CN114904010A - Preparation method and application of layered self-assembly loaded siRNA nano-particles - Google Patents

Preparation method and application of layered self-assembly loaded siRNA nano-particles Download PDF

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CN114904010A
CN114904010A CN202210484895.5A CN202210484895A CN114904010A CN 114904010 A CN114904010 A CN 114904010A CN 202210484895 A CN202210484895 A CN 202210484895A CN 114904010 A CN114904010 A CN 114904010A
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CN114904010B (en
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那娜
关晓文
欧阳津
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Beijing Normal University
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Abstract

The invention belongs to the technical field of biological material preparation, and particularly relates to a preparation method and application of a layered self-assembly loaded siRNA nano particle. The method is realized by the following steps: adding the compound G and siRNA or FAM-siRNA into deionized water, stirring and then carrying out ultrasonic treatment to prepare core G-siRNA of the nano particles; adding a compound WP5, carrying out ultrasonic treatment, and standing to obtain the nano particles. The invention provides a novel method for improving the siRNA carrying capacity and quickly releasing the siRNA in a slightly acidic environment by a layered self-assembly technology aiming at the prior related technology of preparing a nano material with small siRNA carrying capacity and low release efficiency. Realizes the high-efficiency loading of gene drug siRNA and the rapid release in acid environment. In addition, the nano material prepared by the technology can monitor the release of the intracellular medicine under the acidic condition in a fluorescence enhanced manner. Meanwhile, the nano material prepared by the technology can be well applied to tumor treatment.

