CN111068070B - Nano gene medicine for non-alcoholic fatty liver disease and preparation method thereof - Google Patents
Nano gene medicine for non-alcoholic fatty liver disease and preparation method thereof Download PDFInfo
- Publication number
- CN111068070B CN111068070B CN201811221117.7A CN201811221117A CN111068070B CN 111068070 B CN111068070 B CN 111068070B CN 201811221117 A CN201811221117 A CN 201811221117A CN 111068070 B CN111068070 B CN 111068070B
- Authority
- CN
- China
- Prior art keywords
- nano
- metformin
- chitosan
- gene
- fatty liver
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—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
- A61K47/50—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
- A61K47/51—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
- A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—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
- A61K47/50—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
- A61K47/51—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
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—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
- A61K47/50—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
- A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
- A61K47/6931—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
- A61K47/6935—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
Abstract
The invention belongs to the technical field of biological pharmacy, and relates to a novel nano gene medicine for non-alcoholic fatty liver disease and a preparation method thereof. The nano gene medicine is formed by self-assembling metformin grafted chitosan, transmembrane factor and DSPE-PEG2000 which are used as carriers and anion therapeutic genes. The metformin grafted chitosan in the nano gene medicine can simultaneously have high transfection efficiency and internal pharmacological activity for protecting non-alcoholic fatty liver diseases. The nano gene medicine can effectively target liver parts and produce a therapeutic effect by over-expression or interference on synthesis of certain key proteins. The nano gene medicine prepared by the invention has the characteristics of high safety, good stability and controllable quality. The nano gene medicine can effectively treat non-alcoholic fatty liver disease and obesity related metabolic syndrome, and is expected to become a medicine for protecting liver injury and regulating metabolism related diseases.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to a nano gene medicine for non-alcoholic fatty liver disease and a preparation method thereof.
Background
Reports disclose that non-alcoholic fatty liver disease is becoming one of the most common chronic liver diseases with the rapid development of the world economy and the increasing living standard of human beings, affecting nearly one third of the world population [ Issa D, patel V, sanyal AJ. Future therapy for non-alcoholic fatty liver disease. Liver int. Supply.2018.1: 56-63]. Investigation shows that the incidence rate of the non-alcoholic fatty liver disease is nearly 80% in obese people, and the non-alcoholic fatty liver disease is often accompanied by risk factors such as diabetes, cardiovascular and cerebrovascular diseases, metabolic disorder and the like. The main pathological features of the Disease include simple steatosis and steatohepatitis in Liver, and the Disease is very easy to develop into Hepatic fibrosis, cirrhosis and even Liver cancer [ Samuel VT, shulman GI. Nonalcoholic Fatty Liver Disease as a Nexus of Metabolic and Hepatic diseases. Cell Metab.2018.27:22-41]. However, no drug for treating nonalcoholic fatty liver disease exists in clinic at present, so that the development of a high-efficiency and multifunctional drug for treating nonalcoholic fatty liver disease has very important practical significance [ Gluchowski NL, becuwe M, walther TC, farese RV Jr. Lipid complexes and lipid disorders: from basic biology to clinical applications. Nat Rev nutritional alcohol Hepatol.2017.14:343-55].
The development of gene therapy technology provides possibility for realizing targeted gene delivery of liver parts; chitosan is a polysaccharide polymer with positive charge only existing in nature, has been widely researched as a novel non-viral gene vector, and has good biocompatibility, degradability and low immunogenicity; however, they have significant disadvantages, such as low uptake and inefficient escape from lysosomes, and thus low transfection efficiency [ Jiang M, gan L, zhu C, dong Y, liu J, gan Y. Cationic core-shell lipolytices for cellular gene delivery. Biomaterials.2012.33:7621-30]. It has been shown that the use of biguanide modified gene vector can increase the uptake rate of the gene vector, while the Poly-Metformin (Poly-Metformin) side chain formed can retain the intrinsic biological activity of Metformin [ Zhao Y, wang W, guo S, wang Y, miao L, xiong Y, et al, polymetoformin combinations carrier and anti activities for in vivo siRNA delivery. Nat Commin.2016.7: 11822]. Metformin, as the first choice drug for clinical treatment of Diabetes, is currently demonstrated to have significant protective effects on nonalcoholic fatty liver disease [ Mazza a, fruci B, garrinis GA, gigaiano S, malaguarnera R, belfie a. The role of patients in the management of nafld. Exp Diabetes res.2012.716404]; the biguanidine modified Chitosan is used for forming the Chitosan (Chitosan-Metformin) grafted with the dimethyldiguanide, so that higher transfection efficiency can be obtained at the same time, and the gene vector is endowed with inherent pharmacological activity for protecting the non-alcoholic fatty liver disease.
