CN1289142C - Medicinal new dosage form comtaining SA liposome as medicine carrier - Google Patents
Medicinal new dosage form comtaining SA liposome as medicine carrier Download PDFInfo
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- CN1289142C CN1289142C CN 02136437 CN02136437A CN1289142C CN 1289142 C CN1289142 C CN 1289142C CN 02136437 CN02136437 CN 02136437 CN 02136437 A CN02136437 A CN 02136437A CN 1289142 C CN1289142 C CN 1289142C
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Abstract
The present invention relates to a safe and high-efficiency medical carrier, namely an SA liposome. The present invention also provides a medical composition containing the SA liposome used as a medical carrier. The SA liposome used with various medical active components, such as polypeptide, protein, nucleic acid, etc., has little immunogenicity and good stability. To a polypeptide medicine, the SA liposome can extend the half-life period and has certain drug release action. To a nucleic acid medicine, the SA liposome can transfect various mammal eukaryotic cells and insect cells in high efficiency under the existence of blood serum.
Description
Technical field
The present invention relates to medical science and field of pharmacology, relate more specifically to, and contain the pharmaceutical composition of SA liposome as pharmaceutical carrier with the purposes of SA liposome as pharmaceutical carrier.
Background technology
Drug delivery system can greatly improve the safety and the efficient of drug use, and makes new treatment become possibility.The advantage that ideal drug delivery system should have comprises: (1). in pharmaceutically acceptable scope, keep levels of drugs.(2). when reducing targeted delivery to the injury of non-targeted cells or tissue.(3). reduce the effective dose of medication, reduce the medicine side reaction.(4). the application of the medicament (as protein, polypeptide) that the interior half-life of promotion body is short etc.
Drug delivery system is broadly divided into three types by its functional characteristics: polymer-drug delivery system, liposome medicament transmission system and intelligent transmission system.In recent years, drug delivery system has not little development but still has and much demands improvements urgently: (1). discharge the transmission system material of medicine and toxicity and the side effect that catabolite may exist thereof.(2). by system itself or caused inconvenience of introduction method and discomfort.(3). new transmission system costs an arm and a leg.
Along with development of biology, the quantity of polypeptide and pharmaceutical grade protein will increase rapidly.The sales volume of all kinds of in recent years polypeptide drugs in the whole world increased by 20%.Polypeptide and pharmaceutical grade protein generally are drug administration by injection, and concerning this class medicine, the application of its drug delivery system has great potential.
For a long time people dream of can guide effect site, single-minded ground of handlebar medicine always, and control drug release accurately makes the transmission system of drug effect maximum.In recent years, this dream has begun to become the fact and each branch of medical science has been comprised that cardiology, ophthalmology, endocrinology, oncology, lung science, immunology etc. have produced tremendous influence.Drug delivery system has surpassed 1,000,000,000 dollars in the annual sales amount of the U.S., still in growth at full speed.The drug delivery principle also has been applied to a plurality of fields such as pesticide, fertilizer, herbicide, spice, flavoring agent.Drug delivery system has become the main aspect of medicine competition.
Many medicines, cancer therapy drug particularly, when acting on target cell, normal cell had suitable toxicity, can produce stronger side effect to body, thereby it uses and dosage often is limited in certain scope, reduces drug dose, improves drug effect and reduce toxic and side effects to seem very important.Most polypeptide drugs half-life in vivo are short, just need repetitively administered when treatment, and this can make vivo medicine concentration rise and fall, thereby cause the disorder of self-adjusting system.
Medicine with can obvious medicament curative effect enhancement after liposome combines, reduce toxicity, reduce side reaction.Liposome is to be used for the research of genetic defect treatment of diseases as pharmaceutical carrier at first, expands to treatment for cancer antibacterium, fungal infection, the treatment of immune disease, aspects such as metal-chelating therapy afterwards again to.At present existing be that the medicine of carrier enters clinical with the liposome.
Liposome has the following advantages as pharmaceutical carrier: 1) owing to the liposome of mainly being made up of natural phospholipid and cholesterol, it is biodegradable to enter the interior back of body, can not accumulate in vivo, and immunogenicity is little, is easy to be accepted by body.2) water solublity and fat-soluble medicine all can be embedded in the liposome and slowly discharge from liposome, make drug effect continue the long period.3) by with cell endocytic with merge to interact, liposome can directly be sent into medicine in the cell and play a role, thereby avoids using high concentration free drug effect body.4) liposome distribution in vivo can be controlled, thereby makes it discharge medicine at the pathological tissues place, reduces the toxicity to normal structure, reduces side reaction, strengthens drug effect.
Liposome is applied to the report of conveying in the micromolecular body of medicine, thereby it mainly has been the anti-degraded prolong drug intravital half-life of molecule of stable drug molecular structure, medicine and certain slow releasing function has been arranged, its lipid components merges by producing with the cell surface that is subjected to medicine tissue and organ in addition, is beneficial to the release and the absorption of medicine again.
The pharmaceutical preparation of liposome at present has various forms such as injection, oral and topical agent.Compare with other carrier system, do not need covalent cross-linking between liposome and the contained medicine, medicine is without chemical modification.Liposome can merge by film sends contained medicine into cell, makes relatively poor cell of phagocytic activity such as cancerous cell also be easy to ingestion of drugs, and this is the double-deck peculiar function of liposomal lipid.The liposome of various structures can be used for different conditions.
Liposome is the same with other carrier system, after entering in the body, very fast cell by reticuloendothelial system is engulfed, from peripheral blood circulation, be eliminated, and concentrate on liver, spleen tissue, though this is to favourable other therapeutic purposes that are unfavorable for of some disease of treatment liver, spleen tissue.
