CN116440284A - Protein-glucose-containing polymer conjugate and preparation method and application thereof - Google Patents
Protein-glucose-containing polymer conjugate and preparation method and application thereof Download PDFInfo
- Publication number
- CN116440284A CN116440284A CN202310192332.3A CN202310192332A CN116440284A CN 116440284 A CN116440284 A CN 116440284A CN 202310192332 A CN202310192332 A CN 202310192332A CN 116440284 A CN116440284 A CN 116440284A
- Authority
- CN
- China
- Prior art keywords
- glucose
- protein
- conjugate
- containing polymer
- interferon
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229920000642 polymer Polymers 0.000 title claims abstract description 119
- 239000008103 glucose Substances 0.000 title claims abstract description 114
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 51
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 51
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 50
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 38
- 239000003814 drug Substances 0.000 claims abstract description 12
- 230000008878 coupling Effects 0.000 claims abstract description 7
- 238000010168 coupling process Methods 0.000 claims abstract description 7
- 238000005859 coupling reaction Methods 0.000 claims abstract description 7
- 238000002626 targeted therapy Methods 0.000 claims abstract description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 33
- 238000000034 method Methods 0.000 claims description 17
- 239000000178 monomer Substances 0.000 claims description 12
- 239000007870 radical polymerization initiator Substances 0.000 claims description 8
- 108010071942 Colony-Stimulating Factors Proteins 0.000 claims description 7
- 150000001875 compounds Chemical class 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 6
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims description 6
- -1 therapeutic vaccines Proteins 0.000 claims description 6
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 5
- 239000012986 chain transfer agent Substances 0.000 claims description 5
- 102000003390 tumor necrosis factor Human genes 0.000 claims description 5
- 102000055006 Calcitonin Human genes 0.000 claims description 4
- 108060001064 Calcitonin Proteins 0.000 claims description 4
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 claims description 4
- 102000015696 Interleukins Human genes 0.000 claims description 4
- 108010063738 Interleukins Proteins 0.000 claims description 4
- WQZGKKKJIJFFOK-DVKNGEFBSA-N alpha-D-glucose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-DVKNGEFBSA-N 0.000 claims description 4
- 229960004015 calcitonin Drugs 0.000 claims description 4
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 claims description 4
- 239000000122 growth hormone Substances 0.000 claims description 4
- 229940047122 interleukins Drugs 0.000 claims description 4
- 230000008685 targeting Effects 0.000 claims description 4
- 102000007644 Colony-Stimulating Factors Human genes 0.000 claims description 3
- 102000018997 Growth Hormone Human genes 0.000 claims description 3
- 108010051696 Growth Hormone Proteins 0.000 claims description 3
- 102000004877 Insulin Human genes 0.000 claims description 3
- 108090001061 Insulin Proteins 0.000 claims description 3
- 229960000182 blood factors Drugs 0.000 claims description 3
- 229940047120 colony stimulating factors Drugs 0.000 claims description 3
- 239000003102 growth factor Substances 0.000 claims description 3
- 229940125396 insulin Drugs 0.000 claims description 3
- 230000001225 therapeutic effect Effects 0.000 claims description 3
- 229940021747 therapeutic vaccine Drugs 0.000 claims description 3
- 229920002818 (Hydroxyethyl)methacrylate Polymers 0.000 claims description 2
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 claims description 2
- WOBHKFSMXKNTIM-UHFFFAOYSA-N Hydroxyethyl methacrylate Chemical compound CC(=C)C(=O)OCCO WOBHKFSMXKNTIM-UHFFFAOYSA-N 0.000 claims description 2
- 239000000174 gluconic acid Substances 0.000 claims description 2
- 235000012208 gluconic acid Nutrition 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 108091006296 SLC2A1 Proteins 0.000 abstract description 12
- 230000014509 gene expression Effects 0.000 abstract description 8
- 102000058063 Glucose Transporter Type 1 Human genes 0.000 abstract description 5
- 230000006377 glucose transport Effects 0.000 abstract description 4
- 238000003745 diagnosis Methods 0.000 abstract description 3
- 239000012620 biological material Substances 0.000 abstract description 2
- 238000001815 biotherapy Methods 0.000 abstract description 2
- 238000012377 drug delivery Methods 0.000 abstract description 2
- 238000003384 imaging method Methods 0.000 abstract description 2
- 230000001172 regenerating effect Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 59
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 36
- 235000018102 proteins Nutrition 0.000 description 36
- 239000002609 medium Substances 0.000 description 35
- 239000000243 solution Substances 0.000 description 35
- 210000002966 serum Anatomy 0.000 description 28
- 241000699666 Mus <mouse, genus> Species 0.000 description 21
- 238000007920 subcutaneous administration Methods 0.000 description 20
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 18
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 18
- 239000011780 sodium chloride Substances 0.000 description 18
- 108010050904 Interferons Proteins 0.000 description 16
- 102000014150 Interferons Human genes 0.000 description 16
- 238000006243 chemical reaction Methods 0.000 description 15
- 241000699670 Mus sp. Species 0.000 description 14
- 229940079322 interferon Drugs 0.000 description 14
- 239000000872 buffer Substances 0.000 description 13
- 239000003999 initiator Substances 0.000 description 13
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 11
- 238000011033 desalting Methods 0.000 description 11
- 210000001519 tissue Anatomy 0.000 description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 9
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 8
- 238000000338 in vitro Methods 0.000 description 8
- 230000001575 pathological effect Effects 0.000 description 8
- 102100023536 Solute carrier family 2, facilitated glucose transporter member 1 Human genes 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 239000003112 inhibitor Substances 0.000 description 7
- 229920001223 polyethylene glycol Polymers 0.000 description 7
- JXCDTORYGUGXIJ-UHFFFAOYSA-N 2-hydroxyethyl 2-methylprop-2-enoate;pentanedial Chemical compound O=CCCCC=O.CC(=C)C(=O)OCCO JXCDTORYGUGXIJ-UHFFFAOYSA-N 0.000 description 6
- 241000428456 Gluma Species 0.000 description 6
- 239000002202 Polyethylene glycol Substances 0.000 description 6
- 238000010560 atom transfer radical polymerization reaction Methods 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 238000001000 micrograph Methods 0.000 description 6
- 238000000386 microscopy Methods 0.000 description 6
- DWFKOMDBEKIATP-UHFFFAOYSA-N n'-[2-[2-(dimethylamino)ethyl-methylamino]ethyl]-n,n,n'-trimethylethane-1,2-diamine Chemical compound CN(C)CCN(C)CCN(C)CCN(C)C DWFKOMDBEKIATP-UHFFFAOYSA-N 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 238000011729 BALB/c nude mouse Methods 0.000 description 5
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 230000037396 body weight Effects 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 238000005227 gel permeation chromatography Methods 0.000 description 5
- 238000011065 in-situ storage Methods 0.000 description 5
- 210000000265 leukocyte Anatomy 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 4
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 4
- 229920002873 Polyethylenimine Polymers 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 4
- 229960000723 ampicillin Drugs 0.000 description 4
- 230000005587 bubbling Effects 0.000 description 4
- 239000004202 carbamide Substances 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 4
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000001502 gel electrophoresis Methods 0.000 description 4
- 210000002216 heart Anatomy 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 210000004072 lung Anatomy 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 238000006116 polymerization reaction Methods 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- 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 4
- 239000006228 supernatant Substances 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- 102000015790 Asparaginase Human genes 0.000 description 3
- 108010024976 Asparaginase Proteins 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 102000001554 Hemoglobins Human genes 0.000 description 3
- 108010054147 Hemoglobins Proteins 0.000 description 3
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 3
- 241001529936 Murinae Species 0.000 description 3
- 108090000279 Peptidyltransferases Proteins 0.000 description 3
- 229960003272 asparaginase Drugs 0.000 description 3
- DCXYFEDJOCDNAF-UHFFFAOYSA-M asparaginate Chemical compound [O-]C(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-M 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 238000001142 circular dichroism spectrum Methods 0.000 description 3
- 230000004154 complement system Effects 0.000 description 3
- 238000007334 copolymerization reaction Methods 0.000 description 3
- 229910001873 dinitrogen Inorganic materials 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- PMWXGSWIOOVHEQ-UHFFFAOYSA-N pyridine-2,6-dicarbaldehyde Chemical compound O=CC1=CC=CC(C=O)=N1 PMWXGSWIOOVHEQ-UHFFFAOYSA-N 0.000 description 3
- 238000010791 quenching Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 102000055025 Adenosine deaminases Human genes 0.000 description 2
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 2
- 108010082126 Alanine transaminase Proteins 0.000 description 2
- 101710141544 Allatotropin-related peptide Proteins 0.000 description 2
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 2
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 108090000317 Chymotrypsin Proteins 0.000 description 2
- 229910021592 Copper(II) chloride Inorganic materials 0.000 description 2
- 102000004420 Creatine Kinase Human genes 0.000 description 2
- 108010042126 Creatine kinase Proteins 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 108700012941 GNRH1 Proteins 0.000 description 2
- 239000000579 Gonadotropin-Releasing Hormone Substances 0.000 description 2
- 101150103227 IFN gene Proteins 0.000 description 2
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 2
- 102000000589 Interleukin-1 Human genes 0.000 description 2
- 108010002352 Interleukin-1 Proteins 0.000 description 2
- 102000000588 Interleukin-2 Human genes 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 2
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 2
- 108010025020 Nerve Growth Factor Proteins 0.000 description 2
- 102000015336 Nerve Growth Factor Human genes 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 2
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 2
- 102000013275 Somatomedins Human genes 0.000 description 2
- 108010056088 Somatostatin Proteins 0.000 description 2
- 102000005157 Somatostatin Human genes 0.000 description 2
- 102000011923 Thyrotropin Human genes 0.000 description 2
- 108010061174 Thyrotropin Proteins 0.000 description 2
- 108010009583 Transforming Growth Factors Proteins 0.000 description 2
- 102000009618 Transforming Growth Factors Human genes 0.000 description 2
- 108010003205 Vasoactive Intestinal Peptide Proteins 0.000 description 2
- 102400000015 Vasoactive intestinal peptide Human genes 0.000 description 2
- 230000001028 anti-proliverative effect Effects 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000004820 blood count Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000002737 cell proliferation kit Methods 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 229960002376 chymotrypsin Drugs 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000024203 complement activation Effects 0.000 description 2
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 description 2
- 229940109239 creatinine Drugs 0.000 description 2
- 238000004042 decolorization Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000002296 dynamic light scattering Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 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 2
- 239000005457 ice water Substances 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 229940047124 interferons Drugs 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- VBUWHHLIZKOSMS-RIWXPGAOSA-N invicorp Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)C(C)C)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=C(O)C=C1 VBUWHHLIZKOSMS-RIWXPGAOSA-N 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 229940053128 nerve growth factor Drugs 0.000 description 2
- 229910052759 nickel Inorganic materials 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 description 2
- 229960000553 somatostatin Drugs 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- DEQANNDTNATYII-OULOTJBUSA-N (4r,7s,10s,13r,16s,19r)-10-(4-aminobutyl)-19-[[(2r)-2-amino-3-phenylpropanoyl]amino]-16-benzyl-n-[(2r,3r)-1,3-dihydroxybutan-2-yl]-7-[(1r)-1-hydroxyethyl]-13-(1h-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentazacycloicosane-4-carboxa Chemical compound C([C@@H](N)C(=O)N[C@H]1CSSC[C@H](NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](CC=2C3=CC=CC=C3NC=2)NC(=O)[C@H](CC=2C=CC=CC=2)NC1=O)C(=O)N[C@H](CO)[C@H](O)C)C1=CC=CC=C1 DEQANNDTNATYII-OULOTJBUSA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- BIKSKRPHKQWJCW-UHFFFAOYSA-N 3,4-dibromopyrrole-2,5-dione Chemical compound BrC1=C(Br)C(=O)NC1=O BIKSKRPHKQWJCW-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 description 1
- 108700040115 Adenosine deaminases Proteins 0.000 description 1
- 239000012110 Alexa Fluor 594 Substances 0.000 description 1
- 102000004452 Arginase Human genes 0.000 description 1
- 108700024123 Arginases Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 101800001288 Atrial natriuretic factor Proteins 0.000 description 1
- 102400001282 Atrial natriuretic peptide Human genes 0.000 description 1
- 101800001890 Atrial natriuretic peptide Proteins 0.000 description 1
- 229930192334 Auxin Natural products 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 1
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 1
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 1
- 108010036949 Cyclosporine Proteins 0.000 description 1
- 102000000311 Cytosine Deaminase Human genes 0.000 description 1
- 108010080611 Cytosine Deaminase Proteins 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 102000003951 Erythropoietin Human genes 0.000 description 1
- 108090000394 Erythropoietin Proteins 0.000 description 1
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 1
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 1
- 102400000921 Gastrin Human genes 0.000 description 1
- 108010052343 Gastrins Proteins 0.000 description 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 102000002265 Human Growth Hormone Human genes 0.000 description 1
- 108010000521 Human Growth Hormone Proteins 0.000 description 1
- 239000000854 Human Growth Hormone Substances 0.000 description 1
- 108090000172 Interleukin-15 Proteins 0.000 description 1
- 102000003812 Interleukin-15 Human genes 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 102000016267 Leptin Human genes 0.000 description 1
- 108010092277 Leptin Proteins 0.000 description 1
- 102000009151 Luteinizing Hormone Human genes 0.000 description 1
- 108010073521 Luteinizing Hormone Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000192041 Micrococcus Species 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 108010016076 Octreotide Proteins 0.000 description 1
- 102400000050 Oxytocin Human genes 0.000 description 1
- 101800000989 Oxytocin Proteins 0.000 description 1
- XNOPRXBHLZRZKH-UHFFFAOYSA-N Oxytocin Natural products N1C(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CC(C)C)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C(C(C)CC)NC(=O)C1CC1=CC=C(O)C=C1 XNOPRXBHLZRZKH-UHFFFAOYSA-N 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 102000003982 Parathyroid hormone Human genes 0.000 description 1
- 108090000445 Parathyroid hormone Proteins 0.000 description 1
- 229940122907 Phosphatase inhibitor Drugs 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 102000003743 Relaxin Human genes 0.000 description 1
- 108090000103 Relaxin Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 108010086019 Secretin Proteins 0.000 description 1
- 102100037505 Secretin Human genes 0.000 description 1
- 101000677856 Stenotrophomonas maltophilia (strain K279a) Actin-binding protein Smlt3054 Proteins 0.000 description 1
- 102000019197 Superoxide Dismutase Human genes 0.000 description 1
- 108010012715 Superoxide dismutase Proteins 0.000 description 1
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 1
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 1
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108010092464 Urate Oxidase Proteins 0.000 description 1
- 108010004977 Vasopressins Proteins 0.000 description 1
- 102000002852 Vasopressins Human genes 0.000 description 1
- 108010046516 Wheat Germ Agglutinins Proteins 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- KBZOIRJILGZLEJ-LGYYRGKSSA-N argipressin Chemical compound C([C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@@H](C(N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)=O)N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(N)=O)C1=CC=CC=C1 KBZOIRJILGZLEJ-LGYYRGKSSA-N 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- NSQLIUXCMFBZME-MPVJKSABSA-N carperitide Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)=O)[C@@H](C)CC)C1=CC=CC=C1 NSQLIUXCMFBZME-MPVJKSABSA-N 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- AOXOCDRNSPFDPE-UKEONUMOSA-N chembl413654 Chemical compound C([C@H](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](C)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]1N(CCC1)C(=O)CNC(=O)[C@@H](N)CCC(O)=O)C1=CC=C(O)C=C1 AOXOCDRNSPFDPE-UKEONUMOSA-N 0.000 description 1
- 229940015047 chorionic gonadotropin Drugs 0.000 description 1
- 229960001265 ciclosporin Drugs 0.000 description 1
- 238000002983 circular dichroism Methods 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 229930182912 cyclosporin Natural products 0.000 description 1
- 229960003964 deoxycholic acid Drugs 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 229940105423 erythropoietin Drugs 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 229940126864 fibroblast growth factor Drugs 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 230000005917 in vivo anti-tumor Effects 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 229940039781 leptin Drugs 0.000 description 1
- NRYBAZVQPHGZNS-ZSOCWYAHSA-N leptin Chemical compound O=C([C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC(C)C)CCSC)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CS)C(O)=O NRYBAZVQPHGZNS-ZSOCWYAHSA-N 0.000 description 1
- 229940040129 luteinizing hormone Drugs 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000921 morphogenic effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical group OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000012758 nuclear staining Methods 0.000 description 1
- 229960002700 octreotide Drugs 0.000 description 1
- 229960001723 oxytocin Drugs 0.000 description 1
- XNOPRXBHLZRZKH-DSZYJQQASA-N oxytocin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@H](N)C(=O)N1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)NCC(N)=O)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 XNOPRXBHLZRZKH-DSZYJQQASA-N 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229960001319 parathyroid hormone Drugs 0.000 description 1
- 239000000199 parathyroid hormone Substances 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 1
- GCYXWQUSHADNBF-AAEALURTSA-N preproglucagon 78-108 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 GCYXWQUSHADNBF-AAEALURTSA-N 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000010526 radical polymerization reaction Methods 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 239000002464 receptor antagonist Substances 0.000 description 1
- 229940044551 receptor antagonist Drugs 0.000 description 1
- 239000003488 releasing hormone Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000012712 reversible addition−fragmentation chain-transfer polymerization Methods 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 229960002101 secretin Drugs 0.000 description 1
- OWMZNFCDEHGFEP-NFBCVYDUSA-N secretin human Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(N)=O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)C1=CC=CC=C1 OWMZNFCDEHGFEP-NFBCVYDUSA-N 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 231100000057 systemic toxicity Toxicity 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 229960001322 trypsin Drugs 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 229960003726 vasopressin Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/21—Interferons [IFN]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention relates to the technical field of bioengineering, in particular to a conjugate of protein-glucose-containing polymer, a preparation method and application thereof. The conjugate comprises: a protein and a glucose-containing polymer and a biocompatible molecule coupled to the protein; the glucose content of the protein-glucose-containing polymer conjugate is 5-50%. The conjugate obtained by coupling the protein with the glucose-containing polymer and the biocompatible molecule can be combined with the glucose transport receptor 1 with high expression in the tumor on one hand, can efficiently target the tumor, has long circulation capacity on the other hand, can be widely applied to the targeted protein delivery of GLUT1 high-expression tissues, and has wide application in the fields of molecular diagnosis, molecular imaging, biological materials, drug delivery, biological molecule delivery, regenerative medicine, targeted therapy, biological therapy and the like.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a conjugate of protein-glucose-containing polymer, a preparation method and application thereof.
Background
In the prior art, rapid development of biology and chemical biology greatly promotes research and development of protein drugs, more and more protein drugs are used for treating diseases, and meanwhile, protein-conjugates are attracting more and more attention as a novel biomacromolecule derivative. The protein-polymer conjugate refers to a biological hybrid formed by coupling a protein with other functional components through covalent bonds or non-covalent bonds, and can often obtain new characteristics from new components while retaining the characteristics (such as precise structures and biological functions) of the protein, and the bioconjugate, especially the protein-polymer conjugate, which integrates different characteristics can be widely applied to biological medicine fields such as biocatalysis, molecular diagnosis, molecular imaging, biological materials, drug delivery, biological molecular delivery, tissue engineering, regenerative medicine, targeted therapy, biological therapy and the like.
Protein-conjugate is transformed for many generations and applied to biological medicine, but the current protein-macromolecule coupling still has a series of scientific problems which need to be solved urgently in the biological medicine field: the existing polyethylene glycol protein and other protein polymer conjugates do not have targeting capability to focus parts, and the wide application of the protein-conjugate in biological medicine is severely restricted.
Disclosure of Invention
In order to solve at least one existing technical problem, the invention provides a protein-glucose-containing polymer conjugate, and a preparation method and application thereof.
In a first aspect, the present invention provides a protein-glucose-containing polymer conjugate comprising:
a protein and a glucose-containing polymer and a biocompatible molecule coupled to the protein;
the glucose content of the protein-glucose-containing polymer conjugate is 5-50%.
Further, the glucose content of the protein-glucose-containing polymer conjugate is 10-30%.
Further, the glucose content of the protein-glucose-containing polymer conjugate is 25-30%.
The method finds that the proportion of glucose in the conjugate can be regulated and controlled by regulating the proportion of glucose-containing compound monomers in the polymerization process, wherein the conjugate with low glucose content has good biocompatibility but low affinity to glucose transport receptor 1 (GLUT 1); while conjugates with high glucose content have high affinity for the glucose transport receptor 1GLUT1 but activate the complement system in serum, resulting in rapid clearance from the body; and the conjugate with the glucose content of 10% -30% (especially 25% -30%) has long circulation capacity and better affinity to GLUT 1.
Further, the biocompatible molecule comprises an oligoethylene glycol methacrylate and a zwitterionic monomer; and/or, the protein is a therapeutic protein.
Further, the protein comprises:
insulin, monoclonal antibodies, blood factors, colony stimulating factors, growth hormone, interleukins, growth factors, therapeutic vaccines, calcitonin, tumor necrosis factor or enzymes.
Further, the enzyme is an enzyme having a therapeutic function.
Further, the protein includes any one or more of the following:
insulin, monoclonal antibodies, blood factors, colony stimulating factors, growth hormones, interleukins, growth factors, therapeutic vaccines, calcitonin, tumor Necrosis Factor (TNF), enzymes, and the like. Specific examples include, but are not limited to: asparaginase, glutamate, arginase, arginine deaminase, adenosine deaminase ribonuclease, cytosine deaminase, trypsin, chymotrypsin, papain, epidermal Growth Factor (EGF), insulin-like growth factor (IGF), transforming Growth Factor (TGF), nerve Growth Factor (NGF), platelet-derived growth factor (PDGF), bone Morphogenic Protein (BMP), fibroblast growth factor, somatostatin, growth hormone, somatostatin, calcitonin, parathyroid hormone, colony Stimulating Factor (CSF), clotting factors, tumor necrosis factor, interferons, interleukins, gastrointestinal peptides, vasoactive Intestinal Peptide (VIP), chymotrypsin (CCK), gastrin, secretin, erythropoietin, antidiuretic hormone, octreotide, pancreatic enzymes, superoxide dismutase, thyroid stimulating hormone releasing hormone (TRH), thyroid stimulating hormone, colony Stimulating Factor (CSF); luteinizing hormone, luteinizing Hormone Releasing Hormone (LHRH), tissue-type plasminogen activator, interleukin-1, interleukin-15, receptor antagonist (IL-1 RA), glucagon-like peptide-1 (GLP-1), leptin, auxin, granulocyte colony stimulating factor (GM-CSF), interleukin-2 (IL-2), adenosine deaminase, uricase, asparaginase, human growth hormone, asparaginase, chorionic gonadotropin, heparin, atrial natriuretic peptide, hemoglobin, retroviral vectors, relaxin; cyclosporine, oxytocin, vaccine, monoclonal antibody, single chain antibody, ankyrin repeat protein, affibody.
Further, the glucose-containing polymer is prepared from a polymer containing alpha-D-glucose, beta-D-glucose, gluconic acid,
The polymer is polymerized by any compound monomer, wherein n is a positive integer of 1-10.
In a second aspect, the present invention provides a method of preparing a protein-glucose containing polymer conjugate comprising:
forming a functional molecule on the protein; copolymerizing and coupling a glucose-containing compound monomer, a biocompatible molecular monomer, and the protein through the functional molecule; or (b)
Copolymerizing said monomeric glucose-containing compound and said monomeric biocompatible molecule with said functionalized functional molecule to obtain a functionalized polymer, forming said functionalized polymer on said protein;
the functional molecule includes a free radical polymerization initiator and/or a chain transfer agent.
Further, the molar ratio of the protein, the biocompatible molecule, and the glucose-containing polymer is 1: (100-300): (400-700).
The polymerization degree, the size of the protein-polymer conjugate and the glucose content of the protein-glucose-containing polymer conjugate can be controlled by the method and the proportion of each component.
Further, the radical polymerization initiator includes any one or more of the following:
the chain transfer agent comprises any one or more of the following:
further, the copolymerization coupling is aqueous phase in situ copolymerization on the protein, and when the functional molecule is a radical initiator, the copolymerization coupling is free radical polymerization; when the functional molecule is a chain transfer agent, it is a reversible addition-fragmentation chain transfer polymerization.
The invention further provides the conjugate, or application of the conjugate prepared by the preparation method in targeted treatment of tumors.
In a third aspect, the invention provides a method of preparing a medicament for targeting a tumor comprising:
the conjugate is prepared by adopting the conjugate or the conjugate prepared by the preparation method as a raw material.
The invention has the following beneficial effects:
the invention provides a protein-glucose-containing polymer conjugate, wherein a glucose component in the conjugate can be combined with a glucose transport receptor 1 (GLUT 1) with high expression in a tumor, and another component with excellent biocompatibility in the polymer increases the hydration radius of the protein and prolongs the circulation half-life of the protein. Through optimizing the glucose content in the conjugate, the obtained conjugate containing 15% -35% of glucose can effectively target the tumor with high expression of GLUT1 while ensuring the long circulation capacity of the conjugate. The method has great significance for tumor targeting of the drug protein, and has wide application for treatment and diagnosis of specific tumors.
In addition, the preparation method of the conjugate provided by the invention can be widely applied to other protein or small peptide medicaments to improve the pharmacological characteristics of the conjugate.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a gel permeation chromatogram of an interferon-glucose-containing polymer conjugate provided in example 1 of the present invention.
FIG. 2 is a graph showing the particle size distribution of an interferon-glucose-containing polymer conjugate according to example 1 of the present invention.
FIG. 3 is a nuclear magnetic resonance spectrum of an interferon-glucose-containing polymer conjugate provided in example 1 of the present invention.
FIG. 4 is a circular dichroism spectrum of an interferon-glucose-containing polymer conjugate provided in example 1 of the present invention.
FIG. 5 is a gel electrophoresis of an interferon-glucose containing polymer conjugate provided in example 1 of the present invention.
FIG. 6 is a graph showing the antiproliferative activity of interferon-glucose containing polymer conjugates provided in example 1 of the present invention on Daudi cells.
FIG. 7 is a flow chart showing the ability of interferon-glucose containing polymer conjugates provided in example 1 of the present invention to enter cells in SKOV-3 cells in serum-containing medium.
FIG. 8 is a confocal laser microscopy image of interferon-glucose containing polymer conjugate provided in example 1 of the present invention in SKOV-3 cells in the presence of serum-containing medium.
FIG. 9 is a flow chart showing the ability of interferon-glucose containing polymer conjugates provided in example 1 of the present invention to enter cells in SKOV-3 cells in the absence of serum medium.
FIG. 10 is a confocal laser microscopy image of interferon-glucose containing polymer conjugate provided in example 1 of the present invention in SKOV-3 cells in the absence of serum medium.
FIG. 11 is a flow chart showing the ability of interferon-glucose containing polymer conjugates provided in example 1 of the present invention to enter cells in SKOV-3 cells in the presence of serum medium and GLUT1 inhibitor.
FIG. 12 is a confocal laser microscopy image of interferon-glucose containing polymer conjugate provided in example 1 of the present invention in SKOV-3 cells in the presence of serum medium and GLUT1 inhibitor.
FIG. 13 is a flow chart showing the ability of interferon-glucose containing polymer conjugates provided in example 1 of the present invention to enter cells in A2780 cells in the presence of serum-containing medium.
FIG. 14 is a confocal laser microscopy image of interferon-glucose containing polymer conjugate provided in example 1 of the present invention in A2780 cells in the presence of serum-containing medium.
FIG. 15 is a flow chart showing the ability of interferon-glucose containing polymer conjugates provided in example 1 of the present invention to enter cells in A2780 cells without serum medium.
FIG. 16 is a confocal microscope image of interferon-glucose-containing polymer conjugate provided in example 1 of the present invention in A2780 cells in the absence of serum medium.
FIG. 17 is a flow chart showing the ability of interferon-glucose containing polymer conjugates provided in example 1 of the present invention to enter cells in A2780 cells in the presence of serum medium and GLUT1 inhibitor.
FIG. 18 is a confocal microscope image of interferon-glucose-containing polymer conjugate provided in example 1 of the present invention in A2780 cells in the presence of serum medium and GLUT1 inhibitor.
FIG. 19 is a schematic representation of the level of C3a in the serum complement system activated by interferon-glucose containing polymer conjugate provided in example 1 of the present invention.
FIG. 20 is a graph showing the concentration of interferon-glucose-containing polymer conjugate in mice versus time provided in example 1 of the present invention.
FIG. 21 is a graph showing tumor volume change in a mouse SKOV-3 subcutaneous tumor model treatment with an interferon-glucose-containing polymer conjugate as provided in example 1 of the present invention.
FIG. 22 is a schematic representation of the survival of mice in the treatment of the subcutaneous tumor model of mice SKOV-3 with interferon-glucose-containing polymer conjugate as provided in example 1 of the present invention.
FIG. 23 is a graph showing tumor volume change in mouse A2780 subcutaneous tumor model treatment with interferon-glucose-containing polymer conjugate provided in example 1 of the present invention.
FIG. 24 is a schematic representation of the survival of mice in the treatment of a subcutaneous tumor model of mouse A2780 with an interferon-glucose-containing polymer conjugate according to example 1 of the present invention.
FIG. 25 is a diagram showing pathological sections of a mouse tumor after the end of treatment of a mouse SKOV-3 subcutaneous tumor model with an interferon-glucose-containing polymer conjugate according to example 1 of the present invention.
FIG. 26 is a diagram showing pathological sections of a mouse tumor after the end of treatment of a mouse SKOV-3 subcutaneous tumor model with an interferon-glucose-containing polymer conjugate according to example 1 of the present invention.
FIG. 27 is a schematic representation of pathological tissue sections of heart, liver, spleen, lung and kidney after the end of treatment of the murine SKOV-3 subcutaneous tumor model with the interferon-glucose-containing polymer conjugate provided in example 1 of the present invention.
FIG. 28 is a schematic representation of pathological tissue sections of heart, liver, spleen, lung and kidney after the end of treatment of the subcutaneous tumor model of mouse A2780, provided by example 1 of the present invention.
FIG. 29 is a graph showing the results of analysis of the hematology and biochemistry indices of the interferon-glucose-containing polymer conjugate of example 1 of the present invention after the end of treatment in the murine SKOV-3 subcutaneous tumor model; wherein ALT in A is alanine aminotransferase; AST is aspartate aminotransferase; LDH in B is lactate dehydrogenase; CK is creatine kinase; in C, UREA is UREA; CREA is creatinine; RBC in D is red cell count; WBCs are white blood cell counts; HGB in E is hemoglobin concentration; PLT is platelet count.
FIG. 30 is a graph showing the results of analysis of the blood and biochemical indicators of the interferon-glucose-containing polymer conjugate according to example 1 of the present invention after the end of the treatment in the mouse A2780 subcutaneous tumor model; wherein ALT in A is alanine aminotransferase; AST is aspartate aminotransferase; LDH in B is lactate dehydrogenase; CK is creatine kinase; in C, UREA is UREA; CREA is creatinine; RBC in D is red cell count; WBCs are white blood cell counts; HGB in E is hemoglobin concentration; PLT is platelet count.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
This example uses transpeptidase a to attach an atom transfer radical polymerization initiator to the C-terminus of interferon and polymerize oligomeric polyethylene glycol and 6-O-methacryloyl- α -D-glucose in situ to construct an interferon-poly oligomeric polyethylene glycol methyl ether-poly 6 α -D-glucose conjugate, the resulting conjugate was characterized for physicochemical properties in vitro and for evaluation of pharmaceutical properties in vivo and in vitro.
The method specifically comprises the following steps:
1. construction and expression of proteins
1.1 construction and expression of Interferon
From the following using PCR techniqueThe IFN encoding sequence was amplified in the vector and inserted into the pET-25b (+) vector through the Nde I/Eco RI cleavage site. The specific recognition sequence LPETG and the 6 XHis tag of SrtA are connected to the C-terminal of the IFN gene sequence, and the final protein sequence is as follows:
CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGSGGGGSLPETGGHHHHHH。
after verification by DNA sequencing, the constructed IFN-H6 encoding plasmid was transformed into E.coli strain Rosetta-gami (DE 3) pLysS competent cells and cultured overnight at 37℃in ampicillin-resistant LB medium. Cultures were transferred to 1L of sterile ampicillin-resistant TB medium for continued culture, and when the OD600 of the bacterial suspension reached 0.5, IPTG was added to a final concentration of 500. Mu.M for induction, and overexpression was performed overnight at 18 ℃. Cells were harvested by centrifugation, resuspended in 50mM Tris HCl,150mM NaCl,pH 7.4 buffer, sonicated in an ice-water bath and the pellet removed by centrifugation at 14,000Xg for 10min, 2mL of 1% (w/v) Polyethylenimine (PEI) was added to the supernatant and mixed well and centrifuged again. The supernatant containing the soluble protein was purified by passing through a nickel affinity column and through the AKTA Purifier 10 system. The column was equilibrated with 50mM Tris HCl,500mM NaCl,10% glycerol, 25mM imidazole, pH7.4 buffer, the eluted heteroprotein was eluted with 50mM Tris HCl,500mM NaCl,10% glycerol, 50mM imidazole, pH7.4 buffer, and finally G3-IFN-H6 eluted with 50mM Tris HCl,500mM NaCl,10% glycerol, 500mM imidazole, pH7.4 buffer and further purified by HiPrep 26/10 desalting column, replaced with 50mM phosphate, 150mM NaCl,pH 7.4 solution and stored at-80 ℃. IFN-LPETGGH6 purification and purity were assessed by SDS-PAGE gel electrophoresis. The protein concentration was determined by NanoDrop 2000.
1.2 construction and expression of transpeptidase A
The transpeptidase A (Srt A) used in this example was a non-mutated original protein, expressed by cloning according to standard protocols. Samples were sequenced by the gold-only company (GeneWiz). The sequencing results were:
ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGCAAGCTAAACCTCAAATTCCGAAAGATAAATCGAAAGTGGCAGGCTATATTGAAATTCCAGATGCTGATATTAAAGAACCAGTATATCCAGGACCAGCAACACCTGAACAATTAAATAGAGGTGTAAGCTTTGCAGAAGAAAATGAATCACTAGATGATCAAAATATTTCAATTGCAGGACACACTTTCATTGACCGTCCGAACTATCAATTTACAAATCTTAAAGCAGCCAAAAAAGGTAGTATGGTGTACTTTAAAGTTGGTAATGAAACACGTAAGTATAAAATGACAAGTATAAGAGATGTTAAGCCTACAGATGTAGGAGTTCTAGATGAACAAAAAGGTAAAGATAAACAATTAACATTAATTACTTGTGATGATTACAATGAAAAGACAGGCGTTTGGGAAAAACGTAAAATCTTTGTAGCTACAGAAGTCAAA。
the corresponding protein sequences are:
MGSSHHHHHHSSGLVPRGSHMQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGHTFIDRPNYQFTNLKAAKKGSMVYFKVGNETRKYKMTSIRDVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATEVKHHHH。
the Srt a used in this example is all Srt a purified in this batch. Purification was performed using an AKTA protein purifier, the method being as described. After purification of the protein, the concentration was calibrated to 8.5 mg/ml by the Nanodrop 2000UV/Vis method, and the yield per liter of bacteria was 200 mg. And (5) after purification, freezing and preserving in the solution.
2. Interferon-initiator preparation
An ATRP initiator can be attached to the C-terminus of IFN for subsequent polymerization by recognizing the C-terminal near sequence LPETG on the interferon by the transpeptidation ability of Srt a and cleaving, followed by attachment of a triglycine atom transfer radical polymerization initiator (AEBM). The AEBM has the chemical structural formula:
the specific flow is as follows:
100 mu M interferon solution and 100 mu M Srt A solution, the solvent is 50MM Tris,150mM NaCl,pH 7.4 buffer solution, 10mL and 5mL of the above solution are respectively mixed uniformly, and 10.6mg AEBM and 0.15ml 500mM CaCl are added 2 The solution was reacted at 25℃for 16 hours. After the reaction, 4 volumes of 20mM Tris, pH7.4 buffer was added for dilution. The diluted liquid was passed through an akta system equipped with an anion exchange column to which the desired interferon initiator was bound, eluted with a buffer PBS (150mM NaCl,pH 7.4) containing a high concentration of sodium chloride, and the resulting dryThe interferon initiator (IFN-Br) is frozen in solution at-80 ℃ for later use.
3. Preparation of interferon-glucose-containing polymer conjugates
5mL of PBS solution containing IFN-Br (0.5. Mu. Mol) and 4mL of PBS solution containing GluMA and OEGMA monomers were added to the Schlenk reaction tube. The molar ratios of GluMA, OEGMA and IFN were 0:750:1, 118:668:1, 211:635:1, 289:536:1, 492:492:1 and 1400:0:1, respectively, to give interferon-glucose-containing polymer conjugate conjugates with different glucose levels (glucose levels in the resulting conjugates were 0, 18%, 28%, 38%, 56% and 100% in order). High-purity nitrogen is introduced into the reaction tube and bubbling is carried out for 15 minutes at a constant speed. 10. Mu. Mol of CuCl in 1mL of PBS 2 10. Mu. Mol of CuCl and 40. Mu. Mol of 1,1,4,7,10,10-Hexamethyltriethylenetetramine (HMTETA) were added to a 10ml of a grind tube, and after bubbling high purity nitrogen gas at constant speed for 15 minutes, the solution was transferred to protein-Br solution through a bi-directional solvent transfer needle. After stirring and reacting for 4 hours at 4 ℃, air is introduced to quench the reaction to obtain the IFN-polymer conjugate. In situ ATRP polymerization of the interferons in this example all employed similar reaction conditions.
4. Characterization of physicochemical Properties of Interferon-glucose-containing Polymer conjugates
4.1 Gel Permeation Chromatography (GPC)
IFN-polymer conjugates (1 mg/mL) and proteinase K (0.5 mg/mL) were combined in digestion buffer (50 mM Tris-HCl,2mM CaCl) prior to GPC analysis of molecular weights of POEGA and glucose-containing polymer 2 ) pH7.4 was left at 45℃for 12 hours. The molecular weight of the polymer residue was determined by GPC (waters 1510) equipped with Asahipak GS-520HQ column (Shodex). Elution buffer (50 mM Tris-HCl,150mM NaCl,pH 7.4) was introduced at a flow rate of 0.5 mL/min. Calibration curves were created by running PEG standards with different molecular weights on a GPC system. The gel permeation chromatogram of the interferon-glucose-containing polymer conjugate is shown in FIG. 1.
4.2 Dynamic Light Scattering (DLS)
The sample was filtered (0.22 μm pore size, millipore corp.) prior to analysis. The data were analyzed using a Zetasizer software 6.32 using Malvern Zetasizer Nano-zs90 operating at a laser wavelength of 633nm, a scattering angle of 90℃and a temperature of 20 ℃. The particle size of the interferon-glucose-containing polymer conjugate obtained by dynamic light scattering detection is shown in FIG. 2.
4.3 proton Nuclear magnetic resonance (1H NMR) Spectroscopy
The IFN-polymer conjugate was digested with POEGMA and the sugar polymer were dialyzed several times against distilled water and then lyophilized. Dissolving the lyophilized powder of the obtained polymer in D 2 O, and recording them on a JEOL ECX-400 MHz spectrometer 1 H NMR spectrum. The nuclear magnetic pattern of the interferon-glucose-containing polymer conjugate is shown in FIG. 3.
4.4 Circular Dichroism (CD)
Protein and conjugate sample concentrations in 10mM PB were adjusted to 0.15mg/mL prior to CD analysis. CD spectra of IFN and IFN-polymer conjugates were recorded at 187nm to 260nm on a Pistar p-180 (Applied Photophysics Ltd) instrument. The circular dichroism spectrum of the interferon-glucose containing polymer conjugate is shown in FIG. 4.
4.5 agarose gel electrophoresis
Samples of each protein and conjugate were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 20. Mu.L of the sample with a protein concentration of 0.2mg/mL was taken, 5. Mu.L of 5 Xlaemmli sample buffer containing 5% beta-mercaptoethanol was added, after mixing, the sample was loaded into SDS-PAGE gel loaded with 10. Mu.L to a pre-prepared gel content of 8-12%, a vertical electrophoresis tank was subjected to 85V voltage for about 1.5h, protein Maker was developed sufficiently, and then the operation was stopped, after the gel was removed and stained with Coomassie brilliant blue dye for 10min, the bands were observed after full decolorization in a decolorization solution.
The gel electrophoresis pattern of the interferon-glucose-containing polymer conjugate is shown in FIG. 5.
5. In vitro biological Properties of Interferon-glucose-containing Polymer conjugates
5.1Daudi B cell proliferation inhibition assay
Daudi B cells were cultured in RPMI-1640 medium containing 15% (v/v) Fetal Bovine Serum (FBS) and 1% (v/v) penicillin/streptomycin. mu.L of medium containing 7500 cells and 50. Mu.L of medium of diluted sample (2, 4, 10, 20, 40, 100, 200, 1000, 2000, 20000pg IFN equivalent/mL for IFN and IFN-polymer conjugate, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300pM for POEGA and sugar polymer) was added to a 96-well plate and mixed. Wells using empty medium and medium containing 7500 cells were used as background and control, defined as 0% and 100% cell viability, respectively. After 96 hours of incubation, cell proliferation was measured according to the cell proliferation assay kit (Promega) MTS. Data fitting and IC50 calculation were analyzed by GraphPad Prism 8.0 software and expressed as mean ± standard deviation.
The antiproliferative activity of interferon-glucose containing polymer conjugates on Daudi cells is shown in figure 6.
5.2SKOV-3 cell proliferation inhibition assay
The SKOV-3 cancer cell line was grown in 1640 containing 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin, respectively. mu.L of cells (5000 cells per well) and 50. Mu.L of diluted IFN and IFN-polymer conjugate samples (20, 40, 100, 200, 400, 1000, 2000, 10000, 20000, 200000pg IFN equivalents/mL) were added to 96-well plates and then incubated for 96 hours at 37 ℃. Wells using empty medium and medium containing 7500 cells were used as background and control, defined as 0% and 100% cell viability, respectively. Proliferation of cells was determined according to the MTS method of cell proliferation assay kit (Promega).
5.3 cell internalization
SKOV-3 and a2780 cancer cell lines were cultured as described above. For confocal microscopy, cells were plated at 5X 10 per petri dish 4 The density of individual cells was seeded in 35mm glass bottom dishes (NEST) and incubated overnight, followed by 6 hours with FITC-labeled IFN-polymer conjugates (2.5. Mu.M). Incubation was performed under three conditions: serum-containing medium (1640 or DMEM medium), pure medium (1640 or DMEM medium without serum), and serum-containing medium containing BYA87 (GLUT 1 inhibitor). After 3 washes with PBS, cells were fixed with 4% (w/v) cold paraformaldehyde for 10min at room temperature. Cells were then stained with a mixed solution of 5 μg/mL wheat germ lectin (WGA) Alexa Fluor 594 (for membrane staining) and 2.5 μg/mL DAPI (for nuclear staining) for 10 minutes. Washing with PBSAfter 3 times, the cells were imaged with a confocal microscope (Zeiss 780).
For flow cytometry analysis, cells were assayed at 2×10 per well 5 The density of individual cells was seeded in 12-well plates and incubated overnight, followed by incubation with FITC-labeled IFN and IFN-polymer conjugate (2.5 μm) for 6 hours. Incubation was performed under three conditions: serum-containing medium, pure medium and serum-containing medium containing BYA 874. After 3 washes with PBS, cells were collected for flow cytometry analysis.
The results are shown in FIGS. 7-18
Wherein, the flow chart and the confocal laser microscopy chart of the cell-entering ability of the interferon-glucose-containing polymer conjugate in SKOV-3 cells under the condition of containing serum culture medium are shown in FIG. 7 and FIG. 8.
Flow charts and confocal laser microscopy of the ability of interferon-glucose containing polymer conjugates to enter cells in SKOV-3 cells in the absence of serum medium are shown in fig. 9 and 10.
Flow charts and confocal laser microscopy of the ability of interferon-glucose containing polymer conjugates to enter cells in SKOV-3 cells in the presence of serum medium and GLUT1 inhibitor are shown in fig. 11 and 12.
Flow charts and confocal laser microscopy of the ability of interferon-glucose containing polymer conjugates to enter cells in a2780 cells in serum containing medium are shown in fig. 13 and 14.
Flow charts and confocal laser microscopy of the ability of interferon-glucose containing polymer conjugates to enter cells in a2780 cells without serum medium are shown in fig. 15 and 16.
Flow charts and confocal laser microscopy of the ability of interferon-glucose containing polymer conjugates to enter cells in A2780 cells in the presence of serum medium and GLUT1 inhibitor are shown in FIGS. 17 and 18.
5.4 complement activation assay
Blood was collected from female BALB/c nude mice (8 weeks old) through the posterior orbit. After the blood had coagulated at room temperature, the sample was centrifuged at 4500g for 15min at 4℃and serum was collected and mixed. 10. Mu.L of IFN, IFN-polymer conjugate (10. Mu.M) and micrococcus (1 mg/mL, positive control) dissolved in PBS solution were mixed with 40. Mu.L of fresh serum and incubated for 1 hour at 37 ℃. The concentration of complement activation product C3a in the solution was detected using a C3a ELISA kit (cloud cloning).
The results of interferon-glucose containing polymer conjugate activating the C3a level of the serum complement system are shown in figure 19.
6. Biological characteristics in vivo
6.1 pharmacokinetics
Female BALB/c nude mice (8 weeks old) received intravenous injection of IFN and IFN-polymer conjugate at an IFN equivalent dose of 1mg/kg body weight (n=3). Blood samples (15 μl) were collected from tail veins at selected time points (1, 5, 15, 30 minutes, 1, 2, 4, 8, 24, 48, 72, and 96 hours). The samples were centrifuged at 4500g for 15min, and plasma was collected and stored at-80 ℃. The concentration of IFN in plasma was measured using a human IFN-. Alpha.2 ELISA kit (PBL Interferon Source). Pharmacokinetic parameters were generated by fitting the data to a two-compartment model by DAS 2.0 software.
6.2 biodistribution
SKOV-cancer cell lines were cultured in vitro and collected as described above. By 5X 10 suspension in serum-free medium 6 SKOV-3 cells (0.1 mL) were inoculated subcutaneously into female BALB/c nude mice (6 weeks old). Mice with cancer (-100-200 mm) 3 ) An IFN or IFN-polymer conjugate was received i.v. at an IFN equivalent dose of 1mg/kg body weight (n=3). Mice were sacrificed at 96 hours using carbon dioxide and major organs or tissues were collected. The collected tissues and organs were then weighed, homogenized and suspended in a corresponding amount of 10mM PBS extraction buffer containing 1mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 1mM PMSF, phosphatase and protease inhibitor cocktail (1:100 dilution). The concentration of IFN in the sample was calculated as described previously. Data are expressed as IFN equivalent per gram of tissue (pg/g tissue).
6.3 in vivo and ex vivo fluorescence imaging
SKOV-3 cells were cultured and implanted into female BALB/c nude mice (6 weeks old) as described above. Tumor-bearing mice (-100-200 mm) after tumor cell inoculation 3 ) At 1An IFN equivalent dose of mg/kg body weight (n=3) was received intravenous injection of CY 7-labeled IFN and IFN-polymer conjugate. Fluorescence images were taken 6, 24, 48, 72 and 96 hours after injection using an in vivo imaging system (IVIS, perkinElmer). Mice were sacrificed 96 hours after injection. The major organs and tissues were then collected and imaged (excitation and emission wavelengths: 745/800 nm).
FIG. 20 is a graph showing the concentration of interferon-glucose-containing polymer conjugate in mice versus time.
6.4 in vivo anti-tumor efficacy
As previously described, the SKOV-3 cancer cell line and the A2780 cell line were cultured and individually implanted into female BALB/c nude mice (6 weeks old). When the SKOV-3 tumor (or A2780 tumor) volume reached 30mm on day 17 and day 8, respectively, after implantation 3 And 50mm 3 At this time, animals were randomly divided into 8 test groups (n=6 to 8 per group) and 3 intravenous interferon and interferon polymer conjugate equivalent doses of 1mg/kg body weight per 6 days were obtained. Tumor volume and body weight of mice were measured every three days. Tumor volume was determined using the formula ((width x width) x length)/2. Mice were sacrificed if their tumor size was 7 times greater than the initial volume or their weight loss was greater than 15% of their weight.
FIG. 21 is a graph of tumor volume change in a mouse SKOV-3 subcutaneous tumor model treatment with an interferon-glucose containing polymer conjugate.
FIG. 22 is a graphical representation of the survival of mice in the treatment of a murine SKOV-3 subcutaneous tumor model with an interferon-glucose containing polymer conjugate.
FIG. 23 is a graph showing tumor volume change of interferon-glucose containing polymer conjugate in treatment of mouse A2780 subcutaneous tumor model.
FIG. 24 is a graphical representation of mouse survival curves for interferon-glucose containing polymer conjugates in the treatment of mouse A2780 subcutaneous tumor model.
6.5 systemic toxicity
Histomorphology, clinical biochemistry and hematology were examined to assess in vivo toxicity of IFN-polymer conjugates. Mice were sacrificed on day 45 and day 21 after the first treatment in SKOV3 tumor model, respectively. The major organs were collected and fixed with 4% neutral paraformaldehyde, then embedded in paraffin to give 5 μm thick sections, all stained with hematoxylin-eosin (H & E) and imaged with 3DHISTECH panoramic scan. Blood was collected from mice via the posterior orbit and tested to obtain parameters of clinical biochemistry and clinical hematology.
FIG. 25 is a diagram of pathological sections of a mouse tumor after the end of treatment of a mouse SKOV-3 subcutaneous tumor model with an interferon-glucose-containing polymer conjugate.
FIG. 26 is a diagram of pathological sections of a mouse tumor after the end of treatment of a mouse A2780 subcutaneous tumor model with an interferon-glucose-containing polymer conjugate.
FIG. 27 is a schematic representation of pathological tissue sections of heart, liver, spleen, lung and kidney after completion of treatment of the interferon-glucose containing polymer conjugate in a mouse SKOV-3 subcutaneous tumor model.
FIG. 28 is a schematic representation of pathological tissue sections of heart, liver, spleen, lung and kidney after completion of treatment of the interferon-glucose containing polymer conjugate in mouse A2780 subcutaneous tumor model.
FIG. 29 is a graph showing the results of hematology and biochemistry index analyses of interferon-glucose-containing polymer conjugates after the end of treatment in a mouse SKOV-3 subcutaneous tumor model.
FIG. 30 is a graph showing the results of analysis of the hematology and biochemistry indices of interferon-glucose-containing polymer conjugates after the end of treatment in mouse A2780 subcutaneous tumor model.
Example 2
This example uses 2, 6-pyridine dicarboxaldehyde and hydroxylamine functionalized initiator to attach an atom transfer radical polymerization initiator to the N-terminus of interferon and polymerize oligomeric polyethylene glycol and 6-O-methacryloyl- α -D-glucose in situ to construct an interferon-poly oligomeric polyethylene glycol methyl ether-poly 6 α -D-glucose conjugate, the resulting conjugate was characterized for physicochemical properties in vitro and for evaluation of pharmaceutical properties in vivo and in vitro.
The specific flow is as follows:
1. construction and expression of proteins
1.1 construction and expression of Interferon
From the following using PCR techniqueThe IFN encoding sequence was amplified in the vector and inserted into the pET-25b (+) vector through the Nde I/Eco RI cleavage site. The specific recognition sequence LPETG and the 6 XHis tag of SrtA are connected to the C-terminal of the IFN gene sequence, and the final protein sequence is as follows:
GGGCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGSGGGGSLPETGGHHHHHH。
after verification by DNA sequencing, the constructed plasmid encoding G3-IFN-H6 was transformed into E.coli strain Rosetta-gami (DE 3) pLysS competent cells and cultured overnight at 37℃in ampicillin-resistant LB medium. Cultures were transferred to 1L of sterile ampicillin-resistant TB medium for continued culture, and when the OD600 of the bacterial suspension reached 0.5, IPTG was added to a final concentration of 500. Mu.M for induction, and overexpression was performed overnight at 18 ℃. Cells were harvested by centrifugation, resuspended in 50mM Tris HCl,150mM NaCl,pH 7.4 buffer, sonicated in an ice-water bath and the pellet removed by centrifugation at 14,000Xg for 10min, 2mL of 1% (w/v) Polyethylenimine (PEI) was added to the supernatant and mixed well and centrifuged again. The supernatant containing the soluble protein was purified by passing through a nickel affinity column and through the AKTA Purifier 10 system. The column was equilibrated with 50mM Tris HCl,500mM NaCl,10% glycerol, 25mM imidazole, pH7.4 buffer, the eluted heteroprotein was eluted with 50mM Tris HCl,500mM NaCl,10% glycerol, 50mM imidazole, pH7.4 buffer, and finally G3-IFN-H6 eluted with 50mM Tris HCl,500mM NaCl,10% glycerol, 500mM imidazole, pH7.4 buffer and further purified by HiPrep 26/10 desalting column, replaced with 50mM phosphate, 150mM NaCl,pH 7.4 solution and stored at-80 ℃. IFN-LPETGGH6 purification and purity were assessed by SDS-PAGE gel electrophoresis. The protein concentration was determined by NanoDrop 2000.
1.2 Interferon-initiator preparation
100. Mu.M of buffer solution with 50mM Tris,150mM NaCl,pH 7.4 as solvent, 200 times of 2, 6-pyridine dicarboxaldehyde was added and reacted at 25℃for 16 hours. After the reaction, the liquid was passed through an akta system equipped with a desalting column, unreacted 2, 6-pyridine dicarboxaldehyde was removed and the resulting CHO-IFN was replaced in a buffer of 50mM PB,150mM NaCl,pH 5.5, then 30 times of hydroxylamine initiator was added to the solution, after the reaction, the liquid was passed through an akta system equipped with a desalting column, the unreacted hydroxylamine initiator was removed and the desired interferon initiator Br-IFN was obtained, and the resulting interferon initiator was stored in a solution at-80 ℃ for use.
1.3 preparation of Interferon-glucose-containing Polymer conjugates
5mL of PBS solution containing Br-IFN (0.5. Mu. Mol) and 4mL of PBS solution containing GluMA and OEGMA monomers were added to the Schlenk reaction tube. The molar ratios of GluMA, OEGMA and IFN were 0:750:1, 118:668:1, 211:635:1, 289:536:1, 492:492:1 and 1400:0:1, respectively, to give interferon-glucose-containing polymer conjugate conjugates with different glucose levels. High-purity nitrogen is introduced into the reaction tube and bubbling is carried out for 15 minutes at a constant speed. 10. Mu. Mol of CuCl2, 10. Mu. Mol of CuCl and 40. Mu. Mol of 1,1,4,7,10,10-Hexamethyltriethylenetetramine (HMTETA) in 1mL of PBS were added to a 10mL of a grind tube, and after high-purity nitrogen gas was bubbled at a constant rate for 15 minutes, the solution was transferred to a protein-Br solution through a bi-directional solvent transfer needle. After stirring and reacting for 4 hours at 4 ℃, air is introduced to quench the reaction. After the reaction, the liquid passes through an akta system provided with a desalting column to remove unreacted small molecular impurities and interferon, so as to obtain the interferon-glucose-containing polymer conjugate with different glucose contents.
Example 3
In this example, an atom transfer radical polymerization initiator was attached to the surface of atilizumab using a dibromomaleimide functionalized initiator, and oligomeric polyethylene glycol and 6-O-methacryloyl- α -D-glucose were polymerized in situ to construct an interferon-poly (oligopolyethylene glycol) methyl ether-poly (6α -D-glucose) conjugate, which was characterized for physical and chemical properties in vitro and for evaluation of pharmaceutical properties in vivo and in vitro.
The method specifically comprises the following steps:
1. preparation of Abilib-initiator
Ab Li Zhushan antibody buffer was replaced with a desalting column (HiTrap desalting column 5mL, GE) to a final concentration of 4mg/mL in 1mL Tris-HCl (20 mM,150mM sodium chloride, 5mM EDTA, pH 7.4). TCEP (28 μl,5 mM) was added to the solution and incubated at 37 ℃ for 2 hours to produce atenolizumab-SH. The atilizumab-SH was replaced with desalting column (HiTrap desalting column 5mL, GE) to Tris solution (28. Mu.L, 50mM sodium chloride, pH 7.4), and DBEB (28. Mu.L, 50mM DMF solution) was added to the atilizumab-SH solution at 25 ℃. Immediately after addition of DBEB, the solution turned pale yellow. After 16 hours, the reaction mixture was purified with a desalting column to yield atilizumab-Br.
2. Preparation of Abilizumab-glucose-containing polymer conjugates
5mL of PBS solution containing Br-atilizumab (0.125. Mu. Mol) and 4mL of PBS solution containing GluMA and OEGMA monomers were added to a Schlenk reaction tube at molar ratios of GluMA, OEGMA and IFN of 0:750:1, 118:668:1, 211:635:1, 289:536:1, 492:492:1 and 1400:0:1, respectively, to give atilizumab-polymer conjugates with different glucose contents. High-purity nitrogen is introduced into the reaction tube and bubbling is carried out for 15 minutes at a constant speed. 10. Mu. Mol of CuCl2, 10. Mu. Mol of CuCl and 40. Mu. Mol of 1,1,4,7,10,10-Hexamethyltriethylenetetramine (HMTETA) in 1mL of PBS were added to a 10mL of a grind tube, and after high-purity nitrogen gas was bubbled at a constant rate for 15 minutes, the solution was transferred to a protein-Br solution through a bi-directional solvent transfer needle. After stirring and reacting for 4 hours at 4 ℃, air is introduced to quench the reaction. After the reaction, the liquid passes through an akta system provided with a desalting column to remove unreacted small molecular impurities, so as to obtain the atilizumab-glucose-containing polymer conjugate with different glucose contents.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A protein-glucose-containing polymer conjugate comprising:
a protein and a glucose-containing polymer and a biocompatible molecule coupled to the protein;
the glucose content of the protein-glucose-containing polymer conjugate is 5-50%.
2. The conjugate of claim 1, wherein the protein-glucose containing polymer conjugate has a glucose content of 10 to 30%.
3. The protein-glucose-containing polymer conjugate of claim 1 or 2, wherein the biocompatible molecule comprises an oligoethylene glycol methacrylate and/or a zwitterionic monomer; and/or, the protein is a therapeutic protein.
4. A conjugate according to any one of claims 1 to 3, wherein the protein comprises:
insulin, monoclonal antibodies, blood factors, colony stimulating factors, growth hormone, interleukins, growth factors, therapeutic vaccines, calcitonin, tumor necrosis factor or enzymes.
5. The conjugate of any one of claims 1-4, wherein the glucose-containing polymer is composed of a polymer comprising alpha-D-glucose, beta-D-glucose, gluconic acid,
The polymer is polymerized by any compound monomer, wherein n is a positive integer of 1-10.
6. A method of preparing a protein-glucose containing polymer conjugate comprising:
forming a functional molecule on the protein; copolymerizing and coupling a glucose-containing compound monomer, a biocompatible molecular monomer, and the protein through the functional molecule; or (b)
Copolymerizing said monomeric glucose-containing compound and said monomeric biocompatible molecule with said functionalized functional molecule to obtain a functionalized polymer, forming said functionalized polymer on said protein;
the functional molecule includes a free radical polymerization initiator and/or a chain transfer agent.
7. The method of claim 6, wherein the molar ratio of the protein, the biocompatible molecule, and the glucose containing polymer is 1: (100-300): (400-700).
8. The method of claim 6 or 7, wherein the free radical polymerization initiator comprises any one or more of the following:
and/or the number of the groups of groups,
the chain transfer agent comprises any one or more of the following:
9. use of a conjugate according to any one of claims 1 to 5, or a conjugate prepared by a method according to any one of claims 6 to 8, in targeted therapy of a tumor.
10. A method of preparing a medicament for targeting a tumor, comprising:
the conjugate prepared by adopting the conjugate as claimed in any one of claims 1 to 5 or the conjugate prepared by the preparation method as claimed in any one of claims 6 to 8 as a raw material.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310192332.3A CN116440284A (en) | 2023-03-02 | 2023-03-02 | Protein-glucose-containing polymer conjugate and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310192332.3A CN116440284A (en) | 2023-03-02 | 2023-03-02 | Protein-glucose-containing polymer conjugate and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116440284A true CN116440284A (en) | 2023-07-18 |
Family
ID=87129190
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310192332.3A Pending CN116440284A (en) | 2023-03-02 | 2023-03-02 | Protein-glucose-containing polymer conjugate and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116440284A (en) |
-
2023
- 2023-03-02 CN CN202310192332.3A patent/CN116440284A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN116948006A (en) | Interleukin-2polypeptide conjugate and use thereof | |
| US20040171044A1 (en) | Delivery of substances to cells | |
| JP2000506865A (en) | Targeted delivery of genes encoding interferons | |
| US6339139B1 (en) | Receptor-mediated gene transfer system for targeting tumor gene therapy | |
| CN109265512B (en) | Preparation method of protein conjugate based on pyridine dicarbaldehyde | |
| JP2022512746A (en) | Interleukin-10 polypeptide complex, its dimer, and their use | |
| EP3539570B1 (en) | Pegylated endostatin analogue and application thereof | |
| CN102441175A (en) | Hyman serum albumin-siRna nano-sized carrier system | |
| CN110179994A (en) | A kind of temperature and enzyme dual responsiveness protein high molecular conjugate and the preparation method and application thereof | |
| CN108578709A (en) | Thermo-sensitive long-acting slow-releasing medicine carrier and its application | |
| CN113166221A (en) | Process for preparing therapeutically active aldesleukin highly stable in liquid pharmaceutical compositions | |
| MXPA01012802A (en) | Copolymers for the transfer of nucleic acids to the cell. | |
| CN111777667A (en) | Small peptide and application thereof in preparation of immunoregulation medicine | |
| CN116440284A (en) | Protein-glucose-containing polymer conjugate and preparation method and application thereof | |
| CN108383912A (en) | Artificial fusion protein and application thereof | |
| CN108265044B (en) | Arginine deiminase modified by polyethylene glycol at fixed point, preparation method and application thereof | |
| CN110101868B (en) | Environment stimulus responsive protein macromolecular conjugate self-assembly and preparation method and application thereof | |
| CN114452266B (en) | Nucleic acid drug delivery system based on recombinant ribosomal protein and preparation method and application thereof | |
| EP1541135A1 (en) | Hollow nanoparticle having modified cysteine residue and drug with the use thereof | |
| US20060189558A1 (en) | Delivery of substances to cells | |
| CN117547618A (en) | Ferritin nano cage carrier with small nucleic acid medicine loaded in inner cavity and application thereof | |
| JP3938954B2 (en) | Novel graft copolymer, drug using the same, and method for incorporating a drug into specific cells using the same | |
| CN102010461A (en) | Alpha spiral cation polypeptide molecule and preparation method and application thereof | |
| CN112225820A (en) | Recombinant human serum albumin-collagen binding domain fusion protein of tumor specific targeting matrix and application | |
| CN1295338C (en) | Polytheneimine transgened carrier of targeted fibroblast growth factor receptor |
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 |