CN114681619B - Aptamer complex based on Biotin-SA and application thereof - Google Patents
Aptamer complex based on Biotin-SA and application thereof Download PDFInfo
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Abstract
The application relates to a nucleic acid aptamer complex based on Biotin-SA and application thereof, in particular to a Biotin-streptavidin (Biotin-SA) delivery system which is constructed by using at least one streptavidin and a plurality of nucleic acid aptamer drug molecules, wherein the complex formed by combining Biotin (Biotin) modified on the nucleic acid aptamer with Streptavidin (SA) is such as polyhedron, sphere, nanostructure and the like; the application can realize the effective delivery of the aptamer medicine molecules at the tumor part, thereby reducing the degradation and the renal clearance of the aptamer medicine molecules.
Description
Technical Field
The application belongs to the field of targeted drug delivery, and in particular relates to a nucleic acid aptamer compound of Biotin-streptavidin (Biotin-SA) and application thereof, which are targeted delivery systems of nucleic acid aptamer drug molecules.
Background
Targeted treatment of tumors
According to the difference between tumor cells and normal cells, a corresponding target is designed to realize tumor site specific regulation or cell killing and the like, which is called tumor targeted treatment. Targeted therapy has become one of the conventional means of current tumor therapy. The targeted drugs can act alone, such as small molecule inhibitors, monoclonal antibodies; antibody drug conjugates (Antibody Drug Conjugate, ADC) may also be formulated with chemotherapeutic drugs. Currently there are 6 ADC drugs approved by the FDA for targeted treatment of tumors. However, the problems of high cost, batch-to-batch variation, difficult modification, and the like of antibodies themselves limit the wide use of antibody drugs [1].
Aptamer pharmaceutical complexes
A nucleic acid aptamer is a single-stranded DNA or RNA, also known as a chemical antibody. Compared with antibodies, the antibody has low immunogenicity, can be synthesized through solid phase synthesis, has small batch-to-batch difference, is easy to modify and is thermally stable. Currently, there are already the nucleic acid aptamers approved by the FDA for the treatment of age-related macular lesions, confirming the safety of the nucleic acid aptamers themselves. The attachment of nucleic acid aptamers to drugs has been reported in many ways, including non-covalent intercalation, covalent attachment at the ends, and the substitution of drug bases for the original bases. Compared with the antibody, the connection of the aptamer and the drug can realize fixed point and quantification, and the batch-to-batch difference is reduced. However, the simple aptamer is easy to degrade and easy to remove kidney when applied in vivo [2].
Biotin-SA system
There is a strong affinity between Biotin (Biotin) and avidin (avidin) and Streptavidin (strepavidin), and this system is now widely used in various fields including immunopurification, separation, quantitative detection of cell surface proteins, separation of DNA, etc. The existing commercialized kit is used for ELISA, western Blot, immunofluorescence, immunohistochemistry and the like. The system is also used to carry nucleic acids such as siRNA, aptamer et al [3,4], and drugs to achieve targeted treatment of tumors [5,6].
Biotin-SA-based tumor targeted therapy
The current tumor targeting therapy based on Biotin-SA is mainly based on the construction of fusion proteins, so that the immunotherapy, radiotherapy and the like of tumors are realized. If Biotin is connected with nuclide and SA is connected with monoclonal antibody, the targeted radiotherapy of tumor is realized [7]. Among them, there are also reports of aptamer-based. Such as carrying aptamer-doxorubicin complex and polypeptide to achieve dual-targeting drug delivery [5]. It has also been reported that a triangle is formed by strand complementation pairing, doxorubicin is supported, and the triangle end is linked to a cleavable aptamer probe by the action of Biotin-SA for imaging and treatment of tumors [6].
The use of Biotin-SA for targeted delivery of programmably introduced aptamer drug molecules has not been reported so far, and controlled targeted imaging and controlled drug release at specific sites has been achieved through the design of nucleic acids.
Reference to the literature
[1]Dorin Toader.Top Med Chem,2018,28,289-332.
[2]Keith E Maier and Matthew Levy.Molecular Therapy Methods&Clinical Development.2016,5,16014
[3]Ted C.Chu,Karen Y.Twu,Andrew D.Ellington,Matthew Levy.2006,34.
