CN105031653A - Compound preparation of nucleic acid and chemical drugs and preparation method of compound preparation - Google Patents
Compound preparation of nucleic acid and chemical drugs and preparation method of compound preparation Download PDFInfo
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Abstract
The invention discloses a compound preparation of nucleic acid and chemical drugs and a preparation method of the compound preparation. The compound preparation is a mixture of a bio-macromolecular drug and a micromolecular chemico-pharmaceutical preparation, wherein the micromolecular chemico-pharmaceutical preparation is a chemico-pharmaceutical preparation used for treatment of inflammation; the bio-macromolecular drug is a bio-macromolecular drug wrapped with non-viral vectors. The compound preparation provided by the invention has the benefits as follows: the bio-macromolecular nucleic acid drug nano preparation is prepared by utilizing biological materials for physical safety to wrap therapeutic genes, therefore the disadvantages of high toxicity and recurrent attacks due to the single therapeutic means of chemical drugs are avoided, and the defect that the curative effect is slow due to the single therapeutic means of gene treatment is compensated; besides, inflammation can be radically cured on the basis of preventing the inflammation from deteriorating, and vitro and in vivo experiments confirm that the cationic liposome, polypeptide, hyaluromic acid and nucleic acid vector of our own design is a good delivery carrier for genetic materials, and the compound preparation provides a feasible scheme for combined treatment using pharmaceutical chemicals and biological medicines.
Description
Technical field
What the present invention relates to is a kind of medicament delivery method of technical field of pharmaceuticals, particularly a kind of nucleic acid and chemicals compound formulation and preparation method thereof, especially can be used for the biomacromolecule nucleic acid drug preparation of inflammation and the compound formulation of existing small-molecule chemical treated with combined medication for a kind of.
Background technology
Inflammation, particularly arthritis, ubiquity in mid-aged population, bring very big inconvenience with painful to the routine work of patient life, joint is the more inaccessible target organ of Drug therapy, no matter be intravenous injection, intramuscular injection or oral medication, the drug level that intraarticular reaches is always limited, and these Therapeutic Method exist certain side effect.And although the approach of joint puncture administration to a certain degree overcomes above-mentioned Problems existing, but because the half-life of medicine after local injection treatment is short, repeated localised puncture joint is easier to bring more infection chance, arthrocentesiS is treated on widely using, there is certain limitation.And the gene therapy method recently carried out, then can overcome the drawback of above Therapeutic Method, its main advantage show by coding a certain anti-inflammatory substance gene by vector in synovium of joint, reach and efficiently, for a long time, stably express this anti-inflammatory substance to treat the object of rheumatoid arthritis, expect to change therefrom the present situation for the treatment of rheumatoid arthritis at present.Such as biomacromolecule nucleic acid drug interleukin 1 receptor antagonist (interleukin1receptorantagonist, IL-1Ra) can regulate the expression of interleukin-1 (IL-1) on gene level, thus inflammation-inhibiting.
But the electronegative nucleic acid drug of injection is difficult to enter cell separately, so need suitable delivery vehicles to enter nucleus to assist nucleic acid drug, thus reaches the site of curing the disease.Current non-virus carrier become owing to there is no the potential safety hazards such as immunogenicity research focus (people such as YinH, based on the non-virus carrier of gene therapy, nature-summary-hereditism .2014; 15:541-55.), but owing to being difficult to the chemical toxicity that overcomes and low transfection activity is difficult to become pharmaceutical carrier.Therefore, the Main Means for the treatment of at present still adopts small-molecule chemical medicine to be completed by subcutaneous or intramuscular injection.And most chemicals is hormone medicine (Chinese doctor studies magazine for Xiao Zhengyu, the drug treatment of rheumatoid arthritis, the 9th phase, 1993 years).Heavy dose of long term injections easily causes such as infection, femur head necrosis, digestive tract hemorrhage, hypertension, obesity etc. toxic and side effects to patient.
In order to break through this technical bottleneck, the nano-particle that the present invention utilizes cationic polypeptide, classical non-toxic lipid body wraps up interleukin 1 receptor antagonist and existing chemicals injection are mixed to form mix preparation and then carry out joint cavity injection.
