CN103252217A - Novel lipopolysaccharides (LPS) specific adsorption blood purification material and preparation method thereof - Google Patents
Novel lipopolysaccharides (LPS) specific adsorption blood purification material and preparation method thereof Download PDFInfo
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- CN103252217A CN103252217A CN2013101502087A CN201310150208A CN103252217A CN 103252217 A CN103252217 A CN 103252217A CN 2013101502087 A CN2013101502087 A CN 2013101502087A CN 201310150208 A CN201310150208 A CN 201310150208A CN 103252217 A CN103252217 A CN 103252217A
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Abstract
The invention discloses a novel lipopolysaccharides (LPS) specific adsorption blood purification material and a preparation method thereof. The novel LPS specific adsorption blood purification material is characterized by being formed by connecting chloromethylated beads, heparin and a PMB (papillomacular bundle) ligand; the chloromethylated beads are activated by virtue of ethidene diamine to obtain aminated chloromethylated beads, then heparin is introduced as a molecular arm, finally the PMB ligand is fixed on the surfaces of the heparinized chloromethylated beads, and thus a PMB adsorbent is obtained. The preparation method of the novel LPS specific adsorption blood purification material is characterized by comprising the following steps of: 1, aminating a chloromethylated bead carrier; 2, connecting a heparin molecular arm; and 3, fixing the PMB ligand. The novel LPS specific adsorption blood purification material has high safety, no toxic reaction or allergic reaction is produced, blood compatibility is high, and the PMB adsorbent has good adsorption efficiency and selective adsorption property on LPS, so that the novel LPS specific adsorption blood purification material has certain clinical use value.
Description
Technical field
The invention belongs to the blood purification technical field, specifically, relate to blood purification material of the special absorption of a kind of novel endotoxin and preparation method thereof.
Background technology
Medical research shows (LPS) level of endotoxin in the blood and cancer, acute inflammation, wound, the caused multiple organs failures of disease (MODS) such as burn and systemic inflammatory response illnesss such as (SIRS) have substantial connection, be cause endotoxemia main cause [referring to data OReillyM, Newcomb DE, Remick D, etal.Endotoxin sepsis and the primrose path.Shock, 1999,12 (6): 411-419.], high incidence by its endotoxemia of bringing out, problem such as high mortality and high medical expense is threatening human beings'health always and is bringing bigger financial burden.More than 50 ten thousand endotoxemia patient is arranged in the U.S. every year, wherein very most patient is because can not get giving treatment to death effectively and timely [referring to data Keat G.Onga, Joshua M.Leland a, Kefeng Zenga, Gary Barrett a, Mohammed Zourob b, CraigA.Grimes.A rapid highly-sensitive endotoxin detection system.Biosensors and Bioelectronics, 2006 (21): 2270 – 2274.].Therefore numerous experts and the scholar method of attempting to find endotoxin in a kind of safe and effective removal patient body to reach therapeutic purposes has important and profound significance.
Treatment for endotoxemia has at present obtained some progress, use antiendotoxin medicine such as antibiotic, lipoid A antagonist, the antiendotoxin antibody etc. can be directly or the biological effect of remote-effects endotoxin molecule, yet but fail to obtain comparatively inspirer result on the clinical treatment.Along with people's deepening continuously to endotoxin pathogenesis and the research of prophylactic treatment method, utilize modern medical service sophisticated equipment and technology, remove endotoxin by blood purification absorption therapy and receive increasing concern, and become the research focus in fields such as medical science, pharmacy and bioengineering.
Existing adsorbent can't satisfy clinical requirement fully at aspects such as absorption property, security and prices in blood perfusion technique.Japan just will fixedly have the blood perfusion fibre columns of PMB to be applied to endotoxemia as far back as nineteen nineties to treat [referring to data: Chieko Mitaka, Makoto Tomita.PolymyxinB immobilized fiber column hemoperfusion therapy for septicshock.Shock, 2011,36 (4): 332-338.], also obtained certain achievement clinically, yet PMB itself has neurotoxicity and renal toxicity, and being used for fixedly, the spacerarm of aglucon can cause the loss of a large amount of plasma proteins of blood (referring to data: Wicks when carrying out blood perfusion, I.P, etal.Bacteriallipopolysaccharide copurifies with plasmid DNA:Implications for animalmodels and human gene therapy.Hum.Gene.Ther, 1995,6:317-321.), therefore design high adsorption capacity, in the time of the high selectivity endotoxin absorbent, biocompatibility in the time of also will fully taking into account its use, reduce the non-selective absorption to other composition in the blood environment as far as possible, develop the adsorbent with independent intellectual property right.Yet how to improve adsorbance, specific selectivity and the biocompatibility of adsorbent, key is to solve selection and preparation, the selection of aglucon and the problem that the key between them connects these three aspects of mode of its carrier.
The shortcoming of prior art: existing adsorbent can't satisfy clinical requirement fully at aspects such as absorption property, security and prices in blood perfusion technique, and easily toxigenicity reaction and allergic reaction are low with blood compatibility.
Summary of the invention
For solving above technical problem, the object of the present invention is to provide that a kind of absorption property is strong, safe, blood compatibility is high, the blood purification material of the not special absorption of novel endotoxin of toxigenicity reaction and allergic reaction and reasonable price and preparation method thereof.
