CN106046118A - Preparation of siRNA transfer system comprising S-Beta-carboline-3-acyl-RGDV and application of system in SURVIVIN gene silencing - Google Patents
Preparation of siRNA transfer system comprising S-Beta-carboline-3-acyl-RGDV and application of system in SURVIVIN gene silencing Download PDFInfo
- Publication number
- CN106046118A CN106046118A CN201610362271.0A CN201610362271A CN106046118A CN 106046118 A CN106046118 A CN 106046118A CN 201610362271 A CN201610362271 A CN 201610362271A CN 106046118 A CN106046118 A CN 106046118A
- Authority
- CN
- China
- Prior art keywords
- sirna
- survivin
- boc
- crv
- carboline
- 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.)
- Pending
Links
- 108020004459 Small interfering RNA Proteins 0.000 title claims abstract description 63
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 108010002687 Survivin Proteins 0.000 title abstract description 43
- 102000000763 Survivin Human genes 0.000 title abstract description 37
- 238000012546 transfer Methods 0.000 title abstract description 6
- 230000030279 gene silencing Effects 0.000 title description 9
- 238000012226 gene silencing method Methods 0.000 title description 8
- 239000002502 liposome Substances 0.000 claims abstract description 59
- 230000000259 anti-tumor effect Effects 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 23
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims abstract description 18
- 150000001875 compounds Chemical class 0.000 claims abstract description 12
- 235000012000 cholesterol Nutrition 0.000 claims abstract description 9
- 239000000463 material Substances 0.000 claims abstract description 6
- 239000000523 sample Substances 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 206010028980 Neoplasm Diseases 0.000 claims description 16
- 102000007327 Protamines Human genes 0.000 claims description 15
- 108010007568 Protamines Proteins 0.000 claims description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 14
- 229940048914 protamine Drugs 0.000 claims description 14
- MDNRBNZIOBQHHK-KWBADKCTSA-N (2s)-2-[[(2s)-2-[[2-[[(2s)-2-amino-5-(diaminomethylideneamino)pentanoyl]amino]acetyl]amino]-3-carboxypropanoyl]amino]-3-methylbutanoic acid Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCN=C(N)N MDNRBNZIOBQHHK-KWBADKCTSA-N 0.000 claims description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 241000702660 Rice gall dwarf virus Species 0.000 claims description 12
- 239000003814 drug Substances 0.000 claims description 12
- 239000007791 liquid phase Substances 0.000 claims description 12
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 12
- 210000001541 thymus gland Anatomy 0.000 claims description 11
- 244000309466 calf Species 0.000 claims description 10
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 10
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- DYHSDKLCOJIUFX-UHFFFAOYSA-N tert-butoxycarbonyl anhydride Chemical compound CC(C)(C)OC(=O)OC(=O)OC(C)(C)C DYHSDKLCOJIUFX-UHFFFAOYSA-N 0.000 claims description 7
- 239000010408 film Substances 0.000 claims description 6
- 230000002209 hydrophobic effect Effects 0.000 claims description 6
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 5
- 102000044209 Tumor Suppressor Genes Human genes 0.000 claims description 5
- 108700025716 Tumor Suppressor Genes Proteins 0.000 claims description 5
- 229940079593 drug Drugs 0.000 claims description 5
- ZKLRYZNOOWOOMP-UHFFFAOYSA-N 2,2,2-trifluoroacetic acid;trifluoromethanesulfonic acid Chemical compound OC(=O)C(F)(F)F.OS(=O)(=O)C(F)(F)F ZKLRYZNOOWOOMP-UHFFFAOYSA-N 0.000 claims description 4
- 108010016626 Dipeptides Proteins 0.000 claims description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 4
- 238000009833 condensation Methods 0.000 claims description 4
- 230000005494 condensation Effects 0.000 claims description 4
- 238000007327 hydrogenolysis reaction Methods 0.000 claims description 4
- 150000002632 lipids Chemical class 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- JXYACYYPACQCDM-UHFFFAOYSA-N Benzyl glycinate Chemical compound NCC(=O)OCC1=CC=CC=C1 JXYACYYPACQCDM-UHFFFAOYSA-N 0.000 claims description 3
- 238000007796 conventional method Methods 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 238000010189 synthetic method Methods 0.000 claims description 3
- 239000010409 thin film Substances 0.000 claims description 3
- WBIIPXYJAMICNU-AWEZNQCLSA-N (2s)-5-[amino-[(4-methylphenyl)sulfonylamino]methylidene]azaniumyl-2-[(2-methylpropan-2-yl)oxycarbonylamino]pentanoate Chemical compound CC1=CC=C(S(=O)(=O)NC(N)=NCCC[C@H](NC(=O)OC(C)(C)C)C(O)=O)C=C1 WBIIPXYJAMICNU-AWEZNQCLSA-N 0.000 claims description 2
- WFIYPADYPQQLNN-UHFFFAOYSA-N 2-[2-(4-bromopyrazol-1-yl)ethyl]isoindole-1,3-dione Chemical compound C1=C(Br)C=NN1CCN1C(=O)C2=CC=CC=C2C1=O WFIYPADYPQQLNN-UHFFFAOYSA-N 0.000 claims description 2
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 claims description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 150000007942 carboxylates Chemical class 0.000 claims description 2
- 238000006555 catalytic reaction Methods 0.000 claims description 2
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 claims description 2
- 239000012046 mixed solvent Substances 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 238000002390 rotary evaporation Methods 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 239000010936 titanium Substances 0.000 claims description 2
- 229910052719 titanium Inorganic materials 0.000 claims description 2
- 150000001735 carboxylic acids Chemical class 0.000 claims 5
- 239000002253 acid Substances 0.000 claims 1
- 239000002539 nanocarrier Substances 0.000 claims 1
- 229920000136 polysorbate Polymers 0.000 claims 1
- 238000001338 self-assembly Methods 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 17
- 108020004999 messenger RNA Proteins 0.000 abstract description 13
- 230000014509 gene expression Effects 0.000 abstract description 10
- 210000004881 tumor cell Anatomy 0.000 abstract description 6
- 230000004614 tumor growth Effects 0.000 abstract description 4
- 229940126585 therapeutic drug Drugs 0.000 abstract 3
- 150000003904 phospholipids Chemical class 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 67
- 239000000243 solution Substances 0.000 description 30
- 230000000694 effects Effects 0.000 description 19
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 18
- 230000006907 apoptotic process Effects 0.000 description 18
- 239000010413 mother solution Substances 0.000 description 17
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 16
- 239000007788 liquid Substances 0.000 description 16
- 239000003085 diluting agent Substances 0.000 description 14
- 239000001963 growth medium Substances 0.000 description 14
- 238000001890 transfection Methods 0.000 description 14
- 238000001727 in vivo Methods 0.000 description 13
- 210000002966 serum Anatomy 0.000 description 12
- 230000003115 biocidal effect Effects 0.000 description 10
- 239000012071 phase Substances 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 9
- 238000002156 mixing Methods 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 239000012096 transfection reagent Substances 0.000 description 9
- 230000022131 cell cycle Effects 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 230000003834 intracellular effect Effects 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000012467 final product Substances 0.000 description 6
- 238000011068 loading method Methods 0.000 description 6
- 206010008342 Cervix carcinoma Diseases 0.000 description 5
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 239000008346 aqueous phase Substances 0.000 description 5
- 201000010881 cervical cancer Diseases 0.000 description 5
- 238000004140 cleaning Methods 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- 230000037440 gene silencing effect Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000013641 positive control Substances 0.000 description 5
- 239000002244 precipitate Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 230000001629 suppression Effects 0.000 description 5
- 230000001988 toxicity Effects 0.000 description 5
- 231100000419 toxicity Toxicity 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 4
- 230000001093 anti-cancer Effects 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 238000001415 gene therapy Methods 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 108091007065 BIRCs Proteins 0.000 description 3
- 230000006820 DNA synthesis Effects 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 206010019851 Hepatotoxicity Diseases 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 230000018199 S phase Effects 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 230000003828 downregulation Effects 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 230000009368 gene silencing by RNA Effects 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 210000002216 heart Anatomy 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 230000010534 mechanism of action Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 231100000331 toxic Toxicity 0.000 description 3
- 230000002588 toxic effect Effects 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- NWXMGUDVXFXRIG-WESIUVDSSA-N (4s,4as,5as,6s,12ar)-4-(dimethylamino)-1,6,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4,4a,5,5a-tetrahydrotetracene-2-carboxamide Chemical compound C1=CC=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(=O)C(C(N)=O)=C(O)[C@@]4(O)C(=O)C3=C(O)C2=C1O NWXMGUDVXFXRIG-WESIUVDSSA-N 0.000 description 2
- 229930195573 Amycin Natural products 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 235000009161 Espostoa lanata Nutrition 0.000 description 2
- 240000001624 Espostoa lanata Species 0.000 description 2
- 240000002853 Nelumbo nucifera Species 0.000 description 2
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 2
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 108091030071 RNAI Proteins 0.000 description 2
- 206010039491 Sarcoma Diseases 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000007059 acute toxicity Effects 0.000 description 2
- 231100000403 acute toxicity Toxicity 0.000 description 2
- 239000002313 adhesive film Substances 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000012148 binding buffer Substances 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- -1 cation lipid Chemical class 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000012531 culture fluid Substances 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- FFYPMLJYZAEMQB-UHFFFAOYSA-N diethyl pyrocarbonate Chemical compound CCOC(=O)OC(=O)OCC FFYPMLJYZAEMQB-UHFFFAOYSA-N 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000007686 hepatotoxicity Effects 0.000 description 2
