CN85101588A - Merge liposome and merge used acid induced method for liposome - Google Patents

Abstract

Reduce to 7 when following when the pH value of medium, contain PHOSPHATIDYL ETHANOLAMINE, the liposome of PHC or oleic acid or Palmic acid merges apace.The fusion of liposome system borrows (a) as shown in the transfer of resonance energy, all liposome lipids are mixed, (b) glue filtering method and (c) electron microscopy predict.The liposome that contains PHOSPHATIDYL ETHANOLAMINE and PHC (8: 2) is between incorporating period, and nearly all calcein that comprises all can disengage.When including lipidol of gallbladder (40%) in the liposome, then can reduce leakage veritably and do not undermine fusion.Those are merged the sensitive liposome of pH, can be with bioactive molecule, be delivered in the living cells such as DNA etc.

Description

Merge liposome and merge used acid induced method for liposome

The present invention means the fusion liposome, by the liposome fusion of PH control, and the purposes of these liposomes during as the carrier of pharmacy or similar formulations.When the PH of liposome medium was made into acidity, the liposome that contains (for example) PHOSPHATIDYL ETHANOLAMINE and PHC merged apace.

Liposome is the cyst that contains one or more liposome bilayers, and this liposome bilayer is fully around inner aqueous space.They usually system by phospholipid or other aliphatic molecules with simple form or with other molecules such as lipidol, long chain acid or alkali, or the form of combination such as memebrane protein is made.The structure of liposome can be multilamellar macrocyst (0.5 to 5 micron) to monolayer intracellular vesicle (250-750 dust) form.Be according to the size liposome of classifying traditionally, and use triliteral acronym to name the liposome type of being touched upon.The general called after of multilamellar cyst (MLV).The general called after of monolayer intracellular vesicle (SUV).The monolayer macrocyst is called after (LUV) then.Chemical composition that in each following example generally is to be referred to as with acronym.Referring to: DPapahadjopoulos, Ann, NYAcad, Sci308 1(1978) and AnnRptsMedChem, 14 250(1979), it has illustrated

Go into herein for your guidance.

Liposomes preparation can be made by multiple technologies, comprising: ethanol injection method, (Batzri et al, Biochim, Biophys, Acta, 298 1015(1973)); The ether infusion process, (Deamer et al, Biochim, Biophys, Acta, 443 629(1976)) and Schiern et al, Biochim, BiophysACta, 542 137(1978)), detergent is removed method, (Razin, Biochim, BiophysActa, 265 241(1972)), solvent evaporated method, (Matsumato et al, JColloid Interface Sci, 62 149(1977)), evaporation (the SZoka Jret al. of the water in oil (REV) emulsion, Proc NatlAcadSciUSA, 75 4194(1978)), reach MLV and LUV squeezing and pressing method (Olson et al. via nucleopore PC film, Biochim, Biophys, Acta, 557 9(1979).

Liposome can be used for influencing the cellular activity in test tube test and the live test.Wagee and Miller(Nature, 235 399(1972) point out that at first the liposome that has antiviral antibody can protect cell to avoid being subjected to viral infection in the report.Greoriadis and Buckland(Nature, 244 170(1973)) similarly observe the protective effect of liposome pair cell, its viewpoint thinks that the liposome that contains saccharase can cause the storage sucrose cavity in the peritoneal macrophages of mice to disappear.Papa hadjopoulos and Coworkers(BiochimBiophysActa, 323 23(1973) point out in, cell-cytotoxic reaction does not take place but the liposome inducing cell merges.

Liposome is in order to influence the absorbability of cell to temporary molecule (being ordinary nonabsorbable molecule).This effectiveness causes liposome to can be used as the carrier of foreign substance (such as medicine etc.).For example, in the test tube test, ring-type AMP can be by using liposome to strengthen 1000 times of (Papahadjopoulos et al. as carrier to the inhibitory action of STS cell growth, Nature, 252 163(1974) and Papahadjopoulos et al., Biochim BiophysActa, 363 404(1974)).Similar enhancement effect is also at Papahadjopoulos et al., Cancer Res., 36 4406(1976) in address, point out the effectiveness of the vole cell line of the actinomyces D antagonism drug resistance in the liposome.

