WO2012092849A1 - 聚酰胺-胺树枝状聚合物或其衍生物-math1基因纳米微粒及其治疗耳聋的应用 - Google Patents

聚酰胺-胺树枝状聚合物或其衍生物-math1基因纳米微粒及其治疗耳聋的应用 Download PDF

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WO2012092849A1
WO2012092849A1 PCT/CN2012/070005 CN2012070005W WO2012092849A1 WO 2012092849 A1 WO2012092849 A1 WO 2012092849A1 CN 2012070005 W CN2012070005 W CN 2012070005W WO 2012092849 A1 WO2012092849 A1 WO 2012092849A1
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polyamidoamine
mathl
pamam
gene
plasmid
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PCT/CN2012/070005
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English (en)
French (fr)
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杨仕明
吴南
吴雁
韩东
郭维维
赵立东
翟所强
高维强
杨伟炎
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中国人民解放军总医院
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Priority to US13/978,060 priority Critical patent/US20140004196A1/en
Publication of WO2012092849A1 publication Critical patent/WO2012092849A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the present invention relates to the field of nanomaterials, and in particular, the present invention relates to a polyamidoamine, a partial degradation product thereof or a composite thereof - Mathl gene nanoparticle, a preparation method of the same, and its use in the treatment of deafness. Background technique
  • Nanoparticle (NP) Gene carrier is a solid colloidal nanoscale particle carrier synthesized from polymer materials, which can treat gene therapy molecules such as DNA, RNA, PNA (peptide nucleotide) and dsRNA (double-stranded RNA). Wrapped in the nanoparticle or adsorbed on the surface of the nanoparticle, the nanoparticle enters the cell through cell endocytosis, and the gene therapy molecule is gradually released by the degradation of the polymer material, thereby exerting the efficacy of the gene therapy.
  • gene therapy molecules such as DNA, RNA, PNA (peptide nucleotide) and dsRNA (double-stranded RNA).
  • polycations can be combined with genes to form nanoparticles in aqueous solution.
  • a special functional group e.g., galactose, transferrin
  • galactose, transferrin e.g., galactose, transferrin
  • Polycations as gene carriers are still stable nanoparticles in the electrolyte environment of plasma and are not inactivated during freeze-drying storage.
  • Polyamidoamine is a representative synthetic dendrimer which is a common cationic polymer in the physiological pH range where the amino group on the surface carries a positive charge.
  • PAMAM has a unique spherical shape and a highly branched nano-scale dendritic structure. The molecule consists of three parts: a central core, an inner repeating subunit and an outer amino terminal. It has good hydrodynamic properties and is easy to process. It also has low viscosity, high water solubility, miscibility, High anti Characteristics such as sex. In contrast, other cationic polymers such as chitosan, which require lower pH, have higher viscosity and poor water solubility. In terms of bio-performance, polyamidoamine has excellent properties such as no immunity, low toxicity, and excretion through urine and feces.
  • the cochlea is the only organ that humans perceive external sounds. It is also a highly differentiated functional specialized organ.
  • the inner hair cells are mechanical-electrical receptors that sense sound and vibration, and play an important role in maintaining hearing and balance. Degeneration and necrosis of inner ear hair cells caused by any cause can cause hearing and balance dysfunction.
  • the traditional view is that the cochlear hair cells of birds and mammals are differentiated during the embryonic stage and cannot be spontaneously regenerated. Once the deafness caused by the loss of cochlear hair cells is difficult to recover naturally, it must be artificially treated. Recovery, and this has always been a worldwide problem. In recent years, studies have shown that hair cells can be induced to regenerate after ototoxic drugs and noise damage to mammalian inner ear hair cells. Many growth factors play important roles in hair cell regeneration, such as transforming growth factor (TGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), and the like.
  • TGF transforming growth
  • Mathl (Mammalian atonal homolog 1 ) is a basic helix-loop-helix (bHLH) gene that is a mouse homolog of the Drosophila Atohl gene.
  • the Mathl gene is 1.18 kb in length and contains an exon with an mRNA of 1065 bp. It encodes a protein consisting of 354 amino acids, the transcription factor Mathl.
  • the molecular weight is 38.2 kDao Mathl gene is an essential gene for hair cell differentiation and maturation. An important role in the process (Bermingham NA, Hassan BA, Price SD et al, Mathl: an essential gene for the generation of inner ear hair cells. Science, 1999, 284: 1837-1841).
  • Nanoparticles as a novel gene carrier, with its non-immunogenicity, low It has the advantages of toxicity, large loading capacity, easy preparation, stable structure, favorable transformation and modification, and is more conducive to the further research and clinical application of hair cell regeneration.
  • the inner ear introduction route of the nanoparticle gene carrier is mainly a tympanic perforation and a round window membrane injection, and the vector is introduced into the perilymph by a direct injection and a micro-osmotic pump.
  • these two methods are effective, they are all invasive operations, destroying the cochlear tympanos, and there is a risk of inducing inflammation, perilymphatic leakage, and hearing damage.
  • the round window membrane has the characteristics of a semipermeable membrane: secretion and absorption functions
  • the nanoparticle gene carrier has target and permeability, as a new technology, it is expected to realize transmembrane introduction of a round window membrane.
  • the PAMAM-Mathl gene nanoparticle comprises PAMAM and a plasmid as shown in Figure 4, having a particle size of 100-200 nm, a dispersion index of 0.10-0.25, and a zeta potential of about 10-50 mV, The sealing ratio is 90-95%.
  • the PAMAM-Mathl gene nanoparticle has a controlled particle size, uniform size, and surface modification, which can improve the expression and delivery ability of the Mathl gene.
  • Another object of the present invention is to provide a PAMAM partial degradation product, the Mathl gene nanoparticle, for gene delivery.
  • the PAMAM partial degradation product-Mathl gene nanoparticle comprises a PAMAM partial degradation product and a plasmid as shown in Figure 4, having a particle size of 100-200 nm, a dispersion index of 0.10-0.25, and a zeta potential of about 10-50 mV, encapsulation efficiency is 90-95%.
