CN106178160B - Bionical liver and preparation method thereof and purposes - Google Patents
Bionical liver and preparation method thereof and purposes Download PDFInfo
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- CN106178160B CN106178160B CN201610519842.7A CN201610519842A CN106178160B CN 106178160 B CN106178160 B CN 106178160B CN 201610519842 A CN201610519842 A CN 201610519842A CN 106178160 B CN106178160 B CN 106178160B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3679—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by absorption
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M2202/00—Special media to be introduced, removed or treated
- A61M2202/04—Liquids
- A61M2202/0413—Blood
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M2202/00—Special media to be introduced, removed or treated
- A61M2202/04—Liquids
- A61M2202/0468—Liquids non-physiological
- A61M2202/049—Toxic
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Abstract
The invention belongs to technical field of bioengineering, and in particular to a kind of bionical liver and preparation method thereof and purposes.The present invention provides one kind filling bionical liver device made of poly- hexadine (PDA) nano particle de- cell liver holder.De- cell liver holder (DLS) therein remains the shape and three-dimensional structure of liver, and PDA nano particles can selectively neutralize perforation toxin.The device can efficiently remove the perforation toxin in blood without influencing blood constituent or activating complement, have a good application prospect in the antidotal therapy of perforation toxin.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to a kind of preparation method and purposes of bionical liver.
Background technology
Perforation toxin (Pore-forming Toxins) is a toxoid of generally existing in nature, is common in animal
It bites and bacterium infection, such as alpha hemolysis toxin and melittin.Perforating toxin can be by destroying cell membrane, causing cell dissolution
To effectively enter inside host cell, destroy the integrality of host cell.In-vitro detoxification device (such as artificial liver) is to be used for
A kind of important means of blood of human body toxin is removed, they are principle (such as Molecular Adsorption recycling systems based on physics or biology
System MARS, Biotype artificial liver), it is frequently utilized for the treatment of acute hepatic failure, hepatorenal syndrome.But these artificial liver devices have
Expensive, poor selectivity and the shortcomings of body Water-Electrolyte may be caused unbalance, at present for the perforation toxin in blood
There is no effective sweep-out methods.
Invention content
The technical problem to be solved by the present invention is to provide effective removing means for the perforation toxin in blood.
The present invention solve technical problem technical solution provide it is a kind of can be with selective clearing blood middle punch toxin
Bionical liver.The bionical liver is the de- cell liver holder for being filled with poly- hexadine nano particle.I.e. the bionical liver is to use
Poly- hexadine nano particle filling takes off the bionical liver devices of PDA-DLS made from cell liver holder to activation.
Wherein, in above-mentioned bionical liver, the poly- hexadine nano particle (PDA) is by 10,12-, 25 carbon two
Acetylenic acid (PCDA) is with modified 25 carbon diacetylenic acids (PCDA-EDEA-SA-NHS) of 10,12- with mass ratio for 150~200 ︰ 7
After~11 mixing, nano-particle is initially formed by self assembly under ultrasonication, is then crosslinked under the irradiation of 254nm ultraviolet lights
Form the more stable poly- hexadine nano particle of structure.
Further, the preparation process of 10,12-, the 25 carbon diacetylenic acids of the modification is：By n-hydroxysuccinimide
And 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate is added to react in the dichloromethane containing PCDA and generate
Acibenzolar, (2- amino ethoxies) the ethane reactions bis- with 1,2- again of this Acibenzolar generate PCDA-EDEA (intermediate) overnight,
PCDA-EDEA is reacted with succinic acid again obtains modified 10,12-, 25 carbon diacetylenic acids overnight.
It is sub- that modification PDA nano grain surfaces described in above-mentioned bionical liver possess leaving group N- hydroxysuccinimidyls acyl
Amine, this special group are conducive to PDA nano particles and react with the amino in de- cell liver on collagenous fibres.
Wherein, in above-mentioned bionical liver, the PDA nano particles are to pass through amidation process and de- cell liver branch
Collagen connects in frame.
Wherein, in above-mentioned bionical liver, the grain size of the PDA nano particles is 80~100nm.
