CN114099699A - 一种纳米递送系统及其制备方法和应用 - Google Patents
一种纳米递送系统及其制备方法和应用 Download PDFInfo
- Publication number
- CN114099699A CN114099699A CN202111385801.0A CN202111385801A CN114099699A CN 114099699 A CN114099699 A CN 114099699A CN 202111385801 A CN202111385801 A CN 202111385801A CN 114099699 A CN114099699 A CN 114099699A
- Authority
- CN
- China
- Prior art keywords
- nano
- nucleic acid
- delivery system
- bromide
- chitosan
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 229920001661 Chitosan Polymers 0.000 claims abstract description 83
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 61
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 61
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 61
- 239000002502 liposome Substances 0.000 claims abstract description 41
- 150000001875 compounds Chemical class 0.000 claims abstract description 30
- 125000002091 cationic group Chemical group 0.000 claims abstract description 29
- -1 cationic lipid Chemical class 0.000 claims abstract description 20
- 239000002539 nanocarrier Substances 0.000 claims abstract description 11
- 125000002795 guanidino group Chemical group C(N)(=N)N* 0.000 claims abstract description 10
- 230000005934 immune activation Effects 0.000 claims abstract description 10
- 108020004459 Small interfering RNA Proteins 0.000 claims description 78
- 150000002632 lipids Chemical class 0.000 claims description 33
- 108090000623 proteins and genes Proteins 0.000 claims description 28
- 230000014509 gene expression Effects 0.000 claims description 24
- 229920001223 polyethylene glycol Polymers 0.000 claims description 23
- 239000002245 particle Substances 0.000 claims description 22
- QGBSISYHAICWAH-UHFFFAOYSA-N dicyandiamide Chemical compound NC(N)=NC#N QGBSISYHAICWAH-UHFFFAOYSA-N 0.000 claims description 21
- 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 description 18
- 230000019491 signal transduction Effects 0.000 claims description 17
- 230000015788 innate immune response Effects 0.000 claims description 16
- KWVJHCQQUFDPLU-YEUCEMRASA-N 2,3-bis[[(z)-octadec-9-enoyl]oxy]propyl-trimethylazanium Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(C[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC KWVJHCQQUFDPLU-YEUCEMRASA-N 0.000 claims description 13
- 239000013598 vector Substances 0.000 claims description 12
- 108091027967 Small hairpin RNA Proteins 0.000 claims description 11
- 102000004169 proteins and genes Human genes 0.000 claims description 11
- 239000004055 small Interfering RNA Substances 0.000 claims description 11
- 235000012000 cholesterol Nutrition 0.000 claims description 9
- 239000003814 drug Substances 0.000 claims description 9
- 102100031256 Cyclic GMP-AMP synthase Human genes 0.000 claims description 8
- 101710118064 Cyclic GMP-AMP synthase Proteins 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 108020004999 messenger RNA Proteins 0.000 claims description 7
- 102000002227 Interferon Type I Human genes 0.000 claims description 6
- 108010014726 Interferon Type I Proteins 0.000 claims description 6
- 108010050904 Interferons Proteins 0.000 claims description 6
- 108010044012 STAT1 Transcription Factor Proteins 0.000 claims description 6
- 102000004265 STAT2 Transcription Factor Human genes 0.000 claims description 6
- 108010081691 STAT2 Transcription Factor Proteins 0.000 claims description 6
- 102000014150 Interferons Human genes 0.000 claims description 5
- 239000002202 Polyethylene glycol Substances 0.000 claims description 5
- 238000007259 addition reaction Methods 0.000 claims description 5
- 229940079322 interferon Drugs 0.000 claims description 5
- 238000013518 transcription Methods 0.000 claims description 5
- 230000035897 transcription Effects 0.000 claims description 5
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 4
- 101001011382 Homo sapiens Interferon regulatory factor 3 Proteins 0.000 claims description 4
- 101001032341 Homo sapiens Interferon regulatory factor 9 Proteins 0.000 claims description 4
- 102100029843 Interferon regulatory factor 3 Human genes 0.000 claims description 4
- 102100038251 Interferon regulatory factor 9 Human genes 0.000 claims description 4
- 108091046869 Telomeric non-coding RNA Proteins 0.000 claims description 4
- 239000013543 active substance Substances 0.000 claims description 4
- 125000004122 cyclic group Chemical group 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 4
- 230000004721 adaptive immunity Effects 0.000 claims description 3
- 230000036039 immunity Effects 0.000 claims description 3
- 230000001225 therapeutic effect Effects 0.000 claims description 3
- UKAUYVFTDYCKQA-UHFFFAOYSA-N -2-Amino-4-hydroxybutanoic acid Natural products OC(=O)C(N)CCO UKAUYVFTDYCKQA-UHFFFAOYSA-N 0.000 claims description 2
- OOGKYHLYXDOJPR-JDVCJPALSA-M 2,3-bis[(z)-octadec-9-enoxy]propyl-(2-hydroxyethyl)-dimethylazanium;bromide Chemical compound [Br-].CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)CCO)OCCCCCCCC\C=C/CCCCCCCC OOGKYHLYXDOJPR-JDVCJPALSA-M 0.000 claims description 2
- JQDSSKQQSCMUJR-LQDDAWAPSA-M 2,3-bis[(z)-octadec-9-enoxy]propyl-trimethylazanium;bromide Chemical compound [Br-].CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)C)OCCCCCCCC\C=C/CCCCCCCC JQDSSKQQSCMUJR-LQDDAWAPSA-M 0.000 claims description 2
- LDGWQMRUWMSZIU-LQDDAWAPSA-M 2,3-bis[(z)-octadec-9-enoxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)C)OCCCCCCCC\C=C/CCCCCCCC LDGWQMRUWMSZIU-LQDDAWAPSA-M 0.000 claims description 2
