CN109550057B - Active targeting gene delivery nanoparticles and preparation method and application thereof - Google Patents
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- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- 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
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- 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
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Abstract
The invention discloses an active targeting gene delivery nanoparticle and a preparation method and application thereof; the hyaluronic acid-hyaluronic acid composite material consists of a cationic polymer, hyaluronic acid and a negatively charged gene, wherein the cationic polymer and the negatively charged gene form a composite core, the hyaluronic acid is coated on the surface of the composite core through charge action and chemical bond binding action, and the cationic polymer is one of PASP (EDA), PASP (DET), PASP (TET) and PASP (TEP) or one of chemically modified derivatives thereof. The hyaluronic acid adopted by the invention has excellent biocompatibility, not only reduces cytotoxicity caused by cationic polymer, but also can actively target and act on a hyaluronic acid specific receptor highly expressed on the surface of tumor cells, so that exogenous genes are more effectively delivered into the tumor cells, and the cell uptake and transfection efficiency is improved.
Description
Technical Field
The invention belongs to the field of gene therapy of nanotechnology, relates to a nanometer biomaterial and a gene delivery system, and particularly relates to a novel active targeting type gene delivery nanoparticle taking hyaluronic acid as a target molecule, and a preparation method and application thereof.
Background
Gene therapy (gene therapy) refers to the introduction of an exogenous gene of interest into a target cell of a patient to correct or compensate for diseases caused by gene defects and abnormalities, thereby achieving the therapeutic goal. Through the development of more than one and twenty years, the research of gene therapy has made a lot of progress, the development trend is encouraging, especially as a new treatment method of cancer, the human serious disease, has become the research hotspot at home and abroad. Gene delivery vectors are the most critical part of gene therapy and include both viral and non-viral vectors. Although viral vectors have the advantage of high transfection efficiency, they have a series of safety problems (potential mutagenicity, high immunogenicity, etc.), which limit their development and application. Non-viral gene vectors, as a new generation of gene delivery vectors, have incomparable advantages of viral vectors, such as low cytotoxicity, low immunogenicity, unlimited gene fragment size, high gene loading, easy preparation and modification, convenient storage and inspection, etc., and thus have been widely studied and paid attention.
The non-viral vector mainly comprises two categories of cationic polymers and cationic liposomes, wherein the cationic polymers (polycations) can combine and compress nucleic acid through electrostatic action, and can successfully escape from an inclusion body through the action of a proton sponge after entering an acidic inclusion body, so that the premise and guarantee are provided for improving the transfection efficiency of genes. The cationic polymer has the advantages of easy synthesis and modification, no immunogenicity, protection of nucleic acid from degradation, convenience for targeting and biocompatibility modification, and the like.
Generally, after the cationic polymer is compounded with nucleic acid, the surface of the compound has excessive positive charges, so that the adhesion between the compound and a cell membrane with negative charges has no specificity, the positive charges have toxic effect on cells when being excessive, in addition, the excessive positive charges are easy to interact with proteins in blood, so that the proteins are easy to be eliminated by a reticuloendothelial system (RES), and the circulation time in vivo is short. Therefore, the solution of targeting, safety and stability is a problem to be solved urgently.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to provide the preparation and the application of a gene delivery system with hydrophilicity, stability, targeting property and high transfection efficiency and low toxicity.
The technical scheme of the invention is as follows: an active targeting gene delivery nanoparticle comprises a cationic polymer, hyaluronic acid and a negatively charged gene, wherein the cationic polymer and the negatively charged gene form a composite inner core, the hyaluronic acid is coated on the surface of the composite inner core through charge action and chemical bond combination, and the cationic polymer can be PASP (EDA), PASP (DET), PASP (TET) or PASP (TEP) or derivatives thereof. (Hirokuni Uchida, et al, J.am. chem. Soc.2014,136, 12396-12405). The cationic polymers have the advantages of high efficiency, low toxicity, biodegradability and the like when being applied to in vivo and in vitro gene delivery, and can be combined with targeting molecules and/or chemical bonds with tumor microenvironment responsiveness to prepare the active targeting type gene delivery nanoparticles with obviously improved targeting property.
