CN114748640B - 一种pH响应性的siRNA递送系统 - Google Patents
一种pH响应性的siRNA递送系统 Download PDFInfo
- Publication number
- CN114748640B CN114748640B CN202210484674.8A CN202210484674A CN114748640B CN 114748640 B CN114748640 B CN 114748640B CN 202210484674 A CN202210484674 A CN 202210484674A CN 114748640 B CN114748640 B CN 114748640B
- Authority
- CN
- China
- Prior art keywords
- sirna
- delivery system
- peg
- rgdfk
- polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108020004459 Small interfering RNA Proteins 0.000 title claims abstract description 8
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 29
- 229920001184 polypeptide Polymers 0.000 claims abstract description 24
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 24
- 108091081021 Sense strand Proteins 0.000 claims abstract description 14
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims abstract description 13
- 102000040650 (ribonucleotides)n+m Human genes 0.000 claims abstract description 13
- 108020005544 Antisense RNA Proteins 0.000 claims abstract description 13
- 230000000692 anti-sense effect Effects 0.000 claims abstract description 12
- 230000008878 coupling Effects 0.000 claims abstract description 10
- 238000010168 coupling process Methods 0.000 claims abstract description 10
- 238000005859 coupling reaction Methods 0.000 claims abstract description 10
- IUTPJBLLJJNPAJ-UHFFFAOYSA-N 3-(2,5-dioxopyrrol-1-yl)propanoic acid Chemical compound OC(=O)CCN1C(=O)C=CC1=O IUTPJBLLJJNPAJ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000006243 chemical reaction Methods 0.000 claims abstract description 8
- RUVRGYVESPRHSZ-UHFFFAOYSA-N 2-[2-(2-azaniumylethoxy)ethoxy]acetate Chemical compound NCCOCCOCC(O)=O RUVRGYVESPRHSZ-UHFFFAOYSA-N 0.000 claims abstract description 5
- UXWMYMBEWDSDQN-UHFFFAOYSA-N 4-(benzylamino)benzohydrazide Chemical compound C1=CC(C(=O)NN)=CC=C1NCC1=CC=CC=C1 UXWMYMBEWDSDQN-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000003814 drug Substances 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 7
- 238000000137 annealing Methods 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 2
- 239000007859 condensation product Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 34
- 229920001223 polyethylene glycol Polymers 0.000 abstract description 28
- 238000001727 in vivo Methods 0.000 abstract description 14
- 210000003734 kidney Anatomy 0.000 abstract description 12
- 238000009825 accumulation Methods 0.000 abstract description 8
- 239000002202 Polyethylene glycol Substances 0.000 abstract description 6
- NBBJYMSMWIIQGU-UHFFFAOYSA-N Propionic aldehyde Chemical compound CCC=O NBBJYMSMWIIQGU-UHFFFAOYSA-N 0.000 abstract description 6
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 abstract description 5
- ZTQSAGDEMFDKMZ-UHFFFAOYSA-N butyric aldehyde Natural products CCCC=O ZTQSAGDEMFDKMZ-UHFFFAOYSA-N 0.000 abstract description 3
- 230000002503 metabolic effect Effects 0.000 abstract description 3
- 230000001743 silencing effect Effects 0.000 abstract description 3
