CN114159407A - Preparation of self-assembled nano gene targeted delivery system for treating acute myelogenous leukemia - Google Patents
Preparation of self-assembled nano gene targeted delivery system for treating acute myelogenous leukemia Download PDFInfo
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- CN114159407A CN114159407A CN202111438901.5A CN202111438901A CN114159407A CN 114159407 A CN114159407 A CN 114159407A CN 202111438901 A CN202111438901 A CN 202111438901A CN 114159407 A CN114159407 A CN 114159407A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
- A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5169—Proteins, e.g. albumin, gelatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
Abstract
The invention belongs to the technical field of nano delivery drugs, and particularly relates to a preparation method of a self-assembled nano gene targeted delivery system for treating acute myelogenous leukemia. In addition, the nano gene drug delivery system can enter a cell nucleus through the interaction of NLS and a nuclear transport protein, and the gene uptake and expression are enhanced, so that the nano gene drug delivery system can be used for the nuclear targeting and gene therapy of acute myelogenous leukemia cells.
Description
Technical Field
The invention belongs to the technical field of nano delivery drugs, and particularly relates to preparation of a self-assembled nano gene targeted delivery system for treating acute myelogenous leukemia.
Background
Acute Myelogenous Leukemia (AML) is a highly heterogeneous malignant clonal disease caused by abnormal proliferation of myeloid leukocytes, which seriously affects human health. At present, the traditional AML treatment methods (chemotherapy and radiotherapy) have the defects of low specificity, large side effect, easy relapse and the like, and the ideal treatment effect is difficult to obtain clinically. Therefore, finding leukemia cell-specific targets is the key to achieving gene-targeted therapy of AML.
Myeloid cell differentiation antigen 33 (CD 33) is a type I transmembrane receptor protein, specifically expressed on the cell surface of the hematopoietic system. It has been shown that CD33 is a specific antigen of AML cells, and the diagnosis and targeted therapy of AML are realized by the specific binding of CD33 monoclonal antibody (aCD33) to AML tumor cells.
Antisense oligodeoxynucleotide is a molecular drug which inhibits the expression of a target gene by combining the sequence specifically with the DNA or mRNA of the gene and regulates and controls at the gene level, and is a novel preparation for treating solid tumors and hematological malignancies at present. Among them, G3139 is an 18-mer phosphorothioate antisense oligodeoxynucleotide sequence (5'-TCT CCC AGC GTG CGC CAT-3') designed against bcl2 protein, which specifically binds to the first six codons of human bcl2 mRNA, down-regulating bcl2 expression. At present, the experiment of the clinical stage III has been completed by using G3139 as a new drug candidate for the treatment of hematological malignancies, but the ideal therapeutic effect of the intravenous injection of G3139 in clinical trials has not been achieved due to the lack of efficient targeting vectors. Therefore, efficient vectors are searched, the drug specificity is targeted to the drug action target, the drug stability and the transfection rate are improved, and the method has great significance for the clinical application of G3139.
KALA peptide (WEAKLAKALAKALAKHLAKALAKALKACEA), also known as fusogenic peptide, is a synthetic cationic membrane-penetrating peptide with good membrane-penetrating properties. NLS is basic heptapeptide (PKKKRKV) derived from SV40 virus large T antigen, has the capacity of mediating the transmembrane transport of exogenous substances into the nucleus, and can improve the nuclear entry efficiency of anticancer drugs.
At present, no report that the G3139-entrapped nano gene targeting delivery system prepared by KALA peptide, NLS and CD33 is utilized so as to improve the targeting property, the drug stability and the transfection rate is available.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a preparation method of a self-assembled nano gene targeted delivery system, namely, an aCD33-NKSN carrier is used for encapsulating an anti-tumor model drug G3139 to prepare the self-assembled nano gene targeted delivery system, and the constructed aCD33-NKSN/G3139 self-assembled nano targeted delivery system can be used for targeted gene therapy of acute myelogenous leukemia.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a preparation method of a self-assembly nano gene targeting delivery system, which comprises the steps of firstly dispersing aCD33-NKSN into a buffer solution, then adding G3139, and carrying out ultrasonic centrifugation and self-assembly to form the nano gene targeting delivery system (aCD33-NKSN/G3139 nanoparticles).
