CN101269240A - Artificial crystalline len with transforming growth factor resistant beta2 antibody membrane on surface and manufacturing method thereof - Google Patents
Artificial crystalline len with transforming growth factor resistant beta2 antibody membrane on surface and manufacturing method thereof Download PDFInfo
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- CN101269240A CN101269240A CNA2008100615119A CN200810061511A CN101269240A CN 101269240 A CN101269240 A CN 101269240A CN A2008100615119 A CNA2008100615119 A CN A2008100615119A CN 200810061511 A CN200810061511 A CN 200810061511A CN 101269240 A CN101269240 A CN 101269240A
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- intraocular lens
- factor beta
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- lentis
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Abstract
The invention provides an artificial lentis, which contains anti-transforming growth factor beta 2 antibody membrane on the surface and can inhibit intercurrent post-cataract after cataract surgery, and also provides a the production method thereof. The production method includes the steps that: the artificial lentis is charged with positive electricity or negative electricity after the artificial lentis is cleaned, dried, and pretreated on the surface; the artificial lentis is soaked in a polyelectrolyte solution the charge of which is opposite to the surface charge of the artificial lentis for adsorbing, and rinsing the artificial lentis by deionized water, and drying the artificial lentis by nitrogen gas; the artificial lentis is soaked in a phosphate buffering solution of anti-transforming growth factor beta 2 antibody, the pH value of which is 4-10, and the carried charge of which is opposite to that of the polyelectrolyte, for adsorption; finally, the artificial lentis is rinsed by phosphate buffering solution, and the artificial lentis is dried by nitrogen gas; the alternating assembly steps are repeated. The artificial lentis of the invention can inhibit the transformation and differentiation as well as cyst membrane shrinkage of the lentis epithelial cells in a target way, and then interdicts the occurrence of the post-cataract, and has excellent biocompatibility. The production method of the invention is scientific and simple, and can ensure the activity under a dry state and the safety and reliability during medical transplantation of the anti-transforming growth factor beta 2.
Description
Technical field
The present invention relates to a kind of intraocular lens that cataract operation is replaced intra-ocular lens that is used for, especially a kind of surface that can suppress the concurrent after cataract of postoperative has the intraocular lens and the manufacture method thereof of anti-transforming grouth factor beta 2 antibody membrane.
Background technology
After cataract is the muddy complication that forms of part secondary that ECCE does not absorb fully, leaves over because of residual cortex of lens afterwards, so claim posterior capsule opacification or aftercataract again; This ophthalmic will directly have influence on patient's vision restoration.Present raising with ECCE, the incidence rate of after cataract descends by early stage 50%, treats after cataract but still have 14.1～18.8% postoperative patient also need accept Nd-YAG laser operation (being the operation that capsule cuts behind the nd yag doubled-frequency laser); The patient also need take the surgery cost of writing for this reason, and this operation also exists detachment of retina, cystoid macular edema, intraocular pressure rising equivalent risk.Therefore, the postoperative visual effect of the incidence rate of reduction after cataract, raising extracapsular cataract extraction is very urgent.
Existing studies show that, the commentaries on classics of the epithelium that lens epithelial cells took place one mesenchymal cell differentiation, extrtacellular matrix deposition and crystal fibre regeneration etc. residual after the ECCE are the main pathomechanisms that causes after cataract.Therefore set about suppressing after cataract from this generation link at present, suppress the medicament categories, administering mode of after cataract etc. comprising research: for example Chinese patent CN101036804A " nanometer fluorouracil coat artificial crystalloid and preparation method thereof " and CN101053680A " artificial intraocular lenses of the tool antiproliferative agents coating of barrier formation is sent out in the control back " all are distributed in the intraocular lens surface with antiproliferative cell toxicity medicament as coating; Chinese patent CN200973766Y " intraocular lens of control after cataract " and CN2531755Y " slow releasing agent portable type artificial intraocular lenses " are fixed drug slow-released carriers on the outside, intraocular lens ambitus or loop.Though the propagation that they can suppress residual lens epithelial cells by the medicine original position of high local concentrations plays effect, and the effect that in vitro tests is arranged, but find in the test that in vivo they also have following shortcoming: 1) be applied to the antiproliferative cell toxicity medicament or the slow releasing carrier of medication on intraocular lens surface, other tissues of ophthalmic such as their corneal endotheliocytes, iris, ciliary epithelium cell, retina have potential toxic and side effects; In addition, other administering mode also comprises medicine lavation in the capsule bag, ophthalmic implantable bioartificial degradability polymer drug slow-released carrier etc., the former shortcoming is the time weak point that valid density is kept and also easily other tissue of ophthalmic is exerted an influence, and latter Ze Yi influences eye inner tissue or disturb other operation, in case and toxic and side effects occurs then need to perform the operation immediately taking out; 2) medication coat stable poor on the intraocular lens surface, thereby use not too reliable; 3) their manufacturing process more complicated.
