CN116747196A - 脂质体包人血清白蛋白载药纳米粒及其制备方法和用途 - Google Patents
脂质体包人血清白蛋白载药纳米粒及其制备方法和用途 Download PDFInfo
- Publication number
- CN116747196A CN116747196A CN202310950606.0A CN202310950606A CN116747196A CN 116747196 A CN116747196 A CN 116747196A CN 202310950606 A CN202310950606 A CN 202310950606A CN 116747196 A CN116747196 A CN 116747196A
- Authority
- CN
- China
- Prior art keywords
- mtx
- hsa
- serum albumin
- human serum
- liposome
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000003814 drug Substances 0.000 title claims abstract description 41
- 108091006905 Human Serum Albumin Proteins 0.000 title claims abstract description 38
- 102000008100 Human Serum Albumin Human genes 0.000 title claims abstract description 38
- 229940079593 drug Drugs 0.000 title claims abstract description 37
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 35
- 239000002502 liposome Substances 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 108010002159 methotrexate-serum albumin Proteins 0.000 claims abstract description 80
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 claims description 45
- 229960000485 methotrexate Drugs 0.000 claims description 43
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- 239000012071 phase Substances 0.000 claims description 16
- 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 14
- 239000012085 test solution Substances 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 235000012000 cholesterol Nutrition 0.000 claims description 7
- 150000001875 compounds Chemical class 0.000 claims description 7
- 238000002347 injection Methods 0.000 claims description 7
- 239000007924 injection Substances 0.000 claims description 7
- 201000001320 Atherosclerosis Diseases 0.000 claims description 6
- 208000027866 inflammatory disease Diseases 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 6
- JQWAHKMIYCERGA-UHFFFAOYSA-N (2-nonanoyloxy-3-octadeca-9,12-dienoyloxypropoxy)-[2-(trimethylazaniumyl)ethyl]phosphinate Chemical compound CCCCCCCCC(=O)OC(COP([O-])(=O)CC[N+](C)(C)C)COC(=O)CCCCCCCC=CCC=CCCCCC JQWAHKMIYCERGA-UHFFFAOYSA-N 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 3
- 238000000108 ultra-filtration Methods 0.000 claims description 3
- 206010018367 Glomerulonephritis chronic Diseases 0.000 claims description 2
- 206010040047 Sepsis Diseases 0.000 claims description 2
- 239000008346 aqueous phase Substances 0.000 claims description 2
- 239000000969 carrier Substances 0.000 claims description 2
- 239000012295 chemical reaction liquid Substances 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 208000010125 myocardial infarction Diseases 0.000 claims description 2
- 201000008482 osteoarthritis Diseases 0.000 claims description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 claims description 2
- 210000000496 pancreas Anatomy 0.000 claims 1
- 206010061218 Inflammation Diseases 0.000 abstract description 23
- 230000004054 inflammatory process Effects 0.000 abstract description 23
- 230000000694 effects Effects 0.000 abstract description 15
- 238000011282 treatment Methods 0.000 abstract description 10
- 230000008685 targeting Effects 0.000 abstract description 5
- 230000014759 maintenance of location Effects 0.000 abstract description 4
