CN113549132B - 一种基于三联吡啶釕催化短肽共价组装形成的非病毒载体及其制备方法和应用 - Google Patents
一种基于三联吡啶釕催化短肽共价组装形成的非病毒载体及其制备方法和应用 Download PDFInfo
- Publication number
- CN113549132B CN113549132B CN202110818561.2A CN202110818561A CN113549132B CN 113549132 B CN113549132 B CN 113549132B CN 202110818561 A CN202110818561 A CN 202110818561A CN 113549132 B CN113549132 B CN 113549132B
- Authority
- CN
- China
- Prior art keywords
- solution
- dna
- viral vector
- bpy
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000013603 viral vector Substances 0.000 title claims abstract description 47
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- JFJNVIPVOCESGZ-UHFFFAOYSA-N 2,3-dipyridin-2-ylpyridine Chemical compound N1=CC=CC=C1C1=CC=CN=C1C1=CC=CC=N1 JFJNVIPVOCESGZ-UHFFFAOYSA-N 0.000 title claims abstract description 8
- 230000003197 catalytic effect Effects 0.000 title abstract description 9
- 239000005092 [Ru (Bpy)3]2+ Substances 0.000 claims abstract description 26
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 18
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims abstract description 14
- 230000008685 targeting Effects 0.000 claims abstract description 13
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims abstract description 12
- 229910001870 ammonium persulfate Inorganic materials 0.000 claims abstract description 12
- 239000000074 antisense oligonucleotide Substances 0.000 claims abstract description 11
- 238000012230 antisense oligonucleotides Methods 0.000 claims abstract description 11
- 229940079593 drug Drugs 0.000 claims abstract description 11
- 239000003814 drug Substances 0.000 claims abstract description 11
- 238000002156 mixing Methods 0.000 claims abstract description 10
- 108091034117 Oligonucleotide Proteins 0.000 claims abstract description 9
- YBCAZPLXEGKKFM-UHFFFAOYSA-K ruthenium(iii) chloride Chemical compound [Cl-].[Cl-].[Cl-].[Ru+3] YBCAZPLXEGKKFM-UHFFFAOYSA-K 0.000 claims abstract description 5
- 108020004414 DNA Proteins 0.000 claims description 37
- 239000002077 nanosphere Substances 0.000 claims description 24
- 102000004961 Furin Human genes 0.000 claims description 17
- 108090001126 Furin Proteins 0.000 claims description 17
- 102000053602 DNA Human genes 0.000 claims description 12
- 150000007523 nucleic acids Chemical class 0.000 claims description 11