  • the PAMAM partial degradation product is obtained by heat-treating PAMAM. In particular, PAMAM is heated in an aqueous solution at 50 °C-100 °C, more special. It is heated for 2 to 48 hours.
  • Another object of the present invention is to provide a PAMAM complex-Mathl gene nanoparticle for gene delivery.
  • the PAMAM complex-Mathl gene nanoparticle comprises a PAMAM complex and a plasmid as shown in Figure 4, having a particle size of 100-200 nm, a dispersion index of 0.10-0.25, and a zeta potential of about 10- 50 mV, encapsulation efficiency is 90-95%, the PAMAM complex is Obtained by shaking and mixing PAMAM or a part of its degradation product with cyclodextrin in an aqueous solution.
  • the PAMAM or a portion thereof of the degradation product is mixed with the cyclodextrin in a mass ratio of 1:10 to 10:1, more particularly by mixing and mixing for 10 s to 30 s.
  • the PAMAM complex-Mathl gene nanoparticles are significantly less cytotoxic.
  • a further object of the present invention is to provide a process for the preparation of the PAMAM, PAMAM partial degradation products or complexes thereof - Mathl gene nanoparticles of the present invention.
  • the method is simple and easy to operate, and the raw materials are easily obtained. It is not necessary to use an organic solvent and an aldehyde as a crosslinking agent, and the reaction is rapid, the reaction condition is mild, the repeatability is good, the stability is high, the utility is strong, and the utility model has wide applicability.
  • the PAMAM, PAMAM partial degradation product or a complex thereof-Mathl gene nanoparticle is prepared by re-agglomerating one of the above polymers with a plasmid containing a Mathl gene as shown in FIG. .
  • PAMAM, PAMAM partial degradation products or complexes thereof are added to a PBS-dissolved Mathl gene-containing plasmid at room temperature, and under the action of static electricity, the polymer molecule encapsulates the plasmid containing the Mathl gene to generate a nanoparticle suspension. liquid.
  • the method includes the following steps:
  • the molecular weight of PAMAM, PAMAM partial degradation products or PAMAM complex is 500Da ⁇ 1000000Da.
  • PAMAM partial degradation products are obtained by partial degradation (or fragmentation) by heat treatment reaction, which can further improve the transfection level of genes in vitro.
  • the PAMAM complex is obtained by combining PAMAM or PAMAM partial degradation products with cyclodextrin to reduce cytotoxicity, in particular, mixing in an aqueous solution at a mass ratio of 1:10 to 10:1.
  • the plasmid containing the Mathl gene is a plasmid as shown in Fig. 4.
  • the PAMAM, PAMAM partial degradation products prepared by the invention or PAMAM Complex-Mathl Nanoparticles have at least the following characteristics:
  • the selected dendrimer has the advantages of stability, low viscosity, good water solubility, no immunogenicity, protonation in the physiological pH range, and high transport efficiency to biologically active substances.
  • the particle size of the prepared particles can be adjusted and the size is uniform.
  • the surface of the prepared microparticles has a positive charge, which facilitates surface modification.
  • the PAMAM partial degradation product or the PAMAM complex-Mathl gene nanoparticle obtained has higher transfection efficiency and lower cytotoxicity when transfected in vivo or in vitro.
  • Control of gene delivery is easily achieved by adjusting the ratio of various components.
  • a further object of the present invention is to provide a PAMAM, a PAMAM partial degradation product or a PAMAM complex-Mathl gene nanoparticle for transfection in HEK293 cells cultured in vitro.
  • a further object of the present invention is to provide a PAMAM, PAMAM partial degradation product or a PAMAM complex-Mathl gene nanoparticle for use in transfecting cochlear tissue cultured in vitro.
  • a further object of the present invention is to provide a PAMAM, PAMAM partial degradation product or a PAMAM complex-Mathl gene nanoparticle for transfection in a cochlear implant.
  • a further object of the present invention is to provide a PAMAM, a PAMAM partial degradation product or a PAMAM complex-Mathl gene nanoparticle for use in deafness, which can be used for hair cell loss caused by noise, drug poisoning and the like. Sensorineural deafness. DRAWINGS
  • Figure 1 shows a schematic diagram of the formation of PAMAM-Mathl nanoparticles
  • Figure 2 shows a transmission electron micrograph of the PAMAM composite-Mathl nanoparticle
  • Figure 3 shows the particle size distribution of the PAMAM composite nanoparticles
  • Figure 4 shows a map of plasmid PRK5-Mathl-EGFP
  • Figure 5 shows the nucleotide sequence of the Mathl gene
  • Figure 6 shows an electropherogram, wherein lane 1 : marker; 1 : untransfected control group; 2: using the transfection group according to Example 9; 3: using the transfection group according to Example 10; Turn of embodiment 4 Dyeing group
  • Figure 7 shows the expression of EGFP in 293T cells transfected with PAMAM complex-Mathl nanoparticles
  • Figure 8 shows the microinjection of the round window membrane after the auricular approach
  • a the anatomical landmark of the posterior ear to find the occlusion
  • the black arrow shows the facial nerve
  • the blue star shows the sternocleidomastoid muscle
  • b the sternocleidomastoid
  • the muscles are separated and distracted, and the posterior wall of the occipital (blue arrow) and the posterior abdomen of the second abdominal muscle (blue star) are visible
  • C the posterior abdomen of the second abdominal muscle (blue star) is separated backward and exposed to the upper bone wall after the occipital ( The black arrow indicates the direction)
  • d After removing the bubble, the facial nerve enters the posterior bone wall
  • e After opening the ear, the round window (black arrow) and the radial artery (blue arrow) are visible.
  • f ⁇ ⁇ ⁇ ⁇ window film microinjection (black arrow shows the needle);
  • Figure 9 shows the inner ear tissue expressing the Mathl-EGFP protein, 1 : inner hair cell region; 2: column cell region; 3: outer hair cell region.
  • the total RNA of embryonic mouse brain tissue was extracted by Tnzol method, and cDNA was synthesized by reverse transcription.
  • the Math1 gene containing E-box was synthesized by PCR, and the ECOR1 and BamHl restriction sites were added at the 5' and 3' ends.