Wherein, in above-mentioned bionical liver, the de- cell liver holder is to be handled by vitro liver through taking off cell
It arrives.The source of in vitro liver may generally be the liver of mammal.The present invention is but aobvious in embodiment by taking rat liver as an example
So the source of de- cell liver holder is not limited to rat liver, for example can use the in vitro liver of rabbit, monkey, pig, people etc..
The present invention additionally provides the method for preparing above-mentioned bionical liver simultaneously.This approach includes the following steps：A, it prepares de-
Cell liver holder；B, poly- hexadine nano particle is prepared；C, the PDA nano particles of modification are fed into de- cell liver branch
The PDA nano particles of frame, modification are connected on the collagenous fibres of de- cell liver holder, obtain bionical liver.
Wherein, in the above method, the operating procedure of the step a is：By rat liver portal vein and infrahepatic vena cava
It is inserted into Teflon intubation to tighten, ligatures liver superior and inferior vena cava, liver is completely taken out；By portal vein with 2mL/min successively
Pour water 4h, 0.01%SDS (lauryl sodium sulfate) solution 12h, 0.1%SDS solution 12h, 1%SDS solution 12h, distilled water
De- cell liver holder is prepared in 12h.
Wherein, in the above method, the operating procedure of the step b is：It is in the environment of room temperature, N- hydroxysuccinimidyls acyl is sub-
Amine and 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate are added to containing 25 carbon diacetylenic acids of 10,12-
In dichloromethane, agitation obtains Acibenzolar after 2 hours；The activated ester solution of synthesis is added drop-wise to 1,2- bis- (2- amino ethoxies)
In ethane, and stirred overnight in the environment of room temperature；Then the mode of universal vacuum distillation removes dichloromethane, and by product
PCDA-EDEA is obtained after purification by silica gel column chromatography；PCDA-EDEA is mixed with succinic acid and is stirred overnight, column is passed through
PCDA-EDEA-SA-NHS is acquired after purification；The n-hydroxysuccinimide, 1- ethyls-(3- dimethylaminopropyls) carbon
The mass ratio of acyl diimmonium salt hydrochlorate, 25 carbon diacetylenic acids of 10,12-, 1,2- bis- (2- amino ethoxies) ethane and succinic acid
For 9~10 ︰, 10~11 ︰, 6~7 ︰, 19~20 ︰ 2~3.
The mixture of PCDA and PCDA-EDEA-SA-NHS are placed in hot water, sonicated 10min；It was stored in 4 DEG C
After night, 254nm ultraviolet irradiations 10min is generated into blue PDA nano particles；25 carbon diacetylenic acids of the 10,12- and modification
The mass ratio of 25 carbon diacetylenic acids of 10,12- is 150~200 ︰ 7~11；The temperature of the hot water is 70~75 DEG C.
Wherein, in the above method, the operating procedure of the step c is：DLS (de- cell liver holder) is passed through into portal vein
Casing connection is to perfusion equipment, and inferior caval vein is as priming outlet；Poly- hexadine nanoparticle is passed through into door with the speed of 2mL/min
In venous perfusion to DLS, until de- cell liver holder color no longer deepens.
The present invention additionally provides the application of perforation toxin of the above-mentioned bionical liver in removing blood simultaneously.
The beneficial effects of the present invention are：Bionical liver provided by the present invention remains the rete vasculosum of liver complexity itself
Structure, the nano particle filled can selectively capture perforation toxin, and the detoxification device that the two is formed can efficiently remove
Perforation toxin in blood.And the material for preparing bionical liver does not influence blood constituent significantly, will not inducing complement swash
It is living.Bionical liver manufacturing cost provided by the invention is cheap, highly effective and safe, shows in the antidotal therapy for perforation toxin
Good application prospect.
Description of the drawings
The bionical liver of the poly- hexadines of Fig. 1 (PDA) nano particle and DLS compositions is used to remove blood middle punch toxin
Schematic diagram.
Fig. 2 DLS lose cell but keep complete collagen structure and vescular bed completely.(A, B)：In fresh liver
(FL) and the total DNA (A) in de- cell liver holder (DLS) and cell protein (B) amount；(C)：Collagenous fibres scanning in DLS
Electron microscope (SEM)；(D)：The biochemistry of collagen content quantifies (NS in FL and DLS:There was no significant difference)；(E)：After trypan blue is perfused
The vascular tree of leaf among DLS；(F, G)：The scanning electron microscope (SEM) photograph of the scanning electron microscope (SEM) photograph (F) of big blood vessel and portal area (arrow is signified)
(G).Engineer's scale：10 microns (C), 5 millimeters (E), 10 microns (F, G).