Up to the present, the method utilizing merely biomaterial parcel nucleic acid drug to carry out treating is still at the experimental stage.In order to can by this Technology application in practical application, we intend to combine the hydrocortisone acetate suspension injection technique of having gone on the market, the nano-particle formed the parcel IL-RapcDNA optimized and triamcinolone acetonide acetate injection form suspension injection, carry out Arthritis Treated by Injecting in Articular Cavity arthritis.Because joint cavity injection belongs to topical, simple many relative to being administered systemically, if utilize polypeptide safely and efficiently to carry nucleic acid drug to attempt the Regimen Chemotherapy arthritis adopting chemical drugs to carry out therapeutic alliance together with biological medicament together with liposome, wish that the method has better application prospect.
Summary of the invention
The object of the invention is to overcome the deficiency in existing arthritis technology, a kind of nucleic acid and chemicals compound formulation and preparation method thereof are provided.The present invention by the ternary nano granule of nucleic acid, cationic polypeptide (as Fig. 1), cationic-liposome assembling, and wraps up hyaluronic quaternary nano-particle on this basis and forms suspension injection with commercially available triamcinolone acetonide acetate injection respectively: (1) utilizes the ternary nano granule and quaternary nano-particle applied for a patent; (2) commercially available triamcinolone acetonide acetate injection is utilized; (3) (1) and (2) is carried out being mixed with compound formulation.Therefore, we are compared with existing lipopolyplexes (liposome), not only avoid the toxicity (polypeptide is human endogenous's property molecule) of polycation, and effectively breach the drawback being realized long-acting circulation in body by PEGization.Biological responding after taking into account the stability before arrival target cell and entering target cell.
The object of the invention is to be achieved through the following technical solutions:
The present invention relates to the ternary nano granule with nucleic acid, cationic polypeptide, cationic-liposome assembling, and wrap up the suspension injection (see Figure 10) that hyaluronic quaternary nano-particle formed with commercially available triamcinolone acetonide acetate injection respectively on this basis.Be specifically related to a class wrapped up ternary nano granule that nucleic acid formed by cationic polypeptide and cationic-liposome, covered the quaternary nano-particle of the surface band negative charge that hyaluronic acid is formed and commercially available triamcinolone acetonide acetate injection at this ternary nano particle surface.
First aspect, the present invention relates to a kind of compound formulation, and described compound formulation is the mixture of biopharmaceutical macromolecular drug and small-molecule chemical pharmaceutical preparation.
Preferably, described small-molecule chemical pharmaceutical preparation is the chemicals medicine for inflammation treatment; Described biopharmaceutical macromolecular drug is non-virus carrier wrapping biological macromolecular drug.
Preferably, described chemicals medicine is specially commercially available triamcinolone acetonide acetate injection.
Preferably, described non-virus carrier wrapping biological macromolecular drug comprises ternary nano granule or quaternary nano-particle; Described ternary nano granule comprises cationic-liposome, polypeptide, biopharmaceutical macromolecular drug component; Described quaternary nano-particle comprises cationic-liposome, polypeptide, biopharmaceutical macromolecular drug component, hyaluronic acid.
Preferably, the preparation of described ternary nano granule is specially: mix described cationic-liposome and polypeptide, gained mixture is transferred in described biopharmaceutical macromolecular drug component solution, hatches the nano-particle that 15 ~ 35 minutes obtain surface band positive charge under room temperature, be ternary nano granule;
More preferably, the mass ratio of described mixture cationic liposome and polypeptide is (0.5 ~ 2): (2 ~ 10); The mass ratio of described polypeptide and biopharmaceutical macromolecular drug component is (2 ~ 10): (0.5 ~ 2);
Further preferably, the mass ratio of described mixture cationic liposome and polypeptide is 0.75:4 or 1:4; The mass ratio of described polypeptide and biopharmaceutical macromolecular drug component is 4:1.
Preferably, the preparation of described quaternary nano-particle is specially: in described ternary nano granule, add hyaluronate sodium, mix homogeneously, places, obtains quaternary nano-particle;
More preferably, the time of described placement is 5 ~ 20 minutes; The mass ratio adding quality and described polypeptide of described hyaluronate sodium is (5 ~ 35): (2 ~ 10);
Further preferably, the time of described placement is 5 minutes; The mass ratio adding quality and described polypeptide of described hyaluronate sodium is 14.2:4;
Preferably, described biopharmaceutical macromolecular drug component is nucleic acid, specifically comprises DNA, siRNA, shRNA, microRNA;
Preferably, described biopharmaceutical macromolecular drug component is DNA;
Further, described DNA is specially IL-1RapcDNA.
Preferably, described polypeptide comprises the sequence as shown in SEQIDNo.1 or SEQIDNo.2, polycationic polypeptides, and connects the cationic polypeptide of targeting group.