  • the PCR amplification product was digested with ECOR1 and BamHl, purified, and ligated with the PRK5 plasmid (Clontech) digested with ECOR1 and BamHl to construct a PRK5-Mathl plasmid.
  • the Mathl gene has the sequence shown in Figure 5.
  • the amplification primers are as follows:
  • the amplification conditions were: 94 ° C for 5 min; 94 ° C for lmin; 58 ° C for 40 sec ; 72 ° C for 40 sec ; 35 cycles, 72 ° C for 5 min.
  • the plasmid pEGFP-C2 (Invitrogen) containing the EGFP gene was ligated with Hpal and Xball enzymes.
  • the PRK5-Mathl plasmid of Example 1 was double-digested, purified and recovered, and the PRK5-Mathl-EGFP plasmid was constructed by T4 ligase ligation.
  • Example 3 Amplification and Purification of PRK5-Mathl-EGFP
  • the dynamic light dispersion was measured to have a particle size of 105.1 nm and a dispersion index of 0.206; the zeta potential analyzer measured a zeta potential of 39 ⁇ 1.12 (mV).
  • the ⁇ -cyclodextrin was added to the PAMAM solution at a mass ratio of 1:10 and mixed for 10 s. Then, ⁇ 50 ( ⁇ g/ml PAMAM complex was added to ⁇ 720 g/ml of PRK5-Mathl-EGFP plasmid in PBS, mixed rapidly on a vortex mixer for 30 seconds, and then incubated for 0.5 hour at room temperature. PAMAM complex-PRK5-Mathl-EGFP plasmid nanosuspension. The dynamic light dispersion was determined to have a particle size of 129.2 nm and a dispersion index of 0.245. The zeta potential analyzer measured the zeta potential to be 35 ⁇ 1.31 (mV).
  • ⁇ -cyclodextrin Adding ⁇ -cyclodextrin to PAMAM solution partially degraded by heat treatment at 50 ° C for 24 h, its quality The ratio is 10:1 and mixed for 10s. Then, ⁇ 500 g/ml ⁇ complex was added to 100 ⁇ l 720 g/ml PRK5-Mathl-EGFP plasmid in PBS, mixed rapidly on a vortex mixer for 30 seconds, and then incubated for 0.5 hour at room temperature to obtain PAMAM complex.
  • the nano-suspension of the substance-PRK5-Mathl-EGFP plasmid was determined by dynamic light dispersion to have a particle size of 130.2 nm and a dispersion index of 0.247; the zeta potential was determined by a zeta potential analyzer to be 39 ⁇ 1.19 (mV).
  • Example 11 Transfection of PEKM complex-PRK5-Mathl-EGFP nanoparticles in HEK293 cells and expression of Mathl protein in vitro
  • HEK 293T cells were seeded in 35 mm culture dishes and transfected when the cells reached 80% confluence.
  • the transfection was washed twice with DMEM medium containing 10% FBS, and each dish was added with 2 ml of DMEM medium containing 10% FBS preheated at 37 °C.
  • the nanosuspension prepared in the above examples was gently shaken to thoroughly mix (PAMAM complex nano-concentration was 4 ⁇ 3 ⁇ 4/ ⁇ 1), then 300 ⁇ l of nano-solution was added to each dish, and the dish was gently shaken to mix well. Incubate at 37 ° C, 5% C0 2 incubator for 24-48 hours.
  • RNA prepared in the above step was heated at 65 ° C for 5 min to melt the secondary structure and then immediately cooled on ice.
  • reaction at 50 ° C for 60 min reaction at 7 ° C for 5 min, addition of 1 ⁇ of RNase H, and reaction at 37 ° C for 20 min.
  • the obtained reverse transcription product was used as a template for the next PCR amplification reaction, or frozen at -20 °C.
  • PCR reaction procedure pre-denaturation at 95 °C for 5 min, then enter the cycle, denaturation at 95 °C for 45 sec, renaturation at 58 °C for 45 sec, extension at 72 °C for 1 min, 40 cycles, then extension at 72 °C for 5 min, the obtained PCR product Carry out the next reaction or freeze at -20.
  • the RT-PCR reaction product was detected by 1% agarose gel electrophoresis.
  • the Mathl gene can be translated in HEK293 cells to produce Mathl protein. Further, as shown in Fig. 7, 293T cells transfected with PAMAM complex-PRK5-Mathl-EGFP nanoparticles can express the Mathl-EGFP gene, indicating that the PAMAM complex-PRK5-Mathl-EGFP nanoparticles can transport the target gene. Go to living cells and get expression.
  • Example 12 PAMAM complex-Mathl gene nanoparticle transfection of SD rat cochlear tissue in vitro SD rats were immersed in alcohol for 3 days after birth, decapitated, and the vesicles were removed; the removed cochlear tissue was quickly placed in Hank's buffer at 4 °C; the cochlear tissue was separated, and the spiral ligament and vascular striate were removed; The basement membrane is divided into three layers: base layer, middle layer and top layer; DMEM containing 10% FBS is added to the 24-well culture plate; the basement membrane tissue is carefully laid on the culture plate; carefully placed at 37 ° C, 5% CO 2 incubator In the middle; change the liquid every other day.
  • the inner and outer hair cells express Mathl-EGFP, showing green fluorescence, therefore, the PAMAM complex-PRK5-Mathl-EGFP gene Nanoparticles can effectively transfect and express different cells in the inner ear including inner and outer hair cells, promote hair cell regeneration, and can be used for the treatment of sensorineural deafness.
  • the embodiments of the present invention have been specifically described above, but the embodiments are merely exemplary, and those skilled in the art can combine the values of the parameters disclosed in the present invention according to the teachings of the above embodiments. And get the technical solutions.
  • a person skilled in the art can make various changes and modifications to the invention, and they are all within the scope of the invention without departing from the spirit of the invention.