The preparation of Fig. 3 PDA nano particles.(A)：The synthetic schemes schematic diagram of PCDA-EDEA-SA-NHS；(B)：PDA receives
The principle schematic of grain of rice double-layer structure and its assembling process：PCDA and PCDA-EDEA-SA-NHS self assemblies under ultrasonication
Colourless nano particle is formed, then forms alternate 23 by Isosorbide-5-Nitrae addition reaction under 254 nanometers of ultraviolet light
Key, to polymerize；(C)：PDA nano particles are chemically attached to the schematic diagram on the collagenous fibres in DLS by amino；
(D, E)：The size distribution (D) and Potential distribution (E) figure of PDA nano particles；(F)：The images of transmissive electron microscope of PDA nano particles.
Engineer's scale：50 nanometers (F)
Fixation of Fig. 4 PDA nano particles in DLS.(A)：Fixed effect (umPDA of the PDA nano particles in DLS:
Unmodified PDA nano particles, n=3)；(B, C)：PDA nano particles are from the release conditions in DLS.PDA nano-solutions are perfused
Into DLS, measures PDA nano particles in DLS afterwards for 24 hours and be distilled the amount (B) that water flushes out, Freshman blood circulation is poured into and (is held
Continuous 20 minutes) in the DLS devices of the nano particle containing PDA, detect the content (C) of PDA nano particles in blood；(D)：PDA-DLS
The outline drawing of device.Due to being filled with the PDA nano particles of blue, which is in navy blue；(E)：PDA-DLS devices and DLS
The photo of (upper right corner) scanning electron microscope.Engineer's scale：10 millimeters (D), 200 nanometers (E)
The detoxification ability of Fig. 5 PDA-DLS devices.(A)：100mm3Fresh liver or PDA-DLS are to various concentration bee venom
The quantitative assessment of peptide solution neutralising capacity, n=3；(B)：DLS, PDA-DLS device Perfused melittin are used in quantitative assessment respectively
The detoxifying effect of solution (10 μ g/mL), n=3；(C)：Bee venom peptide solution (10 of the quantitative assessment PDA-DLS devices to circumfusion
μ g/mL) detoxifying effect, n=3；(D)：Normal group, the hemolysis rate measurement of three groups of PDA-DLS groups and melittin group, n=3；
(E)：Total bilirubin content in three groups of normal group, PDA-DLS groups and melittin group.N=3；(F)：Normal group, PDA-DLS groups and
Indirect content of bilirubin in three groups of melittin group.N=3；(G)：Normal group (left side), PDA-DLS groups (in) and melittin group (right side)
The form of red blood cell in three groups.Acanthocyte (arrow is signified) has close correlation with haemolysis.Engineer's scale：10 microns (G)
The safety evaluatio of Fig. 6 PDA-DLS devices.(A, B)：Blood smear Wright's staining.(A) and warp in normal blood
The red blood cell (single arrow is signified) of (B), lymphocyte (double-head arrow is signified) and neutral grain in blood after the perfusion of PDA-DLS devices
Cell (three arrows are signified)；(C)：Erythrocyte number before being perfused through PDA-DLS and after perfusion, n=3；(D)：Through PDA-
Quantity of leucocyte before DLS perfusions and after perfusion, n=3；(E)：Blood before being perfused through PDA-DLS and after perfusion is small
Plate quantity, n=3；(F, G)：The amount of total protein (F) and albumin (G) before being perfused through PDA-DLS and after perfusion, n=3；
(H-J)：C3 (H), C4 (I) and SC5b9 (J) in (NC) blood without any processing, by PDA-DLS devices (PDA-
DLS) processed blood is neutralized by the content in the processed blood of FL, n=3, NS:There was no significant difference.Engineer's scale：20
Micron (A, B).
