CN109646675A - A kind of pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule - Google Patents
A kind of pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule Download PDFInfo
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- CN109646675A CN109646675A CN201910085391.4A CN201910085391A CN109646675A CN 109646675 A CN109646675 A CN 109646675A CN 201910085391 A CN201910085391 A CN 201910085391A CN 109646675 A CN109646675 A CN 109646675A
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- Prior art keywords
- tio
- dehydrated alcohol
- centrifuged
- titanium dioxide
- ultrapure water
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- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 title claims abstract description 134
- 239000012528 membrane Substances 0.000 title claims abstract description 42
- 239000004408 titanium dioxide Substances 0.000 title claims abstract description 42
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 34
- 230000003592 biomimetic effect Effects 0.000 title claims abstract description 31
- 230000001413 cellular effect Effects 0.000 title claims abstract description 31
- 239000008187 granular material Substances 0.000 title claims abstract description 31
- JODKFOVZURLVTG-UHFFFAOYSA-N 2-bromo-1-(3,3-dinitroazetidin-1-yl)ethanone Chemical compound [O-][N+](=O)C1([N+]([O-])=O)CN(C(=O)CBr)C1 JODKFOVZURLVTG-UHFFFAOYSA-N 0.000 claims abstract description 43
- 108010054147 Hemoglobins Proteins 0.000 claims abstract description 35
- 102000001554 Hemoglobins Human genes 0.000 claims abstract description 35
- 229910010413 TiO 2 Inorganic materials 0.000 claims abstract description 31
- 210000004027 cell Anatomy 0.000 claims abstract description 28
- 239000002105 nanoparticle Substances 0.000 claims abstract description 27
- 239000007788 liquid Substances 0.000 claims abstract description 17
- 210000000170 cell membrane Anatomy 0.000 claims abstract description 14
- 230000002708 enhancing effect Effects 0.000 claims abstract description 10
- 238000001125 extrusion Methods 0.000 claims abstract description 9
- 238000002360 preparation method Methods 0.000 claims abstract description 8
- 238000005336 cracking Methods 0.000 claims abstract description 7
- 230000009089 cytolysis Effects 0.000 claims abstract description 7
- 238000011938 amidation process Methods 0.000 claims abstract description 3
- 230000004048 modification Effects 0.000 claims abstract 2
- 238000012986 modification Methods 0.000 claims abstract 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 61
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 51
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 51
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 42
- 239000012498 ultrapure water Substances 0.000 claims description 42
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 39
- 238000003756 stirring Methods 0.000 claims description 34
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 19
- 229910052760 oxygen Inorganic materials 0.000 claims description 19
- 239000001301 oxygen Substances 0.000 claims description 19
- 238000002604 ultrasonography Methods 0.000 claims description 19
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- 239000000908 ammonium hydroxide Substances 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 18
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 claims description 15
- 229910052681 coesite Inorganic materials 0.000 claims description 15
- 229910052906 cristobalite Inorganic materials 0.000 claims description 15
- 229910052682 stishovite Inorganic materials 0.000 claims description 15
- 229910052905 tridymite Inorganic materials 0.000 claims description 15
- 150000001875 compounds Chemical class 0.000 claims description 14
- 238000005119 centrifugation Methods 0.000 claims description 11
- 239000011259 mixed solution Substances 0.000 claims description 11
- 229920000515 polycarbonate Polymers 0.000 claims description 11
- 239000004417 polycarbonate Substances 0.000 claims description 11
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 206010006187 Breast cancer Diseases 0.000 claims description 8
- 208000026310 Breast neoplasm Diseases 0.000 claims description 8
- 230000001376 precipitating effect Effects 0.000 claims description 8
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 claims description 7
- 229910052719 titanium Inorganic materials 0.000 claims description 7
- 239000010936 titanium Substances 0.000 claims description 7
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 claims description 6
- 238000013019 agitation Methods 0.000 claims description 6
- 238000001354 calcination Methods 0.000 claims description 6
- 239000006185 dispersion Substances 0.000 claims description 6
- 238000000227 grinding Methods 0.000 claims description 6
- 239000001863 hydroxypropyl cellulose Substances 0.000 claims description 6
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 claims description 6
- 238000010992 reflux Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 230000001464 adherent effect Effects 0.000 claims description 5
- 239000000377 silicon dioxide Substances 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 claims description 4
- YNAVUWVOSKDBBP-UHFFFAOYSA-N Morpholine Natural products C1COCCN1 YNAVUWVOSKDBBP-UHFFFAOYSA-N 0.000 claims description 4
- 210000003743 erythrocyte Anatomy 0.000 claims description 4
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 claims description 4
- 239000012139 lysis buffer Substances 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 239000002244 precipitate Substances 0.000 claims description 4
- 238000009214 sonodynamic therapy Methods 0.000 claims description 4
- 230000000118 anti-neoplastic effect Effects 0.000 claims description 3
- 210000002540 macrophage Anatomy 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 2
- 210000004369 blood Anatomy 0.000 claims description 2
- 239000008280 blood Substances 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 206010017758 gastric cancer Diseases 0.000 claims description 2
- 201000007270 liver cancer Diseases 0.000 claims description 2
- 208000014018 liver neoplasm Diseases 0.000 claims description 2
- 201000005296 lung carcinoma Diseases 0.000 claims description 2
- 201000011549 stomach cancer Diseases 0.000 claims description 2
- 238000010257 thawing Methods 0.000 claims description 2
- -1 3- dimethylamino-propyl Chemical group 0.000 claims 2
- 238000005406 washing Methods 0.000 claims 2
- 229910021529 ammonia Inorganic materials 0.000 claims 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims 1
- 229940072440 bovine lactoferrin Drugs 0.000 claims 1
- 239000007853 buffer solution Substances 0.000 claims 1
- 229910052799 carbon Inorganic materials 0.000 claims 1
- 210000001616 monocyte Anatomy 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 16
- 206010028980 Neoplasm Diseases 0.000 abstract description 13
- 238000002512 chemotherapy Methods 0.000 abstract description 8