Description

聚酰胺-胺树枝状聚合物或其衍生物 -MATH1基因纳米微粒及其治疗耳聋的应用 相关申请的交叉引用
本申请主张于 2011年 1 月 4 日递交的中国专利申请 No.201110005066.6 的优先权， 在此引用该申请将其引入本文。 技术领域
本发明属于纳米材料技术领域， 具体而言， 本发明涉及聚酰胺胺、 其部分 降解产物或其复合物 -Mathl基因纳米微粒、所述基因纳米微粒的制备方法及其 在治疗耳聋上的应用。 背景技术
近年来， 以非病毒材料为基因载体的基因治疗研究引起了广泛的重视， 其 中的一个重要方面是用阳离子聚合物和基因复合形成纳米微粒来模拟类似病 毒的结构作为基因载体。 纳米微粒 （nanoparticle, NP) 基因载体是由高分子 材料合成的一种固态胶体纳米级微粒载体， 其能将 DNA、 RNA、 PNA (肽核 苷酸） 、 dsRNA (双链 RNA) 等基因治疗分子包裹在纳米微粒之中或吸附在 纳米微粒表面， 通过细胞胞吞使纳米微粒进入细胞内， 经高分子材料的降解逐 渐释放出基因治疗分子， 从而发挥其基因治疗的效能。
大量研究表明， 在一定条件下， 聚阳离子可与基因在水溶液中复合形成纳 米微粒。 同时， 可以在聚阳离子链上引入具有特殊功能的基团 （如半乳糖、 转 铁蛋白）， 从而使聚阳离子 /基因纳米微粒具有类似病毒的功能， 如受体调节内 化、进入细胞核等。聚阳离子作为基因载体在血浆的电解质环境中仍是稳定的 纳米微粒， 且在冷冻干燥储存过程中并不失活。
聚酰胺胺 （PAMAM) 是一种代表性的人工合成的树枝状聚合物， 在生理 pH值范围内， 其表面氨基基团会携带正电荷， 是常见的阳离子高分子聚合物。 PAMAM具有独特的球形外形， 以及高度分支的纳米级树枝状结构。 该分子由 三部分组成： 中心核、 内层重复的亚单元和外层的氨基端， 它具有良好的流体 力学性能， 易于加工成型； 同时它还具有低黏度、 高水溶性、 可混合性、 高反 应性等特点。 相比之下， 其它阳离子聚合物如壳聚糖等， 其质子化需要较低的 pH值， 而且黏度较大， 水溶性欠佳。 在生物性能方面， 聚酰胺胺具有无免疫 性、 毒性较低、 可通过尿和粪便排出体外的优异性能。
随着代数的增加， PAMAM树枝状聚合物末端的氨基基团增加。氨基基团 在生理 PH下， 发生质子化， 使 PAMAM具有聚阳离子特征。 带电荷易于与抗 体、 核酸和荧光基团通过静电作用形成稳定的复合物 （Haensler and Szoka, 1993; Bielinska et al., 1996; Wang et al., 2000)。 研究表明， PAMAM可以介导核 酸、 质粒等进入细胞中， 并获得目的基因表达。 其机理是带正电荷的 PAMAM/DNA复合物易于粘附于负电荷的细胞表面， 利于其进入细胞内部并 表达 （Dennig J and Duncan E, 2002)。 PAMAM对于细胞的转染效率和细胞毒 性会随代数的增加而同时增加， 该特性与被转染的靶细胞有关。
耳蜗是人类感受外界声音剌激的唯一器官， 同时也是一种高度分化的功能 特化器官， 其内部的毛细胞是感受声音振动的机械 -电感受器， 对维持听觉及 平衡觉有重要作用。任何原因导致的内耳毛细胞的变性、坏死均可引起听觉和 平衡功能障碍。传统观点认为鸟类及哺乳类动物的耳蜗毛细胞在胚胎期完成分 化， 且不能自发再生， 一旦发生由于耳蜗毛细胞损失而导致的耳聋就很难自然 恢复听力， 必须通过人为的治疗才有可能恢复， 而这一直是个世界性的难题。 近年来有研究表明， 耳毒性药物和噪声损伤哺乳动物内耳毛细胞后， 毛细胞可 以经诱导再生。 许多生长因子在毛细胞再生中起重要作用， 如转化生长因子 (TGF) 、 成纤维细胞生长因子 （FGF) 、 表皮生长因子 （EGF) 、 胰岛素样 生长因子 （IGF) 等等。
Mathl (Mammalian atonal homolog 1 ) 是一种碱性螺旋-环 -螺旋 （bHLH) 基因， 其是果蝇 Atohl基因的小鼠同源基因。 Mathl基因全长 1.18 kb， 含一个外 显子， mRNA 1065 bp， 编码由 354个氨基酸组成的蛋白， 即转录因子 Mathl, 分子量为 38.2 kDao Mathl基因是毛细胞分化成熟的必需基因， 在毛细胞再生 过程中有重要作用 （Bermingham NA, Hassan BA, Price SD et al, Mathl： an essential gene for the generation of inner ear hair cells. Science, 1999, 284:1837-1841 ) 。
由于病毒载体的局限性，利用病毒载体在动物体内进行内耳毛细胞再生的 研究无法应用于临床。 纳米微粒作为一种新型基因载体， 以其无免疫原性， 低 毒性、 装载容量大、 制备容易且结构稳定、 利于改造和修饰等优势， 更有利于 毛细胞再生的进一歩研究及临床应用。
目前， 纳米微粒基因载体的内耳导入途径主要是鼓阶打孔和圆窗膜注射， 通过直接注射和微渗透泵将该载体导入外淋巴液。这两种方式虽然有效， 但都 是侵袭性操作， 破坏耳蜗鼓阶， 存在诱发炎症、 外淋巴漏、 损坏听力的危险。 由于圆窗膜具有半透膜的特性： 分泌和吸收功能， 而纳米微粒基因载体具有靶 向性和通透性， 作为一种新技术， 有望实现圆窗膜跨膜导入。
然而， 至今未见 PAMAM及其衍生物 -Mathl基因纳米微粒的制备以及用 PAMAM-Mathl 基因纳米微粒转染体外培养细胞或在体转染耳蜗并且表达 Mathl基因的报导。 发明内容
