CN114272392A - Lipid nano-gold particle compound and application thereof in delivering ERG and treating cerebral edema diseases - Google Patents
Lipid nano-gold particle compound and application thereof in delivering ERG and treating cerebral edema diseases Download PDFInfo
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Abstract
The invention relates to the technical field of gene therapy, relates to a lipid nano-gold particle compound and application thereof in delivering ERG (epidermal gamma-gene therapy) and treating cerebral edema diseases, and provides a lipid nano-gold particle compound for treating cerebral edema by a gene therapy method. It includes Ang1-Lipo-AuNP-ERG complex; the preparation steps of the compound are as follows: preparation of AuNPs, preparation of cationic AuNPs, preparation of AuNP-ERG, and preparation of Ang 1-Lipo-AuNP-ERG. The invention has the beneficial effects that: the lipide gold nanoparticle compound Ang1-Lipo-AuNP-ERG has good biocompatibility and higher delivery efficiency as a gene vector of the ERG. When Ang1-Lipo-AuNP-ERG is taken up by brain endothelial cells, ERG plasmid is expressed in cells, and expression of Claudin-5, VE-Cadherin and ICAM-1 is regulated, so that the effects of repairing blood brain barrier and treating cerebral edema can be achieved.
Description
Technical Field
The invention relates to the technical field of gene therapy, and particularly provides a lipide gold nanoparticle compound for treating cerebral edema by a gene therapy method.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Cerebral edema is a common clinical non-independent disease, can be secondary or accompanied with various brain diseases, and poses great threat to human health. According to the etiology and pathological mechanism, there are five major categories of cerebral edema, including vasculogenesis, cytotoxicity, osmotic pressure, interstitial property and hydrostatic pressure. Of which vasogenic cerebral edema is the most common type. The basic pathological mechanism is that the blood brain barrier permeability is increased to cause the exudation of plasma protein and water, which causes the extracellular fluid content of brain tissue to be increased. At present, the treatment methods of cerebral edema mainly comprise two main types of operation treatment and drug treatment. The operation treatment is craniotomy, and the risk is extremely high; drug treatments such as the dehydrating agent mannitol, the diuretic furosemide and the glucocorticoid dexamethasone can only reduce intracranial pressure, do not repair damaged blood brain barrier and cannot completely treat cerebral edema. Therefore, the development of a method for completely treating cerebral edema by repairing the damaged blood brain barrier is an urgent problem to be solved at present.
Gene therapy is widely used in the fields of medical treatment and the like because of its characteristics of being direct and efficient. However, the current gene therapy for cerebral edema is less studied.
Disclosure of Invention
In order to solve the above problems, the present invention provides a lipogold nanoparticle complex for the treatment of cerebral edema for gene therapy.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a lipogold nanoparticle complex for treating cerebral edema by means of gene therapy, wherein the complex is Ang1-Lipo-AuNP-ERG complex, and the preparation method comprises:
adding sodium dodecyl sulfate into the AuNPs solution for reaction; adding beta-mercaptoethylamine for reaction; dialyzing and purifying the solution after reaction, wherein the dialyzate is a weakly acidic citric acid solution; filtering after dialysis to obtain a cation AuNPs solution;
adding the cationic AuNPs solution into the ERG plasmid solution, and stirring overnight to obtain an AuNP-ERG solution;
mixing DSPE-PEG2000Reacting MAL and Ang-1 in solution, dialyzing and purifying to obtain Ang-DP2000；
Respectively adding soybean lecithin, cholesterol and 2-dioleoyl hydroxypropyl-3-N, N, N-trimethyl ammonium chloride DOTAP into methanol solution, heating to dissolve the three, mixing, cooling to room temperature, and adding the Ang-DP2000Mixing, evaporating methanol to dryness to obtain liposome;
and carrying out oscillation hydration, ultrasonic disruption, solid-liquid separation and filtration on the AuNP-ERG solution and the liposome to obtain an Ang1-Lipo-AuNP-ERG compound.
