Microspheres, the method of production thereof

 

(57) Abstract:

The invention relates to a pharmacy and concerns of the microspheres. The invention lies in the fact that it contains gilotinirovaniya polar nucleus, which is applied concentrically and alternately n lipid double layers or n water layers in the liquid state and n gelatinising polar layers, and n is an integer equal to or greater than 1, and obtained by dilepididae liposomes, called liposomes with gelatinising polar nucleus, which contain at least one external lipid double layer, and at least one water internal polar phase containing gilotinirovaniya substance. The invention relates also to a method for producing microspheres. The invention provides the possibility of obtaining microspheres with adjustable diameter, can be used as a carrier of active substances, or as a diagnostic reagent, they are stable. 2 C. and 10 C.p. f-crystals, 2 tab., 6 of Fig.

The present invention relates to the gelatin microspheres, method of their production and their use.

Microspheres, which are particles of spherical shape, the size of which varies typically from 1 to 250 μm, consist of metaorganism medicine in the form, which allows you to control the selection of the active component for a certain period of time, if you want to ensure prolonged pharmacological effect, or when necessary to protect the named active component from premature degradation in the gastrointestinal tract.

Depending on the structure of the material carrier, there are two types of microcapsules:

the microcapsules of the capacitive type, when the carrier forms a solid shell of varying thickness containing encapsulated substance,

- microcapsules matrix type, also called microspheres, when the carrier is a continuous lattice, in which dispersed encapsulated substance.

From the point of view of the present invention, the term microcapsule or microsphere includes only microcapsules or microspheres matrix type.

Encapsulated can be a lot of substances: we can talk about chemical products, such as medicinal substance, or macromolecules, such as enzymes and living cells.

Microspheres used in numerous fields such as pharmaceuticals, BioIndustry, cosmetology, agro-industry, associated with presscakes, which are described in the literature, in particular, can be called:

- the method of separation of the phases described in U.S. patent 4675189 and in European patent application EP 52510 describing obtain microcapsules by phase separation method using the agent koatservatsii, such as mineral oil or vegetable.

However, the microcapsules obtained by this method or similar methods have the disadvantage that formed agglutinates /glued together particles upon receipt of the aforementioned microcapsules,

method solvent evaporation, described in particular in U.S. patent 4479911, and in European applications EP 301969 and ER, this method enables the separation of education:

the organic phase by dissolving the corresponding polymer immiscible with water and a volatile solvent, and

the aqueous phase containing the active principle, the introduction of the aqueous phase in the organic phase, mixing both phases by mixing and/or in the presence of the emulsifier, and then evaporation of the solvent, usually under stirring and at ambient temperature to obtain the desired microcapsules.

Application EP 145240 describes, in particular, the microcapsules obtained by cooking pesto, holding the medicine /natural or synthetic vegetable glue or compounds with high molecular weight and, in particular, gelatin/ and one oil layer containing a polymer, preferably polylactic acid, or a copolymer of lactic acid and glycolic acid, or their mixture, in a solvent not miscible with water, such as dichloromethane, then is infilled or utverjdayut inner aqueous layer to achieve a viscosity of greater than 5000 SP, and subsequent preparation of the secondary emulsion/M/in the presence of a suitable surfactant, and finally from the obtained emulsion, the solvent is evaporated. The method described in this application, allows you to receive capsules with a diameter of from 0.5 to 400 microns. In all cases, microcapsules or microspheres of the prior art have a great disadvantage in that their diameter is about 1 μm or above /1-1250 μm/; however, there are many applications that require particles with a significantly smaller diameter, for example of the order of 1 nm or more, for example 20-600 nm.

Therefore, the objective of the invention is to obtain a gelatin microspheres, the diameter of which can be adjusted and can reach in case of no which contain polar gilotinirovaniya core (GPC = gelified polar core), around which is applied concentrically and alternately lipid double layers or water layers in the liquid state and gelatinising polar layers, where is an integer; and these layers obtained by delipidization liposomes, called lipoglikemii(trademark, registered in the name of the company LIPOYL and denoting liposomes with gelatinising polar nucleus), which contain at least one external double lipid layer and at least one internal polar aqueous phase containing gilotinirovaniya substance.