本发明的目的在于提供一种 PAMAM-Mathl基因纳米微粒，实现基因的递 送。
根据本发明的一方面， 该 PAMAM-Mathl基因纳米微粒包括 PAMAM和 如图 4所示的质粒， 其粒径为 100-200nm， 分散指数为 0.10-0.25， zeta电位约 为 10-50 mV，包封率为 90-95%。该 PAMAM-Mathl基因纳米微粒的粒径可控、 尺寸均一、 利于表面修饰、 可提高 Mathl基因的表达和递送能力。
本发明的另一目的在于提供一种 PAMAM部分降解产物 -Mathl基因纳米 微粒， 实现基因的递送。
根据本发明的一方面， 该 PAMAM部分降解产物 -Mathl基因纳米微粒包 括 PAMAM部分降解产物和如图 4所示的质粒， 其粒径为 100-200nm， 分散指 数为 0.10-0.25， zeta电位约为 10-50 mV， 包封率为 90-95%， 该 PAMAM部分 降解产物是通过对 PAMAM进行热处理而获得的， 特别是， 在 50°C-100°C下 将 PAMAM在水溶液中加热， 更特别是加热 2-48小时。
本发明的另一目的在于提供一种 PAMAM复合物 -Mathl基因纳米微粒， 实现基因的递送。
根据本发明的一方面， 该 PAMAM 复合物 -Mathl 基因纳米微粒包括 PAMAM 复合物和如图 4 所示的质粒， 其粒径为 100-200nm， 分散指数为 0.10-0.25， zeta电位约为 10-50 mV， 包封率为 90-95%， 该 PAMAM复合物是 由 PAMAM或其部分降解产物与环糊精在水溶液中震荡混旋获得的。 特别是， 将 PAMAM或其部分降解产物与环糊精以质量比为 1:10到 10:1混合， 更特别 是混旋混合 10s到 30s。该 PAMAM复合物 -Mathl基因纳米微粒的细胞毒性明 显更低。
本发明的又一目的在于提供一种制备本发明的 PAMAM、 PAMAM部分降 解产物或其复合物 -Mathl基因纳米微粒的方法。 该方法简单易行， 原料易得， 无须使用有机溶剂和醛类作为交联剂， 反应迅速， 反应条件温和， 重复性好， 稳定性高， 实用性强， 具有广泛的应用性。
根据本发明的一方面， 所述 PAMAM、 PAMAM部分降解产物或其复合物 -Mathl基因纳米微粒是通过将上述聚合物之一与如图 4所示的含 Mathl基因 的质粒进行复凝聚而制备的。 特别地， 在室温下， 将 PAMAM、 PAMAM部分 降解产物或其复合物加入到 PBS溶解的含 Mathl基因的质粒中， 在静电作用 下， 聚合物分子包裹含 Mathl基因的质粒而生成纳米微粒混悬液。
具体地， 该方法包括下述歩骤：
( 1 )制备浓度为 500-1500μ^πύ的 PAMAM、 PAMAM部分降解产物或其 复合物的水溶液；
(2) 制备浓度为 120-72(^g /ml的含 Mathl基因的质粒的 PBS溶液；
(3 )以 N/P比（PAMAM氨基 /质粒磷酸基）为 30:1到 1 :10混合歩骤（1 )、 (2) 的溶液以进行复凝聚反应得到 PAMAM、 PAMAM部分降解产物或其复 合物 -Mathl基因纳米微粒混悬液。
其中， PAMAM、 PAMAM 部分降解产物或 PAMAM 复合物的分子量是 500Da~1000000Da。
其中， PAMAM的代数是 1-10。
其中， PAMAM部分降解产物是通过热处理反应进行部分降解 （或断裂） 获得的， 其可进一歩提高基因体外转染水平。
其中， PAMAM复合物是由 PAMAM或 PAMAM部分降解产物与环糊精 复合获得的， 以降低细胞毒性， 特别地， 以质量比为 1 :10到 10:1在水溶液中 混旋获得。
其中， 含 Mathl基因的质粒是如图 4所示的质粒。
与现有技术相比， 本发明制备的 PAMAM、 PAMAM 部分降解产物或 PAMAM复合物 -Mathl纳米微粒至少具有以下特点：
( 1 ) 选用的树状分子具有稳定、 黏度低、 水溶性好、 无免疫原性、 在生 理 pH范围内质子化、 对生物活性物质的转运效率高等优点。
(2) 制得微粒的粒径大小可以调节， 尺寸较均一。
(3 ) 制得微粒的表面带有正电荷， 利于进行表面修饰。
(4) 制得的 PAMAM、 PAMAM部分降解产物或 PAMAM复合物与含 Mathl基因的质粒通过静电作用复合制备纳米微粒，一方面能增加 Mathl基因 的稳定性， 使 Mathl基因最终被递送到细胞内， 另一方面， 增强与细胞膜的作 用及保护 Mathl基因免遭核酸酶的降解破坏。
(5)制得的 PAMAM部分降解产物或 PAMAM复合物 -Mathl基因纳米微 粒， 在进行体内或体外细胞转染时， 具有更高的转染效率和更低的细胞毒性。
(6) 通过调节各种成分的比例很容易实现对基因传递的控制。
本发明的又一目的在于提供 PAMAM、 PAMAM部分降解产物或 PAMAM 复合物 -Mathl基因纳米微粒在转染体外培养的 HEK293细胞中的应用。
本发明的又一目的在于提供 PAMAM、 PAMAM部分降解产物或 PAMAM 复合物 -Mathl基因纳米微粒在转染离体培养的耳蜗组织中的应用。
本发明的又一目的在于提供 PAMAM、 PAMAM部分降解产物或 PAMAM 复合物 -Mathl基因纳米微粒在转染在体耳蜗中的应用。
本发明的又一目的在于提供 PAMAM、 PAMAM部分降解产物或 PAMAM 复合物 -Mathl 基因纳米微粒在耳聋方面的应用， 该纳米微粒可以用于由于噪 声、 药物中毒等原因引起的毛细胞缺失而导致的感音神经性耳聋。 附图说明