- 102000039446 nucleic acids Human genes 0.000 claims description 10
- 108020004707 nucleic acids Proteins 0.000 claims description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 238000003776 cleavage reaction Methods 0.000 claims description 4
- 201000010099 disease Diseases 0.000 claims description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 4
- 230000007017 scission Effects 0.000 claims description 4
- 150000001413 amino acids Chemical group 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000004132 cross linking Methods 0.000 claims description 3
- 238000005538 encapsulation Methods 0.000 claims description 3
- 230000001225 therapeutic effect Effects 0.000 claims description 3
- 238000012546 transfer Methods 0.000 claims description 3
- 101150075200 S-2 gene Proteins 0.000 claims description 2
- 101150027674 S1 gene Proteins 0.000 claims description 2
- 238000000137 annealing Methods 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 abstract description 7
- 238000001476 gene delivery Methods 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 42
- 108091093037 Peptide nucleic acid Proteins 0.000 description 21
- 206010028980 Neoplasm Diseases 0.000 description 6
- 102000004196 processed proteins & peptides Human genes 0.000 description 6
- -1 hydroxyl free radical Chemical class 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002428 photodynamic therapy Methods 0.000 description 4
- 150000003254 radicals Chemical class 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 3
- 239000002114 nanocomposite Substances 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 101000971171 Homo sapiens Apoptosis regulator Bcl-2 Proteins 0.000 description 2
- 102100034343 Integrase Human genes 0.000 description 2
- 101710203526 Integrase Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000000799 fluorescence microscopy Methods 0.000 description 2
- 230000030279 gene silencing Effects 0.000 description 2
- 238000012226 gene silencing method Methods 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000001678 irradiating effect Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 239000002539 nanocarrier Substances 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000002834 transmittance Methods 0.000 description 2