Specific implementation mode
Liver is most important removing toxic substances organ in human body, and the special micro-structure of live donor liver can help liver cell efficiently clear
Except the toxin in blood, this provides thinking for the present invention designed for the class liver device effectively to detoxify.The present invention is creatively
Using the de- cell liver of PDA nano particle stuffed animals, the effect of the de- cell liver of " cellularised again " animal, structure are obtained
One bionical liver device is purged the target toxin in blood.The PDA nano particles used in the present invention be by 10,
Vesica shape nano particle made of 12- 25 carbon diacetylenic acid (PCDA) self assembly.PDA nano grain surfaces are handed over by two or three keys
For formation pi-conjugated main chain constitute, it be similar to cell membrane surface, can induce, capture and neutralize perforate toxin.It is de- thin
Born of the same parents' liver holder (DLS) remains the original shape of liver and three-dimensional structure, is a natural accurate microfluidic system.
And toxin of perforating has cytoclasis ability, they can change cell membrane by forming hole on cell membrane
Permeability is to destroy cell.It, can be by being carried out to it since PDA nano particles have many advantages, such as that size is small and it is flexible to prepare
Reasonable design is for the capture and removing of toxin, and therefore, the present invention creatively will be by recyclable PDA nano particles
Combine with the circulatory system of flowing, reach activation and take off cell liver, selectively removes the mesh of the perforation toxin in blood
, this technology has good application value in the removing toxic substances of perforation toxin.
Bionical liver provided by the invention is the de- cell liver holder for being filled with poly- hexadine nano particle.It is with poly-
The bionical liver devices of PDA-DLS are made to which activation takes off cell liver holder in the filling of hexadine nano particle.
Wherein, in above-mentioned bionical liver, the poly- hexadine nano particle (PDA) is by 10,12-, 25 carbon two
Acetylenic acid (PCDA) is with modified 25 carbon diacetylenic acids (PCDA-EDEA-SA-NHS) of 10,12- with mass ratio for 150~200 ︰ 7
After~11 mixing, nano-particle is initially formed by self assembly under ultrasonication, is then crosslinked under the irradiation of 254nm ultraviolet lights
Form the more stable poly- hexadine nano particle of structure.
Further, the preparation process of 10,12-, the 25 carbon diacetylenic acids of above-mentioned modification is：By n-hydroxysuccinimide
And 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate is added to react in the dichloromethane containing PCDA and generate
Acibenzolar, (2- amino ethoxies) the ethane reactions bis- with 1,2- again of this Acibenzolar generate PCDA-EDEA (intermediate) overnight,
PCDA-EDEA is reacted with succinic acid again obtains modified 10,12-, 25 carbon diacetylenic acids overnight.
It is sub- that modification PDA nano grain surfaces described in above-mentioned bionical liver possess leaving group N- hydroxysuccinimidyls acyl
Amine, this special group are conducive to PDA nano particles and react with the amino in de- cell liver on collagenous fibres.Described
PDA nano particles are to be connect with collagen in de- cell liver holder by amidation process.
In above-mentioned bionical liver, the grain size of the PDA nano particles is preferably 80~100nm.
In above-mentioned bionical liver, the de- cell liver holder is handled to obtain by vitro liver through taking off cell.
The source of in vitro liver may generally be the liver of mammal.The present invention is in embodiment by taking rat liver as an example, it is apparent that de-
The source of cell liver holder is not limited to rat liver, for example can use the in vitro liver of rabbit, monkey, pig, people etc..
The present invention additionally provides the method for preparing above-mentioned bionical liver simultaneously.This approach includes the following steps：A, it prepares de-
Cell liver holder；B, poly- hexadine nano particle is prepared；C, the PDA nano particles of modification are fed into de- cell liver branch
The PDA nano particles of frame, modification are connected on the collagenous fibres of de- cell liver holder, obtain bionical liver.