- NEZDNQCXEZDCBI-UHFFFAOYSA-N 2-azaniumylethyl 2,3-di(tetradecanoyloxy)propyl phosphate Chemical compound CCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCCCCCCCC NEZDNQCXEZDCBI-UHFFFAOYSA-N 0.000 claims description 2
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 claims description 2
- CXRFDZFCGOPDTD-UHFFFAOYSA-M Cetrimide Chemical compound [Br-].CCCCCCCCCCCCCC[N+](C)(C)C CXRFDZFCGOPDTD-UHFFFAOYSA-M 0.000 claims description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 2
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 claims description 2
- 101000959820 Homo sapiens Interferon alpha-1/13 Proteins 0.000 claims description 2
- 102100040019 Interferon alpha-1/13 Human genes 0.000 claims description 2
- UKAUYVFTDYCKQA-VKHMYHEASA-N L-homoserine Chemical compound OC(=O)[C@@H](N)CCO UKAUYVFTDYCKQA-VKHMYHEASA-N 0.000 claims description 2
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 claims description 2
- REYJJPSVUYRZGE-UHFFFAOYSA-N Octadecylamine Chemical compound CCCCCCCCCCCCCCCCCCN REYJJPSVUYRZGE-UHFFFAOYSA-N 0.000 claims description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 2
- 239000004202 carbamide Substances 0.000 claims description 2
- 229940106189 ceramide Drugs 0.000 claims description 2
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 claims description 2
- PSLWZOIUBRXAQW-UHFFFAOYSA-M dimethyl(dioctadecyl)azanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCCCCC[N+](C)(C)CCCCCCCCCCCCCCCCCC PSLWZOIUBRXAQW-UHFFFAOYSA-M 0.000 claims description 2
- MWRBNPKJOOWZPW-CLFAGFIQSA-N dioleoyl phosphatidylethanolamine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-CLFAGFIQSA-N 0.000 claims description 2
- XJWSAJYUBXQQDR-UHFFFAOYSA-M dodecyltrimethylammonium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)C XJWSAJYUBXQQDR-UHFFFAOYSA-M 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims description 2
- UGRVUSWSAGRCSJ-UHFFFAOYSA-N ethylammonium trifluoroacetate Chemical compound CC[NH3+].[O-]C(=O)C(F)(F)F UGRVUSWSAGRCSJ-UHFFFAOYSA-N 0.000 claims description 2
- BEBCJVAWIBVWNZ-UHFFFAOYSA-N glycinamide Chemical compound NCC(N)=O BEBCJVAWIBVWNZ-UHFFFAOYSA-N 0.000 claims description 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims description 2
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 claims description 2
- 150000008104 phosphatidylethanolamines Chemical class 0.000 claims description 2
- 229920000729 poly(L-lysine) polymer Polymers 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims 2
- 102100029904 Signal transducer and activator of transcription 1-alpha/beta Human genes 0.000 claims 1
- 239000002105 nanoparticle Substances 0.000 abstract description 88
- 230000000694 effects Effects 0.000 abstract description 55
- 230000004913 activation Effects 0.000 abstract description 35
- 238000001890 transfection Methods 0.000 abstract description 23
- 229920000642 polymer Polymers 0.000 abstract description 14
- 238000001415 gene therapy Methods 0.000 abstract description 13
- 238000009169 immunotherapy Methods 0.000 abstract description 8
- 230000001976 improved effect Effects 0.000 abstract description 7
- 102000007327 Protamines Human genes 0.000 abstract description 6
- 108010007568 Protamines Proteins 0.000 abstract description 6
- 150000001768 cations Chemical class 0.000 abstract description 6
- 229940048914 protamine Drugs 0.000 abstract description 6
- 230000004048 modification Effects 0.000 abstract description 3
- 238000012986 modification Methods 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 99
- 108060001084 Luciferase Proteins 0.000 description 32
- 239000005089 Luciferase Substances 0.000 description 32
- 239000000243 solution Substances 0.000 description 32
- 239000013612 plasmid Substances 0.000 description 28
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 26
- 206010028980 Neoplasm Diseases 0.000 description 20
- 102100023085 Serine/threonine-protein kinase mTOR Human genes 0.000 description 19
- 108010065917 TOR Serine-Threonine Kinases Proteins 0.000 description 19
- 108020004414 DNA Proteins 0.000 description 16
- 210000001165 lymph node Anatomy 0.000 description 16
- 239000006228 supernatant Substances 0.000 description 14
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 13
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 13
- 239000006166 lysate Substances 0.000 description 12
- 238000000034 method Methods 0.000 description 12
- 230000008569 process Effects 0.000 description 11
- 239000013603 viral vector Substances 0.000 description 11
- 238000001476 gene delivery Methods 0.000 description 10
- 210000003712 lysosome Anatomy 0.000 description 10
- 230000001868 lysosomic effect Effects 0.000 description 10
- 238000007920 subcutaneous administration Methods 0.000 description 10
- 241000699670 Mus sp. Species 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 230000003053 immunization Effects 0.000 description 9
- 238000002649 immunization Methods 0.000 description 9
- 238000001727 in vivo Methods 0.000 description 9
- 238000011740 C57BL/6 mouse Methods 0.000 description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 238000002156 mixing Methods 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 230000007365 immunoregulation Effects 0.000 description 7
- 238000007747 plating Methods 0.000 description 7
- 108020003175 receptors Proteins 0.000 description 7
- 102000005962 receptors Human genes 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 230000001939 inductive effect Effects 0.000 description 6