The research shows that: ERG can activate the expression of endothelial cell tight junction, adhesion junction key molecules Claudin-5 and VE-cadherin, and can also regulate the expression of intercellular adhesion molecule ICAM-1, and play a core role in the process of maintaining the integrity of the blood brain barrier. ERG (ETS-related gene) is a specific gene of vascular endothelial cells, belongs to the Ets transcription factor family and is an important regulatory molecule for the generation and differentiation of endothelial progenitor cells. Therefore, the invention tries to carry ERG gene into brain endothelial cells to regulate the expression of Claudin-5, VE-cadherin and ICAM-1, and has the functions of repairing blood brain barrier and treating cerebral edema.
In addition, in order to realize the targeting of gene therapy in vivo, the invention applies angiogenin Ang-1 and a receptor Tie 2 thereof through long-term research and experimental investigation. Angiogenin is a growth factor playing an important role in the development of embryonic blood vessels, and the receptor Tie 2 is a tyrosine kinase type receptor specifically expressed in endothelial cells. Ang-1 is a high affinity ligand for the Tie 2 receptor. The targeting of treatment is realized by the specific combination of Ang-1 and Tie 2 receptor.
The realization of gene therapy cannot leave excellent gene vectors. The lipide gold nanoparticle carrier has the advantages of large capacity, low toxicity and easy synthesis, so the lipide gold nanoparticle is selected as the carrier for carrying ERG.
In a second aspect of the present invention, there is provided a medicament for treating cerebral edema, comprising: the lipidic gold nanoparticle complex described above.
In a third aspect of the invention, the application of the lipide gold nanoparticle compound in preparing a medicament for repairing a blood brain barrier is provided.
In a fourth aspect of the invention, the application of the lipide gold nanoparticle compound in preparing a medicament for treating cerebral edema is provided.
The invention has the beneficial effects that:
(1) the lipide gold nanoparticle compound Ang1-Lipo-AuNP-ERG has good biocompatibility and higher delivery efficiency as a gene vector of the ERG. When Ang1-Lipo-AuNP-ERG is taken up by brain endothelial cells, ERG plasmid is expressed in cells, and expression of Claudin-5, VE-Cadherin and ICAM-1 is regulated, thus playing roles in repairing blood brain barrier and treating cerebral edema.
(2) The operation method is simple, universal and easy for large-scale production.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a schematic diagram of the synthesis process of the lipidic gold nanoparticle complex Ang1-Lipo-AuNP-ERG of the present invention.
Fig. 2 is (a) a cytotoxicity experiment of Ang1-Lipo-AuNP-ERG complex (n-8, p < 0.01). (b) Effect of Ang1-Lipo-AuNP-ERG complex on the expression level of ERG, VE-Cadherin, clsutin-5, ICAM-1 in cells (n-8, p < 0.01). (c) The effect of Ang1-Lipo-AuNP-ERG complex on cell permeability (n-8, p < 0.01).
FIG. 3 is a graph showing the effect of Ang1-Lipo-AuNP-ERG on the protein levels of ERG, VE-Cadherin, Claudin-5 and ICAM1 after addition to TNF- α treated human brain microvascular endothelial cells.
Fig. 4 is (a) the effect of Ang1-Lipo-AuNP-ERG complex treatment on brain tissue hydration in MCAO mouse model (n 6, p < 0.01). (b) Effect on brain tissue water content after Ang1-Lipo-AuNP-ERG complex treatment in LPS mouse model (n 6,. times.p < 0.01).