The transmembrane peptide is a positively charged short peptide under physiological conditions, has strong capability of penetrating cell membranes, can effectively realize efficient endocytosis of cells and lysosome escape, and has the defect that DNA cannot be compressed well; there have been studies to combine the cell-penetrating peptide with other Gene Delivery vehicles, which have been shown to effectively overcome the disadvantages and improve the Gene transfection efficiency [ Liu C, jiang K, tai L, liu Y, wei G, lu W, et al, patient nonactive recombinant Gene Delivery Enabled by peptide and ACS applied Mater interfaces 2016.8:19256-67]. DSPE-PEG2000 can reduce nonspecific binding, increase in vivo circulation time of nano gene drugs, and help nano distribution [ Suk, J.S.; xu, q.; kim, n.; hanes, j.; design, L.M.pegylation as a Stratagene for Improving nanoparticule-Based Drug and Gene Delivery. Adv.drug Delivery Rev.2016,99, 28-51].
Based on the basis of the prior art, the application aims to provide a nano gene drug which is formed by taking Chitosan-Metformin, transmembrane peptide and DSPE-PEG2000 as carriers and carrying out self-assembly with anion therapeutic genes, and has great clinical significance in the treatment of non-alcoholic fatty liver diseases.
Disclosure of Invention
The invention aims to provide a novel nano gene medicine for nonalcoholic fatty liver disease and a preparation method thereof.
The invention further aims to provide application of the novel nano gene medicine for the non-alcoholic fatty liver disease in treating liver diseases such as non-alcoholic fatty liver disease, drug-induced liver injury, autoimmune liver disease, liver cancer, viral hepatitis, alcoholic liver and the like, diabetes, obesity-related metabolic syndrome and cardiovascular and cerebrovascular diseases.
The key technical problems to be solved by the invention are how to improve the safety and stability of the nano gene drug, how to improve the transfection efficiency of the nano gene drug, how to improve the targeting of the nano gene drug at the liver part, and how to realize the accurate and efficient treatment of the non-alcoholic fatty liver disease by the nano gene drug.
The object of the invention is achieved by the following technical solution,
the invention relates to a nano gene medicine for non-alcoholic fatty liver disease, which is prepared by the steps of firstly, taking Metformin grafted Chitosan Chitosan-Metformin, transmembrane factor and DSPE-PEG2000 as carriers, and forming nano particles by self-assembly with anion therapeutic genes; secondly, the nano gene medicine can be highly effectively targeted to the liver part by intravenous injection and over-express or interfere the synthesis of certain proteins.
More specifically, the present invention is directed to a method for producing,
the invention relates to a nano gene medicine for non-alcoholic fatty liver disease, which is characterized in that Metformin grafted Chitosan Chitosan-Metformin, transmembrane peptide and DSPE-PEG2000 are used as carriers, and are formed by self-assembly with anion therapeutic genes;
the Metformin grafted Chitosan Chitosan-Metforin has high transfection efficiency and intrinsic pharmacological activity for protecting non-alcoholic fatty liver diseases, and the structure and the synthesis mode are as follows:
in the invention, the mass ratio of Chitosan-Metformin to the anion therapeutic gene is 1:1 to 30:1;
in the invention, the mass ratio of the transmembrane hormone to the anion therapeutic gene is 4:1 to 30:1;
in the invention, the mass ratio of Chitosan-Metformin to DSPE-PEG2000 is 2:1 to 20:1;
in the invention, the amino acid sequence of the transmembrane peptide comprises RQIKIWFQNRRMKWKKK and RQIKIWFQNRRMKWKK;
in the invention, the anion therapeutic gene is composed of siRNA or DNA;
a novel nano gene medicine for non-alcoholic fatty liver is characterized in that: the particle diameter of the nano gene medicament is between 30 and 500 nM.