At present, Doxoribicin, MTP-PE, the Liposomal formulation of medicines such as Cisplatin and Amphotericin B is obtained better curative effect in the treatment of clinical I phase and II phase, showed the brilliant prospect that liposome is used for the treatment of as pharmaceutical carrier.
Lipidosome drug carrier is used for conventional therapy, also need solve some technical problems.The one, Liposomal formulation is long preservation how.The 2nd, present preparation method often relates to the control of distillation, temperature, even process such as dialysis, is unfavorable for the daily quick preparation of suitability for industrialized production or hospital.The 3rd, the repeatability of preparation must be good.The 4th, how accurately to calculate the embedding rate of medicine.Along with the solution of these problems, drug liposome preparation will become effective routine administration.
Cationic-liposome is the Ceng Zuowei pharmaceutical carrier also.At present, the nucleic acid substances that cationic-liposome generally is used to carry negative charge passes on, and rarely has the carrier that is used as polypeptide drugs.Cationic-liposome is different with common liposome action principle, and the cation lipid surface has positive charge.When medicine and cationic-liposome interact, not only part can be embedded in the vesicle of liposome, also can produce electrostatic interaction, and the hydrophobic part in the polypeptide drug molecule can insert lipid bilayer and stablized owing to the positive charge of cation lipid surface and electronegative drug molecule.Cationic-liposome and electronegative cell surface have very strong affinity interaction, help medicine and enter cell.
Because cationic-liposome is simple to operate, therefore be subjected to increasing attention.But existing commodity cationic-liposome all contains the cation composition of synthetic, and costs an arm and a leg, and the toxicity of pair cell is bigger, is unfavorable for intravital drug conveying.
Ideal drug delivery carrier not only need have very high importing efficient in the cell of isolated culture, suitable intravital turn-over capacity more will be arranged, and anti-degradation capability be arranged, can natural metabolism, and body do not produced toxic and side effects.Although the cationic-liposome that is made of synthetic fat that has has been applied in the cell in vitro transfection and has begun obtaining initial success in the gene therapy in vivo, but they remain in following defective: (1). serum has the obvious suppression effect to most of cationic-liposomes, and the efficient that drug disposition passes on is low.(2). the cation lipid of synthetic is easily discerned by vivo immuning system, produces stronger rejection effect.(3) different cationic-liposome transfection pair cells is selective, and the efficient of the scope of application and transfectional cell is different.
The present invention presses for the new pharmaceutical carrier of exploitation, and described pharmaceutical carrier can carry active constituents of medicine such as polypeptide, albumen or nucleic acid effectively, not suppressed by serum, and immunogenicity is low, and is applied widely.
Summary of the invention
One object of the present invention just provides and a kind ofly effectively, not suppressed by serum, and immunogenicity is low, pharmaceutical carrier applied widely, i.e. SA liposome.
Another object of the present invention provides and contains the pharmaceutical composition of SA liposome as pharmaceutical carrier.
Another object of the present invention provides the purposes of SA liposome at aspects such as gene therapies.
In a first aspect of the present invention, a kind of pharmaceutical composition is provided, it contains active constituents of medicine and as the SA liposome of pharmaceutical carrier.
In a preference, described active constituents of medicine is selected from: polypeptide, protein, nucleic acid.
In another preference, described active constituents of medicine is polypeptide or albumen, and the weight ratio of polypeptide or albumen and SA liposome is 1: 10～1: 300.
In another preference, described active constituents of medicine is a nucleic acid, and the weight ratio of itself and SA liposome is 1: 10～1: 300.
In another preference, described SA liposome is CH
3-(CH
2)
16-CH
2-
+NH
3: C
41H
78NO
8P (DOPE)=0.8: 1～1: 0.8 more preferably is 1: 1 (w/w).
In another preference, described active component is selected from: calcitonin, human calcitonin analogue I, human calcitonin analogue II, osteogenic growth peptide.
In a second aspect of the present invention, the purposes of described SA liposome is provided, it is used to prepare medicine as pharmaceutical carrier.
In a preference, described active ingredient of drugs is selected from polypeptide, protein, nucleic acid.
In a third aspect of the present invention, a kind of transfection method is provided, with nucleic acid and SA liposomal lipid plastid with weight ratio 1: 10.-1: 20 mix, then transfection.
Preferably, described nucleic acid is selected from: DNA, RNA, oligonucleotide, antisensenucleic acids or its mixture.
Description of drawings
The comparison of the time-dose curve of Fig. 1 salmon calcitonin see calcimar (sCT) and SA liposome-sCT complex.
The time graph that falls the blood calcium effect of Fig. 2 human calcitonin analogue I and SA liposome complex.
The blood calcium effect of falling of the complex of Fig. 3 human calcitonin analogue II and SA liposome and the comparison of free human calcitonin analogue II.
The activity curve (with the amount calculating of free osteogenic growth peptide) of the free osteogenic growth peptide of Fig. 4 and itself and SA liposome complex on cell proliferation efficient.
The free SA liposome of Fig. 5 various dose is to the influence of NIH3T3 cell growth.
The ability of Fig. 6 SA liposome and Lipofectin transfection NB-1 cell under whole serum concentration not.
The ability of Fig. 7 SA liposome and Lipofectin transfection P19 cell under whole serum concentration not.
The ability of Fig. 8 SA liposome and Lipofectin transfection PC12 cell under whole serum concentration not.