Summary of the invention
The present invention will overcome above-mentioned shortcoming of the prior art, a kind of visual effect that can effectively suppress the concurrent after cataract of postcataract, improve postcataract is provided, and to human body, particularly the surface that has no side effect of its hetero-organizations of ophthalmic such as endothelial cell, iris, ciliary epithelium cell, retina has the intraocular lens and the manufacture method thereof of anti-transforming grouth factor beta 2 antibody membrane; And also make it have the manufacture method advantages of simple, can suitability for industrialized production, and characteristics such as cost is low, product price is cheap.
Surface of the present invention has the intraocular lens of anti-transforming grouth factor beta 2 antibody membrane, it is characterized in that: have anti-transforming grouth factor beta 2 antibody membrane on the surface of intraocular lens's optic and loop.So that this intraocular lens implants ophthalmic, enters after the capsule bag, its lip-deep anti-transforming grouth factor beta 2 antibody membrane promptly produces the effect that suppresses after cataract, and neither other organizes the toxigenicity effect to ophthalmic, can improve intraocular lens's biocompatibility again, thereby reduced complications after cataract extraction, and helped the recovery of postoperative vision at a specified future date.
Above-mentioned intraocular lens's material is a polymethyl methacrylate, or the silicon gel, or soft hydrophobic polyacrylate.
Surface of the present invention has the intraocular lens's of anti-transforming grouth factor beta 2 antibody membrane manufacture method, it is characterized in that it comprises following step:
1) intraocular lens is cleaned and drying, make this intraocular lens surface lotus with positive electricity or negative electricity by surface preparation again;
2) intraocular lens that above-mentioned surface is charged is immersed in the aqueous solution of polyelectrolyte that the concentration opposite with its surface charge is 0.01-1000mg/mL, adsorbs 1-120 minute, dries up with rinsed with deionized water and nitrogen then; Again this intraocular lens is immersed with polyelectrolyte before this electrically charged opposite concentration be in the phosphate buffered solution of anti-transforming grouth factor beta 2 antibody of 0.01-1000 μ g/mL, this phosphate buffered solution is that 0.01-10mol/L, pH value are 4-10, adsorbed 1-120 minute, use the phosphate buffered solution rinsing then, nitrogen dries up;
3) repeat above-mentioned alternately number of assembling steps and at least once promptly make the intraocular lens that the surface has anti-transforming grouth factor beta 2 antibody membrane; After this at room temperature vacuum drying, pack and get final product.
Surface of the present invention has in intraocular lens's the manufacture method of anti-transforming grouth factor beta 2 antibody membrane, and can make intraocular lens surface lotus is in following three kinds of methods any one with the process for surface preparation of positive electricity or negative electricity:
I) utilize plasma surface treatment, change the gaseous species that feeds and introduce functional group on the intraocular lens surface, amino or carboxyl etc. change pH value then and make surface ionization and be with electric charge.
Ii) make the surface positively charged with the positively charged polyvinyl imines of direct absorption.
Iii) the intraocular lens with polyacrylate carries out surface hydrolysis with aqueous slkali, and the surface produces a large amount of carboxyls and introduces negative charge.
Surface of the present invention has in intraocular lens's the manufacture method of anti-transforming grouth factor beta 2 antibody membrane, used polyelectrolyte is the polyelectrolyte with anti-transforming grouth factor beta 2 antibody oppositely charged, the polyelectrolyte of natural polyelectrolyte or no cytotoxicity preferably, when antibody is electronegative, select positively charged polyelectrolyte for use: PAH hydrochlorate, chitosan, poly-D-lysine, gelatin; When antibody is positively charged, then select electronegative polyelectrolyte for use: kayexalate, sodium alginate, heparin, hyaluronate sodium.
Surface of the present invention has in intraocular lens's the manufacture method of anti-transforming grouth factor beta 2 antibody membrane, used anti-transforming grouth factor beta 2 antibody is that a kind of isoelectric point, IP is 6.8 immunoglobulin, can make it be with negative electricity so be higher than the pH value of isoelectric point, IP, and be lower than the pH value positively charged of isoelectric point, IP.If assembling condition can be similar with pH value and ionic strength under the physiological conditions, as select the phosphate buffered solution (hereinafter to be referred as PBS solution) of pH=7.4, then can keep assembling the consistent of environment and human body environment, thereby can keep its stability and active best.