- 230000000259 anti-tumor effect Effects 0.000 abstract description 5
- 239000003814 drug Substances 0.000 abstract description 4
- 230000001900 immune effect Effects 0.000 abstract description 4
- 239000002246 antineoplastic agent Substances 0.000 abstract description 3
- 229940041181 antineoplastic drug Drugs 0.000 abstract description 3
- 230000006907 apoptotic process Effects 0.000 abstract description 3
- 201000010099 disease Diseases 0.000 abstract description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 2
- 230000001737 promoting effect Effects 0.000 abstract description 2
- 229940124597 therapeutic agent Drugs 0.000 abstract description 2
- 210000004881 tumor cell Anatomy 0.000 abstract description 2
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 description 17
- 235000019441 ethanol Nutrition 0.000 description 6
- 108010025915 Nitrite Reductases Proteins 0.000 description 5
- INGWEZCOABYORO-UHFFFAOYSA-N 2-(furan-2-yl)-7-methyl-1h-1,8-naphthyridin-4-one Chemical compound N=1C2=NC(C)=CC=C2C(O)=CC=1C1=CC=CO1 INGWEZCOABYORO-UHFFFAOYSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 108090000913 Nitrate Reductases Proteins 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 235000013339 cereals Nutrition 0.000 description 3
- 108010002255 deoxyhemoglobin Proteins 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 229960004756 ethanol Drugs 0.000 description 2
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 231100000489 sensitizer Toxicity 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- QQQSFSZALRVCSZ-UHFFFAOYSA-N triethoxysilane Chemical compound CCO[SiH](OCC)OCC QQQSFSZALRVCSZ-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 239000005935 Sulfuryl fluoride Substances 0.000 description 1
- BEVHTVRRVVEMEF-UHFFFAOYSA-N [6'-acetyloxy-4-amino-2',7'-difluoro-5-(methylamino)-3-oxospiro[2-benzofuran-1,9'-xanthene]-3'-yl] acetate Chemical compound C12=CC(F)=C(OC(C)=O)C=C2OC2=CC(OC(C)=O)=C(F)C=C2C21OC(=O)C1=C(N)C(NC)=CC=C21 BEVHTVRRVVEMEF-UHFFFAOYSA-N 0.000 description 1
- HGWOWDFNMKCVLG-UHFFFAOYSA-N [O--].[O--].[Ti+4].[Ti+4] Chemical compound [O--].[O--].[Ti+4].[Ti+4] HGWOWDFNMKCVLG-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- 238000000006 cell growth inhibition assay Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000973 chemotherapeutic effect Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- CWAFVXWRGIEBPL-UHFFFAOYSA-N ethoxysilane Chemical compound CCO[SiH3] CWAFVXWRGIEBPL-UHFFFAOYSA-N 0.000 description 1
- PUSKHXMZPOMNTQ-UHFFFAOYSA-N ethyl 2,1,3-benzoselenadiazole-5-carboxylate Chemical compound CCOC(=O)C1=CC=C2N=[Se]=NC2=C1 PUSKHXMZPOMNTQ-UHFFFAOYSA-N 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000002840 nitric oxide donor Substances 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- OBTWBSRJZRCYQV-UHFFFAOYSA-N sulfuryl difluoride Chemical compound FS(F)(=O)=O OBTWBSRJZRCYQV-UHFFFAOYSA-N 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 210000001364 upper extremity Anatomy 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
- A61K41/0033—Sonodynamic cancer therapy with sonochemically active agents or sonosensitizers, having their cytotoxic effects enhanced through application of ultrasounds
-
- 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
- A61K31/397—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having four-membered rings, e.g. azetidine
-
- 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
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/6445—Haemoglobin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Botany (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
The present invention relates to the pharmaceutical compositions of cellular membrane biomimetic titanium dioxide nano granule, immunologic escape can effectively be solved, long circulating, joint NO chemotherapy is treated by enhancing sound, the problem of promoting apoptosis of tumor cells, its solve technical solution be, hollow mesoporous TiO 2 nanoparticle surface passes through amidation process covalent modification hemoglobin, then physical load NO donator type chemotherapeutics RRx-001, finally handled with cell through hypotonic lysis liquid and cracking, it is homogenized obtained Cell membrane vesicles co-extrusion pressure, up to the pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule, preparation method of the present invention is reliable and stable, producing cost is low, the pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule obtained can play the effect that enhancing sound treats joint NO chemotherapy in terms of preparing anti-tumor drug, enhance antitumous effect, have simultaneously and exempts from Multi-functional, the good biocompatibilities such as epidemic disease escape, long circulating are the innovations in tumor therapeutic agent.
Description
Technical field
The present invention relates to biomedicine field, especially a kind of pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule
Object.
Background technique
Sonodynamic therapy (SonodynamicTherapy, SDT) is that sound sensitiser by ultrasonic excitation generates active oxygen
(ROS), to play the means of anti-tumor effect.Hollow mesoporous TiO 2 nanoparticle (TiO2) has biology as sound sensitiser
The advantages that compatibility is good, chemical stability is high, large specific surface area.But sound treatment is Oxygen Dependence, and the weary oxygen of tumor locus, sternly
The effect of SDT is weakened again.Hemoglobin (Hb) is the key protein that oxygen is transported in red blood cell, has reversible oxygen knot
Conjunction and dissociation capability；Deoxidation Hb also has nitrite reductase activity simultaneously, can be NO by nitrate reductase.Chemotherapeutic
RRx-001 can enhance the nitrite reductase activity of deoxidation Hb, into one as a kind of anti-cancer drugs and a kind of NO donor
Step increases the generation of tumour NO, promotes Apoptosis.Nanoparticle is coated by cell membrane, can completely retain answering for cell membrane surface
Miscellaneous constituent, the function after the film modified nanoparticle of different cells is different, the biocompatibility of nanoparticle can be improved, realization is exempted from
Epidemic disease escape, long circulating, active targeting etc. assign the multi-functional feature of nanoparticle.Therefore, a kind of cellular membrane biomimetic titanium dioxide is invented
The pharmaceutical composition application of titanium nanoparticle is of great significance and is worth in cancer treatment.
Summary of the invention
For above situation, for the defect for solving the prior art, the purpose of the present invention is just to provide a kind of cellular membrane biomimetic
The pharmaceutical composition of titanium dioxide nano granule can effectively solve immunologic escape, long circulating, treat joint NO chemotherapy by enhancing sound,
The problem of promoting apoptosis of tumor cells.
The technical solution that the present invention solves is that hollow mesoporous TiO 2 nanoparticle surface is covalently repaired by amidation process
Adorn hemoglobin, then physical load NO donator type chemotherapeutics RRx-001, finally and cell through hypotonic lysis liquid and cracking at
To get the pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule, partial size is for reason, the obtained Cell membrane vesicles co-extrusion pressure of homogenate
140-200nm。
Specifically includes the following steps:
1) tetraethyl orthosilicate of 0.6-1mL (TEOS) is added to equipped with 0.3-0.5mL ultrapure water, the anhydrous second of 15-25mL
In the flask of pure and mild 0.6-1mL ammonium hydroxide, stirring 10-14h, 10000-15000rpm are centrifuged 10-20min, use dehydrated alcohol strongly
It is washed till neutrality, obtains silica dioxide nano particle (SiO2)；
2) silica dioxide nano particle made from step 1) is dispersed in 10-20mL dehydrated alcohol and 0.05-0.15mL is ultrapure
In solution made of water, the 0.05-0.15g hydroxypropyl cellulose stirring for being dissolved in 2.5-7.5mL dehydrated alcohol is then added
After 0.5-1.5mL tert-butyl alcohol titanium is dissolved in 2.5-7.5mL dehydrated alcohol, it is molten to be slowly dropped to mixing at mixed solution by 30min
In liquid, 80-90 DEG C of reflux 90-110min, 10000-15000rpm centrifugation 10-20min is washed 2-4 times with dehydrated alcohol, is obtained
SiO2@TiO2Compound；
3) by SiO obtained in step 2)2@TiO2Compound is scattered in 15-25mL ultrapure water, adds 0.1-
0.3gPVPk30 stirs 10-14h, and 10000-15000rpm centrifugation 5-10min must be precipitated, and it is anhydrous that precipitating is redispersed in 15-25mL
In ethyl alcohol, 0.3-0.5mL ultrapure water, 0.6-1mLTEOS and 0.6-1mL ammonium hydroxide, magnetic agitation 3-5h, 10000- is added
15000rpm is centrifuged 5-10min, is washed till neutrality with dehydrated alcohol, obtains SiO2@TiO2@SiO2Compound；
4) by SiO obtained in step 3)2@TiO2@SiO2After compound grinding, 400 DEG C of calcining 2-4h in Muffle furnace,
It is redispersed in 30-50mL ultrapure water, adds the sodium hydroxide solution of 1.5-2.5mL2.5M, 80-90 DEG C of return stirring 6-8h,
10000-15000rpm is centrifuged 5-10min, is washed to neutrality with ultrapure, obtains hollow mesoporous TiO 2 nanoparticle (TiO2)；
5) by hollow mesoporous TiO 2 nanoparticle (TiO made from step 4)2) 10-50mg is scattered in the anhydrous second of 4-20mL
80-400 μ L3- aminopropyl triethoxysilane is added in alcohol, adds 0.2-1mL ultrapure water and 0.2-1mL ammonium hydroxide, 25 DEG C of stirrings
10-14h, 10000-15000rpm are centrifuged 5-10min, are washed till neutrality with dehydrated alcohol, obtain amidized hollow meso-porous titanium dioxide
Titanium compound (TiO2-NH2)；
6) 5-50mg hemoglobin (Hb) is dissolved in 10mL solvent, is added 10-50mg1- (3- dimethylamino-propyl)-
3- ethyl-carbodiimide hydrochloride and 8-38mgN- HOSu NHS after 25 DEG C of stirring 30min, instill and contain step (5)
The amidized hollow mesoporous TiO 2 compound (TiO of 5mg obtained2-NH2) 5mL solvent dispersions in, react 4-12h,
5000-10000rpm is centrifuged 5-10min, is washed to neutrality with ultrapure, obtains the hollow mesoporous TiO 2 of modified hemoglobin
(Hb-TiO2)；The hemoglobin is one kind of human hemoglobin, bovine hemoglobin or recombinant hemoglobin；Described is molten
Agent is one kind of formamide, PBS, MES；
7) by the hollow mesoporous TiO 2 (Hb-TiO of modified hemoglobin obtained in 1mg step 6)2) and 0.5-
4mgRRx-001 (NO donator type chemotherapeutics) is scattered in 5mL methanol, ultrasound 2-4h at 25 DEG C, and 40-50 DEG C of revolving, is waved immediately
Dry methanol, then plus 1-2ml methanol ultrasound 2-4s redissolve, 10000-15000rpm is centrifuged 5-10min, obtains the negative of modified hemoglobin
Carry the titanium dioxide medicinal composition (Hb-TiO of RRx-0012/ RRx-001), the drugloading rate of RRx-001 is 20%~60%；
8) adherent cell collecting is digested, cracking processing is carried out to cell after hypotonic lysis liquid is added, is mechanically or manually homogenized
Afterwards, 4 DEG C of 3000-3500g are centrifuged 5min, collect supernatant, are centrifuged 20-40min then at 4 DEG C of 10000-15000g, precipitating adds ultrapure
Water 0.5mL is resuspended, and is squeezed 15-25 times using squeezer by 400nm polycarbonate membrane, obtains cell membrane (CM) vesicle solution；Again
With the titanium dioxide medicinal composition (Hb-TiO of the load RRx-001 of modified hemoglobin made from step 7)2/RRx-001)
By 200nm polycarbonate membrane co-extrusion pressure to get the pharmaceutical composition (CM-Hb- of cellular membrane biomimetic titanium dioxide nano granule
TiO2/RRx-001)；The cell is that breast cancer cell, liver cancer cells, stomach cancer cell, lung carcinoma cell, red blood cell, monokaryon are thin
Born of the same parents, macrophage one kind；The hypotonic lysis liquid is Tris-HCl (20mM), KCl (10mM), MgCl2(2mM) and benzene first
The mixed liquor of base sulfuryl fluoride (PMSF, 1mM), cracking processing be 4 DEG C of 1~3h of placement, liquid nitrogen and 25 DEG C multigelation 2~4 times, often
It is secondary to set 3S in liquid nitrogen, 25 DEG C of thawings.
Preparation method of the present invention is reliable and stable, and producing cost is low, the medicine of cellular membrane biomimetic titanium dioxide nano granule obtained
Compositions can play the effect that enhancing sound treats joint NO chemotherapy in terms of preparing anti-tumor drug, enhance antitumous effect, together
When there is multi-functional, the good biocompatibility such as immunologic escape, long circulating, be the innovation in tumor therapeutic agent, economy and society
Benefit.
Specific embodiment
Specific embodiments of the present invention will be described in further detail with reference to embodiments.
Embodiment 1
1) tetraethyl orthosilicate of 0.8mL (TEOS) is added to equipped with 0.4mL ultrapure water, 20mL dehydrated alcohol and 0.8mL
In the flask of ammonium hydroxide, stirring 12h, 12000rpm are centrifuged 15min, are washed till neutrality with dehydrated alcohol, obtain silica nanometer strongly
Grain (SiO2)；
2) silica dioxide nano particle made from step 1) is dispersed in molten made of 15mL dehydrated alcohol and 0.1mL ultrapure water
In liquid, the 0.1g hydroxypropyl cellulose stirring 30min for being dissolved in 5mL dehydrated alcohol is then added, at mixed solution, by the tertiary fourth of 1mL
After alcohol titanium is dissolved in 5mL dehydrated alcohol, it is slowly dropped in mixed solution, 85 DEG C of reflux 100min, 12000rpm are centrifuged 15min,
Dehydrated alcohol is washed 3 times, and SiO is obtained2@TiO2Compound；
3) by SiO obtained in step 2)2@TiO2Compound is scattered in 20mL ultrapure water, adds 0.2gPVPk30, stirring
12h, 12000rpm centrifugation 8min must precipitate, be redispersed in 20mL dehydrated alcohol, be added 0.4mL ultrapure water, 0.8mLTEOS and
0.8mL ammonium hydroxide, magnetic agitation 4h, 12000rpm are centrifuged 8min, and dehydrated alcohol is washed till neutrality, obtains SiO2@TiO2@SiO2Compound；
4) by SiO obtained in step 3)2@TiO2@SiO2After compound grinding, 400 DEG C of calcining 3h in Muffle furnace, then
It is scattered in 40mL ultrapure water, adds the sodium hydroxide solution of 2mL2.5M, 85 DEG C of return stirrings 7h, 12000rpm are centrifuged 8min, with super
Pure water is washed till neutrality, obtains hollow mesoporous TiO 2 nanoparticle (TiO2)；
5) by hollow mesoporous TiO made from step 4)230mg is scattered in 12mL dehydrated alcohol, and 240 μ L3- aminopropyls are added
Triethoxysilane adds 0.6mL ultrapure water and 0.6mL ammonium hydroxide, and 25 DEG C of stirrings 12h, 12000rpm are centrifuged 8min, and use is anhydrous
Ethyl alcohol is washed till neutrality, and drying obtains amidized hollow mesoporous TiO 2 compound (TiO2-NH2)；
6) 10mL50mM2- (N- morpholine) ethanesulfonic acid (MES) buffer (pH=6.5) is added in 30mg hemoglobin (Hb)
30mg1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and 23mgN- HOSu NHS is added in middle dissolution,
After 25 DEG C of stirring 30min, instill containing 5mgTiO made from step 5)2-NH25mLMES visit oversubscription dispersion liquid in, react 8h,
8000rpm is centrifuged 8min, is washed to neutrality with ultrapure, obtains the hollow mesoporous TiO 2 (Hb-TiO of modified hemoglobin2)；
7) it disperses the hollow mesoporous TiO 2 of modified hemoglobin obtained in 1mg step 6) and 2mgRRx-001 in
In 5mL methanol, the ultrasound 3h at 25 DEG C, 45 DEG C of revolvings, volatilize methanol immediately, then plus 1ml methanol ultrasound 3s redissolve, 12000rpm
8min is centrifuged to get the titanium dioxide medicinal composition (Hb-TiO of the load RRx-001 of modified hemoglobin2/RRx-001)；
8) MCF-7 human breast cancer cell is collected, is resuspended with hypotonic lysis buffer, 4 DEG C of placement 2h, after refiner homogenate, 4
DEG C 3200g is centrifuged 5min, collects supernatant, is centrifuged 30min then at 4 DEG C of 12000g, precipitating plus ultrapure water are resuspended, are squeezed with Avanti
Device is squeezed 20 times by 400nm polycarbonate membrane, obtains Cell membrane vesicles solution；Again with Hb-TiO made from step 7)2/RRx-
001 passes through 200nm polycarbonate membrane co-extrusion pressure to get the pharmaceutical composition (CM-Hb- of cellular membrane biomimetic titanium dioxide nano granule
TiO2/RRx-001)。
Embodiment 2
1) tetraethyl orthosilicate of 0.6mL (TEOS) is added to equipped with 0.3mL ultrapure water, 15mL dehydrated alcohol and 0.6mL
In the flask of ammonium hydroxide, stirring 10h, 10000rpm are centrifuged 10min, are washed till neutrality with dehydrated alcohol, obtain silica nanometer strongly
Grain (SiO2)；
2) by SiO made from step 1)2It is dispersed in solution made of 10mL dehydrated alcohol and 0.05mL ultrapure water, then
The 0.05g hydroxypropyl cellulose stirring 30min for being dissolved in 2.5mL dehydrated alcohol is added, mixed solution is made, by the 0.5mL tert-butyl alcohol
It after titanium is dissolved in 2.5mL dehydrated alcohol, is slowly dropped in mixed solution, 80 DEG C of reflux 90min, 10000rpm are centrifuged 10min, nothing
Water-ethanol is washed 2 times, and SiO is obtained2@TiO2Compound；
3) by SiO obtained in step 2)2@TiO2Compound is scattered in 15mL ultrapure water, adds 0.1gPVPk30, stirring
10h, 10000rpm centrifugation 5min must precipitate, be redispersed in 15mL dehydrated alcohol, be added 0.3mL ultrapure water, 0.6mLTEOS and
0.6mL ammonium hydroxide, magnetic agitation 3h, 10000rpm are centrifuged 5min, and dehydrated alcohol is washed till neutrality, obtains SiO2@TiO2@SiO2Compound；
4) by SiO obtained in step 3)2@TiO2@SiO2After compound grinding, 400 DEG C of calcining 2h in Muffle furnace, then
It is scattered in 30mL ultrapure water, adds the sodium hydroxide solution of 1.5mL2.5M, 80 DEG C of return stirrings 6h, 10000rpm are centrifuged 5min, use
The ultrapure neutrality that is washed to is to get hollow mesoporous TiO 2 nanoparticle (TiO2)；
5) by hollow mesoporous TiO made from step 4)210mg is scattered in 4mL dehydrated alcohol, and 80 μ L3- aminopropyls three are added
Ethoxysilane adds 0.2mL ultrapure water and 0.2mL ammonium hydroxide, and 25 DEG C of stirring 10h, 10000rpm is centrifuged 5min, with anhydrous second
Alcohol is washed till neutrality, obtains amidized hollow mesoporous TiO 2 compound (TiO2-NH2)；
6) 5mg hemoglobin (Hb) is added in 10mL50mM2- (N- morpholine) ethanesulfonic acid (MES) buffer (pH=6.5)
Dissolution, addition 10mg1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and 8mgN- HOSu NHS, 25
After DEG C stirring 30min, instill containing 5mgTiO made from step (5)2-NH25mLMES visit oversubscription dispersion liquid in, react 4h,
5000rpm is centrifuged 5min, is washed to the neutral hollow mesoporous TiO 2 (Hb-TiO to get modified hemoglobin with ultrapure2)；
7) the hollow mesoporous TiO 2 of modified hemoglobin obtained in 1mg step 6) and 0.5mgRRx-001 are dispersed
In 5mL methanol, the ultrasound 2h at 25 DEG C, 40 DEG C of revolvings, volatilize methanol immediately, then plus 1.5ml methanol ultrasound 2s redissolve,
10000rpm is centrifuged 5min to get the titanium dioxide medicinal composition (Hb-TiO of the load RRx-001 of modified hemoglobin2/
RRx-001)；
8) human red blood cells are collected, are resuspended with hypotonic lysis buffer, 4 DEG C of placement 1h, after homogenate, 4 DEG C of 3000g centrifugations
5min collects supernatant；It is centrifuged 20min then at 4 DEG C of 10000g, precipitating plus ultrapure water are resuspended, and pass through 400nm with Avanti squeezer
Polycarbonate membrane squeezes 15 times, obtains Cell membrane vesicles solution；Again with Hb-TiO made from step 7)2/ RRx-001 passes through 200nm
Polycarbonate membrane co-extrusion pressure to get cellular membrane biomimetic titanium dioxide nano granule pharmaceutical composition (CM-Hb-TiO2/RRx-
001)。
Embodiment 3
1) tetraethyl orthosilicate of 1mL (TEOS) is added to equipped with 0.5mL ultrapure water, 25mL dehydrated alcohol and 1mL ammonium hydroxide
Flask in, strongly stirring 14h, 15000rpm be centrifuged 20min, be washed till neutrality with dehydrated alcohol, obtain silica dioxide nano particle
(SiO2)；
2) by SiO made from step 1)2It is dispersed in solution made of 20mL dehydrated alcohol and 0.15mL ultrapure water, then
The 0.15g hydroxypropyl cellulose stirring 30min for being dissolved in 7.5mL dehydrated alcohol is added, mixed solution is made, by the 1.5mL tert-butyl alcohol
After titanium is dissolved in 7.5mL dehydrated alcohol, it is slowly dropped to mixed solution, 90 DEG C of reflux 110min, 15000rpm are centrifuged 20min, nothing
Water-ethanol is washed 4 times, and SiO is obtained2@TiO2Compound；
3) by SiO obtained in step 2)2@TiO2Compound is scattered in 25mL ultrapure water, adds 0.3gPVPk30, stirring
14h, 15000rpm centrifugation 10min must precipitate, be redispersed in 25mL dehydrated alcohol, be added 0.5mL ultrapure water, 1mLTEOS and
1mL ammonium hydroxide, magnetic agitation 5h, 15000rpm are centrifuged 10min, and dehydrated alcohol is washed till neutrality, obtains SiO2@TiO2@SiO2Compound；
4) by SiO obtained in step 3)2@TiO2@SiO2After compound grinding, 400 DEG C of calcining 4h in Muffle furnace, then
It is scattered in 50mL ultrapure water, adds the sodium hydroxide solution of 2.5mL2.5M, 90 DEG C of return stirrings 8h, 15000rpm are centrifuged 10min,
With the ultrapure neutrality that is washed to get hollow mesoporous TiO 2 nanoparticle (TiO2)；
5) by hollow mesoporous TiO made from step 4)250mg is scattered in 20mL dehydrated alcohol, and 400 μ L3- aminopropyls are added
Triethoxysilane adds 1mL ultrapure water and 1mL ammonium hydroxide, and 25 DEG C of stirring 14h, 15000rpm is centrifuged 10min, with anhydrous second
Alcohol is washed till neutrality, obtains amidized hollow mesoporous TiO 2 compound (TiO2-NH2)；
6) 10mL50mM2- (N- morpholine) ethanesulfonic acid (MES) buffer (pH=6.5) is added in 50mg hemoglobin (Hb)
Middle dissolution, is added 50mg1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and 38mg N- hydroxysuccinimidyl acyl is sub-
Amine after 25 DEG C of stirring 30min, is instilled containing 5mgTiO made from step (5)2-NH25mLMES visit oversubscription dispersion liquid in, reaction
12h, 10000rpm are centrifuged 10min, are washed to the neutral hollow mesoporous TiO 2 (Hb- to get modified hemoglobin with ultrapure
TiO2)；
7) it disperses the hollow mesoporous TiO 2 of modified hemoglobin obtained in 1mg step 6) and 4mgRRx-001 in
In 5mL methanol, the ultrasound 4h at 25 DEG C, 50 DEG C of revolvings, volatilize methanol immediately, then plus 2ml methanol ultrasound 4s redissolve, 15000rpm
10min is centrifuged to get the titanium dioxide medicinal composition (Hb-TiO of the load RRx-001 of modified hemoglobin2/RRx-001)；
8) human macrophage is collected, is resuspended with hypotonic lysis buffer, 4 DEG C of placement 3h, after homogenate, 4 DEG C of 3500g centrifugations
5min collects supernatant, is centrifuged 40min then at 4 DEG C of 15000g, and precipitating plus ultrapure water are resuspended, and pass through the poly- carbonic acid of 400nm with squeezer
Ester film squeezes 25 times, obtains Cell membrane vesicles solution；Again with 0.5ml Hb-TiO made from step 7)2/ RRx-001 (1mg/mL) is logical
200nm polycarbonate membrane co-extrusion pressure is crossed to get the pharmaceutical composition (CM-Hb-TiO of cellular membrane biomimetic titanium dioxide nano granule2/
RRx-001)。
Through scientific experimentation, the preparation side of the pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule obtained by the present invention
Method is reliable and stable, Hb oxygen supply enhancing TiO2Sound is treated, and the nitrite reductase activity of RRx-001 enhancing deoxyhemoglobin,
The generation for dramatically increasing NO, greatly enhances antitumous effect.Prepared bionical pharmaceutical composition has immunologic escape, length is followed
Ring, enhancing sound treat the multi-efficiencies such as joint NO chemotherapy, have advantage efficient, that toxic side effect is small compared with classic chemotherapy, related
Experimental data is as follows:
One, the characterization of the pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule
1, in the pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule RRx-001 content measurement
Using ultraviolet specrophotometer, the content of RRx-001 is measured at 202nm wavelength.Sample is calculated with formula (1)
Drugloading rate, carrying drug ratio is up to 50% or so.
2, the measurement of the partial size and current potential of the pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule
The pharmaceutical composition of the cellular membrane biomimetic titanium dioxide nano granule of appropriate load RRx-001 is taken to be scattered in ultrapure water
In, its partial size is measured with Nano-ZS90 type laser nano Particle Size Analyzer and current potential is respectively 145nm and -21.7 ± 1mV.
Two, the pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule generates the detection of ROS in vitro
The rear dosing for 24 hours of MCF-7 human breast cancer cell adherent growth, is followed successively by blank group, CM-TiO2、CM-TiO2+US、CM-
Hb-TiO2+US(TiO2: 10 μ g/mL, RRx-001:3 μM).After 4~6h, it is protected from light the PBS training being added containing DHE activity oxygen probe
Support 30min, ultrasound group ultrasound (1W/cm2, 1MHz, 30s) after, continue to cultivate 30min, cell is collected, with flow cytometer to each
The ROS that group generates is quantitative, CM-TiO2+US、CM-Hb-TiO2+ US active oxygen yield is respectively 23.8%, 58.8%.The result
Show that Hb oxygen supply can enhance TiO2The generation of ROS in Sonodynamic therapy.
Three, the pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule generates the detection of NO in vitro
The rear dosing for 24 hours of MCF-7 human breast cancer cell adherent growth, is followed successively by blank group, CM-TiO2、CM-TiO2+US、CM-
Hb-TiO2+US、CM-Hb-TiO2/RRx-001+US(TiO2: 10 μ g/mL, RRx-001:3 μM).After 4~6h, it is protected from light addition and contains
There are the dilution culture 30min of DAF-FMDA probe, ultrasound group ultrasound (1W/cm2, 1MHz, 30s) after, continue to cultivate 30min,
Cell is collected, the NO generated with flow cytometer to each group is quantitative.Finally obtain CM-Hb-TiO2+US、CM-Hb-TiO2/RRx-
The NO yield of 001+US is respectively 10.2%, 40.0%.This is the result shows that its Asia of the deoxyhemoglobin generated after Hb oxygen supply
The nitrite that nitrate reductase activity can restore tumour enrichment is NO；More NO are generated after adding RRx-001, further
Illustrate that the combination of RRx-001 and Hb can enhance nitrite reductase activity of the Hb under weary oxygen, to generate more NO.
Four, the cell growth inhibition assay under the weary oxygen of the pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule
The rear dosing for 24 hours of MCF-7 human breast cancer cell adherent growth, is followed successively by blank group, CM-TiO2、CM-Hb-TiO2、CM-
Hb-TiO2/RRx-001(TiO2: 10 μ g/mL, RRx-001:3 μM), and point ultrasonic group (1W/cm2, 1MHz, 30s) and non-ultrasound
Group.In weary oxygen environment (pO2: 2%) it after being incubated for for 24 hours, is detected using srb assay, calculates inhibitory rate of cell growth (%)=(1- experiment
Group OD value/control group OD value) × 100%, obtain CM-TiO2、CM-TiO2+US、CM-Hb-TiO2、CM-Hb-TiO2+US、CM-
Hb-TiO2The inhibitory rate of cell growth of/RRx-001+US each group is respectively as follows: 1.7%, 19%, 3%, 28%, 81%.As a result table
It is bright, support C M-TiO2And CM-Hb-TiO2To cell without overt toxicity under test dose, but toxicity is more apparent under ultrasound；
The addition of RRx-001 leads to stronger cytotoxicity after preparation ultrasound, should be that oxygen is treated and the synergistic treatment of NO chemotherapy for enhancing sound
Result.
Five, the pharmacodynamic study of the pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule
Buy nude mice (female, 3~4 week old), right upper extremity inoculates MCF-7 human breast cancer cell, take gross tumor volume >=
100mm3Mouse, be divided into: physiological saline group, CM-TiO2、CM-TiO2+US、CM-Hb-TiO2+US、CM-Hb-TiO2/RRx-
001+US(TiO2: 20mg/kg, RRx-001:5mg/kg), supersonic tumor position (1W/cm after 6h is administered in tail vein2, 1MHz,
30s).It is administered once within every two days, totally 7 times.Using the major diameter (A) and minor axis (B) of electronic digital indicator measurement tumor, by formula V=A
×B2/ 2 calculate gross tumor volume.The results show that CM-TiO2、CM-TiO2+US、CM-Hb-TiO2+US、CM-Hb-TiO2/RRx-001
The tumour inhibiting rate of+US is respectively 4.59%, 17.57%, 33.92%, 84.19%, illustrates the tomour specific targeting drug delivery system
Oxygen, which treats collaboration NO chemotherapy for enhancing sound, can significantly increase antitumous effect.
Experiment shows compared with prior art, the present invention having advantageous effects following prominent:
(1) pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule provided by the invention selects cell membrane package to receive
The grain of rice is conducive to develop multi-functional delivery system.
(2) pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule provided by the invention, hemoglobin can be weary
Oxygen tumour provides oxygen, enhances TiO2Sound is treated.
(3) pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule provided by the invention, RRx-001 can enhance blood red
The nitrite reductase activity of the deoxyhemoglobin generated after albumen oxygen supply, the nitrate reductase by tumour enrichment are
NO significantly increases Antineoplastic effect.
Claims (4)
1. a kind of preparation method of the pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule, which is characterized in that hollow mesoporous
Titanium dioxide nano granule surface passes through amidation process covalent modification hemoglobin, then physical load NO donator type chemotherapeutics
RRx-001 is finally handled through hypotonic lysis liquid and cracking with cell, is homogenized obtained Cell membrane vesicles co-extrusion pressure to get cell
The pharmaceutical composition of the bionical titanium dioxide nano granule of film, partial size 140-200nm, specifically includes the following steps:
1) tetraethyl orthosilicate of 0.6-1mL is added to equipped with 0.3-0.5mL ultrapure water, 15-25mL dehydrated alcohol and 0.6-1mL
In the flask of ammonium hydroxide, stirring 10-14h, 10000-15000rpm are centrifuged 10-20min, are washed till neutrality with dehydrated alcohol, obtain strongly
Silica dioxide nano particle；
2) silica dioxide nano particle made from step 1) is dispersed in 10-20mL dehydrated alcohol and 0.05-0.15mL ultrapure water system
At solution in, then be added be dissolved in 2.5-7.5mL dehydrated alcohol 0.05-0.15g hydroxypropyl cellulose stirring 30min, at
Mixed solution is slowly dropped in mixed solution, 80- after 0.5-1.5mL tert-butyl alcohol titanium is dissolved in 2.5-7.5mL dehydrated alcohol
90 DEG C of reflux 90-110min, 10000-15000rpm centrifugation 10-20min, are washed 2-4 times with dehydrated alcohol, obtain SiO2@TiO2It is compound
Object；
3) by SiO obtained in step 22@TiO2Compound is scattered in 15-25mL ultrapure water, adds 0.1-0.3gPVPk30, is stirred
10-14h is mixed, 10000-15000rpm centrifugation 5-10min must be precipitated, and precipitating is redispersed in 15-25mL dehydrated alcohol, is added
0.3-0.5mL ultrapure water, 0.6-1mLTEOS and 0.6-1mL ammonium hydroxide, magnetic agitation 3-5h, 10000-15000rpm are centrifuged 5-
10min is washed till neutrality with dehydrated alcohol, obtains SiO2@TiO2@SiO2Compound；
4) by SiO obtained in step 3)2@TiO2@SiO2After compound grinding, 400 DEG C of calcining 2-4h in Muffle furnace, then divide
It dissipates in 30-50mL ultrapure water, adds the sodium hydroxide solution of 1.5-2.5mL2.5M, 80-90 DEG C of return stirring 6-8h, 10000-
15000rpm is centrifuged 5-10min, is washed to neutrality with ultrapure, obtains hollow mesoporous TiO 2 nanoparticle；
5) 4-20mL dehydrated alcohol is dispersed by hollow mesoporous TiO 2 nanoparticle 10-50mg made from step 4), 80- is added
400 μ L3- aminopropyl triethoxysilanes, add 0.2-1mL ultrapure water and 0.2-1mL ammonium hydroxide, 25 DEG C of stirring 10-14h,
10000-15000rpm is centrifuged 5-10min, is washed till neutrality with dehydrated alcohol, it is compound to obtain amidized hollow mesoporous TiO 2
Object；
6) 5-50mg hemoglobin is dissolved in 10mL solvent, 10-50mg1- (3- dimethylamino-propyl) -3- ethyl carbon is added
Diimmonium salt hydrochlorate and 8-38mgN- HOSu NHS after 30min is stirred at room temperature, are instilled containing 5mg made from step 5)
In the 5mL solvent dispersions of amidized hollow mesoporous TiO 2 compound, 4-12h is reacted, 5000-10000rpm is centrifuged 5-
10min is washed to neutrality with ultrapure, obtains the hollow mesoporous TiO 2 of modified hemoglobin；The hemoglobin is people's blood
One kind of Lactoferrin, bovine hemoglobin or recombinant hemoglobin；The solvent is one kind of formamide, PBS, MES；
7) it disperses the hollow mesoporous TiO 2 of modified hemoglobin obtained in 1mg step 6) and 0.5-4mgRRx-001 in
In 5mL methanol, ultrasound 2-4h at 25 DEG C, 40-50 DEG C of revolving, volatilizes methanol immediately, then plus 1-2ml methanol ultrasound 2-4s redissolve,
10000-15000rpm is centrifuged 5-10min, obtains the titanium dioxide medicinal composition of the load RRx-001 of modified hemoglobin,
The drugloading rate of RRx-001 is 20%~60%；
8) adherent cell collecting is digested, cracking processing is carried out to cell after hypotonic lysis liquid is added, after being mechanically or manually homogenized, 4
DEG C 3000-3500g is centrifuged 5min, collects supernatant, is centrifuged 20-40min, precipitating plus ultrapure water then at 4 DEG C of 10000-15000g
0.5mL is resuspended, and is squeezed 15-25 times using squeezer by 400nm polycarbonate membrane, obtains Cell membrane vesicles solution；Again with step
7) the titanium dioxide medicinal composition of the load RRx-001 of modified hemoglobin made from passes through 200nm polycarbonate membrane co-extrusion
Press the pharmaceutical composition to get cellular membrane biomimetic titanium dioxide nano granule；The cell be breast cancer cell, liver cancer cells,
One kind of stomach cancer cell, lung carcinoma cell, red blood cell, monocyte, macrophage；The hypotonic lysis liquid is 20mMTris-
HCl、10mMKCl、2mMMgCl2With the mixed liquor of 1mMPMSF, cracking is handled freezes repeatedly for 4 DEG C of 1 ~ 3h of placement, liquid nitrogen and 25 DEG C
Melt 2 ~ 4 times, sets 3S in liquid nitrogen, 25 DEG C of thawings every time.
2. the preparation method of the pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule according to claim 1, special
Sign is, comprising the following steps:
The tetraethyl orthosilicate of 0.8mL is added in the flask equipped with 0.4mL ultrapure water, 20mL dehydrated alcohol and 0.8mL ammonium hydroxide,
Strong stirring 12h, 12000rpm are centrifuged 15min, are washed till neutrality with dehydrated alcohol, obtain silica dioxide nano particle；
Silica dioxide nano particle made from step 1) is dispersed in solution made of 15mL dehydrated alcohol and 0.1mL ultrapure water,
Then the 0.1g hydroxypropyl cellulose that addition is dissolved in 5mL dehydrated alcohol stirs 30min, at mixed solution, 1mL tert-butyl alcohol titanium is molten
It after 5mL dehydrated alcohol, is slowly dropped in mixed solution, 85 DEG C of reflux 100min, 12000rpm are centrifuged 15min, anhydrous second
Alcohol is washed 3 times, and SiO is obtained2@TiO2Compound；
By SiO obtained in step 22@TiO2Compound is scattered in 20mL ultrapure water, adds 0.2gPVPk30, stirs 12h,
12000rpm centrifugation 8min must precipitate, be redispersed in 20mL dehydrated alcohol, be added 0.4mL ultrapure water, 0.8mLTEOS and
0.8mL ammonium hydroxide, magnetic agitation 4h, 12000rpm are centrifuged 8min, and dehydrated alcohol is washed till neutrality, obtains SiO2@TiO2@SiO2Compound；
By SiO obtained in step 3)2@TiO2@SiO2After compound grinding, 400 DEG C of calcining 3h, are redispersed in Muffle furnace
40mL ultrapure water adds the sodium hydroxide solution of 2mL2.5M, and 85 DEG C of return stirring 7h, 12000rpm is centrifuged 8min, with ultrapure washing
To neutrality, hollow mesoporous TiO 2 nanoparticle is obtained；
12mL dehydrated alcohol is dispersed by hollow mesoporous TiO 2 nanoparticle 30mg made from step 4), 240 μ L3- ammonia are added
Propyl-triethoxysilicane, adds 0.6mL ultrapure water and 0.6mL ammonium hydroxide, and 25 DEG C of stirrings 12h, 12000rpm are centrifuged 8min, use
Dehydrated alcohol is washed till neutrality, and drying obtains amidized hollow mesoporous TiO 2 compound；
30mg hemoglobin is added in 10mL50mM2- (N- morpholine) ethanesulfonic acid buffer and is dissolved, 30mg1- (3- diformazan is added
Aminopropyl) -3- ethyl-carbodiimide hydrochloride and 23mgN- HOSu NHS, after 25 DEG C of stirring 30min, instillation contains
5mgTiO made from step 5)2-NH25mLMES visit oversubscription dispersion liquid in, react 8h, 8000rpm be centrifuged 8min, with ultrapure washing
To neutrality, the hollow mesoporous TiO 2 of modified hemoglobin is obtained；
5mL first is dispersed by the hollow mesoporous TiO 2 of modified hemoglobin obtained in 1mg step 6) and 2mgRRx-001
In alcohol, the ultrasound 3h at 25 DEG C, 45 DEG C of revolvings, volatilize methanol immediately, then plus 1ml methanol ultrasound 3s redissolve, 12000rpm centrifugation
8min to get modified hemoglobin load RRx-001 titanium dioxide medicinal composition；
MCF-7 human breast cancer cell is collected, is resuspended with hypotonic lysis buffer, 4 DEG C of placement 2h, after refiner homogenate, 4 DEG C
3200g is centrifuged 5min, collects supernatant, is centrifuged 30min then at 4 DEG C of 12000g, precipitating plus ultrapure water are resuspended, with Avanti squeezer
It is squeezed 20 times by 400nm polycarbonate membrane, obtains Cell membrane vesicles solution；It is negative with modified hemoglobin made from step 7) again
The titanium dioxide medicinal composition for carrying RRx-001 passes through 200nm polycarbonate membrane co-extrusion pressure to get cellular membrane biomimetic titanium dioxide
The pharmaceutical composition of nanoparticle.
3. the pharmaceutical composition of cellular membrane biomimetic titanium dioxide nano granule of any of claims 1 or 2 is based on cell membrane in preparation
Application in the bionical antineoplastic pharmaceutical compositions of package.
4. bionical antineoplastic pharmaceutical compositions as claimed in claim 3 combine NO based on oxygen enhancing Sonodynamic therapy in preparation
Application in donator type chemotherapeutics.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111494639A (en) * | 2020-03-19 | 2020-08-07 | 上海市东方医院（同济大学附属东方医院） | Cell-like structure nano material and preparation method and application thereof |
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| CN111494639A (en) * | 2020-03-19 | 2020-08-07 | 上海市东方医院（同济大学附属东方医院） | Cell-like structure nano material and preparation method and application thereof |
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| CN112121015B (en) * | 2020-08-24 | 2022-03-18 | 四川大学华西医院 | PD-L1 antibody-loaded bionic targeting TiO2Nano particle and its preparing method and use |
| CN112807430A (en) * | 2020-12-28 | 2021-05-18 | 景香香 | Application of nano enzyme-based material |
| CN113428893A (en) * | 2021-07-14 | 2021-09-24 | 陕西科技大学 | Carboxylation modified nano titanium dioxide ultraviolet screening agent and preparation method thereof |
| CN114712501A (en) * | 2022-05-13 | 2022-07-08 | 天津大学四川创新研究院 | Preparation method and application of nano-composite for treating periodontitis by ultrasonic |
| CN114712501B (en) * | 2022-05-13 | 2023-04-25 | 天津大学四川创新研究院 | Preparation method and application of nanocomposite for treating periodontitis by ultrasound |
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