groups, 2-succinimide subunits, 2-thiosuccinimide subunits, amino acid subunits, and nucleotide subunits, or Each R ｉ This is independently a combination of one or two bonds from among a covalent bond, a disulfide bond, a propylene phosphate ester group, a 2-thiosuccinimide subunit, an amino acid subunit, or a GAU trinucleotide subunit, or R ｊ It is a covalent bond, m ０ is 1, or R ｊ is a bonding group, and the bonding group R ｊ It includes a main chain portion, a side chain portion and a composite bonding portion, the main chain portion is connected to the composite bonding portion and the side chain portion, and each of the side chain portions is connected to the main chain portion and the R ＡＰ The base is bonded, and each of the composite bonding portions is bonded to the main chain portion and the functional group A, respectively. ０ Combined, The main chain portion is a linear alkylene group having a length of 1 to 70 carbon atoms, or one or more carbon atoms in the linear alkylene group are C(O), NH, O, S, CH=N, S(O) ２ , OP(O) ２ , C ５ ~C ８ Glycoside group, C ２ ~C １０ Alkenylene group, C ２ ~C １０ Alkynylene group, C ６ ~C １０ Arylene group, C ３ ~C １８ Heterocyclylene group and C ５ ~C １０ The linear alkylene group is substituted with one or more selected from the group consisting of heteroarylene groups, and the linear alkylene group is C １ ~C １０ alkyl group, C ６ ~C １０ Aryl group, C ５ ~C １０ heteroaryl group, C １ ~C １０ Alkyl halogenated groups, -OC １ ~C １０ Alkyl alkyl group, -OC １ ~C １０ Alkylphenyl group, -C １ ~C １０ Alkyl-OH, -OC １ ~C １０ Alkyl halogenates, -SC １ ~C １０ Alkyl alkyl, -SC １ ~C １０ Alkylphenyl group, -C １ ~C １０ Alkyl-SH, -SC １ ~C １０ Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH ２ , -C １ ~C １０ Alkyl-NH ２ , -N(C １ ~C １０ (Alkyl) (C １ ~C １０ Alkyl alkyl group), -NH(C １ ~C １０ Alkyl alkyl group), -N(C １ ~C １０ (Alkyl) (C １ ~C １０ Alkylphenyl group), -NH(C １ ~C １０ Alkylphenyl group, cyano group, nitro group, -CO ２ H, -C(O)O(C １ ~C １０ Alkyl(alkyl group), -CON(C １ ~C １０ (Alkyl) (C １ ~C １０ Alkyl(alkyl group), -CONH(C １ ~C １０ Alkyl(alkyl group), -CONH ２ , -NHC(O)(C １ ~C １０ Alkyl alkyl group), -NHC(O)(phenyl group), -N(C １ ~C １０ Alkyl)C(O)(C １ ~C １０ Alkyl alkyl group), -N(C １ ~C １０ Alkyl)C(O)(phenyl group), -C(O)C １ ~C １０ Alkyl alkyl group, -C(O)C １ ~C １０ Alkylphenyl group, -C(O)C １ ~C １０ Alkyl halogenated groups, -OC(O)C １ ~C １０ Alkyl, -SO ２ (C １ ~C １０ Alkyl(alkyl group), -SO ２ (phenyl group), -SO ２ (C １ ~C １０ (Halogenated alkyl group), -SO ２ NH ２ , -SO ２ NH(C) １ ~C １０ Alkyl(alkyl group), -SO ２ NH (phenyl group), -NHSO ２ (C １ ~C １０ (Alkyl group), -NHSO ２ (phenyl group) and -NHSO ２ (C １ ~C １０ It may have one or more substituents selected from the group consisting of halogenated alkyl groups. Each of the aforementioned side chain portions is independently covalently bonded, or is a linear alkylene group having a length of 1 to 70 carbon atoms, or