CN111265669A - 一种核酸转运用载体组合物及其在制备小干扰rna药物中的应用 - Google Patents
一种核酸转运用载体组合物及其在制备小干扰rna药物中的应用 Download PDFInfo
- Publication number
- CN111265669A CN111265669A CN202010246551.1A CN202010246551A CN111265669A CN 111265669 A CN111265669 A CN 111265669A CN 202010246551 A CN202010246551 A CN 202010246551A CN 111265669 A CN111265669 A CN 111265669A
- Authority
- CN
- China
- Prior art keywords
- sirna
- nucleic acid
- small interfering
- interfering rna
- carrier composition
- 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.)
- Granted
Links
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 72
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 72
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 72
- 239000000203 mixture Substances 0.000 title claims abstract description 40
- 108020004459 Small interfering RNA Proteins 0.000 title claims abstract description 32
- 239000003814 drug Substances 0.000 title claims abstract description 32
- 229940079593 drug Drugs 0.000 title claims abstract description 25
- 239000004055 small Interfering RNA Substances 0.000 title claims abstract description 23
- 238000012546 transfer Methods 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title abstract description 13
- 229920002477 rna polymer Polymers 0.000 title description 4
- 229920001610 polycaprolactone Polymers 0.000 claims abstract description 26
- 239000004632 polycaprolactone Substances 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 18
- 229920000747 poly(lactic acid) Polymers 0.000 claims abstract description 16
- 239000004626 polylactic acid Substances 0.000 claims abstract description 16
- 239000004698 Polyethylene Substances 0.000 claims abstract description 12
- -1 polyethylene Polymers 0.000 claims abstract description 12
- 229920000573 polyethylene Polymers 0.000 claims abstract description 12
- 238000004945 emulsification Methods 0.000 claims abstract description 10
- 238000011282 treatment Methods 0.000 claims description 21
- 229920000962 poly(amidoamine) Polymers 0.000 claims description 19
- 239000002245 particle Substances 0.000 claims description 13
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical group ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 12
- 239000000839 emulsion Substances 0.000 claims description 12
- 239000007864 aqueous solution Substances 0.000 claims description 10
- 238000009210 therapy by ultrasound Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000003960 organic solvent Substances 0.000 claims description 6
- 230000002829 reductive effect Effects 0.000 claims description 6
- 239000002105 nanoparticle Substances 0.000 abstract description 39
- 230000014509 gene expression Effects 0.000 abstract description 18
- 108090000623 proteins and genes Proteins 0.000 abstract description 15
- 238000012377 drug delivery Methods 0.000 abstract description 9
- 206010006187 Breast cancer Diseases 0.000 abstract description 5
- 208000026310 Breast neoplasm Diseases 0.000 abstract description 5
- 238000001727 in vivo Methods 0.000 abstract description 5
- 230000012010 growth Effects 0.000 abstract description 3
- 238000002156 mixing Methods 0.000 abstract description 3
- 230000001717 pathogenic effect Effects 0.000 abstract description 3
- 201000010099 disease Diseases 0.000 abstract description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 33
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 26
- 206010028980 Neoplasm Diseases 0.000 description 18
- 239000000243 solution Substances 0.000 description 17
- 229920000642 polymer Polymers 0.000 description 16
- 229920001223 polyethylene glycol Polymers 0.000 description 13
- 239000002202 Polyethylene glycol Substances 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 238000000527 sonication Methods 0.000 description 11