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The invention provides a lipoid gold nanoparticle compound for treating cerebral edema by a gene therapy mode, wherein the compound is an Ang1-Lipo-AuNP-ERG compound, and the preparation method comprises the following steps:
adding sodium dodecyl sulfate into the AuNPs solution for reaction; adding beta-mercaptoethylamine for reaction; dialyzing and purifying the solution after reaction, wherein the dialyzate is a weakly acidic citric acid solution; filtering after dialysis to obtain a cation AuNPs solution;
adding the cationic AuNPs solution into the ERG plasmid solution, and stirring overnight to obtain an AuNP-ERG solution;
mixing DSPE-PEG2000Reacting MAL and Ang-1 in solution, dialyzing and purifying to obtain Ang-DP2000；
Respectively adding soybean lecithin, cholesterol and 2-dioleoyl hydroxypropyl-3-N, N, N-trimethyl ammonium chloride DOTAP into methanol solution, heating to dissolve the three, mixing, cooling to room temperature, and adding the Ang-DP2000Mixing, evaporating methanol to dryness to obtain liposome;
and carrying out oscillation hydration, ultrasonic disruption, solid-liquid separation and filtration on the AuNP-ERG solution and the liposome to obtain an Ang1-Lipo-AuNP-ERG compound.
In some embodiments, the mass ratio of the sodium dodecyl sulfate to the beta-mercaptoethylamine is 1-5: 3 to 7.
In some embodiments, the mass ratio of the cationic AuNPs to the ERG is 1:1.3 to 1: 1.7.
In some embodiments, the DSPE-PEG2000The mass ratio of-MAL to Ang-1 is 1.3: 1-1.7: 1.
In some embodiments, the mass ratio of soy lecithin, cholesterol, and 2-dioleoyl hydroxypropyl-3-N, N, N-trimethylammonium chloride is 0.2-0.6: 0.05-0.3: 0.01 to 0.09.
In some embodiments, the specific conditions of the ultrasonication are: the power is 80-120W, the working time is 3-7 s, the intermittent time is 3-7 s, and the total time is 3-7 min.
In some embodiments, the AuNPs are prepared by: mixing the gold tetrachloride solution with distilled water, and heating to boil; and then adding a trisodium citrate solution, continuously heating until the solution turns from light yellow to wine red, boiling for 15-30 min, and stopping heating and cooling to room temperature to obtain the gold nanoparticles.
The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.
In the following examples, ERG overexpression plasmids were constructed, amplified by E.coli, and then extracted using a plasmid extraction kit according to the procedure. The concentration of the ERG over-expression plasmid was measured using a Nanodrop Lite spectrophotometer.
Wherein, the sequence table of the ERG overexpression plasmid is as follows:
AACAAAATATTAACGCTTACAATTTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTGATCTATACATTGAATCAATATTGGCAATTAGCCATATTAGTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGAATTTTGTAATACGACTCACTATAGGGCGGCCGGGAATTCGTCGACTGGATCCGGTACCGAGGAGATCTGCCGCCGCGATCGCCATGGCCAGCACTATTAAGGAAGCCTTATCAGTTGTGAGTGAGGACCAGTCGTTGTTTGAGTGTGCCTACGGAACGCCACACCTGGCTAAGACAGAGATGACCGCGTCCTCCTCCAGCGACTATGGACAGACTTCCAAGATGAGCCCACGCGTCCCTCAGCAGGATTGGCTGTCTCAACCCCCAGCCAGGGTCACCATCAAAATGGAATGTAACCCTAGCCAGGTGAATGGCTCAAGGAACTCTCCTGATGAATGCAGTGTGGCCAAAGGCGGGAAGATGGTGGGCAGCCCAGACACCGTTGGGATGAACTACGGCAGCTACATGGAGGAGAAGCACATGCCACCCCCAAACATGACCACGAACGAGCGCAGAGTTATCGTGCCAGCAGATCCTACGCTATGGAGTACAGACCATGTGCGGCAGTGGCTGGAGTGGGCGGTGAAAGAATATGGCCTTCCAGACGTCAACATCTTGTTATTCCAGAACATCGATGGGAAGGAACTGTGCAAGATGACCAAGGACGACTTCCAGAGGCTCACCCCCAGCTACAATGCCGACATCCTTCTCTCACATCTCCACTACCTCAGAGAGACTCCTCTTCCACATTTGACTTCAGATGATGTTGATAAAGCCTTACAAAACTCTCCACGGTTAATGCATGCTAGAAACACAGGGGGTGCAGCTTTTATTTTCCCAAATACTTCAGTATATCCTGAAGCTACGCAAAGAATTACAACTAGGCCAGATTTACCATATGAGCCCCCCAGGAGATCAGCCTGGACCGGTCACGGCCACCCCACGCCCCAGTCGAAAGCTGCTCAACCATCTCCTTCCACAGTGCCCAAAACTGAAGACCAGCGTCCTCAGTTAGATCCTTATCAGATTCTTGGACCAACAAGTAGCCGCCTTGCAAATCCAGGCAGTGGCCAGATCCAGCTTTGGCAGTTCCTCCTGGAGCTCCTGTCGGACAGCTCCAACTCCAGCTGCATCACCTGGGAAGGCACCAACGGGGAGTTCAAGATGACGGATCCCGACGAGGTGGCCCGGCGCTGGGGAGAGCGGAAGAGCAAACCCAACATGAACTACGATAAGCTCAGCCGCGCCCTCCGTTACTACTATGACAAGAACATCATGACCAAGGTCCATGGGAAGCGCTACGCCTACAAGTTCGACTTCCACGGGATCGCCCAGGCCCTCCAGCCCCACCCCCCGGAGTCATCTCTGTACAAGTACCCCTCAGACCTCCCGTACATGGGCTCCTATCACGCCCACCCACAGAAGATGAACTTTGTGGCGCCCCACCCTCCAGCCCTCCCCGTGACATCTTCCAGTTTTTTTGCTGCCCCAAACCCATACTGGAATTCACCAACTGGGGGTATATACCCCAACACTAGGCTCCCCACCAGCCATATGCCTTCTCATCTGGGCACTTACTACAGACGGCCGGCCGCGGTCATAGCTGTTTCCTGAACAGATCCCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTCTGATTTTAAAATAACTATACCAGCAGGAGGACGTCCAGACACAGCATAGGCTACCTGGCCATGCCCAACCGGTGGGACATTTGAGTTGCTTGCTTGGCACTGTCCTCTCATGCGTTGGGTCCACTCAGTAGATGCCTGTTGAATTGGGTACGCGGCCAGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAACCTGAGGCTATGGCAGGGCCTGCCGCCCCGACGTTGGCTGCGAGCCCTGGGCCTTCACCCGAACTTGGGGGGTGGGGTGGGGAAAAGGAAGAAACGCGGGCGTATTGGCCCCAATGGGGTCTCGGTGGGGTATCGACAGAGTGCCAGCCCTGGGACCGAACCCCGCGTTTATGAACAAACGACCCAACACCGTGCGTTTTATTCTGTCTTTTTATTGCCGTCATAGCGCGGGTTCCTTCCGGTATTGTCTCCTTCCGTGTTTCAGTTAGCCTCCCCCTAGGGTGGGCGAAGAACTCCAGCATGAGATCCCCGCGCTGGAGGATCATCCAGCCGGCGTCCCGGAAAACGATTCCGAAGCCCAACCTTTCATAGAAGGCGGCGGTGGAATCGAAATCTCGTGATGGCAGGTTGGGCGTCGCTTGGTCGGTCATTTCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGATCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCGATCTTTGCAAAAGCCTAGGCCTCCAAAAAAGCCTCCTCACTACTTCTGGAATAGCTCAGAGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGTCAGCCATGGGGCGGAGAATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGACTATGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCACAGCTGGTTCTTTCCGCCTCAGGACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTT
example 1
The lipoid gold nanoparticle complex for treating cerebral edema comprises an Ang1-Lipo-AuNP-ERG complex. The preparation steps of the compound are as follows:
(1) preparation of AuNPs
Soaking the glass instrument in aqua regia, washing with distilled water for three times, and oven drying; in a 100mL two-necked flask, 30. mu.L of a 25.6% by mass gold tetrachloride solution and 60mL of distilled water were added, and the mixture was heated to boiling. And then adding 8mL of trisodium citrate solution with the mass fraction of 1%, continuing to heat, changing the solution from light yellow to wine red, boiling for 20min, stopping heating, and cooling to room temperature to obtain the gold nanoparticles with the particle size of 13 +/-2 nm. Stored at 4 ℃ in the dark.