one or more carbon atoms in the linear alkylene group are C(O), NH, O, S, CH=N, S(O) ２ , OP(O) ２ , C ５ ~C ８ Glycoside group, C ２ ~C １０ Alkenylene group, C ２ ~C １０ Alkynylene group, C ６ ~C １０ Arylene group, C ３ ~C １８ Heterocyclylene group and C ５ ~C １０ The linear alkylene group is substituted with one or more selected from the group consisting of heteroarylene groups, and the linear alkylene group is C １ ~C １０ alkyl group, C ６ ~C １０ Aryl group, C ５ ~C １０ heteroaryl group, C １ ~C １０ Alkyl halogenated groups, -OC １ ~C １０ Alkyl alkyl group, -OC １ ~C １０ Alkylphenyl group, -C １ ~C １０ Alkyl-OH, -OC １ ~C １０ Alkyl halogenates, -SC １ ~C １０ Alkyl alkyl, -SC １ ~C １０ Alkylphenyl group, -C １ ~C １０ Alkyl-SH, -SC １ ~C １０ Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH ２ , -C １ ~C １０ Alkyl-NH ２ , -N(C １ ~C １０ (Alkyl) (C １ ~C １０ Alkyl alkyl group), -NH(C １ ~C １０ Alkyl alkyl group), -N(C １ ~C １０ (Alkyl) (C １ ~C １０ Alkylphenyl group), -NH(C １ ~C １０ Alkylphenyl group, cyano group, nitro group, -CO ２ H, -C(O)O(C １ ~C １０ Alkyl(alkyl group), -CON(C １ ~C １０ (Alkyl alkyl group) (C １ ~C １０ Alkyl(alkyl group), -CONH(C １ ~C １０ Alkyl(alkyl group), -CONH ２ , -NHC(O)(C １ ~C １０ Alkyl alkyl group), -NHC(O)(phenyl group), -N(C １ ~C １０ Alkyl)C(O)(C １ ~C １０ Alkyl alkyl group), -N(C １ ~C １０ Alkyl)C(O)(phenyl group), -C(O)C １ ~C １０ Alkyl alkyl group, -C(O)C １ ~C １０ Alkylphenyl group, -C(O)C １ ~C １０ Alkyl halogenated groups, -OC(O)C １ ~C １０ Alkyl, -SO ２ (C １ ~C １０ Alkyl(alkyl group), -SO ２ (phenyl group), -SO ２ (C １ ~C １０ (Halogenated alkyl group), -SO ２ NH ２ , -SO ２ NH(C) １ ~C １０ Alkyl(alkyl group), -SO ２ NH (phenyl group), -NHSO ２ (C １ ~C １０ (Alkyl group), -NHSO ２ (phenyl group) and -NHSO ２ (C １ ~C １０ It may have one or more substituents selected from the group consisting of halogenated alkyl groups. Each of the aforementioned composite bonding portions is independently either a covalent bond or a bond structure C １ ~C １０ A combination of one or more bonds from alkylene groups, phosphate ester bonds, thiophosphate ester bonds, amide bonds, ester bonds, ether bonds, thioether bonds, disulfide bonds, 1,2,3-triazole subunits, polyethylene glycol subunits, pyrrolidine subunits, 2-oxopyrrolidine subunits, phenylene groups, cyclohexylene groups, 2-succinimide subunits, 2-thiosuccinimide subunits, amino acid subunits, and nucleotide subunits, or The aforementioned bonding group R ｊ Each of the aforementioned composite bonding portions within consists of the main chain portion and one of the functional groups A. ０ The side chain portion is connected to n ０ Each side chain portion consists of the main chain portion and one of the R ＡＰ Bonded to the base, or The composite according to claim 7, wherein all of the side chain portions are bonded to the same atom in the main chain portion, or each of the side chain portions is bonded to a different atom in the main chain portion.