The invention provides a preparation method of the nano gene medicine, which comprises the following steps:
1) Dissolving Chitosan-Metformin, transmembrane hormone and DSPE-PEG2000 in ultrapure water to fully disperse the Chitosan-Metformin, the transmembrane hormone and the DSPE-PEG 2000;
2) Slowly dripping the aqueous solution of the therapeutic gene into the solution in the step 1) and uniformly mixing, and forming nano particles through electrostatic adsorption;
3) And (3) incubating the solution obtained in the step 2) for one hour at the temperature of 37 ℃, and finally forming the stable nano gene medicine.
The nano gene medicine can be prepared into a medicine for treating non-alcoholic fatty liver diseases, and the medicine can be used for treating liver diseases such as non-alcoholic fatty liver diseases, drug-induced liver injury, autoimmune liver diseases, liver cancer, viral hepatitis, alcoholic liver diseases and the like, diabetes, obesity-related metabolic syndrome, cardiovascular and cerebrovascular diseases and the like.
The novel nano gene medicine for the non-alcoholic fatty liver disease has the advantages that:
1) The nano gene medicine takes Chitosan-Metformin, transmembrane factor and DSPE-PEG2000 as carriers, and the nano gene medicine and the anion therapeutic gene are self-assembled to form a nano medicine with good particle size, shape and Z potential.
2) The Metformin grafted Chitosan Chitosan-Methformin in the nano gene drug can simultaneously have high transfection efficiency and intrinsic pharmacological activity for protecting non-alcoholic fatty liver diseases;
3) The nano gene medicine has the characteristics of high safety, good stability and controllable quality;
4) The nano gene medicine has the characteristics of high endocytosis efficiency, escape lysosome removal and liver part targeted distribution;
5) The nano gene medicine has high treatment effect on non-alcoholic fatty liver disease and obesity related metabolic syndrome.
Drawings
FIG. 1 shows preparation and characterization of a Nanogene drug (CDPIA);
fig. 2 shows CDPIA uptake by hepatocytes;
fig. 3 shows that CDPIA is able to escape clearance of hepatocyte lysosomes and has low cytotoxicity;
fig. 4 shows that CDPIA can be targeted for distribution at a liver site;
fig. 5 shows that CDPIA can significantly reduce high fat diet-induced hepatic steatosis;
fig. 6 shows that CDPIA significantly reduced metabolic disorders associated with obesity and reduced fat accumulation in vivo;
FIG. 7 shows that CDPIA can significantly activate the intrahepatic STAT3/Erk1/2 and Nrf2/SOD1 signaling pathways and regulate intrahepatic fat metabolism-related gene expression;
fig. 8 shows that CDPIA has no significant toxic side effects on tissues and organs.
Detailed Description
The present invention will be described in detail below with reference to examples and drawings, but the present invention is not limited to the examples.
The kits and starting materials used in the present invention are commercially available or can be prepared according to literature procedures. The following examples are examples of experimental methods not given specific conditions, and may be generally performed according to conventional conditions such as molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), either according to the usual conditions or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.