[4]Zixi Hu,Juntao Tan,Zongqiang Lai,et.al.Nanoscale Research Letters, 2017,12:96.
[5]Kyoungin Min,Hunho Jo,Kyungmi Song,et.al.Biomaterals.2011,32, 2124-2132.
[6]Yanli Lei,Zhenzhen Qiao,Jinlu Tang,et.al.Theranostics.2018,8,4062- 4071.
[7]Jhon H.T.Luong,Sandeep K.Vashist.ACS Omega,2020,5,10-18.
Disclosure of Invention
The application aims to provide a delivery system of a nucleic acid aptamer drug molecule, which is used for solving the problems of poor stability, easiness in renal clearance and short half-life of the existing nucleic acid aptamer drug molecule when the nucleic acid aptamer drug molecule is applied in vivo.
It is another object of the present application to provide a novel method for constructing nanosystems from aptamer drug molecules.
The application relates to a nucleic acid aptamer complex based on Biotin-SA, and in one aspect provides a delivery system of nucleic acid aptamer drug molecules, which comprises,
at least one of the streptavidin-type peptides,
a plurality of nucleic acid aptamer drug molecules, and,
modifying biotin on the aptamer drug molecule to enable binding of the aptamer drug molecule to streptavidin;
wherein the aptamer drug molecule comprises a aptamer sequence specifically recognizing a target and a drug molecule;
the nucleic acid aptamer drug molecule and the streptavidin can quickly form a delivery system of the nucleic acid aptamer drug molecule in a buffer solution.
Preferably, the nucleic acid aptamer drug molecule is formed by introducing a nucleic acid aptamer into the drug molecule in a programmed manner, or the nucleic acid aptamer drug molecule is formed by introducing a nucleic acid molecule in a solid phase synthesis manner through a solid phase synthesis module formed by drug modification.
Preferably, the nucleic acid aptamer drug molecule comprises a nucleic acid aptamer with specific tissue-specific targeting ability, and the biotin modifies one or both ends of the nucleic acid aptamer drug molecule; the drug molecule modifies any position of the aptamer.
Preferably, the drug molecule is selected from the group consisting of base analogues; preferably, the drug molecule is directly accessible to the aptamer by solid phase synthesis; the drug molecule is 5-FU, gemcitabine or camptothecin base analogues.
Preferably, the aptamer is a tumor-targeted aptamer, preferably the aptamer sgc8, XQ2d, DML7 or a combination thereof.
In another aspect, the application relates to a delivery system for a nucleic acid aptamer drug molecule, the delivery system for a nucleic acid aptamer drug molecule forming a complex comprising a nucleic acid aptamer drug molecule, the complex being a nanoparticle, the complex being capable of reaching a tumor site by an EPR effect, and being capable of being retained at the tumor site by a targeting effect of the nucleic acid aptamer after the tumor site has been disassembled in response to the disassembly, to effect targeted imaging or treatment of a tumor, comprising
At least one Streptavidin (SA);
a nucleic acid fragment, one or both ends of which are modified with Biotin to bind to the streptavidin, comprising,
the first nucleic acid fragment is a nucleic acid aptamer, has a specific recognition function and is used as a recognition unit of the delivery system;
the second nucleic acid fragment is a nucleic acid aptamer or other nucleic acid fragment;
wherein the intermediate or terminal positions of the second nucleic acid fragment modify the tissue microenvironment responsive linkage or group to be linked to each other such that the delivery system is capable of breaking down under the specific tissue microenvironment.
Preferably, the drug molecule is selected from the group consisting of base analogues; preferably, the drug molecule is directly accessible to the aptamer by solid phase synthesis; the drug molecule is 5-FU, gemcitabine or camptothecin base analogues, more preferably the drug molecule is nuclide I linked by a phenolic hydroxyl moiety 131 、I 125 。
The aptamer is a aptamer with a tumor targeting effect, preferably the aptamer sgc8, XQ2d, DML7 or a combination thereof.