9. I understand ０ is 1, and the bonding group R ｊ This includes the structure shown in formula (301), 【Chemistry 4】 Formula (301) In the formula, k is an integer from 1 to 3, and L Ｃ L is the main chain portion. Ａ This is the side chain portion, L Ｂ This is the composite joint, 【Transformation 5】 This represents the site where the group is covalently bonded. The main chain portion L Ｃ C is covalent or 2-4 valent, linear or branched. １ ~C ２５ A saturated hydrocarbon group, or one or more carbon atoms in the saturated hydrocarbon group are C(O), NH, O, S, CH=N, S(O) ２ , OP(O) ２ , C ５ ~C ８ Glycoside group, C ２ ~C ５ Alkenylene group, C ２ ~C ５ Alkynylene group, C ６ ~C １０ Arylene group, C ３ ~C ８ Heterocyclylene group and C ５ ~C １０ The saturated hydrocarbon group is substituted with one or more selected from the group consisting of heteroarylene groups, and the saturated hydrocarbon group is C １ ~C ５ alkyl group, C ６ ~C １０ Aryl group, C ５ ~C １０ Heteroaryl group, -O-C １ ~C ５ Alkyl alkyl group, -OC １ ~C ５ Alkylphenyl group, -C １ ~C ５ alkyl-OH, -SC １ ~C ５ Alkyl alkyl groups, nitro, -C(O)O(C １ ~C ５ Alkyl(alkyl group), -CON(C １ ~C ５ (Alkyl) (C １ ~C ５ Alkyl(alkyl group), -CONH(C １ ~C ５ Alkyl(alkyl group), -CONH ２ , -NHC(O)(C １ ~C ５ Alkyl alkyl group), -NHC(O)(phenyl group), -N(C １ ~C ５ Alkyl)C(O)(C １ ~C ５ Alkyl alkyl group), -N(C １ ~C ５ Alkyl)C(O)(phenyl group), -C(O)C １ ~C ５ Alkyl alkyl group, -C(O)C １ ~C ５ Alkylphenyl group, -OC(O)C １ ~C ５ Alkyl, -SO ２ (C １ ~C ５ Alkyl(alkyl group), -SO ２ (phenyl group), -SO ２ NH ２ , -SO ２ NH(C) １ ~C ５ Alkyl(alkyl group), -SO ２ NH (phenyl group), -NHSO ２ (C １ ~C ５ Alkyl(alkyl group) and -NHSO ２ It may have one or more substituents selected from the group consisting of (phenyl groups), Each of the aforementioned side chain portions is independently covalently bonded, or is a linear alkylene group having a length of 1 to 70 carbon atoms, or one or more carbon atoms in the linear alkylene group are C(O), NH, O, S, CH=N, S(O) ２ , OP(O) ２ , C ５ ~C ８ Glycoside group, C ２ ~C １０ Alkenylene group, C ２ ~C １０ Alkynylene group, C ６ ~C １０ Arylene group, C ３ ~C １８ Heterocyclylene group and C ５ ~C １０ The linear alkylene group is substituted with one or more selected from the group consisting of heteroarylene groups, and the linear alkylene group is C １ ~C １０ alkyl group, C ６ ~C １０ Aryl group, C ５ ~C １０ heteroaryl group, C １ ~C １０ Alkyl halogenated groups, -OC １ ~C １０ Alkyl alkyl group, -OC １ ~C １０ Alkylphenyl group, -C １ ~C １０ Alkyl-OH, -OC １ ~C １０ Alkyl halogenates, -SC １ ~C １０ Alkyl alkyl, -SC １ ~C １０ Alkylphenyl group, -C １ ~C １０ Alkyl-SH, -SC １ ~C １０ Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH ２ , -C １ ~C １０ Alkyl-NH ２ , -N(C １ ~C １０ (Alkyl) (C １ ~C １０ Alkyl alkyl group), -NH(C １ ~C １０ Alkyl alkyl group), -N(C １ ~C １０ (Alkyl) (C １ ~C １０ Alkylphenyl group), -NH(C １ ~C １０ Alkylphenyl group, cyano group, nitro group, -CO ２ H, -C(O)O(C １ ~C １０ Alkyl(alkyl group), -CON(C １ ~C １０ (Alkyl) (C １ ~C １０ Alkyl(alkyl group), -CONH(C １ ~C １０ Alkyl(alkyl group), -CONH ２ , -NHC(O)(C １ ~C １０ Alkyl alkyl group), -NHC(O)(phenyl group), -N(C １ ~C １０ Alkyl)C(O)(C １ ~C １０ Alkyl alkyl group), -N(C １ ~C １０ Alkyl)C(O)(phenyl group), -C(O)C １ ~C １０ Alkyl alkyl group, -C(O)C １ ~C １０ Alkylphenyl group, -C(O)C １ ~C １０ Alkyl halogenated groups, -OC(O)C １ ~C １０ Alkyl, -SO ２ (C １ ~C １０ Alkyl(alkyl group), -SO ２ (phenyl group), -SO ２ (C １ ~C １０ (Halogenated alkyl group), -SO ２ NH ２ , -SO ２ NH(C) １ ~C １０ Alkyl(alkyl group), -SO ２ NH (phenyl group), -NHSO ２ (C １ ~C １０ (Alkyl group), -NHSO ２ (phenyl group) and -NHSO ２ (C １ ~C １０ It may have one or more substituents selected from the group consisting of halogenated alkyl groups. Each of the aforementioned composite bonding portions is independently either a covalent bond or a bond structure C １ ~C １０ The complex according to claim 8, wherein the combination of bonds is one or more of the following: alkylene group, phosphate ester bond, thiophosphate ester bond, amide bond, ester bond, ether bond, thioether bond, disulfide bond, 1,2,3-triazole subunit, polyethylene glycol subunit, pyrrolidine subunit, 2-oxopyrrolidine subunit, phenylene group, cyclohexylene group, 2-succinimide subunit, 2-thiosuccinimide subunit, amino acid subunit, and nucleotide subunit.