Preferably, the weight ratio of the aCD33-NKSN to the G3139 is 8-12: 1. Specifically, the weight ratio of the aCD33-NKSN to the G3139 is 10: 1.
Preferably, the concentration of the aCD33-NKSN in the buffer is 2-3mg/5 mL. Specifically, the concentration of the aCD33-NKSN in the buffer solution is 2.5mg/5 mL.
Preferably, the buffer is HEPES buffer. Further, the HEPES buffer had a concentration of 10mM, contained 5% glucose and had a pH of 7.4.
Preferably, the time of ultrasonic centrifugation is 10-30 minutes, the power is 250-. Specifically, the time of ultrasonic centrifugation was 10 minutes, the power was 300w, and 12000 revolutions per minute.
Preferably, the sonication is followed by a filtration step. Further, the filtration is performed by using a filter with a pore size of 0.22 μm.
The invention also provides the self-assembled nano gene targeted delivery system prepared by the preparation method of the self-assembled nano gene targeted delivery system.
The invention also provides application of the self-assembled nano gene targeted delivery system in preparation of a medicine for treating acute myelogenous leukemia.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a preparation method of a self-assembled nano gene targeting delivery system, which is prepared by coating an anti-tumor model drug G3139 on an aCD33-NKSN carrier to obtain the aCD33-NKSN/G3139 self-assembled nano gene targeting delivery system.
The aCD33-NKSN/G3139 self-assembly nano gene targeting delivery system constructed by the invention can effectively encapsulate and compress G3139 through the self-assembly function, can be specifically combined with a target antigen CD33 on the surface of a tumor cell through an aCD33 antibody part in the carrier, realizes endocytosis of the cell by utilizing the membrane penetrating action of KALA, and can escape from an endosome to enter cytoplasm through alpha-helix conformation transition of a polypeptide carrier, thereby rapidly positioning the cell nucleus. In addition, the nano gene drug delivery system can enter cell nucleus through the interaction of NLS and nuclear transport protein to enhance the gene uptake and expression, so that the nano gene drug delivery system can carry out nuclear targeting and gene therapy on acute myelogenous leukemia cells, and brings a new prospect for the clinical application of the antisense oligodeoxynucleotide drug G3139.
Drawings
FIG. 1 shows the physicochemical characteristics of aCD33-NKSN/G3139 nanoparticles (A is a transmission electron micrograph, B is a particle size distribution diagram, and C is a zeta potential diagram);
FIG. 2 shows the uptake of aCD33-NKSN/Cy5-G3139 nanoparticles in Kasumi-1 tumor cells (A is confocal fluorescence image: first column shows blue staining of cell nucleus, second column shows red staining of cell membrane, third column shows green staining of G3139, fourth column shows the superposition of three images; B is flow cytometry image; C is fluorescence signal intensity contrast image, <0.05, <0.01(n ═ 3));
FIG. 3 shows the in vivo antitumor activity of aCD33-NKSN/Cy5-G3139 tumor-bearing nude mice (A is the change curve of tumor volume during treatment; B is the expression of bcl2 protein in Kasumi-1 tumor; C is the survival curve after treatment of tumor-bearing nude mice (n ═ 6)).
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.
Example 1 preparation of self-assembled NanoGene Targeted delivery System (aCD33-NKSN/G3139)
5mg of aCD33-NKSN (i.e., aCD33-NLS-KALA-SA, available from Leon biosciences, Inc., Nanjing, China) was dispersed in 10mL of HEPES buffer (10 mM concentration, 5% glucose, pH 7.4), and then antisense oligodeoxynucleotide G3139 (available from BD Pharmingen, USA) was added, wherein the weight ratio of aCD33-NKSN to G3139 was 10:1(w/w), and self-assembly was performed after 10 minutes of ultrasonic centrifugation (300w, 12000 rotations per minute) to form aCD33-NLS-KALA-SA/G3139 nanoparticles.
After redispersion, the resulting aCD33-NKSN/G3139 nanoemulsion solution was stored at 4 ℃ by pressing 3 times through a polycarbonate filter with a pore size of 0.22. mu.m.
The appearance of the aCD33-NKSN/G3139 nanoparticles was observed by a transmission electron microscope (PHILIPS CM 200), and the particle size distribution and zeta potential thereof were measured by a laser particle sizer (Zetasizer Nano ZS 90). The concentration of free drug in the supernatant after centrifugation was measured and the encapsulation efficiency and drug loading were calculated according to the following formula:
the results in FIG. 1 show that the aCD33-NKSN/G3139 nanoparticles are spherical and uniformly distributed, the average particle size is 66.3 +/-8.2 nm, the polymer dispersion coefficient (PdI) is less than 0.09, and the zeta potential is 30.1 +/-5.5 mV. Meanwhile, the encapsulation efficiency (%) of the drug is 88.7 +/-6.2 and the drug loading (%) is 21.4 +/-4.3 according to the formula.
Experimental example 1 tumor targeting test of aCD33-NKSN/G3139
The internalization and intracellular distribution of aCD33-NKSN/Cy5-G3139 nanoparticles in Kasumi-1 leukemia cells (purchased from Nanjing Leon Biotech, Inc., China) were evaluated by confocal fluorescence microscopy (Zeiss LSM510 META) using Cy 5-labeled G3139 as a probe. Confocal fluorescence images of endocytosis of aCD33-NKSN/Cy5-G3139 nanoparticles were confirmed using flow cytometry (FACScalibur) studies. The method specifically comprises the following steps:
mixing Kasumi-1 leukemia cells (1 × 10)5Per well) were inoculated on a 24-well culture plate, and a DMEM medium containing 10% fetal bovine serum was added thereto for 24 hours, replaced with a fresh medium (containing equal amounts of the drug concentration (1. mu. mol/L) of Cy5-G3139, NKSN/Cy5-G3139, aCD33-NKSN/Cy5-G3139, respectively), incubated for 24 hours, and the nuclear-grade cell membrane was stained with DAPI and WGA-Alexa350, respectively. And (3) observing the fluorescence image by using a confocal fluorescence microscope, and detecting the intracellular fluorescence intensity by using a flow cytometer.
The results in FIG. 2A show that aCD33-NKSN/Cy5-G3139 exhibits the strongest green fluorescence at the nuclear site, observed using confocal fluorescence imaging. The fluorescence signal intensities of aCD33-NKSN/Cy5-G3139 were about 1.6 times that of NKSN/Cy5-G3139 and about 2.9 times that of Cy5-G3139, respectively, as determined by flow cytometry analysis and comparison of fluorescence signal intensities (FIG. 2B, C).
The above results indicate that aCD33-NKSN/Cy5-G3139 can efficiently target delivery of Cy5-G3139 into Kasumi-1 tumor cells and to the nucleus.
Experimental example 2 aCD33-NKSN/G3139 tumor suppression Effect test
Establishing a Kasumi-1 tumor-bearing nude mouse animal model, and investigating the inhibition effect of an aCD33-NKSN/G3139 nano drug delivery system on the tumor growth in an animal body; after treatment, the in vivo tumor suppression effect was assessed by changes in tumor growth curve, survival rate and expression of the relevant target protein. The method specifically comprises the following steps:
establishing a Kasumi-1 leukemia cell tumor-bearing nude mouse animal model, and randomly dividing mice into four groups after the seventh day of tumor inoculation: PBS (control), G3139, NKSN/G3139, aCD33-NKSN/G3139, 6 each. Tail vein injection (5mg/kg) was administered once every 3 days for 2 weeks. Tumor volume was measured in each group of mice before each dose and the number of surviving days was recorded, and tumor tissues were collected 24 hours after the last dose and assayed for protein expression of bcl2 mRNA using RT-PCR.