Example 1 preparation and characterization of Nanogenetic drugs (CDPIA)
The components and the structure of the nano gene medicine are shown in figure 1A; firstly, dissolving a therapeutic gene (pIA plasmid) in ultra-pure water with five mass percent, and quickly adding Chitosan-Metformin cationic polymers with different mass ratios; the mixture was vortexed for 60 seconds and incubated at 37 degrees for one hour; cationic polymer dextran gel electrophoresis with different components is shown in figure 1B, C, indicating a Chitosan-Metformin: pIA can fully wrap therapeutic genes when the mass ratio is 1;
then, dissolving Chitosan-Metformin, transmembrane activator and DSPE-PEG2000 with positive charges into ultra-pure water with five mass percent, and dropwise adding therapeutic genes with negative charges into the mixed solution; the mixture was vortexed for 60 seconds and incubated at 37 degrees for one hour; keeping the Chitosan-Metformin: the proportion of DSPE-PEG2000 is 10, chitosan-Metformin is adjusted: pIA, to form nanocomposites of different ratio components; measuring the particle size, PDI value and Z potential of the nano-drug by using a Malvern particle size analyzer, as shown in D, E and F in figure 1;
as shown in fig. 1G, the CDPIA morphology and particle size distribution are better as observed by transmission electron microscopy; in order to evaluate the stability of the nano gene drug (CDPIA), nano compounds (CDPIA and CMIA) with the same mass and different components are respectively taken and stored at room temperature; simultaneously, measuring the particle size and PDI value of CDPIA and CMIA by a Malvern particle size analyzer at different time points; as shown in fig. 1H, CDPIA has relatively good stability.
Example 2 uptake of CDPIA by hepatocytes
Observing the uptake condition of the liver cells to the nano gene medicine (CDPIA) by using a laser confocal microscope; firstly, marking therapeutic genes pIA and FAM marked transmembrane hormone by fluorescent dye TOTO-3, and preparing a nano gene drug (CDPIA) marked by a fluorescent probe by an autonomous assembly method; then 1X 10 5 The HepG2 cell and the Huh7 cell are inoculated in a confocal small dish, when the cell grows to 70 percent, pIA, CMIA and CDPIA with fluorescent labels are added into the cell for 2 hours; washing with precooled PBS for 3 times, and observing the uptake condition of the nano-gene drug (CDPIA) by the liver cells under a laser confocal microscope; as shown in fig. 2, the results show that the uptake rate of the nano-gene drug (CDPIA) modified by the perforin and formed by self-assembly by the liver cells is remarkably improved, while the uptake rate of the naked plasmid (pIA) and the nano-gene drug (CMIA) without the added perforin is lower; the results of fluorescence co-localization analysis show that the therapeutic gene and the transmembrane hormone have good co-localization, which indicates that the nano-drug carrier can keep the structural integrity and can effectively protect the therapeutic gene from being degraded by lysosome or ribozyme in the process of endocytosis of cells (FIG. 2B, D); the result shows that the uptake rate of the nano gene drug (CDPIA) with the transmembrane hormone modification in the liver cells is obviously improved.
Example 3 CDPIA is able to escape hepatocyte lysosomal clearance and has low cytotoxicity
According to literature reports, lysosome escape is a precondition for good transfection efficiency after nano-gene drug uptake; in this example, the subcellular localization and lysosome escape capacity of nano gene drug (CDPIA) in liver cells are further examined, TOTO-3 dye is adopted to mark CDPIA, lysotracker Green dye is used to mark lysosome, hoechst 33342 is used to mark cell nucleus, and the localization conditions of CDPIA, lysosome and cell nucleus are observed; will be 1 × 10 5 The HepG2 cells and the Huh7 cells are inoculated in a confocal dish; adding CDPIA when the cell grows to have a fusion rate of 70%, and then observing the positioning conditions of CDPIA, lysosome and cell nucleus at different time points; as shown in fig. 3A and BThe experimental result shows that CDPIA is taken up by liver cells within 1 hour and part of nanoparticles and lysosomes are co-localized (yellow); after 4 hours, the green lysosome and the red nano gene drug are separated, and the CDDIA is proved to escape from the phagocytosis of lysosomes; after 8 hours, co-localization of partial nano gene medicine and cell nucleus is carried out; the result shows that the nano gene medicine CDPIA has the function of escaping lysosome phagocytosis;
the MTT method is used for detecting the cytotoxic effect of Chitosan-Metforin with different concentrations on liver cells, as shown in figures 3C and D, the results show that CDPIA has almost no killing effect on liver cells, and the CDPIA is proved to have higher cell safety.