- 239000002086 nanomaterial Substances 0.000 description 6
- 230000037361 pathway Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 102000004082 Calreticulin Human genes 0.000 description 5
- 108090000549 Calreticulin Proteins 0.000 description 5
- 102000006381 STAT1 Transcription Factor Human genes 0.000 description 5
- 230000002378 acidificating effect Effects 0.000 description 5
- 229920006317 cationic polymer Polymers 0.000 description 5
- 239000013592 cell lysate Substances 0.000 description 5
- 238000012512 characterization method Methods 0.000 description 5
- 238000012761 co-transfection Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 230000002601 intratumoral effect Effects 0.000 description 5
- 210000002540 macrophage Anatomy 0.000 description 5
- 239000000178 monomer Substances 0.000 description 5
- 239000013600 plasmid vector Substances 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 102000043138 IRF family Human genes 0.000 description 4
- 108091054729 IRF family Proteins 0.000 description 4
- 241001424413 Lucia Species 0.000 description 4
- 238000011529 RT qPCR Methods 0.000 description 4
- 108700008625 Reporter Genes Proteins 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 239000007795 chemical reaction product Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 230000012202 endocytosis Effects 0.000 description 4
- 230000003832 immune regulation Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 230000000977 initiatory effect Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000002861 polymer material Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- HBZBAMXERPYTFS-SECBINFHSA-N (4S)-2-(6,7-dihydro-5H-pyrrolo[3,2-f][1,3]benzothiazol-2-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid Chemical compound OC(=O)[C@H]1CSC(=N1)c1nc2cc3CCNc3cc2s1 HBZBAMXERPYTFS-SECBINFHSA-N 0.000 description 3
- XNCOSPRUTUOJCJ-UHFFFAOYSA-N Biguanide Chemical group NC(N)=NC(N)=N XNCOSPRUTUOJCJ-UHFFFAOYSA-N 0.000 description 3
- 239000012979 RPMI medium Substances 0.000 description 3
- 102000002689 Toll-like receptor Human genes 0.000 description 3
- 108020000411 Toll-like receptor Proteins 0.000 description 3
- 125000003277 amino group Chemical group 0.000 description 3
- 230000000259 anti-tumor effect Effects 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 230000006907 apoptotic process Effects 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000005538 encapsulation Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000012637 gene transfection Methods 0.000 description 3
- 210000002865 immune cell Anatomy 0.000 description 3
- 210000000987 immune system Anatomy 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 210000004940 nucleus Anatomy 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 230000000770 proinflammatory effect Effects 0.000 description 3
- 230000011664 signaling Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- 102000004091 Caspase-8 Human genes 0.000 description 2
- 108090000538 Caspase-8 Proteins 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 229940126062 Compound A Drugs 0.000 description 2
- NLDMNSXOCDLTTB-UHFFFAOYSA-N Heterophylliin A Natural products O1C2COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC2C(OC(=O)C=2C=C(O)C(O)=C(O)C=2)C(O)C1OC(=O)C1=CC(O)=C(O)C(O)=C1 NLDMNSXOCDLTTB-UHFFFAOYSA-N 0.000 description 2
- 101000617830 Homo sapiens Sterol O-acyltransferase 1 Proteins 0.000 description 2
- 230000004163 JAK-STAT signaling pathway Effects 0.000 description 2
- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 description 2
- 102100021993 Sterol O-acyltransferase 1 Human genes 0.000 description 2
- 101000697584 Streptomyces lavendulae Streptothricin acetyltransferase Proteins 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 230000005975 antitumor immune response Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- UMGXUWVIJIQANV-UHFFFAOYSA-M didecyl(dimethyl)azanium;bromide Chemical compound [Br-].CCCCCCCCCC[N+](C)(C)CCCCCCCCCC UMGXUWVIJIQANV-UHFFFAOYSA-M 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 229920002674 hyaluronan Polymers 0.000 description 2
- 229960003160 hyaluronic acid Drugs 0.000 description 2
- 230000000887 hydrating effect Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000002513 implantation Methods 0.000 description 2
- 210000003000 inclusion body Anatomy 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 201000001441 melanoma Diseases 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 101150087933 rsad2 gene Proteins 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 102100032814 ATP-dependent zinc metalloprotease YME1L1 Human genes 0.000 description 1
- 229940123373 Adenovirus E1A gene Drugs 0.000 description 1
- 229940123208 Biguanide Drugs 0.000 description 1
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 108091062157 Cis-regulatory element Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 1
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 description 1
- 101000844245 Homo sapiens Non-receptor tyrosine-protein kinase TYK2 Proteins 0.000 description 1
- 101000665442 Homo sapiens Serine/threonine-protein kinase TBK1 Proteins 0.000 description 1
- 101000997835 Homo sapiens Tyrosine-protein kinase JAK1 Proteins 0.000 description 1
- 238000004566 IR spectroscopy Methods 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 101710128836 Large T antigen Proteins 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 description 1