The advantage of such microspheres is that they have an adjustable diameter, preferably equal to from 20 to 600 nm,

- they are stable,

- they can capsulitis water-soluble active substances,

- can release immediately the active principle or slow its allocation depending on the melting temperature gelatinising substances,

- can be used as the basis for the suspension of the ligand, substances, malorasprostranennych by the reticuloendothelial system /hidden microspheres/; electrically charged compounds /under electrical influence on the "target" in electrotherapies, and

- able to be hidden according to the invention, gilotinirovaniya substance selected from gelatinising compounds as polymerized and depolymerizing, such as polysaccharides, polypeptides or polyacrylamides.

Preferably polimerizuet gelatinosum substance selected from gelatin, agarose or carragenan, and polymerized gelatinosum substance selected from polyacrylamide gels.

Such liposomes with zeltserman polar core or lipoplasty described in the patent EP 0393049, which indicates that they consist of two-layer interfacial phase, in the case of a single-disk lipogenesis, or of several interfacial double-layer phases superimposed concentric, in the case megaplatinum lipogenesis, and one internal encapsulated gelatinising aqueous polar phase.

This patent describes, in particular, the method of obtaining such a single-disk or megaplatinum lipogenesis: water encapsulated gilotinirovaniya phase formed from the initial liquid aqueous phase in which lipoplasty obtained by a transformation called the aqueous phase in the gel due to the presence in the aqueous phase of one or more gelatinase compounds, polymerized or nacascolo or enzymatic action.

One or more two-layer interfacial phases are, for example, lipid class 4 /phospholipids/, in some cases mixed with lipid class 2 and class 3 /free cholesterol and/ or lipid class 5. This classification of lipids, proposed by Professor Hauton al., based on the partition coefficients KDbetween the polar water phase and the interfacial phase, single layer or double layer, and KCbetween interfacial phase and a hydrophobic phase or non-polar, will be used in the description /Hauton and Lafont, Biochemistry, 1987, 69, 177-204/, it is also described in the patent EP 393049 above.

Also lipoplasty /LGS/ usually classified by the number of double layers:

- small and large lipoplasty single-disk;

SULGS = small unilamellar lipogelosomes;

LULGS = large unilamellar lipogelosomes.

- mnogoplatnye lipoplasty: MLGS = multilamellar lipogelosomes.

According to the invention, the phase dilepididae called lipogenesis can be implemented in different ways and lead to gelatinising the microspheres adjustable diameter:

a/ surface dilepididae a single-disk or megaplatinum lipogenesis carried out by:

1/ extraction surface of the lipid double is(or mixture of organic solvents), not miscible with water;

2/ bi-phase separation of the organic phase and aqueous phase;

3/ Department of the aqueous phase containing gilotinirovaniya microspheres, surface delipidation /remove most of the surface double layer/.

If dilepididae a single-disk lipogenesis (remove only lipid double layer) receive gilotinirovaniya microspheres according to the invention. Called gloomy/GS/ trademark is also registered in the name of the company LIPOYL (small homogeneous gloomy, denoted by SHGS = small homogenous gelosomes, and large homogeneous gloomy denoted LHGS = homogenous gelosomes), or homogeneous polymerisate(trademark, registered in the name of the company LIPOYL and denoting also gilotinirovaniya microspheres according to the invention/, i.e. does not contain any lipid double layer, consisting thus of homogeneous gelatinising water microspheres corresponding gilotinirovaniya polar core /GPC/ indicated above, the cured or not.

In the case of surface dilepididae megaplatinum lipogenesis /paying most of the surface of the lipid double layer/ paleloni /MGS/ hybrid MGS = hibrid multilayered gelesomes/ or multilayer hybrid polymerisable, consisting of gelatinising polar kernel /GPC/, polymerized or not, which caused dual concentric lipid bilayers separated gilotinirovaniya water layers, cured or not, the most outer layer is gelatinising water layer, the cured or not. Such microspheres according to the invention are;

- the single-disk lipoplasty (LGS), when the initial redeliberating lipoplasty serving to receive them, were double-vane or

- mnogoplatnye lipoplasty (LGS), when the initial redeliberating lipogram serving to receive them, were megaplatinum;

these microspheres surrounded by the surface water layer, the cured or not /table 1 and figures 2 and 3/.

Organic solvent not miscible with water, is heptane, but not in a restrictive sense.

b/ Full dilepididae single-disk and megaplatinum lipogenesis by:

1/ lipid extraction a single-disk or megaplatinum lipogenesis using an organic solvent or mixture of organic solvents, which are fully mixed with water or frequent is rites /lei/ from the aqueous phase; and

4/ Department gelatinising and fully deletirovannykh microspheres.