The present invention seeks to realize like this:
The blood purification material of the special absorption of a kind of novel endotoxin, its key is: connected to form by chlorine ball, heparin and PMB aglucon; Described chlorine ball warp ethylenediamine activation is obtained amination chlorine ball (ps), introduce described heparin again as molecular arm, by glutaraldehyde described PMB aglucon is fixed in heparinize chlorine ball (ps-hep) surface at last, obtain the blood purification material.The reaction scheme of chlorine ball fixing heparin molecular arm and PMB aglucon as shown in Figure 1.
The characteristic of utilizing the big molecule excellent biological compatibility of heparin and having a plurality of natural activity groups, it is introduced chloromethylated polystyrene-diethylbenzene olefine resin (being called for short the chlorine ball) as molecular arm, amplify the PMB aglucon of fixing special absorption LPS by chemistry, give full play to the big spatial configuration of molecules effect of heparin and biological effect, and make that strong affinity suction-operated takes place the phosphate group of negative electrical charge among PMB and the LPS molecular activity group L ipid A, prepare the endotoxic adsorbent of special absorption, inquire into this adsorbent LPS is selected absorption property, blood compatibility and potential and meaning aspect blood perfusion treatment endotoxemia.
The blood purification preparation methods of the special absorption of a kind of described novel endotoxin, its key is to be made up of following steps:
(1) amination of chlorine ball carrier:
A, chlorine ball activation pre-treatment: use absolute ethyl alcohol and distilled water alternately washing are removed residual organic or inorganic impurity in the production for several times; Drain standby with the suction filtration device;
B, get the standby chlorine ball of 1～20g in 40～200ml1, swelling 1～5h in the 4-dioxane solution;
C, the mixed solution of above-mentioned chlorine ball and 1,4-dioxane is transferred in the 250ml there-necked flask; 1～20gNaOH, 0.1～5g TBAB (TBAB) add reaction system after being dissolved in 10～100ml distilled water;
D, add 50～100ml ethylenediamine in reaction system, reactant mixture is in 50～90 ℃ of stirring reaction 1～10h;
E, product use absolute ethyl alcohol and distilled water alternately to wash to neutral after filtering, obtain amination chlorine ball (ps), and be standby after the vacuum drying;
Course of reaction is as follows:
(2) connection of heparin molecule arm
The citrate buffer solution of a, preparation 0.01～0.05M is regulated PH to 3.0～6.0 with 0.1M NaOH, and adding molar ratio is the catalyst EDC[1-(3-dimethylamino-propyl of 1:1)-the 3-ethyl carbodiimide] and the NHS(N-HOSu NHS);
B, liquaemin is dissolved in the preparation of described citrate buffer solution obtains the heparin solution that concentration is 1～6mg/ml, then described amination chlorine ball (ps) is immersed in the heparin solution, 37 ℃ of constant temperature oscillating reactions at least 24 hours;
C, then product is carried out suction filtration, use the PBS cyclic washing, obtain heparinize chlorine ball (ps-hep), drain standby;
(3) the PMB aglucon is fixing
The Na of a, preparation 0.1～0.5M
3PO
4-NaCl coupling buffer: regulate PH to 5.0～7.0;
B, with described heparinize chlorine ball (ps-hep) with 5% glutaraldehyde in described Na
3PO
4After the activation, add NaCNBH in the-NaCl coupling buffer
3 Room temperature reaction 2～8 hours;
C, PMB is joined in the described coupling buffer as solvent earlier, preparation obtains the PMB solution that concentration is 0.5～6mg/ml, then with described heparinize chlorine ball (ps-hep) behind the abundant cyclic washing of coupling buffer, with described PMB solution room temperature reaction 1～6 hour under neutrallty condition, use NaCNBH simultaneously
3Catalytic reduction is with the product Na that obtains
3PO
4-NaCl coupling buffer and distilled water washing obtain blood purification material PMB adsorbent (ps-hep-pmb) at last.
One, the determining of various parameters in the above-mentioned blood purification material preparation method:
1, infra-red sepectrometry detects amination chlorine ball (ps)
After 24 hours, adopt infra-red sepectrometry that it is carried out Analysis and Identification amination chlorine ball (ps) vacuum drying for preparing, observe the variation that takes place before and after the activation of chlorine ball warp ethylenediamine, structure as shown in Figure 4 and Figure 5.Can see C-H stretching vibration peak 3025,3059,3081cm on the phenyl ring among Fig. 4
-1, saturated CH and the stretching vibration peak of CH2 2921,2848cm
-1, the vibration of phenyl ring upper skeleton is 1600cm
-1, phenyl ring CH and CH2 in-plane bending vibration are respectively 1494,1452cm
-1In addition, CH2Cl several characteristic peak is: 1428cm
-1Be the CH2 in-plane bending vibration peak among the CH2Cl, 1265cm
-1Be 4 and replaced 1,4-disubstituted benzenes=C-H in-plane bending vibration peak, 676cm by CH2Cl
-1It is the C-Cl stretching vibration peak; Fig. 5 finds that than Fig. 4 obvious variation does not all take place the principal character peak on polystyrene-diethylbenzene alkene, and at 3338.1cm
-1And 3311.3cm
-1Occurred-stretching vibration of NH2, and 1264.9cm
-1-CH2Cl strengthens=and the in-plane bending vibration peak of C-H disappears, and illustrates that reaction has taken place the chloromethyl on ethylenediamine and the phenyl ring, successfully introduces chlorine ball carrier.