The results in FIG. 3 show that aCD33-NKSN/G3139 is able to significantly down-regulate the expression level of bcl2 protein, more effectively inhibit tumor growth, and increase survival of tumor-bearing mice compared to PBS (blank), G3139, NKSN/G3139.
It can be seen from the above experimental examples that the aCD33-NKSN/G3139 can effectively encapsulate and compress the antisense oligodeoxynucleotide drug G3139, can be specifically bound with the target antigen CD33 on the surface of the leukemia tumor cell through the aCD33 antibody part in the carrier, can realize the targeting and endocytosis of the tumor cell by using the membrane-penetrating action of KALA, and can escape from the endosome to the cytoplasm through the transformation of the alpha-helix conformation of the polypeptide carrier, thereby rapidly locating the nucleus. In addition, it is also possible to enter the nucleus by interaction of NLS with nuclear transporters, enhancing the uptake and expression of the G3139 gene. Finally, the nuclear targeting and gene therapy of G3139 on acute myelogenous leukemia are effectively realized, and the clinical application value of the antisense oligodeoxynucleotide drug G3139 is improved.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Claims (10)
1. A preparation method of a self-assembled nano gene targeting delivery system is characterized in that aCD33-NKSN is dispersed into a buffer solution, G3139 is added, and the self-assembly is carried out after ultrasonic centrifugation to form the nano gene targeting delivery system.
2. The method for preparing a self-assembled nano gene targeting delivery system according to claim 1, wherein the weight ratio of aCD33-NKSN to G3139 is 8-12: 1.
3. The method for preparing a self-assembled nano gene targeting delivery system according to claim 1, wherein the concentration of the aCD33-NKSN in the buffer solution is 2-3mg/5 mL.
4. The method for preparing a self-assembled nano gene targeted delivery system according to claim 1, wherein the buffer is HEPES buffer.
5. The method for preparing the self-assembled nano gene targeted delivery system according to claim 4, wherein the HEPES buffer solution has a concentration of 10mM, contains 5% glucose and has a pH of 7.4.
6. The method for preparing a self-assembled nano gene targeting delivery system as claimed in claim 1, wherein the time of ultrasonic centrifugation is 10-30 min, the power is 250-350w, and 10000-15000 r/min.
7. The method for preparing a self-assembled nano gene targeted delivery system according to claim 1, wherein the ultrasound is followed by a filtration step.
8. The method for preparing a self-assembled nano gene targeting delivery system according to claim 7, wherein the filtering is performed by using a filter with a pore size of 0.22 μm.
9. The self-assembled nano gene targeted delivery system prepared by the preparation method of the self-assembled nano gene targeted delivery system according to any one of claims 1 to 8.
10. The use of the self-assembled nano gene targeted delivery system of claim 9 in the preparation of a medicament for treating acute myelogenous leukemia.
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| CN110177544A (en) * | 2016-11-29 | 2019-08-27 | 普尔泰克健康有限公司 | For delivering the excretion body of therapeutic agent |
| CN112587504A (en) * | 2016-10-17 | 2021-04-02 | 南京绿叶制药有限公司 | Lipid nanoparticle of antisense oligonucleotide for inhibiting bcl-2 and preparation method thereof |
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- 2021-11-30 CN CN202111438901.5A patent/CN114159407A/en active Pending
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| CN104884637A (en) * | 2012-11-05 | 2015-09-02 | 普隆奈治疗公司 | Methods of using biomarkers for the treatment of cancer by modulation of BCL2 expression |
| CN112587504A (en) * | 2016-10-17 | 2021-04-02 | 南京绿叶制药有限公司 | Lipid nanoparticle of antisense oligonucleotide for inhibiting bcl-2 and preparation method thereof |
| CN110177544A (en) * | 2016-11-29 | 2019-08-27 | 普尔泰克健康有限公司 | For delivering the excretion body of therapeutic agent |
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