CN103110623B - Use of tryptophan nanometer microspheres in preparation of medicine for treating tumor diseases - Google Patents
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- CN103110623B CN103110623B CN201310029756.4A CN201310029756A CN103110623B CN 103110623 B CN103110623 B CN 103110623B CN 201310029756 A CN201310029756 A CN 201310029756A CN 103110623 B CN103110623 B CN 103110623B
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Abstract
The invention discloses a use of tryptophan nanometer microspheres in preparation of a medicine for treating tumor diseases. According to the invention, tryptophan is embedded by using an emulsification method nanometer microsphere technology to enter tumor cells, so that a large number of exogenous tryptophans are released in the cells to cytoplasm of tumor cells, so as to resist tumors. Researches show that the tryptophan nanometer microspheres have a strong killing effect for human lymphoma cell lines, breast cancer cell lines, lung cancer cell lines, colorectal carcinoma cell lines, cervical cancer cell lines, brain tumor cell lines and human leukemia cell lines. By a manner of local administration, the medicine has an obvious inhibition on the growth of the colorectal cancer cell line nude mice metastatic tumor.
Description
Technical field
The present invention relates to the purposes of tryptophan Nano microsphere in preparation treatment tumor disease medicine, also relate to a kind of method for the preparation of described tryptophan Nano microsphere.The invention belongs to tumour medicine field.
Background technology
The mankind's health and lives in malignant tumor serious threat, though there are in the market a lot of cancer therapy drugs, side effect is large, expensive, brings heavy mental burden and the grief that is difficult to free to patient and family members thereof.Cause very large financial burden also to family numbers of patients and society simultaneously.At present, the whole world reaches more than 5,000,000 because of the number of tumor mortality every year, and its mortality rate of many countries accounts for the first place of the various causes of death.The means such as current existing Drug therapy, chemotherapy, radiotherapy, though there is certain effect, side effect is also very large, therefore find more effectively, have no side effect and the medicine of relatively economical, be physicians' unremitting pursue always.
When tumor growth, can obviously change host's sugar and amino acid metabolism.Aminoacid has many important physiological functions, but is one of tumor tissues conditions on which persons or things depend for existence, and this has brought very large contradiction to aminoacid treatment tumor.At present reach kind more than 100 for the kind of the aminoacid as medicine and amino acid derivativges in the world.Amino acid derivativges has been widely used as antitumor drug, and its model of action has: (1) antitumor drug using aminoacid as carrier, and as phenylalanine mustard gas, Valine, Pidolidone, 1B and phenylenediamine chlormethine be conjugate altogether; (2) utilize amino acid derivativges to reach antineoplastic object as the amino acid needed analog of tumor cell, as S-carbamyl-Cys; (3) amino acid derivativges is as the antitumor drug of enzyme inhibitor, if N-phosphoric acid acetyl-ASPARTIC ACID is the transient condition inhibitor that an aspartic acid turns ammonia cresyl enzyme, utilize the route of synthesis that this inhibitor can interrupt pyrimidine nucleotide to reach antitumor object; (4) amino acid derivativges is as the tumor inhibitor of intermediate product; (5) make the amino acid derivativges of Denucleation.
Tryptophan is one of essential limiting amino acid of humans and animals, and it is relevant with physiogenesis, is widely used in medicine, food and feed interpolation.But the effect of tryptophan in tumor development and whether can be used as medicine tumor disease is treated and be it be unclear that.Only researchs are many from Epidemiological study, there is scholar's research to show in oral squamous cell carcinoma patient body in blood plasma that tryptophan and metabolite thereof significantly increase, prompting tryptophan may be the important substance basis of tumor development, is not also exploited at present using tryptophan and derivant thereof as anti-tumor drug.The growth of all living things, all needs the raw material of aminoacid as synthetic protein.But, in the time that certain seed amino acid enters cytoplasm, not only do not promote the growth of tumor cell to suppress on the contrary its propagation, may become a kind of desirable antitumor drug.
In recent years, feed industrial development is rapid both at home and abroad, the continuous expansion of purposes on medical industry, it is good that tryptophan becomes on a kind of international market development prospect, the product that domestic market demand is larger, and raw material sources are abundant, production technology maturation, with respect to the existing antitumor drug of majority, its cost is lower, sets it as antitumor drug and puts goods on the market and will have wide DEVELOPMENT PROSPECT.
Summary of the invention
One of technical problem to be solved by this invention is to provide the new purposes of a kind of tryptophan Nano microsphere in preparation treatment tumor disease medicine.