More preferably, described polypeptide comprises the sequence as shown in SEQIDNo.1 or SEQIDNo.2.
Preferably, described cationic-liposome is obtained by following steps: be (3 ~ 1) by mass ratio: 1 (2,3-bis-oily oxygen base propyl group) trimethyl ammonium chloride (DOTAP) and 1, the chloroformic solution of 2-bis-oleoyl-sn-glycero-3-phosphoethanolamine (DOPE) rotates steaming, removes solvent, then hydrated overnight, ultrasonic 30 ~ 60 minutes.
More preferably, the mass ratio 1:1 of described DOTAP and DOPE.
Second aspect, the present invention also provides a kind of preparation method of described compound formulation, and described preparation method is specially: by biopharmaceutical macromolecular drug and small-molecule chemical pharmaceutical preparation physical blending.
Preferably, in described blended artifact macromolecular drug and small-molecule chemical pharmaceutical preparation, the concentration ratio of effective ingredient is 40:1.
Compared with prior art, beneficial effect of the present invention is:
1, utilize chemicals and genomic medicine combination formulations, reduce the drawback of toxicity and the state of an illness bounce-back utilizing separately chemicals to bring, and extend drug treating time, overcome the inefficient defect utilizing merely genomic medicine to bring;
2, because the application's lipid used and polypeptide are the medical material of endogenic bio-safety, and this nanometer delivery system by after joint cavity injection by positive and negative charge effect rapidly by synovial cell's endocytosis of intracavity, and the lipid composition in nanometer system realizes endocytosis escape by melting membrane interaction, thus effectively biopharmaceutical macromolecular drug component is discharged in cytoplasm, while chemical drugs works, diminished inflammation gradually by gene regulation from source;
3, hyaluronic acid (HA) is also wrapped in the surface of above-mentioned ternary nano granule by the application further, not only efficiently avoid the high toxicity of polycation, and hyaluronic acid has the therapeutical effect such as antiinflammatory, contributes to the symptom of releasing arthritis.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:
Fig. 1: the structure schematic diagram of ternary nano granule and quaternary nano-particle;
Fig. 2: the gel electrophoresis figure result of ternary nano granule and quaternary nano-particle;
Fig. 3: the Zeta potential figure of ternary nano granule and quaternary nano-particle;
Fig. 4: the grain-size graph of ternary nano granule and quaternary nano-particle;
Fig. 5: the synovial cell C518 luciferase transfection activity of ternary nano granule and quaternary nano-particle is schemed;
Fig. 6: ternary nano granule and quaternary nano-particle wrap up cell conditioned medium IL-1Ra protein level after IL-1RapcDNA transfection synovial cell C518;
Fig. 7: the cell survival rate of synovial cell after the nanoparticle vector parcel IL-1RapcDNA complex solution of different proportion is cultivated;
Fig. 8: rat change of ankle joint girth of 4 days after modeling;
Fig. 9: rat is IL-1Ra protein level in serum after 48h upon administration;
Figure 10: be preparation method schematic diagram of the present invention.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.
The present invention comprises the ternary nano granule of wrapping biological macromole nucleic acid drug and the physico-chemical property characterizing method of quaternary nano-particle: gel electrophoresis, dynamic light scattering and Zeta potential; The DNA plasmid of preliminary transfection activity and toxicity test is luciferase plasmids cell is synovial cell C518; Parcel therapeutic genes is interleukin receptor antagonist (IL-1RapcDNA); The acute arthritis model that experiment in vivo adopts Wistar rat (male, 180-200g, Si Laite) to build.ELISA is utilized to detect the expression (RayBioHumanIL-1RAELISAKit, RayBiotech) of cell conditioned medium liquid IL-1Ra albumen.
embodiment 1
The present embodiment relates to the preparation method (as shown in Figure 1) of ternary nano granule and quaternary nano-particle:
Join in aqueous solution nucleate after a certain amount of cationic-liposome is mixed with polypeptide and continue to mix homogeneously.At room temperature hatch 30 minutes obtained ternary nano granules, then hyaluronic acid to be added in this mixture and abundant mix homogeneously, leave standstill 15 minutes obtained quaternary nano-particle.
The mass ratio of cationic-liposome and polypeptide is (0.5 ~ 2): (2 ~ 10); The mass ratio of described polypeptide and IL-1RapcDNA is (2 ~ 10): (0.5 ~ 2); The mass ratio adding quality and described polypeptide of described hyaluronate sodium is (5 ~ 35): (2 ~ 10).