- 102100032028 Non-receptor tyrosine-protein kinase TYK2 Human genes 0.000 description 1
- 108010077850 Nuclear Localization Signals Proteins 0.000 description 1
- 102100023050 Nuclear factor NF-kappa-B p105 subunit Human genes 0.000 description 1
- 101710082694 Nuclear factor NF-kappa-B p105 subunit Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 102100032709 Potassium-transporting ATPase alpha chain 2 Human genes 0.000 description 1
- 101800000795 Proadrenomedullin N-20 terminal peptide Proteins 0.000 description 1
- 108010083204 Proton Pumps Proteins 0.000 description 1
- 102100038192 Serine/threonine-protein kinase TBK1 Human genes 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102100033438 Tyrosine-protein kinase JAK1 Human genes 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000033289 adaptive immune response Effects 0.000 description 1
- 102000035181 adaptor proteins Human genes 0.000 description 1
- 108091005764 adaptor proteins Proteins 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000004931 aggregating effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- RFCBNSCSPXMEBK-INFSMZHSSA-N c-GMP-AMP Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]3[C@@H](O)[C@H](N4C5=NC=NC(N)=C5N=C4)O[C@@H]3COP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=C(NC2=O)N)=C2N=C1 RFCBNSCSPXMEBK-INFSMZHSSA-N 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000006505 cellular lipid metabolism Effects 0.000 description 1
- 239000005482 chemotactic factor Substances 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000010485 coping Effects 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000002059 diagnostic imaging Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 230000008482 dysregulation Effects 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000037449 immunogenic cell death Effects 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 230000002584 immunomodulator Effects 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000012966 insertion method Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 230000031146 intracellular signal transduction Effects 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 230000005291 magnetic effect Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 230000006676 mitochondrial damage Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 231100000623 nanotoxicology Toxicity 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 230000000242 pagocytic effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 238000002428 photodynamic therapy Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011127 radiochemotherapy Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000005476 size effect Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- 230000014567 type I interferon production Effects 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—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
- A61K47/61—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 the organic macromolecular compound being a polysaccharide or a derivative thereof
-
- 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
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
- A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
- A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
- A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
- A61K47/6939—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nanotechnology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Oncology (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了一种纳米递送系统及其制备方法和应用,所述纳米递送系统包括纳米递送载体和负电荷成分,所述纳米载体包含胍基改性壳聚糖、阳离子脂质体、两亲性化合物。通过对壳聚糖进行胍基改性,使壳聚糖形成带有更多阳离子的聚合物,能够提高核酸分子的包载量和转染效率。此纳米颗粒还具有免疫激活作用,激活效果优于传统的以鱼精蛋白、阳离子脂质制备的纳米颗粒。本发明中,以胍基改性壳聚糖为基础制备的纳米颗粒可为基因治疗和免疫治疗的联合应用提供新策略。
Description
技术领域
本发明属于纳米生物材料领域,具体涉及一种纳米递送系统及其制备方法和应用。
背景技术
纳米材料由于其独特的表面效应、量子尺寸效应,在光学、电学、磁学等方面展现出突出的优势和广泛的应用前景。随着纳米科学的发展,纳米材料所展现出的优异功能催生了其在生物医学领域的研究和应用。目前,纳米生物医学的研究主要集中在纳米生物效应与安全性、纳米毒理学、生物传感、组织工程、医学成像、药物输送、疾病(特别是肿瘤)诊断与治疗等方面。纳米材料在应对癌症等具有复杂病理环境和高度异质性的疾病方面日益显示出独特优势,随着纳米技术的发展与成熟,纳米结构的不断创新与优化,设计具有多重功能的纳米载体、联合多种途径发挥肿瘤预防与治疗作用已经成为当前研究的重要趋势。
基于生物分子设计的纳米材料具有多重优势,其良好的生物相容性、低免疫原性、低毒性等特点使其有别于其他分子,能够作为一种安全性好、稳定性高的载体;此外,天然生物分子生产成本低,环境友好,是一种易于扩大开发和生产的安全材料。生物纳米材料独特的优势为其作为基因递送、小分子化学药物递送的载体提供了保障,为基因治疗手段的发展奠定基础。
基因治疗和免疫治疗是肿瘤、传染病等疾病的主要治疗手段,基因治疗的关键且受限环节是高效且有效的基因递送。目前应用比较广泛的基因递送载体主要包括病毒载体和非病毒载体。病毒载体包括腺病毒载体、慢病毒载体、逆转录病毒、腺相关病毒等类型。病毒载体虽然有极高的转染效率,但是由于某些病毒载体可能会引起机体的炎症和免疫反应,并且采用病毒载体递送基因对基因片段的大小有严格要求,所以现在采取的基因递送手段主要以非病毒载体为主。与病毒载体相比,非病毒载体具有良好的生物相容性和可降解性、低免疫原性、可携带基因片段的大小不受限制等特点,被广泛应用于递送不同类型的核酸分子,极大的推动了基因递送系统的发展与进步。用于基因递送的非病毒载体主要有阳离子聚合物、阳离子脂质体、磷酸钙等形式。非病毒载体虽然在一定程度上提高了核酸分子的稳定性和安全性,但是仍存在转染效率低、转染核酸分子的类型单一等问题。此外,基因治疗作用时间短,对于癌症中晚期治疗效果较差。目前常采取基因治疗与其他治疗手段,如光动力学、放化疗、免疫治疗等联合应用的策略。最受到关注的便是通过基因治疗的策略表达某个抗原,从而引发机体的免疫反应,联合运用基因治疗和免疫治疗。免疫治疗虽然能够克服基因治疗的治疗时间短等局限性,但多数肿瘤抗原不明确,无法联合运用基因治疗。
肿瘤特异性抗原的释放对于联合运用基因治疗和免疫治疗是极其重要的,肿瘤免疫原性细胞死亡可为解决上述问题提供方案。本发明将提供一种纳米递送系统的制备策略,并在此基础上研究了该纳米递送系统诱导细胞免疫原性死亡以联合基因治疗和免疫治疗的多种抗肿瘤功能。
发明内容
本发明的目的在于提供一种既具有较高的基因递送效率,又可同时介导基因的共递送,还可对免疫系统产生强烈激活作用的纳米递送系统。
本发明所采取的技术方案是:
本发明的第一方面,提供一种纳米载体,包含改性壳聚糖、阳离子脂质体和两亲性化合物,其中改性壳聚糖为胍基改性壳聚糖。
在本发明的一些实施方式中,所述胍基改性壳聚糖是通过壳聚糖与双氰胺的加成反应合成的。
在本发明的一些实施方式中,所述壳聚糖与双氰胺的摩尔比为1:(1~10)。
在本发明的一些优选实施方式中,所述壳聚糖与双氰胺的摩尔比为1:(2~5)。
在本发明的一些优选实施方式中,所述壳聚糖与双氰胺的摩尔比为1:2。
在本发明的一些实施方式中,所述阳离子脂质体包括阳离子脂质和辅助脂质。
在本发明的一些实施方式中,所述阳离子脂质和辅助脂质的摩尔比为1:3~8:1。
在本发明的一些实施方式中,所述阳离子脂质和辅助脂质的摩尔比为1:1。