- MJWYCUZCRGLCBD-BGZMIMFDSA-N (4s)-4-amino-5-[[(2s)-1-[[(2s)-1-[[(2s,3s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-3-methyl-1-oxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-oxopentanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCC(O)=O MJWYCUZCRGLCBD-BGZMIMFDSA-N 0.000 abstract description 3
- 238000009825 accumulation Methods 0.000 abstract description 3
- 108010008118 glutamyl-leucyl-valyl-isoleucyl-serine Proteins 0.000 abstract description 3
- 238000010253 intravenous injection Methods 0.000 abstract description 2
- 239000000825 pharmaceutical preparation Substances 0.000 abstract description 2
- 239000002253 acid Substances 0.000 abstract 1
- 241000700159 Rattus Species 0.000 description 18
- 210000002540 macrophage Anatomy 0.000 description 18
- 210000004027 cell Anatomy 0.000 description 16
- 238000011156 evaluation Methods 0.000 description 15
- 210000001519 tissue Anatomy 0.000 description 15
- 238000001727 in vivo Methods 0.000 description 14
- 230000002757 inflammatory effect Effects 0.000 description 14
- 239000002245 particle Substances 0.000 description 11
- 208000009386 Experimental Arthritis Diseases 0.000 description 8
- 230000003110 anti-inflammatory effect Effects 0.000 description 8
- 238000010186 staining Methods 0.000 description 8
- 238000009826 distribution Methods 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 230000006378 damage Effects 0.000 description 6
- 238000009472 formulation Methods 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 208000027418 Wounds and injury Diseases 0.000 description 5
- 206010003246 arthritis Diseases 0.000 description 5
- 208000014674 injury Diseases 0.000 description 5
- 238000011068 loading method Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 230000028327 secretion Effects 0.000 description 5
- 230000002792 vascular Effects 0.000 description 5
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 4
- 206010018910 Haemolysis Diseases 0.000 description 4
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 4
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 208000037976 chronic inflammation Diseases 0.000 description 4
- 230000006020 chronic inflammation Effects 0.000 description 4
- 238000012377 drug delivery Methods 0.000 description 4
- 210000002950 fibroblast Anatomy 0.000 description 4
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 4
- 230000008588 hemolysis Effects 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 230000006676 mitochondrial damage Effects 0.000 description 4
- 230000001575 pathological effect Effects 0.000 description 4
- 230000003285 pharmacodynamic effect Effects 0.000 description 4
- 230000000770 proinflammatory effect Effects 0.000 description 4
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 3
- 102000003777 Interleukin-1 beta Human genes 0.000 description 3
- 108090000193 Interleukin-1 beta Proteins 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 210000002969 egg yolk Anatomy 0.000 description 3
- 230000012202 endocytosis Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000011503 in vivo imaging Methods 0.000 description 3
- 210000004969 inflammatory cell Anatomy 0.000 description 3
- 210000001503 joint Anatomy 0.000 description 3
