CN115957342A - Multifunctional nano delivery platform loaded with drugs/genes and preparation and application thereof - Google Patents
Multifunctional nano delivery platform loaded with drugs/genes and preparation and application thereof Download PDFInfo
- Publication number
- CN115957342A CN115957342A CN202211498632.6A CN202211498632A CN115957342A CN 115957342 A CN115957342 A CN 115957342A CN 202211498632 A CN202211498632 A CN 202211498632A CN 115957342 A CN115957342 A CN 115957342A
- Authority
- CN
- China
- Prior art keywords
- man
- nhac
- cbaa
- dox
- solution
- 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
- 239000003814 drug Substances 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 229940079593 drug Drugs 0.000 title abstract description 26
- 108090000623 proteins and genes Proteins 0.000 title abstract description 26
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 28
- 238000009169 immunotherapy Methods 0.000 claims abstract description 16
- 238000002512 chemotherapy Methods 0.000 claims abstract description 8
- 125000000738 acetamido group Chemical group [H]C([H])([H])C(=O)N([H])[*] 0.000 claims description 101
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 claims description 100
- 239000000243 solution Substances 0.000 claims description 54
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical group CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 30
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims description 27
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims description 23
- 238000011282 treatment Methods 0.000 claims description 23
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 238000006243 chemical reaction Methods 0.000 claims description 19
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 239000002246 antineoplastic agent Substances 0.000 claims description 16
- 239000002904 solvent Substances 0.000 claims description 16
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 15
- 229940041181 antineoplastic drug Drugs 0.000 claims description 15
- 238000004108 freeze drying Methods 0.000 claims description 15
- IZMVGEWQSBMVIU-UHFFFAOYSA-N 2-(2-azaniumyl-1-hydroxycyclobutyl)acetate Chemical compound NC1CCC1(O)CC(O)=O IZMVGEWQSBMVIU-UHFFFAOYSA-N 0.000 claims description 14
- 230000004913 activation Effects 0.000 claims description 14
- 238000000502 dialysis Methods 0.000 claims description 14
- 238000011534 incubation Methods 0.000 claims description 14
- 229920000962 poly(amidoamine) Polymers 0.000 claims description 13
- ORILYTVJVMAKLC-UHFFFAOYSA-N adamantane Chemical compound C1C(C2)CC3CC1CC2C3 ORILYTVJVMAKLC-UHFFFAOYSA-N 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 11
- 238000001338 self-assembly Methods 0.000 claims description 11
- 229920000858 Cyclodextrin Polymers 0.000 claims description 10
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 10
- SENLDUJVTGGYIH-UHFFFAOYSA-N n-(2-aminoethyl)-3-[[3-(2-aminoethylamino)-3-oxopropyl]-[2-[bis[3-(2-aminoethylamino)-3-oxopropyl]amino]ethyl]amino]propanamide Chemical compound NCCNC(=O)CCN(CCC(=O)NCCN)CCN(CCC(=O)NCCN)CCC(=O)NCCN SENLDUJVTGGYIH-UHFFFAOYSA-N 0.000 claims description 10
- 150000001875 compounds Chemical class 0.000 claims description 9
- FFYPMLJYZAEMQB-UHFFFAOYSA-N diethyl pyrocarbonate Chemical compound CCOC(=O)OC(=O)OCC FFYPMLJYZAEMQB-UHFFFAOYSA-N 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- 230000004048 modification Effects 0.000 claims description 9
- 238000012986 modification Methods 0.000 claims description 9
- 108020004459 Small interfering RNA Proteins 0.000 claims description 7
- 230000021736 acetylation Effects 0.000 claims description 7
- 238000006640 acetylation reaction Methods 0.000 claims description 7
- 230000035484 reaction time Effects 0.000 claims description 7
- -1 DOX compound Chemical class 0.000 claims description 6
- 101000744742 Homo sapiens YTH domain-containing family protein 1 Proteins 0.000 claims description 6
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 6
- 239000012498 ultrapure water Substances 0.000 claims description 6
- 239000001116 FEMA 4028 Substances 0.000 claims description 5
- 235000011175 beta-cyclodextrine Nutrition 0.000 claims description 5
- 229960004853 betadex Drugs 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 4
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 4
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims description 4
- 229960003237 betaine Drugs 0.000 claims description 4
- 238000007865 diluting Methods 0.000 claims description 4
- 238000005805 hydroxylation reaction Methods 0.000 claims description 4
- YUWBVKYVJWNVLE-UHFFFAOYSA-N [N].[P] Chemical compound [N].[P] YUWBVKYVJWNVLE-UHFFFAOYSA-N 0.000 claims description 3
- 229940009456 adriamycin Drugs 0.000 claims description 3
- 239000002504 physiological saline solution Substances 0.000 claims description 3
- XVIYCJDWYLJQBG-UHFFFAOYSA-N acetic acid;adamantane Chemical compound CC(O)=O.C1C(C2)CC3CC1CC2C3 XVIYCJDWYLJQBG-UHFFFAOYSA-N 0.000 claims description 2
- 230000000397 acetylating effect Effects 0.000 claims description 2
- 229910021529 ammonia Inorganic materials 0.000 claims description 2
- 238000011284 combination treatment Methods 0.000 claims description 2
- 238000005915 ammonolysis reaction Methods 0.000 claims 2
- 238000004519 manufacturing process Methods 0.000 claims 2
- 230000000694 effects Effects 0.000 abstract description 17
- 206010006187 Breast cancer Diseases 0.000 abstract description 6
- 208000026310 Breast neoplasm Diseases 0.000 abstract description 6
- 230000034994 death Effects 0.000 abstract description 5
- 230000002163 immunogen Effects 0.000 abstract description 5
- 239000002994 raw material Substances 0.000 abstract description 5
- 238000002648 combination therapy Methods 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 3
- 230000002708 enhancing effect Effects 0.000 abstract description 2
- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 238000002619 cancer immunotherapy Methods 0.000 abstract 1
- 230000004041 dendritic cell maturation Effects 0.000 abstract 1
- 238000003786 synthesis reaction Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 89
- 239000000412 dendrimer Substances 0.000 description 25
- 229920000736 dendritic polymer Polymers 0.000 description 25
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 22
- 239000002609 medium Substances 0.000 description 21
- 210000004443 dendritic cell Anatomy 0.000 description 18
- 239000000463 material Substances 0.000 description 17
- 239000011258 core-shell material Substances 0.000 description 14
- 239000007924 injection Substances 0.000 description 14
- 238000002347 injection Methods 0.000 description 14
- 239000000872 buffer Substances 0.000 description 10
- 238000010586 diagram Methods 0.000 description 10
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 239000001963 growth medium Substances 0.000 description 9
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 8
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 8
- 238000007792 addition Methods 0.000 description 8
- 239000000843 powder Substances 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 7
- 210000001744 T-lymphocyte Anatomy 0.000 description 7
- 239000007864 aqueous solution Substances 0.000 description 7
- 201000005389 breast carcinoma in situ Diseases 0.000 description 7
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 description 7
- 230000003833 cell viability Effects 0.000 description 7
- 238000000684 flow cytometry Methods 0.000 description 7
- 238000011068 loading method Methods 0.000 description 7
- 210000001165 lymph node Anatomy 0.000 description 7
- 239000002245 particle Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 210000000952 spleen Anatomy 0.000 description 7
- 108010019160 Pancreatin Proteins 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 229940055695 pancreatin Drugs 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 5
- 102000004082 Calreticulin Human genes 0.000 description 5
- 108090000549 Calreticulin Proteins 0.000 description 5
- 125000003277 amino group Chemical group 0.000 description 5
- 229940097362 cyclodextrins Drugs 0.000 description 5
- 230000002601 intratumoral effect Effects 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 238000005303 weighing Methods 0.000 description 5
- 102100039647 YTH domain-containing family protein 1 Human genes 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000012512 characterization method Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 206010057249 Phagocytosis Diseases 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 230000030279 gene silencing Effects 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000002086 nanomaterial Substances 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 230000008782 phagocytosis Effects 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 206010048610 Cardiotoxicity Diseases 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- HPZOOQSXPMEJBV-ODCFVKFUSA-N Tirilazad mesylate Chemical group CS(O)(=O)=O.O=C([C@@H]1[C@@]2(C)CC=C3[C@@]4(C)C=CC(=O)C=C4CC[C@H]3[C@@H]2C[C@H]1C)CN(CC1)CCN1C(N=1)=CC(N2CCCC2)=NC=1N1CCCC1 HPZOOQSXPMEJBV-ODCFVKFUSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 231100000259 cardiotoxicity Toxicity 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000011748 cell maturation Effects 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- 239000008358 core component Substances 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 238000012226 gene silencing method Methods 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 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 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- HVBDBNBRWGIRLT-UHFFFAOYSA-N 4-nitrosoprocainamide Chemical compound CCN(CC)CCNC(=O)C1=CC=C(N=O)C=C1 HVBDBNBRWGIRLT-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010006189 Breast cancer in situ Diseases 0.000 description 1
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 1
- 101100214180 Drosophila melanogaster Ythdf gene Proteins 0.000 description 1
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- 206010057175 Mass conditions Diseases 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 206010061924 Pulmonary toxicity Diseases 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 238000012288 TUNEL assay Methods 0.000 description 1
- 208000003721 Triple Negative Breast Neoplasms Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 239000000980 acid dye Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000009702 cancer cell proliferation Effects 0.000 description 1
- 230000005907 cancer growth Effects 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 238000012637 gene transfection Methods 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000000865 mononuclear phagocyte system Anatomy 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000000242 pagocytic effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 231100000374 pneumotoxicity Toxicity 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000007047 pulmonary toxicity Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 230000001235 sensitizing effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 208000022679 triple-negative breast carcinoma Diseases 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000013042 tunel staining Methods 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a multifunctional nano delivery platform loaded with drugs/genes and preparation and application thereof. The multifunctional nano delivery platform comprises: CBAA-G5/G3-Man multifunctional nano platform load gene YTHDF1siRNA. The preparation method comprises the following steps: preparing Ad-G3; ad-G3-Man preparation; G5-CD/Ad-G3-Man; preparing CBAA-G5/G3-Man; CBAA-G5/G3-Man/YTHDF1siRNA complex preparation. The preparation method has the advantages of easily obtained raw materials and simple synthesis, and the synthesized multifunctional nano delivery platform promotes dendritic cell maturation, is used for breast cancer immunotherapy, can also be used for breast cancer chemotherapy and causes immunogenic death of tumors, thereby enhancing the immunotherapy effect and having combined therapy performance and potential clinical medical application prospects.
