CN107326028B - Aptamer capable of being combined with CD123 and construction and application of targeting drug-loading system thereof - Google Patents

Abstract

The invention relates to the field of medicine, in particular to a nucleic acid aptamer capable of being combined with CD123 and construction and application of a targeting drug delivery system thereof. The aptamer capable of binding with the CD123 is named as ZW25, and the sequence of the aptamer is represented by SEQ ID No.1, namely 5'-TGCGTGTGTAGTGTGTCTGGGCTACATCGATGAGCTGCCTAGGGTCCCTCTTAGGGATTTGGGCGG-3'. The invention can be synthesized in vitro in a large amount and quickly, has the advantages of simple preparation method, easy modification, easy carrying of carrier drugs, superior stability to antibodies, favorable storage, easy penetration of tumor tissues, low immunogenicity and the like.

Description

Aptamer capable of being combined with CD123 and construction and application of targeting drug-loading system thereof

Technical Field

The invention relates to the field of medicine, in particular to a nucleic acid aptamer capable of being combined with CD123 and construction and application of a targeting drug delivery system thereof.

Background

Acute leukemia in children is the most common one of the pediatric malignancies, the leading cause of death in children, and can be classified into Acute Lymphoblastic Leukemia (ALL) and Acute Myeloid Leukemia (AML). Currently, only about 45% of children's AML acquire long-term disease-free survival with a poor prognosis compared to ALL. The recurrence after chemotherapy, hematopoietic stem cell transplantation and other treatments is a difficult point faced by the modern medical treatment of AML. Studies suggest that Leukemic Stem Cells (LSCs) are a significant cause of relapse. LSCs are self-replicating and renewing, and are not effective with conventional chemotherapeutic drugs, so residual LSCs in patients after AML treatment are a major cause of relapse. Improvements in treatment methods for LSCs are needed. The targeted therapy is a novel treatment means with great prospect, and the development of a treatment strategy for specifically and targeted killing LSCs is very necessary, so that the more effective treatment can be provided for the children suffering from AML. The CD123 molecule is a tumor marker specifically and highly expressed in LSCs and can be a target for LSCs targeted therapy. The Aptamer (Aptamer) is a targeting small molecule with application prospect, is composed of short-chain nucleic acid molecules, can form a complex three-dimensional structure, is combined with a target molecule with high specificity and high affinity, and has the advantages of low immunogenicity, low cost, easy synthesis, easy modification, easy penetration of tumor tissues and the like compared with an antibody, so that the Aptamer can serve as a tumor targeting molecule with excellent performance. At present, no CD123 aptamer report exists at home and abroad. The project aims to screen the first CD123 aptamer so as to provide a new idea for AML treatment.

Disclosure of Invention

In order to overcome the defects, the invention aims to: the invention relates to a nucleic acid aptamer capable of being combined with CD123 and construction and application of a targeting drug-carrying system thereof, which can be synthesized in vitro in a large amount and quickly, has the advantages of simple preparation method, easy modification, easy carrying of carrier drugs, superior stability to antibodies, favorable storage, easy penetration of tumor tissues, low immunogenicity and the like.

The aim of the invention is achieved by the following measures: a nucleic acid aptamer capable of binding to CD123, wherein: the aptamer capable of binding with the CD123 is named as ZW25, and the sequence of the aptamer is shown as SEQ ID No. 1:

5’-TGCGTGTGTAGTGTGTCTGGGCTACATCGATGAGCTGCCTAGGGTCCCTCTTAGGGATTTGGGCGG-3’。

the aptamer capable of binding to CD123 is itself linked to a drug or to a drug-loaded particle.

The drug-loaded particles are drug-loaded nanoparticles.

The target of the aptamer capable of binding to CD123 is a CD 123-positive cell surface membrane protein.

The construction of the aptamer-mediated targeted drug delivery system capable of being combined with CD123 is characterized in that: starting with ZW25, two cytosine and guanine rich probes P1 and P2 form a long chain through base pairing, and doxorubicin Doxycycline is embedded into the formed long chain to form an aptamer-mediated targeted drug delivery system TDT capable of binding with CD 123.

The nucleic acid aptamer capable of being combined with CD123 is applied to preparation of medicines for inhibiting tumor cell proliferation.

The tumor cells are leukemia stem cells.