- 231100000304 hepatotoxicity Toxicity 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000003226 mitogen Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 239000002574 poison Substances 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 210000001550 testis Anatomy 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 231100000167 toxic agent Toxicity 0.000 description 2
- 239000003440 toxic substance Substances 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 239000013603 viral vector Substances 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 description 1
- 108090000672 Annexin A5 Proteins 0.000 description 1
- 102000004121 Annexin A5 Human genes 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 206010048610 Cardiotoxicity Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 230000004668 G2/M phase Effects 0.000 description 1
- 244000283207 Indigofera tinctoria Species 0.000 description 1
- 102000055031 Inhibitor of Apoptosis Proteins Human genes 0.000 description 1
- 101150062031 L gene Proteins 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- 102000000574 RNA-Induced Silencing Complex Human genes 0.000 description 1
- 108010016790 RNA-Induced Silencing Complex Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 108091007602 SLC58A1 Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 244000061458 Solanum melongena Species 0.000 description 1
- 235000002597 Solanum melongena Nutrition 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 231100000215 acute (single dose) toxicity testing Toxicity 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000002390 adhesive tape Substances 0.000 description 1
- 239000005030 aluminium foil Substances 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- 108010091818 arginyl-glycyl-aspartyl-valine Proteins 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000005784 autoimmunity Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000004579 body weight change Effects 0.000 description 1
- 231100000259 cardiotoxicity Toxicity 0.000 description 1
- 230000007681 cardiovascular toxicity Effects 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 125000001142 dicarboxylic acid group Chemical group 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000010224 hepatic metabolism Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 230000036457 multidrug resistance Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 235000010603 pastilles Nutrition 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 150000004965 peroxy acids Chemical class 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 230000032361 posttranscriptional gene silencing Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 229940070353 protamines Drugs 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000011514 reflex Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- 231100000974 spleenotoxicity Toxicity 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 229960002385 streptomycin sulfate Drugs 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1019—Tetrapeptides with the first amino acid being basic
-
- 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
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/07—Tetrapeptides
-
- 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
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Inorganic Chemistry (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a novel method for constructing siRNA transfer system and also discloses application of the transfer system in the preparation of antitumor gene supported therapeutic drugs. Through the research of the method, amphiphilic compound S-Beta-carboline-3-acyl-RGDV with antitumor activity, abbreviated as CRV. A synthetic target compound is used as a film material to replace phospholipid and cholesterol to jointly form liposome outer film, an antitumor gene therapeutic drug is supported to the film to form the novel siRNA transfer system, and the novel siRNA transfer system to which the antitumor gene therapeutic drug is supported may specifically silence the expression of SURVIVIN mRNA, down-regulate the expression of SURVIVIN protein and inhibit the growth of tumor cells.
Description
Technical field
The present invention relates to a kind of novel siRNA delivery system, especially load SURVIVIN-siRNA pharmaceutical carrier and
Its construction method, the invention still further relates to pharmaceutical carrier application in preparation loads Antioncogene medicine, belongs to biological
Medicine.
Background technology
Nowadays, malignant tumor greatly threatens human health.Traditional treatment means such as chemotherapy, its toxic and side effects big and
The cancer patient exceeding half can produce multidrug resistance；Radiotherapy is then without the killing selected to cancerous cell and normal cell,
Patient's autoimmunity is caused great damage；There is many limitations in operative treatment, easily recurrence and transfer.Therefore, spy is found
Different, efficient oncotherapy means are big focuses of clinic study.
SURVIVIN gene is IAP (the inhibitor of apoptosis of discovered in recent years
Protein, IAP) newcomer in family, there is inhibited apoptosis and maintain the dual-use function of cell mitogen,
Normal terminal differentiation tissue is expressed hardly.Under physiological status, SURVIVIN is only expressed in adult secretory phase uterus
In inner membrance, Placenta Hominis, testis, thymus and embryo, but under pathological state, SURVIVIN wide expression is in the various malignant tumor of the mankind
In tissue.SURVIVIN expresses to increase and apoptosis of tumor cells can be suppressed to promote it to breed, and its expression is bright with course advancement
Aobvious relevant, maximum expression values is at G2/ M the phase.Recently, gene therapy is expressed by suppression SURVIVIN, thus reaches to promote tumor
Apoptosis and stop tumor cell differentiation purpose.Therefore SURVIVIN-siRNA transfection or new for oncotherapy offer
Thinking.
The mechanism of action of SURVIVIN-siRNA is to be caused by RNAi (RNA interference, RNA disturb)
SURVIVIN silenced gene expression.RNAi be by siRNA (small interfering RNA, siRNA) cause special
The phenomenon of property sequence PTGS.SiRNA is the double-stranded RNA of 21-23nt, and siRNA induces with RNA as go-ahead sequence
The inactive precursor of silencing complex (RNA induced silencing complex, RISC) combines, under ATP participates in
SiRNA double-strand structure is untwisted the activated RISC of induced synthesis, and specific recognition purpose mRNA is made by endonuclease restriction
Its degraded.The gene therapy of siRNA mediation, is used clinically for the pathology shapes such as cancer, autoimmune, infection and sacred disease
Condition.
Although siRNA gene therapy has a good application prospect, but the siRNA of the most modified or carrier protection is at body
Interior delivery process can run into serious obstruction.Therefore genophore is the major issue of restriction siRNA gene therapy.SiRNA at present
Delivery vector be broadly divided into two big classes: viral vector and non-virus carrier.Viral vector although to have transfection efficiency high
Advantage, but its high immunogenicity so that it is safety enjoys query.In non-virus carrier, liposome is with its good bio-compatible
Property, easy modified are gradually concerned by people.Lipid bilayer and the structure of interior aqueous phase so that liposome has both can carry water solublity
Medicine, the advantage that fat-soluble medicine can be carried again.Cationic-liposome can be on the basis of drug delivery at liposome, due to its positive electricity
Lotus effect, enables it to electronegative nucleic acid molecule by electrostatical binding, thus passenger gene smoothly.Studies have shown that:
Cationic liposomal transfection efficiency be less than viral genetic vector, without antitumor action, it is therefore necessary to develop transfection efficiency height, can be real
The novel gene vector of existing gene silencing effect.
Summary of the invention
Present invention aim at providing a kind of there is anti-tumor activity amphiphilic compound S-B-carboline-3-acyl group-
RGDV, referred to as CRV.
Further object is that the synthetic method that amphiphilic chemical combination CRV is provided, comprise the following steps: by L-color
Propylhomoserin is after PS condensation, Boc protection, with hydrophobic side N-Boc-S-B-carboline carboxylic acid；Main chain side Boc is used to protect aminoterminal
Or OBzl protects c-terminus, the side chain l-amino acid of suitable protection group protection, liquid phase method connects peptide, obtains full guard dipeptides.
Respectively through hydrogenolysis slough OBzl protection or acidolysis slough Boc protection after, liquid phase method connects peptide, obtains full guard tetrapeptide.Through peracid
After solution sloughs Boc protection, finally give the four titanium RGDV that water-wet side amino is exposed；The hydrophobic side N-Boc-S-B-carboline that will obtain
The RGDV liquid phase method that carboxylic acid dissociates with water-wet side amino connects peptide, according to a conventional method with trifluoroacetic acid-trifluoromethanesulfonic acid mixing loss of thick fluid
Deprotection base, finally gives target product CRV.
A kind of novel siRNA delivery system of offer is provided, containing described, there is antitumor
The amphiphilic compound CRV and Antioncogene medicine SURVIVIN-siRNA. of activity
Further, described novel siRNA delivery system, it is made up of following component: Antioncogene medicine
SURVIVIN-siRNA, cholesterol, protamine, calf thymus DNA and the described amphipathic chemical combination with anti-tumor activity
Thing CRV.
Further, described novel siRNA delivery system, it is made up of following component, following percentage ratio is matter
Amount percentage ratio: SURVIVIN-siRNA 8.74-8.80%, cholesterol 3.15-3.17%, protamine 6.35-6.40% is little
Bovine chest gland DNA 3.48-3.50% and the amphipathic film material CRV78.2-78.78%. with anti-tumor activity
Further, antitumor drug is: one or more in CRV, siRNA.
Further, described cation lipid is: cholesterol, protamine, the one or many in calf thymus DNA
Kind.
