CN104825249A - Surface-mediated gene therapy type artificial lens and preparation method for same - Google Patents
Surface-mediated gene therapy type artificial lens and preparation method for same Download PDFInfo
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- CN104825249A CN104825249A CN201510205350.6A CN201510205350A CN104825249A CN 104825249 A CN104825249 A CN 104825249A CN 201510205350 A CN201510205350 A CN 201510205350A CN 104825249 A CN104825249 A CN 104825249A
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Abstract
The invention discloses a surface-mediated gene therapy type artificial lens and a preparation method for the same. A plasmid DNA (Deoxyribonucleic Acid) containing a functional gene or a non-viral gene vector loaded with the plasmid DNA containing the functional gene is fixedly arranged on the surface of the artificial lens. The artificial lens and the preparation method for the same have the advantages that a surface-deviated gene therapy vector is obtained by a surface gene modification method, so that the epithelial cell proliferation and transdifferentiation of the lens are suppressed by a surface-mediated gene therapy method, and the artificial lens capable of effectively suppressing after cataract is obtained.
Description
Technical field
The present invention relates to medical embedded material and equipment surfaces modification field, be specifically related to a kind of surperficial mediated gene therapeutic type intraocular lens and preparation method thereof.The surface of intraocular lens of the present invention is fixed with energy functional gene, the MicroRNA of energy express cell apoptotic proteins and/or interference cell propagation, proliferation of lens epithelial cells or induction lens epithelial cell apoptosis can be suppressed, prevent after cataract from occurring.
Background technology
Cataract is the first diseases causing blindness of China.Current clinical treatment mainly adopts ultrasonic phacoemulsification extracapsular cataract extraction combined with intraocular implantation.But now widely used all kinds of intraocular lens often faces the problem that after cataract height occurs after the implantation after human eye.After cataract, that is, after IOP implantation, crystalline peplos is again muddy, is the major complications having a strong impact on patients' visual recovery after implantation of artificial lens.After cataract not only has a strong impact on postoperative vision restoration, and needs second operation, increases the financial burden of patient.Therefore, the intraocular lens that effectively can prevent and treat after cataract has important potential applicability in clinical practice.
Existing research is thought: stimulate the destruction of crystalline lens top layer lens epithelial cells in cataract surgery process and not exclusively remove, and artificial crystalline lens material surface adhesion, propagation and transdifferentiation cause crystalline peplos that muddy main cause occurs again after surgery to make it.For the generation of after cataract after reduction IOP implantation, usually reduce lens epithelial cells by artificial crystalline lens material surface modification and adhere to, or by pharmaceutical methods blocking-up proliferation of lens epithelial cells or transdifferentiation, and existing Patents.As Chinese patent CN103405807 A " intraocular lens of a kind of surperficial comb-shaped polymer hydrophilic modifying and preparation method thereof ", by the modification on artificial crystalline lens material surface, obtain hydrophilic surface, suppress lens epithelial cells to adhere to, thus reduce the generation of after cataract, Chinese patent CN101036804A " nanometer fluorouracil coat artificial crystalloid and preparation method thereof ", Chinese patent CN101053680A " artificial intraocular lenses of the tool antiproliferative agents coating preventing inverse position method from being formed ", Chinese patent CN2531755Y " slow-releasing agent carried artificial lens ", Chinese patent CN200973766Y " prevents the intraocular lens of after cataract " and Chinese patent CN200810061511 " intraocular lens of surperficial anti-transforming grouth factor beta 2 antibody membrane " etc., all outside intraocular implants surface or ambitus or the position load antiproliferative class chemicals such as loop or antibody drug reach the object suppressing proliferation of lens epithelial cells.
Injected or medication coat method by these anti-proliferative drugs, proliferation of lens epithelial cells or transdifferentiation can be suppressed preferably, but be difficult to avoid anti-increasing class drug osmotic to aqueous humor or its hetero-organization of eye, thus cause the damage of its hetero-organization of eye.
Summary of the invention
For solving the problems of the technologies described above, the object of the invention is by artificial crystalline lens material finishing functional gene or genophore, at intraocular lens's surface in situ load non-viral-based gene carrier, by the method for gene transfection, original position induction lens epithelial cell apoptosis after IOP implantation, thus reduce the incidence rate of after cataract.
The present invention adopts following technical scheme: a kind of surperficial mediated gene therapeutic type intraocular lens, and the finishing of described intraocular lens has containing the plasmid DNA of functional gene or the load non-viral gene vector containing functional gene plasmid DNA.
Described one surface mediated gene therapeutic type intraocular lens, the described plasmid DNA containing functional gene is the one expressed the model plasmid DNA of green fluorescent protein GFP, the plasmid DNA of the apoptotic p53 albumen of induced expression or express in the plasmid DNA of MicroRNA precursor.
Described one surface mediated gene therapeutic type intraocular lens, described load is the one in liposome, chitosan, polylysine, poly arginine, polymine or polyamide-based dendrimers containing the non-viral gene vector of the plasmid DNA of functional gene.
A described surperficial mediated gene therapeutic type process for manufacturing intraocular lenses, comprises following processing step:
(1) get clean intraocular lens, and obtain chemically reactive surface by surface preparation;
(2) intraocular lens after step (1) being processed adopts physics Electrostatic Absorption or chemical graft containing the plasmid DNA of functional gene or the load genophore containing functional gene plasmid DNA, obtains the intraocular lens that surface gene or non-viral gene vector are modified.
Described one surface mediated gene therapeutic type process for manufacturing intraocular lenses, described intraocular lens is hard artificiallens or flexible folding intraocular lens.
Described one surface mediated gene therapeutic type process for manufacturing intraocular lenses, the material of preparing of described hard artificiallens is polymethyl methacrylate, and the material of preparing of described flexible folding intraocular lens is silicone rubber or poly hydroxy ethyl acrylate hydrogel.
Described one surface mediated gene therapeutic type process for manufacturing intraocular lenses, described chemically reactive surface is: the aminated surface that silane coupler or polymine adsorption treatment obtain, the carboxylated surface of NaOH or KOH aqueous slkali soaking process.
Described one surface mediated gene therapeutic type process for manufacturing intraocular lenses, described chemical graft method is the coupling reaction of carboxyl and amido or amido and amido.
Described one surface mediated gene therapeutic type process for manufacturing intraocular lenses, the described plasmid DNA containing functional gene is the one expressed the model plasmid DNA of green fluorescent protein GFP, the plasmid DNA of the apoptotic p53 albumen of induced expression or express in the plasmid DNA of MicroRNA precursor.
Described one surface mediated gene therapeutic type process for manufacturing intraocular lenses, described load is the one in liposome, chitosan, polylysine, poly arginine, polymine or polyamide-based dendrimers containing the non-viral gene vector of the plasmid DNA of functional gene.
Principle of the present invention is: the clinical problem that after cataract that IOP implantation faces below height occurs, and its chief reason is the adhesion of lens epithelial cells on artificial crystalline lens material surface and cyst membrane residual in lens capsule bag in operation process, propagation and transdifferentiation.Therefore, the method for generation of at present conventional minimizing after cataract reduces lens epithelial cells by artificial crystalline lens material surface modification to adhere to, or should be used for realizing suppressing the object of proliferation of lens epithelial cells or transdifferentiation by anti-proliferative drugs or growth factor receptor inhibitor etc.But the application of anti-proliferative drugs unavoidably causes the damage of its hetero-organization of eye.The gene therapy of surface mediation provides a kind of means suppressing after cataract more easily.The generalized concept of gene therapy be some transfer of genetic material in patient body, make it express in vivo, finally reach the method for certain disease for the treatment of.Its syllabus be that genetic material is successfully delivered to target tissue, and transcribing and translating by gene, expressive function protein, finally reaches the object of disease therapy.By at functional genes such as the loading withered gene of material surface or cell inhibitory effect genes, induction lens epithelial cell apoptosis can be reached, suppress the object of proliferation of lens epithelial cells or transdifferentiation.Functional gene or the non-virus carrier containing functional gene are modified in artificial crystalline lens material surface by surface modification method by the present invention, utilize the gene therapy method of surface mediation.By the transfection of function of surface gene to lens epithelial cells, and non-injection anti-proliferative drugs, obtain the Proliferation Ability or apoptosis-induced to lens epithelial cells, realize carrying out situ treatment to after cataract.
The invention has the beneficial effects as follows: the invention provides a kind of surperficial mediated gene therapeutic type intraocular lens and preparation method thereof, by constructing one deck functional gene or the non-virus carrier decorative layer containing functional gene in intraocular implants surface by surface modification method, reach the effect suppressing proliferation of lens epithelial cells or induction lens epithelial cell apoptosis, thus the incidence rate of after cataract can be reduced, avoid drug injection or medication coat intraocular lens application in exist to the cytotropic toxic and side effects of non-target, surface modification method is adopted to build the decorative layer that can suppress after cataract, manufacturing process is simple, with low cost, the three-dimensional implantation instrument surface of various complex shape structure can be used in.
Accompanying drawing explanation
Fig. 1 is the process schematic of artificial crystalline lens material surface construction functional gene coating or the non-virus carrier coating containing functional gene.
Fig. 2 is the schematic diagram that between cationic polyelectrolytes and functional gene DNA molecular, Electrostatic association prepares Nano microsphere type non-viral gene vector.
Fig. 3 is the process schematic that between cationic polyelectrolytes and functional gene DNA molecular, Electrostatic association prepares Nano microsphere type non-viral gene vector.
Fig. 4 is that artificial crystalline lens material surface lens epithelial cells cultivates the photograph via bright field after 3 days.
Fig. 5 is that artificial crystalline lens material surface lens epithelial cells cultivates the fluorescence photo after 3 days.
Detailed description of the invention
Below in conjunction with specification drawings and specific embodiments, the present invention will be further described.
Embodiment 1
First, get clean intraocular lens, and by amino-contained functional group surfactant surface treatment make amine groups on its surface band: select polymethyl methacrylate hard artificiallens, be immersed in the polyethylenimine solution of 3mg/mL, act on 6 hours, artificial crystalline lens material surface adsorption layer of polyethylene imine layer, water cleans, and nitrogen dries up for subsequent use.The intraocular lens that surface amine groups activates is immersed into the plasmid DNA solution of 100 μ g/mL containing coding p53 protein gene subsequently, makes artificial crystalline lens material surface adsorption one deck plasmid DNA layer, as shown in schematic diagram 1 by Electrostatic Absorption.
Embodiment 2
Cationic polyelectrolytes solution and mutually mixing containing the plasmid DNA solution of functional gene fragment, prepare Electrostatic association type nano-gene carrier, preparation principle as shown in Figure 2.The present embodiment selects cationic polyelectrolytes chitosan and the plasmid DNA containing the functional gene fragment of coding p53 albumen to be example.Getting 50mg chitosan (being abbreviated as CHI below) is dissolved in 50mL hac buffer (pH=5.5), after fully dissolving, utilizes the filtering with microporous membrane insoluble matter of 220 μm; Separately getting 10mL concentration is plasmid DNA (be below the abbreviated as pDNA) solution of 100 μ g/mL containing encoding green flourescent protein gene, mix mutually with the above-mentioned CHI solution of 10mL, turbula shaker vortex oscillation 30 seconds, left at room temperature 30 minutes, prepare the non-virus carrier CHI-pDNA nanometer associated complex solution containing functional gene, preparation process as shown in Figure 3.
Embodiment 3
First, get clean intraocular lens, and make amine groups on its surface band by the surfactant surface treatment of amino-contained functional group: select polymethyl methacrylate hard artificiallens, be immersed in the polyethylenimine solution of 3mg/mL, act on 6 hours, artificial crystalline lens material surface adsorption layer of polyethylene imine layer, water cleans, and nitrogen dries up for subsequent use.Subsequently the intraocular lens that surface amine groups activates is immersed in polyelectrolyte heparin (the being abbreviated as HEP below) solution of the anionic property of 1mg/mL, Electrostatic Absorption 30 minutes, the heparin that artificial crystalline lens material surface adsorption one deck is electronegative; Subsequently intraocular lens is immersed into the preparation-obtained non-virus carrier CHI-pDNA nanometer associated complex solution containing functional gene of embodiment 1 30 minutes, the CHI-pDNA nanometer associated complex that intraocular lens's surface adsorption one deck is positively charged; Intraocular lens's alternately immerse is to heparin solution and CHI-pDNA nanometer associated complex solution subsequently, obtains (HEP/CHI-pDNA) surface gene carrier coating of multiple structure.Surface gene carrier coating (HEP/CHI-pDNA) prepared by the present embodiment is 5 bilayers, namely obtains (HEP/CHI-pDNA)
5the intraocular lens that genophore multilayer film is modified.
(HEP/CHI-pDNA) prepared by the present embodiment
5the intraocular lens that genophore multilayer film is modified carries out cell transfection assay.Conventional cell culture processes is utilized to be planted (HEP/CHI-pDNA) by lens epithelial cells
5the artificial crystalline lens material surface that genophore multilayer film is modified.Cell initial seeding density be 5000 cells every/hole, cultivate 3 days under normal conditions, with fluorescence microscope cell interior green fluorescent protein expression.
Embodiment 4
First, get clean poly hydroxy ethyl acrylate soft intraocular lens intraocular lens, be immersed into the KOH solution of 1 mol/L, be hydrolyzed 3 hours, the ester linkage hydrolyzing on artificial crystalline lens material surface makes carboxyl on surface band, subsequently the intraocular lens of surface carboxyl groups is immersed into containing 2mg/mL water-soluble carbodiimide (EDC), 4mg/mL N-hydroxy-succinamide (NHS) and the polylysine-containing in plasmid DNA nanometer associated complex (PLL-pDNA) mixed solution of functional gene for preparing by embodiment 2 method, room temperature Keep agitation 3 hours.The amido on PLL-pDNA nanometer associated complex surface and and the carboxyl of material surface utilize EDC/NHS coupled action to be grafted to material surface, obtain the intraocular lens of the non-virus carrier of area load functional gene.