Description
Technical Field
The invention belongs to the field of functional nano materials and preparation and application thereof, and particularly relates to a multifunctional nano delivery platform loaded with a drug/gene and preparation and application thereof.
Background
With the continuous development of nanotechnology, dendrimer (especially PAMAM) nanomaterials show great application potential in biomedical fields such as gene delivery, drug delivery, molecular imaging, tumor diagnosis and treatment and the like. The core-shell structure dendrimers (CSTDs) constructed based on PAMAM dendrimers not only inherit the advantages of high modifiability, no immunogenicity and the like of single-generation dendrimers, but also overcome the limitations of drug loading capacity, gene transfer efficiency and the like of the single-generation dendrimers, so that the construction of a multifunctional CSTDs nano platform and the application of the multifunctional CSTDs nano platform to the frontier biomedical field become one of the latest research directions of researchers.
Supramolecular host-guest interactions between Cyclodextrins (CD) and adamantane (Ad) have been commonly used to construct supramolecular structures, such as CSTDs for host-guest based supramolecular self-assembly. In our earlier work, the 5 th generation amino-terminated PAMAM dendrimer surface partially modifies beta-CD, the 3 rd generation amino-terminated PAMAM dendrimer surface partially modifies Ad, and G5-CD/Ad-G3 CSTD can be successfully synthesized through host-object recognition of beta-CD and Ad. Compared with the independent G5-CD or Ad-G3 dendrimer, the gene transfer efficiency is respectively improved by 20 times and 170 times (Chen F.et al.J.Mater.chem.B,2017,5,8459). Moreover, the G5-CD/Ad-G3 serving as a nano platform can also load a genotype inhibitor (MicroRNA 21inhibitor, miR 21i) and an anticancer drug, realize gene and drug co-delivery and be used for gene-chemotherapy combined treatment of triple negative breast cancer cells (Song C.et al.J.Mater.Chem.B.2020,8, 2768-2774). Unfortunately, due to the limitations of the nanoplatform itself (e.g., too high surface potential, no active targeting capability, etc.), the nanoplatform is not used for in vivo combination therapy evaluation after drug and gene loading.
Relevant documents and patent results at home and abroad are searched to show that the modified and synthesized G5-CD/Ad-G3 is modified by zwitterions and mannose to construct a novel multifunctional core-shell dendrimer nano delivery platform for targeted delivery of functional gene YTHDF1siRNA for activating dendritic cells and improving the tumor immunotherapy effect, and reports are not found at present. A novel acetylation platform is constructed by performing acetylation treatment on the G5-CD/Ad-G3 which is synthesized in an improved mode so as to load an anti-cancer drug for immunotherapy based on immunogenic death, and reports are not found yet. The two multifunctional nano platforms are used for chemotherapy-immunotherapy of breast cancer in a combined way, so that the breast cancer has the synergistic treatment effect of 'two ways' and '1+1 > 2', and the report is not found at present.
Disclosure of Invention
The invention aims to solve the technical problem of providing a multifunctional nano delivery platform loaded with drugs/genes and preparation and application thereof so as to fill the blank of the prior art.
The invention provides a multifunctional nano delivery platform, comprising: CBAA-G5/G3-Man multifunctional nano platform load gene YTHDF1 siRNA; the CBAA-G5/G3-Man multifunctional nano platform is obtained by reacting G5-CD/Ad-G3-Man formed by self-assembly of G5-CD and Ad-G3-Man with carboxylic betaine CBAA, wherein the G5-CD is a 5 th generation amino-terminated PAMAM dendrimer surface modification beta-cyclodextrin beta-CD, and the Ad-G3-Man is a 3 rd generation amino-terminated PAMAM dendrimer surface modification adamantane Ad and mannose Man.
Preferably, the nitrogen-phosphorus ratio (N/P) of the CBAA-G5/G3-Man multifunctional nano platform to the YTHDF1siRNA is 0.5.
Preferably, the anticancer drug loaded nano-platform is further comprised, and comprises: the anticancer drug adriamycin is wrapped by the G5.NHAc-CD/Ad-G3.NHAc acetylation nano platform;
the G5.NHAc-CD/Ad-G3.NHAc acetylation nano platform is obtained by acetylating Ad-G3 and G5-CD; wherein G5-CD is PAMAM dendrimer surface modification beta-cyclodextrin beta-CD of 5 th generation amino end capping, and Ad-G3 is PAMAM dendrimer surface modification adamantane Ad of 3 rd generation amino end capping.
Preferably, the molar ratio of the G5.NHAc-CD/Ad-G3.NHAc acetylated nano platform to the anticancer drug adriamycin is 1.
The invention also provides a preparation method of the multifunctional nano delivery platform, which comprises the following steps:
(1) Dispersing adamantane acetic acid Ad-COOH in a solvent, adding EDC.HCl and NHS solution for activation, and adding the activated Ad-COOH solution to G3.NH 2 Reacting in the solution, dialyzing, and freeze-drying to obtain Ad-G3;
(2) Dissolving the Ad-G3 in the step (1) in a PBS solution, dropwise adding mannose Man dissolved in the PBS solution, carrying out a first reaction, dialyzing, and carrying out freeze-drying treatment to obtain Ad-G3-Man;
(3) Dispersing beta-cyclodextrin beta-CD in a solvent, dropwise adding N, N' -Carbonyldiimidazole (CDI) solution for activation, and adding the activated beta-CD solution to G5.NH 2 Carrying out ammonia hydroxylation reaction, dialysis and freeze-drying treatment on the solution to obtain G5-CD, dissolving the G5-CD and the Ad-G3-Man in the step (2) by ultrapure water respectively, mixing, carrying out supramolecular self-assembly reaction, and carrying out dialysis and freeze-drying treatment to obtain G5-CD/Ad-G3-Man;
(4) Respectively dissolving the G5-CD/Ad-G3-Man obtained in the step (3) in a solvent 1, dissolving carboxylic betaine CBAA in a solvent 2, mixing the two solutions, reacting, dialyzing, and lyophilizing to obtain CBAA-G5/G3-Man;
(5) And (4) incubating the CBAA-G5/G3-Man and YTHDF1siRNA in the step (4) to obtain a CBAA-G5/G3-Man/YTHDF1siRNA compound, namely the multifunctional nano delivery platform.
Preferably, the step (1) is performed in Ad-COOH, EDC.HCl, NHS and G3.NH 2 1 to 1.5; the solvent is DMSO.
Preferably, the activation temperature in the step (1) is room temperature, and the activation time is 2-4 h.
Preferably, the reaction temperature in the step (1) is room temperature, and the reaction time is 2-4 d.
Preferably, the molar ratio of Ad-G3 to Man in the step (2) is 1.
Preferably, the dropwise addition of mannose Man dissolved in PBS solution in the step (2) is carried out at 85-95 ℃.
Preferably, the reaction temperature in the step (2) is 85-95 ℃, and the reaction time is 1-6 h.
Preferably, the dialysis in the steps (1), (2), (3) and (4) adopts a cellulose dialysis membrane with the molecular weight cutoff of 1000-50000, and the cellulose dialysis membrane is dialyzed in ultra-pure water for 2-3 days.
Preferably, the solvent in step (3) is DMSO; beta-CD, CDI and G5.NH 2 The molar ratio of (b) is 25 to 30.
Preferably, the activation temperature in the step (3) is 25-35 ℃, and the activation time is 5-7 h.
Preferably, the temperature of the ammoniation hydroxylation reaction in the step (3) is 25-35 ℃, and the time of the ammoniation hydroxylation reaction is 58-62 hours.
Preferably, the molar ratio of G5-CD to Ad-G3-Man in the step (3) is 1.
Preferably, the temperature of the supramolecular self-assembly reaction in the step (3) is room temperature, and the time of the supramolecular self-assembly reaction is 20-25 h.
Preferably, the solvent 1in the step (4) is methanol; solvent 2 is physiological saline.
Preferably, the molar ratio of CBAA to G5-CD/Ad-G3-Man in the step (4) is 200-300.
Preferably, the reaction temperature in the step (4) is room temperature, and the reaction time is 45-50 h.
Preferably, the incubation in step (5) is: diluting CBAA-G5/G3-Man with diethyl pyrocarbonate (DEPC) water, then diluting YTHDF1siRNA with DEPC water, mixing the two diluted solutions uniformly, and incubating at 35-40 ℃ for 20-30 min.
Preferably, the YTHDF1siRNA sequence in step (5) is (5 '-3') GGACAUUGGUACUUGGGAUTT.
Preferably, the nitrogen-phosphorus ratio of CBAA-G5/G3-Man to YTHDF1siRNA in the step (5) is 0.5.
