CN1160585A

CN1160585A - Method of removing liposoluble target molecules, dialysis unit for purifying blood, and wash solution

Info

Publication number: CN1160585A
Application number: CN 96114256
Authority: CN
Inventors: 玛丽·卢·拉腾; 奇罗·特塔
Original assignee: Bellco SRL
Current assignee: Bellco SRL
Priority date: 1995-12-22
Filing date: 1996-12-20
Publication date: 1997-10-01

Abstract

The toxins are purified and removed from a colloidal solution by dialysis through an appropriate semipermeable membrane, the opposite sides of which are contacted respectively by the solution to be purified and by a wash solution containing a predetermined concentration of liposomes; the dialysis unit presents a pack of highly permeable, hollow fibers connected internally to a first circuit containing the solution to be purified, and which are immersed in a counterstream of the wash solution containing liposomes; the wash solution circulates in a second circuit presenting means for feeding the liposomes into the wash solution, and possibly a perfusion cartridge in series with the second circuit and in which the wash solution containing the liposomes percolates through a filter of activated-carbon or synthetic resin particles covered with continuous phospholipid membranes.

Description

From colloid solution fast the method for weeding of grease dissolubility target molecule, be used for the wash solution that purifying blood particularly is subjected to the dialysis apparatus of the patients'blood that the organ failure attacks and is used for described method and apparatus

The present invention relates to be used for purifying blood, particularly suffer from efficient, the fast method of the patients'blood of acute and/or chronic renal failure, more fully say to relate to be used for the method for colloid solution that purification has the physicochemical property of similar whole blood or blood plasma.

For the patient who suffers from the organ failure, blood has kept the various materials that are commonly referred to " toxin " of infinite quantity gradually, and for healthy people, relevant organ can be removed these materials usually from blood flow.The accumulation of described toxin finally causes being commonly referred to organ failure's disease, if do not treat, then causes producing various types of infringements and finally causes death.Be used to resist described symptom the system of normal use be by they being adsorbed onto solid matrix (blood and blood plasma perfusion) is gone up or by with suitable semipermeable membrane ultrafiltration blood or blood plasma, or by under the help of barometric gradient (TMP), transmitting this film (blood and plasmapheresis), or the side contacts by will blood to be purified or blood plasma and film, through the suitable wash solution of allocating contact with opposite side spread (hemodialysis is analysed) thus the toxin of purifying blood.

But there is defective in all said system.The blood perfusion is the filter membrane that blood is directly penetrated an adsorbent, so this filter membrane must have the biocompatibility of height.Usually can reach this purpose by covering absorbent particles, but this is with this particulate reservation toxin ability of severe impairment with suitable material.When blood plasma pours into, at first with blood filtration so that separated plasma penetrates adsorbent material with it then.Although this will solve biocompatibility issues between flush phase to a certain extent, the increase of blood viscosity produces many grumeleuses after will causing seeing through this film during filtering, so that in many cases, must use anticoalescent (heparin) processing blood.

On the other hand, blood and plasmapheresis are only removed high-molecular weight toxin, and produce a large amount of loss in weight, must compensate by supplying with primer solution to patients'blood.According to disclosures in Italian patent application No.67168-A/90 (application on March 9 nineteen ninety), by making ultrafiltrate regeneration, by absorption medium high molecular toxin wherein, by using the blood perfusion solution tube of empty activated carbon composition (for example by SORIN BIOMEDICA of Saluggia it, Italy produces DETOXIL2) filter, can address the above problem thereby regenerated ultrafiltrate can be used as primer solution.

Blood dialysis is the most effective if particularly be used in combination with above-mentioned one or more methods, but the purification time is longer relatively, therefore can not remove toxicity fully at acute state.But owing to can only seldom remove some hydrophobic substance for example toxin for example the indoxyl sulphuric acid and the free molecular oxygen base of platelet activating factor (PAF), endotoxin, bilirubin, hippurate, protein bound, finally can only postpone present organ failure and can not stop fully.