According to the invention, when the first step /1/ made with organic phase, miscible with water, add a non-polar organic solvent before step /2/, allowing thus bi-phase separation.

In the case of full dilepididae a single-disk lipogenesis /deletion of a single double lipid layer receive homogeneous gloomy /GS/ or homogeneous polymerisate, such as indicated above.

In the case of full dilepididae double-vane lipogenesis get polar gilotinirovaniya core /GPS/, polymerized or unpolymerized, surrounded by one water layer in the liquid state, and one surface water layer in gilotinirovaniya state polymerized or unpolymerized.

In the case of full dilepididae megaplatinum lipogenesis get gloomy /GS/ multilayer (MGS = or multilayered gelosomes) multilayer polimersomy consisting of gelatinising polar kernel /GPC/ may polymerized, which caused concentric water gilotinirovaniya layers, polymerized or unpolymerized is mainly but not limited to n-butanol.

The object of the present invention is also a method of producing microspheres containing gilotinirovaniya polar core around which is applied if necessary, concentric and alternating lipid double layers or water layers in the liquid state and gelatinising polar layers, and is an integer, characterized in that it includes:

a/ obtaining liposomes, called lipoglikemii containing n+1 double lipid layers, of which one external lipid double layer and at least one dispersed inner aqueous polar phase containing gilotinirovaniya substance, and

b/ dilepididae called lipogenesis.

The phase and/ described in the patent EP 0393049.

According to a preferred variant of the method, before step dilepididae lipoplasty sorted depending on their diameter using ultrasound.

According to another preferred variant of the method, before step dilepididae removed by tangential ultrafiltration unencapsulated public matter.

Then get a concentrate lipogenesis retained on the filter after ultrafiltration.

According to another variant of the method, step b/ dilepididae at surface delipetrov includes:

1/ extraction surface of the lipid double layer of the above-mentioned single-disk or megaplatinum lipogenesis using an organic solvent or mixture of organic solvents not miscible with water;

2/ two-phase separation of the organic phase and the aqueous phase; and

3/ Department of the aqueous phase containing gilotinirovaniya microspheres, after surface dilepididae described above.

According to another variant of the above method, the step /b/ dilepididae includes at full delipetrov:

1/ extraction from a single-disk or megaplatinum lipogenesis using an organic solvent or mixture of organic solvents, miscible with water, or partially miscible with water;

2/ two-phase separation of the organic phase and aqueous phase;

3/ remove the organic solvent from the aqueous phase, and

4/ Department gelatinising and fully deletirovannykh microspheres described above.

In addition to the options mentioned above, the invention also includes other options, atomnogo of the invention, and on the drawings, in which

- Fig. 1 illustrates gelatinising microsphere obtained by delipidization of a single-disk lipoplasty;

- Fig. 2 illustrates gelatinising microsphere obtained surface or full deleterevision double-vane of lipoplasty;

- Fig. 3 illustrates gelatinising microsphere obtained surface and full deleterevision megaplasticheskoi lipoplasty;

- Fig. 4 illustrates the change in molar volume of the single-disk lipogenesis, depending on the radius R /coordinates for a log/log/;

- Fig. 5 illustrates changes of molar volumes of homogeneous Gelos (small and large) depending on the radius R - h /coordinates for a log/log/;

- Fig. 6 illustrates the changes of molar volumes megaplatinum lipogenesis depending on the radius R /coordinates for a log/log/.

These examples are given only as illustrations of the subject matter of the invention and in no way limit the invention.

Different types gelatinising microspheres according to the invention are shown in table 1 and Fig. 1-3.

Fig. 1 illustrates gilotinirovaniya microspheres as polymerized and unpolymerized received ISPs of lipoplasty (SULGS and LULGS) contain gilotinirovaniya polar core /GPC/, either polymerized or unpolymerized /presents white/ with radius RGPCand a double layer of phospholipids /presents black/ thickness "h" equal to 10-8see, in General, the radius is called the single-disk lipogenesis equal to R=RGPC+ h,

By dilepididae of a single-disk lipoplasty get homogeneous gloomy /SHGS and LHGS/ where gilotinirovaniya polar core depolimerizovannogo, or homogeneous polymerisate in which gilotinirovaniya polar core of polymerized.

Radius gelatinising microspheres according to the invention as polymerized and unpolymerized denoted RGPC.

Liquid aqueous phase surrounding lipoplasty and microspheres, represented by broken lines /bar/.

Fig. 2 depicts gilotinirovaniya microspheres, polymerized or unpolymerized obtained by surface or full dilepididae double-vane of lipoplasty.

Fig. 2,And depicts the double-vane lipogenesis containing gilotinirovaniya polar core /GPC/, polymerized or no /presents white with radius RGPCand two lipid double layer /presents che; the whole radius called double-vane lipogenesis /LGS/ equal:

RLGS= RGPC+ 2h + H (1)

Fig. 2 B depicts a multilayer hybrid gloomy or polymerisate obtained surface deleterevision called double-vane lipogenesis /LGS/: extracted only one surface of the lipid double layer of obtaining microspheres that contain gilotinirovaniya polar core, cured or not, surrounded by one of the double lipid layer and one surface gelatinising water layer with thickness H, polymerized or not.

The radius of these microspheres is

R = RGPC+ h + H (2)

Fig. 2 C depicts a multilayer gloomy or polymerisate obtained by full dilepididae called double-vane lipogenesis: lessons both lipid double layer with obtaining inhomogeneous gloomy or polymerisate consisting of gelatinising polar kernel /GPC/, polymerized or not, one water layer in the liquid state with thickness h, and one surface gelatinising water layer, cured or not, with thickness H; the radius of these microspheres is

R = RGPC+ h + H

Fig. 3 depicts a multilayer gloomy or the GS/.

These mnogoplatnye lipoplasty contain gilotinirovaniya polar core /GPC/, polymerized or unpolymerized /presents white with radius RGPCand n lipid double layers /presents black/, where n = 3, and the thickness of each double layer h = 10-8cm; the radius of the named megaplatinum lipogenesis is

RLGS= RGPC+ nh + (n-1)H (3)

in particular, when n = 3, the radius is:

RGPC+ 3h + 2H (4)

By surface dilepididae named trehplastinnaya lipogenesis get a hybrid multilayer gloomy or polymerisate containing gilotinirovaniya polar core /GPC/, polymerized or not, one of the lipid double layer, then the first relatively water layer, the second lipid layer, then the surface of the second relatively water layer, the cured or not.

The radius of such microspheres is:

R = RGPC+ 2h + 2H (5)

By full dilepididae named trehplastinnaya lipogenesis receive multilayer gloomy or polymerisate containing gilotinirovaniya polar core /GPC/, cured or not, the first water layer in the liquid state /presents a bar/, the first heavy water layer, cured or not.

The radius of such microspheres according to the invention is:

R = RGPC+ 2h + 2H

Example 1: Determination of physical parameters of lipogenesis /LGS/ and microspheres according to the invention (at surface or full delipetrov): measurement of the diameters of the microspheres.

In order to follow the standard method of calculation of the physical parameters of a particular type of spherical particles, strict adherence to the system of units LMT.

Among the various individual systems old system of units C. G. S (centimetre - gram - second) best mastered by biologists in practice. However, it is easy to move from one system of units to another through the use of appropriate transition coefficients.

In accordance with the system of C. G. S always Express the length in cm of the surface in cm2the volume in cm3density in g/cm3the concentration in g/cm3or in mol/cm3and time with, these conditions are necessary in order to obtain the standard model, the absence of which creates a discrepancy between the biological Sciences if you do not use a binder system of units (Hauton J. C. et al. Biochimie 1987, 69, 177-204, Biodynamics lipids: a new perspective).

1/ Physical/ or hybrid structure between Lepoglava /LGS/ and gloomy /GS/, with radius R in cm, mean multimolecular unit with volume VXexpressed in cm3with a density of dxin g/cm3with mass mxin g and a surface SXin cm2/physical parameters of the particles represent small letters/.

Thus, one mole of particles X, i.e. N particles X or 6.0231023particles /mole physical parameters expressed in capital letters/ has a molar volume of V, in particular MVxfor VxN cm3equal to 4/3 R3N cm3or 25,221023R3see, molar mass, MM, in particular MMxfor mxNror 25,223dXg, and the molar surface MS, in particular MSXfor sxN cm2equal to 4 R2N cm2or 75,681023R2cm2(4/3 = 4,188; 4/3 N = 25,231023; 4 = 12,5664 and 4 N = 75,691023).

If the concentration of particles X are expressed in mol.cm-3[X] you get on cm3certain etalonnage volume /water phase, in vitro whole blood, plasma, intermediate fluid, bile, etc./ following ratio, indicating the number of nXparticles per cm3:

nX= [X]N or nX= [X]/N-1< / BR>
the inverse of Avogadro number N-1equal 0,16610-23becomes a design is whether the molar chemical parameter of a particle, multiplied by N or divided into N-1gives the value of this parameter on the particle.

2/ Dilepididae a single-disk lipogenesis /LGS/;

In the case of a single-disk lipogenesis /LGS/ with radius R in cm get gelatinising encapsulated aqueous phase, polymerized or not, called PP (polar phase), which is in the form of a sphere with radius /R h/ cm, where h denotes the thickness in cm of the surface of the lipid double layer, called BIP /Bilayer Interfacial Phase). Encapsulated gilotinirovaniya water sphere is denoted by GPC (Gelified Polar Core).

The results are obtained by the method of calculation set forth above, and show the physical characteristics of the single-disk lipogenesis /LGS/ different diameters, and microspheres according to the invention, i.e. gelatinising and received by dilepididae.

Use the following symbols:

R = the radius of lipogenesis /LGS/ cm,

h = thickness in cm of one surface of the lipid double layer /BIP/ approximately about 40 x 10-8cm (i.e., 40 or 4 nm). Half layer BIP approximately 2010-8see

MVLGS= molar volume in cm3lipogenesis /LGS/ with the given radius R.

MVGPCwhat ithin R, i.e., the molar volume of the microspheres obtained by dilepididae a single-disk lipogenesis.

MVBIP= molar volume in cm3only the surface of the lipid double layer /BIP/, surrounding the single-disk lipoplasty /LGS/ with the given radius R.

MVBIP(e)= molar volume of the outer phospholipid monolayer only the surface of the lipid double layer (BIP) surrounding the single-disk lipoplasty /LGS/ with the given radius R.

MVBIP(i)= molar volume of the inner phospholipid monolayer only the surface of the lipid double layer /BIP/, surrounding the single-disk lipoplasty /LGS/ with the given radius R.

MSLGS= molar surface in cm2lipogenesis /LGS/ with the given radius R.

MSGPC= molar surface in cm2gelatinising polar kernel /GPC/ radius /R h/cm, i.e., the molar surface gelatinising microspheres obtained by delipidization.

Presents four examples:

a/ single-disk of lipoplasty /LGS/ with radius R = 10010-8cm /i.e. a diameter of 20 nm/:

MVLGS= 2,55 106cm3< / BR>
MVGPC= 0,54 106cm3/i.e. 22 MMVLGSBIP/

MVBIP(i)= 0,75 106cm3/i.e. 37.8% of MVBIP/

MSLGS= EUR 7.57 1012cm2< / BR>
MSGPC= 2,72 x 1012cm2.

b/ single-disk of lipoplasty /LGS/ with radius R = 500 10-8/i.e. a diameter of 100 nm/:

VLGS= 315 106cm3< / BR>
MVGPC= 247 106cm3/ie 77,9% MVLGS/

MVBIP= 70 106cm3/i.e. 22.1% of MVLGS/

MVBIP(e)= 36 106cm3/i.e., 52% of MVBIP/

MVBIP(i)= 33 106cm3/i.e., 48% of MVBIP/

MSLGS= 189 1012cm2< / BR>
MSGPC= 160 1012cm2.

with/ of a single-disk lipoplasty /LGS/ with radius R = 2500 10-8cm /ie diameter of 50 nm/:

MVLGS= 39413 106cm3< / BR>
MVGPC= 37545 106cm3/i.e. 95.3% of MVLGS/

MVBIP= 1862 106cm3/i.e. 4.7% of MVLGS/

MVBIP(e)= 939 106cm3/ie 50,4% MVBIP/

MVBIP(i)= 923 106cm3/ie 49,6% MVBIP< / BR>
MSLGS= 4730 1012cm2,

MSGPC= 4580 1012cm2< / BR>
n/ a single-disk of lipoplasty /LGS/ with radius R = 10000 10-8/i.e. with a diameter of 2000 nm or 2 μm/:

MVLGS= 2522432 106cm3< / BR>
MVGP
MVBIP(e)= 15108 106cm3(i.e., 50.1% of MVBIP/

MVBIP(i)= 15047 106cm3/ie 49.9% of MVBIP/

MSLGS= 75690 1012cm2< / BR>
MSGPC= 75076 1012cm2< / BR>
By dilepididae small /SULGS/ or large /LULGS/ single-disk by lipogenesis extracted only the surface of the lipid double layer: remain only gilotinirovaniya polar kernel /GPC/, thus forming a homogeneous gloomy /GS/, cured or not cured, as described above.