The specific embodiment
The inventor finds that through extensive and deep research the SA liposome is particularly suitable as the pharmaceutical carrier of active constituents of medicine such as polypeptide, protein and nucleic acid.SA liposome not only immunogenicity is little, and stronger stability is arranged in vivo.For polypeptide drugs, SA lipid physical ability prolongs its half-life and has certain medicament slow release effect.For nucleic acid drug, can be in the presence of serum efficient transfection mammalian species eukaryotic cell of SA liposome and insect cell.Therefore, the SA liposome is a kind of safe, pharmaceutical carrier efficiently.On this basis, finished the present invention
The SA liposome
As used herein, term " SA liposome ", " SA cationic-liposome ", " stearylamine cationic-liposome " or " stearylamine liposome " are used interchangeably, all refer to by stearylamine (Stearylamine, SA) and DOPE (dioleoyl-phosphatidylethanolamine, DOPE) mixture/complex of Gou Chenging.SA was generally about 0.8: 1～1: 0.8 with the ratio of DOPE, preferably was about 1: 1 (w/w).The SA surface of liposome has positive charge, forms complex by electrostatic interaction and electronegative DNA, RNA, oligonucleotide and protein, the electronegative part of polypeptide etc., and cationic-liposome can effectively protect the molecule that is transmitted not to be degraded.Complex can be attracted to cell surface by electrostatic interaction, and fusion by cell and endocytosis are with in the compound molecule transfered cell of institute.
The main cationic hydrophilic fat molecule composition stearylamine (SA) of SA liposome is the hydrophilic polar head that the hydrophobic anchor of the satisfied fatty acid of 18 carbon atoms chain and an amino, and chemical formula is:
CH
3-(CH
2)
16-CH
2-
+NH
3
In the SA liposome, DOPE plays " molecular chaperones " effect, is that the liposome structure of the stable lipid bilayer of formation is necessary.The chemical formula of DOPE (DOPE) is:
A kind of preferred SA liposome is by pressing the complex lipid of about 1: 1 weight ratio (w/w) formation with cationic stearylamine lipid SA (Stearylamine) and DOPE (DOPE).Its molecular structure: CH
3-(CH
2)
16-CH
2-
+NH
3: C
41H
78NO
8P (DOPE)=1: 1 (w/w).
The preparation method of SA liposome is known, for example can be prepared into little monoester lipid bilayer body by the water-bath ultrasonic method.
The SA liposome can be stored in 4 ℃ usually, and stable phase was greater than 6 months.A kind of lipids form commonly used is that water suspension, concentration are 2mg/ml, and is aseptic.
Pharmaceutical composition
Can be used for active constituents of medicine of the present invention and be not particularly limited, can be known in the art or unknown active constituents of medicine.Particularly preferred active component is polypeptide, protein and nucleic acid.
When active component was polypeptide or protein, they can be natural, reorganization or synthetic.In pharmaceutical composition of the present invention, the weight ratio of polypeptide or protein and SA liposome is generally 1: 10～1: 300 (w: w), preferably be 1: 10～1: 100.As used herein, term " nucleic acid " comprises DNA, RNA, oligonucleotide, antisensenucleic acids or its mixture.When active component was nucleic acid, they can be isolating, extractive or synthetic.In pharmaceutical composition of the present invention, the weight ratio of nucleic acid and SA liposome is generally 1: 10～1: 300 (w: w), preferably be 1: 10～1: 100 (w: w), more preferably be 1: 10～1: 50 (w: w).
Preparation of drug combination method of the present invention is not particularly limited, as long as the SA liposome of required ratio is mixed the formation complex with polypeptide drugs.The SA liposome can not cause any destruction to medicine itself.
Certainly, in pharmaceutical composition of the present invention, also can contain other pharmaceutically acceptable carrier and additives, for example water, buffer, glycerol, antioxidant etc.These additives all are to know in the pharmaceutical field.
The dosage form of pharmaceutical composition of the present invention is not particularly limited, as injection, solution, tablet and capsule etc.A kind of optimal way is to make injection, for example liquid solution or suspension; Or be formed in be fit to allocate in solution or the suspension before the injection, the solid form of liquid-carrier.
Medication
In case be made into compositions of the present invention, can directly give object with it.Object to be treated can be an animal; Especially can treat people's object.
The direct conveying of pharmaceutical composition can or be delivered to interstice by subcutaneous, Intradermal, intravenous or intramuscular injection usually and realize.Compositions also can be delivered to focal zone.Other administering mode comprises: blood vessel choked flow administration; The respiratory tract spray delivery; The fixed-point injection transfection of internal organs such as liver, kidney; Inguinal lymph is administered systemically, oral, lung administration, suppository and transdermal or through skin use, with pin, particle gun.The therapeutic dose scheme can be single agent scheme or multi-agent scheme.In the administration operation, should avoid various detergents, strong electrolyte, organic solvent, high temperature, oxidant etc. as far as possible.
Application
The present invention has proved that the SA cationic-liposome not only can be used for polypeptide or protein-based active component, also can be used as non-virus type pharmaceutical carrier and can be applicable to gene therapy.The SA liposome has and transports and protect DNA, RNA, oligonucleotide etc. to enter the ability of cell, and many-sided purposes is arranged.
In addition, experiment of the present invention has also proved can be in the presence of serum efficient transfection mammalian species eukaryotic cell of SA liposome and insect cell.
The effective means that numerous advantages such as therefore, the SA liposome has good stability, anti-degradability is strong, and immunogenicity is weak are in genetic manipulation and gene therapy, the body and external polypeptide transmits.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The preparation of SA cationic-liposome
Get 5mg SA (SIGMA) and 5mg DOPE (SIGMA) (10mg/ml) in the ground round-bottomed flask, high pure nitrogen dries up organic solvent (about 2～3 hours), lyophilization (Labconco, the U.S.) 2～3 hours, add 20ml Millipore sterilized water, inflated with nitrogen is jumped a queue and is sealed, ice-water bath ultrasonic (Shanghai ultrasonic instrument four CQ-50 of factory supersonic generators) 20 minutes, use ultrasonic 30 minutes of probe type ultrasonic instrument (Branson Sonifer CellDisruptorB15 Ultrasound Instrument) probe then, cover nitrogen in the ultrasonic procedure.100,4 ℃ of 000g super centrifugal 30 minutes (Centrikon ultracentrifuge) get supernatant, 159,000g4 ℃ super centrifugal 5 hours, get the intermediate layer, the aseptic membrane filtration of micropore (Millipore) with 0.22 μ m aperture fills the nitrogen packing, in 4 ℃ of preservations.
