CN117919203A - Application of drug-loaded nano particles in preparation of drugs for preventing or treating atherosclerosis - Google Patents
Application of drug-loaded nano particles in preparation of drugs for preventing or treating atherosclerosis Download PDFInfo
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- CN117919203A CN117919203A CN202410097756.6A CN202410097756A CN117919203A CN 117919203 A CN117919203 A CN 117919203A CN 202410097756 A CN202410097756 A CN 202410097756A CN 117919203 A CN117919203 A CN 117919203A
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- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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Abstract
The invention belongs to the field of medicines, and particularly provides an application of a drug-loaded nanoparticle in preparation of a medicine for preventing or treating atherosclerosis, wherein the drug-loaded nanoparticle comprises the following components: (1) A polyamino acid carrier, on which a nitric oxide donor is grafted; (2) A nucleic acid agent that silences Camk2g gene expression, encapsulated within the polyamino acid vector. According to the drug-loaded nanoparticle, nucleic acid drugs for silencing Camk2g gene expression are delivered into plaque macrophages in a targeted manner, so that phagocytic functions of the macrophages are recovered, and the microenvironment inside the plaque is improved; at the same time, the nitric oxide donor can be delivered to the macrophage and react with the inducible nitric oxide synthase in the macrophage to generate nitric oxide. Nitric oxide gas molecules diffuse to the vicinity of damaged endothelial cells, repair the endothelial cell barrier, restore the integrity of the vascular endothelial barrier, play a role in combined therapy and realize the high-efficiency therapy of atherosclerosis.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to application of medicine-carrying nano particles in preparation of medicines for preventing or treating atherosclerosis.
Background
In recent years, the development situation of global cardiovascular diseases is increasingly severe, and the cardiovascular diseases have the characteristics of high prevalence rate, high disability rate and high death rate, and seriously threaten the health of human beings. The types of cardiovascular diseases are numerous, such as: coronary heart disease, myocardial infarction, heart failure, stroke, etc., all of which can lead to serious life health consequences. However, these diseases all have a key and fundamental etiology—atherosclerosis.
The pathogenesis of atherosclerosis is complex, and macrophages play a corresponding physiological function in the pathological development process of atherosclerosis. In the early stages of disease, monocytes differentiate into macrophages, which can phagocytose low density lipoprotein (oxLDL) in the intima layer and expel it. However, when macrophages engulf excessive lipids and cannot be metabolically discharged in time, the macrophages become foam cells, which undergo apoptosis or necrosis, eventually forming an ever-increasing "necrotic core" in the intima layer, consisting of cholesterol esters, cholesterol crystals and cell debris. In the middle and late stages of the disease, the thromboses in the occlusive cavity may be caused, and serious consequences, namely myocardial infarction, apoplexy, sudden cardiac death and the like, are caused. In addition, lymphocytes such as T cells and B cells are involved in various processes in the development of atherosclerosis. In the early stage of disease development, effective drug treatment can well inhibit the development of the disease, but in the middle and later stages, the maintenance of the stability of focal plaque is more important, and is also a key for treating atherosclerosis.
At present, according to clinical treatment means, the medicine is mainly used for reducing blood lipid and resisting inflammation. Among lipid-lowering drugs, statin drugs are clinically commonly used drugs such as atorvastatin, rosuvastatin, lovastatin and the like. They are 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, the main mechanism of action is to lower total cholesterol and medium-low density lipoproteins, and triacylglycerols can be reduced to some extent so as to achieve the anti-atherosclerosis effect. In anti-inflammatory therapies, monoclonal antibodies, protease inhibitors and the like are clinically commonly used drugs, such as Canakinumab (Canakinumab) is an interleukin 1 beta (IL-1 beta) antagonist, and the use of Canakinumab for the dry prognosis of atherosclerosis in patients, and the symptoms of patients are significantly improved. Methanesulfonyl cyanide (Dapansutrile) is an inhibitor of the inflammatory body NLRP3, and phase I randomized trials have demonstrated its safety and tolerability in heart failure patients with reduced left ventricular ejection fraction.
However, after long-term clinical administration, side effects of statin drugs such as myopathy, adverse liver reactions, gastrointestinal reactions and the like are continuously displayed, and in addition, the efficacy of statin drugs is short, and the statin drugs need to be taken for life and cannot be cured. These drawbacks have led to a gradual limitation of the use of statins. Monoclonal antibodies, protease inhibitors and the like also have potential hazards of affecting immune functions in patients, causing inflammatory storms and the like, and are expensive in cost and are not affordable to many patients.
In recent years, researchers have developed new treatments and means to avoid the disadvantages of both lipid lowering and anti-inflammatory treatments. Maintaining plaque stability, and even eliminating plaque, by modulating its microenvironment is a promising research effort. In the atherosclerotic microenvironment, macrophages continue to phagocytose oxLDL due to the constantly accumulating oxLDL barrier, causing macrophages to become foam cells, eventually apoptosis or necrosis, causing the necrotic core to continue to grow. Therefore, regulating macrophages, including inhibiting recruitment of monocytes and their differentiation into macrophages, inhibiting proliferation of macrophages, promoting apoptosis of macrophages, restoring phagocytic function of macrophages, etc., are becoming important targets for treating atherosclerosis. However, modulating macrophages alone does not solve the root cause of oxLDL accumulation. How to design and construct novel drug-loaded nano particles with plaque interior micro-environment remodelling function and improve the effect of treating atherosclerosis is a technical problem to be solved and a target to be realized in urgent need.
Disclosure of Invention
In order to solve the technical problem that the existing medicine has poor therapeutic effect on atherosclerosis, the invention provides application of medicine-carrying nano particles in preparing medicines for preventing or treating atherosclerosis, and the medicine-carrying nano particles can enter plaque macrophages through targeted delivery of nucleic acid medicines for silencing Camk2g gene expression, and the nucleic acid medicines silence Camk2g gene expression, restore phagocytic function of the macrophages and improve micro-environments in the plaque; at the same time, the nitric oxide donor may be delivered to macrophages and react with Inducible Nitric Oxide Synthase (iNOS) in the macrophages to produce nitric oxide. Nitric oxide gas molecules diffuse to the vicinity of damaged endothelial cells to repair the endothelial cell barrier, so that the combined therapy effect is achieved, and the high-efficiency treatment of atherosclerosis is realized.
The invention provides an application of drug-loaded nano-particles in preparing drugs for preventing or treating atherosclerosis, the drug-loaded nano-particles comprise:
(1) A polyamino acid carrier, on which a nitric oxide donor is grafted;
(2) A nucleic acid agent that silences Camk2g gene expression, encapsulated within the polyamino acid vector.
Further, the nitric oxide donor is selected from one or more of arginine, azodiol alkene salt and nitrosothiol.
Wherein the azodiol alkene salt and the isopentyl nitrite are known nitric oxide donors, such as any one defined in CN113244245A specification.
Further, the nucleic acid drug is siRNA, preferably, the nucleic acid drug is selected from one or more of siCamk g, siCCR, siEpsin1/2, siScr and siGFP. These nucleic acid drugs are all commercially available.
Further, the polyamino acid carrier has a structural formula shown in the following formula (I):
wherein m and n are number average polymerization degree, y is selected from integers of 2-16;
preferably, n is 10-500, m is 10-200, and n is not less than m.