Further, the cationic polymer may have protonsThe mol ratio of the chemical nitrogen atom to the phosphate radical in the gene is 0.01-200; protonatable nitrogen atoms in cationic polymer and COO in hyaluronic acid-Is 0.01 to 100. Preferably, the mole ratio of protonatable nitrogen atoms in the cationic polymer to phosphate groups in the gene is 1-60; protonatable nitrogen atoms in cationic polymer and COO in hyaluronic acid-Is 0.1 to 20.
Further, the molecular weight of PASp (EDA) is 1.0-100KDa, the molecular weight of PASp (DET) is 1.0-100KDa, the molecular weight of PASp (TET) is 1.0-100KDa, and the molecular weight of PASp (TEP) is 1.0-100 KDa. Preferably, the molecular weight of PASp (EDA) is 1.0-40kDa, the molecular weight of PASp (DET) is 1.0-40kDa, the molecular weight of PASp (TET) is 1.0-40kDa and the molecular weight of PASp (TEP) is 1.0-40 kDa.
Further, the cationic polymer is poly [ N ' - (N-citric acid-2-aminoethyl) aspartic acid ], poly { N ' - [ N- (2-aminoethyl) -2-aminoethyl ] aspartic acid }, poly (N- { N ' - [ N "- (2-aminoethyl) -2-aminoethyl ] -2-aminoethyl } aspartic acid), or poly [ N- (N ' - { N" - [ N ' "- (2-aminoethyl) -2-aminoethyl ] -2-aminoethyl } -2-aminoethyl) aspartic acid ].
Furthermore, the hyaluronic acid is hyaluronic acid or a derivative of hyaluronic acid after chemical modification, and the molecular weight is 5.0-2000 KDa.
Further, the negatively charged gene is DNA or RNA or oligonucleotide. The DNA or RNA is DNA or RNA or oligonucleotide of various reporter genes, anti-cancer genes, gene editing tool genes or cytokine genes which can be recombined and expressed in eukaryotic cells.
The hyaluronic acid target molecule is connected with the cationic polymer and the inner core of the gene complex with negative charge through chemical bonds. The chemical bonds are ester bonds, amido bonds, azido bonds, triazole bonds, disulfide bonds and the like.
The invention also discloses a preparation method of the active targeting gene delivery nanoparticle, which comprises the following steps:
the method comprises the following steps: preparing a cationic polymer, a negatively charged gene, hyaluronic acid and a cross-linking agent into solutions with proper concentrations by using proper solvents respectively;
step two: and uniformly mixing the cationic polymer solution and the negatively charged gene solution according to a proper proportion to ensure that the cationic polymer and the negatively charged gene form a composite inner core through electrostatic acting force. The surface of the formed complex is positively charged, and the particle size is 10-200 nm;
adding a hyaluronic acid solution or a mixed solution of hyaluronic acid and a cross-linking agent into a compound solution of the cationic polymer and the gene according to a proper proportion, so that the hyaluronic acid is coated on the surface of the inner core of the compound through charge action and chemical bond combination action to form nanoparticles with negative charges on the surface and the particle size of 20-400 nm;
wherein, the concentration of the cationic polymer and the hyaluronic acid is 0.1-10 mug/mug, and the concentration of the gene is 0.01-10 mug/mug; the mole ratio of protonatable nitrogen atoms in the cationic polymer to phosphate radicals in the gene is 0.01-200; the molar ratio of protonatable nitrogen atoms in the cationic polymer to COO-in the hyaluronic acid is 0.01-100.