The preparation method of the bionical liver of the present invention, specifically may include following steps：
A, it prepares and takes off cell liver holder：Rat liver portal vein and infrahepatic vena cava are inserted into Teflon intubation to prick
Tightly, liver superior and inferior vena cava is ligatured, liver is completely taken out；By portal vein with 2mL/min irrigations in their given order 4h, 0.01%SDS
(lauryl sodium sulfate) solution 12h, 0.1%SDS solution 12h, 1%SDS solution 12h, distilled water 12h are prepared de- thin
Born of the same parents' liver holder；
B, poly- hexadine nano particle is prepared：In the environment of room temperature, by n-hydroxysuccinimide and 1- ethyls-
(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate is added in the dichloromethane containing 10,12-, 25 carbon diacetylenic acids, is stirred
Acibenzolar is obtained after 2 hours dynamic；The activated ester solution of synthesis is added drop-wise in bis- (2- amino ethoxies) ethane of 1,2-, and in room
It is stirred overnight under the environment of temperature；Then the mode of universal vacuum distillation removes dichloromethane, and product is passed through silicagel column color
Spectrometry obtains PCDA-EDEA after purification；PCDA-EDEA is mixed with succinic acid and is stirred overnight, by being acquired after column purification
PCDA-EDEA-SA-NHS；The mixture of PCDA and PCDA-EDEA-SA-NHS are placed in hot water, sonicated 10min；In 4
After DEG C storage overnight, 254nm ultraviolet irradiations 10min is generated into blue PDA nano particles；
C, by DLS (de- cell liver holder) by portal vein casing connection to perfusion equipment, inferior caval vein is as perfusion
Outlet；By poly- hexadine nanoparticle with the speed of 2mL/min by portal vein perfusion to DLS, until taking off cell liver holder
Color no longer deepens, and obtains bionical liver.
Wherein, n-hydroxysuccinimide, 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne described in above method step b
Diimmonium salt hydrochlorate, 25 carbon diacetylenic acids of 10,12-, bis- (2- amino ethoxies) ethane of 1,2- and succinic acid mass ratio be 9
10~11 ︰ of~10 ︰, 6~7 ︰, 19~20 ︰ 2~3.25 carbon diacetylenic acids of the 10,12- and modified 25 carbon two of 10,12-
The mass ratio of acetylenic acid is 150~200 ︰ 7~11.The temperature of the hot water is 70~75 DEG C.
The achievement in research of the present invention shows that the bionical liver being composed of PDA nanoparticles and DLS (is known as PDA-DLS dresses
Set) perforation toxin in blood can be efficiently removed in vitro, and blood constituent and the activating complement factor are not influenced.This work
Make that new method and new technology can be provided for the research and development and application of clinical in-vitro detoxification device from now on.
The present invention additionally provides the application of perforation toxin of the above-mentioned bionical liver in removing blood simultaneously.According to above-mentioned
The principle of introduction and following experimental evidences, it is clear that the technology of the present invention can have the perforation toxin such as alpha hemolysis toxin and melittin
Good elimination effect.
Below by way of specific embodiment, the present invention is described in further detail.
The structure of the bionical liver devices of 1 PDA-DLS of embodiment
First using cell technology preparation DLS is taken off, then PDA nano particles are perfused to DLS, PDA nano particles pass through
It is chemically reacted to be fixed on inside DLS with the chemical group on collagenous fibres, finally obtains PDA-DLS devices.
The preparation process of PDA nano particles is：In the environment of room temperature, by n-hydroxysuccinimide (920 milligrams,
7.99 mMs) and 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate (1020 milligrams, 5.32 mMs)
It is added in the dichloromethane containing PCDA (500 milligrams) (100 milliliters), stirring obtains Acibenzolar after 2 hours；By the activation of synthesis
Ester solution is added dropwise in bis- (2- amino ethoxies) ethane (1980 milligrams, 13.35 mMs) of 1,2-, and in the ring of room temperature
It is stirred overnight under border；Dichloromethane is removed by the way of vacuum distillation, and by product by silica gel column chromatography after purification
Obtain PCDA-EDEA；Itself and succinic acid (230 milligrams) are mixed and stirred for overnight, by obtaining PCDA-EDEA- after column purification
SA-NHS；The mixture of PCDA (190 milligrams) and PCDA-EDEA-SA-NHS (10 milligrams) are placed in 70 DEG C of distilled water, sound
After ten minutes, overnight in 4 DEG C of storages, 254 nanometers of ultraviolet irradiations obtain the PDA nanoparticles of blue for 10 minutes for wave processing.
By rat liver by the way that SDS is perfused for a long time from portal vein, the transparent DLS for remaining liver shape can be obtained.