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000006909 anti-apoptosis Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000011319 anticancer therapy Methods 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000000942 confocal micrograph Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000008260 defense mechanism Effects 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical group [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 238000009830 intercalation Methods 0.000 description 1
- 230000002687 intercalation Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229920000724 poly(L-arginine) polymer Polymers 0.000 description 1
- 108010011110 polyarginine Proteins 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 108010085082 sigma receptors Proteins 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000009827 uniform distribution Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
-
- 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
-
- 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
- A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
- A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02B—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO BUILDINGS, e.g. HOUSING, HOUSE APPLIANCES OR RELATED END-USER APPLICATIONS
- Y02B20/00—Energy efficient lighting technologies, e.g. halogen lamps or gas discharge lamps
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Immunology (AREA)
- Nanotechnology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了一种基于三联吡啶釕催化短肽共价组装形成的非病毒载体及其制备方法和应用,所述制备方法包括以下步骤:将反义寡核苷酸与三联吡啶氯化钌溶液混合,孵育得到DNA‑[Ru(bpy)3]2+溶液;将DNA‑[Ru(bpy)3]2+溶液、含酪氨酸的靶向肽溶液、过硫酸铵溶液混合,进行白光照射,离心后即可得到所述非病毒载体;其具有合成简单,绿色,高效等特点;该非病毒载体可在在基因传递或作为靶向药物中进行应用。
Description
技术领域
本发明属于基因输送载体技术领域,具体涉及一种基于三联吡啶釕催化短肽共价组装形成的非病毒载体及其制备方法和应用。
背景技术
药物治疗涉及多种复杂的机制,作一种智能和适应性系统是目前肿瘤研究和临床实践中的一大挑战,癌细胞可以发展多种防御策略来规避治疗药物的影响。这些防御机制通常与耐药相关蛋白表达水平的改变有关,最终导致抗癌治疗的失败。为应对这一挑战,反义寡核苷酸(ASO)和小干扰RNA (sirna)等是能够抑制耐药相关蛋白的表达以恢复癌细胞的药物敏感性的核酸治疗方法,其已与光动力联合使用,达到加法和协同治疗效果。由于光动力治疗药物和核酸具有本质上的不同,常规光动力治疗和基因组合治疗通常采用一种载体来装载光学药物和核酸治疗药物,每个组分分别发挥各自的作用。尽管取得了巨大的进步,但仍然存在局限性。
发明内容
本发明的目的在于提供一种基于三联吡啶釕催化短肽共价组装形成的非病毒载体及其制备方法和应用,本发明通过反义寡核苷酸中的dsDNA双螺旋结构插入[Ru(bpy)3]Cl2形成DNA-[Ru(bpy)3]2+,在白光照射下,DNA-[Ru(bpy)3]2+催化含酪氨酸的靶向肽YY-12共价交联,自组装形成肽-核酸纳米球(RGD-NS)非病毒载体,其具有合成简单,绿色,高效等特点;该非病毒载体可在在基因传递或作为靶向药物中进行应用。
为实现上述目的,本发明采取的技术方案如下:
一种基于DNA-[Ru(bpy)3]2+催化短肽共价组装形成非病毒载体的制备方法,所述制备方法包括以下步骤:
(1)将反义寡核苷酸与三联吡啶氯化钌溶液混合,孵育得到DNA-[Ru(bpy)3]2+溶液;
