CN104306339A - Thymidylate synthase preparation loaded albumin nano-microsphere and preparation method thereof - Google Patents
Thymidylate synthase preparation loaded albumin nano-microsphere and preparation method thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 26
- 108010022394 Threonine synthase Proteins 0.000 title abstract description 11
- 102000005497 Thymidylate Synthase Human genes 0.000 title abstract description 11
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- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a thymidylate synthase preparation loaded albumin nano-microsphere. The nano-microsphere is prepared by the following steps: adding a solution of a thymidylate synthase preparation and an organic solvent into an albumin solution to develop and self-assemble the albumin to form a nano-microsphere; coating the thymidylate synthase preparation in the albumin nano-microsphere to form the thymidylate synthase preparation loaded albumin nano-microsphere, wherein the diameter of the albumin nano-microsphere is 40-500nm, and the thymidylate synthase preparation is 7-20 percent by mass of the albumin nano-microsphere. The nano-microsphere has the advantages of excellent biocompatibility, simple preparation process and stable physiochemical property, is capable of improving the drug stability, has a certain sustained release effect, and has potential application of clinical cancer treatment. The invention further discloses a preparation method of the thymidylate synthase preparation loaded albumin nano-microsphere.
Description
Technical field
The present invention relates to a kind of albumin nano microsphere being loaded with thymidilate synthase inhibitors and preparation method thereof.
Background technology
Antimetabolic antitumor drug is survived by inhibition tumor cell and is copied necessary metabolic pathway, causes death of neoplastic cells.Owing to not yet finding that tumor cell has unique metabolic pathway, so the selectivity of antimetabolite is less, and also presents toxicity to breeding normal structure such as bone marrow, alimentary canal mucous membrane etc. faster at present.The antitumor spectra of antimetabolic antitumor drug is narrow, and majority is used for the treatment of leukemia clinically, but also effective to some solid tumor.Because the application point of antimetabolite is different, general without cross resistance.According to the requirement of antimetabolic basic theories, the structure of antimetabolite is general all very similar to metabolite, and most of antimetabolite take metabolite as lead compound, utilizes the drug design methods such as such as bioisosterism, prepare its antimetabolite.That commonly uses clinically has the medicines such as pyrimidines antimetabolic thing, purines antimetabolite, folic acid class antimetabolite.
Thymidylate synthase is an important target spot of antitumor drug exploitation, relevant to folic acid biological metabolism.The protein dimerization matter that thymidylate synthase is made up of two identical methylene units, each methylene molecular weight is about 36 × 10
3, playing an important role in the synthesis of DNA in vivo, is the methylated key enzyme of catalysis uracil deoxynucleoside, by the biosynthesis suppressing thymidylate synthase can suppress DNA, and then cell growth inhibiting.The structure of thymidilate synthase inhibitors is similar to substrate, can stop or suppress the combination of normal substrates and enzyme with enzyme after being combined, thus the biological activity of AF panel thymidylate synthase, suppress DNA synthesis, reach the effect of Therapeutic cancer.
Nano microsphere makes it have a good application prospect in drug conveying due to its distinctive character.Because the vascular system of tumor locus has permeability (EPR) effect higher than normal portions, polymeric nano medicine carrier can make full use of this effect, in diseased region enrichments such as tumors, reaches the object of passive target; On the basis of killing target cell, the injury of normal tissue can be reduced to greatest extent by the synergy of targeting group or molecule, reduce the system toxicity of medicine.The specific surface area that Nano microsphere is larger can be adsorbed, combine or other small-molecule drug coated as polypeptide, albumen, nucleic acid molecules.Nano microsphere, by changing self composition, increases the water solublity of dewatering medicament, improves small-molecule drug stability in vivo, the half-life that prolong drug molecule circulates in vivo.Natural macromolecular Nano microsphere not only possesses conventional medicament delivery system can improve drug solubility, increase the feature of medicine circulation time in vivo and passive target, also there is good biocompatibility, drug distribution situation can be changed, reduce the advantages such as drug system toxicity.
Albumin is synthesized by hepatic parenchymal cells, and the half-life in blood plasma is about 15 ~ 19 days, is the protein that in blood plasma, content is maximum, accounts for 40% ~ 60% of Total plasma protein.For the single chain polypeptide containing 585 amino acid residues, molecular weight is 66458, containing 17 disulfide bond in molecule, and the component not containing sugar.In the environment of pH=7.4, albumin is anion, and per molecule can with more than 200 negative charges.It is carrier very main in blood plasma, and the material of many poorly water-solubles can by being transported with albuminous combination.These materials comprise bilirubin, long-chain fatty acid (per molecule can in conjunction with 4 ~ 6 molecules), bile salt, prostaglandin, steroid hormone, metal ion (Cu
2+, Ni
2+, Ca
2+) and medicine (aspirin, penicillin etc.).Therefore albumin has a good application prospect as pharmaceutical carrier.
Metal ion has good coordination, has had report metal ion can interactional equilibrium constant between significantly increasing medicament and carrier, can play crosslinking nano microsphere, improve the effect of medicament slow release performance.
Albumin in case of heating can degeneration, have been reported by heating means synthesizing stable albumin microsphere, the method of heating can stablize albumin nano microsphere under part retains the prerequisite of albumin activity structure, improves the stability of Nano microsphere under different pH and salt concentration conditions.
Summary of the invention
One is the object of the present invention is to provide to have biocompatibility, biodegradable, low system toxicity, a kind of albumin nano microsphere being loaded with thymidilate synthase inhibitors that can be used as treatment of cancer and preparation method thereof, solves the problem of sending in hydrophobic type thymidilate synthase inhibitors organism.
Technical scheme of the present invention is as follows:
A kind of albumin nano microsphere being loaded with thymidilate synthase inhibitors, it adds in albumin solution by the solution of thymidilate synthase inhibitors and organic solvent composition, albumin is launched and self assembly formation Nano microsphere, thymidilate synthase inhibitors is coated on the albumin nano microsphere being loaded with thymidilate synthase inhibitors in protein nano microsphere, described albumin nano microsphere diameter 40 ~ 500nm, in described albumin nano microsphere, the mass ratio of thymidylic acid enzyme inhibitor is 7 ~ 20%.
The above-mentioned albumin nano microsphere being loaded with thymidilate synthase inhibitors, described albumin can be bovine serum albumin and human serum albumin.
The above-mentioned albumin nano microsphere being loaded with thymidilate synthase inhibitors, described albumin solution is the aqueous solution of albumin and modifier.
The above-mentioned albumin nano microsphere being loaded with thymidilate synthase inhibitors, described modifier is dithiothreitol, DTT, mercaptoethanol or glutathion, and the mass ratio of albumin and modifier is 25:5.8-8.0.
The above-mentioned albumin nano microsphere being loaded with thymidilate synthase inhibitors, described thymidilate synthase inhibitors can be the compositions of pemetrexed, Raltitrexed or above-mentioned thymidilate synthase inhibitors.
The above-mentioned albumin nano microsphere being loaded with thymidilate synthase inhibitors, described organic solvent can be acetone, ethanol or methanol.