At the acid situation purgation of appropriateness film fusion is to infect most strip of paper used for sealing viruses, comprise Sheng Liji forest (Semliki Forest) virus (Marsh, et al., Cold Spring Harbor SympOuantBiol 46 835(1982) and White et al., JCell Biol89 674(1981), vesicular stomatitis virus and feveret virus (White et al., as above) etc. reason.The correct mechanism of acid induced film adhesive effect is not learnt as yet.Being shown as of the research of relevant liposome, by the obtained liposome of azolectin can under the PH6.5 with grain line body within film merge (Schneider et al., Natl Acad.Sci.USA, 77 442-446(1980)), serum albumin (Schenkman et al., Chem.Phys, Lipids, 649 633(1981) and Schenkman et al., Chem.Phys.Lipids, 28 165(1981) and albuminolysis fragment (Chaimovich et al., Biophys.J., 41 28a(1983)) can under situation about being lower than below the PH4, induce liposome to merge.Blumenthal et al. points out, Clathrin can induce neutral liposome to merge (Blumenthal et al., Biol.Chem., 258 3409(1983) below PH6.5.In all examples, the fusion of liposome all needs certain protein or other macromolecular existence.

People such as Allens (J.Cell Biol.97 10) (a), Abstr, No.419(1983)) in report, point out, contain the liposome of PHOSPHATIDYL ETHANOLAMINE and lipidol of gallbladder hemisuccinic acid ester, in the PH scope of slurry plastid, be sensitive to PH.When these liposomes carry out acidization at the slurry plastid, merge with the film of starching plastid, thereby its content is delivered to Cytoplasm.

Straubinger and Coworkers(J.Cell.Biol.97 109(a), Abstr No.420(1983)) report in point out under subacidity PH, can become the method for making of unsettled liposome.These contain oleic acid, and the liposome of PHOSPHATIDYL ETHANOLAMINE and lipidol of gallbladder (3: 7: 3 molar ratios) is below PH7.0, and (Cacein) has permeability to the anion fluorescent dye.These liposomes impel built-in Calcein to be delivered in the Cytoplasm of CV-1 cell.

The present invention means (1) fusion liposome, the purposes when (2) make these liposomes of method (3) of liposome fusion use for the microscopically injection (promptly transmitting pharmacy or other preparations) of cell.When the medium of liposome was processed into PH acidity, then liposome (making the sensitivity to PH) merged.When these liposomes greater than the amphiphatic molecule that contains one or more weak acid functional group (as carboxyl group), its molar percentage is about at 20 o'clock, liposome becomes to the PH sensitivity.The chemical compound of this kind comprises the long-chain of PHC, (is C ₁₂To C ₃₀, be preferably C ₁₆To C ₂₄) fatty acid such as Palmic acid and oleic acid etc.Existence has amphiphatic molecule such as the PHOSPHATIDYL ETHANOLAMINE of formation six sides phase or convertibility micella trend etc., can strengthen the process of fusion widely.In the following exemplary case, PHOSPHATIDYL ETHANOLAMINE is 8: 2 to the ideal ratio of PHC.

The simple declaration of accompanying drawing:

Fig. 1 describes liposome, the figure that (b) records with Biogel A 50M glue filtering method after (a) and the acid treatment before acid treatment.Bidding shows and the liposome that do not indicate is 200 * 10 at lipid concentration ^-6Mix under the situation of M and n=3.Measuring each several part is lacking (1) and is having N-NBD-PE fluorescent (λ under the situation of (2) 0.2%Triton X-100 _Ex=468nm, λ _Em=530nm).We also draw in the existence of Triton or the fluorescent ratio (3) under lacking.

Fig. 2 describes pH value influences the chart PE-PHC(8 that liposome merges: 2) to tie up to lipid concentration be 200 * 10 to liposome ^-6Merge under the situation of M and n=3.Merging percentage ratio then calculates with equation (1); And

Fig. 3 describes the fusion percentage ratio of liposome, can learn that PH is to reaching the effect of the liposome (5) that process PHOSPHATIDYL ETHANOLAMINE-Palmic acid (8: 2) is handled through the liposome (4) of PHOSPHATIDYL ETHANOLAMINE-oleic acid (8: 2) processing.