Example 4 CDPIA can target distribution at liver site
The therapeutic effect of the nano-gene drug is directly determined based on the distribution condition of the nano-gene drug in vivo, therefore, the TOTO-3 labeled therapeutic gene is utilized in the embodiment, and the distribution condition of CDPIA in a mouse is observed by using a small animal living body imaging system, as shown in fig. 4A and B, after 6 hours of tail vein injection of CDPIA, the liver starts to take the nano-gene drug and reaches the maximum concentration in 12 hours, and compared with other organs, CDPIA has obvious liver targeting property; meanwhile, part of TOTO-3-pIA is found in the kidney, and compared with a control group (CMIA), the accumulation of CDPIA in the liver part is obviously increased, which is related to good nano particle size, shape and cell membrane penetration effect mediated by cell-penetrating peptide;
after 12 hours of tail vein injection, the liver and each organ tissue are frozen and sliced and observed under a laser confocal microscope, as shown in fig. 4C-E, the TOTO-3 marked therapeutic gene is mainly distributed at the liver part, and the accumulation of nano gene medicine (CDPIA) with the modified transmembrane hormone at the liver part is obviously increased, and the result proves that the CDPIA has obvious liver part targeting property in vivo and can be better taken up by liver cells.
Example 5 CDPIA significantly reduces high fat diet-induced hepatic steatosis
Further verifying the protective effect of the medicine on the nonalcoholic fatty liver disease; after experimental mice were fed with 60% high fat diet for 4 weeks, tail vein injection of PBS, CM, CMIA and CDPIA was performed for 12 weeks (fig. 5A), and after 17 weeks of high fat diet, significant liver steatosis was observed in the liver region (fig. 5b, c); HE pathology and oil red O staining results showed significant ballooning of hepatocytes and fat deposition (fig. 5D-F); the triglyceride TG and free fatty acid NEFA content in liver tissue increased significantly (fig. 5g; after CDPIA treatment of mice, the color and appearance of liver can be obviously improved, liver weight can be reduced, and fat accumulation in liver tissues and balloon-like degeneration of liver cells can be obviously reduced (fig. 5B-F); liver homogenate detection results show that CDPIA can remarkably reduce the content of TG and NEFA in liver tissues (fig. 5G, H), and compared with the CDPIA treatment group, the nano gene drug (CMIA) without adding the transmembrane factor has poorer treatment effect, which indicates that the transmembrane factor can improve the treatment effect of the nano gene drug; the empty carrier CM treatment group also has weak treatment effect, which indicates that CM can exert the intrinsic biological activity of metformin, but has no significant difference due to lower administration frequency and administration dosage;
the results show that the nano-gene drug CDPIA added with the transmembrane hormone can obviously reduce the hepatic steatosis induced by high-fat diet, the treatment effect is obviously better than that of the empty carrier CM and the nano-gene drug CMIA not added with the transmembrane hormone, and the synergy between Chitosan-Metformin and the transmembrane hormone and the treatment gene (pIA) is shown.
Example 6 CDPIA significantly reduces metabolic disorders associated with obesity and reduces fat accumulation in the body
Non-alcoholic fatty liver disease based is often accompanied by obesity-related metabolic syndrome; in this example, the weights of the mice in each group were measured during the administration period, and fasting for 8 hours before the material was obtained, and then fasting weights and fasting blood glucose were measured, and the results are shown in fig. 6A-C, the CDPIA treatment significantly reduced the weight gain of the mice, and reduced the fasting weights and fasting blood glucose of the mice, wherein the empty vector CM had a weak effect of reducing weight gain, indicating that the gene vector has an intrinsic protective effect; the therapeutic effect of the nano therapeutic drug (CMIA) without adding the transmembrane hormone is improved relative to that of an empty vector (CM), which indicates that the therapeutic gene can be effectively expressed and play a protective role in the liver part; the regulation effect of the nano therapeutic drug (CDPIA) added with the transmembrane factor on the weight and the blood sugar is further improved compared with the CMIA, probably because the transmembrane factor improves the uptake efficiency of the liver part of the nano therapeutic drug and reduces the phagocytosis of a reticuloendothelial system, thereby exerting better curative effect; the results of insulin tolerance and glucose tolerance tests show that CDPIA can remarkably restore the insulin sensitivity and blood glucose homeostasis of mice, and the effect of CDPIA group is remarkably superior to that of CM and CMIA groups (FIG. 6D, E, G, H); liver function index test results showed that CDPIA significantly attenuated the elevation of ALT, AST levels caused by high fat diet (fig. 6f, i); meanwhile, CDPIA can reduce the epididymis fat weight of mice obviously, and reduce the volume of fat cells obviously, the treatment effect is obviously better than CM and CMIA (figure 6J-L), in addition, the content of triglyceride in serum is also reduced (figure 6M);
the above results indicate that CDPIA is effective in restoring insulin resistance and reducing fat accumulation in the body caused by high fat diet; and the effect of CDPIA is significantly better than CM and CMIA.
Example 7 CDPIA can significantly activate the STAT3/Erk1/2 and Nrf2/SOD1 signal pathways in liver and regulate the expression of genes related to fat metabolism in liver
The main mechanism for interleukin 22 to exert its therapeutic effect is that interleukin 22 binds to its receptor and activates the STAT3 signaling pathway, thereby generating a series of protective cellular mechanisms; in order to further study the mechanism of treating fatty liver by CDPIA, in this embodiment, western blot is used to detect the expression and phosphorylation levels of STAT3/Erk 1/2-related proteins in the downstream signaling pathway of IL-22, as shown in fig. 7A and B, CDPIA can significantly promote the increase of phosphorylation levels of STAT3 and Erk1/2 in liver, and the expression of the downstream proteins Bcl-2 and Cyclin D1 thereof is also significantly up-regulated; CDPIA also significantly promoted expression of intra-hepatic Nrf2 and SOD1 proteins (fig. 7c, d); the expression quantity of genes related to the fat beta-oxidative metabolism, fat export and fat synthesis in liver is detected, and the result shows that the CDPIA can remarkably promote the expression up-regulation of genes related to the fat beta-oxidative metabolism (Acox 1, cpt1 a) and the fat export (Acc 1) in liver, but has no remarkable influence on the genes related to the fat synthesis (CD 36, FAS) (figure 7E), the CDPIA has the most remarkable regulation effect, and the main reason is that the CDPIA has better liver part targeting property and transfection efficiency;
the results prove that the CDPIA effect of protecting the nonalcoholic fatty liver disease is related to activation of STAT3/Erk1/2 and Nrf2/SOD1 signal pathways, and plays a role by regulating the expression of genes related to fat metabolism.
Example 8 CDPIA has no significant toxic side effects on tissues and organs
Based on that NAFLD is a chronic disease and needs long-term administration treatment, the targeted nano gene medicine must have good biological safety and lower toxic and side effects, and research reports show that nanoparticles such as QD or micelles are easy to aggregate in organs such as heart, liver, lung and kidney, and the nonspecific aggregation can often cause the damage of organs such as liver and kidney, and the like; in this example, whether the intravenous CDPIA can cause organ toxicity is studied, pathological detection is performed on each organ of the mouse after 12 weeks of treatment, and the experimental result is shown in fig. 8A, and pathological changes of each organ of the mouse are not caused after 12 weeks of treatment with CM, CMIA and CDPIA; in addition, the results of the detection of mouse renal function indicators BNU and creatinine content show that CDPIA does not cause the renal function injury of mice (FIG. 8B, C), and the results of the detection of serum lactate dehydrogenase show that CDPIA does not cause obvious somatic injury (FIG. 8D); the results show that the CDPIA as a medicine for treating the non-alcoholic fatty liver disease has no obvious toxic or side effect and organ toxicity.
Claims (7)
1. A nanometer gene medicine for non-alcoholic fatty liver disease is characterized in that the nanometer gene medicine is composed of nanometer formed by self-assembling Metformin grafted Chitosan Chitosan-Metformin, transmembrane peptide and DSPE-PEG2000 as carriers and anion therapeutic genes;
the Metformin grafted Chitosan Chitosan-Methformin has high transfection efficiency and intrinsic pharmacological activity for protecting non-alcoholic fatty liver diseases, and the structure and the synthesis mode of the Metformin grafted Chitosan Chitosan-Methformin are as follows:
the amino acid sequence of the transmembrane peptide is represented by RQIKIWFQNRRMKWKKK and RQIKIWFQNRRMKWKK; the anion therapeutic gene is composed of siRNA or DNA.