The invention has the advantages that: the aptamer is a novel targeting small molecule and has superior advantages compared with an antibody. The nucleic acid aptamer can be combined with CD123 with high affinity and high specificity, and meanwhile, because the chemical basis of the nucleic acid aptamer is a nucleic acid molecule, the nucleic acid aptamer is easy to modify and connect with drug-carrying particles without influencing the spatial structure and the function of the drug-carrying particles, so that the aim of targeting drug presentation is fulfilled. The invention screens the first aptamer capable of combining with CD123 by SELEX technology, and the aptamer can combine with CD123 with high specificity and high affinity, so that the aptamer can be used as a targeting small molecule for tumor targeting treatment and provides application prospects for treatment of LSCs.

The present invention will be described in further detail with reference to the drawings and specific examples, but the present invention is not limited thereto.

Drawings

FIG. 1 is a diagram showing the prediction of the secondary structure of aptamer ZW25 using MFold software;

FIG. 2 is a graph of nonlinear regression analysis to calculate Kd values for ZW25 and CD123 polypeptides;

FIG. 3 is an assessment of the binding capacity of ZW25 to CD123 positive cells;

FIG. 4 is a graph of the effect of CD123 expression on ZW25 binding on the surface of CD123 positive cells;

FIG. 5 is a TDT self-assembly process;

FIG. 6 is a confocal microscope showing the uptake of doxorubicin into both CD123 positive cells, Molm-13 and CD123 negative cells, RCH-ACV;

FIG. 7 is the effect of ZW25, ZW25-OCDLS, Dox and TDT on the proliferation of CD123 positive and negative cells;

FIG. 8 shows that TDT reduces the effect of doxorubicin on normal organ damage in vivo;

FIG. 9 shows that TDT can effectively inhibit the growth of CD123+ tumors.

Detailed Description

Screening: screening of aptamers capable of binding to CD123 by SELEX method

a) Principal materials and reagents

Cell lines

Human acute myelocytic leukemia MOLM-13, human blood leukemia stem cell line TF-1, human B cell precursor cell leukemia RCH-ACV and mouse lymphoma cell line EL 4.

Bacteria

Competent cell Trans 1-T1: purchased from Beijing Quanjin Biotechnology Ltd, stored at-80 ℃ for a long period of time, to avoid repeated freeze thawing as much as possible.

b) In vitro construction of DNA libraries and primers:

DNA libraries containing random sequences were designed in vitro using software such as Primer 5 to achieve a clone number of 10¹⁵-10¹⁸Ensuring polymorphism of the aptamer; primers were synthesized to pair with the library and Biotin (Biotin) and Fluorescein Isothiocyanate (FITC) were modified at the 5' ends of the primers, respectively, in preparation for SELEX screening.

c) Immobilization of target CD 123:

fixing the CD123 polypeptide on a micron-sized magnetic bead modified with-COOH through the action of NHS/EDC; and the BCA method is used for detecting the immobilization rate of the polypeptide-magnetic beads.

d) Screening for CD123 aptamers using the SELEX method:

200pmol random library ssDNA was heated at 95 ℃ for 5min, immediately ice-washed for 15min, incubated with BSA-coated microplate for 30min to remove non-specific adsorption, the supernatant was aspirated, incubated with biotin-modified CD123 and streptavidin-coated magnetic beads, and incubated with pretreated ssDNA for 40 min. Magnetically separating and discarding the supernatant, washing the magnetic beads for 3 times by PBS and resuspending, heating for 10min at 95 ℃ after centrifugation, collecting the supernatant, and performing PCR amplification by using primers fitc forward and biotin reverse under the PCR reaction conditions: the PCR products were examined in 1.0% agarose gel electrophoresis at 98 ℃ for 10min, 98 ℃ for 30s, 75 ℃ for 30s, 72 ℃ for 30s, 25 cycles, and used for the next round of screening.

To detect the screening process, the ssDNA mother solution obtained by screening was amplified with primers fitc forward and biotin reverse, single-stranded and reacted with CD 123-coated magnetic beads, and the change in fluorescence intensity on the surfaces of the magnetic beads was detected by flow cytometry. When enrichment was maximized, the mother solution was cloned, and fresh double-stranded DNA product was mixed with 1. mu.L of pEASY-T1 Simple Cloning Vector, reacted at room temperature for 5min and placed on ice. The ligation product was competently mixed with 50. mu.L Trans1-T1 in ice bath for 30min, heat shock at 42 ℃ for 30s, and ice bath for 2 min. And (3) uniformly mixing 250 mu L of LB culture medium with the connection product subjected to ice bath, incubating at the temperature of 37 ℃ at 200rpm/min for 1h, and plating and culturing. White colonies with clear edges were picked and shaken overnight in LB liquid medium (ampicillin/kanamycin). A single oligonucleotide sequence was picked that bound GD 2. After SELEX screening for several circles, FITC labeling is carried out on ssDNA, positive screening and negative screening are carried out in the screening process, the enrichment degree of the affinity of the screening library for CD123 is detected by flow cytometry after several circles, fluorescent signals are observed, and when the signals are maximized and are not further improved, the screening library is cloned, numbered and sequenced.

