CN110075073A - A kind of Cabazitaxel protein nano injection and preparation method thereof - Google Patents
A kind of Cabazitaxel protein nano injection and preparation method thereof Download PDFInfo
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- CN110075073A CN110075073A CN201910478565.3A CN201910478565A CN110075073A CN 110075073 A CN110075073 A CN 110075073A CN 201910478565 A CN201910478565 A CN 201910478565A CN 110075073 A CN110075073 A CN 110075073A
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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Abstract
The present invention is suitable for technical field of medicine, provides a kind of Cabazitaxel protein nano injection and preparation method thereof, the Cabazitaxel protein nano injection includes the raw material of following parts by weight: 50~150 parts of Cabazitaxel；400~4000 parts of protein carrier material；50~300 parts of protein nano decorative material；12~100 parts of freeze drying protectant.The present invention is by being encapsulated in the spherical shape or spheroid shape nanometer of protein carrier material and protein nano decorative material composition or being attached to Nanosurface by Cabazitaxel; and frozen dried is carried out with freeze drying protectant; gained Cabazitaxel protein nano injection has preferable slow release effect; antitumous effect with higher simultaneously; the use bring hypersensitivity of the cosolvent such as surfactant is effectively prevented, formulations toxic is reduced, improves bioavilability; dosage is reduced, administration time is extended.
Description
Technical field
The invention belongs to technical field of medicine more particularly to a kind of Cabazitaxel protein nano injection and its preparations
Method.
Background technique
Cabazitaxel belongs to taxanes series antineoplastic medicament, is the semi-synthetic purple of the compound that extracts in the needle by Japanese yew
China fir 01 derivatives, the mechanism of action is similar with other taxones, by promoting micro-pipe dimer to be assembled into micro-pipe, together
When prevent polymerisation process and make microtubule stabilization, inhibit cell further divide, thus inhibit cancer cell mitosis and
Increment.
Commercialized product is Cabazitaxel injection at present, for the dissolubility and preparation stability for increasing Cabazitaxel, note
It penetrates in agent prescription added with surfactants such as a large amount of polyoxyethylene sorbitan monoleates, dewatered ethanols as cosolvent, clinical research confirmation can be led
Cause serious hypersensitivity, in some instances it may even be possible to including generality fash/erythema, low blood pressure and bronchial spasm, give sufferers themselves and family
Category all brings very big pain；Meanwhile dosage is big, the time is long, the injection after dilution need to use in a short time, no
Precipitating will be then generated, stability is poor, and there are the security risks in terms of medication.
It can be seen that existing Cabazitaxel injection toxic side effect is big, easily causes serious hypersensitivity, also, it is administered
Dosage is big, the time is long and stability is poor, there are problems that the security risk in terms of medication.
Summary of the invention
The embodiment of the present invention is designed to provide a kind of Cabazitaxel protein nano injection, it is intended to solve existing card
Ba Tasai injection toxic side effect is big, easily causes serious hypersensitivity, also, dosage is big, the time is long and stability
It is poor, there are problems that the security risk in terms of medication.
The embodiments of the present invention are implemented as follows, a kind of Cabazitaxel protein nano injection, including following parts by weight
Raw material:
50~150 parts of Cabazitaxel；400~4000 parts of protein carrier material；50~300 parts of protein nano decorative material；
12~100 parts of freeze drying protectant.
The another object of the embodiment of the present invention is a kind of preparation method of Cabazitaxel protein nano injection, feature
It is, comprising:
Cabazitaxel and protein nano decorative material are weighed according to quantity, are dissolved in organic solvent, it is spare as oily phase；
Protein carrier material is weighed according to quantity, is dissolved in pure water, and adjusting pH value is spare as water phase to 7~10；
The oil is mutually subjected to ice-bath ultrasonic process with water phase, forms colostric fluid, ultrasonic power 20%~60%, when ultrasonic
Between 3~12min；
By the colostric fluid continue at room temperature 1~4h of stirring or by high pressure homogenizer pressure be 500~
Under conditions of 800bar, recycle 6~10 times；
By treated, lotion carries out vacuum rotary steam processing, removes organic solvent, obtains Cabazitaxel protein nano material；
Freeze drying protectant is weighed according to quantity, and the freeze drying protectant and Cabazitaxel protein nano material are placed in freeze-drying
In machine, carry out sublevel segmentation cooling frozen dried to get.
A kind of Cabazitaxel protein nano injection provided in an embodiment of the present invention, by Cabazitaxel, protein carrier material,
Protein nano decorative material and freeze drying protectant composition, i.e., by the way that Cabazitaxel is encapsulated in protein carrier material and albumen
In the spherical shape or spheroid shape nanometer of nano-modified material composition or it is attached to Nanosurface, and is lyophilized with freeze drying protectant
Processing, gained Cabazitaxel protein nano injection have preferable slow release effect, while antitumous effect with higher,
The use bring hypersensitivity of the cosolvent such as surfactant is effectively prevented, formulations toxic is reduced, improves biological utilisation
Degree reduces dosage, extends administration time.
Detailed description of the invention
Fig. 1 is a kind of Cabazitaxel protein nano material particle size distribution map；
It in pH value is that cumulative release is bent in 6.5 dissolution mediums that Fig. 2, which is Cabazitaxel protein nano injection and free KBTS,
Line chart；
Fig. 3 is cell survival rate curve graph of the Cabazitaxel protein nano injection under various concentration；
Fig. 4 is cell survival rate curve graph of the blank protein nano under various concentration；
Fig. 5 is BSA concentration (A) and cholesterol dosage (B) response surface interaction diagram；
Fig. 6 is selection (C) response surface interaction diagram of BSA concentration (A) with oily phase；
Fig. 7 is selection (C) response surface interaction diagram of cholesterol dosage (B) with oily phase；
Fig. 8 is HSA concentration (A), cholesterol dosage (B), pH value (C), carries the effect song that medicine ratio (D) influences encapsulation rate
Face figure；
Fig. 9 is another Cabazitaxel protein nano material particle size distribution map.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention,
It is not intended to limit the present invention.
Cabazitaxel is lipophilic drugs, not soluble in water to dissolve in ethyl alcohol.Due to the multiple binding sites of protein carrier material
Presence, albumin can be in conjunction with multiple compounds, such as Doxorubicin, methotrexate (MTX), exendin-4
(exendin-4) etc., the solubility of hydrophobic molecule in blood can be enhanced.As the transport protein of compound, pass through enhancing
Compound is delivered to body by the permeability of drug, the transcytosis that retention and tissue gp60 are mediated in cytoplasm
Interior specific organization simultaneously influences the distribution of compound molecule in blood, realizes targeting transhipment, allows more drug accumulations swollen
Tumor tissue enters in tumour cell.To which effectively its content is discharged into cytoplasm.The stimulation of drug will be reduced
Property and toxicity, preferably play its antitumor action.
Therefore, the embodiment of the present invention, which focuses on, researches and develops a kind of Cabazitaxel protein nano injection, by Cabazitaxel, albumen
Carrier material, protein nano decorative material and freeze drying protectant composition, i.e., by the way that Cabazitaxel is encapsulated in protein carrier material
In the spherical shape or spheroid shape nanometer of material and protein nano decorative material composition or it is attached to Nanosurface, and with freeze drying protectant
Frozen dried is carried out, gained Cabazitaxel protein nano injection has preferable slow release effect, while with higher anti-swollen
Tumor effect effectively prevents the use bring hypersensitivity of the cosolvent such as surfactant, reduces formulations toxic, improves life
Object availability reduces dosage, extends administration time.
In embodiments of the present invention, protein carrier material includes zeins, bovine serum albumin(BSA), human seralbumin egg
It is one or more of white.
In embodiments of the present invention, protein nano decorative material includes soybean lecithin, hydrogenated soya phosphatide, cholesterol, egg
One or more of yellow lecithin, natural phospholipid and phosphatidyl-ethanolamine.
In embodiments of the present invention, freeze drying protectant includes bovine serum albumin(BSA), mannitol, trehalose, Sodium Caprylate, cream
One or more of sugar, acetyl-l-tryptophan sodium.
In embodiments of the present invention, the preparation method of the Cabazitaxel protein nano injection is 50 including taking parts by weight
~150 parts of Cabazitaxel, 50~300 parts of protein nano decorative material are dissolved in organic solvent, mutually spare as oil；Separately
400~4000 parts of parts by weight of protein carrier material is taken to be dissolved in pure water, it is spare as water phase；During the oil phase is added to the aqueous phase
It carries out ice-bath ultrasonic and forms colostric fluid, ultrasonic power 20%~60%, 3~10min of ultrasonic time；Colostric fluid is displaced through height
Homogenizer is pressed, 500~800bar of pressure is recycled 6~10 times, or colostric fluid is continued to 1~4h of stirring at room temperature；It will most
The lotion that obtains eventually carries out 35 DEG C of vacuum rotary steams removal organic solvents, under the conditions of 22000r/min (34700 × g), 4 DEG C from
Heart 30min removes supernatant, and precipitating plus appropriate ultrapure water, which redissolve, obtains Cabazitaxel protein nano material；It in turn, will be described
Cabazitaxel protein nano material and 12~100 parts of freeze drying protectant are placed in freeze dryer, are carried out the cooling of sublevel segmentation and are frozen
Dry-cure to get.
In embodiments of the present invention, sublevel segmentation cooling frozen dried process specifically: under the conditions of -75 DEG C~-65 DEG C
2~4h of pre-freeze, then be warming up to -50 DEG C~-40 DEG C with 0.1~0.2 DEG C/min, and after constant temperature keeps 0.5~1h, then with 0.1~
After 0.2 DEG C DEG C/min is warming up to -35~-25 DEG C, and constant temperature keeps 9~11h, continue to be warming up to -20 with 0.1~0.2 DEG C/min
DEG C~-15 DEG C, and after 9~11h of constant temperature holding, it is warming up to 0 DEG C with 0.2~0.3 DEG C/min, keeps 4~6h；Continue with 0.5~
0.6 DEG C/min is warming up to 20 DEG C~30 DEG C, sampling.
In embodiments of the present invention, organic solvent is chloroform and dehydrated alcohol mixed liquor, and through of the invention a large amount of real
Verifying, with the increase of dehydrated alcohol in oily phase, nanoparticle partial size is obviously reduced, comprehensive consideration its to entrapment efficiency, carry
The influence of dose selects chloroform and entrapment efficiency, drugloading rate equal highest when dehydrated alcohol volume ratio 11:1, and partial size
It is relatively small.
In embodiments of the present invention, through lot of experiment validation, grease Phase Proportion influences entrapment efficiency, drugloading rate opposite
It is smaller, there is certain influence to nanometer particle size, there is minimum grain size in when grease Phase Proportion 1:10, and encapsulation rate, drugloading rate are also higher, because
This preparation condition of preferred oil watr-proportion 1:10 as protein nano grain, while the use of organic oil phase can be reduced.
It is described in further details that (grease phase volume ratio is equal in all embodiments to the present invention below in conjunction with specific embodiment
For 1:10, final sample volume is 100ml).
Embodiment 1
Cabazitaxel protein nano injection, including following raw material:
Cabazitaxel 100mg；Cholesterol 150mg；Bovine serum albumin(BSA) 1.5g (concentration 1.5%)；Sodium Caprylate 12mg.
Preparation process:
Cabazitaxel and cholesterol are dissolved in organic solvent (chloroform: dehydrated alcohol=11:1, total 10ml), made
It is mutually spare for oil；Bovine serum albumin(BSA) is dissolved in ultrapure water, pH to 10 is adjusted, it is spare as water phase；The oil is added
Enter in water phase, carry out ice-bath ultrasonic 12min under 30% power, forms colostric fluid；The colostric fluid is existed by high pressure homogenizer
Under conditions of pressure is 800bar, recycle 8 times；By treated, lotion carries out 35 DEG C of vacuum rotary steams processing, removes organic molten
Agent is centrifuged 30min under the conditions of 22000r/min (34700 × g), 4 DEG C, removes supernatant, and precipitating plus appropriate ultrapure water redissolve
Obtain Cabazitaxel protein nano material；Freeze drying protectant is added in Cabazitaxel protein nano material, is placed in freeze dryer,
Pre-freeze 3h under the conditions of -70 DEG C, then be warming up to -45 DEG C with 0.15 DEG C/min, keeps 0.5h at this temperature, then with 0.15 DEG C/
Min is warming up to -30 DEG C, keeps 10h at this temperature, continues to be warming up to -20 DEG C with 0.15 DEG C/min, keeps 10h at this temperature
Afterwards, be warming up to 0 DEG C with 0.2 DEG C/min, keep 5h, continue to be warming up to 25 DEG C with 0.5 DEG C/min, sampling to get.
Embodiment 2
Cabazitaxel protein nano injection, including following raw material:
Cabazitaxel 150mg；Cholesterol 200mg；Bovine serum albumin(BSA) 1.5g (concentration 1.5%)；Sodium Caprylate 12mg.
Preparation process:
Cabazitaxel and cholesterol are dissolved in organic solvent (chloroform: dehydrated alcohol=11:1, total 10ml), made
It is mutually spare for oil；Bovine serum albumin(BSA) is dissolved in ultrapure water, pH to 8 is adjusted, it is spare as water phase；The oil is added
Enter in water phase, carry out ice-bath ultrasonic 8min under 40% power, forms colostric fluid；The colostric fluid is existed by high pressure homogenizer
Under conditions of pressure is 500bar, recycle 8 times；By treated, lotion carries out 35 DEG C of vacuum rotary steams processing, removes organic molten
Agent is centrifuged 30min under the conditions of 22000 r/min (34700 × g), 4 DEG C, removes supernatant, and precipitating plus appropriate ultrapure water redissolve
Obtain Cabazitaxel protein nano material；Freeze drying protectant is added in Cabazitaxel protein nano material, is placed in freeze dryer,
Pre-freeze 3h under the conditions of -70 DEG C, then be warming up to -45 DEG C with 0.15 DEG C/min, keeps 0.5h at this temperature, then with 0.15 DEG C/
Min is warming up to -30 DEG C, keeps 10h at this temperature, continues to be warming up to -20 DEG C with 0.15 DEG C/min, keeps 10h at this temperature
Afterwards, be warming up to 0 DEG C with 0.2 DEG C/min, keep 5h, continue to be warming up to 25 DEG C with 0.5 DEG C/min, sampling to get.