Do not detect remaining DNA and cell protein (Fig. 2A, B) in DLS, but remain collagen fiber structure (Fig. 2 C) and collagen at
Divide (Fig. 2 D).Trypan Blue dye is injected from portal vein, the complete microvasculatures of DLS can be shown (Fig. 2 E).
The big blood vessel and thin vessels being also visually observed that under scanning electron microscope in DLS be same as in former liver portal vein thribble (Fig. 2 F,
G).It is indicated above that de- cell processes can selectively remove the cell component in liver, and retain extracellular matrix components and
Complete blood vessel network in DLS.
It after obtaining DLS, is filled with PDA nano particles, the function of the alternative part of hepatocytes of PDA nanoparticles.Pass through
50 milligrams of PDA nanoparticles are perfused into rat DLS, produce PDA-DLS devices, its appearance is as shown in Figure 4 D.
PDA nano particles by modification, so that its surface is possessed leaving group n-hydroxysuccinimide (NHS), it easily and
Chemical reaction (Fig. 3 C) occurs for the amino on collagen.The nano particle average grain diameter of synthesis is about 100 nanometers (Fig. 3 D), Zeta electricity
Gesture is -20mV (Fig. 3 E).Under transmission electron microscope, nano particle shows imitated vesicle structure, and grain size is at 80-100 nanometers (Fig. 3 F).
It cannot effectively be retained by DLS if PDA nanoparticles are without modification.With PDA in colorimetric determination efflux
Content find modification after PDA nanoparticles be retained in DLS (Fig. 4 A) by 100%.PDA-DLS devices be distilled water or
Blood further rinses, it can be seen that the PDA nanoparticles of modified will not be eluted out, and PDA nanometers unmodified
Grain can be eluted out (Fig. 4 B, C).By transmission electron microscope it is observed that PDA nanoparticles are retained in the collagen in PDA-DLS
On fiber (Fig. 4 E).Therefore, the chemical modification of PDA nanoparticles is essential step for it to be efficiently retained in DLS
Suddenly.
The in-vitro detoxification ability of the bionical liver devices of 2 PDA-DLS of embodiment
Prove that preparation-obtained PDA-DLS devices are to the removing toxic substances energy for toxin of perforating in embodiment one by hemolytic experiment
Power.
The perforation toxin using melittin as pattern.As a control group with fresh liver, detection PDA-DLS devices are to melittin
The detoxification ability of solution.First, we test the removing toxic substances of PDA-DLS devices and fresh liver in the bee venom peptide solution of standing
Ability.PDA-DLS devices and fresh liver are individually cut to 100mm3Fritter, the melittin (200 μ L) of various concentration is set
The tissue block cut is incubated 60 minutes, then will take supernatant after the centrifugation of the solution of incubation by group respectively therewith, by supernatant plus
Enter into the red blood cell liquid of rat and carry out erythrocyte splitting experiment, centrifuged after being incubated 30 minutes, detects the OD values of each group supernatant.
As shown in Figure 5A, PDA-DLS devices and fresh liver are respectively 80 μ g/mL and 10 μ g/mL. to the maximum removing toxic substances concentration of melittin
Therefore, PDA-DLS is almost 8 times or more of fresh liver for the detoxification ability of melittin.
Then PDA-DLS devices evaluate the detoxification ability of bee venom peptide solution under perfusion state.Melittin
Solution (10 μ g/mL, 100mL) trans-portal vein is poured into PDA-DLS and DLS respectively with the flow velocity of 2mL/min.It was received every 5 minutes
The liquid that collection is once flowed out from inferior caval vein carries out erythrocyte splitting experiment, is centrifuged after being incubated 30 minutes, detects each group supernatant
OD values.As a result, it has been found that PDA-DLS can be with the melittin in fully erased Perfused solution, but DLS can not significantly subtract
Toxin in few perfusate.Detoxification ability is more as shown in Figure 5 B.Solution of the PDA-DLS devices in Perfused bee venom peptide solution
Malicious ability is 100%, and the detoxification ability of DLS is almost 0.This result means that the detoxification ability of PDA-DLS devices is main
It is the effect by PDA nano particles.