A kind of method preparing above-mentioned novel siRNA delivery system of offer is provided, including: will
Cholesterol and have the mixed solvent of the amphiphilic compound CRV chloroform of anti-tumor activity and methanol and dissolve, rotary evaporation goes
Except solvent, form homogeneous thin film.Dried in vacuum overnight, removes trace organic reagent.Add 1mL concentration be 1% tween-
The ultrasonic 10min of 80DEPC water hydratable, washes from bottle wall completely by liposome membrane, then uses Probe Ultrasonic Searching 15min, obtains blank liposomes
Body.SURVIVIN-siRNA is mixed according to the ratio of 2.5:1 (w:w) with calf thymus DNA, repeatedly blows and beats, left at room temperature
10min.Blank liposome is mixed with appropriate protamine simultaneously, repeatedly blow and beat, left at room temperature 10min.After by lipid
Body/protamine complex joins in SURVIVIN-siRNA/DNA complex, the most quickly blows and beats, and room temperature stands 15min,
Obtain.
The present invention relates to described CRV application in preparing novel siRNA delivery system.
The present invention relates to the various pharmaceutical carriers such as described novel siRNA delivery system in the medicine of preparation treatment tumor
Application.
The present invention relates to Cervical Cancer HeLa Cells strain as model, with the novel siRNA delivery system described in mtt assay evaluation
Anti tumor activity in vitro, siRNA can be deliverrf into cell by result display novel siRNA delivery system, and himself is also
There is anti-tumor activity.
The present invention relates to Cervical Cancer HeLa Cells strain as model, the gene silencing being measured protein level by ELISA is imitated
Rate, measures the gene silencing efficiency of mRNA level in-site by RT-PCR, and result shows, novel siRNA delivery system and commercially available transfection
Reagent is compared, and gene silencing efficiency there was no significant difference.
The present invention relates to lotus sarcoma S180Mice is model, evaluates internal anti-swollen by the way of tail vein injection is administered
Tumor activity, result shows that novel siRNA delivery system shows obvious anti-tumor in vivo activity, and toxicity is less, application prospect
Preferably.
The present invention relates to ICR male mice as model, evaluate by the way of intraperitoneal injection and there is antitumor work
Property the safety of amphipathic film material CEV, result shows that under CRV urgency toxic agent amount, (in vivo test dosage 100 times) performance is light
Micro-hepatotoxicity, toxicity is less, and safety is high.
The present invention relates to Cervical Cancer HeLa Cells strain as model, use AnnexinV-FITC/PI double dye standard measure apoptosis
The incidence rate of cell, uses PI staining to investigate Cell Cycle change, and result shows that novel siRNA delivery system can be by
SiRNA is deliverrf into cell, and the suppression cell entrance S phase carries out DNA synthesis, and with G2The effect that/M the phase blocks, thus draw
Play apoptosis, and the peak period of apoptosis is 24-48h upon administration.
The present invention is for existing product, and beneficial effect is mainly manifested in:
S-B-carboline-3-acyl group-RGDV is used for the preparation of genophore by the present invention first, and successfully have developed one newly
SiRNA delivery system: CRV liposomes, SURVIVIN-siRNA can be loaded and realize gene silencing.Cell in vitro poison is tested
Result shows, SURVIVIN-siRNA/100%CRV liposomes is compared with commercially available transfection reagent, and cytotoxicity has significantly
Property improve；Gene silencing effect experimental result shows, SURVIVIN-siRNA/100%CRV liposomes and commercially available transfection examination
Agent is compared, and gene silencing effect there was no significant difference；Experiment in vivo result shows, SURVIVIN-siRNA/100%CRV
Liposomes is compared with amycin, and anti-tumor activity there was no significant difference, but toxicity is less, and application prospect is preferable；Cell withers
Experiment of dying shows with cell cycle result, and SURVIVIN-siRNA/100%CRV liposomes mechanism of action is suppression cell
Entering the S phase carries out DNA synthesis, and the effect blocked with the G2/M phase, thus inducing cell apoptosis, and the peak period of apoptosis is
24-48h upon administration.
Relational language described in the present invention is further explained:
PS: dehydration
Boc: tertbutyloxycarbonyl
OBzl: benzyl ester
RGDV: essence-sweet-Radix Asparagi-valine sequence (Arg-Gly-Asp-Val)
DMF: dimethylformamide
Boc2O: Bis(tert-butoxycarbonyl)oxide
SURVIVIN-siRNA: survivin siRNA
NC: the negative control of survivin siRNA
CH: cholesterol
CRV liposomes (is called for short 100%lipo): do not carry the genes delivery system of SURVIVIN-siRNA
SURVIVIN-siRNA/100%CRV liposomes (is called for short S/lipo): carry the gene of SURVIVIN-siRNA
Delivery system
NC/lipo: carry the genes delivery system of NC
S/R: carry the commercially available transfection reagent of SURVIVIN-siRNA
N/R: carry the commercially available transfection reagent of NC
Zeta-Potential:Zeta current potential, is again electrokinetic potential or eletrokinetic potential
The abbreviation of OD:optical density (optical density), detection unit OD value represents
DEPC water: processed with DEPC (diethyl pyrocarbonate, pyrocarbonic acid diethyl ester) and go out through High Temperature High Pressure
The pure water of bacterium.
RNase-free: without RNase
Accompanying drawing explanation
Fig. 1: 24h Cyto toxic experiment showed；☆ represents there is significant difference relative to NC group；
Represent there is statistically-significant difference relative to NC group；Δ represents there is significant difference relative to NC/R group；▲ table
Show there is statistically-significant difference relative to NC/R group；Abscissa initialism represents meaning；◇ represents to be had relative to 100%lipo group
Significant difference；◆ represent there is statistically-significant difference relative to 100%lipo group: zero represents there is statistics relative to S/R group
Difference；● represent there is statistically-significant difference relative to S/R group.
Fig. 2: protein level gene silencing effect result；P ＞ 0.05 represents no difference of science of statistics；P ＜ 0.01 indicates system
Notable difference learned by meter.
Fig. 3: mRNA level in-site gene silencing effect result；P ＞ 0.05 represents no difference of science of statistics；P ＜ 0.01 indicates system
Notable difference learned by meter.
Fig. 4: anti-tumor in vivo activity experiment result；P ＞ 0.01 indicates without statistically-significant difference；P ＜ 0.01 indicates
Statistically significant notable difference.
Fig. 5: anti-tumor in vivo is tested dirty body and is compared result；P ＜ 0.05 indicates statistics notable difference；P ＜ 0.01 represents
There is statistically significant notable difference.
The dirty body of Fig. 6: acute toxicity testing compares result；P ＜ 0.05 indicates statistics notable difference.
Fig. 7: early apoptosis of cells experimental result.
Fig. 8: cell late apoptic experimental result.
Fig. 9: cell cycle distribution figure.
Detailed description of the invention
In order to the present invention is expanded on further, a series of embodiment is given below.These embodiments are entirely illustrative, it
Only be used for the present invention is specifically described, be not construed as limitation of the present invention.
The preparation of embodiment 1 isoquinilone derivatives
The preparation method of S-B-carboline-3-acyl group-RGDV (referred to as CRV).
1) preparation of N-Boc-S-carboline carboxylate
Under the catalysis of dilute sulfuric acid, tryptophan (L-Trp) occurs PS to react at normal temperatures with formaldehyde, and condensation generates S-β-click
Dicarboxylic acid moiety；By the S-B-carboline carboxylic acid of gained in DMF, in the basic conditions with Boc2O react, obtain N-Boc-S-β-
Carboline carboxylate.
2) preparation of RGDV
Boc-Arg (Tos)-OH and H-Gly-OBzl HCl is connect peptide by liquid phase method, obtains full guard dipeptides Boc-
Arg (Tos)-Gly-OBzl, in like manner obtains Boc-Asp (the OBzl)-Val-OBzl of full guard.Respectively by Boc-Arg (Tos)-
The exposed carboxyl of Gly-OBzl hydrogenolysis, the exposed amino of Boc-Asp (OBzl)-Val-OBzl acidolysis, connect peptide by liquid phase method, obtain complete
Protection tetrapeptide.The RGDV that water-wet side amino is exposed is finally given through acidolysis.
3) preparation of S-B-carboline-3-acyl group-RGDV
RGDV liquid phase method exposed with water-wet side amino for the hydrophobic side N-Boc-S-B-carboline carboxylic acid obtained is connect peptide, prepares
Intermediate N Boc-S-B-carboline-3-acyl group-Arg (Tos)-Gly-Asp (the OBzl)-Val-OBzl of full guard.Side routinely
Protection group sloughed by method trifluoroacetic acid-trifluoromethanesulfonic acid mixed liquor, product Sephadex G10 column purification, finally gives target
Product S-B-carboline-3-acyl group-RGDV, for light yellow solid powder.
ESI-MS(m/z)+30V 644.36[M+H]+.