- 229940067606 lecithin Drugs 0.000 description 3
- 235000010445 lecithin Nutrition 0.000 description 3
- 239000000787 lecithin Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000004065 mitochondrial dysfunction Effects 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 102000004506 Blood Proteins Human genes 0.000 description 2
- 108010017384 Blood Proteins Proteins 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 206010023232 Joint swelling Diseases 0.000 description 2
- -1 MTX compound Chemical class 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 108010022394 Threonine synthase Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 229960004104 amiloride hydrochloride Drugs 0.000 description 2
- LTKVFMLMEYCWMK-UHFFFAOYSA-N amiloride hydrochloride dihydrate Chemical compound [H+].O.O.[Cl-].NC(=N)NC(=O)C1=NC(Cl)=C(N)N=C1N LTKVFMLMEYCWMK-UHFFFAOYSA-N 0.000 description 2
- 210000003423 ankle Anatomy 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 238000004624 confocal microscopy Methods 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 102000004419 dihydrofolate reductase Human genes 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 238000005538 encapsulation Methods 0.000 description 2
- 230000002121 endocytic effect Effects 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 230000037406 food intake Effects 0.000 description 2
- 210000002683 foot Anatomy 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 208000018937 joint inflammation Diseases 0.000 description 2
- 210000005067 joint tissue Anatomy 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 238000003760 magnetic stirring Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000012982 microporous membrane Substances 0.000 description 2
- 210000001700 mitochondrial membrane Anatomy 0.000 description 2
- 239000002086 nanomaterial Substances 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 238000011552 rat model Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229950003937 tolonium Drugs 0.000 description 2
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 description 2
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 2
- MSTNYGQPCMXVAQ-RYUDHWBXSA-N (6S)-5,6,7,8-tetrahydrofolic acid Chemical compound C([C@H]1CNC=2N=C(NC(=O)C=2N1)N)NC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 MSTNYGQPCMXVAQ-RYUDHWBXSA-N 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- 206010065553 Bone marrow failure Diseases 0.000 description 1
- 208000000668 Chronic Pancreatitis Diseases 0.000 description 1
- 102100024746 Dihydrofolate reductase Human genes 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 206010049119 Emotional distress Diseases 0.000 description 1
- 206010015866 Extravasation Diseases 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 206010019851 Hepatotoxicity Diseases 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- NPGIHFRTRXVWOY-UHFFFAOYSA-N Oil red O Chemical compound Cc1ccc(C)c(c1)N=Nc1cc(C)c(cc1C)N=Nc1c(O)ccc2ccccc12 NPGIHFRTRXVWOY-UHFFFAOYSA-N 0.000 description 1