Carrying out in aqueous for the system surveying physico-chemical property of ternary nano granule and quaternary nano-particle; Carrier system serum-free medium for cell experiment replaces ultra-pure water.
embodiment 2
The present embodiment relates to the gel electrophoresis of ternary nano granule prepared by method described in embodiment 1 and quaternary nano-particle, specifically comprises following operation:
Take the agarose powder of 0.3g, pour in conical flask, 30ml1 × TAE buffer is added in conical flask, under the condition of heating, it is fully dissolved, after temperature drops to 65 DEG C, add Gelredinwarter3 μ L, pour into rapidly in glue groove, insert sample comb, at room temperature place 0.5 ~ 1h and wait for that glue becomes and solidifies state.Then sample comb is extracted, then TAE buffer is poured in sample cell make its submergence gel.Prepare ternary nano granule and the quaternary nano-particle 10 μ L of different proportion, 6 × sample-loading buffer 2 μ L and complex are mixed, loading 10 μ L, loading 10 μ LnakedIL-1RapcDNA in contrast simultaneously.Electrophoresis 45min under 110mV voltage, turns off electrophoresis subsequently, and taking-up gel is placed in ultraviolet gel imaging system and observes and record electrophoretic image.
Result is as Fig. 2, band is followed successively by NakedDNA from left to right, sample L:P1:D=1:4:1 (1), L:P2:D=1:4:1 (2), L:P1:D=0.75:4:1 (3), L:P2:D=0.75:4:1 (4), L:P1:D:H=1:4:1:14.2 (H-1), L:P2:D:H=1:4:1:14.2 (H-2), L:P1:D:H=0.75:4:1:14.2 (H-3), L:P2:D:H=0.75:4:1:14.2 (H-4), wherein, H represents hyaluronic acid; L represents cationic-liposome; D representation DNA; The polypeptide of P1 to be aminoacid sequence be RRRRRRRRRRRRRRRRKRPTMRFRYTWNPMK (SEQIDNo.1); The polypeptide of P2 to be aminoacid sequence be RRRRRRRRRRRRRRRRKMPNWTYRFRMTPRK (SEQIDNo.2); Each data represent the mass ratio (following chart is same) of each component.
| Sequence number | L (cationic-liposome) | P1/P2 (polypeptide) | D(DNA) | H (hyaluronic acid) |
| 1 | 1 | P1:4 | 1 | - |
| 2 | 1 | P2:4 | 1 | - |
| 3 | 0.75 | P1:4 | 1 | - |
| 4 | 0.75 | P2:4 | 1 | - |
| H-1 | 1 | P1:4 | 1 | 14.2 |
| H-2 | 1 | P2:4 | 1 | 14.2 |
| H-3 | 0.75 | P1:4 | 1 | 14.2 |
| H-4 | 0.75 | P2:4 | 1 | 14.2 |
The position that we can observe ternary nano granule and quaternary nano-particle from electrophoretogram is parked in starting point, illustrates that our carrier has wrapped up IL-1RapcDNA completely, thus available protecting DNA, avoid DNA to be degraded before cytophagy.
embodiment 3
The Zeta potential of the present embodiment to granule prepared by embodiment 2 is tested: the current potential of complex uses Particle Size Analyzer to measure.
Adopt zetasizer2000 to measure the surface charge of granule, sample determination set of time is automatically, each sample determination 3 times, mapping of averaging.
The zeta potential measurement result of complex as shown in Figure 3.The zeta potential of these 8 different nano-particle is more stable as seen from the figure: PLD is positively charged, and HPLD is electronegative, and the absolute value of its electric charge is greater than 25mV, illustrates that the dispersion that they are formed is also relatively stable.
embodiment 4
The particle diameter of the granule (the complex 200 μ L of different proportion) that the present embodiment is prepared embodiment 2 is tested, specific as follows: under room temperature condition, the hydrated radius of Q-complexes is measured with MalvernNanoZS laser particle analyzer, refractive medium is set to water, refractive index is 1.33, viscosity is 0.8872cP, each sample determination 3 times, mapping of averaging.
First, at room temperature preheating Particle Size Analyzer 30min, particle solution is added in micro-example pond, 450 μ L are diluted to ultra-pure water, micro-example pond is inserted in the test trough of Particle Size Analyzer, each sample record three test results, observe and record the meansigma methods of sample particle diameter, also will note the dispersibility (PDI) of sample simultaneously.