- JYXLDXBKFBWKOV-UPHRSURJSA-N (z)-4-(2-carboxyethylamino)-4-oxobut-2-enoic acid Chemical compound OC(=O)CCNC(=O)\C=C/C(O)=O JYXLDXBKFBWKOV-UPHRSURJSA-N 0.000 abstract 1
- 102000009618 Transforming Growth Factors Human genes 0.000 abstract 1
- 108010009583 Transforming Growth Factors Proteins 0.000 abstract 1
- 102000005962 receptors Human genes 0.000 abstract 1
- 108020003175 receptors Proteins 0.000 abstract 1
- 239000002924 silencing RNA Substances 0.000 description 34
- 210000004027 cell Anatomy 0.000 description 14
- 238000000034 method Methods 0.000 description 13
- 239000000243 solution Substances 0.000 description 12
- 239000007789 gas Substances 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 102000039446 nucleic acids Human genes 0.000 description 9
- 108020004707 nucleic acids Proteins 0.000 description 9
- 150000007523 nucleic acids Chemical class 0.000 description 9
- 238000011580 nude mouse model Methods 0.000 description 9
- 239000002504 physiological saline solution Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 241000699660 Mus musculus Species 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 210000004185 liver Anatomy 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 7
- 229940079593 drug Drugs 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- 230000006378 damage Effects 0.000 description 6
- 230000014509 gene expression Effects 0.000 description 6
- 230000030279 gene silencing Effects 0.000 description 6
- 238000001819 mass spectrum Methods 0.000 description 6
- 208000032612 Glial tumor Diseases 0.000 description 5
- 206010018338 Glioma Diseases 0.000 description 5
- 239000012043 crude product Substances 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 150000003573 thiols Chemical class 0.000 description 5
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000012226 gene silencing method Methods 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 239000002502 liposome Substances 0.000 description 4
- 238000004949 mass spectrometry Methods 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000008685 targeting Effects 0.000 description 4
- 125000003396 thiol group Chemical group [H]S* 0.000 description 4
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 3
- 108010082126 Alanine transaminase Proteins 0.000 description 3
- 102000001189 Cyclic Peptides Human genes 0.000 description 3
- 108010069514 Cyclic Peptides Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- PMZXXNPJQYDFJX-UHFFFAOYSA-N acetonitrile;2,2,2-trifluoroacetic acid Chemical compound CC#N.OC(=O)C(F)(F)F PMZXXNPJQYDFJX-UHFFFAOYSA-N 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 3
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 3
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 239000003755 preservative agent Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 3
- 239000003381 stabilizer Substances 0.000 description 3
- PXQLVRUNWNTZOS-UHFFFAOYSA-N sulfanyl Chemical compound [SH] PXQLVRUNWNTZOS-UHFFFAOYSA-N 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 108090000174 Interleukin-10 Proteins 0.000 description 2