图 1示出 PAMAM-Mathl纳米微粒的形成示意图；
图 2示出 PAMAM复合物 -Mathl纳米微粒的透射电镜图；
图 3示出 PAMAM复合物纳米颗粒的粒径分布；
图 4示出质粒 PRK5-Mathl -EGFP的图谱；
图 5示出 Mathl基因的核苷酸序列；
图 6示出电泳图谱， 其中泳道 1 : 标记； 1 : 未经转染对照组； 2: 利用根据 实施例 9的转染组； 3: 利用根据实施例 10的转染组； 4: 利用根据实施例 4的转 染组；
图 7示出 EGFP在转染有 PAMAM复合物 -Mathl纳米颗粒的 293T细胞中 的表达；
图 8示出耳后入路圆窗膜显微注射导入， a: 耳后入路寻找听泡的解剖标 志， 黑箭头示面神经、 蓝星示胸锁乳突肌； b: 将胸锁乳突肌向上分离牵开， 可见听泡后壁（蓝色箭头）及二腹肌后腹（蓝星）； C: 二腹肌后腹（蓝星） 向 后分离后暴露听泡后上骨壁（黑箭头指示方向）； d: 磨除此处听泡后上、 面神 经入听泡处后方骨壁； e: 打开听泡后可见圆窗龛（黑箭头示）、 镫骨动脉（蓝 箭头示）； f: 剌破圆窗膜显微注射 （黑箭头示针头）； 以及
图 9示出表达了 Mathl-EGFP蛋白的内耳组织， 1 : 内毛细胞区域； 2: 柱 细胞区域； 3: 外毛细胞区域。 具体实施方式
下面通过实施例对本发明作详细阐述。
除非特别指出， 否则下面实例中的试剂药品材料均为市售可得， 是实例中 使用的方法参考 《分子克隆实验指南》 （Sambrook和 Russell, 2001 )。 实施例 1 : PRK5-Mathl质粒的构建
用 Tnzol法提取胚胎小鼠 16天脑组织的总 RNA, 反转录合成 cDNA, 利用 PCR法合成含有 E盒的 Mathl基因， 且 5'和 3'末端加入 ECOR1和 BamHl酶切位 点。 将 PCR扩增产物经 ECOR1和 BamHl酶切， 纯化后与经 ECOR1和 BamHl酶 切的 PRK5质粒 （Clontech公司） 连接， 构建 PRK5-Mathl质粒。 其中的 Mathl 基因具有图 5所示序列。
扩增引物如下：
F: 5， -GGAATT AAAAT AGTTGGGGGACC-3 '；
R: 5，-TGGACAGCTTCTTGTTGGCTT-3，，
扩增条件为： 94 °C 5min; 94 °C lmin; 58 °C 40sec; 72 °C 40sec; 35个循 环， 72°C延伸 5min。
实施例 2: PRK5-Mathl-EGFP质粒的构建
用 Hpal和 Xball酶对含有 EGFP基因的质粒 pEGFP-C2 (Invitrogen公司） 和 实施例 1的 PRK5-Mathl质粒分别进行双酶切， 纯化回收， 经 T4连接酶连接构建 PRK5-Mathl -EGFP质粒。 实施例 3： PRK5-Mathl-EGFP的扩增和纯化
取 5 μΐ质粒 PRK5-Mathl-EGFP， 加入 100 μΐ感受态大肠杆菌 DH5a细菌， 混 匀， 冰浴 30分钟， 42°C热休克 1分钟， 冰浴 2分钟， 加入 800 μΐ LB培养基， 37 °C培养 1小时。 取 100 μΐ菌液涂布于含有氨苄青霉素的平板， 37°C倒置培养 16 小时。 挑取平板中的单菌落， 接种于 5ml含有氨苄青霉素的 LB液体培养基中， 37°C恒温振荡过夜，使细菌生长至对数晚期。按照质粒提取试剂盒（QIAGEN) 说明书提取质粒。
取质粒 0. 5〜1μ 加入内切酶 5 U (不超过总反应体积 1/10)，反应体积 20μ1， 适温水浴 2 h， 取少量样品进行琼脂糖凝胶电泳检测酶切结果。 实施例 4:
将 ΙΟΟμΙ 的 500 g/ml 的 PAMAM 溶液加入到 ΙΟΟμΙ 720 g/ml 的 PRK5-Mathl-EGFP质粒的 PBS溶液中，迅速在旋涡混合器上混合 30秒后，室 温下继续孵育 0.5小时得到 PAMAM-PRK5-Mathl-EGFP质粒的纳米混悬液。 动态光散色测定其粒径为 118.6nm， 分散指数 0.187; zeta 电位分析仪测定其 zeta电位为 42±1.17 (mV)。 实施例 5:
将 50°C热处理 24h而部分降解的 ΙΟΟμΙ的 500 g/ml的 PAMAM溶液加到 ΙΟΟμΙ 720 g/ml的 PRK5-Mathl-EGFP质粒的 PBS溶液中，迅速在旋涡混合器 上混合 30秒后，室温下继续孵育 0.5小时得到 PAMAM-PRK5-Mathl-EGFP质 粒的纳米混悬液。 动态光散色测定其粒径为 105.1nm， 分散指数为 0.206; zeta 电位分析仪测定其 zeta电位为 39±1.12 (mV)。
实施例 6:
将 50°C热处理 24h而部分降解的 ΙΟΟμΙ的 500 g/ml的 PAMAM溶液加到 50μ1 720 g/ml的 PRK5-Mathl-EGFP质粒的 PBS溶液中， 迅速在旋涡混合器 上混合 30秒后，室温下继续孵育 0.5小时得到 PAMAM-PRK5-Mathl-EGFP质 粒的纳米混悬液。 动态光散色测定其粒径为 104.2nm， 分散指数为 0.198; zeta 电位分析仪测定其 zeta电位为 41.6±1.19 (mV)。 实施例 7:
将 100°C热处理 24h而部分降解的 ΙΟΟμΙ的 500 g/ml的 PAMAM溶液加 到 25μ1 720 g/ml的 PRK5-Mathl-EGFP质粒的 PBS溶液中， 迅速在旋涡混合 器上混合 30秒后， 室温下继续孵育 0.5小时得到 PAMAM-PRK5-Mathl-EGFP 质粒的纳米混悬液。 动态光散色测定其粒径为 102.9nm， 分散指数为 0.202; zeta电位分析仪测定其 zeta电位为 42.9±1.23 (mV)。 实施例 8:
将 50°C热处理 48h而部分降解的 ΙΟΟμΙ的 500 g/ml的 PAMAM溶液加到 ΙΟΟμΙ 720 g/ml的 PRK5-Mathl-EGFP质粒的 PBS溶液中，迅速在旋涡混合器 上混合 30秒后，室温下继续孵育 0.5小时得到 PAMAM-PRK5-Mathl-EGFP质 粒的纳米混悬液。 动态光散色测定其粒径为 101.5nm， 分散指数为 0.211 ; zeta 电位分析仪测定其 zeta电位为 37± 1.28 (mV)。 实施例 9:
将 β-环糊精加入到 PAMAM溶液，其质量比为 1 :10，混合 10s。然后将 ΙΟΟμΙ 的 50(^g/ml PAMAM复合物加到 ΙΟΟμΙ 720 g/ml的 PRK5-Mathl-EGFP质粒 的 PBS溶液中， 迅速在旋涡混合器上混合 30秒后， 室温下继续孵育 0.5小时 得到 PAMAM复合物 -PRK5-Mathl-EGFP质粒的纳米混悬液。动态光散色测定 其粒径为 129.2nm， 分散指数为 0.245; zeta 电位分析仪测定其 zeta 电位为 35士 1.31 (mV) o
实施例 10:
将 β-环糊精加入到 50°C热处理 24h而部分降解的 PAMAM溶液， 其质量 比为 10:1，混合 10s。然后将 ΙΟΟμΙ的 500 g/ml ΡΑΜΑΜ复合物加到 100μ1 720 g/ml的 PRK5-Mathl-EGFP质粒的 PBS溶液中， 迅速在旋涡混合器上混合 30 秒后， 室温下继续孵育 0.5小时得到 PAMAM复合物 -PRK5-Mathl-EGFP质粒 的纳米混悬液动态光散色测定其粒径为 130.2nm，分散指数为 0.247; zeta电位 分析仪测定其 zeta电位为 39± 1.19 (mV)。 实施例 11 : PAMAM复合物 -PRK5-Mathl-EGFP纳米微粒在体外培养 HEK293 细胞的转染及 Mathl蛋白的表达
转染前一天， 将 HEK 293T细胞接种于 35mm培养皿， 待细胞达 80%融合时 进行转染。转染时使用含 10% FBS的 DMEM培养基冲洗两遍， 每皿加入 37°C预 热的 2ml含 10% FBS的 DMEM培养基。 将按上述实施例制备的纳米混悬液轻轻 晃动以充分混匀 （PAMAM复合物纳米浓度为 4ι¾/μ1) ， 然后向每培养皿加入 300μ1纳米溶液，轻轻晃动培养皿以充分混匀，于 37°C， 5% C02培养箱培养 24-48 小时。
收集于 36.5°C±0.5°C培养 48小时的 293T细胞 （约 107个） ， 用 Trizol法提取 RNA。加入 3(^1 DEPC水溶解 RNA， 取 2 μΐ用紫外分光光度计测定 RNA含量后， 于 -80 V的冰箱中冻存 RNA。
PCR扩增
模板变性：于 65°C将上述歩骤中制备的 RNA加热 5min以熔化二级结构，然 后在冰上立即冷却。
模板变性反应体系：
组分 体积
RNA 5.0μ1
Oligo dT (20) Ι.ΟμΙ
dNTPs 2.0μ1
模板 3.0μ1 (200ng)
dd¾0 4.0μ1 反转录反应体
组分 体积 5x反应缓冲液 5.0μ1
0.1M DTT Ι.ΟμΙ
RNAseOUT™ Ι.ΟμΙ
反转录酶 III 3.0μ1
dd¾0 Ι.ΟμΙ
充分混匀后进行下述反应： 50°C反应 60min， 7 °C反应 5min， 加入 1 μΐ的 RNase H， 于 37°C反应 20min。 将获得的反转录产物作为模板进行下一歩 PCR 扩增反应， 或者于 -20 °C冻存。
—里 ——————— L—
10χ反应缓冲液 （-Mg2+) 5.0μ1
50mM MgCl2 1.5μ1
lOmM dNTP Ι.ΟμΙ
ΙΟμΜ引物 1 Ι.ΟμΙ
ΙΟμΜ引物 2 Ι.ΟμΙ
Taq酶 0.4μ1
cDNA 2.0μ1
DEPC ¾0 Ι.ΟμΙ
PCR反应程序： 95°C预变性 5min后进入循环， 95°C变性 45sec， 58°C复性 45sec， 72°C延伸 lmin, 共 40个循环， 然后 72°C延伸 5min， 将获得的 PCR产物 进行下一歩反应或者于 -20 冻存。
1%琼脂糖凝胶电泳检测 RT-PCR反应产物。
如图 6可见， Mathl基因能够在 HEK293细胞中进行翻译， 产生 Mathl蛋白。 进一歩地， 如图 7可见， 经过 PAMAM复合物 -PRK5-Mathl-EGFP纳米颗粒 转染的 293T细胞能够表达 Mathl-EGFP基因， 表明 PAMAM复合物 -PRK5-Mathl-EGFP纳米颗粒能够把目的基因运送到活细胞中去并获得表达。
实施例 12: PAMAM复合物 -Mathl基因纳米微粒转染体外培养的 S-D大鼠耳蜗 组织 将出生后 3天的 S-D大鼠迅速浸过酒精， 断头， 取出听泡； 将取下的耳蜗 组织迅速放入 4°C Hank's缓冲液中； 分离耳蜗组织， 去除螺旋韧带及血管纹； 将基底膜分为基层、 中层及顶层三段； 24 孔培养板上加入含 10%FBS 的 DMEM; 小心将基底膜组织平铺于培养板上； 小心放置于 37°C， 5%C02培养 箱中； 隔日换液。
