CN113350512B - PEG 2,n Lipid derivative modified nano-carrier, preparation method and application - Google Patents
PEG 2,n Lipid derivative modified nano-carrier, preparation method and application Download PDFInfo
- Publication number
- CN113350512B CN113350512B CN202110629173.XA CN202110629173A CN113350512B CN 113350512 B CN113350512 B CN 113350512B CN 202110629173 A CN202110629173 A CN 202110629173A CN 113350512 B CN113350512 B CN 113350512B
- Authority
- CN
- China
- Prior art keywords
- peg
- dspe
- injection
- carrier
- molecular weight
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000002539 nanocarrier Substances 0.000 title claims abstract description 53
- 238000002360 preparation method Methods 0.000 title claims abstract description 29
- 150000002632 lipids Chemical class 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 12
- 229920001223 polyethylene glycol Polymers 0.000 claims description 398
- 239000002502 liposome Substances 0.000 claims description 88
- 239000000839 emulsion Substances 0.000 claims description 87
- 150000003904 phospholipids Chemical class 0.000 claims description 71
- 239000002202 Polyethylene glycol Substances 0.000 claims description 21
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 238000005303 weighing Methods 0.000 claims description 17
- 238000003756 stirring Methods 0.000 claims description 13
- 229920001427 mPEG Polymers 0.000 claims description 12
- 239000012528 membrane Substances 0.000 claims description 12
- 235000012000 cholesterol Nutrition 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- 239000008227 sterile water for injection Substances 0.000 claims description 9
- 239000003995 emulsifying agent Substances 0.000 claims description 8
- 239000012071 phase Substances 0.000 claims description 8
- 238000001132 ultrasonic dispersion Methods 0.000 claims description 6
- 210000003022 colostrum Anatomy 0.000 claims description 5
- 235000021277 colostrum Nutrition 0.000 claims description 5
- 150000001875 compounds Chemical class 0.000 claims description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 4
- 239000004472 Lysine Substances 0.000 claims description 4
- 239000012141 concentrate Substances 0.000 claims description 4
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 4
- JQWAHKMIYCERGA-UHFFFAOYSA-N (2-nonanoyloxy-3-octadeca-9,12-dienoyloxypropoxy)-[2-(trimethylazaniumyl)ethyl]phosphinate Chemical group CCCCCCCCC(=O)OC(COP([O-])(=O)CC[N+](C)(C)C)COC(=O)CCCCCCCC=CCC=CCCCCC JQWAHKMIYCERGA-UHFFFAOYSA-N 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 125000003277 amino group Chemical group 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 50
- 229940079593 drug Drugs 0.000 abstract description 45
- 239000000463 material Substances 0.000 abstract description 14
- 230000006320 pegylation Effects 0.000 abstract description 11
- 230000004087 circulation Effects 0.000 abstract description 9
- 230000009466 transformation Effects 0.000 abstract description 4
- 230000007547 defect Effects 0.000 abstract description 3
- 230000036571 hydration Effects 0.000 abstract description 2
- 238000006703 hydration reaction Methods 0.000 abstract description 2
- 238000002347 injection Methods 0.000 description 119
- 239000007924 injection Substances 0.000 description 119
- 239000004698 Polyethylene Substances 0.000 description 52
- 238000010253 intravenous injection Methods 0.000 description 38
- 210000004369 blood Anatomy 0.000 description 32
- 239000008280 blood Substances 0.000 description 32
- 239000000203 mixture Substances 0.000 description 26
- 239000000243 solution Substances 0.000 description 25
- 230000000694 effects Effects 0.000 description 24
- 210000003462 vein Anatomy 0.000 description 24
- 241000700157 Rattus norvegicus Species 0.000 description 22
- 238000009472 formulation Methods 0.000 description 16
- 230000004048 modification Effects 0.000 description 15
- 238000012986 modification Methods 0.000 description 15
- 238000003556 assay Methods 0.000 description 13
- 238000010171 animal model Methods 0.000 description 12
- 238000005406 washing Methods 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 9
- 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 description 9
- 239000008355 dextrose injection Substances 0.000 description 9
- 229940088598 enzyme Drugs 0.000 description 9
- 239000008103 glucose Substances 0.000 description 9
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 description 8
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 8
- 238000004140 cleaning Methods 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 239000011248 coating agent Substances 0.000 description 6
- 238000000576 coating method Methods 0.000 description 6
- 230000007423 decrease Effects 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- ACTIUHUUMQJHFO-UHFFFAOYSA-N Coenzym Q10 Natural products COC1=C(OC)C(=O)C(CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UHFFFAOYSA-N 0.000 description 5
- 241000700159 Rattus Species 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 235000017471 coenzyme Q10 Nutrition 0.000 description 5
- 229940110767 coenzyme Q10 Drugs 0.000 description 5
- ACTIUHUUMQJHFO-UPTCCGCDSA-N coenzyme Q10 Chemical compound COC1=C(OC)C(=O)C(C\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UPTCCGCDSA-N 0.000 description 5
- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 description 5
- 229960004768 irinotecan Drugs 0.000 description 5
- 238000002372 labelling Methods 0.000 description 5
- 150000003839 salts Chemical class 0.000 description 5
- -1 sphingomyelin Chemical compound 0.000 description 5
- 210000000952 spleen Anatomy 0.000 description 5
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 description 4
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 4
- 206010020751 Hypersensitivity Diseases 0.000 description 4
- 210000003719 b-lymphocyte Anatomy 0.000 description 4
- 239000000337 buffer salt Substances 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000005538 encapsulation Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- LVNGJLRDBYCPGB-UHFFFAOYSA-N 1,2-distearoylphosphatidylethanolamine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(COP([O-])(=O)OCC[NH3+])OC(=O)CCCCCCCCCCCCCCCCC LVNGJLRDBYCPGB-UHFFFAOYSA-N 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000013270 controlled release Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000693 micelle Substances 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- SRLOHQKOADWDBV-NRONOFSHSA-M sodium;[(2r)-2,3-di(octadecanoyloxy)propyl] 2-(2-methoxyethoxycarbonylamino)ethyl phosphate Chemical compound [Na+].CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCCNC(=O)OCCOC)OC(=O)CCCCCCCCCCCCCCCCC SRLOHQKOADWDBV-NRONOFSHSA-M 0.000 description 3
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 2
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 208000026935 allergic disease Diseases 0.000 description 2
- 230000007815 allergy Effects 0.000 description 2
- 230000006399 behavior Effects 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 229960004679 doxorubicin Drugs 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 102000035118 modified proteins Human genes 0.000 description 2
- 108091005573 modified proteins Proteins 0.000 description 2
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 2
- 150000003905 phosphatidylinositols Chemical class 0.000 description 2
- 229920000573 polyethylene Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000002047 solid lipid nanoparticle Substances 0.000 description 2
- 239000012089 stop solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- CITHEXJVPOWHKC-UUWRZZSWSA-N 1,2-di-O-myristoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCC CITHEXJVPOWHKC-UUWRZZSWSA-N 0.000 description 1
- MLKLDGSYMHFAOC-AREMUKBSSA-N 1,2-dicapryl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCC MLKLDGSYMHFAOC-AREMUKBSSA-N 0.000 description 1
- IBUKXRINTKQBRQ-KCKFLZCVSA-N 1,2-dihexadecanoyl-sn-glycero-3-phospho-D-myo-inositol Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@@H](OC(=O)CCCCCCCCCCCCCCC)COP(O)(=O)O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O IBUKXRINTKQBRQ-KCKFLZCVSA-N 0.000 description 1
- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 description 1
- SLKDGVPOSSLUAI-PGUFJCEWSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine zwitterion Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)CCCCCCCCCCCCCCC SLKDGVPOSSLUAI-PGUFJCEWSA-N 0.000 description 1
- SNKAWJBJQDLSFF-NVKMUCNASA-N 1,2-dioleoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC SNKAWJBJQDLSFF-NVKMUCNASA-N 0.000 description 1
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 description 1
- BIABMEZBCHDPBV-MPQUPPDSSA-N 1,2-palmitoyl-sn-glycero-3-phospho-(1'-sn-glycerol) Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@@H](O)CO)OC(=O)CCCCCCCCCCCCCCC BIABMEZBCHDPBV-MPQUPPDSSA-N 0.000 description 1
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 1
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 1
- IJFVSSZAOYLHEE-UHFFFAOYSA-N 2,3-di(dodecanoyloxy)propyl 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CCCCCCCCCCCC(=O)OCC(COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCC IJFVSSZAOYLHEE-UHFFFAOYSA-N 0.000 description 1
- NEZDNQCXEZDCBI-UHFFFAOYSA-N 2-azaniumylethyl 2,3-di(tetradecanoyloxy)propyl phosphate Chemical compound CCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCCCCCCCC NEZDNQCXEZDCBI-UHFFFAOYSA-N 0.000 description 1
- ZLGYVWRJIZPQMM-HHHXNRCGSA-N 2-azaniumylethyl [(2r)-2,3-di(dodecanoyloxy)propyl] phosphate Chemical compound CCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)CCCCCCCCCCC ZLGYVWRJIZPQMM-HHHXNRCGSA-N 0.000 description 1
- VHSHLMUCYSAUQU-UHFFFAOYSA-N 2-hydroxypropyl methacrylate Chemical compound CC(O)COC(=O)C(C)=C VHSHLMUCYSAUQU-UHFFFAOYSA-N 0.000 description 1
- AOJJSUZBOXZQNB-VTZDEGQISA-N 4'-epidoxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-VTZDEGQISA-N 0.000 description 1
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 1
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 102000016918 Complement C3 Human genes 0.000 description 1
- 108010028780 Complement C3 Proteins 0.000 description 1
- SBJKKFFYIZUCET-JLAZNSOCSA-N Dehydro-L-ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(=O)C1=O SBJKKFFYIZUCET-JLAZNSOCSA-N 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- HTIJFSOGRVMCQR-UHFFFAOYSA-N Epirubicin Natural products COc1cccc2C(=O)c3c(O)c4CC(O)(CC(OC5CC(N)C(=O)C(C)O5)c4c(O)c3C(=O)c12)C(=O)CO HTIJFSOGRVMCQR-UHFFFAOYSA-N 0.000 description 1
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 1
- 241000282560 Macaca mulatta Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241001028048 Nicola Species 0.000 description 1
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- FVJZSBGHRPJMMA-IOLBBIBUSA-N PG(18:0/18:0) Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@@H](O)CO)OC(=O)CCCCCCCCCCCCCCCCC FVJZSBGHRPJMMA-IOLBBIBUSA-N 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 206010033553 Palmar-plantar erythrodysaesthesia syndrome Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108700023158 Phenylalanine ammonia-lyases Proteins 0.000 description 1
