CN111617233A - Material loaded with anti-tumor drug and preparation method thereof - Google Patents
Material loaded with anti-tumor drug and preparation method thereof Download PDFInfo
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- CN111617233A CN111617233A CN202010631018.7A CN202010631018A CN111617233A CN 111617233 A CN111617233 A CN 111617233A CN 202010631018 A CN202010631018 A CN 202010631018A CN 111617233 A CN111617233 A CN 111617233A
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- Prior art keywords
- phycobiliprotein
- folic acid
- tumor
- drug
- loaded
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- 229940041181 antineoplastic drug Drugs 0.000 title claims abstract description 55
- 239000000463 material Substances 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 31
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims abstract description 88
- 239000002105 nanoparticle Substances 0.000 claims abstract description 50
- 108060006184 phycobiliprotein Proteins 0.000 claims abstract description 49
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 claims abstract description 45
- 229960000304 folic acid Drugs 0.000 claims abstract description 45
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- 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 claims description 10
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 10
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- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical compound C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-FQEVSTJZSA-N 0.000 claims description 6
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- KLWPJMFMVPTNCC-UHFFFAOYSA-N Camptothecin Natural products CCC1(O)C(=O)OCC2=C1C=C3C4Nc5ccccc5C=C4CN3C2=O KLWPJMFMVPTNCC-UHFFFAOYSA-N 0.000 claims description 4
- 229940127093 camptothecin Drugs 0.000 claims description 4
- VSJKWCGYPAHWDS-UHFFFAOYSA-N dl-camptothecin Natural products C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)C5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-UHFFFAOYSA-N 0.000 claims description 4
- 229960004528 vincristine Drugs 0.000 claims description 4
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 claims description 4
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- 229930012538 Paclitaxel Natural products 0.000 claims description 2
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 claims description 2
- 229960004316 cisplatin Drugs 0.000 claims description 2
- 229960001592 paclitaxel Drugs 0.000 claims description 2
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 claims description 2
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- 231100000252 nontoxic Toxicity 0.000 abstract description 4
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 abstract description 4
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- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
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- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 2
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- 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
- A61K47/54—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 an organic compound
- A61K47/55—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 an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0045—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent agent being a peptide or protein used for imaging or diagnosis in vivo
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- A61K49/0013—Luminescence
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- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0052—Small organic molecules
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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Abstract
The invention provides a preparation method of a material loaded with an anti-tumor drug, which comprises the following steps: s1, preparing folic acid active lipid; s2, preparing phycobiliprotein nanoparticles; s3, preparing the folic acid-phycobiliprotein coupling nanoparticles. Under alkaline conditions, carboxyl on the active folic acid ester can react with amino on the surface amino acid residue of phycobiliprotein to be coupled to obtain the folic acid-phycobiliprotein conjugate, phycoerythrin or phycocyanin in the phycobiliprotein not only has good anti-tumor effect, but also is safe and nontoxic, and meanwhile, has fluorescence, excellent fluorescence effect and long duration, and can realize long-acting targeted imaging in tumor treatment.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a material loaded with an anti-tumor medicine and a preparation method thereof.
Background
The tumor intratumoral injection treatment is a new tumor treatment scheme developed in recent years, and the medicine is directly injected into the tumor to play a role in site-specific treatment, so that the first-pass effect and systemic toxic and side effects and the like required by means of oral administration, intravenous injection and the like can be avoided or alleviated to a certain extent. Treatment regimens employing intratumoral injection of drugs have unique advantages in sites that are accessible without open surgical instrumentation, as well as sites that are not conducive to surgical resection, or in view of post-operative aesthetics, among other things.
The approved marketed virus therapy for the polyoma virus, such as IMLYGIC from Anjin, domestic new drugs from today and Ankeri, all adopt the intratumoral injection method. In addition, many drug development studies have also adopted intratumoral injection, such as the MultiVir company used intratumoral injection to deliver the drug Ad-IL-24 for the treatment of head and neck tumors. Furthermore, the existing clinical related technical means are mature, can realize the intratumoral injection of multiple parts, for example, can realize the minimally invasive treatment of tumor puncture under the guidance of B ultrasonic, CT, magnetic resonance and other images, and the clinical means such as argon-nitrogen knife freezing, radio frequency heating and the like can be used.