Further, the preparation method of the polyamino acid carrier comprises the following steps:
(1) Reacting lysine with an amino acid protecting group with triphosgene or phosgene in the presence of a solvent to obtain L-lysine-N-carboxyl cyclic anhydride with the amino acid protecting group;
(2) Ring-opening polymerizing L-lysine-N-carboxyl cyclic anhydride with amino acid protecting group and monomer shown in formula (II) in the presence of solvent, and removing amino acid protecting group to obtain polylysine with alkyl chain;
(3) Arginine with an amino acid protecting group is used as a grafting monomer to react with an amidation catalyst in the presence of a solvent, the reaction product is subjected to graft polymerization reaction with polylysine with an alkyl chain in the presence of an acid binding agent, and then the amino acid protecting group is removed;
(4) Modifying the polymerization product with the amino acid protecting group removed, which is prepared in the step (3), by using small molecular anhydride shown in a formula (III) to prepare a polyamino acid carrier;
Wherein y is selected from an integer from 2 to 16, R 2、R3 is independently selected from H or C1-C6 alkyl;
Is that Or/>Preferably, the small molecule anhydride is selected from one or more of Maleic Anhydride (MA), succinic anhydride (SSA) or Dimethyl Maleic Anhydride (DMA).
Further, the small molecule anhydride is Dimethyl Maleic Anhydride (DMA); wherein n is 60-120, and the grafting rate of the arginine is more than 0 and less than or equal to 100 percent; preferably 25-100%, the modification ratio of the DMA is more than 0 and less than or equal to 100%; preferably 10-100%; more preferably, n is 90, the grafting ratio of the arginine is 50%, and the modification ratio of the DMA is 100%.
The grafting rate of the arginine is more than 0 and less than or equal to 100 percent, including but not limited to 10 percent, 20 percent, 30 percent, 50 percent, 80 percent and 100 percent. The modification proportion of the dimethyl maleic anhydride is more than or equal to 0 and less than or equal to 100 percent, including but not limited to 10 percent, 20 percent, 30 percent, 50 percent, 80 percent and 100 percent.
Further, the molar ratio between the nitrogen element in the polyamino acid carrier and the phosphorus element in the nucleic acid drug is 0.25-16:1 (preferably 4:1); and/or the average particle size of the polyamino acid carrier-based nucleic acid drug delivery system is 40-80nm (preferably 50-60 nm).
Further, the preparation method of the drug-loaded nanoparticle comprises the steps of mixing a polyamino acid carrier and a nucleic acid drug in the presence of an aqueous solution; preferably, the mass of the polyamino acid carrier and the molar ratio of the nucleic acid drug are 0.25 mug-0.03 mg:80-500pmol; preferably, the mixing time is 30s-5min.
Further, the aqueous solution is water, PBS solution, glucose solution or culture medium.
Further, the medicament also comprises other pharmaceutically acceptable carriers; preferably, the pharmaceutically acceptable auxiliary materials are selected from at least one of pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrating agents, permeation promoters, pH regulators, buffers, plasticizers, surfactants, thickeners, coating agents, moisturizers, absorbents, diluents, flocculating agents, deflocculants, filter aids, release retarders, polymeric matrix materials and film forming materials.
Further, the dosage form of the medicine is selected from injection, oral preparation or external preparation; preferably, the injection is selected from injection or powder injection; the oral preparation is selected from tablet, solution, capsule, powder, pill, granule, syrup, suspension or oral sustained and controlled release preparation; the external preparation is selected from ointment, spray or patch.
Further, the molecular weight of the polyamino acid carrier is 1500 to 8000g/mol, for example, the molecular weight of the polyamino acid carrier obtained in example 1 below is 32397g/mol.
Compared with the prior art, the technical scheme provided by the invention has the following advantages:
1. The invention provides an application of drug-loaded nano-particles in preparing drugs for preventing or treating atherosclerosis, wherein the drug-loaded nano-particles comprise: (1) A polyamino acid carrier, on which a nitric oxide donor is grafted; (2) A nucleic acid agent that silences Camk2g gene expression, encapsulated within the polyamino acid vector. The drug-loaded nanoparticle can restore phagocytic function of macrophages by targeted delivery of nucleic acid drugs silencing the expression of Camk2g genes into plaque macrophages, and the nucleic acid drugs silence the expression of Camk2g genes. Macrophages that restore phagocytic capacity can clear necrotic cores, thereby improving plaque internal microenvironment; at the same time, the nitric oxide donor may be delivered to macrophages and react with Inducible Nitric Oxide Synthase (iNOS) in the macrophages to produce nitric oxide. Nitric oxide gas molecules diffuse to the vicinity of damaged endothelial cells, repair the endothelial cell barrier, restore the integrity of the vascular endothelial barrier, play a role in combined therapy and realize the high-efficiency therapy of atherosclerosis.
2. The polyamino acid carrier has a structural formula shown in the following formula (I), and the polyamino acid carrier not only has higher drug carrying capacity, but also provides the capability of releasing nitric oxide in macrophages by taking arginine as a nitric oxide donor, so that the drug carrying nanoparticle has good capability of preventing or treating atherosclerosis.
3. Compared with other choices of the invention, when R 1 is selected from the group consisting of the application of the drug-loaded nano-particles in the preparation of drugs for preventing or treating atherosclerosis
When the small molecule anhydride is DMA, the transfection efficiency is obviously improved; the reason is that succinic anhydride lacks acid responsiveness, maleic anhydride has weaker acid responsiveness, dimethyl maleic anhydride has strongest acid responsiveness, and finally DMA modified PLL-Arg lysosome has strongest escape ability and optimal transfection performance.
4. The invention provides application of drug-loaded nano particles in preparation of drugs for preventing or treating atherosclerosis, and researches on the influence of polylysine number average polymerization degree, arginine grafting rate and dimethyl maleic anhydride modification ratio on polylysine gene carrier transfection efficiency show that the polylysine gene carrier transfection efficiency is optimal, wherein the L-lysine polymerization degree is 90, the arginine grafting rate is 50%, and the DMA modification ratio is 100%.
The average particle size of the polyamino acid carrier-based nucleic acid drug delivery system of the invention is 40-80nm (preferably 50-60 nm), significantly smaller than the particle size reported in the literature (> 100 nm).