The particle diameter of granule is shown in Fig. 4, and the particle diameter of these 8 different nano-particle is all more stable as seen from the figure, and substantially at about 100nm, its size is conducive to cell endocytic.
embodiment 5
The present embodiment is specially the luciferase transfection activity experiment that embodiment 2 prepares the synovial cell C518 of ternary nano granule and quaternary nano-particle, comprises the steps:
Be inoculated in by cultured cell in 96 orifice plates, every porocyte number is 2 × 10
4individual, be placed in cell culture incubator and cultivate 24h.Add different complex: the quality of the DNA that every hole adds is 250ng, every hole adds the complex solution (namely the quality of every 200 μ L nanoparticles solution DNA is 250ng) of 200 μ L, and parallel 3 multiple holes, diluent media is Opti-MEM culture medium, and with the complex of commercialization transfection reagent PEI25KDa and DNA as a control group.After hatching 24h, take out 96 orifice plates and discard culture fluid, after every hole rinses one time with 200 μ L1 × PBS buffer solution, every hole adds the complex solution of 200 μ L, be placed in after cell culture incubator cultivates 4h, take out 96 orifice plates, suck culture fluid, every hole adds the DMEM/f12 culture medium containing 10% hyclone of 200 μ L, is placed in cell culture incubator and cultivates 24h.Detect instantaneous light emission intensity: after spending the night, take out 96 orifice plates, from each hole, sucking-off 20 μ L lysate is in the streaming pipe of correspondence, open illumination meter, preheating 20min, 20 μ L luciferase substrate are added in each streaming pipe during test, quick piping and druming is even, detects its instantaneous light emission intensity (RLU).Detect Tot Prot: employing MicroBCA algoscopy determines the Tot Prot in lysate.Carry out measure of cell transfection efficiency (i.e. the expression of luciferase protein) by the relative luminous intensity of sample divided by institute total (RLU/mgprotein) after the total protein concentration of sample, each sample parallel surveys 3 times, mapping of averaging.
Testing result as shown in Figure 5, therefrom can be found out, adopt ternary nano granule suitable with conventional transfection reagent PEI25kDa to the effect of C518 transfection, and the effect of corresponding quaternary nano-particle transfection synovial cell C518 is far above these two groups.
embodiment 6
The present embodiment relate to embodiment 2 prepare ternary nano granule and quaternary nano-particle transfection synovial cell C518 after IL-1Ra protein level test, specifically comprise:
By the complex transfection synovial cell of the carrier of IL-1RapcDNA and different proportion composition, step is with the process of the in-vitro transfection of luciferase reporter gene plasmid, every hole is put into cell culture incubator after adding the complete medium of 200 μ L and is cultivated 48h, afterwards by the cell culture supernatant sucking-off in each experimental group and matched group hole, be transferred in EP pipe, etc. to be determined.This test measures on 96 orifice plates, and concrete operations detect description according to RayBioHumanIL-1RAELISA test kit.
Testing result as shown in Figure 6, ternary nano granule and quaternary nano-particle are to the efficiency of synovial cell C518 transfection IL-1RapcDNA all higher than naked IL-1RapcDNA and PEIkDa, and particularly its IL-1RapcDNA protein expression of sample L:P1:D=1:4:1 and L:P1:D:H=1:4:1:14.2 is far above other group.
embodiment 7
The present embodiment relates to the toxicity test of synovial cell C518, is specially and adopts CCK-8CellProliferationandCytotoxicityAssayKit to investigate the nano-particle complex of parcel IL-1RapcDNA to the toxicity of synovial cell, comprise the steps:
Before toxicity test starts, first seed cells in 96 holes, the cell number in every hole is 10
4individual, after inoculation 24h, take out 96 orifice plates and discard culture fluid, after every hole rinses one time with 200 μ L1 × PBS buffer solution, every hole is to the complex solution of 200 μ L of different proportion, the multiple hole of each sample parallel 3, is placed in after cell culture incubator cultivates 4h, the complex solution of sucking-off 100 μ L, CCK-8 reagent 10 μ L is added in the every porocyte of 96 orifice plate, after lucifuge cultivates 1h in cell culture incubator, finally, by the absorbance OD value of multi-functional microplate reader working sample at 450nm place.Cell survival rate (percentage ratio) compares Normocellular absorbance to represent with the absorbance of testing sample.
