CN117757087A - Sulfonated gelatin, preparation method and sulfonated gelatin embolic microsphere, preparation method and application - Google Patents
Sulfonated gelatin, preparation method and sulfonated gelatin embolic microsphere, preparation method and application Download PDFInfo
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- CN117757087A CN117757087A CN202311795814.4A CN202311795814A CN117757087A CN 117757087 A CN117757087 A CN 117757087A CN 202311795814 A CN202311795814 A CN 202311795814A CN 117757087 A CN117757087 A CN 117757087A
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- Prior art keywords
- gelatin
- sulfonated
- microspheres
- microsphere
- embolic
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Links
- 108010010803 Gelatin Proteins 0.000 title claims abstract description 161
- 229920000159 gelatin Polymers 0.000 title claims abstract description 161
- 235000019322 gelatine Nutrition 0.000 title claims abstract description 161
- 235000011852 gelatine desserts Nutrition 0.000 title claims abstract description 161
- 239000008273 gelatin Substances 0.000 title claims abstract description 155
- 239000004005 microsphere Substances 0.000 title claims abstract description 149
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 230000003073 embolic effect Effects 0.000 title claims description 58
- 239000003814 drug Substances 0.000 claims abstract description 95
- 238000011068 loading method Methods 0.000 claims abstract description 56
- 208000005189 Embolism Diseases 0.000 claims abstract description 20
- 239000002245 particle Substances 0.000 claims abstract description 9
- 125000000542 sulfonic acid group Chemical group 0.000 claims abstract description 9
- 239000000463 material Substances 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims description 38
- 239000000243 solution Substances 0.000 claims description 32
- 150000002148 esters Chemical class 0.000 claims description 23
- 238000006243 chemical reaction Methods 0.000 claims description 20
- 239000002904 solvent Substances 0.000 claims description 18
- 229920000642 polymer Polymers 0.000 claims description 17
- 238000004140 cleaning Methods 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- 239000003431 cross linking reagent Substances 0.000 claims description 12
- 238000006277 sulfonation reaction Methods 0.000 claims description 12
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 claims description 11
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 11
- 239000007864 aqueous solution Substances 0.000 claims description 11
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 claims description 11
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 claims description 10
- XHZPRMZZQOIPDS-UHFFFAOYSA-N 2-Methyl-2-[(1-oxo-2-propenyl)amino]-1-propanesulfonic acid Chemical compound OS(=O)(=O)CC(C)(C)NC(=O)C=C XHZPRMZZQOIPDS-UHFFFAOYSA-N 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000000178 monomer Substances 0.000 claims description 9
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 8
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 8
- 229910052717 sulfur Inorganic materials 0.000 claims description 8
- 239000011593 sulfur Substances 0.000 claims description 8
- 239000003054 catalyst Substances 0.000 claims description 7
- 239000003999 initiator Substances 0.000 claims description 7
- 239000004094 surface-active agent Substances 0.000 claims description 7
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 6
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 239000012043 crude product Substances 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 claims description 5
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 claims description 5
- 235000010958 polyglycerol polyricinoleate Nutrition 0.000 claims description 5
- 239000003996 polyglycerol polyricinoleate Substances 0.000 claims description 5
- 235000012424 soybean oil Nutrition 0.000 claims description 5
- 239000003549 soybean oil Substances 0.000 claims description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 4
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000011203 carbon fibre reinforced carbon Substances 0.000 claims description 4
- 238000000502 dialysis Methods 0.000 claims description 4
- 239000001257 hydrogen Substances 0.000 claims description 4
- 229910052739 hydrogen Inorganic materials 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 238000002390 rotary evaporation Methods 0.000 claims description 4
- 239000011734 sodium Substances 0.000 claims description 4
- 229910052708 sodium Inorganic materials 0.000 claims description 4
- XFNJVJPLKCPIBV-UHFFFAOYSA-N trimethylenediamine Chemical compound NCCCN XFNJVJPLKCPIBV-UHFFFAOYSA-N 0.000 claims description 4
- YXMISKNUHHOXFT-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) prop-2-enoate Chemical group C=CC(=O)ON1C(=O)CCC1=O YXMISKNUHHOXFT-UHFFFAOYSA-N 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 claims description 3
- -1 allyl dimethyl (3-sulfopropyl) ammonium Chemical compound 0.000 claims description 3
- 229910052786 argon Inorganic materials 0.000 claims description 3
- 235000019400 benzoyl peroxide Nutrition 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
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- ACGJEMXWUYWELU-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)ON1C(=O)CCC1=O ACGJEMXWUYWELU-UHFFFAOYSA-N 0.000 claims description 2
- JHUFGBSGINLPOW-UHFFFAOYSA-N 3-chloro-4-(trifluoromethoxy)benzoyl cyanide Chemical compound FC(F)(F)OC1=CC=C(C(=O)C#N)C=C1Cl JHUFGBSGINLPOW-UHFFFAOYSA-N 0.000 claims description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 2
- OMPJBNCRMGITSC-UHFFFAOYSA-N Benzoylperoxide Chemical compound C=1C=CC=CC=1C(=O)OOC(=O)C1=CC=CC=C1 OMPJBNCRMGITSC-UHFFFAOYSA-N 0.000 claims description 2
- 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 claims description 2
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- HVUMOYIDDBPOLL-XWVZOOPGSA-N Sorbitan monostearate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O HVUMOYIDDBPOLL-XWVZOOPGSA-N 0.000 claims description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 2
- 229910001870 ammonium persulfate Inorganic materials 0.000 claims description 2
- 230000000973 chemotherapeutic effect Effects 0.000 claims description 2
- 238000007710 freezing Methods 0.000 claims description 2
- 230000008014 freezing Effects 0.000 claims description 2
- 229940057995 liquid paraffin Drugs 0.000 claims description 2
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical group O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 claims description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 2
- 239000003208 petroleum Substances 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 229920000136 polysorbate Polymers 0.000 claims description 2