Further, the cross-linking agent may be selected from one or more of the general class of carbodiimide/NHS esters, preferably 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS); the solvent used for preparing the solution is a buffer salt solution or water, and the buffer salt solution is selected from any one or a mixture of Tris-HCl, HEPES or phosphate solution.
The delivery nanoparticles are applied to gene transfection of cells in vivo and in vitro and gene drug delivery in human or animal bodies.
Compared with the prior art, the invention has the following beneficial effects:
(1) the novel non-viral gene delivery nanoparticle provided by the invention has hydrophilicity and low cytotoxicity, has stability under physiological conditions, and greatly improves the safety and stability of a gene delivery system.
(2) The novel gene delivery system provided by the invention has wide gene adaptation types, can be used for delivering gene fragments with different sizes from dozens of bp to tens of thousands of bp, and has wide application prospects.
(3) The novel gene delivery system provided by the invention has active targeting property and high transfection efficiency, and can efficiently introduce exogenous target genes into CD44 receptor highly-expressed tumor cells, thereby greatly improving the effectiveness of the gene delivery system and providing guarantee for the effectiveness of tumor gene therapy.
(4) The novel gene delivery system provided by the invention can more effectively introduce exogenous therapeutic genes into tumor tissues (such as lung cancer, liver cancer and melanoma) with high expression of CD44 receptor, can inhibit the growth of malignant tumors, and has tumor targeting property and tumor treatment effectiveness. The novel gene delivery system can also be used for preparing combined delivery of multiple genes to treat tumors, can also be used as a platform technology for gene therapy of other diseases, and has wide clinical application prospect.
Drawings
FIG. 1: PASp (DET)/DNA/HA nanoparticle size and Zeta potential results;
FIG. 2: PASp (DET)/DNA/HA nanoparticle TEM image;
FIG. 3: PASp (DET)/DNA/HA nanoparticle cytotoxicity test results;
FIG. 4: PASp (DET)/DNA/HA nanoparticle cell transfection experiment result;
FIG. 5: PASp (DET)/DNA/HA nanoparticle cell uptake assay results;
FIG. 6: transgenic tumor inhibition experiment results of the PASp (DET)/DNA/HA nanoparticles in a mouse subcutaneous solid tumor model;
FIG. 7: the results of transgenic tumor inhibition experiments of the PASp (DET)/DNA/HA nanoparticles in a mouse lung metastasis tumor model.
Detailed Description
In the examples below, PASP (EDA) is poly [ N '- (N-citric acid-2-aminoethyl) aspartic acid ], PASP (DET) is poly { N' - [ N- (2-aminoethyl) -2-aminoethyl ] aspartic acid }, PASP (TET) is poly (N- { N '- [ N "- (2-aminoethyl) -2-aminoethyl ] -2-aminoethyl } aspartic acid), and PASP (TEP) is poly [ N- (N' - { N '- [ N' (2-aminoethyl) -2-aminoethyl ] -2-aminoethyl } -2-aminoethyl) aspartic acid ]. HEPES is 4-hydroxyethyl piperazine ethanesulfonic acid, and Tris is Tris (hydroxymethyl) aminomethane. The EGFP plasmid is a plasmid (plasmid DNA, pDNA) for coding strong green fluorescent protein, and the sFlt-1 plasmid is a plasmid for coding a soluble vascular growth factor receptor.
Example 1: preparation of cationic Polymer/Gene complexes
The PASP (DET) and sFlt-1 plasmids were dissolved in HEPES (10mM, pH7.4) at a concentration of 0.05. mu.g/. mu.l, and the concentration of the PASP (DET) solution was adjusted as required for the N/P ratio. The PASP (DET) solution and the sFlt-1 plasmid solution with 2 times volume are mixed and then vortexed for 30 seconds to prepare a PASP (DET)/pDNA complex solution with the final concentration of the sFlt-1 plasmid of 33.3 mu g/mL. Using the same methods and procedures, cationic polymer/pDNA complexes can be constructed from PASP (EDA), PASP (TET) and PASP (TEP) or their derivatives.