The application of the aptamer complex based on Biotin-streptavidin-SA in preparing medicaments and/or imaging for treating cell proliferative diseases.
Drawings
FIG. 1 is a schematic diagram of the operation of the present application;
FIG. 2 is a further possible form of the application;
FIG. 3 is an electron microscope image demonstrating nanoparticle formation in accordance with the present application;
FIG. 4 is a PAGE gel diagram verifying the formation of nanoparticles in accordance with the present application;
FIG. 5 is a graph verifying the in vitro targeting of the present application;
FIG. 6 is a graph verifying in vivo stability of the present application;
FIG. 7 is a graph verifying in vitro toxicity of the present application;
FIG. 8 is a graph demonstrating the effects of in vivo targeted therapies of the application;
Detailed Description
The application will be further described in connection with the accompanying drawings and specific embodiments to facilitate a better understanding of the application, but are not to be construed as limiting the application.
The present application relates to a nucleic acid aptamer complex comprising a nucleic acid aptamer and a streptavidin protein; the aptamer is connected with Biotin so as to be combined into a aptamer complex according to the binding action between the Biotin and the streptomycin protein; in the aptamer complexes formed herein, biotin is modified at both ends of some aptamers to allow ligation of two SAs to form larger molecules or particles, while other aptamers may be free to perform targeting or imaging drug delivery functions (FIG. 1).
Specifically, based on the structure of the aptamer complex, the embodiment of the application comprises a delivery system for a aptamer drug molecule, wherein the delivery system comprises streptavidin, the aptamer drug molecule and biotin, and the aptamer drug molecule is combined with the streptavidin through the biotin; the aptamer drug molecule may be a aptamer having a sequence that specifically recognizes a target and a drug molecule modified with a drug property.
The aptamer complex according to the specific embodiment of the application comprises a plurality of streptavidin proteins and a plurality of aptamers, wherein both ends of one type of aptamer are modified with Biotin, the aptamers can be in a form of single or multiple aptamers connected end to end, and the aptamers can be in a form of a microenvironment response group or a connecting bond in the middle, and in some embodiments, the microenvironment response group or the connecting bond can be decomposed under a specific tissue microenvironment, such as a tumor microenvironment. The aptamer of the aptamer complex specifically comprises two types of nucleic acid fragments, wherein the first type of nucleic acid fragment can be a aptamer and can be used for identifying a specific target; the second nucleic acid fragment is a nucleic acid aptamer or other nucleic acid fragments, and is used for connecting each streptavidin protein and stabilizing a nucleic acid aptamer complex; in more specific embodiments, the fluorescent/emissive substance, pharmaceutical compound, or the like is the opposite end of the aptamer to the biotin-binding end, to be able to better exert its effect; the second class of nucleic acid aptamers may also be modified with fluorescent/emissive substances and/or pharmaceutical compounds, wherein the second class of nucleic acid fragments have a tissue microenvironment responsive linkage or group for interconnection in order to be able to break down under specific circumstances.
The modified drug molecules in the above-described nucleic acid aptamers or nucleic acid fragments may be formed by programmatically introducing drug molecules.In a further embodiment of the present application, the drug molecule is a modified solid phase synthesis module formed by introducing a nucleic acid molecule by solid phase synthesis, wherein the drug molecule may be 5-FU, gemcitabine (document 1), a camptothecin base analog (document 2), a phenolic hydroxyl-linked nuclide I 131 Can be connected with I through phenolic hydroxyl 125 Is described in (3).