The slow release of SA liposome-calcitonin complex and prolong half-life effect
2.1SA the formation of liposome-calcitonin complex
1 μ g salmon calcitonin see calcimar (SIGMA) is diluted to 300 μ l with normal saline, and the SA liposome that contains 60 μ g is diluted to 300 μ l with normal saline, both mixings, and room temperature was placed 20～30 minutes, and the reuse normal saline is diluted to 24ml.
2.2SA the formation of liposome-calcitonin-like I or II complex
With 1 μ g human calcitonin analogue I (hCT-I) or human calcitonin analogue II (hCT-II), be diluted to 300 μ l with normal saline respectively.The SA liposome that will contain 60 μ g is diluted to 300 μ l with normal saline, both mixings, and room temperature was placed 20～30 minutes, and the reuse normal saline is diluted to 24ml.
2.3 method of testing
Human calcitonin analogue I, II before and after the effect of intraperitoneal injection comparison SA liposome tire and half-life fasting 18hr, only the male Wistar rat of feeding distilled water is by the body weight random packet, every group 8, SA liposome (matched group), salmon or human calcitonin analogue solution and the SA liposome-salmon or the human calcitonin analogue complex solution of the dilution of difference intraperitoneal injection of saline, every Mus injection 0.4ml (16.6ng).Press the different time blood sampling, (OCPC method) measures the blood calcium value by the following method.
Every Mus is got the about 1ml of blood, and 4000rpm/min is centrifugal 10 minutes behind the blood coagulation.Get serum 50 μ l, add calcium developer 4.0ml, mixing is measured trap (Hitachi, U-2000 spectrophotometer spectrophotometer) immediately at 570nm wavelength place.The standard calcium curve is all accompanied in each test.
2.4 experimental result
(a) comparison of half-life in the calcitonin body behind Wistar rats by intraperitoneal injection salmon calcitonin see calcimar (sCT) and injection SA liposome-salmon calcitonin see calcimar (sCT) complex
In this experiment, respectively and in contrast with the SA liposome of normal saline dilution with normal saline.
Experimental result is as seeing Table 1 and shown in Figure 1.1 hour rat blood calcium value drops to minimumly behind the injection salmon calcitonin see calcimar, finishes fully but fall the calcium effect 4 hours the time.Relatively inject found that of SA-sCT complex, the smaller of sCT injected in 1 hour the calcium effect of falling more merely, and 2 hours effect is remarkable slightly, and the SA-sCT complex still had at 4 hours and necessarily falls the calcium effect.
The calcium level of different time rat behind table 1. lumbar injection salmon calcitonin see calcimar (sCT) and the SA liposome-salmon calcitonin see calcimar complex
| Matched group | 1.0hr | 2.0hr | 4.0hr | |||||
| NS | SA+NS | SCT | sCT+SA | sCT | sCT+SA | sCT | sCT+SA | |
| Blood calcium value mg/100ml | 9.30± 0.44 | 9.31± 0.07 | 5.92± 0.39 | 6.37± 0.64 | 7.66± 0.40 | 7.52± 0.69 | 9.43± 0.37 | 8.54± 0.40 |
| Average weight (g) | 84 | 78 | 78 | 78 | ||||
| NS: injection normal saline; SCT: salmon calcitonin see calcimar; The SA:SA cationic-liposome | ||||||||
(b) drug effect and the timeliness behind Wistar rats by intraperitoneal injection SA liposome-human calcitonin analogue I (hCT-I) complex
In this experiment, in contrast with the SA liposome of normal saline dilution.
Experimental result is as seeing Table 2 and shown in Figure 2.Rat blood calcium concentration behind injection SA-hCT-I complex promptly descends, and reaches minimum in 2 hours, and there were significant differences (P＜0.01) with the matched group comparison in the blood calcium value of injection after 4 hours for the SA-hCT-I complex, and in 6 little fashion certain calcium effect of falling is arranged.
The human calcitonin analogue I (hCT-I) of table 2. and the effect of SA liposome is to the comparison of different time blood calcium Mus level in the rat body
| Matched group | 1.0hr | 2.0hr | 4.0hr | 6.0hr | |
| SA+NS | hCT-I+SA | ||||
| Blood calcium value mg/100ml | 10.09±0.86 | 7.57±0.64 | 7.19±0.58 | 7.85±0.82 | 9.61±0.88 |
| Average weight (g) | 82 | 76 | 76 | 78 | 80 |
| NS: injection normal saline; HCT-I: human calcitonin analogue-I; The SA:SA cationic-liposome | |||||
(c) comparison that calcium level changes behind Wistar rats by intraperitoneal injection human calcitonin analogue II (hCT-II) and the SA-hCT-II complex
In this experiment, in contrast with the SA liposome of normal saline dilution.
Experimental result is as seeing Table 3 and shown in Figure 3.1 hour rat blood calcium value drops to minimumly behind the free human calcitonin analogue II of injection, but the blood calcium value after 5 hours recovers normal.The slightly poor of hCT-II injected in 1 hour the blood calcium effect of falling more merely behind the injection SA-hCT-II complex, and 2 hours identical, and still can keep the calcium effect of necessarily falling when injecting back 5 hours.Relatively there were significant differences (P＜0.0015) with matched group for the blood calcium value of injection SA-hCT-II complex after 3 hours, and dissociate with injection that more also there were significant differences (P＜0.005) for blood calcium value hCT-II3 hour the time.