Description

Preparation method and application of layered self-assembly loaded siRNA nano-particles
Technical Field
The invention belongs to the field of preparation of biological materials, and particularly relates to a preparation method and application of a layered self-assembled siRNA-loaded nanoparticle.
Background
At present, the clinical tumor treatment means is mainly to carry out local and specific treatment on tumors by technologies such as radiotherapy, chemotherapy, surgical operation and the like, and the problems of easy relapse and rapid metastasis of the tumors, indirect damage of nonspecific tissues of organisms and the like are inevitable on the advantages of direct and efficient treatment. Specific biomolecules, such as genes, protein molecules or certain links in organelles, and the like in the process of generating and developing tumors are used as targets, and the tumor is treated accurately, targetedly, efficiently and thoroughly by using specially designed medicines, so that the method becomes the key point of the modern tumor biological research. Small interfering RNA (siRNA), sometimes referred to as short interfering RNA (short interfering RNA) or silencing RNA (silencing RNA), is a relatively short double-stranded nucleic acid molecule (typically 20-25 nucleotides) that has many different biological uses. Currently, siRNA is known to be mainly involved in RNA interference (RNAi) phenomenon, and is a drug with the function of specifically regulating gene expression in cells. In recent years, the application of RNA interference (RNAi) to the treatment of viral infectious diseases has received great attention, and application studies in the treatment of hepatitis b, hepatitis c, and the like have been most actively conducted. In addition, protein molecules, nucleic acid molecules and the like which are specifically and highly expressed in malignant tumor cells such as lung cancer, breast cancer, colon cancer, melanoma and the like and tumor intravascular cells can be targeted for treatment through the RNAi technology. Therefore, the siRNA is a specific tumor treatment drug with great development potential.
However, many of the currently studied nanomaterials loaded with siRNA suffer from a number of deficiencies. The method comprises the following steps: 1. the existing nano material adsorbs siRNA through electrostatic attraction, and has small carrying capacity; 2. the efficiency of the siRNA-loaded nano material in stimulating response and release in cells is low, and the treatment effect is seriously influenced; 3. although the carrying capacity is improved, after the nano material is injected into a body through an intravenous injection, a plurality of hydrolytic enzymes and shearing enzymes can decompose the material in the body, and less effective medicines reach tumor parts. Therefore, it is urgently needed to develop a preparation method of a nano-carrier with good biocompatibility, high drug loading amount, quick release and excellent treatment effect.
Disclosure of Invention
Aiming at the defects of small drug loading capacity, low release efficiency, poor biocompatibility, unsatisfactory treatment effect and the like in the prior art, the invention provides a preparation method of a layered self-assembly loaded siRNA nano particle. The siRNA release process is observed and monitored by utilizing the real-time imaging function of weak acidic stimulus response. It is noteworthy that the nanoparticles can not only release siRNA rapidly, but also fluorescence enhancement for biological imaging at slightly acidic PH = 6.8.
The invention also provides an application of the layered self-assembly loaded siRNA nano-particle in cell imaging.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the invention provides a preparation method of a layered self-assembly loaded siRNA nano particle, which comprises the following steps:
(1) adding the compound G and siRNA or FAM-siRNA into deionized water, stirring and then carrying out ultrasonic treatment to prepare core G-siRNA of the nano particles;
(2) adding a compound WP5, performing ultrasonic treatment, and standing for 12-16 hours to obtain nano-particle WP5 ⊃ G-siRNA or WP5 ⊃ G-FAM-siRNA.
Further, in the step (1), the adding amount of the compound G in every 3mL of deionized water is 50-70 mu mol; the adding amount of the siRNA or WP5 ⊃ G-FAM-siRNA in each 3mL of deionized water is 10-25 mu mol.
The sequence of the siRNA is as follows:
sense, shown as SEQ ID NO. 1: 5 '-GGCUACAUUCAGUGACDCDTdT-3',
antisense, as shown in SEQ ID NO. 2: 5 '-GUGUGUCAUCUGAAUGUAGCCdTdT-3';
the sequence of the FAM-siRNA is as follows:
sense, shown in SEQ ID NO. 3: 5 '-FAM-GGCUACAUUCAGUGACDCDTdT-3',
antisense, as shown in SEQ ID NO. 4: 5 '-GUGUGUCAUCUGAAUGUAGCCdTdT-3'.
Further, in the step (1), the stirring time is 5 min; the ultrasonic conditions are as follows: 100 Hz megasonic at 25 deg.C for 15 min.
The structural formula of the compound G used in the invention is as follows:
Figure RE-525280DEST_PATH_IMAGE001
。
further, in the step (2), the adding amount of WP5 in every 3mL of deionized water is 100-200 mu mol.
Further, in the step (2), the ultrasonic conditions are as follows: 100 Hz megasonic at 25 deg.C for 15 min.
The structural formula of WP5 used in the invention is:
Figure RE-DEST_PATH_IMAGE002
；
Figure RE-301475DEST_PATH_IMAGE003
。
the invention also provides application of the nano particles prepared by the preparation method in serving as cell imaging drugs.
The nanoparticles can rapidly release siRNA at pH 6.8 and 4.5, and fluorescence enhancement is used for biological imaging.
The nanometer material preparation part provided by the invention is prepared by a two-step layering self-assembly method. Firstly, adding a compound G with cations and siRNA into deionized water, stirring for 5 minutes, and then carrying out ultrasonic treatment for 15 minutes to prepare core G-siRNA of nanoparticles; then adding the compound WP5, and standing overnight after ultrasonic treatment for 15 minutes to obtain the nano-particle WP5 ⊃ G-siRNA. The nanoparticle morphology and particle size were then characterized by transmission electron microscopy and dynamic light scattering. In addition, the nano particle shows high siRNA loading capacity, and simultaneously can quickly release siRNA in a slightly acidic environment. Finally, the nanoparticles can be used for tumor treatment, and the result shows excellent treatment effect.
The present invention utilizes a variety of supramolecular forces including: electrostatic attraction, pi-pi accumulation, hydrophilic and hydrophobic effects and host-guest effects, mixing a quaternary ammonium salt cation modified compound G and siRNA with phosphate anions in deionized water, stirring for 5 minutes, and preparing the core G-siRNA of the nanoparticles by ultrasonic treatment for 15 minutes; then adding the compound WP5, and standing overnight after ultrasonic treatment for 15 minutes to obtain the nano-particle WP5 ⊃ G-siRNA. In the acidic environment of tumor cells, the compound G can be protonated, the fluorescence is rapidly enhanced, and the targeted imaging of tumor parts is realized. Meanwhile, WP5 can also be protonated to form WP5H with very poor water solubility, which enables the nanoparticles to be quickly disassembled, and the rapid release of siRNA is realized. It is worth noting that due to the double protonation in the acidic environment, the nanoparticle can not only realize siRNA release, but also monitor the drug release and image tumor tissues.