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, in which:
FIG. 1 is a nuclear magnetic spectrum of polyamino acid carriers LAD with different DMA grafting ratios prepared in experimental example 1;
FIG. 2 is a graph showing particle size distribution of drug-loaded nanoparticles LAD@siCamk2g; the ordinate Concentration is: concentration;
FIG. 3 is a transmission electron microscope image of the drug-loaded nanoparticle LAD@siCamk2g;
FIG. 4 is an ultraviolet absorbance spectrum of polyamino acid carrier LAD, nucleic acid drug siCamk g and drug-loaded nanoparticle LAD@siCamk2g; ordinate absorpance: absorption degree; abscissa Wavelength: a wavelength;
FIG. 5 is a gel electrophoresis diagram of drug-loaded nanoparticles prepared by loading nucleic acid drugs with different N/P ratios based on polyamino acid carriers LAD with different DMA grafting ratios in experimental example 2;
FIG. 6 is a confocal imaging of nitric oxide production in macrophages by drug-loaded nanoparticles of Experimental example 3;
FIG. 7 shows the detection result of the Western-blot detection of the gene silencing efficiency of drug-loaded nanoparticles;
FIG. 8 shows the results of a test for the effect of drug-loaded nanoparticles on promoting endothelial cell migration;
FIG. 9 is a graph showing the results of detection of in vivo targeting effects of drug-loaded nanoparticles on atherosclerosis mice;
FIG. 10 shows the results of in vivo therapeutic efficacy detection of drug-loaded nanoparticles in atherosclerosis mice;
FIG. 11 is a test result of in situ editing of atherosclerotic mouse macrophages by drug-loaded nanoparticles;
FIG. 12 is a graph showing the results of detection of the repair effect of drug-loaded nanoparticles on the endothelial barrier in vivo in atherosclerotic mice; where ordinate INTENSITY OF EB IN AORTA in panel B is the intensity of EB in the aorta, p <0.05 is indicated.
Detailed Description
The following description of the embodiments of the present invention will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the invention are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The preparation method of the polyamino acid carrier provided by the invention comprises the following steps:
(1) Taking 0.5-20g of N epsilon-benzyloxycarbonyl-L-lysine, preferably 8g; dissolved in 4-100mL anhydrous tetrahydrofuran, preferably 40mL; followed by taking 0.4-15g, preferably 4g, of triphosgene; dissolved in 4-100mL anhydrous tetrahydrofuran, preferably 20mL; dropping at constant speed in a constant pressure dropping funnel, and condensing and refluxing at 50-60 ℃, preferably 50 ℃; after the reaction solution became clear and transparent, it was precipitated with n-hexane, and the resulting solid was recrystallized from THF/n-hexane=1:1-4, preferably 1:2; the obtained recrystallized solid was dried under reduced pressure at normal temperature to obtain N ε -benzyl-benzyloxycarbonyl-L-lysine- (N-carboxyanhydride) (i.e., ZLL (NCA)).
(2) Taking 1-100mg of hexadecylamine solid into a round bottom flask, preferably 7mg; subsequently 0.3-30g of ZLL (NCA) solids, preferably 1g, are added thereto; and 1-50mL of anhydrous DMF, preferably 5mL; reacting at 30-40 ℃ for 48-96h, preferably 37 ℃ for 72h; the reaction was then precipitated twice with glacial diethyl ether to give a solid which was dried overnight in vacuo to give hexadecylamine-PZLL 90. Subsequently, adding 1-50mL of trifluoroacetic acid to the hexadecylamine-PZLL 90 to dissolve the hexadecylamine-PZLL 90 sufficiently, preferably 5mL; 2-20mL of a 33% HBr/acetic acid solution, preferably 3mL, is then added thereto; hydrolyzing in ice water bath for 3-6h, preferably 4h; the resulting solution was centrifuged with ice-diethyl ether precipitate to give hexadecylamine-PLL 90 as a white solid which was dried overnight in vacuo.
(3) Synthesis of PLL 90 -Arg
Taking 0.2-40g of nα -Boc-nω - (2, 4,6, 7-pentamethyl-dihydrobenzofuran-5-sulfonyl) -L-arginine, preferably 1g;0.3-30g EDCI, preferably 0.75g; and from 0.2 to 20g of HOBT, preferably 0.5g; dissolved in 5-800mL DMF, preferably 20mL. Subsequently taking 0.1-20g of hexadecylamine-PLL 90, preferably 0.5g; and 0.2 to 20g of N, N-diisopropylethylamine, preferably 0.5g; dissolved in 4-400mL deionized water, preferably 10mL, and then added and maintained at room temperature for reaction for 12-96 hours, preferably 72 hours; the reacted solution is permeated with deionized water (dialysis bag cutoff molecular weight 3500 Da) for 24-96 hours, preferably 72 hours; lyophilization afforded a white solid. The white solid was then passed through trifluoroacetic acid to give PLL 90 -Arg.
(4) Synthesis of PLL 90 -Arg-DMA (abbreviated as LAD)
0.1-10G of PLL 90 -Arg, preferably 0.5g; dissolved in 2-200mL of NaOH at ph=8.0, preferably 10mL; subsequently, a DMA of greater than 0 and less than or equal to 30g, preferably 1.5g, is removed; dissolved in 1-20mL of 2,4 dioxane, preferably 5mL, and reacted for 4-6h, preferably 5h. Taking out dialysis (dialysis bag with molecular weight cut-off of 3500 Da) for 24-96 hours, preferably 72 hours; lyophilization afforded PLL 90 -Arg-DMA as a white solid (polyamino acid carrier).
By a similar method, maleic Anhydride (MA) or succinic anhydride (SSA) can be used instead of DMA to prepare maleic anhydride or succinic anhydride modified polymers PLL 90 -Arg-MA (abbreviated as LAM) and PLL 90 -Arg-SSA (abbreviated as LAS).
SiCamk2g of the invention was from Sharpo biotechnology Co., ltd; the sequences (5 '-3') are as follows: 5'-AAC GUG GUA CAU AAU GCU ACA-3'.
EXAMPLE 1 preparation of polyamino acid Carrier
The embodiment provides a preparation method of a polyamino acid carrier, which comprises the following steps:
(1) Synthesis of ZLL (NCA)
8G of N epsilon-benzyloxycarbonyl-L-lysine was dissolved in 40mL of anhydrous tetrahydrofuran to obtain a reaction base solution. Then 4g of triphosgene is taken and dissolved in 20mL of anhydrous tetrahydrofuran, a constant pressure dropping funnel is added into the reaction base solution at a constant speed, and the mixture is condensed and refluxed at 50 ℃; after the reaction solution became clear and transparent, it was precipitated with n-hexane, and the resulting solid was recrystallized in THF/n-hexane=1:2 (v/v) by filtration; the obtained recrystallized solid is dried under normal temperature and reduced pressure to obtain ZLL (NCA), which is fully known in Chinese: n epsilon-benzyl-benzyloxycarbonyl-L-lysine-N-carboxylmethylanhydride.
(2) Synthesis of hexadecylamine-PLL
Taking 7mg of hexadecylamine solid into a round-bottom flask; subsequently, 1g of ZLL (NCA) solid and 5mL of anhydrous DMF were added thereto and reacted at 37℃for 72 hours; the reaction was then precipitated twice with glacial diethyl ether to give a solid which was dried overnight in vacuo to give hexadecylamine-PLL 90. The resulting hexadecylamine-PLL 90 was then taken and 5mL of trifluoroacetic acid was added thereto for sufficient dissolution. Then 3mL of 33% HBr/acetic acid solution is added, and the mixture is put into ice water bath for hydrolysis for 4 hours; the resulting solution was centrifuged with ice-diethyl ether precipitate to give hexadecylamine-PLL 90 as a white solid which was dried overnight in vacuo.