(2)将步骤(1)得到的DNA-[Ru(bpy)3]2+溶液、含酪氨酸的靶向肽溶液、过硫酸铵溶液混合,进行白光照射,离心后即可得到所述非病毒载体;
所述含酪氨酸的靶向肽的氨基酸序列为YYRRVRRRGDYY;其中,Y为酪氨酸,RVRR为具有弗林酶切割位点多肽,RGD是可与αvβ3整合素和sigma受体过表达的肿瘤可以主动结合的序列肽。
步骤(1)中,所述反义寡核苷酸的制备方法为:将DNA S1溶液和DNA S2溶液混合,90℃退火10min,并在4~10℃静置至形成双链DNA结构;
所述DNA S1基因序列为:
3’-GGA TTG GAG TTC CTC CAG CGT GCG CCA TCC TTC CCA TCC TCCTCC-5’;划横线的部分为可降低抗凋亡蛋白Bcl-2表达的ASO的序列。
所述DNA S2基因序列为:3’-GAG GAA CTC CAA TCC-5’。
所述DNA S1、DNA S2的摩尔比为1:1。
步骤(1)中,反应体系中,反义寡核苷酸、三联吡啶氯化钌的摩尔比为1:800~1200,优选为1:1000。
步骤(1)中,所述孵育的条件为34~37℃孵育6.5~7.5h,优选为37℃孵育7.0h。
步骤(2)中,DNA-[Ru(bpy)3]2+溶液、短肽溶液、过硫酸铵溶液的体积之比为1:0.8~1.2:2.5~3.2,优选为1:1:3;所述短肽溶液的浓度为0.5~1.5mg/mL,优选为1.0mg/mL;所述过硫酸铵溶液的浓度为8~12mM,优选为10mM。
步骤(2)中;所述照射的时间为5~10min,优选为6min。
步骤(2)中,使用海门其林贝尔GL-800白光透射仪进行白光照射。
按照本发明所述的制备方法制备得到的非病毒载体,其为平均粒径200nm的纳米球,且纳米球中的C、Ru、P等元素均匀分布,所述非病毒载体实现了将核酸包封在交联体内,实现对核酸的运载。
本发明还提供了所述的非病毒载体在基因传递或作为靶向药物中的应用,通过非病毒载体实现了对核酸的包封,其在弗林酶的作用下可释放出包封的核酸,进而实现基因的传递及疾病的靶向治疗。
本发明还提供了一种可释放所述非病毒载体中的DNA的方法,将弗林酶加入到所述非病毒载体中进行切割。
具体为:将弗林酶溶液加入到所述非病毒载体中,消化1h,加入EDTA终止弗林酶的活性。在白光照射下,将酶解产物通过ESR检测羟基自由基。
所述弗林酶溶液的制备方法为:将弗林酶溶解在pH=7.4的tris-HCl溶液中得到质量浓度为0.1%弗林酶溶液。
本发明提供的基于DNA-[Ru(bpy)3]2+催化短肽共价组装成非病毒载体的制备方法中,首先,利用反义寡核苷酸的dsDNA双螺旋结构插入[Ru(bpy)3]Cl2形成DNA-[Ru(bpy)3]2+,在白光照射下,DNA-[Ru(bpy)3]2+催化含酪氨酸的靶向肽形成含有酪氨酸自由基,两个酪氨酸自由基共价交联,自组装形成肽-核酸纳米球(RGD-NS)非病毒载体,该载体是直径约为200nm肽-核酸纳米球非病毒载体。该非病毒载体可实现对于DNA的良好包封,并在弗林酶的作用下水解,释放DNA-[Ru(bpy)3]2+,其中被保护的核酸ASO(Anti-sense single-strandedDNA oligonucleotide),其可通过碱基互补配对与靶标的mRNA结合,使得靶标RNA被RNaseH切割引发基因沉默,提高细胞对于药物的敏感性,另一方面释放的[Ru(bpy)3]2+在光照下产生羟基自由基,两者协同进行抗肿瘤治疗。该方法具有合成简单,高效且是毒性低等特点。
附图说明
图1为基于DNA-[Ru(bpy)3]2+催化短肽共价组装的非病毒载体的制备方法示意图;
图2为实施例1中的肽-核酸纳米球非病毒载体的形态和特性,其中,a)肽-核酸纳米球非病毒载体的SEM图;b)肽-核酸纳米球非病毒载体的TEM图;c)为肽-核酸纳米球非病毒载体的元素分布图像;d)肽-核酸纳米球的粒径分布图;
图3为(a)dsDNA与[Ru(bpy)3]Cl2孵育生成DNA-[Ru(bpy)3]2+复合物荧光图;(b)短肽溶液与过硫酸铵及DNA-[Ru(bpy)3]Cl2孵育生成纳米复合物过程中Zeta电势变化图;
图4为短肽溶液与过硫酸铵及DNA-[Ru(bpy)3]2+孵育生成荧光图及(b)不同时间下测试体系的紫外吸收图;
图5为纳米复合物及纳米复合物经弗林酶处理后的产物在200mw·cm-2的532nm光照射后产生羟基自由基的图;
图6为PBS、YY-12、DNA-[Ru(bpy)3]2+、肽-核酸纳米球分别处理在A549细胞,经AM/PI染色后的荧光成像图。
具体实施方式
下面结合实施例对本发明进行详细说明。
实施例中的各溶液是通过以下方法制备的:
DNA S1溶液和DNA S2溶液:分别将通过将DNA S1、DNA S2溶解在pH=7.4的10mM的Tris-HCl缓冲溶液中得到;
[Ru(bpy)3]Cl2溶液:将三联吡啶氯化釕溶解在超纯水溶液中得到;
短肽溶液:是将短肽溶解在超纯水中得到;
过硫酸铵溶液:将过硫酸铵溶解在超纯水中得到的。
实施例1
一种基于DNA-[Ru(bpy)3]2+催化短肽共价组装形成非病毒载体的制备方法,所述制备方法包括以下步骤:
(1)将等体积的浓度均为2μM的DNA S1溶液和DNA S2溶液加热到90℃,10分钟后取出,冷却至室温,随后保存在温度为4~10℃的冰箱上层至形成dsDNA结构,得到dsDNA溶液;