Similarly, cationic polymer/mRNA complexes, cationic polymer/siRNA complexes, cationic polymer/oligonucleotide complexes, etc. can be formed using PASP (EDA), PASP (DET), PASP (TET), and PASP (TEP) or their derivatives and mRNA, siRNA, oligonucleotides, etc. according to the same procedures and methods as described above.
Example 2: preparation of PASp (DET)/DNA/HA nanoparticles
In this example, PASp (DET) is poly { N' - [ N- (2-aminoethyl) -2-aminoethyl ] aspartic acid }, HA is a transparent acid, and a cross-linking agent is used to chemically bond HA to the core of the complex. EDC is 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and NHS is N-hydroxysuccinimide.
The PASP (DET) and EGFP plasmids were prepared with Tris-HCl (10mM, pH7.4) into solutions at concentrations of 2.0. mu.g/. mu.l and 0.05. mu.g/. mu.l, respectively. A mixed solution of HA, EDC and NHS was prepared with water at concentrations of 1.0. mu.g/. mu.l, 0.15. mu.g/. mu.l and 0.25. mu.g/. mu.l, respectively. The solution of PASp (DET)/DNA complex is prepared by adjusting the solution of PASp (DET) and EGFP plasmids to the appropriate concentration with Tris-HCl, mixing the solution of PASp (DET) and EGFP plasmids according to the appropriate proportion and vortexing for 30 seconds. After the compound solution is placed at room temperature for 30 minutes, the mixed solution of hyaluronic acid obtained by sequentially adding the EDC solution and the NHS solution in sequence is added according to a proper proportion, and after 30 seconds of vortex, the mixed solution is placed at room temperature for 8 hours to obtain the nano-particle solution of hyaluronic acid and cationic polymer amino residue crosslinking. The solution is placed in an ultrafiltration tube, and after Tris-HCl buffer solution is added, the solution is centrifugally purified to obtain the nanoparticle solution which can be used for in vitro and in vivo experiments.
By the above preparation method, according to N/P/COO-(Nitrogen atom protonatable in cationic Polymer/phosphate radical in EGFP plasmid/COO in hyaluronic acid-In a molar ratio) of 20:1:20 and N/P of 20: 1. Using the same method and procedure, cationic polymer/DNA/HA nanoparticles can be constructed from PASP (EDA), PASP (TET) and PASP (TEP) or their derivatives. Also, cationic polymer/mRNA/HA, cationic polymer/siRNA/HA, cationic polymer/oligonucleotide/HA nanoparticles, etc. can be formed using PASP (EDA), PASP (DET), PASP (TET), and PASP (TEP) or their derivatives and mRNA, siRNA, oligonucleotide, etc. according to the same steps and methods as described above.
50 μ l of the nanoparticles PASp (DET)/DNA/HA were measured by a laser particle sizer. The results are shown in FIG. 1. The result shows that the hydration grain diameter of the nano-particle is about 150nm, and the Zeta potential is about-24 mV.
By the above preparation method, according to N/P/COO-(Nitrogen atom protonatable in cationic Polymer/phosphate radical in EGFP plasmid/COO in hyaluronic acid-In a molar ratio) of 20:1:20 and N/P of 20:1, preparing nanoparticles and a compound, and observing the shape and size of the compound by a transmission electron microscope after negative dyeing. Observation was performed by a transmission electron microscope. The results are shown in fig. 2, where the nanoparticles are in the form of round particles.
