CN102335139B - Adriamycin sustained-release nano particles and preparation method thereof - Google Patents
Adriamycin sustained-release nano particles and preparation method thereof Download PDFInfo
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Abstract
The invention relates to adriamycin sustained-release nano particles which comprise 6-16% of adriamycin and 83-93% of cucumber mosaic virus in mass percent, wherein the adriamycin penetrates the capsid protein of the cucumber mosaic virus through micropores on the surface of the capsid protein and is absorbed on the RNA (Ribonucleic Acid) of the cucumber mosaic virus. The adriamycin sustained-release nano particles provided by the invention not only greatly reduce the release rate of adriamycin, but also can further improve the concentration effect of the sustained-release nano particles on tumor tissues after being in key joint with target group folic acid.
Description
Technical field
The present invention relates to the medicinal preparation take used non-active ingredients as feature, be specifically related to sustained releasing type amycin medicine.
Background technology
Amycin is a kind of anthracene nucleus antineoplastic antibiotic, can suppress the synthetic of RNA and DNA, thereby stops the growth of tumor cell, antitumor spectra is wider, kinds of tumors all there is effect, belongs to cell cycle nonspecific agent (CCNSA), the tumor cell of various growth cycles is had killing action.All effective in cure to other various cancers such as acute leukemia, malignant lymphoma, breast carcinoma, sarcoma, pulmonary carcinoma, bladder cancer.At present clinical administration mostly is intravenous drip, but amycin can not see through blood brain barrier, and this medicine whole body that distributes rapidly behind the quiet notes, strong toxic and side effects is arranged, be mainly manifested in: cardiac toxicity, the lighter shows as arrhythmia, and severe one carrying out property cardiomyopathy occurs and congestive heart failure occurs; Cause leukocyte and thrombocytopenia, bone marrow depression; Alopecia; Digestive tract reaction, nauseating, loss of appetite; Medicine overflows outside the blood vessel can cause tissue ulcer and necrosis.These toxic and side effects have limited the extensive use of amycin at clinical chemotherapy, although dosage is very large, the ratio that can reach lesions position performance curative effect is very low.For reducing its toxicity, improving curative effect, the research of amycin novel form becomes problem demanding prompt solution.
The Patent Application Publication of publication number CN101234205A a kind of " high molecule adriamycin bonding medicine nano capsule with target function ", this Nano capsule is formed by two kinds of polyethylene glycol-polylactic acid block copolymer Hybrid assemblings, wherein a kind of polylactic acid chain termination of block copolymer has amycin, and the molar ratio in Nano capsule is 98%-70%; The polyglycol chain termination of another kind of block copolymer has lactose, and the molar ratio in Nano capsule is 2-30%.In the described scheme of above-mentioned patent application, the amycin that is connected on the polylactic acid chain segment is in the capsule kernel, is subjected to the duplicate protection of polylactic acid and Polyethylene Glycol, has slow-release function; The lactose that is connected to the Polyethylene Glycol end of the chain is in outer capsule layer, has target function, makes Nano capsule preferentially enter the cell that carries the lactose receptor.But, since described polyethylene glycol-polylactic acid block copolymer not only molecular weight is large but also be a kind of chain structure, therefore the segment of the overwhelming majority is, a winding is embedded in the inside of nano-particle, the other end is emerging in the surface of nano-particle.So, when amycin is emerging in nano-particle surperficial, just lose slow releasing function, when the lactose group is wound the inside that is embedded in nano-particle, just do not had target function.In addition, owing to formed by a molecule-OH and another molecule-COOH dehydrating condensation between the lactic acid molecules of polylactic acid, and this ester bond links to each other and to be connected fragility with respect to amycin with amido link between the polylactic acid, chain rupture occurs easily in biodegradable process, tend in succession one section polylactic acid and affect drug effect on the amycin of this moment.
Summary of the invention
Technical problem to be solved by this invention provides a kind of adriablastina target sustained-release nano, and this nano-particle has the effective advantage of target controlling and releasing.
The technical scheme that the present invention addresses the above problem is as described below:
A kind of adriamycin sustained-release nano particles, this nano-particle comprises amycin 6-16% and cucumber mosaic virus 83-93% by mass percentage, and the micropore of wherein said amycin through the capsid protein surface of cucumber mosaic virus sees through described capsid protein and be adsorbed on the RNA of cucumber mosaic virus.
Adriamycin sustained-release nano particles of the present invention can also further be modified into the sustained-release nano with target function; this improved sustained-release nano comprises that also mass percent is the targeting group folic acid of 1-2%, and described folic acid is connected to the surface of the capsid protein of cucumber mosaic virus.
In the technique scheme, described amycin can be amycin isomers or amycin derivant; Described cucumber mosaic virus can adopt the phytovirology method to extract from the Folium Cucumidis sativi that infects cucumber mosaic virus and obtain, concrete extracting method can be implemented (Howard Scott with reference to the disclosed method of Howard Scott, Purification of Cucumber Mosaic Virus, Virology, 1963,20,103-106.).
The preparation method of above-mentioned adriamycin sustained-release nano particles is comprised of following steps:
(1) from the Folium Cucumidis sativi that infects cucumber mosaic virus, extracts cucumber mosaic virus by the phytovirology method, then it is joined in the stabilizing solution, making the weight concentration of cucumber mosaic virus in stabilizing solution is 0.5-5mg/mL, obtains containing the cucumber mosaic virus buffer;
(2) get amycin and be dissolved in water, making the weight concentration of amycin in water is 0.10-1.25mg/mL, gets amycin solution;
(3) the prepared cucumber mosaic virus buffer that contains is mixed by 1: 1 volume ratio with amycin solution, 2-8 ℃ of lower temperature bathed 12-16 hour; Then be that 10%～40% sucrose solution carries out density gradient centrifugation with mass concentration, be loaded with amycin cucumber mosaic virus contaminated area band with the absorption of minute hand head, at last with stabilizing solution washing, centrifugalize, collecting precipitation thing, lyophilization namely get adriamycin sustained-release nano particles;
Stabilizing solution described in above-mentioned steps (1) and (3) is comprised of the buffer of ethylenediaminetetraacetic acid and pH6-8, and the weight concentration of ethylenediaminetetraacetic acid in stabilizing solution is 1.5-3mg/mL.
Above-mentioned preparation method with sustained-release nano of target function is comprised of following steps:
(I) folic acid and the 1-(3-dimethylaminopropyl) that is respectively folic acid 5-10 times quality-3-ethyl phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide are dissolved in the reaction dissolvent, and to make folic acid mass concentration in reaction dissolvent be 0.5-1mg/mL, reaction is 4 hours under the room temperature, gets activated folic acid solution; Wherein, described reaction dissolvent is that volume ratio is stabilizing solution: DMSO=4: 1 mixed solution;
(II) getting in the preparation method of above-mentioned adriamycin sustained-release nano particles prepared adriamycin sustained-release nano particles, to add stabilizing solution resuspended, making the concentration of cucumber mosaic virus in stabilizing solution that is loaded with amycin is 0.5-5mg/mL, add the prepared folic acid solution of isopyknic step (I), 2-8 ℃ of lower temperature bathed reaction 12-16 hour, be that 10%～40% sucrose solution carries out density gradient centrifugation with mass concentration, be loaded with amycin cucumber mosaic virus contaminated area band with what the minute hand head was drawn modified with folic acid, wash with stabilizing solution at last, centrifugalize, collecting precipitation thing, lyophilization namely get the sustained-release nano with target function;
Above-mentioned steps (I) and (II) described stabilizing solution formed by the buffer of ethylenediaminetetraacetic acid and pH 6-8, and the concentration of ethylenediaminetetraacetic acid in stabilizing solution is 1.5-3mg/mL.
Adriamycin sustained-release nano particles of the present invention can method routinely be made injection and various oral formulations, with the treatment malignant tumor.When the sustained-release nano with target function of the present invention was prepared into injection, the treatment malignant ascite can adopt the intraperitoneal injection administration, but the administration for the treatment of entity tumor local injection.
Since the capsid protein of cucumber mosaic virus be a kind of have certain rigidity naturally divide submounts, mean diameter 29nm (particle size distribution 28-30nm), its surface also is densely covered with micropore, therefore utilize the sucking action of positive and negative charge, amycin is easy to see through described capsid protein physical absorption or be entrenched on the RNA of cucumber mosaic virus.Under the dual function that attraction and the capsid protein of described positive and negative charge are protected, not only amycin can not come off at the RNA of cucumber mosaic virus, and has greatly delayed the rate of release of amycin.In addition, compare and prior art, the technical problem of the high molecular polymerization carrier of keyed jointing medicine adhesion chain rupture can not occur, therefore can keep the pharmacologically active of amycin not impaired fully.
Because the molecular volume of folic acid is little and the affinity of folacin receptor is high, and folacin receptor density is significantly higher than normal structure on the tumor tissues, therefore, the further improvement of the present invention scheme is bonded in described sustained-release nano surface with folic acid, given the target function of described sustained-release nano, by folacin receptor mediated endocytosis path, the amycin in the sustained-release nano of folic acid bonding is absorbed by tumor-selective ground, has significantly reduced the toxic and side effects of amycin.Especially, the capsid protein of cucumber mosaic virus of the present invention be a kind of have certain rigidity naturally divide submounts, and the surface of the three-D space structure that it is spherical is densely covered with amino group, therefore, after the carboxylic group of folate molecule is activated, just be easy to intensive and be bonded in equably the surface of the capsid protein of cucumber mosaic virus, under the effect of " boundling effect ", the concentration effect of described sustained-release nano on tumor tissues is able to further raising.
Below further specify the technique effect that adriamycin sustained-release nano particles of the present invention can reach by release experiment and curative effect and toxicity contrast experiment.
One, release experiment
1, experimental drug
Sample: get embodiment 1 gained adriamycin sustained-release nano particles, adding water, to make the concentration of amycin in water be 100 μ g/mL.
Reference substance: get amycin and be diluted with water to 100 μ g/mL.
2, experimental technique
Sample thief 50 μ L place Dialysis tubing; Get 25mL pH and be 7.4 PBS solution and be the dialysis medium, Dialysis tubing is put into the dialysis medium, under 37 ℃, stir dialysis with 100 rev/mins; With 1-8 hour interval, each 200uL dialysis solution of regularly getting adopted the amycin content that discharges in the fluorescence spectrophotometry dialysis solution.Getting reference substance 50 μ L adopts above-mentioned identical dialysis and assay method to carry out the release experiment.The amycin release profiles of above-mentioned sample and reference substance as shown in Figure 3.As seen from Figure 3, described reference substance was sent out with regard to quick-fried property in 4 hours and is discharged 50%, and sustained release only is 24 hours, and the sustained release of described sample is for up to more than 5 days, and curative effect is lasting, does not need frequent intermittent administration.
Two, myocardial cell toxicity test
1, experimental drug
Sample: get embodiment 1 gained adriamycin sustained-release nano particles, adding water, to make the concentration of amycin in water be 100 μ g/mL.
Reference substance: get amycin and be diluted with water to 100 μ g/mL.
2, experimental technique
Get in the DMEM culture fluid that mouse cardiac myocytes joins 20% hyclone, adjust mouse cardiac myocytes to 1.5 * 10
4Individual/mL; The mouse cardiac myocytes culture fluid of preparing is inoculated in the burnt culture dish of copolymerization, and every hole 2mL is in 37 ℃, 5%CO
2And cultivated 24 hours under the saturated humidity.The well of the burnt culture dish of copolymerization is divided into sample sets and matched group, establishes 5 multiple holes for every group; In each well of sample sets, add the 100uL sample, in 37 ℃, 5%CO
2And continue under the saturated humidity to cultivate 3 hours.Equally, in each well of matched group, add 50 μ L reference substances, continue to cultivate 3 hours with same condition.Behind the cell culture 3 hours, remove cell culture fluid, behind PBS liquid washed cell three times, fix 20 minutes with 90% ethanol, use again PBS liquid washed cell three times, then with DAPI liquid transfect cell nuclear, use PBS liquid washed cell three times again, place under the Laser Scanning Confocal Microscope and observe, experimental result is seen Fig. 4.As seen from Figure 4, free amycin enters in the mouse cardiac myocytes nuclear (left figure) in a large number in the reference substance, makes nucleus be purple with the blue-fluorescence stack of DAPI; Compare with reference substance, the cucumber mosaic virus nano-particle in the sample (right figure) has significantly reduced the gathering of amycin in cardiac cell nucleus, thereby has reduced cardiac toxicity.
Three, pharmacodynamic experiment
1, experimental drug
Sample: treating excess syndrome is executed the sustained-release nano that example 4 gained have target function, thin up to amycin in water concentration be 450-550ug/mL.
Reference substance: commercially available amycin injection.
2, experimental technique
Getting body weight is 18 of 18-22g/ mices only, and will contain OVCAR-3 oncocyte number is 5*10
6The physiological saline solution 200 μ L of individual/mL are injected to nude mice abdominal cavity and inoculate, and make up pernicious Ascite of Ovarian Cancer tumor model, and ascites tumor just forms after 18 days.18 above-mentioned pernicious Ascite of Ovarian Cancer tumor mices are divided into negative control group, treatment group and matched group at random; Wherein, negative control group, not administration, treatment group is given sample by 5mg/kg, and matched group is given the doxorubicin injection agent by 5mg/kg.After dosing interval is 5 days, periodic measurement mice abdominal circumference, the result is as shown in Figure 6.
As seen from Figure 6, sample has better tumor-inhibiting action to ovarian cancer cell OVCAR-3 cell line ascites tumor model.
Description of drawings
Fig. 1 makes up target controlling and releasing system sketch map with cucumber mosaic virus.
Fig. 2 is sucrose density gradient centrifugation contrast figure, and wherein, left figure is cucumber mosaic virus, and right figure is the cucumber mosaic virus of parcel amycin.
Fig. 3 is for measuring the curve chart that adriamycin sustained-release nano particles of the present invention discharges with dialysis.
Fig. 4 is the burnt microphotograph of the copolymerization of mouse cardiac myocytes toxicity test, and wherein, left figure is the toxicity test result of amycin, and right figure is the toxicity test result of adriamycin sustained-release nano particles.
Fig. 5 is flight time mass spectrum figure, and wherein, A figure is the flight time mass spectrum figure of cucumber mosaic virus, and B figure is the flight time mass spectrum figure of the sustained-release nano of target function that the present invention has.
The sustained-release nano of Fig. 6 target function that the present invention has suppresses the effect curve figure of nude mice OVCAR-3 ascites tumor.
The specific embodiment
Embodiment 1:
At first ethylenediaminetetraacetic acid is dissolved in the Tris buffer of 10mM pH 7.4, is ready to operate according to the following steps after the stabilizing solution that ethylenediaminetetraacetic acid concentration is 2.8mg/mL:
(1) from the Folium Cucumidis sativi that infects cucumber mosaic virus, extracts cucumber mosaic virus by known phytovirology method, then it is joined in the stabilizing solution, making the concentration of cucumber mosaic virus in stabilizing solution is 5mg/mL, obtains containing the cucumber mosaic virus buffer;
(2) get amycin and be dissolved in water, making the concentration of amycin in water is 1.25mg/m L, gets amycin solution;
(3) will contain cucumber mosaic virus buffer and each 1mL of amycin solution bathed 14 hours 4 ℃ of lower temperature of mixing; Then be that 10%～40% sucrose solution carries out density gradient centrifugation with mass concentration, draw with the minute hand head and be loaded with amycin cucumber mosaic virus contaminated area band, last stabilizing solution washing, centrifugalize, the collecting precipitation thing, lyophilization namely gets adriamycin sustained-release nano particles.
In order to make the public further understand the effect that sucrose solution described in the above-mentioned steps (3) carries out density gradient centrifugation, the inventor puts the centrifuge tube of above-mentioned steps (3) together and has taken a photo (seeing Fig. 2) with the centrifuge tube of adopting the density gradient centrifugation cucumber mosaic virus solution that uses the same method.
Embodiment 2:
At first ethylenediaminetetraacetic acid is dissolved in the phosphate buffer of 10mM pH6, is ready to operate according to the following steps after the stabilizing solution that ethylenediaminetetraacetic acid concentration is 2mg/mL:
(1) from the Folium Cucumidis sativi that infects cucumber mosaic virus, extracts cucumber mosaic virus by the phytovirology method by known, then it is joined in the stabilizing solution, making the concentration of cucumber mosaic virus in stabilizing solution is 4mg/mL, obtains containing the cucumber mosaic virus buffer;
(2) get amycin and be dissolved in water, making the concentration of amycin in water is 0.5mg/mL, gets amycin solution;
(3) will contain cucumber mosaic virus buffer and each 2mL of amycin solution bathed 16 hours 2 ℃ of lower temperature of mixing; Then be that 10%～40% sucrose solution carries out density gradient centrifugation with mass concentration, be loaded with amycin cucumber mosaic virus contaminated area band with the absorption of minute hand head, at last with stabilizing solution washing, centrifugalize, collecting precipitation thing, lyophilization namely get adriamycin sustained-release nano particles.
Embodiment 3:
At first ethylenediaminetetraacetic acid is dissolved in the Tris buffer of 10mM pH8.0, is ready to operate according to the following steps after the stabilizing solution that ethylenediaminetetraacetic acid concentration is 1.6mg/mL:
(1) by from the Folium Cucumidis sativi that infects cucumber mosaic virus, extracting cucumber mosaic virus by known phytovirology method, then it is joined in the stabilizing solution, making the concentration of cucumber mosaic virus in stabilizing solution is 2.4mg/mL, obtains containing the cucumber mosaic virus buffer;
(2) get amycin and be dissolved in water, making the concentration of amycin in water is 0.2mg/mL, gets amycin solution;
(3) will contain cucumber mosaic virus buffer and each 1mL of amycin solution bathed 12 hours 8 ℃ of lower temperature of mixing.Then be that 10%～40% sucrose solution carries out density gradient centrifugation with mass concentration, draw with the minute hand head and be loaded with amycin cucumber mosaic virus contaminated area band, last stabilizing solution washing, centrifugalize, the collecting precipitation thing, lyophilization namely gets adriamycin sustained-release nano particles.
Embodiment 4:
1. the preparation that has the sustained-release nano of target function
Ethylenediaminetetraacetic acid is dissolved in 10mM, in the Tris buffer of pH7.4, makes the stabilizing solution that ethylenediaminetetraacetic acid concentration is 2.8mg/mL, for subsequent use; Get stabilizing solution, the DMSO that adds 1/4 stabilizing solution volume obtains reaction dissolvent, and is for subsequent use;
5mg folic acid and 50mg 1-(3-dimethylaminopropyl)-3-ethyl phosphinylidyne diimmonium salt hydrochlorate and 50mg N-hydroxy-succinamide are dissolved in the 5mL reaction dissolvent, and reaction is 4 hours under the room temperature, gets activated folic acid solution;
It is resuspended to get embodiment 1 prepared adriamycin sustained-release nano particles 4.56mg adding stabilizing solution 4mL, the folic acid solution 4mL that adds above-mentioned activation, 5 ℃ of lower temperature were bathed reaction after 14 hours, be that 10%～40% sucrose solution carries out density gradient centrifugation with mass concentration, be loaded with amycin cucumber mosaic virus contaminated area band with what the minute hand head was drawn modified with folic acid, at last with stabilizing solution washing, centrifugalize, collecting precipitation thing, lyophilization namely get the sustained-release nano with target function.
2. the mensuration that has sustained-release nano Folic Acid, amycin and the cucumber mosaic virus content of target function
(1) folate content is measured
Precision takes by weighing 0.0020g folic acid reference substance, with 10mM pH6.8 phosphate buffer dissolving and be settled to scale, is mixed with the folic acid reference substance stock solution of 200 μ g/ml in the 10mL volumetric flask.Getting respectively above-mentioned reference substance storing solution 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.75mL, 1mL puts in the 10mL volumetric flask, use 10mM, pH6.8 phosphate buffer standardize solution shakes up, and is made into the reference substance solution that concentration is respectively 1,2,4,6,8,10,15,20 μ g/ml.With 10mM, the pH6.8 phosphate buffer is reference, measures respectively the absorbance of every a reference substance solution at the 360nm place with 1 centimetre of quartz cell, then with absorbency Y concentration X is carried out linear regression, gets regression beeline equation and is:
Y＝0.0120X+0.0521，R
2＝0.9962。
Getting the sustained-release nano 4mg that gained has target function is sample, be dissolved in the phosphate buffer of 4mL 10mM pH6.8, recording its absorbance at the 360nm place with 1 centimetre of quartz cell is 0.2441, and the folate content that calculates in the sample according to regression beeline equation again is 16 μ g/mg.
(2) amycin assay
Precision takes by weighing 0.0020g amycin reference substance, with 10mM pH6.8 phosphate buffer dissolving and be settled to scale, is mixed with the amycin reference substance stock solution of 200 μ g/ml in the 10mL volumetric flask.Getting respectively above-mentioned reference substance storing solution 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.75mL, 1mL puts in the 10mL volumetric flask, shake up with 10mM pH6.8 phosphate buffer standardize solution, be made into the reference substance solution of 1,2,4,6,8,10,15,20 μ g/ml.With 10mM, the pH6.8 phosphate buffer is reference, measures respectively the absorbance of every a reference substance solution at the 495nm place with 1 centimetre of quartz cell, then with absorbency Y concentration X is carried out linear regression, gets regression beeline equation and is:
Y＝0.0134X+0.0696，R
2＝0.9916。
Getting the sustained-release nano 1mg that gained has target function is sample, be dissolved in the 10mL 10mMpH6.8 phosphate buffer, recording absorbance at the 495nm place with 1 centimetre of quartz cell is 0.2572, and the amycin content that calculates in the sample according to regression beeline equation again is 140 μ g/mg.
(3) Lowry method cucumber mosaic virus assay
Be reference substance with the standard protein in the improvement Lowry protein quantification test kit of U.S. Pierce company, and its to carry out to specifications experimental implementation preparation standard protein concentration range be the different reference substance solution of a series of concentration of 10-1500 μ g/ml, measure respectively the absorbance of every a reference substance solution at the 750nm place with 1 centimetre of quartz cell, then with absorbency Y concentration X is carried out linear regression, gets regression beeline equation and be:
Y＝0.0016X+0.1871，R
2＝0.9807。
Get the sustained-release nano 0.4mg that gained has target function and be dissolved in the 0.4mL 10mM pH6.8 phosphate buffer, add mixing behind the modification Lowry reagent in the 2mL test kit, left standstill under the room temperature 10 minutes; Then add 1.0N phenol reagent mixing in the 0.1mL test kit, left standstill under the room temperature 30 minutes; Record absorbance 1.5311 with 1 centimetre of quartz cell at the 750nm place, the cucumber mosaic virus content that calculates in the sample according to regression beeline equation again is 840 μ g/mg..
Above-mentioned mass ratio is converted to gained has in the sustained-release nano of target function in visible this example of quality percentage composition, the mass content of amycin is 14%, and the mass content of folic acid is 1.6%, and the mass content of cucumber mosaic virus is 84%.
3. flying time mass spectrum analysis
Be connected on the cucumber mosaic virus for further proving conclusively folic acid, this example is carried out the cucumber mosaic virus flying time mass spectrum analysis as follows: the sustained-release nano that will have target function is resuspended in the water, regulating wherein, the concentration of cucumber mosaic virus is 1mg/mL, get the guanidine hydrochloride aqueous solution mixing of 24 μ L and 6 μ L6M, use Ziptip
C18Directly with mass spectrometer matrix liquid eluting, carry out mass spectral analysis after the desalination, the gained spectrogram is seen Fig. 5.As seen from Figure 5, the mass-to-charge ratio m/z of cucumber mosaic virus virus capsid protein subunit is 24140 (seeing the A curve), and cucumber mosaic virus virus capsid protein subunit mass-to-charge ratio m/z is 24563 (seeing the B curve) behind the modified with folic acid, and conclusive evidence folic acid is connected on the cucumber mosaic virus virus capsid protein subunit.
Embodiment 5:
Ethylenediaminetetraacetic acid is dissolved in the phosphate buffer of pH6.0, makes the stabilizing solution that ethylenediaminetetraacetic acid concentration is 2mg/mL, for subsequent use; Get stabilizing solution, the DMSO that adds 1/4 stabilizing solution volume obtains reaction dissolvent, and is for subsequent use;
4mg folic acid and 32mg 1-(3-dimethylaminopropyl)-3-ethyl phosphinylidyne diimmonium salt hydrochlorate and 32mg N-hydroxy-succinamide are dissolved in the 5mL reaction dissolvent, and reaction is 4 hours under the room temperature, gets activated folic acid solution;
It is resuspended to get embodiment 2 prepared adriamycin sustained-release nano particles 6mg adding stabilizing solution 3mL, add in the folic acid solution of above-mentioned activation and add 3mL, 2 ℃ of lower temperature were bathed reaction after 16 hours, be that 10%～40% sucrose solution carries out density gradient centrifugation with mass concentration, be loaded with amycin cucumber mosaic virus contaminated area band with what the minute hand head was drawn modified with folic acid, at last with stabilizing solution washing, centrifugalize, collecting precipitation thing, lyophilization namely get the sustained-release nano with target function.
Adopt method similarly to Example 4 to carry out Uv-visible Spectrophotometric Analysis resulting sustained-release nano with target function, record wherein, contain amycin 11%, cucumber mosaic virus 87%, folic acid 1.3%.
Embodiment 6:
Ethylenediaminetetraacetic acid is dissolved in the Tris buffer of pH8.0, makes the stabilizing solution that ethylenediaminetetraacetic acid concentration is 1.6mg/mL, for subsequent use; Get stabilizing solution, the DMSO that adds 1/4 stabilizing solution volume obtains reaction dissolvent, and is for subsequent use;
3mg folic acid and 15mg 1-(3-dimethylaminopropyl)-3-ethyl phosphinylidyne diimmonium salt hydrochlorate and 15mg N-hydroxy-succinamide are dissolved in the 5mL reaction dissolvent, and reaction is 4 hours under the room temperature, gets activated folic acid solution;
It is resuspended to get embodiment 3 prepared adriamycin sustained-release nano particles 4mg adding stabilizing solution 1mL, add in the folic acid solution of above-mentioned activation and add 1mL, 8 ℃ of lower reactions are after 12 hours, be that 10%～40% sucrose solution carries out density gradient centrifugation with mass concentration, be loaded with amycin cucumber mosaic virus contaminated area band with what the minute hand head was drawn modified with folic acid, at last with stabilizing solution washing, centrifugalize, collecting precipitation thing, lyophilization namely get the sustained-release nano with target function.
Adopt method similarly to Example 4 to carry out Uv-visible Spectrophotometric Analysis resulting sustained-release nano with target function, record wherein, contain amycin 7%, cucumber mosaic virus 92%, folic acid 1.1%.
Claims (2)
1. adriamycin sustained-release nano particles, this nano-particle is comprised of amycin 6-16%, cucumber mosaic virus 83-93% and targeting group folic acid 1-2% by mass percentage, wherein, described amycin is adsorbed on the RNA of cucumber mosaic virus through described capsid protein through the micropore on the capsid protein surface of cucumber mosaic virus; Described folic acid is connected to the surface of the capsid protein of cucumber mosaic virus.
2. method for preparing the described adriamycin sustained-release nano particles of claim 1, the method is comprised of following steps:
(1) from the Folium Cucumidis sativi that infects cucumber mosaic virus, extracts cucumber mosaic virus by the phytovirology method, then it is joined in the stabilizing solution, making the weight concentration of cucumber mosaic virus in stabilizing solution is 0.5-5mg/mL, obtains containing the cucumber mosaic virus buffer;
(2) get amycin and be dissolved in water, making the weight concentration of amycin in water is 0.10-1.25mg/m L, gets amycin solution;
(3) the prepared cucumber mosaic virus buffer that contains is mixed by the volume ratio of 1:1 with amycin solution, 2-8 ℃ of lower temperature bathed 12-16 hour; Then be that 10%～40% sucrose solution carries out density gradient centrifugation with mass concentration, draw with the minute hand head and be loaded with amycin cucumber mosaic virus contaminated area band, at last with the stabilizing solution washing, centrifugalize, the collecting precipitation thing, lyophilization namely gets adriamycin sustained-release nano particles;
(4) folic acid and the 1-(3-dimethylaminopropyl) that is respectively folic acid 5-10 times quality-3-ethyl phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide are dissolved in the reaction dissolvent, and to make folic acid mass concentration in reaction dissolvent be 0.5-1mg/mL, reaction is 4 hours under the room temperature, gets activated folic acid solution; Wherein, described reaction dissolvent is that volume ratio is that Wen is Dinged the mixed solution of Ye ︰ DMSO=4:1;
(5) getting the prepared adriamycin sustained-release nano particles of step (3), to add stabilizing solution resuspended, making the concentration of cucumber mosaic virus in stabilizing solution that is loaded with amycin is 0.5-5mg/mL, add the prepared folic acid solution of isopyknic step (4), 2-8 ℃ of lower temperature bathed reaction 12-16 hour, be that 10%～40% sucrose solution carries out density gradient centrifugation with mass concentration, be loaded with amycin cucumber mosaic virus contaminated area band with what the minute hand head was drawn modified with folic acid, wash with stabilizing solution at last, centrifugalize, collecting precipitation thing, lyophilization namely get the sustained-release nano with target function;
Stabilizing solution described in above-mentioned steps (1) and (3) is comprised of the buffer of ethylenediaminetetraacetic acid and pH6-8, and the weight concentration of ethylenediaminetetraacetic acid in stabilizing solution is 1.5-3mg/mL;
Above-mentioned steps (4) and (5) described stabilizing solution are comprised of the buffer of ethylenediaminetetraacetic acid and pH6-8, and the concentration of ethylenediaminetetraacetic acid in stabilizing solution is 1.5-3mg/mL.
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| US20060216238A1 (en) * | 2005-02-28 | 2006-09-28 | Marianne Manchester | Compositions and methods for targeting or imaging a tissue in a vertebrate subject |
| WO2008153725A2 (en) * | 2007-05-25 | 2008-12-18 | North Carolina State University | Viral nanoparticle cell-targeted delivery platform |
| US8535727B2 (en) * | 2009-02-25 | 2013-09-17 | North Carolina State University | Biodegradable non-woven fabric having plant virus encapsulated actives for drug delivery |
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2011
- 2011-10-08 CN CN201110303974.3A patent/CN102335139B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| Folic acid-conjugated protein cages of a plant virus: a novel delivery platform for doxorubicin;Yupeng Ren et al.;《Bioconjugate chem》;20070531;第18卷(第3期);第839页右栏第4-7行,第837页右栏第4-5段,第1-2行 * |
| Yupeng Ren et al..Folic acid-conjugated protein cages of a plant virus: a novel delivery platform for doxorubicin.《Bioconjugate chem》.2007,第18卷(第3期),参见第839页右栏第4-7行. |
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