One object of the present invention by provide from blood, more conventional be from colloid solution soon bundle, effectively remove any and all toxin, particularly water miscible and fat-soluble toxin overcomes defective above-mentioned.

According to the present invention, the method of removing fat-soluble target molecule from colloid solution fast is provided, particularly remove toxin and free group from whole blood or from the ultrafiltrate of whole blood or plasmapheresis liquid, thereby a side contacts with the semipermeable membrane of purification solution and preset aperture, and contact with the opposite side of this film by the wash solution that will be scheduled to component and to use the dialysis of this film, be characterised in that in wash solution, add predetermined concentration usually greater than the liposome in the aperture of semipermeable membrane.

According to the present invention, also provide to be used for the dialysis apparatus that purifying blood particularly suffers from organ failure's patients'blood, this device comprise wherein flow through come from patient's blood flow be used for first of the purification solution closed circuit that surges; Wherein flow through second of the wash solution closed circuit that surges; And the filter membrane that comprises the semipermeable membrane of preset aperture successively, surge and link to each other by means of this film first closed circuit and second closed circuit that surges that surges, it is characterized in that second closed circuit that surges comprises to described second closed circuit that surges and supplies with the device of liposome so that the toxin that is used in the solution of purification is diffused into wash solution.

According to the present invention, the wash solution that is used for the blood penetration device also is provided, be characterised in that it contains water-based solvent, wherein liposome is scattered in the suspension.Therefore this invention also relates to the use of the wash solution that contains liposome of the performance that is used to strengthen dialysis apparatus.

By means of embodiment, with reference to accompanying drawing many non-limiting examples will be described, wherein:

Accompanying drawing 1 diagram has shown the operating principle of dialysis process of the present invention;

Accompanying

drawing

2 and 3 diagrams have shown two examples of a test dialysis circuit of the present invention;

Accompanying drawing 4 diagrams have shown the ultimate principle of the improved form of the inventive method;

Accompanying drawing 5 shows dialysis apparatus figure of the present invention;

The testing result of accompanying drawing 6 and 7

expression accompanying drawings

2 and 3 tests.

Accompanying drawing 8 diagrams show the operating principle of existing dialysis process.

Referring to accompanying drawing 8, be used for the treatment of the known devices of suffering from the organ failure patient based on known thoroughly Analysis method, briefly, this dialysis process is to remove through semipermeable membrane A by diffusion to comprise (to the right of film A) water soluble molecules (being shown by black circle) in blood (or blood plasma), and with it Transfer to the permeate flow (to the left side of film A) that contains the suitable aqueous solution. But, film A the right Harmful lipophilic molecule and the molecule of the protein bound in the blood flow are left over the blood in the patient In.

Referring to accompanying drawing 1, dialysis process of the present invention is similar to known method: will be for the glue of purifying Liquid solution flows 2 (being shown by arrow) (for example ultrafiltrate of whole blood or whole blood or plasma filtrates) and partly oozes One side 4 of permeable membrane 1 contacts, and has the many holes 5 to sizing and arrangement on this film; And Another of the dialysis fluid flow 3 (being shown by arrow) that will contain the known water washing lotion of given component and film 1 Opposite side 6 contacts are preferably along opposite approach and flow to flow of solution 2, so that in the colloidal solution 2 Water-soluble target molecule 8 for example the low-molecular-weight toxin pass the hole 5 of film 1 (they formed two Interface between the contact mutually) and from solution 2 be diffused into dislysate 3.

The method according to this invention is with usually greater than the liposome of the predetermined concentration in the hole 5 of film 1 10 supply in the wash solution 3. According to the present invention, dialysis is carried out according to a conventional method, still Utilized the dislysate that contains a kind of wash solution, described wash solution contains water-based solvent and (contains Or do not contain suitable solute), wherein liposome 10 is scattered in the suspension, preferably, relatively Be 1-20g/l in film 1 its concentration. The liposome 10 that uses among the present invention contains diameter Circle between 100nm-200nm bubble, and be defined as immobilized artificial membrane, for example with lecithin alkali or Its equivalent (phosphatidylserine, two palm phosphatid ylcholines etc.).