所述DNA S1的基因序列为:3’-GGA TTG GAG TTC CTC CAG CGT GCG CCA TCC TTC CCA TCCTCC TCC-5’;划横线的部分为可降低抗凋亡蛋白Bcl-2表达的ASO的序列,如果将其替换为可治疗其他疾病的核酸序列,同样可实现其他疾病的靶向治疗;
所述DNA S2的基因序列为:3’-GAG GAA CTC CAA TCC-5’;
(2)将1mL 2mM[Ru(bpy)3]Cl2溶液加入1mL的dsDNA溶液中,37℃孵育7h,形成DNA-[Ru(bpy)3]2+溶液;
(3)将0.2mL 1mg/ml短肽(YY-12)溶液与0.2mL DNA-[Ru(bpy)3]2+溶液及0.6mL10mM过硫酸铵溶液混合混匀,然后白光下照射6min,使用海门其林贝尔GL-800白光透射仪进行白光照射,得到肽-核酸纳米球(RGD-NS)溶液;所述短肽的氨基酸序列为YYRVRRRGDYY,其中Y为酪氨酸,RVRR为具有弗林酶切割位点多肽,RGD是一种靶向为多肽;
本实施例制备得到的非病毒载体的TEM、SEM、mapping图如图2所示,从其SEM、TEM,DLS图中可以看出其约为200nm肽-核酸纳米球。进一步对元素映射图像进行了探究,C、Ru和P等元素的也显示均匀分布。
实施例2
其他同实施例1,在进行步骤(2)时测试[Ru(bpy)3]Cl2、DNA-[Ru(bpy)3]2+的荧光光谱,如图3a)所示,生成DNA-[Ru(bpy)3]2+时625nm特征荧光峰强度的增加,证明DNA-[Ru(bpy)3]2+小分子的形成;同时在进行步骤(2-4)时进行Zeta电势的测量,如图3b)所示,当向[Ru(bpy)3]Cl2溶液中添加dsDNA溶液制备DNA-[Ru(bpy)3]2+时,DNA-[Ru(bpy)3]2+的Zeta电位绝对值变得比dsDNA小,这是由于[Ru(bpy)3]2+带正电荷,也进一步证明了[Ru(bpy)3]2+在dsDNA里的成功嵌入得到了DNA-[Ru(bpy)3]2+;当DNA-[Ru(bpy)3]2+与YY-12混合经光照射得到非病毒载体后,zeta电位进一步变的更正,这可能是由于酪氨酸共价交联聚精氨酸更多暴露在球外所致。
实施例3
其他同实施例1,在进行步骤(4)时测试YY-12、及反应得到的非病毒载体的荧光光谱,如图3a)所示,生成的肽-核酸纳米球的荧光峰为410nm,证明非病毒载体的形成;反应物混合均匀后,照射不同时间下取样测试体系的紫外吸收图,测试时间截止在600s,6min后峰值趋于平缓不再上升,此时的紫外吸光度值达到最大,也证明了短肽共价交联的成功,成功制备得到了非病毒载体。
实施例4
一种可释放所述非病毒载体中的DNA的方法,包括以下步骤:
将50μL质量浓度为0.1%弗林酶溶液加入到实施例1制备得到的肽-核酸纳米球溶液中,消化1h,加入EDTA终止弗林酶的活性。在532nm白光照射下,通过ESR分别检测非病毒载体、酶解产物中的羟基自由基。结果如图5所示,结果显示经弗林酶处理的溶液,有明显的羟基自由基信号;而对于相同浓度、相同辐照时间的肽-核酸纳米球溶液,信号微弱几乎可忽略不计。再次表明本发明制备得到的非病毒载体肽-核酸纳米球是一种弗林酶响应的纳米载体。
实施例5
为了研究肽-核酸纳米球的抗癌作用,进行了荧光成像展示。
将实施例1制备的非病毒载体肽-核酸纳米球中DNA的浓度调整为原来的10倍而其他原料的浓度保持不变进行包裹加入A549细胞中,用AM-PI对活/死细胞染色来进一步证实肽-核酸纳米球对A549细胞的光动力及基因治疗作用,以同等浓度的PBS、YY-12、NS为对照。共聚焦显微镜图像清楚地表明,在532nm光辐射下,经RGD-NS处理的细胞几乎全部凋亡,而DNA-[Ru(bpy)3]2+处理细胞的仅有部分细胞凋亡,这表明肽-核酸纳米球(RGD-NS)是一个良好的非病毒纳米载体。
上述实验中,肽-核酸纳米球与A549细胞共孵育,A549细胞中的高表达的弗林酶切割肽-核酸纳米球,释放出DNA-[Ru(bpy)3]2+复合物,其中与靶标的mRNA结合,使得靶标RNA被RNase H切割引发基因沉默,恢复癌细胞对于药物的敏感性,同时用白光照射,[Ru(bpy)3]2+产生羟基自由基损伤细胞,两者协同作用使得癌细胞凋亡。
上述参照实施例对一种基于三联吡啶釕催化短肽共价组装形成的非病毒载体及其制备方法和应用进行的详细描述,是说明性的而不是限定性的,可按照所限定范围列举出若干个实施例,因此在不脱离本发明总体构思下的变化和修改,应属本发明的保护范围之内。
Claims (8)
1.一种基于DNA-[Ru(bpy)3]2+催化短肽共价组装形成非病毒载体的制备方法,其特征在于,所述制备方法包括以下步骤:
(1)将反义寡核苷酸与三联吡啶氯化钌溶液混合,孵育得到DNA-[Ru(bpy)3]2+溶液;
(2)将步骤(1)得到的DNA-[Ru(bpy)3]2+溶液、含酪氨酸的靶向肽溶液、过硫酸铵溶液混合,进行白光照射,离心后即可得到所述非病毒载体;
所述非病毒载体为纳米球,所述非病毒载体实现了将核酸包封在交联体内,实现对核酸的运载;
所述含酪氨酸的靶向肽的氨基酸序列为YYRRVRRRGDYY;
步骤(1)中,所述反义寡核苷酸的制备方法为:将DNA S1溶液和DNA S2溶液混合,90℃退火10 min,并在4~10℃静置至形成双链DNA结构;
所述DNA S1基因序列为:
3’-GGA TTG GAG TTC CTC CAG CGT GCG CCA TCC TTC CCA TCC TCC TCC-5’;