Two of technical problem to be solved by this invention is to provide a kind of preparation method of tryptophan Nano microsphere and prepares tryptophan Nano microsphere by the method.
The present invention solves the problems of the technologies described above taked technical scheme, utilizes emulsion process Nano microsphere technology to make it enter tumor cell tryptophan embedding, and then in cell, discharges a large amount of external source tryptophans to tumor cell slurry, thereby reaches antineoplastic object.
Research shows, tryptophan Nano microsphere has stronger lethal effect to people's lymphocytic cancer cell system, breast cancer cell line, lung cancer cell line, rectum cancer cell system, cervical cancer cell system, cerebroma tumor cell line and human leukemia cell line, by topical mode, this medicine can have obvious inhibitory action to the growth of rectum cancer cell strain nude mice metastatic tumor.
Therefore, the present invention proposes the purposes of tryptophan Nano microsphere in treatment tumor disease.
In the present invention, preferred, a kind of method of preparing described tryptophan Nano microsphere, is characterized in that comprising following steps:
(1) tryptophan solution is distributed in the dichloromethane solution that contains PLGA under ultrasound condition, obtains milky homogeneous emulsion;
(2) aqueous solution of bovine serum albumin is joined under ultrasound condition in the emulsion that step (1) obtains, ultrasonic, form the emulsion of homogeneous;
(3) in the emulsion obtaining in step (2), put into rotor, stir, emulsion is centrifugal, centrifugal speed 2500-3500rpm, time 8-12min, gets supernatant, by supernatant recentrifuge, centrifugal speed 2500-3500rpm, time 8-12min, gets centrifugal precipitate 2 times;
(4) after precipitate being added in distilled water and disperseing, then carry out centrifugally, and then disperse, repeat 3 times, last precipitate is dispersed in distilled water, and lyophilization to obtain final product.
In the present invention, preferred, the concentration of the tryptophan solution described in step (1) is 0.1g/ml-0.5g/ml, more preferably 0.2g/ml.
In the present invention, preferred, the concentration of the dichloromethane solution that contains PLGA described in step (1) is 0.05g/ml, and the volume ratio of the dichloromethane solution that contains PLGA and tryptophan solution is 10:1.
In the present invention, preferred, the concentration of the Bovine Serum Albumin in Aqueous Solution described in step (2) is 0.001g/ml-0.01g/ml, more preferably 0.005g/ml, the volume ratio 5-6:1 of the emulsion that the aqueous solution of bovine serum albumin and step (1) obtain.
In the present invention, preferred, step is put into rotor in (3) in emulsion, stirs 4 hours under electromagnetic agitation condition, emulsion is centrifugal, centrifugal speed 3000rpm, time 10min, gets supernatant (solution above), supernatant is centrifugal again, centrifugal speed 3000rpm, time 10min.
Further, the present invention also provides the tryptophan Nano microsphere that described method prepares.
Wherein, the drug loading of described tryptophan Nano microsphere is [(27.18 ± 0.51) × 10
-3] more than %.
Further, the invention allows for a kind of medicine that is used for the treatment of tumor disease, it is characterized in that the effective ingredient in described medicine is tryptophan Nano microsphere.
Under normal circumstances, comprise that human cell in tumor cell, tryptophan all can not directly enter cytoplasm from extracellular.Therefore effectively tryptophan to be passed to intracellular technology be also another large characteristic of the present invention to one.The present invention utilizes emulsion process Nano microsphere technology to make tryptophan enter tumor cell, adopt topical mode to make thing of the present invention enter tumor cell, and then in cell, discharge a large amount of external source tryptophans to tumor cell slurry, disturb the DNA of tumor cell synthetic, thereby reach antineoplastic object.
The inventor finds through development test, and tryptophan enters after cell inhibited to kinds of tumors.
Tryptophan described in the present invention refers to the compound with trade name same structure, and tryptophan can be bought by commercial channel, also can prepare by the method for synthetic technology.
The present invention uses Nano microsphere technology to carry out the medicine of embedding as treatment tumor disease to tryptophan.Dosage is tryptophan 25mg/kg, 3 times weekly.
Brief description of the drawings
Fig. 1 is transplanted tumor in nude mice growth curve.
Detailed description of the invention
Below by experiment, also the present invention will be further described in conjunction with the embodiments, it should be understood that these embodiment, only for the object of illustration, never limit the scope of the invention.
The preparation of embodiment 1 tryptophan Nano microsphere
1, material:
1. Poly(D,L-lactide-co-glycolide (poly (lactic-co-glycolic acid), PLGA), molecular weight 100000, source: Shandong medical device research institute
2. L-Trp analytical pure is bought from Shanghai Jingchun Industrial Co., Ltd.'s lot number: 42217
3. dichloromethane analytical pure is bought from Tianjin Kermel Chemical Reagent Co., Ltd.'s lot number: 20110328
2, method
0.2g tryptophan is dissolved in and in 1ml water, obtains tryptophan solution (concentration is 0.2g/ml), 0.5gPLGA is dissolved in to (PLGA concentration is 0.05g/ml) in 10ml dichloromethane, obtain the dichloromethane solution that contains PLGA, tryptophan solution is distributed in the dichloromethane solution that contains PLGA under ultrasound condition, ultrasonic dispersion 3min, obtain milky homogeneous emulsion solution, then the 50ml aqueous solution that is dissolved with 0.25g bovine serum albumin is joined in emulsion under ultrasound condition, ultrasonic 3min, the emulsion of formation homogeneous.In emulsion, put into rotor, under electromagnetic agitation condition, stir 4 hours.
After stirring finishes, emulsion is centrifugal, centrifugal speed 3000rpm, centrifugal 10min, gets solution supernatant (solution above) above, by centrifugal again supernatant (solution above), centrifugal speed 3000rpm, centrifugal 10min, gets centrifugal sediment 2 times, adds distilled water to disperse centrifugal again, and then disperse, repeat 3 times, last precipitate is dispersed in distilled water, lyophilization.Products obtained therefrom is the present invention.Drug loading of the present invention is [(27.18 ± 0.51) × 10
-3] %.
The preparation of embodiment 2 tryptophan Nano microspheres
1, material: with embodiment 1
2, method
0.5g tryptophan is dissolved in and in 1ml water, obtains tryptophan solution (concentration is 0.5g/ml), 0.5gPLGA is dissolved in to (PLGA concentration is 0.05g/ml) in 10ml dichloromethane, obtain the dichloromethane solution that contains PLGA, tryptophan solution is distributed in the dichloromethane solution that contains PLGA under ultrasound condition, ultrasonic dispersion 5min, obtain milky homogeneous emulsion, then the 55ml aqueous solution that is dissolved with 0.5g bovine serum albumin is joined in emulsion under ultrasound condition, ultrasonic 5min, the emulsion of formation homogeneous.In emulsion, put into rotor, under electromagnetic agitation condition, stir 5 hours.
After stirring finishes, emulsion is centrifugal, centrifugal speed 3500rpm, centrifugal 8min, gets supernatant (solution above), and supernatant is centrifugal again, centrifugal speed 3500rpm, centrifugal 8min, gets centrifugal sediment 2 times, adds distilled water to disperse centrifugal again, and then disperse, repeat 3 times, last precipitate is dispersed in distilled water, lyophilization.Products obtained therefrom is the present invention.
The preparation of embodiment 3 tryptophan Nano microspheres
1, material: with embodiment 1
2, method
0.1g tryptophan is dissolved in and in 1ml water, obtains tryptophan solution (concentration is 0.1g/ml), 0.25g PLGA is dissolved in to (PLGA concentration is 0.025g/ml) in 10ml dichloromethane, obtain the dichloromethane solution that contains PLGA, tryptophan solution is distributed in the dichloromethane solution that contains PLGA under ultrasound condition, ultrasonic dispersion 2min, obtain milky homogeneous emulsion, then the 50ml aqueous solution that is dissolved with 0.05g bovine serum albumin is joined in emulsion under ultrasound condition, ultrasonic 3min, the emulsion of formation homogeneous.In emulsion, put into rotor, under electromagnetic agitation condition, stir 3 hours.
After stirring finishes, emulsion is centrifugal, centrifugal speed 3000rpm, centrifugal 10min, gets supernatant (solution above), and supernatant is centrifugal again, centrifugal speed 3000rpm, centrifugal 10min, gets centrifugal sediment 2 times, adds distilled water to disperse centrifugal again, and then disperse, repeat 3 times, last precipitate is dispersed in distilled water, lyophilization.Products obtained therefrom is the present invention.