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- UIIIBRHUICCMAI-UHFFFAOYSA-N prop-2-ene-1-sulfonic acid Chemical compound OS(=O)(=O)CC=C UIIIBRHUICCMAI-UHFFFAOYSA-N 0.000 claims description 2
- 125000001273 sulfonato group Chemical group [O-]S(*)(=O)=O 0.000 claims description 2
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 2
- 239000012498 ultrapure water Substances 0.000 claims description 2
- OPRIWFSSXKQMPB-UHFFFAOYSA-N 2-methyl-2-(prop-2-enoylamino)propane-1-sulfonic acid;sodium Chemical compound [Na].OS(=O)(=O)CC(C)(C)NC(=O)C=C OPRIWFSSXKQMPB-UHFFFAOYSA-N 0.000 claims 1
- 230000000118 anti-neoplastic effect Effects 0.000 claims 1
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- MWWSFMDVAYGXBV-RUELKSSGSA-N Doxorubicin hydrochloride Chemical compound Cl.O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 MWWSFMDVAYGXBV-RUELKSSGSA-N 0.000 description 10
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- AOJJSUZBOXZQNB-VTZDEGQISA-N 4'-epidoxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-VTZDEGQISA-N 0.000 description 1
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 1
- HTIJFSOGRVMCQR-UHFFFAOYSA-N Epirubicin Natural products COc1cccc2C(=O)c3c(O)c4CC(O)(CC(OC5CC(N)C(=O)C(C)O5)c4c(O)c3C(=O)c12)C(=O)CO HTIJFSOGRVMCQR-UHFFFAOYSA-N 0.000 description 1
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- XDXDZDZNSLXDNA-UHFFFAOYSA-N Idarubicin Natural products C1C(N)C(O)C(C)OC1OC1C2=C(O)C(C(=O)C3=CC=CC=C3C3=O)=C3C(O)=C2CC(O)(C(C)=O)C1 XDXDZDZNSLXDNA-UHFFFAOYSA-N 0.000 description 1
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- XSMVECZRZBFTIZ-UHFFFAOYSA-M [2-(aminomethyl)cyclobutyl]methanamine;2-oxidopropanoate;platinum(4+) Chemical compound [Pt+4].CC([O-])C([O-])=O.NCC1CCC1CN XSMVECZRZBFTIZ-UHFFFAOYSA-M 0.000 description 1
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- DWAFYCQODLXJNR-BNTLRKBRSA-L oxaliplatin Chemical compound O1C(=O)C(=O)O[Pt]11N[C@@H]2CCCC[C@H]2N1 DWAFYCQODLXJNR-BNTLRKBRSA-L 0.000 description 1
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Landscapes
- Materials For Medical Uses (AREA)
Abstract
The invention discloses sulfonated gelatin, a preparation method, sulfonated gelatin embolism microspheres, a preparation method and application, wherein the sulfonated gelatin embolism microspheres are macromolecular sulfonated gelatin rich in sulfonic acid groups and are obtained through amidation reaction, and the sulfonated gelatin embolism microspheres are obtained through a microfluidic technology and have the advantages of good elasticity, degradability and high drug loading, and the preparation process is simple and efficient, and the prepared microspheres are uniform in particle size and controllable in particle size. Compared with the nondegradable drug-loaded microspheres in the market, such as DC Beads and CalliSpheres, equalSpheres, the sulfonated gelatin microsphere can realize gradient slow release of the loaded chemotherapeutic drugs and release the loaded chemotherapeutic drugs to the greatest extent along with the degradation of the microsphere. Compared with artificially synthesized degradable materials such as polylactic acid, the sulfonated gelatin has the advantages of abundant and cheap raw material sources, capability of being processed and molded by a conventional method, good biocompatibility, capability of being degraded by biological enzymes, difficulty in causing foreign body reaction, predictable degradation period and the like.
Description
Technical Field
The invention relates to the technical field of interventional medical devices, in particular to sulfonated gelatin and a preparation method thereof, and embolic microspheres prepared from the sulfonated gelatin and a preparation method and application thereof.
Background
In the new cases of cancer, the death rate is ranked second, and liver cancer is ranked next to pancreatic cancer, and then esophageal cancer, skin cancer, nerve tumor and lung cancer are respectively ranked. The world health organization predicts that by 2040 liver cancer patients alone may grow to 1276679. The best treatment for liver cancer patients is early detection of liver cancer and treatment by surgical excision or transplantation, but most liver cancer patients find late stage, and alternative treatment methods can select embolic treatment besides ablation, radiotherapy/chemotherapy. The drug-loaded embolic microsphere is three interventional medical devices used for blocking blood flowing to main blood vessels of tumor cells in Transcatheter Arterial Chemoembolization (TACE) so as to block blood oxygen supply of the tumor cells and reduce or necrose the tumor. The chemotherapy medicine is added into the embolism microsphere to block blood oxygen supply and release the chemotherapy medicine at the same time, so as to accelerate the death of tumor cells.
Drug-loaded embolic microspheres that are currently marketed, such as: DC (direct current)Embozene/> DC load LUMI (TM) adopts negatively charged groups to graft and modify non-degradable high polymer materials, such as: polyethylene glycol, polyvinyl alcohol, polyacrylic acid and the like, and the medicine loading is realized by utilizing the electrostatic action of negative groups on positively charged medicines. However, permanent embolism of non-degradable microspheres has a certain potential safety hazard, for example, when ectopic embolism occurs, the permanent embolism microspheres easily cause irreversible permanent injury of normal organs and generate long-term foreign body reaction; for tumor tissues, new blood vessel branches are easy to generate after permanent embolism, the original blood vessel is damaged, and the subsequent secondary blood vessel treatment is blocked; drug-loaded microspheres for permanent embolization are difficult to fully release drug over a long period of timeAnd (3) an object.
At present, a plurality of degradable embolic microspheres are published, and natural degradable polymer materials are mainly used as base materials, such as composite gelatin-sodium alginate microspheres, for example, patent CN114748680A, CN114887109A; composite gelatin-polyethylene glycol microspheres, such as patent CN116271186a; composite chitosan-hyaluronic acid microspheres, such as patent CN115845120a. Although the degradable microsphere is prepared by compounding various functional materials to realize functions such as degradability and drug loading, the problems of poor stability and compatibility of two phases exist, the uniformity of the particle size of the microsphere prepared by an emulsion method is difficult to control, the heterogeneous microsphere is difficult to reach the end point of embolism, and embolism gaps are easy to form.
Therefore, it is necessary to develop a novel degradable material and achieve high drug loading and uniform sulfonated gelatin embolic microspheres.