2. The nanogene drug for nonalcoholic fatty liver disease according to claim 1, wherein: the particle size of the nano gene medicine is between 30 and 500 nM.
3. The method of preparing a nano gene drug for nonalcoholic fatty liver disease according to claim 1, comprising the steps of,
1) Dissolving Chitosan-Metformin, transmembrane activator and DSPE-PEG2000 in ultrapure water to fully disperse the Chitosan-Metformin, the transmembrane activator and the DSPE-PEG 2000;
2) Slowly dripping the aqueous solution of the therapeutic gene into the solution in the step 1) and uniformly mixing, and forming nano particles through electrostatic adsorption;
3) And (3) incubating the solution obtained in the step 2) for one hour at the temperature of 37 ℃, and finally forming the stable nano gene medicine.
4. The method of claim 3, wherein the mass ratio of Chitosan-Metformin to the anion therapeutic gene is 1:1 to 30:1.
5. the method according to claim 3, wherein the mass ratio of the transmembrane peptide to the anion therapeutic gene is 4:1 to 30:1.
6. the preparation method of claim 3, wherein the mass ratio of Chitosan-Metformin to DSPE-PEG2000 is 2:1 to 20:1.
7. use of the nano-genetic drug for non-alcoholic fatty liver disease of claim 1 in the preparation of a drug for treating non-alcoholic fatty liver disease, drug-induced liver injury, autoimmune liver disease, liver cancer, viral hepatitis, alcoholic liver disease, diabetes, obesity-related metabolic syndrome, or cardiovascular and cerebrovascular disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811221117.7A CN111068070B (en) | 2018-10-19 | 2018-10-19 | Nano gene medicine for non-alcoholic fatty liver disease and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811221117.7A CN111068070B (en) | 2018-10-19 | 2018-10-19 | Nano gene medicine for non-alcoholic fatty liver disease and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111068070A CN111068070A (en) | 2020-04-28 |
| CN111068070B true CN111068070B (en) | 2022-11-08 |
Family
ID=70309155
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811221117.7A Active CN111068070B (en) | 2018-10-19 | 2018-10-19 | Nano gene medicine for non-alcoholic fatty liver disease and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111068070B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111249468B (en) | 2020-03-18 | 2022-06-24 | 湖南大学 | Adipocyte-targeted DNA nano-drug and preparation method and application thereof |
| CN113930454A (en) * | 2021-11-26 | 2022-01-14 | 南通大学 | Application of chitosan-metformin in aspect of being used as gene transfection reagent |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103977422A (en) * | 2014-06-11 | 2014-08-13 | 中国药科大学 | Guanidine hypoglycemic drug-polysaccharide conjugate, as well as preparation method and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7429575B2 (en) * | 2006-01-10 | 2008-09-30 | Yu Ruey J | N-(phosphonoalkyl)-amino acids, derivatives thereof and compositions and methods of use |
-
2018
- 2018-10-19 CN CN201811221117.7A patent/CN111068070B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103977422A (en) * | 2014-06-11 | 2014-08-13 | 中国药科大学 | Guanidine hypoglycemic drug-polysaccharide conjugate, as well as preparation method and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| Biocompatible polymeric nanocomplexes as an intracellular stimuli-sensitive prodrug for type-2 diabetes combination therapy;Wang等;《Biomaterials》;20150911;第73卷;摘要、第150页左栏第2段 * |
| In vivo hepatocellular expression of interleukin-22 using penetratin-based hybrid nanoparticles as potential anti-hepatitis therapeutics;Chen等;《Biomaterials》;20181003;第187卷;第67页右栏第2段，第67页第2.1节，第69-70页第3.1节 * |
| Microwave-assisted synthesis of chitosan biguanidine hydrochloride and its regulation on InsR and GLUT2 in insulin resistant HepG2 cells;Zhang等;《RSC Advances》;20170206;第7卷;摘要，第10109页图1a * |