The invention has the beneficial effects that: the invention provides a method for preparing layered self-assembled drug-loaded nanoparticles and a method for applying the same to cancer treatment, aiming at the defects of the existing preparation and application fields of nano materials with high drug loading amount and rapid in-vivo drug release. The method can realize high loading amount and quick release of the drug, and can also realize fluorescence enhancement in the slightly acidic environment of tumor cells, thereby realizing the treatment and targeted imaging of tumors.
Drawings
FIG. 1 illustrates nanoparticle preparation and its acid-responsive release of siRNA;
FIG. 2 is the transmission electron microscope of nanoparticle WP5 ⊃ G-siRNA;
FIG. 3 is dynamic light scattering of WP5 ⊃ G-siRNA of nanoparticles;
FIG. 4 is the elemental analysis (HAADF-STEM) of nanoparticle WP5 ⊃ G-siRNA by high angle annular dark field scanning transmission electron microscope.
FIG. 5 shows the intracellular imaging of nanoparticles and their drug release.
FIG. 6 is a graph showing the effect of tumor treatment in tumor mice.
Detailed Description
The technical solution of the present invention is further explained and illustrated by the following specific examples.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
As shown in FIG. 1, compound G (50. mu. mol) and siRNA (10. mu. mol) were added to 3ml of deionized water, stirred and mixed well in the dark, and then subjected to ultrasound for 15 minutes (100 Hz at 25 ℃) to prepare nanoparticle core G-siRNA. Then adding the compound WP5 (100 micromoles), and standing overnight after 10 minutes of ultrasonic treatment (100 Hertz at 25 ℃) to obtain the nano-particle WP5 ⊃ G-siRNA.
Example 2
Referring to fig. 1, compound G (50 μmol) and fluorescein (FAM, green fluorescence) labeled siRNA (FAM-siRNA, 10 μmol) were added to 3ml of deionized water, stirred and mixed well in the dark, and then subjected to ultrasound for 15 minutes (100 hz, temperature 25 ℃) to prepare nanoparticle core G-FAM-siRNA. Then adding compound WP5 (100 micromoles), and standing overnight after 10 minutes of ultrasonic treatment (100 Hertz at 25 ℃) to obtain the WP5 ⊃ G-FAM-siRNA nanoparticle.
Results and discussion
1. Nano particle transmission electron microscope test:
the freshly prepared aqueous solution of nanoparticles was dropped onto a copper mesh for a transmission electron microscope with 2.5. mu.l of a relative using a pipette, allowed to stand, left to air-dry, and then tested. The morphology of the nanoparticles as in fig. 2 was obtained, spherical with a diameter of approximately 180 nm. High angle circular dark field scanning transmission electron microscopy elemental analysis (HAADF-STEM) was also performed, and in FIG. 4 the phosphorus (p) element is mainly derived from siRNA, which can be seen to be uniformly distributed in the nanoparticles.
2. And (3) testing a nanoparticle dynamic light scattering experiment:
the freshly prepared nanoparticle aqueous solution was dialyzed for 12 hours using a treated dialysis bag (molecular weight cut-off: 20000) to remove the monomers of WP5, G and siRNA and small-sized nanoparticles. Then 2.5 ml of the dialyzed nanoparticle aqueous solution was transferred to a cuvette and then subjected to a dynamic light scattering test. The diameter of the obtained nanoparticles is normally distributed as shown in FIG. 3, and the particle diameter is mainly concentrated around 180 nm.
3. Intracellular imaging and drug release thereof
SKOV-3 cancer cells were seeded in 35mm glass-bottomed dishes for 24 hours. Complete Dulbecco's Modified Eagle Medium (DMEM) was used in place of the medium. The cells were then incubated with WP5 ⊃ G-FAM-siRNA nanoparticles for 30 minutes. The plates were washed 3 times with sterile PBS and the cells were stained for 10 minutes in total with nuclear Hoechst 33258. After washing again with PBS, and incubation with 4.0% paraformaldehyde fixing for 15min at room temperature. Finally, the cells were washed 3 times with PBS and then observed under a confocal fluorescence microscope (Nikon A1R). As shown in fig. 5, the red fluorescence is the fluorescence of protonation of molecule G, the green fluorescence is the fluorescence of siRNA modified Fluorescein (FAM), and the blue is the nucleus, showing that siRNA can be endocytosed into cells while imaging.
4. Tumor treatment of tumor mice
When the tumor volume of the mouse reaches 150-200 mm 3 In time, tumor therapy was performed. Mice were divided into 3 groups depending on the treatment method: (1) control group: PBS group, (2) WP5 ⊃ G, (3) WP5 ⊃ G-siRNA, five mice in each group. Three groups of mice were injected with PBS, WP5 ⊃ G and WP5 ⊃ G-siRNA nanoparticles (100. mu.g kg) -1 ) Repeated every three days for a total of 4 injections. And measuring the length and the length of the tumor by using a vernier caliper at a fixed time point, and calculating the volume of the tumor: volume = length x width 2 And/2, drawing a relative change curve of the tumor volume. FIG. 6 analysis of results shows that only mice injected with nanoparticles loaded with siRNA showed minimal tumor volume increase, indicating significant inhibition of tumor growth.
As a result: the constructed supramolecular nanoparticle medicine carrying system is used for preparing nanoparticles in a layered self-assembly mode. The preparation method is different from the traditional simple siRNA adsorption method, mainly uses siRNA as raw materials to carry out assembly, and obtains the nano particles with high loading capacity and high release efficiency through two-step synthesis process. The nano particles have low toxicity, good biocompatibility and excellent treatment effect. Meanwhile, siRNA can be realized in an acidic environment, and the self-assembled small molecules can be protonated in the acidic environment to enhance fluorescence for cell imaging. The technology is not only beneficial to the high-efficiency loading and the in-vivo accurate and quick release of the medicine, but also can image the tumor part. Simultaneously provides a simple, convenient, rapid, high-efficiency and controllable nano drug-loaded material synthesis technology.
<110> university of Beijing teachers
<120> preparation method and application of layered self-assembly loaded siRNA nano-particle
<160>4
<210> 1
<211>21
<212>RNA
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GGCUA CAUUC AGAUG ACACdT dT 21
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<211>21
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GUGUC AUCUG AAUGU AGCCdT dT 21
<210> 3
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GGCUA CAUUC AGAUG ACACdT dT 21
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GUGUC AUCUG AAUGU AGCCdT dT 21