Surface of the present invention has the intraocular lens's of anti-transforming grouth factor beta 2 antibody membrane manufacture method, also can be in surface preparation so that intraocular lens surface lotus with positive electricity or negative electricity after and the immersion concentration opposite with its surface charge be before the aqueous solution of polyelectrolyte of 0.01-1000mg/mL, also increase and to improve this intraocular lens's surface charge density together with the static that the strengthens follow-up antibody step of self assembly absorbability layer by layer, this step is that surperficial lotus is alternately immersed in the strong polyelectrolyte aqueous solution that two kinds of opposite concentration of charge property are 0.01-1000mg/mL with the intraocular lens of positive electricity or negative electricity, adsorption time is 1-120 minute, after finishing, absorption all need use rinsed with deionized water, nitrogen dries up, and the number of times that alternately repeats this step at least once; Wherein positively charged strong polyelectrolyte is selected PAH hydrochlorate (hereinafter to be referred as PAH) for use, or PDDA (hereinafter to be referred as PDDA), and electronegative strong polyelectrolyte is selected kayexalate (hereinafter to be referred as PSS) for use.Thereby form the assembling bottom of the high strong polyelectrolyte of charge density on intraocular lens's surface.
Adsorption time length in the said method is to select according to the concentration of used solution, the concentration of the solution desirable low value of adsorption time of greatly then taking for example, otherwise then the time of absorption long.In addition, in the said method to the anti-transforming grouth factor beta 2 antibody of intraocular lens's surface-assembled with the precedence of the polyelectrolyte of opposite charge can be according to the process for surface preparation that is adopted with it, the different conditions such as solution that had select that (for example the anti-transforming grouth factor beta 2 antibody in the PBS solution of pH=7.4 is electronegative, if it is electronegative after intraocular lens's surface preparation, the polyelectrolyte that then adsorbs positively charged thereafter earlier, adsorb electronegative anti-transforming grouth factor beta 2 antibody again, if positively charged after intraocular lens's surface preparation, the also above-mentioned order of interchangeable), and just do not influence, attraction between the negative electricity, thus guarantee in the effect of the anti-transforming grouth factor beta 2 antibody membrane of intraocular lens's surface layer assembly identical.Given example is all adsorbed polyelectrolyte earlier among the embodiment, adsorbs anti-transforming grouth factor beta 2 antibody again, omits the example of transposing order.
Beneficial effect of the present invention: 1) intraocular lens of the present invention is after implanting ophthalmic, entering the capsule bag, the anti-transforming grouth factor beta 2 antibody on its surface is directly with the interaction of transforming grouth factor beta 2 generation specificity and do not influence its hetero-organization of ophthalmic, and targeting suppresses the commentaries on classics differentiation and the cyst membrane shrinkage of lens epithelial cells, effectively blocked the key link that after cataract takes place, thereby suppressed after cataract and help the recovery of postoperative vision at a specified future date; 2) because of the used assembling composition of assembling process, comprise that used anti-transforming grouth factor beta 2 antibody is the polyelectrolyte of hypotoxicity and immunogenic natural polyelectrolyte or no cytotoxicity, so intraocular lens of the present invention has no side effect to its hetero-organization of ophthalmic, and also have excellent biological compatibility, can work in coordination with and suppress after cataract; 3) the present invention is by the method for the feasible static self assembly layer by layer of science, can make biomolecule replace dip-coating with more weak electrostatic interaction and be fixed on the intraocular lens surface, realized the layer structure of nanometer, submicron-scale, its result neither influences intraocular lens's ontological property, realized that again anti-transforming grouth factor beta 2 antibody firmly deposits on the intraocular lens surface, and can keep active and the security reliability in medical transplanting of anti-transforming grouth factor beta 2 antibody under dry state; 4) manufacture method of the present invention is because of the equal avirulence of solvent for use, so it is environmentally friendly, but also because of the manufacture method advantages of simple, and also can be by regulating assembling condition, the pH value, ionic strength, concentration etc. that for example change polyelectrolyte simply and are effectively controlled the fine structure and the character thereof of thin film; In addition, the range of choice of assembling molecule is wider, can be the polyelectrolyte of synthesis type, also can be charged bioactive macromolecules such as protein, polysaccharide, DNA etc.; 5) by evidence, manufacture method of the present invention is scientific and reasonable, practical, and its manufacturing process cost comparatively simple, that spent is lower, and therefore the intraocular lens that can make surface of the present invention have anti-transforming grouth factor beta 2 antibody membrane becomes a kind of product of cheap suitability for industrialized production.
Description of drawings
The intraocular lens surface that Fig. 1 is illustrated in soft hydrophobic polypropylene acid esters makes up (poly-D-lysine/anti-transforming grouth factor beta 2 antibody)
2The static of multilayer film is self assembling process layer by layer, and the surface of the present invention of promptly having assembled two bilayers (poly-D-lysine/anti-transforming grouth factor beta 2 antibody) has the intraocular lens's of anti-transforming grouth factor beta 2 antibody membrane sketch map; Only on intraocular lens's a surface, express the structure that it is assembled out among the figure, also have same package assembly on remaining surface and the loop; But for simplify and clear for the purpose of, on remaining surface and loop, drawn.