As showing that in accompanying drawing 1 existence of the liposome 10 of dislysate 3 not only can be from solution The middle water soluble molecules 8 of removing, and can be with common and respective egg white matter 12 knots that exist in the blood flow The many lipoprotein solution target molecules (toxin) that close are toxin 11 (for example indoxylsulfuric acid) for example, and in low the branch The Hemodialysis material 13 of son amount (bilirubin, hippurate, PAF, the endotoxin that may exist, The lipoprotein solution medicine of overtreatment) and episome. In fact, contact with film 1, corresponding with hole 5 Liposome 10 (owing to can not see through flow of solution 2 too greatly) improved the film 1 in flow of solution 3 one sides Hydrophobicity.

Therefore liposome 10 can be caught all or in part and mix in its adipose membrane safely and be connect Any molecule 13 in nearly hole 5, and they can be extracted in the dialysis fluid flow 3 from liquid stream 2. Similarly, based on the hydrophobicity increase of film 1, with protein 12 and film close contact, therefore can Make the lipophilic molecule 11 that is attached on the protein near hole 5, they are caught by liposome 10 at this Obtain (top of Fig. 1) and from liquid stream 2, be drawn out of. Similarly operating principle also is applied to liquid stream In 2 the free group.

In order more effectively from liquid stream 2, to remove free group, perhaps at least fully with they neutralizations, according to the preferred embodiment of the invention, also can be in known manner one or more antioxidants, particularly vitamin E or ubiquinone/ubiquinol or single form or mixed form be incorporated in the liposome 10.For the situation that even free group can not be entered liquid stream 3, also can see through the liposome 10 in the hole 5 of film 1 by contact, they can be neutralized by the antioxidant of the liposome of for example immobilized artificial membrane or inner antioxidant itself, may neutralize to capturing the aqueous phase solution form in the bubble that limits by immobilized artificial membrane.

The further preferred embodiment according to the present invention, with contain entering the wash solution 3 that the liquid stream rightabout is added on the film 1 and can filtering (accompanying drawing 4) by the filter membrane 20 that height permeates doughnut subsequently of colloid solution 2, filled adsorbent in the capillary tube outside in the gas fiber in described, be hidden in this filter membrane in the tube 21, the immobilized artificial membrane that wherein is similar to or is same as liposome 10 according to the present invention with group categories covers adsorbent.

Preferably, adsorbent contains the active carbon particle 22 (or microspheric synthetic plastic resin) that is covered by successive immobilized artificial membrane 23 fully.The liposome 10 that therefore will carry the target molecule (toxin) of catching contacts closely with filter membrane 20, wherein also be because the absorbability of granule/microgranule spheroid 22, to transfer on the immobilized artificial membrane 23 and be collected in the inside of filter membrane 20 by the liposoluble substance that liposome is caught, and the liposome 10 that discharges from the molecule that keeps has " reproducibility " and is used to form " cleaning " liquid stream 3, is used for recirculation on film 1.

Accompanying

drawing

2 and 3 diagrams show two kinds of devices that are used to implement, and can be used as " external " test detection of the inventive method.More particularly, accompanying drawing 2 devices contain and are useful on first of the purification solution closed circuit 30 that surges; Be used for second of the wash solution closed circuit that surges; With a filter 32, exerciser 32 contains the semipermeable membrane (not shown) in the sleeve pipe 33 of being hidden in of a sleeve pipe 33 and the preset aperture identical with film 1, communicate with loop 31 by surging Circulation by this loop 30, be diffused in the wash solution to be used in target molecule (for example toxin) predetermined in the purification solution.In the embodiment that shows.This embodiment as described is a kind of " external " assay device, and loop 30 and 31 is closed-loop paths, and various have a corresponding pump 34; Loop 30 is provided for for example jar 35 of human or animal's blood plasma or blood of solution to be purified; The wash solution that loop 31 has in loop 31 provides the device of liposome, and contains the jar 36 that is suspended in the liposome in the aqueous solution that has preferably contained desired concn in the embodiment shown.Therefore, in this case, the device of liposome being provided for loop 31 also can provide liposome to wash solution simultaneously, therefore includes the liposome in the suspension that is scattered in the starting point right side.Change a kind of method, irritate 36 and only contain liposome (or its concentration in suspension is very high), and equipped and be used for controlling the known devices (for example Electronic Control metering valve and/or pump) that does not show that liposome is discharged into loop 31.