2, toluidine blue colorimetric determination heparin
2.1 make the heparin content calibration curve:
2.1.1 preparation liquaemin standard liquid: get ten test tubes, number 1～10 respectively, add 10ug/ml heparin solution 0,0.1,0.2,0.3,0.4,0.5,0.6,0.7,1.0,1.2ml respectively; Adding 0.2%NaCl solution respectively in 1～No. 10, to make cumulative volume be 5ml; The 0.2%NaCl solution that adds 5ml in the sample tube.
2.1.2 prepare 0.005% toluidine blue (TB) solution: 0.005g TB is dissolved in 100ml0.2%NaCl solution, mixes, and is standby.
2.1.3 add 1ml TB solution respectively in 1～No. 10 and the sample tube, mix, in 37 ℃ of reactions of constant temperature vibration case 2h.
2.1.4 respectively get 1～No. 10 and sample tube supernatant liquid 2ml, add in the separatory funnel, respectively add the 2ml n-hexane then, thermal agitation is surveyed its absorbance and drafting calibration curve Fig. 2 at ultraviolet specrophotometer 631nm place behind the extraction certain hour.
2.2 the heparin of variable concentrations and the heparin fixed amount under the differential responses time are analyzed
2.2.1 amination chlorine ball (ps) is under 1mg/ml, 2mg/ml, 3mg/ml, 5mg/ml, the 6mg/ml in heparin solution concentration respectively, behind the reaction 24h, heparin grafting amount by toluidine blue colorimetric method for determining chlorine ball surface, the absorbance that records is with reference to the heparin content calibration curve, calculate the content of heparin in the unit mass chlorine ball sample, determine that heparin solution concentration is to the influence of heparin reaction, as shown in Figure 6.The result shows: heparin solution concentration is between 1～6mg/ml the time, fixation reaction has all taken place to heparin in amination chlorine ball (ps), and along with the increase of heparin reaction density, the fixed amount of heparin on scavenging material also increases thereupon, and its fixed amount is tending towards saturated gradually.
2.2.2 amination chlorine ball (ps) is under the 5mg/ml in heparin solution concentration, behind the reaction different time, heparin grafting amount by toluidine blue colorimetric method for determining chlorine ball surface, the absorbance that records is with reference to the heparin content calibration curve, calculate the content of heparin in the unit mass chlorine ball sample, determine that the reaction time is to the influence of heparin reaction, as shown in Figure 7.The result shows: from the increase along with the heparin reaction time, the fixed amount of heparin on scavenging material also increases thereupon, and its fixed amount tends towards stability saturatedly gradually behind reaction 24h, and in order to guarantee the fully fixing of heparin, its reaction time remains on 24h or more than the 24h.
3, PMB aglucon assay
3.1PMB the drafting of solution calibration curve
At first under ultraviolet specrophotometer, measure the maximum corresponding wavelength of PMB absorbance, compound concentration is 0.5mg/ml, 1mg/ml, 2mg/ml, 3mg/ml, 4mg/ml, 6mg/ml PMB solution, be blank with water, under maximum wavelength, measure its absorbance respectively, and the drawing standard curve, as shown in Figure 3.The PMB content that has neither part nor lot in reaction after fixing obtains by the calibration curve analytical calculation.
3.2PMB PMB concentration, reaction time and pH value determines in the aglucon fixation procedure
3.2.1 heparinize chlorine ball (ps-hep) is the PMB solution reaction 6 hours of 7 variable concentrations 0.5mg/ml, 1mg/ml, 2mg/ml, 4mg/ml, 6mg/ml in PH, PMB concentration is to its influence in chlorine ball carrier fixed amount, as shown in Figure 8.The result shows: PMB solution is between 0.5～6mg/ml the time, and the chlorine ball to the PMB aglucon fixation reaction has taken place all, and along with the increase of PMB concentration, its fixed amount also increases thereupon, and is tending towards saturated gradually.
3.2.2 heparinize chlorine ball (ps-hep) and 4mg/ml PMB solution after reacting different time respectively under the PH=7 condition, the variation tendency of its fixed amount, as shown in Figure 9.The result shows: the reaction time, fixation reaction all took place to the PMB aglucon in the chlorine ball between 1～6h the time, and the PMB fixed amount in time increase and increase, and fixed amount tends towards stability gradually.
3.2.3 heparinize chlorine ball (ps-hep) and 4mg/ml PMB solution behind the coupling buffer reaction 6h of different PH, the influence of the PMB fixed amount of PH, as shown in figure 10.The result shows: the PMB fixed amount is that the increase with PH increases before 7 at PH, and the coupling efficiency in this stage is than higher, and pH value reaches maximum and basicly stable greater than 7 later fixed amounts.This shows that PMB aglucon coupling reaction reagent dosage under neutrallty condition is few, effective.
Two, the mensuration of PMB adsorbents adsorb performance
1, rat plasma preparation
Rat plasma research is all adopted in the outer adsorption test of this experiment sorbent body.The healthy adult rat adopts the apyrogeneity needle tubing by the blood sampling of jugular vein intubate behind yellow Jackets 30mg/kg intraperitoneal injection of anesthesia, anticoagulant heparin, and it is centrifugal that to obtain blood plasma standby.