10. The composite according to claim 9, having the structure shown in formula (305). 【Transformation 6】 Formula (305)

11. The aforementioned bonding group R ｊ It has the structure shown in formula (306), 【Transformation 7】 Formula (306) In the formula, n ３０６ is an integer from 0 to 3, and each p ３０６ These are independent integers from 1 to 6, 【Transformation 8】 This represents the site where the group is covalently bonded. ＊ The oxygen atom marked with R ＡＰ The group forms a phosphate ester bond, an ether bond, or an ester bond, and at least one of the oxygen atoms marked with # is the functional group A ０ The remaining oxygen atoms, marked with #, are bonded to an ether bond, ester bond, or phosphate ester bond, and bond to a hydrogen atom to form a hydroxyl group, or C １ ~C ３ Bonded with an alkyl group, C １ ~C ３ The composite according to claim 9, which forms an alkoxy group.

12. The composite according to claim 11, having the structure shown in formula (307a), (307b), or (307c). 【Chemistry 9】

13. Having the structure shown in formula (308), 【Chemistry 10】 Formula (308) During the ceremony, n ３０８ is an integer selected from 1 to 10, each m ３０８ These are independent integers selected from 2 to 10. Each R ３０８ These are H and C, which are independent of each other. １ ~C １０ alkyl group, C １ ~C １０ Halogenated alkyl group or C １ ~C １０ It is an alkoxy group, Each R ３ Independently, the functional group A ０ is or R ＡＰ It is a base and has at least one R ３ The functional group A ０ and at least one R ３ is the aforementioned R ＡＰ It is a base, or one R ３ The functional group A ０ And the remaining R ３ is the aforementioned R ＡＰ It is the basis, The aforementioned functional group A ０ Each L that is combined １ represents the composite joint, and R ＡＰ Each L that is combined １ represents the side chain portion, or Each L １ These are independently selected from the group consisting of groups L4 to L23 and any combination thereof, or Each L １ The group is independently selected from the group consisting of combinations of at least two bonds from groups L4-L9, L13, L14, and L18, or each L １ This is independently a combination of at least two bonds from groups L4, L5, L7, L9, L13, L14, and L18, or Each L １ The length is independently 3 to 25 atoms, or each L １ The composite according to claim 9, wherein the length of each atom is independently 4 to 15 atoms.

14. n ３０８ R is an integer between 2 and 6, and has 2 to 4 values. ３ is the aforementioned R ＡＰ It is the base, and the remaining R ３ is the functional group, or each m ３０８ Each of these is an integer between 2 and 5, and / or each m ３０８ They are all equal, or n ３０８ m is an integer selected from 2 to 4, and each m ３０８ These are independent integers selected from 2 to 4, and each R ３０８ Both are H, or One R ３ The functional group A ０ And the remaining R ３ is the aforementioned R ＡＰ The composite according to claim 13, which is the base.

15. Each L １ It includes a bonding site that is bonded to the N atom on the nitrogen-containing skeleton, and the functional group A ０ or R ＡＰ The base contains both a bonding site and a site that bonds to the N atom on the nitrogen-containing skeleton, and the site that bonds to the N atom forms an amide bond with the N atom, or one or more L １ The format is selected from B5, B6, B5', or B6'. 【Chemistry 11】 During the ceremony, 【Chemistry 12】 represents the site where the group is covalently bonded, and q ２ is an integer from 1 to 10, or q ２ The composite according to claim 14, wherein is an integer from 1 to 5.