Fig. 4 and 5 show in coordinates log/log, with one hand, changes in the magnitude MVLGSin cm3depending on the radius R in cm, and on the other hand, changes MVGSdepending on the radius /R-h/ - denominated see

The results, expressed in V /molar volume/ cm3. mol-1can be expressed in MM /molar mass/ gmol-1when MV multiplication by the density d in Pol-3. Knowing the density of dPPgelatinising encapsulated aqueous phase, cured or not, and the density of the surface of the lipid double layer dBIPyou can calculate the density of dLGSlipogenesis /LGS/ or the density of dGS/ enamel /GS/.

3/ Delimiting polar core (GPC), cured or not, with radius RGPCexpressed in cm, which is concentric to put the first lipid double layer thickness "h" of approximately 4010-8see, then relatively water layer, cured or not, with thickness H of the order of 100 10-8cm, then the second lipid layer, etc.

The symbol n denotes the number of lipid double layers, and the radius R megaplasticheskoi lipoplasty /MLGS/ is determined by the equation /3/ above.

Table II shows as an example the changes MVLGSand the corresponding volumetric content in % gelatinising encapsulated aqueous phase, polymerized or no (% MVPP) and the lipid phase lipogenesis (% MVBIP), with RGPC= 210 10-8cm and n lipid double layers and, thus, /n-1/ gelatinising water layers, cured or not, of a thickness of 100 10-8see

Fig. 6 shows in coordinates log/log/ those same parameters for megaplatinum lipogenesis. In practice it is necessary to determine the parameters RGPC, n, h and H each megaplasticheskoi lipoplasty /MLGS/ to elaborate on their physical characteristics.

According to the invention, when the surface delipetrov extrage the th h. Then get a hybrid structure between lipoglikemii /LGS/ and gelsomini /GS/ containing relatively water layer of thickness H, cured or not, and containing /n-1/ lipid double layers surrounding gilotinirovaniya polar core /GPC/, polymerized or not. The radius of these hybrid structures is equal to RLGS- h, where RLGSis the radius megaplatinum lipogenesis /MLGS/ before delipidization.

According to the invention, with full delipetrov extracted all the lipid double layers megaplatinum lipogenesis /MLGS/, leaving a space that will be filled with water in the polar phase in the liquid state in the separation of the phases the organic/water. So, get inhomogeneous structures called multilayer gloomy /MGS/, in which the original gilotinirovaniya encapsulated aqueous phase originating from megaplatinum lipogenesis /MLGS/, can be polymerized or unpolymerized. The radius of these inhomogeneous Gelos /GS/ is equal to RLGS-h, where RLGSmean radius megaplatinum lipogenesis before delipidization. After full dilepididae molar volume in percent of the space freed thus from MVBIPfrom megaplatinum redeliberating lipogenesis.

Example 2: obtain the microspheres according to the invention.

A. Products:

and/ phospholipids:

The lipid phase consists of phospholipids degreased salt (STERN - France) dry and used at a concentration of 7.5% weight/volume.

b/ polimerizuet gelatinases agents:

- gelatin type B Blooms firm Sanofi-BioIndustry, France;

it has a melting point equal to 30-35oC, and used at a concentration of 7.5% weight/volume.

- carragenan company Sanofi-BioIndustry, France. Mix gelatin/carragenan 80/20 (V/C) has a melting point 50oC. This mixture is used at a concentration of 7.5 % weight/volume.

Gelatin or a mixture of gelatin/carragenan when the above ratio and the concentration of unpolymerized form gelatinising the aqueous phase at temperatures below their melting points, respectively, below 30-35oC and 50oC.

with/ Polymerized relativiely agent:

The acrylamide/bis-acrylamide SIGMA.

Polymerized gelatinising the aqueous phase obtained by mixing ingredients: acrylamide/ACS-acrylamide Sigma, Temedcompany Bio-Rad Ives/the amount of acrylamide/bis-acrylamide.

The polyacrylamide gel is not restrictive example, be selected from the polymerized compounds.

d/ Crotamiton substance:

- sucrose SIGMA is used in a 7.5 % weight/volume.

B. Methods of work.

These concentrations are the concentrations used for the initial aqueous phase in which the dispersed phospholipids.

Among the various methods used to obtain liposomes and lipogenesis suitable for industrial implementation, preferred is the following method, because it involves the step of ultrasonic treatment (used in industry), which allows you to get vast number of lipogenesis /LGS/, having a diameter of about 100 nm, i.e., the radius R = 500 10-8see By quasi-elastic diffusion, measured with a laser granulometer company Sema - Tech /Unit/, 81% lipogenesis LGS/ have a diameter of about of 92.7 nm /radius R = 413 10-8cm and 19% of the diameter of 312 nm is the radius R = 1560 10-8cm/.

This method includes:

1/ Slow mechanical mixing phospholipids degreased soybean (Stern - France) at a concentration of 7.5% /in/about/ for 3 h, dispersed in an aqueous phase containing a gelling agent or a 7.5 % gelatin or condition. If necessary, add 7.5 per cent /in/about/ sucrose as critising agent.

a/ for neprimirimyh gelling substances/gelatin or a mixture of gelatin/carrageenate/ this stage is mechanical mixing is carried out above the melting point of these gelling agents.

used for polyacrylamide as polymerized gelling agents, mechanical stirring is carried out without adding Temedand of ammonium persulfate in order to prevent the polymerization process.

At this stage mechanical mixing receive mnogoplatnye liposomes /haven't crossed in lipoplasty/, which has a diameter that ranges from 297 nm to 2084 nm with an average diameter of 504 nm /i.e., radius R = 2520 10-8cm/.

2/ Ultrasonic treatment is carried out using ultrasonic device SONROREATOR (Undation Ultrasonics, Louvain-La-Neuve, Belgium with zvukovedenie titanium, suitable for the volume of the liposomal suspension. Operate at a frequency of 20 kHz and with a power corresponding to the volume of the suspension. For volumes of the order of 10 cm3processing time is 3-4 min and volumes 700-750 cm3it increases up to 10 minutes

a/ For depolymerizing gelling substances specified the/ for the polymerized polyacrylamide gelling add at the beginning of the ultrasonic treatment Temedand ammonium persulfate to initiate polymerization. At the end of the ultrasonic treatment phase was diluted with water, the resulting product on 1/10 - 1/20-th part, to prevent polymerization of the unencapsulated public water phase in the liposomes, while the encapsulated aqueous phase of the liposomes will dry out.

3/ Remove the unencapsulated public connections by tangential ultrafiltrable, liposomes and/or lipoplasty remain withheld in part. The size of the used equipment manufactured by Filtron /France/, is determined depending on the volume that must be processed. Used membranes have a threshold gap equal to 300 kDa, 1000 kDa /i.e. more precisely the 300,000 - 1 million gmol-1/.

If you do not carry out the dilution water, tangential ultrafiltration of liposomes should be carried out at a temperature higher than the melting temperature depolymerizing gelling substances /gelatin or a mixture of gelatin/carrageenan/. If you carry out the dilution water after ultrasonic treatment, it can be tangential ultrafiltration at ambient temperature. To increase the efficiency of dialysis depolymerize the frame of gelatin. Then lower the temperature for the conversion of the liposomes in lipoplasty, which then condenses retained on the filter part.

When using polyacrylamide as polymerized gelling agents mentioned dilution and ultrafiltration, which follow immediately after the ultrasonic treatment, allows you to remove the polymerized unencapsulated public matter, and the polymerized encapsulated substance turns liposomes in lipoplasty.

4, Surface or full dilepididae lipogenesis /LGS/.

Lipoplasty /LGS/ found in greater or lesser concentrations on the filter, depending on the selected operating conditions is subjected to surface delipidation solvent or mixture of solvents that are not miscible with water. The solvent is not miscible with water using heptane. Full dilepididae carried out using a solvent miscible with water, or mostly partially miscible with water, such as n-butanol. After bi-phase separation of the organic phase and the aqueous phase is different kinds of the obtained microspheres Recuperat in an aqueous polar phase, in particular small /SHCS is consistent of gloomy (homogeneous polymerisate), mnogoplatnye lipoplasty /MLGS/ surrounded unpolymerized gelatinising water layer (hybrid structure between lipoglikemii and gelsomini), mnogoplatnye lipoplasty /MLGS/ surrounded by polymerized gelatinising water layer /hybrid structure between lipoglikemii and gelsomini) and polymerized or unpolymerized multilayer gloomy.

Carried out by means of laser granulometry and electron microscopy monitoring of the reduction of the diameters of the resulting surface or full dilepididae a single-disk lipogenesis, allows you to determine what method to use is properly. In addition, quantitative analysis of lipids in the organic phase obtained after separation of the solvent/water, allows to determine the effectiveness of dilepididae.

The invention is not limited to the above-described methods of execution, but includes all variations that may occur at the level of specialist knowledge, without leaving the scope of the invention.

1. Microspheres, characterized in that they contain gilotinirovaniya polar core, on which is superimposed concentrically and alternately or n lipid bilayer is s, and n denotes an integer equal to or greater than 1, thus the name of the microspheres can be obtained by dilepididae liposomes, called liposomes with gelatinising polar nucleus, which contain at least one external lipid bilayer and at least one internal polar aqueous phase containing gilotinirovaniya substance.

2. Microspheres under item 1, characterized in that gilotinirovaniya substance selected from gelatinising compounds, curable or not, such as polysaccharides, polypeptides or polyacrylamides.

3. Microspheres under item 2, characterized in that gilotinirovaniya substance selected from gelatin, agarose or Karaganov and gilotinirovaniya curable substance selected from polyacrylamide gels.

4. Microspheres according to one of paragraphs.1 to 3, characterized in that they have a diameter of 20 to 600 nm.

5. Microspheres according to one of paragraphs.1 to 3, characterized in that dilepididae carried out by surface dilepididae pallamallawa liposomes with gelatinising polar nucleus by extraction surface of a lipid bilayer called pallamallawa liposomes with gelatinising polar nucleus with organic ical phase and aqueous phase, and separating the aqueous phase containing surface delipidation gilotinirovaniya microspheres.

6. Microspheres according to one of paragraphs.1 to 4, characterized in that dilepididae carried out by full dilepididae pallamallawa liposomes with gelatinising polar nucleus by extraction of lipids from pallamallawa liposomes with gelatinising polar nucleus with an organic solvent or mixture of organic solvents, miscible with water or partially miscible with water, bi-phase separation of the organic phase and the aqueous phase, and separating the organic(solvent(s) from the aqueous phase, and separating gelatinising microspheres, fully deletirovannykh.

7. Microspheres under item 6, characterized in that the stage of extraction of the lipids are carried out with the organic phase, miscible with water, and before the stage of the bi-phase separation type apolar organic solvent.

8. A method of producing microspheres according to one of paragraphs.1 - 7 containing gilotinirovaniya polar nucleus, which is applied concentrically and alternately or n lipid bilayers and n gelatinising polar layers, or n water layers in the liquid state and n jelatinizirovanny polar core, containing n + 1 lipid bilayers, including one outside the lipid bilayer, and at least one internal aqueous polar phase containing gilotinirovaniya substance, and delipetrov called liposomes with gelatinising polar core.

9. The method according to p. 8, characterized in that the front of the stage dilepididae carry out selection of liposomes with gelatinising polar nucleus depending on their diameter, the selection is carried out mainly with the help of ultrasound.

10. The method according to p. 8 or 9, characterized in that the front of the stage dilepididae remove unencapsulated public substances, preferably by tangential ultrafiltration.

11. The method according to one of paragraphs.8 to 10, characterized in that stage dilepididae includes for surface dilepididae extraction surface of the lipid bilayer of pallamallawa liposomes with gelatinising polar nucleus with an organic solvent or mixture of organic solvents not miscible with water, bi-phase separation of the organic phase and the aqueous phase, and separating the aqueous phase containing gilotinirovaniya microspheres, delipidation surface.

12. The method according to one of paragraphs.8 - 10, otlichalis the lamellar liposomes with gelatinising polar nucleus with an organic solvent or mixture of organic solvents, miscible with water or partially miscible with water, bi-phase separation of the organic phase and the aqueous phase, separating the organic(solvent(s) from the aqueous phase, and separating gelatinising and fully deletirovannykh microspheres.

 

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