(3) Synthesis of PLL 90 -Arg
1G of N.alpha. -Boc-N.omega- (2, 4,6, 7-pentamethyl-dihydrobenzofuran-5-sulfonyl) -L-arginine, 0.75g of EDCI (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride) and 0.5g of HOBT (1-hydroxybenzotriazole) were dissolved in 20mL of DMF and reacted at room temperature for 30 minutes to obtain a reaction solution. Subsequently, 0.5g of hexadecylamine-PLL 90 and 0.5g of n, n-diisopropylethylamine were dissolved in 10mL of deionized water, followed by addition to the above reaction solution and reaction was carried out at room temperature for 72 hours. Dialyzing the reacted solution with deionized water (dialysis bag with molecular weight cutoff of 3500 Da) for 72h; lyophilization afforded a white solid. The white solid was then added to 6mL of trifluoroacetic acid; stirring is carried out at room temperature for 4h. Then the mixture is subjected to sedimentation and centrifugation by using anhydrous diethyl ether, the obtained solid is redissolved by using DMSO, and dialysis (dialysis bag cut-off molecular weight 3500 Da) is carried out for 72 hours; then 2mL of mercaptoethanol was added thereto; stirring overnight at room temperature, and dialyzing (dialysis bag cut-off molecular weight 3500 Da) for 72h; followed by lyophilization gives PLL 90 -Arg.
(4) Synthesis of PLL 90 -Arg-DMA (abbreviated as LAD)
0.5G of PLL 90 -Arg was dissolved in 10mL of NaOH solution having ph=8.0 to obtain a reaction solution, and then 1.5g of Dimethyl Maleic Anhydride (DMA) was taken out and dissolved in 5mL of 2,4 dioxane, and the reaction solution was added thereto to react for 5 hours. Taking out for dialysis (dialysis bag molecular weight cut-off 3500 Da) for 72h; lyophilization yielded PLL 90 -Arg-DMA as a white solid (polyamino acid carrier, abbreviated as "LAD carrier").
EXAMPLE 2 preparation of polyamino acid Carrier
This example provides a method for preparing a polyamino acid carrier, which is substantially the same as that of example 1, except that 1.5g of Maleic Anhydride (MA) is used instead of Dimethyl Maleic Anhydride (DMA) in step (4), and finally PLL 90 -Arg-MA is prepared as a white solid (abbreviated as LAM carrier).
EXAMPLE 3 preparation of polyamino acid Carrier
This example provides a method for preparing a polyamino acid carrier, which is substantially the same as that of example 1, except that 1.5g of succinic anhydride (SSA) is used instead of Dimethyl Maleic Anhydride (DMA) in step (4), and finally PLL 90 -Arg-SSA white solid (abbreviated as LAS carrier) is prepared.
EXAMPLE 4 preparation of drug-loaded nanoparticles
The embodiment provides a preparation method of drug-loaded nano particles, which comprises the following steps:
The LAD carrier prepared in example 1 was dissolved in water to prepare a dispersion having a LAD concentration of 1 mg/mL. The 20. Mu.L of LAD carrier dispersion was added with 460pmol of siCamk g of nucleic acid drug and mixed for 1min to obtain drug-loaded nanoparticles, which were then diluted into 1mL of water to prepare a suspension containing drug-loaded nanoparticles LAD@siCamk2g (hereinafter referred to as "LAD@SiCamk2g").
The particle size distribution and morphology of the drug-loaded nanoparticles LAD@siCamK2g were characterized by a nanofluidic instrument and a transmission electron microscope, as shown in FIGS. 2-3. The result shows that the average particle diameter of the drug-loaded nano particles is 54nm, and the PDI is 0.12.
And respectively detecting ultraviolet spectrums of the polyamino acid carrier, the nucleic acid drug and the drug-carrying nano particles by an ultraviolet-visible light spectrophotometer. As can be seen from FIG. 4, there is a characteristic absorption of both LAD vector alone and siCamk g nucleic acid drug alone in the UV spectrum of LAD@siCamk2g. Indicating that the drug-loaded nanoparticle can successfully load nucleic acid drugs.
EXAMPLE 5 preparation of drug-loaded nanoparticles
This example provides a method for preparing drug-loaded nanoparticles, which is substantially the same as example 4, except that the LAM carrier of example 2 is used instead of the polyamino acid carrier prepared in example 1, to prepare a suspension of drug-loaded nanoparticles lam@sicam k2g (hereinafter referred to as "lam@sicam k2 g").
EXAMPLE 6 preparation of drug-loaded nanoparticles
This example provides a method for preparing drug-loaded nanoparticles, which is substantially the same as example 4, except that the LAS vector of example 3 is used instead of the polyamino acid vector prepared in example 1 to prepare a suspension of drug-loaded nanoparticles and drug-loaded nanoparticles LAS@siCamk2g (hereinafter simply referred to as "LAS@SiCamk2g").
Comparative example 1
SiCamk2g was entrapped in a commercial liposome carrier (specific name: lipofectamine2000, available from Thermo Fisher Co., ltd.; cat. 11668019) by: 8. Mu.L of the liposome was mixed with 250. Mu.L of serum-free medium for 5min to prepare a dispersion of the liposome carrier. 460pmol of nucleic acid drug siCamk g was mixed with 250. Mu.L of serum-free medium for 5min to prepare siCamk g of dispersion of nucleic acid drug. The dispersion of the liposome carrier was mixed with a dispersion of siCamk g of a nucleic acid drug for 10 minutes, and then diluted into 1mL of water to prepare a suspension containing drug-loaded liposome particles (hereinafter referred to as "lipo@sicamk2g").
The particle size distribution of Lipo@siCamk2g was measured by a particle sizer, and the result showed that the average particle size of Lipo@siCamk2g was 110nm and PDI was 0.24.
Experimental example 1 Synthesis and characterization of polyamino acid Carrier with different grafting ratios
1. Preparation of polyamino acid vectors
PLL 90 -Arg was prepared according to the method of example 1. DMA was not grafted to the polyamino acid support, which was designated LAD 0.
0.5G of PLL 90 -Arg was dissolved in 10mL of NaOH solution having ph=8.0 to obtain a reaction solution.
0.1G, 0.2g, 0.5g and 1.5g of Dimethyl Maleic Anhydride (DMA) were taken out and dissolved in 5mL of 2,4 dioxane, and the solution was added to the reaction solution to react for 5 hours. Taking out for dialysis (dialysis bag molecular weight cut-off 3500 Da) for 72h; and freeze-drying to obtain LAD carriers with different grafting ratios (which are named as LAD 10、LAD20、LAD50、LAD100 from low to high).
2. Characterization of
The synthesized polyamino acid carrier is characterized by adopting a nuclear magnetic resonance spectrum, the result is shown in fig. 1, and compared with the polyamino acid carrier LAD 0,LAD10、LAD20、LAD50、LAD100, the peak area of the synthesized polyamino acid carrier LAD 0,LAD10、LAD20、LAD50、LAD100 in chemical shift of 1.6-2.0ppm is gradually increased as shown in fig. 1. The characteristic peak with chemical shift of 1.6-2.0ppm is the characteristic peak of dimethyl maleic acid group in the polyamino acid carrier, and is marked as a peak.
The modification ratio of DMA in each group of polyamino acid carriers was calculated as follows, modification ratio = 1/6 (integrated area of a peak-area of a 0 peak)/integrated area of peak at chemical shift of 4.2ppm x 100%, wherein: the area of the a 0 peak is the integrated area of the LAD 0 at the a peak. The modification ratios of the polyamino acid vector LAD 0、LAD10、LAD20、LAD50、LAD100 were calculated to be 0, 10, 20, 50 and 100%, respectively. It was confirmed that polyamino acid vectors of different modification ratios were successfully synthesized.