在本发明的一些实施方式中,所述阳离子脂质选自1,2-二油酰-3-三甲基铵盐丙烷、氯化三甲基-2,3-二油烯氧基丙基铵、溴化三甲基-2,3-二油酰氧基丙基铵、三氟乙酸二甲基-2,3-二油烯氧基丙基-2-(2-精胺甲酰氨基)乙基铵、溴化三甲基十二烷基铵、溴化三甲基十四烷基铵、溴化三甲基十六烷基铵、溴化二甲基双十八烷基铵、溴化二甲基-2-羟乙基-2,3-二油酰氧基丙基铵、溴化二甲基-2-羟乙基-2,3-二油烯氧基丙基铵、溴化二甲基-3-羟丙基-2,3-二油烯氧基丙基铵、溴化二甲基-4-羟丁基-2,3-二油烯氧基丙基铵、溴化二甲基-5-羟戊基-2,3-二油烯氧基丙基铵、溴化二甲基-2-羟乙基-2,3-双十六烷氧基丙基铵、溴化二甲基-2-羟乙基-2,3-双十八烷氧基丙基铵、溴化二甲基-2-羟乙基-2,3-双十四烷氧基丙基铵、N-(2-精胺甲酰基)-N’,N’-双十八烷基甘氨酰胺、1,2-二油酰-3-琥珀酰-sn-甘油胆碱酯、3β-[N-(N’,N’-二甲基胺乙基胺基甲酰基)]胆固醇、脂质多聚-L-赖氨酸和硬脂胺中的至少一种。
在本发明的一些优选实施方式中,所述阳离子脂质为1,2-二油酰-3-三甲基铵盐丙烷。
在本发明的一些实施方式中,所述辅助脂质选自胆固醇、磷脂酰乙醇胺、磷脂酰胆碱、二油酰基磷脂酰乙醇胺、1,2-二油酰基-sn-丙三基-3-胆碱磷酸、1,2-双(10,12-三甲苯二酰基)-sn-甘油-3-磷酸胆碱、2-油酰-1-棕榈锡甘油-3-磷酸胆碱、1,2-二棕榈酰-sn-甘油-3--O-4'-(N,N,N-三甲基)高丝氨酸和尿素神经酰胺中的至少一种。
在本发明的一些优选实施方式中,所述辅助脂质为胆固醇。
在本发明的一些实施方式中,所述两亲性化合物选自DSPE-PEG、1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇、二肉豆蔻酰基磷脂酰乙醇胺PEG、二硬脂酰基磷脂酰乙醇胺-聚乙二醇和二月桂酰基磷脂酰乙醇胺-聚乙二醇中的至少一种。
在本发明的一些实施方式中,所述两亲性化合物为DSPE-PEG。
本发明的第二方面,提供本发明第一方面所述纳米载体在递送货物分子系统中的应用。
在本发明的一些实施方式中,所述货物分子选自核酸、肽、蛋白质、脂质、化学化合物、药物活性剂、其生物类似物、其生物优化物、其生物衍生物及其生物等效物中的至少一种。
在本发明的一些优选实施方式中,所述货物分子为核酸或核酸类似物。
在本发明的一些优选实施方式中,所述核酸或核酸类似物为siRNA、mRNA、shRNA、lnc RNA、pDNA、poly IC、CpG或环状二核苷酸。
本发明的第三方面,提供一种纳米递送系统,包含本发明第一方面所述载体和负电荷成分。
胍基改性壳聚糖是一种以壳聚糖分子为骨架、偶联多个双胍基的聚合物,由于含有丰富的氮(N)元素,在酸性环境下,能够质子化形成带有大量正电荷的阳离子聚合物,其能够与负电荷成分相结合,通过静电相互作用形成稳定复合物,有利于提高负电荷分子的稳定性和载量。
在本发明的一些实施方式中,所述负电荷成分为货物分子,所述货物分子选自核酸、肽、蛋白质、脂质、化学化合物、药物活性剂、其生物类似物、其生物优化物、其生物衍生物及其生物等效物中的至少一种。
在本发明的一些实施方式中,所述货物分子为核酸或核酸类似物。
在本发明的一些实施方式中,所述核酸或核酸类似物为siRNA、mRNA、shRNA、lncRNA、pDNA、poly IC、CpG或环状二核苷酸。
在本发明的一些实施方式中,所述pDNA或siRNA递送的基本原理如下:本发明主要将拟递送的负电荷成分集中于核酸分子上,如pDNA、siRNA等,根据前述,胍基改性壳聚糖能够与带负电荷的核酸分子形成复合物,提高其稳定性和包载量,在复合物的基础上再加入阳离子脂质体,将核酸-胍基改性壳聚糖复合物包裹在脂质体内部,不仅降低了核酸分子被降解的风险,还能通过促进阳离子脂质体与负电性的细胞膜的融合,将胍基改性壳聚糖-核酸分子内核释放到细胞内。该复合物被吞噬泡吞噬后,最终运送到溶酶体,在溶酶体酸性条件下,含有丰富双胍基的聚合物材料将会引发”质子海绵效应",提高核酸分子的体内释放和转染效率。且胍基改性壳聚糖丰富的阳离子能够发挥类似于“核定位信号”的作用,介导pDNA入核转录,翻译成蛋白质发挥生物学功能。siRNA直接递送到细胞质中,与靶基因的mRNA形成RISC复合体,介导靶基因mRNA的降解,从而发挥敲低特定基因表达的生物学作用。
在本发明的一些实施方式中,所述纳米递送系统的粒径为10~350nm。
在本发明的一些实施方式中,所述胍基改性壳聚糖与货物分子的N/P的比例为(1~48):1;所述阳离子脂质体与货物分子的N/P的比例为(1~36):1,所述两亲性化合物占阳离子脂质体的含量百分比为0.5%~30%。
在本发明的一些实施方式中,所述胍基改性壳聚糖与货物分子的N/P的比例为(1~28):1;所述阳离子脂质体与货物分子的N/P的比例为(4~16):1,所述两亲性化合物占阳离子脂质体的含量百分比为1%~10%。
在本发明的一些实施方式中,所述胍基改性壳聚糖与货物分子的N/P的比例为(1~28):1;所述阳离子脂质体与货物分子的N/P的比例为(8~16):1,所述两亲性化合物占阳离子脂质体的含量百分比为2%~10%。
本发明的第四方面,提供本发明第三方面所述纳米递送系统在制备产品或免疫激活中的应用。
在本发明的一些实施方式中,所述产品为药物。
在本发明的一些实施方式中,所述免疫为固有免疫或适应性免疫。
在本发明的一些实施方式中,所述固有免疫信号通路包括:STING、TLR诱导I型干扰素的产生和I型干扰素与其受体结合诱导干扰素刺激基因的转录。
在本发明的一些实施方式中,所述诱导I型干扰素的产生是通过STING、TLR激活TBK1-IRF3信号轴达到诱导I型干扰素产生的效果。
在本发明的一些实施方式中,所述I型干扰素与其受体结合通过激活JAK-STAT信号通路诱导干扰素刺激基因的转录。
在本发明的一些实施方式中,上述信号通路主要涉及cGAS、IRF3、IFNβ、STAT1、STAT2、IRF9和Rsad2的基因表达变化。
本发明的第五方面,提供一种药物,包含本发明第三方面所述纳米递送系统。
在本发明的一些实施方式中,所述药物还包括药学上可接受的载体或赋形剂。
本发明的第六方面,提供本发明第三方面所述纳米递送系统的制备方法,包含以下步骤:
S1:将阳离子脂质和辅助脂质溶于有机溶剂混合,挥发有机溶剂并抽真空,20~75℃水化,超声均质并挤压粒径,得到阳离子脂质体;
S2:通过壳聚糖与双氰胺的加成反应合成胍基改性壳聚糖,分离纯化;
S3:将胍基改性壳聚糖与货物分子混合,室温静置不低于10min,得复合物A;
S4:将复合物A与阳离子脂质体混合,室温静置不低于10min,得复合物B;
S5:将复合物B与两亲性化合物混合,20~75℃静置不低于10min。
其中步骤S1中温度的范围主要跟应用的脂质类型有关,准确的来说,是跟脂质的相变温度有关。只有温度高于脂质的相变温度,才能增加脂质的分散度和流动性,才有利于脂质体的形成。
步骤S3和步骤S4中纳米颗粒制备过程中,主要是通过电荷相互作用形成复合物,室温静置不低于10min主要是为了让电荷作用更充分形成复合物,优选为10~20min,只要不出现沉淀形成都是合理的。
由于siRNA的大小一般是19~23nt,该分子不易与阳离子聚合物结合形成复合物,需要有一个带大量负电荷的分子作为siRNA的载体,起聚集凝缩siRNA的作用,针对siRNA小分子双链RNA片段,对步骤S3做出适应性调整:先将透明质酸HA或poly IC与siRNA按照质量比为2:1~1:8混合,使得透明质酸或poly IC作为siRNA的载体,再将HA-siRNA复合物或poly IC-siRNA与胍基改性壳聚糖混合。
本发明的有益效果是:
本发明通过对壳聚糖的氨基进行胍基改性合成了一种聚双胍基修饰的壳聚糖聚合物分子,并基于该聚合物分子制备了一种由胍基改性壳聚糖-核酸分子及其类似物-阳离子脂质体-两亲性化合物组成的纳米颗粒。壳聚糖的生物可降解性和低免疫原性使得该聚合物的生物安全性得到保障,并且壳聚糖对固有免疫以及适应性免疫均具有良好的调节和激活作用。通过对壳聚糖进行胍基改性,使壳聚糖形成带有更多阳离子的聚合物,理论上能够提高核酸分子的包载量并提高核酸分子的稳定性。由于该纳米颗粒中含有丰富的壳聚糖和聚双胍基阳离子,具有多重生理功能,本发明将主要阐述一种纳米递送系统的制备方案以及对其作为基因递送载体、作为免疫调节剂发挥抗肿瘤功能的疗效进行评估和验证。
实验结果说明基于该聚合物制备的纳米递送系统介导pDNA转染的效率不亚于市售PEI,极大地提升了核酸分子的转染效率;基于该聚合物制备的纳米递送系统能够介导siRNA转染,且以poly IC作为siRNA的载体时,比HA作为siRNA载体,能够显著提高siRNA的敲低作用并能增强其相关功能活性以及下游级联信号反应;基于该基因递送系统递送polyIC能够显著激活固有免疫信号通路,瘤内注射上述poly IC纳米递送系统能够诱导强烈的ICD效应;基于该聚合物的纳米递送系统还具有一定的免疫调节与激活作用,激活效果优于传统的以鱼精蛋白作为载体的纳米颗粒的激活效果、优于常见的阳离子脂质制备的纳米颗粒的激活效果。
本发明中通过化学加成反应合成了胍基改性壳聚糖,并以此为基础制备的纳米颗粒是一种具有多重功能的基因载体,既可有效介导基因的转染,又可对免疫系统产生强烈激活作用,从而为联合基因治疗和免疫治疗的手段发挥多重抗肿瘤作用提供新的指导。
附图说明
图1为胍基改性壳聚糖的结构示意图。
图2为纳米颗粒的制备流程图。
图3为纳米颗粒的制备示意图。
图4为不同N/P纳米颗粒的粒径和电位表征。其中图4A为DNA与胍基改性壳聚糖按照不同N/P比混合后,粒径和电位的情况;图4B为在图4A的基础上筛选N/P为1:1~8:1组的胍基改性壳聚糖-DNA复合物与阳离子脂质体按照不同的N/P混合后,粒径和电位情况;图4C为在图4B筛选出来的复合物的基础上加入10%DSPE-PEG时,粒径和电位的情况。
图5为纳米颗粒细胞水平转染示意图。
图6为不同分组的纳米颗粒向293T细胞转染pcDNA3.1-Luc-GFP质粒36h后,细胞裂解液中luciferase的表达情况。
图7为不同的脂质体纳米颗粒包裹不同浓度pcDNA3.1-Luc-GFP质粒转染RAWLucia细胞24h时,培养液上清中luciferase化学发光情况。
图8为不同的脂质体纳米颗粒包裹不同浓度poly IC转染RAW Lucia细胞24h时,培养液上清中luciferase化学发光情况。
图9为包裹不同质粒的纳米颗粒转染RAW Lucia细胞24h时,培养液上清中luciferase化学发光情况。
图10为制备包裹pcDNA3.1-Luc-GFP质粒的纳米颗粒,皮下免疫小鼠,24h时,淋巴结细胞固有免疫信号通路的激活情况。其中图10A为对INFβ、STAT1、IRF9和IRF3的表达情况;图10B为对Rsad2、STAT2和cGAS的表达情况。
图11为制备包裹poly IC的纳米颗粒,皮下免疫小鼠24h后,淋巴结细胞固有免疫信号通路的激活情况。图11A为对cGAS和IRF3的表达情况;图11B为对INFβ和Rsad2的表达情况。
图12为制备包裹luciferase siRNA的纳米颗粒转染B16F10-GFP-Luc稳转细胞系后细胞裂解液中luciferase表达情况。
图13为包裹HA和luciferase siRNA、poly IC和luciferase siRNA的纳米颗粒转染B16F10-GFP-Luc稳转细胞系后细胞裂解液中luciferase的表达情况。
图14为包裹HA和mTOR siRNA、poly IC和mTOR siRNA的纳米颗粒诱导B16F10肿瘤细胞产生ICD的情况。
图15为包裹HA和mTOR siRNA、poly IC和mTOR siRNA的纳米颗粒瘤内注射小鼠2天后淋巴结细胞内固有免疫激活情况。
图16为包裹HA和mTOR siRNA、poly IC和mTOR siRNA的纳米颗粒瘤内注射小鼠2天后Western Blot检测肿瘤组织中mTOR信号通路抑制情况以及ICD产生相关蛋白表达水平。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述,以充分地理解本发明的目的、特征和效果。显然,所描述的实施例只是本发明的一部分实施例,而不是全部实施例,基于本发明的实施例,本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例,均属于本发明保护的范围。
DOTAP:1,2-二油酰-3-三甲基铵盐丙烷,一种阳离子脂质体材料。
Chol:胆固醇,脂质体制备过程中可用作辅助脂质,起到稳定脂质体结构、降低阳离子毒性、提高脂质体跨膜效率等作用。