Preferably, the multifunctional nano delivery platform further comprises an anticancer drug-loaded nano platform; the preparation method of the anticancer drug loaded nano platform comprises the following steps:
(a) Dissolving Ad-G3 in the step (1) and G5-CD in the step (3) with ultrapure water respectively, mixing, dropwise adding triethylamine and acetic anhydride solution, reacting, dialyzing, and lyophilizing to obtain G5.NHAc-CD/Ad-G3.NHAc, wherein the molar ratio of G5-CD, ad-G3, triethylamine and acetic anhydride is 1;
(b) Dissolving G5.NHAc-CD/Ad-G3.NHAc in the step (a) in water, mixing with deprotonated DOX solution, carrying out open reaction, centrifuging, taking supernate and carrying out freeze-drying treatment to obtain a G5.NHAc-CD/Ad-G3.NHAc/DOX compound, namely a loaded anticancer drug nano platform; wherein the molar ratio of G5.NHAc-CD/Ad-G3.NHAc to DOX is 1-20, and the open reaction is as follows: stirring for 8-16h at room temperature in the dark.
The invention also provides application of the multifunctional nano delivery platform in preparation of tumor immunotherapy drugs or tumor immunotherapy and chemotherapy combined therapy drugs.
According to the invention, on one hand, mannose is modified by utilizing the shell component dendrimer at the tail end of the amino group, the core-shell dendrimer is formed by virtue of supramolecular self-assembly and the core component dendrimer, then zwitterions are modified on the surface, and a multifunctional nano gene complex is constructed by virtue of electrostatic compression YTHDF1siRNA to activate dendritic cells, so that the aim of immunotherapy of in-situ breast cancer is achieved. On the other hand, the invention utilizes a supermolecule self-assembly method to lead the shell component dendrimer at the tail end of the amino group and the core component dendrimer to form the core-shell dendrimer and carry out acetylation treatment, thereby further physically coating the anticancer drug DOX for chemotherapy of the breast cancer in situ and causing immunogenic death of tumors, further activating a mouse immune system and sensitizing the immunotherapy effect of the multifunctional core-shell dendrimer constructed based on YTHDF1siRNA. Through rational design and application of the core-shell dendrimer nano material, the invention overcomes some defects of the application of the dendrimer, has potential guiding significance for two problems of limited clinical drug side effects and immunotherapy effects, and has potential application prospects in the aspects of realizing tumor chemotherapy, gene therapy and immunotherapy.
Advantageous effects
(1) The preparation method has the advantages of low cost, commercialized raw material source, mild condition, easy operation, high transfection efficiency, high drug loading rate and the like, and has good application prospect in the aspects of tumor chemotherapy and gene-based immunotherapy;
(2) The zwitterion and mannose modified core-shell structure dendritic macromolecule prepared by the invention can resist the elimination of a reticuloendothelial system and target dendritic cells, can cause the silencing of specific genes of the dendritic cells and the activation of the dendritic cells after loading genes, and is further used for the immunotherapy of tumors.
(3) The acetylated core-shell dendrimer prepared by the invention has drug slow release performance, can slowly release drugs at tumor sites after loading the drugs, continuously release chemotherapeutic drugs and cause immunogenic death of tumors, and is used for chemotherapy and sensitization immunotherapy of tumors.
(4) The two multifunctional nano-platforms based on the core-shell dendrimer, which are prepared by the invention, share the synergistic treatment effects of 'two tubes' and '1+1 > 2', and provide a new idea for further research on tumor combination treatment.
Drawings
FIG. 1 is a schematic process flow diagram of the present invention for preparing a carrier/siRNA complex (A) and a carrier/drug complex (B);
FIG. 2 shows Ad-G3.NH prepared according to the present invention 2 (A) And G5.NH 2 of-CD (B) 1 H NMR spectrum;
FIG. 3 is a schematic representation of the Ad-G3-Man (A), G5-CD/Ad-G3-Man (B) and CBAA-G5-CD made according to the present invention
of/Ad-G3-Man (C) 1 An H NMR spectrum;
FIG. 4 shows the G5.NH prepared by the present invention 2 -CD/Ad-G3.NH 2 (A) And G5.NHAc-CD/Ad-G3.NHAc (B) 1 H NMR spectrum;
FIG. 5 is an Atomic Force Microscope (AFM) picture (A) and a height picture (B) of G5.NHAc-CD/Ad-G3.NHAc prepared by the present invention, and an Atomic Force Microscope (AFM) picture (C) and a height picture (D) of G5.NHAc-CD/Ad-G3.NHAc/DOX complex prepared by the present invention;
FIG. 6 is an Atomic Force Microscope (AFM) picture (A) and a height map (B) of CBAA-G5/G3-Man prepared according to the present invention;
FIG. 7 is a 7-day hydrodynamic diameter variation graph (A) of CBAA-G5/G3-Man prepared by the present invention and an anti-protein adsorption experiment data graph (B) of CBAA-G5/G3-Man under different mass conditions;
FIG. 8 is the gel retardation test electrophoresis of the CBAA-G5/G3-Man/YTHDF1siRNA complex prepared by the present invention, wherein lane 1 is marker 2000, lane 2 is naked YTHDF1siRNA, lanes 3-8 correspond to 0.5, 1, 2, 4, 6, 8:1 nitrogen to phosphorus (N/P) ratio;
FIG. 9 is a hydrodynamic diameter map (A) and a surface potential map (B) of CBAA-G5/G3-Man/YTHDF1siRNA prepared by the present invention at different N/P ratios;
FIG. 10 is a diagram of cell viability of the CBAA-G5/G3-Man/YTHDF1siRNA prepared in the present invention after treating Dendritic (DC) cells for 24 hours under different G5/G3 concentration conditions;
FIG. 11 is a histogram and fluorescence intensity quantitative data chart of DC cell phagocytosis ability measured by flow cytometry after DC cells are treated with CBAA-G5/G3-Man/Cy3-YTHDF1siRNA complexes prepared by the present invention at different N/P ratios for 4 hours;
FIG. 12 is a schematic diagram of a confocal microscope detecting DC cells after the CBAA-G5/G3-Man/Cy3-YTHDF1siRNA complex prepared by the invention is processed for 4 hours under the optimal N/P ratio;
FIG. 13 is a flow cytometric histogram (A) and a fluorescent intensity quantitative data graph for DC fluorescent intensity analysis after treating DC cells with CBAA-G5/G3-Man/Cy3-YTHDF1siRNA complexes prepared in the present invention at the optimal N/P ratio for 4 hours. Wherein, PBS, free siRNA and DC cells are treated by Man in advance and then added with CBAA-G5/G3-Man/Cy3-YTHDF1siRNA compound as a control group;
FIG. 14 is a Western blot (Westernblot) test result chart of the effect of CBAA-G5/G3-Man/Cy3-YTHDF1siRNA complex prepared by the present invention on YTHDF1 protein expression in DC cells;
FIG. 15 is a data diagram of flow detection of DC cell maturation using CD80 and CD86 fluorescent antibodies after treating DC cells with CBAA-G5/G3-Man/Cy3-YTHDF1siRNA complexes prepared in the present invention at the optimal N/P ratio for 24 hours;
FIG. 16 is a graph showing in vitro drug release profile of G5.NHAc-CD/Ad-G3.NHAc/DOX complex prepared in the present invention (A) and a graph showing cell viability of 4T1 cells treated with G5.NHAc-CD/Ad-G3.NHAc/DOX complex for 24 hours (B);
FIG. 17 is a histogram (A) and a fluorescence intensity quantitative data chart (B) of phagocytic capacity of 4T1 cells detected by a flow cytometer after 4T1 cells are treated by the prepared G5.NHAc-CD/Ad-G3.NHAc/DOX complex of the invention for 6 hours under different DOX concentrations;
FIG. 18 is a diagram of the CRT expression on the surface of 4T1 cells detected by a confocal microscope after 4T1 cells are treated by a G5.NHAc-CD/Ad-G3.NHAc/DOX complex prepared by the invention and a control group thereof under the condition of different DOX concentrations for 24 hours;
FIG. 19 is a data chart of flow detection of DC cell maturation using CD80 and CD86 fluorescent antibodies after 4T1 cells were treated with the G5.NHAc-CD/Ad-G3.NHAc/DOX complexes prepared in accordance with the present invention at different DOX concentrations for 24 hours;
FIG. 20 is a diagram of experimental evaluation of biosafety performed after extraction of the heart, liver, spleen, lung and kidney of a mouse in situ breast cancer model (i.e., a diagram of effect of hematoxylin-eosin (HE) staining after slicing each tissue) after lymph node injection of CBAA-G5/G3-Man/Cy3-YTHDF1siRNA complex prepared by the present invention and intratumoral injection of G5.NHAc-CD/Ad-G3.NHAc/DOX complex;
FIG. 21 is the diagram of the anti-tumor effect of the mouse in situ breast cancer model after lymph node injection of CBAA-G5/G3-Man/Cy3-YTHDF1siRNA complex prepared by the present invention and intratumoral injection of G5.NHAc-CD/Ad-G3.NHAc/DOX complex, including the tumor volume change diagram (A) and the mouse weight change diagram (B);
FIG. 22 shows a flow chart (A) of spleen tissue extracted from a mouse in situ breast cancer model after lymph node injection of CBAA-G5/G3-Man/Cy3-YTHDF1siRNA complex prepared by the present invention and intratumoral injection of G5.NHAc-CD/Ad-G3.NHAc/DOX complex, and detection of CD4+/CD8+ T cells using fluorescence labeled CD4 and CD8 antibodies, a statistical chart (B) of CD4+ T cell ratios corresponding to statistics, and a statistical chart (C) of CD8+ T cell ratios corresponding to statistics, wherein I, II, III, IV, V, VI represent PBS group, DOX.HCl group, G5.NHAc/G3.NHAc/DOX group, CBAA-G5/G3-Man/HDF 1siRNA group, DOX.HCl + CBAA-G5/G3-Man/YTF 1siRNA group, and G5.NHAc/G3. NHAc/G5 + GYTF 1siRNA group, respectively.