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 9
- 239000008194 pharmaceutical composition Substances 0.000 description 9
- 108010056274 polo-like kinase 1 Proteins 0.000 description 9
- 238000003756 stirring Methods 0.000 description 9
- 108091030071 RNAI Proteins 0.000 description 8
- 230000009368 gene silencing by RNA Effects 0.000 description 8
- 239000012071 phase Substances 0.000 description 8
- 239000000463 material Substances 0.000 description 7
- 108020004999 messenger RNA Proteins 0.000 description 7
- 101150073897 plk1 gene Proteins 0.000 description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- 108091034117 Oligonucleotide Proteins 0.000 description 6
- 125000002091 cationic group Chemical group 0.000 description 6
- 230000002209 hydrophobic effect Effects 0.000 description 6
- 108091070501 miRNA Proteins 0.000 description 6
- 239000002861 polymer material Substances 0.000 description 6
- 230000004614 tumor growth Effects 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 230000030279 gene silencing Effects 0.000 description 5
- 239000000693 micelle Substances 0.000 description 5
- 238000011580 nude mouse model Methods 0.000 description 5
- RKDVKSZUMVYZHH-UHFFFAOYSA-N 1,4-dioxane-2,5-dione Chemical compound O=C1COC(=O)CO1 RKDVKSZUMVYZHH-UHFFFAOYSA-N 0.000 description 4
- 229920001400 block copolymer Polymers 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 230000002452 interceptive effect Effects 0.000 description 4
- CTSLXHKWHWQRSH-UHFFFAOYSA-N oxalyl chloride Chemical compound ClC(=O)C(Cl)=O CTSLXHKWHWQRSH-UHFFFAOYSA-N 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 102000053642 Catalytic RNA Human genes 0.000 description 3
- 108090000994 Catalytic RNA Proteins 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108091093037 Peptide nucleic acid Proteins 0.000 description 3
- 238000010806 PrimeScriptTM RT Reagent kit Methods 0.000 description 3
- 239000000074 antisense oligonucleotide Substances 0.000 description 3
- 238000012230 antisense oligonucleotides Methods 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000001647 drug administration Methods 0.000 description 3
- 239000003999 initiator Substances 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- JJTUDXZGHPGLLC-UHFFFAOYSA-N lactide Chemical compound CC1OC(=O)C(C)OC1=O JJTUDXZGHPGLLC-UHFFFAOYSA-N 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 108091092562 ribozyme Proteins 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- JJTUDXZGHPGLLC-IMJSIDKUSA-N 4511-42-6 Chemical compound C[C@@H]1OC(=O)[C@H](C)OC1=O JJTUDXZGHPGLLC-IMJSIDKUSA-N 0.000 description 2
- OEOIWYCWCDBOPA-UHFFFAOYSA-N 6-methyl-heptanoic acid Chemical compound CC(C)CCCCC(O)=O OEOIWYCWCDBOPA-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 108700016481 Acute Hepatic Porphyria Proteins 0.000 description 2
- 208000003914 Acute hepatic porphyria Diseases 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 239000013614 RNA sample Substances 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 239000012736 aqueous medium Substances 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 229920001577 copolymer Polymers 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 210000004292 cytoskeleton Anatomy 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000009509 drug development Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000009881 electrostatic interaction Effects 0.000 description 2
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 208000033552 hepatic porphyria Diseases 0.000 description 2
- 239000005457 ice water Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 230000001743 silencing effect Effects 0.000 description 2