2FITC-LPS plasma solutions adsorption experiment
In order to observe the situation that LPS adsorbs at adsorbent more intuitively, we adopt and have the LPS of FITC mark as research object, carry out external adsorption test, under same experimental conditions, analyzed the variation that the adsorbent surface fluorescence intensity reflects its adsorbance qualitatively by the Image-Pro software statistics.Concrete grammar is as follows:
2.1 the FITC-LPS plasma solutions of preparation variable concentrations, the lucifuge operation;
2.2 get respectively 0.2g chlorine ball (ps), 0.2g heparinize chlorine ball (ps-hep) and 0.2g PMB adsorbent (ps-hep-pmb) in 2ml variable concentrations FITC-LPS plasma solutions in room temperature lucifuge oscillating reactions certain hour;
2.3 the polymeric adsorbent after the adsorption reaction is taken pictures in observing under identical exposure intensity and time for exposure condition under the inverted fluorescence microscope with PBS washing three times.
2.4 adopt and have the LPS of FITC mark as research object, directly observe the situation that LPS adsorbs at adsorbent by inverted fluorescence microscope, under identical exposure intensity and time for exposure condition, observe and take pictures, and use Image-Pro software to calculate the variation that the adsorbent surface fluorescence intensity reflects its adsorbance qualitatively.
2.5 experimental result:
2.5.1FITC-LPS initial concentration is to the influence of absorption property, result such as Figure 12 and shown in Figure 13: the chlorine ball (ps-hep) of PMB adsorbent (ps-hep-pmb), heparinize and chlorine ball (ps) be fluoroscopic image (seeing Figure 12) and the fluorescence intensity statistical comparison (seeing Figure 13) behind the absorption 2h in the FITC-LPS of variable concentrations rat plasma solution respectively.
The result shows: along with the increase of FITC-LPS initial concentration, the fluorescence intensity on the surface of three kinds of different materials all strengthens thereupon, and it is saturated to reach absorption more than concentration 40ug/ml substantially, and its fluorescence intensity does not have obvious variation; Under same concentrations, the adsorbent of PMB will be higher than other two kinds of adsorbents that do not have grafting PMB to the absorption property of LPS, illustrated that the LPS of PMB has the effect of special antagonism, the PMB aglucon can improve adsorbent to the special selection adsorptivity of LPS, simultaneously heparin molecule forms " arm " at material surface, has strengthened the LPS seizure of PMB effect in the moving of adsorbent surface; Simultaneously we find, under the same concentrations, grafting the adsorbent of heparin molecule arm than the chlorine ball adsorbent through modification absorption property is not increased, namely ps-hep surface fluorescence intensity is higher than ps,
2.5.2FITC-LPS adsorption time is to the influence of absorption property, as Figure 14 and shown in Figure 15: PMB adsorbent (ps-hep-pmb), heparinize chlorine ball (ps-hep) and chlorine ball (ps) are in the fluoroscopic image (seeing Figure 14) of 40 μ g/ml FITC-LPS plasma solutions absorption different time and fluorescence intensity relatively (seeing Figure 15).
The result shows: three kinds of materials are along with the increase of the time adsorbance to LPS all increases thereupon, and under the identical time situation of absorption, the absorption property of ps-hep-pmb is better than ps and ps-hep, significant change does not take place to its fluorescence intensity of the 2h left and right sides in adsorption time again, illustrate that the PMB adsorbent has higher special selection adsorptivity to LPS, and adsorption efficiency is higher, can reach adsorption equilibrium at 2h, has certain clinical use value.
3, the external adsorption experiment of LPS water and plasma solutions
Carry out external Staticadsorption experiment respectively at water and plasma solutions, study adsorbent to the LPS absorption property, and adsorbance and adsorption rate have been carried out quantitative study, concrete steps are as follows:
3.1 prepare LPS water and the plasma solutions of variable concentrations respectively with apirogen water, in whirlpool vortex mixer vibration mixing;
3.2 get 0.2g chlorine ball (ps), heparinize chlorine ball (ps-hep), PMB adsorbent (ps-hep-pmb) 37 ℃ of vibration adsorption reaction certain hours behind 2ml variable concentrations LPS water and plasma solutions mixing respectively;
3.3 adsorb front and back LPS concentration with colour developing matrix tachypleus amebocyte lysate box by the terminal point determination of color, and calculate adsorption rate and the adsorbance of adsorbent according to following formula:
AR=（Cb–Ca/Cb）×100% AC=[（Cb-Ca）×V]/M
AR is adsorption rate (%) in the following formula, and AC is adsorbance (EU/g), and Cb, Ca are respectively the endotoxin concns (EU/ml) before and after the absorption, and V is the endotoxin solution volume, and M is adsorbent mass (g).
3.4 colour developing matrix standard measure detects LPS
Colour developing matrix TAL is used for external detection of bacterial endotoxins, its basic principle is under optimum conditions, bacterial endotoxin activates the C factor in the TAL, cause a series of enzymatic reactions, and then the activation proclotting enzyme forms coagulase, the colour developing matrix that coagulase will manually synthesize is decomposed into polypeptide and yellow paranitroanilinum (pNA, λ
Max=405nm), in the certain hour scope, the growing amount of pNA becomes positive correlation with endotoxin concns, and pNA can dye rose-colored (λ with azo reagent simultaneously
Max=545nm), avoid the interference of test sample intrinsic colour, accordingly, can survey its absorbance at the 545nm place, the drawing standard curve quantitatively detects endotoxin concns, and experimental technique is as follows:
3.4.1 the preparation of endotoxin standard liquid: get one of endotoxin work product, it is diluted to 1EU/ml, the according to the form below preparing standard solution;
3.4.2 TAL, colour developing matrix, azo reagent are prepared according to labelled amount;
3.4.3 the endotoxin testing sample is according to the following table operation that experimentizes before and after the absorption:
3.4.4 draw the endotoxin calibration curve, as shown in figure 11.
3.5 experimental result
3.5.1LPS initial concentration is to the influence of absorption property:
3.5.1.1PMB adsorbent adsorbs the absorption property of 2h with plasma solutions in the LPS of the variable concentrations aqueous solution, see Table 1; 16 figure are seen in the comparison of its adsorbance behind the water of variable concentrations and plasma solutions absorption 2h respectively of PMB adsorbent.
Table 1 is the PMB adsorbent adsorbs 2h with plasma solutions in the LPS of the variable concentrations aqueous solution absorption property
Found that: along with the increase of water and plasma levels of LPS concentration, adsorbance is also along with increase, and this stage LPS molecule is monolayer absorption at adsorbent, and is more good to the special selection absorption property of LPS; Under the same concentrations, LPS adsorbance and adsorption rate all are higher than plasma solutions in the aqueous solution, adsorption efficiency is higher, may be because there are competitive relation in plasma protein and LPS in the blood plasma in the adsorption process of material surface, plasma protein may be covered the binding site of adsorbent LPS on the one hand, cause on the one hand certain sterically hindered, thereby influence LPS in the suction-operated of material surface.
3.5.1.2 variable concentrations LPS water and plasma solutions to the influence of the adsorption rate of PMB adsorbent, are seen 17 figure.The result shows: the PMB adsorbent is higher to the adsorption rate of the LPS in the aqueous solution, substantially all can reach more than 96%; The LPS adsorption rate is then substantially more than 70% in the plasma solutions, and along with the increase of LPS concentration, adsorption rate descends to some extent, and this may have relation with the coherent condition of LPS molecule and the competitive Adsorption of plasma protein.
3.5.2, the LPS adsorption time is to the influence of absorption property:
3.5.2.1PMB adsorbent is the absorption property of absorption different time in the LPS water of 0.5EU/ml and the plasma solutions at initial concentration, sees Table 2; Adsorption time is seen 18 figure to the influence of PMB adsorbents adsorb amount.
Table 2 is that the time is to the adsorbents adsorb performance impact
The result shows: along with the increase of time, adsorbent increases very fast in starting stage LPS adsorbance, slow down gradually then, and remain unchanged substantially and reach balance in its adsorbance of the absorption 2h left and right sides, this phenomenon may cause in solution coherent condition difference owing to the LPS molecule, starting stage mainly is that the LPS unimolecule is adsorbed in material surface, along with adsorbing generation LPS concentration reduces, the LPS molecule that is adsorbed in the surface mainly is to be subjected to congeries to be dissociated into monomolecular speed to control, the formation of congeries simultaneously makes the free phosphoric acid group that exposes reduce, weaken with the PMB aglucon effect of adsorbent surface, thereby influenced its adsorbance.
3.5.2.2 when initial concentration was 0.5EU/ml, the time was seen 19 figure for the influence of the LPS adsorption rate of PMB adsorbent.The result shows, along with its adsorption rate of increase of time increases thereupon, and the adsorption rate in the blood plasma of the 2h left and right sides can reach more than 70%, and this shows that this adsorbent has good adsorption efficient to LPS at short notice, and potential use value is being arranged aspect the removal of LPS.
4, in sum:
4.1 external FITC-LPS adsorption experiment observes directly the PMB adsorbent to the adsorption effect of LPS, the absorption property of discovering the PMB adsorbent is better than heparinize chlorine ball and chlorine ball: under the identical LPS initial concentration, the adsorbance of PMB adsorbent and adsorption rate all will be higher than other two kinds; Under the identical time situation of adsorption reaction, the adsorption efficiency of PMB adsorbent will be higher than other two kinds.
4.2PMB adsorbent is under the aqueous conditions of same concentrations LPS, its adsorbance and adsorption rate all are better than the suction-operated in blood plasma.Can reach more than 96% in the adsorption rate of the aqueous solution to LPS, then can reach more than 70% in the blood plasma.
4.3LPS in the time of in the finite concentration scope, the LPS initial concentration is more big, PMB adsorbents adsorb amount is more many, demonstrates its selection absorption property good to LPS.
4.4PMB adsorbent can reach adsorption equilibrium about 2h, can reach more than 70% the clearance of plasma levels of LPS, shows that this adsorbent has good adsorption efficient to LPS at short notice, has certain clinical use value.
Three, PMB adsorbent blood compatibility experiment
1, experimental procedure:
1.1 rat is behind yellow Jackets (30mg/kg) intraperitoneal injection of anesthesia, femoral artery is got rat blood, anti-freezing.
1.2 make blood perfusion device by oneself with disposable syringe, use liquaemin physiological saline washing pipe before the 0.5gPMB adsorbent of packing into, perfusion three times, control suitable flow velocity, the blood perfusion that exsomatizes experiment.
1.3 absorption carries out 30,60, during 120min, get 20 μ l and 2ml respectively and measure haemocyte and blood parameters variation in blood cell analyzer and conventional automatic clinical chemistry analyzer.
2, experimental result:
The haemocyte of this research perfusion different time and the front and back of plasma protein change shown in table 3 and table 4.Found that, blood constituent is overall before and after the experiment changes not quite, the haemocyte relative change rate is below 10%, the relative change rate of plasma protein is then below 20%, be that plasma protein can have the rate of recovery more than 80%, wherein blood platelet front and back rate of change is below 10%, in endotoxin absorbent research at present, the result is still more considerable, in clinical allowed band.Aspect advantages such as its molecular structure and anticoagulation, high-hydrophilic have been brought into play in the introducing that heparin molecule is described, help to improve the blood compatibility of material surface, thereby improve adsorbent to endotoxic specific adsorption.
The variation of haemocyte composition before and after table 3 absorption
Beneficial effect: the present invention is the blood purification material of the special absorption of a kind of novel endotoxin, have safe, not toxigenicity reaction and allergic reaction, with the blood compatibility height, the price material benefit, the PMB adsorbent has good adsorption efficient and selects absorption property LPS, has certain clinical use value.
Description of drawings
Fig. 1 is chlorine ball fixing heparin molecular arm and PMB aglucon reaction scheme figure;
Fig. 2 is the heparin content calibration curve;
Fig. 3 is PMB solution calibration curve;
Fig. 4 is the infrared spectrogram of chlorine ball;
Fig. 5 is the infrared spectrogram of amination chlorine ball;
Fig. 6 is that heparin concentration is to the influence curve figure of heparin fixed amount;
Fig. 7 is that the reaction time is to the influence curve figure of heparin fixed amount;
Fig. 8 is that PMB concentration is to the influence curve figure of PMB fixed amount;
Fig. 9 is that the reaction time is to the influence curve figure of PMB fixed amount;
Figure 10 is the influence curve figure of the PMB fixed amount of PH;
Figure 11 is endotoxin mark curve;
Figure 12 is the fluoroscopic images of three kinds of materials behind the FITC-LPS of variable concentrations rat plasma solution absorption 2h, and inverted fluorescence microscope * 400(wherein a is the PMB adsorbent; B is heparinize Lee chlorine ball; C is the chlorine ball);
Figure 13 is the column diagram of the fluorescence intensity of three kinds of materials behind the FITC-LPS of variable concentrations rat plasma solution absorption 2h;
Figure 14 is three kinds of materials adsorb different time in FITC-LPS rat plasma solution fluoroscopic image, and inverted fluorescence microscope * 200(wherein d is the PMB adsorbent; E is heparinize chlorine ball; F is the chlorine ball);
Figure 15 is three kinds of materials adsorb the fluorescence intensity of different time in FITC-LPS rat plasma solution column diagram;
Figure 16 is the PMB adsorbent reacts adsorbance behind the 2h in the LPS of variable concentrations solution column diagram;
Figure 17 is the PMB adsorbent reacts adsorption efficiency behind the 2h in the LPS of variable concentrations solution column diagram;
Figure 18 is that the time is to the column diagram of PMB adsorbents adsorb amount influence;
Figure 19 is that the time is to the broken line graph of PMB adsorbents adsorb effectiveness affects.
The specific embodiment
Explain the present invention in more detail below with reference to embodiment, embodiments of the invention only are used for explanation technical scheme of the present invention, and non-limiting essence of the present invention.
Embodiment 1:
The blood purification material of the special absorption of a kind of novel endotoxin is connected to form by chlorine ball, heparin and PMB aglucon; The blood purification preparation methods of the special absorption of this a kind of novel endotoxin, formed by following steps:
(1) amination of chlorine ball carrier:
A, chlorine ball activation pre-treatment: use absolute ethyl alcohol and distilled water alternately washing are removed residual organic or inorganic impurity in the production for several times; Drain standby with the suction filtration device;
B, get the standby chlorine ball of 1g in 40ml1, swelling 1h in the 4-dioxane solution;
C, the mixed solution of above-mentioned chlorine ball and 1,4-dioxane is transferred in the 250ml there-necked flask; 1gNaOH, 0.1g TBAB (TBAB) add reaction system after being dissolved in 10ml distilled water;
D, add the 50ml ethylenediamine in the reaction system, reactant mixture is in 50 ℃ of stirring reaction 1h;
E, product use absolute ethyl alcohol and distilled water alternately to wash to neutral after filtering, obtain amination chlorine ball (ps), and be standby after the vacuum drying;
Course of reaction is as follows:
(2) connection of heparin molecule arm
The citrate buffer solution of a, preparation 0.01M is regulated PH to 3.0 with 0.1M NaOH, and adding molar ratio is the catalyst EDC[1-(3-dimethylamino-propyl of 1:1)-the 3-ethyl carbodiimide] and the NHS(N-HOSu NHS);
B, liquaemin is dissolved in the preparation of described citrate buffer solution obtains the heparin solution that concentration is 1mg/ml, then described amination chlorine ball (ps) is immersed in the heparin solution, 37 ℃ of constant temperature oscillating reactions at least 24 hours;
C, then product is carried out suction filtration, use the PBS cyclic washing, obtain heparinize chlorine ball (ps-hep), drain standby;
(3) the PMB aglucon is fixing
The Na of a, preparation 0.1M
3PO
4-NaCl coupling buffer: regulate PH to 5.0;
B, with described heparinize chlorine ball (ps-hep) with 5% glutaraldehyde in described Na
3PO
4After the activation, add NaCNBH in the-NaCl coupling buffer
3 Room temperature reaction 2 hours;
C, PMB is joined in the described coupling buffer as solvent earlier, preparation obtains the PMB solution that concentration is 0.5mg/ml, then with described heparinize chlorine ball (ps-hep) behind the abundant cyclic washing of coupling buffer, with described PMB solution room temperature reaction 1 hour under neutrallty condition, use NaCNBH simultaneously
3Catalytic reduction is with the product Na that obtains
3PO
4-NaCl coupling buffer and distilled water washing obtain blood purification material PMB adsorbent (ps-hep-pmb) at last.
Embodiment 2:
(1) amination of chlorine ball carrier:
A, chlorine ball activation pre-treatment: use absolute ethyl alcohol and distilled water alternately washing are removed residual organic or inorganic impurity in the production for several times; Drain standby with the suction filtration device;
B, get the standby chlorine ball of 20g in 200ml1, swelling 5h in the 4-dioxane solution;
C, the mixed solution of above-mentioned chlorine ball and 1,4-dioxane is transferred in the 250ml there-necked flask; 20gNaOH, 5g TBAB (TBAB) add reaction system after being dissolved in 100ml distilled water;
D, add the 100ml ethylenediamine in the reaction system, reactant mixture is in 90 ℃ of stirring reaction 10h;
E, product use absolute ethyl alcohol and distilled water alternately to wash to neutral after filtering, obtain amination chlorine ball (ps), and be standby after the vacuum drying;
Course of reaction is as follows:
(2) connection of heparin molecule arm
The citrate buffer solution of a, preparation 0.05M is regulated PH to 6.0 with 0.1M NaOH, and adding molar ratio is the catalyst EDC[1-(3-dimethylamino-propyl of 1:1)-the 3-ethyl carbodiimide] and the NHS(N-HOSu NHS);
B, liquaemin is dissolved in the preparation of described citrate buffer solution obtains the heparin solution that concentration is 6mg/ml, then described amination chlorine ball (ps) is immersed in the heparin solution, 37 ℃ of constant temperature oscillating reactions at least 24 hours;
C, then product is carried out suction filtration, use the PBS cyclic washing, obtain heparinize chlorine ball (ps-hep), drain standby;
(3) the PMB aglucon is fixing
The Na of a, preparation 0.5M
3PO
4-NaCl coupling buffer: regulate PH to 7.0;
B, with described heparinize chlorine ball (ps-hep) with 5% glutaraldehyde in described Na
3PO
4After the activation, add NaCNBH in the-NaCl coupling buffer
3 Room temperature reaction 8 hours;
C, PMB is joined in the described coupling buffer as solvent earlier, preparation obtains the PMB solution that concentration is 6mg/ml, then with described heparinize chlorine ball (ps-hep) behind the abundant cyclic washing of coupling buffer, with described PMB solution room temperature reaction 6 hours under neutrallty condition, use NaCNBH simultaneously
3Catalytic reduction is with the product Na that obtains
3PO
4-NaCl coupling buffer and distilled water washing obtain blood purification material PMB adsorbent (ps-hep-pmb) at last.
Embodiment 3:
The blood purification material of the special absorption of a kind of novel endotoxin is connected to form by chlorine ball, heparin and PMB aglucon; The blood purification preparation methods of the special absorption of this a kind of novel endotoxin, formed by following steps:
(1) amination of chlorine ball carrier:
A, chlorine ball activation pre-treatment: use absolute ethyl alcohol and distilled water alternately washing are removed residual organic or inorganic impurity in the production for several times; Drain standby with the suction filtration device;
B, get the standby chlorine ball of 10g in 100ml1, swelling 3h in the 4-dioxane solution;
C, the mixed solution of above-mentioned chlorine ball and 1,4-dioxane is transferred in the 250ml there-necked flask; 10gNaOH, 2.5g TBAB (TBAB) add reaction system after being dissolved in 60ml distilled water;
D, add the 75ml ethylenediamine in the reaction system, reactant mixture is in 85 ℃ of stirring reaction 6h;
E, product use absolute ethyl alcohol and distilled water alternately to wash to neutral after filtering, obtain amination chlorine ball (ps), and be standby after the vacuum drying;
Course of reaction is as follows:
(2) connection of heparin molecule arm
The citrate buffer solution of a, preparation 0.03M is regulated PH to 4.7 with 0.1M NaOH, and adding molar ratio is the catalyst EDC[1-(3-dimethylamino-propyl of 1:1)-the 3-ethyl carbodiimide] and the NHS(N-HOSu NHS);
B, liquaemin is dissolved in the preparation of described citrate buffer solution obtains the heparin solution that concentration is 3mg/ml, then described amination chlorine ball (ps) is immersed in the heparin solution, 37 ℃ of constant temperature oscillating reactions 24 hours;
C, then product is carried out suction filtration, use the PBS cyclic washing, obtain heparinize chlorine ball (ps-hep), drain standby;
(3) the PMB aglucon is fixing
The Na of a, preparation 0.3M
3PO
4-NaCl coupling buffer: regulate PH to 6.0;
B, with described heparinize chlorine ball (ps-hep) with 5% glutaraldehyde in described Na
3PO
4After the activation, add NaCNBH in the-NaCl coupling buffer
3 Room temperature reaction 6 hours;
C, PMB is joined in the described coupling buffer as solvent earlier, preparation obtains the PMB solution that concentration is 4mg/ml, then with described heparinize chlorine ball (ps-hep) behind the abundant cyclic washing of coupling buffer, with described PMB solution room temperature reaction 4 hours under neutrallty condition, use NaCNBH simultaneously
3Catalytic reduction is with the product Na that obtains
3PO
4-NaCl coupling buffer and distilled water washing obtain blood purification material PMB adsorbent (ps-hep-pmb) at last.
