CN114681619A - Aptamer compound based on Biotin-SA and application thereof - Google Patents
Aptamer compound based on Biotin-SA and application thereof Download PDFInfo
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Abstract
The invention relates to a Biotin-SA-based aptamer complex and application thereof, in particular to a delivery system for constructing Biotin-streptavidin (Biotin-SA) into aptamer drug molecules, which comprises at least one streptavidin and a plurality of aptamer drug molecules, wherein the aptamer drug molecules are combined with the Biotin (Biotin) modified on the aptamer to form a complex, such as a polyhedron, a sphere, a nanostructure and the like; the invention can realize the effective delivery of the aptamer drug molecules at the tumor part, thereby reducing the degradation of the aptamer drug molecules and the renal clearance.
Description
Technical Field
The invention belongs to the field of targeted drug delivery, and particularly relates to a Biotin-streptavidin (Biotin-SA) aptamer compound and application thereof, which are a targeted delivery system of aptamer drug molecules.
Background
Targeted treatment of tumors
According to the difference between tumor cells and normal cells, corresponding targets are designed to realize the tumor site specific regulation or cell killing and the like, which are called the targeted therapy of tumors. Targeted therapy has become one of the conventional approaches to current tumor therapy. The targeted drug can play a role independently, such as a small molecule inhibitor and a monoclonal antibody; antibody Drug Conjugates (ADCs) may also be used in combination with chemotherapeutic drugs. Currently, 6 ADC drugs are approved by the FDA for targeted therapy of tumors. However, the problems of high cost of the antibody itself, lot-to-lot variation, difficulty in modification, etc. limit the widespread use of antibody drugs [1 ].
Aptamer drug complex
An aptamer is a single-stranded DNA or RNA, also known as a chemical antibody. Compared with antibodies, the antibodies have low immunogenicity, can be synthesized by solid phase, have small batch-to-batch difference, are easy to modify and are thermostable. The existing aptamer is approved by FDA to be used for treating age-related macular lesions, and the safety of the aptamer is proved. There have been many reports on the connection of aptamers to drugs, including non-covalent intercalation, terminal covalent linkage, and the way of replacing original base with drug base. Compared with the antibody, the connection of the aptamer and the medicine can realize fixed-point and quantitative determination, and the difference between batches is reduced. However, the simple aptamer has the problems of easy degradation, easy renal clearance and the like when applied in vivo [2 ].
Biotin-SA system
Biotin (Biotin), avidin (avidin) and Streptavidin (Streptavidin) have strong affinity, and the system is widely used in various fields, including immune purification, separation, quantitative detection of cell surface protein, separation of DNA, and the like. At present, the commercialized kit is used for ELISA, Western Blot, immunofluorescence, immunohistochemistry and the like. The system can also be used to carry nucleic acids such as siRNA, aptamer and the like [3,4], and drugs to achieve targeted therapy of tumors [5,6 ].
Biotin-SA-based tumor targeted therapy
Currently, the targeted therapy of tumors based on Biotin-SA is mainly based on the construction of fusion proteins, so as to realize immunotherapy, radiotherapy and the like of tumors. For example, Biotin is connected with nuclide, SA is connected with monoclonal antibody, and the target radiotherapy of tumor is realized [7 ]. Among them, those based on nucleic acid aptamers have been reported in the literature. For example, it carries aptamer-adriamycin complex and polypeptide to realize double target drug carrying [5 ]. It has also been reported that a triangle is formed by strand complementary pairing, loaded with doxorubicin, and the end of the triangle is ligated with a cleavable aptamer probe by the action of Biotin-SA for imaging and treatment of tumors [6 ].
So far, there is no literature report that Biotin-SA is used for targeted delivery of nucleic acid aptamer drug molecules which can be introduced in a programmed mode, and controllable targeted imaging and controllable drug release of specific positions are achieved through 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 invention aims to provide a delivery system of nucleic acid aptamer drug molecules, which is used for solving the problems of poor stability, easy renal clearance and short half-life of the existing nucleic acid aptamer drug molecules in-vivo application.
Another objective of the invention is to provide a novel method for constructing a nano system by using the aptamer drug molecule.
The invention relates to a nucleic acid aptamer complex based on Biotin-SA, and in one aspect provides a delivery system of a nucleic acid aptamer drug molecule, which comprises,
at least one of the streptavidin (S) molecules,
a plurality of 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 that specifically recognizes a target and a drug molecule;
the aptamer drug molecule and the streptavidin can quickly form a delivery system of the aptamer drug molecule in a buffer solution.