- 229940126586 small molecule drug Drugs 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 239000012581 transferrin Substances 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- NGNBDVOYPDDBFK-UHFFFAOYSA-N 2-[2,4-di(pentan-2-yl)phenoxy]acetyl chloride Chemical compound CCCC(C)C1=CC=C(OCC(Cl)=O)C(C(C)CCC)=C1 NGNBDVOYPDDBFK-UHFFFAOYSA-N 0.000 description 1
- KDELTXNPUXUBMU-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid boric acid Chemical compound OB(O)O.OB(O)O.OB(O)O.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KDELTXNPUXUBMU-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 239000012103 Alexa Fluor 488 Substances 0.000 description 1
- 206010006189 Breast cancer in situ Diseases 0.000 description 1
- 241000238860 Chrysiogenetes <class> Species 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- LCGLNKUTAGEVQW-UHFFFAOYSA-N Dimethyl ether Chemical compound COC LCGLNKUTAGEVQW-UHFFFAOYSA-N 0.000 description 1
- 241001411320 Eriogonum inflatum Species 0.000 description 1
- 101150112014 Gapdh gene Proteins 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000835093 Homo sapiens Transferrin receptor protein 1 Proteins 0.000 description 1
- 101000772194 Homo sapiens Transthyretin Proteins 0.000 description 1
- 239000012098 Lipofectamine RNAiMAX Substances 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 206010036105 Polyneuropathy Diseases 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 102000007238 Transferrin Receptors Human genes 0.000 description 1
- 108010033576 Transferrin Receptors Proteins 0.000 description 1
- 102100026144 Transferrin receptor protein 1 Human genes 0.000 description 1
- 102100029290 Transthyretin Human genes 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- GCYZRQQEBNFQTK-UHFFFAOYSA-N [2,3-dihydroxypropoxy(hydroxy)phosphoryl] phosphono hydrogen phosphate Chemical compound OCC(O)COP(O)(=O)OP(O)(=O)OP(O)(O)=O GCYZRQQEBNFQTK-UHFFFAOYSA-N 0.000 description 1
- YUWBVKYVJWNVLE-UHFFFAOYSA-N [N].[P] Chemical compound [N].[P] YUWBVKYVJWNVLE-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 150000008065 acid anhydrides Chemical class 0.000 description 1
- 150000001263 acyl chlorides Chemical class 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- SMZOGRDCAXLAAR-UHFFFAOYSA-N aluminium isopropoxide Chemical compound [Al+3].CC(C)[O-].CC(C)[O-].CC(C)[O-] SMZOGRDCAXLAAR-UHFFFAOYSA-N 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 229920000469 amphiphilic block copolymer Polymers 0.000 description 1
- 206010002022 amyloidosis Diseases 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 238000010533 azeotropic distillation Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 201000005389 breast carcinoma in situ Diseases 0.000 description 1
- 230000005907 cancer growth Effects 0.000 description 1
- DUEPRVBVGDRKAG-UHFFFAOYSA-N carbofuran Chemical compound CNC(=O)OC1=CC=CC2=C1OC(C)(C)C2 DUEPRVBVGDRKAG-UHFFFAOYSA-N 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000000412 dendrimer Substances 0.000 description 1
- 229920000736 dendritic polymer Polymers 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 229950010941 givosiran Drugs 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 229920001519 homopolymer Polymers 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 229920001427 mPEG Polymers 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000002539 nanocarrier Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- ZPIRTVJRHUMMOI-UHFFFAOYSA-N octoxybenzene Chemical compound CCCCCCCCOC1=CC=CC=C1 ZPIRTVJRHUMMOI-UHFFFAOYSA-N 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229950005564 patisiran Drugs 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 description 1