- 206010033649 Pancreatitis chronic Diseases 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000006538 anaerobic glycolysis Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 208000022531 anorexia Diseases 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940124599 anti-inflammatory drug Drugs 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000003143 atherosclerotic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 230000004579 body weight change Effects 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- ZPEIMTDSQAKGNT-UHFFFAOYSA-N chlorpromazine Chemical compound C1=C(Cl)C=C2N(CCCN(C)C)C3=CC=CC=C3SC2=C1 ZPEIMTDSQAKGNT-UHFFFAOYSA-N 0.000 description 1
- 229960001076 chlorpromazine Drugs 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 230000006395 clathrin-mediated endocytosis Effects 0.000 description 1
- 229940126523 co-drug Drugs 0.000 description 1
- 230000006957 competitive inhibition Effects 0.000 description 1
- 230000002380 cytological effect Effects 0.000 description 1
- 206010061428 decreased appetite Diseases 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 108020001096 dihydrofolate reductase Proteins 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 230000009429 distress Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 230000028023 exocytosis Effects 0.000 description 1
- 230000036251 extravasation Effects 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 230000007686 hepatotoxicity Effects 0.000 description 1
- 231100000304 hepatotoxicity Toxicity 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 230000004898 mitochondrial function Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 239000002539 nanocarrier Substances 0.000 description 1
- 239000002114 nanocomposite Substances 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000006036 negative regulation of mitochondrial membrane potential Effects 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000001991 pathophysiological effect Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000009919 sequestration Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 210000005222 synovial tissue Anatomy 0.000 description 1
- 239000005460 tetrahydrofolate Substances 0.000 description 1
- 230000002992 thymic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
-
- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- 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
-
- 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
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Nanotechnology (AREA)
- Epidemiology (AREA)
- Rheumatology (AREA)
- Urology & Nephrology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Biotechnology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Pain & Pain Management (AREA)
- Immunology (AREA)
- Vascular Medicine (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Biophysics (AREA)
- Physical Education & Sports Medicine (AREA)
- General Engineering & Computer Science (AREA)
- Medical Informatics (AREA)
- Molecular Biology (AREA)
- Inorganic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- General Physics & Mathematics (AREA)
Abstract
本发明属于药物制剂技术领域，具体涉及脂质体包人血清白蛋白载药纳米粒及其制备方法和用途。所述脂质体包人血清白蛋白载药纳米粒是将小尺寸甲氨蝶呤‑白蛋白复合物(MTX‑HSA)包载在酸敏感脂质体中获得。本发明提供的脂质体包人血清白蛋白载药纳米粒能显著提高药物在炎症微环境中积累和保留时间，并且通游离药物相比，具有更高的治疗效果和更小的副作用。此外，脂质体包人血清白蛋白载药纳米粒可利用炎症微环境具有ELVIS效应，静脉注射给药后可以实现被动靶向，在炎症部位释放出更小的人血清白蛋白载药复合物，增强在炎症部位的蓄积，提高治疗效果。
Description
技术领域
本发明属于药物制剂技术领域，具体涉及脂质体包人血清白蛋白载药纳米粒及其制备方法和用途。
背景技术