Example 3: comparison of cytotoxicity
The preparation method is used for preparing the PASp (DET)/DNA/HA nanoparticles, and different N/P/COO are investigated by CCK-8 experiment-The gene delivers the cytotoxicity of the nanoparticles at the ratio. HUVEC/B16F10 cells in logarithmic growth phase were trypsinized at 5X 103The density of each well was seeded in 96-well plates with 50. mu.l of each wellCell suspension, 5% CO at 37 ℃2After 24 hours of incubation in an incubator, sample solutions adjusted to 50 μ l volume with culture medium were added, four solutions with different N/P ratios were prepared with N/P of 5:1, 10:1, 20:1 and 40:1 for each sample solution, the sample solution added to each well contained 1.0 μ g of EGFP plasmid, and low-toxicity PEG-PASP (DET) and more toxic 25K PEI were used as controls. After 24 hours of incubation, 10. mu.l of CCK-8 solution was added to each well, incubation was continued for 2 hours, and the absorbance value (A) at 450nm was measured with a microplate reader. Calculating the cell survival rate according to the value A and the following formula:
survival rate%sample-A0/ABlank-A0) 100, in the formula AsampleRepresents the average absorbance value, A, of each sample setBlankRepresents the average absorbance value of the blank cell group without the addition of the sample, A0Represents the mean absorbance value of the medium-only group. The results are shown in FIGS. 3A and B. The results show that under the same N/P ratio condition, the toxicity of the PASp (DET)/DNA complex is obviously lower than that of the PEI/DNA complex, and the cytotoxicity of the cation is obviously lower than that of PEI with high transfection efficiency but larger toxicity; the modification of the PASp (DET)/DNA complex with hyaluronic acid further reduces the cytotoxicity of the cationic polymer. The experiment proves that the PASp (DET)/DNA compound and the PASp (DET)/DNA/HA prepared by the invention effectively reduce the cytotoxicity of the existing product, and the invention is a technical progress.
Example 4: pasp (DET)/DNA/HA nanoparticles for gene transfection of cells
By the above preparation method, with N/P/COO-Pasp (DET)/DNA/HA nanoparticles were prepared at a ratio of 20:1:20 and cellular uptake in vitro was examined. PEG-pasp (det) not actively targeted was used as a control. The N/P ratio of the PEG-PASP (DET)/DNA nanoparticles and the PASP (DET)/DNA complex is 20: 1. B16F10 cells in logarithmic growth phase were trypsinized and cultured at 3X 104One/well density was seeded in 24-well plates, 400. mu.l cell suspension per well, 5% CO at 37 ℃2After 24 incubations in the incubator, the medium was replaced with fresh RPMI1640 medium containing 10% fetal bovine serum, the prepared sample solution was added, and 5% CO was added at 37 deg.C2Incubator inner cultureAfter 24 days, cells were collected and examined by flow cytometry. The experimental results are shown in figure 4. The experimental result shows that the PASp (DET)/DNA complex and the PASp (DET)/DNA/HA nanoparticles have very high cellular uptake rate, the former is due to the electrostatic action between the positively charged complex and the cell membrane, the latter is due to the active targeting action of hyaluronic acid molecules, and the cellular uptake rate of the two nanoparticles is remarkably higher than that of the non-active targeting PEG-PASp (DET)/DNA nanoparticles reported in the prior art.
By the above preparation method, with N/P/COO-Pasp (DET)/DNA/HA nanoparticles were prepared at 20:1:20 and the in vitro cell transfection efficiency was examined. PEG-pasp (det) not actively targeted was used as a control. The N/P ratio of PEG-PASp (DET)/DNA nano-particles and PASp (DET)/DNA complex is 20:1, and the N/P/COO ratio of PASp (DET)/DNA/HA is-The ratio was 20:1: 20. B16F10 cells in logarithmic growth phase were trypsinized and cultured at 3X 104One/well density was seeded in 24-well plates, 400. mu.l cell suspension per well, 5% CO at 37 ℃2After 24 incubations in the incubator, the medium was replaced with fresh RPMI1640 medium containing 10% fetal bovine serum, and the prepared sample solutions (each containing 1.0. mu.g of EGFP plasmid) were added at 37 ℃ with 5% CO2After incubation in the incubator 48, observation and photographing were performed with a fluorescence microscope. Cells were then collected and detected using a flow cytometer. The results are shown in FIG. 5.