Claims (10)

1. A preparation method of a layered self-assembly loaded siRNA nanoparticle is characterized by comprising the following steps:
(1) adding the compound G and siRNA or FAM-siRNA into deionized water, stirring and then carrying out ultrasonic treatment to prepare core G-siRNA of the nano particles;
(2) adding a compound WP5, carrying out ultrasonic treatment, and standing to obtain nano-particle WP5 ⊃ G-siRNA or WP5 ⊃ G-FAM-siRNA.
2. The preparation method according to claim 1, wherein in the step (1), the compound G is added in an amount of 50 to 70 μmol per 3mL of deionized water; the adding amount of the siRNA or WP5 ⊃ G-FAM-siRNA in each 3mL of deionized water is 10-25 mu mol.
3. The production method according to claim 1 or 2,
the sequence of the siRNA is as follows:
sense ：5'-GGCUACAUUCAGAUGACACdTdT-3',
antisense： 5'-GUGUCAUCUGAAUGUAGCCdTdT-3'；
the sequence of the FAM-siRNA is as follows:
5'-FAM-GGCUACAUUCAGAUGACACdTdT-3',
antisense 5'-GUGUCAUCUGAAUGUAGCCdTdT-3'。
4. the production method according to claim 1 or 2, wherein in the step (1), the stirring time is 5 min; the ultrasonic conditions are as follows: 100 Hz megasonic at 25 deg.C for 15 min.
5. The process according to any one of claims 1 to 3, wherein the compound G has the formula:
Figure RE-RE-RE-DEST_PATH_IMAGE001
。
6. the preparation method of claim 1, wherein in the step (2), the WP5 is added in an amount of 100-200 μmol per 3mL of deionized water.
7. The production method according to claim 1 or 4, wherein in the step (2), the conditions of the ultrasonication are: 100 Hz megasonic at 25 deg.C for 15 min; the standing time is 12-16 h.
8. The method of claim 1, 4 or 5, wherein WP5 has the formula:
Figure RE-RE-DEST_PATH_IMAGE003
；
Figure RE-RE-DEST_PATH_IMAGE005
。
9. use of nanoparticles prepared according to the method of any one of claims 1 to 8 as a medicament for cell imaging.
10. The use according to claim 9, wherein the nanoparticles are capable of rapid release of siRNA at pH 6.8 and 4.5 and fluorescence enhancement for biological imaging.
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Citations (6)

* Cited by examiner, † Cited by third party
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