The comparison of different time calcium level in human calcitonin analogue II (hCT-II) the rat body of table 3. and the effect of SA liposome
| Matched group | 1.0hr | 2.0hr | 3.0hr | 5.0hr | |||||
| SA+NS | hCT-II | hCT- II+SA | hCT-II | hCT- II+SA | hCT-II | HCT- II+SA | hCT-II | hCT- II+SA | |
| Blood calcium value mg/100ml | 10.31± 0.51 | 7.64± 0.27 | 8.22± 0.60 | 7.62± 0.34 | 7.77± 0.72 | 9.40± 0.51 | 8.27± 0.81 | 10.25± .24 | 9.85± 0.37 |
| Average weight (g) | 105 | 104 | 104 | 101 | 101 | 108 | 108 | 117 | 117 |
| NS: injection normal saline; HCT-II: human calcitonin analogue-II; The SA:SA cationic-liposome | |||||||||
The above results shows that the SA liposome can prolong the half-life of calcitonin or its analog, and plays slow releasing function.
The SA liposome is to the influence of osteogenic growth peptide (OGP) effect
3.1SA the preparation of liposome
By with embodiment 1 in identical method prepare the SA liposome.
3.2 become the synthetic of bone peptide
With Boc system in the conventional solid-phase synthesis; proportionately the bone peptide sequence is synthetic one by one to the N end from the C end with the aminoacid of various side chains and N end protection; with dry fluohydric acid gas cutting peptide and slough protecting group down, desalt and the HPLC purification then through Sephadex D10, product aminoacid is formed and is met theoretical value.
3.3 cell culture
Cell NIH3T3 (Embryo, contact-inhibited, NIH Swiss) cell strain is that (American Type Culture Collection ATCC) obtains from American type culture collection.Cell is with containing the DMEM/F12 culture fluid of 10%FBS at 37 ℃, 5%CO
2Cell culture incubator (Herarus) in cultivate.Changed liquid in two days, went down to posterity in three days.
3.4 method of testing
Measure the effect of bone peptide that dissociate into by the following method: inoculating cell about 5 * 10 to the NIH3T3 cell
3In 24 hole plastic culture plates (Corning), every hole adds the DMEM/F12 culture fluid that 500ul contains the 10%FBS hyclone and cultivated 24 hours.OGP is diluted to the different concentration (10 of series with PBS
-5To 10
-15M).Before NIH3T3 and the OGP effect, change the DMEM/F12 training liquid that 450ul contains the 10%FBS hyclone, add the OGP solution 50ul of variable concentrations, making the OGP of final and cytosis is 10 in the concentration of training liquid
-6～10
-16M changed the training liquid that once contains corresponding original OGP concentration in per 2 days.
Measure the effect of SA liposome-skeletonization peptide complexes to the NIH3T3 cell by the following method: the OGP that SA liposome (2ug/ul) 1ug and variable concentrations are got in every hole is incubated at room 30 minutes, and is diluted to 50ul with 1 * PBS, and the series concentration that makes OGP is 10
-6To 10
-16M.NIH3T3 is incubated with the DMEM/F12 training liquid that 450ul contains the 10%FBS hyclone, adds the SA-OGP solution of variable concentrations, and making the concentration of OGP in training liquid of final and cytosis is 10
-6～10
-16M changed the SA-OGP solution that once contains respective concentration in per 2 days.
Measure cell growth value with the crystal violet method, method is as follows: absorb the cell culture fluid in 24 orifice plates to be measured, add 250ul, 4% formalin (is assigned among 1 * PBS, pH7.2) fixes 1 hour, use ddH
2O gives a baby a bath on the third day after its birth inferior, air drying.Every hole adds 200ul 0.01% violet staining liquid again, and (be assigned in the 0.2M phosphate buffer, pH6.8) dyeing is 30 minutes, uses ddH
2O gives a baby a bath on the third day after its birth inferior, air drying.Every hole adds 500ul 10%HAC, dissolves to survey OD after 2 hours
590Value (Beckman DU650 spectrophotometer).
3.5 experimental result
The result is shown in Figure 4 and 5.
Fig. 4 is free OGP and the variable concentrations OGP warp and the 3rd day cell growth curve of lugSA liposome effect post processing NIH3T3 cell enlargement of variable concentrations.Experiment is found, when free OGP concentration is 10
-11～10
-7Help lend some impetus to cell proliferation during M, and 10
-9Be best during M, when further strengthening OGP concentration, the growth of cell is suppressed on the contrary.After the OGP of variable concentrations and SA liposome were hatched, the growth curve of cell obviously changed, and peak value is subjected to displacement, with the bonded OGP concentration of SA liposome 10
-12～10
-16Help lend some impetus to cell proliferation during M, 10
-14Be best during M, along with the increase cell of OGP concentration, it is gradually slow again to grow then.With the bonded OGP of SA liposome promote the cell growth reach peak value the just free OGP of concentration 100,000/.
Fig. 5 is the influence of the free SA liposome of various dose to NIH3T3 cell growth in 24 orifice plates, every hole inoculation 5 * 10
4Cell.The result shows that growth has no adverse effects the free SA liposome of various dose to the NIH3T3 cell.
The SA liposome is applied to gene therapy
4.1SA the optimal condition of liposome
Under different condition, the SA liposome is applied to the DNA transfecting eukaryotic cells, obtained the optimal condition of the external mediated dna transfection of SA liposome shown in the table 4.In SA liposome-DNA complex, preferred SA liposome is about 10-50/1 with the ratio of DNA.
The optimum condition of the external mediated dna transfection of table 4.SA liposome
| Culture dish diameter (mm) | The dilution volume of liposome and DNA (μ l) | SA liposome consumption (μ l) | DNA consumption (μ g) | Cell number (every hole) | Culture fluid volume (ml) |
| 35 60 100 | 100 300 800 | 2-25 6-75 16-200 | 1-2 3-6 8-16 | 1×10 5-3×10 5 3×10 5-9×10 5 8×10 5-24×10 5 | 0.8 2.4 6.4 |
4.2SA the step of liposome in-vitro transfection (in vitro)
1) cell inoculation:
Inoculating cell is cultivated after 24 hours in culture bottle/culture dish, and cell density reaches 80%～90%.
2) formation of SA-DNA complex:
With transfection 2.5 * 10
5Individual cell is an example, and 0.1～10 μ g DNA is diluted to 50 μ l with ultra-pure water, and 5～30 μ g liposomees are diluted to 50 μ l with ultra-pure water, both mix homogeneously, and room temperature was placed 25～30 minutes.
3) change training liquid:
Cell changes serum-free medium.The cell strain that has is used the training liquid better effects if that contains an amount of calf serum instead.
4) transfection:
The SA-DNA complex is added dropwise to culture dish, shakes up gently, cultivated 18 hours for 37 ℃, change and normally contain serum training liquid and cultivated 48 hours, survey it then and decide the transfection vigor.
4.3SA the step of transfection (in vivo) in the liposome body
The mixture of SA: DNA=10: 1 (w/w) is added 10 times of volumes the ultrafiltration water or all kinds of adjuvant of (looking experiment needs and can suitably increase and decrease), mixing, room temperature was placed 25 minutes, formed the SA-DNA complex.Carry out zoopery then.
Embodiment 5
The liposome-mediated DNA of SA under the serum condition is arranged to the transfection of insect cell
Adopt SA liposome (2mg/ml) mediated dna that insect cell is carried out transfection.According to the method for document, from virus in cells and supernatant granule of infective virus, extract Autographa californica multicapsid nucleopolyhedrosisvirus (Autographa californica) nuclear polyhedrosis virus (AcMNPV) genomic DNA.Transfer vector pBlueBacEPO plasmid DNA gets through Sepharose 2B column separating purification after pressing the extraction of document alkaline process.Noctuid (Spodoptera frugiperda) Sf-21 cell strain is coveted with the TC-100 training liquid (GIBCO-BRL Co.) that contains 10% hyclone (FBS) (Sigma Co.), in 27 ℃ of constant temperature culture in the meadow.
The wild type AcMNPV of 138kb contains polyhedron gene, polyhedrin can reach more than 17% of total alkaline soluble protein in the whole cell at later period of infection, the formed viral polyhedral body of later period of infection is full of whole nucleus, is very easy to identification at microscopically.
Inoculation about 1 * 10 in the plastic culture dish of diameter 35mm
5The Sf-21 cell, cell density is about 70%, and adherent growth is spent the night.Change 1ml in second day and contain 10% hyclone TC-100 training liquid.Viral DNA and SA liposome dilute respectively with sterilized water, both mixings, and totally 100 μ l make transfection liquid, and room temperature leaves standstill 15min, dropwise adds in the culture dish of cultured cell, behind 27 ℃ of cultivation 6h, adds training liquid to 3ml.Continue to put 27 ℃ and cultivate 72h, under 200 times of inverted microscopes, 10 visuals field of every culture dish casual inspection, statistics contains polyhedrosis cell quantity.As seen have and occur tangible polyhedral body in the nuclear of 2796 cells, account for 69% of cell total amount.
Presentation of results, the SA liposome can still can mediate the virus genom DNA transfection insect cell that reaches 138kb effectively under 10% serum condition.And Lipofectim reagent can only be under the serum-free existence condition mediated dna transfectional cell.
With the inhibition of the vEGF antisensenucleic acids of SA liposome to nude mice people liver cancer growth
1. animal: the Balb/cA nude mice in 4 ages in week.Make an about 1.0cm leather bag of diameter in its flank portion in advance, with 5 * 10
6LC1-D
20The human liver cancer cell suspension inoculation is in leather bag.
2. antisensenucleic acids: 5 '-GCA GTA GCT GCG CTG ATA AGT GC-3 '
3.SA liposome 300 μ g in normal saline and 60 μ g antisensenucleic acidses in mixed at room temperature, place 15min.
4. animal and grouping, dosage:
The A group, totally 8, injecting normal saline
The B group, is injected 300 μ g SA liposomees by totally 6
The C group, totally 10, injection antisensenucleic acids 100 μ g
The D group, is injected 60 μ g antisensenucleic acidses+300 μ g SA liposomees by totally 8
5. injecting method and effect observation
The inoculation animal next day, begin oligonucleotide is injected in the leather bag, the next day once, totally 7 times.After 5 weeks of inoculation, put to death animal, take by weighing tumor and weigh.
After 5 weeks of inoculation, the tumor of injecting antisensenucleic acids merely heavily is 53% of a matched group, and the tumor of injection SA liposome-antisensenucleic acids complex heavily is 37% of a matched group, 3/5ths when the antisensenucleic acids consumption of SA-liposome-antisensenucleic acids group is simple the injection.
The result shows with the vEGF antisensenucleic acids of SA liposome merging administration more remarkable to the inhibition of nude mice people liver cancer growth.
Embodiment 7
The SA liposome is rotaring redyeing COS cell when having serum to exist
1.pCH110 pressing document [Birnboim, H.C., and Doly, J.1979, Nucleic Acids Research, 7:1513], the preparation of plasmid DNA carries out.
2. cell
The COS cell strain obtains from American type culture collection (American Type CultureCollection).