- 201000011252 Phenylketonuria Diseases 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 229940123237 Taxane Drugs 0.000 description 1
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 1
- FQZQXPXKJFOAGE-KICCZPNWSA-N [(2r)-3-[hydroxy-[(5r)-2,3,4,5,6-pentahydroxycyclohexyl]oxyphosphoryl]oxy-2-octadecanoyloxypropyl] octadecanoate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@@H](OC(=O)CCCCCCCCCCCCCCCCC)COP(O)(=O)OC1C(O)C(O)C(O)[C@@H](O)C1O FQZQXPXKJFOAGE-KICCZPNWSA-N 0.000 description 1
- PSVRFUPOQYJOOZ-MEYQAEQNSA-N [2-[(5z,8z,11z,14z)-icosa-5,8,11,14-tetraenoyl]oxy-3-octadecanoyloxypropyl] 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC PSVRFUPOQYJOOZ-MEYQAEQNSA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- LYBDVVBIMGTZMB-HVIJGSDCSA-N [3-[hydroxy-[(2s,3r,5s,6s)-2,3,4,5,6-pentahydroxycyclohexyl]oxyphosphoryl]oxy-2-tetradecanoyloxypropyl] tetradecanoate Chemical compound CCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCC)COP(O)(=O)OC1[C@@H](O)[C@@H](O)C(O)[C@@H](O)[C@@H]1O LYBDVVBIMGTZMB-HVIJGSDCSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000004154 complement system Effects 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- LHCZDUCPSRJDJT-UHFFFAOYSA-N dilauroyl phosphatidylglycerol Chemical compound CCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCCCCCC LHCZDUCPSRJDJT-UHFFFAOYSA-N 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 229960003724 dimyristoylphosphatidylcholine Drugs 0.000 description 1
- 229960005160 dimyristoylphosphatidylglycerol Drugs 0.000 description 1
- MWRBNPKJOOWZPW-CLFAGFIQSA-N dioleoyl phosphatidylethanolamine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-CLFAGFIQSA-N 0.000 description 1
- ZGSPNIOCEDOHGS-UHFFFAOYSA-L disodium [3-[2,3-di(octadeca-9,12-dienoyloxy)propoxy-oxidophosphoryl]oxy-2-hydroxypropyl] 2,3-di(octadeca-9,12-dienoyloxy)propyl phosphate Chemical compound [Na+].[Na+].CCCCCC=CCC=CCCCCCCCC(=O)OCC(OC(=O)CCCCCCCC=CCC=CCCCCC)COP([O-])(=O)OCC(O)COP([O-])(=O)OCC(OC(=O)CCCCCCCC=CCC=CCCCCC)COC(=O)CCCCCCCC=CCC=CCCCCC ZGSPNIOCEDOHGS-UHFFFAOYSA-L 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229940042317 doxorubicin liposome Drugs 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- 210000002969 egg yolk Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229960001904 epirubicin Drugs 0.000 description 1
- 229940049769 erwinia asparaginase Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 229940099578 hydrogenated soybean lecithin Drugs 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 230000009610 hypersensitivity Effects 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000008076 immune mechanism Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- KLEAIHJJLUAXIQ-JDRGBKBRSA-N irinotecan hydrochloride hydrate Chemical compound O.O.O.Cl.C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 KLEAIHJJLUAXIQ-JDRGBKBRSA-N 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 201000011649 lymphoblastic lymphoma Diseases 0.000 description 1
- 210000003826 marginal zone b cell Anatomy 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 229960001156 mitoxantrone Drugs 0.000 description 1
- KKZJGLLVHKMTCM-UHFFFAOYSA-N mitoxantrone Chemical compound O=C1C2=C(O)C=CC(O)=C2C(=O)C2=C1C(NCCNCCO)=CC=C2NCCNCCO KKZJGLLVHKMTCM-UHFFFAOYSA-N 0.000 description 1
- 210000000865 mononuclear phagocyte system Anatomy 0.000 description 1
- 125000001419 myristoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 229940042880 natural phospholipid Drugs 0.000 description 1
- 229940049964 oleate Drugs 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 229940048191 onivyde Drugs 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 229960001744 pegaspargase Drugs 0.000 description 1
- 108010001564 pegaspargase Proteins 0.000 description 1
- 229950011036 pegnivacogin Drugs 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 229920002939 poly(N,N-dimethylacrylamides) Polymers 0.000 description 1
- 229920006289 polycarbonate film Polymers 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- OLBCVFGFOZPWHH-UHFFFAOYSA-N propofol Chemical compound CC(C)C1=CC=CC(C(C)C)=C1O OLBCVFGFOZPWHH-UHFFFAOYSA-N 0.000 description 1
- 229960004134 propofol Drugs 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 229940083466 soybean lecithin Drugs 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- DKPFODGZWDEEBT-QFIAKTPHSA-N taxane Chemical class C([C@]1(C)CCC[C@@H](C)[C@H]1C1)C[C@H]2[C@H](C)CC[C@@H]1C2(C)C DKPFODGZWDEEBT-QFIAKTPHSA-N 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- 229940045997 vitamin a Drugs 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Abstract
PEG 2,n The invention provides a lipid derivative modified nano-carrier, a preparation method and application thereof, belonging to the technical field of medicines, and the method for eliminating ABC phenomenon caused by PEG nano-carrier, wherein the modified material used by the nano-carrier is PEG 2,n PEG of (2) 2,n Lipid derivatives, which form a dense hydration layer on the surface of the carrier, improving the physical and biological stability of the carrier. In addition, the present invention selects PEG 2,n PEG of (2) 2,n The lipid derivative is used for modifying the nano-carrier, so that the ABC phenomenon caused by the PEGylation nano-carrier can be eliminated, and the serious problem faced by the existing PEGylation carrier is greatly solved; the internal circulation time of the nano-carrier is ensured, the defect of insufficient circulation time of a plurality of PEG substituted materials is overcome, and a firmer foundation is laid for clinical transformation of the PEGylated nano-preparation.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a novel method for eliminating the phenomenon that a PEGylated nano-carrier accelerates blood clearance.
Background
In order to overcome the defects that the traditional nano-carrier is easy to be identified and absorbed by a mononuclear-macrophage system (mononuclear phagocyte system, MPS), has short blood circulation half-life period, poor targeting property and the like, researchers modify the surface of the nano-carrier by utilizing PEG lipid derivatives, so that the physical, chemical and biological stability of the carrier and the medicine is greatly improved, and the process is called PEGylation. Since the 90 s of the 20 th century, this technology has been widely used in the field of nano-carrier surface modification. Although more and more studies have shown that other synthetic hydrophilic polymers for surface modification may exhibit long-circulating properties similar to pegylation, the unique advantages of low toxicity, low immunogenicity, and amphiphilicity of PEG still make it a difficult place to replace in the pharmaceutical formulation field. At present, the most widely used PEG-lipid derivative is linear mPEG 2000 DSPE has become a "gold standard" for extending the cycle time of nanocarriers.
However, in recent years, research has shown that there is a non-negligible problem with PEGylated nanocarriers, namely the generation of accelerated blood clearance (accelerated blood clearance, ABC) phenomena. The ABC phenomenon refers to repeated injections (spaced apart by days) of pegylated liposomes in the same animal, and causes abnormal changes in pharmacokinetics and tissue distribution of the secondarily injected liposomes, which are generally manifested by an increase in blood clearance rate and a significant increase in the amount of liver and spleen aggregates. This phenomenon occurs in repeated administration of various PEGylated formulations, such as PEGylated micelles (Koide H, asai T, hatanaka K, et al particle size-dependent triggering of accelerated blood clearancephenomenon [ J ]. Int J Pharm,2008,362 (1-2): 197-200.), solid lipid nanoparticles (Zhao Y, wang L, yan M, et al repeated injection of PEGylated solid lipid nanoparticles inducesaccelerated blood clearance in mice and beagles [ J ]. Int J Nanomedicine,2012,7:2891-2900 ]), emulsions (Wang C, cheng X, sui Y, et al A noticeable phenomenon: thiol terminal PEG enhances theimmunogenicity of PEGylated emulsions injected intravenously or subcutaneously into rats [ J ]. Eur JPharmBiopharm,2013,85 (3 Pt A): 744-751 ], lactic acid bodies (Hara E, makino A, kurilara K, et al pharmacokinetic change of nanoparticulate formulation "Lactume" on multiple administrations [ J ]. International Immunopharmacology,2012,14 (3): 261-266 ]), and the like. Furthermore, it was found that the first injection of non-PEGylated normal liposomes (Conventional liposomes, CL) also induced the ABC phenomenon (Ishida T, harada M, wang XY, et al accepted blood clearance of PEGylated liposomes followingpreceding liposome injection: effects of lipid dose and PEG surface-density and chain length of thefirst-dose liposomes [ J ]. Journal of controlled release,2005,105 (3): 305-317.). This suggests that we, the ABC phenomenon, may be a problem commonly faced by nano-formulations. Furthermore, it has been reported that the ABC phenomenon has been found in animal models such as mice, rats, guinea pigs, rabbits, rhesus monkeys, beagle dogs, etc. More seriously, several studies indicate that anti-PEG antibodies (Kinam, park. Image of anti-PEG antibodies on PEGylated nanoparticles fate in vivo. Journal of Controlled Release Official Journal of the Controlled Release society. 2018.) are detectable in the blood of 40% -72% healthy subjects, meaning that the generation of ABC phenomenon is also unavoidable in humans.
The generation of ABC phenomenon not only greatly weakens the long circulation advantage of the pegylated nanocarrier, but also causes serious toxic and side effects of the encapsulated drug in vivo due to the change of pharmacokinetics and tissue distribution behaviors, which seriously hampers the clinical transformation of the nano-preparation, because as more and more pegylated preparations gradually step into clinical research links, the hidden serious problems and risks related to the ABC phenomenon in clinical trials or after-market use are increasingly highlighted, so a practical and feasible method for solving the ABC phenomenon is urgently needed.
Researchers have attempted to replace PEG (Hoang Thi, thai Thanks, et al Importance of Poly (ethylene glycol) Alternatives for Overcoming PEG Immunogenicity in Drug Delivery and bioconjugation. Polymers12.2 (2020): 298.) with materials such as PVP, PDMA, HPMA, although these materials modified nanoformulations do not induce ABC, these materials suffer from other drawbacks such as short half-life in blood circulation. Moreover, in view of the great amount of time and effort and financial resources put into the field of PEG modification for many years, finding a PEG substitute is an almost always an approach, but is not yet completely uneconomical, so that the immunogenicity of PEG is directly urgent.