(2) Preparation of cationic AuNPs
To the synthesized AuNPs solution, 0.3g of sodium dodecyl sulfate SDS was added and stirred for 2 hours. 0.5g of beta-mercaptoethylamine was subsequently added and stirring was continued for 4 h. Putting the solution into a dialysis bag with a cut-off of 3500Da, wherein the dialysate is a weakly acidic citric acid solution (Ph is 2-4). And (4) filtering the solution in the dialysis bag by using a 0.22 mu m filter membrane after dialysis for two days to obtain a cation AuNPs solution, and storing the solution at 4 ℃ in a dark place.
(3) Preparation of AuNP-ERG
Adding ERG plasmid solution into the cationic AuNPs solution with the mass ratio of 1:1.5 (AuNPs: ERG), and stirring overnight to obtain AuNP-ERG solution.
(4) Preparation of Ang1-Lipo-AuNP-ERG
Firstly DSPE-PEG2000Dissolving MAL and Ang-1 in methanol solution at ratio of 1.5:1, stirring at room temperature for 48 hr, dialyzing and purifying to obtain Ang-DP2000。
Putting 0.4g of soybean lecithin, 0.1g of cholesterol and 0.05g of 2-dioleoyl hydroxypropyl-3-N, N, N-trimethyl ammonium chloride DOTAP into a 500mL eggplant-shaped bottle, adding 200mL of methanol solution, heating to 70-80 ℃ to dissolve the three, and stirring to uniformly mix the three. After cooling to room temperature, 0.05g of Ang-DP was added2000And (4) stirring uniformly. Methanol was spin-dried on a rotary evaporator to form a uniform layer of liposomes on the wall of the vial. Adding AuNP-ERG solution into a solanaceous bottle, and shaking to hydrate for 2 h. The sonicator breaks large liposomes, 100W, work 5s, pause 5s, for a total of 5 min. Followed by centrifugal purification. 10000rpm, 3 min. Filtering with 0.22 μm filter membrane for three times to obtain Ang1-Lipo-AuNP-ERG complex.
Example 2
The lipoid gold nanoparticle complex for treating cerebral edema comprises an Ang1-Lipo-AuNP-ERG complex. The preparation steps of the compound are as follows:
(1) preparation of AuNPs
All the used glass instruments are soaked in aqua regia, then washed for three times by distilled water and dried. 10. mu.L of a gold tetrachloride solution having a mass fraction of 25.6% and 40mL of distilled water were placed in a 100mL two-necked flask and heated to boiling. And then adding 6mL of trisodium citrate solution with the mass fraction of 1%, continuing to heat, changing the solution from light yellow to wine red, boiling for 15min, stopping heating, and cooling to room temperature to obtain the gold nanoparticles with the particle size of 13 +/-2 nm. Stored at 4 ℃ in the dark.
(2) Preparation of cationic AuNPs
To the synthesized AuNPs solution, 0.1g of sodium dodecyl sulfate SDS was added and stirred for 1 hour. 0.3g of beta-mercaptoethylamine was then added and stirring was continued for 2 h. Putting the solution into a dialysis bag with a cut-off of 3000-4000 Da, wherein the dialysate is a weakly acidic citric acid solution. And (4) filtering the solution in the dialysis bag by using a 0.22 mu m filter membrane after dialysis for two days to obtain a cation AuNPs solution, and storing the solution at 4 ℃ in a dark place.