The specific embodiment of the application relates to the use of a nucleic acid aptamer compound, which can be sgc8, a nucleic acid aptamer XQ-2D and a nucleic acid aptamer DML-7, wherein the nucleic acid aptamer adopted in the specific embodiment of the application is sgc8, and the nucleic acid aptamer comprises the following sequence:
5’-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3’ (SEQ ID No.:1)
the aptamer was screened and named in 2006 (reference: dihua Shangguan, ying Li, zhiwen Tang, et al PNAS,2006,103,11838-11843), and identified in 2008 that the target was PTK7. The specific embodiment of the application is to construct a nucleating acid aptamer complex based on the nucleic acid aptamer or the modified nucleic acid aptamer by using streptomycin protein as a connection carrier (adapter). A nucleic acid aptamer complex formed comprising a streptomycin protein (at least one) and a nucleic acid aptamer (comprising the sequence set forth in SEQ ID No.: 1); the nucleic acid aptamer of the double-end modified Biotin can form a network by combining Biotin and streptavidin, and can be used for constructing polyhedrons, even larger nano particles and the like. Wherein the linkages or linking groups in the middle of the aptamer are tissue environment-responsive, i.e., the linkages or linking groups are cleaved under microenvironment, e.g., in tumor microenvironment; in certain embodiments of the application, the linkage or linking group is a disulfide bond. To achieve the functions of visualization or patency of the aptamer, the aptamer may be modified with a fluorescent/radioactive substance, or the aptamer may be modified with a pharmaceutical compound, e.g., the aptamer is modified with a nucleotide analog.
According to the functions of the aptamer, the aptamer related to the application is divided into two types, namely, the first type of aptamer is used for targeted development or administration of tumors, namely, one end of the aptamer is modified with Biotin and the other end of the aptamer is modified with fluorescein or radioactive substances; the second kind of aptamer has biotins modified at both ends, the aptamer can be in the form of single or multiple aptamer first connected, and the intermediate modification can be in groups responding in microenvironment. In an alternative embodiment of the application, both types of nucleic acid aptamers (first type of aptamer and second type of aptamer) are modified (e.g. nucleotide analogues) with a pharmaceutical compound. In alternative embodiments of the application, the second class of nucleic acid aptamers may be modified with fluorescent groups and/or quenching groups, or groups of the fluorescence resonance energy transfer effect, i.e. after cleavage of the tissue microenvironment responsive linkage or group, the quenching groups are separated from the fluorescent groups, either luminescent or non-luminescent, resulting in a dynamic change of development. In a specific embodiment of the application, one position of four binding sites of streptomycin protein is a nucleic acid aptamer with fluorescent/radioactive substances and the like for imaging or medicines, the other three positions are nucleic acid aptamer complexes with both ends modified by Biotin, and the nucleic acid aptamer can be in a form of connecting the first positions of single or multiple nucleic acid aptamers, and the middle modification can be a group responding in a microenvironment; alternatively, the aptamer bound at the four binding sites of streptavidin is a aptamer modified with Biotin at both ends, and the aptamer may be in the form of a single or multiple aptamers connected at their first sites, and the intermediate modification may be a group that responds in the microenvironment. The micro-environment response connection bond is a linker which is broken under a specific environment, such as a disulfide bond, and the like.
The aptamer compound based on Biotin-SA can protect the aptamer to a certain extent, reduce the possibility of being attacked by enzyme, reduce the renal excretion rate compared with pure nucleic acid, and solve the problems of poor stability and short half-life of the current aptamer drug compound in vivo application. In addition, the aptamer itself has targeting properties, and the aptamer complex based on Biotin-SA can be targeted.
The nucleic acid aptamer compound of the Biotin-SA constructed by the application has good biocompatibility as biomacromolecule, drug base or terminal covalent modification drugs can realize fixed point and quantitative drug modification of nucleic acid aptamer, and can realize controllable drug release of tumor microenvironment based on linker of tumor microenvironment response, which is different from the current carrying method of nucleic acid aptamer drug compound based on the system, the current carrying method mostly only has one SA and four Biotin modified nucleic acid aptamer or nucleic acid aptamer drugs, the triangle formed by the construction also only has three groups of SA-Biotin combination, and the drug release has uncontrollable load and release problems.
The Biotin-SA aptamer complex can be used as a larger nano assembly, a micro-environment response connecting bond is broken in a tumor micro-environment, a simple Biotin-SA complex or a simple monomer can be formed, the complex is retained at a tumor part by virtue of the targeting effect of the aptamer, and the nucleic acid is degraded due to the disintegration of a nano system and the exposure of the tail end of the aptamer, so that drug molecules are released, and multiple effects such as targeted delivery, fixed-point administration, drug slow release and the like are achieved (refer to figure 1).
The present application will be described in detail with reference to specific examples.
Referring to fig. 1 and 2, in the construction of the aptamer complex of the application, both the aptamer for targeting and for ligation can be modified, the drug base (nucleotide analogue) can be ligated to the aptamer in a fixed ratio and position by means of solid phase synthesis, and the non-base drug (non-nucleotide analogue) can be terminally ligated to the aptamer. One end of the targeting aptamer is modified with Biotin, and the other end is modified with fluorescein/radioactive substances or medicines. The two ends of the connective nucleic acid aptamer are modified by Biotin and contain disulfide bonds and other groups which can respond in a specific microenvironment. Then, streptavidin and the aptamer (modified aptamer and disulfide-linked aptamer) are mixed, and self-assembled into a aptamer complex.
EXAMPLE 1 construction of Biotin-SA-aptamer complexes
Biotin-SA-aptamer pharmaceutical construction
The end of the aptamer for targeting is modified with a Biotin molecule (sgc 8) or the aptamer with targeting function and modified with a drug molecule (5-FU) sgc8. Both ends of the aptamer used for connection are modified with a Biotin molecule (sgc 8-sgc 8) or a drug molecule (5-FU) aptamer (sgc 8-sgc8-5 FU), and disulfide bonds (iHS-SH) are formed in the aptamer.
The sequences are shown in Table 1
TABLE 1
2. Nano-assembly construction
By virtue of the affinity between SA protein and Biotin, and the designability of the DNA strands, it is theoretically possible to construct different nanostructures by controlling the length of the DNA strands, or by complementary pairing between DNA strands (FIG. 2). SA protein and double Biotin modified DNA and single Biotin modified aptamer drug complex were combined according to 1:3:1,1:2:2,1:1:3, proportioning and mixing. Incubate at 37℃for 30 min. The constructed complex can be identified by electron microscopy (FIG. 3) and agarose gel mapping (FIG. 4). As shown in FIG. 3, SA and Biotin can form nanoparticles (left) or nanoplatelets (right) through the ligation of aptamer by different ratios. As shown in FIG. 4, the nanostructure formed is significantly larger, and the system can be disassembled by disrupting the aptamer for ligation by addition of DTT or GSH.
3. Stability of nanosystems
The stability of the system was significantly improved compared to the nucleic acid strand of the pure aptamer and the end-modified Biotin (FIG. 4). The pure aptamer or Biotin-SA-aptamer complex was incubated with a medium containing 10% FBS for 0-72 hours, after which the amount of remaining nucleic acid was detected by agarose gel, and the complex was found to remain more at the same time.
Example 2 Biotin-SA-aptamer complex construction for Targeted imaging
1. Complex construction
Fluorescent molecules or groups with similar functions are attached to the aptamer for targeting. Biotin (sgc 8) was modified at the other end of the aptamer. DNA (sgc 8-sgc 8) with disulfide bond modification in the middle and Biotin modification at both ends was synthesized. Or a fluorescent group or a quenching group is contained on both sides of the disulfide bond, or a group capable of changing fluorescence such as a group capable of generating FRET effect.
2. Nano-assembly construction
SA protein and acid response moiety and targeting moiety were combined according to 1:3:1, mixing the materials in proportion. Incubate at 37℃for 30 min.
3. Targeted imaging
The targeting of the complex was examined at the cellular level, and the results showed that the system was able to maintain the targeting of the aptamer itself. Whereas pure proteins are not targeted (fig. 5). Collecting colon cancer cells HCT116, taking 30 ten thousand cells to respectively incubate with Biotin-sgc8, SA protein and a complex system for 30 minutes, removing unbound chains, detecting signals on nucleic acid aptamer on the surface of the cells, and detecting signals of the sgc8 and the complex (left), wherein through flow detection of fluorescent signals bound to SA on the surface of the cells, fluorescent signals of the complex are visible, and pure SA is not bound to the cells, so that no fluorescent signals are generated (right).
Subcutaneous engrafting tumor or orthotopic tumor mice for constructing colon cancer cell HCT116, and treating tumor growth to 200mm 3 The system was followed by tail vein injection of Biotin-sgc8, biotin-SA-sgc 8. The imaging effect of the tumor site was observed within 0-6 hours. The distribution of each organ was examined by mouse dissection (FIG. 6), and it was found that the Biotin-SA-sgc8 system was superior to Biotin-sgc8 itself.
EXAMPLE 3 Biotin-SA-aptamer complex construction for targeted therapy
1. Complex construction
Biotin and a drug molecule (5-FU-sgc 8) are respectively modified at the ends of the aptamer.
DNA (sgc 8-sgc8-5 FU) with disulfide bond modification in the middle and Biotin modification at both ends was synthesized.
2. Nano-assembly construction
SA protein and acid response moiety and targeting moiety were combined according to 1:3:1, mixing the materials in proportion. Incubate at 37℃for 30 min.
3. Targeted therapy
At the cellular level, the cytotoxicity of the system was examined. It was found that the system alone was not toxic, whereas the drug-carrying system had targeted therapeutic effects (fig. 7). HCT116 cells were seeded into 96-well plates, 5FU was added, and the Biotin-SA-sgc8-5FU complex was added, and its cytotoxicity was examined, and the complex was found to retain the toxicity of 5-FU itself.
Subcutaneous engrafting tumor mice of colon cancer cells HCT116 were constructed by tail vein injection of 5-FU (10 mg/kg) and equivalent Biotin-SA-sgc8-5FU system. Once weekly dosing, tumor size and survival of mice were observed (fig. 8). The complex is seen to be superior to 5-FU in therapeutic effect.
Sequence listing
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<120> Biotin-SA-based aptamer complex and application thereof
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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atctaactgc tgcgccgccg ggaaaatact gtacggttag a 41
Claims (5)
1. A delivery system for a nucleic acid aptamer drug molecule comprising,
at least one Streptavidin (SA),
a plurality of nucleic acid aptamer drug molecules, and,
modifying Biotin (Biotin) on the aptamer drug molecule to enable binding of the aptamer drug molecule to streptavidin;
wherein the aptamer drug molecule comprises a aptamer sequence specifically recognizing a target and a drug molecule;
the nucleic acid aptamer drug molecule and the streptavidin can quickly form a delivery system of the nucleic acid aptamer drug molecule in a buffer solution; the biotin modifies one or both ends of the aptamer drug molecule; the drug molecule is 5-FU; the aptamer is sgc8.
2. The delivery system of a nucleic acid aptamer drug molecule of claim 1, wherein the nucleic acid aptamer drug molecule is formed by programmatically introducing a nucleic acid aptamer to the drug molecule, or wherein the nucleic acid aptamer drug molecule is formed by introducing a nucleic acid molecule by means of solid phase synthesis using a solid phase synthesis module modified from the drug.
3. A delivery system for a nucleic acid aptamer drug molecule according to claim 2, wherein said drug molecule is directly accessible to the aptamer by solid phase synthesis.
4. A delivery system of a nucleic acid aptamer drug molecule is characterized in that the delivery system of the nucleic acid aptamer drug molecule forms a complex comprising the nucleic acid aptamer drug molecule, the complex is nanoparticle, can reach a tumor site through an EPR effect, can be retained at the tumor site through a targeting effect of the nucleic acid aptamer after responding to disintegration at the tumor site, and realizes targeted imaging or treatment of tumors, and comprises
At least one Streptavidin (SA);
a nucleic acid fragment, one or both ends of which are modified with Biotin (Biotin) to bind to the streptavidin, comprising,
the first nucleic acid fragment is a nucleic acid aptamer, has a specific recognition function and is used as a recognition unit of the delivery system;
the second nucleic acid fragment is a nucleic acid aptamer, wherein the middle or end position of the second nucleic acid fragment modifies the tissue microenvironment responsive linkage or group to be linked to each other so that the delivery system is capable of decomposing under a specific tissue microenvironment;
the drug molecules are directly connected into the aptamer through solid phase synthesis; the drug molecule is 5-FU; the aptamer is sgc8.
5. Use of a delivery system of a nucleic acid aptamer drug molecule according to any one of claims 1-4 for the preparation of a medicament and/or an imaging agent for the treatment of a cell proliferative disease.
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