质子海绵效应:带有阳离子的颗粒与细胞膜结合,通过细胞内吞作用进入细胞形成内吞体,内吞体与溶酶体融合。阳离子颗粒上不饱和的氨基螯合由质子泵(V-ATPase)提供的质子,质子泵持续开放,每个质子导致一个氯离子和一个水分子潴留在溶酶体内,引发溶酶体肿胀破裂,颗粒释放,进入细胞质,进一步导致线粒体损伤和细胞凋亡。
DSPE-PEG:二硬脂酰基磷脂酰乙醇胺-聚乙二醇,一种两亲性化合物。
293T细胞:由人胚胎肾细胞系293细胞通过腺病毒E1A基因的转染派生出的细胞株,能表达SV40大T抗原,含有SV40复制起始点与启动子区,比较容易转染,是一个很常用的研究外源基因表达的细胞株。
Raw Lucia细胞:由小鼠RAW 264.7巨噬细胞系通过稳定整合干扰素调节因子(IRF)诱导的荧光素酶报告基因构建物产生的细胞株。
pcDNA3.1-Luc-GFP:能够在哺乳动物细胞中表达的质粒载体,在其多克隆位点插入luciferase和GFP基因。
TLR:Toll样受体,一种病原体相关分子模式(PAMP)识别受体,可以识别入侵机体的病原微生物的蛋白质、核酸和脂类及其在反应过程中合成的中间产物和代谢产物。TLR通过对PAMP的识别,快速激活包括接头蛋白、信号复合体和转录因子复合体负责的细胞内信号级联反应,最终导致机体产生促炎性细胞因子、抗炎症细胞因子及趋化因子。TLR通过不同的识别途径活化多种免疫细胞,启动非特异性免疫应答并激起适应性免疫应答以清除病原体。
为了便于筛选合适的纳米颗粒分组,我们根据纳米颗粒的组成对其进行简单的命名。名称主要由两部分组成“X:1-Y:1(X、Y为任意数)”,“X:1”指的是胍基改性壳聚糖与核酸分子之间的N/P,“Y:1”指的是阳离子脂质体与核酸分子之间的N/P,如不做其他说明,默认DSPE-PEG的含量占总脂质的2%。下文中出现的名称均符合上述命名规则。
实施例1阳离子脂质体的制备
(1)称取一定质量的DOTAP和胆固醇,分别将其溶于氯仿中,制备成浓度为10mg/mL的储存液;
(2)按照摩尔比为1:1的比例将DOTAP与胆固醇混合;
(3)氮气挥发氯仿,过夜抽真空,形成脂膜;
(4)50℃蒸馏水水化脂膜,超声均质;
(5)依次用200nm和100nm的滤膜挤压粒径,得到DOTAP/chol阳离子脂质体。
实施例2胍基改性壳聚糖的制备
(1)配制1%盐酸溶液,以30mL为例,向29.7mL蒸馏水中加入0.3mL浓盐酸;
(2)称取0.1g壳聚糖(Mr=30000~300000),加入10mL 1%盐酸溶液溶解,配制成1%壳聚糖溶液;
(3)为提高反应效率,将壳聚糖与双氰胺的摩尔比调整为1:2。壳聚糖单体的分子量为161g/mol,故壳聚糖单体的摩尔量为n(壳聚糖单体)=0.1g/161(g/mol)=0.000621nmol,则双氰胺的摩尔量应该为0.001242nmol,m(双氰胺)=104.447mg。将双氰胺加入到1%壳聚糖溶液中,搅拌促溶。
(4)待双氰胺与壳聚糖完全溶解后,将反应温度提高到100℃,反应3h,冷却至室温,即得反应产物,见图1。
实施例3胍基改性壳聚糖的纯化及物理表征
(1)将冷却得到的反应产物置于3500Da透析袋中,为避免壳聚糖在水溶液中溶解性减小析出,透析在弱酸性环境下进行(1%盐酸溶液),透析3h以除去未参与反应的双氰胺。
(2)向透析后的溶液中加入2倍体积的无水乙醇以沉淀壳聚糖,65℃加热挥发乙醇即可得到目标反应产物。
(3)取出部分固体反应产物进行红外光谱物理表征以鉴定其结构。
实施例4胍基改性壳聚糖含N量的计算
根据反应物与产物之间的质量差计算双氰胺与壳聚糖的连接率,进而计算该聚合物材料的含N量。具体计算步骤如下:
取冷却后的产物2mL置于3500Da透析袋中(即透析除去未反应的双氰胺),在弱酸性环境下,透析3h,随后加入2倍体积的无水乙醇沉淀壳聚糖,65℃挥发乙醇,得到干燥的固体,进行称重,质量为W1(如:某次样品的质量W1为22.9mg)。在反应及透析过程中,壳聚糖由于分子量较大,不会被透析除去,所以忽略壳聚糖的损失,2mL反应物中壳聚糖的量仍为W2(由于采用2ml体系,故W2质量为20mg),(W1-W2)mg即为壳聚糖与双氰胺加成反应过程中连接的双氰胺的质量W3(则W3为2.9mg)。W3/84.08(g/mol)即为偶联的双氰胺的摩尔量,将其与壳聚糖上氨基的摩尔量相比较,可以计算出该聚合物材料的连接率,与双氰胺连接的一个壳聚糖单体上N为5个,未与双氰胺连接的一个壳聚糖单体上N为1个,从而可以计算出该聚合物材料总的含N量(以上述例子为例,2ml产物的质量为22.9mg时,该聚合物的含N摩尔量为262.307μmol)。将上述材料溶于0.2mL pH为4的柠檬酸盐缓冲液中,可以计算出N的浓度。
实施例5纳米颗粒递送系统的制备及表征
根据聚合物上N的含量和核酸分子上P的含量设计不同N/P的胍基改性壳聚糖和核酸分子的复合物,并通过测定该复合物的粒径和电位等数据筛选较为合适的N/P比例,用于后续纳米颗粒的设计。在此基础上,根据DOTAP/chol脂质体上N的含量和核酸分子上P的含量设计不同N/P的纳米递送系统,并通过测定该聚合物的粒径和电位等数据筛选较为合适的N/P比例用于后续纳米颗粒的设计。在此基础上,为了提高纳米颗粒在水溶液中的稳定性,采用“后插法”向纳米颗粒中加入不同含量的DSPE-PEG分子,以提高在水中的分散度,纳米颗粒的制备过程如图2、图3所示。根据DOTAP/chol脂质体的含量以及拟加入DSPE-PEG的比例计算DSPE-PEG分子的用量,DSPE-PEG的比例以DSPE-PEG的摩尔量占总脂质摩尔量的百分比表示。
不同N/P纳米颗粒的粒径和电位表征见图4,图4中是pDNA与胍基改性壳聚糖、DOTAP/chol脂质体按照不同N/P结合形成的复合物的粒径电位表征图。其中横坐标的1:1、2:1等代表的是胍基改性壳聚糖与核酸分子的N/P比,8:1、16:1代表的是DOTAP/chol脂质体与核酸分子的N/P比,10%DSPE-PEG是指DSPE-PEG的摩尔量占总脂质摩尔量的10%。
其中图4A图为DNA与胍基改性壳聚糖按照不同N/P比混合后,粒径和电位的情况;图4B为在图4A图的基础上选出合适的几组(即1:1~8:1),按照不同的N/P比与阳离子脂质体混合,粒径和电位情况;图4C图为在图4B基础上加入10%DSPE-PEG时,粒径和电位的情况。
结果表明:从纳米颗粒物理表征来讲,当DNA与胍基改性壳聚糖的比例为1:1~8:1时,粒径和电位情况适宜用于纳米颗粒的制备;当外层阳离子脂质体与核酸的比例为8:1~16:1时,粒径和电位情况适宜用于纳米颗粒的制备;加入10%的DSPE-PEG会改善纳米颗粒的粒径和电位情况。
实施例6包载pcDNA3.1-Luc-GFP质粒的纳米递送系统转染293T细胞
当核酸分子为pcDNA3.1-Luc-GFP质粒时,构建纳米递送系统转染至293T细胞:
构建合成了pcDNA3.1-Luc-GFP报告基因质粒,该质粒能在真核细胞中正常表达,使用纳米载体包裹该质粒构建纳米递送系统转染293T细胞,以检测该纳米载体作为质粒载体介导基因转染的效果。
其中纳米颗粒细胞水平转染示意图如图5所示,通过细胞的内吞作用形成内含体将纳米颗粒摄取到细胞中,在细胞内内含体逐渐与溶酶体融合,将纳米颗粒释放到溶酶体中,溶酶体是一个酸性环境的细胞器,氨基会解离成带正电荷的阳离子脂质体形式,引发溶酶体的“质子海绵样效应”,帮助纳米颗粒溶酶体逃逸,将胍基改性壳聚糖与核酸分子的复合物释放到细胞质中。当该纳米载体递送质粒DNA分子时,还需要进入到细胞核,DNA在细胞核内转录成mRNA,才能进行后续的翻译、表达过程。
具体操作过程如下:
(1)细胞种板:将293T细胞种96孔板,DMEM培养液。
(2)制备纳米颗粒转染细胞:待细胞密度达到80%~85%左右时,制备不同N/P包裹80ng DNA的纳米颗粒递送系统(具体计算前已说明),转染细胞,调整加入培养液的体积,使不同组间药物浓度基本保持一致。以PEI作为阳性对照。
(3)分别于12h、24h和36h用荧光显微镜观察细胞,观察质粒表达情况;于转染36h后,从培养箱取出细胞,弃上清,PBS洗涤一遍并弃去,加入luciferase裂解液,冰上30min,将裂解液转移到1.5mL EP管中,13000g,4℃,5min。收集裂解液上清,加入luciferase底物(检测试剂盒)测定裂解液中luciferase的表达情况。转染293T细胞36h后,细胞裂解液中luciferase的表达情况如图6所示。
结果表明:以胍基改性壳聚糖为基础制备的DNA纳米递送系统能够介导质粒的转染,且转染效果优于市售试剂PEI,并且通过荧光显微镜观察,20h时,胍基改性纳米颗粒转染的DNA能够表达。
实施例7包裹pcDNA3.1-Luc-GFP质粒转染RAW Lucia细胞
当核酸分子为pcDNA3.1-Luc-GFP质粒时,构建纳米递送系统转染至RAW Lucia细胞:
构建合成了pcDNA3.1-Luc-GFP报告基因质粒,使用纳米载体包裹该质粒转染RAWLucia细胞构建纳米颗粒递送系统,以检测递送系统对巨噬细胞的免疫调节与激活效果。具体操作过程如下:
(1)细胞种板:将Raw Lucia细胞种96孔板,DMEM培养液。
(2)制备纳米颗粒转染细胞:待细胞密度达到80%~85%左右时,制备不同N/P纳米载体,转染细胞,调整加入培养液的体积,使不同组间药物终浓度分别为4μg/ml和10μg/ml。
(3)分别于12h、24h和36h取出10μL培养液上清,置于白色96孔板中,并向其中加入25μL底物溶液,检测luciferase的化学发光情况,以检测包裹DNA的纳米载体对免疫调节和激活的作用效果。
本实施例主要将纳米递送系统与传统的脂质递送系统进行对比,传统的LPH纳米递送主要是将鱼精蛋白作为纳米颗粒的阳离子聚合物成分;传统的LNP脂质体主要将阳离子脂质、辅助脂质、DSPE-PEG按照一定的摩尔比混合形成脂质体,包裹核酸分子。本发明主要与LPH以及以DDAB、MVL5等作为阳离子脂质的LNP作为对比,检测包裹DNA的纳米递送系统对免疫调节和激活的作用效果。
结果见图7。结果表明:以胍基改性壳聚糖为基础制备的包裹DNA的纳米颗粒递送系统能够介导体外固有免疫的激活,然而用其他阳离子脂质制备的纳米颗粒未能介导体外固有免疫的激活。说明以胍基改性壳聚糖为基础制备的纳米颗粒可对固有免疫具有显著的激活作用。
实施例8包裹poly IC转染RAW Lucia细胞
当核酸分子为核酸类似物poly IC时,构建纳米递送系统转染至RAW Lucia细胞:
使用纳米颗粒包裹核酸分子的类似物市售poly IC构建纳米递送系统转染RAWLucia细胞,以检测该纳米递送系统对巨噬细胞的免疫调节与激活效果。具体操作过程如下:
(1)细胞种板:将Raw Lucia细胞种96孔板,DMEM培养液;
(2)制备纳米颗粒转染细胞:待细胞密度达到80%~85%左右时,制备不同N/P、包裹不同质量的poly IC的纳米递送系统,转染细胞,调整加入培养液的体积,使不同组间药物浓度分别为4μg/mL、10μg/mL。
(3)分别于12h、24h和36h取出10μL培养液上清,向置于白色96孔板中,并向其中加入25μL底物溶液,检测luciferase的化学发光情况,以检测包裹poly IC的纳米载体递送系统对免疫调节和激活的作用效果,并与传统的以鱼精蛋白作为载体的纳米颗粒包裹polyIC进行比较。
本实施例主要将纳米递送系统与传统的脂质递送系统进行对比,传统的LPH纳米递送主要是将鱼精蛋白作为纳米颗粒的阳离子聚合物成分;传统的LNP脂质体主要将阳离子脂质、辅助脂质、DSPE-PEG按照一定的摩尔比混合形成脂质体,包裹核酸分子。本发明主要与LPH以及以DDAB、MVL5等作为阳离子脂质的LNP作为对比,检测包裹poly IC的纳米递送系统对免疫调节和激活的作用效果。
结果见图8。结果表明:以胍基改性壳聚糖为基础制备的包裹双链RNA类似物——poly IC的纳米颗粒能够在细胞水平明显激活固有免疫,且激活效果优于传统的脂质递送系统。
实施例9包裹不同类型的shRNA质粒转染RAW Lucia细胞
当核酸分子为shRNA质粒时,构建纳米递送系统转染至RAW Lucia细胞:
常用的用于构建shRNA质粒的载体包括pGIPZ、MIG、pTRIPZ,构建不同类型的shRNA质粒,使用纳米载体包裹该质粒转染RAW Lucia细胞构建纳米颗粒递送系统,以检测递送系统对巨噬细胞的免疫调节与激活效果是否与不同的载体类型有关、是否与特定的基因序列有关,其中,以pcDNA3.1-Luc-GFP质粒作为对照。具体操作过程如下:
(1)细胞种板:将Raw Lucia细胞种96孔板,DMEM培养液。
(2)制备纳米颗粒转染细胞:待细胞密度达到80%~85%左右时,制备不同N/P、不同类型质粒载体的纳米递送系统,转染细胞,调整加入培养液的体积,使不同组间药物终浓度分别为4μg/ml和10μg/ml。