- 108090001005 Interleukin-6 Proteins 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000008499 blood brain barrier function Effects 0.000 description 2
- 210000001218 blood-brain barrier Anatomy 0.000 description 2
- -1 cationic cholesterol derivative Chemical class 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical group 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 description 2
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000009368 gene silencing by RNA Effects 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 229940088592 immunologic factor Drugs 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 125000001805 pentosyl group Chemical group 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 230000004962 physiological condition Effects 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 210000004926 tubular epithelial cell Anatomy 0.000 description 2
- 230000004614 tumor growth Effects 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical class [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000001301 EGF receptor Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 238000003820 Medium-pressure liquid chromatography Methods 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 206010029155 Nephropathy toxic Diseases 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- NWHWRJIOZRGVRW-UHFFFAOYSA-N OC([Pt])=O Chemical compound OC([Pt])=O NWHWRJIOZRGVRW-UHFFFAOYSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229940124350 antibacterial drug Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 229940109239 creatinine Drugs 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 239000002274 desiccant Substances 0.000 description 1
- 230000025009 detection of wounding Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 201000011243 gastrointestinal stromal tumor Diseases 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000000367 immunologic factor Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 229920001427 mPEG Polymers 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000002114 nanocomposite Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000007694 nephrotoxicity Effects 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000001208 nuclear magnetic resonance pulse sequence Methods 0.000 description 1
- 238000001668 nucleic acid synthesis Methods 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 231100000683 possible toxicity Toxicity 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000006453 vascular barrier function Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K47/60—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
- 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)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明涉及生物技术领域,特别的涉及一种针对肿瘤生长因子受体的pH响应性的siRNA递送系统。本发明的递送系统包括由多肽衍生物修饰的正义链ssRNA和PEG衍生物修饰的反义链asRNA所形成的分子;本发明的递送系统中多肽为c(RGDfK),PEG衍生物修饰的反义链asRNA由甲氧基聚乙二醇丙醛(mPEG‑CHO)分子、偶联分子和asRNA连接反应而成,mPEG‑CHO中的PEG分子量为500‑20000,mPEG‑CHO与偶联分子之间形成腙键,偶联分子包括8‑氨基‑3,6‑二氧杂辛酸、4‑(苄基氨基)苯甲酰肼、N‑马来酰基‑β‑丙氨酸、3‑马来酰亚胺基丙酸中的一种或多种。该递送系统能够延长siRNA的体内循环时间,同时抑制siRNA在肾脏的代谢累积,提高肿瘤靶向递送效率,从而更好地实现siRNA的沉默效应。