炎症(Inflammation)是具有血管系统的活体组织对各种损伤因子的刺激所发生的以防御反应为主的基本病理过程。炎症是损伤、抗损伤和修复的动态过程，包括如下步骤：①各种损伤因子对机体的组织和细胞造成损伤；②在损伤周围组织中的前哨细胞(例如巨噬细胞)，识别损伤因子及组织坏死物，产生炎症介质；③炎症介质激活宿主的血管反应及白细胞反应，使损伤局部的血液循环中的白细胞及血浆蛋白渗出到损伤因子所在部位，稀释、中和、杀伤及清除有害物质；④炎症反应的消退与终止；⑤实质细胞和间质细胞增生,修复受损伤的组织。致炎因子持续存在并且损伤组织是发生慢性炎症的根本原因。炎症灶内主要是巨噬细胞、淋巴细胞和浆细胞浸润。激活的巨噬细胞分泌多种生物活性产物，是造成慢性炎症中的组织破坏和纤维化的重要介质。因此，促进炎症型巨噬细胞的凋亡是一种有希望治疗慢性炎症的策略。
目前临床用于治疗炎症的药物包括抗炎药和抗生素。甲氨蝶呤(MTX)具有与叶酸相似的化学结构，对二氢叶酸还原酶(Dihydrofolate Reductase，DHFR)具有竞争性抑制作用，可导致四氢叶酸的生成受阻。除此之外，MTX还可以抑制胸腺核苷酸合成酶，同时可以抑制TNF-α等促炎细胞因子的产生，延缓炎症的发展。但是MTX在体内不具有靶向性，若患者长期服用，可能会导致比较严重的毒副作用，包括胃肠道窘迫、骨髓抑制、食欲不振和肝毒性等。同时，体内给药时，药物制剂也会分布在全身各处，导致在靶组织的治疗效果受损和副作用风险增加。最重要的是，即使药物制剂到达炎症微环境处，它仍然会从炎症部位迅速被清除。
根据文献报道，在炎症组织中增强渗透性和保留EPR效应也类似存在，这被称为通过泄漏血管的外渗和随后的炎症细胞介导的隔离(ELVIS)效应，这是因为随着炎症的发展，导致血管渗漏，炎症部位内皮细胞间的间隙高达600nm。当与患病部位微环境的特征性病理生理特征相结合时，NMs也能实现主动靶向策略。
与外源性纳米递药系统相比，天然仿生纳米递药系统具有更好的生物相容性、低细胞毒性和非免疫原性等优点。人血清白蛋白(HSA)是血清蛋白的主要成分，具有天然的生物相容性、可降解性、易生产及成本低等优点，是一种具有炎症靶向能力的多功能治疗和诊断药物载体。但由于炎症组织中纤维组织增生，致密的成纤维细胞阻止了大粒径载体的渗透，降低了治疗效果。最近，一种小尺寸的白蛋白-紫杉醇纳米复合物(10nm)被用于有效穿透肿瘤成纤维细胞，这与炎症中纤维性细胞的特点是一致的。脂质体作为应用最广泛的纳米颗粒之一，具有生物相容性高、易于获得和表面修饰等特点，在临床中得到了广泛的应用。此外，由于炎症细胞的快速增殖导致厌氧糖酵解和乳酸水平升高，酸性环境是炎性滑膜组织的典型特征。因此，特定的病理环境为设计治疗慢性炎症的按需给药系统提供了潜在的靶点。
综上，如何解决小尺寸载药纳米颗粒在血液中的滞留，有效将纳米颗粒积累到炎症组织中是本发明所要解决的技术问题。
发明内容
有鉴于上述技术问题，本发明提供了脂质体包人血清白蛋白载药纳米粒及其制备方法和用途。
第一方面，本发明提供一种脂质体包人血清白蛋白载药纳米粒的制备方法，包括以下步骤：
用甲醇将甲氨蝶呤MTX溶解为1-5mg/mL的试液I，用PBS缓冲液将人血清白蛋白HSA溶解为3-6mg/mL的试液II，以摩尔比HSA:MTX为1:1-6将试液I和试液II混合，反应2-4h，然后除去游离MTX与甲醇，再通过冷冻干燥得到MTX-HSA复合物；
2)用无水乙醇将蛋黄卵磷脂、胆固醇琥珀酸单酯溶解成油相，MTX-HSA复合物溶于PBS溶液中作为水相；将油相注入水相，并在38-42℃下反应2-3h，将反应液过滤，即得所述脂质体包人血清白蛋白载药纳米粒。
进一步的，步骤1)，通过超滤离心除去游离MTX与甲醇，参数为5000-7000r/min，20-40min。
更进一步的，步骤2)，油相中蛋黄卵磷脂和胆固醇琥珀酸单酯的质量比为3-5:1。
更进一步的，步骤2)，油相注入水相的速率为0.5-2mL/min。
更进一步的，步骤2)，MTX-HSA复合物与胆固醇琥珀酸单酯的质量比为1-2:1。
第二方面，本发明提供所述方法制备得到的脂质体包人血清白蛋白载药纳米粒。
第三方面，本发明提供所述脂质体包人血清白蛋白载药纳米粒在制备治疗炎症性疾病的药物中的用途。
进一步的，所述炎症性疾病包括类风湿性关节炎、骨关节炎、动脉粥样硬化、心肌梗死、脓毒症、慢性肾炎和胰腺炎。
第四方面，本发明提供一种用于治疗炎症性疾病的药物，其包括所述脂质体包人血清白蛋白载药纳米粒，以及药学上可接受的辅料或载体。
与现有技术相比，本发明具有以下有益效果：
1、本发明得到粒径为10nm左右的人血清白蛋白MTX复合物，载药量为2.4％。
2、本发明用脂质体包人血清白蛋白MTX复合物(Lipo/MTX-HSA)，包封率为85.1％，载药量为8.2％。此外，该纳米粒形态圆整规则，大小均一，粒径基本在100nm左右，Zeta电位为-14.43±0.39mV。Lipo/MTX-HSA可以作为一种优异的药物递送纳米载体。
3、本发明制备了脂质体包人血清白蛋白载MTX纳米粒，利用炎症部位具有通透性增加的脉管系统的渗漏作用和随后的炎症细胞介导的滞留效应(ELVIS)，静脉注射给药后可以实现被动靶向性，在炎症部位释放出小粒径的MTX-HSA，增强在炎症部位的蓄积，提高治疗效果。
4、本发明选用人血清白蛋白载MTX和脂质体包裹作为载体材料，制备出高载药量、高生物相容性、低毒性以及低免疫原性的靶向炎症微环境的纳米药物。体外试验结果显示，该纳米药物能够靶向炎症部位，被活化的巨噬细胞靶向摄取，表现出降低促炎因子TNF-α、IL-1β，升高抗炎因子IL-10的作用，将巨噬细胞表型从M1型转化为M2型的能力，调节炎症微环境，进一步抑制炎症的发展，并且通过JC-1可发现MTX相关制剂可导致线粒体膜电位下降，诱导巨噬细胞的线粒体功能障碍；在类风湿性关节炎大鼠模型中，活体成像研究显示出：该纳米药物可以延长循环时间，表现出良好的炎症关节靶向性；体内试验显示：该纳米药物对疾病大鼠表现出优异的疗效，共载药纳米粒可以有效改善关节炎症微环境，调节炎症因子的分泌，同时显著改善疾病组织的病理状况；体内安全性评价显示：该纳米药物未发生任何溶血情况，具备良好的安全性。
附图说明
图1：本发明实施例提供的纳米粒紫外可见光光谱、荧光共振能量转移。A、MTX、HSA、MTX-HSA、Lipo/MTX-HSA的紫外可见光光谱图；B、DIO-Lipo/DIL-HSA在1:1/2:1的荧光共振能量转移图。
图2：本发明实施例提供的粒子的马尔文粒径图。A、MTX-HSA；B、Lipo/MTX-HSA。
图3：本发明实施例提供的粒子的透射电镜图。A、MTX-HSA；B、Lipo/MTX-HSA。
图4：本发明实施例提供的药物释放能力评价。Lipo/MTX-HSA在不同pH条件下的释放效率。
图5：本发明实施例提供的稳定性评价。A、Lipo/MTX-HSA一周内的粒径变化；B、Lipo/MTX-HSA一周内的PDI变化。
图6：本发明实施例提供的体外生物相容性评价。MTX、MTX-HSA、Lipo/MTX-HSA在0.5-30μg/mL浓度下在RAW264.7细胞中的生物相容性。
图7：本发明实施例提供的巨噬细胞细胞摄取评价。A、用Cy5代替MTX，用共聚焦显微镜进行巨噬细胞摄取研究；B、用共聚焦显微镜进行药物内吞机制研究；C、流式检测活化前后的RAW264.7对荧光制剂的摄取情况；D、流式检测不同内吞抑制剂对荧光制剂的摄取方式情况。
图8：本发明实施例提供的滑膜成纤维细胞摄取评价。A、用Cy5代替MTX，用共聚焦显微镜进行FLS细胞摄取研究；B、A图中摄取实验放大倍数图；C、采用JC-1研究MTX相关制剂对FLS细胞的线粒体损伤情况；D、对FLS进行AM/PI钙黄蛋白染色的定量分析。E、AM/PI钙黄蛋白染色不同处理的FLS细胞的荧光显微镜图像。