(3)分别于12h、24h和36h取出10μL培养液上清,置于白色96孔板中,并向其中加入25μL底物溶液,检测luciferase的化学发光情况,以检测包裹不同类型shRNA质粒的纳米载体对免疫调节和激活的作用效果。
结果见图9。结果表明:以胍基改性壳聚糖为基础制备的包裹shRNA质粒的纳米颗粒递送系统能够介导体外固有免疫的激活,并且激活作用与质粒载体类型无关,与特定的基因序列无关。
综上,图7、图8、图9表明:以胍基改性壳聚糖为基础的纳米颗粒包裹核酸分子及其类似物(pDNA、poly IC)对固有免疫具有强烈的激活效果,激活效果明显优于传统的鱼精蛋白制备的纳米颗粒、优于常见的阳离子脂质制备的纳米载体的激活作用;且上述激活效果与DNA序列无关,CRISPR-Cas9、shRNA等质粒载体也展现对免疫存在激活作用。由此,上述制备的纳米颗粒可联合免疫激活和基因调控共同发挥作用。
实施例10包裹pcDNA3.1-Luc-GFP质粒皮下免疫小鼠
当核酸分子为pcDNA3.1-Luc-GFP质粒时,构建纳米递送系统皮下免疫C57BL/6雌鼠:
使用纳米颗粒载体包裹核酸分子——pcDNA3.1-Luc-GFP报告基因质粒构建纳米颗粒递送系统皮下免疫C57BL/6小鼠,免疫24h后,取淋巴结,qPCR检测淋巴结细胞中固有免疫信号通路,如STING、TLR等通路的激活效果,以检测该纳米颗粒递送系统体内对固有免疫信号通路的激活效果。具体操作过程如下:
(1)制备包裹pcDNA3.1-Luc-GFP报告基因质粒皮下免疫C57BL/6小鼠;
(2)免疫24h后,取小鼠两侧腹股沟淋巴结,胶原酶消化成单细胞,冻存于-80℃;
(3)Trizol试剂提取淋巴结细胞的RNA;
(4)qPCR检测cGAS、STAT1、STAT2等固有免疫信号通路分子的RNA水平。
结果见图10。结果表明:包裹pDNA的纳米颗粒皮下免疫小鼠,在体内能够显著激活TLR、STING等信号通路,从而激活固有免疫信号通路,发挥免疫激活和调节作用,诱导I型干扰素和促炎因子的分泌,引发机体的抗肿瘤免疫反应。
其中:
(1)STAT参与调控细胞的生长、分化和凋亡。IFN-I是JAK-STAT信号通路的重要调节因子,IFN-I受体受到IFN刺激后形成异二聚体,导致JAK1和TYK2激活,随后STAT1和STAT2蛋白磷酸化。磷酸化的STATs从受体异二聚体上解离,并与IRF9结合,形成主要干扰素基因因子的三聚体,称为ISGF3复合物。该复合物转运至细胞核,并与顺式作用元件ISRE结合,从而启动多个IFN诱导性基因的转录。
(2)当DNA转染到细胞中,免疫系统会将DNA识别为病原体相关分子模式,启动一系列级联反应引发强烈的先天免疫反应。DNA与胞质DNA传感器cGAS结合,导致cGAS活化,继而催化合成cGAMP,cGAMP作为第二信使与STING结合并使STING激活,活化的STING激活TBK1,继而激活IRF3,IRF3进入细胞核并诱导包括IFN-I在内的多种免疫及炎症基因产物。
(3)Rsad2编码的蛋白是一种干扰素诱导的抗病毒蛋白,属于酶的s-腺苷-l-蛋氨酸(SAM)超家族。该蛋白可通过抑制病毒RNA复制、干扰分泌途径、与病毒蛋白结合以及细胞脂质代谢失调等方式在细胞抗病毒反应和先天免疫信号传导中发挥作用。
实施例11包裹poly IC皮下免疫小鼠
当核酸分子为核酸类似物poly IC时,构建纳米递送系统皮下免疫C57BL/6雌鼠:
使用纳米颗粒载体包裹核酸分子的类似物——市售佐剂poly IC皮下免疫C57BL/6小鼠,免疫24h后,取淋巴结,qPCR检测淋巴结细胞中固有免疫信号通路,如STING、TLR等通路的激活效果,以检测该纳米颗粒递送系统体内对固有免疫细胞通路的激活效果。具体操作过程如下:
(1)制备包裹poly IC的纳米颗粒皮下免疫C57BL/6小鼠;
(2)免疫24h后,去小鼠两侧腹股沟淋巴结,胶原酶消化成单细胞,冻存于-80℃;
(3)Trizol试剂提取淋巴结细胞的RNA;
(4)qPCR检测cGAS、STAT1、STAT2等固有免疫信号通路分子的RNA水平。
结果见图11。结果表明:包裹poly IC的纳米颗粒皮下免疫小鼠,在体内能够显著激活TLR、STING等固有免疫信号通路,发挥免疫激活和调节作用,诱导I型干扰素和促炎因子的分泌,引发机体的抗肿瘤免疫反应。
实施例12包裹luciferase siRNA转染B16F10-GFP-Luc细胞
当核酸分子为luciferase siRNA时,构建纳米递送系统转染至luciferase稳转细胞系B16F10-GFP-Luc:
使用纳米颗粒载体包裹核酸分子——luciferase siRNA构建纳米颗粒递送系统转染luciferase的稳转细胞株B16F10-GFP-Luc细胞,通过检测转染后细胞表达luciferase的水平,检测该纳米载体作为siRNA载体介导基因转染的效果。具体操作过程如下:
(1)细胞种板:将B16F10-GFP-Luc细胞种96孔板,RPMI培养液。
(2)制备纳米颗粒转染细胞:待细胞密度达到80%~85%左右时,制备不同N/P(1:1-8:1、2:1-8:1、4:1-8:1、8:1-8:1、12:1-8:1、1:1-16:1、2:1-16:1)、HA与siRNA质量比为1:1,包裹3pmol和10pmol luciferase siRNA的纳米载体,转染细胞。
(3)于转染36h后,从培养箱取出细胞,弃上清,PBS洗涤一遍并弃去,加入裂解液,冰上30min,将裂解液转移到1.5mL EP管中,13000g,4℃,5min。收集裂解液上清,加入luciferase底物(检测试剂盒)测定裂解液中luciferase的表达情况。
结果见图12。结果表明:以胍基改性壳聚糖为基础制备的纳米颗粒能够介导siRNA的转染并展现较好的敲低效果,且敲低效果展现出剂量依赖性。
实施例13共转染poly IC和luciferase siRNA敲低B16F10-GFP-Luc细胞中luciferase表达
当核酸分子为poly IC及luciferase siRNA时,构建纳米递送系统转染至luciferase稳转细胞系B16F10-GFP-Luc:
使用纳米颗粒载体分别包裹HA和siRNA、poly IC和siRNA给药B16F10-GFP-Luc细胞,通过检测细胞裂解液中luciferase表达情况,检测纳米颗粒体外共转染poly IC和siRNA的效果。具体操作过程如下:
(1)细胞种板:将B16F10-GFP-Luc细胞种96孔板,RPMI培养液。
(2)制备纳米颗粒转染细胞:待细胞密度达到80%~85%左右时,制备不同N/P、HA与siRNA或poly IC与siRNA质量比为1:1的纳米颗粒递送系统,转染细胞,使得siRNA的剂量分别为3pmol和10pmol。
(3)于转染36h后,从培养箱取出细胞,弃上清,PBS洗涤一遍并弃去,加入裂解液,冰上30min,将裂解液转移到1.5mL EP管中,13000g,4℃,5min。收集裂解液上清,加入luciferase底物(检测试剂盒)测定裂解液中luciferase的表达情况。
结果见图13。结果表明:在低浓度时,以poly IC作为luciferase siRNA的载体能够增强siRNA的敲低效果;在高浓度时并未展现明显差异。
实施例14共转染poly IC和mTOR siRNA诱导B16F10细胞产生免疫原性死亡(ICD)
当核酸分子为poly IC及mTOR siRNA时,构建纳米递送系统转染至B16F10细胞:
使用纳米颗粒载体分别包裹HA和siRNA、poly IC和siRNA构建纳米颗粒递送系统,给药B16F10细胞,通过检测肿瘤细胞钙网蛋白(CRT)的外翻情况,检测纳米颗粒诱导肿瘤细胞产生ICD的效果。具体操作过程如下:
(1)细胞种板:将B16F10细胞种48孔板,RPMI培养液。
(2)制备纳米颗粒转染细胞:待细胞密度达到80%~85%左右时,制备不同N/P、HA与siRNA或poly IC与siRNA质量比为1:1的纳米颗粒递送系统,转染细胞,调整加入培养液的体积,使得各组间siRNA的浓度为2.5μg/ml。
(3)于转染24h后,从培养箱取出细胞,弃上清,PBS洗涤一遍并弃去,胰酶消化,收集细胞并用预冷的PBS洗涤一遍,弃上清。
(4)Fc block:CD16/32抗体室温孵育10min。
(5)CRT流式抗体染色,洗涤并上机检测。
(6)Flow jo分析CRT的比例。
结果见图14。结果表明:以胍基改性壳聚糖为基础制备的纳米颗粒包裹HA和siRNA、poly IC和siRNA均能诱导肿瘤细胞产生ICD,引发肿瘤疫苗样效应,并且以poly IC作为siRNA的负载分子诱导肿瘤细胞免疫原性死亡的效果优于以HA作为siRNA的负载分子。实施例15体内共转染poly IC和mTOR siRNA能够提高siRNA的敲低效果,并激活固有免疫
当核酸分子为poly IC及mTOR siRNA时,构建纳米递送系统瘤内注射荷B16F10黑色素瘤C57BL/6雌鼠:
制备1:1-8:1组纳米颗粒载体包裹HA和siRNA、poly IC和siRNA构建纳米颗粒递送系统,瘤内注射荷B16F10黑色素瘤的C57BL/6雌鼠,瘤内注射2天后,取肿瘤和淋巴结,通过流式检测淋巴结中固有免疫细胞的激活情况以及Western Blot检测肿瘤组织中mTOR信号通路的敲低情况,检测poly IC和siRNA共转染的效果。具体操作过程如下:
(1)C57BL/6雌鼠皮下植瘤:对小鼠进行双侧皮下植瘤B16F10细胞,每侧0.5Million。
(2)待肿瘤长到100cm3时,分别瘤内注射HA+mTOR siRNA(10μg HA,10μg mTORsiRNA)、poly IC+mTOR siRNA(10μg poly IC,10μg mTOR siRNA)。
(3)瘤内注射2天后,取肿瘤和淋巴结,流式检测淋巴结细胞的固有免疫激活情况,结果见图15;WB检测肿瘤组织中mTOR信号通路的敲低效果,结果见图16。
其中:p-4EBP-1是mTOR信号通路的下游,mTOR的抑制剂能够降低p-4EBP-1的表达水平;caspase-8是凋亡相关通路的关键蛋白,caspase-8的表达上调能够促进CRT由内质网向细胞膜的移位。
图15结果表明:以胍基改性壳聚糖为基础制备的纳米颗粒包裹HA和siRNA、polyIC和siRNA在体内均能诱导固有免疫的激活,并且以poly IC作为siRNA的负载分子诱导巨噬细胞、树突状细胞激活的效果比以HA作为siRNA负载分子的效果更明显。
图16结果表明:以胍基改性壳聚糖为基础制备的纳米颗粒包裹HA和siRNA、polyIC和siRNA在体内均能发挥siRNA的敲低作用,并且能够影响下游信号诱导ICD产生,并且以poly IC作为siRNA的负载分子比以HA作为siRNA负载分子的效果更明显。
综上,图15、16表明:以胍基改性壳聚糖为基础制备的纳米颗粒包裹HA和siRNA、poly IC和siRNA在体内能发挥多种作用,如诱导固有免疫的激活、发挥siRNA的敲低作用,并且以poly IC作为siRNA的负载分子比以HA作为siRNA负载分子的效果更明显。
上述具体实施方式对本发明作了详细说明,但是本发明不限于上述实施例,在所属技术领域普通技术人员所具备的知识范围内,还可以在不脱离本发明宗旨的前提下作出各种变化。此外,在不冲突的情况下,本发明的实施例及实施例中的特征可以相互组合。
Claims (10)
1.一种纳米载体,包含改性壳聚糖、阳离子脂质体、两亲性化合物,其中改性壳聚糖为胍基改性壳聚糖。
2.根据权利要求1所述的载体,其特征在于,所述胍基改性壳聚糖是通过壳聚糖与双氰胺的加成反应合成的;
优选地,所述壳聚糖与双氰胺的摩尔比为1:(1~10);
优选地,所述阳离子脂质体包括阳离子脂质和辅助脂质;
优选地,所述阳离子脂质和辅助脂质的摩尔比为1:3~8:1;
优选地,所述阳离子脂质选自1,2-二油酰-3-三甲基铵盐丙烷、氯化三甲基-2,3-二油烯氧基丙基铵、溴化三甲基-2,3-二油酰氧基丙基铵、三氟乙酸二甲基-2,3-二油烯氧基丙基-2-(2-精胺甲酰氨基)乙基铵、溴化三甲基十二烷基铵、溴化三甲基十四烷基铵、溴化三甲基十六烷基铵、溴化二甲基双十八烷基铵、溴化二甲基-2-羟乙基-2,3-二油酰氧基丙基铵、溴化二甲基-2-羟乙基-2,3-二油烯氧基丙基铵、溴化二甲基-3-羟丙基-2,3-二油烯氧基丙基铵、溴化二甲基-4-羟丁基-2,3-二油烯氧基丙基铵、溴化二甲基-5-羟戊基-2,3-二油烯氧基丙基铵、溴化二甲基-2-羟乙基-2,3-双十六烷氧基丙基铵、溴化二甲基-2-羟乙基-2,3-双十八烷氧基丙基铵、溴化二甲基-2-羟乙基-2,3-双十四烷氧基丙基铵、N-(2-精胺甲酰基)-N’,N’-双十八烷基甘氨酰胺、1,2-二油酰-3-琥珀酰-sn-甘油胆碱酯、3β-[N-(N’,N’-二甲基胺乙基胺基甲酰基)]胆固醇、脂质多聚-L-赖氨酸和硬脂胺中的至少一种;