Description
技术领域
本发明涉及生物技术领域,特别的涉及一种针对肿瘤的pH响应性的siRNA递送系统。
背景技术
小干扰核酸分子(siRNA)有时称为短干扰RNA或沉默RNA,是一类双链RNA分子,一般为19到23个碱基对(bp)组成的双链RNA,siRNA主要参与RNA干扰(RNAi)现象,以带有专一性的方式调节基因的表达,其具有序列同源性驱动的基因敲除能力。正是基于这种能力,其在临床应用前景广阔,但是由于siRNA具有分子量大、带负电荷等生物大分子结构特性,体内应用siRNA分子会存在稳定性差(易被血清中的核酸酶降解)、脱靶效应、免疫原性、易在肝肾代谢累积以及难以被细胞摄取等问题。这些问题严重阻碍了siRNA临床开发。
近年来随着多功能脂质体、高分子聚合物、无机纳米粒等靶向递送系统取得了飞速进展,CN202210009042.6 公开了一种阳离子胆固醇衍生物,含有天然胆固醇骨架和赖氨酸作为头基,选择了最适于siRNA结合的Linker链长,确保该单一脂质能与siRNA形成稳定的纳米复合物,无需辅助脂质即可实现基因递送。CN202111610856.7公开了以具有抗肿瘤作用的羧基铂为交联分子,将含有精氨酸与赖氨酸的多肽交联形成铂肽共聚物,再利用静电相互作用与具有诱导细胞凋亡的siRNA混合组装,最后用负电荷脂质体包裹,形成稳定的纳米复合物,最终制成化学/基因协同治疗的纳米传递系统。等等,上述相关专利技术部分解决了siRNA的靶向递送、肿瘤细胞摄取等难题,但siRNA进入体内后,生物分布不均匀、肝肾累积量高、肿瘤靶区浓度低等靶向递送效率不足的问题,对于siRNA肿瘤靶向递送系统的设计提出了更高的要求。
发明内容
要解决的问题
针对肿瘤靶向siRNA递送系统效率不高、生物分布不佳、肿瘤部位浓度低的问题,本发明人通过潜心研发,开发了一种全新的pH响应性的siRNA递送系统,该递送系统能够延长siRNA的体内循环时间,同时抑制siRNA在肾脏的代谢累积,提高肿瘤靶向递送效率,从而更好地实现siRNA的沉默效应。
技术方案
本发明公开了一种pH响应性的siRNA递送系统,所述系统包括由多肽衍生物修饰的正义链ssRNA和PEG衍生物修饰的反义链asRNA所形成的分子。
本发明的一个方面,本发明的多肽衍生物修饰的正义链ssRNA由多肽、偶联分子和ssRNA连接反应而成。
本发明的一个方面,本发明的mPEG衍生物修饰的反义链asRNA由甲氧基聚乙二醇丙醛(mPEG-CHO)分子、偶联分子和asRNA连接反应而成。
本发明的一个方面,本发明中多肽为短肽,优先所述多肽由小于10个氨基酸分子组成的短肽,所述短肽可以是9个、8个、7个、6个、5个、4个、3个氨基酸分子组成的。
本发明的一个方面,本发明中多肽为环肽,由多个氨基酸分子形成环状肽链结构,优选的由小于10个氨基酸分子形成的环肽;
本发明的一个方面,本发明中多肽为肿瘤靶向肽,优选所述多肽为RGD肿瘤靶向肽,更优选所述多肽为c(RGDfK)
本发明的一个方面,本发明中偶联分子为易于与多肽的末端或侧链发生氨基、羧基缩合反应的试剂,优选4-(苄基氨基)苯甲酰肼、3-马来酰亚胺基丙酸、1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)、N,N′-二环己基碳二亚胺(DCC)、N-羟基琥珀酰亚胺(NHS)、8-氨基-3,6-二氧杂辛酸中的一种或多种。
本发明的一个方面,本发明中mPEG-CHO, 聚乙二醇的分子量可以是500、1000、1500、2000、4000、6000、8000、20000,所述mPEG-CHO与偶联分子之间形成pH敏感的腙键。PEG可以通过增强药物的EPR效应更好地靶向肿瘤部位,较小的PEG分子可以提高肾脏清除率加快代谢从而降低其潜在的毒性,而较大的PEG分子可以更有效地延长siRNA的体内循环时间,从而提高其在肿瘤组织内的浓度,但是过高的PEG会导致分子容易在肝脏聚集可能会造成潜在的毒性,因此,在本发明中选择500-20000分子量的PEG。
本发明的一个方面,本发明提供了一种制备siRNA递送系统的方法,所述方法包括如下步骤:
(1)将mPEG-CHO和4-(苄基氨基)苯甲酰肼反应制备获得含有腙键的PEG衍生物mPEG-hdy,然后将mPEG-hdy与3-马来酰亚胺基丙酸、纯化获得含有腙键(hdy)和马来酰亚胺基(Mal)的PEG衍生物mPEG-hdy-Mal;
(2)将多肽分子与8-氨基-3,6-二氧杂辛酸脱水缩合,然后,将缩合产物与3-马来酰亚胺基丙酸进一步偶联,纯化获得含有马来酰亚胺基(Mal)的多肽-Mal;
(3)将多肽-Mal、mPEG-hdy-Mal分别与siRNA的正义链、反义链连接,形成多肽衍生物修饰的正义链ssRNA、PEG衍生物修饰的反义链asRNA;
(4)将多肽衍生物修饰的正义链ssRNA、PEG衍生物修饰的反义链asRNA溶于退火缓冲液中,退火反应、纯化获得所述siRNA递送系统。
本发明的一个方面,本发明中所述mPEG-CHO分子中聚乙二醇的分子量为2000,所述多肽分子为c(RGDfK)。
本发明的一个方面,本发明还提供了pH响应性的siRNA递送系统在制备试剂、药物、试剂盒中应用,其所述试剂、药物、试剂盒中包括所述siRNA递送系统。
本发明的一个方面,本发明中的试剂除含有本发明的siRNA递送系统以外还含有必要保持递送系统活性、稳定的物质,例如缓冲液、稳定剂、防腐剂、水、生理盐水等物质。本发明中的药物除含有本发明的siRNA递送系统以外还含有必要保持递送系统活性、稳定的物质,例如缓冲液、稳定剂、防腐剂、水、生理盐水、以及药物上可接受的辅料;本发明的药物可以制备为注射剂、冻干剂、喷剂、凝胶制剂以及其他任意可以方便药物递送和使用的剂型。本发明的含有siRNA递送系统的药物还可以与其他用于预防和/或治疗疾病的药物共同使用,例如与抗病毒药物一起使用、与抗细菌药物一起使用、与抗肿瘤药物一起使用。本发明中的试剂盒除含有本发明的siRNA递送系统以外还含有必要保持递送系统活性、稳定的物质或其他与本递送系统配合使用的物质,例如缓冲液、稳定剂、防腐剂、水、生理盐水等物质,所述试剂盒还含有纸质或电子的说明书以指导试剂盒的使用、指示相关结果的判断和/或分析。
本发明的一个方面,本发明还提供了本发明所述的pH响应性的siRNA递送系统在基因沉默中的应用,所述应用包括使用包含所述递送系统的试剂、药物、试剂盒进行目的基因沉默,目的基因可以是任意的人们感兴趣或有需求的想要改变基因表达水平的基因。
本发明的一个方面,本发明针对已有的靶向神经胶质瘤特异性沉默表皮生长因子受体EGFR mRNA表达的cRGD-siEGFR偶联分子所存在的递送效率不足、生物分布不佳、肿瘤部位浓度低的问题,提供了一种pH响应性的siRNA递送系统,特别涉及了一种c(RGDfk)-PEG-siRNA递送系统,,递送系统中PEG分子量为2000,所述递送系统的结构如式I所示。