The effect of test example 1 tryptophan Nano microsphere of the present invention to cancerous cell
1, experiment material
People's lymphocytic cancer cell system (M-9), breast cancer cell line (MCF-7), lung cancer cell line (H125), rectum cancer cell system (SW480), cervical cancer cell system (A431), cerebroma tumor cell line (U373) and human leukemia cell line (HL-60) are provided by institute of oncology of Harbin Medical University;
Tryptophan Nano microsphere: the method according to embodiment 1 prepares
2, test method
With the positive contrast medicine of 5-Fu, by medicine of the present invention (tryptophan Nano microsphere, method according to embodiment 1 prepares) join in Tissue Culture Dish people's lymphocytic cancer cell system (M-9), breast cancer cell line (MCF-7), lung cancer cell line (H125), rectum cancer cell system (SW480), cervical cancer cell system (A431), cerebroma tumor cell line (U373) and human leukemia cell line (HL-60), carry out MTT test and detect cell viability, MTT can be generated crystalloid darkviolet product formazan by more Intramitochondrial dehydrogenase reduction.The in the situation that of dimethyl sulfoxide, can be dissolved completely.Then can measure near absorbance 595nm wavelength by microplate reader.Cell viability is larger, and absorbance is higher; Cytoactive is less, and absorbance is lower.According to above-mentioned principle, obtain the IC of this medicine
50result is as shown in table 1:
Table 1 tryptophan Nano microsphere of the present invention is to various tumor cell IC
50(mg/ml)
As can be seen from Table 1, the present invention all has lethal effect to various tumor cell strains, we adopt common tryptophan and blank Nano microsphere respectively as negative control simultaneously, find that tryptophan and common Nano microsphere do not have lethal effect to tumor cell, with 5-Fu as positive control, the T that matches inspection, proves that this medicine fragmentation effect is better than traditional antineoplastic thing 5-Fu.
The inhibitory action of test example 2 tryptophan Nano microsphere of the present invention to transplanted tumor in nude mice growth
1, laboratory animal
BALB/C (nu/nu) nude mouse is purchased from Beijing laboratory animal company limited of dimension tonneau China, SPF level, 3～4 weeks ages of Mus, body weight 16～18g, male, raise in ultra-clean biological laminar-flow rack, the regular disinfection by ultraviolet light of environment, keep constant temperature (25 ± 2 DEG C), constant humidity (45%～50%), cage tool, drinking-water are all through high pressure steam sterilization, and experimental implementation is all carried out in aseptic cover.
2, experimental technique
By rectum cancer cell (SW480) amplification of recovering according to a conventional method, after digestion, serum-free medium centrifuge washing 2 times, counts and adjusts cell number, Trypan Blue viable count >99%.With being with, No. 6 needle applicator extraction cancerous cell suspension inoculations are subcutaneous in the right front armpit of nude mice, and each position inoculation 0.2mL is (containing viable count 1 × 10
6individual), after inoculation, observe and have or not tumor formation and injection point to have or not ulceration redness every day, measure in required time gross tumor volume, through 3～7d incubation period, the subcutaneous canescence tuberosity that occurs of visible inoculation position, and grow up gradually, the rounded or oval body surface that protrudes from, with diameter of tumor 0.5cm for becoming tumor.Meticulously raise, observe 1-2 every day, claims every other day nude mice body weight, measures tumor size (with 2 mutually perpendicular diameters of vernier caliper measurement tumor, including skin thickness), and substitution following formula calculates: V=1/2 × AB
2, V is gross tumor volume, A, B are respectively the line of apsides of tumor; It is all orthogonal while measuring 2 diameters of tumor body, will making slide gauge at every turn.
Each nude mice tumor weight and tumour inhibiting rate mensuration organized after treatment: after last medication, 24h puts to death nude mice in dislocation of cervical vertebra mode, and the tumor stripping out is removed the non-tumor tissues such as blood stains, fat, measures tumor weight, calculates each group of average tumor and weighs and tumour inhibiting rate (IR).
The average tumor of IR=(the average tumor weight of the average tumor weight-treatment group of tumor matched group)/matched group heavy × 100%.
Tumor-bearing mice is divided into 3 groups at random, 8 every group: (1) invention group: each lumbar injection prepares tryptophan Nano microsphere 25mg/kgbw according to method described in the embodiment of the present invention 1; (2) 5-Fu group: each lumbar injection 5-Fu20mg/kgbw; (3) matched group: each intraperitoneal injection of saline reagent 10mL/kgbw, is 3 times/week, totally 4 weeks.
3, experimental result
When experiment finishes, the body weight of each group nude mice is without significant difference, though increase but difference is not remarkable with relatively having before experiment, as shown in Figure 1, the tumor of invention group tumor weighs and volume is starkly lower than other groups (P<0.05), tumour inhibiting rate is that the tumour inhibiting rate of 65.85%, 5-Fu group is 57.21%.
Experimental result shows that this medicine can have obvious inhibitory action to the growth of rectal cancer SW480 cell transplanted tumor in nude mice.
The foregoing is only the preferred embodiments of the present invention, is only illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skill in the art understand, and in the spirit and scope that limit, can carry out many changes to it in the claims in the present invention, amendment, and even equivalence is changed, but all will fall within the scope of protection of the present invention.
Claims (10)
1. prepare a method for tryptophan Nano microsphere, it is characterized in that comprising following steps:
(1) tryptophan solution is distributed in the dichloromethane solution that contains PLGA under ultrasound condition, obtains milky homogeneous emulsion;
(2) aqueous solution of bovine serum albumin is joined under ultrasound condition in the emulsion that step (1) obtains, ultrasonic, form the emulsion of homogeneous;
(3) in the emulsion obtaining in step (2), put into rotor, stir, emulsion is centrifugal, centrifugal speed 2500-3500rpm, time 8-12min, gets supernatant, by supernatant recentrifuge, centrifugal speed 2500-3500rpm, time 8-12min, gets centrifugal precipitate 2 times;
(4) after precipitate being added in distilled water and disperseing, then carry out centrifugally, and then disperse, repeat 3 times, last precipitate is dispersed in distilled water, and lyophilization to obtain final product.
2. method as claimed in claim 2, is characterized in that, in step (1), in described tryptophan solution, the concentration of tryptophan is 0.1g/ml-0.5g/ml.
3. method as claimed in claim 3, is characterized in that, in step (1), in described tryptophan solution, the concentration of tryptophan is 0.2g/ml.
4. the method for claim 1, is characterized in that, in step (1), in the described dichloromethane solution that contains PLGA, the concentration of PLGA is 0.05g/ml, and the volume ratio of the dichloromethane solution that contains PLGA and tryptophan solution is 10:1.
5. the method for claim 1, it is characterized in that in step (2), the concentration of described Bovine Serum Albumin in Aqueous Solution is 0.001g/ml-0.01g/ml, the volume ratio 5-6:1 of the emulsion that Bovine Serum Albumin in Aqueous Solution and step (1) obtain.
6. method as claimed in claim 5, is characterized in that, in step (2), the concentration of described Bovine Serum Albumin in Aqueous Solution is 0.005g/ml.
7. the method for claim 1, it is characterized in that in step (3), in emulsion, put into rotor, under electromagnetic agitation condition, stir 4 hours, emulsion is centrifugal, centrifugal speed 3000rpm, time 10min, gets supernatant, by supernatant recentrifuge, centrifugal speed 3000rpm, time 10min.
8. the tryptophan Nano microsphere preparing according to the method described in claim 1-7 any one.
9. tryptophan Nano microsphere as claimed in claim 8, the drug loading that it is characterized in that tryptophan Nano microsphere is [(27.18 ± 0.51) × 10
-3] more than %.
10. be used for the treatment of a medicine for tumor disease, the effective ingredient in the medicine described in it is characterized in that is the tryptophan Nano microsphere described in claim 8 or 9.
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| CN113615827A (en) * | 2021-07-08 | 2021-11-09 | 武汉万德瑞生物技术股份有限公司 | Tryptophan-deficient combined food with anticancer function and preparation method thereof |
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Non-Patent Citations (3)
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| "西洋参化学成分及药理活性研究进展";王蕾 等;《特产研究》;20071231(第3期);第73-77页 * |
| 王蕾 等."西洋参化学成分及药理活性研究进展".《特产研究》.2007,(第3期),第73-77页. |
| 田燕 等.2.特点.《药剂学》.清华大学出版社,2011,第376页. * |
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Inventor after: Zhang Zhiren Inventor after: Ma Heping Inventor after: Wang Qiushi Inventor after: Chu Wenfeng Inventor after: Zhao Dan Inventor after: Yang Baofeng Inventor before: Zhang Zhiren Inventor before: Ma Heping Inventor before: Yang Baofeng Inventor before: Liu Xiaoyan Inventor before: Chu Wenfeng Inventor before: Zhao Dan Inventor before: Wang Qiushi |
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Free format text: CORRECT: INVENTOR; FROM: ZHANG ZHIREN MA HEPING YANG BAOFENG LIU XIAOYAN CHU WENFENG ZHAO DAN WANG QIUSHI TO: ZHANG ZHIREN MA HEPING WANG QIUSHI CHU WENFENG ZHAO DAN YANG BAOFENG |