培养 6天后， 基底膜组织贴壁良好， 转染时用含 10%FBS的 DMEM洗两 遍，每皿加入 37°C预热的 3ml含 10%FBS的 DMEM;将实施例制备的 PAMAM 复合物纳米溶液充分轻轻混匀 （PAMAM复合物纳米浓度为 4ι¾/μ1); 每培养 皿加入 300μ1纳米溶液轻轻晃动培养皿使充分混匀； 37°C， 5%C02培养箱培养 24-48小时观察结果。 实施例 13： PAMAM复合物 -Mathl基因纳米微粒经圆窗膜穿剌纤维注射转染在 体的 S-D大鼠耳蜗组织
取健康的 3周龄 S-D大鼠， 雌雄不限， 体重 120g〜130g， 耳廓反射灵敏， 双 耳鼓膜正常， 无感染。 使用动物用水合氯醛（北京） 按 4.5 ml/kg体重对大鼠进 行麻醉， 于 37°C保温袋保温。 麻醉后， 在严格无菌条件下， 右侧耳经腹侧进路 显露听泡， 在手术显微镜下用电钻打开听泡， 暴露耳蜗， 在底周鼓阶以穿剌针 打孔至外淋巴液流出。根据体外细胞转染水平的实验优化条件， 制定活体转染 最佳浓度；稀释液为人工外淋巴液；将 5 μΐ PAMAM复合物 -PRK5-Mathl -EGFP 基因纳米微粒溶液经鼓阶打孔缓慢 （约 5分钟） 注入， 然后取一小块肌肉填塞 封闭听泡打孔处， 分层缝合切口。 经鼓阶打孔纤维注射给药， 给药方法简便易 行、 转染可靠、 高效、 对内耳骚扰比较小。 实施例 14: PAMAM复合物 -PRK5-Mathl-EGFP基因纳米微粒转染 S-D大鼠内耳 组织的免疫组化
PAMAM复合物 -PRK5-Mathl-EGFP基因纳米微粒转染 7天后， 对大鼠断头 处理， 然后迅速取出听泡， 用 4%的多聚甲醛固定 1 h， 进行耳蜗基底膜铺片； 免疫组化染色后于共聚焦荧光显微镜下观察结果， 激发光 488nm ; 表达 Mathl-EGFP的内外毛细胞为绿色荧光。 如图 9所示， 内外毛细胞表达 Mathl -EGFP, 显示绿色荧光， 因此， PAMAM复合物 -PRK5-Mathl-EGFP基因 纳米微粒可以有效的转染内耳包括内外毛细胞在内的不同细胞并表达，促进毛 细胞再生， 可以用于感音神经性耳聋的治疗。 需指出， 上面对本发明的实施方式进行了具体的描述， 然而所述实施例仅 为示例性的， 本领域技术人员可以根据上述实施例的教导， 对本发明公开的各 参数的取值范围进行组合而得出各技术方案。更近一歩地， 本领域技术人员还 可对本发明做出多种改变和变形， 在不脱离本发明精神的前提下， 它们均在本 发明的范围内。

Claims

权利要求书
1、一种聚酰胺胺 -Mathl基因纳米微粒，包括聚酰胺胺和图 4所示的质粒， 其粒径为 100-200nm， 分散指数为 0.10-0.25， zeta电位约为 10-50 mV， 包封率 为 90-95%。
2、 一种聚酰胺胺部分降解产物 -Mathl基因纳米微粒， 包括聚酰胺胺部分 降解产物和图 4所示的质粒，其粒径为 100-200nm，分散指数为 0.10-0.25， zeta 电位约为 10-50 mV， 包封率为 90-95%， 该部分降解产物是通过对完整聚酰胺 胺分子热处理获得的。
3、 一种聚酰胺胺复合物 -Mathl基因纳米微粒， 包括聚酰胺胺复合物和图 4所示的质粒，其粒径为 100-200nm，分散指数为 0.10-0.25， zeta电位约为 10-50 mV， 包封率为 90-95%， 该复合物是由聚酰胺胺或其部分降解产物与环糊精混 合获得的， 该部分降解产物是通过对完整聚酰胺胺分子热处理获得的。
4、 一种纳米微粒的制备方法， 其通过在室温下将聚酰胺胺、 聚酰胺胺部 分降解产物或聚酰胺胺复合物的水溶液与含 Mathl基因质粒的 PBS溶液进行 复凝聚而获得纳米微粒混悬液，所述部分降解产物是通过对完整聚酰胺胺分子 热处理获得的，所述复合物是由聚酰胺胺或其部分降解产物与环糊精混合获得 的， 所述含 Mathl基因的质粒为图 4所示的质粒。
5、 根据权利要求 4的方法， 其特征在于， 包括：
( 1 ) 制备浓度为 500-150(^g/ml的聚酰胺胺或其部分降解产物的水溶液 或聚酰胺胺复合物的水溶液；
(2) 制备浓度为 120-72(^g /ml的含 Mathl基因的质粒的 PBS溶液；
(3 ) 以聚酰胺胺氨基 /质粒磷酸基比为 30:1至 1 :10混合歩骤 （1 )、 (2 ) 的溶液以进行复凝聚反应得到聚酰胺胺 -Mathl基因纳米微粒混悬液、聚酰胺胺 部分降解产物 -Mathl基因纳米微粒混悬液， 或聚酰胺胺复合物 -Mathl基因纳 米微粒混悬液。
6、 根据权利要求 4或 5的方法， 其特征在于， 所述聚酰胺胺的分子量是 500Da~1000000Da。
7、 根据权利要求 4或 5的方法， 其特征在于， 所述热处理是在 50-100°C 下在水溶液中加热 2-48小时， 所述聚酰胺胺或其部分降解产物与环糊精以质
8、 权利要求 4的方法制备的纳米颗粒， 其特征在于， 所述纳米颗粒的粒 径为 100-200nm， 分散指数为 0.10-0.25， zeta电位约为 10-50 mV， 包封率为 90-95o/o。
9、 根据权利要求 1、 2、 3或 8的纳米颗粒在转染体外培养的 HEK293细 胞、 转染离体培养的耳蜗组织或转染在体耳蜗中的应用。
PCT/CN2012/070005 2011-01-04 2012-01-04 聚酰胺-胺树枝状聚合物或其衍生物-math1基因纳米微粒及其治疗耳聋的应用 WO2012092849A1 (zh)

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