Preferably, the aptamer drug molecule is formed by programming introduction of a aptamer into a drug molecule, or the aptamer drug molecule is formed by introducing a solid-phase synthesis module formed by drug modification into a nucleic acid molecule by means of solid-phase synthesis.
Preferably, the aptamer drug molecule comprises a aptamer with specific tissue-specific targeting ability, and the biotin modifies one or both ends of the aptamer drug molecule; the drug molecule modifies any position of the aptamer.
Preferably, the drug molecule is selected from base analogues; preferably, the drug molecule is directly accessed to the aptamer by solid phase synthesis; the drug molecule is 5-FU, gemcitabine or camptothecin base analogues.
Preferably, the aptamer is a tumor-targeting aptamer, preferably the aptamer sgc8, XQ2d, DML7 or a combination thereof.
The invention also relates to a delivery system of the aptamer drug molecules, the delivery system of the aptamer drug molecules forms a compound comprising the aptamer drug molecules, the compound is nanoparticles and can reach a tumor part through an EPR effect, and the compound can be retained at the tumor part through the targeting effect of the aptamer after the tumor part responds to disintegration to realize the targeted imaging or treatment of the tumor, and the delivery system of the aptamer drug molecules comprises
At least one Streptavidin (SA);
a nucleic acid fragment, one or both ends of which are Biotin-modified to bind to the streptavidin, comprising,
the first nucleic acid fragment is an aptamer, has a specific recognition function and serves as a recognition unit of the delivery system;
the second nucleic acid fragment is aptamer or other nucleic acid fragments;
wherein the intermediate or terminal positions of the second type of nucleic acid segment modify the tissue microenvironment responsive linkage or group to interconnect so as to enable the delivery system to disintegrate under a specific tissue microenvironment.
Preferably, the drug molecule is selected from base analogues; preferably, the drug molecule is directly accessed to the aptamer through solid phase synthesis; the drug molecule is 5-FU, gemcitabine or camptothecin base analogue, and more preferably the drug molecule is nuclide I connected through a phenolic hydroxyl module131、I125。
The aptamer has a tumor targeting effect, and preferably the aptamer sgc8, XQ2d, DML7 or a combination thereof.
The nucleic acid aptamer complex based on the Biotin-streptavidin Biotin-SA is used for preparing medicines for treating cell proliferative diseases and/or imaging.
Drawings
FIG. 1 is a schematic diagram of the functioning of the present invention;
FIG. 2 is another possible form of the present invention;
FIG. 3 is an electron micrograph demonstrating the formation of nanoparticles according to the present invention;
FIG. 4 is a PAGE gel demonstrating nanoparticle formation according to the present invention;
FIG. 5 is a graph demonstrating in vitro targeting of the present invention;
FIG. 6 is a graph demonstrating the in vivo stability of the present invention;
FIG. 7 is a graph demonstrating in vitro toxicity of the present invention;
FIG. 8 is a graph demonstrating the efficacy of the in vivo targeted therapy of the present invention;
Detailed Description
The invention is further described in connection with the drawings and the detailed description of the invention for the purpose of facilitating a better understanding of the invention, but is not to be construed as limiting the invention.
The present invention relates to a nucleic acid aptamer complex comprising a nucleic acid aptamer and streptavidin; the aptamer is connected with Biotin (Biotin) so as to be capable of being combined into an aptamer complex according to the binding effect between the Biotin-streptomycin proteins; in the aptamer complexes formed, some aptamers are modified with biotin at both ends, so that two SAs can be linked to form larger molecules or particles, while others can 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 invention comprises a delivery system for the aptamer drug molecules, wherein the delivery system comprises streptavidin, the aptamer drug molecules and biotin, and the aptamer drug molecules are combined with the streptavidin through the biotin; the aptamer drug molecule can be aptamer, which has a sequence for specifically recognizing a target and is modified with a drug molecule with drug properties.
The invention relates to a nucleic acid aptamer compound, which comprises a plurality of streptavidin proteins and a plurality of nucleic acid aptamers, wherein the two ends of one type of nucleic acid aptamers are modified with Biotin, the nucleic acid aptamers can be in a form that single or a plurality of nucleic acid aptamers are connected end to end, and a micro-environment response group or a connection bond can be contained in the nucleic acid aptamers, and in some embodiments, the micro-environment response group or the connection bond can be decomposed under a specific tissue micro environment, such as a tumor micro environment. The aptamer of the aptamer complex specifically comprises two types of nucleic acid fragments, wherein the first type of nucleic acid fragment can be the aptamer and can be used for identifying a specific target; the second nucleic acid fragment is an aptamer or other nucleic acid fragments, and is used for connecting each streptavidin protein and stabilizing the aptamer compound; in more specific examples, a fluorescent/emissive substance, a pharmaceutical compound, or the like is located at the end opposite to the end where the aptamer binds to biotin, to enable better exertion of 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 tissue microenvironment responsive linkages or groups for interconnection, in order to be able to break down under specific circumstances.
The modified drug molecule in the aptamer or the nucleic acid fragment can be formed by programmed introduction of the drug molecule. In another embodiment of the present invention, the drug molecule is formed by introducing a nucleic acid molecule into a solid phase synthesis module formed by modifying a drug, wherein the drug molecule can be 5-FU, gemcitabine (reference 1), a camptothecin base analog (reference 2), or a nuclide I linked to a phenolic hydroxyl group131May be linked to I via the phenolic hydroxyl group125The module of (document 3).
Embodiments of the invention relate to the use of a nucleic acid aptamer complex, which may be sgc8, aptamer XQ-2D, aptamer DML-7, and in one embodiment of the invention the nucleic acid aptamer used is sgc8, which comprises the sequence shown below:
5’-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3’ (SEQ ID No.:1)
this aptamer was screened and named 2006 (ref: Dihua Shangguan, Ying Li, Zhiwen Tang, et al. PNAS,2006,103, 11838-containing 11843), and its target was identified as PTK7 in 2008. The specific embodiment of the application is based on the aptamer or the aptamer modified by the aptamer, and streptomycin protein is used as a connecting carrier (adapter) to construct the aptamer complex. Forming a nucleic acid aptamer complex comprising streptomycin protein (at least one) and a nucleic acid aptamer (comprising a sequence shown in SEQ ID No.: 1); the nucleic acid aptamer of the double-end modified Biotin forms a network with SA protein through the combination of Biotin and streptavidin, and can be used for constructing polyhedra, even larger nanoparticles and the like. Wherein the connection bonds or connection groups in the nucleic acid aptamer are tissue environment response type, that is, the connection bonds or connection groups are broken under the microenvironment, such as the tumor microenvironment; in certain embodiments of the invention, the linkage or linking group is a disulfide bond. In order to realize the functions of developing or pharmaceutical aptamer, the aptamer may be modified with a fluorescent/radioactive substance, or the aptamer may be modified with a pharmaceutical compound, for example, the aptamer may be modified with a nucleotide analog.
According to the function of the aptamer, the aptamer related by the application is divided into two types, namely, the first type of aptamer is used for targeted development or drug delivery of tumors, namely, one end is modified with Biotin and the other end is modified with fluorescein or radioactive substances; the second kind of aptamer has Biotin modified at both ends, and may be in the form of one or several first connected aptamers with middle modified group capable of responding in micro environment. In an alternative embodiment of the present invention, both of the above-described classes of nucleic acid aptamers (the first class of nucleic acid aptamers and the second class of nucleic acid aptamers) are modified with a pharmaceutical compound (e.g., a nucleotide analog). In alternative embodiments of the present invention, the second type of aptamer may be modified with a fluorophore and/or a quencher, or a group with fluorescence resonance energy transfer effect, that is, after the tissue microenvironment response linkage bond or the group is broken, the quencher is separated from the fluorophore, and the fluorophore emits light or does not emit light, forming a dynamic change of visualization. In a specific embodiment of the present invention, one of the four binding sites of streptomycin protein is an aptamer for imaging or drugs, which has a fluorescent/radioactive substance or the like, and the other three sites are aptamer complexes, both ends of which are modified by Biotin, wherein the aptamer can be in the form of a single or multiple aptamers linked together at the head, and the middle of the aptamer is modified to be a group that can respond in a microenvironment; alternatively, the aptamers combined at the four binding positions of the streptavidin protein are all aptamers both ends of which are modified with Biotin, and the aptamers can be in a form that single or multiple aptamers are connected in the first position, and the aptamers can be modified in the middle to be groups capable of responding in a microenvironment. The microenvironment-responsive linkage is a linker that breaks under specific environments, such as disulfide bonds.
The aptamer compound based on Biotin-SA provided by the invention can protect the aptamer to a certain extent, reduces the possibility of being attacked by enzyme, reduces the renal excretion rate compared with simple nucleic acid, and solves the problems of poor stability and short half-life of the existing aptamer drug compound in-vivo application. In addition, the aptamer has targeting property, and the aptamer compound based on Biotin-SA can have targeting property.
The constructed aptamer complex of Biotin-SA has good biocompatibility as a biological macromolecule, the base or terminal covalent modification of a drug can realize fixed-point and quantitative drug modification of the aptamer, and the controllable drug release of a tumor microenvironment can be realized based on a linker responding to the tumor microenvironment.