This paper use hereinafter to be referred as:

The PHC-PHC

The PE-PHOSPHATIDYL ETHANOLAMINE

N-NBD-PE-N-(nitro-2 ,-1,3-Ben Bing Evil diazonium-4-yl)-PE

N-RH-PE=N-(lissamine rhodamine B-sulfonyl)-PE

The PC-phosphatidylcholine

The PA-Palmic acid

PS-phosphatidyl silk amino acid

OA-oleic acid

The Chol-lipidol of gallbladder

PBS-phosphate-buffered saline

The present invention at first is that proof is a kind of not with protein or other macromole acid induced liposome fusion as medium.Discuss facilitating property of protein liposome fusion aspect, as serum albumin (Schenkman et al., Biochim.Biophy S.ACta as above reaches Chaimovich, as above), Clathrin(Blumenthal, as above) and viral glucoproteid (White, as above, White et al., Proc.Natl.Acad Sci.USA.77 3272(1980)); And Marsh et al, J.Cell Biol., 96 455(1983)) etc., obviously the change of protein structure is the main motive force of fusion as can be known.In the present invention's the method, motive force is then from lipid itself.Homocysteine forms thiolactone ring (Baernstein J.Biol.Chem., 106 451(1934) under acid PH).This mechanism is considered to cause to contain the reason of the PH-sensitivity leakage in the liposome of two saturated PC and PHC in the past.（YatVin et al.，Science 210 1253（1980））。Yet nearest proof has been refuted this saying; The effect of PH can be interpreted as the change of " acid/alkali " balance, that is the ratio of electrically charged and not charged N-acyl group amino acid, changes because they produce the static reciprocal action between a group of lipid.In addition, the double-deck dissolubility of protonated PHC may be low so that form the magnetic domain of PHC under acid PH.The main cause (Cestaro et., J.Biochem., 133 229(1983) that the side of this lipid bilayer is separated and can constitutes fusion; Yatvin et al., in Liposome Technology, Gregoriadis, G.ed.) (CRC Press Boca Raton 1984); And Sundler et al., Biochim Biophys.Acta, 649 751(1981)).Latter's mechanism has special captivation, and we find that the existence of PE can strengthen acid induced liposome fusion widely to this point just.Any amphiphatic molecule that has formation six sides phase or form convertibility micella trend, such as unsaturated pure PE, two oleoyl PE especially used in the present invention etc. are suitably.PE easily forms six sides and reaches convertibility micella (Cullis et al. mutually, Nature(Lond), 271 672(1978), Cullis et al., Biochim Biophys Acta 559 399(1979), (Cullis et al. Nature(Lond) as above reaches Lucy Nature(Lond), 277 815(1970)).Even other long-chain parent amphoteric compounds that contain carboxyl group also can be used for the fusion of inductivity liposome such as (see figure 3)s such as fatty acids.

Studied the cross reaction of liposome and zooblast widely.Though think that at first the fusion of liposome-cell is that dominant mechanism is recently at (Huang et al., J.Biol.Chem., and other laboratorys (Heath et al. 258 14034(1983)), Proc Natl.Acad.Sci.USA, 80 1377(1983); Leserman et al., Nature(Lond.), 293226(1981); And Machy et al., J Immunol., 129 2098(1982)) in research point out that the cell imbibition effect of liposome is the absorbed main cause of liposome.Even people (Cell32 1069(1983) such as Straubinger) points out, in a single day be under the sour environment, then can enter in the slurry plastid through the liposome of cell imbibitionization.The PH of slurry plastid is measured as about 5 (Tycko et al., Cell, 28 643(1980)).So, the PH(in the slurry plastid scope is about 5) and sensitive liposome will be fused on the film of slurry plastid, and its content will be released in the Cytoplasm.

When medium PH reduces to 7 when following, the sound wave liposome that then contains PE and PHC merges.Reduce the method for liposome medium PH, any those skilled in the art can accomplish that all prevailing method is to add acid.Liposome medium used herein is generally the phosphate-buffered saline solution of PH7.6.Desire reduces this PH, then need add inorganic salt, example hydrochloric acid etc.This sour concentration can change with the final PH that requires and the amount of processed medium.For example, as mentioned above, when 5 microlitre 1N Hcl being added in the 2ml liposome suspension (at the phosphate-buffered saline of PH7.6), then PH reduces to 4.8, thereby liposome merges.This fusion can be with the negativity electron microscope technique that dyes, and whether observe the liposome size increases.Original, the diameter of unprocessed sound wave liposome is 4.9 ± 2.3 * 10 ²Dust.With (to PH4.8) after the acid treatment, about 1/3 liposome does not change its size, that is diameter is in the scope of 300-700 dust.It is bigger that all the other liposomes then become, and promptly diameter is 2.9 ± 1.1 * 10 ³Dust.The amalgamation liposome is not assembled.Since the liposome diameter increases about about 6 times, so the essential fusion that ring of numbers takes place.