Fig. 2 is illustrated in soft hydrophobic polypropylene acid esters intraocular lens surface to make up (PAH/PSS)
2The static of/(poly-D-lysine/anti-transforming grouth factor beta 2 antibody) multilayer film is layer by layer self assembling process, and the surface of the present invention of promptly having assembled two bilayers (PAH/PSS), a bilayer (poly-D-lysine/anti-transforming grouth factor beta 2 antibody) has the intraocular lens's of anti-transforming grouth factor beta 2 antibody membrane sketch map; Only on intraocular lens's a surface, express the structure that it is assembled out among the figure, also have same package assembly on remaining surface and the loop; But for simplify and clear for the purpose of, on remaining surface and loop, drawn.
Fig. 3 follows the tracks of intraocular lens surface (PAH/PSS) by the frequency change of QCM
3/ (poly-D-lysine/anti-transforming grouth factor beta 2 antibody)
3The static of multilayer film is self assembling process layer by layer, has promptly assembled three bilayers (PAH/PSS), three bilayers (poly-D-lysine/anti-transforming grouth factor beta 2 antibody).
Fig. 4 shows with the photo of inverted phase contrast microscope to cultivate the adhesion photo of macrophage on the intraocular lens surface that exsomatize after 24 hours, wherein (a) is existing intraocular lens, (b) is the intraocular lens that surface of the present invention has anti-transforming grouth factor beta 2 antibody membrane.
Fig. 5 is that the photo with inverted phase contrast microscope is presented under the inducing of transforming grouth factor beta 2, cultivate the adhesion of lens epithelial cells of exsomatizing after 24 hours on the intraocular lens surface, wherein (c) is existing intraocular lens, (d) is the intraocular lens that surface of the present invention has anti-transforming grouth factor beta 2 antibody membrane.
The specific embodiment
Embodiment 1:
The present embodiment surface has the intraocular lens of anti-transforming grouth factor beta 2 antibody membrane, be a kind of the intraocular lens optic and the surface of loop on have the intraocular lens of anti-transforming grouth factor beta 2 antibody membrane.
Anti-transforming grouth factor beta 2 antibody in the present embodiment is a kind of immunoglobulin, it can suppress after cataract by the biological approach of immunocyte, because causing the key factor of after cataract is transforming grouth factor beta 2, so adopt anti-transforming grouth factor beta 2 antibody then can suppress the effect of transforming grouth factor beta 2 with the specificity of its height, suppress extrtacellular matrix deposition and fibrosis, thereby play the targeting that other drug is short of to turn to main after cataract with fiber, and this humanized's antibody do not have immunogenicity and toxicity.Therefore, the intraocular lens that the present embodiment surface has anti-transforming grouth factor beta 2 antibody membrane implants ophthalmic, enters after the capsule bag, its lip-deep anti-transforming grouth factor beta 2 antibody membrane promptly produces the effect that suppresses after cataract, and neither other organizes the toxigenicity effect to ophthalmic for it, can improve intraocular lens's biocompatibility again, thereby reduced complications after cataract extraction, and helped the recovery of postoperative vision at a specified future date.
Confirm by numerous tests, the present embodiment intraocular lens should not make by physisorphtion or chemical bonding, the former is that anti-transforming grouth factor beta 2 antibody membrane can not be stabilized in the intraocular lens surface for a long time, and the latter can make the biological activity of anti-transforming grouth factor beta 2 antibody decline to a great extent even lose; But the method for a kind of static self assembly layer by layer is feasible, this method is to utilize the characteristic that protein energy under specific pH condition is electrically charged, have polyelectrolyte, and, can make anti-transforming grouth factor beta 2 antibody deposit to intraocular lens's surface with the form of one deck, thin film by alternately using positively charged, electronegative polyelectrolyte; Therefore this intraocular lens's surface modification, can in use have safe targeting concurrently, and suppresses the effect of after cataract stably and lastingly.
The manufacture method that the present embodiment surface has the intraocular lens of anti-transforming grouth factor beta 2 antibody membrane comprises the steps:
Take the intraocular lens, a kind of in the desirable polymethyl methacrylate of its material, silicon gel, the soft hydrophobic polypropylene acid esters etc., the intraocular lens that it is material that present embodiment is taken soft hydrophobic polypropylene acid esters; After earlier using dehydrated alcohol, washed with de-ionized water respectively, reuse ultrasonic cleaning instrument cleaned 1-3 minute, then with after the rinsed with deionized water 60 ℃ of vacuum dryings 24 hours; Then put it in the low-temperature plasma generator, generator power is 60W, radio frequency is 13.5kHz, gas is carbon dioxide in it, gas pressure maintains 50-60Pa, handled 2～5 minutes with plasma glow discharge, take out this intraocular lens then, its surface has had carboxyl functional group; Again it was immersed in the sodium hydroxide solution of 0.01mol/L 15 minutes, and made carboxyl be converted into carboxylic ions, rinsed with deionized water, nitrogen dries up, and obtains electronegative intraocular lens surface; Then should the electronegative intraocular lens in surface immerse solution concentration and be in the poly-D-lysine aqueous solution of positively charged of 5mg/mL after 60 minutes, with rinsed with deionized water, nitrogen dries up; After this immerse solution concentration again and be the electronegative anti-transforming grouth factor beta 2 antibody of 500 μ g/mL PBS solution (PBS solution concentration 0.05mol/L, pH=7.4) in, adsorbed 30 minutes, the rinsing of reuse PBS solution, nitrogen dries up; Repeat above alternate steps and can obtain the intraocular lens that the surface has the anti-transforming grouth factor beta 2 antibody of 1-20 layer; At last it is taken out, and use washed with de-ionized water, nitrogen dries up, and vacuum drying packs under the room temperature.