所述DNA S2基因序列为:3’-GAG GAA CTC CAA TCC-5’。
2.根据权利要求1所述的制备方法,其特征在于,步骤(1)中,反应体系中,反义寡核苷酸、三联吡啶氯化钌的摩尔比为1:800~1200。
3.根据权利要求1所述的制备方法,其特征在于,步骤(1)中,所述孵育的条件为34~37℃孵育6.5~7.5 h。
4.根据权利要求1所述的制备方法,其特征在于,步骤(2)中,DNA-[Ru(bpy)3]2+溶液、含酪氨酸的靶向肽溶液、过硫酸铵溶液的体积之比为1:0.8~1.2:2.5~3.2;所述含酪氨酸的靶向肽溶液的浓度为0.5~1.5mg/mL;所述过硫酸铵溶液的浓度为8~12 mM。
5.根据权利要求1所述的制备方法,其特征在于,步骤(2)中,所述照射的时间为5~10min。
6.如权利要求1-5任意一项所述的制备方法制备得到的非病毒载体。
7.根据权利要求6所述的非病毒载体在制备基因传递药物或靶向药物中的应用。
8.一种基于非疾病诊断或治疗目的的释放权利要求6所述的非病毒载体中的DNA的方法,其特征在于,将弗林酶加入到所述非病毒载体中进行切割。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110818561.2A CN113549132B (zh) | 2021-07-20 | 2021-07-20 | 一种基于三联吡啶釕催化短肽共价组装形成的非病毒载体及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110818561.2A CN113549132B (zh) | 2021-07-20 | 2021-07-20 | 一种基于三联吡啶釕催化短肽共价组装形成的非病毒载体及其制备方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113549132A CN113549132A (zh) | 2021-10-26 |
CN113549132B true CN113549132B (zh) | 2023-11-24 |
Family
ID=78103780
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110818561.2A Active CN113549132B (zh) | 2021-07-20 | 2021-07-20 | 一种基于三联吡啶釕催化短肽共价组装形成的非病毒载体及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113549132B (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101353647A (zh) * | 2008-09-10 | 2009-01-28 | 中国人民解放军第三军医大学 | 靶向肿瘤的模拟逆转录病毒及其制备方法和应用 |
CN103710383A (zh) * | 2013-12-11 | 2014-04-09 | 深圳先进技术研究院 | 一种非病毒转基因载体、制备方法及其应用 |
CN103788211A (zh) * | 2012-11-01 | 2014-05-14 | 中国科学院上海药物研究所 | 双功能肽、所述双功能肽与核酸分子形成的复合物以及治疗肿瘤的药物组合物 |
-
2021
- 2021-07-20 CN CN202110818561.2A patent/CN113549132B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101353647A (zh) * | 2008-09-10 | 2009-01-28 | 中国人民解放军第三军医大学 | 靶向肿瘤的模拟逆转录病毒及其制备方法和应用 |
CN103788211A (zh) * | 2012-11-01 | 2014-05-14 | 中国科学院上海药物研究所 | 双功能肽、所述双功能肽与核酸分子形成的复合物以及治疗肿瘤的药物组合物 |
CN103710383A (zh) * | 2013-12-11 | 2014-04-09 | 深圳先进技术研究院 | 一种非病毒转基因载体、制备方法及其应用 |
Non-Patent Citations (1)
Title |
---|
Peptide–Ruthenium Conjugate as an Efficient Photosensitizer for the Inactivation of Multidrug-Resistant Bacteria;Scott Pierce等;《Inorganic Chemistry》;20200929;第14866–14870页 * |
Also Published As
Publication number | Publication date |
---|---|
CN113549132A (zh) | 2021-10-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107614685B (zh) | Rna纳米颗粒及其使用方法 | |
Joo et al. | Porous silicon–graphene oxide core–shell nanoparticles for targeted delivery of siRNA to the injured brain | |