Compared to linear PEG, studies have shown that branched PEG (PEG 2 ) The modified protein medicine can raise its stability and prolong its circulation time. PEG at present 2 Has been widely used in the modification of enzymes, monoclonal antibodies, oligonucleotides and small molecule drugs, such as the above market preparationsEtc. When PEG is used 2 When modified, PEG chains are distributed on the surface of the modified protein medicine in an umbrella-shaped structure, so that the coverage is wider, the medicine is prevented from being identified and ingested by MPS, the stability is further improved, and the circulation time (Monfardini, cristina, et al, "A branched monomethoxypoly (ethylene glycol) for protein modification)" Bioconjugate chemistry 6.1.6.1 (1995): 62-69.H.Nishimura.T.Takahashi.K.Sakurai.A.Matcushima and Y.Inada,Gann,73 (I982) 470.) is prolonged. It has been reported that "brush-like" branched PEGs do not bind to anti-PEG IgM and do not bind non-specifically to proteins and cells. Thus, we speculate that branched PEG may have great potential in reducing formulation antigenicity, thereby reducing or even eliminating ABC phenomenon.
In conclusion, we consider the use of PEG 2 Modification of nanocarriers may break the dilemma of pegylated nanocarriers. At present, there is little concern about the use of PEG 2 Related reports of modified nano-carriers are not found by researchers to examine PEG 2 ABC phenomenon of the nano-carrier is functionalized. Our research just fills the gap in this area. We used PEG of different molecular weights 2 Lipid derivative, DSPE-mPEG 2,n Modification of nano-carriers such as emulsion, liposome and micelle, and research on repeated injection of PEG with different molecular weights 2 The ABC phenomenon induction condition of the PEGylated nano-carrier provides a new method for eliminating the ABC phenomenon of the PEGylated nano-carrier.
The classical immune mechanism of the ABC phenomenon is currently widely recognized as: the first injected PEGylated nano-carrier stimulates the proliferation and differentiation of spleen marginal zone B cells in a TI-2 type antigen mode and generates anti-PEG IgM, after the second injection of the PEGylated nano-carrier, the anti-PEG IgM is quickly identified and combined with the PEGylated nano-carrier, and a complement system is activated, so that the conditioning effect of a complement C3 fragment on particles is caused, the ingestion of the PEGylated nano-carrier by MPS is accelerated, the PEGylated nano-carrier is quickly cleared from blood, and then an ABC phenomenon occurs.
It follows that anti-PEG IgM is the main cause of the ABC phenomenon, and it is known that PEGylation therapy induces anti-PEG antibodies in both animals and humans. Clinical experience with pegylation therapy has shown that anti-PEG antibodies not only increase clearance and lose efficacy, but also cause severe allergies or anaphylactic reactions. [ POVSIC, thomas J., et al Pre-exposing anti-PEG antibodies are associated with severe immediate allergic reactions to pegnivacogin, a PEGylated adapter.journal of Allergy and Clinical Immunology,2016, 138.6:1712-1715; RAU, rachel E., et al Outcome of pediatric patients with acute lymphoblastic leukemia/lymphoblastic lymphoma with hypersensitivity to pegaspargase treated with PEGylated Erwinia asparaginase, pepercritisaspase A report from the Children's Oncology group &cancer,2018,65.3:e26873.；LONGO,Nicola,et al.Single-dose,subcutaneous recombinant phenylalanine ammonia lyase conjugated with polyethylene glycol in adult patients with phenylketonuria:an open-label,multicentre,phase 1dose-escalation trial.The Lancet,2014,384.9937:37-44.]Notably, the current FDA requires that patients detect anti-PEG antibodies prior to treatment with experimental PEGylated compounds. Laboratory studies have shown that compared to PEG 2k (straight-chain PEG) the first in vivo injection of PEG into rats 2,n Modified nanocarriers, significantly reduced anti-PEG IgM production, suggesting that we use PEG 2,n Modifying the nanocarrier can reduce the generation of anti-PEG antibodies; furthermore, studies have shown that the first injection of PEG 2,n The emulsion was modified and injections were repeated seven days later, with the formulation varying in binding level to the antibody. With PEG 2,n Molecular weight increase, formulation and anti-cancer agentThe slower the body binding, wherein PE 2,40k The binding to the antibody was the slowest. This also suggests that we, high molecular weight PEG 2,n The modification of the nano-carrier is more beneficial to prolonging the circulation time of the nano-carrier, and provides a foundation for the research and development of nano-preparations.
Disclosure of Invention
In view of the above problems, the primary object of the present invention is to provide a PEG 2,n The lipid derivative modified nano-carrier and the preparation method thereof are used for eliminating ABC phenomenon caused by PEGylation nano-carrier and solving the problems that the blood clearance speed of the PEGylation nano-carrier is increased, the aggregation of liver and spleen is obviously increased and the like caused by the ABC phenomenon.
The invention is realized by adopting the following technical scheme:
The method for eliminating ABC phenomenon caused by PEG nano-carrier provided by the invention uses branched PEG (PEG) 2,n ) PEG of (2) 2,n -lipid derivatives to modify the nanocarriers.
The nano-carrier comprises: emulsions, liposomes, micelles.
The PEG 2,n Is a compound of two linear PEG chains with methoxy ends and two amino groups of lysine are respectively connected with covalent bonds, wherein n represents the total molecular weight of the two linear PEG chains. Theoretically, such PEG 2,n There can be countless kinds, two linear PEG chains (PEG 2 ) Has a molecular weight of 200-400000Da. Preferably, PEG 2,n The molecular weight of (C) is 20000-40000Da.
The branched PEG of the invention also comprises branched PEG comprising three or more PEG chains.
PEG according to the invention 2,n PEG of (2) 2,n The lipid in the lipid derivative is natural phospholipid, semisynthetic phospholipid or synthetic phospholipid. Comprising the following steps: soybean lecithin, egg yolk lecithin (E80), phosphatidylglycerol, EPG (egg yolk phosphatidylglycerol), phosphatidic acid, cardiolipin, sphingomyelin, phosphatidic acid serine, phosphatidylinositol, phosphatidylethanolamine, hydrogenated soybean lecithin, hydrogenated egg yolk lecithin, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, and dimyristoyl phosphatidylcholine Myristoyl phosphatidylcholine, dilauryl phosphatidylcholine, didecanoyl phosphatidylcholine, dioctyl phosphatidylcholine, dihexanoyl phosphatidylcholine, distearoyl phosphatidylglycerol and salts thereof, dipalmitoyl phosphatidylglycerol and salts thereof, L-alpha-dimyristoyl phosphatidylglycerol and salts thereof, dilauroyl phosphatidylglycerol, didecanoyl phosphatidylglycerol, dioctanoyl phosphatidylglycerol, dihexanoyl phosphatidylglycerol, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dilauroyl phosphatidylethanolamine, bisdistearoyl phosphatidylglycerol and salts thereof, bisdimyristoyl phosphatidylglycerol and salts thereof, bisdilauroyl phosphatidylglycerol, distearoyl phosphatidylinositol, dipalmitoyl phosphatidylinositol, dioleoyl phosphatidylinositol, dimyristoyl phosphatidylinositol, palmitoyl phosphatidylcholine, palmitoyl linoleoyl phosphatidylcholine, stearoyl arachidonyl phosphatidylcholine, distearoyl phosphatidylethanolamine (DSPE).
In the present invention, PE means a linear or branched PEG modified emulsion, PE 2k Represents a linear PEG of molecular weight 2000; PE (polyethylene) 2,2k ，PE 2,10k ，PE 2,20k PE (polyethylene) 2,40k A compound obtained by covalent bonding 2 linear PEG chains with methoxy ends and lysine, wherein the total molecular weight of the 2 PEG chains is 2000, 10000, 20000 and 40000 respectively.
In the present invention, PL means a linear or branched PEG-modified liposome, PL 2k Represents a linear PEG of molecular weight 2000; PL (PL) 2,2k ，PL 2,10k PL (Poly L) 2,20k A compound obtained by covalent bonding 2 linear PEG chains with methoxy ends and lysine, wherein the total molecular weight of the 2 PEG chains is 2000, 10000 and 20000.
PEG according to the invention 2,n -lipid derivative modified emulsions, the components and preparation method thereof comprising:
the components are as follows: oil phase, emulsificationAgents, PEG 2,n -lipid derivatives, sterile water for injection; wherein the mass ratio of the oil phase to the emulsifier is (3-5) 1, PEG 2,n The molar ratio of the lipid derivative to E-80 is 1 (10-100).
The preparation method comprises the following steps:
weighing membrane material (oil phase, emulsifier, PEG) 2,n -lipid derivative) is completely dissolved in a water bath at 55-65 ℃, and sterilized water for injection heated to the same temperature is injected under stirring;
continuously stirring and incubating in a water bath at 55-65deg.C for 10-30min to obtain colostrum;
Performing ultrasonic dispersion treatment (for reducing particle size) on the obtained colostrum, and sequentially filtering with microporous membranes of 0.80, 0.45 and 0.22 μm to obtain PEG 2,n -lipid derivative modified emulsions.
Further, DSPE-mPEG with different molecular weights 2,n Modified emulsion, prescription and preparation method:
TABLE 1 different molecular weights DSPE-mPEG 2,n Modified emulsion prescription
The preparation method comprises the following steps: weighing the membrane material with the prescription amount, completely dissolving in a water bath at 55 ℃, and injecting the sterilizing injection water heated to the same temperature under the stirring condition. Stirring and incubating in a water bath at 55 ℃ for 20min to obtain the colostrum. Performing ultrasonic dispersion treatment (power and time: 200w×4min+400w×6min, work for 1s intermittently for 1 s), and sequentially filtering with microporous membrane of 0.80, 0.45 and 0.22 μm to obtain emulsion.
PEG according to the invention 2,n -lipid derivative modified liposomes, the components and preparation method thereof comprising:
the components are as follows: phospholipid, cholesterol, PEG 2,n -lipid derivatives and sterile water for injection; wherein the mass ratio of the phospholipid to the cholesterol is (3-5) 1, PEG 2,n The molar ratio of lipid derivative to (phospholipid+cholesterol) is 1 (10-100).
The preparation method comprises the following steps:
weighing membrane material (phospholipid, cholesterol, and PEG) 2,n -lipid derivative), adding absolute ethyl alcohol, stirring and dissolving in a water bath at 55-75 ℃, volatilizing 50-80% of absolute ethyl alcohol to obtain a concentrate;
Dripping sterilized water for injection preheated to the same temperature into the concentrate;
continuously stirring in 55-75deg.C water bath for 15-30min to obtain liposome primary product;
the obtained liposome primary product is treated by ultrasonic dispersion and sequentially passes through microporous filter membranes of 0.80, 0.45 and 0.22 mu m to obtain PEG 2,n -lipid derivative modified liposomes.
