CN112472664B - Preparation method of gene targeted therapy nano motor-hydrogel system - Google Patents
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- 238000002626 targeted therapy Methods 0.000 title claims description 19
- 239000000758 substrate Substances 0.000 claims abstract description 43
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- 108020004459 Small interfering RNA Proteins 0.000 claims abstract description 14
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- 239000004055 small Interfering RNA Substances 0.000 claims abstract description 14
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 10
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Abstract
The invention relates to a preparation method of a nano motor-hydrogel system capable of injecting high cell uptake for gene targeting therapy. Dripping the platinum nano particle solution on a substrate, and drying; soaking the substrate in a polyethyleneimine solution, taking out the substrate, washing the substrate for multiple times by using ultrapure water, soaking the substrate in a sodium polystyrene sulfonate solution, taking out the substrate, and washing the substrate for multiple times by using the ultrapure water; repeating the steps repeatedly, assembling polyethyleneimine and sodium polystyrene sulfonate on the surface of the platinum nanoparticles, and assembling folic acid modified polyethyleneimine on the outermost layer; dispersing the nano-carrier in mould-free sterile water by utilizing ultrasound, and connecting the small interfering RNA to the nano-carrier through electrostatic action to obtain a nano motor; mixing the chitosan solution and the dialdehyde starch solution containing the nanomotor to finish the preparation. The invention improves the cell uptake performance and the cell retention time, and achieves better gene targeting treatment effect. The method is simple and effective, is simple and convenient to operate, and requires short time.
Description
Technical Field
The invention relates to the technical field of gene targeted therapy, in particular to a preparation method of an injectable high-cell uptake nano motor-hydrogel system for gene targeted therapy.
Background
According to the investigation of the international cancer research institute, cancer is likely to be the leading cause of death worldwide in the 21 st century. Today, the main treatment methods for cancer include surgical treatment, radiotherapy and chemotherapy. However, these therapies have limitations such as postoperative tumor cell retention and drug resistance. Gene therapy is one approach to solve these problems. Small interfering RNA is a commonly used therapeutic agent in gene therapy. It can reduce gene expression leading to apoptosis. But naked small interfering RNA is difficult to enter cells due to its hydrophilicity and electronegativity. Therefore, various nanocarriers have been developed for transporting small interfering RNA.
As a special nano-carrier, a nano-motor is attracting attention. The nano motor is a nano material which can convert other forms of energy into kinetic energy so as to realize self-driving. Various kinds of driving nanomotors, such as optical driving, chemical driving, field driving, etc., have been developed. The self-driving of the nanomotor can increase the contact probability of the nanomotor and the cell and reduce the time required for the contact. Therefore, the use of nanomotors for the transport of small interfering RNA molecules can increase their cellular uptake efficiency.
The invention utilizes the injectability of the hydrogel to ensure that the nano motor can directly reach the tumor part, thereby reducing the possibility that small molecule interfering RNA is decomposed. While the responsive release of the hydrogel increases the residence time of the nanomotor. And secondly, the self-driving performance of the nano motor enables the nano motor to enter cells more quickly, so that a better cell uptake effect is achieved. Therefore, the nano motor-hydrogel system prepared by the method can realize better gene targeting treatment effect under the conditions of injectability and high cell uptake.
Disclosure of Invention
The technical problem is as follows: the invention aims to provide a preparation method of an injectable high-cell uptake nano motor-hydrogel system for gene targeted therapy, which combines the injectability and the responsiveness release of hydrogel, the self-drive of a nano motor and the like to improve the cell uptake performance and the cell retention time of small-molecule interfering RNA and can achieve better gene targeted therapy effect.
The technical scheme is as follows: the invention discloses a preparation method of an injectable high-cell uptake nano motor-hydrogel system for gene targeting therapy. The injectability of the hydrogel enables the nanomotor to directly reach the tumor site, reducing the possibility of the decomposition of small interfering RNA. While the responsive release of the hydrogel increases the residence time of the nanomotor. And secondly, the self-driving performance of the nano motor enables the nano motor to enter cells more quickly, so that a better cell uptake effect is achieved. Therefore, the nano motor-hydrogel system prepared by the method can realize better gene targeting treatment under the conditions of injectability and high cellular uptake.
The preparation method of the injectable high-cell uptake nano motor-hydrogel system for gene targeted therapy comprises the following steps:
firstly, dripping a platinum nano particle solution on a substrate, and drying;
soaking the substrate in a polyethyleneimine solution, taking out the substrate, washing the substrate for a plurality of times by using ultrapure water, soaking the substrate in a sodium polystyrene sulfonate solution, taking out the substrate, and washing the substrate for a plurality of times by using the ultrapure water;
step three, repeating the step two for a plurality of times, assembling polyethyleneimine and sodium polystyrene sulfonate on the surface of the platinum nano particle, and assembling folic acid modified polyethyleneimine on the outermost layer to obtain a nano carrier;
dispersing the nano-carrier in mould-free sterile water by using ultrasound, and connecting the small interfering RNA to the nano-carrier through electrostatic action to obtain a nano motor;
and step five, mixing the chitosan solution with the dialdehyde starch solution containing the nano motor to prepare the injectable high-cell uptake nano motor-hydrogel system for gene targeted therapy.