However, in the scheme of intratumoral injection, two risks need to be particularly concerned, one is that the intratumoral pressure is higher than that of normal tissues, so that liquid medicines are easy to overflow after being injected into tumors, and the other is that blood vessels in the tumors are rich, so that the medicines are easy to diffuse to other tissues from the tumor part quickly after being administered, and the purpose of long-acting administration cannot be achieved.
Therefore, a new preparation or a new material for intratumoral injection is provided, the risk of drug overflow is avoided, and the long-time intratumoral fixed-point slow-release drug delivery is realized, so that the method has practical application value.
Disclosure of Invention
The invention aims to provide a material loaded with an anti-tumor drug and a preparation method thereof, wherein under alkaline conditions, carboxyl on active lipid of folic acid can react with amino on amino acid residues on the surface of phycobiliprotein to be coupled to obtain a folic acid-phycobiliprotein conjugate, phycoerythrin or phycocyanin in the phycobiliprotein not only has good anti-tumor effect, but also is safe and nontoxic, and simultaneously has fluorescence, excellent fluorescence effect and long duration, and long-acting targeted imaging can be realized in tumor treatment.
The technical scheme of the invention is realized as follows:
the invention provides a preparation method of a material loaded with an anti-tumor drug, which comprises the following steps:
s1 preparation of folic acid active lipid: weighing folic acid, dissolving the folic acid in DMSO (dimethyl sulfoxide), adding ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride and N-hydroxysuccinimide, controlling the temperature at 20-30 ℃, continuously stirring, reacting for 3-7h in the dark, performing suction filtration to remove precipitates, adding 1-3 times of diethyl ether into residual liquid, stirring to generate precipitates, performing suction filtration, washing the obtained precipitates with diethyl ether, and drying to obtain folic acid active ester;
s2 preparation of phycobiliprotein nanoparticles: weighing phycobiliprotein, dissolving the phycobiliprotein in PBS buffer solution with pH =7.2-7.5, dropwise adding absolute ethyl alcohol, adding 0.1-0.2% propylene glycol, stirring in the dark for reaction for 1-2h, centrifuging, repeatedly washing with deionized water for 1-3 times, and ultrasonically dispersing the precipitate with equal volume of deionized water to obtain phycobiliprotein nanoparticle suspension;
s3 preparation of folic acid-phycobiliprotein coupling nanoparticles: weighing folic acid active ester, dissolving the folic acid active ester in a buffer solution, dropwise adding phycobiliprotein nanoparticle suspension drops, continuously stirring and reacting for 15-30min after mixing, transferring reactants into a dialysis bag with the relative molecular mass of 3000-4000 after the reaction is finished, using the buffer solution as dialysate, monitoring by using an ultraviolet spectrophotometer in the dialysis process until the concentration of folic acid in the dialysate is not increased any more, collecting the solution in the dialysis bag, and freeze-drying to obtain folic acid-phycobiliprotein nanoparticles, namely the material loaded with the antitumor drug.
As a further improvement of the invention, the mass ratio of the folic acid, the N-hydroxysuccinimide and the ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride in the step S1 is 1: (0.1-0.3): (0.7-1.2); the drying method is drying at 40-50 ℃ to constant weight.
As a further improvement of the present invention, in step S2, the mass ratio of the phycobiliprotein to propylene glycol is 10: (0.1-1); the ultrasonic power is 1000-1200W.
As a further improvement of the present invention, the buffer solution in step S3 is selected from one of carbonate buffer with pH =9-10, PBS buffer with pH =7.2-7.5, Tris-HCl buffer with pH = 7-7.5; the mass ratio of the folic acid active ester to the phycobiliprotein is 1: (0.2-0.5); the phycobiliprotein is phycocyanin or phycoerythrin, and the purity of the phycobiliprotein is more than 90%.
As a further improvement of the invention, the method for loading the anti-tumor drug loaded material comprises the following steps: reacting N, N' -dicyclohexylcarbodiimide with N-hydroxysuccinimide for 10-30 min to obtain a mixed solution, carrying out amination on an anti-tumor drug, dissolving the anti-tumor drug and folic acid-phycobiliprotein nanoparticles loaded with the anti-tumor drug in DMF, adding the solution into the reaction mixed solution, and carrying out crosslinking reaction to obtain the anti-tumor drug.
As a further improvement of the invention, the antitumor drugs include, but are not limited to, adriamycin, cisplatin, vincristine, paclitaxel, and camptothecin.
As a further improvement of the invention, the method for amination of the antitumor drug comprises the steps of dissolving the antitumor drug in toluene, dropwise adding a toluene solution of DBU under an ice bath condition, heating to room temperature after dropwise adding, reacting for 2-5h, filtering, washing the precipitate with deionized water for 1-3 times, and obtaining the aminated antitumor drug.
The invention further protects the material loaded with the antitumor drug prepared by the method.
The invention further protects the application of the material loaded with the anti-tumor drug in preparing the anti-tumor drug.
As a further improvement of the invention, the folic acid-phycobiliprotein nanoparticle loaded with the antitumor drug has a fluorescence characteristic and is applied to targeted imaging.
The invention has the following beneficial effects: under alkaline conditions, carboxyl on the active folic acid ester can react with amino on the surface amino acid residue of phycobiliprotein to be coupled to obtain the folic acid-phycobiliprotein conjugate, phycoerythrin or phycocyanin in the phycobiliprotein not only has good anti-tumor effect, but also is safe and nontoxic, and meanwhile, has fluorescence, excellent fluorescence effect and long duration, and can realize long-acting targeted imaging in tumor treatment.
(1) The invention provides an application of a HAND-YE ACID-phycobiliprotein nanoparticle in preparing a tumor inhibiting medicine.
(2) The phycobiliprotein in the functionalized folic acid-phycobiliprotein nanoparticles obtained by the invention is used as an active component for inhibiting tumor and a carrier for loading anti-tumor drugs, and has the synergistic effects of inhibiting the growth of multidrug-resistant tumor cells and realizing the high-efficiency anti-tumor effect.
(3) The folic acid-phycobiliprotein nanoparticle prepared by the invention is a fluorescent nano material, no other auxiliary reagent is needed to be added in the preparation process, the product system is simple, and the product is convenient and quick to store and use.
(4) The invention effectively regulates and controls the shape and size of nano selenium by controlling the coupling of the phycobiliprotein suspension and folic acid, is beneficial to increasing the drug intake of cells and reducing the efflux, thereby ensuring that the intracellular drug is maintained at a higher level.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is an SEM photograph of a folic acid-phycobiliprotein nanoparticle suspension prepared by dissolving folic acid-phycobiliprotein nanoparticles prepared in example 1 in water to obtain a 10% folic acid-phycobiliprotein nanoparticle suspension by mass fraction;
FIG. 2 is an image of a confocal fluorescence microscope after the drug-loaded system in test example 1 of the present invention is cultured in HeLa cells for 6 hours;
FIG. 3 is a graph showing a comparison of tumor growth (n =6) in the groups of tumor-bearing mice in test example 2 of the present invention after administration.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the examples, the Tris-HCl buffer with pH =7.2 is 0.01mol/L Tris-HCl with pH =7.2, and the preparation method is as follows: 50mL of 0.1mol/L Tris solution was mixed with 44.7mL of 0.1mol/L hydrochloric acid and diluted to 100 mL.
In the examples, the PBS buffer with pH =7.2 is 0.01mol/L PBS buffer with pH =7.2, and the preparation method is as follows: 50mL of 0.2mol/L potassium dihydrogen phosphate solution was added to 35mL of 0.2mol/L sodium hydroxide solution, and the mixture was diluted with water to 200 mL.
EXAMPLE 1 preparation of antitumor drug-loaded Material
The method comprises the following steps:
s1 preparation of folic acid active lipid: weighing 1g of folic acid, dissolving the folic acid in 20mL of DMSO, adding 0.7g of ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride and 0.1g N-hydroxysuccinimide, controlling the temperature at 20 ℃, continuously stirring, reacting for 3 hours in a dark place, removing precipitates by suction filtration, adding 1-time volume of diethyl ether into residual liquid, stirring to generate precipitates, carrying out suction filtration, washing the obtained precipitates with diethyl ether, and drying at 40 ℃ to constant weight to obtain folic acid active lipid;
s2 preparation of phycobiliprotein nanoparticles: weighing 10g of phycobiliprotein, dissolving the phycobiliprotein in 100mL of PBS buffer solution with pH =7.2, dropwise adding 10mL of anhydrous ethanol, adding 100g of 0.1% propylene glycol, stirring in the dark for reaction for 1h, centrifuging at 3000r/min for 5min, repeatedly washing with 50mL of deionized water for 1 time each time, and ultrasonically dispersing 1000W of the precipitate with equal volume of deionized water to obtain phycobiliprotein nanoparticle suspension with the mass percent of 2%;
s3 preparation of folic acid-phycobiliprotein coupling nanoparticles: weighing 1g of active folic ester, dissolving the active folic ester in 10mL of Tris-HCl buffer solution with pH =7.2, dropwise adding 10g of suspension drops of phycobiliprotein nanoparticles, mixing, continuously stirring for reaction for 15min, transferring a reactant into a dialysis bag with a relative molecular mass of 3000 after the reaction is finished, using Tris-HCl buffer solution with pH =7.2 as dialysate, monitoring by using an ultraviolet spectrophotometer during the dialysis process until the concentration of folic acid in the dialysate is not increased any more, collecting the solution in the dialysis bag, and freeze-drying to obtain folic acid-phycobiliprotein nanoparticles, namely a material loaded with an antitumor drug, wherein FIG. 1 is an SEM image of 10% folic acid-phycobiliprotein nanoparticle suspension prepared by dissolving the prepared folic acid-phycobiliprotein nanoparticles in water;
s4 camptothecin amination: dissolving 1g of camptothecin in 10mL of toluene, dropwise adding 10mL (containing DBU0.5g) of toluene solution of DBU under an ice bath condition, heating to room temperature after dropwise adding, reacting for 2h, filtering, washing the precipitate with deionized water for 1-3 times to obtain aminated camptothecin;
s5, a method for loading anti-tumor drug material medicine, which comprises the following steps: dissolving 0.82g N, N' -dicyclohexylcarbodiimide and 0.4g of N-hydroxysuccinimide in 20mL of dichloromethane, reacting for 10min to obtain a mixed solution, dissolving 1g of aminated camptothecin and 5g of material folic acid-phycobiliprotein nanoparticles loaded with antitumor drugs in DMF, adding into the reaction mixed solution, carrying out cross-linking reaction for 1g, and filtering to obtain the anti-tumor drug.
In this example, phycobiliprotein is phycocyanin with a purity of 95%, and was purchased from alatin biochemicals.
Example 2 preparation method of antitumor drug-loaded Material
The method comprises the following steps:
s1 preparation of folic acid active lipid: weighing 1g of folic acid, dissolving the folic acid in 20mL of DMSO, adding 1.2g of ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride and 0.3g N-hydroxysuccinimide, controlling the temperature at 30 ℃, continuously stirring, reacting for 7 hours in a dark place, removing precipitates by suction filtration, adding diethyl ether with the volume of 3 times of the residual liquid, stirring to generate precipitates, carrying out suction filtration, washing the obtained precipitates with diethyl ether, and drying at 50 ℃ to constant weight to obtain folic acid active lipid;
s2 preparation of phycobiliprotein nanoparticles: weighing 10g of phycobiliprotein, dissolving the phycobiliprotein in 100mL of PBS buffer solution with pH =7.2, dropwise adding 10mL of anhydrous ethanol, adding 50g of 0.2% propylene glycol, stirring in the dark for reaction for 2h, centrifuging at 3000r/min for 5min, repeatedly washing with 50mL of deionized water for 3 times each time, and ultrasonically dispersing the precipitate with equal volume of deionized water at 1200W to obtain phycobiliprotein nanoparticle suspension with the mass percent of 5%;
s3 preparation of folic acid-phycobiliprotein coupling nanoparticles: weighing 1g of folic acid active ester, dissolving the folic acid active ester in 10mL of Tris-HCl buffer solution with pH =7.2, dropwise adding 10g of suspension drops of phycobiliprotein nanoparticles, mixing, continuously stirring for reaction for 30min, transferring reactants into a dialysis bag with the relative molecular mass of 4000 after the reaction is finished, using Tris-HCl buffer solution with pH =7.2 as dialysis solution, monitoring by using an ultraviolet spectrophotometer in the dialysis process until the folic acid concentration in the dialysis solution is not increased any more, collecting the solution in the dialysis bag, and freeze-drying to obtain folic acid-phycobiliprotein nanoparticles, namely the material loaded with the antitumor drug;
s4. doxorubicin amination: dissolving 1g of adriamycin in 10mL of toluene, dropwise adding 10mL (containing 1g of DBU) of toluene solution of DBU under an ice-bath condition, heating to room temperature after dropwise adding, reacting for 5 hours, filtering, washing precipitates with deionized water for 3 times to obtain aminated adriamycin;
s5, a method for loading anti-tumor drug material medicine, which comprises the following steps: dissolving 0.82g N, N' -dicyclohexylcarbodiimide and 0.4g of N-hydroxysuccinimide in 20mL of dichloromethane, reacting for 30min to obtain a mixed solution, dissolving 1g of aminated adriamycin and 5g of material folic acid-phycobiliprotein nanoparticles loaded with antitumor drugs in DMF, adding into the reaction mixed solution, carrying out cross-linking reaction for 1g, and filtering to obtain the product.
In this example, phycobiliprotein is phycoerythrin, 95% pure, and purchased from alatin biochemical reagents.
EXAMPLE 3 preparation of antitumor drug-loaded Material
The method comprises the following steps:
s1 preparation of folic acid active lipid: weighing 1g of folic acid, dissolving the folic acid in 20mL of DMSO, adding 1g of ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride and 0.2g N-hydroxysuccinimide, controlling the temperature at 25 ℃, continuously stirring, reacting for 5 hours in a dark place, performing suction filtration to remove precipitates, adding 1-3 times of diethyl ether into residual liquid, stirring to generate precipitates, performing suction filtration, washing the obtained precipitates with diethyl ether, and drying at 40-50 ℃ to constant weight to obtain folic acid active lipid;
s2 preparation of phycobiliprotein nanoparticles: weighing 10g of phycobiliprotein, dissolving the phycobiliprotein in 100mL of PBS buffer solution with pH =7.2, dropwise adding 10mL of anhydrous ethanol, adding 70g of 0.15% propylene glycol, stirring in the dark for reaction for 1.5h, centrifuging at 3000r/min for 5min, repeatedly washing with 50mL of deionized water for 2 times each time, precipitating with equal volume of deionized water, and performing ultrasonic dispersion at 1100W to obtain phycobiliprotein nanoparticle suspension with the mass percentage of 3%;
s3 preparation of folic acid-phycobiliprotein coupling nanoparticles: weighing 1g of folic acid active ester, dissolving the folic acid active ester in 10mL of Tris-HCl buffer solution with pH =7.2, dropwise adding 10g of suspension drops of phycobiliprotein nanoparticles, mixing, continuously stirring for reaction for 20min, transferring reactants into a dialysis bag with the relative molecular mass of 3500 after the reaction is finished, using Tris-HCl buffer solution with pH =7.2 as dialysate, monitoring by using an ultraviolet spectrophotometer in the dialysis process until the folic acid concentration in the dialysate is not increased any more, collecting the solution in the dialysis bag, and freeze-drying to obtain the folic acid-phycobiliprotein nanoparticles, namely the material loaded with the antitumor drug;
s4 vincristine amination: dissolving 1g of vincristine in 10mL of toluene, dropwise adding 10mL (containing DBU0.7 g) of toluene solution of DBU under ice bath condition, heating to room temperature after dropwise adding, reacting for 3h, filtering, washing precipitate with deionized water for 2 times to obtain aminated vincristine;
s5, a method for loading anti-tumor drug material medicine, which comprises the following steps: dissolving 0.82g N, N' -dicyclohexylcarbodiimide and 0.4g of N-hydroxysuccinimide in 20mL of dichloromethane, reacting for 20min to obtain a mixed solution, dissolving 1g of aminated vincristine and 5g of antitumor drug-loaded folic acid-phycobiliprotein nanoparticles in DMF, adding the solution into the reaction mixed solution, carrying out crosslinking reaction for 1g, and filtering to obtain the antitumor drug-loaded folic acid-phycobiliprotein nanoparticles.
In this example, phycobiliprotein is phycocyanin with a purity of 95%, and was purchased from alatin biochemicals.
Test example 1 cellular uptake
The drug uptake condition of the carbon-point targeted adriamycin-drug-loaded material folic acid-phycobiliprotein nanoparticle targeted drug-loading system by the HeLa cells is observed by imaging with a laser confocal fluorescence microscope, and the result is shown in figure 2. the HeLa cells are respectively cultured with the adriamycin-drug-loaded material folic acid-phycobiliprotein nanoparticles for 6h, and can receive blue and red fluorescence signals, which indicates that the blue and red fluorescence signals are taken by the HeLa cells of the system, so that the adriamycin-drug-loaded material folic acid-phycobiliprotein nanoparticle targeted drug-loading system can enter the cells through folic acid mediated targeting, and the uptake of the system by the cells is increased. After 6h incubation, pictures A, B and C were observed to find that the blue fluorescence signal was only present in the cell membrane and cytoplasmic region, but not in the nucleus. The red fluorescent signal is obviously present in the cell nucleus area except the cell membrane and cytoplasm area, which shows that after 6 hours of incubation, the material folic acid-phycobiliprotein nano-particle loaded with the adriamycin medicine releases a large amount of adriamycin medicine to enter the cell nucleus area, so that DNA is damaged and the growth of the cell is inhibited.
Test example 2 in vivo pharmacodynamic experiment
The inoculation concentration is 3.6 × 106cell·mL-1KB cell to the subcutaneous part of right front limb right axilla of BALB/c nude mouse, and selecting the KB cell with good tumor growth and tumor volume of about 80-150 mm after about one week of tumor planting3The left and right tumor-bearing mice are used as the experimental animal model. The mice were randomly divided into 5 groups (control group, 10. mu.M, 20. mu.M, 40. mu.M, 80. mu.M) according to the principle of average distribution of tumor size to medium and small, and each group had 6 tumor-bearing mice. Each tumor-bearing mouse is injected with 10 mg/kg once-1The drug prepared in example 3 was administered in a dose of 0.3 mL at different concentrations, 1 injection at 1 d intervals, and 5 consecutive administrations were carried out. At the time of first administration, the length of the longest diameter (a) of the tumor-bearing nude mice and the length of the short diameter (b) perpendicular to the longest diameter are measured and recorded at intervals of 1 d by a digital vernier caliper. Tumor volume was calculated and tumor volume growth curves were plotted after 24 d according to the following formula: v = pi ab2The results are shown in FIG. 3.
On the basis of successfully establishing a human KB tumor-bearing mouse model, the initial research of in vivo pharmacodynamics is carried out. The tumor volume of 1 tumor-bearing mouse was measured every 1 d from the first administration to 24 d. The tumor volume of the control group is continuously increased along with the prolonging of the administration time, and is in a continuously accelerated trend, and the activity of the tumor-bearing mice is influenced to a certain degree due to the overlarge tumor body, so that the hypokinesia is shown; the tumor volume of the administration group is increased along with the time, but the increase speed is obviously reduced, and the tumor-bearing mice show better mental state, can freely move, and have normal physiological functions of appetite and excretion. As shown in FIG. 3 (note: P < 0.05 compared with blank group), the tumor growth curve was significantly lower in the 40. mu.M group than in the other groups, and the tumor growth was inhibited. ANOVA analysis of variance 40. mu.M group reduced tumor volume by nearly 50% (P < 0.05) compared to the blank group.
Compared with the prior art, under the alkaline condition, carboxyl on the active lipid of folic acid can react with amino on the surface amino acid residue of phycobiliprotein to be coupled to obtain the folic acid-phycobiliprotein conjugate, phycoerythrin or phycocyanin in the phycobiliprotein not only has better anti-tumor effect, is safe and nontoxic, but also has fluorescence, excellent fluorescence effect and long duration, and can realize long-acting targeted imaging in tumor treatment.
(1) The invention provides an application of a HAND-YE ACID-phycobiliprotein nanoparticle in preparing a tumor inhibiting medicine.
(2) The phycobiliprotein in the functionalized folic acid-phycobiliprotein nanoparticles obtained by the invention is used as an active component for inhibiting tumor and a carrier for loading anti-tumor drugs, and has the synergistic effects of inhibiting the growth of multidrug-resistant tumor cells and realizing the high-efficiency anti-tumor effect.
(3) The folic acid-phycobiliprotein nanoparticle prepared by the invention is a fluorescent nano material, no other auxiliary reagent is needed to be added in the preparation process, the product system is simple, and the product is convenient and quick to store and use.