The aptamer capable of binding with CD123 is obtained by screening and is named as ZW25, and the sequence of the aptamer is shown as SEQ ID No. 1:

5’-TGCGTGTGTAGTGTGTCTGGGCTACATCGATGAGCTGCCTAGGGTCCCTCTTAGGGATTTGGGCGG-3’。

the aptamer capable of binding to CD123 is itself linked to a drug or to a drug-loaded particle. The drug-loaded particles are drug-loaded nanoparticles.

Identification of aptamers capable of binding to CD123 and evaluation of polypeptide binding performance thereof

ZW25 specifically binds to the extracellular structure of CD 123:

an ideal aptamer needs to have a high specific binding ability to a target, which is a hard indicator for evaluating the performance of aptamers. To demonstrate the effectiveness of the screen and whether the aptamer ZW25 specifically binds to CD123, the binding of ZW25 to CD123 polypeptides and other proteins such as albumin, IgG, trypsin was tested. Albumin (BSA) is most abundant in blood and immunoglobulin G (IgG) is more abundant in serum, and therefore is often used together with Trypsin (Trypsin) as a negative control for detecting aptamer binding specificity. The binding of the aptamer ZW25 to the three proteins was examined. Firstly, respectively connecting CD123 polypeptide, BSA, trypsin and IgG to a quantitative UF magnetic bead modified with carboxyl through EDC/NHS catalysis, respectively, then respectively incubating ZW25 marked with FITC with the magnetic beads, and detecting and analyzing the binding condition of the aptamer and the protein on the magnetic beads by a flow cytometer. To exclude the effect of non-specific adsorption of nucleic acids to the magnetic beads themselves on the detection, random pool DNA was used as a negative control. The results were: the fluorescence intensity of ZW25 after reaction with BSA, trypsin and IgG is basically consistent with that of a random control, while the fluorescence intensity of ZW25 after reaction with a target molecule CD123 polypeptide is obviously higher than that of the random control, which shows that ZW25 has weak cross reaction with BSA, trypsin and IgG and strong binding with the target CD123 polypeptide.

ZW25 Secondary Structure prediction

ZW25 can form a space three-dimensional structure due to the matching of purine and pyrimidine in DNA molecules and the force such as electrostatic action, the secondary structure of ZW25 is predicted by MFold software, as shown in FIG. 1, ZW25 is composed of three short stem regions and opposite loop regions. Presumably, from the nature and structure of the aptamer, the structure may be associated with specific binding of the aptamer to the target.

Determination of the affinity constant of ZW25 for CD123 structures

Although an ideal aptamer should bind with high specificity to a target, if the affinity for the target is not high, the application value is not high, and therefore, the affinity for the target is also one of important indicators for evaluating the quality of the aptamer. To detect the affinity of aptamer ZW25 for CD123 polypeptide, CD123 polypeptide was immobilized on UF beads via EDC/NHS catalysis, different concentrations of FITC-labeled ZW25 were bound to the beads, and at each DNA concentration, to remove the effect of the beads themselves on the affinity assay, the same concentration of random pool ssDNA was incubated with the beads. Flow cytometry was used to measure the mean fluorescence intensity values on the beads under different conditions, and nonlinear regression analysis was used to calculate the Kd of ZW25, giving a Kd of 29.41nM for the polypeptides ZW25 and CD123 (FIG. 2). Generally speaking, the affinity range between antibody antigens is 10 < -5 > to 10 < -12 > M, and the binding force of ZW25 and target molecules reaches 10 < -6 > M, which indicates that the relative affinity of ZW25 and target proteins is better.

Binding specificity of ZW25 to CD123+ cells

In the screening process, since highly pure CD123 protein is not readily available, experiments have shown that aptamer ZW25 specifically binds to CD123 polypeptides using a segment of the extracellular exposure of CD123 as a target. However, whether the conformation of the CD123 polypeptide is consistent with the actual conformation of the surface of the CD123 protein, whether the aptamer to be screened can also recognize the CD123 protein on CD123+ cells while binding to the CD123 polypeptide, and whether the aptamer still has certain specificity, in order to evaluate the problem, Molm-13 and TF-1 cells highly expressing CD123 molecules, and EL4 and RCH-ACV cells not expressing CD123 molecules were selected. FITC-labeled ZW25 was incubated with each of the above four cells, and changes in fluorescence intensity on the cell surface were detected by flow cytometry. To exclude non-specific adsorption of nucleic acids by the cells themselves, we used random pool ssDNA as a negative control. The results showed that in both positive cells, the fluorescence intensity of the added aptamer ZW25 was significantly higher than that of the random pool DNA group (FIG. 4A & B), while in the negative cells, the fluorescence intensity of the ZW25 group was substantially identical to that of the random pool DNA group (FIG. 4C & D). The results indicate that ZW25 has strong binding force with CD123 positive cells and weak cross binding with CD123 negative cells, and indicate that ZW25 can selectively identify CD123 expressing positive cells.