式 I
该递送系统能够延长siEGFR的体内循环时间,同时抑制siEGFR在肾脏的代谢累积,提高肿瘤靶向递送效率,从而更好地实现siEGFR的沉默效应。
有益效果
基于本发明的技术方案,本发明具有如下有益效果:
(1)直接利用多肽分子、PEG分子对siRNA分子进行修饰制备共轭化合物,通过对siRNA分子直接进行修饰,无需借助脂质体、高分子载体等材料即可实现siRNA的靶向递送,是一种全新的设计方法。本发明的设计方法与将突破各级递送屏障的各种基团叠加、制备复杂的多功能药物递送系统的设计相比,本发明的递送分子结构清楚,合成简单,工艺稳定,更容易实现 GMP 产业化。
(2)采用pH敏感性PEG衍生化修饰,既能抑制非特异性吸附,又能增强靶细胞摄取。
总之,采用本发明的制备方法和递送系统解决了目前siRNA靶向递送系统普遍存在递送效率低、生物分布不理想、肿瘤靶区浓度低的难题。
附图说明
图1:本发明中mPEG-hdy-Mal的合成路径;
图2:本发明合成的mPEG-hdy-Mal核磁共振氢谱;
图3:本发明中c(RGDfk)-Mal的合成路径;
图4:本发明中c(RGDfk)-Mal的HPLC纯化分析;
图5:本发明中c(RGDfk)-Mal的质谱分析;
图6:本发明中c(RGDfk)-PEG-siRNA的合成路径,c(RGDfk)-Mal与ssRNA合成c(RGDfk)-ssRNA,mPEG-hdy-Mal与asRNA合成PEG-asRNA,c(RGDfk)-ssRNA与PEG-asRNA反应形成c(RGDfk)-PEG-siRNA;
图7:本发明中c(RGDfk)-ssRNA的质谱图;
图8:本发明中c(RGDfk)-PEG-siRNA的琼脂糖凝胶电泳,泳道1:naked siRNA;泳道2:c(RGDfk)-PEG-siRNA;泳道3:在pH6.5条件孵育后的c(RGDfk)-PEG-siRNA,c(RGDfk)-PEG-siRNA分子在pH6.5条件孵育后PEG与siRNA之间对酸敏感的腙键发生断裂,缺少PEG的“延迟效应”后,条带便与第一泳道的 naked siRNA基本持平;
图9:本发明中c(RGDfk)-PEG-siRNA对肾小管上皮细胞(HK2)的细胞毒性(p>0.05);
图10:本发明中qRT-PCR验证c(RGDfk)-PEG-siRNA的基因沉默效率;
图11: c(RGDfk)-PEG-siRNA递送系统的在荷瘤裸鼠的体内分布;
图12:施用不同浓度的c(RGDfk)-PEG-siRNA递送系统对小鼠胶质瘤荷瘤体积的影响。
图13:施用c(RGDfk)-PEG-siRNA递送系统对荷瘤小鼠的肝肾毒性;
图14:ELISA检测c(RGDfk)-PEG-siRNA递送系统对小鼠血清中免疫因子的含量;
图15:HE染色检测c(RGDfk)-PEG-siRNA递送系统对裸鼠脏器的损伤。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。
实施例1mPEG-hdy-Mal的制备
mPEG-hdy-Mal的合成反应路径如图1所示,具体的:第一步:将甲氧基聚乙二醇丙醛(mPEG-CHO)和4-(苄基氨基)苯甲酰肼按1:2的摩尔比在无水乙醇体系中搅拌,添加当量的乙酸作为催化剂;在氮气保护下将反应混合物在80℃回流6-8小时后将反应混合物冷却到室温,通过过滤收集沉淀物,通过使用二氯甲烷进行简单洗涤,去除残留的起始材料和杂质得到含有腙键(hdy)PEG衍生物(mPEG-hdy)。第二步,将上述所得的mPEG-hdy和3-马来酰亚胺基丙酸按1:5的摩尔比添加到二氯甲烷溶液中,搅拌混合物4小时。用饱和氯化铵溶液洗涤混合物,然后用饱和碳酸氢钠溶液洗涤两次。收集有机层,用无水硫酸钠进行干燥,并在旋转蒸发仪上浓缩。将粗产物溶解于含0.05%乙酸的水/乙腈(2:1)体系中。通过高效液相色谱法对粗产物进行纯化,得到mPEG-hdy-Mal(色谱条件:进样体积50μL;柱温40℃;流速2.5mL/min;流动相为A相0.1%TFA乙腈,B相0.1%水;洗脱程序为0~30min,5%~50%A;30~32min,50%~5%A)。采用1H NMR进行表征。(使用600MHz型Bruker-AVANCE Ⅲ NMR仪进行检测,调用Noesyprld脉冲序列,具体设置如下:采样温度298K,采样时间间隔2s,累加次数为64次,混合时间为80ms,90°的脉冲宽度为11μs,谱宽为12kHz,采用32K傅里叶变换方法将自由感应衰减信号转为1H-NMR谱图)
实施例2 c(RGDfk)-Mal的制备
将c(RGDfk)多肽(12.1 mg)和8-氨基-3,6-二氧杂辛酸(3.02 mg)在聚乙二醇溶液中在氮气室温下搅拌4-8h,直至所有的起始肽被消耗完,加入3-马来酰亚胺基丙酸(6.76mg)继续搅拌反应4-8h。所得的粗产物通过RP-MPLC纯化。(色谱条件:进样体积50μL;柱温40℃;流速2.5mL/min;流动相为A相0.1%TFA乙腈,B相0.1%水;洗脱程序为0~30min,5%~50%A;30~32min,50%~5%A)。
得到的c(RGDfk)-Mal采用质谱进行表征(应用UHPLC-Q Exactive Orbitrap四级杆组合静电轨道阱液质联用仪进行检测,采用电喷雾离子源(H-ESI),鞘气流量30L/min,气帘气流量10L/min,喷嘴电压+4000V,毛细管温度320ºC,气帘气温度350ºC,扫描模式FullMS-ddMS2,扫描质量范围(m/z)100~1050,一级质谱分辨率为70,000 FWHM,二级质谱分辨率为17,500 FWHM,碎裂窗口0.4 m/z),c(RGDfk)-Mal HPLC纯化及质谱二级图如图4-5所示。
实施例3c(RGDfk)-PEG-siRNA的合成
c(RGDfk)-PEG-siRNA的合成路径如图6所示。具体的:通过上述实施例1和实施例2成功合成了中间体及mPEG-hdy-Mal、c(RGDfk)-Mal,以常规的核酸化学合成法构建巯基(-SH)修饰的核酸单链。核酸链5’端单体修饰:在siRNA正义链5’端的核苷酸单元内戊糖5位上羟基磷酸化后,连接巯基(-SH)基团。核酸链3’端单体修饰:在siRNA反义链3’端的核苷酸单元内戊糖5位上羟基磷酸化后,连接巯基(-SH)基团。按照常规核酸合成方法合成单链的正义链ssRNA和反义链asRNA,当需要2’-OMe修饰时,则采用2’-OMe修饰核酸单体合成即可。巯基(-SH)修饰的核酸单链正义链(20 nmol)同过量的c(RGDfk)-Mal(400nmol),溶于1 ml0.1M HEPES缓冲溶液中,4℃均匀混合震荡反应12小时,溶液中巯基修饰的siRNA正义链变为c(RGDfk)-ssRNA。将巯基(-SH)修饰的核酸单链反义链(20 nmol)同过量的mPEG-hdy-Mal(1000nmol),溶于1 ml 0.1M HEPES缓冲溶液中,4℃均匀混合震荡反应12小时,溶液中巯基修饰的siRNA反义链变为PEG-asRNA。将偶联后的核酸单链在20%聚丙烯酰胺凝胶上进行电泳检测。将c(RGDfk)-ssRNA溶液以及PEG-asRNA溶液超滤浓缩:利用XX8200230小型切向流超滤系统分别对(c(RGDfk)-ssRNA、PEG-asRNA)进行超滤处理,得到<10KD的组分,将粗产物溶解于含0.05%乙酸的水/乙腈(2:1)体系中。
通过高效液相色谱法对粗产物进行纯化,使用以下参数:仪器:e2695高效液相色谱仪,配备光电二极管列阵检测器(制备型,美国Waters公司)色谱条件:色谱柱采用Shim-pack GIST C18(250×10mm,5μm,日本岛津公司);进样体积50μL;柱温40℃;流速2.5mL/min;流动相为A相0.1%TFA乙腈,B相0.1%水;洗脱程序为0~30min,5%~50%A;30~32min,50%~5%A ;检测波长260nm下。冻干后得到白色粉末状固体c(RGDfk)-ssRNA和PEG-asRNA。
c(RGDfk)-PEG-siRNA制备:将等量c(RGDfk)-ssRNA(50μM)和PEG-asRNA(50 μM),溶于退火缓冲液(10 mM Tris-HCl pH 7.4, 50 mM NaCl, and 1mM EDTA),95℃加热3分钟,缓慢恢复至室温,除去过量盐分,真空冻干得c(RGDfk)-PEG-siRNA分子冻干粉。
使用高效液相色谱法、质谱(参见图7)、和琼脂糖凝胶电泳(参见图8)等纯化分离化合物以及验证化合物结构和纯度。质谱表征参数:应用UHPLC-Q Exactive Orbitrap四级杆组合静电轨道阱液质联用仪进行检测,采用电喷雾离子源(H-ESI),鞘气流量30L/min,气帘气流量10L/min,喷嘴电压+4000V,毛细管温度320ºC,气帘气温度350ºC,扫描模式FullMS-ddMS2,扫描质量范围(m/z)100~1050,一级质谱分辨率为70,000 FWHM,二级质谱分辨率为17,500 FWHM,碎裂窗口0.4 m/z。琼脂糖凝胶电泳:调节DEPC水pH=6.5及pH=7.4,用于溶解c(RGDfk)-PEG-siRNA分子,在37℃条件下进行孵育48小时,用1.2%琼脂糖凝胶电泳,120V,30min,用凝胶电泳成像仪观察。
根据实施例1-3的方法,发明人构建了靶向神经胶质瘤特异性沉默表皮生长因子受体EGFR mRNA表达的pH响应性的siRNA递送系统c(RGDfk)-PEG-siEGFR,其中PEG分子量为2000。
实施例4使用CCK8检测c(RGDfk)-PEG-siRNA分子的体外正常细胞毒性
在细胞培养皿的每孔加入100μL 5000个肾小管上皮细胞HK-2细胞,置于37℃,5%CO2培养箱内培养24小时。24 h后,进行试验分组处理,实验组分别加入100nM、200nM、500nM、1000nM、1500nM、2000nM浓度梯度的c(RGDfk)-PEG-siEGFR分子,对照组即为未处理组(NC),以及空白对照组,每组设定5个复孔,于CO2培养箱内继续培养24h。然后每孔加入10μl CCK-8溶液,用加了相应量细胞培养液和CCK-8 溶液但没有加入细胞的孔作为空白对照。在细胞培养箱内继续孵育1小时,然后在450nm 检测各孔吸光度A值。计算细胞毒性即:细胞增值率(%)= (A给药组-A空白孔)/(A阴性对照组-A空白孔)]×100﹪。结果如图9显示:在不同浓度水平c(RGDfk)-PEG-siEGFR处理后,c(RGDfk)-PEG-siRNA与对照相比对人肾小管上皮细胞HK-2细胞无明显肾毒性,没有显著的差异。
实施例5 qRT-PCR验证c(RGDfk)-PEG-siRNA的基因沉默效率
选择人源胶质瘤细胞U87 MG细胞系作为实验模型,细胞消化传代铺12孔板约24小时后,弃去完全培养基,用PBS洗一遍,加入不含血清的基础培养基,细胞周期同步化培养24小时。更换OPTI-MEM培养基,分别加入c(RGDfk)-PEG-siEGFR、lipo3000/siEGFR、lipo3000/siNC加入细胞培养基中 ,c(RGDfk)-PEG-siEGFR分子浓度梯度为20 nM、50 nM、100 nM、200nM。同时调节培养基pH 7.4和6.5,6小时后,更换新鲜的完全培养基培养。
如图10结果显示,在没有额外载体材料或者脂质体等转染试剂的辅助下,在正常生理条件(pH 7.4)的条件下由于PEG衍生物分子中的腙键不在中性和弱碱性的条件下不断裂,能“屏蔽”细胞对c(RGDfk)-PEG-siEGFR的摄取,因此其不能有效地抑制U87 MG细胞EGFRmRNA的表达水平;而模拟肿瘤弱酸微环境(pH约为6.5)的条件下,PEG衍生物分子中的腙键断裂,可有效被细胞摄取,因此c(RGDfk)-PEG-siEGFR可显著抑制U87 MG细胞EGFR mRNA的表达水平。
实施例6 c(RGDfk)-PEG-siRNA递送系统的体内分布和体内疗效
(1)c(RGDfk)-PEG-siRNA递送系统体内分布:
建立颅内原位荷瘤裸鼠模型,裸鼠在接种肿瘤之后的7天后,对裸鼠按照分组进行尾静脉注射给药,实验分为3组,每组3只,A组为连接了Cy5荧光基团的裸siRNA(Cy5-siNC):剂量为1.5 nmol/20 g,B组为连接Cy5荧光基团的c(RGDfk)-siRNA(Cy5-c(RGDfk)-siNC):剂量为1.5 nmol/20 g,C组为连接Cy5荧光基团的c(RGDfk)-PEG-siRNA(Cy5-c(RGDfk)-PEG-siNC):剂量为1.5 nmol/20 g。给药后在不同的时间点(12h、24h、48h)利用荧光成像系统,对Cy-5标记的siRNA进行检测,以没有给药的裸鼠作为对照,激发波长和发射波长分别为640nm和680nm。对获得的荧光图像进行分析,评价c(RGDfk)-PEG-siRNA在体内各器官中的分布。如图11所示,与siRNA相比,c(RGDfk)-siRNA和c(RGDfk)-PEG-siRNA具有透过血脑屏障及脑肿瘤血管屏障,同时能延长体内循环时间,在24h和48h后仍在脑部聚集;而相较于c(RGDfk)-siRNA,本发明的c(RGDfk)-PEG-siRNA可以明显减少在非肿瘤组织(如肝肾)中的积累,增强肿瘤病灶中的浓度。
(2)c(RGDfk)-PEG-siRNA递送系统的体内疗效:
建立小鼠胶质瘤荷瘤模型,每三天测量肿瘤体积以及荷瘤鼠体重变化。待肿瘤生长到可触摸时用游标卡尺定期测量肿瘤体积,当体积长到约为50 mm3 时开始给药;实验分为4组,每组8只,A组为正常对照组,给予生理盐水(125μl/20 g)、B组为c(RGDfk)-PEG-siEGFR(1.5 nmol/20 g)、C组为c(RGDfk)-PEG-siEGFR(5 nmol/20 g)、D组 c(RGDfk)-PEG-siNC(5 nmol/20 g)。每3天给药一次,一共6次。定期测量荷瘤鼠体重和肿瘤体积,体积计算公式 :V=ab2×0.5 (a为长直径,b为短直径)。如图12所示:与生理盐水组和c(RGDfk)-PEG-siNC组相比,c(RGDfk)-PEG-siEGFR分子能明显抑制肿瘤生长,且浓度越大,抑制作用越强。
实施例7、血液化学检测连续给药后的肝肾毒性
建立荷瘤裸鼠模型,实验分为5组,每组8只,A组为正常对照组,给予生理盐水(125μl/20 g)、B组为含siRNA无意义序列的阴性对照组c(RGDfk)-PEG-siNC(5 nmol/20 g)、C组为c(RGDfk)-siEGFR(5 nmol/20 g)、D组 c(RGDfk)-PEG-siEGFR(1.5 nmol/20 g)、E组为c(RGDfk)-PEG-siEGFR(5 nmol/20 g)。尾静脉注射7次,每隔48h给药一次。最后一次给药后3天,摘眼球采血,分离血清,用全自动血液分析仪检测各组血清中丙氨酸转氨酶(ALT)和肌酐(Cr)的含量。检测结果如图13所示:与A组正常对照组相比,D组和E组注射c(RGDfk)-PEG-siEGFR分子后ALT和Cr的含量均无显著性差异。表明连续多次尾静脉注射c(RGDfk)-PEG-siEGFR分子,对裸鼠的肝肾功能无明显影响。
实施例8 ELISA检测c(RGDfk)-PEG-siRNA递送系统对小鼠血清中免疫因子的含量
采用免疫系统完整的野生型Balb/c小鼠作为实验模型,实验分为5组给药,每组8只,A组为正常对照组,给予生理盐水(125μl/20 g)、B组为siRNA无意义序列的阴性对照组c(RGDfk)-PEG-siNC(5 nmol/20 g)、C组为c(RGDfk)-siEGFR(5 nmol/20 g)、D组 c(RGDfk)-PEG-siEGFR(1.5 nmol/20 g)、E组为c(RGDfk)-PEG-siEGFR(5 nmol/20 g),单次注射,6小时后,取血清,用ELISA的方法检测细胞因子IL-1β、IL-6、IL-10、TNF-α的变化。检测结果见图14,与生理盐水组相比,使用c(RGDfk)-PEG-siEGFR分子不同浓度处理组处理,其IL-1β、IL-6、IL-10及TNF-α含量均无显著性差异。该结果表明c(RGDfk)-PEG-siEGFR不会引起动物系统性免疫反应。
实施例9 HE染色检测c(RGDfk)-PEG-siRNA递送系统对裸鼠脏器的损伤
建立荷瘤裸鼠模型,实验分为3组给药,每组8只,A组为生理盐水组、B组为c(RGDfk)-PEG-siEGFR(5nmol/20g )和C组为 c(RGDfk)-siEGFR(5nmol/20g),在连续高剂量给药7次(间隔48小时一次),最后一次给药3天后,解剖取材。分别选取了的心、肝、脾、肾进行HE染色观察。结果如图15所示:与A组(生理盐水)相比,c(RGDfk)-siEGFR和c(RGDfk)-PEG-siEGFR分子在高剂量连续给药的条件下,对小鼠的肝脏和脾脏无明显损伤;而C组(c(RGDfk)-siEGFR)的肾脏切片张可看出存在肾间质充血的损伤(图15中黑色箭头所示),而B组(c(RGDfk)-PEG-siEGFR)则无明显的肾损伤。结果表明与c(RGDfk)-siEGFR相比,高剂量的c(RGDfk)-PEG-siEGFR也能避免对肾脏的损伤。
综上所述,本发明成功的构建了一种新型的siRNA递送系统,并且能够在肿瘤环境中发生pH响应性断裂分解发挥作用,而在正常生理条件性保持较高的稳定性,同时,本发明的递送系统在体内体外对于肝肾细胞、正常细胞并没有明显的毒性和损伤,也不会引起体内的免疫反应,在体内能够通过血脑屏障,明显减少在非肿瘤组织(如肝肾)中的积累,增强肿瘤病灶中的浓度,较长时间驻留在肿瘤部位,延长siRNA分子的作用时间,取得了有益的效果和显著的进步。
以上内容是结合具体实施方式对本发明作进一步详细说明,不能认定本发明具体实施只局限于这些说明,对于本发明所属技术领域的普通技术人员来说,在不脱离本发明的构思的前提下,还可以做出若干简单的推演或替换,都应当视为属于本发明所提交的权利要求书确定的保护范围。
Claims (3)
1.一种pH响应性的siRNA递送系统,其特征在于,所述系统包括由多肽衍生物修饰的正义链ssRNA和PEG衍生物修饰的反义链asRNA所形成的分子,其中所述多肽为c(RGDfK);所述分子的结构如下式所示:
。
2.一种制备权利要求1所述的递送系统的方法,其特征在于,所述方法包括如下步骤:
(1)将mPEG-CHO和4-(苄基氨基)苯甲酰肼反应制备获得含有腙键的PEG衍生物mPEG-hdy,然后将mPEG-hdy与3-马来酰亚胺基丙酸、纯化获得含有腙键(hdy)和马来酰亚胺基(Mal)的PEG衍生物mPEG-hdy-Mal;
(2)将多肽分子与8-氨基-3,6-二氧杂辛酸脱水缩合,然后,将缩合产物与3-马来酰亚胺基丙酸进一步偶联,纯化获得含有马来酰亚胺基(Mal)的多肽-Mal,其中所述多肽为c(RGDfK);
(3)将多肽-Mal、mPEG-hdy-Mal分别与siRNA的正义链、反义链连接,形成多肽衍生物修饰的正义链ssRNA、PEG衍生物修饰的反义链asRNA;
(4)将多肽衍生物修饰的正义链ssRNA、PEG衍生物修饰的反义链asRNA溶于退火缓冲液中,退火反应、纯化获得所述递送系统。
3.根据权利要求1所述的递送系统在制备试剂、药物、试剂盒中应用,其特征在于所述试剂、药物、试剂盒中包括所述递送系统。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210484674.8A CN114748640B (zh) | 2022-05-06 | 2022-05-06 | 一种pH响应性的siRNA递送系统 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210484674.8A CN114748640B (zh) | 2022-05-06 | 2022-05-06 | 一种pH响应性的siRNA递送系统 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114748640A CN114748640A (zh) | 2022-07-15 |
CN114748640B true CN114748640B (zh) | 2024-02-09 |
Family
ID=82333603
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210484674.8A Active CN114748640B (zh) | 2022-05-06 | 2022-05-06 | 一种pH响应性的siRNA递送系统 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114748640B (zh) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1253507A (zh) * | 1997-04-23 | 2000-05-17 | 柏林自由大学诊断研究所有限公司 | 用于用近红外光诊断和用于治疗的酸不稳定的及可酶裂解的染料结构 |
CN103232531A (zh) * | 2013-03-05 | 2013-08-07 | 武汉泽智生物医药有限公司 | 一种癌细胞靶向性结构分子及其应用 |
CN104027816A (zh) * | 2014-06-16 | 2014-09-10 | 西安交通大学 | 一种可去聚乙二醇化的共装载阿霉素和siRNA载体及其合成方法 |
CN107029243A (zh) * | 2017-06-08 | 2017-08-11 | 厦门大学 | 一种多肽桥连的双酰腙连接键应用于醛衍生药物的递送 |
CN107261156A (zh) * | 2017-06-07 | 2017-10-20 | 华侨大学 | 一种基于SANH的共载siRNA和阿霉素的MSR‑1磁小体复合物及其制备方法 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5529895B2 (ja) * | 2009-02-04 | 2014-06-25 | ソンギュングヮン ユニバーシティ ファウンデーション フォー コーポレート コラボレーション | 細胞内の伝達能が増加した低分子干渉rna複合体 |
-
2022
- 2022-05-06 CN CN202210484674.8A patent/CN114748640B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1253507A (zh) * | 1997-04-23 | 2000-05-17 | 柏林自由大学诊断研究所有限公司 | 用于用近红外光诊断和用于治疗的酸不稳定的及可酶裂解的染料结构 |
CN103232531A (zh) * | 2013-03-05 | 2013-08-07 | 武汉泽智生物医药有限公司 | 一种癌细胞靶向性结构分子及其应用 |
CN104027816A (zh) * | 2014-06-16 | 2014-09-10 | 西安交通大学 | 一种可去聚乙二醇化的共装载阿霉素和siRNA载体及其合成方法 |
CN107261156A (zh) * | 2017-06-07 | 2017-10-20 | 华侨大学 | 一种基于SANH的共载siRNA和阿霉素的MSR‑1磁小体复合物及其制备方法 |
CN107029243A (zh) * | 2017-06-08 | 2017-08-11 | 厦门大学 | 一种多肽桥连的双酰腙连接键应用于醛衍生药物的递送 |
Non-Patent Citations (1)
Title |
---|
A Diazonium Reagent Which Will Introduce Maleimide Groups into Proteins and Peptides;R.J.S. Duncan et al.;《Journal oflmmunologicaIMethods》;第第80卷卷(第1985期期);第137-140页 * |
Also Published As
Publication number | Publication date |
---|---|
CN114748640A (zh) | 2022-07-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Yang et al. | Dual-modified liposomes with a two-photon-sensitive cell penetrating peptide and NGR ligand for siRNA targeting delivery | |
EP0861228B1 (fr) | Lipopolymamines comme agents de transfection et leurs applications pharmaceutiques | |
Yuan et al. | Dendrimer-triglycine-EGF nanoparticles for tumor imaging and targeted nucleic acid and drug delivery | |
CA2742842A1 (en) | Releasable conjugates for nucleic acids delivery systems | |
WO2023029928A1 (zh) | 一种氨基脂质及其应用 | |
CN109453187B (zh) | 具有双重酶敏感特性的抗体核酸药物偶联物及其制备方法和应用 | |
US20100279408A1 (en) | Polymeric short interfering rna conjugates | |
CN114748640B (zh) | 一种pH响应性的siRNA递送系统 | |
KR20220110174A (ko) | 핵산 치료제를 위한 종양-표적화 폴리펩티드 나노입자 전달 시스템 | |
US20220054645A1 (en) | Targeted Delivery of Therapeutic Molecules | |
US20210214726A1 (en) | Peptide Docking Vehicle for Targeted Nucleic Acid Delivery | |
CN115521220A (zh) | 长链烷基酯胺类化合物及其制备方法和在核酸递送方面的应用 | |
CN106188223B (zh) | 一种含有二肽类脂质阳离子的化合物及其制备方法与应用 | |
CN115212185A (zh) | pH敏感型阿霉素-脂肪酸前药的白蛋白纳米粒 | |
CN114364404A (zh) | 寡核苷酸-聚合物多手臂偶联物以及使用方法 | |
CN115872893B (zh) | 阳离子脂质化合物及其制备方法和应用 | |
CN111195353B (zh) | 一种美登素类抗体药物偶联物及其应用 | |
AU2002257904B2 (en) | Polythiourea lipid derivatives | |
CN115475252A (zh) | 一种基于非天然核酸适体的protac的制备方法及其在三阴性乳腺癌治疗中的应用 | |
CN117624302A (zh) | 一种用于有效递送核酸的分支链结构多肽载体及其变化形式 | |
CN116615246A (zh) | 包含单取代的同-二价连接基的多缀合物 | |
CN117615789A (zh) | 一类可用于活性分子递送的可降解脂质体及其纳米复合物 | |
CN117598980A (zh) | 一种可注射核酸水凝胶原位肿瘤疫苗及其应用 | |
MXPA02004449A (es) | Derivados de oligobencimidazoles y su uso como agentes de transfeccion del acido desoxirribunucleico (adn).. | |
CN117563014A (zh) | 一种抗体-药物基因偶联物及其应用 |
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 |