The increase of liposome size also can record (see figure 1) by glue filtering method.In this measurement method, liposome that will indicate (containing not ear %N-Rh-PE of 1 mole of %N-NBD-PE and 1) and the liposome that do not indicate more than 3 times mix.Take out a part, make it place that (under PH7.4) accepts chromatographic separation on the Biogel A50M post.Heterogeneous liposome group causes having broad peak in the elution elevation.Other parts make PH return back to PH7.4(again and use NaoH then with acid treatment (to PH4.8)) after, place and give chromatographic separation on the same column.The exit dose of measuring N-NBD-PE is examined and determine the liposome of elution under 530nm.That is thoroughly dissolve liposome at 0.2%Triton X-100() in the presence of measure the fluorescent of each part.As finding, sound wave liposome (elution in the volume part within first in post) with after the acid treatment, has been transferred to void volume part (the 9th part), and this is to refer to that the liposome size increases.And the ratio of the N-NBD-PE fluorescent in these liposomes is in the existence of Triton X-100 or be very different under lacking.Before the fusion, ratio is usually greater than 5, and this is indication, causes highly quenching of N-NBD-PE fluorescent owing to effective energy shifts.After acid treatment, its ratio is reduced to below 5 significantly, and this is to refer to that the usefulness of energy transfer is lower.If can cause indicating and not indicating between liposome merging with acid treatment, then this result is that we are desired.This is because the cause of dilution takes place in nexus the fluorescent probe.

But the initial resonance energy transfer methods of discussing of people (Biochem., 20 4093(1981) such as acid induced liposome fusion mat Struck give quantitatively.In this analytic process, the degree of fusion is that mat is embedded in the dilution factor of the fluorescent probe in the film and records.As shown in Figure 2, use the PE of equation (1) calculating: PHC(8: 2) liposome fusion percentage ratio system decides according to the PH of medium.When PH is lower than 7.0, merge, when PH=4.8-5.0, reach at utmost, during PH=6.2, then reach mid point.

The lipid constituent of liposome determines the usefulness of acid induced fusion consumingly.Table 1 demonstration is various merges percentage ratio by the obtained liposome of different lipid constituents in the PH4.8 purgation.Shown in data, we determine fusion to need the existence of PHC.PE and PHC(8: combination 2) is very effective to fusion; Yet PC added so far then lower fusion in the liposome.When the PE(PE when 25%: PC: PHC=6: 2: 2) replacing, then merge power and reduce to 36% by 89 with PC.We observe, and contain a large amount of PE and have maximum fusion power when not having the liposome of PC.Merge percentage ratio be lower than 20% be considered as not remarkable.

Except the liposome of PE-PHC constituent, we also estimate in other lipids, and PH sensitivity is to the effectiveness of liposome fusion.For example, as shown in Figure 3, the liposome that contains PHOSPHATIDYL ETHANOLAMINE (PE) and Palmic acid (PA) (molar ratio is 8: 2) is at PH about 6.3 and be lower than 6.3 times and show to have fusion speed and the percentage ratio that makes one notice.Show among Fig. 2 and contain PE-PHC(8: the fusion percentage ratio of liposome 2) and PH function system are stable and nearly straight line to be increased.Again the two is contrasted.

Except containing PE/PA(8: the liposome 2), also studied the liposome (see figure 3) that contains PE and oleic acid (OA) (8: 2), these liposomes are as the liposome that contains PE/PA, and wherein PH6.3 and 6.3 has when following and merges speed and percentage ratio fast.

Each liposome type to the PH sensitivity (merge gradually or merge fast) as the used agent delivery aspect of microscopically injection for cell, all has its advantage and shortcoming.For example, among the PH on a large scale that the liposome of fusion can exist in the cytoplasm matter of various differences, carry its content gradually.Yet, owing to fusion is limited under the higher pH value, so injection rate (under higher PH) is low.2) and PE/OA(8 on the other hand, contain PE/PA(8:: liposome 2) (only merging under some PH) is invalid fully under the situation of (pH value that does not promptly have certain cell fully) more than its valve point PH.

Table 1

The acid induced fusion of the liposome that various different constituent formed ^*

The lipid constituent merges power ⁺%

PE∶PHC （8∶2） 89.0±2.14

PE∶PHC （6∶4） 53.0±1.58

PE∶PHC （3∶7） 62.0±1.87

PE∶PC∶PHC （6∶2∶2） 36.0±1.29

PE∶PC∶PHC （4∶4∶2） 21.0±2.01

PC∶PHC （8∶2） 8.0±1.00

PS∶PHC （8∶2） 16.0±1.29

PE∶PC （7∶3） 6.0±1.87

PE∶PS （7∶3） 13.0±1.29

PS∶PC （7∶3） 5.3±1.58

^*At lipid concentration is 200 * 10 ^-6Fusion PH=4.8 under the situation of M and n=3

+ calculate it according to equation (1), represent with meansigma methods ± standard deviation.

Though PE: PHC(8: 2) liposome can get best fusion, points out that with the Calcein experiment this fusion process is very easily missed.Under the Calcein of high concentration, fluorescent is to quench automatically effectively, however when dyestuff by liposome in leakage and when making dyestuff be subjected to diluting, fluorescent strengthens (Allen et al. significantly, Biochim.Biophys.Acta, 597 418(1980)).The table II shows that between incorporating period, nearly all PE: PHC(8: 2) the built-in Calcein in the liposome all disengages fast.Yet,, can be observed the leakage less (55% the amount of hiding) of Calcein if contain 40% lipidol of gallbladder in these liposomes.Shown in resonance energy transfer analysis method, the liposome that contains lipidol of gallbladder still can merge effectively.

Can provide effective Cytoplasm transmission system by film fusion with liposome that method of the present invention forms with the slurry plastid.The liposome that is difficult for leakage that contains lipidol of gallbladder is particularly useful in its inclusions is drained in the Cytoplasm.For example, can be with foreign substance such as medicine, ferment, hormone, nutrient, antigen, antibody (individual plant or habitual person), RNA, the natural or reorganization person of DNA() or above any conjugate and similar substance etc. be filled in the present invention's the PH susceptiveness liposome, again its is inserted in living cells at last.

Above what is called it " living cells " means active matter, the cell of plant or animal.For example, unicellular organism is such as yeast, algae, and mycete antibacterial etc., multicellular organism or system comprise cell culture (pernicious or optimum) etc., and all animals such as mammals (comprising the mankind), reptile class, birds etc. all are.

Material packed into, and method system in the liposome is detailed to be known.For example, Szoka, people such as Jr. be in United States Patent (USP) the 4th, 394, describes in No. 448 DNA is inserted in the lipid cyst, and use this liposome DNA is inserted the method in the living cells.The alternative genealogy of law of packing into is used technology (the Philippot et al. of dialysis, Biochim.Biophys.Acta, 716 140(1982)) .Szoka, Jr. wait the method for people institute teachings such as people and Philippot can be successfully with DNA, RNA and other macromolecular complex such as peptide and hormone be filled in the present invention's the PH susceptiveness liposome.Szoka, people's such as Jr. and Philippot explanation has been inserted herein for your guidance.

The first step of envelope material is that the mixture that seals the aqueous mixture of material in the constituent (in organic solvent) that contains a kind of formation liposome and a kind of the treating is provided in the preparation.Can make the material (comply with its dissolution characteristics and decide) of envelope in treating be dissolved in the organic solvent (as chloroform etc.) in addition.Then evaporate organic solvent, re-use aqueous buffer (as phosphate-buffered saline) and, can make liposome thus by removing thin lipid film in the container for evaporation.The constituent of formation liposome and method for making thereof are detailed usually to be known.In United States Patent (USP) the 4th, 235, shown in No. 871, it has illustrated referring to people such as Papahadjopoulos

Go into herein for your guidance.

As shown here, PH susceptiveness liposome system is made with various molar ratios by PHOSPHATIDYL ETHANOLAMINE (PE) and the high half amine amino acid of palmityl, and molar ratio can account in per nine moles of PHC between the ratio that one mole of PE accounts for nine moles of PE to every mole of PHC and change.The molar ratio of ideal is to have 3 moles PE at least among 1 mole of PHC.The most desirable PE: the PHC mol ratio is 4: 1.

Material and method

Material:

PHC system utilizes the detailed method of knowing synthesize to reach purification (Yatvin et al., Science, the same).Commercial dioleoyl PE, dioleoyl PC and Medulla Bovis seu Bubali PS also can use.All phospholipid comprise that N-NBD-PE and N-Rh-PE etc. by Avanti(Birmingham, AL) sell.Lipidol of gallbladder and Calcein system obtain (St.Louis.No) by Sigma.

Liposomes preparation:

Make solvent-free lipid film be suspended in PBS(PH7.4 with 10 little ear/ml that rub) in, and under room temperature to bathe sound wave (laboratory supply) sound waveization 15 minutes.The lipid constituent of various differences is to use according to indication.The fluorescent that respectively contains a molar percentage N-NBD-PE and N-Rh-PE indicates liposome system and does not make by indicating the identical method of liposome with preparation.

The liposome fusion:

The sign liposome and the various not commensurability liposome that do not indicate of 10 microlitres are added among the 2mlPBS.After measuring relative fluorescent, the aqueous 1N Hcl of 5-20 microlitre is added the concentration that makes it to become various differences, again sample is given strong agitation to reach the PH of expectation.Behind about 2min under the room temperature, an amount of aqueous 1N NaOH is added, make PH return back to 7.4.Then measure the relative fluorescent of sample once more.

The fluorescent measurement method:

Use a kind of Perkin-ElmerLS5 fluorescent one spectrophotometer and assert the emission spectrum of each sample (under 468nm, being excited).Wide 5nm of being respectively of the seam that excites and launch and 3nm.The light emission amount is about the 5-6% of whole fluorescent mark.N-NBD-PE is sensitive measurement method (Struck et al., the same) that between N-NBD-PE and N-Rh-PE resonance energy shift usefulness to N-Rh-PE in the ratio (R) of exit dose under the 580nm in 530nm purgation exit dose.The R value that does not merge liposome is 0.20, and this is that it has the high-effect event of energy transfer.When indicating liposome and not indicating liposome when merging, the fluorescent lipid dilutes, thereby causes the reduction of energy transfer usefulness and the increase of R value (Blumenthal et al., the same and Struck et al., the same).Thereby the recruitment of R value is the quantitative measurement that liposome merges degree, and all the lipid of mixing can get maximum R value (R value is the ratio that does not indicate in the mensuration integration material the sign liposome) through merging fully.The fusion percentage ratio of liposome is to calculate with following equation:

The % fusion=(Rf/Ri-1)/(n) * 100

Wherein Ri and Rf reach the R value of reacting the back before being respectively fusion reaction.Not indicating liposome is n with the concentration rate that indicates liposome.The correctness of desire verification equation (1) is with sign and do not indicate liposome mixes with different ratios, and surveys its Ri value.When Rf/Ri-1 was the straight line of 45 ° of gradients to the point of n before n reaches 3, represent that then equation (1) is effective.Because n＞3 o'clock, it is lower than in theory that fluorescent adds epistasis, so most experiment is all carried out when n=3.The regeneration range of R value is ± 5%.

The leakage of liposome:

Desire is measured the leakage degree of liposome between incorporating period, is use the aqueous free token (Allen et al., the same) of the automatic quenching fluorescent dye (Calcein) of water solublity as inside.Liposome system is made by sound waveization among the PBS that contains 140mM Calcein by the lipoprotein mixture that lacks fluorescent phospholipid.Not built-in Calcein is that mat makes liposome pass through Sepharose-4B post (reaching equilibrium state in PBS) removing.The liposome that contains Calcein is then with above-mentioned acid and alkali treatment.Respectively at before the measurement fusion under the radiation wavelength of the excitation wavelength of 490nm and 518nm and after merging, and add 0.2%Triton X-100(and break in order to liposome) after the Calcein fluorescent.The latent form of Calcein fluorescent is calculated with following equation:

%=(1-(F)/(Ft) hides) * 100 (2)

Wherein F and Ft are respectively the existence of Triton or lack purgation Calcein fluorescence intensity.

Electron microscope technique:

Liposome gives negativity dyeing with 0.5% uranium acetate, observes down in Hitachi 600 ultramicroscope (in 75KV operation down) again.Picked-up amplifies 35, and the photo of 000X further amplifies photo again.Desire is measured the size distribution of liposome, be with take the photograph in the liposomes preparation by three kinds of differences microphotograph measure liposome more than 200, and draw block diagram.

Example 1

The deutero-monoclonal antibody of fatty acid is (anti--H2K ^k) with people such as Shen in Biochim.Biophys, Acta, 689 31(1982) method described in is inserted and is contained suitable substance P E-PHC(8 of the present invention: 2) merge in the reverse-phase evaporation cyst (REV) (Szoka Jr.et al., the same) of liposome.Then these immune liposomes are served as a mark to contain 140mM Calcein() PBS handle.

With mice L929 cell (K single set type) with these the immune liposome of marking be incubated jointly, and learn that according to the fluorescent inspection technique fluorescent of diffusion is to spread all over cell, this means that the Calcein dyestuff releases to Cytoplasm.

Under contrasting,, only show the fluorescent of fleck with the mouse cell that contains Calcein to the insensitive common insulation of immune liposome of PH.

In the control experiment, the result of the common insulation of mice A31 cell (d single set type) and the present invention's immune liposome, there is no fluorescent can detect.

Example 2

People's such as use-case 1 or Philippot method will contain in a kind of recombinant DNA (as plastid PBR322 etc.) the present invention's that packs into who is translated into the gene of nitrogenase password the PH susceptiveness liposome.Then Rhizoma Solani tuber osi oleo stock (it can make cell absorption liposome effectively) is added in the liposome.The slurry plastid film of the liposome of cell absorptionization and oleo stock merges, and its DNA is released to Cytoplasm.After this DNA enters host's genosome, reform a period of time, can show the effectiveness of nitrogenase.Then, make oleo stock in whole potato plants, regenerate.Because of its fixing nitrogen in the atmosphere, therefore can increase the output of frumentum.

Example 3

People's such as use-case 1 or Philippot method, to contain HGH structural gene such as bovine growth hormone (referring to Miller et al., European Patent Appln.No 47.600 is with 26,08.80 US 181,348 is basis or Praser et al., European Patent Appln.No.68,646, with 08.06.81US271,449 is the basis) or the mankind's former growth hormone (referring to Baxt-er et al., Eurropean Patent Appln No.20,147 with 01.06.79 US44,647 are the basis) etc. recombinant DNA (as the plastid PBR322 etc.) the present invention's that packs into PH susceptiveness liposome (except that containing PE and PHC(8: 2), also contain lactose acyl cerebrogalactoside (1 to 10 molar percentage)) in.These liposomes are injected kinetoplast by intravenous, in calves, selectively absorb by hepatocyte again.(Szoka, Jr.et al., Biochim Biophys.Res.Comm., 110 140(1983),

Go into herein for your guidance.The DNA of reformation a period of time after entering group, can produce excessive growth hormone.Growth rate of animal thereby increase.

Example 4

People such as use-case 1 or Philippot, the same method, to contain be translated into viral antiinflammatory or its partial password gene (as hepatitis B genosome etc., referring to Tiollais et al., U.K.Patent Appln.No.2,034,323: or Galibert et al., U.S.Patent No.4,428,941 recombinant DNA (as plastid PBR322 etc.) is filled in the present invention's the PH susceptiveness liposome (except that 3PE and PHC(8: 2), still have in the lactose acyl brain glycosides (1 to 10 molar percentage), these liposomes are injected animal (comprising mammals such as people etc.) body young or that grow up by intravenous.Hepatocyte is immunoreation (making antibody with antagonism antigen) in the primosome to the expression way of virus antigen or its part.Therefore, animal can inoculate the infection of this vaccine with antiviral.

Claims (32)

1, a kind of method of fusion liposome, comprising:

(1) manufacturing contains at least 20 molar percentage amphiphatic molecules liposome of (containing one or more weak acid functional groups);

(2) these PH from the liposome of step (a) are brought down below PH7.

2, be one or more carboxyl group according to the wherein above-mentioned amphiphilic weak acid functional group of the method for claim 1.

3, according to the method for claim 2, wherein above-mentioned amphiphatic molecule is long-chain (C ₁₂To C ₃₀) fatty acid.

4, according to the method for claim 3, wherein above-mentioned long-chain fatty acid is selected by Palmic acid and oleic acid to decide.

5, according to the method for claim 2, wherein above-mentioned amphiphatic molecule is the high half Guang acid of palmityl.

6, according to the process of claim 1 wherein that pH value is reduced to below 6.0 in step (b).

7, according to the process of claim 1 wherein that pH value is reduced to below 5.0 in step (b).

8, according to the process of claim 1 wherein, step (b) is to use aqueous inorganic acid to carry out.

9, method according to Claim 8, wherein, aqueous inorganic acid is a hydrochloric acid.

10, this liposome contains the amphiphatic molecule that can form six side's phase trend according to the process of claim 1 wherein the method to comprise the formation interior liposome of step (a) again.

11, according to the method for claim 10, wherein above-mentioned six side's phase amphiphatic molecules are PHOSPHATIDYL ETHANOLAMINE or its sour additivity long-chain (C ₁₆-C ₂₄) ester.

12, according to the method for claim 11, wherein above-mentioned long-chain ester is a DOPE.

13, according to the method for claim 12, wherein DOPE is about 8: 2 to the above-mentioned mol ratio that contains the amphiphatic molecule of one or more weak acid functional groups.

14, according to the method for claim 13, the wherein above-mentioned amphiphatic molecule system that contains one or more weak acid functional groups is by PHC, and Palmic acid and oleic acid are selected fixed.

15, external material is inserted to method in the living cells, wherein the method comprises:

(a) external material is filled to by in the made PH susceptiveness liposome of amphiphatic molecule (containing more than or a plurality of weak acid functional group); And

(b) make above-mentioned living cells contact, thereby the insertion effect takes place with the PH susceptiveness liposome of above-mentioned interior envelope.

16, according to the method for claim 15, wherein the PH susceptiveness liposome of step (a) further contains the amphiphatic molecule that can form six side's phase trend.

17, according to the method for claim 16, wherein six side's phase amphiphatic molecules system is by PHC, and it is fixed to be selected by acid and Palmic acid.

18, according to the method for claim 16, wherein the amphiphatic molecule of step (a) is a PHOSPHATIDYL ETHANOLAMINE.

19, according to the method for claim 18, wherein the mol ratio that contains of PH susceptiveness liposome was by 9: 1 to 1: 9 PHOSPHATIDYL ETHANOLAMINE (PE) and PHC (PHC).

20, according to the method for claim 19, wherein, the PE/PHC molar ratio is by 5: 1 to 1: 5.

21, according to the method for claim 19, wherein, the PE/PHC molar ratio is 8: 2.

22, according to the method for claim 15, wherein, foreign substance has: medicine, ferment, hormone, nutrient, antigen, antibody, RNA, DNA and conjugate thereof.

23, according to the method for 15 of claim the, wherein, the living cells born of the same parents are by yeast, algae, and mycete, antibacterial, it is fixed to select in the cell of the cell culture of mammal and all animals.

24, contain merging and not coalescent liposome of amphiphatic molecule (containing one or more acid functional groups).

25, according to the fusion liposome of 25 of claim the, wherein amphiphatic molecule system is selected from PHC, in Palmic acid and the oleic acid.

26, according to the fusion liposome of 25 of claim the, this liposome further contains the amphiphatic molecule that tool forms six side's phase trend.

27, according to the fusion liposome of 26 of claim the, wherein, six side's phase amphiphatic molecules are PHOSPHATIDYL ETHANOLAMINE.

28, according to the merging of 27 of claim the, do not assemble liposome, it contains mol ratio by 1: 9 to 9: 1 PHOSPHATIDYL ETHANOLAMINE (PE) and PHC (PHC).

29, according to the fusion liposome of 28 of claim the, wherein PE to the mol ratio of PHC by 5: 1 to 1: 5.

30, according to the fusion liposome of 28 of claim the, wherein PE is 8: 2 to the mol ratio of PHC.

31, containing mol ratio is 8: 2 PHOSPHATIDYL ETHANOLAMINE and oleic fusion liposome.

32, containing molar ratio is 8: 2 the PHOSPHATIDYL ETHANOLAMINE and the fusion liposome of Palmic acid.

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