The intraocular lens's of present embodiment structure and method for making can be seen from Fig. 1; This figure is the present embodiment intraocular lens surface electrostatic sketch map of self assembling process layer by layer, and it is that intraocular lens with soft hydrophobic polypropylene acid esters is an example, makes up on the surface (poly-D-lysine/anti-transforming grouth factor beta 2 antibody)
2Multilayer film, that is assembled two bilayers (poly-D-lysine/anti-transforming grouth factor beta 2 antibody); 4 steps representing manufacture process among the figure with 1-4, a represents soft hydrophobic polypropylene acid esters intraocular lens, b, c represents used reagent in each step, + expression positive charge,-expression negative charge, wherein, make the intraocular lens surface produce carboxyl after the 1 expression carbon dioxide Cement Composite Treated by Plasma and electronegative, the positively charged poly-D-lysine of 2 expressions is assembled into the intraocular lens surface, 3 are illustrated in anti-transforming grouth factor beta 2 antibody electronegative under the pH=7.4 condition is assembled into the intraocular lens surface, 4 are illustrated in the intraocular lens surface alternately assembles poly-D-lysine/anti-transforming grouth factor beta 2 antibody once more, b represents positively charged poly-D-lysine, and c is illustrated in anti-transforming grouth factor beta 2 antibody electronegative under the pH=7.4 condition.
Embodiment 2:
The used intraocular lens of present embodiment is a kind of in polymethyl methacrylate or the soft hydrophobic polypropylene acid esters material, with different among the embodiment 1 be the method that intraocular lens's surface preparation obtains negative charge, thereafter the used reagent of assembling process is also identical with embodiment 1 with method, is the poly-D-lysine and the electronegative anti-transforming grouth factor beta 2 antibody of positively charged.Be with the difference of the process for surface preparation of embodiment 1: the intraocular lens press that embodiment 1 method is cleaned and oven dry after, it was immersed in 30% sodium hydrate aqueous solution 80 ℃ of hydrolysis after 30 minutes, its surface can produce a large amount of carboxylic ions, and this surface is elecrtonegativity under aqueous conditions.
The something in common of present embodiment and embodiment 1 is that the intraocular lens surface all has negative charge after surface preparation, table 1 is after this to select positively charged other polyelectrolyte (PAH that is different from poly-D-lysine for use on the intraocular lens surface, or chitosan, or gelatin) and the electronegative anti-transforming grouth factor beta 2 antibody example that carries out static self assembly layer by layer, each example all can produce the intraocular lens that surface of the present invention has anti-transforming grouth factor beta 2 antibody membrane.The polyelectrolyte of used positively charged is other polyelectrolyte that is different from poly-D-lysine used among embodiment 1 and the embodiment 2 in the table 1; The used PBS pH value of solution of the anti-transforming grouth factor beta 2 antibody of dissolving all is higher than 6.8 in the table 1, and anti-with this understanding transforming grouth factor beta 2 antibody is electronegative.
Table 1
Embodiment 3:
Take the intraocular lens, a kind of in the desirable polymethyl methacrylate of its material, silicon gel, the soft hydrophobic polypropylene acid esters etc., as the silicon gel is the intraocular lens of material, after pressing cleaning of embodiment 1 method and oven dry, put it in the low-temperature plasma generator, generator power is 60W, radio frequency is 13.5kHz, gas is ammonia in it, gas pressure maintains 50-60Pa, handled 2～5 minutes with plasma glow discharge, take out this intraocular lens then, its surface has amido functional group; It was immersed in the hydrochloric acid solution that concentration is 1mol/L 30 minutes, and making the amide acidify is ammonium ion, rinsed with deionized water, and nitrogen dries up, and obtains positively charged intraocular lens surface; Then the intraocular lens of above-mentioned surperficial positively charged was immersed in the electronegative heparin solution of 0.01mg/mL 120 minutes, rinsed with deionized water, nitrogen dries up; After this immerse 0.01 μ g/mL positively charged anti-transforming grouth factor beta 2 antibody PBS solution (0.05mol/L, pH=4) in, adsorbed 120 minutes, with the rinsing of PBS solution, nitrogen dries up; Repeat above step and can obtain the intraocular lens that the surface has the anti-transforming grouth factor beta 2 antibody of 1-20 layer.Take out the intraocular lens at last and use washed with de-ionized water, nitrogen dries up, and vacuum drying packs under the room temperature.
Embodiment 4:
Identical among the used intraocular lens of present embodiment and the embodiment 3, with different among the embodiment 3 be the method that intraocular lens's surface preparation obtains positive charge, and identical among the used reagent of assembling process thereafter and method and the embodiment 3, be the anti-transforming grouth factor beta 2 antibody of electronegative heparin and positively charged.Process for surface preparation in the present embodiment is: the intraocular lens was immersed in the polyethyleneimine: amine aqueous solution of positively charged of 3mg/mL 15 minutes, rinsed with deionized water, nitrogen dry up obtain the polymine physical absorption the surface, this surface is electropositive under aqueous conditions.
The something in common of present embodiment and embodiment 3 is that the intraocular lens surface all has positive charge after surface preparation, table 2 is examples of after this selecting for use other the electronegative polyelectrolyte (PSS, sodium alginate, hyaluronate sodium) that is different from heparin and positively charged anti-transforming grouth factor beta 2 antibody to carry out static self assembly layer by layer on the intraocular lens surface, and each example all can produce the intraocular lens that surface of the present invention has anti-transforming grouth factor beta 2 antibody membrane.Used electronegative polyelectrolyte is other polyelectrolyte that is different from heparin used among embodiment 3 and the embodiment 4 in the table 2; The used PBS pH value of solution of the anti-transforming grouth factor beta 2 antibody of dissolving all is lower than 6.8, anti-with this understanding transforming grouth factor beta 2 antibody positively charged in the table 2.
Table 2
Embodiment 5:
The difference of present embodiment and embodiment 1 is after the carbon dioxide Cement Composite Treated by Plasma makes that the intraocular lens surface is electronegative, immerse concentration and be before the PBS solution of the aqueous solution of positively charged poly-D-lysine of 5mg/mL and the electronegative anti-transforming grouth factor beta 2 antibody of 500 μ g/mL, also increase step one, the intraocular lens that the surface is electronegative was dipped in the strong electrolyte PAH solution of positively charged of 5mg/mL absorption 15 minutes, use rinsed with deionized water, nitrogen dries up, and obtains positively charged surface; Then it is dipped in the strong electrolyte PSS aqueous solution of 5mg/mL 15 minutes, rinsed with deionized water, nitrogen dries up, and obtains electronegative surface; Repeat above step and can obtain the bottom that 2-6 layer PAH/PSS alternately exists on the intraocular lens surface.In intraocular lens's surface-assembled behind the bottom of the high strong electrolyte of charge density, help the assembling of follow-up anti-transforming grouth factor beta 2 antibody.
The intraocular lens's of present embodiment structure and method for making can be seen from Fig. 2; This figure is the present embodiment intraocular lens surface electrostatic sketch map of self assembling process layer by layer, and it is that intraocular lens with the hydrophobic polypropylene acid esters is an example, makes up on the surface (PAH/PSS)
2/ (poly-D-lysine/anti-transforming grouth factor beta 2 antibody) multilayer film, that is the surface of the present invention of having assembled two bilayers (PAH/PSS), a bilayer (poly-D-lysine/anti-transforming grouth factor beta 2 antibody) has the intraocular lens's of anti-transforming grouth factor beta 2 antibody membrane sketch map; Among Fig. 2 with 1 '-6 ' expression manufacture process six steps, a represents soft hydrophobic polypropylene acid esters intraocular lens, b-e represents used reagent in each step, + expression positive charge,-expression negative charge, wherein, make the intraocular lens surface produce carboxyl after 1 ' the expression carbon dioxide Cement Composite Treated by Plasma and electronegative, the PAH of 2 ' expression positively charged is assembled into the intraocular lens surface, the electronegative PSS of 3 ' expression is assembled into the intraocular lens surface, 4 ' is illustrated in the intraocular lens surface alternately assembles PAH/PSS once more and forms the assembling that the high bottom of electrically charged density is beneficial to next step antibody, the positively charged poly-D-lysine of 5 ' expression is assembled into the intraocular lens surface, and electronegative anti-transforming grouth factor beta 2 antibody is assembled into the intraocular lens surface under 6 ' the expression pH=7.4 condition.B represents the poly-D-lysine of positively charged, and c represents electronegative anti-transforming grouth factor beta 2 antibody under the pH=7.4 condition, and d represents the PAH of positively charged, and e represents electronegative PSS.
The strong electrolyte PAH of the positively charged in the present embodiment can use variable concentrations instead or use the strong electrolyte of other positively charged instead, for example with the strong electrolyte PDDA of the positively charged shown in the table 3, the electronegative intraocular lens surface that the surface is pretreated is assembled, with the example (only listing the related parameter that has of this bottom of assembling in the table 3) that forms the high bottom of charge density; Two examples in the table 3 all can produce the intraocular lens that surface of the present invention has anti-transforming grouth factor beta 2 antibody membrane.
The strong electrolyte PAH of the positively charged in the present embodiment also can use the strong electrolyte of other positively charged beyond the table 3 instead to form the high bottom of charge density to assembling through pretreated electronegative intraocular lens surface.
Table 3
The intraocular lens that the present invention also has an anti-transforming grouth factor beta 2 antibody membrane to the surface that makes in the various embodiments described above has made following mensuration:
1) follows the tracks of intraocular lens surface (PAH/PSS) with the frequency change of QCM
3/ (poly-D-lysine/anti-transforming grouth factor beta 2 antibody)
3The static self assembling process, as shown in Figure 3: it is to be example with embodiment 5, assemble three bilayers (PAH/PSS), three bilayers (poly-D-lysine/anti-transforming grouth factor beta 2 antibody), the abscissa among the figure represents to assemble the number of plies, vertical coordinate is represented frequency change, wherein in the odd- level 1,3,5 layer expression PAH, 7,9,11 layers of expression poly-D-lysine, 2,4,6 layers of expression PSS in the even level, 8,10,12 layers of anti-transforming grouth factor beta 2 antibody of expression.Its result shows that the frequency change of multilayer film assembling increases along with the increase of assembling the number of plies is linear, and frequency change and weight change are proportional, thereby has proved (PAH/PSS)
3/ (poly-D-lysine/anti-transforming grouth factor beta 2 antibody)
3The successful structure of multilayer film, just the step with embodiment 5 can make intraocular lens of the present invention.
2) observe the present embodiment surface with the macrophages in vitro adherence test and have the influence of the intraocular lens of anti-transforming grouth factor beta 2 antibody membrane macrophage adhesion behavior.The intraocular lens who makes with embodiment 1 and the intraocular lens of unmodified all after the method sterilization with epoxyethane fumigation, lie in them in 24 well culture plates, with macrophage with 1.0 * 10
6The density of/mL is inoculated in each artificial lens surface, and every hole 30 μ L carry out cell culture.Be divided into two groups, every group of three intraocular lenss, one group is the intraocular lens of unmodified, the intraocular lens that another group is modified for multilayer film.Cultivate and compare with the macrophage of inverted phase contrast microscope after 24 hours two groups of intraocular lens's surface adhesion.Its result as shown in Figure 4, wherein Fig. 4 (a) is the intraocular lens of unmodified, Fig. 4 (b) is the intraocular lens of present embodiment, the former obviously is less than the latter at the macrophage quantity of surface adhesion, that is the intraocular lens of present embodiment significantly reduced the adhesion of inflammatory cell, improved biocompatibility.
3) cultivate the observation surface with external lens epithelial cells and have the influence of the intraocular lens of anti-transforming grouth factor beta 2 antibody membrane the inductive lens epithelial cells behavior of transforming grouth factor beta 2.Get the intraocular lens that embodiment 2 makes and the intraocular lens of unmodified, and all after the method sterilization with epoxyethane fumigation, it lain in 24 well culture plates, with lens epithelial cells with 6.0 * 10
4The density of/mL is inoculated in each artificial lens surface, and every hole 30 μ L carry out cell culture.Be divided into two groups, every group of three intraocular lenss, one group is the intraocular lens of unmodified, another group is the intraocular lens in the present embodiment, two groups all added the 10ng/mL transforming grouth factor beta 2, observed lens epithelial cells with inverted phase contrast microscope after 24 hours in culture plate.Intercellular substance takes place under the inducing of transforming grouth factor beta 2 the lens epithelial cells on unmodified intraocular lens surface increases, and divides a word with a hyphen at the end of a line obviously, becomes tangible spindle shape, presents the trend that transforms to fibroblast, shown in Fig. 5 (c); The intraocular lens of present embodiment is normal, and the lens epithelial cells of cultivating is polygon, and cell is bright, and kytoplasm is abundant, keeps polygonal normal morphology mostly, does not show Fibrotic trend, and it is not obvious to divide a word with a hyphen at the end of a line, shown in Fig. 5 (d); From above-mentioned epithelial cell explanation, the present embodiment intraocular lens can obviously suppress the caused epithelium of transforming grouth factor beta 2-mesenchymal cell changes harmful acts such as differentiation and cell migration, keep the normal morphology and the behavior of lens epithelial cells, thereby suppress the cyst membrane shrinkage after the intraocular lens implants, complication such as after cataract improve postoperative vision at a specified future date.
Employed anti-transforming grouth factor beta 2 antibody is buied from the cambridge antibody technology company of Britain in the foregoing description.Other used in the foregoing description reagent all can be buied from domestic and international chemical reagents corporation.Used cell is a Turnover of Mouse Peritoneal Macrophages in the stripped macrophage adherence test, and from the 6-8 BALB/c mouse abdominal cavity extraction in age in week, used cell behaviour lens epithelial cells was HLEB-3 during the lens epithelial cells that exsomatizes was cultivated, and buied from Japan.