Experimental example 2 nucleic acid Loading Capacity of polyamino acid vectors with different grafting ratios
1. Preparation of drug-loaded nanoparticles
The method comprises the steps of (1) respectively mixing a polyamino acid carrier and a nucleic acid medicine according to the molar ratio of nitrogen element in the polyamino acid carrier to phosphorus element in the nucleic acid medicine (nitrogen-phosphorus ratio, namely N/P) of 0:1 and 0.5:1;1:1,2:1,4:1,8:1 and 16:1 (specifically, LAD 0、LAD10、LAD20、LAD50、LAD100 carriers prepared in experimental example 1 are taken as LAD carriers, and 0g, 0.5 mug, 0.9 mug, 1.9 mug, 3.7 mug, 7.4 mug, 14.8 mug and 92pmol of nucleic acid drug siCamk g are mixed for 1 min) to obtain drug-loaded nano particles with different N/P.
2. Preparation of agarose gel
2G of agarose was dissolved in 100mL of a1 xTAE solution; then placing the mixture into a microwave oven, and heating the mixture for 1min by medium and high fire; it was completely dissolved, then after it was cooled slightly, nucleic acid dye (10204 ES76, division of Saint Biotechnology (Shanghai)) was added to 1/10000 of the total volume of the solution, and then poured into a mold, and after it was completely cooled to be molded.
3. Electrophoresis
And adding 5 mu L of loading buffer solution into the prepared drug-loaded nano particles with different N/P, and mixing for 1min to obtain a loading system.
Adding 2L into the electrophoresis tank; 1 xTAE solution, immersing gel in the TAE solution, adding the prepared sample system into a gel hole, starting electrophoresis, and selecting 140V voltage; electrophoresis time was selected to be 30min. After electrophoresis, the sample was placed in an imager for imaging, and the result is shown in FIG. 5.
As can be seen from FIG. 5, when N/P is 4:1, the electronegativity of the nucleic acid drug is weakened due to the complete binding with the polyamino acid carrier, and the nucleic acid drug cannot move downwards; the polyamino acid carrier can be used for efficiently loading nucleic acid medicines, so that 100% full loading of the nucleic acid medicines is realized.
Experimental example 3 evaluation of nitric oxide production efficiency of drug-loaded nanoparticles in macrophages
1. Preparation of test drugs
The LAD vector prepared in example 1, the LAM vector prepared in example 2, and the LAS vector prepared in example 3 were assembled with control siRNA (abbreviated as "scr" from Sharp Biotech Inc. of Guangzhou, sequence (5 '-3') as follows: 5'-UUGGGAAAAAGUUGAGUGGUU-3') to form a polyamino acid vector-based nucleic acid drug delivery system LAD@scr, LAM@scr, LAS@scr, respectively, as follows. The preparation method comprises the following steps: the polyamino acid carrier is dissolved in water to prepare an aqueous polyamino acid carrier solution with the concentration of 1 mg/mL. mu.L of the aqueous polyamino acid carrier solution was added to 460pmol of scr and mixed for 1min to obtain the polyamino acid carrier-based nucleic acid drug delivery systems LAD@scr, LAM@scr and LAS@scr, respectively.
2. Cell culture and administration
Raw 264.7 cells were seeded in 8-well cell culture chambers at a density of 5X 10 4 cells/well; after cell attachment, the cells were resuspended in cell culture medium and the cell suspensions were randomly divided into 6 groups, respectively PBS, LPS, LPS+Arg, LPS+LAS@scr, LPS+LAM@scr and LPS+LAD@scr, each group being 3 wells in parallel and 200. Mu.L per well.
PBS group: adding 2. Mu.L of PBS buffer to the cells;
LPS group: 0.2. Mu.L of LPS (lipopolysaccharide) at 1mg/mL was added to the cells;
Lps+arg group: mu.L of arginine solution (solvent: PBS, concentration 0.7 mg/mL) was added to the cells, followed by 1mg/mL of LPS, 0.2. Mu.L;
LPS+LAS@scr group: mu.L of a nucleic acid drug delivery system based on a polyamino acid carrier LAS@scr was added to the cells followed by 1mg/mL of LPS 0.2. Mu.L.
LPS+LAM@scr group: mu.L of the polyamino acid vector-based nucleic acid drug delivery system LAM@scr was added to the cells followed by 1mg/mL LPS 0.2. Mu.L.
Lps+lad@scr group: mu.L of the polyamino acid vector-based nucleic acid drug delivery system LAD@scr was added to the cells, followed by 1mg/mL LPS 0.2. Mu.L.
3. Detection of nitric oxide production efficiency in macrophages of nucleic acid drug delivery systems based on polyamino acid vectors
Each of the above groups was incubated at 37℃for 24 hours, 2. Mu.L DAF-FM DA (green fluorescence, nitric oxide indicator, beyotime, S0019S) was added to each well, incubated at 37℃for 0.5 hours, washed with PBS, stained with 1. Mu.g DAPI (nuclear dye) for 10 minutes, washed with PBS, and then observed by confocal microscopy.
4. Test results
As shown in FIG. 6, the fluorescence intensity of the PBS group was (1.52.+ -. 0.15). Times.10 3, the fluorescence intensity of the LPS group was (1.47.+ -. 0.14). Times.10 3, the fluorescence intensity of the LPS+Arg group was (6.01.+ -. 0.54). Times.10 3, the fluorescence intensity of the LPS+LAS@scr group was (1.53.+ -. 0.01). Times.10 3, the fluorescence intensity of the LPS+LAM@scr group was (2.5.+ -. 0.58). Times.10 3, and the fluorescence intensity of the LPS+LAD@scr group was (6.16.+ -. 1.35). Times.10 3), which significantly improved (p < 0.0001) compared with the fluorescence intensity of the PBS group and the LPS group; compared with the LPS group, the fluorescent intensity of the LPS+LAM@scr group and the LPS+LAS@scr group is slightly improved, but no significant difference exists. From the results, the LAD@scr treatment group was found to react more effectively with Inducible Nitric Oxide Synthase (iNOS) in macrophages to produce nitric oxide.
Experimental example 4 Gene silencing efficiency
1. Cell culture and administration
Inoculating 7×10 5 Raw 264.7 cells in a cell 6 well plate; after cell attachment, the complete medium was changed to serum-free medium to resuspend cells, and the cell suspension was randomly divided into two groups of 3 wells in parallel with 2mL per well. 40. Mu.L of LAD@siCamk2g and Lipo@siCamk2g prepared in example 4 and comparative example 1 were added to each of the two groups, incubated at 37℃for 8 hours, the supernatant was removed after incubation and replaced with 2 mL/well of complete medium, and the culture was continued for 40 hours at 37℃and the medium was discarded, and the cells were collected.
2. Western-blot experimental detection
A proper amount of lysate is added into the hole, and the cells are blown down by a gun for several times. Centrifuge at 12000rpm at 4℃for 10min. After centrifugation, the protein was aspirated, placed in an EP tube, quantified with BCA kit (protein loading 30. Mu.g), and Loading buffer (loading buffer) was added, and the protein was denatured by heating at 100℃for 10min in a metal bath, and further DNA bound to the protein was isolated and degraded, and should not be sticky after boiling.