1HNMR (300MHz, DMSO-d6) δ/ppm=11.04 (s, 1H), 10.89 (s, 1H), 9.62 (s, 1H), 8.83
(m, 1H), 8.33 (d, J=6.6Hz, 2H), 7.95 (m, 1H), 7.49 (m, 1H), 7.37 (d, J=11.1Hz, 1H), 7.30 (d,
J=10.8Hz, 1H), 7.22 (m, 1H), 7.19 (m, 1H), 7.12 (m, 1H), 4.75 (m, 1H), 4.43 (m, 2H), 4.28 (d, J
=12.6Hz, 2H), 4.11 (m, 1H), 3.81 (s, 2H), 3.13 (s, 2H), 2.81 (m, 1H), 2.65 (m, 1H), 2.12 (m,
1H),1.91(s,1H),1.57(s,3H),1.27(m,4H),1.17(m,1H),0.87(s,6H).
13CNMR (75MHz, DMSO-d6) δ/ppm=173.16,172.05,171.34,171.22,168.99,168.60,
157.09,136.82,135.26,126.80,126.15 123.30,119.72,119.03,105.26,60.85,57.74,
55.64,53.00,49.76,42.07,39.18,34.72,32.28,32.11,29.80,25.43,19.53,18.39.
IR: ν NH-:3350.08cm-1
ν C-H:2967.78cm-1
ν C=O (amide): 1660.07cm-1
ν C=C (phenyl ring): 1538.97cm-1 1454.05cm-1
δ O-H: 1172.62cm in face-11028.07cm outside face-1
δ C-C (phenyl ring skeleton): 761.68cm-1,638.24cm-1
HPLC purity=(98.92 ± 1.08) %.
The preparation of embodiment 2. blank liposome
Film material and the preparation of drug solution:
The chloroform of 10mM CRV (MW=643): methanol=1:1 (v:v) mixed liquor: accurately weigh 64.3mg CRV with appropriate
Chloroform: after methanol=1:1 (v:v) mixed liquor dissolves, proceed to 10mL brown volumetric flask, use mixed liquor constant volume；
The chloroformic solution of 10mM CH (MW=387): accurately weigh 38.7mg CH and dissolve with appropriate chloroform, proceeds to 10mL palm fibre
Color tolerance measuring bottle, with chloroform constant volume；
Measure appropriate film material solution in corresponding ratio, add eggplant bottle, 37 DEG C of water-baths, 120rpm, the rotations of vacuum 250mbar
After turning evaporation 30min, every 5min reduces 50mbar until 1mbar, removes organic solvent, forms homogeneous thin film.It was vacuum dried
At night, remove trace organic reagent.Add the ultrasonic 10min of aquation after 1mL 1% tween 80 solution dissolves, lipid membrane is complete
Wash from bottle wall, then with Probe Ultrasonic Searching 15min (general power 130W, 90%, super 2s stop 2s), cool down and i.e. obtain 100%CRV
liposomes。
Between particle diameter 150-200nm, Zeta-Potential about 25mV.
The preparation of embodiment 3. novel siRNA delivery system
After the SURVIVIN-siRVIVIN dry powder of the DEPC solution of 10 μMs of SURVIVIN-siRNA: 1OD is centrifugal, use 250 μ
After the DEPC water of L fully dissolves and get final product.
The DEPC solution of 0.1 μ g/ μ L calf thymus DNA: take the calf thymus DNA dry powder of 1mg centrifugal after, use 1mL DEPC
Water fully dissolves and obtains storing solution.Take the storing solution of 100 μ L during use, add after 900 μ L DEPC water dilute and get final product.
The DEPC solution of 2 μ g/ μ L protamines: accurately weigh the protamine of 4mg, after fully dissolving with 2mL DEPC water
Obtain.
SURVIVIN-siRVIVIN is mixed according to the ratio of 2.5:1 (w:w) with calf thymus DNA, repeatedly blows and beats, room
10min is stood under temperature.Blank liposome is mixed with suitable protamine simultaneously, repeatedly blow and beat, left at room temperature 10min.
After liposome/protamine complex is joined in SURVIVIN-siRVIVIN/DNA complex, the most quickly blow and beat, room
Gentle and quiet put 15min, obtain SURVIVIN-siRNA/100%CRV liposomes.Omnidistance use RNase-free rifle head, EP
Pipe.
Between particle diameter 150-200nm, Zeta-Potential about 0mV.
The anti tumor activity in vitro evaluation of embodiment 4. novel siRNA delivery system
Cell is cultivated
Cultivation cell: human cervical cancer cell line's HeLa cell,
Culture fluid: containing 10% hyclone, the final concentration of 100U/mL of penicillin, the final concentration of 100 μ g/ of streptomycin sulfate
The DMEM culture fluid of mL is cultivated；
Culture environment: 37 DEG C, volume fraction and the CO of 5%2In the constant incubator of saturated humidity；
Propagating method: often pass on according to the ratio of 1:2 or 1:3 for 1-2 days
Under inverted microscope, observe its form and upgrowth situation, find that cell attachment grows, triangular in shape or polygon,
Edge clear, is uniformly distributed.
Medicine is prepared
I experimental group
Take the DEPC solution of 10 μMs of SURVIVIN-siRNA of 20 μ L and the calf thymus DNA of the 0.1 μ g/mL of 10.4 μ L
DEPC solution in RNase-free tube, repeatedly blow and beat, left at room temperature 10min；The C blank liposome of 39.2 μ L simultaneously
It is placed in another RNase-free tube with the protamine of 19.2 μ L, repeatedly blows and beats, left at room temperature 10min.After by lipid
Body/protamine complex joins in siRNA/DNA complex, the most quickly blows and beats, and room temperature stands 15min, to obtain final product.
Being diluted to 2mL respectively by the DMEM culture medium without serum and antibiotic, obtaining SURVIVIN-siRNA concentration is
The mother solution of 100nM.Take the mother solution of 200,400,600 μ L respectively, dilute respectively by the DMEM culture medium without serum and antibiotic
To 800 μ L, obtain mother solution that SURVIVIN-siRNA concentration is 100nM and 25,50, the SURVIVIN-siRNA/100% of 75nM
The diluent of CRV liposomes, standby.
II preparation group
Take the blank liposome of same volume required during preparation novel siRNA delivery system mother solution in RNase-free
Tube, dilutes 2mL respectively by the DMEM culture medium without serum and antibiotic, obtains the mother solution that CRV concentration is 100nM.Respectively
Take the mother solution of 200,400,600 μ L, be diluted to 800 μ L respectively by the DMEM culture medium without serum and antibiotic, obtain CRV dense
Degree for the mother solution of 100nM and 25,50, the diluent of the 100%CRV liposomes of 75nM, standby.
III naked siRNA group
Take the DEPC solution of 10 μMs of SURVIVIN-siRNA of 20 μ L in RNase-free tube, with without serum and anti-
The DMEM culture medium of raw element is diluted to 2mL, obtains the mother solution that SURVIVIN-siRNA concentration is 100nM, take 200 respectively, 400,
The mother solution of 600 μ L, is diluted to 800 μ L respectively by the DMEM culture medium without serum and antibiotic, obtains SURVIVIN-siRNA
Concentration be the mother solution of 100nM and 25,50, the Naked SURVIVIN-siRNA diluent of 75nM, standby.
IV positive controls
Take the DEPC solution of 10 μMs of SURVIVIN-siRNA of 20 μ L in RNase-free tube, with 1mL without serum
Dilute with the DMEM culture medium of antibiotic, separately take 60 μ L RNAiMAX, dilute by the 1mL DMEM culture medium without serum and antibiotic
Release, respectively after dilution uniformly, after the DMEM liquid of SURVIVIN-siRNA joined in the DMEM liquid of RNAiMAX, repeatedly blow and beat
After, room temperature stands 5min, to obtain final product.
Being diluted to 2mL respectively by the DMEM culture medium without serum and antibiotic, obtaining SURVIVIN-siRNA concentration is
The mother solution of 100nM.Take the mother solution of 200,400,600 μ L respectively, dilute respectively by the DMEM culture medium without serum and antibiotic
To 800 μ L, obtain mother solution that SURVIVIN-siRNA concentration is 100nM and 25,50, the SURVIVIN-siRNA/ of 75nM
RNAiMAX diluent, standby.
V negative control group
The DEPC solution taking 20 10 μMs of NC-siRNA of μ L, obtains with III in RNase-free tube, step thereafter
NC-siRNA concentration be the mother solution of 100nM and 25,50, the NC-siRNA diluent of 75nM, standby.
The DEPC solution taking 20 10 μMs of NC-siRNA of μ L, obtains with IV in RNase-free tube, step thereafter
NC-siRNA concentration be the mother solution of 100nM and 25,50, the NC/RNAiMAX diluent of 75nM, standby.
VI blank group (control)
The normal HeLa cell cultivated, and experimental group same operation, only give when being administered equivalent without serum and anti-
The DMEM culture medium culturing of raw element.
VIII zeroing group
Not planting cell in hole, other process same experimental group.