(4) The invention effectively regulates and controls the shape and size of nano selenium by controlling the coupling of the phycobiliprotein suspension and folic acid, is beneficial to increasing the drug intake of cells and reducing the efflux, thereby ensuring that the intracellular drug is maintained at a higher level.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A preparation method of a material loaded with an anti-tumor drug is characterized by comprising the following steps:
s1 preparation of folic acid active lipid: weighing folic acid, dissolving the folic acid in DMSO (dimethyl sulfoxide), adding ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride and N-hydroxysuccinimide, controlling the temperature at 20-30 ℃, continuously stirring, reacting for 3-7h in the dark, performing suction filtration to remove precipitates, adding 1-3 times of diethyl ether into residual liquid, stirring to generate precipitates, performing suction filtration, washing the obtained precipitates with diethyl ether, and drying to obtain folic acid active ester;
s2 preparation of phycobiliprotein nanoparticles: weighing phycobiliprotein, dissolving the phycobiliprotein in PBS buffer solution with pH =7.2-7.5, dropwise adding absolute ethyl alcohol, adding 0.1-0.2% propylene glycol, stirring in the dark for reaction for 1-2h, centrifuging, repeatedly washing with deionized water for 1-3 times, and ultrasonically dispersing the precipitate with equal volume of deionized water to obtain phycobiliprotein nanoparticle suspension;
s3 preparation of folic acid-phycobiliprotein coupling nanoparticles: weighing folic acid active ester, dissolving the folic acid active ester in a buffer solution, dropwise adding phycobiliprotein nanoparticle suspension drops, continuously stirring and reacting for 15-30min after mixing, transferring reactants into a dialysis bag with the relative molecular mass of 3000-4000 after the reaction is finished, using the buffer solution as dialysate, monitoring by using an ultraviolet spectrophotometer in the dialysis process until the concentration of folic acid in the dialysate is not increased any more, collecting the solution in the dialysis bag, and freeze-drying to obtain folic acid-phycobiliprotein nanoparticles, namely the material loaded with the antitumor drug.
2. The method for preparing the material loaded with the anti-tumor drug according to claim 1, wherein the mass ratio of the folic acid, the N-hydroxysuccinimide and the ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride in the step S1 is 1: (0.1-0.3): (0.7-1.2); the drying method is drying at 40-50 ℃ to constant weight.
3. The method for preparing a material loaded with an anti-tumor drug according to claim 1, wherein the mass ratio of the phycobiliprotein to the propylene glycol in step S2 is 10: (0.1-1); the ultrasonic power is 1000-1200W.
4. The method of claim 1, wherein the buffer solution in step S3 is selected from one of carbonate buffer with pH =9-10, PBS buffer with pH =7.2-7.5, Tris-HCl buffer with pH = 7-7.5; the mass ratio of the folic acid active ester to the phycobiliprotein is 1: (0.2-0.5); the phycobiliprotein is phycocyanin or phycoerythrin, and the purity of the phycobiliprotein is more than 90%.
5. The method for preparing the material loaded with the antitumor drug according to claim 1, wherein the method for loading the antitumor drug with the material is as follows: reacting N, N' -dicyclohexylcarbodiimide with N-hydroxysuccinimide for 10-30 min to obtain a mixed solution, carrying out amination on an anti-tumor drug, dissolving the anti-tumor drug and folic acid-phycobiliprotein nanoparticles loaded with the anti-tumor drug in DMF, adding the solution into the reaction mixed solution, and carrying out crosslinking reaction to obtain the anti-tumor drug.
6. The method for preparing the material loaded with the antitumor drug according to claim 5, wherein the antitumor drug includes, but is not limited to, adriamycin, cisplatin, vincristine, paclitaxel, and camptothecin.
7. The method for preparing the antitumor drug-loaded material according to claim 5, wherein the method for amination of the antitumor drug is to dissolve the antitumor drug in toluene, dropwise add a toluene solution of DBU under ice bath conditions, raise the temperature to room temperature after completion of the dropwise addition, react for 2-5h, filter, and wash the precipitate with deionized water for 1-3 times to obtain the aminated antitumor drug.
8. An anti-tumor drug loaded material prepared by the method of any one of claims 1 to 7.
9. Use of the anti-tumor drug loaded material of claim 8 in the preparation of an anti-tumor drug.
10. The use of claim 9, wherein the loaded anti-tumor drug folic acid-phycobiliprotein nanoparticle has fluorescence characteristics and is used in targeted imaging.
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