(3.Lipofectin GIBCO BRL Co.) mediated dna transfection:
The transfection of Lipofectin reagent is pressed document [Whitt, M.A.et al., 1991, Focus, 13:8-12] and is carried out; The COS cell is with containing DMEM/F12 (GIBCO BRL the Co.) culture fluid of 10%FCS (GIBCO BRL Co.) at 37 ℃, 5%CO
2Cell culture incubator in cultivate.
4.SA liposome-mediated DNA transfection:
Inoculating cell about 7.5 * 10
5Individual in Φ 60mm plastic culture dish (Corning), the DMEM/F12 training liquid that contains the 10%FBS hyclone was cultivated 24 hours.Change serum-free and the DMEM/F12 training liquid 3ml that contains variable concentrations (being respectively 1% to 20%) hyclone (FBS), this moment, cultured cells density should be 80～90%.3 μ l concentration are that the plasmid DNA of 1mg/ml is diluted to 150 μ l with sterilized water, the SA liposome that contains 60 μ l (concentration 2mg/ml) lipids is diluted to 150 μ l with sterilized water, both mixings, and room temperature is placed and was formed the SA-DNA complex in 15～30 minutes, be added dropwise in the culture dish 37 ℃, CO
2The training case was cultivated 18 hours.Change 4ml 10%FCS DMEM/F12 culture fluid, cultivated 48 hours.
5. the mensuration of gene expression
Harvesting, cell PBS rinsing three times, add the PBS digestion that contains 10mM EDTA, the cell of collection is at liquid nitrogen-37 ℃ water-bath freeze thawing three times, cell lysis, centrifugal, press document [Hall, C.V.et al., 1983, J.Mol.Appl.Genet., 2:101-109] get the beta galactosidase vigor (OD that extraction liquid of cell is surveyed the pCH110 gene expression change over to
420).
6. protein content is measured [Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J., 1951, J.Biol.Chem., 193:265] with Folin phenol method (Lowry method)
7. result
Transfection is to carry out in the culture dish of diameter 60mm, and cell number is represented with the extraction liquid of cell total protein in the culture dish.Transfection is carried out under the best lipid/DNA concentration of SA liposome and Lipofectin reagent respectively.Transfection efficiency is represented with beta galactosidase vigor in the extraction liquid of cell.
Serum is distinct to the influence of SA liposome or Lipofectin reagent rotaring redyeing COS cell efficient.The SA liposome for the transfection efficiency of COS cell increase along with increasing of serum-concentration (be optimum during 20%FBS, for serum-free condition 230%); The efficient of Lipofectin rotaring redyeing COS cell is suppressed significantly in the presence of serum, contrast when transfection efficiency has only serum-free when 20%FBS 11%.
Embodiment 8
The SA liposome is transfection NB-1 cell when having serum to exist
1.pCH110 plasmid DNA: with embodiment 7.
2. (Neuroblastoma, Human) cell strain obtains from American type culture collection (American Type Culture Collection) cell: NB-1.
(3.Lipofectin GIBCO BRL Co.) mediated dna transfection: with embodiment 7.
4.SA liposome-mediated DNA transfection: with embodiment 7, difference only is to replace the COS cell with NB-1.
5. the mensuration of gene expression: with embodiment 7.
6. protein content is measured with Folin phenol method (Lowry method): with embodiment 7.
7. result
NB-1 belongs to the neuroglial cytoma strain, and it is difficult transfected usually.Serum has diverse influence to SA liposome and Lipofectin transfection NB-1 cell, the trend of the dose-response curve of two kinds of methods is opposite, the ten transfection vigor that are five times in serum-free condition are arranged, 20% when Lipofectin then drops to serum-free in the presence of 20%FBS SA liposome.(see figure 6).
Embodiment 9
The SA liposome is transfection P19 cell when having serum to exist
1.pCH110 plasmid DNA: with embodiment 7.
2. (Embryonic carcinoma, mouse) cell strain obtains from American type culture collection (American Type Culture Collection) cell: P19.
(3.Lipofectin GIBCO BRL Co.) mediated dna transfection: with embodiment 7.
4.SA liposome-mediated DNA transfection: with embodiment 7, difference only is to have replaced the COS cell with the P19 cell.
5. the mensuration of gene expression: with embodiment 7.
6. protein content is measured with embodiment 7 with Folin phenol method (Lowry method).
7. result
P19 is a strain embryonal carcinoma cell, is the stem cell in the teratoma, has the pluripotency of growth, by inducing the embryo development procedure that can simulate multiple tissue, by the in vitro study model of many laboratorys as the nervous system generation.The SA liposome in the presence of different serum-concentration gradients and the comparative experiments of Lipofectin transfection find that the SA liposome has obtained optimum when 10%FBS, and all transfection values when containing serum are all greater than serum-free; And the transfection of Lipofectin is with the increasing of serum-concentration, transfection vigor (60% when the being serum-free during 20%FBS) (see figure 7) that descends.
The SA liposome is transfection PC12 cell when having serum to exist
Test by embodiment 7 described methods, difference only is that (Adrenalpheochromocytoma, rat) cell strain (obtaining from American type culture collection (American TypeCulture Collection)) is replaced the COS cell with PC12.
PC12 is a kind of rat adrenal chromaffin cancerous cell, is often used as cell model.In the transfection of PC12 cell, the SA liposome obtains best transfection value when 6% FBS+HS pooled serum, and the transfection value under all serum conditions is all greater than serum-free; And Lipofectin successively decreases (during 20%FBS be serum-free condition 10%) (Fig. 8) with serum-concentration transfection vigor.