FIG. 3 evaluation of the binding capacity of ZW25 to CD123 positive cells. Wherein A is the binding of ZW25 to TF-1 cells. B is the binding of ZW25 to Molm-13 cells. C is the binding of ZW25 to RCH-ACV cells. D is the binding of ZW25 to EL4 cells.

Effect of CD 123-Positive cell surface Membrane protein on the binding of ZW25 to cells

Previous experiments have demonstrated that aptamer ZW25 can specifically bind to CD123 positive cells, but have not demonstrated whether aptamer ZW25 binds to membrane proteins on the surface of CD123 positive cells. To investigate this problem, CD123 positive lymphoma cells, Molm-13 cells, trypsinized for 10min and non-trypsinized Molm-13 cells, were incubated with FITC labeled ZW25, respectively, and cell surface fluorescence intensity was measured by flow cytometry, and Molm3 cells without any treatment served as a negative control. As shown in FIG. 4, the binding of 5min trypsinized Molm-13 cells to ZW25 was significantly reduced compared to undigested cells, indicating that the target of aptamer ZW25 is likely to be a CD123 positive cell surface membrane protein.

Construction of ZW 25-mediated Targeted drug delivery System

In order to prove that the CD123 aptamer ZW25 has the functions of targeting presentation and targeted killing of an anti-tumor drug to CD123+ tumor cells, a self-assembled ZW 25-guided targeted drug delivery system (TDT) is constructed. As shown in fig. 5, TDT is ZW 25-initiated, long chain formed by base pairing of two cytosine and guanine rich probes P1 and P2. Doxycyline (Doxycyline) can be embedded in the formed long chain.

FIG. 5. self-assembly process of TDT. 1. Nucleic acid aptamer ZW 25; ZW25 is connected with the probe; ZW25-ligand paired with P1; continued pairing of P2; continuously pairing P1 with P2 to form ZW 25-OCDLS; 6. the antitumor drug Doxycyline is embedded into double chains to form TDT.

ZW 25-mediated TDT Targeted binding to CD123+ cells

Because the chemotherapy drug adriamycin Dox enters cells in a passive diffusion mode, the adriamycin Dox has no selectivity on normal cells and tumor cells, can kill tumor cells and a large number of normal cells, and has large toxic and side effects. The aptamer ZW25 can be specifically combined with CD123 positive tumor cells, and the constructed TDT can be selectively taken by the CD123 positive cells, so that the drug concentration in the tumor cells is increased, and the drug concentration in the negative cells is reduced, thereby playing the role of targeted killing. To verify the above hypothesis, uptake of TDT by CD123 positive and negative cells was observed separately using confocal microscopy. Respectively incubating free Dox and TDT with CD123 positive lymphoma cells Molm-13 and CD123 negative cells RCH-ACV, and observing the content of Dox in the cells by using a confocal microscope. As shown in FIG. 6, the fluorescence intensity of doxorubicin in the two cells treated with free Dox was very high, and the doxorubicin concentration was very high, thus confirming that Dox was not selective for cells. In TDT treated cells, the fluorescence intensity in the Molm-13 cells is obviously stronger than that in RCH-ACV cells, and the TDT uptake in the RCH-ACV cells of CD123 negative cells is obviously lower than that in the free Dox group. The results show that the TDT compound is more taken up by CD123 positive tumor cells and less taken up by CD123 negative tumor cells, the TDT compound can be selectively taken up by the CD123 positive cells, and the ZW25 can bring the medicine into the CD123 positive cells by utilizing targeting.

TDT can inhibit proliferation of CD123+ tumor cells in vitro and selectively reduce killing of CD123 negative cells by cytotoxic drugs

The optimal effect of targeted therapy is that the drug only kills tumor cells, while having little or no effect on normal cells. It has been demonstrated that TDT can be selectively taken up by CD123 positive cells and take up is reduced in CD123 negative cells due to the targeting effect of aptamer ZW25, and thus it is speculated that TDT will further reduce the toxic side effects of cytotoxic drug Dox on CD123 negative cells. To test the above hypothesis, we established ZW25 group, ZW25-OCDLS group, TDT group and free Dox group, incubated these materials with CD123 positive cells Molm-13 and TF-1, CD123 negative cells RCH-ACV and EL4, respectively, and after a certain period of time, the proliferation of the two cells was examined by CCK8 method under different treatment conditions. As shown in FIG. 7, the survival rates of the four cells in the ZW25 group and the ZW25-OCDLS group were close to 100%, indicating that ZW25 and ZW25-OCDLS themselves were not toxic to the cells. In CD123 positive cells, the cell survival rate of the free Dox group is not obviously different from that of the TDT group, and the TDT does not influence the killing effect of Dox on the CD123 positive cells; cell survival in the free Dox group was significantly lower than the TDT group in CD123 negative cells, showing that TDT kills CD 123-cells significantly lower than free Dox (P < 0.01). The above results demonstrate that TDT can lead to targeted killing of CD123 positive cells and significantly reduce killing of CD123 negative cells.

TDT can inhibit and reduce the damage of the antitumor drug Doxycyline to normal tissues and cells in vivo

To demonstrate that TDT can reduce doxorubicin damage to normal tissues in vivo, TDT and saline were injected into C57 mice from the tail vein, respectively, and after 25 days, the mice were sacrificed and their hearts, kidneys and livers were H & E stained. As shown in FIG. 8, the organs of the mice in the TDT group were not significantly damaged, and were not significantly different from the normal saline group in the control group, indicating that TDT can reduce the toxic side effects of doxorubicin on normal tissues.

TDT can prolong the survival period of CD123+ tumor-bearing mice

To verify whether TDT could inhibit the growth of CD123+ tumor cells in vivo, a CD123+ tumor-bearing mouse model was constructed. Free doxycline, TDT and saline were injected into mice separately via tail vein, 6 mice per group. As shown in fig. 9, mice in the saline group all died on day nine, mice in the TDT group all died on day 28, and mice in the free doxorubicin group all died on day 33, indicating that TDT can effectively inhibit the growth of CD123+ tumors in the mice.

The invention screens the first aptamer capable of combining with CD123 by SELEX technology, and the aptamer can combine with CD123 with high specificity and high affinity, can be used as a targeting small molecule for tumor targeting treatment, and provides application prospects for treatment of LSCs. Therefore, the nucleic acid aptamer capable of being combined with CD123 provided by the invention is applied to preparation of medicines for inhibiting tumor cell proliferation. The tumor cells are leukemia stem cells.

The parts of the embodiment not described in detail and the english abbreviations are common general knowledge in the industry and can be searched on the internet, which is not described herein.

Sequence listing

SEQUENCE LISTING

<110> Children hospital in xi' an city

<120> nucleic acid aptamer capable of being combined with CD123 and construction and application of targeting drug delivery system thereof

<130>2017

<160>1

<170>PatentIn version 3.3

<210>1

<211>66

<212>DNA

<213> Artificial sequence

<400>1

tgcgtgtgta gtgtgtctgg gctacatcga tgagctgcct agggtccctc ttagggattt

gggcgg 66

Claims (6)

1. A nucleic acid aptamer capable of binding to CD123, wherein: the aptamer capable of binding with the CD123 is named as ZW25, and the sequence of the aptamer is shown as SEQ ID No. 1:

5’-TGCGTGTGTAGTGTGTCTGGGCTACATCGATGAGCTGCCTAGGGTCCCTCTTAGGGATTTGGGCGG-3’。

2. the aptamer capable of binding to CD123 according to claim 1, wherein: the aptamer capable of binding to CD123 is itself linked to a drug or to a drug-loaded particle.

3. The aptamer capable of binding to CD123 according to claim 2, wherein: the drug-loaded particles are drug-loaded nanoparticles.

4. The aptamer capable of binding to CD123 according to claim 1, wherein: the target of the aptamer capable of binding to CD123 is a CD 123-positive cell surface membrane protein.

5. The construction of the aptamer-mediated targeted drug delivery system capable of binding to CD123 according to claim 1, wherein the aptamer-mediated targeted drug delivery system comprises: starting with ZW25, two cytosine and guanine rich probes P1 and P2 form a long chain through base pairing, and doxorubicin Doxycycline is embedded into the formed long chain to form an aptamer-mediated targeted drug delivery system TDT capable of binding with CD 123.

6. Use of a CD123 binding aptamer of claim 1 in the preparation of a medicament for inhibiting tumor cell proliferation, wherein: the tumor cells are leukemia stem cells.

Images