Ding et al. | A self-assembled RNA-triple helix hydrogel drug delivery system targeting triple-negative breast cancer | |
Lee et al. | Stability and cellular uptake of polymerized siRNA (poly-siRNA)/polyethylenimine (PEI) complexes for efficient gene silencing | |
CN102666879B (zh) | 模板化的纳米缀合物 | |
Yang et al. | Gold nanoparticle based fluorescent oligonucleotide probes for imaging and therapy in living systems | |
US11155830B2 (en) | Preparation and use of nanoparticle-doped RNA hydrogel targeting to triple negative breast cancer | |
JP5969164B2 (ja) | 電荷結合と生分解性共有結合により同時に連結された高分子−siRNAナノ粒子担体及びその製造方法 | |
US20130101512A1 (en) | Crosslinked polynucleotide structure | |
Kovtun et al. | Calcium phosphate nanoparticles for the transfection of cells | |
Xu et al. | DNA Origami Frameworks Enabled Self‐Protective siRNA Delivery for Dual Enhancement of Chemo‐Photothermal Combination Therapy | |
CN106929508B (zh) | 一种激活PTPRO基因表达的saRNA及其转运载体 | |
Cui et al. | DNA‐based pH‐responsive core–shell drug nanocarrier for tumor‐targeted chemo‐photodynamic therapy | |
US20140128451A1 (en) | Compositions and Methods for Delivery of MicroRNA to Cells | |
Gazori et al. | Inhibition of EGFR expression with chitosan/alginate nanoparticles encapsulating antisense oligonucleotides in T47D cell line using RT-PCR and immunocytochemistry | |
Luo et al. | A dynamic DNA nanosponge for triggered amplification of gene-photodynamic modulation | |
Chen et al. | Polyethylenimine modified graphene oxide for effective chemo-gene-photothermal triples therapy of triple-negative breast cancer and inhibits metastasis | |
Asakiya et al. | Current progress of miRNA-derivative nucleotide drugs: modifications, delivery systems, applications | |
CN106467915A (zh) | 小干扰RNA及其应用和抑制plk1基因表达的方法 | |
Zhou et al. | Upconverting nanoparticles based nanodevice for DNAzymes amplified miRNAs detection and artificially controlled chemo-gene therapy | |
CN113549132B (zh) | 一种基于三联吡啶釕催化短肽共价组装形成的非病毒载体及其制备方法和应用 | |
CN113941010A (zh) | 一种协同no气体治疗并增强声动力治疗效果的纳米粒及其制备方法与应用 | |
CN111249469B (zh) | 一种能够溶酶体逃逸的肽纳米颗粒及其制备方法和应用 | |
CN105267983B (zh) | 一种iNGR修饰的脑胶质瘤靶向自组装RNAi纳米给药系统及其制备方法 | |
Li et al. | Construction of Smart DNA‐Based Drug Delivery Systems for Cancer Therapy |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |