CN106265513B - A kind of effect of nano-paclitaxel and preparation method thereof - Google Patents
A kind of effect of nano-paclitaxel and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of preparation methods of effect of nano-paclitaxel, and the mixed polymer of PEG-PLA or PEG-PLA and folate-PEG-PLA is dissolved in tetrahydrofuran；Taxol is dissolved in methylene chloride；Above-mentioned solution is uniformly mixed；2% polyvinyl alcohol of 1.5 times of volumes, ultrasonic mixing is added；Mixed solution is instilled in water with 20-30 drop/minute speed, it is ultrasonic under ice bath；By stirring in water bath under resulting lotion normal temperature and pressure, volatile organic solvent, centrifugation is collected precipitating, is washed with water, again aquation；Again the micellar solution after aquation is dialysed；The freeze drying protectant F-68 that mass fraction is 2.5% is added to the nano-micelle dialysed, freezes to obtain effect of nano-paclitaxel；Method operating procedure of the invention is simple, easy to operate.Drug micelles solution is placed one week, no drug release at normal temperature, further confirms that drug is wrapped in the core of nano particle, and property is relatively stable.
Description
Technical field
The present invention relates to a kind of preparation method of biopolymer nanoparticles more particularly to a kind of effect of nano-paclitaxel and its systems
Preparation Method.
Background technique
Taxol (Paclitaxel) is to extract the two of separation from Pacific yew (Taxus brevifolia) bark
Terpenoid is insoluble in water, and taxol antitumor mechanism, which mainly passes through, inhibits cellular tubulin activity to have carefully
Cellular toxicity is the fiest-tire medication of oophoroma and breast cancer treatment, however multidrug resistance, and dissolubility difference and chemotherapeutic toxicity limit
Its clinical efficacy.
The injection of taxol clinical use is that taxol is solubilized into Emulsifier EL-60 (Cremophor mostly
EL) in dehydrated alcohol (1: 1), it is diluted to administration concentration before use.But Emulsifier EL-60 can cause serious allergic reaction,
Before patient's administration Claritin is taken in advance to mitigate adverse reaction.Limit its application clinically.It is purple to improve
The dissolubility of China fir alcohol, at present there are mainly two types of mode, first is that chemical modification, second is that physically encapsulation；Wherein, chemical modification can't
It does not reduce taxol in the case that toxicity do not enhance in therapeutic effect, increases the dissolubility of its own, improve antitumor
Efficiency.
Nanosecond science and technology experienced development at full speed from rising in early 1990s till now, achieves and attracts people's attention
Achievement.Nanotechnology has been widely used in the multiple fields such as aerospace, biomedicine, material at present.In field of medicaments
Aspect, the emergence and development of medicament-carried nano carrier provide new approach for the development of anti-tumor drugs targeting.It is carried using nanometer
Body packaging medicine can increase the solubility of insoluble drug, reinforce the stability of water soluble drug, improve the biology benefit of drug
Expenditure reduces the toxic side effect of drug.
Taxol is wrapped up in nano-carrier system, and biodistribution in vivo will change, wherein with load
The biodistribution of medicine nano-carrier is closely related.After nano-carrier enters blood by the circulatory system, in most of and blood
Albumen opsonin combine, identified by reticuloendothelial system (RSE), absorbed at first by organs such as liver spleens,
Concentration decline in blood, only small part can reach in tumor tissues by blood circulation system.Research shows that due to swollen
Tumor tissue growth is fast, and blood supply is sufficient, and the permeability of tumor tissues medium vessels is compared with normal blood vessels height, while tumor tissues shortage has
The lymphatic drainage of effect, therefore medicament-carried nano carrier has the permeability and anelasticity effect (EPR effect) of enhancing to tumor tissues,
To reach the passive target and slow releasing function to tumor tissues.HPH sensitive controlled-release (physical chemistry targeting) tumor tissues without
Oxygen glycolysis increases, and acidic environment is presented in tumor by local, and the nano-carrier prepared using the biodegradable material of PH sensibility is made
Degrading in tumor locus for nano-carrier selectivity discharges drug, and keeps stablizing the target, it can be achieved that tumour in the normal tissue
To.Polylactic acid can degrade in tumor tissues acidic environment, discharge drug, to realize the physical chemistry targeting of nano-carrier
Property.The property of the metabolism of nano-carrier in vivo and nano-carrier itself enters the chemical modification and nanometer of partial size, charge, surface
Structure and composition etc. factor it is related.Ideal nano-carrier partial size should be maintained at 100nm or so, and current potential is in 10mV
Within, and certain chemical modification is carried out on surface, tumour-specific or highly expressed receptor are further screened, antigen will
The monoclonal antibody of corresponding ligand either specificity is mutually coupled by way of physics or chemical bond with nano-carrier, according to
By targeted molecular specific recognition tumor tissues, to improve the selectivity to tumor tissues, medicament-carried nano carrier is reduced in week
The aggregation in tissue is enclosed, will realize that the active targeting transmitting to drug acts on, make paclitaxel nano carrier in tumor tissues
Accumulation.
At present common targeted molecular be mainly the specificity either highly expressed monoclonal antibody of tumor tissues and by
Body aglucon.But be that antibody is confined on cell membrane mostly, it cannot achieve the intracellular delivery of drug, while easily causing in vivo
Immune exclusion reaction, plus antibody is at high price so that antibody using being limited by very large.Receptor-ligand modification
Nano-carrier, due to high specificity between ligand and receptor, affinity is high, and the advantages that be not easy to cause immune response, causes
The extensive concern of people, wherein most representational is folic acid.Folic acid (FA) is a kind of water-soluble B vitamin, is people
A kind of necessary nutriment of body participates in the synthesis and metabolism of the important substance of multiple physical body.Nucleotide is participated in eukaryocyte
Biosynthesis, be cells survival and be proliferated necessary to nutriment.Folacin receptor great expression in tumour cell, such as ovary
Cancer, breast cancer, colon cancer, lung cancer, prostate cancer and nose larynx and the tumour of brain etc., be significantly higher than normal tissue cell folic acid by
Body expression.Folic acid is woven with specificity and targeting to tumor group, and this as small-molecule substance, good water solubility can be each
It is stabilized in kind solution, easily stores, and there is no toxic side effect to human body, be an ideal targeted molecular.
Nano-carrier can change the water solubility of hydrophobicity taxol, preferably meet the mode of intravenously administrable, improve purple
The bioavilability of China fir alcohol.In addition, folate-targeted nano-carrier can be received by active targeting, passive target and PEG-PLA
The Physical Target tropicrole of meter Zai Ti improves the accumulation ability of tumor tissues, increases the content of taxol in tumour.However nanometer
The slow releasing function of carrier itself can also maintain the content of taxol in tumor tissues in effective range.Therefore, there is targeting
Property nano-carrier and traditional chemotherapeutic paclitaxel plus, can preferably embody the targeted therapy of tumour, improve treatment
Effect.
Generally existing following problems of the preparation method of nano-paclitaxel particle at present: taxol polymer nanometer in order to obtain
The suspension of particle needs repeated multiple times high speed centrifugation-resuspension, complex steps；During preparing water phase, need to be added
Surfactant, the addition of surfactant will lead to nano particle and increase irritation to gastrointestinal tract during use；
Without removing organic solvent is frozen after medicine preparation success, it is directly injected into vivo, organic solvent damages body, and
It is not easy to store；The disadvantages of poor, form that nanoparticle after freezing redissolves that there are dissolubilities and particle size can change.
Summary of the invention
An object of the present invention is to provide a kind of preparation method of effect of nano-paclitaxel.
The second object of the present invention is to provide a kind of effect of nano-paclitaxel.
The third object of the present invention is to provide a kind of preparation method of drug for treating tumour, including heretofore described
The preparation method of effect of nano-paclitaxel, preferably: the tumour is oophoroma, breast cancer, colon cancer, lung cancer, prostate cancer, nose
The tumour of larynx or brain.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of preparation method of effect of nano-paclitaxel, steps are as follows:
(1) by nanometer block polymer polyethylene glycol-polylactic acid (PEG-PLA) either polyethylene glycol-polylactic acid (PEG-
PLA) and the mixed polymer of folic acid-polyethylene glycol-polylactic acid (folate-PEG-PLA) is dissolved in organic solvent tetrahydrofuran
In, solution A is made；
(2) taxol is dissolved in methylene chloride, solution B is made；
(3) solution A and solution B are sufficiently mixed, the uniform pharmaceutical polymer organic solution of concentration is made；
(4) polyvinyl alcohol that mass fraction is 2% will be added in the mixed solution of step (3) preparation, ultrasound makes to mix molten
Liquid is sufficiently mixed；
(5) mixed solution is instilled in water dropwise with 20-30 drop/minute speed, it is ultrasonic under ice bath；
(6) by stirring in water bath under resulting lotion normal temperature and pressure, volatile organic solvent is centrifuged, and is collected precipitating, is washed with water
It washs, again aquation；
(7) micellar solution after aquation again is put into bag filter, bag filter is placed in water and is dialysed to get arriving
Paclitaxel nano micelle；
(8) freeze drying protectant that mass fraction is 2.5% is added to the nano-micelle dialysed is F-68, freezes to obtain purple
China fir alcohol nanoparticle；
The mass ratio of the nanometer block polymer and taxol are as follows: 6:1.
It is preferred: when the mixing that nanometer block polymer is PEG-PLA and folate-PEG-PLA is poly- in the step (1)
When closing object, the mass percent of folate-PEG-PLA is 8%-12%, more preferably 10%.
It is preferred: when the molecular weight that nanometer block polymer is polyethylene glycol-polylactic acid is 10000- in the step (1)
13000, ethylene glycol in polymer: the molar ratio of lactic acid monomer is (4-5): 1.
Preferred: in the step (4), the volume ratio of polyvinyl alcohol and mixed solution is 1.5:1；Ultrasound power be
30-40W, ultrasonic time are 10min.Advantage: two kinds of solution can be mixed adequately under this condition, and prepared by requirement
Nano-micelle degree of scatter is good, and particle is in uniform spherical.
It is preferred: in the step (6), stirs and use magnetic stirrer, mixing speed 500-1000r/min,
Mixing time is 4h.Advantage: stirring can change the distribution of each constituent concentration in solution, is conducive to the dispersion of various composition, stirs
Mix speed it is lower when solution cannot adequately react, nanoparticle forming it is bad, be easy to produce thick film, mixing speed is excessively high, nanometer
Grain is easy to be destroyed, so suitable mixing speed can make the ingredient in solution relatively uniform, the nanoparticle size of formation
Uniformly, partial size is suitable.So it is 500-1000rpm/min that the present invention, which selects optimum mixing speed,.Mixing time is to nanometer
The form of grain is related, and mixing time is too short, and not exclusively, nanoparticle form is irregular, molecular weight less for organic solvent volatilization.Stirring
Overlong time, nanoparticle are easy to be destroyed, and are unfavorable for the production of nanoparticle technique.Therefore, optimal mixing time is 4h.
Preferred: in the step (6), the condition of centrifugation is that 14000r/min centrifugation time is 30min.Advantage: pass through
Centrifugation can effectively collect nanoparticle, and centrifugal rotational speed is too slow, and centrifugation time is too short then to be collected not exclusively, be centrifuged too fast, centrifugation
Time is long, then nanoparticle form is destroyed, and can not achieve effective load medicine, so by screening, centrifugation time of the invention
Both guarantee to have collected nanoparticle completely for 30min, revolving speed 14000r/min, in turn avoids the destruction of nanoparticle form.
Preferred: in the step (7), the molecular cut off of bag filter is 3500KDa.
Beneficial effects of the present invention:
1, protective agent freeze-drying is added in the paclitaxel nano micelle of preparation by the present invention, is in loose powdered shape after freeze-drying, multiple
No change has taken place for resulting nanoparticle property after molten, partial size 100-150nm, good, the taxol packet of nanoparticle dispersion spherical in shape
It is rolled among nanoparticle, reduces intake of its hetero-organization for taxol, thus reduce taxol bring toxic side effect,
The release in vitro situation of drug is detected after redissolution as before freeze-drying, illustrates the effect of nano-paclitaxel property of the method for the present invention preparation
Stablize, drug micelles solution is placed one week at normal temperature, no drug release, further confirms that drug is wrapped in nano particle
Core, and property is relatively stable.
2, effect of nano-paclitaxel drugloading rate prepared by method of the invention be 40%, encapsulation rate 83%, drugloading rate and
Encapsulation rate is higher, reduces the dosage of taxol, has high application value.
3, when nanometer block polymer is the mixed polymer of PEG-PLA and folate-PEG-PLA, by different folic acid
After the nanoparticle of content and ovarian cancer cell SKOV3 are incubated for, with fluorescence microscope, when folate-PEG-PLA mass percentage
When than less than 8%, with the increase of folate content, the intensity of intracellular Fluorescence is consequently increased, as folate- in mixture
When PEG-PLA mass percent is greater than 12%, with the increase of folate content, fluorescence intensity not will increase, and thus be illustrated, be mixed
Folic acid mass percent can achieve optimal therapeutic effect in 8-12% in object.
4, polyethylene glycol-polylactic acid is amphiphilic polymer, and the nanoparticle of composition both can effectively be dissolved in aqueous solution,
Its inner hydrophobic region can provide space for fat-soluble medicine, to improve the water solubility of taxol, change it in vivo
Distribution, to improve the curative effect of drug.Polylactic acid can be decomposed into monomer lactic acid in vivo, and grape is converted into liver
Sugar provides energy.And polyethylene glycol is the high-molecular compound that Food and Drug Adminstration of the US (FDA) approval can be used for human body.Leaf
Acid is a kind of nutriment that human body is important, participates in a variety of metabolic responses of human body.Therefore, the drug-carrying nanometer particle of above-mentioned material preparation
It is smaller to human body toxic side effect.In addition, the polyethylene glycol-polylactic acid polymer that the present invention uses has Amino End Group, it can passing through
It learns key and links folic acid group, increase the tumor-targeting effect of nanometer.
The molecular weight of polyethylene glycol-polylactic acid is 10000-13000, ethylene glycol in polymer: the molar ratio of lactic acid monomer
Example is (4-5): 1, the proportionate relationship of the quality of water delivery part and hydrophilic segment, meeting in the molecular weight and polymer of polymer
Directly influence the micellar conformation of polymer, the present invention passes through to polymer molecular weight and to the excellent of ethylene glycol and lactic acid ratios
Change, the nanoparticle form prepared is uniform spherical in shape, scattered.
5, of the invention by being investigated to a variety of different organic dissolutions, as a result, it has been found that, it is received using tetrahydrofuran dissolution
Rice block polymer, prepared nano-micelle form rule, good dispersing state, the partial size of gained nanoparticle in 120mn or so,
It is good compared with the micellar solution state that other organic solvents obtain.Taxol is dissolved with methylene chloride, the dissolution of taxol can be improved
Property, increase the encapsulation rate and drugloading rate of medicament-carried nano carrier.In step (4), organic solvent is that mass fraction is 2% polyethylene
Alcohol can be such that mixed solution adequately mixes.
6, the rate of addition of pharmaceutical polymer organic solvent generates important influence, the speed mistake of dropwise addition to the formation of micella
Fastly, not exclusively, the micellar particle size of formation is big, and size distribution is wide, and unstable, Chang Youyi thickness film for reaction.Rate of addition mistake
Slowly, nanoparticle negligible amounts can be made, the production cycle extends.The rate of addition for grasping drug aggregation organic solution makes guarantee
The technology stability and properties of product of standby micella have important influence.Therefore the present invention is filtered out best by constantly optimizing
Drop speed be 20-30 drop/minute.
7, the main effect of nanoparticle freeze drying protectant is the complete of protection nanoparticle structure, prevents nanoparticle from protecting in freeze-drying
Reunite during depositing, increases the dissolubility of nanoparticle after freeze-drying.By constantly screening discovery, protective agent is not added and either adds
Enter other kinds of protective agent such as mannitol etc., the nanoparticle dissolubility after freeze-drying, form and particle size change.Cause
This, the freeze drying protectant that the present invention selects is the F-68 that mass fraction is 2.5%, avoids the appearance of the above problem.
8, taxol and polymer are not readily dissolved in water, respectively with organic solvent PEG-PLA either PEG-PLA and
The mixed polymer of folate-PEG-PLA carries out dissolution respectively with methylene chloride and forms oily phase, adds insoluble in organic solvent
Polyvinyl alcohol formation water phase (soluble easily in water) be conducive to the formation of nanoparticle using uniform lotion is ultrasonically formed.
Detailed description of the invention
Fig. 1 is paclitaxel nano micelle transmission electron microscope picture；
Fig. 2 is paclitaxel nano micelle grain size distribution；
Fig. 3 is the transmission electron microscope picture after effect of nano-paclitaxel freeze-drying is redissolved；
Fig. 4 is the grain size distribution after effect of nano-paclitaxel freeze-drying is redissolved.
Specific embodiment
The invention will be further described with embodiment with reference to the accompanying drawing.
Embodiment 1: the optimization of effect of nano-paclitaxel preparation condition
1. the investigation of preparation method
The preparation of nanoparticle can take rotary evaporation, dialysis and rotary evaporation dialysis.Rotary evaporation be by
Drug and the mixed solution of nano material are added drop-wise in water, with certain speed stirring to obtain micellar solution.And rule of dialysing
Drug and nanometer block are directly attached in bag filter, obtain nano micellar solution through dialysis.Rotary evaporation dialysis rule be
The two is combined, the resulting solution of rotary evaporation is fitted into bag filter, dialyses in water with aforementioned by the method for rotary evaporation,
To obtain micellar solution.The advantage of rotary evaporation is that reaction is more thorough, obtains more pure nanoparticle.
It is observed by the nanoparticle that transmission electron microscope prepares distinct methods, the results are shown in Table 1.
Influence of the different preparation methods of table 1 to nanoparticle form
| Preparation method | Nanoparticle form |
| Rotary evaporation | Particle size is uneven, easily formation thick film |
| Dialysis | Form is irregular, disperses good |
| Rotary evaporation dialysis | Form rule is spherical in shape, uniform in size, disperses good |
2. the investigation of organic solvent
Accurate totally 4 four parts of PEG-PLA nanometer block for weighing equivalent respectively, use is dissolved in the different organic molten of equivalent respectively
In liquid acetone, tetrahydrofuran, DMSO and dimethylformamide.Prepare 4 50ml beakers, be separately added into the ultrapure water of equivalent,
Under identical mixing speed, the different organic solutions containing nano material are added drop-wise to dropwise with 20-30 drop/minute speed
In ultrapure water, continue to stir, the micellar solution being stirred is put into bag filter.It dialyses the identical time, takes suitable prosthese
By its form of transmission electron microscope observing, suitable organic solvent is filtered out.The nanometre glue prepared using tetrahydrofuran organic solvent
Beam form is uniform, good dispersion, and partial size is in 100nm or so, and the micellar particle size that other organic solvents obtain is smaller, or cannot
Nanoparticle is formed, therefore can choose organic solvent of the tetrahydrofuran as nanometer block, the results are shown in Table 2
Influence of 2 different organic solvents of table to nanoparticle form
| Organic solvent | Nanoparticle form |
| Acetone | Particle size is 50nm or so, scattered, even particle size |
| Tetrahydrofuran | Particle shape rule is spherical in shape, and partial size is scattered in 100nm or so |
| DMSO | It is not easy to form nano particle |
| Dimethylformamide | Shape bad, partial size with adhesion, forms thick film in 40nm or so |
3. influence of the dosage of polyvinyl alcohol to nano shape
Precision weighs PEG-PLA polymer 300mg respectively, is dissolved in 1ml tetrahydrofuran, and being prepared into concentration is 300mg/
The polymer organic solution of ml weighs taxol 50mg and is dissolved in 1ml methylene chloride, 2ml, 3ml is added after the two is mixed,
4ml, 5ml polyvinyl alcohol, ultrasound sufficiently, are added drop-wise in 30ml pure water with 20-30 drop/minute speed, and it is two small to continue stirring
When, the solution after stirring is put into bag filter, the form of transmission electron microscope observing nanoparticle, when the amount of polyvinyl alcohol is 2ml,
Uniform solution can not be formed after ultrasound, has block not react sufficiently, and the nanoparticle of preparation forms thick film, polyvinyl alcohol
Dosage is too many, and resulting nanoparticle amount is few, disperses bad.Influence of the dosage of polyvinyl alcohol to nano shape see the table below.
| Polyvinyl alcohol dosage | Nano shape |
| 2ml | Form thick film |
| 3ml | Nanometer is spherical in shape, easily disperses |
| 4ml | Nanoparticle is few spherical in shape |
| 5ml | Nanoparticle is few spherical in shape |
4. influence of pharmaceutical polymer organic solution/water ratio to nano shape
Precision weighs PEG-PLA polymer 300mg respectively, is dissolved in 1ml tetrahydrofuran, and being prepared into concentration is 300mg/
The polymer organic solution of ml weighs taxol 50mg and is dissolved in 1ml methylene chloride, and 3ml polyvinyl alcohol is added, and ultrasound is sufficiently mixed
It closes.Respectively in 4 beakers be added 10ml, 20ml, 30ml, 40ml ultrapure water, guarantees organic solution/water ratio be 1:2,1:4,
1:6,1:8, in the case where certain speed magnetic agitation, by the organic solution of the various concentration containing nano material with 20-30
Drop/minute speed is added dropwise in pure water, is continued to be put into bag filter after stirring and be dialysed, takes after obtaining medicament-carried nano micelle
Suitable nano-micelle is through its form of transmission electron microscope observing.According to form as it can be seen that different organic solution/water ratios are to nanometer particle shape
The influence of state be not it is very big, as organic solvent/water ratio < 1:8, it is spherical in shape to prepare nano-micelle form rule, disperses good.
It is shown in Table 4
Influence of 4 pharmaceutical polymer organic solution of table/water ratio to nanoparticle form
| Pharmaceutical polymer organic solution/water | Nanoparticle form |
| 1:2 | Form rule, there is thick film |
| 1:4 | Form rule is spherical in shape, and adhesion is more |
| 1:6 | Form rule, particle is more, disperses good |
| 1:8 | Shape bad, for partial size in 40nm or so, particle is less |
5. influence of the mixing speed to nanoparticle form
Totally three parts of PEG-PLA are weighed respectively, is dissolved in a certain amount of tetrahydrofuran, are added one in another 50ml beaker
Quantitative ultrapure water, in the case where friction speed magnetic stirring speed, by the organic solvent containing nano material with 20-30
Drop/minute speed is added drop-wise in pure water dropwise, continues to be put into bag filter after stirring a period of time, the nanometre glue dialysed
Beam observes its form by transmission electron microscope detection.According to morphological analysis, influence of the different mixing speeds to nanoparticle form is very
Greatly, select the nano-carrier form of suitable mixing speed preparation uniform, good dispersion.
Influence of 5 mixing speed of table to nanoparticle
6. influence of the freeze drying protectant to nanoparticle form by the above-mentioned nano-micelle being successfully prepared be not added protective agent or
Different freeze drying protectants is added, transmission electron microscope results are as shown in table 5 after observing dissolubility after being lyophilized and redissolving.
Influence of the different protective agents to nanoparticle form is added in table 6
| Different freeze drying protectants | Appearance after freeze-drying | Nanoparticle form |
| Protective agent is not added | Color milky, does not redissolve | It is not soluble in water, it is dissolved in organic solvent |
| 5% mannitol | Colours white, good water solubility | Nanoparticle morphologic change, more adhesion |
| 2.5%F-68 | Color milky, good water solubility | Nanoparticle form does not change |
Embodiment 2: the preparation without folic acid effect of nano-paclitaxel
Precision weighs PEG-PLA 300mg polymer, is dissolved in 1ml tetrahydrofuran, is configured to the organic molten of 300mg/ml
Liquid.It is protected from light and weighs taxol 50mg, be dissolved in the methylene chloride of 1ml, be made into the organic solution that concentration is 50mg/ml.By the two
Solution, which is sufficiently mixed, is made uniform solution, and 3ml polyvinyl alcohol is added into the mixed solution, and 30-40W ultrasound 10min makes medicine
Object polymer solution adequately mixes, and the organic solution mixed is added drop-wise to 30ml ultrapure water with 20-30 drop/minute speed
In, ice bath 300W ultrasound 20min.By stirring in water bath 4h under resulting lotion normal temperature and pressure, volatile organic solvent, centrifugation
(14000r/min) 30min collects precipitating, is washed with distilled water 4 times, again aquation.Again the micellar solution after aquation is put
Enter to be placed in water bag filter in dialysis band (3500KD) and dialyse, the organic dissolution that do not react completely and drug are dialysed
Out to get arrive paclitaxel nano micelle.2.5% F-68 is added to the nano-micelle dialysed, freezes to obtain without folic acid
Effect of nano-paclitaxel.For nanoparticle prepared by the present invention through transmission electron microscope observing, form rule is spherical in shape, and scattered, size is equal
It is even.
The transmission electron microscope of effect of nano-paclitaxel manufactured in the present embodiment without folic acid is shown in Fig. 1, the taxol without folic acid
The grain size distribution of nanoparticle is shown in Fig. 2
Embodiment 3: the preparation of folic acid effect of nano-paclitaxel
Polymer P EG-PLA 270mg is weighed, polymer polate-PEG-PLA 30mg is weighed, makes the polymerization with folic acid
Object ratio accounts for the 10% of total polymer, is dissolved in 1ml tetrahydrofuran, is configured to the organic solution of 300mg/ml.It is protected from light and weighs purple
China fir alcohol 50mg, is dissolved in the methylene chloride of 1ml, is made into the organic solution that concentration is 50mg/ml.The two solution is sufficiently mixed system
At uniform solution, 3ml polyvinyl alcohol is added into the mixed solution, 30-40W ultrasound 10min fills pharmaceutical polymer solution
The mixing divided, the organic solution mixed is added drop-wise in 30ml ultrapure water with 20-30 drop/minute speed, and ice bath 300W is super
Sound 20min.By stirring in water bath 4h under resulting lotion normal temperature and pressure, volatile organic solvent is centrifuged (14000r/min) 30min,
Precipitating is collected, is washed with distilled water 4 times, again aquation.Again the micellar solution after aquation is put into dialysis band (3500KD)
Bag filter is placed in water and is dialysed, the organic dissolution that do not react completely and drug are given to get purple to folic acid
China fir alcohol nano-micelle.2.5% F-68 is added to the nano-micelle dialysed, what is frozen arrives the effect of nano-paclitaxel containing folic acid.
For nanoparticle prepared by the present invention through projection Electronic Speculum observation, form rule is spherical in shape, scattered, uniform in size.
Effect of nano-paclitaxel physiological saline after freeze-drying is redissolved, the transmission electron microscope after resulting effect of nano-paclitaxel redissolution
Figure is shown in Fig. 3, and the grain size distribution of the effect of nano-paclitaxel after redissolution is shown in Fig. 4.Fig. 1 and Fig. 3 is respectively the transmission electricity that front and back is lyophilized
Mirror figure, comparison it can be concluded that, the shape for freezing front and back effect of nano-paclitaxel is spherical shape, uniform in size, and dispersion is good, is frozen to purple
China fir alcohol nanoparticle does not have morphologic change, but freezes the storage for being conducive to nanoparticle, and will be to body in frozen storage process
Harmful organic solvent removes, and reduces damage of the organic solvent to body, provides condition for clinic for nanoparticle.Fig. 2
With Fig. 4 be respectively be lyophilized front and back grain size distribution, by two figure as can be seen that freeze-drying before and after partial size 100-150nm it
Between, there is no biggish variations for partial size, remain the distinctive nanoparticle characteristic of effect of nano-paclitaxel.
Above-mentioned, although the foregoing specific embodiments of the present invention is described with reference to the accompanying drawings, not protects model to the present invention
The limitation enclosed, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art are not
Need to make the creative labor the various modifications or changes that can be made still within protection scope of the present invention.
Claims (11)
1. a kind of preparation method of taxol micelle nano grain, it is characterized in that: steps are as follows:
(1) by nanometer block polymer polyethylene glycol-polylactic acid either polyethylene glycol-polylactic acid and folic acid-polyethylene glycol
The mixed polymer of lactic acid is dissolved in organic solvent tetrahydrofuran, and solution A is made；
(2) taxol is dissolved in methylene chloride, solution B is made；
(3) solution A and solution B are sufficiently mixed, the uniform pharmaceutical polymer organic solution of concentration is made；
(4) polyvinyl alcohol that mass fraction is 2% will be added in the mixed solution of step (3) preparation, ultrasound fills mixed solution
Divide mixing；
(5) mixed solution is instilled in water dropwise with 20-30 drop/minute speed, it is ultrasonic under ice bath；
(6) by stirring in water bath under resulting lotion normal temperature and pressure, volatile organic solvent, centrifugation is collected precipitating, is washed with water, heavy
New aquation；
(7) micellar solution after aquation again is put into bag filter, bag filter is placed in water and is dialysed to get Japanese yew is arrived
Alcohol nano-micelle；
(8) the freeze drying protectant F-68 that mass fraction is 2.5% is added to the nano-micelle dialysed, freezes to obtain taxol and receives
The grain of rice；The mass ratio of the nanometer block polymer and taxol are as follows: 6:1.
2. preparation method as described in claim 1, it is characterized in that: when nanometer block polymer is poly- second in the step (1)
When glycol-polylactic acid and folic acid-polyethylene glycol-polylactic acid mixed polymer, folic acid-polyethylene glycol-polylactic acid quality hundred
Divide than being 8%-12%.
3. preparation method as described in claim 1, it is characterized in that: the mass percent of the folic acid-polyethylene glycol-polylactic acid
It is 10%.
4. preparation method as described in claim 1, it is characterized in that: when nanometer block polymer is poly- second in the step (1)
Glycol-polylactic acid molecular weight is 10000-13000, and ethylene glycol in polymer: the molar ratio of lactic acid monomer is (4-5): 1.
5. preparation method as described in claim 1, it is characterized in that: in the step (4), the body of polyvinyl alcohol and mixed solution
Product is than being 1.5:1, and ultrasonic power is 30-40W, and the ultrasonic time is 10min.
6. preparation method as described in claim 1, it is characterized in that: stirring is stirred using magnetic stirring apparatus in the step (6)
It mixes, mixing speed 500-1000r/min, mixing time 4h.
7. preparation method as described in claim 1, it is characterized in that: the condition of centrifugation is 14000r/min in the step (6)
Centrifugation time is 30min.
8. preparation method as described in claim 1, it is characterized in that: in the step (7), the molecular cut off of bag filter is
3500KDa。
9. the taxol micelle nano grain that any preparation method of claim 1-8 is prepared.
10. a kind of preparation method for the drug for treating tumour, characterized in that including any preparation side claim 1-8
Method.
11. preparation method as claimed in claim 10, which is characterized in that the tumour is oophoroma, breast cancer, colon
Cancer, lung cancer, prostate cancer, the tumour of nose larynx or brain.
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| CN105126102A (en) * | 2015-07-31 | 2015-12-09 | 山东大学齐鲁医院 | Hypocrellin B nanoparticle and preparation method thereof |
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| CN105126102A (en) * | 2015-07-31 | 2015-12-09 | 山东大学齐鲁医院 | Hypocrellin B nanoparticle and preparation method thereof |
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