(3) Preparation of AuNP-ERG
Adding ERG plasmid solution into the cationic AuNPs solution with the mass ratio of 1:1.3 (AuNPs: ERG), and stirring overnight to obtain AuNP-ERG solution.
(4) Preparation of Ang1-Lipo-AuNP-ERG
Firstly DSPE-PEG2000Dissolving MAL and Ang-1 in methanol solution at ratio of 1.3:1, stirring at room temperature for 24 hr, dialyzing and purifying to obtain Ang-DP2000。
Putting 0.2g of soybean lecithin, 0.05g of cholesterol and 0.01g of 2-dioleoyl hydroxypropyl-3-N, N, N-trimethyl ammonium chloride DOTAP into a 500mL eggplant-shaped bottle, adding 100mL of methanol solution, heating to 60 ℃ to dissolve the three, and stirring to uniformly mix the three. After cooling to room temperature, a small amount of Ang-DP was added2000And (4) stirring uniformly. Methanol was spin-dried on a rotary evaporator to form a uniform layer of liposomes on the wall of the vial. Adding AuNP-ERG solution into eggplant-shaped bottle, and shaking to hydrate for 1 h. The sonicator breaks large liposomes, 80W, work for 3s, pause for 3s, for a total of 3 min. Followed by centrifugal purification. 8000rpm, 2 min. Filtering with 0.22 μm filter membrane for three times to obtain Ang1-Lipo-AuNP-ERG complex.
Example 3
The lipoid gold nanoparticle complex for treating cerebral edema comprises an Ang1-Lipo-AuNP-ERG complex. The preparation steps of the compound are as follows:
(1) preparation of AuNPs
All the used glass instruments are soaked in aqua regia, then washed for three times by distilled water and dried. A100 mL two-necked flask was charged with 50. mu.L of a 25.6% by mass gold tetrachloride solution and 70mL of distilled water, and heated to boiling. And then adding 10mL of trisodium citrate solution with the mass fraction of 1%, continuing to heat, changing the solution from light yellow to wine red, boiling for 30min, stopping heating, and cooling to room temperature to obtain the gold nanoparticles with the particle size of 13 +/-2 nm. Stored at 4 ℃ in the dark.
(2) Preparation of cationic AuNPs
To the synthesized AuNPs solution, 0.5g of sodium dodecyl sulfate SDS was added and stirred for 3 hours. 0.7g of beta-mercaptoethylamine was subsequently added and stirring was continued for 6 h. Putting the solution into a dialysis bag with a cut-off of 3000-4000 Da, wherein the dialysate is a weakly acidic citric acid solution. And (4) filtering the solution in the dialysis bag by using a 0.22 mu m filter membrane after dialysis for two days to obtain a cation AuNPs solution, and storing the solution at 4 ℃ in a dark place.
(3) Preparation of AuNP-ERG
Adding ERG plasmid solution into the cationic AuNPs solution with the mass ratio of 1:1.7 (AuNPs: ERG), and stirring overnight to obtain AuNP-ERG solution.
(4) Preparation of Ang1-Lipo-AuNP-ERG
Firstly DSPE-PEG2000Dissolving MAL and Ang-1 in methanol solution at ratio of 1.7:1, stirring at room temperature for 72h, and dialyzing to purify to obtain Ang-DP2000。
Putting 0.6g of soybean lecithin, 0.3g of cholesterol and 0.09g of 2-dioleoyl hydroxypropyl-3-N, N, N-trimethyl ammonium chloride DOTAP into a 500mL eggplant-shaped bottle, adding 300mL of methanol solution, heating to 90 ℃ to dissolve the three, and stirring to uniformly mix the three. After cooling to room temperature, a small amount of Ang-DP was added2000And (4) stirring uniformly. Methanol was spin-dried on a rotary evaporator to form a uniform layer of liposomes on the wall of the vial. Adding AuNP-ERG solution into a solanaceous bottle, and shaking to hydrate for 4 h. The sonicator disrupted large liposomes, 20W, work for 7s, pause for 7s, for a total of 7 min. Followed by centrifugal purification. 13000rpm, 3 min. Filtering with 0.22 μm filter membrane for three times to obtain Ang1-Lipo-AuNP-ERG complex.
Examples of the experiments
After the Ang1-Lipo-AuNP-ERG complex is synthesized in the embodiment 1, in-vivo and in-vitro experiments are respectively carried out to prove the effects of the complex on repairing blood brain barrier and treating cerebral edema.
The invention selects Human Brain Microvascular Endothelial Cells (HBMEC) to carry out cell experiments. Firstly, the invention researches the cytotoxicity of Ang1-Lipo-AuNP-ERG complex, and the cell activity is detected by culturing HBMEC, adding 0, 10, 100, 1000, 10000ng/ml (10ug/ml) Ang1-Lipo-AuNP-ERG complex to mediate ERG over-expression, collecting cells after 6, 12, 24 hours. Cell activity was found to decrease after 24 hours of treatment at a concentration of 10ug/ml (p <0.01), whereas other concentrations had no significant effect on cell activity (a in figure 2). Subsequently, the present inventors investigated the effect of adding Ang1-Lipo-AuNP-ERG complex on the expression levels of ERG, VE-Cadherin, Cludin-5, and ICAM-1 in cells. As shown in b in FIG. 1, after adding 100ng/mL of Ang1-Lipo-AuNP-ERG complex, the expression levels of ERG and the cell connexins VE-Cadherin and Cludin-5 in the cells were increased, while the expression level of the proinflammatory leukocyte adhesion protein ICAM-1 was decreased (b in FIG. 2). Besides, the present invention also investigated the effect of Ang1-Lipo-AuNP-ERG complex on cell permeability using the Transwell-FTTC-Dexran permeation method (c in FIG. 2). First stimulated with TNF- α and then treated with 100ng/mL of Ang1-Lipo-AuNP-ERG complex for a period of time. The experimental results show that the permeability of the cells in the experimental group is obviously reduced compared with the cells in the control group. In addition, the present inventors also investigated the effect of Ang1-Lipo-AuNP-ERG on the protein levels of ERG, VE-Cadherin, Claudin-5 and ICAM-1 after addition to TNF- α treated HBMEC. As shown in FIG. 3, ERG, VE-Cadherin, Claudin-5 had an increased protein level, while ICAM-1 had a significantly decreased protein level. The experimental results show that the Ang1-Lipo-AuNP-ERG complex can reduce the permeability of cerebrovascular endothelial cells by adjusting the expression quantity of VE-Cadherin, Cludin-5 and ICAM-1 in vitro, thereby playing a role in repairing the damaged blood brain barrier.
After the in vitro experiment is completed, the invention also carries out the in vivo experiment. The invention adopts two methods to construct a cerebral edema model of a mouse, namely a Middle Cerebral Artery Occlusion (MCAO) method and a lipopolysaccharide injection (LPS) method. Ang1-Lipo-AuNP-ERG complex was injected intravenously at 0.1 mg/kg. One week later, the mice were sacrificed and brain tissue was removed to calculate their water content. As shown in FIG. 2, the water content of brain tissue was decreased in both animal models treated with Ang1-Lipo-AuNP-ERG complex (a, b in FIG. 4). This result indicates that the Ang1-Lipo-AuNP-ERG complex can play a role in treating cerebral edema.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Hospital of Qianfishan in Shandong province
<120> lipid nano-gold particle compound and application thereof in delivering ERG and treating cerebral edema diseases
<130> 2022.01.28
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 6224
<212> DNA
<213> Artificial sequence
<400> 1
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ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 5940
taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 6000
aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 6060
ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 6120
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Claims (10)
1. The lipide gold nanoparticle complex for treating cerebral edema through a gene therapy mode is an Ang1-Lipo-AuNP-ERG complex, and the preparation method comprises the following steps:
adding sodium dodecyl sulfate into the AuNPs solution for reaction; adding beta-mercaptoethylamine for reaction; dialyzing and purifying the solution after reaction, wherein the dialyzate is a weakly acidic citric acid solution; filtering after dialysis to obtain a cation AuNPs solution;
adding the cationic AuNPs solution into the ERG plasmid solution, and stirring overnight to obtain an AuNP-ERG solution;
mixing DSPE-PEG2000Reacting MAL and Ang-1 in solution, dialyzing and purifying to obtain Ang-DP2000；
Respectively adding soybean lecithin, cholesterol and 2-dioleoyl hydroxypropyl-3-N, N, N-trimethyl ammonium chloride DOTAP into methanol solution, heating to dissolve the three, mixing, cooling to room temperature, and adding the Ang-DP2000Mixing, evaporating methanol to dryness to obtain liposome;
and carrying out oscillation hydration, ultrasonic disruption, solid-liquid separation and filtration on the AuNP-ERG solution and the liposome to obtain an Ang1-Lipo-AuNP-ERG compound.
2. The lipogold nanoparticle complex for treating cerebral edema through gene therapy according to claim 1, wherein the mass ratio of the sodium dodecyl sulfate to the beta-mercaptoethylamine is 1-5: 3 to 7.
3. The lipogold nanoparticle complex for treating cerebral edema through gene therapy according to claim 1, wherein the mass ratio of the cationic AuNPs to the ERG is 1: 1.3-1: 1.7.
4. The lipogold nanoparticle complex for the treatment of cerebral edema by gene therapy as claimed in claim 1, wherein DSPE-PEG2000The mass ratio of-MAL to Ang-1 is 1.3: 1-1.7: 1.
5. The lipogold nanoparticle complex for the treatment of cerebral edema by gene therapy according to claim 1, wherein the mass ratio of soybean lecithin, cholesterol and 2-dioleoyl hydroxypropyl-3-N, N-trimethylammonium chloride is 0.2-0.6: 0.05-0.3: 0.01 to 0.09.
6. The lipogold nanoparticle complex for the treatment of cerebral edema by means of gene therapy according to claim 1, wherein the specific conditions of the ultrasonication are: the power is 80-120W, the working time is 3-7 s, the intermittent time is 3-7 s, and the total time is 3-7 min.
7. The lipogold nanoparticle complex for treating cerebral edema by gene therapy according to claim 1, wherein the AuNPs are prepared by: mixing the gold tetrachloride solution with distilled water, and heating to boil; and then adding a trisodium citrate solution, continuously heating until the solution turns from light yellow to wine red, boiling for 15-30 min, and stopping heating and cooling to room temperature to obtain the gold nanoparticles.
8. A medicament for treating cerebral edema, comprising: the lipidic gold nanoparticle complex of any one of claims 1-7.
9. Use of the lipidic gold nanoparticle complex of any one of claims 1-7 for the preparation of a medicament for repairing the blood brain barrier.
10. Use of the lipidic gold nanoparticle complex of any one of claims 1-7 for the preparation of a medicament for the treatment of cerebral edema.
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| WO2024075089A1 (en) * | 2022-10-08 | 2024-04-11 | Universidade De Coimbra | Hybrid nanoparticles as multifunctional platform for brain tumor therapy |
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| US20190030190A1 (en) * | 2005-08-05 | 2019-01-31 | Gholam A. Peyman | Methods to regulate polarization and enhance function of cells |
| WO2018187287A1 (en) * | 2017-04-03 | 2018-10-11 | Charles Jeffrey Brinker | Porous nanoparticle-supported lipid bilayer delivery of transcriptional gene modulators |
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| WO2024075089A1 (en) * | 2022-10-08 | 2024-04-11 | Universidade De Coimbra | Hybrid nanoparticles as multifunctional platform for brain tumor therapy |
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