In order to simulate the detoxification processes of liver under physiological condition, we have investigated bee of the PDA-DLS devices to circumfusion
The detoxification ability of phallotoxins solution.The bee venom peptide solution (10 μ g/mL) for being placed on solution pool is filled by portal vein with the flow velocity of 2mL/min
Enter into PDA-DLS devices, the solution flowed out from inferior caval vein is recycled to solution pool again, is fed into portal vein again.Every 25
Minute extracts the bee venom peptide solution in solution pool and carries out erythrocyte splitting experiment, and as shown in Figure 5 C, PDA-DLS will can be recycled almost
The melittin of perfusion is fully erased.Simultaneously it was found that circumfusion bee venom peptide solution, detoxifying effect can reach after 75 minutes
100% (1.5 times of the Perfused time) (Fig. 5 C).This result proves that PDA-DLS can be neutralized efficiently in circumfusion
The toxicity of melittin.
The bionical liver devices of 3 PDA-DLS of embodiment remove the ability of people's blood middle punch toxin
In order to evaluate detoxification abilities of the PDA-DLS to blood, the PDA-DLS prepared in embodiment one is subjected to bee in blood
The removing of phallotoxins is tested.
Melittin (10 μ g/mL) is added in advance in normal person's blood, after twenty minutes through PDA-DLS circumfusions, to normal
People's blood (normal group), after PDA-DLS is recycled blood (PDA-DLS groups), the blood containing melittin that is recycled without PDA-DLS
Liquid (melittin group) carries out blood routine and the detection of blood biochemistry index of correlation.
Red blood cell count(RBC) is the result shows that the erythrocyte splitting rate of PDA-DLS groups is significantly lower than melittin group (Fig. 5 D).Total courage
Red pigment and indirect bilirubin testing result show the cleavage rate of PDA-DLS device groups well below melittin group (Fig. 5 E, F).Together
When, the blood cell shape each organized is observed with blood smear, normocyte has the shape of smooth two-sided recess, melittin will
Normal red blood cell becomes acanthocyte, has close association with haemolysis.In PDA-DLS device groups, the ratio of acanthocyte
Example is significantly lower than melittin group (Fig. 5 G).Thus illustrate that PDA-DLS devices can effectively remove the melittin in blood circulation, from
And red blood cell is protected to be destroyed from melittin.
The safety evaluatio of the bionical liver devices of 4 PDA-DLS of embodiment
In order to assess influence of the bionical livers of PDA-DLS to normal person's blood, by normal person's blood (100 milliliters) trans-portal vein with
In the flow velocity circumfusion to PDA-DLS devices of 2mL/min, as a result, it has been found that the haemocyte after perfusion maintains normal cell shape
State (Fig. 6 A, B), and the quantity of haemocyte does not have significant change (Fig. 6 C, D, E).In addition, blood biochemistry is analysis shows that in blood
Total protein and albumin also do not significantly reduce (Fig. 6 F, G) in filling process.
For the ability of the activation of complement, as a comparison by PDA-DLS and fresh rat liver, blank is done with normal blood
It compares (NC groups).After the perfusion of rat fresh liver, wherein Complement C_3 and C4 significantly reduces the blood of people, and complement SC5b9
Content increases, it means that the complement in human blood is by the heterogeneous activation (Fig. 6 H, I, J) of fresh liver.And it is filled through PDA-DLS
Note Complement C_3 in processed blood, C4 (Fig. 6 H, I, J) almost consistent with the content in normal blood with the content of SC5b9.Cause
This, PDA-DLS when being purged to the perforation toxin in blood, mend by the ingredient and activation that will not significantly affect normal blood
The body factor.
Claims (9)
1. bionical liver, it is characterised in that：To be filled with the de- cell liver holder of poly- hexadine nano particle；
Wherein, the poly- hexadine nano particle is by 10,12-, 25 carbon diacetylenic acids and modified 10,12-, 25 carbon
Diacetylenic acid is with mass ratio for 150 ~ 200:After 7 ~ 11 mixing, nano-particle is initially formed by self assembly under ultrasonication, then
The more stable poly- hexadine nano particle of structure is cross-linked to form under the irradiation of 254nm ultraviolet lights；
The structure of the modification 10,25 carbon diacetylenic acids of 12- is：
。
2. bionical liver according to claim 1, it is characterised in that：10,12-, the 25 carbon diacetylenic acids of the modification
Preparation process is：By n-hydroxysuccinimide and 1- ethyls-（3- dimethylaminopropyls）Phosphinylidyne diimmonium salt hydrochlorate adds
Acibenzolar, (2- amino ethoxies) the ethane reactions bis- with 1,2- again of this Acibenzolar are generated to reaction in the dichloromethane containing PCDA
PCDA-EDEA is generated overnight, and PCDA-EDEA is reacted with succinic acid again obtains modified 10,12-, 25 carbon diacetylenic acids overnight.
3. bionical liver according to claim 1, it is characterised in that：The poly- hexadine nano particle passes through amidation
Reaction is connect with collagen in de- cell liver holder.
4. bionical liver according to claim 1, it is characterised in that：The grain size of the poly- hexadine nano particle be 80 ~
100nm。
5. bionical liver according to claim 1, it is characterised in that：The de- cell liver holder is by vitro liver
Dirty warp takes off what cell was handled.
6. preparing the bionical liver method of Claims 1 to 5 any one of them, it is characterised in that include the following steps：A, it prepares
De- cell liver holder；B, poly- hexadine nano particle is prepared；C, poly- hexadine nano particle is poured into de- cell liver holder,
Poly- hexadine nano particle is connected on the collagenous fibres of de- cell liver holder, obtains bionical liver.
7. according to the method described in claim 6, it is characterized in that：The operating procedure of the step b is：In the environment of room temperature
Under, n-hydroxysuccinimide and 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate are added to containing 10,
In the dichloromethane of 25 carbon diacetylenic acids of 12-, agitation obtains Acibenzolar after 2 hours；The activated ester solution of synthesis is added drop-wise to
In 1,2- bis- (2- amino ethoxies) ethane, and stirred overnight in the environment of room temperature；Then the mode of universal vacuum distillation will
Dichloromethane removes, and product is obtained PCDA-EDEA after purification by silica gel column chromatography；By PCDA-EDEA and succinic acid
It mixes and stirs overnight, by acquiring modified 10,12-, 25 carbon diacetylenic acids after column purification；The N- hydroxysuccinimidyls acyl
Imines, 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate, 25 carbon diacetylenic acids of 10,12-, the bis- (2- of 1,2-
Amino ethoxy) mass ratio of ethane and succinic acid is 9 ~ 10:10~11：6~7:19~20：2~3；
The mixture of 10,12-, 25 carbon diacetylenic acids and modified 10,12-, 25 carbon diacetylenic acids is placed in hot water, sound wave
Handle 10min；After 4 DEG C of storages overnight, 254nm ultraviolet irradiations 10min is generated into the poly- hexadine nano particle of blue；It is described
The mass ratio of 25 carbon diacetylenic acids of 10,12- and modified 25 carbon diacetylenic acids of 10,12- is 150 ~ 200:7~11；The heat
The temperature of water is 70 ~ 75 DEG C.
8. according to the method described in claim 6, it is characterized in that：The operating procedure of the step c is：By de- cell liver branch
Frame is by portal vein casing connection to perfusion equipment, and inferior caval vein is as priming outlet；By poly- hexadine nanoparticle with 2mL/min
Speed by portal vein perfusion to de- cell liver holder, until de- cell liver holder color no longer deepens.
9. the bionical liver of Claims 1 to 5 any one of them answering in the device of the perforation toxin during structure removes blood
With.
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|---|---|---|---|---|
| CN101850136A (en) * | 2010-06-11 | 2010-10-06 | 四川大学华西医院 | Implantable bioartificial liver |
| CN104841017A (en) * | 2015-05-28 | 2015-08-19 | 四川大学华西医院 | Decellularized liver biological scaffold with anticoagulant property and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101850136A (en) * | 2010-06-11 | 2010-10-06 | 四川大学华西医院 | Implantable bioartificial liver |
| CN104841017A (en) * | 2015-05-28 | 2015-08-19 | 四川大学华西医院 | Decellularized liver biological scaffold with anticoagulant property and preparation method thereof |
Non-Patent Citations (1)
| Title |
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| "Bio-inspireddetoxificationusing3D-printedhydrogelnanocomposites";MalingGou etal.;《nature communications》;20140508;第5卷;第1-9页 * |
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