Experimental technique
Human cervical cancer cell lines HeLa cell is adherent growth cell, is inoculated in culture bottle with debita spissitudo, and addition contains
10% hyclone, the penicillin of 100U/mL, the DMEM complete culture solution of streptomycin of 100 μ g/mL, be placed in 37 DEG C of constant temperature,
5%CO2The incubator of saturated humidity is cultivated.Take the logarithm trophophase HeLa cell, after 0.25% trypsinization completely, add suitable
When complete medium terminate digestion, be transferred to after piping and druming in the centrifuge tube of 15mL, 1500rpm is centrifuged 3min, after supernatant discarded,
With fresh complete culture solution Eddy diffusion, cell counter counts, is diluted to 4 × 104The cell suspending liquid of cells/mL.100μ
L/well by cell suspension inoculation in 96 well culture plates, 37 DEG C, 5%CO2Incubator cultivates 24h, to be administered.
Carefully suck supernatant, after washing 3 times with PBS liquid, add the pastille culture medium 100 μ L for preparing, zeroing hole and right
Adding isopyknic DMEM culture medium without serum and antibiotic according to hole, often group sets 6 multiple holes.Put 37 DEG C, 5%CO2Incubator
Suck supernatant after cultivating 4h, after washing 3 times with PBS liquid, change complete medium into and continue to cultivate 20h.
Every hole adds the 20 freshly prepared serum-free mediums containing 5mg/mLMTT of μ L, 37 DEG C, 5%CO2Incubator continues training
Support 4h.After terminating cultivating, 96 orifice plate high speed centrifuge 1000rpm are centrifuged 5min, make cell sink at the bottom of hole.Carefully suck
Clear liquid, and add 150 μ L DMSO, shake 500rpm, 10min with microoscillator.After mixing, to detect ripple in microplate reader
A length of 490nm measures absorbance (OD) value, calculates tumor cell survival rate.Cells survival rate (%)=(experimental group OD value-blank
Group OD value)/(matched group OD value-blank group OD value) × 100%.
Experimental result
By accompanying drawing 1 it can be seen that in the range of experimental concentration:
Naked SURVIVIN-siRNA group and Naked NC group do not have significant difference (P ＞ 0.05), illustrate with negative
Comparison Naked NC compares, and Naked SURVIVIN-siRNA, almost without cytotoxicity, also demonstrates that free siRNA not simultaneously
Can be by cellular uptake；NC/RNAiMAX group and Naked NC group do not have significant difference (P ＞ when NC concentration is 25nM and 50nM
0.05), but there is significant difference (P ＜ 0.05) when NC concentration is 75nM, have statistically significant when NC concentration is 100nM
Difference, illustrates when the NC of high concentration also can produce toxic action to cell.
100%CRV liposomes group and NC group have significant difference (P ＜ 0.05) when CRV concentration is 100nM, say
Bright have obvious antitumor action as 100%CRV liposomes, and presents dose dependent.
SURVIVIN-siRNA/RNAiMAX group is not united when siRNA concentration is 25nM and 50nM with NC/RNAiMAX group
Difference (P ＞ 0.05) learned by meter, but have significant difference (P ＜ 0.05) when siRNA concentration is 75nM and 100nM, illustrates to work as
Cell can not be caused significant toxic action when SURVIVIN-siRNA concentration is relatively low.
SURVIVIN-siRNA/100%CRV liposomes and 100%CRV liposomes in siRNA concentration is
There is significant difference (P ＜ 0.05) during 50nM and 75nM, have statistically-significant difference (P ＜ when siRNA concentration is 100nM
0.01), illustrate that SURVIVIN-siRNA/100%CRV liposomes resisting under siRNA low concentration or middle and high concentration swells
Tumor effect is all from the summation of gene silencing and carrier self, i.e. 100%CRV liposomes transfection efficiency is preferable, can be by
Major part siRNA transfection enters cell.
SURVIVIN-siRNA/100%CRV liposomes Yu SURVIVIN-siRNA/RNAiMAX is in siRNA concentration
There is significant difference (P ＜ 0.05) during for 50nM and 100nM, have statistically-significant difference (P ＜ when siRNA concentration is 75nM
0.01), illustrate at SURVIVIN-siRNA/100%CRV liposomes body under siRNA low concentration or middle and high concentration
Interior anti-tumor activity is superior to commercially available transfection reagent.
The evaluation of experimental example 5. novel siRNA delivery system protein level silence efficiency
The extraction of total protein
Take the Tissue Culture Plate that transfection terminates, discard culture medium, 6 orifice plates are placed on ice, wash 3 times with ice PBS, add
The ice PBS of 1mL/well, scrapes with cell and carefully fully scrapes cell.The PBS liquid containing cell scraped is transferred to 1.5mL
Tube in, 3000rpm, 4 DEG C of centrifugal 5min, abandoning supernatant, add the mixed pyrolysis liquid of 250 μ L/well, slightly vortex oscillation
Ultrasonic it is placed on ice, reacts 30min.After completion of the reaction, 12000rpm, 4 DEG C of centrifugal 10min, take supernatant ,-80 DEG C of preservations
Standby.
Total protein is quantitative
The mensuration of standard curve
Taking the standard sample 25 μ L prepared in 96 orifice plates, every hole adds the BCA working solution of 200 μ L, after by 96 orifice plates
It is placed on agitator vibration 30s, is allowed to be sufficiently mixed.It is transferred to 96 orifice plates at 37 DEG C hatch 30min.After completion of the reaction by 96
Orifice plate is cooled to room temperature, measures its absorbance by microplate reader under 562nm wavelength
The mensuration of sample
Taking the sample 25 μ L of extraction in 96 orifice plates, step is ibid thereafter.The total protein concentration of sample is calculated according to formula.
ELISA
The preparation of solution
Wash Buffer: take Wash Buffer concentrated solution (25 ×) of 2mL in 50mL centrifuge tube, dilute with pure water
To 50mL, to obtain final product.
Substrate Solution: Color Reagent A and B equal-volume being mixed, lucifuge, after mixing in 15min
Use.
Calibrator Diluent RD5-33 (1 ×): take 2mLCalibrator Diluent RD5-33 in 15mL from
In heart pipe, it is diluted to 12mL with pure water, to obtain final product.
SURVIVIN Standard: with 1mL DEPC water resuspended SURVIVIN standard substance, be sufficiently mixed uniformly, stand
15min, obtains the SURVIVIN mother solution that concentration is 20000pg/mL.
The preparation of SURVIVIN standard curve
Prepare 2mL RNase-free tube 8, take the SURVIVIN mother solution of 100 μ L in pipe 1, use Calibrator
Diluent RD5-33 (1 ×) is diluted to 1000 μ L, and remaining often pipe adds 500 μ L Calibrator Diluent RD5-33
(1 ×), takes 500 μ L pipe 1 solution and adds pipe 2, takes the solution after 500 μ L mixings the most successively and adds pipe below, and multiple proportions is dilute
Release.Obtain concentration be 31.2,62.5,125,250,500,1000, the SURVIVIN standard solution of 2000pg/mL, use
Calibrator Diluent RD5-33 (1 ×) is as comparison.
Test kit detects
1. will balance 30min under various reagent and sample room temperature and prepare reagent and see 2.5.1；
2. remove microwell plate packaging, unnecessary aluminium foil dress of putting into is repacked；
3. every hole adds the Assay Diluent RD1-9 (mensuration diluent) of 100 μ L；
Set criterion group, matched group and sample sets the most respectively.Matched group adds 100 μ L Calibrator Diluent RD5-33
(1×)；Criterion group adds the standard sample prepared in 100 μ L 2.5.2；Sample sets adds the sample that total protein is 50 μ g, not enough
100 μ L persons, supply with DEPC water.After rocking mixing gently, seal microwell plate with the adhesive tape provided, be then transferred to vortex
Instrument 500 ± 50rpm, vortex 2h under room temperature；
5. discarding liquid in hole, dry, 400 μ L/ time wash buffer wash 3 times, tip upside down on for the last time on clean filter paper
Pat to clean no liquid；
6. every hole adds 200 μ L Survivin Conjugate, reseals microwell plate, incubated at room 2h；
7. repeat the 5th step；
8. every hole adds 200 μ LSubstrate Solution, reseals microwell plate, and room temperature lucifuge hatches 30min；
9. every hole adds the Stop Solution termination reaction of 50 μ L, and solution colour is turned yellow by indigo plant, if green can
Rap or low speed vortex microwell plate, to guarantee to mix completely；
10. in reaction terminating 30min, under 450nm wavelength, measure the absorbance in each hole by microplate reader, then make with 540nm
For calibration wavelength, deduct the suction after the absorbance recorded under 540nm wavelength i.e. obtains calibration with the absorbance recorded under 450nm wavelength
Luminosity.