By surface preparation and functional gene or build finishing coat containing functional gene on the surface at artificial crystalline lens material containing functional gene non-virus carrier surface modification method subsequently, process as shown in Figure 1.Wherein, containing the non-virus carrier preparation principle of functional gene and process as shown in Figures 2 and 3.Fig. 4 and Fig. 5 is respectively artificial crystalline lens material surface lens epithelial cells and cultivates the photograph via bright field after 3 days and fluorescence photo, shows surface and can transfer the possession of lens epithelial cells containing the artificial crystalline lens material surface of the non-virus carrier coating modifying of green fluorescent protein gene and express green fluorescent protein.By surface preparation and functional gene or carry out intraocular lens's finishing containing the non-virus carrier of functional gene, intraocular implants surface is loaded to containing functional gene or containing the non-virus carrier of functional gene by various, the gene transfection to the lens epithelial cells on its surface can be realized and expressive function albumen, to after cataract, there is potential good gene therapy effect.
Detailed description of the invention is only used to further illustrate the present invention; can not as limiting the scope of the present invention; person skilled in art makes some nonessential improvement and adjustment according to the content of foregoing invention to the present invention simultaneously; all be positioned at protection scope of the present invention, protection scope of the present invention is as the criterion with claims.
Claims (10)
1. a surperficial mediated gene therapeutic type intraocular lens, is characterized in that: the finishing of described intraocular lens has containing the plasmid DNA of functional gene or the load non-viral gene vector containing functional gene plasmid DNA.
2. one according to claim 1 surperficial mediated gene therapeutic type intraocular lens, is characterized in that: the described plasmid DNA containing functional gene is the one expressed the model plasmid DNA of green fluorescent protein GFP, the plasmid DNA of the apoptotic p53 albumen of induced expression or express in the plasmid DNA of MicroRNA precursor.
3. one according to claim 1 surperficial mediated gene therapeutic type intraocular lens, is characterized in that: described load is the one in liposome, chitosan, polylysine, poly arginine, polymine or polyamide-based dendrimers containing the non-viral gene vector of plasmid DNA of functional gene.
4. a surperficial mediated gene therapeutic type process for manufacturing intraocular lenses as claimed in claim 1, is characterized in that: comprise following processing step:
(1) get clean intraocular lens, and obtain chemically reactive surface by surface preparation;
(2) intraocular lens after step (1) being processed adopts physics Electrostatic Absorption or chemical graft containing the plasmid DNA of functional gene or the load genophore containing functional gene plasmid DNA, obtains the intraocular lens that surface gene or non-viral gene vector are modified.
5. one according to claim 4 surperficial mediated gene therapeutic type process for manufacturing intraocular lenses, is characterized in that: described intraocular lens is hard artificiallens or flexible folding intraocular lens.
6. one according to claim 5 surperficial mediated gene therapeutic type process for manufacturing intraocular lenses, it is characterized in that: the material of preparing of described hard artificiallens is polymethyl methacrylate, the material of preparing of described flexible folding intraocular lens is silicone rubber or poly hydroxy ethyl acrylate hydrogel.
7. one according to claim 4 surperficial mediated gene therapeutic type process for manufacturing intraocular lenses, it is characterized in that: described chemically reactive surface is: the aminated surface that silane coupler or polymine adsorption treatment obtain, the one in the carboxylated surface of NaOH or KOH aqueous slkali soaking process.
8. one according to claim 4 surperficial mediated gene therapeutic type process for manufacturing intraocular lenses, is characterized in that: described chemical graft method is the coupling reaction of carboxyl and amido or amido and amido.
9. one according to claim 4 surperficial mediated gene therapeutic type process for manufacturing intraocular lenses, is characterized in that: the described plasmid DNA containing functional gene is the one expressed the model plasmid DNA of green fluorescent protein GFP, the plasmid DNA of the apoptotic p53 albumen of induced expression or express in the plasmid DNA of MicroRNA precursor.
10. one according to claim 4 surperficial mediated gene therapeutic type process for manufacturing intraocular lenses, is characterized in that: described load is the one in liposome, chitosan, polylysine, poly arginine, polymine or polyamide-based dendrimers containing the non-viral gene vector of plasmid DNA of functional gene.
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| CN106215236A (en) * | 2016-07-20 | 2016-12-14 | 国家纳米科学中心 | Intraocular lens and its production and use is modified in a kind of gold nanorods region |
| CN107754018A (en) * | 2017-09-21 | 2018-03-06 | 温州医科大学 | A kind of intraocular lens with hydrophilic drugs sustained release synergistic 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 |
| CN113750294A (en) * | 2021-08-31 | 2021-12-07 | 四川大学 | Nerve repair stent loaded with multiple gene vector microspheres and preparation method thereof |
| CN114224822A (en) * | 2022-01-28 | 2022-03-25 | 复旦大学附属眼耳鼻喉科医院 | Sustained-release drug delivery implant for eyes and manufacturing method thereof |
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