More particularly, filter 32 contains the high osmosis doughnut of known quantity, inner they circulate for surging with loop 30 and be connected and be hidden in 33 inside, so that wash solution in tube 33, the doughnut outer circulation, and solution to be purified at the doughnut internal recycle to regenerate according to the mode of accompanying drawing 1, wherein semipermeable membrane contains the sidewall of doughnut.

In order to operate according to the scheme of accompanying drawing 4, loop 31 is installed in series with perfusion tube 38, keeps the filter membrane 20 of adsorbing material in the tube 38; Perhaps filter membrane 20 directly be inserted into the tube 33 in (so that forming suitable tube) and with formation semipermeable membrane 1 the doughnut adjacency.For another kind of situation, loop 31 can not be a kind of closed-loop path with changing, loop 30 obviously can be the open to the outside world loop by the human body closure, so that having the accompanying drawing 2 of above-mentioned variation not only can " external " operate, and can " in the body " operate, can directly solution to be purified be taken out from patient's blood flow and after being purified, promptly flow back in patient's body in this case from filter membrane 32.

The device of accompanying drawing 3 has pointed out or has been same as the details of describing content that for the identical numbering system of easy usefulness, this device is inequality with accompanying drawing 2 similar, and it can provide and the directly opposite two filtrations (diafiltration) of dialysis.In other words, loop 30,31 such sizes of accompanying drawing 2 devices can make two kinds of solution (being purified solution and wash solution) circulation of equal volume, for example 100,200ml/ branch, so that the both sides of film 1 do not have pressure reduction, two kinds of solution that the loop 30a of accompanying drawing 3 devices and the such size of 31a can make different volumes, for example in 30a the solution to be purified of 300ml and in 31a the liposome wash solution of 200ml, circulate.

Therefore, on the film of filter 32, form barometric gradient (TMP), this gradient not only can be by film diffusion but also can convection current, and the solution among the 30a of loop is because its solute (with the part of solvent) is transferred to wash solution and bodies lost weight.Different with loop 31, loop 31a links to each other with 40 series connection of second filter, the feature of described filter 40 is the semipermeable membranes that are similar to film 1, and its hole is less than the liposome in the wash solution, and extends at the downstream of filter 32 and the branch road 41 of loop 30a close proximity from this loop.Like this, because the partial purification solution that flows among the 31a of loop of barometric gradient reclaims on filter 32, purification and in filter 40, remove liposome, and flow back into loop 30a as reperfusion solutions along branch road 41, so that remedy the loss in weight fully.In this case, loop 31a also provides the tube 38 of the liposome that is used to regenerate, and perhaps the filter 20 of adsorbent is hidden in the tube of filter 32 as described before.

Accompanying drawing 5 diagrams show and to be used for purifying blood, particularly to suffer from organ failure's patient's the major part of dialysis apparatus 100 of blood, wherein contain an open loop, the ground that surges between input pipe 104 that is used for this device is connected with known loop (not shown) and output duct 105 is contained in this loop, and mutually the blood filter element 101 and the hemodialysis of series connection (being connected in series) are analysed element 103, described known loop draw blood and purifying blood got back in patient's body in patient's body.Other structure of describing element 101,103 in detail and hereinafter point out in Italian utility model patent No.198528 and disclosures in Italian patent application No.67168-A/90 is incorporated herein these two pieces of documents for referencial use as required.

Blood to be purified 104 flows along the loop, flows through

series element

101 and 103, and along conduit 105 flow out, purification, the hemodialysis product (ultrafiltrate) that forms in element 101 flows out along output duct 107; Conduit 108 can be supplied with the conduit 106 that blood osmotic component 103 is connected with element 101, the loss in weight that produces in solution (blood) process with part or all of compensation ultrafiltration solution in element 101 with reperfusion solutions.More particularly, form infusion liquid again by the ultrafiltrate that will be extracted along conduit 107 regeneration, for this reason, device 100 contains and connecting duct 107,108 placed in-line regenerating units 110, and comprise free hemoperfusion carbon tube, for example by SorinBiomedica S.P.A.of Saluggia, the trade mark that Italy produces is Detoxil2 tube (describing at disclosures in Italian patent application No.67168-A/90), or contain the device B that is same as accompanying

drawing

2 or 3, it is different that just it is a closo, corresponding loop 30 or 30a are open, for example irritate 35 and are removed, and the other end in loop is connected with 108 with loop 107 respectively.

According to the version of device 100, the square frame in the chain-dotted line rectangle of the 120 usefulness accompanying drawings 3 of the element square frame in the chain-dotted line rectangle 121 can be substituted, so that part 101 usefulness filters 32 substitute, its input and output are connected with 108 with conduit 107 respectively.

Hemodialysis element 103 is connected with the wash solution loop 120 of the square frame indication that is formed by dotted line.According to another kind of version, conventional loop 120 can be substituted by the liposome loop 31 of accompanying drawing 2, is perhaps substituted by the loop 31a of accompanying drawing 3, forms complete loops with loop 41, and

element

101 and 111 is applied to the upstream of element 103 significantly in this case.

Therefore, device 100 always contains at least by dialysis or hemodialysis and analyses separated two closed circuits that surge of filter (101,32 and 103); The purification solution that reclaims (being connected filter with one by another loop directly or indirectly for example 101 is connected) is contained in first loop from patient's blood flow; The wash solution that has liposome 10 suspensions is contained in second loop.31a is similar to the loop, in

series equips device

40,41 also can for second loop, be used for isolating the ultrafiltrate that does not contain liposome or toxin in a large number from cleaning mixture, and first loop that is used for it is joined the filter downstream.

Successively, the blood device that is positioned at dialysis (or two filtration) filter upstream is contained in first loop, and for example 101, be used for a large amount of ultrafiltrates is separated from patient's blood flow, also can pick out a shunt, be connected to the device that is used to supply reperfusion solutions from the downstream of hemofilter.This device can contain the device that is connected on the hemofilter, for use in reclaiming ultrafiltrate and it for example 111 being fed in raw material by an activated carbon blood perfusion tube continuously, then it is added back to first loop, as reperfusion solutions.Another kind method, hemoperfusion tube 111 can in series be connected on first loop.By means of embodiment many embodiments of the present invention will be described.

Embodiment 1

-liposome preparation

With following material preparation liposome:

-100% the purity that obtains from Ovum Gallus domesticus album and Semen sojae atricolor is the L-α-phosphatidylcholine (PC) more than 99%.Adopt GMP to obtain employed phospholipid, this phospholipid is pharmaceutical grade, show with the spectrophotometer analysis result and do not contain lysophosphatide, hydrogen peroxide, hydroxide and fatty acid, and by Avanti Polar Lipids (Alabama, USA) supply.

-by Avanti Polar Lipids (Alabama, USA) synthetic ubiquinone/Ubiquinol antioxidant of making and supplying.

With phospholipid be incorporated into antioxidant in the phospholipid with the amount that accounts for gross weight 0.5% and be dissolved in the ethanol in the flask, cover with a cover and make solvent evaporation; Before solvent evaporates fully, flask is contacted nitrogen current to help solvent evaporation and assurance phospholipid and antioxidant not oxidized; After the evaporation, form skim, it is detected to see whether have bubble or other solvent residues at drag phospholipid and antioxidant; Add methanol or ethanol or chloroform during detection and make the thin layer dissolving again, the repeated evaporation process is up to forming good thin layer, then this thin layer is added to several milliliters through the improvement phosphate-buffered saline (PBS) in, this buffer saline contains sodium phosphate 0.008M, potassium phosphate 0.002M, sodium chloride 0.14M and 7.4pH potassium chloride 0.01M (from Pierce, Rockford Illinois, USA buys)-common several milliliters of foots are in phospholipid hydration and dissolving thin layer; In case sealed flask is operated in the thin layer dissolving in constant nitrogen current, make wherein to keep nitrogen, stirred intensely 10 minutes, check, stir if still show the thin layer or the phospholipid " group " of trace, adding PBS acquisition concentration then is the phospholipid concentration that contains 3-10mg among every ml PBS again; And collect phospholipid with many thin layers vesicle (MLV).

Pass through liposome extruder (the LIPEX BiomembranesInc. of a 10ml capacity then, Vancouver, British Columbia Canada) adds MLV solution, and it is 100 or the disposable 25mm PC filter of 200nm that described extruder has been equipped two apertures.So that guaranteeing uniform liposome forms, formed be the monolithic thin layer that diameter is similar to filter aperture (100 or 200nm) to the MLV solution of being supplied with by extruder extruding 10 times.

The carbon preparation of-coating:

The XEN546 (Supelco) of about 50gr is placed the flask of 250ml; To be dissolved in 10 in 100% chloroform gram lecithin and add in the flask, agitating solution lentamente in nitrogen current; When drying, carbon placed contain K ₂SO ₄In the exsiccator of solution, thereby phospholipid is gathered into the biplate layer; In the PBS of 200ml, add carbon then and pass through a poly-vitriol HFT-02 filter (BELLCO) suction; After filter is full of, remove excessive PBS.

Example II

With the blood of cow in PBS liposome or only PBS solution be incubated, as shown in table 1:

A 1ml liposome+1ml blood

The PBS of B 0.5ml liposome+1ml blood+O.5ml

C 1ml blood+1ml PBS

D 1ml liposome+1ml blood+2 μ l chloroforms

E 1ml liposome+1ml blood+5 μ l chloroforms

F 1ml liposome+1ml blood+10 μ l chloroforms

G 1ml blood+1ml PBS+2 μ l methanol

H 1ml liposome+1ml blood+5 μ l methanol

I 1ml liposome+1ml blood+10 μ l methanol

J 1ml liposome+1ml blood+2 μ l ethanol

K 1ml liposome+1ml blood+5 μ l ethanol

L 1ml liposome+1ml blood+10 μ l ethanol

M 1ml PBS+1ml blood+2 μ l chloroforms

N 1ml PBS+1ml blood+2 μ l methanol

O 1ml PBS+1ml blood+2 μ l ethanol

In the 10ml polypropylene tube under the room temperature with 2,500rpm was with centrifugal 15 minutes of blood plasma; The spectrum (200-800nm) that utilizes the score of BECKMAN7400 two utmost point medium beam split photometries to analyse plasma component; And in O, 5,15,30 minutes and sampling in 1,6,24 hour.In containing the sample of liposome (A and B), in 234nm wavelength (peroxidating of indication phospholipid), do not observe blood dissolves or light absorption value increase, and contain all samples demonstration adequate blood dissolving of chloroform.

EXAMPLE III

Utilize as shown in Figure 2 the loop to carry out the loop dialysis and detect test, and in the loop 30 (blood surveys) with following material circulation:

A: bovine blood

B: cow blood plasma

C:PBS+4 grams per liter albumin

Obtain fresh blood from the slaughterhouse, place 5 liters of bags of the ordinary salt aqueous solution that contains the 500ml heparinization, described saline solution is that to transfer to final volume be that 5 liters of two deionized waters prepare by add 45 gram NaCl in 10ml heparin sodium stock solution (5 of the two deionized waters of every ml, 000U heparin); With porous fibre film (use in advance the ordinary salt aqueous solution of heparinization saturated) filtering blood; By filterable blood transfer is prepared blood plasma in the 50ml centrifuge tube that is dipped in advance in the heparinized saline solution, and with 2, centrifugal 15 minutes of 700rpm; Then blood plasma is transferred in another 50ml centrifuge tube and or fresh use or be stored in-20 ℃.(SIGMA, Milan Italy) prepare solution C, slowly stir then to prevent that foam from forming or protein denaturation to add 4 gram V-type albumin by every 100mlPBS.