Disclosure of Invention
In order to solve the technical problems, the invention provides sulfonated gelatin, sulfonated gelatin embolic microspheres and corresponding preparation methods thereof. The sulfonated gelatin embolic microsphere is the macromolecular sulfonated gelatin rich in sulfonic acid groups and obtained through amidation reaction, and the sulfonated gelatin embolic microsphere is obtained through a microfluidic technology, and has the advantages of good elasticity, degradability and high drug loading capacity, and the preparation process is simple and efficient, and the prepared microsphere is uniform in particle size and controllable in particle size.
The aim of the invention is achieved by the following technical scheme:
a first object of the present invention is to provide: a sulfonated gelatin having the structural formula shown in (i):
wherein m and n are positive integers; n is m=10-48:1;
R 2 is a sulfonic acid group or a sodium sulfonate group;
R 3 is methyl or hydrogen;
R 1 the four kinds of knots are as followsOne of the following:
the index system of the sulfonated gelatin meets the following conditions:
sulfonation rate: 10-50%;
sulfur element content: 8-12.5%;
molecular weight: 54000-110000Da.
A second object of the present invention is to provide: a method for preparing sulfonated gelatin, comprising the following steps:
1) Adding a sulfonic acid or sulfonate monomer P1 with a polymerizable carbon-carbon double bond, an active ester P2 with a polymerizable carbon-carbon double bond and an initiator into a container, adding a solvent to prepare a reaction solution with the mass concentration of 5-30%, and reacting the reaction solution for 24-48h at 65-70 ℃ under the protection of argon to obtain an active ester polymer P3 rich in sulfonic acid or sulfonate groups;
2) Concentrating the active ester polymer P3 obtained in the step 1) to remove the solvent, and then adding a buffer solution with the pH value of 3.0-6.0 for dissolving to obtain an active ester polymer P3 solution for later use; then weighing gelatin, adding buffer solution to dissolve, then slowly adding active ester polymer P3 solution, and reacting at 40-50 ℃ for 24-48 hours to obtain sulfonated gelatin P4 crude product;
3) Carrying out rotary evaporation concentration, dialysis and freeze drying treatment on the sulfonated gelatin P4 crude product in the step 2) to obtain a sulfonated gelatin P4 product with the sulfonation rate of 10-50%;
the sulfur content of the sulfonated gelatin is obtained through organic element analysis, the sulfonation rate is the proportion of active ester groups to amino groups of the gelatin, and the calculation formula is the amount of P2 substances/the amount of amino substances in the gelatin.
The specific reaction equation for synthesizing sulfonated gelatin is shown below:
further, in the step 1), the active ester P2 is acrylic acid-N-succinimidyl ester (NAS) or methacrylic acid-N-succinimidyl ester (MNAS);
further, in the step 1), the sulfonic acid or sulfonate monomer P1 is one of allylsulfonic acid, sodium allylsulfonate, 2-acrylamido-2-methyl-1-propane sulfonic Acid (AMPS), sodium 2-acrylamido-2-methyl-1-propane sulfonate (AMPS-Na), allyldimethyl (3-sulfopropyl) ammonium, and sodium 12-acrylamidodecyl-1-sulfonate;
further, in the step 1), the initiator is one of dibenzoyl peroxide (BPO), azobisisobutyronitrile (AIBN), azobisisoheptonitrile, ammonium persulfate and potassium persulfate;
further, in the step 1), the solvent is ultrapure water or N, N-Dimethylformamide (DMF);
further, in the step 1), the mass ratio of the active ester P2 to the sulfonic acid or sulfonate monomer P1 is 1:10-48, and the mass ratio of the initiator to the sulfonic acid or sulfonate monomer P1 is 0.2-1:100;
further, in the step 2), the buffer solution is phosphate aqueous solution or morpholine ethane sulfonic acid aqueous solution;
further, in the step 2), the gelatin freezing force is 180-250g Bloom, and the molecular weight is 50000-100000Da;
further, in the step 2), the mass ratio of the active ester polymer P3 to the amino group in the gelatin is 0.1-0.5:1;
a third object of the present invention is to provide: a sulfonated gelatin embolism microsphere,
the sulfonated gelatin embolic microspheres have a size of 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm, or 1100 μm;
the CV value of the sulfonated gelatin embolism microsphere is less than 10%;
the Young modulus of the sulfonated gelatin embolic microspheres is 40-3000KPa;
the sphericity of the sulfonated gelatin embolic microsphere is 0.75-0.95.
A fourth object of the present invention is to provide: a preparation method of sulfonated gelatin embolism microsphere comprises the following steps:
a. preparing a sulfonated gelatin aqueous solution with the mass concentration of 10-50%, and using a catalyst to adjust the pH value to 7.0-9.0 to be used as a disperse phase;
b. preparing a continuous phase and a receiving liquid, wherein the continuous phase is a mixture of an oily solvent and a surfactant, and the receiving liquid is a mixture of an oily solvent and a cross-linking agent;
c. connecting the continuous phase and the disperse phase with a microfluidic chip, mechanically shearing the continuous phase and the disperse phase to obtain droplets with uniform size, and collecting and solidifying the droplets by using a receiving liquid;
d. pouring the upper layer receiving liquid after the liquid drops are solidified, and cleaning by deionized water after cleaning by using a cleaning solvent;
e. and after the cleaning is finished, freeze-drying to obtain the sulfonated gelatin embolism microsphere.
Further, in the step a, the catalyst is one of tetramethyl ethylenediamine, 1, 3-propane diamine, ammonia water and sodium hydroxide, preferably tetramethyl ethylenediamine or sodium hydroxide.
Further, in the step b, the oily solvent is mineral oil, such as liquid paraffin, or vegetable oil, such as soybean oil;
further, in the step b, the surfactant is one or more of span 60, span 80, tween 85 and polyglycerol polyricinoleate (PGPR); preferably span 80 or PGPR;
further, in the step b, the mass concentration of the surfactant in the continuous phase is 1-10%;
further, in the step b, the cross-linking agent is formaldehyde, glutaraldehyde or calcium chloride, preferably glutaraldehyde;
further, in the step b, the mass concentration of the cross-linking agent in the receiving liquid is 0.55%;
further, in the step c, the microfluidic chip includes: the device comprises a disperse phase pipe, a continuous phase pipe, a collecting pipe and a receiving container, wherein the inner diameter of the continuous phase pipe is larger than the outer diameters of the mobile phase pipe and the collecting pipe, one ends of the continuous phase pipe and the collecting pipe are respectively and partially inserted into the mobile phase pipe, one end of the continuous phase pipe inserted into the mobile phase pipe is provided with a cone opening, the end part of the cone opening is aligned with the front end of the collecting pipe, the receiving container is arranged right below one end of the collecting pipe far away from the mobile phase pipe, a stirring device is arranged in the receiving container, the flow velocity of the mobile phase in the mobile phase pipe is 50-1000 mu L/min, the flow velocity of the disperse phase in the mobile phase pipe is 1-500 mu L/min, and the size of the cone opening of the disperse phase pipe is 30-600 mu m; the inner diameter of the receiving tube is 300-1000 mu m; the size of the microsphere is controlled by the diameter of the conical mouth of the disperse phase tube; by adjusting the flow rates of the mobile phase and the disperse phase, the disperse phase liquid drops are sheared out at the cone opening of the disperse phase pipe, so that uniform microspheres, namely microspheres with CV values within 10 percent, can be obtained; controlling the Young's modulus of the microspheres by controlling the concentration of the dispersed phase; controlling the Young's modulus of the microspheres by adjusting the concentration of the receiving liquid cross-linking agent;
further, in the step d, the cleaning solvent is ethyl acetate, petroleum ether, isopropanol, ethanol or methanol, preferably isopropanol;
a fifth object of the present invention is to provide: an application of embolic microspheres for loading positively charged anti-tumor chemotherapeutic drugs;
the anti-tumor chemotherapeutic medicine with positive charges is doxorubicin hydrochloride, epirubicin, pirarubicin, daunorubicin, idarubicin, irinotecan, oxaliplatin or lobaplatin, preferably doxorubicin hydrochloride or irinotecan.
The invention has the beneficial effects that:
1. the sulfonated gelatin has excellent biocompatibility and water solubility, the preparation method is simple, the yield is high, the gelatin is degradable, and the aqueous solution can be stably stored at normal temperature and does not gel.
2. The sulfonated gelatin microsphere can be prepared easily at normal temperature by using a microfluidic device, and the prepared microsphere has uniform particle size, good elasticity and good catheter trafficability.
3. The sulfonated gelatin is obtained by amidation reaction of active ester sulfonic acid or sulfonate polymer and gelatin, sulfonated gelatin droplets are crosslinked by aldehyde crosslinking agents to obtain sulfonated gelatin microspheres, and a large number of sulfonic acid groups are contained in the sulfonated gelatin microspheres, so that the rapid and large-scale loading of negatively charged anticancer chemotherapeutics can be realized.
4. Compared with the nondegradable drug-loaded microspheres in the market, the sulfonated gelatin microspheres such as DC Beads and CalliSpheres, equalSpheres can realize gradient slow release of the loaded chemotherapeutic drugs and release the loaded chemotherapeutic drugs to the greatest extent along with the degradation of the microspheres.
5. The invention can conveniently control the characteristic parameters such as the size, the shape, the degradation speed and the like of the microsphere by flexibly adjusting the parameters such as the concentration of the disperse phase, the concentration of the mobile phase, the flow rate, the crosslinking time and the like, so as to meet various application requirements.
6. The invention prepares the sulfonated gelatin microsphere by using a microfluidic technology, has simple and efficient process, and the prepared microsphere has uniform particle size and controllable particle size.
7. Compared with artificially synthesized degradable materials such as polylactic acid, the sulfonated gelatin has the advantages of abundant and cheap raw material sources, capability of being processed and molded by a conventional method, good biocompatibility, capability of being degraded by biological enzymes, difficulty in causing foreign body reaction, predictable degradation period and the like.
8. According to the invention, the microsphere with the required Young modulus can be prepared more accurately by controlling the concentration of the disperse phase and the concentration of the cross-linking agent of the receiving liquid.
9. In the invention, due to the micro-fluidic technology, the liquid drops are uniformly and controllably formed, the liquid drops in the receiving liquid are uniformly dispersed, and the prepared microsphere has good sphericity.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of an active ester polymer P3 of group 1 in example 1 of the present invention.
FIG. 2 is a nuclear magnetic resonance spectrum of gelatin in example 1 of the present invention.
FIG. 3 is a nuclear magnetic resonance spectrum of sulfonated gelatin of group 1 of example 1 of the present invention.
Fig. 4 is a schematic diagram of a microfluidic chip for preparing microsphere droplets according to the present invention.
FIG. 5 is a graph showing the drug loading of sulfonated gelatin embolic microspheres in example 12 of the present invention.
FIG. 6 is a graph showing the release profile of sulfonated gelatin embolic microspheres in example 12 of the present invention.
FIG. 7 is an optical micrograph of sulfonated gelatin embolic microspheres of group 10-G of example 12 of the present invention prior to drug loading.
FIG. 8 is an optical micrograph of a sulfonated gelatin embolic microsphere of group 10-G of example 12 of the present invention after drug loading.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto.
Example 1
The embodiment provides a method for preparing sulfonated gelatin, in the embodiment, the sulfonated gelatin is prepared by the following steps:
1) Adding 2-acrylamido-2-methyl-1-propane sulfonic Acid (AMPS), acrylic acid-N-succinimidyl ester (NAS) and Azodiisobutyronitrile (AIBN) into a container, adding N, N-Dimethylformamide (DMF), preparing a reaction solution with the mass concentration of 10%, and reacting the reaction solution for 48 hours at 65 ℃ under the protection of argon to obtain an active ester polymer P3 rich in sulfonic acid groups;
2) Concentrating the active ester polymer P3 obtained in the step 1) to remove the solvent, and then adding morpholine ethane sulfonic acid buffer solution with the pH value of 5.5 for dissolution to obtain active ester polymer P3 solution for later use; then weighing gelatin (nuclear magnetic resonance hydrogen spectrum is shown as figure 2), adding morpholine ethanesulfonic acid aqueous solution (MES) buffer solution for dissolution, then slowly adding active ester polymer solution, and reacting at 45 ℃ for 24 hours to obtain sulfonated gelatin crude product;
3) Concentrating the sulfonated gelatin crude product obtained in the step 2) by rotary evaporation, dialyzing for 3 days at normal temperature by using an 8-14kDa dry dialysis bag, and replacing water for three times; after dialysis is completed, rotary evaporation concentration and freeze drying are carried out, thus obtaining the sulfonated gelatin product with the following structural formula,
the specific reaction equation is as follows:
step one
Step two
In this example, sulfonated gelatin was prepared according to the types and amounts of raw materials in each group shown in table 1 below, and the index parameters are shown in table 2:
TABLE 1 types and amounts of raw materials in each group in example 1
Raw materials | Substance (B) | Group 1 (mol) | Group 2 (mol) | Group 3 (mol) | Group 4 (mol) | Group 5 (mol) |
P1 | AMPS | 0.056 | 0.056 | 0.057 | 0.058 | 0.059 |
P2 | NAS | 0.0012 | 0.0023 | 0.0035 | 0.0047 | 0.0059 |
Initiator(s) | AIBN | 0.00036 | 0.00037 | 0.00038 | 0.00039 | 0.00040 |
Table 2 index parameters for sulfonated gelatins for each panel of example 1
FIG. 1 shows the nuclear magnetic resonance spectrum of the active ester polymer P3 rich in sulfonic acid groups prepared in group 1 of this example;
FIG. 3 shows the nuclear magnetic resonance spectrum of the sulfonated gelatin prepared in group 1 of this example.
Example 2
This example provides a method for preparing sulfonated gelatin, which is different from example 1 in that AMPS is replaced with sodium 2-acrylamido-2-methyl-1-propanesulfonate (AMPS-Na), and the rest of the steps are the same as example 1.
In this example, sulfonated gelatin was prepared according to the types and amounts of raw materials in each group shown in table 3 below, and the index parameters are shown in table 4:
TABLE 3 types and amounts of raw materials in each group in example 2
Table 4 index parameters of the corresponding sulfonated gelatins for each panel of example 2
Example 3
This example provides a process for preparing sulfonated gelatin, which differs from example 1 in that the raw material types and amounts of each group in this example are shown in Table 5, and the remaining steps are the same as in example 1.
In this example, sulfonated gelatin was prepared according to the types and amounts of raw materials in each group shown in Table 5, and the index parameters are shown in Table 6;
TABLE 5 types and amounts of raw materials in each group in example 3
TABLE 6 index parameters for sulfonated gelatins for each panel of example 3
Example 4
This example provides a method for preparing sulfonated gelatin, which is different from the 1 group in example 1 in that in this example, the reaction condition parameters in the reaction process are shown in Table 7, and the rest steps are the same as in example 1.
TABLE 7 reaction condition parameters for each subgroup in example 4
The index parameters of the prepared sulfonated gelatin are shown in table 8,
TABLE 8 index parameters for sulfonated gelatins for each panel in example 4
Example 5
The embodiment provides a sulfonated gelatin embolic microsphere, which is prepared by the following steps:
a. the sulfonated gelatin of 1-5 groups prepared in example 1 was respectively prepared into 30% concentration aqueous solutions of sulfonated gelatin, and the pH was adjusted to 7.5 using tetramethyl ethylenediamine as a catalyst as a dispersed phase;
b. preparing a continuous phase and a receiving liquid, and preparing PGPR soybean oil solution with the concentration of 5 percent as the continuous phase; preparing glutaraldehyde soybean oil solution with the concentration of 3% as receiving solution;
c. as shown in fig. 5, the continuous phase and the disperse phase are connected with a microfluidic chip, droplets with uniform size are obtained by mechanical shearing of the continuous phase and the disperse phase, the droplets are collected for 1h by using a receiving liquid and crosslinked in the receiving liquid for 24h to solidify,
d. pouring the upper layer receiving liquid after the liquid drops are solidified, and cleaning by using deionized water after cleaning by using cleaning solvent isopropanol;
e. and after the cleaning is finished, freeze-drying to obtain the sulfonated gelatin embolism microsphere.
As shown in fig. 4, in the step c, the microfluidic chip includes: the inner diameter of the continuous phase pipe is larger than the outer diameters of the mobile phase pipe and the collecting pipe, one ends of the continuous phase pipe and the collecting pipe are respectively and partially inserted into the mobile phase pipe, one end of the continuous phase pipe inserted into the mobile phase pipe is provided with a cone opening, the end part of the cone opening is aligned with the front end of the collecting pipe, the receiving container is arranged right below one end of the collecting pipe far away from the mobile phase pipe, a stirring device is arranged in the receiving container, the flow velocity of the mobile phase in the mobile phase pipe is 150 mu L/min, the flow velocity of the mobile phase in the mobile phase pipe is 15 mu L/min, the diameter of the cone opening of the mobile phase pipe is 150 mu m, and the inner diameter of the receiving pipe is 450 mu m;
drug loading experiment:
the drug loading capacity of the microsphere is tested by the drug loading capacity of the sulfonated gelatin embolism microsphere to doxorubicin hydrochloride, and the specific steps are as follows:
A. firstly, preparing doxorubicin hydrochloride standard curve solution, and drawing standard curve;
B. preparing doxorubicin hydrochloride solution with the concentration of 2.5mg/mL, adding 10mL of doxorubicin hydrochloride solution into 25mg of sulfonated gelatin embolism microsphere, starting drug loading in a 20mL penicillin bottle, respectively taking 50 mu L of drug loading supernatant with the concentration of 0min,5min,10min,15min,20min,25min and 30min, and fixing the volume to 20mL, and sucking all the solution out of a syringe after 30min of drug loading.
C. Testing ultraviolet absorbance of the diluted drug-loaded liquid at 233.2nm, and calculating the drug-loaded amount for 30min by using a standard curve; the calculation formula of the drug loading Lx of the drug loading xth min is as follows:
Lx=(A 0 -A x )*V x /m
wherein A is 0 The concentration of the liquid medicine is 0min, the unit is mg/mL, A x The concentration of the liquid medicine is x min, the unit mg/mL, the volume of Vx is x min, and the mass of the dry sulfonated gelatin microspheres is m.
Wherein the Young modulus of the sulfonated gelatin after drug loading is tested by a special compressive strength tester (KP-20Y microstress tester). After the sulfonated gelatin microsphere is loaded with medicine, the microsphere sphericity is analyzed by using Image analyzers such as Digital Micrograph, FLUOCA FCSnap, image-Pro Plus, image J, beckman Coulter RapidVUE and the like, and the pixel area and the pixel circumference are calculated, so that the microsphere sphericity is calculated, the microsphere sphericity P is calculated according to the following calculation formula:
wherein A is the pixel area, ρ is the pixel perimeter, if P is greater than 0.8, it is spherical, and the larger the P value, the better the sphericity.
The performance index of the sulfonated gelatin embolic microspheres in this example is shown in Table 9
Table 9 performance index of sulfonated gelatin embolic microspheres for each panel in example 5
In this example, compared with group 1, groups 2 to 5 only changed the sulfonation rate of the sulfonated gelatin, and tested the drug loading and mechanical properties thereof, as shown in table 9, it can be seen that as the sulfonation rate increases, the drug loading of the microspheres increases, the highest drug loading of 0.34mg/mg is found at 30% of the sulfonation rate, and then the sulfonation rate is increased, and the drug loading of the microspheres decreases; this is because the increase in the sulfonation rate hinders the crosslinking of the sulfonated gelatin microspheres, so that the structural strength of the microspheres is reduced, the sulfonic acid group segments not fixed to the microsphere net structure are removed during the pure water washing, and thus the drug loading of the microspheres is reduced.
It is noted that the problem of balancing the drug carrying properties and mechanical properties of sulfonated gelatin is that as the Young's modulus increases, the hardness of the microspheres increases and the elasticity decreases, which results in poor passage of the microspheres through the catheter; while too low a Young's modulus indicates that the strength of the microspheres is poor and they are easily broken, which results in deterioration of the embolic properties of the microspheres.
Example 6
This example provides a sulfonated gelatin embolic microsphere, which differs from example 5 in that in this example, 6-12 sets of sulfonated gelatin prepared in example 2 are used to prepare the sulfonated gelatin embolic microsphere, and the rest of the steps are the same as in example 5.
The performance index of the sulfonated gelatin embolic microspheres in this example is shown in table 10:
table 10 Performance index of sulfonated gelatin embolic microspheres for each panel of example 6
The only difference between the groups 6-8 in this example and the groups 2-4 in example 5 is that the raw material P1 in preparing sulfonated gelatin is replaced by AMPS (AMPS-Na) which is sodium salt of AMPS, and as can be seen from tables 8 and 10, the drug loading of the sulfonated gelatin microsphere is obviously improved by replacing more stable sulfonate in aqueous solution, and the drug loading is improved to be 0.45mg/mg at the maximum when the sulfonation rate is 30%.
As can be seen from tables 3, 4 and 10, 9-12 groups in this example showed higher sulfur content (i.e., sulfonic acid group content) of sulfonated gelatin and higher drug loading and mechanical properties than 7 groups, and as shown in Table 10, when sulfur content was increased to 10.42%, the sulfonated gelatin microspheres had higher drug loading and more suitable mechanical strength.
Example 7
The difference between the 10 th group of sulfonated gelatin embolic microsphere and the 10 th group of the embodiment 6 is that in the embodiment, 4 groups of sulfonated gelatin aqueous solutions with different concentrations are respectively prepared by the sulfonated gelatin prepared in the 10 th group of the embodiment 2, and the pH is regulated to 7.5 by using a catalyst tetramethyl ethylenediamine to be used as a disperse phase; a total of 4 different sulfonated gelatin embolic microspheres of groups 10-A, 10-B, 10-C and 10-D were prepared, respectively, with the remainder of the procedure being the same as in group 10 of example 6.
The performance index of the sulfonated gelatin embolic microspheres in this example is shown in table 11:
TABLE 11 sulfonated gelatin embolic microsphere Performance index for each panel in example 7
The difference between this example and the 10 groups in example 6 is that the concentration of the dispersed phase in the step a is different, and it can be seen from tables 10 and 11 that an appropriate increase in the concentration of the dispersed phase can improve the drug-carrying performance, but the increase in the Young's modulus is small, and the hardening of the microspheres is caused.
Example 8
The difference between the sulfonated gelatin embolic microsphere provided in this example and the 10-B group in the example 7 is that glutaraldehyde soybean oil solutions with different concentrations of 4 groups of 10-E group, 10-F group, 10-J group and 10-H group are respectively prepared as receiving solutions; the remaining procedure was the same as in example 7, group 10-B.
The performance index of the sulfonated gelatin embolic microspheres in this example is shown in table 12:
table 12 performance index of sulfonated gelatin embolic microspheres for each panel in example 8
In this example, compared with the 10-B group in example 7, the difference is only that the concentration of the receiving liquid in the step B is different, and as can be seen from Table 11 and Table 12, the Young's modulus of the sulfonated gelatin embolic microspheres is also reduced with the reduction of the concentration of the receiving liquid, and the drug loading of the sulfonated gelatin embolic microspheres is increased and then reduced, so that the sulfonated gelatin microspheres with better balance of drug loading performance and mechanical properties can be obtained by properly reducing the concentration of the cross-linking agent of the receiving liquid.
Example 9
This example is different from example 5 in that the sulfonated gelatin embolic microspheres were prepared using the 13-20 groups of sulfonated gelatins prepared in example 3, in which the reaction condition parameters in each group are shown in Table 13, and the rest of the steps are the same as in example 5.
TABLE 13 reaction condition parameters for each subgroup in example 9
The performance index of the sulfonated gelatin embolic microspheres in this example is shown in table 14:
table 14 performance index of sulfonated gelatin embolic microspheres for each panel in example 9
As can be seen from Table 14, the sulfonate monomer with smaller molecular weight has lower drug loading rate, but the prepared sulfonated gelatin microsphere has better strength and sphericity, the sulfonated gelatin microsphere of AMPS and sodium salt thereof has higher drug loading rate as a whole, and the drug loading rate of the microsphere can be improved by using the combined cross-linking agent of glutaraldehyde and calcium chloride, and the concentration of the cross-linking agent can be improved to a small extent without influencing the drug loading rate of the microsphere. The type and concentration of the surfactant, the type of the catalyst, the pH value and the type of the cleaning solvent have no obvious influence on the drug loading of the microspheres.
Example 10
This example is different from example 5 in that the sulfonated gelatin embolic microspheres were prepared using 21-25 sets of sulfonated gelatins prepared in example 4, in which the reaction condition parameters in each set are shown in Table 15, and the rest of the steps are the same as in example 5.
TABLE 15 reaction condition parameters for each subgroup in example 10
The performance index of the sulfonated gelatin embolic microspheres in this example is shown in table 16:
table 16 Performance index of sulfonated gelatin embolic microspheres for each panel of example 10
As can be seen from Table 16, the Young's modulus and sphericity of the sulfonated gelatin microspheres with low sulfonation rate are both higher; in the preparation process of the sulfonated gelatin, the concentration, the reaction temperature and the reaction time of the reaction solution in the step 1) can be increased to increase the sulfur content of the sulfonated gelatin and the drug loading rate of the sulfonated gelatin microspheres, but the increase is limited; the sulfur content is reduced after the concentration of the reaction solution is increased to 30%, namely the sulfur content is highest when the concentration of the reaction solution is required to be between 10 and 20 percent, and the reaction temperature and the reaction time in the step 2) are improved, so that the effect on the drug loading of the microspheres is not great.
Example 11
The difference between this example and the 10-G group in example 8 is that in this example, a plurality of groups of sulfonated gelatin embolic microspheres with different sizes are prepared by changing the parameter conditions of the microfluidic chip, and the rest steps are the same as those of the 10-G group in example 8.
TABLE 17 example 11 microfluidic chip parameter conditions and sulfonated gelatin embolic microsphere Performance index
As shown in table 17, to prepare small-sized microspheres, the required cone diameter is small, and the size of the microspheres is 2-4 times of the cone size under the condition of a certain concentration of the dispersed phase; the preparation of small-sized microspheres requires a low concentration of the dispersed phase and a low flow rate of the dispersed phase, so that the yield is low; the size error (CV value) of the sulfonated gelatin microspheres can be controlled to be lower and less than 10 percent due to the micro-fluidic chip.
Example 12
In this example, the sulfonated gelatin embolic microspheres prepared in the 3 groups in example 5, 10 groups in example 6 and 10-G groups in example 8 were selected for drug-loading experiments, respectively, to obtain drug-loading curves of the sulfonated gelatin embolic microspheres.
1. Drug loading experiment
(1) Firstly, preparing doxorubicin hydrochloride standard curve solution, and drawing standard curve;
(2) Preparing doxorubicin hydrochloride solution with the concentration of 2.5mg/mL, adding 10mL of doxorubicin hydrochloride solution into 25mg embolic microspheres, starting drug loading in a 20mL penicillin bottle, respectively taking 50 mu L of drug loading supernatant with the concentration of 0min,5min,10min,15min,20min,25min and 30min, and fixing the volume to 20mL, and sucking all the solution out of a syringe after 30min of drug loading.
(3) Testing ultraviolet absorbance of diluted drug-loaded liquid at 233.2nm, calculating drug-loaded amount by using a standard curve, and drawing a drug-loaded curve; drug loading rate L of drug loading x min x The calculation formula is as follows:
L x =(A 0 -A x )*V/m
wherein A is 0 The concentration of the liquid medicine is 0min, the unit is mg/mL, A x The concentration of the liquid medicine is x min, the unit mg/mL, V is the volume of the liquid medicine, and m is the dry ball mass of the sulfonated gelatin microspheres.
2. Drug release experiment
(1) Adding 10mL PBS buffer solution with pH of 7.5 into sulfonated gelatin microspheres 30min after drug loading, and carrying out drug release experiment in a shaking table with constant temperature of 37 ℃;
(2) Taking 1mL of drug release supernatant of 5min, 15min, 30min, 1h, 6h, 12h, 24h, 3d, 4d, 5d, 6d, 9d, 12d, 15d, 18d, 21d, 28d, 35d, 42d, 49d, 56d, 63d, 70d, 77d, 84d, 91d, 98d, 105d, 112d and 119d, fixing the volume to 10mL, and then supplementing 1mL of PBS buffer solution into the drug release liquid.
(3) Testing ultraviolet absorbance of the drug release solution at 233.2nm, calculating drug release amount by standard curve, and drawing drug release curve to give drug release amount m at n time n The calculation formula is as follows:
wherein A is n The concentration of the liquid medicine at the time n is V n The liquid medicine volume at time n is V 0 Is the volume of the liquid;
the drug loading curves of the sulfonated gelatin embolic microspheres prepared in the 3 groups in the example 5, the 10 groups in the example 6 and the G groups in the example 8 are shown in the figure 5, and it can be seen that the drug loading speed of the sulfonated gelatin embolic microspheres is very fast, and the equilibrium point of drug loading can be basically reached after 30 minutes of drug loading. The drug release curve is shown in fig. 6, and it can be seen that the release amount of the sulfonated gelatin embolic microsphere is very rapid 6 hours before drug release, mainly because the drug adsorbed and swelled on the microsphere surface has weak acting force and the microsphere is easier to elute from the microsphere; the medicine loaded in the microsphere is gradually released along with the ion exchange action along with the progress of medicine release, and the release speed is slower than that of the medicine on the surface; and as the sulfonated gelatin microspheres degrade in PBS solution, the medicine inside the microspheres is almost completely released, and the medicine release is obviously increased when the microspheres degrade. As shown in fig. 7, which is an optical micrograph of the 10-G group microsphere before drug loading in the present embodiment, and as shown in fig. 8, which is an optical micrograph of the 10-G group microsphere after drug loading in the present embodiment, it can be seen that the diameter of the microsphere after drug loading is smaller than that before drug loading, mainly because the osmotic pressure of the drug loading solution is higher, so that swelling of the microsphere in the liquid medicine becomes difficult; the microspheres are dyed after absorbing doxorubicin hydrochloride after carrying medicine, the color is deepened, and the sphericity of the microspheres is not changed due to the medicine carrying process.
It will be understood that the invention has been described in terms of several embodiments, and that various changes and equivalents may be made to these features and embodiments by those skilled in the art without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (10)
1. A sulfonated gelatin, characterized in that: the sulfonated gelatin has a structural formula shown in (I):
wherein m and n are positive integers; n is m=10-48:1;
R 2 is a sulfonic acid group or a sodium sulfonate group;
R 3 is methyl or hydrogen;
R 1 is one of the following four structures:
the index system of the sulfonated gelatin meets the following conditions:
sulfonation rate: 10-50%;
sulfur element content: 8-12.5%;
molecular weight: 54000-110000Da.
2. A method for preparing sulfonated gelatin, which is characterized by comprising the following steps:
1) Adding a sulfonic acid or sulfonate monomer with a polymerizable carbon-carbon double bond, an active ester with a polymerizable carbon-carbon double bond and an initiator into a container, adding a solvent to prepare a reaction solution with the mass concentration of 5-30%, and reacting the reaction solution for 24-48 hours at 65-70 ℃ under the protection of argon to obtain an active ester polymer rich in sulfonic acid or sulfonate groups;
2) Concentrating the active ester polymer obtained in the step 1) to remove the solvent, and then adding a buffer solution with the pH value of 5.0-6.0 for dissolution to obtain an active ester polymer solution for later use; then weighing gelatin, adding buffer solution for dissolving, then slowly adding active ester polymer solution, and reacting at 40-50 ℃ for 24-48 hours to obtain sulfonated gelatin crude product;
3) And (3) carrying out rotary evaporation concentration, dialysis and freeze drying treatment on the sulfonated gelatin crude product in the step (2) to obtain a sulfonated gelatin product with the sulfonation rate of 10-50%.
3. The method for producing sulfonated gelatin according to claim 2, wherein: in the step 1), the active ester is acrylic acid-N-succinimidyl ester or methacrylic acid-N-succinimidyl ester; the sulfonic acid or sulfonate monomer is one of allylsulfonic acid, sodium allylsulfonate, 2-acrylamido-2-methyl-1-propane sulfonic acid, sodium 2-acrylamido-2-methyl-1-propane sulfonic acid, allyl dimethyl (3-sulfopropyl) ammonium and sodium 12-acrylamidodeane-1-sulfonate; the initiator is one of dibenzoyl peroxide, azodiisobutyronitrile, azodiisoheptonitrile, ammonium persulfate and potassium persulfate; the solvent is ultrapure water or N, N-dimethylformamide; the amount ratio of the initiator to the sulfonic acid or sulfonate monomer material is 0.2-1:100; the ratio of the active ester to the sulfonic acid or sulfonate monomer material is 1:10-48.
4. The method for producing sulfonated gelatin according to claim 2, wherein: in the step 2), the buffer solution is phosphate aqueous solution or morpholine ethane sulfonic acid aqueous solution; the gelatin has a freezing force of 180-250g Bloom and a molecular weight of 50000-100000Da; the mass ratio of the active ester polymer to the gelatin amino matter is 0.1-0.5:1.
5. A sulfonated gelatin embolic microsphere, characterized in that: a sulfonated gelatin produced from the sulfonated gelatin according to any one of claims 1-4, said sulfonated gelatin embolic microspheres having particle size dimensions comprising: 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1000 μm or 1100 μm;
the CV value of the sulfonated gelatin embolism microsphere is less than 10%;
the Young modulus of the sulfonated gelatin embolic microspheres is 40-3000Kpa;
the sphericity of the sulfonated gelatin embolic microsphere is 0.75-0.95.
6. The preparation method of the sulfonated gelatin embolism microsphere is characterized by comprising the following steps:
a. preparing a sulfonated gelatin aqueous solution with the mass concentration of 10-50%, and using a catalyst to adjust the pH value to 7.0-9.0 to be used as a disperse phase;
b. preparing a continuous phase and a receiving liquid, wherein the continuous phase is a mixture of an oily solvent and a surfactant, and the receiving liquid is a mixture of an oily solvent and a cross-linking agent;
c. connecting the continuous phase and the disperse phase with a microfluidic chip, mechanically shearing the continuous phase and the disperse phase to obtain droplets with uniform size, and collecting and solidifying the droplets by using a receiving liquid;
d. pouring an upper layer receiving liquid after the liquid drops are solidified, and cleaning by using a cleaning solvent, namely ethyl acetate, petroleum ether, isopropanol, ethanol or methanol, and then cleaning by using deionized water;
e. and after the cleaning is finished, freeze-drying to obtain the sulfonated gelatin embolism microsphere.
7. The method for preparing sulfonated gelatin embolic microspheres according to claim 6, wherein: in the step a, the catalyst is one of tetramethyl ethylenediamine, 1, 3-propylene diamine, ammonia water and sodium hydroxide.
8. The method for preparing sulfonated gelatin embolic microspheres according to claim 6, wherein: in the step b, the oily solvent is liquid paraffin or soybean oil; the surfactant is one or a combination of more of span 60, span 80, tween 85 and polyglycerol polyricinoleate, and the mass concentration of the surfactant in the continuous phase is 1-10%; the cross-linking agent is formaldehyde, glutaraldehyde or calcium chloride, and the mass concentration of the cross-linking agent in the receiving liquid is 0.5-5%.
9. The method for preparing sulfonated gelatin embolic microspheres according to claim 6, wherein: in the step c, the microfluidic chip includes: the device comprises a disperse phase pipe, a continuous phase pipe, a collecting pipe and a receiving container, wherein the inner diameter of the continuous phase pipe is larger than the outer diameters of the mobile phase pipe and the collecting pipe, one ends of the continuous phase pipe and the collecting pipe are respectively and partially inserted into the mobile phase pipe, one end of the continuous phase pipe inserted into the mobile phase pipe is provided with a cone opening, the end part of the cone opening is aligned with the front end of the collecting pipe, the receiving container is arranged right below one end of the collecting pipe far away from the mobile phase pipe, a stirring device is arranged in the receiving container, the continuous phase flow velocity in the continuous phase pipe is 50-1000 mu L/min, the disperse phase flow velocity in the disperse phase pipe is 1-500 mu L/min, and the diameter of the disperse phase pipe is 30-600 mu m; the inner diameter of the receiving tube is 300-1000 mu m.
10. The sulfonated gelatin embolic microsphere according to claim 5, wherein: it is used for loading antineoplastic chemotherapeutic medicine with positive charges.
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