Preparing 8% polyacrylamide gel, placing the gel into an established electrophoresis tank, sequentially adding samples into gel holes, pouring electrophoresis liquid, and starting electrophoresis. 80V constant pressure, electrophoresis for 30min, then 120V constant pressure, electrophoresis for 1.5h. And taking out the gel after electrophoresis, putting the gel into a built film transfer system for film transfer, and starting film transfer. Constant current is 200mA, and film transferring is carried out for 40min. After the transfer, the membrane is closed by using 5% of skimmed milk by mass fraction for 2 hours. After blocking, washing with TBST was performed, and after washing, the primary antibodies to GAPDH and CaMKII gamma protein were added and incubated overnight at 4 ℃. After the primary antibody incubation is finished, the primary antibody is washed by TBST, and after the washing is finished, the secondary antibody is added, and the primary antibody is incubated for 2 hours at room temperature. After the secondary antibody incubation is completed, development is performed with a developer.
3. Test results
The results are shown in FIG. 7. From the results, it can be seen that both lipo@siCamk2g and LAD@siCamk2g drug delivery systems can efficiently silence CaMKII gamma protein expression in macrophages.
Experimental example 5 cell migration
1. Test method
Positioning the plate with a Mark pen line on the back of the plate, and inoculating 6×10 5 HUVEC cells per well; inoculating 1×10 5 cells/well Raw 264.7 cells; after cell attachment, the wells were scored with 200 μl of pipette tips, the dropped cells were washed clean with PBS, replaced with fresh complete medium to resuspended cells, and the cell suspensions were randomly divided into PBS, LPS, lps+arg, and lps+lad@sicamk2g groups, 3 wells in parallel, and 2mL per well.
PBS group: adding 20. Mu.L of PBS buffer to the cells;
LPS group: adding 1mg/mL LPS 2. Mu.L to the cells;
Lps+arg group: mu.L of arginine solution (solvent: PBS, concentration 0.7 mg/mL) was added to the cells, followed by 1mg/mL of LPS 2. Mu.L;
LPS+LAD@siCamk2g group: 40. Mu.L of LAD@siCamk2g prepared in example 4 was added to the cells followed by 1mg/mL of LPS 2. Mu.L.
Each of the above groups was incubated at 37 ℃ for 24 hours during which time scratch photographs were taken of the same location at time points of 0h,8h,24 h.
2. Test results
As a result, as shown in FIG. 8, the cell migration area of the PBS group was (6.7.+ -. 0.39). Times.10 4 pixels (pixels), the cell migration area of the LPS group was (8.42.+ -. 0.33). Times.10 4 pixels, the cell migration area of the LPS+Arg group was (11.45.+ -. 1.35). Times.10 4 pixels, and the cell migration area of the LPS+LAD@siCamk2g group was (12.36.+ -. 1.85). Times.10 4 pixels. P <0.0001 between PBS group and lps+lad@sicamk2g group. From the results, it can be seen that the migration area of cells in the lps+arg group and the lps+lad@sicamk2g group was significantly increased (p < 0.0001) compared to the PBS group, indicating that the cells of both groups were significantly migrated. The drug-loaded nano particles can effectively generate nitric oxide gas in cells to promote cell migration.
Experimental example 6 investigation of the Targeted accumulation Capacity of drug-loaded nanoparticles in mice
1. Preparation of the sample to be tested
① The polyamino acid carrier prepared in example 1 was dissolved in water to prepare an aqueous polyamino acid carrier solution having a polyamino acid carrier concentration of 1mg/mL, and 62.5. Mu.L of the aqueous polyamino acid carrier solution was mixed with 62.5. Mu.L of Cyanine 5-labeled siCamk g (available from Sharpbo Biotechnology Co., ltd. In Guangzhou, solvent: DEPC water, molar concentration of 230 pmol/. Mu.L) for 30s-2min (1 min in this experimental example), to obtain a Cyanine 5-labeled LAD@SiCamk2g.
② 31.25. Mu.L of liposome (specific name: lipofectamine2000, available from Thermo Fisher Co., ltd., model Lipo 2000) was incubated with 31.25. Mu.L of PBS solution at normal temperature for 5 minutes, and then mixed with 62.5. Mu.L of Cyanine 5-labeled siCamk g (available from Ruibo Biotechnology Co., ltd., guangzhou, solvent: DEPC water, molar concentration: 230 pmol/. Mu.L) for 10 minutes to obtain Cyanine 5-labeled Lipo@siCamk2g.
2. Grouping, administration and testing of animals
6 Apoe -/- mice (model group) at 12 weeks of age. 3 wild type C57BL/6J mice at 9 weeks of age were used as blank treatment groups (WT). Model groups were randomly divided into two groups of 3 models each, two model groups: 125. Mu.L of Cyanine 5-labeled LAD@siCamk2g and Cyanine 5-labeled lipo@siCamk2g were injected into mice via tail vein, respectively. Blank treatment group: 125. Mu.L of Cyanine 5-labeled LAD@siCamk2g was injected into mice via the tail vein, and the three groups were kept for 24h. The mouse aorta was removed and imaged by a small animal biopsy imager.
3. Test results
As a result, as shown in FIG. 9, the fluorescence intensity of the WT group was (1.21.+ -. 0.86). Times.10 6, the fluorescence intensity of the lipo@siCamk2g group was (7.63.+ -. 3.21). Times.10 6, and the fluorescence intensity of the LAD@siCamk2g group was (18.07.+ -. 4.88). Times.10 6. P <0.01 between the lipo@siCamk2g group and the LAD@siCamk2g group. Compared with the WT group and the model treatment group injected with Lipo@siCamk2g, the LAD@SiCamk2g provided by the invention has significantly improved fluorescence intensity (p < 0.01) of the aorta of the mice after being injected into the model group, which proves that the drug-loaded nano particles provided by the invention can be accumulated at focal plaques more accurately without affecting normal tissue cells, and have excellent targeting ability on atherosclerosis plaques.
Experimental example 7 investigation of therapeutic Effect of drug-loaded nanoparticles on atherosclerosis mice
1. Grouping and administration of animals
9 Apoe -/- mice (model group) aged 12 weeks. Model groups were randomly divided into three groups of 3 each: PBS group, LAD@siCamk2g treatment group and Lipo@siCamk2g treatment group.
Lad@sicamk2g treatment group: 125 μl of the LAD@siCamk2g prepared in example 4 was injected into mice via tail vein. lipo@siCamk2g treatment group: 125. Mu.L of Lipo@siCamk2g prepared in comparative example 1 was injected into mice via tail vein. Injections were given 2 times per week for a total of 4 weeks. PBS groups were injected in parallel with equal volumes of PBS solution.
2. Test method
After the treatment, the aorta of each group of mice was removed, immersed 2 times in PBS, and the blood vessel was carefully dissected longitudinally along the wall of the blood vessel with dissecting scissors. Immersing the dissected blood vessel into oil red O dye solution for dyeing at 37 ℃ for 60min; taking out, differentiating with 75% ethanol until the fat plaque in the lumen turns to orange or bright red, and other parts are nearly colorless, and washing with distilled water for 2 times. The staining results were photographed with a camera.
3. Test results
As a result, as shown in FIG. 10, the percentage of the total area of the oil red O area of the PBS group was 61.79.+ -. 18.73%, the percentage of the total area of the oil red O area of the Lipo@siCamk2g group was 27.73.+ -. 11.46%, and the percentage of the total area of the oil red O area of the LAD@siCamk2g group was 8.47.+ -. 3.17%. P <0.05 between PBS group and lad@sicamk2g group. Compared with the PBS group and the Lipo@siCamk2g treatment group, the LAD@siCamk2g treatment group has significantly smaller area (red area) of fat plaque in the lumen (p < 0.05) by injecting the LAD@siCamk2g treatment group, and the treatment effect is significantly higher than that of the other two groups, and shows that the in-situ gene editing macrophage combined with nitric oxide gas therapy can realize good treatment effect of atherosclerosis.
Experimental example 8 in-situ editing Effect of drug-loaded nanoparticles on macrophages in vivo
1. Grouping and administration of animals
9 Apoe -/- mice (model group) aged 12 weeks. Model groups were randomly divided into three groups of 3 each: PBS group, LAD@siCamk2g treatment group and Lipo@siCamk2g treatment group.
Lad@sicamk2g treatment group: 125 μl of the LAD@siCamk2g prepared in example 4 was injected into mice via tail vein. lipo@siCamk2g treatment group: 125. Mu.L of Lipo@siCamk2g prepared in comparative example 1 was injected into mice via tail vein. Injections were given 2 times per week for a total of 4 weeks. The PBS group was injected in parallel with the same volume of PBS solution.
2. Test method
After the end of the treatment, the aorta of each group of mice was removed, embedded with OTC, and then frozen into sections. Frozen sections were subjected to TUNEL (red) and F8/40 (green) immunofluorescent staining. The staining results were observed with a slice scanner. The fluorescence staining imaging results are shown in fig. 11. White arrows indicate co-localization of macrophages (green) with apoptotic cells (red).
3. Test results
As a result, as shown in FIG. 11, the co-localized fluorescence intensity of the PBS group was (3.95.+ -. 0.68). Times.10 5, the co-localized fluorescence intensity of the Lipo@siCamk2g group was (6.44.+ -. 0.7). Times.10 5, and the co-localized fluorescence intensity of the LAD@siCamk2g group was (14.65.+ -. 2.85). Times.10 5. P <0.001 between the lipo@siCamk2g group and the LAD@siCamk2g group. The degree of co-localization of macrophages (green) with apoptotic cells (red) was significantly higher (p < 0.001) in the lad@sicamk2g treated group by injection of lad@sicamk2g in vivo compared to the PBS group and the lipo@sicamk2g treated group. The method shows that the macrophage can be remarkably genetically modified, the phagocytic capacity of the macrophage is recovered, apoptotic cells are cleared, and the treatment effect of the macrophage is obviously higher than that of other two groups.
Experimental example 9 investigation of the repair Effect of drug-loaded nanoparticles on endothelial Barrier in vivo
Evan Blue (EB) dye is commonly used for vascular endothelial cell barrier permeability testing. Under physiological conditions, the endothelium is impermeable to albumin; whereas under pathological conditions, dysfunction of endothelial intercellular junction proteins leads to an increase in barrier permeability, at which time leakage of evans blue-albumin can be detected in the monolayer endothelial cell model.
1. Grouping and administration of animals
9 Apoe -/- mice (model group) aged 12 weeks. Model groups were randomly divided into three groups of 3 each: PBS group, LAD@siCamk2g treatment group and Lipo@siCamk2g treatment group.
Lad@sicamk2g treatment group: 125 μl of the LAD@siCamk2g prepared in example 4 was injected into mice via tail vein. lipo@siCamk2g treatment group: 125. Mu.L of Lipo@siCamk2g prepared in comparative example 1 was injected into mice via tail vein. Injections were given 2 times per week for a total of 4 weeks. The PBS group was injected in parallel with the same volume of PBS solution.
2. Test method
After the end of treatment, 2% of evans blue dye was injected into each group of mice via the tail vein. After 30min of injection, perfusion was performed with 4% paraformaldehyde solution, and then the aorta was harvested and opened longitudinally for photography and staining intensity statistics. The heart was then embedded with OTC and the aortic root sections were cut using the frozen section technique to observe the staining.
3. Test results
As a result, as shown in FIG. 12, the blood vessels of the PBS-treated atherosclerosis model mice were significantly colored by Evan blue, the Lipo@siCamk2g treatment group was shallowly colored, and the LAD@siCamk2g treatment group was minimally colored. Therefore, compared with lipo@siCamk2g, namely the group without nitric oxide gas treatment, the LAD@siCamk2g group can obviously promote proliferation and migration of vascular endothelial cells through gene editing and nitric oxide gas treatment, and finally realizes repair of vascular endothelial barriers.
Experimental example 10 reprogrammed macrophage gene silencing efficacy test
(1) Test method
Samples of different DMA modification rates and L-lysine grafting rates and L-lysine polymerization degrees were prepared.
Samples 1-3: the polyamino acid vectors corresponding to examples 1-3, respectively, are shown in Table 2.
Sample 4-5: the preparation was substantially the same as in example 1 except that the amount of Dimethyl Maleic Anhydride (DMA) used in step (4) was adjusted from 1.5g to 0.1g, 0.2g and 0.5g in this order.
Comparative samples 2-5: the preparation was carried out in substantially the same manner as in example 1 except that step (4) was not carried out, and in step (3), the amount of nα -Boc-nω - (2, 4,6, 7-pentamethyl-dihydrobenzofuran-5-sulfonyl) -L-arginine was sequentially adjusted from 1g to 0.5g, unregulated, 1.4g and 2g.
Comparative samples 6-8: the preparation was essentially the same as in example 1, except that steps (3) and (4) were not carried out, and the amount of ZLL (NCA) solids in step (2) was adjusted from 1g to 0.67g, unregulated, 1.34g in this order.
The number average polymerization degree, the arginine grafting ratio and the DMA modification ratio of the polyamino acid carrier sample are shown in Table 1, and in the invention, the grafting ratio or the modification ratio of DMA is tested and calculated according to the item (1) of experimental example 1; the grafting ratio of arginine was determined by nuclear magnetic resonance hydrogen spectrometry, and the number average polymerization degree was determined by nuclear magnetic resonance hydrogen spectrometry.
① Preparing a sample to be tested: and respectively dissolving each group of polyamino acid carriers into PBS solution to prepare 1mg/mL of polyamino acid carrier PBS solution. mu.L of 1mg/mL of a PBS solution of the polyamino acid carrier is taken, 460pmol of siRNA (i.e. siCamk g) of the nucleic acid drug is added, and the mixture is mixed for 1min to obtain a nucleic acid drug delivery system based on the polyamino acid carrier.
② Cell treatment: inoculating Raw 264.7 cells growing in logarithmic phase into a cell 6 pore plate according to the density of 7×10 5 cells/pore, and standing for cell attachment; the complete medium was replaced with serum-free medium to resuspend cells, and the cell suspensions were randomly divided into 14 groups, test groups 1-13 and PBS groups, respectively, with 3 wells in parallel and 2mL of cell suspension added per well. Test groups 1-13 respectively adding 40 mu L of each group of samples to be tested into the cell suspension, and adding the PBS group into the PBS solution with the same volume; incubate for 8h. Removing the supernatant after incubation, changing the supernatant into a complete culture medium, and continuing to culture for 16 hours; the cells were collected.
③ And (3) testing: culture medium was removed before collection of cultured adherent cells, cells were lysed by adding TRIzol 1mL, repeatedly blown with a pipette until no significant precipitation was observed in the lysate, and placed on ice for 5min. To 1mL of the sample was added 200. Mu.L of chloroform; shaking for 15s, and standing on ice for 3min. The sample was carefully removed and placed on a test tube rack by centrifugation at 12000g for 15min at 4 ℃. Taking 500 mu L of supernatant into a new centrifuge tube, taking care that marks are made on a tube cover, and adding 500 mu L of isopropanol; gently mixing, and standing on ice for 10min; centrifuging at 12000g for 10min at 4 ℃; slowly discarding the supernatant, and adding 1mL of 75% ethanol; gently flick the pellet by hand, centrifuge for 5min at 7500g at 4deg.C, slowly discard supernatant; then 7500g is centrifuged for 30s; sucking residual liquid as much as possible; the centrifuge tube was inverted over a clean PE glove and cooled until the pellet edge became clear. Adding 40 mu L of RNase-Free H 2 O; standing at room temperature for 10min; dissolving RNA precipitate, and repeatedly blowing with a pipetting gun for several times to mix uniformly; the quality of the extracted RNA was checked by agarose gel electrophoresis, and three ribosomal RNA bands (28S, 18S, 5S) were used to evaluate the RNA quality. 1.5. Mu.L of total RNA was aspirated and the RNA concentration of the sample was determined with a micro-spectrophotometer. Then marking the tube wall and the cover, and placing the tube wall and the cover at-80 ℃ for freezing storage. Reverse transcription was performed according to the procedure of RT-qPCR kit to synthesize cDNA. And finally, performing qPCR experiments according to qPCR kit operation to detect the content of target mRNA in the cells. The delta CT method is used for calculating the silencing efficiency of siRNA, and the calculation formula is as follows:
ΔCT Sample of =CT Target gene -CT Reference gene
ΔCT Control =CT Target gene -CT Reference gene
ΔΔCT=ΔCT Sample of -ΔCT Control
Target gene expression = 2- ΔΔct
(2) Test results
The results are shown in Table 1 below:
TABLE 1 Effect of different lysine polymerization degree, arginine grafting ratio, DMA modification ratio on siRNA silencing Effect results
/>
As shown in the table above, compared with the method without grafting DMA, the silencing efficiency of the prepared polyamino acid carrier by grafting DMA is obviously improved, especially the polymerization degree of L-lysine is 90, the grafting rate of arginine is 50%, and the modification proportion of DMA is 100%.
TABLE 2 Effect of different small molecule anhydrides on siRNA silencing efficacy results
As can be seen from the above table, compared with other small molecule anhydrides grafted, the preferred technical scheme of the invention significantly improves the silencing efficiency of the polyamino acid carrier prepared by grafting DMA.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (10)
1. Use of a drug-loaded nanoparticle for the preparation of a medicament for the prevention or treatment of atherosclerosis, the drug-loaded nanoparticle comprising:
(1) A polyamino acid carrier, on which a nitric oxide donor is grafted;
(2) A nucleic acid agent that silences Camk2g gene expression, encapsulated within the polyamino acid vector.
2. The use according to claim 1, wherein the nitric oxide donor is selected from one or more of arginine, azodiol alkene salts, nitrosothiols.
3. The use according to claim 1, wherein the nucleic acid agent is an siRNA, preferably the nucleic acid agent is selected from one or more of siCamk g, siCCR2, siEpsin1/2, siscr, sifp.
4. Use according to any one of claims 1 to 3, wherein the polyamino acid carrier has the structural formula (I):
wherein m and n are number average polymerization degree, y is selected from integers of 2-16;
preferably, n is 10-500, m is 10-200, and n is not less than m.
5. The use according to claim 4, wherein the preparation method of the polyamino acid carrier comprises the following steps:
(1) Reacting lysine with an amino acid protecting group with triphosgene or phosgene in the presence of a solvent to obtain L-lysine-N-carboxyl cyclic anhydride with the amino acid protecting group;
(2) Ring-opening polymerizing L-lysine-N-carboxyl cyclic anhydride with amino acid protecting group and monomer shown in formula (II) in the presence of solvent, and removing amino acid protecting group to obtain polylysine with alkyl chain;
(3) Arginine with an amino acid protecting group is used as a grafting monomer to react with an amidation catalyst in the presence of a solvent, the reaction product is subjected to graft polymerization reaction with polylysine with an alkyl chain in the presence of an acid binding agent, and then the amino acid protecting group is removed;
(4) Modifying the polymerization product with the amino acid protecting group removed, which is prepared in the step (3), by using small molecular anhydride shown in a formula (III) to prepare a polyamino acid carrier;
Wherein y is selected from an integer from 2 to 16, R 2、R3 is independently selected from H or C1-C6 alkyl;
Is that Or/>Preferably, the small molecule anhydride is selected from one or more of Maleic Anhydride (MA), succinic anhydride (SSA) or Dimethyl Maleic Anhydride (DMA).
6. The use according to claim 5, wherein the small molecule anhydride is Dimethyl Maleic Anhydride (DMA); wherein n is 60-120, and the grafting rate of the arginine is more than 0 and less than or equal to 100 percent; preferably 25-100%, the modification ratio of the DMA is more than 0 and less than or equal to 100%; preferably 10-100%; more preferably, n is 90, the grafting ratio of the arginine is 50%, and the modification ratio of the DMA is 100%.
7. The use according to any one of claims 1 to 6, wherein the molar ratio between the nitrogen element in the polyamino acid carrier and the phosphorus element in the nucleic acid drug is 0.25 to 16:1, a step of; and/or the average particle size of the nucleic acid drug delivery system based on the polyamino acid carrier is 40-80nm.
8. The use according to any one of claims 1 to 7, wherein the method of preparation of the drug-loaded nanoparticle comprises mixing a polyamino acid carrier and a nucleic acid drug in the presence of an aqueous solution; preferably, the mass of the polyamino acid carrier and the molar ratio of the nucleic acid drug are 0.25 mug-0.03 mg:80-500pmol; preferably, the mixing time is 30s-5min.
9. The use according to any one of claims 1 to 8, wherein the medicament further comprises a further pharmaceutically acceptable carrier; preferably, the pharmaceutically acceptable auxiliary materials are selected from at least one of pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrating agents, permeation promoters, pH regulators, buffers, plasticizers, surfactants, thickeners, coating agents, moisturizers, absorbents, diluents, flocculating agents, deflocculants, filter aids, release retarders, polymeric matrix materials and film forming materials.
10. The use according to claim 9, wherein the pharmaceutical dosage form is selected from the group consisting of injection, oral preparation or external preparation; preferably, the injection is selected from injection or powder injection; the oral preparation is selected from tablet, solution, capsule, powder, pill, granule, syrup, suspension or oral sustained and controlled release preparation; the external preparation is selected from ointment, spray or patch.
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