Experimental results show that the PASp (DET)/DNA/HA nanoparticles prepared by modifying the PASp (DET)/DNA complex with hyaluronic acid can improve the transfection efficiency, and the transfection efficiency is obviously higher than that of a PEG-PASp (DET)/DNA nanoparticle control group with non-active targeting.
Example 5: tumor inhibition experiment of PASp (DET)/DNA/HA nanoparticles in mouse subcutaneous solid tumor model
B16F10 cell strain is inoculated under the skin of Balb/C mice to establish a melanoma subcutaneous tumor model, and the tumor grows to about 30 mm3The gene therapy is divided into groups, the mouse tail vein is injected with PASp (DET)/DNA/HA nano-particles carrying the sFlt-1 plasmid gene for gene therapy, the single administration dosage is 20 mu g of the sFlt-1 plasmid, the administration frequency is 3 times, and the administration interval time is 2 days, namely 1 time is respectively administered on the 0 th day, the 3 th day and the 6 th day. The morphological change of the tumor is observed,tumor volumes were measured every 1 day and mice monitored for weight change. In addition, PBS (0.1M phosphate buffer pH7.4) group, sFlt-1 plasmid group, and PASP (DET)/sFlt-1 plasmid complex group and PEG-PASP (DET)/DNA nanoparticle group were set to compare the tumor-suppressing effect. The tumor growth curves are shown in FIG. 6A, compared with the other three groups, the tumor growth of the PASp (DET)/sFlt-1 plasmid/HA nanoparticle group is obviously inhibited, and the tumor inhibition rate of the PASp (DET)/sFlt-1 plasmid/HA nanoparticle group reaches 65%, and is obviously higher than that of the non-actively targeted PASp (DET)/sFlt-1 plasmid complex group and the PEG-PASp (DET)/DNA nanoparticle group. The body weight change curve of the mice is shown in figure 6, and no obvious body weight change is seen in the PASp (DET)/sFlt-1 plasmid/HA nanoparticle group, which indicates that the PASp (DET)/sFlt-1 plasmid/HA nanoparticle group HAs safety when being applied in vivo.
Example 6: tumor inhibition experiment of PASp (DET)/DNA/HA nanoparticles in mouse lung metastasis tumor model
B16F10-luc cell strain is inoculated to Balb/C mice through tail vein injection to establish a melanoma lung metastasis tumor model, whether lung metastasis tumor is formed or not and the growth condition are detected by a small animal living body imaging system (IVIS) after injecting a fluorescein substrate into an abdominal cavity, the mice are injected with PASp (DET)/DNA/HA nanoparticles carrying sFlt-1 plasmid genes in tail vein for gene therapy, the single administration dose is 20 mu g of sFlt-1 plasmid, the administration times are 3 times, and the administration interval time is 1 day, namely 1 administration is carried out on 0 th, 2 th and 4 th days respectively. The lung tumor growth was monitored by IVIS measurements of bioluminescence and mouse weight changes were monitored. In addition, PBS (0.1M phosphate buffer pH7.4), sFlt-1 plasmid, PASP (DET)/sFlt-1 plasmid complex, and PEG-PASP (DET)/DNA nanoparticle were prepared, and the antitumor effects of the respective groups were compared. The bioluminescence intensity values positively correlated to tumor growth are shown in fig. 7 a. The bioluminescence intensity values for the PASp (DET)/sFlt-1 plasmid/HA nanoparticle group were significantly lower than those for the other groups, and tumor growth was significantly inhibited. Tumor growth was significantly inhibited in mice treated with pasp (det)/sFlt-1 plasmid/HA nanoparticles compared to the non-actively targeted PEG-pasp (det)/DNA nanoparticle group. The body weight change curve of the mice is shown in B in figure 7, and no obvious body weight change is seen in the PASp (DET)/sFlt-1 plasmid/HA nanoparticle group, which indicates the safety of the application of the PASp (DET)/sFlt-1 plasmid/HA nanoparticles in vivo.