The Biotin-SA aptamer compound 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 compound or a simple monomer can be formed, the compound is retained at a tumor part by virtue of the targeting action of the aptamer, and the nucleic acid is degraded based on the disintegration of a nano system and the end exposure of the aptamer, so that drug molecules are released, and multiple effects of targeted delivery, fixed-point drug delivery, drug slow release and the like are achieved (refer to fig. 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 invention, the aptamers for targeting and for ligation can be modified, the drug bases (nucleotide analogs) can be ligated to the aptamers at a fixed ratio and position by solid phase synthesis, and the non-base drugs (non-nucleotide analogs) can be terminally ligated to the aptamers. One end of the targeting aptamer is modified with Biotin, and the other end is modified with fluorescein/radioactive substances or drugs. The two ends of the connecting aptamer are modified by Biotin, and the middle of the connecting aptamer contains disulfide bonds and other groups capable of responding in a specific microenvironment. Then, streptavidin and aptamers (the modified aptamers and the aptamers linked by disulfide bonds) are mixed and self-assembled into aptamer complexes.
Example 1 construction of Biotin-SA-aptamer complexes
Biotin-SA-aptamer drug construction
The aptamer used for targeting is end-modified with a Biotin molecule (sgc8) or aptamer sgc8 with targeting function and modified with a drug molecule (5-FU). The aptamer used for connection is modified with Biotin molecules (sgc8-sgc8) or modified with drug molecules (5-FU) aptamer (sgc8-sgc8-5 FU) at both ends, and has disulfide bonds (iHS-SH) in the molecule.
The sequences are shown in Table 1
TABLE 1
2. Nano-assembly construction
By affinity between the SA protein and Biotin, and 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 the DNA strands (fig. 2). The SA protein, double-Biotin modified DNA and single-Biotin modified aptamer drug complex are mixed according to the proportion of 1: 3: 1,1: 2: 2,1: 1: 3, mixing the components in proportion. Incubate at 37 ℃ for 30 minutes. The constructed complexes 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 nanosheets (right) by ligation of aptamers at different ratios. As shown in fig. 4, the formed nanostructures were significantly larger, and the system could be disassembled by destroying the aptamer used for ligation by adding DTT or GSH.
3. Stability of the nanosystem
Compared with the pure aptamer and the nucleic acid chain of the end-modified Biotin, the stability of the system is obviously improved (figure 4). The aptamer alone or the Biotin-SA-aptamer complex was incubated with a medium containing 10% FBS for 0 to 72 hours, after which the amount of remaining nucleic acid was checked by agarose gel, and it was found that the complex remained more in 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 aptamers for targeting. Biotin (sgc8) was modified at the other end of the aptamer. DNA with disulfide bond modification in the middle and Biotin modification at both ends was synthesized (sgc8-sgc 8). Or groups capable of changing fluorescence, such as fluorescent groups or quenching groups, or groups capable of generating FRET effect, and the like, are arranged on two sides of the disulfide bond.
2. Nano-assembly construction
The SA protein is conjugated to an acidic response moiety and a targeting moiety according to a 1: 3: 1, mixing. Incubate at 37 ℃ for 30 minutes.
3. Targeted imaging
The targeting property of the compound is inspected at a cellular level, and the result shows that the system can maintain the targeting property of the aptamer. Whereas the pure protein did not target (figure 5). Collecting the colon cancer cell HCT116, taking 30 ten thousand cells, respectively incubating with Biotin-sgc8, SA protein and the complex system for 30 minutes, removing the unbound chains, detecting the signal on the aptamer on the cell surface, and seeing the signal of sgc8 and the complex (left), while the fluorescent signal of the complex can be seen by detecting the fluorescent signal bound to the SA on the cell surface by flow, and the fluorescent signal of the complex is not bound to the cell by simple SA (right).
Constructing a mouse with colon cancer cell HCT116, a subcutaneous transplantation tumor or an orthotopic tumorThe tumor grows to 200mm3When the system is used, Biotin-sgc8 is injected through the tail vein, and Biotin-SA-sgc8 conforms to the system. The imaging effect of the tumor site was observed for 0-6 hours. The mice were dissected and the distribution of each organ was examined (FIG. 6), and it was found that the Biotin-SA-sgc8 system was superior to the Biotin-sgc8 itself.
Example 3 Biotin-SA-aptamer Complex construction for Targeted therapy
1. Complex construction
Biotin and a drug molecule (5-FU-sgc8) were modified at the ends of the aptamer, respectively.
DNA with disulfide bond modification in the middle and Biotin modification at both ends was synthesized (sgc8-sgc8-5 FU).
2. Nano-assembly construction
The SA protein is conjugated to an acidic response moiety and a targeting moiety according to a 1: 3: 1, mixing. Incubate at 37 ℃ for 30 minutes.
3. Targeted therapy
At the cellular level, the cytotoxicity of this system was examined. It was found that this system alone was not toxic, whereas the drug-carrying system had the effect of targeted therapy (fig. 7). HCT116 cells were seeded in a 96-well plate, and 5FU and the Biotin-SA-sgc8-5FU complex were added to examine the cytotoxicity thereof, and it was found that the complex retained the toxicity of 5-FU itself.
A mouse with subcutaneous tumor transplantation of colon cancer cell HCT116 was constructed by tail vein injection of 5-FU (10mg/kg) and an equivalent amount of Biotin-SA-sgc8-5FU system. Once weekly dosing, tumor size and mouse survival were observed (figure 8). The therapeutic effect of the complex was seen to be superior to that of 5-FU.
Sequence listing
<110> Shanghai university of traffic medical college affiliated renji hospital
<120> nucleic acid aptamer complex based on Biotin-SA and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atctaactgc tgcgccgccg ggaaaatact gtacggttag a 41
Claims (10)
1. A delivery system of a nucleic acid aptamer drug molecule comprises,
at least one of the streptavidin (S) molecules,
a plurality of 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 that specifically recognizes a target and a drug molecule;
the aptamer drug molecule and the streptavidin can quickly form a delivery system of the aptamer drug molecule in a buffer solution.
2. The delivery system of claim 1, wherein the aptamer drug molecule is formed by programming introduction of a aptamer drug molecule, or the aptamer drug molecule is formed by introduction of a solid-phase synthesis module with drug modification by means of solid-phase synthesis.
3. The delivery system of claim 2, wherein the aptamer drug molecule comprises a aptamer with specific tissue-specific targeting ability, and the biotin modifies one or both ends of the aptamer drug molecule; the drug molecule modifies any position of the aptamer.
4. The delivery system of claim 2, wherein the drug molecule is selected from the group consisting of a base analog; preferably, the drug molecule is directly accessed to the aptamer by solid phase synthesis; the drug molecule is 5-FU, gemcitabine or camptothecin base analogues.
5. The delivery system of claim 2, wherein the drug molecule is a linker moiety formed by artificially modifying a drug molecule, preferably a nuclide I that can be linked via a phenolic hydroxyl group131(ii) a Preferably I may be linked via the phenolic hydroxyl group125The module of (1).
6. The delivery system of claim 3, wherein the aptamer is a tumor-targeting aptamer, preferably the aptamer sgc8, XQ2d, DML7 or a combination thereof.
7. A delivery system of nucleic acid aptamer drug molecules is characterized in that the delivery system of the nucleic acid aptamer drug molecules forms a compound including the nucleic acid aptamer drug molecules, the compound is nanoparticles and can reach a tumor part through an EPR (ethylene propylene rubber) effect, the compound can be retained at the tumor part through the targeting effect of the nucleic acid aptamer after the tumor part is disintegrated in response to the EPR effect, and targeted imaging or treatment of tumors is achieved
At least one Streptavidin (SA);
a nucleic acid fragment, which is Biotin-modified at one end or both ends thereof to bind to the streptavidin, comprising,
the first nucleic acid fragment is an aptamer, has a specific recognition function and serves as a recognition unit of the delivery system;
the second nucleic acid fragment is aptamer or other nucleic acid fragments;
wherein the intermediate or terminal positions of the second type of nucleic acid segment modify the tissue microenvironment responsive linkage or group to interconnect so as to enable the delivery system to disintegrate under a specific tissue microenvironment.
8. The delivery system of aptamer drug molecules of claim 7,
the drug molecule is selected fromA base analog; preferably, the drug molecule is directly accessed to the aptamer through solid phase synthesis; the drug molecule is 5-FU, gemcitabine or camptothecin base analogue, and more preferably the drug molecule is nuclide I connected through a phenolic hydroxyl module131、I125。
9. The delivery system of aptamer drug molecules according to claim 7, wherein the aptamer is a tumor targeting aptamer, preferably an aptamer sgc8, XQ2d, DML7 or a combination thereof.
10. Use of a Biotin-streptavidin Biotin-SA-based aptamer complex as claimed in any of claims 1 to 9 for the preparation of a medicament and/or imaging for the treatment of cell proliferative diseases.
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