Prepare the above-mentioned method being loaded with the albumin nano microsphere of thymidilate synthase inhibitors, it comprises the steps:
Step 1. uses the aqueous dissolution albumin containing modifier, and albumin and modifier mass ratio are 25:5.8-8.0, and in solution, reactant total concentration is 18.5 ~ 38.2mg/mL, at 37 DEG C, react 0.1 ~ 2h;
Step 2. adds hydrochloric acid or sodium hydroxide solution in the system of step 1, adjustment system pH=5-9;
The thymidilate synthase inhibitors solution that thymidilate synthase inhibitors obtains 1.5mg/mL is dissolved in step 3. organic solvent, join in the albumin solution that step 2 obtains, control adds medication amount 2mg, then evaporating organic solvent, and described organic solvent can be acetone, ethanol or methanol;
Step 4. adds the slaine of system gross mass 0.1 ‰ ~ 1 ‰, after stirring 0.25 ~ 3h, and 60 ~ 70 DEG C of heating Nano microsphere 0 ~ 5min;
Step 5. ultracentrifugation, frozen centrifugation or ultrafiltration centrifugal method are separated Nano microsphere and unreacting material, add excipient substance mannitol or the glucose of system gross mass 1 ~ 20%, lyophilization obtains the albumin nano microball preparation being loaded with thymidilate synthase inhibitors, Nano microsphere diameter is 50 ~ 500nm, and thymidilate synthase inhibitors accounts for 7 ~ 20% of albumin medicament-carrying nano-microsphere quality.
The above-mentioned method preparing albumin nano microsphere, described albumin is bovine serum albumin or human serum albumin.
The above-mentioned method preparing albumin nano microsphere, described modifier is dithiothreitol, DTT, mercaptoethanol or glutathion.
The above-mentioned method preparing albumin nano microsphere, the metal salt solution described in step 4 can be the aqueous solution of calcium sulfate, calcium chloride, iron chloride, zinc sulfate or zinc nitrate.
The modification albumin nano microspherulite diameter being loaded with thymidilate synthase inhibitors of the present invention is homogeneous, and good dispersion, dissolubility is high, has performance in good slow release effect and body.
The present invention is cross-linked Nano microsphere by two kinds of methods, and the Nano microsphere after Action of Metal Ions can improve medicament slow release performance, improves the Interaction Force of thymidilate synthase inhibitors and albumin; Be heating and curing and can strengthen the albuminous structural stability of Nano microsphere main body.
The present inventor enables albumin with the mol ratio load thymidilate synthase inhibitors class medicine of 1:1 ~ 1:30 by the means of modification, final recleaner flotation mol ratio is that 1:15 ~ 1:25 thymidilate synthase inhibitors and modification albumin interact, and under the effect of hydrophobic force, while balling-up, coating medicine forms the modification albumin nano microsphere being loaded with thymidilate synthase inhibitors.
Nano microsphere authentication method of the present invention comprises: dynamic light scattering, uv-vis spectra, MTT cell toxicity test, and vitro drug release is tested, and demonstrates the existence of modification albumin nano microsphere and and exists and interact between thymidilate synthase inhibitors.
The present invention relates to the preparation of the modification albumin nano microball preparation being loaded with thymidilate synthase inhibitors, comprising: a) preparation is loaded with the modification albumin nano microsphere of thymidilate synthase inhibitors; B) the modification albumin nano microsphere being loaded with thymidilate synthase inhibitors is mixed according to a certain percentage in excipient substance; C) by mixture lyophilization, powder is obtained.
The invention provides a kind of method being loaded with the modification albumin nano microsphere of thymidilate synthase inhibitors that mean diameter is 40 ~ 500nm.Modification albumin medicament-carrying nano-microsphere good biocompatibility of the present invention, structural property is stablized.Medicine carrying composite nano-microsphere of the present invention well improves the performance of existing thymidilate synthase inhibitors.
Accompanying drawing illustrates:
Fig. 1 is the modification serum albumin DLS data of load Raltitrexed
Fig. 2 is the modification serum albumin transmission electron microscope data of load Raltitrexed
Detailed description of the invention
Illustrate content of the present invention further below in conjunction with embodiment, but these embodiments do not limit the scope of the invention.
The preparation of embodiment 1 load pemetrexed modification bovine serum albumin medicament-carrying nano-microsphere
In 4mL reactor, 25mg bovine serum albumin and 6.3mg dithiothreitol, DTT are dissolved in 1mL distilled water, 0.5h is reacted at 37 DEG C, add sodium hydroxide solution regulation system pH=8.0, drip 1.5mg/mL pemetrexed acetone soln 1.5mL, form Nano microsphere, add after 0.5mg/mL calcium sulphate soln 20 μ L reacts 15 minutes, 60 DEG C are reacted 5 minutes, 37 DEG C of rotary evaporations 15 minutes, Nano microsphere is through molecular cut off 100, and the ultra-filtration centrifuge tube of 000 is centrifugal obtains modification bovine serum albumin medicament-carrying nano-microsphere aqueous dispersion liquid.Add the mannitol lyophilization of system gross mass 2%, obtain medicament-carrying nano-microsphere powder.
The preparation of embodiment 2 load Raltitrexed modification bovine serum albumin medicament-carrying nano-microsphere
In 4mL reactor, 15mg bovine serum albumin and 4.8mg dithiothreitol, DTT are dissolved in 1mL distilled water, 0.5h is reacted at 37 DEG C, add hydrochloric acid solution regulation system pH=5.0, drip 1.5mg/mL Raltitrexed ethanol solution 1.1mL, form Nano microsphere, add after 0.5mg/mL solution of zinc sulfate 20 μ L reacts 15 minutes, add 0.02mM calcium sulphate soln 100 μ L, stir 2h, 37 DEG C of rotary evaporations 15 minutes, Nano microsphere is 12, ultracentrifugation 3min under 000 rotating speed, redissolves and obtains modification bovine serum albumin medicament-carrying nano-microsphere aqueous dispersion liquid.Add the mannitol lyophilization of system gross mass 2%, obtain medicament-carrying nano-microsphere powder.
The preparation of embodiment 3 load Raltitrexed modification bovine serum albumin medicament-carrying nano-microsphere
In 4mL reactor, 15mg bovine serum albumin and 3.5mg mercaptoethanol are dissolved in 1mL distilled water, 0.1h is reacted at 37 DEG C, add sodium hydroxide solution regulation system pH=9.0, drip 1.5mg/mL Raltitrexed ethanol solution 1.5mL, form Nano microsphere, after adding 0.25mg/mL calcium chloride solution 20 μ L reaction 2h, 70 DEG C are reacted 2 minutes, 37 DEG C of rotary evaporations 15 minutes, Nano microsphere is 100, and frozen centrifugation 5min under 000 rotating speed, redissolves and obtain modification bovine serum albumin medicament-carrying nano-microsphere aqueous dispersion liquid.Add the glucose lyophilization of system gross mass 5%, obtain medicament-carrying nano-microsphere powder.
The preparation of embodiment 4 load Raltitrexed modification bovine serum albumin medicament-carrying nano-microsphere
In 4mL reactor, 25mg bovine serum albumin and 7.3mg glutathion are dissolved in 1mL distilled water, at 37 DEG C, react 2h, add sodium hydroxide regulation system pH=6.0, drip 1mg/mL Raltitrexed methanol solution 2mL and form Nano microsphere, after adding 0.5mg/mL zinc nitrate solution 20 μ L reaction 2h, 70 DEG C of reactions 3 minutes, 37 DEG C of rotary evaporations 15 minutes, Nano microsphere is 12, ultracentrifugation 3min under 000 rotating speed, redissolves and obtains modification bovine serum albumin medicament-carrying nano-microsphere aqueous dispersion liquid.Add the lactose lyophilization of system gross mass 10%, obtain medicament-carrying nano-microsphere powder.
The preparation of embodiment 5 load pemetrexed modification human serum albumin medicament-carrying nano-microsphere
In 4mL reactor, 25mg human serum albumin and 6.3mg dithiothreitol, DTT are dissolved in 1mL distilled water, 0.5h is reacted at 37 DEG C, add sodium hydroxide solution regulation system pH=8.0, drip 1.5mg/mL pemetrexed acetone soln 1.5mL, form Nano microsphere, add after 0.5mg/mL calcium sulphate soln 20 μ L reacts 15 minutes, 60 DEG C are reacted 5 minutes, 37 DEG C of rotary evaporations 15 minutes, Nano microsphere is through molecular cut off 100, and the ultra-filtration centrifuge tube of 000 is centrifugal obtains modification human serum albumin medicament-carrying nano-microsphere aqueous dispersion liquid.Add the mannitol lyophilization of system gross mass 2%, obtain medicament-carrying nano-microsphere powder.
The preparation of embodiment 6 load Raltitrexed modification human serum albumin medicament-carrying nano-microsphere
In 4mL reactor, 15mg human serum albumin and 4.8mg dithiothreitol, DTT are dissolved in 1mL distilled water, 0.5h is reacted at 37 DEG C, add hydrochloric acid solution regulation system pH=5.0, drip 1.5mg/mL Raltitrexed ethanol solution 1.5mL, form Nano microsphere, add after 0.5mg/mL solution of zinc sulfate 20 μ L reacts 15 minutes, add 0.02mM calcium sulphate soln 100 μ L, stir 2h, 37 DEG C of rotary evaporations 15 minutes, Nano microsphere is 12, ultracentrifugation 3min under 000 rotating speed, redissolves and obtains modification human serum albumin medicament-carrying nano-microsphere aqueous dispersion liquid.Add the mannitol lyophilization of system gross mass 2%, obtain medicament-carrying nano-microsphere powder.
The preparation of embodiment 7 load Raltitrexed modification human serum albumin medicament-carrying nano-microsphere
In 4mL reactor, 15mg bovine serum albumin and 3.5mg mercaptoethanol are dissolved in 1mL distilled water, 0.1h is reacted at 37 DEG C, add sodium hydroxide solution regulation system pH=9.0, drip 1.5mg/mL Raltitrexed ethanol solution 1.5mL, form Nano microsphere, after adding 0.25mg/mL calcium chloride solution 20 μ L reaction 2h, 70 DEG C are reacted 2 minutes, 37 DEG C of rotary evaporations 15 minutes, Nano microsphere is 100, and frozen centrifugation 5min under 000 rotating speed, redissolves and obtain modification human serum albumin medicament-carrying nano-microsphere aqueous dispersion liquid.Add the glucose lyophilization of system gross mass 5%, obtain medicament-carrying nano-microsphere powder.
The preparation of embodiment 8 load Raltitrexed modification human serum albumin medicament-carrying nano-microsphere
In 4mL reactor, 25mg human serum albumin and 7.3mg glutathion are dissolved in 1mL distilled water, 2h is reacted at 37 DEG C, add sodium hydroxide solution regulation system pH=6.0, drip 1mg/mL Raltitrexed methanol solution 2mL and form Nano microsphere, after adding 0.5mg/mL zinc nitrate solution 20 μ L reaction 2h, 70 DEG C are reacted 3 minutes, 37 DEG C of rotary evaporations 15 minutes, Nano microsphere is 12, ultracentrifugation 3min under 000 rotating speed, redissolves and obtains modification human serum albumin medicament-carrying nano-microsphere aqueous dispersion liquid.Add the lactose lyophilization of system gross mass 10%, obtain medicament-carrying nano-microsphere powder.
The preparation of embodiment 9 load pemetrexed and Raltitrexed modification bovine serum albumin medicament-carrying nano-microsphere
In 4mL reactor, 25mg bovine serum albumin and 6.3mg dithiothreitol, DTT are dissolved in 1mL distilled water, 0.5h is reacted at 37 DEG C, add sodium hydroxide solution regulation system pH=8.0, drip 1.5mg/mL pemetrexed/Raltitrexed (mol ratio 1:1) acetone soln 1.5mL, form Nano microsphere, add after 0.5mg/mL calcium sulphate soln 20 μ L reacts 15 minutes, 60 DEG C are reacted 5 minutes, 37 DEG C of rotary evaporations 15 minutes, Nano microsphere is through molecular cut off 100, the ultra-filtration centrifuge tube of 000 is centrifugal obtains modification bovine serum albumin medicament-carrying nano-microsphere aqueous dispersion liquid.Add the mannitol lyophilization of system gross mass 2%, obtain medicament-carrying nano-microsphere powder.
Claims (9)
1. one kind is loaded with the albumin nano microsphere of thymidilate synthase inhibitors, it is characterized in that: it adds in albumin solution by the solution of thymidilate synthase inhibitors and organic solvent composition, albumin is launched and self assembly formation Nano microsphere, thymidilate synthase inhibitors is coated on the albumin nano microsphere being loaded with thymidilate synthase inhibitors in protein nano microsphere, described albumin nano microsphere diameter 40 ~ 500nm, in described albumin nano microsphere, the mass ratio of thymidylic acid enzyme inhibitor is 7 ~ 20%.
2. the albumin nano microsphere being loaded with thymidilate synthase inhibitors according to claim 1, is characterized in that: described albumin can be bovine serum albumin or human serum albumin.
3. the albumin nano microsphere being loaded with thymidilate synthase inhibitors according to claim 1, is characterized in that: described albumin solution is the aqueous solution of albumin and modifier.
4. the albumin nano microsphere being loaded with thymidilate synthase inhibitors according to claim 1, is characterized in that: described modifier is dithiothreitol, DTT, mercaptoethanol or glutathion, the mass ratio of albumin and modifier is 25:5.8-8.0.
5. the albumin nano microsphere being loaded with thymidilate synthase inhibitors according to claim 1, is characterized in that: described thymidilate synthase inhibitors is the compositions of pemetrexed, Raltitrexed or above-mentioned thymidilate synthase inhibitors.
6. prepare the method being loaded with the albumin nano microsphere of thymidilate synthase inhibitors according to claim 1, it is characterized in that it comprises the steps:
Step 1. uses the aqueous dissolution albumin containing modifier, and albumin and modifier mass ratio are 25:5.8-8.0, and in solution, reactant total concentration is 18.5 ~ 38.2mg/mL, at 37 DEG C, react 0.1 ~ 2h;
Step 2. adds hydrochloric acid or sodium hydroxide solution in the system of step 1, adjustment system pH=5-9;
The thymidilate synthase inhibitors solution that thymidilate synthase inhibitors obtains 1.5mg/mL is dissolved in step 3. organic solvent, add in the albumin solution that 0.5 ~ 3mL obtains to step 2, control adds medication amount and is about 2mg, then evaporating organic solvent, described organic solvent is acetone, ethanol or methanol;
Step 4. adds the slaine of system gross mass 0.1 ‰ ~ 1 ‰, after stirring 0.25 ~ 3h, and 60 ~ 70 DEG C of heating Nano microsphere 0 ~ 5min;
Step 5. ultracentrifugation, frozen centrifugation or ultrafiltration centrifugal method are separated Nano microsphere and unreacting material, add excipient substance mannitol or the glucose of system gross mass 1 ~ 20%, lyophilization obtains the albumin nano microball preparation being loaded with thymidilate synthase inhibitors, Nano microsphere diameter is 50 ~ 500nm, and thymidilate synthase inhibitors accounts for 7 ~ 20% of albumin medicament-carrying nano-microsphere quality.
7. the method preparing albumin nano microsphere according to claim 6, is characterized in that: described albumin is bovine serum albumin or human serum albumin.
8. the method preparing albumin nano microsphere according to claim 6, is characterized in that: described modifier is dithiothreitol, DTT, mercaptoethanol or glutathion.
9. the method preparing albumin nano microsphere according to claim 6, is characterized in that: the metal salt solution described in step 4 is the aqueous solution of calcium sulfate, calcium chloride, iron chloride, zinc sulfate or zinc nitrate.
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