Further, DSPE-mPEG with different molecular weights 2,n The prescription and the preparation method of the modified liposome comprise the following steps:
TABLE 2 different molecular weights DSPE-mPEG 2,n Modified liposome formulations
The preparation method comprises the following steps: weighing membrane material (hydrogenated soybean phospholipid, cholesterol and DSPE-mPEG) 2,n ) Placing into a 10mL penicillin bottle, adding 300-500 mu L of absolute ethyl alcohol, stirring in a water bath at 65 ℃ for dissolution, and volatilizing most of the absolute ethyl alcohol. At 4 mL/min -1 Is injected into the membrane material with sterile water for injection (5 mL) preheated to the same temperature. Stirring in water bath at 65deg.C for 20min to obtain liposome primary product. And (3) performing ultrasonic dispersion treatment (power and time: 200W multiplied by 4min+400W multiplied by 6min, work for 1s and interval for 1 s) on the obtained primary product, and sequentially passing through microporous filter membranes of 0.80, 0.45 and 0.22 mu m to obtain the liposome.
The invention has the advantages that:
the method can eliminate ABC phenomenon caused by PEGylation nano-carriers. The modification material used by the nano-carrier is PEG 2,n PEG of (2) 2,n Lipid derivatives, which form a dense hydration layer on the surface of the carrier, improving the physical and biological stability of the carrier. In addition, the present invention selects PEG 2,n PEG of (2) 2,n -lipid derivatives for modificationThe nano carrier can not only eliminate ABC phenomenon caused by PEGylation nano carrier, but also greatly solve the serious problem faced by the existing PEGylation carrier; the internal circulation time of the nano-carrier is ensured, the defect of insufficient circulation time of a plurality of PEG substituted materials is overcome, and a firmer foundation is laid for clinical transformation of the PEGylated nano-preparation.
Drawings
FIG. 1 shows DSPE-PEG with a total molecular weight of 2000Da for the first and second intravenous injections of PEG in example 1 of the present invention 2000 DSPE-PEG with molecular weight of 2000Da for secondary tail vein injection of modified emulsion 2000 Modifying the effect of the emulsion drug time profile;
PE in the figure 2k DSPE-PEG representing PEG total molecular weight of 2000Da 2000 Modified emulsions; 5 mu mol/kg PE 2k -1 represents a first injection of 5% dextrose injection, a second injection of DSPE-PEG with a phospholipid concentration of 5. Mu. Mol/kg and a total PEG molecular weight of 2000Da 2000 Modified emulsions; 5 mu mol/kg PE 2k -2 represents DSPE-PEG with a total PEG molecular weight of 2000Da, with a phospholipid concentration of 5. Mu. Mol/kg for both the first and second injections 2000 Modified emulsions.
FIG. 2 shows DSPE-PEG with a total molecular weight of 2000Da for the first and second intravenous injections of PEG in example 2 of the present invention 2,2k DSPE-PEG with molecular weight of 2000Da for secondary tail vein injection of modified emulsion 2,2k Modifying the effect of the emulsion drug time profile;
PE in the figure 2,2k DSPE-PEG representing PEG total molecular weight of 2000Da 2,2k Modified emulsions; 5 mu mol/kg PE 2,2k -1 represents a first injection of 5% dextrose injection, a second injection of DSPE-PEG with a phospholipid concentration of 5. Mu. Mol/kg and a total PEG molecular weight of 2000Da 2,2k Modified emulsions; 5 mu mol/kg PE 2,2k -2 represents DSPE-PEG with a total PEG molecular weight of 2000Da, with a phospholipid concentration of 5. Mu. Mol/kg for both the first and second injections 2,2k Modified emulsions.
FIG. 3 shows DSPE-PEG with a total molecular weight of 10000Da for the first and second intravenous injections of PEG in example 3 of the present invention 2,10k DSPE-PEG with 10000Da total molecular weight of modified emulsion for secondary tail intravenous injection of PEG 2,10k Modifying the effect of the emulsion drug time profile;
PE in the figure 2,10k DSPE-PEG representing PEG total molecular weight of 10000Da 2,10k Modified emulsions; 5 mu mol/kg PE 2,10k -1 represents a first injection of 5% dextrose injection, a second injection of DSPE-PEG with a phospholipid concentration of 5. Mu. Mol/kg and a total PEG molecular weight of 10000Da 2,10k Modified emulsions; 5 mu mol/kg PEG 2,10k -2 represents DSPE-PEG with a total PEG molecular weight of 10000Da, with a phospholipid concentration of 5. Mu. Mol/kg for both the first and second injections 2,10k Modified emulsions.
FIG. 4 shows DSPE-PEG with a total molecular weight of 20000Da for first and second intravenous injections of PEG in example 4 of the present invention 2,20k DSPE-PEG with 20000Da total molecular weight of modified emulsion for secondary tail vein injection of PEG 2,20k Modifying the effect of the emulsion drug time profile;
PE in the figure 2,20k DSPE-PEG representing PEG total molecular weight of 20000Da 2,20k Modified emulsions; 5 mu mol/kg PE 2,20k -1 represents a first injection of 5% dextrose injection, a second injection of DSPE-PEG with a phospholipid concentration of 5. Mu. Mol/kg and a total PEG molecular weight of 20000Da 2,20k Modified emulsions; 5 mu mol/kg PEG 2,20k -2 represents DSPE-PEG with a total PEG molecular weight of 20000Da, with a phospholipid concentration of 5. Mu. Mol/kg for both the first and second injections 2,20k Modified emulsions.
FIG. 5 shows DSPE-PEG with total molecular weight of 40000Da for first and second intravenous injection of PEG in example 5 of the present invention 2,40k DSPE-PEG with 40000Da total molecular weight of modified emulsion for secondary tail intravenous injection of PEG 2 Modifying the effect of the emulsion drug time profile;
PE in the figure 2,40k DSPE-PEG representing PEG total molecular weight of 40000Da 2,40k Modified emulsions; 5 mu mol/kg PE 2,40k -1 represents a first injection of 5% dextrose injection, a second injection of DSPE-PEG with a phospholipid concentration of 5. Mu. Mol/kg and a total PEG molecular weight of 40000Da 2,40k Modified emulsions; 5 mu mol/kg PEG 2,40k -2 represents DSPE-PEG with total molecular weight of 40000Da of 5. Mu. Mol/kg of PEG by first and second injection of phospholipid 2,40k Modified emulsions.
FIG. 6 shows the total molecular weight of PEG for the first and second intravenous injections in example 6 of the present inventionDSPE-PEG of 2000Da 2000 Modified liposome DSPE-PEG with molecular weight of 2000Da for secondary tail intravenous injection of PEG 2000 The effect of modifying the profile of liposomal drug;
PL in the figure 2k DSPE-PEG representing PEG total molecular weight of 2000Da 2000 Modified liposomes; 5 mu mol/kg PL 2k -1 represents a first injection of 5% dextrose injection, a second injection of DSPE-PEG with a phospholipid concentration of 5. Mu. Mol/kg and a total PEG molecular weight of 2000Da 2000 Modified liposomes; 5 mu mol/kg PE 2k -2 represents DSPE-PEG with a total PEG molecular weight of 2000Da, with a phospholipid concentration of 5. Mu. Mol/kg for both the first and second injections 2000 Modified liposomes.
FIG. 7 shows DSPE-PEG with a total molecular weight of 2000Da for the first tail intravenous injection of PEG in example 7 of the present invention 2,2k Modified liposome DSPE-PEG with molecular weight of 2000Da for secondary tail intravenous injection of PEG 2,2k The effect of modifying the profile of liposomal drug;
PL in the figure 2,2k DSPE-PEG representing PEG total molecular weight of 2000Da 2,2k Modified liposomes; 5 mu mol/kg PL 2,2k -1 represents a first injection of 5% dextrose injection, a second injection of DSPE-PEG with a phospholipid concentration of 5. Mu. Mol/kg and a total PEG molecular weight of 2000Da 2,2k Modified liposomes; 5 mu mol/kg PL 2,2k -2 represents DSPE-PEG with a total PEG molecular weight of 2000Da, with a phospholipid concentration of 5. Mu. Mol/kg for both the first and second injections 2,2k Modified liposomes.
FIG. 8 shows DSPE-PEG with a total molecular weight of 10000Da for the first and second intravenous injections of PEG in example 8 of the present invention 2,10k Modified liposome DSPE-PEG with molecular weight of 10000Da for secondary tail intravenous injection of PEG 2,10k The effect of modifying the profile of liposomal drug;
PL in the figure 2,10k DSPE-PEG representing PEG total molecular weight of 10000Da 2,10k Modified liposomes; 5 mu mol/kg PL 2,10k -1 represents a first injection of 5% dextrose injection, a second injection of DSPE-PEG with a phospholipid concentration of 5. Mu. Mol/kg and a total PEG molecular weight of 10000Da 2,10k Modified liposomes; 5 mu mol/kg PL 2,10k -2 represents the total PEG of phospholipid concentration of 5. Mu. Mol/kg for both the first and second injectionsDSPE-PEG with molecular weight of 10000Da 2,10k Modified liposomes.
FIG. 9 shows DSPE-PEG with a total molecular weight of 20000Da for first and second intravenous injections of PEG in example 9 of the present invention 2,20k Modified liposome DSPE-PEG with molecular weight of 20000Da for secondary tail vein injection of PEG 2,20k The effect of modifying the profile of liposomal drug;
PL in the figure 2,20k DSPE-PEG representing PEG total molecular weight of 20000Da 2,20k Modified liposomes; 5 mu mol/kg PL 2,20k -1 represents a first injection of 5% dextrose injection, a second injection of DSPE-PEG with a phospholipid concentration of 5. Mu. Mol/kg and a total PEG molecular weight of 20000Da 2,20k Modified liposomes; 5 mu mol/kg PL 2,20k -2 represents DSPE-PEG with a total PEG molecular weight of 20000Da, with a phospholipid concentration of 5. Mu. Mol/kg for both the first and second injections 2,20k Modified liposomes.
FIG. 10 shows the effect of first tail intravenous injection of different branched PEG modified PE on anti-PEG IgM levels in examples 1-5 of the present invention.
FIG. 11 shows the effect of first tail intravenous injection of different branched PEG-modified PE on anti-PEG IgM levels in examples 6-9, 11 of the present invention.
FIG. 12 is a first injection of PE in example 12 of the invention 2,n PE injection was repeated 7 days after 2,n Measurement of anti-PEG IgM levels in plasma at various time points later.
FIG. 13 shows the first injection PL of example 13 of the present invention 2,n Repeated injection of PL 7 days after 2,n Measurement of anti-PEG IgM levels in plasma at various time points later.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
It should be understood that the practice of the invention is not limited to the following examples, but is intended to be within the scope of the invention in any form and/or modification thereof.
Example 1:
DSPE-PEG with total molecular weight of 2000Da by first tail intravenous injection 2000 Modified emulsion for PEG total molecular weight of secondary tail vein injectionDSPE-PEG of 2000Da 2000 Effects of modifying the time profile of an emulsion drug
Experimental animals:
wistar rats (180-210 g,lei Yun from the laboratory animal center of the pharmaceutical industry
Dosing regimen:
Male Wistar rats weighing 180-210 g were randomly divided into 2 groups, one group being control group and the other group being experimental group, 6 animals each, and were given by tail vein injection. The control group is firstly injected with 5% glucose solution, the experimental group is firstly injected with DSPE-PEG with total molecular weight of 2000Da according to phospholipid dosage of 5 mu mol/kg 2000 Modifying the emulsion. After the first injection, after 7 days apart, all groups were intravenously injected with 5. Mu. Mol phospholipids/kg of DSPE-PEG with a total PEG molecular weight of 2000Da 2000 Modifying the emulsion, taking blood from orbital venous plexus at 1min, 5min, 15min, 30min, 1h, 4h and 8h after secondary administration, centrifuging at 4500rpm for 10min, separating plasma, and detecting the drug concentration by using an enzyme-labeling instrument.
The processing specific operation is as follows: 0.5mL of plasma is placed in a 1.5mL EP tube, 0.9mL of absolute ethanol is added, and the mixture is vortexed for 5min to mix uniformly. The resulting suspension was centrifuged at 10000rpm for 10min, the resulting supernatant (0.6 mL) was transferred to another 1.5mL EP tube and centrifuged at 10000rpm for 10min, 200 μl of the supernatant was loaded into a 96-well plate, and fluorescence intensity F was measured at λex=750 nm, λem=790 nm wavelength. The treatment in the following experiment was the same as in example 1.
Results:
DSPE-PEG with total PEG molecular weight of 2000Da and phospholipid concentration of 5 mu mol/kg by first tail intravenous injection 2000 The emulsion was modified by injecting a second injection of 5. Mu. Mol/kg of phospholipid in a concentration of 5. Mu. Mol/kg of PEG with a total molecular weight of DSPE-PEG of 2000Da at 7 days intervals 2000 Modified emulsion with its drug time curve shown in 5. Mu. Mol/kg PE in figure 1 2k -2, ABC index (0-0.5h) The results are shown in Table 1, and the results of the anti-PEG IgM assay are shown in FIG. 10, wherein the anti-PEG IgM content is higher, and the OD value is 3.27.+ -. 0.07.
The results show that the first injectionDSPE-PEG with total molecular weight of 2000Da 2000 Modifying the emulsion, inducing double repeated injection of DSPE-PEG with total molecular weight of 2000Da 2000 Modified emulsion rapidly eliminates in blood, its ABC index (0-0.5h) A strong ABC phenomenon is induced at 0.17.+ -. 0.01.
Example 2:
DSPE-PEG with total molecular weight of 2000Da by first tail intravenous injection 2,2k DSPE-PEG with total molecular weight of 2000Da for secondary tail intravenous injection of PEG by modified emulsion 2,2k Effects of modifying the time profile of an emulsion drug
Experimental animals:
wistar rat (180-210 g, male, lei Yun experimental animal center for pharmaceutical industry)
Dosing regimen:
male Wistar rats weighing 180-210 g were randomly divided into 2 groups and given by tail vein injection. The control group is firstly injected with 5% glucose solution, the experimental group is firstly injected with DSPE-PEG with total molecular weight of 2000Da according to phospholipid dosage of 5 mu mol/kg 2,2k Modifying the emulsion. After the first injection, after 7 days apart, all groups were intravenously injected with 5. Mu. Mol phospholipids/kg of DSPE-PEG with a total PEG molecular weight of 2000Da 2,2k Modifying the emulsion, taking blood from orbital venous plexus at 1min, 5min, 15min, 30min, 1h, 4h and 8h after secondary administration, centrifuging at 4500rpm for 10min, separating plasma, and detecting the drug concentration by using an enzyme-labeling instrument.
Results:
DSPE-PEG with total PEG molecular weight of 2000Da and phospholipid concentration of 5 mu mol/kg by first tail intravenous injection 2,2k The emulsion was modified by injecting a second injection of 5. Mu. Mol/kg of phospholipid in a concentration of 5. Mu. Mol/kg of PEG with a total molecular weight of DSPE-PEG of 2000Da at 7 days intervals 2,2k Modified emulsion with its drug time curve shown in 5. Mu. Mol/kg PE in figure 2 2,2k -2, ABC index (0-0.5h) The results are shown in Table 1, and the results of the anti-PEG IgM assay are shown in FIG. 10, wherein the anti-PEG IgM content is high, and the OD value is 1.97.+ -. 0.10.
The results show that the first injection of DSPE-PEG with the total molecular weight of 2000Da 2,2k Modifying the emulsion, and repeatedly injecting DSPE-PEG with total molecular weight of 2000Da into the emulsion 2,2k The removal rate of the modified emulsion in blood is almost not influenced, and ABC index is obtained (0-0.5h) The ABC phenomenon is obviously eliminated when the temperature is 1.00+/-0.03.
Example 3:
DSPE-PEG with 10000Da total molecular weight of PEG injected from first tail vein 2,10k DSPE-PEG with 10000Da total molecular weight of modified emulsion for secondary tail intravenous injection of PEG 2,10k Effects of modifying the time profile of an emulsion drug
Experimental animals:
wistar rat (180-210 g, male, lei Yun experimental animal center for pharmaceutical industry)
Dosing regimen:
male Wistar rats weighing 180-210 g were randomly divided into 2 groups and given by tail vein injection. The control group is firstly injected with 5% glucose solution, the experimental group is firstly injected with DSPE-PEG with 10000Da of total molecular weight of PEG according to phospholipid dosage of 5 mu mol/kg 2,10k Modifying the emulsion. 7 days after the first injection, all groups were intravenously injected with 5. Mu. Mol phospholipids/kg of DSPE-PEG with a total PEG molecular weight of 10000Da 2,10k Modifying the emulsion, taking blood from orbital venous plexus at 1min, 5min, 15min, 30min, 1h, 4h and 8h after secondary administration, centrifuging at 4500rpm for 10min, separating plasma, and detecting the drug concentration by using an enzyme-labeling instrument.
Results:
DSPE-PEG with 5 mu mol/kg PEG total molecular weight of 10000Da for first tail intravenous injection of phospholipid 2,10k The emulsion was modified by injecting a second injection of 5. Mu. Mol/kg of phospholipid in DSPE-PEG with a total molecular weight of 10000Da at 7 days intervals 2,10k Modified emulsion with its drug time curve shown in 5. Mu. Mol/kg PEG in figure 3 2,10k -2, ABC index (0-0.5h) The results of the anti-PEG IgM assay are shown in Table 1 in FIG. 10, with higher anti-PEG IgM content and OD value of 1.06.+ -. 0.09.
The results show that the first injection of DSPE-PEG with the total molecular weight of 10000Da 2,10k Modifying the emulsion, and repeatedly injecting DSPE-PEG with 10000Da total molecular weight into the emulsion 2,10k The removal rate of the modified emulsion in blood is almost not influenced, and ABC index is obtained (0-0.5h) 1.01+/-0.02, obvious eliminationExcept for the ABC phenomenon.
Example 4:
DSPE-PEG with 20000Da total molecular weight of first tail intravenous injection PEG 2,20k DSPE-PEG with 20000Da total molecular weight of modified emulsion for secondary tail vein injection of PEG 2,20k Effects of modifying the time profile of an emulsion drug
Experimental animals:
wistar rat (180-210 g, male, lei Yun experimental animal center for pharmaceutical industry)
Dosing regimen:
male Wistar rats weighing 180-210 g were randomly divided into 2 groups and given by tail vein injection. The control group is firstly injected with 5% glucose solution, the experimental group is firstly injected with DSPE-PEG with the total molecular weight of 20000Da according to the phospholipid dosage of 5 mu mol/kg 2,20k Modifying the emulsion. 7 days after the first injection, all groups were intravenously injected with 5. Mu. Mol phospholipids/kg of DSPE-PEG having a total PEG molecular weight of 20000Da 2,20k Modifying the emulsion, taking blood from orbital venous plexus at 1min, 5min, 15min, 30min, 1h, 4h and 8h after secondary administration, centrifuging at 4500rpm for 10min, separating plasma, and detecting the drug concentration by using an enzyme-labeling instrument.
Results:
DSPE-PEG with 5 mu mol/kg PEG total molecular weight of 20000Da for first tail intravenous injection of phospholipid 2,20k The emulsion was modified by injecting a second injection of DSPE-PEG with a phospholipid concentration of 5. Mu. Mol/kg and a total PEG molecular weight of 20000Da for 7 days 2,20k Modified emulsion with its drug time curve shown in 5. Mu. Mol/kg PEG in figure 4 2,20k -2, ABC index (0-0.5h) The results of the anti-PEG IgM assay are shown in Table 1 in FIG. 10, with higher anti-PEG IgM content and OD value of 0.68.+ -. 0.06.
The results show that the first injection of DSPE-PEG with the total molecular weight of 20000Da 2,20k Modifying emulsion, and repeatedly injecting DSPE-PEG with total molecular weight of 20000Da 2,20k The removal rate of the modified emulsion in blood is almost not influenced, and ABC index is obtained (0-0.5h) The ABC phenomenon is obviously eliminated when the temperature is 1.03+/-0.04.
Example 5:
first tail veinInjection of DSPE-PEG with total PEG molecular weight of 40000Da 2,40k DSPE-PEG with 40000Da total molecular weight of modified emulsion for secondary tail intravenous injection of PEG 2,40k Effects of modifying the time profile of an emulsion drug
Experimental animals:
wistar rats (180-210 g,lei Yun from the laboratory animal center of the pharmaceutical industry
Dosing regimen:
male Wistar rats weighing 180-210 g were randomly divided into 2 groups and given by tail vein injection. The control group is firstly injected with 5% glucose solution, the experimental group is firstly injected with DSPE-PEG with total molecular weight of 40000Da according to phospholipid dosage of 5 mu mol/kg 2,40k Modifying the emulsion. 7 days after the first injection, all groups were intravenously injected with 5. Mu. Mol phospholipids/kg of DSPE-PEG with total PEG molecular weight of 40000Da 2,40k Modifying the emulsion, taking blood from orbital venous plexus at 1min, 5min, 15min, 30min, 1h, 4h and 8h after secondary administration, centrifuging at 4500rpm for 10min, separating plasma, and detecting the drug concentration by using an enzyme-labeling instrument.
Results:
DSPE-PEG with total PEG molecular weight of 40000Da and phospholipid concentration of 5 mu mol/kg by first tail intravenous injection 2,40k Modified emulsion, 7 days apart, two injections of 5. Mu. Mol/kg of phospholipid concentration of DSPE-PEG with total molecular weight of 40000Da 2,40k Modified emulsion with its drug time curve shown in 5. Mu. Mol/kg PEG of figure 5 2,40k -2, ABC index (0-0.5h) The results of the anti-PEG IgM assay are shown in Table 1 in FIG. 10, with higher anti-PEG IgM content and OD value of 0.38.+ -. 0.08.
The results show that the total molecular weight of the injected PEG is 40000Da when DSPE-PEG is injected for the first time 2,40k Modifying emulsion, and repeatedly injecting DSPE-PEG with total molecular weight of 40000Da 2,40k The removal rate of the modified emulsion in blood is almost not influenced, and ABC index is obtained (0-0.5h) The ABC phenomenon is obviously eliminated when the ratio is 1.01 plus or minus 0.01.
Example 6:
first tailDSPE-PEG with total molecular weight of 2000Da by intravenous injection of PEG 2000 Modified Liposome DSPE-PEG with total molecular weight of 2000Da for secondary tail intravenous injection of PEG 2000 Effects of modifying the Curve of Liposome drugs
Experimental animals:
wistar rat (180-210 g, male, lei Yun experimental animal center for pharmaceutical industry)
Dosing regimen:
male Wistar rats weighing 180-210 g were randomly divided into 2 groups and given by tail vein injection. The control group is firstly injected with 5% glucose solution, the experimental group is firstly injected with DSPE-PEG with total molecular weight of 2000Da according to phospholipid dosage of 5 mu mol/kg 2000 And (3) modifying the liposome. 7 days after the first injection, all groups were intravenously injected with 5. Mu. Mol phospholipids/kg of DSPE-PEG with a total PEG molecular weight of 2000Da 2000 Modifying liposome, taking blood from orbital venous plexus at 1min, 5min, 15min, 30min, 1h, 4h, 8h after secondary administration, centrifuging at 4500rpm for 10min, separating plasma, and detecting drug concentration by enzyme-labeled instrument.
Results:
DSPE-PEG with total PEG molecular weight of 2000Da and phospholipid concentration of 5 mu mol/kg by first tail intravenous injection 2000 Modified liposomes, at 7 days intervals, were twice injected with 5. Mu. Mol/kg of phospholipid at a concentration of 5. Mu. Mol/kg of PEG and DSPE-PEG having a total molecular weight of 2000Da 2000 Modified liposome with its drug time curve shown in 5. Mu. Mol/kg PE in figure 6 2k -2, ABC index (0-0.5h) The results of the anti-PEG IgM assay are shown in Table 2, and FIG. 11 shows that the anti-PEG IgM content is high and the OD value is 3.28.+ -. 0.06.
The results show that the first injection of DSPE-PEG with the total molecular weight of 2000Da 2000 Modification of liposomes, induction of double repeated injection of DSPE-PEG with total molecular weight of 2000Da 2000 Modified liposome rapidly eliminates in blood, its ABC index (0-0.5h) A strong ABC phenomenon is induced at 0.21.+ -. 0.02.
Example 7:
DSPE-PEG with total molecular weight of 2000Da by first tail intravenous injection 2,2k The total molecular weight of PEG injected into the secondary tail vein by the modified liposome is 2000DaDSPE-PEG of (C) 2,2k Effects of modifying the Curve of Liposome drugs
Experimental animals:
wistar rats (180-210 g,lei Yun from the laboratory animal center of the pharmaceutical industry
Dosing regimen:
male Wistar rats weighing 180-210 g were randomly divided into 2 groups and given by tail vein injection. The control group is firstly injected with 5% glucose solution, the experimental group is firstly injected with DSPE-PEG with total molecular weight of 2000Da according to phospholipid dosage of 5 mu mol/kg 2,2k And (3) modifying the liposome. 7 days after the first injection, all groups were intravenously injected with 5. Mu. Mol phospholipids/kg of DSPE-PEG with a total PEG molecular weight of 2000Da 2,2k Modifying liposome, taking blood from orbital venous plexus at 1min, 5min, 15min, 30min, 1h, 4h, 8h after secondary administration, centrifuging at 4500rpm for 10min, separating plasma, and detecting drug concentration by enzyme-labeled instrument.
Results:
DSPE-PEG with total PEG molecular weight of 2000Da and phospholipid concentration of 5 mu mol/kg by first tail intravenous injection 2,2k Modified liposomes, at 7 days intervals, were twice injected with 5. Mu. Mol/kg of phospholipid at a concentration of 5. Mu. Mol/kg of PEG and DSPE-PEG having a total molecular weight of 2000Da 2,2k Modified liposome with its drug time curve shown in 5. Mu. Mol/kg PL of figure 7 2,2k -2, ABC index (0-0.5h) The results of the anti-PEG IgM assay are shown in Table 2, and FIG. 11 shows that the anti-PEG IgM content is high and the OD value is 1.77.+ -. 0.07.
The results show that the first injection of DSPE-PEG with the total molecular weight of 2000Da 2,2k Modified liposome, and double repeated injection of DSPE-PEG with total molecular weight of 2000Da 2,2k Modified liposome has almost no influence on the clearance rate in blood, and ABC index thereof (0-0.5h) The ABC phenomenon is obviously eliminated when the ratio is 1.02+/-0.03.
Example 8:
DSPE-PEG with 10000Da total molecular weight of PEG injected from first tail vein 2,10k Modified liposomes for second tail intravenous PEG total fractionDSPE-PEG with molecular weight of 10000Da 2,10k Effects of modifying the Curve of Liposome drugs
Experimental animals:
wistar rats (180-210 g,lei Yun from the laboratory animal center of the pharmaceutical industry
Dosing regimen:
male Wistar rats weighing 180-210 g were randomly divided into 2 groups and given by tail vein injection. The control group is firstly injected with 5% glucose solution, the experimental group is firstly injected with DSPE-PEG with 10000Da of total molecular weight of PEG according to phospholipid dosage of 5 mu mol/kg 2,10k And (3) modifying the liposome. 7 days after the first injection, all groups were intravenously injected with 5. Mu. Mol phospholipids/kg of DSPE-PEG with a total PEG molecular weight of 10000Da 2,10k Modifying liposome, taking blood from orbital venous plexus at 1min, 5min, 15min, 30min, 1h, 4h, 8h after secondary administration, centrifuging at 4500rpm for 10min, separating plasma, and detecting drug concentration by enzyme-labeled instrument.
Results:
DSPE-PEG with 5 mu mol/kg PEG total molecular weight of 10000Da for first tail intravenous injection of phospholipid 2,10k Modified liposome, interval of 7 days, and secondary injection of phospholipid concentration 5 mu mol/kg of PEG with DSPE-PEG total molecular weight of 10000Da 2,10k Modified liposome with its drug time curve shown in 5. Mu. Mol/kg PL of figure 8 2,10k -2, ABC index (0-0.5h) The results of the anti-PEG IgM assay are shown in Table 2, and FIG. 11 shows that the anti-PEG IgM content is high and the OD value is 0.95.+ -. 0.04.
The results show that the first injection of DSPE-PEG with the total molecular weight of 10000Da 2,10k Modified liposome, and repeatedly injecting DSPE-PEG with total molecular weight of 10000Da into the liposome 2,10k Modified liposome has almost no influence on the clearance rate in blood, and ABC index thereof (0-0.5h) The ABC phenomenon is obviously eliminated when the ratio is 1.10 plus or minus 0.05.
Example 9:
DSPE-PEG with 20000Da total molecular weight of first tail intravenous injection PEG 2,20k Modified liposome pairDSPE-PEG with 20000Da total molecular weight of PEG by secondary tail intravenous injection 2,20k Effects of modifying the Curve of Liposome drugs
Experimental animals:
wistar rats (180-210 g,lei Yun from the laboratory animal center of the pharmaceutical industry
Dosing regimen:
male Wistar rats weighing 180-210 g were randomly divided into 2 groups and given by tail vein injection. The control group is firstly injected with 5% glucose solution, the experimental group is firstly injected with DSPE-PEG with the total molecular weight of 20000Da according to the phospholipid dosage of 5 mu mol/kg 2,20k And (3) modifying the liposome. 7 days after the first injection, all groups were intravenously injected with 5. Mu. Mol phospholipids/kg of DSPE-PEG having a total PEG molecular weight of 20000Da 2,20k Modifying liposome, taking blood from orbital venous plexus at 1min, 5min, 15min, 30min, 1h, 4h, 8h after secondary administration, centrifuging at 4500rpm for 10min, separating plasma, and detecting drug concentration by enzyme-labeled instrument.
Results:
DSPE-PEG with 5 mu mol/kg PEG total molecular weight of 20000Da for first tail intravenous injection of phospholipid 2,20k Modified liposome, interval of 7 days, and secondary injection of phospholipid concentration 5 mu mol/kg of PEG with total molecular weight of 20000Da DSPE-PEG 2,20k Modified liposome with its drug time curve shown in 5. Mu. Mol/kg PL of figure 9 2,20k -2, ABC index (0-0.5h) The results of the anti-PEG IgM assay are shown in Table 2, and FIG. 11 shows that the anti-PEG IgM content is high and the OD value is 0.46.+ -. 0.06.
The results show that the first injection of DSPE-PEG with the total molecular weight of 20000Da 2,20k Modified liposome, and double repeated injection of DSPE-PEG with total molecular weight of 20000Da 2,20k Modified liposome has almost no influence on the clearance rate in blood, and ABC index thereof (0-0.5h) The ABC phenomenon is obviously eliminated when the ratio is 1.08 plus or minus 0.03.
In order to quantitatively compare the intensity of the occurrence of the ABC phenomenon, the concept of ABC index is introduced, namely, the concept of secondary injectionAUC ratio to the first-injection formulation served as a measure of this extent. The ABC index calculation method comprises the following steps: ABC index = AUC of secondary injection (0～t) AUC of first injection (0～t) . The larger ABC index value indicates that the first formulation causes less change in pharmacokinetic behavior of the second injection, i.e., the weaker ABC phenomenon. Earlier work in the laboratory showed that ABC index (0-0.5h) In the range of 0.9 to 1.0, no ABC phenomenon is considered to occur, and 0.7 to 0.9 is a weak ABC phenomenon. As can be seen from Table 3, branched PEG modified ABC index of PE groups (0-0.5h) It is considered that the repeated injection of each group of branched PE did not induce ABC phenomenon, both of which were above 0.95. As can be seen from Table 4, branched PEG modified ABC index of each group PL (0-0.5h) It is considered that the repeated injection of each group of branched chain PL did not induce ABC phenomenon, both of which were 0.95 or more.
The ABC phenomenon is an important reason that hampers the clinical transformation of pegylated nanomaterials, and therefore exploring the mechanism of ABC phenomenon generation and finding an effective way to reduce or eliminate this phenomenon is an unavoidable problem for drug delivery system researchers. The branched chain PEG is adopted to modify the nano carrier, so that a new method is provided for eliminating the ABC phenomenon of the PEGylated nano preparation. It is notable that modification of branched PEG not only eliminates ABC phenomenon for pegylated nanoformulations, but also has good circulation time. In conclusion, the branched PEG modified nano-carrier can improve the curative effect of the preparation, reduce toxic and side effects, greatly increase the clinical conversion probability of the PEGylated nano-preparation, and has good application prospect in the field of nano-preparation modification.
TABLE 3 first and second injections of branched PEABC index groups (0-0.5h) Value of
TABLE 4 first and second injection of branched PLABC index groups (0-0.5h) Value of
Example 10:
determination of anti-PEG IgM
After the first tail vein injection of PE with different branched chains, blood is taken from the orbital venous plexus before the second administration (7 d after the first injection), and serum is separated for later use.
Coating: the coating solution (50. Mu.L) was added to a 96-well plate and allowed to air dry overnight at room temperature.
The coating liquid comprises the following components: 2.80mg of DSPE-mPEG2000 was dissolved in 5mL measuring flask, diluted to scale with absolute ethanol and shaken well to obtain 0.56mg/mL of DSPE-mPEG2000 solution.
Closing: 150. Mu.L of the blocking solution was added to a 96-well plate, and incubated at room temperature for 1 hour.
The sealing liquid comprises: 1g BSA was diluted to the scale with PBS buffer salt solution in a 100mL volumetric flask, and shaken well to obtain a blocking solution (PBS buffer salt solution containing 1% BSA).
Cleaning: firstly, the 96-well plate is patted dry, each well is filled with washing liquid, the mixture is stood for 30 to 60 seconds, the washing liquid is poured out, the mixture is patted dry gently, and the washing is repeated for 5 times.
Washing liquid: 0.5g BSA was diluted to the scale with PBS buffer salt solution in a 500mL volumetric flask, and shaken well to obtain a washing solution (PBS buffer salt solution containing 0.1% BSA).
Sample adding: 100. Mu.L of diluted (1:1000) serum samples were added to 96-well plates (except for blank wells), 3 wells of each serum sample were run in parallel and incubated for 1h at room temperature.
Cleaning: the washing step is the same as above.
Adding enzyme-labeled conjugate: 100. Mu.L of diluted conjugate was incubated in 96-well plates (excluding blank wells) at room temperature for 1h.
Cleaning: the washing step is the same as above.
Color development: 100 mu L of newly prepared color reagent is precisely added into each hole, and the mixture is incubated for 15min at room temperature.
Color-developing agent: 0.184g of disodium hydrogen phosphate and 0.047g of citric acid are diluted to the scale by redistilled water in a 10mL volumetric flask, and are uniformly shaken to obtain the pH 5.0 phosphoric acid-citric acid buffer solution. In addition, accurately weighing 10.00mg of o-phenylenediamine in a 10mL volumetric flask, diluting to a scale with a pH 5.0 phosphoric acid-citric acid buffer solution, adding 10 mu L of 30% hydrogen peroxide, and shaking uniformly to obtain the color reagent. It should be noted that the preparation is carried out just before use and stored in a dark place.
Terminating the reaction: 100. Mu.L of stop solution was added precisely to each well.
Measuring the absorption degree: the absorbance was measured with an enzyme-labeled instrument within 5min after termination of the reaction, and the wavelength was 490nm and 630nm in the dual wavelength assay.
The results show that: the first injection of 5. Mu. Mol phospholipids/kg of different branched PEG modified PE stimulated rats to produce anti-PEG IgM, but at different levels. As can be seen from FIG. 10, as the molecular weight of PEG increases in the first injection preparation, the secretion of anti-PEG IgM decreases, and the production amount is PE in order from large to small 2,2k >PE 2,10k >PE 2,20k >PE 2,40k Wherein PE is injected for the first time 2,40k Minimal amount of anti-PEG IgM generated (×p)<0.05,**P<0.01,***P<0.001.). This is probably due to the high molecular weight branched PEG modification that results in too high an epitope density, which makes it difficult to effectively stimulate spleen B cells, resulting in reduced B cell secretion of antibodies.
Example 11:
determination of anti-PEG IgM
After the first tail vein injection of different branched chain PLs, blood is taken from the orbital venous plexus before the second administration (7 d after the first injection), and serum is separated for later use.
The coating solution, the sealing solution, the color-developing agent, the washing solution and the cleaning process in the following steps were the same as in example 10.
Coating: the coating solution (50. Mu.L) was added to a 96-well plate and allowed to air dry overnight at room temperature.
Closing: 150. Mu.L of the blocking solution was added to a 96-well plate, and incubated at room temperature for 1 hour.
Cleaning: firstly, the 96-well plate is patted dry, each well is filled with washing liquid, the mixture is stood for 30 to 60 seconds, the washing liquid is poured out, the mixture is patted dry gently, and the washing is repeated for 5 times.
Sample adding: 100. Mu.L of diluted (1:1000) serum samples were added to 96-well plates (except for blank wells), 3 wells of each serum sample were run in parallel and incubated for 1h at room temperature.
Cleaning: as in example 10.
Adding enzyme-labeled conjugate: 100. Mu.L of diluted conjugate was incubated in 96-well plates (excluding blank wells) at room temperature for 1h.
Cleaning: as in example 10.
Color development: 100 mu L of newly prepared color reagent is precisely added into each hole, and the mixture is incubated for 15min at room temperature.
Terminating the reaction: 100. Mu.L of stop solution was added precisely to each well.
Measuring the absorption degree: the absorbance was measured with an enzyme-labeled instrument within 5min after termination of the reaction, and the wavelength was 490nm and 630nm in the dual wavelength assay.
The results show that: the first injection of 5. Mu. Mol phospholipids/kg of different branched PEG modified PL stimulated rats to produce anti-PEG IgM, but at different levels. As can be seen from FIG. 11, as the molecular weight of PEG increases in the first injection preparation, the secretion of anti-PEG IgM decreases, and the amount of production is PL from large to small 2,2k >PL 2,10k >PL 2,20k Wherein PL is injected for the first time 2,20k Minimal amount of anti-PEG IgM generated (×p)<0.05,**P<0.01,***P<0.001.). This is probably due to the high molecular weight branched PEG modification that results in too high an epitope density, which makes it difficult to effectively stimulate spleen B cells, resulting in reduced B cell secretion of antibodies. The above data indicate that branched PEG is selected 20k The modification of nano-formulations may be of good prospect.
Example 12:
first tail intravenous injection DSPE-PEG 2,n Modified emulsion, repeated tail vein injection DSPE-PEG after 7 days 2,n The emulsion was modified and antibody levels were measured over different time periods.
Experimental animals:
wistar rats (180-210 g,lei Yun from the laboratory animal center of the pharmaceutical industry
Dosing regimen:
male Wistar rats weighing 180-210 g were randomly divided into 4 groups and administered by tail vein injection. Head part DSPE-PEG was injected at a dose of 5. Mu. Mol/kg of phospholipid 2,n Modified emulsion (the PEG) 2,n Is PE 2k ，PE 2,2k ，PE 2,10k ，PE 2,20k ，PE 2,40k ). After 7 days, all groups were repeatedly injected with the corresponding DSPE-PEG 2,n The emulsion was modified, blood was collected from the orbital venous plexus at 0.016, 0.5, 1, 4 and 8 hours after the second administration, and after standing for 2 hours, the serum was separated by centrifugation at 5000rpm for 20 minutes, and the sample was processed as in example 5 to determine the antibody content.
Results:
the anti-PEG IgM assay results are shown in FIG. 12.
As can be seen from fig. 12, PE 2,2k ，PE 2,10k ，PE 2,20k After 1min of repeated injections, the amount of anti-PEG IgM was rapidly decreased, indicating that the preparation rapidly bound to the antibody after repeated injections; while PE 2,40k Within 0.5h of repeated injection, the amount of the anti-PEG IgM does not change significantly, and the content of the anti-PEG IgM begins to be reduced after 0.5h, which indicates PE 2,40k The binding rate to the antibody was slow after repeated injections.
At the first injection of PE 2,n PE injection was repeated 7 days later 2,n The results of the measurements of the rate of decrease in anti-PEG IgM levels in plasma at various time points are shown in Table 5.
Table 5 at first injection PE 2,n PE injection was repeated 7 days later 2,n Measurement of the rate of decrease in anti-PEG IgM levels in plasma at various time points
Example 13:
first tail intravenous injection DSPE-PEG 2,n Modified liposome, and repeated tail vein injection DSPE-PEG after 7 days 2,n Modified liposomes, determination of antibody levels over different time periods.
Experimental animals:
wistar rat (180-210 g, male, lei Yun experimental animal center for pharmaceutical industry)
Dosing regimen:
male Wistar rats weighing 180-210 g were randomly divided into 4 groups and administered by tail vein injection. DSPE-PEG is injected for the first time according to phospholipid dosage of 5 mu mol/kg 2,n And (3) modifying the liposome. After 7 days, all groups were repeatedly injected with the corresponding DSPE-PEG 2,n The liposomes were modified and blood was collected via orbital venous plexus at 0.016, 0.5, 1, 4, 8h after the second administration, and after standing for 2h, the serum was separated by centrifugation at 5000rpm for 20min, and the samples were processed as in example 5 to determine the antibody content.
Results:
the anti-PEG IgM assay results are shown in FIG. 13.
As can be seen from FIG. 13, PL 2,2k ，PL 2,10k ，PL 2,20k After 1min of repeated injections, the amount of anti-PEG IgM was rapidly decreased, indicating that the formulation rapidly bound to the antibody after repeated injections.
To accurately evaluate the binding of the formulation to anti-PEG IgM, the antibody reduction rate was calculated and the results are shown in table 4.
At the first injection of PL 2,n PL was repeatedly injected 7 days later 2,n The results of the measurements of the rate of decrease in anti-PEG IgM levels in plasma at various time points are shown in Table 6.
TABLE 6 first injection of PL ,n PL was repeatedly injected 7 days later 2,n Measurement of the rate of decrease in anti-PEG IgM levels in plasma at various time points
Example 14:
application of branched PEG in irinotecan and other liposomes
Irinotecan liposomes were prepared according to the recipe composition of the product irinotecan liposome (ONIVYDE) marketed in the united states. The different prescription compositions are shown in Table 7.
TABLE 7 different molecular weights DSPE-mPEG 2,n Modified liposome formulations
Remarks: the equi-proportion substitution is carried out according to the molar dosage of DSPE-PEG (straight-chain PEG) to be branched-chain PEG (DSPE-mPEG) 2,n ；PL 2,2k 、PL 2,10k 、PL 2,20k )
The liposome membrane material with the prescription amount is weighed, absolute ethyl alcohol with the final volume of 20% (v/v) of the blank preparation is added, and the mixture is stirred and dissolved in a water bath with the temperature of 65 ℃. And (3) injecting a citric acid-sodium citrate solution (200 mM, pH 4.0) preheated to the same temperature into the membrane material at a speed of 3mL/min, and stirring in a water bath for 0.5h to obtain a blank liposome primary product. And (3) sequentially extruding the blank liposome primary product through polycarbonate films of 5 times 400nm, 10 times 200nm, 10 times 100nm and 5 times 80nm to obtain blank liposome suspension with different prescriptions. The pH of the outer aqueous phase was adjusted to 7.0 by using 500mM sodium phosphate solution as a pH adjuster, and after the gradient liposome with delta pH of 3.0 was established, the drug was added at a drug-to-lipid ratio of 1:10, incubated at 70℃for 30min, and drug loading was performed, and the ice water bath was left for 5min to terminate drug loading, and the encapsulation efficiency was measured and placed in a refrigerator (4.+ -. 2 ℃) to examine the stability of the encapsulation efficiency, and the results are shown in Table 8.
Table 8 irinotecan liposome encapsulation efficiency stability
Remarks: the equi-proportion substitution is carried out according to the molar feeding amount of DSPE-PEG (straight-chain PEG) to be branched-chain PEG
It is evident that branched PEG is superior to linear PEG, especially in terms of placement stability, PL 2,10k Optimally.
Similarly, doxorubicin liposomes were prepared and the procedure was consistent with irinotecan liposomes, with the results shown in table 9.
Table 9 stability of encapsulation efficiency of doxorubicin liposomes
It is evident that branched PEG is superior to linear PEG, especially in terms of placement stability, PL 2,2k 、PL 2,10k And PL (PL) 2,20k Are all superior to straight-chain PEG (DSPE-PEG). The prescription of doxorubicin liposome is superior to the commercial onePEG density is low, hand and foot syndromes are not generated, and ABC phenomenon is not generated even if the PEG is administrated in small doses.
In addition, other antitumor drugs (epirubicin, mitoxantrone, pitaxolone and vincristine) and antibiotic drug liposome are prepared, and better stability is shown.
Example 15:
coenzyme Q10 injection
The existing coenzyme Q10 injection can be precipitated after being placed for 6 months, and the problem is not solved up to the present. And (3) feeding the coenzyme Q10 and tween 80 according to the mass ratio of 1:100 to prepare the coenzyme Q10 injection. Branched PEG (DSPE-PEG 2000) and branched PEG were added to the injections, and the values of the branched PEG were examined and compared, and the results are shown in Table 10.
TABLE 10 formulation and shelf stability of different coenzyme Q10 injections
It is clear that the linear PEG does not solve the problem of drug precipitation, PEG in branched PEG 2,10k Preferably, the problem of precipitation of the drug is completely solved.
The formulation can be used for medicaments such as vitamin A, D, E, K.
In addition, we also made propofol injection emulsion, which appeared yellowish after one year in ordinary emulsion, whereas PEG 2,10k Without improving the chemical stability of the formulation.
The method also has good value in preparing taxol/taxane and derivatives thereof (including oleate, DHA, disulfide S-S and monosulfide-S-derivatives) by using branched PEG, and has great development prospect.
Claims (3)
1. PEG (polyethylene glycol) 2,n -lipid derivative modified nanocarriers characterized by the use of PEG of different molecular weights 2,n PEG of (2) 2,n -lipid derivatives to modify the nanocarriers; the nano-carrier is emulsion or liposome; the PEG 2,n The lipid derivative is DSPE-mPEG 2,2k ，DSPE-mPEG 2,10k ，DSPE-mPEG 2,20k Or DSPE-mPEG 2,40k The method comprises the steps of carrying out a first treatment on the surface of the The PEG 2,n A compound which is formed by covalent bond connection of more than two linear PEG chains with methoxy ends and two amino groups of lysine respectively, wherein n represents the total molecular weight of the two linear PEG chains, and n is 2000-40000 Da; when the nano-carrier is emulsion, the components of the nano-carrier comprise oil phase, emulsifying agent and PEG 2,n -lipid derivatives, sterile water for injection; wherein the mass ratio of the oil phase to the emulsifier is (3-5) 1, PEG 2,n The molar ratio of the lipid derivative to the emulsifier is 1 (10-100), the oil phase is MCT, and the emulsifier is egg yolk lecithin; the preparation method comprises the following steps:
weighing oil phase, emulsifier and PEG 2,n -the lipid derivative is completely dissolved in the temperature of 55-65 ℃, and the sterilized water for injection heated to the same temperature is injected under stirring;
continuously stirring and incubating at 55-65deg.C for 10-30 min to obtain colostrum;
performing ultrasonic dispersion treatment on the obtained colostrum, and sequentially filtering with microporous membranes of 0.80, 0.45 and 0.22 μm to obtain PEG 2,n -lipid derivative modified emulsions;
when the nano carrier is liposome, the components of the nano carrier comprise phospholipid, cholesterol and PEG 2,n -lipid derivatives and sterile water for injection; wherein the mass ratio of the phospholipid to the cholesterol is (3-5) 1, PEG 2,n -the molar ratio of lipid derivative to phospholipid+cholesterol is 1 (10-100); the preparation method comprises the following steps:
phospholipid, cholesterol and PEG 2,n Mixing the lipid derivatives, adding absolute ethyl alcohol, stirring at 55-75 ℃ for dissolution, and volatilizing 50-80% of absolute ethyl alcohol to obtain a concentrate;
Dripping sterilized water for injection preheated to the same temperature into the concentrate;
continuously stirring at 55-75deg.C for 15-30min to obtain liposome primary product;
after the obtained liposome primary product is subjected to ultrasonic dispersion treatment, sequentially passing through microporous filter membranes of 0.80, 0.45 and 0.22 mu m to obtain PEG 2,n -lipid derivative modified liposomes.
2. A PEG according to claim 1 2,n -lipid derivative modified nanocarriers, characterized in that the PEG 2,n The molar ratio of lipid derivative to emulsifier is 1:10.
3. A PEG according to claim 1 2,n -lipid derivative modified nanocarriers, characterized in that the phospholipid is hydrogenated soy phospholipid and the PEG is 2,n -the molar ratio of lipid derivative to phospholipid+cholesterol is 1:10.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110629173.XA CN113350512B (en) | 2021-06-07 | 2021-06-07 | PEG 2,n Lipid derivative modified nano-carrier, preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110629173.XA CN113350512B (en) | 2021-06-07 | 2021-06-07 | PEG 2,n Lipid derivative modified nano-carrier, preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113350512A CN113350512A (en) | 2021-09-07 |
| CN113350512B true CN113350512B (en) | 2023-10-27 |
Family
ID=77532535
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110629173.XA Active CN113350512B (en) | 2021-06-07 | 2021-06-07 | PEG 2,n Lipid derivative modified nano-carrier, preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113350512B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102813929A (en) * | 2011-06-09 | 2012-12-12 | 沈阳药科大学 | Low-concentration polyethylene glycol-lipid (PEG-lipid) derivative and application thereof |
| WO2017052255A1 (en) * | 2015-09-23 | 2017-03-30 | 세종대학교 산학협력단 | Liposome for delivering taxane-based drug and preparation method therefor |
| KR20170035782A (en) * | 2015-09-23 | 2017-03-31 | 세종대학교산학협력단 | Liposome for taxane family drug delivery and preparation method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11684584B2 (en) * | 2016-12-30 | 2023-06-27 | Genevant Sciences Gmbh | Branched peg molecules and related compositions and methods |
-
2021
- 2021-06-07 CN CN202110629173.XA patent/CN113350512B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102813929A (en) * | 2011-06-09 | 2012-12-12 | 沈阳药科大学 | Low-concentration polyethylene glycol-lipid (PEG-lipid) derivative and application thereof |
| WO2017052255A1 (en) * | 2015-09-23 | 2017-03-30 | 세종대학교 산학협력단 | Liposome for delivering taxane-based drug and preparation method therefor |
| KR20170035782A (en) * | 2015-09-23 | 2017-03-31 | 세종대학교산학협력단 | Liposome for taxane family drug delivery and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| "Branched PEG-modification: A new strategy for nanocarriers to evade of the accelerated blood clearance phenomenon and enhance anti-tumor efficacy";Min Liu et al.;《Biomaterials》;20220221;第283卷;文献编号121415第1-13页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113350512A (en) | 2021-09-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dal Magro et al. | ApoE-modified solid lipid nanoparticles: A feasible strategy to cross the blood-brain barrier | |
| Ibrahim et al. | Polyethylene glycol (PEG): The nature, immunogenicity, and role in the hypersensitivity of PEGylated products | |
| JP5438317B2 (en) | Novel liposome composition | |
| JP5645954B2 (en) | Irinotecan or its hydrochloride liposome and method for producing the same | |
| US11357724B2 (en) | Pharmaceutical composition, preparation and uses thereof | |
| Riggio et al. | Nano-oncology: clinical application for cancer therapy and future perspectives | |
| CN110755387B (en) | Immune adjuvant-coated nanoparticle and application thereof | |
| US20120164065A1 (en) | Method and composition for treating cancer | |
| JP2001510451A (en) | Ion carrier carrying weakly basic drug-liposome in the middle | |
| KR20150100903A (en) | Nanoparticle compositions of albumin and paclitaxel | |
| US10391058B2 (en) | Pharmaceutical composition combining at least two distinct nanoparticles and a pharmaceutical compound, preparation and uses thereof | |
| CN112716915A (en) | Bionic nano-carrier and application thereof in preparing medicament for treating brain glioma | |
| US20170258718A1 (en) | Pharmaceutical composition, preparation and uses thereof | |
| WO2008080369A1 (en) | Steady liposomal composition | |
| CN108379230A (en) | A kind of oral granule of bile acid modification | |
| CN115006368B (en) | Cell membrane coated nano-drug and application thereof | |
| Dan | Core–shell drug carriers: liposomes, polymersomes, and niosomes | |
| WO2009101614A1 (en) | Casein particles encapsulating therapeutically active agents and uses thereof | |
| CN113350512B (en) | PEG 2,n Lipid derivative modified nano-carrier, preparation method and application | |
| Perrie | Pharmaceutical nanotechnology and nanomedicines | |
| Marcato | Pharmacokinetics and pharmacodynamics of nanomaterials | |
| Qi et al. | Research Progress of SN38 Drug Delivery System in Cancer Treatment | |
| WO2024075089A1 (en) | Hybrid nanoparticles as multifunctional platform for brain tumor therapy | |
| EP4237091A1 (en) | A silicasome nanocarrier for metal-based drug delivery | |
| Abouelmagd et al. | Nanoparticle-based drug delivery to solid tumors |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |