CN116178493B - Calcium ion nano regulator for tumor radiotherapy sensitization and preparation method and application thereof - Google Patents
Calcium ion nano regulator for tumor radiotherapy sensitization and preparation method and application thereof Download PDFInfo
- Publication number
- CN116178493B CN116178493B CN202310081442.2A CN202310081442A CN116178493B CN 116178493 B CN116178493 B CN 116178493B CN 202310081442 A CN202310081442 A CN 202310081442A CN 116178493 B CN116178493 B CN 116178493B
- Authority
- CN
- China
- Prior art keywords
- cap
- gffpy
- radiotherapy
- pen
- calcium
- 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
- 238000001959 radiotherapy Methods 0.000 title claims abstract description 49
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 206010070834 Sensitisation Diseases 0.000 title claims abstract description 20
- 230000008313 sensitization Effects 0.000 title claims abstract description 20
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 title abstract description 4
- 229910001424 calcium ion Inorganic materials 0.000 title abstract description 4
- 210000004881 tumor cell Anatomy 0.000 claims abstract description 26
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 24
- 229920001184 polypeptide Polymers 0.000 claims abstract description 13
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 13
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims abstract description 10
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims abstract description 10
- 230000004094 calcium homeostasis Effects 0.000 claims abstract description 8
- 238000005232 molecular self-assembly Methods 0.000 claims abstract description 5
- YKPUWZUDDOIDPM-SOFGYWHQSA-N capsaicin Chemical compound COC1=CC(CNC(=O)CCCC\C=C\C(C)C)=CC=C1O YKPUWZUDDOIDPM-SOFGYWHQSA-N 0.000 claims description 50
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 26
- 229960002504 capsaicin Drugs 0.000 claims description 25
- 235000017663 capsaicin Nutrition 0.000 claims description 25
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 22
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 11
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 239000007787 solid Substances 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- IOGXOCVLYRDXLW-UHFFFAOYSA-N tert-butyl nitrite Chemical compound CC(C)(C)ON=O IOGXOCVLYRDXLW-UHFFFAOYSA-N 0.000 claims description 5
- 239000012414 tert-butyl nitrite Substances 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 4
- 239000011541 reaction mixture Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- 239000002840 nitric oxide donor Substances 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims 1
- 229940126585 therapeutic drug Drugs 0.000 claims 1
- 239000011575 calcium Substances 0.000 abstract description 80
- 230000003834 intracellular effect Effects 0.000 abstract description 10
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 abstract description 7
- 229910052791 calcium Inorganic materials 0.000 abstract description 7
- 230000008901 benefit Effects 0.000 abstract description 5
- 239000002086 nanomaterial Substances 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 238000006555 catalytic reaction Methods 0.000 abstract description 3
- 230000001276 controlling effect Effects 0.000 abstract description 3
- 230000001105 regulatory effect Effects 0.000 abstract description 3
- 108090000312 Calcium Channels Proteins 0.000 abstract 1
- 102000003922 Calcium Channels Human genes 0.000 abstract 1
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 45
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 40
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 26
- 210000004027 cell Anatomy 0.000 description 23
- 229960003180 glutathione Drugs 0.000 description 13
- 239000000017 hydrogel Substances 0.000 description 13
- 238000006243 chemical reaction Methods 0.000 description 12
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 239000000047 product Substances 0.000 description 9
- 238000010532 solid phase synthesis reaction Methods 0.000 description 9
- 238000005406 washing Methods 0.000 description 9
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 229930182555 Penicillin Natural products 0.000 description 6
- 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 6
- 239000003054 catalyst Substances 0.000 description 6
- 230000006378 damage Effects 0.000 description 6
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 6
- 210000003470 mitochondria Anatomy 0.000 description 6
- 229940049954 penicillin Drugs 0.000 description 6
- 230000005855 radiation Effects 0.000 description 6
- 239000011347 resin Substances 0.000 description 6
- 229920005989 resin Polymers 0.000 description 6
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 5
- 210000000805 cytoplasm Anatomy 0.000 description 5
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 5
- 201000002528 pancreatic cancer Diseases 0.000 description 5
- 208000008443 pancreatic carcinoma Diseases 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- XSGMGAINOILNJR-PGUFJCEWSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methyl-3-tritylsulfanylbutanoic acid Chemical compound CC(C)([C@H](NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C(O)=O)SC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 XSGMGAINOILNJR-PGUFJCEWSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 230000005540 biological transmission Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000007822 coupling agent Substances 0.000 description 4
- 230000001186 cumulative effect Effects 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- VANNPISTIUFMLH-UHFFFAOYSA-N glutaric anhydride Chemical compound O=C1CCCC(=O)O1 VANNPISTIUFMLH-UHFFFAOYSA-N 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 230000002438 mitochondrial effect Effects 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000002121 nanofiber Substances 0.000 description 4
- 102000005962 receptors Human genes 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 238000002390 rotary evaporation Methods 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- 102000003563 TRPV Human genes 0.000 description 3
- 108060008564 TRPV Proteins 0.000 description 3
- 102000003566 TRPV1 Human genes 0.000 description 3
- 101150016206 Trpv1 gene Proteins 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 230000001086 cytosolic effect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- AMXUJIHSMANWDW-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-(4-phosphonooxyphenyl)propanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=C(OP(O)(O)=O)C=C1 AMXUJIHSMANWDW-QFIPXVFZSA-N 0.000 description 2
- SJVFAHZPLIXNDH-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-phenylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=CC=C1 SJVFAHZPLIXNDH-QFIPXVFZSA-N 0.000 description 2
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- -1 Hexafluorophosphate Chemical compound 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 125000003963 dichloro group Chemical group Cl* 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 239000012065 filter cake Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- VOFUROIFQGPCGE-UHFFFAOYSA-N nile red Chemical compound C1=CC=C2C3=NC4=CC=C(N(CC)CC)C=C4OC3=CC(=O)C2=C1 VOFUROIFQGPCGE-UHFFFAOYSA-N 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- YQUVCSBJEUQKSH-UHFFFAOYSA-N protochatechuic acid Natural products OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- 238000001338 self-assembly Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- TUUBOHWZSQXCSW-UHFFFAOYSA-N vanillic acid Natural products COC1=CC(O)=CC(C(O)=O)=C1 TUUBOHWZSQXCSW-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- RMOGWMIKYWRTKW-UONOGXRCSA-N (S,S)-paclobutrazol Chemical compound C([C@@H]([C@@H](O)C(C)(C)C)N1N=CN=C1)C1=CC=C(Cl)C=C1 RMOGWMIKYWRTKW-UONOGXRCSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 102000000584 Calmodulin Human genes 0.000 description 1
- 108010041952 Calmodulin Proteins 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 239000005985 Paclobutrazol Substances 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 238000011284 combination treatment Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 239000011258 core-shell material Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000005251 gamma ray Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000004941 influx Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004065 mitochondrial dysfunction Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000009635 nitrosylation Effects 0.000 description 1
- 238000010534 nucleophilic substitution reaction Methods 0.000 description 1
- 238000013379 physicochemical characterization Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- FWYUJENICVGSJH-UHFFFAOYSA-M sodium;2-[bis[2-[2-(2-methyl-5-nitroimidazol-1-yl)ethoxy]-2-oxoethyl]amino]acetate Chemical compound [Na+].CC1=NC=C([N+]([O-])=O)N1CCOC(=O)CN(CC([O-])=O)CC(=O)OCCN1C([N+]([O-])=O)=CN=C1C FWYUJENICVGSJH-UHFFFAOYSA-M 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a calcium ion nano regulator for tumor radiotherapy sensitization, and a preparation method and application thereof. The self-assembled peptide GFFpY is taken as a framework to synthesize a polypeptide derivative CAP-GA-GFFpY-Pen-NO, and the polypeptide derivative CAP-P-NO can be formed into a calcium nano regulator CAP-P-NO which can destroy the calcium steady state in tumor cells through molecular self-assembly under the catalysis of alkaline phosphatase and is used for reversing the tolerance of tumors to radiotherapy. The invention aims to realize the imbalance of intracellular calcium homeostasis by utilizing the endogenous calcium of the organism and specifically regulating and controlling the calcium channel switch of tumor cells, and compared with the traditional calcium-based nanomaterial, the invention avoids the introduction of exogenous calcium, obviously improves the biological safety, obviously enhances the sensitivity of the tumor cells to radiotherapy after the calcium homeostasis is damaged, and provides an effective new radiotherapy sensitization strategy. The calcium nano regulator disclosed by the invention has the advantages of simple preparation process, good biocompatibility, good sensitization effect on tumor radiotherapy and bright clinical application prospect.
Description
Technical Field
The invention relates to the field of nano biological medicine materials, in particular to a preparation method of a peptide-based calcium ion (Ca 2+) nano regulator and application thereof in tumor radiotherapy sensitization.
Background
In recent years, the number of cancer attacks and deaths in China rise year by year, and the estimated death cases of the cancers in 2022 China reach five times in the United states, so that the increase of cancer burden seriously affects the social public health safety and the increase of national economy. Radiotherapy is one of three traditional treatments, and plays a significant role in clinical treatment of tumors, and more than 70% of cancer patients receive radiotherapy or radiotherapy combination treatment. However, the problems of radiation resistance caused by tumor tissue heterogeneity, normal tissue damage caused by radiation, and the like are always bottlenecks that are difficult to break through by various radiation treatment means. Therefore, development of a radiotherapy sensitizer capable of specifically improving the radiosensitivity of tumor cells and reducing damage of normal tissues has important significance for improving the curative effect of radiotherapy.
Tumor cells can develop tolerance through a variety of mechanisms during radiotherapy to circumvent direct and indirect damage caused by radiotherapy. Among them, endoplasmic reticulum stress caused by irradiation is one of the main causes of radiation therapy resistance in tumorigenesis. Specifically, calmodulin on the endoplasmic reticulum binds to tyrosine kinase in the presence of Ca 2+ causing a downstream series of signaling, ultimately protecting cells from ROS induced damage by radiotherapy by up-regulating the apoptosis regulator molecule MCL. The endoplasmic reticulum, which serves as the most important storage site for Ca 2+ within cells, is tightly regulated in tumor cells to maintain Ca 2+ homeostasis within the cytoplasm. Therefore, disruption of Ca 2+ homeostasis in tumor cells would theoretically amplify the ROS damage caused by irradiation, hopefully reversing tumor tolerance to radiation.
At present, a plurality of research teams design nano materials loaded with Ca 2+, and the purpose of destroying Ca 2+ steady state in tumor cells is achieved by transporting exogenous Ca 2+ to the tumor. For example, tan et al report a core-shell structured nanomaterial (ti 2 @cap) that specifically releases Ca 2+ in an ultrasonically activated acidic tumor microenvironment, inducing mitochondrial dysfunction (ANGEW CHEM INT ED ENGL 2021, 60:14051), and Zheng et al report a multi-channel Ca 2+ nano-modulator (CaNM CUR+CDDP) that, upon entry into tumor cells, significantly inhibited proliferation of tumors by burst release of Ca 2+ while inhibiting Ca 2+ efflux in combination with cisplatin-induced multi-stage destruction of mitochondria (Adv Mater 2021, 33: 2007426.). However, this transport of exogenous Ca 2+ not only places special demands on the carrier, but often causes abnormal activation of normal tissues such as muscles, nerves, etc. In fact, the gradient of extracellular Ca 2+ concentration (1-1.5 mM) versus cytoplasmic Ca 2+ concentration (about 100 nM) differs by more than 10000 times. Therefore, the disruption of Ca 2+ steady state by introducing endogenous high-concentration extracellular Ca 2+ into the cell is a safe and convenient strategy, and can avoid the damage to the organism caused by the introduction of the calcium-based nanomaterial. On the one hand, the maintenance of the tumor cytoplasm Ca 2+ steady-state depends on Ca 2+ channels such as transient receptor potential vanillic acid receptor 1 (TRPV 1) and the like which are highly expressed on the surface of a cell membrane to regulate the inflow of Ca 2+. On the other hand, tumor cells can also regulate Ca 2+ content in the cytoplasm by precisely controlling Ca 2+ release channels on the endoplasmic reticulum, such as fish ni Ding Danbai (RyR). Therefore, designing tumor intervention strategies targeting TRPV1 and RyR is expected to jointly achieve disruption of cytoplasmic Ca 2+ homeostasis by "intracellular Ca 2+ influx" in combination with "endoplasmic reticulum Ca 2+ release" to effectively reverse radiotherapy tolerance. Vanilloid, such as Capsaicin (CAP), is a typical TRPV1 agonist that can cause Ca 2+ to flow into cells by activating TRPV 1. RyR is very sensitive to intracellular redox due to its enrichment with cysteine, and Nitric Oxide (NO) can be inactivated by nitrosylation modification of the thiol group of cysteine, causing Ca 2+ to be released from the endoplasmic reticulum to the cytoplasm. In addition, the peptide-based nanomaterial has the advantages of simple preparation, easiness in multi-module modification, good biocompatibility and the like, and has a wide application prospect in drug delivery. Therefore, the invention aims to take self-assembled polypeptide as a framework, simultaneously adopts tumor microenvironment responsive chemical bonds to respectively connect CAP and NO, and prepares the Ca 2+ nanometer regulator capable of inducing Ca 2+ in tumor cells to have unbalanced steady state through a molecular self-assembly technology, and the regulator is used as a radiotherapy sensitizer to reverse the tolerance of tumors to radiotherapy.
Disclosure of Invention
The present invention aims to develop a peptide-based Ca 2+ nm modulator (CAP-P-NO) co-delivered by CAP and NO and use it to specifically disrupt Ca 2+ homeostasis in tumor cells to reverse tumor radiotherapy tolerance. The Ca 2+ nanometer regulator has the advantages of (1) being economical and easily available in raw materials and simple in preparation process, (2) being stable in nanofiber microstructure, (3) being capable of releasing CAP and Glutathione (GSH) in response to release NO, wherein for CAP release, the CAP-P-NO can release about 36% of CAP after 16 h treatment at pH 7.4, the release rate of CAP is obviously accelerated at pH 6.5, the CAP cumulative release rate is up to 96% after incubation for the same time, for NO release, under the condition of GSH concentration (2 mu M), only 16% of NO is released after 12 h, and 86% of NO is released after 12 h in tumor cells (10 mM), and (4) being capable of effectively breaking Ca 2+ steady state in tumor cells and in mitochondria, the content of Ca 2+ in the cells is 1.66 times of that of a blank control group after the treatment of ICP-MS result, the content of the CAP-P-NO is 2+ in the blank group is 1.07, compared with that of the blank control group, and the sensitivity of the Ca-P-NO can be obviously enhanced by the contrast group after the radiation of the radiotherapy is calculated to be almost 17, compared with that of the paclobutrazol in the experiment, and the experiment is remarkably lost after the experiment is carried out by the contrast to the contrast of the 1.17, and the experiment is calculated to have the contrast to be remarkably lost, and the contrast to the contrast of the contrast group and the contrast has been 1.
In order to achieve the purpose of the invention, the technical scheme of the invention is as follows:
A Ca 2+ nanometer regulator CAP-P-NO for tumor radiotherapy sensitization is characterized in that self-assembled peptide GFFpY is taken as a framework, capsaicin (CAP) and nitric oxide donor (Pen-NO) are respectively bonded at two ends of the framework, the obtained polypeptide derivative CAP-GA-GFFpY-Pen-NO forms a Ca 2+ nanometer regulator capable of inducing Ca 2+ steady-state unbalance in tumor cells through molecular self-assembly under the catalysis of alkaline phosphatase (ALP), and the chemical structure of the CAP-GA-GFFpY-Pen-NO is shown as a structural formula I:
Structural formula I.
The invention further discloses a preparation method of the Ca 2+ nanometer regulator for tumor radiotherapy sensitization, which is characterized by comprising the following preparation steps:
Dissolving 5-15 mg CAP-GA-GFFpY-Pen-NO in 1-3 mL PBS, adding Na 2CO3 to adjust pH to 7-8 to make it fully dissolved, then adding 5-15U ALP, mixing uniformly, standing at room temperature for 1-2h to obtain light green clear transparent hydrogel-like Ca 2+ nano regulator CAP-P-NO.
The preparation method of the polypeptide derivative CAP-GA-GFFpY-Pen-NO comprises the following steps:
(1) Dissolving 100-200 mg CAP-GA-GFFpY-Pen in 2-4 mL anhydrous N, N-Dimethylformamide (DMF), and pre-cooling at-10-0deg.C under nitrogen protection for 0.5-1 hr;
(2) Slowly dripping 100-200 mu L of tert-butyl nitrite into the system (1) under the condition of avoiding light, and carrying out light-resistant reaction at-10-0 ℃ under the protection of nitrogen for 2-4 h;
(3) Dropwise adding the reaction mixture into 30-60mL of glacial ethyl ether, separating out light green precipitate, centrifuging at 8000-10000 rpm for 10-15 min, and repeatedly washing with glacial ethyl ether for three times to remove residual DMF, wherein the whole process is protected from light;
(4) And (3) drying the solid obtained by centrifugation in vacuum to obtain a light green powdery CAP-GA-GFFpY-Pen-NO pure product.
The CAP-GA-GFFpY-Pen is characterized in that,
(1) It is prepared by solid phase synthesis;
(2) The chemical structure is shown as a structural formula II;
Structural formula II.
The invention also discloses an application of the Ca 2+ nanometer regulator CAP-P-NO for tumor radiotherapy sensitization in preparing drugs for specifically destroying Ca 2+ steady state in tumor cells to reverse tumor radiotherapy tolerance. The invention forms a Ca 2+ nano regulator capable of inducing the imbalance of Ca 2+ in tumor cells to realize the reversion of the tolerance of tumors to radiotherapy by molecular self-assembly, and breaks the steady state of cytosolic Ca 2+ by specifically regulating and controlling transient receptor potential vanillic acid receptor 1 (TRPV 1) of Ca 2+ in a channel on tumor cell membranes and releasing channel Raney Ding Danbai (RyR) by Ca 2+ on endoplasm, thereby promoting the overload of Ca 2+ in mitochondria and further reversing the tolerance of tumors to radiotherapy, thus being a brand-new radiotherapy sensitization strategy. Experimental results show that the CAP-P-NO is a light green, clear and transparent hydrogel macroscopically, and the microstructure is a compact three-dimensional network nanofiber structure. The CAP-P-NO can effectively break up Ca 2+ in tumor cells and mitochondria, ICP-MS results show that after being treated by CAP-P-NO, the content of intracellular Ca 2+ is 1.66 times that of a blank control group, the content of intracellular Ca 2+ is 3.07 times that of the blank control group, the CAP-P-NO can obviously enhance the sensitivity of the tumor cells to radiotherapy, pancreatic cancer Panc-1 cells which are tolerant to radiotherapy are taken as models, experiments are cloned and formed, after being pretreated by CAP-P-NO, 6Gy dose irradiation is given, and compared with a pure irradiation group, the clone groups almost completely disappear, and the radiotherapy sensitization ratio is 1.58 which is far higher than that of commercially available sodium glycidazole (1.17) through calculation of a survival curve.
The invention is described in more detail below:
The method comprises the steps of synthesizing CAP-GA through esterification reaction, synthesizing polypeptide derivatives CAP-GA-GFFpY-Pen through classical solid phase synthesis, further carrying out nucleophilic substitution reaction with tert-butyl nitrite to generate polypeptide derivatives CAP-GA-GFFpY-Pen-NO loaded with NO, and self-assembling under ALP catalysis to form the peptide-based Ca 2+ nano regulator CAP-P-NO with three-dimensional network nano fiber microstructure. CAP-P-NO can release CAP and NO in cells under the slightly acidic and high GSH microenvironment of the tumor respectively, so that TRPV1 is activated simultaneously to cause Ca 2+ to flow into cells and inactive RyR to release Ca 2+ from the endoplasmic reticulum to cytoplasm, and the steady-state unbalance of Ca 2+ in tumor cells is realized, so that the tolerance of the tumor to radiotherapy is reversed.
A preparation method of a Ca 2+ nanometer regulator for tumor radiotherapy sensitization comprises the following steps:
Dissolving 5-15 mg CAP-GA-GFFpY-Pen-NO in 1-3 mL PBS, adding Na 2CO3 to adjust pH to 7-8 to make it fully dissolved, then adding 5-15U ALP, mixing uniformly, standing at room temperature for 1-2h to obtain light green clear transparent hydrogel-like Ca 2+ nano regulator CAP-P-NO. The preparation method of the CAP-GA-GFFpY-Pen-NO comprises the following steps:
(1) Dissolving 100-200 mg CAP-GA-GFFpY-Pen in 2-4 mL anhydrous N, N-Dimethylformamide (DMF), and pre-cooling at-10-0deg.C under nitrogen protection for 0.5-1 hr;
(2) Slowly dripping 100-200 mu L of tert-butyl nitrite into the system (1) under the condition of avoiding light, and carrying out light-resistant reaction at-10-0 ℃ under the protection of nitrogen for 2-4 h;
(3) Dropwise adding the reaction mixture into 30-60mL of glacial ethyl ether, separating out light green precipitate, centrifuging at 8000-10000 rpm for 10-15 min, and repeatedly washing with glacial ethyl ether for three times to remove residual DMF, wherein the whole process is protected from light;
(4) And (3) drying the solid obtained by centrifugation in vacuum to obtain a light green powdery CAP-GA-GFFpY-Pen-NO pure product.
The preparation method of the CAP-GA-GFFpY-Pen comprises the following steps:
(1) Weighing 0.5-1g of dichloro resin in a solid phase synthesis tube, adding 10-20 mL Dichloromethane (DCM) into the solid phase synthesis tube, soaking for 5-10min to fully swell the resin, and extruding the DCM in the synthesis tube by using an ear washing ball;
(2) Fmoc-S-Trityl-L-PENICILLAMINE (0.5-1 mmol,306-712 mg) is weighed in a penicillin bottle, 10-20 mLDCM and catalyst N, N-Diisopropylethylamine (DIEA) (1-2 mmol,200-400 mu L) are sequentially added, and after full dissolution, a solid phase synthesis tube is added for reaction at room temperature of 2-4 h;
(3) The reaction solution was extruded, washed five times with DCM, and 10-20 mL blocking solution (DCM: CH 3 OH: diea=17:2:1) was added to react 0.5-1 h to block the remaining active reaction sites;
(4) Sequentially cleaning with DCM and DMF for five times, adding 20% piperidine (15-30 mL) to remove Fmoc protecting group on Fmoc-S-Trityl-L-PENICILLAMINE, and exposing active amino group;
(5) Fmoc-Tyr (H 2PO3) -OH (2-4 mmol,966-1932 mg), coupling agent O-benzotriazol-tetramethyluronium Hexafluorophosphate (HBTU) (2-4 mmol,758-1516 mg) and catalyst DIEA (4-8 mmol, 800-1600. Mu.L) were weighed five times and dissolved in DMF in a penicillin bottle and added to a solid phase synthesis tube for reaction 2-4H;
(6) Repeating the steps (4) and (5), and coupling Fmoc-Phe-OH, fmoc-Gly-OH and CAP-GA in sequence. Unreacted amino acid, catalyst and coupling agent were removed by washing sequentially with DMF and DCM, after which 95% trifluoroacetic acid (TFA) was added (H 2 O: TIS: tfa=1:1:95) to cleave the peptide chain from the resin;
(7) TFA was removed by rotary evaporation, and to the resulting viscous liquid was added anhydrous diethyl ether to precipitate and collect crude polypeptide. Finally, the CAP-GA-GFFpY-Pen pure product is obtained by High Performance Liquid Chromatography (HPLC) separation and purification.
Furthermore, the CAP-GA is prepared by the following steps:
(1) Glutaric Anhydride (GA) (5-10 mmol,570-1140 mg) and Capsaicin (CAP) (1.5-3 mmol,440-880 mg) are added into 20-40 mL anhydrous pyridine, and reacted at room temperature for 18-24 h;
(2) After the reaction is finished, the solvent is removed by rotary evaporation, 1N hydrochloric acid is added under stirring to precipitate solid, a filter cake is obtained after filtration, and finally, the CAP-GA pure product is obtained by cold water washing and vacuum drying.
The raw materials or the reagents involved in the invention are all common commercial products, and the related operations are all routine operations in the field unless specified.
The invention has the beneficial effects that:
the invention prepares a peptide-based Ca 2+ nanometer regulator co-delivered by CAP and NO and uses the peptide-based Ca 2+ nanometer regulator to specifically destroy Ca 2+ in tumor cells to reverse the tolerance of the tumor to radiotherapy, has the advantages of simple preparation, easily obtained raw materials, NO introduction of exogenous Ca 2+, small toxic and side effects on normal tissues, and (3) dual-responsiveness drug release of weak acid and GSH to precisely regulate and control Ca 2+ inflow and release channels of the tumor cells so as to specifically break Ca 2+ in the cells, and (4) improves the sensitivity of the tumor to radiotherapy by breaking Ca 2+ in the cells, thereby being a novel radiotherapy sensitization strategy and hopefully providing a new paradigm for clinical tumor radiotherapy.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention;
For a clearer description of an embodiment of the invention or of the solutions of the prior art, reference will be made to the accompanying drawings, which are used in the description of the embodiment or of the prior art, it being obvious to a person skilled in the art that other drawings can be obtained from these without inventive effort;
FIG. 1 shows the results of high-resolution mass spectrum (A) and nuclear magnetic resonance hydrogen spectrum (B) of CAP-GA-GFFpY-Pen-NO prepared in example 3 of the present invention;
FIG. 2 is a photograph of a hydrogel-like Ca 2+ nm modulator formed by the polypeptide derivative CAP-GA-GFFpY-Pen-NO by an enzymatic self-assembly process;
FIG. 3 is a physicochemical characterization of the hydrogel-like Ca 2+ nm modulator CAP-P-NO. Wherein A is the critical assembly concentration measurement of the hydrogel, and B is the transmission electron microscope photo of the hydrogel;
FIG. 4 shows the drug release profile of CAP-P-NO. Wherein A is the release curve of CAP-P-NO under different GSH concentrations, and B is the release curve of CAP-P-NO under different pH conditions;
FIG. 5 shows the intracellular and mitochondrial Ca 2+ content after ICP-MS measurement with CAP-P-NO. Wherein A is the intracellular Ca 2+ content, and B is the mitochondrial Ca 2+ content;
FIG. 6 shows images of clones from pancreatic cancer Panc-1 cells incubated with CAP-P-NO or PBS and given different doses of radiation (A) and cell survival curves (B).
Detailed Description
In order that the above objects, features and advantages of the invention will be more clearly understood, a further description of the invention will be made.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced otherwise than as described herein, and it is apparent that the embodiments in the specification are only some, rather than all, of the embodiments of the present invention.
Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
The preparation method of CAP-GA comprises the following steps:
(1) Glutaric Anhydride (GA) (5 mmol,570 mg) and Capsaicin (CAP) (1.5 mmol,440 mg) were added to 20 mL anhydrous pyridine solution and reacted at room temperature 18 h;
(2) After the reaction is finished, the solvent is removed by rotary evaporation, 1N hydrochloric acid is added under stirring to precipitate solid, a filter cake is obtained after filtration, and finally, the CAP-GA pure product is obtained by cold water washing and vacuum drying.
Example 2
The preparation method of the CAP-GA-GFFpY-Pen comprises the following steps:
(1) Weighing 0.5g of dichloro resin in a solid phase synthesis tube, adding 10 mL Dichloromethane (DCM) to soak 5min to fully swell the resin, and extruding the DCM in the synthesis tube by using an ear washing ball;
(2) Fmoc-S-Trityl-L-PENICILLAMINE (0.5 mmol,306 mg) was weighed into a penicillin bottle, 10 mL DCM and catalyst N, N-Diisopropylethylamine (DIEA) (1 mmol, 200. Mu.L) were added sequentially, after complete dissolution, the mixture was added into a solid phase synthesis tube, and reacted at room temperature for 2h;
(3) The reaction was squeezed out, washed five times with DCM, and 10 mL blocking solution (DCM: CH 3 OH: diea=17:2:1) was added to react 0.5: 0.5 h to block the remaining active reaction sites;
(4) Sequentially cleaning with DCM and DMF for five times, adding 20% piperidine (15 mL) to remove Fmoc protecting group on Fmoc-S-Trityl-L-PENICILLAMINE, and exposing active amino group;
(5) Fmoc-Tyr (H 2PO3) -OH (2 mmol,966 mg), coupling agent O-benzotriazol-tetramethyluronium Hexafluorophosphate (HBTU) (2 mmol,758 mg) and catalyst DIEA (4 mmol, 800. Mu.L) were weighed out in a vial and added to a solid phase synthesis tube after complete dissolution in DMF for reaction 2H;
(6) Repeating the steps (4) and (5), and coupling Fmoc-Phe-OH, fmoc-Gly-OH and CAP-GA in sequence. Unreacted amino acid, catalyst and coupling agent were removed by washing sequentially with DMF and DCM, after which 95% trifluoroacetic acid (TFA) was added (H 2 O: TIS: tfa=2.5:2.5:95) to cleave the peptide chain from the resin;
(7) TFA was removed by rotary evaporation, and to the resulting viscous liquid was added anhydrous diethyl ether to precipitate and collect crude polypeptide. Finally, the CAP-GA-GFFpY-Pen pure product is obtained by High Performance Liquid Chromatography (HPLC) separation and purification.
The chemical structure of CAP-GA-GFFpY-Pen is confirmed by nuclear magnetic resonance hydrogen spectrum, and the nuclear magnetic data are as follows:
1HNMR (300 MHz, DMSO) δ 8.37-8.09 (m, 4H), 7.99 (dd, J=13.6, 7.1Hz, 2H), 7.30-7.13 (m, 12H), 7.06 (d, J=8.1Hz, 2H), 6.99 (d, J=7.9Hz, 2H), 6.80 (d, J=8.2Hz,1H), 4.76-4.66 (m, 1H), 4.58-4.41 (m, 3H), 4.24 (d, J=5.7Hz, 2H), 3.72 (s, 3H), 3.67-3.47 (m, 2H), 3.09-2.60 (m, 8H), 2.21 (t, J=7.2Hz, 2H), 2.13 (t, J=7.3Hz,2H), 1.81 (dt, J=14.2, 7.1Hz, 2H), 1.55-1.46 (m, 2H), 1.39 (d, J=10.4Hz, 6H), 1.28-1.18 (m, 10H), 0.85 (t, J = 6.5 Hz, 3H).
example 3
The preparation method of CAP-GA-GFFpY-Pen-NO comprises the following steps:
(1) 100 mg CAP-GA-GFFpY-Pen is dissolved in 2 mL anhydrous N, N-Dimethylformamide (DMF), and then precooled for 0 ℃ for 0.5h under the protection of nitrogen;
(2) Slowly dropwise adding 100 mu L of tert-butyl nitrite into the system (1) under the dark condition, and carrying out dark reaction at 0 ℃ under the protection of nitrogen for 2 h;
(3) Dropwise adding the reaction mixture into 30mL of glacial ethyl ether, separating out a light green precipitate, centrifuging 8000 rpm, 10min, and repeatedly washing three times by using the glacial ethyl ether to remove residual DMF, wherein the whole process is carefully protected from light;
(4) And (3) drying the solid obtained by centrifugation in vacuum to obtain a light green powdery CAP-GA-GFFpY-Pen-NO pure product.
Referring to FIG. 1, the detection of the obtained product by using high resolution mass spectrum (A) and nuclear magnetic resonance hydrogen spectrum (B) verifies that the obtained polypeptide derivative has correct chemical structure, and the nuclear magnetic data is as follows ：1H NMR (300 MHz, DMSO) δ 8.37–8.32 (m, 1H), 8.18 (dd, J=17.4, 7.2 Hz, 2H), 8.05–7.94 (m, 3H), 7.24–7.14(m, 12H), 7.05–6.96 (m, 4H), 6.80 (d, J=7.8 Hz, 1H), 5.09 (d, J=9.0 Hz, 1H), 4.66 (d, J=6.8 Hz, 1H), 4.57–4.44 (m, 2H), 4.24 (d, J=5.0 Hz, 2H), 3.72 (s, 3H), 3.59–3.33 (m, 2H), 3.03–2.62(m, 8H), 2.21 (t, J=6.6 Hz, 2H), 2.13 (t, J=7.0 Hz, 2H), 1.92 (d, J=3.4 Hz, 6H), 1.84–1.75 (m, 2H), 1.56–1.46 (m, 2H), 1.28–1.18(m, 10H), 0.85 (t, J=6.2 Hz, 3H).
Example 4
A preparation method of a Ca 2+ nanometer regulator for tumor radiotherapy sensitization comprises the following steps:
Dissolving 5 mg CAP-GA-GFFpY-Pen-NO in 1 mL PBS, adding Na 2CO3 to adjust pH to 7.4 to dissolve thoroughly, then adding 5U ALP, mixing well, standing at room temperature for 1h to obtain light green clear transparent hydrogel-like Ca 2+ nanometer regulator CAP-P-NO (figure 2).
Example 5
The physicochemical property of CAP-P-NO is characterized by the following steps:
(1) Critical assembly concentration determination 10. Mu.L of Nile Red working solution (1X 10 -6 mol/L) was added to the bottom of the brown EP tube and dried in a 60℃oven for 2 h. Then hydrogel with the concentration of 2 mg/mL is prepared according to the method of example 4, the hydrogel is subjected to gradient dilution, the hydrogel solution 1 mL after gradient dilution is respectively taken and added into a dried EP tube containing nile red, the ultrasound is 30 and min, and the mixture is left to stand at normal temperature for overnight, so that the hydrogel and the dye are fully mixed. The next day uses a fluorescence spectrophotometer to detect the solutions with various concentrations from low to high, the detection wavelength is 560-720 nm, the step length is 5 nm, the voltage is 650V, and the solution concentration corresponding to the change of the absorption peak is recorded.
(2) Microcosmic appearance observation A hydrogel with a concentration of 5 mg/mL was prepared according to the method of example 4, 10. Mu.L of the prepared hydrogel was pipetted and dropped onto a transmission electron microscope copper mesh and left to stand for 2min, then the excess gel was blotted with filter paper, and the negative dye was performed for 2min with uranyl acetate, and after drying, the microcosmic appearance was observed under a transmission electron microscope.
Referring to FIG. 3A, the critical assembly concentration of CAP-GA-GFFpY-Pen-NO is about 181.5 mu M, which indicates that the assembly can be carried out at a lower concentration, and the assembly has good enzymatic self-assembly performance. Further observing the microscopic morphology by a transmission electron microscope, as shown in fig. 3B, the microscopic morphology of CAP-P-NO is a compact three-dimensional network nanofiber structure.
Example 6
GSH-responsive NO release and weak acid-responsive CAP release, as follows:
(1) GSH-responsive NO Release A hydrogel CAP-P-NO was prepared at a concentration of 5mg/mL according to the method of example 4, and 100. Mu.L was pipetted into a clear penicillin bottle as the sample to be tested. 50. Mu.L of PBS containing GSH (0. Mu.M, 2. Mu.M, 10 mM) was added to the hydrogel surface and the penicillin bottles were placed in a 37℃incubator. 50. Mu.L of the supernatant was pipetted into a 96-well plate at a predetermined time point, 50. Mu. LGriess reagent 1 and 50. Mu. LGriess reagent 2 were sequentially added, and after thoroughly mixing, the absorbance of the solution at 540 nm was measured with an enzyme-labeled instrument, and the NO release rate was calculated from the concentration-absorbance standard curve. Note that the same volume of fresh PBS containing GSH was added after each supernatant removal.
(2) Weak acid responsive CAP Release hydrogel CAP-P-NO was prepared at a concentration of 5mg/mL according to the procedure of example 4 and 200. Mu.L was pipetted into a clear penicillin bottle as the sample to be tested. 50. Mu.L of PBS of different pH (7.4, 6.5) was added to the hydrogel surface and the vial was placed in a 37℃incubator. After 50. Mu.L of supernatant was pipetted at a predetermined time point and thoroughly mixed with 50. Mu.L of methanol, HPLC detection was performed, and the CAP release rate was calculated from the concentration-peak area standard curve. Note that fresh PBS at ph7.4 or 6.5 was added to the same volume after each supernatant removal.
FIG. 4A shows the cumulative release profile of NO under various conditions, showing that NO is gradually released from CAP-P-NO over time, and that the release rate has a significant GSH concentration dependence. After 24h incubation with PBS alone or GSH (2. Mu.M), CAP-P-NO released less than 20% of the NO. Under GSH (10 mM) condition, the NO release speed is obviously accelerated, and the accumulated NO release rate of CAP-P-NO after 24 hours is up to 95%.
The CAP cumulative release profile of CAP-P-NO is shown in FIG. 4B, which shows that about 36% of CAP is released after 16 h of CAP-P-NO at pH 7.4. And at pH of 6.5, the release speed of CAP is obviously accelerated, and the cumulative release rate of CAP after 16 h is up to more than 96%.
Example 7
ICP-MS measures intracellular and mitochondrial Ca 2+ content as follows:
After incubating pancreatic cancer Panc-1 cells with CAP-P-NO (50 μm) or pure PBS for 4h, the cells were digested, centrifuged, collected and counted, and the intracellular Ca 2+ content was determined by ICP-MS after the same number of cells were taken out from each group. For determination of intra-mitochondrial Ca 2+ content, pancreatic cancer Panc-1 cells were incubated with CAP-P-NO (50 μm) or pure PBS for 4: 4h, and after digestion, centrifugation, cell collection and counting, the same number of cells were taken from each group to extract mitochondria using a mitochondrial extraction kit, after which digestion was performed and the Ca 2+ content was determined by ICP-MS.
Referring to FIG. 5, after the CAP-P-NO was reacted, the Ca 2+ content in the cells and mitochondria was significantly increased, 1.66 times and 3.07 times, respectively, compared to the blank group, demonstrating that CAP-P-NO has significant intracellular Ca 2+ steady state destructive power.
Example 8
The clone formation experiment evaluates the radiotherapy sensitization effect of CAP-P-NO, and the steps are as follows:
(1) Pancreatic cancer Panc-1 cells were seeded at a density of 500 per well in 12-well plates and incubated 24: 24h in a CO 2 incubator;
(2) The medium was replaced with fresh serum-free medium with/without CAP-P-NO (25. Mu.M) and incubation was continued for 12 h;
(3) The culture medium is replaced by fresh complete culture medium, then different doses of gamma-ray irradiation (0, 2, 4, 6 Gy) are given, and then the culture is continued for about 7 days;
(4) When the colony grows to the macroscopic size, dyeing the colony with 0.25% crystal violet ethanol solution;
(5) Shooting and counting the clone groups, and calculating the radiotherapy sensitization ratio according to the number of the clone groups.
As shown in FIG. 6, which shows the clone formation image (A) and the cell survival curve (B), the number of the clone clusters of CAP-P-NO and PBS groups was almost unchanged without irradiation, and a significant decrease in the clone cluster number was observed after CAP-P-NO pretreatment, and the proliferation of cells was significantly inhibited after low dose (2 Gy) irradiation, demonstrating that the sensitivity of the tumor cells to radiotherapy was significantly improved after the Ca 2+ steady state was disrupted by CAP-P-NO. Further, the radiotherapy sensitization ratio of CAP-P-NO is 1.58 which is far higher than that of the commercially available radiotherapy sensitizer sodium glycididazole (1.17) through calculation of a cell survival curve.