16. A composite according to any one of claims 13 to 15, having the structure shown in formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), (422), (423), (424), (425), (426), or (427). 【Chemistry 13】 Formula (403) 【Chemistry 14】 Formula (404) 【Chemistry 15】 Formula (405) 【Chemistry 16】 Formula (406) 【Chemistry 17】 Formula (407) [Chemistry 18] Formula (408) 【Chemistry 19】 Formula (409) 【Chemistry 20】 Formula (410) 【Chemistry 21】 Formula (411) 【Chemistry 22】 Formula (412) 【Chemistry 23】 Formula (413) 【Chemistry 24】 Formula (414) 【Chemistry 25】 Formula (415) 【Chemistry 26】 Formula (416) 【Chemistry 27】 Formula (417) 【Chemistry 28】 Formula (418) 【Chemistry 29】 Formula (419) 【Transformation 30】 Formula (420) 【Chemistry 31】 Formula (421) 【Chemistry 32】 Formula (422) 【Transformation 33】 Formula (423) 【Transformation 34】 Formula (424) 【Chemistry 35】 Formula (425) 【Transformation 36】 Formula (426) 【Chemistry 37】 Formula (427)

17. R ｊ It comprises nucleotide sequence I and nucleotide sequence II, each comprising 5 to 25 modified or unmodified nucleotides, nucleotide sequence I and nucleotide sequence II being at least partially inversely complementary, the delivery group being bound to nucleotide sequence I, the functional group being bound to nucleotide sequence II, and nucleotide sequence I and nucleotide sequence II not inducing an immune or toxic response in the body of a subject, or The composite according to claim 8, wherein R j is digestible.

18. The complex according to claim 17, wherein the 3' end of the delivery group is bonded to the 5' position of the ribose of the 5' terminal nucleotide of the nucleotide sequence I via a phosphate ester bond, and the functional group is bonded to the 5' position of the ribose of the 5' terminal nucleotide of the nucleotide sequence II, or the functional group comprises a nucleotide sequence, and the 3' end of the nucleotide sequence is bonded to the 5' position of the ribose of the 5' terminal nucleotide of the nucleotide sequence II via a phosphate ester bond.

19. The nucleotide sequence I and the nucleotide sequence II are substantially inversely complementary or completely inversely complementary, or the nucleotide sequence I and the nucleotide sequence II are of equal length and each consists of 10 to 20 modified or unmodified nucleotides, or the nucleotide sequence I and the nucleotide sequence II each consist of 17 nucleotides and are completely inversely complementary, or the nucleotide sequence I and the nucleotide sequence II are the sequences shown in SEQ ID NO: 40 and SEQ ID NO: 41, respectively. 5'-GUACAUUCUAGAUAGCC-3' (Sequence ID 40) 5'-GGCUAUCUAGAAUGUAC-3' (Sequence ID 41) It has, Alternatively, nucleotide sequence I and nucleotide sequence II are the sequences shown in SEQ ID NO: 42 and SEQ ID NO: 43, respectively. 5'-GmUfAmCfAmUfUfCfUfAmGmAmUfAmGmCfCf-3' (Sequence ID 42) 5'-GmGmCfUfAmUfCfUfAmGmAmAmUfGmUfAmCf-3' (Sequence ID 43) The composite according to claim 18, having the following characteristics.

20. At least one or all of the functional groups are diagnostic groups, and each of the diagnostic groups is independently selected from contrast groups or fluorescence imaging groups, or At least one or all of the functional groups are low molecular weight therapeutic groups having a therapeutic effect against tumors, and each of the low molecular weight therapeutic groups is independently selected from cytotoxic groups, antibiotic groups, angiogenesis inhibitors, or antibody drug groups, or At least one or all of the functional groups are functional oligonucleotide groups, and the functional oligonucleotide groups can regulate the expression levels of cancer-related genes in tumor cells, or At least one of the functional groups is a delivery aid group, and the delivery aid group is C １０ ~C ３０ The composite according to any one of claims 7 to 19, wherein the composite is one or more selected from hydrocarbon groups, cholesterol groups, and phospholipid groups.

21. A pharmaceutical composition for the diagnosis and / or treatment of tumors and tumor-related diseases or symptoms, comprising the complex described in any one of claims 7 to 20 and a pharmaceutically acceptable carrier.

22. A kit comprising the complex according to any one of claims 7 to 20 and / or the pharmaceutical composition according to claim 21.

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