FIG. 23 shows TUNEL (TdT-mediated dUTP Nickel-End Labeling) of tumor sections after tumor tissues are extracted from a mouse in situ breast cancer model after lymph nodes are injected with CBAA-G5/G3-Man/Cy3-YTHDF1siRNA complexes prepared by the present invention and intratumoral injection of G5.NHAc-CD/Ad-G3.NHAc/DOX complexes, wherein I, II, III, IV, V, and VI represent a PBS group, a DOX.HCl group, a G5.NHAc/G3.NHAc/DOX group, a CBAA-G5/G3-Man/YTHDF1siRNA group, a DOX.HCl + CBAA-G5/G3-Man/YTHDF1siRNA group, and a G5. NHAc/DOX + CBAA-G5/G3-YTHDF 1siRNA group, respectively.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
In the following examples, all chemicals were commercially available and were used without further purification unless otherwise specified. Wherein, the third generation of amino-terminal polyamide-amine dendrimer is G3.NH 2 And a fifth generation amino-terminated polyamidoamine dendrimer G5.NH 2 Purchased from Dendritech (Midland, MI) and mannose Man from Sigma-Aldrich (St. Louis, mo.). YTHDF1siRNA is synthesized by Shanghai Ji Ma pharmaceutical technology Limited, and the sequence is (5 '-3') GGACAUUGGUACUUGGGAUTT; and the rest of the raw materials or processing techniques which are not specifically described indicate that the raw materials or the processing techniques are all conventional and commercially available raw materials or conventional processing techniques in the field.
Example 1
A preparation method of a multifunctional core-shell dendrimer nano platform loaded with genes or medicines and a compound thereof is shown in figure 1, and specifically comprises the following steps:
(1) 8.44mg Ad-COOH,83.24mg EDC.HCl and 43.96mg NHS were weighed out separately and dissolved in 5mL DMSO solutions, and the EDC.HCl and NHS solutions were added dropwise to the Ad-COOH solution and stirred at room temperature for 3h. Then 200mg of G3.NH is weighed 2 Dissolving the activated Ad-COOH solution in 5mL of DMSO solution, and gradually dropping the activated Ad-COOH solutionInto G3.NH 2 After further reaction for 3 days in the solution, the obtained product was transferred to a dialysis bag having a molecular weight cut-off of 1000, dialyzed in distilled water for three days (2 L.times.3), and subjected to freeze-drying to obtain Ad-G3 powder, which was stored at-20 ℃ for further use.
(2) 100mg of Ad-G3.NH were weighed out separately 2 50.60mg of Man, respectively dissolved in 5mL of PBS solution, and then the Man solution is added dropwise to Ad-G3.NH at 90 DEG C 2 The solution was stirred for 2h. The resulting product was then transferred to a dialysis bag with a molecular weight cut-off of 1000, dialyzed in distilled water for 1 day (2L. Times.3), and lyophilized to give Man-G3-Ad powder, which was stored at-20 ℃ until use.
(3) 43.64mg of beta-CD and 62.34mg of CDI are weighed out and dissolved in 5mL of DMSO solution, respectively, and then the CDI solution is added dropwise to the beta-CD solution and stirred for 6h at room temperature. Weighing 40mg of G5.NH 2 Dissolved in 5mL of DMSO solution, and the resulting activated beta-CD solution was added dropwise to G5.NH 2 And continuing to react in the solution for 60 hours, transferring the obtained product into a dialysis bag with the molecular weight cutoff of 5000, dialyzing the product in distilled water for three days (2L multiplied by 3), and then performing freeze drying treatment to obtain G5-CD, and storing the G5-CD at the temperature of-20 ℃ for later use.
(4) 61.67mg of Man-G3-Ad obtained in the step (2) and 22.60mg of G5-CD obtained in the step (3) are respectively weighed and respectively dissolved in 5mL of DMSO solution, then the Man-G3-Ad solution is dropwise added into the G5-CD solution, the reaction is continued for 24 hours, the obtained product is transferred into a dialysis bag with the molecular weight cutoff of 50000 and dialyzed in distilled water for three days (2L multiplied by 3), and then freeze drying treatment is carried out, so that G5-CD/Ad-G3-Man powder, namely G5/G3-Man powder, is obtained, and the powder is stored at the temperature of minus 20 ℃ for standby.
(5) Respectively weighing 40mg of G3-Ad obtained in the step (1) and 15.16mg of G5-CD obtained in the step (3), respectively dissolving the G3-Ad and the G5-CD in 5mL of DMSO solutions, then dropwise adding the G3-Ad solution into the G5-CD solution, continuously stirring for 24h, dropwise adding 400 mu L of triethylamine, dropwise adding 200 mu L of acetic anhydride after half an hour, continuously stirring for 24h, transferring the obtained product into a dialysis bag with the molecular weight cutoff of 50000, dialyzing in distilled water for three days (2L multiplied by 3), and then carrying out freeze drying treatment to obtain G5.NHAc-CD/Ad-G3.NHAc powder, namely G5.NHAc/G3.NHAc powder, and storing at-20 ℃ for later use.
(6) Respectively weighing 20mg of the G5/G3-Man obtained in the step (4) above to dissolve in 5mL of methanol, weighing 10mg of CBAA to dissolve in 5mL of physiological saline, then dropwise adding the CBAA into the G5/G3-Man solution, stirring at room temperature for 2 days, transferring the obtained product into a dialysis bag with the molecular weight cutoff of 1000, dialyzing in distilled water for three days (2L multiplied by 3), and then carrying out freeze drying treatment to obtain CBAA-G5/G3-Man powder, and storing at-20 ℃ for later use.
(7) Dissolving the CBAA-G5/G3-Man obtained in the step (6) with DEPC water to prepare a 2mg/mL aqueous solution, dissolving YTHDF1siRNA with DEPC water to prepare a 264 mu G/mL aqueous solution, mixing the CBAA-G5/G3-Man aqueous solution and 1 mu G YTHDF1siRNA according to different N/P ratios (0.5, 1, 2, 4, 6, 8, 10, 15 and 30), putting the mixture into a 37 ℃ incubator for incubation, and obtaining a CBAA-G5/G3-Man/YTHDF1siRNA compound after 20-30 min, wherein the compound is prepared for use.
(8) Weighing 1.389mg of anticancer drug DOX.HCl and 20mg of G5.NHAc/G3.NHAc obtained in the step (5) respectively. Then, G5.NHAc/G3.NHAc was dissolved in 5mL of ultrapure water, DOX. HCl was dissolved in 500. Mu.L of methanol and 5. Mu.L of triethylamine was added to give deprotonated DOX. Subsequently, the DOX solution and the G5.NHAc/G3.NHAc solution were mixed, the mixed solution was stirred overnight with being left to stand out from the light by being opened to evaporate methanol therein, the resulting mixed solution was centrifuged (7000 rpm, 10min) to remove the uncomplexed deprotonated DOX precipitate, and then the supernatant was lyophilized to obtain G5.NHAc/G3.NHAc/DOX, which was stored at-20 ℃ for further use.
Example 2
The nuclear magnetic characterization was performed on each material prepared in step (1) to step (6) of example 1, 1 the characterization results of H NMR are shown in the attached figure 2, figure 3 and figure 4 of the specification: 2.2-3.4ppm in FIG. 2A are G3.NH 2 1.6-1.9ppm are characteristic proton peaks of Ad, and 1.2 Ad molecules are connected to each G3 according to the ratio of the integral areas; in FIG. 2B, 2.2-3.4ppm are characteristic proton peaks of G5, 3.4-4.0ppm and 5.0ppm are characteristic proton peaks of beta-CD, and each G5 link was calculated from the ratio of their integrated areas14.0 CD molecules; 2.2-3.4ppm in FIG. 3A are characteristic proton peaks for G3 and 3.5-3.8ppm are characteristic proton peaks for Man, and 2.4 Man molecules are linked to each Ad-G3 calculated from their ratio of integrated areas; in FIG. 3B, 1.6-1.9ppm are characteristic proton peaks of Ad, 5.0ppm are characteristic proton peaks of β -CD, and according to the ratio of their integrated areas, 0.91 Ad per β -CD is calculated, and further, an average of 12.8 Ad-G3-Man linked to each G5-CD is calculated; in FIG. 3C, 2.0ppm is the characteristic proton peak of CBAA, 5.0ppm is the characteristic proton peak of beta-CD, and according to the ratio of their integrated areas, an average of 2.8 CBAA per CD is calculated, and an average of 39.2 CBAA per G5-CD/Ad-G3-Man is further calculated; in FIG. 4A, 1.6-1.9ppm are characteristic proton peaks of Ad, 3.4-4.0ppm and 5.0ppm are characteristic proton peaks of β -CD, and 0.94 Ad per CD is calculated according to the ratio of their integrated areas, and further 13.2 Ad-G3 is calculated to be connected to each G5-CD on average; 1.9ppm in FIG. 4B is the methyl proton peak of the acetyl group, indicating that the remaining amino groups on the surface of material G5/G3 were successfully acetylated.
Example 3
The materials G5.NHAc/G3.NHAc, CBAA-G5/G3-Man and G5.NHAc/G3.NHAc/DOX prepared in step 5, step 6 and step 8 of example 1 were characterized by an Atomic Force Microscope (AFM), and the results are shown in FIG. 5 and FIG. 6 of the specification, wherein the average height of the test of the material G5.NHAc/G3.NHAc on the AFM was 11.8 + -0.32 nm (FIG. 5A-B), the average height of the test of the material G5.NHAc/G3.NHAc/DOX was 14.4 + -0.53 nm (FIG. 5C-D), and the average height of the test of the material CBAA-G5/G3-Man prepared in step 5, step 6 was 5.7 + -0.50 nm (FIG. 6).