Claims (3)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310081442.2A CN116178493B (en) | 2023-02-08 | 2023-02-08 | Calcium ion nano regulator for tumor radiotherapy sensitization and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310081442.2A CN116178493B (en) | 2023-02-08 | 2023-02-08 | Calcium ion nano regulator for tumor radiotherapy sensitization and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116178493A CN116178493A (en) | 2023-05-30 |
| CN116178493B true CN116178493B (en) | 2025-03-14 |
Family
ID=86439772
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310081442.2A Active CN116178493B (en) | 2023-02-08 | 2023-02-08 | Calcium ion nano regulator for tumor radiotherapy sensitization and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116178493B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN120025400A (en) * | 2025-04-24 | 2025-05-23 | 中国医学科学院放射医学研究所 | Peptide-based iron death inducer for improving curative effect of melanoma radiotherapy and preparation method and application thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6093743A (en) * | 1998-06-23 | 2000-07-25 | Medinox Inc. | Therapeutic methods employing disulfide derivatives of dithiocarbamates and compositions useful therefor |
| WO2007087452A2 (en) * | 2006-01-27 | 2007-08-02 | Theraquest Biosciences, Llc | Abuse resistant and extended release formulations and method of use thereof |
| EP3056902B1 (en) * | 2015-02-16 | 2020-04-01 | Universite De Bordeaux | Novel voltage dependent ion channel fusions and method of use thereof |
| TW201815395A (en) * | 2016-10-27 | 2018-05-01 | 德瑪製藥公司 | Use of dianhydrogalactitol or derivatives or analogs thereof for treatment of pediatric central nervous system malignancies |
| CN113082222B (en) * | 2021-04-20 | 2022-09-20 | 中国医学科学院放射医学研究所 | Peptide-based nano-drug targeting tumor cell mitochondria and preparation method and application thereof |
| CN113788878B (en) * | 2021-09-24 | 2023-08-01 | 成都赛恩贝外科学研究院 | Self-assembled short peptides, their use in broad-spectrum vaccines and biomedical science |
-
2023
- 2023-02-08 CN CN202310081442.2A patent/CN116178493B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| A Dual-Channel Ca2+ Nanomodulator Induces Intracellular Ca2+ Disorders via Endogenous Ca2+ Redistribution for Tumor Radiosensitization;Dianyu Wang等;ADVANCED MATERIALS;20240501;第36卷(第29期);第2401222页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116178493A (en) | 2023-05-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ding et al. | A NO/ROS/RNS cascaded-releasing nano-platform for gas/PDT/PTT/immunotherapy of tumors | |
| CN114044898B (en) | Lysine grafted polyethyleneimine cationic gene vector and preparation method and application thereof | |
| CN116178493B (en) | Calcium ion nano regulator for tumor radiotherapy sensitization and preparation method and application thereof | |
| CN114306282B (en) | A diagnosis and treatment integrated drug-loaded nanoparticle that enhances the effect of ferroptosis and its preparation method and application | |
| CN111544596A (en) | A kind of GSH-responsive nanodiamond targeting drug and its preparation method and application | |
| Yang et al. | Multifunctional organic nanomaterials with ultra-high photothermal conversion efficiency for photothermal therapy and inhibition of cancer metastasis | |
| Liu et al. | Curcumin copolymerized drugs mediated by enteric-coated polymers: Their design, synthesis and biocompatibility cell imaging studies | |
| Luo et al. | A pH/ROS dual-responsive nanoparticle system for tumor targeting combined chemotherapy/phototherapy | |
| Wu et al. | Preparation of a chlorophyll derivative and investigation of its photodynamic activities against cholangiocarcinoma | |
| CN113817164B (en) | Hypoxic sensitive drug carrier polymer and preparation method and application thereof | |
| Kang et al. | A water-dependent reversible photoacidity strategy for cancer treatment | |
| Bu et al. | Cocrystallization-driven self-assembly with vanillic acid offers a new opportunity for surmounting fast and excessive absorption issues of antifungal drug 5-fluorocytosine: a combined theoretical and experimental research | |
| CN114853810A (en) | A kind of curcumin derivative and its preparation method and application | |
| He et al. | Nanobubble-mediated co-delivery of siTRIM37 and IR780 for gene and sonodynamic combination therapy against triple negative breast cancer | |
| CN113549143A (en) | Anti-tumor polypeptide Bax-BH3, fluorescent polymer nano micelle and preparation method and application thereof | |
| Liang et al. | Activatable Photosensitizer Prodrug for Self‐Amplified Immune Therapy Via Pyroptosis | |
| CN116102622A (en) | Supermolecule polypeptide radiotherapy sensitizer for inhibiting tumor apoptosis evasion and preparation method and application thereof | |
| CN114788875A (en) | Supermolecule nano-drug for activating Hippo pathway and preparation method and application thereof | |
| CN103304569A (en) | Erlotinib-phthalocyanine conjugate and preparation method thereof | |
| CN116063354B (en) | Reduction-response chemotherapy-immunotherapy prodrug, nano micelle and preparation method | |
| CN114470229B (en) | Preparation and application of carrier-free double-drug self-assembled nanoparticle | |
| CN116120390B (en) | Tripterine derivative and preparation method and application thereof | |
| CN115920036A (en) | Amphiphilic ROS-responsive photosensitizer-drug conjugate, ROS-responsive carrier-free nanomedicine, preparation method and application | |
| CN114404364A (en) | Construction of photosensitive nano-micelle capable of inducing polarization of tumor-associated macrophage M1 type and anti-tumor application thereof | |
| CN110734429B (en) | Usnea nicotinamide compound and preparation method and application thereof |
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 |