In the second step, the soaking time is 2-10 min.
The concentration of the platinum nanoparticle solution is as follows: 0.1 mg/mL-20 mg/mL, and the concentration of the polyethyleneimine solution and the sodium polystyrene sulfonate solution is as follows: 1 mg/mL-5 mg/mL.
The repetition times in the third step are 3-20 cycles.
And in the fourth step, the ligation reaction is carried out for 1-4 hours.
The concentration of the chitosan solution in the step five is as follows: 30 mg/mL-80 mg/mL, the concentration of dialdehyde starch solution is: 15 mg/mL-35 mg/mL, and the concentration of the nanomotor is 1 mg/mL-5 mg/mL.
Has the advantages that:
1. the injectability of the hydrogel enables the nanomotor to directly reach the tumor site, reducing the possibility of the decomposition of small interfering RNA. While the responsive release of the hydrogel increases the residence time of the nanomotor.
2. The self-driving performance of the nano motor enables the nano motor to enter cells more quickly, and a better cell uptake effect is achieved.
3. The nano motor-hydrogel system can achieve better gene targeting treatment effect under the conditions of injectability and high cell uptake.
4. The selected materials are all materials with good biocompatibility, and the prepared nano motor-hydrogel system has low cytotoxicity and certain biodegradability.
Drawings
FIG. 1 is a scanning electron microscope image of the nanomotor-hydrogel system prepared in example 1.
Fig. 2 is a graph showing the anticancer activity results of the nanomotor-hydrogel system prepared in example 1.
Detailed Description
The present invention is explained further below. The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.
The invention relates to a preparation method of an injectable high-cell uptake nano motor-hydrogel system for gene targeted therapy, which comprises the following steps:
firstly, dripping a platinum nano particle solution on a substrate, and drying;
soaking the substrate in a polyethyleneimine solution, taking out the substrate, washing the substrate for multiple times by using ultrapure water, soaking the substrate in a sodium polystyrene sulfonate solution, taking out the substrate, and washing the substrate for multiple times by using ultrapure water;
step three, repeating the step two repeatedly, assembling polyethyleneimine and sodium polystyrene sulfonate on the surface of the platinum nanoparticle, and assembling folic acid modified polyethyleneimine on the outermost layer to obtain a nano carrier;
dispersing the nano-carrier in mould-free sterile water by using ultrasound, and connecting the small interfering RNA to the nano-carrier through electrostatic action to obtain a nano motor;
and step five, mixing the chitosan solution and the dialdehyde starch solution containing the nano motor to prepare the injectable high-cell uptake nano motor-hydrogel system for gene targeted therapy.
And step six, detecting the cell uptake efficiency and the anticancer activity of the nano motor-hydrogel system.
Example 1:
dripping 3 mg/mL of platinum nanoparticle solution on a substrate, and drying; soaking the substrate in 3 mg/mL polyethyleneimine solution for 5 min, taking out the substrate, washing with ultrapure water for 3 times, soaking the substrate in 3 mg/mL sodium polystyrene sulfonate solution for 5 min, taking out the substrate, and washing with ultrapure water for 3 times; repeating the step for 15 times, assembling polyethyleneimine and sodium polystyrene sulfonate on the surface of the platinum nanoparticle, and assembling folic acid modified polyethyleneimine on the outermost layer to obtain a nano carrier; dispersing the nano-carrier in mildew-free sterile water (with the concentration of 3 mg/mL) by utilizing ultrasound, and reacting the nano-carrier with 5 mM small interfering RNA in ice bath for 1 hour to obtain a nano motor; mixing 40 mg/mL chitosan solution and 17 mg/mL dialdehyde starch solution (containing 3 mg/mL nanomotor) to prepare the injectable high-cell uptake nanomotor-hydrogel system for gene targeted therapy. And detecting the cellular uptake efficiency and the anticancer activity of the nanomotor-hydrogel system.
Fig. 1 is a scanning electron microscope image of the nanomotor-hydrogel system prepared in this example. From the figure, it can be observed that the nanomotor is about 70 nm in size and is loaded into the hydrogel, successfully producing a nanomotor-hydrogel system.
Fig. 2 is a graph showing the anticancer activity of the nanomotor-hydrogel system prepared in this example. From the figure, it can be found that compared with the pure hydrogel and the hydrogel directly loaded with the small interfering RNA, the nanomotor-hydrogel system has the highest anticancer activity. After 72 h of co-incubation with cancer cells, only 25.2% of the cancer cells survived.
Example 2:
dripping 1 mg/mL platinum nanoparticle solution on a substrate, and drying; soaking the substrate in 2 mg/mL polyethyleneimine solution for 5 min, taking out the substrate, washing with ultrapure water for 3 times, soaking the substrate in 2 mg/mL sodium polystyrene sulfonate solution for 5 min, taking out the substrate, and washing with ultrapure water for 3 times; repeating the step for 10 times, assembling polyethyleneimine and sodium polystyrene sulfonate on the surface of the platinum nanoparticle, and assembling folic acid modified polyethyleneimine on the outermost layer to obtain a nano carrier; dispersing the nano-carrier in mildew-free sterile water (with the concentration of 3 mg/mL) by utilizing ultrasound, and reacting the nano-carrier with 5 mM small interfering RNA in ice bath for 1 hour to obtain a nano motor; mixing 40 mg/mL chitosan solution and 20 mg/mL dialdehyde starch solution (containing 3 mg/mL nanomotor) to prepare the injectable high-cell uptake nanomotor-hydrogel system for gene targeted therapy. And detecting the cellular uptake efficiency and the anticancer activity of the nanomotor-hydrogel system.
Example 3:
dripping 5 mg/mL of platinum nanoparticle solution on a substrate, and drying; soaking the substrate in 4 mg/mL polyethyleneimine solution for 10 min, taking out the substrate, washing with ultrapure water for 3 times, soaking the substrate in 4 mg/mL sodium polystyrene sulfonate solution for 10 min, taking out the substrate, and washing with ultrapure water for 3 times; repeating the step for 8 times, assembling polyethyleneimine and sodium polystyrene sulfonate on the surface of the platinum nanoparticles, and assembling folic acid modified polyethyleneimine on the outermost layer to obtain a nano carrier; dispersing the nano-carrier in mildew-free sterile water (with the concentration of 5 mg/mL) by utilizing ultrasound, and reacting the nano-carrier with 10 mM small interfering RNA in ice bath for 2 hours to obtain a nano motor; mixing 30 mg/mL chitosan solution and 17 mg/mL dialdehyde starch solution (containing 5 mg/mL nanomotor) to prepare the injectable high-cell uptake nanomotor-hydrogel system for gene targeted therapy. And detecting the cellular uptake efficiency and the anticancer activity of the nanomotor-hydrogel system.
Claims (6)
1. A preparation method of a gene targeting therapy nanomotor-hydrogel system is characterized by comprising the following steps:
firstly, dripping a platinum nano particle solution on a substrate, and drying;
soaking the substrate in a polyethyleneimine solution, taking out the substrate, washing the substrate for a plurality of times by using ultrapure water, soaking the substrate in a sodium polystyrene sulfonate solution, taking out the substrate, and washing the substrate for a plurality of times by using the ultrapure water;
step three, repeating the step two for a plurality of times, assembling polyethyleneimine and sodium polystyrene sulfonate on the surface of the platinum nano particle, and assembling folic acid modified polyethyleneimine on the outermost layer to obtain a nano carrier;
dispersing the nano-carrier in mould-free sterile water by using ultrasound, and connecting the small interfering RNA to the nano-carrier through electrostatic action to obtain a nano motor;
and step five, mixing the chitosan solution with the dialdehyde starch solution containing the nano motor to prepare the injectable high-cell uptake nano motor-hydrogel system for gene targeted therapy.
2. The method for preparing the gene targeted therapy nanomotor-hydrogel system according to claim 1, wherein the soaking time in the second step is 2-10 min.
3. The method for preparing the gene targeted therapy nanomotor-hydrogel system according to claim 1, wherein the concentration of the platinum nanoparticle solution is as follows: 0.1 mg/mL-20 mg/mL, and the concentration of the polyethyleneimine solution and the sodium polystyrene sulfonate solution is as follows: 1 mg/mL-5 mg/mL.
4. The method for preparing the gene targeted therapy nanomotor-hydrogel system according to claim 1, wherein the number of repetitions in step three is 3 to 20 cycles.
5. The method for preparing the gene targeted therapy nanomotor-hydrogel system according to claim 1, wherein the ligation reaction is performed for 1-4 hours in the fourth step.
6. The method for preparing the gene targeted therapy nanomotor-hydrogel system according to claim 1, wherein the concentration of the chitosan solution in the fifth step is: 30 mg/mL-80 mg/mL, the concentration of dialdehyde starch solution is: 15 mg/mL-35 mg/mL, and the concentration of the nanomotor is 1 mg/mL-5 mg/mL.
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