Claims (2)
1. the blood purification material of the special absorption of a novel endotoxin is characterized in that: connected to form by chlorine ball, heparin and PMB aglucon; Described chlorine ball warp ethylenediamine activation is obtained amination chlorine ball, introduce described heparin again as molecular arm, by glutaraldehyde described PMB aglucon is fixed in heparinize chlorine ball surface at last, obtain blood purification material PMB adsorbent.
2. the blood purification preparation methods of the special absorption of the described novel endotoxin of claim 1 is characterized in that being made up of following steps:
(1) amination of chlorine ball carrier:
A, chlorine ball activation pre-treatment: use absolute ethyl alcohol and distilled water alternately washing are removed residual organic or inorganic impurity in the production for several times; Drain standby with the suction filtration device;
B, get the standby chlorine ball of 1～20g in 40～200ml1, swelling 1～5h in the 4-dioxane solution;
C, the mixed solution of above-mentioned chlorine ball and 1,4-dioxane is transferred in the 250ml there-necked flask; 1～20gNaOH, 0.1～5g TBAB add reaction system after being dissolved in 10～100ml distilled water;
D, add 50～100ml ethylenediamine in reaction system, reactant mixture is in 50～90 ℃ of stirring reaction 1～10h;
E, product use absolute ethyl alcohol and distilled water alternately to wash to neutral after filtering, obtain amination chlorine ball, and be standby after the vacuum drying;
Course of reaction is as follows:
(2) connection of heparin molecule arm
The citrate buffer solution of a, preparation 0.01～0.05M is regulated PH to 3.0～6.0 with 0.1M NaOH, and adding molar ratio is catalyst EDC and the NHS of 1:1;
B, liquaemin is dissolved in the preparation of described citrate buffer solution obtains the heparin solution that concentration is 1～6mg/ml, then described amination chlorine ball is immersed in the heparin solution, 37 ℃ of constant temperature oscillating reactions at least 24 hours;
C, then product is carried out suction filtration, use the PBS cyclic washing, obtain heparinize chlorine ball, drain standby;
(3) the PMB aglucon is fixing
The Na of a, preparation 0.1～0.5M
3PO
4-NaCl coupling buffer: regulate PH to 5.0～7.0;
B, described heparinize chlorine ball activated in described coupling buffer with 5% glutaraldehyde after, add NaCNBH
3Room temperature reaction 2～8 hours;
C, PMB is joined in the described coupling buffer as solvent earlier, preparation obtains the PMB solution that concentration is 0.5～6mg/ml, then with behind the abundant cyclic washing of described heparinize chlorine ball warp coupling buffer, with described PMB solution room temperature reaction 1～6 hour under neutrallty condition, use NaCNBH simultaneously
3Catalytic reduction is with the product Na that obtains
3PO
4-NaCl coupling buffer and distilled water washing obtain blood purification material PMB adsorbent at last.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108311121A (en) * | 2018-01-25 | 2018-07-24 | 珠海健帆生物科技股份有限公司 | A kind of blood perfusion absorption resin and preparation method thereof and perfusion device |
| CN109174022A (en) * | 2018-08-22 | 2019-01-11 | 武汉瑞法医疗器械有限公司 | A kind of process for fixation of blood purification adsorbent material |
| CN111744464A (en) * | 2020-07-03 | 2020-10-09 | 北京中科太康科技有限公司 | Adsorbent with bifunctional ligand and preparation method thereof |
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| CN1493368A (en) * | 2003-09-02 | 2004-05-05 | 南开大学 | Endotoxin absorbing agent for blood perfusion and its preparation method |
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| CN1493368A (en) * | 2003-09-02 | 2004-05-05 | 南开大学 | Endotoxin absorbing agent for blood perfusion and its preparation method |
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| 王菲菲: "一种新型内毒素特异吸附的血液净化材料的制备与性能研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108311121A (en) * | 2018-01-25 | 2018-07-24 | 珠海健帆生物科技股份有限公司 | A kind of blood perfusion absorption resin and preparation method thereof and perfusion device |
| CN108311121B (en) * | 2018-01-25 | 2021-05-14 | 健帆生物科技集团股份有限公司 | Adsorption resin for blood perfusion, preparation method thereof and perfusion apparatus |
| CN109174022A (en) * | 2018-08-22 | 2019-01-11 | 武汉瑞法医疗器械有限公司 | A kind of process for fixation of blood purification adsorbent material |
| CN109174022B (en) * | 2018-08-22 | 2021-06-11 | 武汉瑞法医疗器械有限公司 | Method for immobilizing adsorption material for blood purification |
| CN111744464A (en) * | 2020-07-03 | 2020-10-09 | 北京中科太康科技有限公司 | Adsorbent with bifunctional ligand and preparation method thereof |
| CN111744464B (en) * | 2020-07-03 | 2023-03-24 | 北京中科太康科技有限公司 | Adsorbent with bifunctional ligand and preparation method thereof |
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