Discuss
In order to illustrate the mechanism and the scope of application of SA liposome in-vitro transfection, in tests such as embodiment 7-10, the representational 20 various kinds of cell strains (partial data is not shown) commonly used of separate sources have been selected, mediate plasmid PCH110 respectively in conjunction with coprecipitation of calcium phosphate method, Lipofectin method or plasmid PSK110 enters cell with the SA liposome, the vigor of measuring its beta galactosidase is to compare, found that different transfection methods has visibly different cell selective, every kind of transfection method all has the cell strain of suitable high expressed.Total result shows that the SA liposome has comparatively ideal transfection ability.
In general, the stearylamine liposome comes well with the effect of Lipofectin reagent than calcium phosphate.And SA liposome range of application and better effects if some.The absolute optimum selection rate of SA liposome and relative optimum selection rate are all than Lipofectin method and coprecipitation of calcium phosphate method height, some cells such as cells such as CV-1, CHO there is specific high expressed, and cell such as PA-1, NRK49F can only be used the transfection of SA method, consider complementarity, stability and the potential price factor of different transfection methods, the SA cationic-liposome is that mediated gene enters eukaryotic a kind of effectively new rotaring dyeing technology.
External SA liposome is to the influence of oligonucleotide transfection efficiency
1. the preparation of oligonucleotide is by the method in " molecular cloning experiment guide ".
2.SA the preparation of liposome (2mg/ml).
Method for making is with embodiment 1.
3. cell: 7721 human hepatoma cell strains obtain from American type culture collection (American TypeCulture Collection).
4. transfection method
People's hepatocarcinoma 7721 cells that are in exponential phase of growth are with 1 * 10
4In/every milliliter of 24 well culture plate that are seeded in the RPMI-1640 (GIBCO Co.) that 1000 microlitres contain 10% hyclone (FBS) (GIBCO Co.), 37 ℃, 5% carbon dioxide were cultivated 24 hours.Wash 2 times with the RPMI-1640 of serum-free.Respectively with 1: 5,1: 10,1: 15,1: 20 ratio (weight ratio of oligonucleotide and SA liposome) parcel 5 * 10
4Dpm
32The oligonucleotide of P labelling and the unlabelled oligonucleotide of 4 micrograms continue at 37 ℃, 5%CO
2Cultivated 12 hours.Wash 3 times with cold Hank ' s liquid, add 1000 microlitre 1%SDS (SIGMA Co.) dissolved cell.Through LS-6500 liquid scintillation instrument counting dpm value.
5. result:
| The total DPM counting in every hole is | 63331dpm |
| (not adding the SA liposome) control wells is | 149dpm |
| 1: 5w/w (oligonucleotide and SA liposome) | 1169dpm |
| 1: 10w/w (oligonucleotide and SA liposome) | 5339dpm |
| 1: 15w/w (oligonucleotide and SA liposome) | 9003dpm |
| 1: 20w/w (oligonucleotide and SA liposome) | 9132dpm |
The result shows that SA lipid physical ability significantly improves the transfection efficiency of oligonucleotide, at 1: 15 o'clock near reaching high transfection efficiency.External SA liposome shows that to the result that influences of oligonucleotide transfection efficiency the transfection efficiency that SA cation lipid physical ability improves oligonucleotide reaches 60 times.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (14)
1. the purposes of a SA liposome, it is characterized in that, be used to prepare the pharmaceutical composition that carries out transfection under the serum condition having, described pharmaceutical composition contains active constituents of medicine and as the SA liposome of pharmaceutical carrier, wherein active constituents of medicine is a nucleic acid, and described the serum condition is arranged is that serum-concentration is 0-20% and is not equal to 0%.
2. purposes as claimed in claim 1 is characterized in that, described nucleic acid is selected from DNA, RNA, oligonucleotide, antisensenucleic acids or its mixture.
3. purposes as claimed in claim 1 is characterized in that, the weight ratio of nucleic acid and SA liposome is 1: 10～1: 300.
4. purposes as claimed in claim 1 is characterized in that, the weight ratio of nucleic acid and SA liposome is 1: 10～1: 100.
5. purposes as claimed in claim 1 is characterized in that, described SA liposome is CH
3-(CH
2)
16-CH
2-
+NH
3: C
41H
78NO
8P (DOPE)=0.8: 1～1: 0.8 (w/w).
6. purposes as claimed in claim 1 is characterized in that, the weight ratio of nucleic acid and SA liposome is 1: 10～1: 50.
7. purposes as claimed in claim 1 is characterized in that, described the serum condition is arranged is that serum-concentration is 5-20%.
8. purposes as claimed in claim 1 is characterized in that, described the serum condition is arranged is that serum-concentration is 5%, 6%, 10% or 20%.
9. the method for an in-vitro transfection cell is characterized in that, with nucleic acid and SA liposome with weight ratio 1: 10-1: 20 mix, and then at the transfectional cell that has under the serum condition, wherein said the serum condition is arranged is that serum-concentration is 0-20% and is not equal to 0%.
10. method as claimed in claim 9 is characterized in that, described nucleic acid is selected from: DNA, RNA, oligonucleotide, antisensenucleic acids or its mixture.
11. method as claimed in claim 9 is characterized in that, described the serum condition is arranged is that serum-concentration is 5-20%.
12. method as claimed in claim 9 is characterized in that, described the serum condition is arranged is that serum-concentration is 5%, 6%, 10% or 20%.
13. method as claimed in claim 9 is characterized in that, described cell is insect cell, COS cell, stem cell, cancerous cell.
14. method as claimed in claim 13 is characterized in that, described cancerous cell is neuroglial cytoma NB-1, adrenal chromaffin cancerous cell PC12 or hepatocarcinoma 7721 cells.
15. method as claimed in claim 9 is characterized in that, described SA liposome is CH
3-(CH
2)
16-CH
2-
+NH
3: C
41H
78NO
8P (DOPE)=0.8: 1～1: 0.8 (w/w).
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