Experimental result
By accompanying drawing 2 it can be seen that
The intracellular SURVIVIN content of Naked SURVIVIN-siRNA group and Naked NC group is relative to control group not
Having significant difference, Naked SURVIVIN-siRNA group is not the most added up relative to Naked NC group intracellular SURVIVIN content
Learning difference, illustrate under administration concentration, Naked siRNA can not play and lower the effect that target protein is expressed；
The intracellular SURVIVIN content of SURVIVIN-siRNA/RNAiMAX group is relative to Naked NC and control group all
There is significant difference, and the intracellular SURVIVIN content of NC/RNAiMAX group does not all have relative to Naked NC and control group
Significant difference, illustrates that SURVIVIN-siRNA transfection can be entered cell by commercially available transfection reagent RNAiMAX really, and special
The expression of specific down-regulation SURVIVIN albumen, can be as positive control；
The intracellular SURVIVIN content of SURVIVIN-siRNA/100%CRV liposomes group relative to Naked NC and
Control group has a statistically-significant difference, and the intracellular SURVIVIN content of NC/100%CRV liposomes group relative to
Naked NC and control group all do not have significant difference, illustrate that 100%CRV liposomes can be by SURVIVIN-
SiRNA transfection enters cell, and the expression of specific downregulation SURVIVIN albumen；
The intracellular SURVIVIN content of SURVIVIN-siRNA/100%CRV liposomes group is relative to SURVIVIN-
SiRNA/RNAiMAX group does not has significant difference, illustrates under administration concentration, SURVIVIN-siRNA/100%CRV
The effect of the expression of liposomes specific downregulation SURVIVIN albumen there was no significant difference with commercially available transfection reagent.
The evaluation of experimental example 6. novel siRNA delivery system mRNA level in-site silence efficiency
The extraction of the full RNA of cell
1. cell cracking:. take and transfect 6 orifice plates terminated, supernatant discarded (abandon clean, but not washed cell, in order to avoid mRNA fall
Solve), add the Trizol lysate of 1mL/well, repeatedly blow and beat with liquid-transfering gun, until cell cracks completely, be transferred to EP pipe,
Stand 5min so that nucleic acid-protein complex decomposes completely；
2. it is separated: adding 0.2mL chloroform in often pipe, build lid, acutely vibrate 15s, and ambient temperatare puts 2-3min,
Then 12000 × g, 4 DEG C of centrifugal 15min.Being divided into three layers after Li Xin, the bottom is red phenol chloroform phase, and the superiors are colourless
Aqueous phase, and marginal intermediate layer.RNA exists only in aqueous phase, aqueous phase the most of the total volume 50%；
3. precipitation RNA: the careful often pipe that is transferred in RNase-free tube by colourless for upper strata aqueous phase adds 0.5mL isopropyl
Alcohol, rear chamber is gentle and quiet puts 10min in mixing, then 12000 × g, 4 DEG C of centrifugal 10min..RNA precipitate is not seen, after being centrifuged before Li Xin
Cotton-shaped glue sample precipitation is occurring at the bottom of tube side and pipe, is being the RNA precipitated；
4. washing RNA: take supernatant, often pipe adds 75% ethanol that 1mL has prepared, after agitator mixing, 7500 × g, 4 DEG C
Centrifugal 5min；
5.RNA re-dissolved: go supernatant, ambient temperatare to put 5-10min, is dried RNA precipitate.Noting can not be complete by RNA precipitate
White drying, because so can greatly reduce its dissolubility.With the resuspended RNA precipitate of DEPC water of 25 μ L, use RNase-free
Tips repeatedly blows and beats and is allowed to mix, and places 10-15min, until RNA precipitate is completely dissolved under 55-60 DEG C of water-bath.-20 DEG C of guarantors
Deposit；
6. survey concentration: after cleaning with DEPC water and calibrate Nanodrop-1000, take the RNA solution sample after 2 μ L mixings successively
Product, drip in loading pond, close loading pond, select RNA-40, click on measure and start to measure and record, note measuring difference
Sample before clean loading pond.
Reverse transcription
1. all of sample and reagent are placed in 4 DEG C of thawings；
2. preparation of samples: the applied sample amount ensureing each sample is 2 μ g, according to the RNA concentration recorded, the RNA needed for calculating
Volume；
3. take a MicroAmpTMOptical 96-Well Reaction, each reacting hole adds 10 μ L 2 × RT
Buffer, 1 μ L20 × RT EnzymeMix and the RNA solution of proper volume, if less than 20 μ L, supplying with DEPC water, then using
Plank is sealed by Optical adhesive film.4 DEG C, 1000rmp is centrifuged 1min, makes liquid accumulation in bottom and drive gas away
Bubble；
4. put into PCR System 9700, condition is set.94 DEG C of 5min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 72 DEG C
7min, 4 DEG C of ∞.Totally 30 circulations.
5. survey concentration: after cleaning with DEPC water and calibrate Nanodrop-1000, take the cDNA solution after 2 μ L mixings successively
Sample, drips in loading pond, closes loading pond, selects DNA-50, clicks on measure and starts to measure and record, notes measuring not
Loading pond is cleaned before same sample.
RT-PCR
1.4 DEG C of thawingsGene ExpressionMaster Mix、Gene Expression
BIRC5 primer in Assays and GAPDH primer；
2. preparation of samples: the applied sample amount ensureing each sample is 20ng, according to the cDNA concentration recorded, needed for calculating
CDNA volume；
3. take a MicroAmpTMOptical 96-Well Reaction, each reacting hole adds 10 μ L
Gene ExpressionMaster Mix, 1 μ L primer (BIRC5 or GAPDH) and the cDNA sample of proper volume, if less than 20 μ
L then supplies with DEPC water, is then sealed by plank with Optical adhesive film.4 DEG C, 1000rpm is centrifuged 1min, makes
Liquid accumulation is in bottom and drives bubble away
4. put into Applied Biosystems 7500 Real-Time PCR System, condition is set.Hold:50 DEG C
2min, 95 DEG C of 10min.Cycle (40cycles): 95 DEG C 15s, 60 DEG C of 1min.
5. Data processing, the threshold of Ct value selects Applied Biosystems Sequence detection
The automatically calculated of software (7500FastSystem SDS Software version 1.4).
With GAPDH as internal reference, use the amount of 2^-Δ Δ Ct method relative quantification each experimental group mRNA.
Experimental result
By accompanying drawing 3 it can be seen that
The SURVIVIN mRNA relative amount of Naked SURVIVIN-siRNA group and Naked NC group is for control
Group does not has significant difference, and the SURVIVIN mRNA relative amount of Naked SURVIVIN-siRNA group is for Naked NC
Group does not has significant difference yet, illustrates that, under administration concentration, Naked siRNA can not play the effect of gene silencing.
The SURVIVIN mRNA relative amount of SURVIVIN-siRNA/RNAiMAX group is for Naked NC and control
Group has a statistically-significant difference, and NC/RNAiMAX group content is not the most added up for Naked NC and control group
Learn difference, illustrate that SURVIVIN-siRNA transfection can be entered cell, and specificity by commercially available transfection reagent RNAiMAX really
Reticent SURVIVIN gene, can be as positive control.
The SURVIVIN mRNA relative amount of SURVIVIN/100%CRV liposomes group for Naked NC and
Control group has a statistically-significant difference, and the SURVIVIN mRNA relative amount of NC/100%CRV liposomes group
All there is no significant difference relative to Naked NC and control group, illustrate that 100%CRV liposomes can be by
SURVIVIN-siRNA transfection enters cell, and specificity silence SURVIVIN gene.
The SURVIVIN mRNA relative amount of SURVIVIN-siRNA/100%CRV liposomes group relative to
SURVIVIN-siRNA/RNAiMAX group does not has significant difference, illustrates under administration concentration, SURVIVIN-siRNA/100%
The effect of CRV liposomes specificity silence SURVIVIN gene there was no significant difference greatly with commercially available transfection reagent.
The anti-tumor in vivo activity rating of embodiment 7. novel siRNA delivery system
Model is set up
By S180Lotus sarcoma mouse peritoneal tumor cell inoculation in an ICR mouse peritoneal, continuous passage, when the 8th day without
Bacterium extraction mouse peritoneal tumor cell, in addition normal saline adjustment cell suspension, cell density is to 1.5 × 107Cell/mL, uses
In inoculation.With 2% iodine tincture cotton balls and 75% cotton ball soaked in alcohol, is sterilized in right side of mice oxter, inject 0.2mL oncocyte liquid.By this side
Method gives every ICR mouse inoculation, and mice is randomly divided into 5 groups, and often group 12, body weight zero difference between each group, put into barrier and raise
Support.
Antitumor activity evaluation
Plant rising for the 8th day after tumor to start to be administered, the medicinal liquid prepared by 0.2mL/ tail vein injection, successive administration 5 days.
Sacrifice, dissection were taken out tumor tissues and weighed in after first administration the 6th day；Dissect take out heart, liver, spleen, kidney,
Brain is weighed respectively.After kind of tumor the 7th day is denoted as the 0th day, in 0, within 1,2,3,4,5 day, weighs Mouse Weight with electronic scale.Record
Mouse Weight changes, and calculates tumour inhibiting rate, organ index.
Experimental result
By accompanying drawing 4 it can be seen that
The tumor heavy phase of Naked SURVIVIN-siRNA group and 100%CRV liposomes group has statistics for NS group
Significant difference, illustrates under administration concentration, and Naked SURVIVIN-siRNA and 100%CRV liposomes is respectively provided with necessarily
Anticancer effect in vivo, suppress tumor growth to a certain extent.
The tumor heavy phase of DOX group has statistics pole significant difference for NS group, illustrates that DOX has clearly under administration concentration
Anticancer effect in vivo, hence it is evident that suppression tumor growth, can be as positive control.SURVIVIN-siRNA/100%CRV
The tumor heavy phase of liposomes group has statistics pole significant difference to NS group, illustrates under administration concentration, SURVIVIN-siRNA/
100%CRV liposomes has clear and definite Anticancer effect in vivo, hence it is evident that suppression tumor growth.
The tumor heavy phase of SURVIVIN-siRNA/100%CRV liposomes group is to positive controls DOX group the most substantially
Diversity, illustrates under administration concentration, the Anticancer effect in vivo of SURVIVIN-siRNA/100%CRV liposomes and city
The antitumor drug DOX selling determined curative effect is roughly the same.
The tumor heavy phase of SURVIVIN-siRNA/100%CRV liposomes group is for Naked SURVIVIN-siRNA group
Or 100%CRV liposomes group has statistics pole significant difference, illustrate under administration concentration, 100%CRV
SURVIVIN-siRNA transfection can be entered cell, and specificity silence SURVIVIN gene, water in vivo by liposomes
The obvious anti-tumor activity of flat performance.
By accompanying drawing 5 it can be seen that
DOX group, under administration concentration, has obvious cardiac toxicity, liver spleen toxicity and slight Toxicity of Kidney；SURVIVIN-
SiRNA/100%CRV liposomes group, under administration concentration, has slight hepatotoxicity.SURVIVIN-siRNA/ is described
100%CRV liposomes is through liver metabolism, but toxicity is less compared with amycin, and application prospect is preferable.
Embodiment 8.CRV acute toxicity is evaluated
Acute toxicity is evaluated
Mice is randomly divided into 2 groups, often group 10, body weight zero difference between each group, puts into barrier and raise.
After adaptability is raised 1 day, within second day, start to be administered, according to 0.2mL/ lumbar injection urgency toxic agent amount (experiment in vivo
100 times of dosage) medicinal liquid, conventional after administration raise, observe 7 days, put to death, dissect taking-up heart, liver, spleen, kidney
Dirty, brain, testis are weighed respectively.Administration is denoted as first day the 1st day, in 1, within 2,3,4,5,6,7 days, weighs Mice Body with electronic scale
Weight.Observe the action of mice appearance, reflex activity, drink amount, survival condition, body weight change etc., and calculate organ index.
Experimental result
By accompanying drawing 6 it can be seen that
Compared with matched group (C.g), the dirty body ratio of experimental group (E.g) liver and spleen has significant difference.Anxious poison is described
Under dosage (100 times of experiment in vivo dosage), CRV has slight hepatotoxicity.
Embodiment 9. cell apoptosis assay
The process of cell and labelling
After being administered, 6,12,24,48h collect cell.The trypsinization of the 0.25% of every hole 0.3mL is complete
After, adding 1mL complete medium and terminate digestion, be transferred to after piping and druming in the centrifuge tube of 15mL, 1500rpm is centrifuged 3min, discards
After supernatant, obtain cell mass.Washing with 1mL ice PBS, 1500rpm is centrifuged 3min, discards cleaning mixture.Washed cell is used
1 × Binding Buffer of 200 μ L is resuspended, measures cell density, is diluted to 1 × 106cell/mL
Taking 100 μ L Cell saps, count 5 μ L FITC annexin V and the PI working solutions of 1 μ L, under room temperature, lucifuge is hatched
15min, mends 1 × Binding Buffer of 400 μ L again before upper machine testing.
Cross film, use flow cytomery as early as possible.
Experimental result
By accompanying drawing 7 and 8 it can be seen that
Relatively naked siRNA group and blank group, find that between two groups, each time point apoptosis rate is basically identical, explanation
Free SURVIVIN-siRNA can not enter cell, causes apoptosis；Comparative experiments group and blank group, find two groups
Between each time point apoptosis rate there were significant differences, wherein SURVIVIN-siRNA/100%CRV liposomes has induction
The effect of HeLa early apoptosis of cells but and inconspicuous, the effect inducing HeLa cell late apoptic is the most fairly obvious, from
24h plays the peak period that 48h is late apoptic.Illustrate 100%CRV liposomes SURVIVIN-siRNA can be transfected into
Entering cell, causing apoptosis, apoptosis peak period is 24-48h upon administration.
Embodiment 10. cell cycle is tested
Cell is fixed
After being administered, 6,12,24,48h collect cell.The trypsinization of the 0.25% of every hole 0.3mL is complete
After, adding 1mL complete medium and terminate digestion, be transferred to after piping and druming in the centrifuge tube of 15mL, 1500rpm is centrifuged 3min, discards
After supernatant, obtain cell mass.Washing with 1mL ice PBS, 1500rpm is centrifuged 3min, discards cleaning mixture, and cell mass is complete
Being suspended in 0.5mL PBS, dropwise instill in off-the-shelf 9.5mL 70% ethanol solution ,-20 DEG C overnight, preserves stand-by.
PI dyes
Taking out fixing cell, 1000rpm, centrifugal 10min, outwell ethanol, ice PBS washs 2 times, 1000rpm, centrifugal
10min, discards cleaning mixture.Cell mass is resuspended in the PBS liquid of the RNase A that 1mL prepares, adds the PI solution of 50 μ L
(1mg/mL), 37 DEG C of water-bath lucifuge dyeing 30min.Using flow cytomery cell cycle, Cytosoft software is carried out point
Analysis.
Experimental result
By accompanying drawing 9 it can be seen that
Relatively naked siRNA group and blank group, finds that between two groups, Cell Cycle change is basically identical, illustrates free
SURVIVIN-siRNA can not enter cell, cause Cell Cycle to change；Comparative experiments group and blank group, find
Between two groups, Cell Cycle there are differences, and experimental group can suppress the HeLa cell entrance S phase to carry out DNA synthesis after being administered, and
And with G2The effect that/M the phase blocks, this with document on SURVIVIN gene inhibition apoptosis and maintenance cell mitogen
The mechanism of action matches.Illustrate that SURVIVIN-siRNA transfection can be entered cell by 100%CRV liposomes, cause thin
Born of the same parents' apoptosis.
Claims (10)
1. an amphiphilic compound S-B-carboline-3-acyl group-RGDV, referred to as CRV with anti-tumor activity.
2. the compound described in claim 1, it is characterised in that this compound is little molecule the energy self assembly of anti-tumor activity
Form nano-carrier.
3. prepare a synthetic method of amphiphilic compound CRV described in claim 1, comprise the following steps: by L-Trp
Hydrophobic side N-Boc-S-B-carboline carboxylic acid is obtained after PS condensation, Boc protection；Use main chain side with Boc protection aminoterminal or
OBzl protects c-terminus, the side chain l-amino acid of suitable protection group protection, and liquid phase method connects peptide, obtains full guard dipeptides, point
Not after hydrogenolysis sloughs OBzl protection or Boc protection is sloughed in acidolysis, liquid phase method connects peptide, obtains full guard tetrapeptide, through acidolysis
After sloughing Boc protection, finally give the four titanium RGDV that water-wet side amino is exposed；The hydrophobic side N-Boc-S-B-carboline carboxylic that will obtain
The acid RGDV liquid phase method exposed with water-wet side amino connects peptide, sloughs with trifluoroacetic acid-trifluoromethanesulfonic acid mixed liquor according to a conventional method
Protection group, finally gives target product CRV.
Synthetic method the most according to claim 3, it is characterised in that comprise the following steps:
1) preparation of N-Boc-S-carboline carboxylate
Under the catalysis of dilute sulfuric acid, tryptophan (L-Trp) occurs PS to react at normal temperatures with formaldehyde, and condensation generates S-B-carboline carboxylic
Acid；By the S-B-carboline carboxylic acid of gained in DMF, in the basic conditions with Boc2O reacts, and obtains N-Boc-S-B-carboline
Carboxylic acid,
2) preparation of RGDV
Boc-Arg (Tos)-OH and H-Gly-OBzl HCl is connect peptide by liquid phase method, obtains full guard dipeptides Boc-Arg
(Tos)-Gly-OBzl, in like manner obtains Boc-Asp (the OBzl)-Val-OBzl of full guard, respectively by Boc-Arg (Tos)-Gly-
The exposed carboxyl of OBzl hydrogenolysis, the exposed amino of Boc-Asp (OBzl)-Val-OBzl acidolysis, connect peptide by liquid phase method, obtain full guard
Tetrapeptide, finally gives, through acidolysis, the RGDV that water-wet side amino is exposed,
3) preparation of S-B-carboline-3-acyl group-RGDV
RGDV liquid phase method exposed with water-wet side amino for the hydrophobic side N-Boc-S-B-carboline carboxylic acid obtained is connect peptide, prepares all risk insurance
Intermediate N Boc-S-B-carboline-3-acyl group-Arg (Tos)-Gly-Asp (the OBzl)-Val-OBzl protected, uses according to a conventional method
Protection group sloughed by trifluoroacetic acid-trifluoromethanesulfonic acid mixed liquor, product Sephadex G10 column purification, finally gives target product
S-B-carboline-3-acyl group-RGDV, for light yellow solid powder.
