Nucleic acid aptamer derivative and application thereof in preparation of medicament carrier

A technology of nucleic acid aptamers and derivatives, applied in biochemical equipment and methods, microbial measurement/testing, drug combination, etc., can solve problems such as difficult chemical modification and transformation, easy to cause immune response, and limit wide application, etc., to achieve Reduced toxicity, no immune activity and toxicity, high application value effect

Active Publication Date: 2011-01-12

谭蔚泓

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AI-Extracted Technical Summary

Problems solved by technology

However, the antibodies used for targeted delivery of drugs are derived from animals or fused monoclonal cells, which need to be produced through biological processes. The preparation is cumbersome, high cost, poor stabilit...

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Abstract

The invention discloses a nucleic acid aptamer derivative and application thereof in preparation of a medicament carrier. The derivative is the single-chain DNA represented by formula (I); a nucleic acid aptamer is represented by the sequence 1 of a sequence list; and n is equal to 1 to 50. The nucleic acid aptamer derivative provided by the invention is liver cancer cell targeted, so that the nucleic acid aptamer derivative can serve as the medicament carrier for targeted administration of liver cancer cells in a human body to specifically kill the liver cancer cells and greatly reduce the toxicity of an anti-cancer medicament to the organism. The nucleic acid aptamer derivative provided by the invention can be chemically synthesized in a large scale and has the advantages of easy connection with medicament molecules, low cost, relatively low molecular weight, no immune activity or toxicity, and good stability. The prepared medicinal composition is stable. The medicinal composition (targeted medicament) provided by the invention has a strong bonding force with the liver cancer cells, can greatly reduce the toxic and side effects during chemical treatment of liver cancer and has very high application value. The formula (I) is: 5'-(CG)n-nucleic acid aptamer-3'.

Application Domain

Organic active ingredientsMicrobiological testing/measurement +3

Technology Topic

Chemical synthesisSide effect +15

Image

Examples

Experimental program(4)
Effect test(1)

Example Embodiment

[0041] Example 1. Characterization of the affinity between nucleic acid aptamer derivatives and human hepatoma cells

[0042] 1. Preparation of nucleic acid aptamers and their derivatives

[0043] Three nucleic acid aptamer derivatives were synthesized artificially, and 1 GC, 29 GC and 50 GC were introduced into the 5' end of the nucleic acid aptamer TLS11a, respectively. TLS11a-(GC) 1 As shown in Sequence 3 of the Sequence Listing. TLS11a-(GC) 29 As shown in Sequence 4 of the Sequence Listing. TLS11a-(GC) 50 As shown in Sequence 5 of the Sequence Listing.

[0044] Three kinds of control nucleic acid aptamer derivatives were artificially synthesized, and 1 GC, 29 GCs and 50 GCs were introduced into the 5' end of the nucleic acid aptamer TD05, respectively. TD05-(GC) 1 As shown in Sequence 6 of the Sequence Listing. TD05-(GC) 29 As shown in Sequence 7 of the Sequence Listing. TD05-(GC) 50 As shown in Sequence 8 of the Sequence Listing.

[0045] The nucleic acid aptamer TLS11a shown in SEQ ID NO: 1 and the control nucleic acid aptamer TD05 shown in SEQ ID NO: 2 in the sequence listing were synthesized.

[0046] 2. Fluorescent labeling of nucleic acid aptamer derivatives

[0047] Three nucleic acid aptamer derivatives, three control nucleic acid aptamer derivatives, nucleic acid aptamer TLS11a and control nucleic acid aptamer TD05 synthesized in step 1 were labeled with fluorescent dye Phycoerythrin-Cy5.5 (phycoerythrin/cyanine dye 5.5). .

[0048] 3. Characterization of the affinity of aptamer derivatives with human hepatoma cells

[0049] The affinity of three nucleic acid aptamer derivatives, three control nucleic acid aptamer derivatives, nucleic acid aptamer TLS11a and control nucleic acid aptamer TD05 and human hepatoma cells (or human normal liver cells Hu1082) were detected respectively. The detection methods are as follows:

[0050] Human cancer cells LH86 were lysed using enzyme-free cell lysate (Cellgro) and washed with wash buffer. Liver cancer cells LH86 (approximately 10 5 each) with 0.1mL of 25nM aptamer derivative (TLS11a-(GC) 1 , TLS11a-(GC) 29 , TLS11a-(GC) 50 , TD05-(GC) 1 , TD05-(GC) 29 , TD05-(GC) 50 , TLS11a or TD05) were mixed and incubated under ice bath for 0.5 hours. Cells were then washed twice with wash buffer and suspended in 0.2 mL of binding buffer for flow cytometry.

[0051] see the results figure 1; A: TLS11a and its three derivatives; B: TD05 and its three derivatives.

[0052] The results showed that: TLS11a and its derivatives had significant affinity for human hepatoma cells (LH86), but normal human hepatocytes (Hu1082) did not interact with TLS11a and its derivatives. The binding force of normal liver cells is significantly increased, and it has high specificity and high binding ability to bind to human liver cancer cells, and can be used as a probe to distinguish normal liver cells from liver cancer cells; TD05 and its three derivatives are highly specific to human liver cancer cells. (LH86) and human normal hepatocytes (Hu1082) had no significant differences in affinity.

Example Embodiment

[0053] Example 2. Preparation of nucleic acid aptamer derivative-drug complex

[0054] 1. Nucleic acid aptamer TLS11a and nucleic acid aptamer derivative TLS11a-(GC) 29 preparation

[0055] In order to insert more doxorubicin, a long GC sequence (composed of 29 GCs connected in sequence) was introduced at the 5' end of the nucleic acid aptamer TLS11a, thereby obtaining a nucleic acid aptamer derivative with an extended sequence TLS11a-(GC ) 29. TLS11a-(GC) 29 As shown in Sequence 4 of the Sequence Listing.

[0056] The nucleic acid aptamer TLS11a shown in SEQ ID NO: 1 of the Sequence Listing was synthesized. The nucleic acid aptamer derivative TLS11a-(GC) shown in SEQ ID NO: 4 of the synthetic sequence listing 29.

[0057] 2. Preparation and characterization of nucleic acid aptamer derivative-drug complexes

[0058] 1. Preparation of nucleic acid aptamer derivatives-drug complexes

[0059] In binding buffer, the nucleic acid aptamer derivative TLS11a-(GC) 29 It is mixed with adriamycin at a ratio of 1:25 (molar ratio), and incubated with stirring at room temperature for 3 hours to obtain a nucleic acid aptamer derivative-drug complex.

[0060] 2. Purification of nucleic acid aptamer derivatives-drug complexes

[0061] The nucleic acid aptamer derivative-drug complex prepared in step 1 was purified by HPLC.

[0062] The column length of the chromatographic column is 250mm, and the inner diameter is 4.6mm. The filler is silica, and the particle size is 5um. The mobile phase is This-HCl buffer solution (10mM, pH7.4) and acetonitrile (gradient elution: 0-3min, 0% acetonitrile, 3-30min: acetonitrile concentration linearly increases to 60%, 30-35min: acetonitrile concentration linearly increases to 90%, 35-45 min acetonitrile 90%, after 45 min 0% acetonitrile). The flow rate was 1 mL/min. The detection wavelength is 260 nm. Target product aptamer derivative-drug complex (TLS11a-(GC) 29 ) peaked at 35 min.

[0063] 3. Characterization of aptamer derivative-drug complexes

[0064] The purified aptamer derivative-drug complex was detected by UV light. The concentration of doxorubicin was obtained from the UV absorbance at 495 nm wavelength. TLS11a-(GC) 29 Concentrations were obtained from UV absorbance at 260 nm wavelength. Due to the obvious UV absorption of doxorubicin at 260nm, it interferes with TLS11a-(GC) 29 Concentration determination, for accurate determination of TLS11a-(GC) 29 The concentration was measured by ultraviolet spectrometer at 495nm and 260nm. The UV absorption coefficients of doxorubicin at 495nm and 260nm are ε, respectively 495 =12000Gm -1 M -1 , ε 260 =24000cm -1 M -1. The concentration of doxorubicin measured at 495nm, after deducting the interference of doxorubicin at 260nm, finally obtained TLS11a-(GC) 29 concentration. TLS11a-(GC) 29 The concentration is calculated by the following formula:

[0065] A is the UV absorption value of the corresponding wavelength.

[0066] On average, each aptamer derivative-drug complex contains 1 aptamer derivative and 25 doxorubicin.

[0067] Nucleic acid aptamer derivative-drug complex (TLS11a-(GC) 29 -DOX) schematic diagram such as figure 2 shown.

[0068] 3. Preparation and characterization of nucleic acid aptamer-drug complexes

[0069] 1. In the binding buffer, mix the nucleic acid aptamer TLS11a and doxorubicin at a ratio of 1:25 (molar ratio), and incubate with stirring at room temperature for 3 hours to obtain the nucleic acid aptamer-drug complex.

[0070] 2. Purification of nucleic acid aptamer-drug complexes

[0071] The nucleic acid aptamer-drug complex prepared in step 1 was purified by HPLC. The liquid chromatography parameters were the same as in step two.

[0072] The peak of aptamer-drug complex appeared at 24 min.

[0073] 3. Purification of nucleic acid aptamer-drug complexes

[0074] The nucleic acid aptamer-drug complex prepared in step 2 is characterized by ultraviolet, and the detection parameters are the same as in step 2.

[0075] The results showed that, on average, each aptamer-drug complex contained 1 aptamer and 2 doxorubicin.

[0076] Therefore, the use of nucleic acid aptamer derivatives can improve the drug loading.

Example Embodiment

[0077] Example 3. Effects of nucleic acid aptamer derivatives-drug complexes on liver cancer cells

[0078] Determination of chemosensitivity of liver cancer cells LH86 to doxorubicin or aptamer derivative-drug complexes using CellTiter AQueous Single Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA) was performed. CellTiter AQueous single solution reagents: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS and an electron coupling reagent ( thiophenazine, PES). The details are as follows: 100 μL liver cancer cells LH86 (about 5×10 4 cells/mL) were seeded in 96-well plates (n=3) and grown overnight at 37°C (5% CO 2 , FBS-free DMEM medium), then 100 μL of the following DNA strands were added:

[0079] (1) TLS11a; set up four concentration gradients, respectively with TLS11a-(GC) in the nucleic acid aptamer derivative-drug complex in (4) 29 The concentrations were the same, namely 40, 100, 200, 300 nM, respectively.

[0080] (2)TLS11a-(GC) 29; set up four concentration gradients, respectively with TLS11a-(GC) in the nucleic acid aptamer derivative-drug complex in (4) 29 The concentrations were the same, namely 40, 100, 200, 300 nM, respectively.

[0081] (3) Doxorubicin; set up four concentration gradients, 1.0, 2.5, and 5.0.7.5 μM, respectively.

[0082] (4) TLS11a-(GC) purified in Example 2 29 -DOX; set up four concentration gradients, based on the concentration of doxorubicin, the concentrations were 1.0, 2.5, 5.0.7.5 μM; corresponding TLS11a-(GC) 29 The concentrations were 40, 100, 200, and 300 nM, respectively.

[0083] Washed after 1 hour and cultured in fresh DMEM medium (10% FBS) for 48h.

[0084] In the cytotoxicity assay, the medium in each well was first removed, and then 20 uL of cell titration reagent (CellTiter) was added to each well. AQueous single solution reagent) and DMEM medium (100 μL), cultured for 3 h. Absorbance values were recorded at 490 nm using a microplate reporter (Tecan Safire microplate reader, AG, Switzerland). Cell viability (number of viable cells) was calculated by comparing the viability of cells treated with TLS11a, TLS11a-GC, doxorubicin, aptamer derivative-drug complexes and blank cells.

[0085] see the results image 3. TLS11a-GC had no apparent effect on cell viability with TLS11a, doxorubicin and TLS11a-(GC) 29 -DOX can significantly reduce the activity of liver cancer cells. The results showed that the nucleic acid aptamer derivative-drug complex had a killing ability similar to that of doxorubicin on liver cancer cells at different concentrations.

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