Claims (1)
1. An active targeting gene delivery nanoparticle is characterized in that the nanoparticle consists of a cationic polymer, hyaluronic acid and a negatively charged gene, the cationic polymer and the negatively charged gene form a compound core, the hyaluronic acid is coated on the surface of the compound core through the charge action and the chemical bond combination action,
the active targeting gene delivery nanoparticle is prepared by the following method:
the method comprises the following steps: preparing the cationic polymer, the negatively charged gene, the hyaluronic acid and the cross-linking agent into solutions with proper concentrations by using proper solvents respectively;
step two: uniformly mixing the cationic polymer solution and the negatively charged gene solution according to a proper proportion to ensure that the cationic polymer and the negatively charged gene form a composite inner core through electrostatic acting force, wherein the particle size of the composite inner core is 10-200 nm;
adding a mixed solution of hyaluronic acid and a cross-linking agent into a compound solution of the cationic polymer and the gene according to a proper proportion, so that the hyaluronic acid is coated on a compound core through charge action and chemical bond combination action to form nanoparticles with the particle size of 20-400nm, wherein the concentrations of the cationic polymer and the hyaluronic acid are both 0.1-10 mu g/mu l, and the concentration of the gene is 0.01-10 mu g/mu l; the mole ratio of nitrogen atoms capable of being protonated in the cationic polymer to phosphate radicals in the genes is 1-60; the molar ratio of nitrogen atoms which can be protonated in the cationic polymer to COO-in the hyaluronic acid is 0.1-20; (ii) a
The cationic polymer is one of PASP (EDA), PASP (DET), PASP (TET) or PASP (TEP); the molecular weight of PASp (EDA) is 1.0-100KDa, the molecular weight of PASp (DET) is 1.0-100KDa, the molecular weight of PASp (TET) is 1.0-100KDa, and the molecular weight of PASp (TEP) is 1.0-100 KDa;
the cross-linking agent is carbodiimide/NHS, the solvent for preparing the solution is buffer salt solution or water, and the buffer salt solution is any one or more mixed solution of Tris-HCl, HEPES or phosphate solution;
the molecular weight of the hyaluronic acid is 5.0-2000 KDa;
the gene with negative charge is DNA or RNA.
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| CN101417134A (en) * | 2008-05-13 | 2009-04-29 | 中国药科大学 | Hyaluronic acid decorated novel tertiary structure non-virogene transmission system and use thereof |
| JP5661626B2 (en) * | 2009-07-23 | 2015-01-28 | 国立大学法人 東京大学 | Anionic polymer, polyion complex and ternary polymer complex using the anionic polymer, and pharmaceutical composition |
| WO2011096408A1 (en) * | 2010-02-02 | 2011-08-11 | 国立大学法人 東京大学 | Fine composite particles and method of producing same, and pharmaceutical composition using fine composite particles |
| WO2014078399A1 (en) * | 2012-11-13 | 2014-05-22 | Baylor College Of Medicine | Multi-arm biodegradable polymers for nucleic acid delivery |
| CN103255174B (en) * | 2013-05-02 | 2015-10-28 | 天津大学 | With liquid and application that the hyaluronic acid of polyoxyethylene glycol grafting is outer field ternary complex and ternary complex |
| CN103665384B (en) * | 2013-05-20 | 2016-10-05 | 中国药科大学 | Novel cation graft copolymer and MULTIPLE COMPOSITE non-viral gene vector preparation method and application |
| CN104258416B (en) * | 2014-09-25 | 2018-05-25 | 山东大学 | Deliver the nano-carrier and preparation method of drug and gene altogether based on oligonucleotide |
| WO2016138528A1 (en) * | 2015-02-27 | 2016-09-01 | Wayne State University | Methods and compositions relating to biocompatible implants |
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