The following toxin that in above-mentioned solution, adds various concentration:

PAF (20ng/ml); Bilirubin (10ng/ml)

In initial substance, add this toxin, whole material be divided into sample to detect following wash solution in loop 31:

-contrast solution (only PBS);

-liposome solutions;

The liposome solutions of-tube 38, described wound packages have been equipped with the activated carbon filter that phospholipid coats.Can guarantee that like this toxin concentration is identical in all tests.

Loop 30 and 31 test are carried out as follows:

Employed filter (32) is the poly-vitriol 0.44m2 filter of BELLCO HFT-02 high flow rate; The loop is dipped in 2 liters the ordinary salt aqueous solution, afterwards, load the dialysis solution (PBS) of full 300ml or the liposome solutions (the 600mg liposome is dissolved among the PBS of 300ml) of 300ml in dialysis fluid side (loop 31), and blood side (loop 30) is loaded full isopyknic substance A (blood), B (blood plasma) and C (PBS+ albumin solution).Only carry out the PAF test with C solution, wherein blood plasma and blood all contain the acid (acetolysis enzyme) of the platelet activating factor of any adding of degrading.Utilize blood, blood plasma and dissolving C to carry out BIL, obtain similar results.

In this loop, circulate in (loop 30) with 75ml/ minute in the blood side, and divide kind of a circulation with 135ml/ in dialysis fluid side (loop 31); When off-test, the liposome filling is placed on the analytical balance to check that ultrafiltration does not take place reacts.

After

pre-dialysis period

1,10 and 30 minute, and from two loops 30,31, took a sample in 1,3,4,6 and 24 hour.

Then sample is analyzed to measure:

_ platelet activating factor:

By preparing following species analysis PAF content:

The gelatin buffer of _ PH6.5 (utilizing HCl1M to turn down pH value) (does not have Ca ⁺);

The EDTA0.2M of _ PH7.2.

The platelet suspension of the rabbit blood (being exempted to obtain by 2.5kg) of preparation 50ml in the preparation 50ml test tube in the 50ml of the EDTA that contains 1.3ml test tube detects platelet activating factor (PAF) by the gelatin buffer that adds 250ml, the platelet suspension of 20ml and the test specimen of 10-30ml.Contrast comprises and only contains liposome solutions (concentration is 0.1-1mg/ml), only dialysis solution (PBS) and standard P AF sample (2-20ng/ml).

Utilize ELVI (Midan Italy) detector monitors platelet aggregation response; Change according to the platelet shape, utilization detects platelet activating factor (C.Tetta, G.Segoloni, A.Pacitti by the method measured quantity that people such as TETTA describe, G.Regis, M.Salomone, E.Turello, G.Camussi, A.Vercellone, " The production of platelet activating factorduring hemodialysis. " Int.J.Artif.Organs12:766-772,1989, Milan).

_ bilirubin detects:

According to the explanation of manufacturer, utilize commodity SIGMA Chemicals550-A to detect bilirubin.

Result of the test is shown in Fig. 6 and 7.Just as can be seen, regenerate filter (tube 38) no matter be used in combination liposome separately or with the carbon that phospholipid coats, can improve the removal of control toxin effectively from absolute magnitude and speed.