| PolyMetformin combines carrier and anticancer activities for in vivo siRNA delivery;Zhao等;《NATURE COMMUNICATIONS》;20160606;全文 * |
| 非病毒性载体在肝脏疾病基因治疗中的研究进展;谭平萍等;《现代生物医学进展》;20090330(第06期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111068070A (en) | 2020-04-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | The use of antibody modified liposomes loaded with AMO-1 to deliver oligonucleotides to ischemic myocardium for arrhythmia therapy | |
| Xue et al. | Delivery of microRNA-1 inhibitor by dendrimer-based nanovector: An early targeting therapy for myocardial infarction in mice | |
| CN107789632A (en) | A kind of active Brain targeting nanoscale medicine delivery system of T7 peptides modification and preparation method thereof | |
| CN111068070B (en) | Nano gene medicine for non-alcoholic fatty liver disease and preparation method thereof | |
| Zhao et al. | Tumor acidity-induced sheddable polyethylenimine-poly (trimethylene carbonate)/DNA/polyethylene glycol-2, 3-dimethylmaleicanhydride ternary complex for efficient and safe gene delivery | |
| CN107184987B (en) | Lipoic acid modified targeted integrin alpha v beta 3 nano-polypeptide carrier and preparation method and application thereof | |
| CN111632153B (en) | Chemical gene drug co-loaded targeting nano drug delivery system and preparation method thereof | |
| Chen et al. | Poly (N-isopropylacrylamide) derived nanogels demonstrated thermosensitive self-assembly and GSH-triggered drug release for efficient tumor Therapy | |
| Cheng et al. | pH-responsive multifunctional theranostic rapamycin-loaded nanoparticles for imaging and treatment of acute ischemic stroke | |
| CN109568594A (en) | Polycation macromolecular prodrug is used for the preparation and application of gene delivery, nano composite system | |
| Wang et al. | Acid-and reduction-sensitive micelles for improving the drug delivery efficacy for pancreatic cancer therapy | |
| Rastegari et al. | Inhibiting hepatic gluconeogenesis by chitosan lactate nanoparticles containing CRTC2 siRNA targeted by poly (ethylene glycol)-glycyrrhetinic acid | |
| He et al. | In situ self-assembly of near-infrared-emitting gold nanoparticles into body-clearable 1D nanostructures with rapid lysosome escape and fast cellular excretion | |
| Cheng et al. | One-pot synthesis of acid-induced in situ aggregating theranostic gold nanoparticles with enhanced retention in tumor cells | |
| Wu et al. | Glucose-sensitive nanoparticles based on poly (3-acrylamidophenylboronic acid-block-n-vinylcaprolactam) for insulin delivery | |
| WO2015188423A1 (en) | Guanidine hypoglycemic drug-polysaccharide conjugate, preparation method therefor and use thereof | |
| Wang et al. | Mitochondria-targeting folic acid-modified nanoplatform based on mesoporous carbon and a bioactive peptide for improved colorectal cancer treatment | |
| WO2022262050A1 (en) | Non-viral vector, and preparation method therefor and use thereof | |
| Wang et al. | In vivo pharmacokinetics, biodistribution and the anti-tumor effect of cyclic RGD-modified doxorubicin-loaded polymers in tumor-bearing mice | |
| CN107693505B (en) | Oil-water double-soluble ROS sensitive nanoparticle and preparation method thereof | |
| CN104546709B (en) | Deliver the pH sensitive polymer micella of drug | |
| CN111135312B (en) | Preparation and application of mixed nano preparation based on tumor pan-metabolism regulation | |
| Chen et al. | Drug-Free Liposomes Containing Mannosylated Ligand for Liver-Targeting: Synthetic Optimization, Liposomal Preparation, and Bioactivity Evaluation | |
| CN106512020B (en) | Accurate diagnosis and treatment system of nanometer microRNA of target ischemia myocardial | |
| CN104368009B (en) | A kind of Alzheimer disease target gene of bifunctional peptide modification, which is passed, releases compound and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |