CN1829498A - Lipid particles having asymmetric lipid coating and method of preparing same - Google Patents

Abstract

A method of preparing lipid particles having an asymmetric lipid coating is described. The lipid composition of the outer lipid coating of the particles varies from the inner to outer surfaces. The asymmetric lipid particles are formed by preparing a lipid composition containing a charged lipid and a therapeutic agent, where the particles each have an outer lipid coating with an external lipid leaflet and an internal lipid structure. The particles are then incubated under conditions effective to remove the charged lipid from the external lipid leaflet, thus rendering the lipid coating asymmetric. The particles have the ability to their regain surface charge via translocation of the lipids.

Description

Lipid particles and the method for preparing it with asymmetric lipid coating

Invention field

The present invention relates to a kind of lipid particles compositions, be used for to the individual with asymmetric lipid coating, more specifically, to the cell delivering therapeutic agents.

Background of invention

Lipid vesicle, or liposome have demonstrated for the purposes to target tissue and organ delivering therapeutic agents and diagnostic agent.Lipid vesicle has the aqueous interior that is surrounded by one or more lipid bilayer, and wherein said therapeutic agent bag is stated from the inner space of aqueous or in lipid bilayer.Thereby water solublity and water-insoluble drug can both be transported by lipid vesicle in the aqueous space and lipid bilayer respectively.

The effect of many medicines relates to the direct interaction in site in they and the cell.In order to play a role, medicine must pass cell membrane and arrive Cytoplasm.Owing to multiple reason, the success of sending in the cell of the reagent of realization liposome entrapment is restricted.A kind of reason is that liposome is removed from circulation by reticuloendothelial system rapidly after general is applied in the blood flow.Another reason is to send a kind of molecule, and particularly big and/or charged molecule enters the intrinsic difficulty in Cytoplasm and/or the nuclear.

Make it does not discerned and absorb by the hydrophilic polymer pan coating that is added on the liposome to cover up vesicle, overcome to a great extent by reticuloendothelial system and absorb the restriction that causes rapidly by reticuloendothelial system.The bag quilt (U.S. Patent number 5,013,556) that the liposome blood circulation life-span that prolongs provides bigger chance, described liposome to have Polyethylene Glycol (PEG) polymer chain for cell absorbs.

Send in the cell of charged molecule and remain a kind of technological challenge.Particularly, because the electric charge and the size of molecule, nucleic acid comprises the two send of DNA and RNA, all is challenging.Albumen, peptide and charged pharmaceutical composition relate to the technology barrier of identical cross-cell membrane transhipment.A kind of electronegative reagent, the delivering method that particularly carries out the nucleic acid fragment of gene therapy are that DNA or RNA are mixed mutually with cation lipid.The electrostatic interaction of cation lipid and nucleic acid allows to form the lipid-nucleic acid microgranule, and its magnitude range is suitable for carrying out using in the body.Externally the cation lipid of positively charged is useful for the interaction with electronegative cell membrane on the microparticle surfaces, thereby promotion lipid-nucleic acid microgranule merges or is absorbed in the cell.

But the existence of the positive charge on the outer surface of the lipid particles for preparing with cation lipid is deleterious for obtaining the long blood circulation life-span with the target of carrying out extensive bio distribution (biodistribution).Electric charge on the microgranule causes combining with tissue surface immediately near using site or its, limited microgranule basically and circulated and be distributed to availability on the target site.It is that neutral lipid vesicle composition is so that allow to carry out bio distribution using the back that people wish to design a kind of, its after date in the time of one section in addition, that is, be with electric charge after the bio distribution of microgranule, interacting to send in the cell that combines and wrap the reagent that carries with cell membrane so that allow.

Summary of the invention

Therefore, an object of the present invention is to provide a kind of lipid particles compositions, it comprises that a kind of charged lipid is used for interacting with charged therapeutic agent, and it has minimum outer surface electric charge after formation in addition.

Another object of the present invention provides a kind of lipid particles compositions, and it comprises a kind of charged lipid and have minimum outer surface electric charge after microgranule forms, but it can produce electric charge as time passes, as carrying out between incubation period at physiological temp.

On the one hand, the present invention includes the method that a kind of preparation has the lipid particles of external lipid bag quilt.This method comprises that preparation comprises the lipid composition of charged lipids and the (ii) lipid particles formed of therapeutic agent by (i).Each microgranule all has external lipid bag quilt, and described lipid coating has external lipid lobule and internal lipids structure.Subsequently microgranule is carried out incubation under the condition that described charged lipids is effectively removed from the external lipid lobule.

In one embodiment, described lipid particles is made up of the lipid composition that comprises at least a cation lipid.

In another embodiment, preparation process comprises that (i) forms the lipid vesicle be made up of lipid composition and (ii) that lipid vesicle and therapeutic agent is compound.

In one embodiment, the incubation of lipid particles is included in the medium that contains charged lipid vesicle not and carries out incubation.In another embodiment, a kind of lipid-polymer-ligand conjugate can be joined in the incubation medium.In other embodiment, described incubation medium may further include a kind of with the deutero-lipid of hydrophilic polymer.A kind of exemplary be by the deutero-phospholipid of Polyethylene Glycol with the deutero-lipid of hydrophilic polymer.

In other embodiment, the incubation of microgranule is less than about 15 ℃ temperature and/or continue to carry out greater than about 5 hours time.

In one embodiment, described lipid particles is a liposome.

Still in other embodiment, described lipid particles is prepared as has a kind of therapeutic agent that is carried by bag, described therapeutic agent is selected from charged medicine, albumen, peptide and nucleic acid.

On the other hand, the present invention includes a kind of compositions that comprises the lipid particles with lipid coating, described lipid coating is made up of external lipid lobule and internal lipids structure.The lipid composition that described lipid coating comprises charged lipids by (i) and (ii) has gel-crystal form phase transition temperature forms, wherein said lipid particles has a little or does not have tangible electric charge in the temperature that is lower than the lipid composition phase transition temperature, but has the electric charge that can measure after being higher than described phase transition temperature incubation.

In one embodiment, described lipid composition has about 34-38 ℃ phase transformation.

Also in yet another aspect, the present invention includes a kind of method for preparing lipid particles, in its external lipid bag quilt, have asymmetric charged lipids compositions before described lipid particles is used in body.This method comprises that preparation comprises the lipid composition of charged lipids and the (ii) lipid particles formed of therapeutic agent by (i), and wherein each microgranule all has outside lipid coating, and described lipid coating has external lipid lobule and internal lipids structure.Microgranule is carried out incubation under the condition that charged lipids is effectively removed from the external lipid lobule.

In one embodiment, incubation is carrying out less than about 15 ℃ temperature.In another embodiment, incubation period is for surpassing about 5 hours time.In another embodiment, incubation medium is made up of the neutral fat vesicle.

When reading following detailed Description Of The Invention in conjunction with the accompanying drawings, these and other objects that invention will be more fully understood and feature.

The accompanying drawing summary

Figure 1A-1E is the diagram of lipid particles described herein;

Fig. 2 A shows the flow chart that forms the lipid particles step;

Fig. 2 B is each sketch map that forms the exemplary lipid-DNA microgranule on the step;

Fig. 3 is the figure of the zeta potential represented with mV, the function of its cation lipid concentration as cationic-liposome (DOTPA, mole percent);

Fig. 4 A by DMTAP/DOPE/ cholesterol/mPEG-DS (50: 24: 24: the figure of the zeta potential of representing with mV of the asymmetric lipid particles of 2) forming, it is as at buffer (rhombus) with containing in the buffer (triangle) of neutral fat vesicle in 37 ℃ with hour function of the incubation time of expression;

Fig. 4 B by DMTAP/DOPE/ cholesterol/mPEG-DS (50: 25.5: 22.5: the figure of the zeta potential of representing with mV of the asymmetric lipid particles of 2) forming, it is as at buffer (rhombus) with containing in the buffer (triangle) of neutral fat vesicle in 37 ℃ with hour function of the incubation time of expression;

Fig. 5 by DMTAP/DOPE/ cholesterol/mPEG-DS (50: 25.5: 22.5: 2) form and in 4 ℃ of figure that preserve the zeta potential of representing with mV of bimestrial asymmetric lipid particles, it is as at buffer (rhombus) with containing in the buffer (triangle) of neutral fat vesicle in 37 ℃ with hour function of the incubation time of expression; And

Fig. 6 is the figure that shows luciferase expression in the cell of representing with pg/mg albumen for six kinds of lipid particles preparations, described lipid particles preparation was by DMTAP/DOPE/ cholesterol/mPEG-DS (50: 25.5: 22.5: 2) form, wherein preparation 1-3 does not have the contrast microgranule of asymmetric bilayer and preparation 4-6 is the lipid particles with asymmetric outside bilayer, wherein before transfection all preparations all at 37 ℃ of incubations 0 hour (preparation 1,3), the time of 48 hours (preparation 2,5) and 60 hours (preparation 3,6).

Detailed Description Of The Invention

I. definition

" lipid particles " means Any shape or the big or small microgranule with at least one lipid bilayer as used herein.That is, this term comprises monolayer, multilamellar (plurilamellar) and multilamellar (multilamellar) vesicle.In some microgranules, the part of microgranule can be that the other parts of monolayer then can be multiwalled.Microgranule can be spheric, or can be in shape more like spherical.Be included in the complex in the term " lipid particles " for liposome and lipid and other microgranule composition.Microgranule can have definite aqueous space, that is, liposome perhaps can have the spatial zone of pocket or aqueous, that is, and and lipid complex.

Abbreviation: DMTAP:1,2-two myristoyl-3-trimethyl ammonium-propane; DOPE: DOPE; PEG: Polyethylene Glycol; DS: distearyl acyl group; MPEG-DS: methoxyl group (Polyethylene Glycol)-distearyl acyl group.

II. lipid particles and preparation method

On the one hand, the present invention relates to a kind of method for preparing lipid particles, it has external lipid bag quilt and the internal lipids part that has outer surface, and the compositions gradient of crossing over described lipid coating, and described lipid coating extends between outer surface and internal lipids part.This microgranule with the compositions gradient of crossing over all or part of lipid coating has the material that is called asymmetric lipid composition, and it will illustrate in Figure 1A-1E, wherein show the idealized diagram of these lipid particles.Figure 1A has shown to have single external lipid bag by 12 monolayer lipid particles 10.The lipid coating of a part shows to illustrate the arrangement of described lipid better with exploded view.Described bag is made up of by surface 16 by surface 14 and inner bag external packets.As shown here, bag is taked the form of lipid bilayer; But this figure is that Utopian and described lipid coating can have complicated more lipid arrangement.External packets is by the external lipid lobule of surface corresponding to the bag quilt, and wherein the polarity headgroup to the lipid in the lobule carries out orientation, so that contact with outside a large amount of media of the microgranule that suspended.Corresponding to the internal lipids structure, it can be that the lipid lobule maybe can be that complicated more lipid is arranged to inner bag by the surface, and wherein the polarity headgroup to the lipid in the inner lobule carries out orientation, so that contact in the aqueous space of microgranule.Because mainly by not formed with the lipid of just (cationic) or negative (anionic) electric charge, lipid or other neutral lipid that for example neutral vesicle forms, external lipid lobule have electric charge low or minimum.In a preferred embodiment, the external lipid lobule is made up of neutrality or anion lipid; Promptly owing to the existence of cation lipid, the external lipid lobule comprises a little or does not have significant cationic charge.The internal lipids structure is made up of charged lipids.This feature is illustrated by the positively charged lipid on 18,20 in the indication internal lipids structure in Figure 1A, and described positively charged lipid provides positive charge on wrapping by the surface in inside.For this lipid particles with monolayer lipid coating, lipid is formed between inside and outside lipid lobule difference has formed the structure that this paper is referred to as asymmetric lipid coating.More specifically, stride across the external lipid bag and be called asymmetric external lipid bag quilt by the difference of the charged lipids of thickness composition.

Figure 1B-1C is the diagram of multilamellar lipid vesicle, and it also is to be prepared by describing the method that produces asymmetric outside lipid bilayer or bag quilt.At first, with reference to Figure 1B, vesicle 24 has lipid coating layers a plurality of nido, concentric, demarcates to be Utopian bilayer 26,28,30.In practice, the number of lipid bilayer can be than three shown in the figure much more and on lipid is arranged can than shown in simplification bilayer complexity many.The part of the lipid coating that will be made up of lipid bilayer 26,28,30 shows to be convenient to observe the arrangement of lipid with exploded view.Lipid coating 30 be outmost lipid layer and with the suspension of outside the medium of microgranule contact.The external lipid bag is had outer surface 32 and inner surface 34 by 30, and its outer surface is limited by external lipid lobule 36 and inner surface is limited by internal lipids structure 38.Lipid with respect to internal lipids structure 38 is formed, and it is different that the lipid of external lipid lobule 36 is formed, and its difference is that the external lipid lobule has less charged lipids, and is more preferably less cation lipid.On the contrary, the internal lipids structure comprises the cation lipid that causes along the positive surface charge of inner surface 38, shown in the plus sige on 40,42.Thereby in the multilamellar lipid particles shown in Figure 1B, the external lipid bag is asymmetric by forming about its lipid.

Fig. 1 C shows the lipid particles 44 similar to the lipid particles 24 of Figure 1B, and similar elements is determined by similar Digital ID.Microgranule 44 comprises a plurality of lipid layers, as layer 26,28,30.The external lipid bag is had outer surface 32 and inner surface 34 by 30.In this embodiment, the external lipid bag by 30 lipid to form be constant relatively between the surface 32 and inner surface 34 outside, wherein the external lipid bag is had minimum charged lipids.Yet, in it is formed, comprise charged lipid at the lipid layer of most external lipid coating 30 inside, shown in plus sige 43,45,47.Thereby, in this embodiment of lipid particles, asymmetric lipid coating about the external lipid bag by 30 and internal lipids structure or lipid layer.That is, form about its lipid, lipid coating is asymmetric owing to extend to inner particle areas from the outer particle surface so do as a whole.

Fig. 1 D shows another embodiment to multilamellar lipid particles 46 similar described in Figure 1B.But microgranule 46 comprises a kind of with hydrophilic polymer deutero-lipid, and as lipid 48,50,52, it is the representative of lipid of deriving in the every kind of lipid bilayer 54,56,58 shown in the figure.As discussed below, the microgranule with the lipid that crosses the polymer-derived that the microgranule lipid coating distributes is to form by during microgranule forms the lipid of polymer-derived being included in the lipid mixture.External lipid bag in the microgranule 46 is had asymmetric lipid by 58 to be formed, and this is to have charged lipids because externally do not have charged lipids in the lipid lobule 60 in internal lipids structure 66, as lipid 62,64.In the internal lipids lobule, exist charged lipids to cause into charged internal lipids bag by the surface, as along inside bag by shown in the lip-deep plus sige.

The lipid particles of Fig. 1 D comprises a kind of additional features that can randomly be comprised.Lipid particles can also be prepared into and comprise the targeting part, and as part 70,72, it works as targeting (homing) device so that lipid particles brought into the required site of carrying out therapeutical effect.The targeting part that is attached on the lipid particles exists, for example, and U.S. Patent number 5,891,468; 6,056,973 and 6,180, introduce to some extent in 134, be incorporated herein by reference about the part that is suitable for use as the targeting part, the preparation of lipid conjugates of carrying part and the disclosure of preparation that comprises the lipid particles of lipid-ligand conjugates in these patents at this.Can after formation, the targeting part be mixed in the lipid particles by incubation preformed liposome microgranule in the micelle solution of lipid-part or lipid-joint-ligand conjugates.Can also to form microgranule the targeting part be incorporated in the lipid particles by lipid-ligand conjugates being included in the lipid composition.

Another kind of exemplary lipid particles is shown among Fig. 1 E.As mentioned above, the diagram among Figure 1A-1D is highly Utopian, has shown globular microgranule and clear and definite lipid layer and clear and definite aqueous space.In practice, micrograined texture may be complicated many, illustrated as part among Fig. 1 E.Fig. 1 E has shown a kind of lipid-DNA microgranule 51 of being made up of cation lipid (being represented by solid headgroup, as lipid 53,55) and neutral lipid (being represented by hollow headgroup, as lipid 57,59).DNA 61 is placed the inside of microgranule, and by charge interaction to wrap quilt than the more cation lipid of neutral lipid.The internal lipids structure that microgranule has definite external lipid lobule 63 and is made up of the lipid layer of the little intralobar part of external lipid.Thereby the internal lipids structure comprises the inside lobule 65 relative with outside lobule 63 and surrounds the inside bilayer of DNA, as bilayer 67,69.Microgranule 51 has the spatial pocket of aqueous, as pocket 71, but do not have in conventional liposome general determine the aqueous interior compartment.

A. lipid particles preparation

As discussed above, particularly about Figure 1B-1C, microgranule as herein described about the single lipid layer in the microgranule (for example, Figure 1B, the lipid components of wherein said external lipid layer is asymmetric) or have asymmetric lipid coating about lipid coating (because it extends to inner particle areas from the outer particle surface) (Fig. 1 C).To the preparation of this asymmetric lipid particles be described now.

According to the lipid particles that in Fig. 2 A, has asymmetric external lipid bag quilt, wherein provide the flow chart of the basic step that relates to lipid particles formation with the method preparation of general term explaination.Fig. 2 B provides the sketch map of exemplary lipid particles character on each step in formation.On the meaning of broad, the first step is by a kind of charged lipids that comprises, prepares lipid vesicle as the lipid composition of cation lipid.Next, lipid vesicle is mixed with therapeutic agent,, form lipid particles thus so that make reagent mutually compound with charged lipids.A kind of exemplary microgranule corresponding to this step is shown as microgranule 80 in Fig. 2 B.Microgranule 80 is made up of cation lipid (being represented by solid headgroup, as lipid 82,84) and neutral lipid (being represented by hollow headgroup, as lipid 86,88).DNA 90 is placed the inside of microgranule and use than the more cation lipid of neutral lipid and wrap quilt by charge interaction.

Then with reference to figure 2A, subsequently with lipid particles in medium and realizing being carried out incubation under the condition of extraction charged lipids from external lipid bag quilt or from the outside lobule of the outermost layer lipid coating of lipid particles.After carrying out incubation with the incubation medium that comprises the neutral lipid liquid suspension, the character of lipid-DNA microgranule is shown as microgranule 100 in Fig. 2 B.Represented in the diagram as the lipid that is had solid headgroup by the minority with respect to the microgranule before the incubation 80, the incubation of microgranule causes the removal of cation lipid from external lipid lobule 102.Externally the difference of forming between lipid lobule and the internal lipids structure is that microgranule has added asymmetric lipid composition.Asymmetric microgranule 100 has the surface charge that reduces with respect to the microgranule before the incubation 80.In one embodiment of the invention, incubation is enough to charged lipids is removed from the external lipid lobule with from the internal lipids structure of adjacency external lipid lobule.In another embodiment, carry out incubation in such a way, for example, by changing incubation time, temperature and/or medium, so that mainly from the external lipid lobule, remove charged lipids.This after the microgranule 110 of embodiment in Fig. 2 B in illustrate, and can discuss more fully below.In brief, this after embodiment in, charged lipids is present in the internal lipids structure, as the lipid lobule 104 of microgranule 100, and can be used for transposition or " upset " goes to the external lipid lobule.Described transposition is by microgranule is carried out incubation in the temperature that is enough to make lipid move, and generally is the temperature in the phase transition temperature that is higher than lipid composition.The higher concentration of charged lipids permission charged lipids inserts to the low concentration district in the external lipid lobule in the internal lipids floor.As with respect to microgranule 100 in microgranule 110 by as shown in the cation lipid (representing) that increases by solid polarity headgroup, described transposition causes forming surface charge again on lipid-DNA microgranule.

Also might have the lipid vesicle of asymmetric external lipid bag quilt and generate asymmetric lipid particles with asymmetric vesicle and charged medicine are compound subsequently by preparation at first.In this embodiment, the lipid particles of forming by charged lipids be suitable for from the external lipid bag by or lobule remove under the condition of most of charged lipids and carry out incubation, make asymmetric vesicle thus.After asymmetric lipid coating forms, that asymmetric lipid vesicle and medicine is compound to form asymmetric lipid-drug microparticles.

1. the preparation of lipid vesicle

Prepare the lipid particles that is generally the single or multiple lift liposome by lipid composition, described lipid composition comprises a kind of charged lipids, preferably cation lipid.Described cation lipid can be that vesicle unique in the compositions forms the property lipid, perhaps can be that vesicle in the compositions forms one of them of two or more lipids that property or non-vesicle form property.Exemplary monolayer vesicle prepared in accordance with the present invention is by lipid composition preparation, and described lipid composition is formed by cation vesicle formation property lipid, neutral lipid with the deutero-vesicle formation property of hydrophilic polymer lipid.

Cation vesicle formation property lipid is at effective pH, and for example, pH 4-9 has the polarity headgroup with clean positive charge.Exemplary cation lipid comprises 1,2-two oleoyl oxygen-3-(trimethyl amino) propane (DOTAP); N-[1-(2,3 ,-two tetradecyloxyanilines) propyl group]-N, N-dimethyl-N-ethoxy ammonium bromide (DMRIE); N-[1-(2,3 ,-two oleoyl oxygen) propyl group]-N, N-dimethyl-N-ethoxy ammonium bromide (DORIE); N-[1-(2,3-two oleoyl oxygen) propyl group]-N, N, N-trimethyl ammonium chloride (DOTMA); 1,2-two myristoyl-3-trimethyl ammonium-propane (DMTAP); Dioleoyl phospholipid phatidylcholine (DOPC); 3 β [N-(N ', N '-dimethylamino ethane) carbamoyl] cholesterol (DC-Chol); Dimethyl dioctadecyl ammonium DDA N,N-Dimethyl-N-octadecyl-1-octadecanaminium (DDAB), cationic surfactant, sterin amine etc.Also might be by provide neutral or electronegative lipid cation with deriving of cationic moiety.For example, can be with a kind of phospholipid, as PHOSPHATIDYL ETHANOLAMINE, on its polarity headgroup, use the positive electricity part, for example lysine carries out derivatization, for example for illustrated (Guo, L. etc., Journal of LiposomeResearch 3 (1): the 51-70 (1993)) of lipid DOPE (LYS-DOPE) with L-lysine derivatization.This cationoid lipid also comprises glycolipid, as has the cerebroside and the ganglioside of cation polarity headgroup.The another kind of cation vesicle that can adopt formation property lipid is Cholesterylamines and relevant cation sterin.

Be to be understood that the charged lipids during being included in lipid vesicle forms can be the anion lipid, as GLYCEROL,DIMYRISTOYL PHOSPHATIDYL (DMPG); DOPG (DOPG); DOPE (DOPE); Dioleoyl phospholipid phatidylcholine (DOPC); Deng.

Except charged lipid matter, the lipid composition of preparation lipid vesicle can also comprise other lipid.Generally, said composition will comprise that a kind of vesicle forms the property lipid, its mean a kind of can be in aqueous medium the lipid of spontaneous formation bilayer vesicle, for example phospholipid.Described lipid composition can also comprise the stable lipid that mixes lipid bilayer, and its hydrophobic parts contacts with the inner hydrophobic zone of bimolecular tunic, and its headgroup partly is oriented to the outside polar surfaces of bilayer lipid membranes.Vesicle formation property lipid preferably has two hydrocarbon chains, is generally the fatty acyl group chain, and polarity or nonpolar headgroup.Multiple synthetic vesicle formation property lipid and naturally occurring vesicle formation property lipid are arranged, comprise phospholipid, as phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, phosphatidic acid, the pure and mild sphingomyelins of phosphatidyl-4, wherein two hydrocarbon chain length are generally about 14-22 carbon atom, and have the unsaturation of intensity of variation.Having the phospholipid that has the fatty acyl group chain of the unsaturation of intensity of variation can be commercially available or prepare according to disclosed method.

In one embodiment, select vesicle to form the property lipid obtaining specific flowability or rigidity, with the condition of controlling effective insertion targeting lipid-ligand conjugates and/or allow charged lipids after the body lipid microgranule that will introduce is used internally lipid conformation be displaced in the external lipid lobule.By mixing the lipid of relative stiffness, for example, have high relatively phase transition temperature, for example, be up to the lipid particles that 60 ℃ lipid obtains to have more inflexible lipid coating.Inflexible, that is, saturated lipid helps membrane rigidity bigger in the lipid coating.Also known other lipid composition as cholesterol, also helps the membrane rigidity in the lipid conformation.Exemplary rigidity lipid comprises distearoyl phosphatidylcholine (DSPC) with 62 ℃ of phase transition temperatures and the hydrogenated soya phosphatide phatidylcholine (HSPC) with 58 ℃ of phase transition temperatures.

Have the lipid of relatively low liquid to liquid-crystal form phase transition temperature by mixing mobile relatively lipid, being typically, the lipid that for example is equal to or less than about 37-38 ℃ body temperature obtains mobile more bilayer.Example with the lipid that is lower than 38 ℃ of phase transition temperatures is lecithin phatidylcholine (15 to-7 ℃), dimyristoyl phosphatidyl choline (23 ℃), 1-myristoyl-2-palmitoylphosphatidyl choline (27 ℃), 1-palmityl-2-myristoyl phosphatidylcholine (35 ℃), GLYCEROL,DIMYRISTOYL PHOSPHATIDYL (23 ℃), brain sphingomyelins (32 ℃) (Szoka, F. etc., Ann.Rev.Biophys.Bioeng., 9:467 (1980)).The phase transition temperature of many lipids is tabulated in multiple source, as Szoka ﹠amp; Papahadjopoulos, Ann.Rev.Biophys.Bioeng., 9:467-508 (1980), Avanti polar lipid catalogue, with LipidThermotropic Phase Transition Database (LIPIDAT, NIST StandardReference Database 34).

As above addressed about Fig. 1 D, lipid particles can randomly comprise the pan coating of the targeting conjugate of hydrophilic polymer chain and/or lipid-anchored.By being included in the lipid composition that is used to form lipid vesicle, any of these features all can be incorporated in the lipid particles at the initial step of described method lipid and/or the deutero-lipid of part with polymer-derived.Also may be in the 3rd step of method, when lipid particles during incubation, is incorporated into these features in the lipid particles in medium.In this case, the deutero-lipid of the lipid of polymer-derived and/or part is included in the incubation medium, and is inserted between incubation period in the external lipid bag quilt of lipid particles.This so-called " insertion " method is at U.S. Patent number 5,891, carries out description in 468,6,056,973 and 6,210,707.

Described with the deutero-lipid of hydrophilic polymer and the liposome (U.S. Patent number 5,013,556 that comprise the lipid of polymer-derived; U.S. Patent number 5,395,619).The lipid that is incorporated into the polymer-derived in the lipid coating forms the pan coating of a hydrophilic polymer chain around lipid vesicle.When comparing with the lipid particles that lacks this bag quilt, the pan coating of described hydrophilic polymer chain has effectively increased the body inner blood cycle life of lipid particles.Be suitable for forming the property lipid with the vesicle of hydrophilic polymer derivatization and comprise any above listed lipid, phospholipid particularly is as DSPE (DSPE).

Be suitable for comprising polyvinylpyrrolidone, polyvinyl methyl ether, Ju Jia oxazolin, Ju ethyl oxazoline, poly-Qiang Bing oxazolin, poly-hydroxypropyl methyl acrylamide, PMAm, polydimethylacrylamiin, polymethylacrylic acid hydroxypropyl acrylate, Poly(Hydroxyethyl Methacrylate), hydroxy methocel, hydroxyethyl-cellulose, Polyethylene Glycol, poly-asparagine and hydrophilic peptide sequence with the hydrophilic polymer of vesicle formation property lipid derivatization.Above-mentioned polymer can be used as homopolymer or block or random copolymer.

A kind of preferred hydrophilic chain is Polyethylene Glycol (PEG), and preferred molecular weight is 500-10,000 daltonian PEG chain, more preferably 1,000-5,000 dalton.Can commercially availablely buy what have the multiple polymers size also is preferred hydrophilic with the end capped PEG analog of methoxy or ethoxy, 120-20 for example, 000 dalton.

Preparation with hydrophilic polymer derivatization vesicle formation property lipid was for example being carried out description in the U.S. Patent number 5395619.Preparation comprises also existing description of method of the liposome of this kind derivatization lipid, wherein usually comprises the described derivatization lipid of 1-20 mole percent in the Liposomal formulation.

Lipid with targeting part derivatization had also carried out description (U.S. Patent number 5,891,468,6,056,973 and 6,210,707).The targeting part generally is as the part of receptor-ligand in conjunction with a paired part, and wherein said paired part is attached on the lipid particles so that microgranule can specificity be attached to have on the paired particular target of its receptor.Exemplary part is at U.S. Patent number 5,891, provides in 468, at this it is incorporated herein by reference.Particularly preferred part is those parts by cell internalizing after being attached to cell receptor.Send in the cell of this part permission lipid particles content.

2. the lipid particles that is compounded to form by lipid vesicle and therapeutic agent

Then, after forming charged lipid vesicle, vesicle is mixed with therapeutic agent to form lipid particles with reference to figure 2.As used herein, " lipid vesicle " refers to lipid conformation, and it can be little or big single or multiple lift liposome, perhaps can be the lipid conformation with less definite lipid layer." lipid particles " refers to and therapeutic agent, more specifically with the compound lipid vesicle of charged therapeutic agent.

As mentioned above, lipid vesicle comprises a kind of charged lipids, to give vesicle total electric charge.Described total electric charge is by comprising that the anion lipid can be minus, perhaps by comprising that cation lipid can be positive.With the blended therapeutic agent of charged lipid vesicle also be charged, more specifically, carry the electric charge opposite with lipid vesicle.The cationic lipid vesicle mixes with electronegative therapeutic agent complex to form lipid particles.Similarly, the anion resin vesicle mixes with the treatment complex of positively charged to form lipid particles.

Positively charged and electronegative therapeutic agent are known in the art.Preferred electronegative therapeutic agent is a nucleic acid, can be DNA or RNA, strand or two strands.In one embodiment, described nucleic acid is a kind of by the antisense DNA oligonucleotide of forming with the complementary sequence of its target, and described target is generally messenger RNA (mRNA) or mRNA precursor.Described mRNA comprises function or hereditary information in justice, the orientation is arranged, and antisense oligonucleotide in conjunction with the mRNA of deactivation expection and stop it to translate into albumen.Based on biochemical test these antisense molecules are measured, shown that albumen translate by specific RNAs, and the sequence of known RNA in a single day, just can design by complementary Wo Sen-Ke Like base pair and be attached to the antisense molecule on it.This antisense molecule generally comprises 10-30 base pair, more preferably is 10-25, most preferably 15-20.Antisense oligonucleotide can be modified to thiophosphate, methyl phosphonate, di-phosphate ester and to the ethyoxyl oligonucleotide to improve resistance (WO97/07784) for the nuclease hydrolysis.

Nucleic acid can include but not limited to as the therapeutic agent of multiple therapy, the treatment of viral, pernicious and inflammatory diseases and situation, as, cystic fibrosis, ADA Adenosine deaminase defective and AIDS.Be intended to by using tumor suppressor gene, as APC, DPC4, NF-1, NF-2, MTS1, RB, p53, WT1, BRCA1, BRCA2, VHL or use oncogene, carry out treatment of cancer as PDGF, erb-B, erb-B2, RET, ras (comprising Ki-ras, N-ras), c-myc, N-myc, L-myc, Bcl-1, Bcl-2 and MDM2.Also be intended to the situation that shows be treated: HLA-B7, tumor, colorectal carcinoma, melanoma by using following nucleic acid; IL-2, cancer, particularly breast carcinoma, pulmonary carcinoma and tumor; IL-4, cancer; TNF, cancer; Antisense IGF-1, cerebroma; IFN, neuroblastoma; GM-CSF, renal cell carcinoma; MDR-1, cancer, particularly terminal cancer, mammary gland and ovary cancer; Factor IX, hemophilia and HSV thymidine kinase, cerebroma, head and neck tumor, mesothelioma and ovarian cancer.

Except nucleic acid, charged organic drug molecule also is suitable for lipid vesicle compound.Multiple charged medicine is known in the art, and is easy to be discerned by those skilled in the art.

3. the incubation lipid particles is to produce asymmetric external lipid bag quilt

Then, after lipid vesicle and therapeutic agent is compound, lipid particles is effectively being carried out incubation under the condition of extraction at least a portion charged lipids from external lipid bag tegillum or from the external lipid lobule of external lipid bag tegillum with reference to figure 2.Discuss in Fig. 1 as top, described lipid particles comprises the external lipid bag quilt by internal lipids structure and external lipid surface composition.External lipid surface or lobule contact with the incubation medium of outside.Lipid particles is carried out incubation to realize from outside hour or remove whole basically charged lipids from outmost lipid coating, obtain asymmetric external lipid bag quilt thus.As will be described further, measure the removal degree of charged lipids, and by controlling such as the factor of incubation time, temperature and medium.

Distribute medium into to realize charged lipids is removed from external lipid bag quilt or lobule by lipid particles being placed charged lipids.The condition that influences the distribution of lipid in from the microgranule to the medium changes, and comprises the selection of incubation medium, the temperature and the incubation time of incubation medium.In the research of carrying out supporting the present invention, the incubation medium of being made up of the waterborne suspension of neutral fat vesicle can effectively cause the lobule distribution of cation lipid from external lipid bag quilt or microgranule lipid coating.Comprise the incubation medium of neutral fat vesicle and work, cause cation lipid and move to low concentration the incubation medium from the high concentration of lipid particles external packets quilt as the storehouse (sink) of cation lipid.Preferred incubation medium comprise be present in lipid particles in identical neutral lipid, thereby a large amount of transfers of neutral lipid from the microgranule to the incubation medium do not take place.Other exemplary incubation medium is to comprise that electronegative lipid, surfactant, polymer particles or other can pull out the medium of the material of charged lipids from lipid particles.

In another embodiment, at the incubation lipid particles with after reducing the cation form surface charge, subsequently with the microgranule incubation in second kind of medium, it comprises a kind of electronegative lipid matter so that negative charge is imported in the external lipid lobule of lipid particles.

As mentioned above, lipid particles can randomly comprise the pan coating of hydrophilic polymer chain and/or the targeting conjugate of lipid-anchored.Be included in the incubation medium and the lipid or the deutero-lipid of part of polymer-derived can be mixed in the lipid particles by one of them or both these conjugates.Between incubation period, conjugate is inserted in the external lipid bag quilt of lipid particles.The insertion of lipid-polymer conjugate and/or lipid-targeting ligand conjugates can be adjusted according to composition, targeting part and the other factors of lipid bilayer between the incubation period of lipid particles.For example, inserting speed fast can be obtained by higher heated culture temperature, but must carry out balance with respect to temperature, and described part can be heated to this temperature safely and can not influence its activity.The phase transition temperature of lipid also can be determined the temperature that is suitable for inserting in the lipid composition.It is also understood that insertion can change with the existence of solvent,, comprise Polyethylene Glycol and ethanol as two solvophilics, or detergent.

B. the sign of lipid particles

Preparation lipid particles as described in example 1 above.In brief, prepare the little monolayer vesicle of cation (SUVs) by DMTAP, DOPE, cholesterol and mPEG-DSPE.Cationic lipid vesicle and the DNA plasmid that carries the luciferase reporter gene is mutually compound to form lipid particles.Carry out the compound of kation S UVs and nucleic acid in about 0 ℃ temperature.To separate in never compound kation S UVs of lipid particles and/or the nucleic acid.Subsequently, with lipid particles in the incubation medium of forming by neutral SUVs (POPC, cholesterol and mPEG-DSPE) in 4 ℃ temperature incubation 24 hours.Behind the incubation, will have now by sucrose density gradient ultra-centrifugation in lipid particles other lipid composition from incubation medium of asymmetric outside lipid bilayer and separate, to carry out the charge analysis of zeta potential.

The zeta potential value provides measuring of apparent charge on the microgranule outer surface.More specifically, zeta potential is measuring of the electromotive force that takes place of face transboundary, described interface between with contacted liquid boundary layer of solid and liquid in removable diffusion layer, for example, between the sliding surface.Utilize commercially available instrument, shown in following method part, the zeta potential value is measured.Fig. 3 has shown for by DOTAP (xmol%), POPC (55-xmol%), cholesterol (40mol%) and the deutero-distearyl acyl group of Polyethylene Glycol (PEG-DS; the cationic lipid vesicle of 5mol%) being formed; relation between the zeta potential of representing with mV, wherein the amount of DOTAP shows with mol% along the x axle of figure.Composition by indication prepares lipid vesicle and is squeezed into the size of about 100nm.In 5mM NaCl, measure zeta potential in 25 ℃.Zeta potential is as the function of cation lipid DOTAP and improve, and wherein observing zeta potential when concentration when 0 brings up to 10 mole percents improves rapidly, then observes slower raising for the compositions that has greater than 10 mole percent DOTAP.

In another research, according to embodiment 1 preparation lipid particles.Described lipid composition is made up of DMTAP (50mol%), DOPE (24mol%), cholesterol (24mol%) and PEG-DS (2mol%).After lipid particles and DNA is compound, but carrying out incubation, keeping a lipid particles sample and carry out zeta potential and analyze property contrast as a comparison with before generating asymmetric lipid coating.With remaining microgranule in the medium that comprises neutral lipid microgranule (embodiment 1) incubation repeatedly to generate asymmetric lipid coating.To separate in asymmetric lipid particles other lipid vesicle from incubation medium by sucrose density gradient ultra-centrifugation, and measure zeta potential.The result is shown among the table 1A with the particle size of measuring by dynamic light scattering.

Table 1A

The zeta potential of lipid particles of Chu Liing and asymmetric lipid particles under various conditions

Lipid particles	In 90 ° particle size (nm)	Zeta potential (mV)
			The lipid particles that does not have asymmetric lipid coating, contrast	272	17.27
Lipid particles with asymmetric lipid coating, it by forming in 25 ℃ of incubations in the medium of neutral vesicle is arranged in 24 hours	311	8.30
			Lipid particles with asymmetric lipid coating, it by forming in 37 ℃ of incubations in the medium of neutral vesicle is arranged in 3.5 hours	295	8.63

The zeta potential that does not have the lipid particles of asymmetric lipid coating is 17.27mV, shows the positive charge on the microgranule outer surface.Microgranule was reduced to zeta potential 8.30mV and 8.63mV respectively in 3.5 hours effectively in 25 ℃ of incubations 24 hours with in 37 ℃ of incubations in the incubation medium that comprises the neutral fat vesicle, the display surface electric charge is significantly weakened.

Utilization has been carried out similar research by the lipid particles that DMTAP (50mol%), POPC (24mol%), cholesterol (24mol%) and PEG-DS (2mol%) form.After lipid particles and DNA is compound, but carrying out incubation to measure zeta potential in contrast on the sample at lipid particles before generating asymmetric lipid coating.Microgranule is carried out incubation to generate asymmetric lipid coating in different time and temperature in incubation medium.Zeta potential is measured and is shown among the table 1B by the particle size that dynamic light scattering is measured.

Table 1B

The zeta potential of lipid particles of Chu Liing and asymmetric lipid particles under various conditions

Lipid particles	In 90 ° particle size (nm)	Zeta potential (mV)
			The lipid particles that does not have asymmetric lipid coating, contrast	299	23.07
Lipid particles with asymmetric lipid coating, it by forming in 25 ℃ of incubations in the medium of neutral vesicle is arranged in 24 hours	349	4.50
			Lipid particles with asymmetric lipid coating, it by forming in 37 ℃ of incubations in the medium of neutral vesicle is arranged in 3.5 hours	294	7.23

As reduce in asymmetric lipid particles with respect to the contrast microgranule zeta potential proved, the incubation lipid particles extracts cation lipid effectively from the external lipid lobule in the medium that comprises the neutral fat vesicle.

In a word, measuring as the zeta potential of asymmetric lipid particles is proved, makes the lipid particles incubation in medium that has charged lipids in the lipid coating compositions, with charged lipids from external packets by extract.During microgranule forms charged lipids exist favourable because the electric charge-charge interaction between lipid and the charged therapeutic agent allows effective formation of microgranule.It is favourable to remove charged lipids from external lipid bag quilt, because send the long blood circulation time of the surface charge permission of that reduce the back or disappearance in the body to carry out bio distribution more widely.

Whether use back maintenance neutral in vivo in order to measure the above-mentioned lipid particles that closes with showing among 1A, 1B and the embodiment 1, asymmetric lipid particles was placed 37 ℃ of temperature 15 hours, with the condition after using in the analogue body.After 15 hour time period, measured the zeta potential of microgranule, will the results are shown among table 2A, the 2B.Equally, lipid coating surrounds and the degree of the DNA that the protection bag carries in order to measure, with a kind of dyestuff (PicoGreen ^The dsDNA quantitative reagent) join in the aliquot of every part of preparation, described dyestuff is emitting fluorescence when contact DNA.By the fluorescent emission of lipid particles is recently measured the percentage ratio that DNA protects mutually with the naked DNA of handling with dyestuff.The percentage ratio of DNA protection also is shown among table 2A, the 2B.

Table 2A

The percentage of the particle size of the asymmetric lipid particles of Chu Liing, DNA protection under various conditions

Ratio and zeta potential

Lipid particles 1	In 90 ° particle size (nm)	DNA protects (%)	Zeta potential (mV)
				The lipid particles that does not have asymmetric lipid coating, contrast	272	93.8	17.27
The lipid particles that does not have asymmetric lipid coating, contrast, simulation back in the body (in 37 ℃ 15 hours)	319	-	25.93
				Lipid particles with asymmetric lipid coating, it by forming in 25 ℃ of incubations in the medium of neutral vesicle is arranged in 24 hours	311	82.4	8.30
Lipid particles with asymmetric lipid coating, it is by forming in 25 ℃ of incubations in the medium of neutral vesicle is arranged in 24 hours, simulation back in the body (in 37 ℃ 15 hours)	314	-	9.63
				Lipid particles with asymmetric lipid coating, it by forming in 37 ℃ of incubations in the medium of neutral vesicle is arranged in 3.5 hours	295	81.0	8.63
Lipid particles with asymmetric lipid coating, it is by forming in 37 ℃ of incubations in the medium of neutral vesicle is arranged in 3.5 hours, simulation back in the body (in 37 ℃ 15 hours)	291	-	9.73

¹The lipid particles of forming DMTAP/DOPC/ cholesterol/mPEG-DS (50/24/24/2) preparation by lipid.

Table 2B

The percentage of the particle size of the asymmetric lipid particles of Chu Liing, DNA protection under various conditions

Ratio and zeta potential

Lipid particles 1	In 90 ° particle size (nm)	DNA protects (%)	Zeta potential (mV)
				The lipid particles that does not have asymmetric lipid coating, contrast	299	97.2	23.07
The lipid particles that does not have asymmetric lipid coating, contrast, simulation back in the body (in 37 ℃ 15 hours)	432	-	26.37
				Lipid particles with asymmetric lipid coating, it by forming in 25 ℃ of incubations in the medium of neutral vesicle is arranged in 24 hours	349	78.4	4.50
Lipid particles with asymmetric lipid coating, it is by forming in 25 ℃ of incubations in the medium of neutral vesicle is arranged in 24 hours, simulation back in the body (in 37 ℃ 15 hours)	329	-	4.40
				Lipid particles with asymmetric lipid coating, it by forming in 37 ℃ of incubations in the medium of neutral vesicle is arranged in 3.5 hours	294	78.7	7.23
Lipid particles with asymmetric lipid coating, it is by forming in 37 ℃ of incubations in the medium of neutral vesicle is arranged in 3.5 hours, simulation back in the body (in 37 ℃ 15 hours)	307	-	7.40

¹The lipid particles of forming DMTAP/POPC/ cholesterol/mPEG-DS (50/24/24/2) preparation by lipid.

The zeta potential that externally has the lipid particles (contrast microgranule) of cation lipid in vivo during the simulated conditions in the lipid lobule improves, and is presented on the outer particle surface electric charge and exists and increase.The zeta potential of asymmetric lipid particles does not have significant change after the simulated conditions in being exposed to 37 ℃ of bodies of 15 hours.For example, after forming, has the zeta potential of 8.30mV by DMTAP, DOPE, cholesterol and mPEG-DS (table 2A) the asymmetric lipid particles of forming.Behind 37 ℃ of incubations the zeta potential of microgranule change minimum, point out in 25 ℃ of initial incubation 24 hours or in 37 ℃ of initial incubation be enough in 3.5 hours with cation lipid from the external lipid bag by remove.

The ability of right title lipid particles in-vitro transfection cell is assessed.According to the method described in the embodiment 2 above-mentioned lipid particles compositions is contacted external with cell.The luciferase expression of cell is determined as the indication of transfection.Table 3A and 3B show the luciferase expression with the asymmetric microgranule cells transfected for preparing as described in example 1 above.

Table 3A

The expression of luciferase behind the plasmid in-vitro transfection of coding luciferase, described plasmid bag is stated from

Under various conditions in the asymmetric lipid particles of Chu Liing

Lipid particles 1	In 90 ° particle size (nm)	Zeta potential (mV)	Luciferase expression (pg/mg)
				The lipid particles that does not have asymmetric lipid coating, contrast	272	17.27	98,778
The lipid particles that does not have asymmetric lipid coating, contrast, simulation back in the body (in 37 ℃ 15 hours)	319	25.93	755,671
				Lipid particles with asymmetric lipid coating, it by forming in 25 ℃ of incubations in the medium of neutral vesicle is arranged in 24 hours	311	8.30	3,274
Lipid particles with asymmetric lipid coating, it is by forming in 25 ℃ of incubations in the medium of neutral vesicle is arranged in 24 hours, simulation back in the body (in 37 ℃ 15 hours)	314	9.63	1,319
				Lipid particles with asymmetric lipid coating, it by forming in 37 ℃ of incubations in the medium of neutral vesicle is arranged in 3.5 hours	295	8.63	981
Lipid particles with asymmetric lipid coating, it is by forming in 37 ℃ of incubations in the medium of neutral vesicle is arranged in 3.5 hours, simulation back in the body (in 37 ℃ 15 hours)	291	9.73	1,757

¹The lipid particles of forming DMTAP/DOPC/ cholesterol/mPEG-DSPE (50/24/24/2) preparation by lipid.

Table 3B

The expression of luciferase behind the plasmid in-vitro transfection of coding luciferase, described plasmid bag is stated from

Under various conditions in the asymmetric lipid particles of Chu Liing

Lipid particles 1	In 90 ° particle size (nm)	Zeta potential (mV)	Luciferase expression (pg/mg)
				The lipid particles that does not have asymmetric lipid coating, contrast	299	23.07	146,957
The lipid particles that does not have asymmetric lipid coating, contrast, simulation back in the body (in 37 ℃ 15 hours)	432	26.37	206,723
				Lipid particles with asymmetric lipid coating, it by forming in 25 ℃ of incubations in the medium of neutral vesicle is arranged in 24 hours	349	4.50	739
Lipid particles with asymmetric lipid coating, it is by forming in 25 ℃ of incubations in the medium of neutral vesicle is arranged in 24 hours, simulation back in the body (in 37 ℃ 15 hours)	329	4.40	274
				Lipid particles with asymmetric lipid coating, it by forming in 37 ℃ of incubations in the medium of neutral vesicle is arranged in 3.5 hours	294	7.23	120
Lipid particles with asymmetric lipid coating, it is by forming in 37 ℃ of incubations in the medium of neutral vesicle is arranged in 3.5 hours, simulation back in the body (in 37 ℃ 15 hours)	307	7.40	147

¹The lipid particles of forming DMTAP/DOPC/ cholesterol/mPEG-DSPE (50/24/24/2) preparation by lipid.

Table 3A and 3B show that the transfection ability of asymmetric lipid particles weakens with respect to the contrast microgranule with positive surface charge.Low transfection efficiency provides the further evidence of the surface charge that reduces on the asymmetric lipid particles.

In a word, the data show among table 1A-1B, 2A-2B and the 3A-3B first aspect of the present invention, wherein prepare lipid composition, and, microgranule carried out incubation to reduce surface charge with respect to the surface charge before the incubation by charged lipids.Under the condition that the suitable major part of charged lipids is removed, form microgranule from external lipid bag quilt.Described microgranule is formed with respect to identical lipid but is handled so that for the microgranule of removing all or part of charged lipids the external lipid bag quilt, have the electric charge that weakens.

On the other hand, the invention provides asymmetric lipid particles, it has low or minimum surface charge after formation, but can recover or generate the outer surface electric charge in being exposed to body after the condition.Above this has been carried out concise and to the point discussion on the one hand about the microgranule among Fig. 2 B 110.After distributing in the body of lipid particles, the existence of surface charge can be useful.For example, after distributing and entering in the tumor, need surface charge and exist to cause combining of lipid particles and cell membrane.Zhi Bei the lipid particles with asymmetric lipid coating can carry out cation lipid displacement after microgranule forms as mentioned above, and it is charged and the internal lipids structure is charged wherein to form the external lipid lobule of back bag quilt at asymmetric lipid particles.Because the Concentraton gradient of charged lipids in lipid particles, wherein on the outer surface of microgranule inside, there is the charged lipids of higher concentration, so in the transfer gradually of body temperature generation charged lipids than microgranule.Charged lipids lipid conformation internally shifts gradually, or is displaced in the external lipid lobule has obtained confirmation in a plurality of researchs that will introduce now.

In this one side of the present invention, a kind of lipid particles compositions is provided, wherein prepare lipid particles, but described microgranule does not have significant surface charge in first temperature by charged lipids, described first temperature generally is the temperature that is lower than the lipid coating phase transition temperature.Yet when being exposed to the temperature that is higher than the lipid coating phase transition temperature, microgranule has the surface charge that can measure.Above about Fig. 2 B to charged lipids internally lipid conformation be displaced in the external lipid lobule and discuss, and illustrate by the microgranule among Fig. 2 B 100,110.About the microgranule for preparing for the research among table 2A, the 2B, lipid composition is made up of 50 mole percent DMTAP, it has gel-liquid crystal form phase transformation (Tc) (Zelphati etc., Proc.Natl.Acad.Sci.USA, 93:11493 (1996)) of about 20-24 ℃.Thereby DMTAP is characterized as being the fluid lipid, and adds cholesterol and make lipid composition rigidity more.DOPE is the hexagonal phase being higher than about 11 ℃ temperature.Be expected at 37 ℃ of following incubation microgranules and bring DMTAP/DOPE/ cholesterol/mPEG-DS compositions on its phase transformation, wherein lipid is a fluid.When lipid is in this fluid state that is higher than its phase transformation, be easy to take place lipid displacement from a lipid lobule to another.Thereby, as for example by zeta potential measure prove that when the lipid in making lipid coating was in temperature on its phase transformation, the asymmetric microgranule that does not have remarkable electric charge became charged after formation.

Cation lipid lipid lobule internally is shown among Fig. 4 A-4B to the displacement in the external lipid lobule.As described in example 3 above, lipid particles is prepared into had DMTAP/DOPE/ cholesterol/mPEG-DS (50: 24: 24: lipid coating 2), and by forming unsymmetry in 24 hours in 0-4 ℃ of incubation in medium, described medium is formed the neutral fat vesicle of DOPE/ cholesterol/mPEG-DS and is formed by having lipid.Subsequently asymmetric lipid particles is remained in 37 ℃ to maximum 90 hours, and in the selected time, sample thief carries out zeta potential and measures.At the medium of 37 ℃ of asymmetric lipid particles of incubation are suspensions of independent water or the neutral fat vesicle that is formed by POPC/ cholesterol/mPEG-DS (58: 40: 2).As being shown among Fig. 4 A with hour zeta potential measurement of the incubation time function of expression.The asymmetric lipid particles (triangle) of incubation has the initial zeta potential of about 11mV in the medium that comprises the neutral fat vesicle.After 20 hours, zeta potential is brought up to about 16mV in 37 ℃ of incubations.By 42 hours incubation, zeta potential was brought up to 18mV, did not observe further raising.The asymmetric lipid particles (rhombus) of incubation also shows the raising of zeta potential along with the incubation passage of time in independent buffer, shows that cation lipid lipid lobule displacement internally or " upset " are in the external lipid lobule.

Fig. 4 B is the similar graph of the asymmetric lipid particles of slightly different lipid composition; DMTAP/DOPE/ cholesterol/mPEG-DS (50: 25.5: 22.5: 2).Here, the asymmetric lipid particles (square) of incubation interim zeta potential when 75 hours incubation improves when having the neutral fat vesicle.Asymmetric microgranule (rhombus) for incubation in independent buffer is observed similar result.

In another kind research, analyzed the stability of asymmetric lipid particles.Preparation as described in example 1 above lipid particles and be stored in 4 ℃ two months.Preserving after two months, microgranule is being remained in 37 ℃ having and do not have in the medium of neutral fat vesicle lasting about 100 hours.The zeta potential of incubation right title lipid particles on the time of 100 hours assess with the monitoring cation lipid internally the lipid lobule to the displacement of external lipid lobule.The results are shown among Fig. 5.

When at independent buffer (rhombus) or when containing in the buffer (square) of neutral fat vesicle incubation, the zeta potential of asymmetric lipid particles improves along with the incubation passage of time.The raising of zeta potential is the cation lipid indication of lipid leaflet movement in the external lipid lobule internally, is presented at that asymmetric lipid coating is stable in 2 months the storage life.

Carry out in-vitro transfection research to a kind of at 4 ℃ of bimestrial asymmetric lipid particles of preservation.Prepare a kind of as described in example 1 above by DMTAP/DOPE/ cholesterol/mPEG-DS (50: 24: 24: the asymmetric lipid particles compositions of 2) forming.Subsequently asymmetric lipid particles was kept two months in 4 ℃.The reference composition that preparation is made up of identical lipid, but it does not carry out the incubation setting to generate asymmetric lipid bilayer.Reference composition was also preserved two months at 4 ℃.After the preservation, with two kinds of preparations in 37 ℃ of incubations.Sample with preparation after 37 ℃ of incubations 0 hour, 48 hours were with 60 hours contacts external with cell, and measures luciferase expression.The results are shown among Fig. 6.

In Fig. 6, preparation numbers 1,2 and 3 is corresponding to the contrast lipid particles that lacks asymmetric lipid coating.Preparation numbers 4,5 and 6 is corresponding to asymmetric lipid particles.Preparation 1 and 4 showed luciferase expression (in 37 ℃ of incubations 0 hour) before 37 ℃ of incubations.Because the disappearance of positive charge on the outer particle surface, asymmetric lipid particles has lower luciferase expression, thereby has lower transfection ability.Preparation 2 and 5 is corresponding in control formulation and the asymmetric lipid particles preparation of 37 ℃ of incubations after 48 hours.Since between 48 hours incubation period cation lipid internally lobule be displaced in the outside lobule, so with respect to preparation 4, the luciferase expression of preparation 5 has improved, condition in the described incubation analogue body.Preparation 3 and 6 is corresponding in control formulation and the asymmetric lipid particles preparation of 37 ℃ of incubations after 60 hours.Since between 60 hours incubation period cation lipid further internally lobule be displaced in the outside lobule, so with respect to preparation 4 and 5, the luciferase expression of preparation 6 has improved.As in the result of 37 ℃ of incubations, control formulation 2 and 3 luciferase expression have reduced.

Because the disappearance of electric charge on the microparticle surfaces, the asymmetric lipid particles compositions of the data show that provides in Fig. 6 begins to have low transfection efficiency.The temperature that microgranule is exposed to is approaching, be equal to or higher than the lipid composition phase transition temperature allows charged lipids lipid lobule displacement internally, or " upset " in the external lipid lobule, thereby generates surface charge on asymmetric lipid particles.Transfection has been promoted in the existence of electric charge, because electric charge strengthens the combination between microgranule and the cell.

III. embodiment

Further explaination invention as herein described of the following example, and will never limit the scope of the invention.

Method

The measurement of zeta potential: the zeta potential value of measuring cationic-liposome and lipid-DNA microgranule by Zetasizer 2000 (Malvern Ins.).Particularly, according to the given operation sequence of instrument supplier the liposome of 50 μ L is joined 5mL and comprise in the aqueous solution of 5mM NaCl (making), and be injected in the sample room by 30 times of diluents of the brinish Milli-Q water of USP.In 25 ℃ every duplicate samples being carried out 3 times measures.

Dynamic light scattering:, utilize Coulter N4MD instrument by the mensuration of dynamic light scattering (DLS) acquisition to the lipid particles size according to manufacturer's description operation.The standard deviation of the microgranule Gauss distribution that the result is expressed as the average diameter represented with nm and represents by relative volume.

Embodiment 1

The preparation of asymmetric lipid particles

Form DMTAP/DOPE/CHOL/PEG-DS (50/24/24/2mol/mol) preparation cationic-liposome (little monolayer vesicle) by lipid.The various lipid mother solutions of preparation in chloroform/methanol (90: 10 v/v); DMTAP is 10mg/mL (Avanti Polar Lipids; 890860; DOPE is 20mg/mL (Avanti Polar Lipids; 850725), cholesterol is 20mg/mL, and mPEG-DS is 10mg/mL (methoxyl group-Polyethylene Glycol-distearyl acyl group; the mPEG molecular weight is 2000 dalton, Shearwater Polymers Inc).Utilize the positive displacement suction pipe to get the solvent solution that comprises an amount of lipid of aliquot, obtain the final lipid suspension of 2mL 20mM lipid concentration, and be mixed in the round-bottomed flask of 10mL.By slowly removing solvent in about 45 ℃ rotary evaporation so that form thin film around the flask.Spend the night by vacuum and to remove residual solvent.By the deionized water that adds 2mL in 50 ℃ lipid was stirred hydration 0.5-1 hour subsequently.Subsequently by having double focusing carbonic ester filter (0.8 μ m is on 0.1 μ m) Lipex ^(10mL volume, Northern Lipids Inc.) are pressed through lipid suspension 10 times in 50 ℃ extruder.As measured by Coulter submicron particlesizer (model N4MD), final liposome diameter is 117 ± 30nm.After the extruding, join the 2mL deionized water in the extruder and pass filter and release to wash remaining liposome.This cleanout fluid and liposome are mixed to the final volume of 4mL.Final lipid concentration is about 10mM.

A. prepare the neutral lipid body and be used to carry out incubation

Has the liposome that POPC/DOPE/CHOL/PEG-DS (58/40/2mol/mol) forms by being similar to the method preparation that provides in top 1..In 40mg/mL POPC (Avanti Polar Lipids, 850457) preparation solvent mother liquor.Final lipid concentration is 47.1mM and the microgranule average diameter is 108 ± 40nm.

B. prepare lipid-plasmid dna complex compound

The DNA plasmid soluble in water (pCC-luciferase) of 1mg/mL concentration slowly is injected into the cation lipid liquid suspension that places the frozen water container (as the prepared 10mM neutral lipid/DOPE/ cholesterol/mPEG-DS of embodiment 1,50/24/24/2mol/mol, wherein said neutral lipid are DOPE or POPC).The charge pump that utilization is set in 10 μ L/ branch clock rate is accompanied by to continue to stir and carries out the DNA injection.The final volume of complex is 1.6mL, and DNA concentration is 0.5mg/mL, and lipid concentration is 5mM, i.e. the DNA/ drugrlipid ratio of 1 μ g/20nmoles lipid.

C. prepare asymmetric lipid-DNA microgranule

By being mixed mutually with 10 times of excessive neutral fat plastids (" storehouse liposome ") of preparation described in top 2., lipid-DNA compound particles prepares asymmetric lipid-DNA complex.1.28mL lipid-DNA the complex and the neutral incubation liposome of the 2mL that comprises 64 μ moles lipids (POPC/CHOL/mPEG-DS, 58/40/2mol/mol) slowly mixing that will comprise particularly, the preparation described in top 3. of 6.4 μ moles lipids.As shown in Fig. 4-6 in before the incubation mixture being placed ice-water bath under the various conditions.

After incubation finishes, separate the storehouse liposome by the sucrose density gradient centrifugation method subsequently.Particularly, with the application of sample of gradient step by step (4.0mL 11 * 60mm, Beckman cat 344062 are used for SW 60 Ti rotors) in clean super centrifuge tube of sucrose.Described gradient be 25,20,15,10 and 5wt% sucrose (by the end to last).The sample size that loads is generally 0.8-1mL.Centrifugal generally in 20 ℃ 40,000rpm carried out 3 hours.Generally lipid-DNA complex is loaded in the weak band on the 10-15% interface.The storehouse liposome remains on the 10% sucrose district usually.Utilize suction pipe carefully to pipette DNA complex band subsequently.In order to prepare the asymmetric lipid-DNA microgranule of large volume, use the centrifuge tube of 40mL with rotor SW28 (Beckman).The sample volume that is added in the test tube is 4mL.Centrifugal generally 20 ℃ in 40,000rpm carried out 15-20 hour.

(Molecular Probes P-7581) determines final DNA concentration in asymmetric lipid-DNA microgranule by fluoremetry to utilize PicoGreen  dsDNA quantitative reagent.Generate standard curve by a series of plasmid DNA solutions, described plasmid DNA solution is up to 2000ng/mL DNA (pCC-Luc), and it is dissolved in 10mM Tris HCl and 1mM EDTA, among the pH 7.0.Find that the range of linearity is up to 1000ng/mL.

Embodiment 2

In-vitro transfection

Compare in external asymmetric lipid-DNA microgranule and multiple contrast preparation as described in example 1 above.With 1.13 * 104 cells/well bhk cell is inoculated in 6 orifice plates, and cultivates based on 37 ℃ 5%CO2 incubation 48 hours with complete MEM.Before transfection, cell is cleaned twice with the MEM culture medium of 1.0mL serum-free.The transfection sample of one five equilibrium sample is mixed plasmid DNA (general 60-200 μ g/mL pCC-Luc) to obtain desired concn with the MEM culture medium of serum-free.In order to carry out transfection, subsequently 1mL is covered on the cell after the cleaning, then 37 ℃ of incubations 5 hours.Behind the incubation, sucking-off comprises the culture medium of sample and replaces with the complete MEM culture medium of 1.0mL, and proceeds incubation under the same conditions 16.5 hours.

Utilize Promega Luciferase Assay System (cat#E1500) to measure luciferase activity.Cell is cleaned twice and adds subsequently the Cell Culture Lysis 1X Reagentlysis of 250 μ L with phosphate buffered saline(PBS) (PBS).Two inferior to room temperature incubation after 8 minutes (whirlpool culture plate simultaneously), with cracked cell transfer in microcentrifugal tube.To manage rotation 10 minutes with 14K subsequently.Utilize the sample of 20 μ L immediately luciferase activity to be measured by luminometer (100 μ L luciferins and the mensuration buffer that comprises ATP were measured in 10 seconds).By the protein content in the extract relative light unit is standardized.

Utilize the BioRad protein reagent to measure protein content.With the cracked cell transfer of 10 microlitres to the flat culture plate in 96 holes and add 200 μ L reagent.Utilize Molecular Devices culture plate reader to measure absorbance on 595nm.

Embodiment 3

Prepare asymmetric lipid particles

Form lipid particles as described in example 1 above, only (temperature maintenance of seeing the step 4) among the embodiment 1 is between 0-4 ℃ except the incubation solution that will be made up of neutral SUVs with ice bath.

Although invention has been described about particular, to those skilled in the art, to carry out multiple changes and improvements under the prerequisite of the present invention be conspicuous not deviating from.

Claims (21)

1. method for preparing the lipid particles with external lipid bag quilt comprises:

Preparation comprises the lipid composition of charged lipids and the (ii) lipid particles formed of therapeutic agent by (i), and each of described microgranule all has external lipid bag quilt, and described lipid coating has external lipid lobule and internal lipids structure; With

The described microgranule of incubation under the condition that described charged lipids is effectively removed from the external lipid lobule.

According to the process of claim 1 wherein described preparation by the preparation lipid particles form, described lipid particles is made up of the lipid composition that comprises at least a cation lipid.

3. according to the process of claim 1 wherein that described preparation comprises that (i) forms the lipid vesicle be made up of described lipid composition and (ii) that described lipid vesicle and described therapeutic agent is compound.

4. according to each method of claim 1-3, wherein said incubation is included in the described lipid particles of incubation in the medium that comprises charged lipid vesicle not.

5. according to the method for claim 4, wherein said incubation further comprises add lipid-polymer-ligand conjugate in medium.

6. according to the method for claim 4, wherein said incubation further comprises and adding in medium with the deutero-lipid of hydrophilic polymer.

7. the method for claim 6, wherein said adding is formed with the deutero-phospholipid of Polyethylene Glycol by adding.

8. according to each method of aforementioned claim, wherein said lipid particles is a liposome.

9. according to each method of claim 1-7, wherein said incubation is carrying out less than about 15 ℃ temperature.

10. according to each method of claim 1-7, the wherein said incubation persistent period was above about 5 hours.

11. according to each method of claim 1-7, wherein said preparation is made up of the lipid particles that preparation has the therapeutic agent that bag carries, described therapeutic agent is selected from charged medicine, albumen, peptide and nucleic acid.

12. a compositions, it comprises

Lipid particles with lipid coating, described lipid coating is made up of external lipid lobule and internal lipids structure, the lipid composition that described lipid coating comprises charged lipids by (i) and (ii) has gel-crystal form phase transition temperature forms, described lipid particles does not have significant electric charge in the temperature that is lower than described phase transition temperature, but has the electric charge that can measure after being higher than the temperature incubation of described phase transition temperature.

13. according to the compositions of claim 12, wherein said lipid composition comprises a kind of cation lipid.

14. according to the compositions of claim 12, wherein said lipid particles further comprises the therapeutic agent with electric charge.

15. according to the compositions of claim 14, wherein said therapeutic agent is a nucleic acid.

16. according to each compositions of claim 12-15, wherein said lipid composition has about 34-38 ℃ phase transformation.

17. according to each compositions of claim 12-16, wherein said lipid particles is a liposome.

18. have asymmetric charged lipids compositions before a method for preparing lipid particles, described lipid particles are used in body in its external lipid bag quilt, this method comprises:

Preparation comprises the lipid composition of charged lipids and the (ii) lipid particles formed of therapeutic agent by (i), and each of described microgranule all has external lipid bag quilt, and described lipid coating has external lipid lobule and internal lipids structure; With

The described microgranule of incubation under the condition that described charged lipids is effectively removed from the external lipid lobule.

19. according to the method for claim 18, wherein said incubation is included in less than about 15 ℃ temperature and carries out incubation.

20. according to the method for claim 18 or claim 19, wherein said incubation comprises that the time surpasses about 5 hours incubation.

21. according to each method of claim 18-20, wherein said incubation is included in and carries out incubation in the medium of being made up of the neutral fat vesicle.

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