优选地,所述辅助脂质为中性辅助脂质;
优选地,所述中性辅助脂质选自胆固醇、磷脂酰乙醇胺、磷脂酰胆碱、二油酰基磷脂酰乙醇胺、1,2-二油酰基-sn-丙三基-3-胆碱磷酸、1,2-双(10,12-三甲苯二酰基)-sn-甘油-3-磷酸胆碱、2-油酰-1-棕榈锡甘油-3-磷酸胆碱、1,2-二棕榈酰-sn-甘油-3--O-4'-(N,N,N-三甲基)高丝氨酸和尿素神经酰胺中的至少一种;
优选地,所述两亲性化合物选自DSPE-PEG、1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇、二肉豆蔻酰基磷脂酰乙醇胺PEG、二硬脂酰基磷脂酰乙醇胺-聚乙二醇和二月桂酰基磷脂酰乙醇胺-聚乙二醇中的至少一种。
3.权利要求1~3中任一项所述载体在递送货物分子系统中的应用。
4.根据权利要求3所述的应用,其特征在于,所述货物分子选自核酸、肽、蛋白质、脂质、化学化合物、药物活性剂、其生物类似物、其生物优化物、其生物衍生物及其生物等效物中的至少一种;
优选地,所述货物分子为核酸或核酸类似物;
优选地,所述核酸或核酸类似物为siRNA、mRNA、shRNA、lnc RNA、pDNA、poly IC、CpG或环状二核苷酸。
5.一种纳米递送系统,包含权利要求1~3任一项所述载体和负电荷成分。
6.根据权利要求5所述的纳米递送系统,其特征在于,所述负电荷成分为货物分子,所述货物分子选自核酸、肽、蛋白质、脂质、化学化合物、药物活性剂、其生物类似物、其生物优化物、其生物衍生物及其生物等效物中的至少一种;
优选地,所述货物分子为核酸或核酸类似物;
优选地,所述核酸、核酸类似物为siRNA、mRNA、shRNA、lnc RNA、pDNA、poly IC、CpG或环状二核苷酸。
7.根据权利要求6所述的纳米递送系统,其特征在于,所述纳米递送系统的粒径为10~350nm。
8.根据权利要求6所述的纳米递送系统,其特征在于,所述改性壳聚糖与货物分子的N/P的比例为1:1~48:1;所述阳离子脂质体与货物分子的N/P的比例为1:1~36:1,所述两亲性化合物占阳离子脂质体的含量百分比为0.5%~30%。
9.权利要求5~8任一项所述纳米递送系统在制备产品或非治疗目的的免疫激活中的应用;
优选地,所述产品为药物;
优选地,所述免疫为固有免疫或适应性免疫;
优选地,所述固有免疫信号通路包括:STING、TLR诱导I型干扰素的产生和I型干扰素与其受体结合诱导干扰素刺激基因转录;
优选地,所述固有免疫信号通路涉及cGAS、IRF3、IFNβ、STAT1、STAT2、IRF9和Rsad2的基因表达变化。
10.一种药物,其特征在于,包含权利要求所述5~8任一项所述纳米递送系统,优选地,所述药物还包括药学上可接受的载体或赋形剂。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111385801.0A CN114099699B (zh) | 2021-11-22 | 2021-11-22 | 一种纳米递送系统及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111385801.0A CN114099699B (zh) | 2021-11-22 | 2021-11-22 | 一种纳米递送系统及其制备方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114099699A true CN114099699A (zh) | 2022-03-01 |
CN114099699B CN114099699B (zh) | 2024-03-19 |
Family
ID=80439148
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111385801.0A Active CN114099699B (zh) | 2021-11-22 | 2021-11-22 | 一种纳米递送系统及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114099699B (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2024109612A1 (zh) * | 2022-11-23 | 2024-05-30 | 中国科学院化学研究所 | 一种用于高效递送核酸药物的脂质纳米颗粒的制备方法和应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20190117799A1 (en) * | 2016-04-01 | 2019-04-25 | The Brigham And Women's Hospital, Inc. | Stimuli-responsive nanoparticles for biomedical applications |
KR20190121613A (ko) * | 2018-04-18 | 2019-10-28 | 순천대학교 산학협력단 | 인테그린 αvβ₃ 표적용 RGD 덴드리머 펩타이드를 갖는 폴리에틸렌이민 그래프트된 키토산의 공중합체 및 이를 포함하는 복합체 |
CN112704742A (zh) * | 2019-10-24 | 2021-04-27 | 华东理工大学 | 一种用于恶性肿瘤治疗的包载质粒的阳离子脂质体复合物 |
-
2021
- 2021-11-22 CN CN202111385801.0A patent/CN114099699B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20190117799A1 (en) * | 2016-04-01 | 2019-04-25 | The Brigham And Women's Hospital, Inc. | Stimuli-responsive nanoparticles for biomedical applications |
KR20190121613A (ko) * | 2018-04-18 | 2019-10-28 | 순천대학교 산학협력단 | 인테그린 αvβ₃ 표적용 RGD 덴드리머 펩타이드를 갖는 폴리에틸렌이민 그래프트된 키토산의 공중합체 및 이를 포함하는 복합체 |
CN112704742A (zh) * | 2019-10-24 | 2021-04-27 | 华东理工大学 | 一种用于恶性肿瘤治疗的包载质粒的阳离子脂质体复合物 |
Non-Patent Citations (1)
Title |
---|
王婧等: "胍基化非病毒载体研究进展", 中南药, vol. 18, no. 4, pages 80 - 84 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2024109612A1 (zh) * | 2022-11-23 | 2024-05-30 | 中国科学院化学研究所 | 一种用于高效递送核酸药物的脂质纳米颗粒的制备方法和应用 |
Also Published As
Publication number | Publication date |
---|---|
CN114099699B (zh) | 2024-03-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Yonezawa et al. | Recent advances in siRNA delivery mediated by lipid-based nanoparticles | |
Li et al. | The nano delivery systems and applications of mRNA | |
Rezaee et al. | Progress in the development of lipopolyplexes as efficient non-viral gene delivery systems | |
Howard | Delivery of RNA interference therapeutics using polycation-based nanoparticles | |
KR102198736B1 (ko) | 생체 내 약물 전달을 위한 지질 나노입자 및 이의 용도 | |
Kwon et al. | Engineering approaches for effective therapeutic applications based on extracellular vesicles | |
Wang et al. | Design of multifunctional non-viral gene vectors to overcome physiological barriers: dilemmas and strategies | |
US6217912B1 (en) | Polyester analogue of poly-L-lysine as a soluble, biodegradable gene delivery carrier | |
Song et al. | Assembly strategy of liposome and polymer systems for siRNA delivery | |
JP2004522809A (ja) | 生物学的適合遺伝子送達剤としての新規カチオンリポポリマー | |
JPH09508530A (ja) | 核酸を含む組成物、その調製および使用 | |
Tao et al. | Brain-targeting gene delivery using a rabies virus glycoprotein peptide modulated hollow liposome: bio-behavioral study | |
Karim et al. | Scope and challenges of nanoparticle-based mRNA delivery in cancer treatment | |
Wang et al. | Strategies for improving the safety and RNAi efficacy of noncovalent peptide/siRNA nanocomplexes | |
CN116744979A (zh) | 包含甘露糖的脂质纳米颗粒或其应用 | |
CN114099699B (zh) | 一种纳米递送系统及其制备方法和应用 | |
Mahmoudi et al. | Preparation and in-vitro transfection efficiency evaluation of modified cationic liposome-polyethyleneimine-plasmid nanocomplexes as a novel gene carrier | |
Bartsch et al. | Cell-specific targeting of lipid-based carriers for ODN and DNA | |
CN115487306B (zh) | 一种药物递送载体及其制备方法、应用、糖尿病治疗药物 | |
WO2023093596A1 (zh) | 一种用于有效递送核酸的环状多肽载体及其变化形式 | |
Nelson et al. | Gene delivery into cells and tissues | |
Duvall et al. | Gene Delivery into Cells and Tissues | |
Wang et al. | Gene delivery for lung cancer using nonviral gene vectors | |
KR100986604B1 (ko) | 신규한 아미노지질을 함유하는 에스아이알엔에이 수송용 유전자 조성물 및 제조방법 | |
Wan et al. | Construction of α-Cyclodextrin-Based Stimuli-Responsive Gene Delivery Nanovectors: In Vitro, Ex Vivo, and In Vivo Investigations |
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 |