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DNA fixed nano hydrogel microsphere and preparation and application of DNA fixed nano hydrogel microsphere and nucleic acid aptamer compound

A nano-hydrogel and nucleic acid aptamer technology is applied in the directions of medical preparations without active ingredients, medical preparations containing active ingredients, and pharmaceutical formulas, which can solve complex and expensive, complicated and complicated post-processing and purification, combined with Insufficient stability and other problems, to achieve the effect of simple preparation process, good tolerance to different temperatures, and mild reaction conditions

Active Publication Date: 2019-05-14
SHENYANG PHARMA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among the above nucleic acid aptamer ligation methods, the first two methods are complex and expensive, and their application has certain limitations
The third method can only be used on some cationic carriers, and the binding is not stable enough
The fourth method is a more commonly used method at present, but this method still has major defects, such as amidation organic reaction on the surface of the preparation material, complicated and complicated post-treatment purification, etc.

Method used

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  • DNA fixed nano hydrogel microsphere and preparation and application of DNA fixed nano hydrogel microsphere and nucleic acid aptamer compound
  • DNA fixed nano hydrogel microsphere and preparation and application of DNA fixed nano hydrogel microsphere and nucleic acid aptamer compound
  • DNA fixed nano hydrogel microsphere and preparation and application of DNA fixed nano hydrogel microsphere and nucleic acid aptamer compound

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] (1) Synthesis of Acrydite-DNA

[0066] Weigh 6-amino-1-hexanol (1.0g, 8.1mmol) into a 50mL round bottom flask, then add 10mL of anhydrous CH 2 Cl 2 Dissolve 6-amino-1-hexanol, measure anhydrous triethylamine (2.4mL, 17mmol), add it dropwise to the reaction solution, put the round bottom flask in ice bath for 15min, slowly add methacryloyl chloride (2.5 mL, 25mmol), reacted for 4 hours, and used saturated NaHCO 3 Extract with saturated NaCl three times each. The lower organic phase was removed by rotary evaporation to remove the solvent, then 10 mL of absolute ethanol was added to dissolve the sample, and then newly prepared NaOH solution (4 mL 15%, 15 mmol) was added to continue the reaction for 2 hours. After the reaction was completed, the solvent was removed by rotary evaporation and 25mL CH 2 Cl 2 The solution was dissolved, respectively, with 8 mL of saturated NaHCO 3 Extract with saturated NaCl three times, and then remove the solvent by rotary evaporation t...

Embodiment 2

[0076] Example 2 Investigation on the degradation performance of reduction-sensitive PMAA nano-hydrogels

[0077] First, 12 mg of DNA-PMAA nanohydrogel was weighed, dispersed and dissolved in 40 mL of PBS pH 7.4 buffer solution, and then 6 parts of the above solution were weighed, each 6 mL in a vial. Number the six vials 1, 2, 3, 4, 5, and 6 respectively. Then add reducing agent DTT 3mg, 5mg, 8mg, 10mg, 15mg, 20mg into the vials numbered 1, 2, 3, 4, 5, 6 respectively. After dispersing and dissolving, take a sample of 100 μL every 5 minutes in a water bath at 37°C, and use an ultraviolet spectrophotometer UV-2700 to detect its light transmittance T%. The experimental results are shown in Figure 4 .

Embodiment 3

[0078] Example 3 Fluorescence detection experiment after connecting complementary DNA strands to the surface of PMAA nanohydrogel

[0079]In this experiment, the DNA fluorescent dye SYBR combined with single-stranded DNA to display pink fluorescence, combined with the principle of double-stranded DNA to display green fluorescence, were used to analyze the single-stranded DNA modified PMAA nano-hydrogel and the T-DNA complementary to the gel surface DNA (sequence Validation for 5'-TTT TTT TTT TTT TTT TTT TT-3') bound hydrogel microspheres. Firstly, 3 parts of PMAA nanohydrogels covalently modified by single-stranded DNA were weighed, 3 mg each, and 140 μL of PBS pH7.4 buffer was added respectively, and named as: Group A, Group B, and Group C. Then add 20 μL PBS buffer to group A and group B respectively, add 20 μL 0.05 mM T-DNA complementary to DNA on the gel surface to group C, then continue to add 20 μL PBS buffer to group A, and add 20 μL PBS buffer to group B and C respecti...

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Abstract

The invention belongs to the technical field of chemical and biochemical medicines, particularly relates to a DNA fixed nano hydrogel microsphere, a preparation method of the DNA fixed nano hydrogel microsphere and a nucleic acid aptamer compound and an application of the DNA fixed nano hydrogel microsphere as a tumor targeting preparation. The preparation method is simple, only a segment of single-stranded DNA is synthesized at one end of the nucleic acid aptamer as a connecting arm when the nucleic acid aptamer is synthesized, and the sequence of the DNA connecting arm and the DNA sequence on the surface of the nano material of the nano hydrogel microsphere can be complemented and paired. One or a plurality of nucleic acid can be matched and connected on the surface of the nano materialat one time by co-incubation in aqueous solution so as to obtain the drug delivery carrier with the specific targeting function. According to the method, one or more nucleic acid aptamers can be connected on the surface of the drug delivery carrier at one time in a base complementary pairing mode; the base complementary pairing can be carried out in aqueous solution; reaction conditions are mild and easy to control; and the preparation process is very simple.

Description

technical field [0001] The invention belongs to the technical field of chemical and biochemical medicines, and in particular relates to a preparation method of a DNA immobilized nano hydrogel microsphere and a complex with a nucleic acid aptamer and its application as a tumor targeting preparation. Background technique [0002] Cancer is one of the major diseases that seriously endanger human health today, and has been listed as the "No. 2 killer" (second only to cardiovascular disease) facing human beings. At present, clinical cancer treatment methods mainly include surgical treatment, radiotherapy and chemotherapy. However, except for early-stage cancers, surgical treatment can achieve good results, and the curative effect on advanced-stage cancers is not satisfactory, and small-molecule chemotherapy drugs are used It is the most conventional method to deal with patients in the middle and advanced stages clinically. As we all know, small-molecule chemotherapeutic drugs ha...

Claims

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Application Information

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IPC IPC(8): A61K47/58A61K31/704A61P35/00C08F220/06C08F222/38C08F230/02C08J3/075
CPCY02A50/30
Inventor 刘明哲吴春福蒋刚锋杨静玉王立辉王楠楠
Owner SHENYANG PHARMA UNIVERSITY
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