- 230000007824 polyneuropathy Effects 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000007142 ring opening reaction Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000012265 solid product Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- IUTCEZPPWBHGIX-UHFFFAOYSA-N tin(2+) Chemical compound [Sn+2] IUTCEZPPWBHGIX-UHFFFAOYSA-N 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- PAPBSGBWRJIAAV-UHFFFAOYSA-N ε-Caprolactone Chemical compound O=C1CCCCCO1 PAPBSGBWRJIAAV-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- 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
- A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/595—Polyamides, e.g. nylon
-
- 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
- A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Inorganic Chemistry (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Gynecology & Obstetrics (AREA)
- Pregnancy & Childbirth (AREA)
- Endocrinology (AREA)
- Reproductive Health (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
本发明公开了一种核酸转运用载体组合物及其在制备小干扰RNA药物中的应用。该组合物是由聚乙二醇‑聚乳酸及(聚酰胺‑胺)‑2‑丙酸‑3‑甲基马来酸酐‑聚己内酯两者混合均匀得到的。本发明将siRNA加入所述载体组合物溶液中,通过双乳化的方法得到siRNA与纳米颗粒的复合体,得到小干扰RNA药物。本发明不但可以制备出能够高效结合siRNA的纳米颗粒,降低siRNA与纳米颗粒完全结合所需最低NP比。而且该小干扰RNA药物能有效进入细胞,有效沉默致病靶基因的表达,并在体内抑制乳腺癌的生长。因而此给药体系在siRNA和类似小核酸药物的给药用于疾病治疗中具有良好的前景。
Description
技术领域
本发明属于小干扰RNA药物领域,具体涉及一种核酸转运用载体组合物及其在制备小干扰RNA药物中的应用。
背景技术
小干扰RNA能够简单高效地沉默靶基因的表达,因此成为研究基因功能与疾病治疗的重要工具。和小分子药物相比,siRNA能够特异性下调致病基因的表达,并不影响细胞中正常基因表达,这与传统的小分子药物相比更具有治疗潜力。2018年8月,Alnylam公司的Onpattro(patisiran)获美国和欧盟批准,用于遗传性ATTR(hATTR)淀粉样变性多发性神经病的治疗,成为RNAi现象被发现整整20年以来获准上市的首款RNAi药物,这标志着基因药物开发的一个重大里程碑。2019年11月,Alnylam公司的另一款RNAi药物Givlaari(givosiran)成为获得FDA批准的全球第二款RNAi药物,该药通过皮下注射给药,治疗急性肝卟啉症(AHP)成人患者,鼓舞了RNAi相关药物研发领域的热情。
然而,siRNA在人体内的给药面临着巨大挑战。由于siRNA分子在血液中不稳定,容易被机体清除,穿透细胞膜的能力极差等缺点,如何设计siRNA的给药系统是RNAi疗法相关研究的焦点。RNAi传递的细胞内障碍。siRNA的载体设计要求可分为三个部分:1)有效荷载并保护siRNA;2)将荷载siRNA的系统递送到靶点细胞;3)siRNA在细胞质中的有效释放。
目前用于siRNA给药系统构建的材料主要包括阳离子胶束、阳离子脂质体和阳离子高分子聚合物等大类。这些载体与天然电负性的siRNA通过电荷相互作用形成复合物,从而实现荷载siRNA的目的。然而为实现高效的siRNA荷载率,一般研究中的氮磷比(NP比),即阳离子材料和siRNA的电荷比会比较高。而高NP比会增加阳离子材料消耗,升高给药系统表面正电性而降低体内稳定性,使载体更容易被免疫系统清除,并导致一定的毒性作用。如何使载体系统能够高效荷载siRNA且降低阳离子材料与siRNA的比例,是相关研究的一个重难点。例如由Calando制药公司开发的siRNA药物CALAA-01,是第一个用于RNAi疗法肿瘤临床试验的聚合物类纳米载体。在CALAA-01的几个主要成分中,CDP能提供正电荷与siRNA通过电荷作用形成颗粒内核;金刚烷-PEG(AD-PEG)与金刚烷-PEG-转铁蛋白(AD-PEG-Tf)形成颗粒主要结构,其中PEG能够提高颗粒在体内循环中的稳定性,转铁蛋白Tf能够与肿瘤表面高表达的转铁蛋白受体CD71特异性结合,增强递送体系的肿瘤靶向能力。但是,由于少数患者的毒性反应,该体系已于2014年终止临床实验(KanastyR,Dorkin J R,Vegas A,etal.Delivery materials for siRNA therapeutics[J].Nature Materials,2013,12(11):967-977)。
发明内容
为了克服现有技术存在的上述不足,本发明的目的是提供一种核酸转运用载体组合物及其在制备小干扰RNA药物中的应用。
本发明的目的至少通过如下技术方案之一实现。
本发明的目的是提供一种核酸药物给药载体,具体涉及一种利用两种两亲性高分子聚合物材料共同制备的核酸药物的给药载体、载体制备方法和药物组合物。本发明提供的给药载体组合物具有很高的siRNA结合效率。
本发明提供的核酸转运用载体组合物是由两种两亲性高分子聚合物PEG-PLGA(聚乙二醇-聚乳酸)和PAMAM-CDM-PCL((聚酰胺-胺)-2-丙酸-3-甲基马来酸酐-聚己内酯)共同制备的小干扰RNA(siRNA)的给药系统和制剂。
本发明将siRNA加入到两种两亲性高分子材料PEG-PLGA和PAMAM-CDM-PCL的溶液中,通过双乳化的方法得到siRNA与纳米颗粒的复合体。这种方法不但可以制备高效结合siRNA纳米颗粒,而且纳米颗粒能有效进入细胞,有效沉默致病靶基因的表达,并在体内抑制乳腺癌的生长。
本发明提供的核酸转运用载体组合物是一种利用两亲性聚合物通过双乳化制备得到能够包载siRNA的给药系统。
本发明提供的一种核酸转运用载体组合物,按重量份数计,包括:
聚乙二醇-聚乳酸 100份;
(聚酰胺-胺)-2-丙酸-3-甲基马来酸酐-聚己内酯 11-55份。
进一步地,所述聚乙二醇-聚乳酸中,聚乙二醇的嵌段分子量为3000-7000,聚乳酸的嵌段分子量为9000-13000。
进一步地,所述(聚酰胺-胺)-2-丙酸-3-甲基马来酸酐-聚己内酯中,聚酰胺-胺的代数为四代,聚酰胺-胺的分子量为14215,聚己内酯的分子量为3000-5000。
进一步地,其粒径为90-140纳米。所述核酸转运用载体组合物为纳米颗粒,可以在其纳米颗粒表明进行化学修饰、抗体修饰或配体修饰。
本发明提供的小干扰RNA药物,包含所述的核酸转运用载体组合物和小干扰RNA(siRNA)。
本发明提供的一种制备所述小干扰RNA药物的方法,包括如下步骤:
(1)将聚乙二醇-聚乳酸和(聚酰胺-胺)-2-丙酸-3-甲基马来酸酐-聚己内酯加入油相中,混合均匀,得到所述核酸转运用载体组合物的溶液,然后加入siRNA水溶液,超声处理,得到初始乳液;
(2)将步骤(1)初始乳液加入水相中,超声乳化处理,然后减压去除有机溶剂,得到所述药物组合物的水溶液。
进一步地,步骤(1)所述聚乙二醇-聚乳酸和(聚酰胺-胺)-2-丙酸-3-甲基马来酸酐-聚己内酯的质量比为100:11-55;所述油相为氯仿;所述聚乙二醇-聚乳酸与油相的质量体积比为3-5:1mg/mL。
进一步地,步骤(1)所述siRNA水溶液的浓度为2-3mg/mL;所述油相与siRNA水溶液的体积比为15-25:1。
进一步地,步骤(1)所述超声处理的时间为0.5-1.5分钟,超声处理的功率为60-100W。
进一步地,步骤(2)所述超声乳化处理的时间为0.5-1.5分钟,超声乳化处理的功率为60-100W。
优选地,步骤(2)所述减压去除有机溶剂的压力为1000帕。
优选地,步骤(2)所述初始乳液与水相的体积比为1:8
本发明提供的药物组合物,可以通过与细胞接触,使核酸导入到细胞内。
所述核酸为小干扰核酸(siRNA)。
本发明提供的药物组合物(两种两亲性高分子聚合物的组合)可以应用在制备核酸转运用载体组合物中。
所述核酸转运用载体组合物可以应用在制备抗肿瘤药物中的应用。所述肿瘤优选为乳腺肿瘤。
本发明提供的核酸药物给药载体由两种两亲性高分子聚合物材料共同制备而成。所述两亲性高分子聚合物是指在一个大分子链上同时含有亲水性和疏水性链段。其中一种两亲性共聚物为PEG-PLGA,其中PEG(聚乙二醇)的数均分子量为5000g/mol,PLGA(聚乳酸)数均分子量为11000g/mol。这种嵌段共聚物能够在水介质中自组装成胶束或纳米粒,相对疏水性的PLGA聚集成疏水性的核,PEG嵌段组装成亲水性的壳,具有稳定胶束、有效躲避生物体内质网系统的捕捉和蛋白质吸附的作用。另一种两亲性聚合物为PAMAM-CDM-PCL,PAMAM(聚酰胺-胺)的代数为四代,PCL(聚己内酯)的数均分子量为4000g/mol,PAMAM和PCL通过一个小分子CDM相结合。PAMAM的表面具有许多氨基,在水溶液中能够质子化从而带正电,与带负电的siRNA通过静电相互作用形成复合物,在此给药系统中的主要作用是通过提高核酸药物与载体的结合效率,降低siRNA与纳米颗粒完全结合所需最低NP比。
由上述两种两亲性高分子材料共同制备的给药载体能够形成纳米颗粒,所述纳米颗粒的直径约为130nm。
本发明的核酸转运用载体组合物适用的核酸,对其种类或结构没有特殊的限定。作为该核酸的具体例,可以为siRNA、mRNA、tRNA、rRNA、cDNA、miRNA(微RNA)、核酶、反义寡核普酸、质粒DNA、肽核酸、三链形成型寡核普酸(Triplex Forming Oligonucleotide,TFO)、基因等。其中,本发明的核酸转运用载体组合物在将siRNA向细胞内转运方面特别有效。本发明的核酸转运用载体适用的核酸,可以是来自人、动物、植物、细菌、病毒等的核酸,另外,也可以是通过化学合成制备的核酸。进而,上述核酸可以是单链、双链、三链中的任一种,并且对其分子量也没有特殊的限定。另外,本发明中,核酸可以为被化学、酶或肽修饰的核酸。
本发明中,核酸可以单独使用1种,也可以2种以上适当地组合使用。在一较佳的实施方式中,本发明的核酸转运用载体组合物优选转运小干扰核酸(siRNA)或其类似物。
本发明还提供了一种药物组合物,该组合物包含上述的核酸转运用载体组合物及核酸。药物组合物中适用的核酸,对其种类或结构没有特殊的限定。作为该核酸的具体例,可以为siRNA、mRNA、tRNA、rRNA、cDNA、miRNA(微RNA)、核酶、反义寡核普酸、质粒DNA、肽核酸、三链形成型寡核普酸(Triplex Forming Oligonucleotide,TFO)、基因等。在一较佳的实施方式中,优选为小干扰核酸(siRNA)。
本发明还提供了上述药物组合物的制备方法,所述方法包括如下步骤:将两种两亲性高分子材料溶于油相(氯仿等)中,加入siRNA水溶液后超声(80瓦,1分钟)形成初始乳液,将初始乳液加入到水相中并再次超声(80瓦,1分钟)乳化,将乳液减压(1000帕)除去有机溶剂,得到纳米颗粒水溶液。
本发明还提供了一种核酸导入方法,通过使上述药物组合物与细胞接触,将核酸导入到细胞内。所述细胞优选为哺乳动物细胞,更优选为病理状态下或非正常生理状态下的哺乳动物细胞,所述核酸优选为小干扰核酸(siRNA)。
与现有技术相比,本发明具有如下优点和有益效果:
(1)本发明提供的核酸转运用载体组合物,其具有良好的稳定性,制备方法简单,siRNA结合效率高,能保护siRNA免于降解,并能将核酸药物高效率地转运至细胞内,沉默靶点基因,达到相应治疗效果;
(2)本发明提供的核酸转运用载体组合物,能够输送特异性siRNA,在细胞和动物水平证明了其沉默靶基因表达的功效,以及沉默癌基因Plk1表达并抑制乳腺癌生长的效果。
附图说明
图1为实施例1中用双乳化方法和纳米沉淀法制备得到的核酸转运用载体组合物的电泳图;
图2为实施例1的核酸转运用载体组合物粒径图;
图3为荷载Cy5-siRNA的核酸转运用载体组合物与MDA-MB-231细胞培养5小时后在细胞内分布的激光共聚焦显微镜照片;
图4为荷载siPlk1的核酸转运用载体组合物进入MDA-MB-231细胞后下调Plk1的mRNA水平的效果图;
图5为尾静脉注射包载siPlk1的的靶向给药系统抑制小鼠原位植入乳腺癌生长的效果图;
图6为治疗实验结束后降低肿瘤组织中Plk1的mRNA水平的效果图。
具体实施方式
以下结合实例对本发明的具体实施作进一步说明,但本发明的实施和保护不限于此。需指出的是,以下若有未特别详细说明之过程,均是本领域技术人员可参照现有技术实现或理解的。所用试剂或仪器未注明生产厂商者,视为可以通过市售购买得到的常规产品。
本发明所述的核酸转运用载体组合物由两种两亲性高分子聚合物材料共同制备而成。所述两亲性高分子聚合物是指在一个大分子链上同时含有亲水性和疏水性链段。其中一种两亲性共聚物为PEG-PLGA,其中PEG(聚乙二醇)的数均分子量为5000g/mol,PLGA(聚乳酸)数均分子量为11000g/mol。这种嵌段共聚物能够在水介质中自组装成胶束或纳米粒,相对疏水性的PLGA聚集成疏水性的核,PEG嵌段组装成亲水性的壳,具有稳定胶束、有效躲避生物体内质网系统的捕捉和蛋白质吸附的作用。另一种两亲性聚合物为PAMAM-CDM-PCL,PAMAM(聚酰胺-胺)的代数为四代,PCL(聚己内酯)的数均分子量为4000g/mol,PAMAM和PCL通过一个小分子CDM(2-丙酸-3-甲基马来酸酐)相结合。PAMAM的表面具有许多氨基,在水溶液中能够质子化从而带正电,与带负电的siRNA通过静电相互作用形成复合物,在此给药系统中的主要作用是通过提高核酸药物与载体的结合效率,降低siRNA与纳米颗粒完全结合所需最低NP比(PAMAM表面正电荷与siRNA负电荷之比)。
本发明的核酸转运用载体组合物适用的核酸,对其种类或结构没有特殊的限定。作为该核酸的具体例,可以为siRNA、mRNA、tRNA、rRNA、cDNA、miRNA(微RNA)、核酶、反义寡核普酸、质粒DNA、肽核酸、三链形成型寡核酸(Triplex Forming Oligonucleotide,TFO)、基因等。其中,本发明的核酸转运用载体组合物在将siRNA向细胞内转运方面特别有用。本发明的核酸转运用载体组合物适用的核酸,可以是来自人、动物、植物、细菌、病毒等的核酸,另外,也可以是通过化学合成制备的核酸。进而,上述核酸可以是单链、双链、三链中的任一种,并且对其分子量也没有特殊的限定。另外,本发明中,核酸可以为被化学、酶或肽修饰的核酸。本发明中,核酸可以单独使用1种,也可以2种以上适当地组合使用。
通过双乳化方法制备负载siRNA的纳米颗粒(即所述小干扰RNA药物),具体方法包括:将聚合物PEG5000-PLGA(2.25mg)和不同质量(参考表1)的PAMAM-CDM-PCL溶于0.5mL氯仿中,加入FAM-siRNA(0.025mL,0.055mg)溶液后超声下(超声功率80瓦,每超声五秒停止两秒,共超声一分钟)形成初始乳液后,将初始乳液加入到5mL DEPC水溶液中并再次超声乳化(声功率80瓦,每超声十秒停止两秒,共超声一分钟),减压下(1000帕)挥发有机溶剂,收集颗粒溶液。
实施例中所用原料来源及处理方法:
丙交酯(D,L-Lactide;CAS:4511-42-6),购自济南岱罡生物工程有限公司,使用前用升华瓶在真空下加热至90℃升华纯化,40℃下抽真空干燥12小时;
乙交酯(Glycolide;CAS:02-97-6)购自济南岱罡生物工程有限公司,使用前使用乙酸乙酯重结晶3次,溶剂质量比依次为70%,90%,130%,40℃下抽真空干燥12小时;
异辛酸亚锡(Tin(II)2-ethylhexanoate;CAS:301-10-0)购自Sigma-Aldrich,于手套箱中保存和取用。
单甲醚的聚乙二醇5000(mPEG5K;CAS:9004-74-4)购买自百灵威,使用前经甲苯135℃常压共沸除水(1g/10mL),随后降温至70℃抽真空搅拌干燥12h;
二氯甲烷(DCM;CAS:75-09-2),购自国药试剂,HPLC级别DCM经MB-SPS溶剂净化系统纯化后接入手套箱内直接取用PEG5K-PLGA11K为本发明合成,在手套箱内称取单体和引发剂固体于干燥圆底烧瓶中,丙交酯和乙交酯物质的量之比为3:1,由目标聚合物的分子量计算投料比,考虑单体活性及反应损失,相对引发剂丙交酯投入1.2倍过量,乙交酯投入1.1倍过量。将烧瓶置于油浴中125℃加热,并缓慢搅拌约20min后引发剂和单体完全熔化混匀,搅拌过程中避免混合液飞溅到瓶壁上;确认无固态反应物后,用玻璃滴管取一滴(5-10mg)异辛酸亚锡,于烧瓶口正上方小心滴入,塞上瓶塞。反应持续约30min,在搅拌过程中体系粘稠度逐渐增大,可以适当减小转速以维持体系搅拌,待搅拌子无法正常搅拌后,取下反应体系,迅速取出手套箱并置于液氮中淬冷以终止反应。向冷却的体系中加入适量二氯甲烷,震荡溶解,旋蒸浓缩体系至20mL左右,将粘稠的浓缩液滴入0℃,5%甲醇的无水乙醚中搅拌沉淀,过滤,干燥,最终得到白色固体聚合物。
羟基末端聚己内酯(PCL-OH)均聚物为本发明合成,三异丙醇铝(0.15g,1.0eqv)溶解于2mL无水甲苯中,加入到己内酯(10.0g,125.0eqv)中并在室温下反应1h,然后加入1mL冰醋酸并继续搅拌12小时。将得到的产物用二氯甲烷溶解并沉淀至冰乙醚中,真空下干燥12h后得到白色固体产物,
PCL-CDM为本发明合成。合成方法包括:将CDM(2-丙酸-3-甲基马来酸酐,60mg,1.0eqv)溶解于4mL无水二氯甲烷中,再依次加入草酰氯(52mg,1.25eqv)、N,N-二甲基甲酰胺(DMF,80μL)。上述反应先置于冰水浴中反应10min,然后转移至室温反应2h。在真空条件下除去二氯甲烷、N,N-二甲基甲酰胺和过量的草酰氯,得到酰氯化CDM中间产物。将酰氯化CDM溶解于4mL无水二氯甲烷中,将预先通过甲苯共沸除水干燥后的PCL-OH(0.20g,0.33eqv)溶解于3mL无水二氯甲烷,置于干燥的恒压滴液漏斗于冰水浴中缓慢加入,滴加完毕后转移至室温继续反应2h。加入饱和氯化铵水溶液(20mL)消耗过量的酰氯化CDM后,用氯仿萃取三次(20mL×3),干燥浓缩有机相后,沉淀至冰乙醚中,真空下干燥12h后得到浅棕色固体产物。
第四代聚酰胺-胺树枝状高分子(PAMAM,G4,Mn=14,215g/mol)购自美国Dendritech公司。
PCL-CDM-PAMAM通过PCL-CDM中酸酐与PAMAM/Pt表面氨基的开环反应合成。PAMAM(142mg,1.0eqv)与PCL-CDM(40mg,1.0eqv)溶解于5mL二甲亚砜中,并在避光条件下于室温下搅拌2h。于搅拌条件下加入20mL超纯水使产物组装成纳米颗粒,进而通过超滤(MWCO=100,000)进行纯化,收集上层溶液并冻干,得到白色固体。
Alexa 488-phalloidin、LipofectamineRNAiMAX购于Invitrogen公司。
RNAiso Plus、PrimeScriptTM RT reagent Kit(Perfect Real Time)购自TaKaRa公司。
FastStart Universal SYBR Green Master(ROX)购自Roche公司。
siRNA是一种由二十多个核苷酸组成的双链小分子RNA,带有负电荷。
以下实验使用中的siPlk1,对应反义链序列为:
UAAGGAGGGUGAUCUUCUUCAdTdT。
对照阴性siRNA(siN.C.),对应反义链序列为:
AACCACUCAACUUUUUCCCAAdTdT。
Cy5-siRNA为荧光染料Cy5标记的siN.C.。FAM-siRNA为荧光染料FAM标记的siN.C.。
以上siRNA均由苏州瑞博制药技术有限公司合成。
未经说明的其它试剂直接使用。
下述实施例中的实验方法,如无特殊说明,均为常规方法。
实施例一.siRNA给药系统的制备与表征
使用两亲性嵌段共聚物PEG-PLGA和两亲性聚合物PAMAM-CDM-PCL,利用超声双乳化的方法制备负载siRNA的纳米颗粒。
1.不同NP比下颗粒与siRNA结合能力
制备负载siRNA的纳米颗粒,具体方法为:将聚合物PEG-PLGA(2.25mg)和不同质量(参考表1)的PAMAM-CDM-PCL分别溶于0.5mL氯仿中,加入siRNA(0.025mL,0.055mg)溶液后超声下(超声功率80瓦,每超声五秒停止两秒,共超声一分钟)形成初始乳液后,将初始乳液加入到5mL DEPC水中并再次超声乳化(声功率80瓦,每超声十秒停止两秒,共超声一分钟),减压下(1000帕)挥发有机溶剂,并将颗粒溶液浓缩至1ml。
表1.不同NP比的纳米颗粒制备中各组分的投料量
得到不同NP比的颗粒后,通过凝胶电泳检测siRNA的结合。每种颗粒取20μL加入4μL6×loading buffer(擎科,TSJ010),混匀后将含20pmol siRNA的上述溶液于2%琼脂糖凝胶、100V下和三羟甲基氨基甲烷-硼酸-乙二胺四乙酸缓冲液中电泳15分钟。siRNA条带在凝胶成像仪下检测。
从图1可见,纳米颗粒(即所述核酸转运用载体组合物)可以在NP比等于4的时候完全结合siRNA。
2.纳米颗粒表征
以NP比为4的纳米颗粒(即所述核酸转运用载体组合物)为实例说明其性质。利用马尔文Malvern Zetasizer Nano ZSE纳米粒度电位仪测量颗粒的粒径。
从图2可见,得到的纳米颗粒粒径强度统计在130nm左右。
实施例二.此给药系统在细胞水平的效应评价
NP比为4时,纳米颗粒(即所述核酸转运用载体组合物)可以完全结合siRNA,并且此NP比下使用的材料最少,故将NP比为4的纳米颗粒命名为NCN(Nano-confinedNanoparticle),并以此实例比来说明此给药系统的生物学效应。
1.纳米颗粒进入细胞的能力
用实施例1中所述方法制备包载Cy5-siRNA的NCN来研究此给药系统的细胞摄取。
将包载FAM-siRNA纳米颗粒(Cy5-siRNA终浓度为100nM)与人乳腺癌MDA-MB-231细胞(24孔板,5×104细胞/孔)在37℃共同培养5小时后,用冰PBS洗涤细胞3次,使用4wt%的多聚甲醛固定细胞15分钟。去除4wt%多聚甲醛,PBS洗涤细胞3次。随后用0.1%的TritonX-100(聚乙二醇辛基苯基醚)穿膜5分钟,PBS缓冲液洗涤细胞3次。加入含1wt%BSA的PBS进行封闭,室温避光放置20分钟。以1:100倍稀释的Alexa Fluor 488 phalloidin(Invitrogen,A12379)标记细胞骨架,室温标记20分钟,PBS缓冲液洗3次。用含1μg/mL 4',6-二脒基-2-苯基吲哚(DAPI)的PBS缓冲液染色5分钟,PBS缓冲液洗3次。最后用抗荧光淬灭剂进行封片,封片后使用型号为Zeiss LSM880的激光共聚焦显微镜进行观察。结果见图3。
图3为荷载Cy5-siRNA的核酸转运用载体组合物(包载Cy5-siRNA的NCN)与MDA-MB-231细胞培养5小时后在细胞内分布的激光共聚焦显微镜照片;图3中,细胞内绿色荧光来源于Alexa 488-phalloidin标记的细胞骨架;红色荧光来源于Cy5-siRNA;蓝色荧光来源于DAPI标记的细胞核。通过三种颜色叠加的结果可知5小时培养后,纳米颗粒能有效进入细胞胞浆中,呈现出颗粒状分布。
2.细胞水平包载Plk1 siRNA的纳米颗粒对靶基因的沉默效果。
用实施例1中所述方法制备包载siRNA的NCN来研究此给药系统的体外沉默水平。使用的siRNA分别为siPlk1和siN.C.,Plk1有助于促进和加速哺乳动物细胞有丝分裂,并在多种肿瘤细胞中高表达。通过沉默其表达,可以抑制肿瘤生长。
将MDA-MB-231细胞以1×105细胞/孔的密度接种于12孔板,37℃培养12小时后,分别进行如下处理:
处理一(对照组):加入等体积PBS溶液。
处理二(裸siPlk1组):加入等体积siPlk1溶液,siPlk1终浓度为100nM。
处理三(RNAiMAX组):用载siPlk1的RNAiMAX溶液处理细胞,siPlk1终浓度为20nM。
处理四(NCN组):用携载siPlk1的纳米颗粒(包载siPlk1的NCN)处理细胞,siPlk1的终浓度为100nM。
在转染培养24h后,用RNAiso Plus(TaKaRa)提取细胞中的总RNA,用Nanodrop测定提取的RNA样品浓度,然后用PrimeScriptTM RT reagent Kit(Perfect Real Time)合成cDNA,每个样品使用2μg总mRNA。在合成cDNA后,按照FastStart Universal SYBR GreenMaster(ROX)试剂盒进行实时荧光定量PCR反应。其中Plk1和甘油三磷酸脱氢酶GAPDH基因的PCR引物如下:
Plk1-上游引物5'-AGCCTGAGGCCCGATACTACCTAC-3',
Plk1-下游引物5'-ATTAGGAGTCCCACACAGGGTCTTC-3';
GAPDH-上游引物5'-TTCACCACCATGGAGAAGGC-3',
GAPDH-下游引物5'-GGCATGGACTGTGGTCATGA-3'。
PCR反应如下:
1)95℃预热600秒。
2)95℃加热15秒,57℃加热30秒,72℃加热30秒。此步循环45次。
3)37℃加热30秒。
利用2-ΔΔCT对不同实验组中Plk1基因表达差异进行了分析,其中以GAPDH为内参,分析不同实验组中Plk1基因表达水平。以PBS实验组为100%,其他实验组表达表示为相对于PBS组表达量,实验结果见图4。
图4中,不经过处理的PBS组细胞内Plk1表达量高,包载siN.C.的实验对照组对细胞中Plk1基因表达没有明显影响,而包载siPlk1的实验组(处理四组)可以有效抑制Plk1基因的表达。
实施例三.此给药系统在动物水平的生物效应评价
1.携载Plk1 siRNA的纳米颗粒对肿瘤生长的抑制作用
在裸鼠第二个乳腺的脂肪垫下原位接种MDA-MB-231细胞(0.5×107),9天左右形成可见肿瘤,肿瘤体积约50mm3,随机分成三组,进行尾静脉注射治疗,每两天注射一次。肿瘤的体积按公式计算:V=0.5×a×b2计算,其中a指肿瘤长径,b指肿瘤短径。
处理一(PBS对照组):每只裸鼠注射200μL PBS。
处理二(裸siPlk1组):每只裸鼠注射200μL裸siPlk1,siPlk1剂量为40μg。
处理三(NCN/siN.C.组):每只裸鼠注射包载siN.C.的纳米颗粒NCN,聚合物质量为2.2mg,siN.C.剂量为40μg。
处理四(NCN/siPlk1组):每只裸鼠注射包载siPlk1的纳米颗粒NCN,聚合物质量为2.2mg,siPlk1剂量为40μg。
在治疗开始后,每隔一天对肿瘤体积进行测量。实施例结果如图5所示,所有阴性对照组中肿瘤生长速度均较快,而在使用包载siPlk1纳米颗粒治疗组中,肿瘤生长速度与阴性对照组相比受到明显抑制。说明本发明提供的包载siRNA纳米颗粒(所述小干扰RNA药物)在体内能够有效沉默致癌基因,从而抑制肿瘤生长。
2.携载Plk1 siRNA的纳米颗粒对肿瘤部位Plk1基因的沉默效果
收集治疗结束后各组荷瘤小鼠的肿瘤组织。取部分肿瘤组织,用RNAiso Plus(TaKaRa)提取细胞中的总RNA,用Nanodrop测定提取的RNA样品浓度,然后用PrimeScriptTMRT reagent Kit(Perfect Real Time)合成cDNA,每个样品使用2μg总mRNA。在合成cDNA后,按照FastStart Universal SYBR Green Master(ROX)试剂盒进行实时荧光定量PCR反应。相关引物序列与实时荧光定量PCR反应具体步骤与细胞水平实验相同。结果如图6所示,阴性对照组中肿瘤部位Plk1基因的表达水平与PBS组相比无明显区别,而使用包载siPlk1纳米颗粒治疗组中,肿瘤部位Plk1基因的表达受到明显抑制。
由上述结果可知,此siRNA给药系统在动物水平能够有效抑制肿瘤生长,并且这种抑制效果是由于沉默肿瘤部位靶基因的表达导致的。
以上实施例仅为本发明较优的实施方式,仅用于解释本发明,而非限制本发明,本领域技术人员在未脱离本发明精神实质下所作的改变、替换、修饰等均应属于本发明的保护范围。
Claims (10)
1.一种核酸转运用载体组合物,其特征在于,按重量份数计,包括:
聚乙二醇-聚乳酸 100份;
(聚酰胺-胺)-2-丙酸-3-甲基马来酸酐-聚己内酯 11-55份。
2.根据权利要求1所述的核酸转运用载体组合物,其特征在于,所述聚乙二醇-聚乳酸中,聚乙二醇的嵌段分子量为3000-7000,聚乳酸的嵌段分子量为9000-13000。
3.根据权利要求1所述的核酸转运用载体组合物,其特征在于,所述(聚酰胺-胺)-2-丙酸-3-甲基马来酸酐-聚己内酯中,聚酰胺-胺的代数为四代,聚酰胺-胺的分子量为14215,聚己内酯的分子量为3000-5000。
4.根据权利要求1所述的核酸转运用载体组合物,其特征在于,其粒径为90-140纳米。
5.一种小干扰RNA药物,其特征在于,包含权利要求1-3任一项所述的核酸转运用载体组合物和小干扰RNA。
6.一种制备权利要求5所述的小干扰RNA药物的方法,其特征在于,包括如下步骤:
(1)将聚乙二醇-聚乳酸、(聚酰胺-胺)-2-丙酸-3-甲基马来酸酐-聚己内酯加入油相中,然后加入siRNA水溶液,超声处理,得到初始乳液;
(2)将步骤(1)所述初始乳液加入水相中,超声乳化处理,然后减压去除有机溶剂,得到所述小干扰RNA药物的水溶液。
7.根据权利要求6所述的小干扰RNA药物的制备方法,其特征在于,步骤(1)所述聚乙二醇-聚乳酸和(聚酰胺-胺)-2-丙酸-3-甲基马来酸酐-聚己内酯的质量比为100:11-55;所述油相为氯仿;所述聚乙二醇-聚乳酸与油相的质量体积比为3-5 :1mg/mL。
8.根据权利要求6所述的小干扰RNA药物的制备方法,其特征在于,步骤(1)所述siRNA水溶液的浓度为2-3 mg/mL;所述油相与siRNA水溶液的体积比为15-25:1。
9.根据权利要求6所述的小干扰RNA药物的制备方法,其特征在于,步骤(1)所述超声处理的时间为0.5-1.5分钟,超声处理的功率为60-100 W。
10.根据权利要求6所述的小干扰RNA药物的制备方法,其特征在于,步骤(2)所述超声乳化处理的时间为0.5-1.5分钟,超声乳化处理的功率为60-100 W。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010246551.1A CN111265669B (zh) | 2020-03-31 | 2020-03-31 | 一种核酸转运用载体组合物及其在制备小干扰rna药物中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010246551.1A CN111265669B (zh) | 2020-03-31 | 2020-03-31 | 一种核酸转运用载体组合物及其在制备小干扰rna药物中的应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111265669A true CN111265669A (zh) | 2020-06-12 |
CN111265669B CN111265669B (zh) | 2022-08-16 |
Family
ID=70991892
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010246551.1A Active CN111265669B (zh) | 2020-03-31 | 2020-03-31 | 一种核酸转运用载体组合物及其在制备小干扰rna药物中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111265669B (zh) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113082001A (zh) * | 2021-05-25 | 2021-07-09 | 中国科学院广州生物医药与健康研究院 | 一种核酸递送系统及其制备方法和应用 |
CN113116855A (zh) * | 2021-04-13 | 2021-07-16 | 华南理工大学 | 联合递送化疗药物和免疫检查点抗体药物的纳米颗粒 |
CN113440621A (zh) * | 2021-04-01 | 2021-09-28 | 中山大学孙逸仙纪念医院 | 一种共轭树枝状分子纳米核酸载体及其应用 |
CN114796157B (zh) * | 2022-05-12 | 2024-01-05 | 苏州大学 | 一种氟化纳米胶囊及其制备方法和应用 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103764076A (zh) * | 2011-06-30 | 2014-04-30 | 万能医药公司 | 可生物降解的内置假体及其制造方法 |
AU2016247059A1 (en) * | 2007-12-04 | 2016-11-03 | Arbutus Biopharma Corporation | Targeting lipids |
CN106905532A (zh) * | 2015-12-18 | 2017-06-30 | 天津国际生物医药联合研究院 | 侧链带pamam的聚合物胶束及其制备方法 |
CN107260706A (zh) * | 2017-06-21 | 2017-10-20 | 广州博徕斯生物科技有限公司 | 一种肿瘤靶向双载药纳米载体及其制备方法 |
CN110823979A (zh) * | 2019-11-22 | 2020-02-21 | 重庆大学 | 一种超敏电化学生物传感器及其制备方法和应用 |
-
2020
- 2020-03-31 CN CN202010246551.1A patent/CN111265669B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2016247059A1 (en) * | 2007-12-04 | 2016-11-03 | Arbutus Biopharma Corporation | Targeting lipids |
CN103764076A (zh) * | 2011-06-30 | 2014-04-30 | 万能医药公司 | 可生物降解的内置假体及其制造方法 |
CN106905532A (zh) * | 2015-12-18 | 2017-06-30 | 天津国际生物医药联合研究院 | 侧链带pamam的聚合物胶束及其制备方法 |
CN107260706A (zh) * | 2017-06-21 | 2017-10-20 | 广州博徕斯生物科技有限公司 | 一种肿瘤靶向双载药纳米载体及其制备方法 |
CN110823979A (zh) * | 2019-11-22 | 2020-02-21 | 重庆大学 | 一种超敏电化学生物传感器及其制备方法和应用 |
Non-Patent Citations (2)
Title |
---|
刘晶: "肿瘤微环境调控的纳米载体用于药物", 《中国优秀博硕士学位论文全文数据库(博士) 工程科技Ⅰ辑》 * |
梅兴国 等: "《微载体药物递送系统》", 30 November 2009, 华中科技大学出版社 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113440621A (zh) * | 2021-04-01 | 2021-09-28 | 中山大学孙逸仙纪念医院 | 一种共轭树枝状分子纳米核酸载体及其应用 |
CN113116855A (zh) * | 2021-04-13 | 2021-07-16 | 华南理工大学 | 联合递送化疗药物和免疫检查点抗体药物的纳米颗粒 |
CN113082001A (zh) * | 2021-05-25 | 2021-07-09 | 中国科学院广州生物医药与健康研究院 | 一种核酸递送系统及其制备方法和应用 |
CN113082001B (zh) * | 2021-05-25 | 2023-05-26 | 中国科学院广州生物医药与健康研究院 | 一种核酸递送系统及其制备方法和应用 |
CN114796157B (zh) * | 2022-05-12 | 2024-01-05 | 苏州大学 | 一种氟化纳米胶囊及其制备方法和应用 |
Also Published As
Publication number | Publication date |
---|---|
CN111265669B (zh) | 2022-08-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111265669B (zh) | 一种核酸转运用载体组合物及其在制备小干扰rna药物中的应用 | |
Lee et al. | MicroRNA delivery through nanoparticles | |
Elsabahy et al. | Non-viral nucleic acid delivery: key challenges and future directions | |
Abbasi et al. | Co-encapsulation of Cas9 mRNA and guide RNA in polyplex micelles enables genome editing in mouse brain | |
Yoshinaga et al. | Induced packaging of mRNA into polyplex micelles by regulated hybridization with a small number of cholesteryl RNA oligonucleotides directed enhanced in vivo transfection | |
JP2021522248A (ja) | 粒子状製剤用の凍結保護剤 | |
CN102727907B (zh) | 一种小干扰rna药物的给药系统和制剂 | |
van den Brand et al. | siRNA in ovarian cancer–Delivery strategies and targets for therapy | |
Li et al. | Nanoparticle depots for controlled and sustained gene delivery | |
JP7252132B2 (ja) | 核酸送達試薬の調製における化合物または伝統的漢方薬抽出物の応用およびその関連生成物 | |
Gaspar et al. | Gas-generating TPGS-PLGA microspheres loaded with nanoparticles (NIMPS) for co-delivery of minicircle DNA and anti-tumoral drugs | |
Han et al. | Responsive disassembly of nucleic acid nanocomplex in cells for precision medicine | |
WO2017105138A1 (ko) | 음이온성 약물을 함유하는 고분자 미셀의 제조방법 | |
WO2015125147A9 (en) | Anionic polyplexes for use in the delivery of nucleic acids | |
Gozuacik et al. | Anticancer use of nanoparticles as nucleic acid carriers | |
Oieni et al. | Nano-ghosts: Novel biomimetic nano-vesicles for the delivery of antisense oligonucleotides | |
Sarisozen et al. | Lipid-based siRNA delivery systems: challenges, promises and solutions along the long journey | |
Xiong et al. | Engineered Aptamer‐Organic Amphiphile Self‐Assemblies for Biomedical Applications: Progress and Challenges | |
Asakiya et al. | Current progress of miRNA-derivative nucleotide drugs: modifications, delivery systems, applications | |
Chen et al. | The Hydrophobicity of AIE Dye Facilitates DNA Condensation for Carrier‐Free Gene Therapy | |
Zhang et al. | Ionizable drug delivery systems for efficient and selective gene therapy | |
KR20220092363A (ko) | 지질 나노입자를 포함하는 암 예방 또는 치료용 조성물 | |
AU2018280191A1 (en) | Cationic liquid crystalline nanoparticles | |
CN113968968B (zh) | 氨基脂质化合物、其制备方法和应用 | |
CN104031268B (zh) | 聚乙烯亚胺三嵌段共聚物及其在基因载体中的用途 |
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 | ||
TR01 | Transfer of patent right | ||
TR01 | Transfer of patent right |
Effective date of registration: 20231115 Address after: Room 1703-A-133, Shenya Building, No. 47 Guomao Road, Jinmao Street, Longhua District, Haikou City, Hainan Province, 570100 Patentee after: Haikou Kehua Zhicheng Biotechnology Co.,Ltd. Address before: 510640 No. five, 381 mountain road, Guangzhou, Guangdong, Tianhe District Patentee before: SOUTH CHINA University OF TECHNOLOGY |