图9：本发明实施例提供的体外细胞抗炎评价。A-C、采用RT-qPCR检测促炎因子TNF-α、IL-1β，以及抗炎因子IL-10的表达。
图10：本发明实施例提供的线粒体损伤评价。采用JC-1研究MTX相关制剂对RAW264.7细胞的线粒体损伤情况。
图11：本发明实施例提供的体内组织分布评价。CIA大鼠给DID荧光制剂后，进行活体成像。A、分别在1、6、12、24h拍摄了尾静脉注射Cy5-HSA、Lipo/Cy5-HSA后的荧光照片；B、给荧光制剂24h后，牺牲大鼠，对心、肝、脾、肺、肾、关节、血液进行荧光拍照；C、对A的荧光定量分析。
图12：本发明实施例提供的体内药效学评价。经normal组、control组、MTX、MTX-HSA、Lipo/MTX-HSA组治疗前后CIA大鼠，A、关节肿胀程度变化；B、足体积变化；C、关节评分；D、踝关节直径/足掌厚度；E、大鼠体重变化。
图13：本发明实施例提供的体内抗炎评价。A-C：经normal组、control组、MTX、MTX-HSA、Lipo/MTX-HSA组治疗前后CIA大鼠，取血清进行ELISA检测促炎因子TNF-α、IL-1β；以及抗炎因子IL-10。
图14：本发明实施例提供的关节组织病理学评价。对关节组织进行病理学研究，A-B、H&E染色；C-D、SO染色；E-F、甲苯胺蓝染色。
图15：本发明实施例提供的体内安全性评价。MTX、MTX-HSA、Lipo/MTX-HSA组的溶血实验。
图16：本发明实施例提供的动脉粥样硬化小鼠模型的体内分布研究。
图17：本发明实施例提供的动脉粥样硬化小鼠血管油红O染色。
图18：本发明实施例提供的动脉粥样硬化小鼠血管HE染色。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明，但不应理解为本发明的限制。如未特殊说明，下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段，下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1：脂质体包人血清白蛋白载药(Lipo/MTX-HSA)纳米粒制备和表征
本实施例的Lipo/MTX-HSA纳米粒制备方法如下：
步骤1：合成MTX-HSA复合物，具体地，①精密称取处方量的MTX，用甲醇/无水乙醇/水溶解为2mg/mL的试液；②精密称取处方量的HSA，用PBS缓冲液溶解为4mg/mL的试液；③按摩尔比HSA:MTX＝1:1/1:2/1:4/1:6量取两试液，混合搅拌3h后，通过超滤离心除去游离MTX与甲醇(6000r/min，30min)；④再通过冷冻干燥即可制得MTX-HSA复合物(MTX-HSA使用前溶解在PBS溶液中)。
步骤2：合成Lipo/MTX-HSA纳米粒，具体地，①精密称取处方中所述的蛋黄卵磷脂E80、胆固醇琥珀酸单酯(CHS)，无水乙醇作为溶剂涡旋溶解制成3mL的油相(蛋黄卵磷脂:CHS＝3:1/4:1/5:1，w/w)，处方中所述的MTX-HSA复合物溶于5mL pH 7.4的PBS溶液中作为水相；②将注入速度设置为0.5/1/2mL/min，使用针孔注射器将制备的油相缓慢注入水相中(边搅拌边注入，MTX-HSA:CHS＝1:1/1.5:1/2:1，w/w)，在40℃水浴条件下以200/300/400/500r/min的转速缓慢搅拌反应2h；③将搅拌2h的反应液先后用0.45μm与0.22μm的微孔滤膜过滤，则可以得到所需Lipo/MTX-HSA溶液。
通过粒径(Size)、Zeta电位和分散指数(PDI)对MTX-HSA复合物及Lipo/MTX-HSA纳米粒进行表征。
表1显示，步骤1所制得的MTX-HSA复合物，制备的优选有机溶剂为甲醇，摩尔比HSA:MTX为1:4。
表2、3、4、5显示，步骤2所制得的Lipo/MTX-HSA纳米粒制备的优选MTX-HSA:CHS质量比为1.5:1，蛋黄卵磷脂E80:CHS质量比为4:1，磁力搅拌速度的选择为300r/min，针孔注射器注入速度的选择0.5mL/min。
表6为最终的实验处方。
表1MTX和HSA剂量比的单因素研究
表2MTX-HSA与CHS比值(w/w)的单因素研究
表3蛋黄卵磷脂E80和CHS剂量比(w/w)的单因素研究
表4磁性搅拌速度的单因素研究
表5针孔注射器注射速度的单因素研究
表6最终的实验处方
图1A通过紫外可见光光谱全波长扫描Lipo/MTX-HSA，观察到MTX在302nm处有明显的吸收峰，证实了脂质体中成功包载了MTX。并且HSA和Lipo/MTX-HSA的蛋白特定吸收峰也存在，表明蛋白结构不受载药的影响。图1B通过荧光共振能量转移(FRET)可以确认脂质体上装载了HSA。DIO作为供体与脂质体结合，而DIL作为受体与HSA结合。随着DIL-HSA浓度的增加，受体(DIL，569nm)的荧光强度增加，而供体(DIO)在501nm处的荧光强度降低，表明存在FERT效应。
图2A显示MTX-HSA粒径约为10nm，图2B显示Lipo/MTX-HSA的粒径约为100nm。此外，Lipo/MTX-HSA的PDI为0.171±0.013，Zeta电位为-14.43±0.39mV。通过MTX的浓度可计算得到MTX-HSA复合物的载药量约为2.4％，所制备的Lipo/MTX-HSA的包封率约为85.1％，载药量约为8.2％。
图3A显示，通过透射电镜观察MTX-HSA为20nm的圆形纳米粒，图3B可以看出Lipo/MTX-HSA的形态圆整规则，大小均一，粒径基本在100nm左右。
图4显示Lipo/MTX-HSA在不同pH条件下的释放。与pH＝7.4组相比，Lipo/MTX-HSA在pH＝5.5的释放速度较快。在12h内pH＝5.5组的释放速率较快，pH＝5.5和pH＝7.4分别释放了70％和57％的MTX。表明Lipo/MTX-HSA能响应炎症的微酸性环境，加速MTX的释放。
图5A、B显示，Lipo/MTX-HSA的粒径和PDI连续7天几乎没有变化，在体外表现出良好的稳定性。
实施例2：Lipo/MTX-HSA纳米粒的体外细胞学研究
(1)采用MTT法评价不同剂型的细胞毒性。从图6可以看出，与RAW264.7细胞孵育24h后，MTX和MTX-HSA表现出浓度依赖性的抑制作用(0.5～30μg/mL)。在浓度为30μg/mL时，MTX和MTX-HSA的存活率分别低于50％和60％。然而，通过Lipo/MTX-HSA处理，我们可以看到细胞活力显著增加，在高浓度(30μg/mL)下达到70％以上(*P<0.05)。证实脂质体包被提高了纳米颗粒对巨噬细胞的生物相容性和选择性。
(2)研究荧光制剂在细胞内的分布。图7A显示，LPS激活的巨噬细胞中红色荧光强度明显高于未激活的巨噬细胞。在无LPS刺激的RAW264.7中，Lipo/Cy5-HSA组Cy5荧光强度与Cy5-HSA组相近，且强于游离Cy5组。图7B显示，在LPS刺激的RAW264.7细胞中，Lipo/Cy5-HSA组与Cy5-HSA组的荧光强度存在差异，与流式细胞仪(图7C)检测的荧光强度数据一致。这可能是由于较小的Cy5-HSA纳米颗粒(7nm)比较大的Lipo/Cy5-HSA(80nm)更有利于胞吐。与Cy5-HSA相比，Lipo/Cy5-HSA在一定程度上降低了Cy5-HSA的胞外分泌，延长了LPS激活RAW264.7细胞中Cy5-HSA的停留时间。
图8A显示在FLS细胞中，Lipo/Cy5-HSA组Cy5荧光强度与Cy5-HSA组相近，且强于游离Cy5组。如图8B所示，MTX制剂组JC-1单体的绿色荧光强度明显高于对照组，证实MTX相关制剂可诱导巨噬细胞和FLSs线粒体功能障碍。图8C，D显示，MTX-HSA处理后的细胞活力显著低于相同浓度的MTX或Lipo/MTX-H SA(**P<0.01)。图8E所示，经MTX-HSA和Lipo/MTX-HSA处理的CIA-FLS红色荧光最强。相反，经MTX处理的CIA-FLS发出大量绿色荧光，表明细胞死亡水平较低。这些数据和结果表明，MTX-HSA可以通过抑制FLSs的活力来减轻炎症。
(3)探究制剂的细胞内吞途径。采用甲基-β-CD(小孔介导的内吞抑制剂)、盐酸阿米洛利(大胞饮介导的内吞抑制剂)、氯丙嗪(网格蛋白介导的内吞抑制剂)研究了纳米材料的细胞进入途径。如图7D所示，盐酸阿米洛利显著降低了活化巨噬细胞对Lipo/Cy5-HSA的摄取效率。结果表明，大部分Lipo/Cy5-HSA通过大胞饮进入细胞，这种摄取方式有助于绕过溶酶体。
实施例3：Lipo/MTX-HSA的体外细胞药效学研究
(1)研究制剂的细胞抗炎药效学。通过研究Lipo/MTX-HSA对炎症因子分泌的影响，发现Lipo/MTX-HSA可以抑制活化巨噬细胞中这些细胞因子的分泌，这与正常巨噬细胞的分泌相当(图9A-B)。值得注意的是，Lipo/MTX-HSA增加了抗炎因子IL-10的表达(图9C)。这些数据表明Lipo/MTX-HSA具有较强的抑制炎症因子和增加抗炎因子表达的能力，这对于缓解RA的炎症至关重要。
(2)研究制剂的细胞线粒体功能。为研究MTX相关纳米制剂是否会损伤巨噬细胞的线粒体，使用JC-1作为探针来检测线粒体膜电位的变化。当线粒体膜电位高时，JC-1在线粒体基质中聚集为聚合物，产生红色荧光。相反，JC-1形成的单体在膜电位较低时发出绿色荧光。通过从红色荧光切换到绿色荧光来检测膜电位的降低，这被认为是线粒体损伤的标志。如图10所示，MTX组JC-1单体的绿色荧光强度明显高于对照组，证实MTX相关制剂可诱导巨噬细胞的线粒体功能障碍。
实施例4：Lipo/MTX-HSA的体内分布研究
(1)CIA大鼠模型方法建立。SD大鼠适应饲养一周后于第0天，在大鼠尾根部皮下注射等体积的牛二型胶原蛋白与完全弗式佐剂(Complete Freund'sadjuvant,CFA)充分乳化后的乳剂，每只大鼠注射100μL。初次免疫后第7天，每只大鼠第二次注射等体积的牛二型胶原蛋白与不完全弗式佐剂(Incomplete Freund's adjuvant,IFA)充分乳化后的乳剂100μL，建立SD大鼠胶原蛋白诱导的关节炎(CIA)模型，CIA大鼠关节炎临床症状评分如表7所示。
表7CIA大鼠关节炎临床症状评分系统
(2)荧光活体成像研究。将6只CIA大鼠随机分为Cy5-HSA和Lipo/Cy5-HSA两组(每组n＝3)，静脉注射Cy5剂量为5μg，观察NPs在炎症关节中的生物分布。在注射后1、6、12和24小时麻醉大鼠，使用Carestream MI对其脚掌成像。荧光成像24h后处死大鼠进行体外组织分布分析。采集的器官和血浆的荧光也成像。
结果如图11所示，Lipo/Cy5-HAS组荧光强度在注射6小时后明显高于Cy5-HAS组。
实施例5：Lipo/MTX-HSA的体内药效学研究
(1)在诱发关节炎、初次免疫后的第16、19、22、25、28天，各组分别静脉注射生理盐水、MTX、MTX-HSA或Lipo/MTX-HSA，三只正常大鼠作为对照组。给药剂量按MTX为0.8mg/kg，每三天给药一次，共五次。每次给药前拍照并记录关节(图12A)、足体积(图12B)、关节评分(图12C)、踝关节直径/足掌厚度(图12D)、大鼠体重(图12E)。
(2)给药完成后，取血清完成炎症因子检测(图13)，并对组织病理学研究，进行H&E染色(图14A-B)、SO染色(图14C-D)、甲苯胺蓝染色(图14E-F)
图12-15结果证明，Lipo/MTX-HSA可以有效抑制关节肿胀，改善关节炎症微环境，调节炎症因子的分泌，同时显著改善关节组织的病理状况。
实施例6：Lipo/MTX-HSA的体内安全性评价
图15溶血实验结果表明，即便在500μg/mL如此高浓度情况下，Lipo/MTX-HSA仍然未见溶血情况的发生，证明Lipo/MTX-HSA是一种安全有效的纳米粒药物。
实施例7：在动脉粥样硬化疾病模型中的体内分布及药效学评价
(1)动脉粥样硬化模型中的体内血管分布实验表明，Lipo/MTX-HSA组的分布显著高于游离药物组(图16)。
(2)油红染色结果显示，Lipo/MTX-HSA组的斑块显著低于其他组(图17)。
(3)HE染色结果显示，Lipo/MTX-HSA组的斑块显著低于其他组(图18)。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
Claims (9)
1.一种脂质体包人血清白蛋白载药纳米粒的制备方法，其特征在于，包括以下步骤：
1)用甲醇将甲氨蝶呤MTX溶解为1-5mg/mL的试液I，用PBS缓冲液将人血清白蛋白HSA溶解为3-6mg/mL的试液II，以摩尔比HSA:MTX为1:1-6将试液I和试液II混合，反应2-4h，然后除去游离MTX与甲醇，再通过冷冻干燥得到MTX-HSA复合物；
2)用无水乙醇将蛋黄卵磷脂、胆固醇琥珀酸单酯溶解成油相，MTX-HSA复合物溶于PBS溶液中作为水相；将油相注入水相，并在38-42℃下反应2-3h，将反应液过滤，即得所述脂质体包人血清白蛋白载药纳米粒。
2.根据权利要求1所述的制备方法，其特征在于，步骤1)，通过超滤离心除去游离MTX与甲醇，参数为5000-7000r/min，20-40min。
3.根据权利要求2所述的制备方法，其特征在于，步骤2)，油相中蛋黄卵磷脂和胆固醇琥珀酸单酯的质量比为3-5:1。
4.根据权利要求3所述的制备方法，其特征在于，步骤2)，油相注入水相的速率为0.5-2mL/min。
5.根据权利要求4所述的制备方法，其特征在于，步骤2)，MTX-HSA复合物与胆固醇琥珀酸单酯的质量比为1-2:1。
6.权利要求5所述方法制备得到的脂质体包人血清白蛋白载药纳米粒。
7.权利要求6所述脂质体包人血清白蛋白载药纳米粒在制备治疗炎症性疾病的药物中的用途。
8.根据权利要求7所述的用途，其特征在于，所述炎症性疾病包括类风湿性关节炎、骨关节炎、动脉粥样硬化、心肌梗死、脓毒症、慢性肾炎和胰腺。
9.一种用于治疗炎症性疾病的药物，其特征在于，其包括权利要求6所述脂质体包人血清白蛋白载药纳米粒，以及药学上可接受的辅料或载体。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310950606.0A CN116747196A (zh) | 2023-07-31 | 2023-07-31 | 脂质体包人血清白蛋白载药纳米粒及其制备方法和用途 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310950606.0A CN116747196A (zh) | 2023-07-31 | 2023-07-31 | 脂质体包人血清白蛋白载药纳米粒及其制备方法和用途 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116747196A true CN116747196A (zh) | 2023-09-15 |
Family
ID=87959262
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310950606.0A Pending CN116747196A (zh) | 2023-07-31 | 2023-07-31 | 脂质体包人血清白蛋白载药纳米粒及其制备方法和用途 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116747196A (zh) |
-
2023
- 2023-07-31 CN CN202310950606.0A patent/CN116747196A/zh active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | Cancer nanomedicines stabilized by π-π stacking between heterodimeric prodrugs enable exceptionally high drug loading capacity and safer delivery of drug combinations | |
| CN102740895B (zh) | 纳米轭合物以及纳米轭合物配制品 | |
| Li et al. | Synergistic effect of all-trans-retinal and triptolide encapsulated in an inflammation-targeted nanoparticle on collagen-induced arthritis in mice | |
| Long et al. | Alendronate-functionalized hypoxia-responsive polymeric micelles for targeted therapy of bone metastatic prostate cancer | |
| AU2006221448A1 (en) | Microparticle and pharmaceutical composition | |
| Li et al. | Reduction breakable cholesteryl pullulan nanoparticles for targeted hepatocellular carcinoma chemotherapy | |
| Wang et al. | Highly uniform ultrasound-sensitive nanospheres produced by a pH-induced micelle-to-vesicle transition for tumor-targeted drug delivery | |
| US11154514B2 (en) | Quinic acid-modified nanoparticles and uses thereof | |
| CN107050040A (zh) | Hifu控释的脑胶质瘤靶向纳米递药系统及其制备方法和用途 | |
| Fattal et al. | Nanomedicine technology: current achievements and new trends | |
| AU2018384096B2 (en) | Amphiphilic block copolymers, micelles, and methods for treating or preventing heart failure | |
| CN104324384A (zh) | 透明质酸-槲皮素结合物自组装胶束制剂及其制备方法 | |
| Gao et al. | Ultrasound-assisted C3F8-filled PLGA nanobubbles for enhanced FGF21 delivery and improved prophylactic treatment of diabetic cardiomyopathy | |
| Lin et al. | Self-targeted, bacillus-shaped, and controlled-release methotrexate prodrug polymeric nanoparticles for intratumoral administration with improved therapeutic efficacy in tumor-bearing mice | |
| Ma et al. | Hierarchical responsive micelle facilitates intratumoral penetration by acid-activated positive charge surface and size contraction | |
| Li et al. | Polysialic acid-functionalized liposomes for efficient honokiol delivery to inhibit breast cancer growth and metastasis | |
| CN109432049B (zh) | 一种具有肾脏靶向分布特性的大黄酸脂质囊纳米粒及应用 | |
| CN107126425A (zh) | 一种丹参酮iiapeg‑plga‑peg纳米粒及其制备方法 | |
| CN116747196A (zh) | 脂质体包人血清白蛋白载药纳米粒及其制备方法和用途 | |
| Yuan et al. | In vivo metabolizable branched poly (ester amide) based on inositol and amino acids as a drug nanocarrier for cancer therapy | |
| Hong et al. | Cancer‐cell‐biomimetic nanoparticles for enhanced prostate cancer therapy | |
| CN108498485A (zh) | 二氢青蒿素修饰的药物传递载体及其在药学中的应用 | |
| Wu et al. | Effects of liposomal simvastatin nanoparticles on vascular endothelial function and arterial smooth muscle cell apoptosis in rats with arteriosclerotic occlusive disease of lower limb via p38 mitogen-activated protein kinase nuclear factor kappa-b pathway | |
| Zhang et al. | PEGylation of ginsenoside rg3-entrapped bovine serum albumin nanoparticles: Preparation, characterization, and in vitro biological studies | |
| EP3355947A1 (en) | Compositions for an injectable, in situ forming neuroscaffold and methods of using the same |
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 |