5. a novel siRNA delivery system, it is characterised in that: containing the anti-tumor activity that has described in claim 1 or 2
Amphiphilic compound CRV and Antioncogene medicine SURVIVIN-siRNA.
Delivery system the most according to claim 5, it is characterised in that be made up of following component: Antioncogene is treated
Medicine SURVIVIN-siRNA, cholesterol, protamine, calf thymus DNA and described there is the amphipathic of anti-tumor activity
Compound CRV.
Delivery system the most according to claim 5, it is characterised in that be made up of following component, following percentage ratio is
Mass percent: SURVIVIN-siRNA 8.74-8.80%, cholesterol 3.15-3.17%, protamine 6.35-6.40%,
Calf thymus DNA 3.48-3.50% and the amphipathic film material CRV 78.2-78.78% with anti-tumor activity.
8. the method preparing novel siRNA delivery system described in any one of claim 5-7, comprises the following steps: will
Cholesterol and have the mixed solvent of the amphiphilic compound CRV chloroform of anti-tumor activity and methanol and dissolve, rotary evaporation goes
Except solvent, form homogeneous thin film, dried in vacuum overnight, remove trace organic reagent, add tween that 1mL concentration is 1%-
The ultrasonic 10min of 80DEPC water hydratable, washes from bottle wall completely by liposome membrane, then uses Probe Ultrasonic Searching 15min, obtains blank liposomes
Body, mixes SURVIVIN-siRNA according to the ratio of 2.5:1 (w:w) with calf thymus DNA, repeatedly blows and beats, left at room temperature
10min, mixes blank liposome with appropriate protamine simultaneously, repeatedly blows and beats, left at room temperature 10min, after by lipid
Body/protamine complex joins in SURVIVIN-siRNA/DNA complex, the most quickly blows and beats, and room temperature stands 15min,
Obtain.
9. the application in preparing siRNA delivery system of the S-B-carboline-3-acyl group-RGDV described in any one of claim 1-2.
10. the application in the medicine of preparation treatment tumor of the novel siRNA delivery system described in claim 5-7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610362271.0A CN106046118A (en) | 2016-05-26 | 2016-05-26 | Preparation of siRNA transfer system comprising S-Beta-carboline-3-acyl-RGDV and application of system in SURVIVIN gene silencing |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610362271.0A CN106046118A (en) | 2016-05-26 | 2016-05-26 | Preparation of siRNA transfer system comprising S-Beta-carboline-3-acyl-RGDV and application of system in SURVIVIN gene silencing |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106046118A true CN106046118A (en) | 2016-10-26 |
Family
ID=57176016
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610362271.0A Pending CN106046118A (en) | 2016-05-26 | 2016-05-26 | Preparation of siRNA transfer system comprising S-Beta-carboline-3-acyl-RGDV and application of system in SURVIVIN gene silencing |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106046118A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103539837A (en) * | 2012-07-15 | 2014-01-29 | 彭莉 | 1-methyl-beta-carbolinyl-3-formyl RGD peptides, and synthesis, nano structure, antithrombotic action and application thereof |
| CN104211768A (en) * | 2013-06-05 | 2014-12-17 | 首都医科大学 | Conjugates of beta-carboline-3-carboxylic acid and oligopeptides, preparation, nano structure, and application thereof as antitumor agent |
| CN104531709A (en) * | 2014-12-24 | 2015-04-22 | 葛银林 | siRNA dual-interference composition for inhibiting growth and metastasis of tumors and application thereof |
| CN105002183A (en) * | 2015-08-13 | 2015-10-28 | 吉林大学 | siRNA molecule for inhibiting survivin gene expression and application of siRNA molecule |
| CN105152939A (en) * | 2008-11-10 | 2015-12-16 | 阿尔尼拉姆医药品有限公司 | Lipids and compositions for the delivery of therapeutics |
-
2016
- 2016-05-26 CN CN201610362271.0A patent/CN106046118A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105152939A (en) * | 2008-11-10 | 2015-12-16 | 阿尔尼拉姆医药品有限公司 | Lipids and compositions for the delivery of therapeutics |
| CN103539837A (en) * | 2012-07-15 | 2014-01-29 | 彭莉 | 1-methyl-beta-carbolinyl-3-formyl RGD peptides, and synthesis, nano structure, antithrombotic action and application thereof |
| CN104211768A (en) * | 2013-06-05 | 2014-12-17 | 首都医科大学 | Conjugates of beta-carboline-3-carboxylic acid and oligopeptides, preparation, nano structure, and application thereof as antitumor agent |
| CN104531709A (en) * | 2014-12-24 | 2015-04-22 | 葛银林 | siRNA dual-interference composition for inhibiting growth and metastasis of tumors and application thereof |
| CN105002183A (en) * | 2015-08-13 | 2015-10-28 | 吉林大学 | siRNA molecule for inhibiting survivin gene expression and application of siRNA molecule |
Non-Patent Citations (1)
| Title |
|---|
| NA LIN, ET AL.: "Synthesis and antithrombotic activity of carbolinecarboxyl RGD sequence", 《BIOORG MED CHEM LETT.》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106137968B (en) | Inner membrance has reversible crosslink Biodegradable polymer vesicles of positive electricity and preparation method thereof and application in preparation of anti-tumor drugs | |
| CN107446019A (en) | Antibacterial peptide derivatives and application thereof | |
| CN107400180B (en) | Redox responds chitosan-liposome preparation method and purposes | |
| CN106344924B (en) | It is a kind of combine metabolic block nano-formulation and its drug resistance inversion application | |
| US11364232B2 (en) | Polymer of gamma-glutamyl transpeptidase catalyzing hydrolysis-induced charge reversal and its application in the field of drug delivery | |
| CN110054660A (en) | A kind of preparation and application of the breast cancer targeting lipids material of fructose modification | |
| CN110772645A (en) | Functionalized cell-penetrating peptide modified drug delivery system | |
| CN108478802A (en) | A kind of the administering drug combinations system and construction method of high stability use for cancer treatment | |
| CN105859990A (en) | Polymer with side chains containing lipoyl, preparation method of polymer, polymer vesica prepared from polymer and application of polymer vesica | |
| CN106967717A (en) | A kind of aptamers mediation adenovirus targeting drug delivery system and its construction method | |
| CN107233332A (en) | Preparation, activity and the application of GO PLL RGDS/VEGF siRNA target gene medicines | |
| CN103626846A (en) | Ligand polypeptide specifically combined with MDSCs (Myeloid-Derived Suppressor Cells) and drug delivery system | |
| CN107320731A (en) | The preparation and evaluation of the new Ru-polypyridine complex carrier of RGDF targetings | |
| CN107320736A (en) | The new Ru-polypyridine complex of cancer target carries siRNA preparation and evaluation | |
| CN107998081A (en) | A kind of application for targeting reduction response vesica Nano medication in treatment of brain tumor medicine is prepared | |
| CN109422801A (en) | Multifunctional targeted polypeptide RAP and its preparing the purposes in cancer target delivery system | |
| CN106046118A (en) | Preparation of siRNA transfer system comprising S-Beta-carboline-3-acyl-RGDV and application of system in SURVIVIN gene silencing | |
| CN105521497B (en) | Embedded cationic liposomal gene carrier system of sucrose fatty ester and its preparation method and application | |
| CN109276570A (en) | The Nano medication and preparation method thereof of biological source macrocycle molecule | |
| CN101317835B (en) | Application of cantharidin and its derivant in preparing sensitization medicament for tumour chemotherapy | |
| CN104788391B (en) | Cinnamoyl dimethyl diaminophenazine chloride amide (CA PZ) and its preparation and application | |
| CN102210668B (en) | Application of cantharidin and derivants thereof in preparation of tumor chemotherapy sensitivity enhancing medicine | |
| CN108403665B (en) | Prostate cancer targeted drug delivery carrier modified by EpDT3 aptamer, delivery system, preparation method and application thereof | |
| CN104971350A (en) | Efficacy-enhancing nanometre medicine composition for treating liver cancer and preparation method of efficacy-enhancing nanometre medicine composition | |
| CN102532262B (en) | Preparation of fatty acid-RGD-fatty alcohol conjugate mediated epirubicin targeted liposome and evaluation on anticancer activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20161026 |