Claims

One kind from colloid solution fast weeding of grease dissolubility target molecule, particularly from whole blood or from the ultrafiltrate of whole blood or plasma filtrate, remove the method for toxin and free residue, wherein with a side contacts of the semipermeable membrane of solution to be purified and preset aperture, and the opposite side of wash solution by will being scheduled to component and film contacts and uses described film dialysis; It is characterized in that in wash solution, adding the liposome of predetermined concentration, common aperture greater than semipermeable membrane.
2. according to the described method of claim 1, it is characterized in that to contain diameter be the circle bubble of 100nm-200nm to described liposome and be defined as immobilized artificial membrane.
3. according to claim 1 or 2 described methods, it is characterized in that in described liposome, mixing at least a antioxidant substance, preferably be selected from by vitamin E and ubiquinone/ubiquinol group.
4. according to the described method of the arbitrary claim in front, it is characterized in that the concentration of liposome described in the wash solution is the 1-20g/l scope.
5. according to the described method of aforementioned arbitrary claim, it is characterized in that the described wash solution that contains described liposome is used to form a scrub stream, this scrub stream is added on the described film with the direction opposite with the inflow stream that contains colloid solution to be purified; Subsequently the filter of described scrub stream by an adsorbent filtered, described adsorbent has covered immobilized artificial membrane.
6. according to the described method of claim 5, it is characterized in that described adsorbent is the activated carbon that has covered the same or similar immobilized artificial membrane in liposome membrane of component.
7. according to the described method of claim 5, it is characterized in that described adsorbent contains synthetic resin, it is same or similar in the immobilized artificial membrane of liposome membrane that described synthetic resin has covered component.
8. be used for purifying blood, particularly suffer from the device of organ failure's patients'blood dialysis, this device contains first closed circuit that surges, and the solution to be purified that derives from patient's blood flow flows through therein; With second closed circuit that surges that makes that wash solution flows through therein; And filter, this filter contains the semipermeable membrane of preset aperture, by this filter, first loop communicates with the method for surging with second loop, so that the toxin that is used in the solution of purification is diffused in the wash solution, it is characterized in that second loop comprises the device that is used for supplying with to described second loop liposome.
9. as dialysis apparatus as described in the claim 8, the device that it is characterized in that being used for supplying with to second loop liposome can supply with described liposome for simultaneously described wash solution, so as with the liposome suspended dispersed in cleaning mixture.
10. according to Claim 8 or 9 described dialysis apparatuss, it is characterized in that second loop and a perfusion tube are connected in series with surging, described tube contains the filter of the adsorbent that is covered by the compatible immobilized artificial membrane of the liposome that is limited; Described wash solution and described liposome suspended dispersed are in the described cleaning mixture that flows through described filter.
11. dialysis apparatus according to claim 10 is characterized in that described perfusion tube contains the active carbon particle filter that is covered by successive immobilized artificial membrane, described immobilized artificial membrane contains the composition in a kind of immobilized artificial membrane that is usually used in described liposome.
12. each described dialysis apparatus according to Claim 8-11 is characterized in that described second loop provides to be used for from isolating first ultrafiltrate that does not have described liposome and described toxin of described wash solution; And a series of devices in first loop that are used for described first ultrafiltrate is supplied to the downstream of described filter.
13. each described dialysis apparatus is characterized in that described first loop in series comprises a hemoperfusion tube according to Claim 8-12.
14. each described dialysis apparatus according to Claim 8-13, it is characterized in that described first loop contains is useful on from patient's blood flow the hemofilter that separates a large amount of second ultrafiltration liquid, and connects out branch in the downstream of hemofilter and link to each other with the device that is used to supply with reperfusion solutions.
15. dialysis apparatus according to claim 14, the described device that it is characterized in that being used to supplying with described reperfusion solutions is connected with hemofilter, and can receive described second ultrafiltrate, and supply with this filtrate continuously by the activated carbon hemoperfusion tube of a sky, get back to first loop then as reperfusion solutions.
16. be used for the wash solution of haemodialysis equipment, it is characterized in that including water-based solvent, wherein liposome is scattered in suspension.
17. wash solution according to claim 16 is characterized in that the liposome content that it provides is the 1-20 grams per liter.
18., it is characterized in that including size and be the liposome of 100-200nm according to claim 16 or 17 described wash solutions.
19. according to the described wash solution of claim 16-18, it is characterized in that mixing a kind of antioxidant substance in the liposome at least, and be preferable over vitamin E and ubiquinone/ubiquinol group.
20. contain the dialysis performance that the wash solution of liposome is used to improve dialysis apparatus.