A stability experiment was performed with respect to CBAA-G5/G3-Man prepared in step (6) of example 1. The resulting aqueous solution was prepared at 2mg/mL and left at room temperature for seven days, and the particle size was tested for change at 1,3,5,7 days. As a result, as shown in FIG. 7A, the hydrodynamic diameter of CBAA-G5/G3-Man was between 207 and 244nm at different time conditions. This shows that the overall particle size of the prepared CBAA-G5/G3-Man is in a stable state, and the stability of the particle size of the material is beneficial to the next gene transfection experiment.
An anti-protein adsorption experiment was performed on CBAA-G5/G3-Man prepared in step (6) of example 1. An aqueous solution of bovine serum albumin (2 mg/mL) was mixed with aqueous solutions of G5/G3 at different concentrations (0.25, 1 and 4 mg/mL) in a volume ratio of 1:1, respectively, and after standing at 37 ℃ for 4 hours, the supernatant was collected by centrifugation (8000rpm, 5 min), and the protein content in the supernatant was measured using a BCA quantification kit according to standard specifications. The results are shown in FIG. 7B, where the protein content in the supernatant decreases with increasing material concentration for the group G5/G3-Man without CBAA modification, which indicates that the ability of G5/G3-Man to adsorb non-specific proteins increases with increasing material concentration due to the presence of surface amino groups. After the CBAA is modified, the protein content in the supernatant of the CBAA-G5/G3-Man group is always higher than 0.8mg/mL, and the protein content is slightly increased along with the increase of the material concentration, which shows that the CBAA-G5/G3-Man group has good capacity of resisting non-specific protein adsorption due to the modification of the CBAA.
Example 4
A nitrogen determination experiment was performed on CBAA-G5/G3-Man prepared in step (6) of example 1. Configured as a 2mg/mL aqueous solution, using a primary amino Nitrogen (NOPA) detection kit following standard specifications, the average number of amino groups per CBAA-G5/G3-Man surface was found to be 88.
Gel retardation experiments were performed on the CBAA-G5/G3-Man/YTHDF1siRNA complexes prepared in step (7) of example 1.8 wells of the agarose gel containing the nucleic acid dye were prepared (1.0% w/v), and the agarose gel was left at room temperature to coagulate, and then left in an electrophoresis chamber to carry out the sample application operation. Wherein, the sample comprises CBAA-G5/G3-Man solution (2 mg/mL) and YTHDF1siRNA (1 mu G/hole) which are mixed according to different N/P ratios (0.5, 1, 2, 4, 6 and 8) and incubated for 20-30 min to prepare CBAA-G5/G3-Man/YTHDF1siRNA compound, DNA marker and single (naked) HDYTF 1siRNA. Electrophoresis was performed at a voltage of 80V for 40min, and then the migration ability of the CBAA-G5/G3-Man/YTHDF1siRNA complexes in the gel was analyzed by a gel imager. The results are shown in FIG. 8, CBAA-G5/G3-Man can completely compress YTHDF1siRNA and block YTHDF1siRNA migration when the N/P ratio is more than or equal to 1.
Example 5
Hydrodynamic diameter and surface potential characterization of the CBAA-G5/G3-Man/YTHDF1siRNA complexes prepared in step (7) of example 1, i.e., CBAA-G5/G3-Man/YTHDF1siRNA complexes of different N/P ratios (1, 5, 10, 15, 20, 30) were prepared and diluted to a final volume of 1mL with DEPC water and characterized with a Malvern laser particle sizer (Malvern, M K,633nm laser). As shown in FIG. 9, under different N/P ratio conditions, the hydrodynamic particle size of the complex is approximately 160-176 nm (FIG. 9A), the surface potential is 32-37 mV (FIG. 9B), which indicates that the change of the N/P ratio in a certain range can not obviously change the particle size and the potential of the complex, the particle size and the potential are in a stable state overall and are in a proper gene transfer range, and the absorption, the endocytosis and the gene transfer of the cell are facilitated.
Example 6
And (3) detecting the cell viability of the CBAA-G5/G3-Man and CBAA-G5/G3-Man/YTHDF1siRNA complexes under different concentration conditions by taking the dendritic cells as model cells. Dendritic cells were plated at 5X 10 3 Cell density per well was seeded in 96-well plates, and 100. Mu.L of DMEM medium (designated DMEM + +) containing 100U/mL penicillin, 100U/mL streptomycin, and 10% FBS was added per well. Cells were placed in 5% CO 2 After overnight incubation in an incubator at 37 ℃ the medium was changed to 100. Mu.L of DMEM + + containing different G5/G3 molar concentrations, with the G5/G3 concentrations being 0, 50, 100, 500, 1000, 2000 and 3000nM respectively, and with YTHDF1siRNA additions of 1. Mu.g/well, and the cells were then cultured for 24h. The culture medium was discarded and the cell viability was determined using the CCK-8 kit according to the instructions. The results are shown in FIG. 10, the cell viability is slightly reduced with the increase of the material concentration, but the cell viability is still above 80% even at the concentration of 3000nM, which indicates the good cell compatibility of CBAA-G5/G3-Man. Meanwhile, the toxicity of the CBAA-G5/G3-Man compounded with YTHDF1siRNA is reduced to a certain extent, and the good cell compatibility of the compound is further verified.
Example 7
The dendritic cells are used as model cells, cy3 (a fluorescent dye) labeled YTHDF1siRNA is selected, and the efficiency of delivering YTHDF1siRNA in the cells by CBAA-G5/G3-Man/YTHDF1siRNA complexes under different N/P ratio conditions is detected by a flow cytometer. 1% of dendritic cells10 5 Cell density per well was seeded in 12-well plates, 1mL DMEM + + medium was added per well, and the plates were placed in 5% CO 2 Incubate overnight at 37 ℃ in an incubator. Then the culture medium is changed into 1mL DMEM + + culture medium containing CBAA-G5/G3-Man/YTHDF1siRNA complexes with different N/P ratios, wherein the N/P ratios are respectively 0, 5, 10, 15, 20 and 30, and the addition amount of YTHDF1siRNA is 1 mu G/hole. After 4h incubation, the cells were washed 2 times with PBS buffer, digested with pancreatin, harvested by centrifugation (1000rpm, 5 min) and resuspended in the appropriate amount of PBS. Finally, the fluorescence intensity of the cells was measured by flow cytometry, and the results are shown in FIG. 11, in which the fluorescence intensity of the naked YTHDF1siRNA group was slightly increased but still very low compared to the normal cell group (control group). While the fluorescence intensity of the material group increases with the increase of the N/P ratio, and the red fluorescence intensity is strongest when the N/P ratio is 15. Therefore, under the condition of the experimentally designed N/P ratio, CBAA-G5/G3-Man shows good efficiency of delivering YTHDF1siRNA, and the delivery efficiency is highest when the N/P ratio is 15.
Dendritic cells are used as model cells, cy3 (a fluorescent dye) labeled YTHDF1siRNA is selected, and the efficiency of delivering YTHDF1siRNA in the cells by CBAA-G5/G3-Man/YTHDF1siRNA complexes under the condition of optimal N/P ratio (N/P = 15) is detected by a laser confocal microscope. Dendritic cells were plated at 1X 10 5 Cell density per well was seeded in confocal microscope dishes with 1mL DMEM + + media per dish and the dishes placed in 5% CO 2 Incubate overnight at 37 ℃ in an incubator. Then the culture medium was changed to 1mL DMEM + + medium containing CBAA-G5/G3-Man/YTHDF1siRNA complex with N/P =15, wherein the addition amount of YTHDF1siRNA was 1 μ G/dish. After 4h incubation, cells were washed 2 times with PBS buffer, fixed with glutaraldehyde by standard specifications and stained for nuclei with DAPI. Finally, 300. Mu.L of PBS was added to each well, and phagocytosis of the cells was observed by confocal laser microscopy. As shown in FIG. 12, no significant red fluorescence was observed in the naked YTHDF1siRNA group and the CBAA-G5/G3-Man/YTHDF1siRNA complex group showed strong red fluorescence mostly around or inside the nucleus, compared to the control group. Therefore, CBAA-G5/G3-Man can successfully deliver YTHDF1siRNA to cytoplasmAnd nucleus, and is favorable for realizing subsequent gene silencing.
The dendritic cells are used as model cells, cy3 (a fluorescent dye) labeled YTHDF1siRNA is selected, and the capability of CBAA-G5/G3-Man/YTHDF1siRNA complex targeting DC cells to deliver YTHDF1siRNA under the condition of optimal N/P ratio is detected by a flow cytometer. Dendritic cells were plated at 1X 10 5 Cell density per well was seeded in 12-well plates, 1mL DMEM + + medium per well and placed in 5% CO 2 Incubate overnight at 37 ℃ in an incubator. The well plates were then divided into 4 groups of 3 wells each, control (PBS), naked YTHDF1siRNA, CBAA-G5/G3-Man/YTHDF1siRNA (N/P = 15), man + CBAA-G5/G3-Man/YTHDF1siRNA, respectively. Then, the culture medium of each group is changed into 1mL of DMEM + + culture medium containing the corresponding group content, the YTHDF1siRNA addition amount is 1 mu G/hole, and the Man + CBAA-G5/G3-Man/YTHDF1siRNA group needs to be added with Man (5 mu M/hole) for 4 hours in advance and then added with CBAA-G5/G3-Man/YTHDF 1siRNA. After 4h incubation, the cells were washed 2 times with PBS buffer, digested with pancreatin, centrifuged (1000rpm, 5 min), harvested and resuspended in the appropriate amount of PBS. Finally, the fluorescence intensity of the cells was measured by flow cytometry. As shown in FIG. 13, compared with the control group, no significant red fluorescence was observed in the naked YTHDF1siRNA group, the red fluorescence of the CBAA-G5/G3-Man/YTHDF1siRNA complex group was the strongest, and the red fluorescence of the CBAA-G5/G3-Man/YTHDF1siRNA complex was decreased after blocking by adding Man in advance. This indicates that CBAA-G5/G3-Man/YTHDF1siRNA has the ability to target DC cells because of Man, which is more beneficial for the realization of subsequent gene silencing.
Example 8
The expression of intracellular YTHDF1 protein was detected by Western Blot using dendritic cells as model cells. Dendritic cells were cultured at 2X 10 5 Cell density per well was seeded in 6-well plates, 2mL DMEM + + medium per well was added and the plates were placed in 5% CO 2 Incubate overnight at 37 ℃ in an incubator. Then, each well of culture medium is changed into a culture solution containing PBS buffer solution, naked YTHDF1siRNA, CBAA-G5/G3-Man and CBAA-G5/G3-Man/YTHDF1siRNA, wherein the N/P ratio of the compound is 15, the addition amount of YTHDF1siRNA is 1 mu, and the concentration of the siRNA is lower than that of the siRNAg/well, 1mL of culture medium was added per well and incubated for 4h. The medium was then discarded, 1mL of fresh DMEM + + medium was added to each well, and the incubation continued in the incubator for 48h. The cells were washed 2 times with PBS buffer, digested with pancreatin, centrifuged (1000rpm, 5 min) to collect cells and then subjected to Western Blot assay according to standard specifications. The results are shown in FIG. 14, in which PBS was used as the blank control group, GADPH was used as the reference protein, the expression of YTHDF1 protein was normal in the blank control group and CBAA-G5/G3-Man group, the expression of YTHDF1 protein was downregulated in the CBAA-G5/G3-Man/YTHDF1siRNA group, and the downregulation efficiency was 44%.
Example 9
Dendritic cells and breast cancer cells (4T 1) are used as model cells, fluorescence labeled CD80 and CD86 antibodies are utilized, and CBAA-G5/G3-Man/YTHDF1siRNA complexes are detected by a flow cytometer under the condition of optimal N/P ratio to improve the antigen presentation efficiency of DC cells. 4T1 cells were plated at 1X 10 5 Cell density per well was seeded in the upper chamber of a transwell-12 well plate (0.4 μm) with 1mL of DMEM + + medium per well, while dendritic cells were plated at 1X 10 5 Cell density per well was seeded in the lower chamber of a transwell-12 well plate, and the cells seeded in the upper and lower chambers were placed separately in 5% CO 2 And cultured in an incubator at 37 ℃. After overnight incubation, the upper and lower chambers were combined and the lower chamber medium was changed to 1mL of DMEM + + medium containing PBS or Free YTHDF1siRNA or CBAA-G5/G3-Man/NC siRNA complex or CBAA-G5/G3-Man/YTHDF1siRNA complex with an N/P ratio of 15, YTHDF1siRNA or NC siRNA addition of 1. Mu.g/well. After 4h incubation, the lower chamber was replaced with fresh DMEM + + medium and cultured for another 24h, then the lower chamber DC cells were washed 2 times with PBS buffer solution, digested with pancreatin, centrifuged (1000rpm, 5 min) to collect the cells and resuspended with an appropriate amount of PBS. The intensities of both fluorescence on the cell surface were measured by flow cytometry using CD80 and CD86 antibodies or isotype controls for labeling according to standard specifications. The results are shown in FIG. 15, and the fluorescence intensity was compared with other groups, it was found that the expression of CD80 and CD86 in the CBAA-G5/G3-Man/YTHDF1siRNA complex group was significantly up-regulated, which indicates that the CBAA-G5/G3-Man/YTHDF1siRNA complex still has the effect of promoting the growth of cancer cellsCross-antigen presenting ability into DC cells.
Example 10
And (3) performing drug loading and slow release effect characterization on the G5.NHAc/G3.NHAc/DOX compound prepared in the step (8) in the embodiment 1. The deprotonated DOX precipitate obtained by centrifugation in step (8) of example 1 was collected and dissolved in methanol for ultraviolet spectrophotometer (UV-vis) testing to obtain its absorbance at 490 nm. And correspondingly measuring a standard curve of DOX in methanol, further calculating the DOX precipitation amount, and then subtracting the DOX precipitation amount from the DOX addition amount in the reaction to obtain the DOX loading amount in the G5.NHAc/G3.NHAc/DOX compound. The result shows that the encapsulation rate of the material to DOX is 78.5%, and further calculation shows that 11.8 DOX are encapsulated in 1 average of G5.NHAc/G3.NHAc.
The complex of g5.Nhac/g3.Nhac/DOX prepared in step (8) of example 1 was dissolved in water (2mg, 1ml), placed in cellulose dialysis membranes having a molecular weight of 14000, respectively, suspended in 9mL of PBS (pH = 7.4) or acetic acid buffer (pH = 5.5) after binding, and then placed in a 37 ℃ constant temperature shaker with shaking. At each time point 1mL of the buffered external solution was removed and tested with UV-vis. At the same time, 1mL of fresh buffer corresponding to the pH was added. For comparison, DOX · HCl at the corresponding concentration was dissolved in water and placed in an external solution of PBS (pH = 7.4) to test its sustained release effect. Meanwhile, a standard curve of DOX under the conditions of pH =5.0 and pH =7.4 is obtained by using UV-vis, absorbance values of the obtained buffered external liquid under different pH conditions (pH =5.5 and pH = 7.4) are obtained by UV-vis test, and the concentration of the slow-release DOX is calculated from the standard curve, thereby calculating the slow-release curve of the g5.Nhac/g3.Nhac/DOX complex under different pH conditions (pH =5.5 and pH = 7.4). The results are shown in fig. 16A, where the release rate of DOX in the g5.Nhac/g3.Nhac/DOX complex is significantly slowed compared to DOX alone, demonstrating the excellent drug release characteristics of the carrier. And at pH =5.5, the release rate of DOX in the g5.Nhac/g3.Nhac/DOX complex was faster than pH =7.4, indicating that the release of the drug was more favorable in an acidic microenvironment.
Example 11
Using murine breast cancer (4T 1) cells as model cells toAnd G5.NHAc/G3.NHAc and DOX are used as controls, and the cytotoxicity of the G5.NHAc/G3.NHAc/DOX compound is detected under the conditions of different DOX concentrations. 4T1 cells were plated at 2X 10 3 Cell density per well was seeded in 96-well plates, and 100. Mu.L of DMEM + + medium was added per well. Cells were placed in 5% CO 2 After overnight incubation in an incubator at 37 deg.C, the medium was changed to 100. Mu.L of DMEM + + containing materials at different molar concentrations of DOX, 0, 0.25, 0.5, 2.5, 5, 10, 25 and 50. Mu.g/mL respectively, and then the cells were cultured for a further 24h. The culture medium was discarded and the activity of the cells was determined using the CCK-8 kit according to the protocol. The results are shown in fig. 16B, with increasing DOX concentration, the cell viability of the group of the g5.Nhac/g3.Nhac/DOX complex decreased as in the free DOX group, which indicates that the DOX-loaded g5.Nhac/g3.Nhac material had the effect of inhibiting cancer cell proliferation, while the group of the non-drug-loaded g5.Nhac/g3.Nhac material had no effect on cell viability.
Example 12
The efficiency of phagocytosis of G5.NHAc/G3. NHAc-loaded DOX by cells was evaluated by flow cytometry using 4T1 cells as model cells and the red fluorescence of DOX itself. At 1 × 10 5 Density per well 4T1 was seeded in 12-well plates with 1mL DMEM + + media per well. Cells were placed in 5% CO 2 After overnight incubation in an incubator at 37 deg.C, the medium was changed to 1mL DMEM + + containing materials at different molar concentrations of DOX, 2.5, 5, 10 and 20. Mu.g/mL respectively. After 4h incubation, the cells were washed 2 times with PBS buffer, digested with pancreatin, harvested by centrifugation (1000rpm, 5 min) and resuspended in the appropriate amount of PBS. Finally, the fluorescence intensity of the cells was measured by flow cytometry, and the results are shown in FIG. 17. Compared with the normal cell group (control group), the fluorescence intensity of the G5.NHAc/G3.NHAc/DOX group is enhanced along with the increase of DOX concentration, which indicates that the prepared G5.NHAc/G3.NHAc/DOX complex can be phagocytized by cells and is dose-dependent.
Example 13
4T1 cells are used as model cells, immunofluorescence staining is carried out by using Calreticulin (CRT) antibodies, and the induced immunity after DOX is loaded on G5.NHAc/G3.NHAc is evaluated through laser confocal microscopy researchA lethal effect. 4T1 cells were plated at 1X 10 5 The cell density of each well was inoculated into a special dish for a confocal microscope, 500. Mu.L of DMEM + + medium was added to each dish, and the dish was placed in 5% CO 2 Incubate overnight at 37 ℃ in an incubator. Then, the medium was changed to 500. Mu.L of DMEM + + medium containing G5.NHAc/G3.NHAc/DOX complex (IV) at a DOX concentration of 10. Mu.g/mL, and PBS group (I), corresponding concentration of G5.NHAc/G3.NHAc group (II), and corresponding concentration of DOX group (III) were used as controls. After 12h of cell culture, cells were washed 2 times with PBS buffer and fixed with glutaraldehyde by standard protocols, then treated with immunostaining blocking buffer for 60min according to standard procedures, incubated with anti-CRT (primary antibody) for 60min, washed with PBS and incubated with FITC-labeled secondary antibody for 60min. Finally, cells were stained with DAPI at 37 ℃ for 15min and CRT expression was observed with a confocal laser microscope. The results are shown in FIG. 18, and compared with the PBS group and the G5.NHAc/G3.NHAc group, the CRT expression of the DOX group and the G5.NHAc/G3.NHAc/DOX complex group is increased, which shows that the prepared G5.NHAc/G3.NHAc/DOX complex can cause the immunogenic death effect of cancer cells due to the presence of DOX.
Example 14
4T1 is taken as a model cell, fluorescence labeled CD80 and CD86 antibodies are utilized, and after a G5.NHAc/G3.NHAc/DOX compound is added into a cancer cell culture solution, the antigen presenting capacity of the DC cell is detected by a flow cytometer after ICD stimulation. 4T1 cells were plated at 1X 10 5 Cell density per well was seeded in the upper chamber of a transwell-12 well plate (0.4 μm) with 0.5mL of DMEM + + medium per well, and dendritic cells were plated at 1X 10 5 Cell density per well was seeded in the lower chamber of a transwell-12 well plate, and cells seeded in the upper and lower chambers were placed separately in 5% CO 2 And cultured in an incubator at 37 ℃. After overnight incubation, the upper chamber medium was changed to 0.5mL DMEM + + medium containing PBS, 5. Mu.g/mL DOX of the G5.NHAc/G3.NHAc/DOX complex or 50. Mu.g/mL DOX of the G5.NHAc/G3.NHAc/DOX complex. The upper and lower chambers were combined and incubated for 24h. Then, the dendritic cells were washed 2 times with PBS buffer, digested with pancreatin, centrifuged (1000rpm, 5 min) to collect the cells, and then resuspended with an appropriate amount of PBS. According to the standard specification, liLabeled with CD80 and CD86 antibodies or isotype controls, and the intensity of both fluorescences on the cell surface was detected by flow cytometry. The results are shown in fig. 19, and it can be seen that the expression of CD80 and CD86 in the group of the g5.Nhac/g3.Nhac/DOX complex is significantly up-regulated by comparing the fluorescence intensity with the other groups, and the up-regulation is more obvious when the concentration of DOX is higher, which indicates that the g5.Nhac/g3.Nhac/DOX complex can promote the antigen presenting ability of DC cells.
Example 15
Female ICR healthy mice (purchased from Shanghai Si Laike laboratory animal center) of 4-6 weeks were selected and injected according to established protocols (all animal experiments were performed strictly according to the animal protection Association standards) to evaluate the biosafety of the designed multifunctional nucleocapsid dendrimer loaded with drugs or genes. Firstly, selecting 18 ICR mice, randomly dividing the ICR mice into six groups, namely a PBS group, a DOX.HCl group, a G5.NHAc/G3.NHAc/DOX group, a CBAA-G5/G3-Man/YTHDF1siRNA group, a DOX.HCl + CBAA-G5/G3-Man/YTHDF1siRNA group, a G5.NHAc/G3.NHAc/DOX + CBAA-G5/G3-Man/YTHDF1siRNA group, wherein the group containing the DOX needs to be ensured to be injected intravenously, and the injection dose of the DOX is 5mg/kg; the group containing YTHDF1siRNA should be guaranteed to be injected into inguinal lymph node, and the injection dose of YTHDF1siRNA is 0.3mg/kg (about 10 μ g/body). 15 days after injection, mice were euthanized and the major organs were dissected and H & E stained. The results are shown in fig. 20, and the preparations of the other groups did not produce any significant cardiotoxicity, liver and kidney damage, pulmonary toxicity and spleen infiltration, except that the group containing free DOX had significant cardiotoxicity. This shows that the gene or drug complex prepared by using the multifunctional core-shell dendrimer has good biocompatibility.
Example 16
The 4T1 is used as a model cell to construct an in-situ breast cancer mouse model, and injection administration is carried out according to a formulated scheme to evaluate the treatment effect of the designed multifunctional core-shell dendrimer loaded with drugs or genes. Cells were first cultured and harvested at 1X 10 6 Dose of 4T1 cells/mouse tumor cells were injected at the breast pad of the white mouse when the tumor volume reached about 350mm 3 When selecting the model mouse36, were randomly divided into six groups, PBS group (I), DOX.HCl group (II), G5.NHAc/G3.NHAc/DOX group (III), CBAA-G5/G3-Man/YTHDF1siRNA group (IV), DOX.HCl + CBAA-G5/G3-Man/YTHDF1siRNA group (V), and G5.NHAc/G3.NHAc/DOX + CBAA-G5/G3-Man/YTHDF1siRNA group (VI). Wherein, the group containing DOX needs to be ensured to be intratumoral injection, and the injection dosage of DOX is 5mg/kg; the group containing YTHDF1siRNA needs to be guaranteed to be injected into inguinal lymph node, and the injection dosage of YTHDF1siRNA is 0.3mg/kg (about 10 mu g/body). At the same time, the tumor volume reaches about 350mm 3 Times as the day 1 of the experiment, groups containing DOX were intratumorally injected on day 1, and groups containing YTHDF1siRNA were injected in the inguinal lymph node on day 2. Each group was dosed once every 3 days for a total of 5 doses, tumor volume was measured every 2 days, and mouse weight was weighed every 2 days. The results are shown in FIG. 21, the tumor volume of each group of mice is reduced compared with that of the blank control group, especially the group of G5.NHAc/G3.NHAc/DOX + CBAA-G5/G3-Man/YTHDF1siRNA has the most obvious treatment effect. The body weight of the mice did not change much during the treatment period, only the individual DOX groups lost slightly after the treatment period. This shows that the gene or drug complex prepared by using the multifunctional core-shell dendrimer has good therapeutic effect.
After 15 days of treatment, mice were euthanized and their spleens and tumor sites were extracted by dissection. CD4 labeling with fluorescence + And CD8 + The antibody is used for detecting the content of various T cells in each group of spleen tissues through a flow cytometer and evaluating the activation degree of the T cells; in addition, the extracted tumor sites were sectioned and immunofluorescent stained and TUNEL experiments using CD4 + And CD8 + Antibodies, activated T cell infiltration assessed by immunofluorescence staining; apoptosis at the tumor site was assessed by fluorescence microscopy using TUNEL assay. The results are shown in FIG. 22, CD4 in spleen of G5.NHAc/G3.NHAc/DOX + CBAA-G5/G3-Man/YTHDF1siRNA group + And CD8 + The proportion of T cells was the highest, which indicated that T cells in the spleen of the G5.NHAc/G3.NHAc/DOX + CBAA-G5/G3-Man/YTHDF1siRNA group were significantly activated. TUNEL staining results showed (FIG. 23), cells at tumor sites of the G5.NHAc/G3.NHAc/DOX + CBAA-G5/G3-Man/YTHDF1siRNA groupThe degree of apoptosis is most pronounced. These phenomena also fully illustrate the use of multifunctional core-shell dendrimers to prepare gene or drug complexes for chemotherapy-immunotherapy combination therapy, and can achieve the goal of enhancing therapeutic effect.
Claims (10)
1. A multifunctional nano-delivery platform, comprising: CBAA-G5/G3-Man multifunctional nano platform load gene YTHDF1 siRNA; the CBAA-G5/G3-Man multifunctional nano platform is obtained by reacting G5-CD/Ad-G3-Man formed by self-assembly of G5-CD and Ad-G3-Man with carboxylic betaine CBAA, wherein the G5-CD is a 5 th generation amino-terminated PAMAM dendrimer surface modification beta-cyclodextrin beta-CD, and the Ad-G3-Man is a 3 rd generation amino-terminated PAMAM dendrimer surface modification adamantane Ad and mannose Man.
2. The multi-functional nano delivery platform according to claim 1, further comprising a loaded anticancer drug nano platform comprising: g5, NHAc-CD/Ad-G3, NHAc acetylation nanometer platform wraps anticancer drug adriamycin;
the G5.NHAc-CD/Ad-G3.NHAc acetylation nanometer platform is obtained by acetylating Ad-G3 and G5-CD; wherein G5-CD is a 5 th generation amino-terminated PAMAM dendrimer surface modified beta-cyclodextrin beta-CD, and Ad-G3 is a 3 rd generation amino-terminated PAMAM dendrimer surface modified adamantane Ad.
3. A method of making the multifunctional nano-delivery platform of claim 1, comprising:
(1) Dispersing adamantane acetic acid Ad-COOH in a solvent, adding EDC.HCl and NHS solution for activation, and adding the activated Ad-COOH solution to G3.NH 2 Reacting in the solution, dialyzing, and freeze-drying to obtain Ad-G3;
(2) Dissolving the Ad-G3 in the step (1) in a PBS solution, dropwise adding mannose Man dissolved in the PBS solution, reacting, dialyzing, and performing freeze-drying treatment to obtain Ad-G3-Man;
(3) Dispersing beta-cyclodextrin beta-CD in a solvent, dropwise adding CDI solution for activation, and addingThe activated beta-CD solution was added to G5.NH 2 In the solution, carrying out ammonia hydroxylation reaction, dialysis and freeze-drying treatment to obtain G5-CD, dissolving the G5-CD and the Ad-G3-Man in the step (2) by ultrapure water respectively, mixing, carrying out supramolecular self-assembly reaction, and carrying out dialysis and freeze-drying treatment to obtain G5-CD/Ad-G3-Man;
(4) Respectively dissolving the G5-CD/Ad-G3-Man obtained in the step (3) in a solvent 1, dissolving the carboxylic betaine CBAA in a solvent 2, mixing the two solutions, reacting, dialyzing, and lyophilizing to obtain CBAA-G5/G3-Man;
(5) And (4) incubating the CBAA-G5/G3-Man and YTHDF1siRNA in the step (4) to obtain a CBAA-G5/G3-Man/YTHDF1siRNA compound, namely the multifunctional nano delivery platform.
4. The method according to claim 3, wherein the step (1) comprises Ad-COOH, EDC.HCl, NHS and G3.NH 2 1 to 1.5; the solvent is DMSO; the activation temperature is room temperature, and the activation time is 2-4 h; the reaction temperature is room temperature, and the reaction time is 2-4 d.
5. The preparation method according to claim 3, wherein the molar ratio of Ad-G3 to Man in the step (2) is 1; the reaction temperature is 85-95 ℃, and the reaction time is 1-6 h.
6. The method according to claim 3, wherein the solvent in the step (3) is DMSO; beta-CD, CDI and G5.NH 2 The molar ratio of (1) to (25) is; the activation temperature is 25-35 ℃, and the activation time is 5-7 h; the temperature of the ammonolysis reaction is 25-35 ℃, and the time of the ammonolysis reaction is 58-62 h; the molar ratio of the G5-CD to the Ad-G3-Man is 1; the reaction temperature of the supermolecule self-assembly is room temperature, and the reaction time of the supermolecule self-assembly is 20-25 h.
7. The method according to claim 3, wherein the solvent 1in the step (4) is methanol; the solvent 2 is physiological saline; the molar ratio of CBAA to G5-CD/Ad-G3-Man is 200-300; the reaction temperature is room temperature, and the reaction time is 45-50 h.
8. The method according to claim 3, wherein the incubation in the step (5) is: diluting CBAA-G5/G3-Man with diethyl pyrocarbonate (DEPC) water, then diluting YTHDF1siRNA with DEPC water, mixing the two diluted solutions uniformly, and incubating at 35-40 ℃ for 20-30 min; the nitrogen-phosphorus ratio of CBAA-G5/G3-Man to YTHDF1siRNA is 0.5.
9. The method of manufacturing of claim 3, wherein the multifunctional nano delivery platform further comprises a anticancer drug loaded nano platform; the preparation method of the anticancer drug loaded nano platform comprises the following steps:
(a) Dissolving Ad-G3 in the step (1) and G5-CD in the step (3) with ultrapure water respectively, mixing, dropwise adding triethylamine and acetic anhydride solution, reacting, dialyzing, and lyophilizing to obtain G5.NHAc-CD/Ad-G3.NHAc, wherein the molar ratio of G5-CD, ad-G3, triethylamine and acetic anhydride is 1;
(b) Dissolving G5.NHAc-CD/Ad-G3.NHAc in the step (a) in water, mixing with deprotonated DOX solution, carrying out open reaction, centrifuging, taking supernate, and carrying out freeze-drying treatment to obtain a G5.NHAc-CD/Ad-G3.NHAc/DOX compound, namely a loaded anticancer drug nano platform; wherein the molar ratio of G5.NHAc-CD/Ad-G3.NHAc to DOX is 1-20, and the open reaction is as follows: stirring for 8-16h at room temperature in the dark.
10. Use of the multifunctional nano-delivery platform of claim 1in the preparation of a tumor immunotherapy medicament or a tumor immunotherapy and chemotherapy combination treatment medicament.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211498632.6A CN115957342B (en) | 2022-11-28 | 2022-11-28 | Multifunctional nano delivery platform for loading medicine/gene and preparation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211498632.6A CN115957342B (en) | 2022-11-28 | 2022-11-28 | Multifunctional nano delivery platform for loading medicine/gene and preparation and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115957342A true CN115957342A (en) | 2023-04-14 |
| CN115957342B CN115957342B (en) | 2024-08-09 |
Family
ID=87360717
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211498632.6A Active CN115957342B (en) | 2022-11-28 | 2022-11-28 | Multifunctional nano delivery platform for loading medicine/gene and preparation and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115957342B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104870464A (en) * | 2012-07-24 | 2015-08-26 | 国家科研中心 | Multimeric mannosides, a process for preparing the same and their uses as a drug |
| US20180028647A1 (en) * | 2016-07-27 | 2018-02-01 | Partikula Llc | Modifications of therapeutic agents for enhanced delivery to target sites |
| CN110302178A (en) * | 2019-07-24 | 2019-10-08 | 东华大学 | A kind of delivering nano platform of carrying medicament and its preparation method and application |
| US20200078471A1 (en) * | 2018-08-27 | 2020-03-12 | Board Of Regents, The University Of Texas System | Renal clearable drug delivering nanoparticles and methods of use therefor |
| CN113350516A (en) * | 2021-05-21 | 2021-09-07 | 东华大学 | Functional dendrimer simultaneously loaded with dexamethasone and gene drugs and preparation and application thereof |
| CN113663086A (en) * | 2021-07-19 | 2021-11-19 | 东华大学 | Dendritic cell targeted hybrid dendrimer/YTHDF 1siRNA compound and preparation and application thereof |
-
2022
- 2022-11-28 CN CN202211498632.6A patent/CN115957342B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104870464A (en) * | 2012-07-24 | 2015-08-26 | 国家科研中心 | Multimeric mannosides, a process for preparing the same and their uses as a drug |
| US20180028647A1 (en) * | 2016-07-27 | 2018-02-01 | Partikula Llc | Modifications of therapeutic agents for enhanced delivery to target sites |
| US20200078471A1 (en) * | 2018-08-27 | 2020-03-12 | Board Of Regents, The University Of Texas System | Renal clearable drug delivering nanoparticles and methods of use therefor |
| CN110302178A (en) * | 2019-07-24 | 2019-10-08 | 东华大学 | A kind of delivering nano platform of carrying medicament and its preparation method and application |
| CN113350516A (en) * | 2021-05-21 | 2021-09-07 | 东华大学 | Functional dendrimer simultaneously loaded with dexamethasone and gene drugs and preparation and application thereof |
| CN113663086A (en) * | 2021-07-19 | 2021-11-19 | 东华大学 | Dendritic cell targeted hybrid dendrimer/YTHDF 1siRNA compound and preparation and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| CONG SONGA: "Core-shell tecto dendrimer-mediated cooperative chemoimmunotherapy of breast cancer", 《JOURNAL OF CONTROLLED RELEASE》, vol. 358, 22 May 2023 (2023-05-22), pages 601 - 611 * |
| LONGGANG WANG: "Reducing the Cytotoxity of Poly(amidoamine) Dendrimers byModification of a Single Layer of Carboxybetaine", 《LANGMUIR》, vol. 29, no. 28, 18 June 2013 (2013-06-18), pages 8914 * |
| MENGYUE PEI: "Mannose-functionalized antigen nanoparticles for targeted dendritic cells, accelerated endosomal escape and enhanced MHC-I antigen presentation", 《COLLOIDS AND SURFACES B: BIOINTERFACES》, vol. 197, 31 January 2011 (2011-01-31), pages 111378 * |
| 赵俊义: "糖树状大分子在生物医学方面的应用研究进展", 《重庆医科大学学报》, vol. 32, no. 8, 31 December 2007 (2007-12-31), pages 894 - 896 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115957342B (en) | 2024-08-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ke et al. | Matrix metalloproteinase-responsive multifunctional peptide-linked amphiphilic block copolymers for intelligent systemic anticancer drug delivery | |
| Zhang et al. | Dendrimer grafted persistent luminescent nanoplatform for aptamer guided tumor imaging and acid-responsive drug delivery | |
| Singh et al. | Folate and folate− PEG− PAMAM Dendrimers: synthesis, characterization, and targeted anticancer drug delivery potential in tumor bearing mice | |
| Yuan et al. | A novel self-assembly albumin nanocarrier for reducing doxorubicin-mediated cardiotoxicity | |
| Ying et al. | Macrophage membrane-biomimetic adhesive polycaprolactone nanocamptothecin for improving cancer-targeting efficiency and impairing metastasis | |
| Wang et al. | Reversion of multidrug resistance by tumor targeted delivery of antisense oligodeoxynucleotides in hydroxypropyl-chitosan nanoparticles | |
| Yu et al. | Enzyme sensitive, surface engineered nanoparticles for enhanced delivery of camptothecin | |
| JP2011524446A (en) | Chitosan oligosaccharide fatty acid graft product modified with polyglycol, its preparation method and use thereof | |
| Song et al. | Erythrocyte-biomimetic nanosystems to improve antitumor effects of paclitaxel on epithelial cancers | |
| Nicholas et al. | Exploiting a Rose Bengal-bearing, oxygen-producing nanoparticle for SDT and associated immune-mediated therapeutic effects in the treatment of pancreatic cancer | |
| CN110559448A (en) | Target delivery siRNA bionic nanoparticle, preparation method and application thereof | |
| Wang et al. | cRGD mediated redox and pH dual responsive poly (amidoamine) dendrimer-poly (ethylene glycol) conjugates for efficiently intracellular antitumor drug delivery | |
| CN108339124B (en) | Preparation method and application of two-stage brain-targeted polymer micelle drug delivery system | |
| CN110302178B (en) | Drug-loaded delivery nano platform and preparation method and application thereof | |
| Gao et al. | Self-amplified ROS production from fatty acid oxidation enhanced tumor immunotherapy by atorvastatin/PD-L1 siRNA lipopeptide nanoplexes | |
| CN113663086B (en) | Dendritic cell targeted hybrid dendrimer/YTHDF 1siRNA complex and preparation and application thereof | |
| Lu et al. | Preparation and characterization of a gemini surfactant-based biomimetic complex for gene delivery | |
| WO2008007932A1 (en) | Chitosan complex containing ph sensitive imidazole group and preparation method thereof | |
| Chen et al. | Construction of multi-program responsive vitamin E succinate-chitosan-histidine nanocarrier and its response strategy in tumor therapy | |
| Shi et al. | Fabrication and characterization of a folic acid-bound 5-fluorouracil loaded quantum dot system for hepatocellular carcinoma targeted therapy | |
| Gu et al. | Construction of multifunctional targeted nano-prodrugs based on PAMAM dendrimers for tumor therapy | |
| Li et al. | Recent developments of polymeric delivery systems in gene therapeutics | |
| Guo et al. | β-glucan-modified nanoparticles with different particle sizes exhibit different lymphatic targeting efficiencies and adjuvant effects | |
| CN115957342B (en) | Multifunctional nano delivery platform for loading medicine/gene and preparation and application thereof | |
| CN114869858B (en) | Nucleic acid-chemotherapeutic drug composite nano-particles coated by homologous cancer cell membrane |
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 |