As shown in Figure 7, most ternary nano granule and the toxicity of quaternary nano-particle to synovial cell C518 are less than PEI25kDa to testing result.The cytoactive of sample segment group reaches more than 90%.
embodiment 8
The present embodiment relates to foundation and the experiment in vivo of rat acute arthritis model, comprises the steps:
Get rat 30, every complete Freund's adjuvant only injecting 100 μ L at its joint, observes the arthroncus degree of rat, is equally divided into 5 groups for after modeling second day, often organize 6, be respectively blank group, non-treatment group, chemical medicine treatment group, chemical medicine gene mentation treatment group, gene therapy group.Rat was treated in after modeling second day, what we selected is that L:P1:D=1:4:1 ratio carrys out compound IL-1RapcDNA formation nano-particle complex to carry out arthritic treatment, in the medicine 100 μ L that the articular cavity local injection of rat is different, chemical medicine treatment group is 100 μ L triamcinolone acetonide acetate injections (concentration is 10mg/mL), containing 10mg/mL triamcinolone acetonide acetate injection 50 μ L and nano-particle complex 50 μ L (containing 12.5 μ gIL-1RapcDNA) in chemical medicine gene mentation treatment group, in 100 μ L solution of gene therapy group (containing 25 μ gIL-1RapcDNA), negative genes treatment group carrys out compound PGL3DNA by L:P1:D=1:4:1 ratio to form nano-particle complex (containing 25 μ gPGL3DNA), a medicine is given again every one day, the 48h expression of the IL-1Ra albumen in ELISA method detection bodies after second time administration.
Testing result as shown in figs. 8 and 9, in chemical medicine treatment group and the curative effect that identical improvement arthroncus degree can be seen in chemical medicine gene mentation treatment group, although do not see weakening of obvious Articular swelling in therapeutic gene group, but from the expression of IL-1RapcDNA albumen, see there is certain increase, illustrate that genome inherently improves the arthritic state of an illness, just curative effect is slower.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (10)
1. a compound formulation, is characterized in that, described compound formulation is the mixture of biopharmaceutical macromolecular drug and small-molecule chemical pharmaceutical preparation.
2. compound formulation according to claim 1, is characterized in that, described small-molecule chemical pharmaceutical preparation is the chemicals medicine for inflammation treatment; Described biopharmaceutical macromolecular drug is non-virus carrier wrapping biological macromolecular drug.
3. compound formulation according to claim 2, is characterized in that, described non-virus carrier wrapping biological macromolecular drug comprises ternary nano granule or quaternary nano-particle;
Described ternary nano granule comprises cationic-liposome, polypeptide, biopharmaceutical macromolecular drug component;
Described quaternary nano-particle comprises cationic-liposome, polypeptide, biopharmaceutical macromolecular drug component, hyaluronic acid.
4. compound formulation according to claim 3, it is characterized in that, the preparation of described ternary nano granule is specially: mix described cationic-liposome and polypeptide, gained mixture is transferred in described biopharmaceutical macromolecular drug component solution, hatch the nano-particle that 25 ~ 35 minutes obtain surface band positive charge under room temperature, be ternary nano granule.
5. compound formulation according to claim 3, is characterized in that, the preparation of described quaternary nano-particle is specially: in described ternary nano granule, add hyaluronate sodium, mix homogeneously, places, obtains quaternary nano-particle.
6. compound formulation according to claim 3, is characterized in that, described biopharmaceutical macromolecular drug component is nucleic acid, specifically comprises DNA, siRNA, shRNA or microRNA.
7. compound formulation according to claim 3, is characterized in that, described polypeptide comprises the sequence as shown in SEQIDNo.1 or SEQIDNo.2, polycationic polypeptides, and connects the cationic polypeptide of targeting group.
8. compound formulation according to claim 3, it is characterized in that, described cationic-liposome is obtained by following steps: be (3 ~ 1) by mass ratio: 1 (2,3-bis-oily oxygen base propyl group) trimethyl ammonium chloride and 1, the chloroformic solution of 2-bis-oleoyl-sn-glycero-3-phosphoethanolamine rotates steaming, removes solvent, then hydrated overnight, ultrasonic 30 ~ 60 minutes, to obtain final product.
9. a preparation method for the compound formulation according to any one of claim 1 ~ 8, is characterized in that, described preparation method is specially: by biopharmaceutical macromolecular drug and small-molecule chemical pharmaceutical preparation by physical blending.
10. the preparation method of compound formulation according to claim 9, is characterized in that, in described blended artifact macromolecular drug and small-molecule chemical pharmaceutical preparation, the concentration ratio of effective ingredient is 40:1.
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