Claims (8)
1, a kind of surface has the intraocular lens of anti-transforming grouth factor beta 2 antibody membrane, it is characterized in that: have anti-transforming grouth factor beta 2 antibody membrane on the surface of intraocular lens's optic and loop.
2, surface according to claim 1 has the intraocular lens of anti-transforming grouth factor beta 2 antibody membrane, it is characterized in that: said intraocular lens's material is polymethyl methacrylate or silicon gel or soft hydrophobic polypropylene acid esters.
3, a kind of surface according to claim 1 has the intraocular lens's of anti-transforming grouth factor beta 2 antibody membrane manufacture method, and it comprises the steps:
1) intraocular lens is cleaned and drying, make this intraocular lens surface lotus with positive electricity or negative electricity by process for surface preparation again;
2) intraocular lens that above-mentioned surface is charged is immersed in the aqueous solution of polyelectrolyte that the concentration opposite with its surface charge is 0.01-1000mg/mL, adsorbs 1-120 minute, dries up with rinsed with deionized water and nitrogen then; Again this intraocular lens is immersed with polyelectrolyte before this electrically charged opposite concentration be in the phosphate buffered solution of anti-transforming grouth factor beta 2 antibody of 0.01-1000 μ g/mL, this phosphate buffered solution is that 0.01-10mol/L, pH value are 4-10, adsorbed 1-120 minute, use the phosphate buffered solution rinsing then, nitrogen dries up;
3) repeat above-mentioned alternately number of assembling steps and at least once promptly make the intraocular lens that the surface has anti-transforming grouth factor beta 2 antibody membrane; After this at room temperature vacuum drying, pack and get final product.
4, surface according to claim 3 has the intraocular lens's of anti-transforming grouth factor beta 2 antibody membrane manufacture method, it is characterized in that: the said intraocular lens of making surface lotus is to utilize plasma surface treatment with the process for surface preparation of positive electricity or negative electricity, change the gaseous species that feeds and introduce functional group, change pH value then and make surface ionization and be with electric charge on the intraocular lens surface.
5, surface according to claim 3 has the intraocular lens's of anti-transforming grouth factor beta 2 antibody membrane manufacture method, it is characterized in that: the said intraocular lens of making surface lotus is that directly the positively charged polyvinyl imines of absorption makes the surface positively charged with the process for surface preparation of positive electricity.
6, surface according to claim 3 has the intraocular lens's of anti-transforming grouth factor beta 2 antibody membrane manufacture method, it is characterized in that: the said intraocular lens of making surface lotus is that the polyacrylate intraocular lens is carried out surface hydrolysis with aqueous slkali with the process for surface preparation of negative electricity, and the surface produces a large amount of carboxyls and introduces negative charge.
7, surface according to claim 3 has the intraocular lens's of anti-transforming grouth factor beta 2 antibody membrane manufacture method, it is characterized in that: said polyelectrolyte is the polyelectrolyte of natural polyelectrolyte or no cytotoxicity, it comprises positively charged polyelectrolyte PAH hydrochlorate, chitosan, poly-D-lysine, gelatin, and electronegative polyelectrolyte kayexalate, sodium alginate, heparin, hyaluronate sodium.
8, the manufacture method that has the intraocular lens of anti-transforming grouth factor beta 2 antibody membrane according to claim 3 or 4 or 5 or 6 or 7 described surfaces, it is characterized in that: it is after intraocular lens surface lotus is with positive electricity or negative electricity and before the immersion concentration opposite with its surface charge is the aqueous solution of 0.01-1000mg/mL polyelectrolyte, also increase and to improve this intraocular lens's surface charge density together with the static that the strengthens follow-up antibody step of self assembly absorbability layer by layer, this step is earlier surperficial lotus alternately to be immersed in the strong polyelectrolyte aqueous solution that two kinds of opposite concentration of charge property are 0.01-1000mg/mL with the intraocular lens of positive electricity or negative electricity, adsorption time is 1-120 minute, after finishing, absorption all need use rinsed with deionized water, nitrogen dries up, and the number of times that alternately repeats this step at least once; Wherein positively charged strong polyelectrolyte is selected the PAH hydrochlorate for use, or PDDA; Electronegative strong polyelectrolyte is selected kayexalate for use.
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| CN112402098A (en) * | 2020-11-19 | 2021-02-26 | 浙江大学 | Drug-eluting intraocular lens with slow release function and preparation method thereof |
| CN112402098B (en) * | 2020-11-19 | 2022-03-08 | 浙江大学 | Drug-eluting intraocular lens with slow release function and preparation method thereof |
| CN113018508A (en) * | 2021-03-15 | 2021-06-25 | 西安交通大学医学院第一附属医院 | Surface-modified artificial lens and preparation method thereof |
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