Embodiment 3
Cabazitaxel protein nano injection, including following raw material:
Cabazitaxel 100mg；Cholesterol 100mg；Human serum albumins 2.0g (concentration 2%)；Sodium Caprylate 16mg.
Preparation process:
Cabazitaxel and cholesterol are dissolved in organic solvent (chloroform: dehydrated alcohol=11:1, total 10ml), made
It is mutually spare for oil；Human serum albumins is dissolved in ultrapure water, pH to 8 is adjusted, it is spare as water phase；The oil is added
Enter in water phase, carry out ice-bath ultrasonic 3min under 40% power, forms colostric fluid；The colostric fluid is continued to stir at room temperature
2h；Treated lotion is subjected to 35 DEG C of vacuum rotary steams processing, removes organic solvent, at 22000r/min (34700 × g), 4
It is centrifuged 30min under the conditions of DEG C, removes supernatant, precipitating plus appropriate ultrapure water redissolve to obtain Cabazitaxel protein nano material；It will card
Freeze drying protectant is added in Ba Tasai protein nano material, is placed in freeze dryer, pre-freeze 3h under the conditions of -70 DEG C, then with 0.15
DEG C/min is warming up to -45 DEG C, 0.5h is kept at this temperature, then be warming up to -30 DEG C with 0.15 DEG C/min, keep at this temperature
10h continues to be warming up to -20 DEG C with 0.15 DEG C/min, after keeping 10h at this temperature, is warming up to 0 DEG C with 0.2 DEG C/min, keeps
5h continues to be warming up to 25 DEG C with 0.5 DEG C/min, sampling to get.
Embodiment 4
Cabazitaxel protein nano injection, including following raw material:
Cabazitaxel 100mg；Cholesterol 100mg；Human serum albumins 2.0g (concentration 2%)；Sodium Caprylate 16mg.
Preparation process:
Cabazitaxel and cholesterol are dissolved in organic solvent (chloroform: dehydrated alcohol=11:1, total 10ml), made
It is mutually spare for oil；Human serum albumins is dissolved in ultrapure water, pH to 8 is adjusted, it is spare as water phase；The oil is added
Enter in water phase, carry out ice-bath ultrasonic 8min under 60% power, forms colostric fluid；The colostric fluid is continued to stir at room temperature
2h；Treated lotion is subjected to 35 DEG C of vacuum rotary steams processing, removes organic solvent, at 22000r/min (34700 × g), 4
It is centrifuged 30min under the conditions of DEG C, removes supernatant, precipitating plus appropriate ultrapure water redissolve to obtain Cabazitaxel protein nano material；It will card
Freeze drying protectant is added in Ba Tasai protein nano material, is placed in freeze dryer, pre-freeze 3h under the conditions of -70 DEG C, then with 0.15
DEG C/min is warming up to -45 DEG C, 0.5h is kept at this temperature, then be warming up to -30 DEG C with 0.15 DEG C/min, keep at this temperature
10h continues to be warming up to -20 DEG C with 0.15 DEG C/min, after keeping 10h at this temperature, is warming up to 0 DEG C with 0.2 DEG C/min, keeps
5h continues to be warming up to 25 DEG C with 0.5 DEG C/min, sampling to get.
Embodiment 5
Cabazitaxel protein nano injection, including following raw material:
Cabazitaxel 50mg；Cholesterol 100mg；Human serum albumins 1.0g (concentration 1%)；Trehalose 100mg.
Preparation process:
Cabazitaxel and cholesterol are dissolved in organic solvent (chloroform: dehydrated alcohol=11:1, total 10ml), made
It is mutually spare for oil；Human serum albumins is dissolved in ultrapure water, pH to 8 is adjusted, it is spare as water phase；The oil is added
Enter in water phase, carry out ice-bath ultrasonic 8min under 60% power, forms colostric fluid；The colostric fluid is continued to stir at room temperature
2h；Treated lotion is subjected to 35 DEG C of vacuum rotary steams processing, removes organic solvent, at 22000r/min (34700 × g), 4
It is centrifuged 30min under the conditions of DEG C, removes supernatant, precipitating plus appropriate ultrapure water redissolve to obtain Cabazitaxel protein nano material；It will card
Freeze drying protectant is added in Ba Tasai protein nano material, is placed in freeze dryer, pre-freeze 3h under the conditions of -70 DEG C, then with 0.15
DEG C/min is warming up to -45 DEG C, 0.5h is kept at this temperature, then be warming up to -30 DEG C with 0.15 DEG C/min, keep at this temperature
10h continues to be warming up to -20 DEG C with 0.15 DEG C/min, after keeping 10h at this temperature, is warming up to 0 DEG C with 0.2 DEG C/min, keeps
5h continues to be warming up to 25 DEG C with 0.5 DEG C/min, sampling to get.
Embodiment 6
Cabazitaxel protein nano injection, including following raw material:
Cabazitaxel 100mg；Cholesterol 100mg；Human serum albumins 1.5g (concentration 1.5%)；Mannitol 60mg.
Preparation process:
Cabazitaxel and cholesterol are dissolved in organic solvent (chloroform: dehydrated alcohol=11:1, total 10ml), made
It is mutually spare for oil；Human serum albumins is dissolved in ultrapure water, pH to 8 is adjusted, it is spare as water phase；The oil is added
Enter in water phase, carry out ice-bath ultrasonic 8min under 60% power, forms colostric fluid；The colostric fluid is continued to stir at room temperature
2h；Treated lotion is subjected to 35 DEG C of vacuum rotary steams processing, removes organic solvent, at 22000r/min (34700 × g), 4
It is centrifuged 30min under the conditions of DEG C, removes supernatant, precipitating plus appropriate ultrapure water redissolve to obtain Cabazitaxel protein nano material；It will card
Freeze drying protectant is added in Ba Tasai protein nano material, is placed in freeze dryer, pre-freeze 3h under the conditions of -70 DEG C, then with 0.15
DEG C/min is warming up to -45 DEG C, 0.5h is kept at this temperature, then be warming up to -30 DEG C with 0.15 DEG C/min, keep at this temperature
10h continues to be warming up to -20 DEG C with 0.15 DEG C/min, after keeping 10h at this temperature, is warming up to 0 DEG C with 0.2 DEG C/min, keeps
5h continues to be warming up to 25 DEG C with 0.5 DEG C/min, sampling to get.
Embodiment 7
Cabazitaxel protein nano injection, including following raw material:
Cabazitaxel 100mg；Cholesterol 100mg；Human serum albumins 1.5g (concentration 1.5%)；Trehalose 80mg.
Preparation process:
Cabazitaxel and cholesterol are dissolved in organic solvent (chloroform: dehydrated alcohol=11:1, total 10ml), made
It is mutually spare for oil；Human serum albumins is dissolved in ultrapure water, pH to 8 is adjusted, it is spare as water phase；The oil is added
Enter in water phase, carry out ice-bath ultrasonic 8min under 60% power, forms colostric fluid；The colostric fluid is continued to stir at room temperature
2h；Treated lotion is subjected to 35 DEG C of vacuum rotary steams processing, removes organic solvent, at 22000r/min (34700 × g), 4
It is centrifuged 30min under the conditions of DEG C, removes supernatant, precipitating plus appropriate ultrapure water redissolve to obtain Cabazitaxel protein nano material；It will card
Freeze drying protectant is added in Ba Tasai protein nano material, is placed in freeze dryer, pre-freeze 3h under the conditions of -70 DEG C, then with 0.15
DEG C/min is warming up to -45 DEG C, 0.5h is kept at this temperature, then be warming up to -30 DEG C with 0.15 DEG C/min, keep at this temperature
10h continues to be warming up to -20 DEG C with 0.15 DEG C/min, after keeping 10h at this temperature, is warming up to 0 DEG C with 0.2 DEG C/min, keeps
5h continues to be warming up to 25 DEG C with 0.5 DEG C/min, sampling to get.
Embodiment 8
Cabazitaxel protein nano injection, including following raw material:
Cabazitaxel 100mg；Cholesterol 100mg；Human serum albumins 0.4g (concentration 0.4%)；Trehalose 80mg.
Preparation process:
Cabazitaxel and cholesterol are dissolved in organic solvent (chloroform: dehydrated alcohol=11:1, total 10ml), made
It is mutually spare for oil；Human serum albumins is dissolved in ultrapure water, pH to 8 is adjusted, it is spare as water phase；The oil is added
Enter in water phase, carry out ice-bath ultrasonic 8min under 60% power, forms colostric fluid；The colostric fluid is continued to stir at room temperature
2h；Treated lotion is subjected to 35 DEG C of vacuum rotary steams processing, removes organic solvent, at 22000r/min (34700 × g), 4
It is centrifuged 30min under the conditions of DEG C, removes supernatant, precipitating plus appropriate ultrapure water redissolve to obtain Cabazitaxel protein nano material；It will card
Freeze drying protectant is added in Ba Tasai protein nano material, is placed in freeze dryer, pre-freeze 3h under the conditions of -70 DEG C, then with 0.15
DEG C/min is warming up to -45 DEG C, 0.5h is kept at this temperature, then be warming up to -30 DEG C with 0.15 DEG C/min, keep at this temperature
10h continues to be warming up to -20 DEG C with 0.15 DEG C/min, after keeping 10h at this temperature, is warming up to 0 DEG C with 0.2 DEG C/min, keeps
5h continues to be warming up to 25 DEG C with 0.5 DEG C/min, sampling to get.
Embodiment 9
Cabazitaxel protein nano injection, including following raw material:
Cabazitaxel 100mg；Cholesterol 100mg；Human serum albumins 0.45g (concentration 0.45%)；Trehalose 80mg.
Preparation process:
Cabazitaxel and cholesterol are dissolved in organic solvent (chloroform: dehydrated alcohol=11:1, total 10ml), made
It is mutually spare for oil；Human serum albumins is dissolved in ultrapure water, pH to 8 is adjusted, it is spare as water phase；The oil is added
Enter in water phase, carry out ice-bath ultrasonic 8min under 60% power, forms colostric fluid；The colostric fluid is continued to stir at room temperature
2h；Treated lotion is subjected to 35 DEG C of vacuum rotary steams processing, removes organic solvent, at 22000r/min (34700 × g), 4
It is centrifuged 30min under the conditions of DEG C, removes supernatant, precipitating plus appropriate ultrapure water redissolve to obtain Cabazitaxel protein nano material；It will card
Freeze drying protectant is added in Ba Tasai protein nano material, is placed in freeze dryer, pre-freeze 3h under the conditions of -70 DEG C, then with 0.15
DEG C/min is warming up to -45 DEG C, 0.5h is kept at this temperature, then be warming up to -30 DEG C with 0.15 DEG C/min, keep at this temperature
10h continues to be warming up to -20 DEG C with 0.15 DEG C/min, after keeping 10h at this temperature, is warming up to 0 DEG C with 0.2 DEG C/min, keeps
5h continues to be warming up to 25 DEG C with 0.5 DEG C/min, sampling to get.
Cabazitaxel protein nano material obtained by embodiment 1-9 is subjected to partial size, encapsulation rate index is investigated, gained knot
Fruit is as shown in table 1:
Table 1
| Cabazitaxel | Albumin | Cholesterol | Partial size | Encapsulation rate | |
| Embodiment 1 | 100mg | 1.5g | 150mg | 145nm | 87.82% |
| Embodiment 2 | 150mg | 1.5g | 200mg | 180.2nm | 84.61% |
| Embodiment 3 | 100mg | 2.0g | 100mg | 141nm | 94.75% |
| Embodiment 4 | 100mg | 2.0g | 100mg | 122nm | 94.75% |
| Embodiment 5 | 50mg | 1.0g | 100mg | 128nm | 91.57% |
| Embodiment 6 | 100mg | 1.5g | 100mg | 156nm | 89.31% |
| Embodiment 7 | 100mg | 1.5g | 100mg | 156nm | 89.31% |
| Embodiment 8 | 100mg | 0.4g | 100mg | 105nm | 73.22% |
| Embodiment 9 | 100mg | 0.45g | 100mg | 117nm | 79.53% |
To sum up, as shown in Table 1, Cabazitaxel protein nano material is successfully made in 1-9 of the embodiment of the present invention.And embodiment
The carrier material of 1-2 is bovine serum albumin(BSA), and the carrier material of embodiment 3-9 is human serum albumins, can speculate human serum
Albumin is better than bovine serum albumin(BSA) to the Drug loading capacity of Cabazitaxel；In addition, embodiment 1-9 partial size is solid with main ingredient dosage, gallbladder
The increase of alcohol dosage and carrier material and increase, illustrate that ultrasonic power, ultrasonic time and homogenization pressure have a fixing to partial size
It rings, but influences smaller.Meanwhile load medicine ratio, albumin concentration, cholesterol have larger impact to encapsulation rate, carry medicine ratio and increase to one
After certainty ratio, encapsulation rate is substantially reduced.
In addition, being carried out respectively to cholesterol, egg yolk lecithin, soybean lecithin during the test of protein nano decorative material
Screening, egg yolk lecithin and soybean lecithin are bad as decorative material effect as the result is shown, and lotion colour changed into yellow has been precipitated for 24 hours
Precipitating.Therefore following all examples is the preferred preparation parameter of further research Cabazitaxel protein nano material, is selected
Cholesterol is as protein nano decorative material.
Further, by Cabazitaxel protein nano injection obtained by embodiment 1-9, (injection Cabazitaxel protein nano freezes
Dry powder) the variation detection of encapsulation rate index is carried out, measured as shown in table 2:
Table 2
| Cabazitaxel | Albumin | Cholesterol | Freeze drying protectant | Encapsulation rate | |
| Embodiment 1 | 100mg | 1.5g | 150mg | 12mg Sodium Caprylate | 74.60% |
| Embodiment 2 | 150mg | 1.5g | 200mg | 12mg Sodium Caprylate | 76.15% |
| Embodiment 3 | 100mg | 2.0g | 100mg | 16mg Sodium Caprylate | 77.50% |
| Embodiment 4 | 100mg | 2.0g | 100mg | 16mg Sodium Caprylate | 89.92% |
| Embodiment 5 | 50mg | 1.0g | 100mg | 100mg trehalose | 82.41% |
| Embodiment 6 | 100mg | 1.5g | 100mg | 60mg mannitol | 78.59% |
| Embodiment 7 | 100mg | 1.5g | 100mg | 80mg trehalose | 80.11% |
| Embodiment 8 | 100mg | 0.4g | 100mg | 80mg trehalose | 65.16% |
| Embodiment 9 | 100mg | 0.45g | 100mg | 80mg trehalose | 69.19% |
Further, Cabazitaxel protein nano injection obtained by embodiment 1-9 is subjected to preliminary quality evaluation, partial size point
Cloth is as shown in Figure 1, Zeta potential between -10mv~-40mv, is placed 30 days at 4 DEG C, and appearance is that white loose is blocky, specifically
As a result as shown in table 3 below.
Table 3
| Redispersibility | Average grain diameter | Zeta potential | Encapsulation rate | |
| Embodiment 1 | It is good | 158nm | -24.4mv | 73.51% |
| Embodiment 2 | It is excellent | 203nm | -26.9mv | 76.38% |
| Embodiment 3 | It is excellent | 167nm | -14.1mv | 77.62% |
| Embodiment 4 | It is excellent | 137nm | -13.4mv | 87.33% |
| Embodiment 5 | It is excellent | 143nm | -15.8mv | 82.33% |
| Embodiment 6 | It is excellent | 169nm | -16.3mv | 77.65% |
| Embodiment 7 | It is excellent | 163nm | -16.7mv | 79.13% |
| Embodiment 8 | It is excellent | 119nm | -15.2mv | 63.58% |
| Embodiment 9 | It is excellent | 131nm | -16.7mv | 70.23% |
To sum up, it can be seen that from table 2-3, Cabazitaxel protein nano injection (injection card can be increased by freeze-drying
Ba Tasai protein nano freeze-dried powder) stability, but the processes such as the pre-freeze phase of freeze-drying process, cycle in time can make protein nano
Performance is lost, and therefore, encapsulation rate is declined slightly.The result shows that, placed 30 days at 4 DEG C, Cabazitaxel egg through actually detected
Redispersibility is good in 5mL ultrapure water for white nano injection agent (injection Cabazitaxel protein nano freeze-dried powder), wraps in 30 days
Envelope rate is increased without significant change, partial size, but still less than 220nm.That is Cabazitaxel protein nano obtained by the embodiment of the present invention
Injection (injection Cabazitaxel protein nano freeze-dried powder) is under the conditions of 4 DEG C, 30 days Storage period outers, redispersibility, grains
Diameter, encapsulation rate have good stability.
Further, Cabazitaxel protein nano injection is made in above-described embodiment 2 and carries out body in phosphate buffer
Outer release test.It prepares the phosphate buffer 1 00mL (containing 5% ethyl alcohol) that pH is 6.5 and is used as dissolution medium, then take certain
Cabazitaxel protein nano material is measured in the bag filter that molecular cut off is 2000, places it in above-mentioned 6.5 phosphate of pH
In buffer release medium, using constant temperature oscillation method, respectively at 0.5h, 1h, 4h, 6h, 8h, 12h, for 24 hours, 48h sample 2mL,
Fluid infusion 2mL calculates Accumulation dissolution, and it is as shown in Figure 2 to draw release profiles.Show free Cabazitaxel and carries medicine protein nano
The difference of release rule in 6.5 phosphate buffer dissolution medium of pH.
Further, Cabazitaxel protein nano injection is made in above-described embodiment 2 and carries out cytotoxicity experiment test.It takes
The source of mouse breast cancer 4T1 cell of logarithmic growth phase, is diluted postdigestive cell suspension according to 2000, every hole cell.
In 96 orifice plate inoculating cell suspensions, every hole 100uL is cultivated 24 hours as in cell incubator.Configuring drug concentration is
The Cabazitaxel protein nano solution of 100ug/mL makes the drug concentration point for carrying medicine protein nano group using decimal dilution method
Not Wei 100ug/mL, 10ug/mL, 1ug/mL, 0.1ug/mL, 0.01ug/mL, control group be corresponding free Cabazitaxel
The solution of concentration, blank group are that (for the protein nano that main ingredient is not added, remaining forms and carries medicine protein nano blank protein nano
It is identical, select bovine serum albumin(BSA)), compound concentration is the blank protein nano solution of 1000ug/mL, is diluted using ten times
Method makes the drug concentration of blank protein nano group be respectively 1000ug/mL, 100ug/mL, 10ug/mL, 1ug/mL, 0.1ug/
mL.Every hole is added 100uL, parallel 3 hole of each concentration, cultivated in cell incubator for 24 hours, 48h, 72h.It is every after culture
10uL CCK-8 (Cell Counting Kit-8) solution is added in hole, and 2h is cultivated in cell incubator, is measured with microplate reader
Absorption value at 450nm, calculates its cell survival rate, and cell survival rate trend is as shown in Figure 3-4.The result shows that one
Determine in concentration range, blank protein nano is non-toxic, and the anti-source of mouse of Cabazitaxel protein nano material (KBTS-BSANPs)
The effect of breast cancer cell is obvious.
To sum up, a kind of Cabazitaxel protein nano injection provided in an embodiment of the present invention, by Cabazitaxel, protein carrier
Material, protein nano decorative material and freeze drying protectant composition, i.e., by by Cabazitaxel be encapsulated in protein carrier material and
In the spherical shape or spheroid shape nanometer of protein nano decorative material composition or it is attached to Nanosurface, and is carried out with freeze drying protectant
Frozen dried, gained Cabazitaxel protein nano injection have preferable slow release effect, while antitumor effect with higher
Fruit effectively prevents the use bring hypersensitivity of the cosolvent such as surfactant, reduces formulations toxic, improves biology benefit
Expenditure reduces dosage, extends administration time.
Further, the present invention found in preliminary experiment albumin concentration difference, to entrapment efficiency and nano shape have compared with
Big to influence, excessive concentration, nano particle is easy aggregation, and partial size increases.For the best albumin concentration of determination, contrived experiment scheme
It is as follows:
This experiment fixed main ingredient (Cabazitaxel) and albumin (bovine serum albumin(BSA)) ratio (1:20), albumin solution pH
It is 7.0, grease Phase Proportion is 1:20, cholesterol dosage 150mg, and Probe Ultrasonic Searching (30%) 10min, high-pressure homogeneous condition is constant,
Three batches of samples of preparation in parallel, with encapsulation rate (EE%), drugloading rate (DL%) for main inspection target, measurement result such as 4 institute of table
Show.
The selection of 4 albumin of table (BSA) solution concentration
To sum up, as seen from the results in Table 4, when BSA solution concentration is 1.5%, encapsulation rate is more satisfactory.Therefore preferably 1.5%
BSA concentration prepares Cabazitaxel protein nano.
Further, the present invention has found albumin isoelectric point is 4.4 or so, and albumin solution pH is closer etc. in preliminary experiment
Electric, albumin, which is easily assembled, agglomerating to be not easy to form protein nano.For the best albumin solution pH value of determination, contrived experiment scheme
It is as follows:
Fixed BSA concentration is 1.5%, and other conditions are constant, with the citric acid solution of 0.2mol/L and the hydrogen of 0.2mol/L
Sodium hydroxide solution, the pH for adjusting BSA solution is respectively 6.0,7.0,8.0,9.0,10.0, other preparation conditions are same as above, in each pH
Under the conditions of prepare three batches of samples, with encapsulation rate, drugloading rate for main inspection target, select optimal ph, measurement result is shown in Table 5.
The selection of 5 BSA pH value of solution of table
To sum up, as seen from the results in Table 5, Cabazitaxel protein nano material, pH 6.0 is successfully made when pH value is 7~10
When, BSA conglomerate, and pH is bigger, nanoparticle partial size has reduction trend, and when pH > 8.0, encapsulation rate, drugloading rate are obvious
It reduces, it is therefore preferable that the BSA solution of pH=8.0 prepares Cabazitaxel protein nano material.
Further, the present invention is to determine the ratio between optimal coagulant dose and albumin (carrying medicine ratio), designs following experimental program:
Fixed aqueous phase solution pH is 8.0, and albumin solution concentration is 1.5%, other preparation conditions are same as above, by KBTS (card
It is Ba Tasai) that 1:20,1:13.3,1:10,1:8,1:6.67 offer medicine with BSA ratio, prepares three batches of samples, in parallel with packet
Envelope rate, drugloading rate partial size, current potential are inspection target, and measurement result is shown in Table 6.
The selection of the load medicine ratio of table 6
To sum up, as known from Table 6, with the increase for carrying medicine ratio, nanoparticle partial size is significantly increased, comprehensive consideration its to encapsulating
The influence of rate, drugloading rate, partial size, although the load medicine of 1:20, than corresponding encapsulation rate highest, encapsulation rate is not to investigate
Single factor, and drugloading rate is increased to 6.14% from 4.07% by the load medicine ratio of 1:10, it is therefore preferable that the load medicine ratio of 1:10, is obtained
The protein nano obtained has compared with high encapsulation rate and drugloading rate KBTS, can avoid the waste of drug.
It finds that the addition of cholesterol facilitates KBTS and stablizes in conjunction with albumin in preliminary experiment, has to entrapment efficiency bright
It is aobvious to improve.Therefore, the present invention further investigates cholesterol dosage to Cabazitaxel protein nano material encapsulation rate and drugloading rate
It influencing, that is, fixes other preparation conditions, loads medicine ratio is 1:10, and change cholesterol dosage is respectively 100mg, 150mg, 200mg,
250 mg, 300mg prepare three batches of samples under each variable respectively, and measurement result is shown in Table 7.
The selection of 7 cholesterol dosage of table
As seen from the results in Table 7, cholesterol dosage has larger impact to the encapsulation rate of Cabazitaxel protein nano material, works as gallbladder
Sterol dosage has maximum encapsulation rate when increasing to 200mg, continue growing cholesterol dosage, encapsulation rate is without increase, nanoparticle partial size
Have and slightly increase, therefore preferably cholesterol dosage is 200mg (0.2%).
Further, the present invention is to determine the use of best organic oil phase, is related to following experimental program:
Organic oil is mutually respectively chloroform, chloroform-dehydrated alcohol volume ratio 9:1, chloroform-dehydrated alcohol body
For product than 11:1, chloroform-dehydrated alcohol volume ratio 13:1, fixed each organic oil phase and water phase volume ratio are 1:20, each to prepare
Three batches of samples, measurement result are shown in Table 8.
The selection of 8 organic oil phase of table
It is shown from 8 experimental result of table, when selecting chloroform as oily phase, the protein nano grain partial size of preparation is maximum.
With the increase of dehydrated alcohol in oily phase, nanoparticle partial size is obviously reduced, comprehensive consideration its to entrapment efficiency, drugloading rate
It influences, entrapment efficiency, drugloading rate equal highest when selecting chloroform-dehydrated alcohol volume ratio 11:1, and partial size is relatively
It is small.
Previous experiments are the results show that grease Phase Proportion (ratio of organic solvent and pure water) influences relatively entrapment efficiency
It is smaller, there is larger impact to nanoparticle partial size.Further determine that optimum oil watr-proportion, i.e., fixed to carry medicine ratio 1:10, other systems
Standby condition is constant, changes grease Phase Proportion, prepares three batches of samples under the conditions of each ratio, investigates using nanoparticle partial size as main
Index, measurement result are shown in Table 9.
The selection of 9 grease Phase Proportion of table
Table 9 has one to nanometer particle size the results show that grease Phase Proportion is relatively small on entrapment efficiency, drugloading rate influence
Fixing is rung, and there is minimum grain size in when grease Phase Proportion 1:10, and encapsulation rate, drugloading rate are also higher, therefore selects grease Phase Proportion 1:10
As the preparation condition of protein nano grain, while the use of organic oil phase can be reduced.
Preliminary result of the present invention shows that ultrasonic power has certain influence to nanoparticle partial size, to encapsulation rate, drugloading rate shadow
Sound is unobvious.For the best ultrasonic power of determination, the present invention designs following experimental program:
Fixed other conditions are constant, setting ultrasonic power (general power 650W) is respectively 20%, 30%, 40%, 50%,
60%, three batches of samples are prepared respectively under each power condition, and with partial size, Zeta potential for main inspection target, measurement result is shown in Table
10。
The selection of 10 ultrasonic power of table
As seen from the results in Table 10, with the increase of ultrasonic power, nanoparticle partial size has reduction trend, when power is more than
40%, partial size reduction is unobvious, and power is bigger, acting of the kinetic energy to protein nano, it is possible to cause nanoparticle to be assembled, sample
A possibility that product are contaminated by heavy metals is also bigger.Therefore preferably ultrasonic power is the 40% of equipment general power, i.e. 260W.
Further, preliminary experiment early period investigation show that ultrasonic time length has certain influence to nanoparticle partial size, to encapsulating
Rate, the influence of drugloading rate are unobvious.Therefore the ultrasonic power of selection 40%, it is constant to fix other preparation conditions, when setting ultrasound
Between be respectively 3min, 5min, 8min, 10min, 12min, three batches of samples are prepared respectively under the conditions of each, using partial size as mainly examining
Index is examined, measurement result is shown in Table 11.
The selection of 11 ultrasonic time of table
As shown in Table 11, with the increase of ultrasonic time, partial size is on a declining curve, and when ultrasound is more than 8min, albumen is received
It is unobvious that grain of rice diameter reduces trend, therefore selects ultrasonic time for 8min, prepares protein nano colostrum.
By the above single factor exploration result it is found that in conjunction with bovine serum albumin(BSA) to the encapsulation rate, drugloading rate and egg of KBTS
The partial size of white nanometer, Zeta potential etc. comprehensive consideration determine that Cabazitaxel protein nano material forms are as follows: albumin is dense
Degree 1.5%, albumin solution pH are 8.0, and loads medicine ratio is 1:10, and cholesterol dosage 200mg (0.2%, quality and volume ratio w/
V, prescription dosage are formed into 100ml preparation), chloroform and dehydrated alcohol volume ratio 11:1, grease Phase Proportion 1:10 should
Composition is in ultrasonic power 40%, ultrasonic time 8min, homogeneous 8 circulations, the protein nano encapsulation rate of preparation under 500bar
Good, drugloading rate is high, and partial size is relatively small.
It further, is further optimization Cabazitaxel protein nano material composition, according to single factor experiment as a result, BSA
The selection (C) of concentration (A), cholesterol dosage (B) and oily phase is to influence the principal element of encapsulation rate (EE%)；With its difference
Dosage carries out the optimization of 3 factor, 3 horizontal respone face contrived experiment.Using the Box-Behnken in Design-Export software
Design, abbreviation BBD and the common experimental design method of response surface optimization method, the basic, normal, high experimental level of each factor
Be separately encoded is -1,0,1.This experiment is chosen encapsulation rate (EE%) and is worth in response, keeps the amount of main ingredient KBTS in each prescription to be
150mg, protein solution pH value are 8.0, and oil is mutually the mixed solvent of chloroform and dehydrated alcohol, and grease Phase Proportion is 1:10,
Ultrasonic power 40%, ultrasonic time 8min, homogenization pressure 500bar carry out 8 circulations.
Establish the horizontal model of fit of 3 factor 3 using Design-Expert 8.0.6 software, in experiment factor and it is horizontal such as
Table 12.
12 model of fit design factor of table and coding
Experimental design is carried out with Design-Expert 8.0.6, totally 17 groups of compositions, prepares Cabazitaxel protein nano
Material and the encapsulation rate (being shown in Table 13) and response surface optimization variance point for measuring Cabazitaxel protein nano material obtained by each prescription
It analyses result (being shown in Table 14).
13 contrived experiment result of table
14 response surface optimization variance analysis of table
" * * * " indicates p<0.0001, and " * " indicates p<0.05, and " * " indicates p>0.05.
Table data are analyzed by software, discovery quadratic polynomial is optimal models, fit equation EE%=
85.45+3.51A+3.58B+3.57C+2.27AB+2.44AC+2.81BC-9.52A2-9.28B2-10.53C2(R2=0.9865；
P=0.0001, it is significant；Lose quasi- item: P=0.9305, it is not intended to justice).There were significant differences for fit equation model (P < 0.05), loses
Intend item without significant difference (P > 0.05), show that 98.65% experimental result can be explained with the model of fit, there is statistics meaning
Justice.
The reciprocation, BSA concentration (A) and oily phase of the BSA concentration (A) that regression equation obtains and cholesterol dosage (B)
Select (C) reciprocation and cholesterol dosage (B) with oil phase selection (C) reciprocation to entrapment efficiency
(EE%) Fig. 5-Fig. 7 is shown in influence.According to Design-Expert 8.0.6 the results of analysis of variance, the optimal place of forecast of regression model
Side is BSA concentration 1.58%, and cholesterol dosage 195mg, oil is accordingly chloroform: dehydrated alcohol=11.3:1 (v/v), model
Predict encapsulation rate 84.60%.
The present invention further using human serum albumins (HSA) as research object, has equally carried out following research:
Since in preliminary experiment, albumin concentration is different, there are larger impact, concentration mistake to entrapment efficiency and nano shape
Height, nano particle are easy aggregation, and partial size increases.The fixed main ingredient of this experiment and HSA ratio (1:20), the dosage of cholesterol are
10mg, HSA pH value of solution are 7.0, and organic solvent is the mixed solution (11:1) of chloroform and dehydrated alcohol, and other conditions are protected
Hold it is constant, using encapsulation rate, the two factors of partial size as main inspection target, measurement result such as table 15.
The selection of 15 HSA solution concentration of table
As shown in Table 15, with the increase of human serum albumin solution's concentration, partial size is gradually increased, and concentration is wrapped when being 2%
Envelope rate is maximum, comprehensively considers, and 2%HSA solution is selected to prepare KBTS-HSA-NPS。
Albumin isoelectric point is 4.4 or so, and for albumin solution pH closer to isoelectric point, albumin, which is easily assembled, agglomerating is not easy shape
At protein nano.HSA solution concentration is fixed as 2%, keeps remaining condition constant, with the citric acid solution of 0.2mol/L and
The sodium hydroxide solution of 0.2 mol/L, respectively adjust HSA solution pH value to 5.0,6.0,7.0,8.0,9.0 this five value,
Other preparation conditions are same as above, with supersound method prepare KBTS-HSA-NPs, using encapsulation rate, two factors of particle size as
Main inspection target determines that the optimal value of pH, measurement result are shown in Table 16.
As can be seen from Table 16, with the increase of pH value, the partial size of albumin nano granular reduces therewith, and pH value is white when being 7
The encapsulation rate of protein nano particle is relatively large, comprehensively considers, and pH value is selected to prepare KBTS-HSA- for 7 HSA solution
NPs。
The selection of 16 HSA pH value of solution of table
The pH of fixed HSA solution is 7.0, and concentration 2%, other preparation conditions are same as above, by KBTS and HSA usage ratio
It offers medicine for 1:8,1:10,1:13.3,1:20, while still being tied using two encapsulation rate, partial size factors as inspection target
Fruit is listed in table 17 in detail.
The selection of the load medicine ratio of table 17
Can see by 17 result of table, the partial size of albumin nano granular increase with the increase for carrying medicine ratio but amplitude compared with
It is small, it is more maximum than the encapsulation rate of the nanoparticle in 1:10 to carry medicine.Comprehensively consider, load medicine ratio is 1:10, the human seralbumin being prepared
Protein nano particle can avoid the waste of drug to KBTS encapsulation rate with higher.
In preliminary experiment, the addition of cholesterol facilitates Cabazitaxel and stablizes in conjunction with albumin.Investigate cholesterol dosage pair
The influence of KBTS-HSA-NPs encapsulation rate, the concentration of fixed HSA solution are 2%, pH 7, and load medicine ratio is 1:10, other conditions
Constant, changing cholesterol dosage is respectively 0mg, 10mg, 20mg, 30mg, still using two encapsulation rate, partial size factors as examining
Index is examined, measurement result is shown in Table 18.
As shown in Table 18, cholesterol dosage is in inverse ratio with the partial size of KBTS-HSA-NPs, when cholesterol dosage is 10mg
When, the encapsulation rate of albumin nanoparticle is larger, and when cholesterol dosage is 0mg, encapsulation rate is smaller, dosage 20mg, 30 mg
When, the encapsulation rate difference of protein nano grain is little, and therefore, final choice 10mg cholesterol is to prepare albumin nano granular.
The selection of 18 cholesterol dosage of table
In terms of prescription screening, by single factor exploration, choosing influences significant four items to albumin nano graininess matter
Part: HSA concentration (A), the pH value (C) of HSA, carries medicine ratio (D) for investigation factor, with encapsulation rate (EE%) at cholesterol dosage (B)
As evaluation index, KBTS-HSA-NP is optimized using the horizontal box-behnken contrived experiment of four factor threeSPrescription.
Box-Behnken Design in Design-Export software, abbreviation BBD are in response to the common experimental design of face optimization
Method, it is -1,0,1 that the basic, normal, high experimental level of each factor, which is separately encoded,.It the results are shown in Table 19, be then described in detail in table 20
Formulation optimization experimental design, table 21 list the result of variance analysis.
19 model of fit design factor of table and coding
Experimental design is carried out with Design-Expert 8.0.6, totally 29 groups of compositions, prepares KBTS-HSA-NPSAnd it surveys
Fixed each resulting encapsulation rate of prescription, the results are shown in Table 20.
The experimental design table and result of 20 optimization formulation of table
The variance analysis of 21 response surface optimization of table
Note: *≤0.0001 shows result highly significant；* < 0.05, show that result is more significant.
Formulation optimization test the data obtained is handled, multiple linear is carried out to each factor using encapsulation rate as evaluation index
It returns and binomial equation fitting, discovery quadratic polynomial is optimal models.Equation is EE%=90.10+2.48A+1.89B
+2.04C-1.51D+2.03AB-3.31AC-3.15AD-2.26BC+0.37BD+1.53CD-7.2 2A2-7.57B2-4.91C2-
8.19D2(R2=0.9771；P=0.0001；Lose quasi- item: P=0.1785 > 0.05).Variance analysis shows that P value is 0.0001,
Far smaller than 0.05, show that this model reliability is higher；The R of equation2Value is 0.9771, illustrates to have 97.71% data available
The model explanation；Losing analog values is 0.1785, is greater than 0.05, loses analog values and does not also show the good predictability of model significantly, has
It is statistically significant.To sum up, our regression equation is higher to the degree of fitting of experimental data, can determine KBTS- with this model
The optimal prescription of HSA-NPs.
In order to more intuitively observe 4 factors: HSA concentration (A), cholesterol dosage (B), the pH (C) of HSA, load medicine ratio
(D) the effect of influencing each other draws response surface curve graph and carrys out vivider explanation, as shown in Figure 8.Response surface is by fixing four
Any two factor in factor, ordinate indicate the size of response, the two orthogonal abscissa tables on horizontal plane
Show that other two factor, three reference axis collectively form the threedimensional model of response surface.
It is shown by Fig. 8, encapsulation rate (%) increases with HSA concentration and cholesterol dosage and increased, and reaches after a peak value again
It reduces.Fig. 8 is directed to KBTS-HSA-NPSFour factors of prescription, encapsulation rate have similar variation tendency.It is provided from Fig. 8
Three-dimensional response curve can be seen that when the value of tetra- factors of A, B, C, D is in intermediate range, respond surface curve in Y-axis
There is peak value in direction, shows that influence of tetra- factors of A, B, C, D to encapsulation rate at this time is more significant.
The software also gives prescription optimal selection simultaneously: HSA solution concentration is 2.2%, and cholesterol dosage is 21.4mg,
PH value is 7.1, and load medicine ratio is 1:10.4.Under this condition, forecast of regression model value is encapsulation rate 90.65%.
In order to which the accuracy predicted response surface model carries out experimental verification, carry out KBTS-HSA-NPs's under this condition
Preparation, the encapsulation rate for measuring HSA nano particle is 88.70%, it may be possible to which albumin is easy to foaming during the preparation process, will lead to
Certain error.So obtained actual value and theoretical value have the difference of a bit, but use the KBTS- of optimal conditions preparation
HSA-NPs encapsulation rate is still higher than the encapsulation rate under single factor test screening conditions, therefore the optimal conditions is feasible reliable.Partial size is
153.2nm, PDI 0.178, the results showed that nano particle distribution is good, the visible Fig. 9 of particle diameter distribution.
In such optimised conditions, continuous 3 crowdes of KBTS-HSA-NPs of preparation carry out encapsulation rate and particle size determination, as a result such as table 22
Shown, human serum albumins is 88.70% to the encapsulation rate of Cabazitaxel drug, partial size 148.9nm.
22 KBTS-HSA-NPs measurement result of table
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to of the invention
Protection scope.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. a kind of Cabazitaxel protein nano injection, which is characterized in that the raw material including following parts by weight:
50~150 parts of Cabazitaxel；400~4000 parts of protein carrier material；50~300 parts of protein nano decorative material；Freeze-drying
12~100 parts of protective agent.
2. Cabazitaxel protein nano injection according to claim 1, which is characterized in that the freeze drying protectant includes
One or more of bovine serum albumin(BSA), mannitol, trehalose, Sodium Caprylate, lactose, acetyl-l-tryptophan sodium.
3. Cabazitaxel protein nano injection according to claim 1, which is characterized in that the protein carrier material packet
Include one or more of bovine serum albumin(BSA), human serum albumins, zeins.
4. Cabazitaxel protein nano injection according to claim 1, which is characterized in that the protein nano modifies material
Material includes one of soybean lecithin, hydrogenated soya phosphatide, cholesterol, egg yolk lecithin, natural phospholipid and phosphatidyl-ethanolamine
Or it is several.
5. Cabazitaxel protein nano injection according to claim 1, which is characterized in that the Cabazitaxel and albumen
The weight ratio of carrier material is 1:10.
6. a kind of preparation method of Cabazitaxel protein nano injection characterized by comprising
Cabazitaxel and protein nano decorative material are weighed according to quantity, are dissolved in organic solvent, it is spare as oily phase；
Protein carrier material is weighed according to quantity, is dissolved in pure water, and adjusting pH value is spare as water phase to 7~10；
The oil is mutually subjected to ice-bath ultrasonic process with water phase, forms colostric fluid, ultrasonic power 20%~60%, ultrasonic time 3
~12min；
The colostric fluid is continued into stirring 1~4h at room temperature or it is 500~800bar in pressure by high pressure homogenizer
Under the conditions of, it recycles 6~10 times；
By treated, lotion carries out vacuum rotary steam processing, organic solvent is removed, in 22000r/min (34700 × g), 4 DEG C of items
It is centrifuged 30min under part, removes supernatant, precipitating plus appropriate ultrapure water redissolve up to Cabazitaxel protein nano material；
Freeze drying protectant is weighed according to quantity, and the freeze drying protectant and Cabazitaxel protein nano material are placed in freeze dryer
In, carry out sublevel segmentation cooling frozen dried to get.
7. the preparation method of Cabazitaxel protein nano injection according to claim 6, which is characterized in that the sublevel
Segmentation cooling frozen dried mode are as follows:
2~4h of pre-freeze under the conditions of -75 DEG C~-65 DEG C, then -50 DEG C~-40 DEG C, and constant temperature are warming up to 0.1~0.2 DEG C/min
After keeping 0.5~1h, then with 0.1~0.2 DEG C DEG C/min be warming up to -35~-25 DEG C, and after constant temperature keeps 9~11h, continue with
After 0.1~0.2 DEG C/min is warming up to -20 DEG C~-15 DEG C, and constant temperature keeps 9~11h, 0 is warming up to 0.2~0.3 DEG C/min
DEG C, keep 4~6h；Continue to be warming up to 20 DEG C~30 DEG C with 0.5~0.6 DEG C/min, sampling.
8. the preparation method of Cabazitaxel protein nano injection according to claim 6, which is characterized in that described organic
Solvent is chloroform and dehydrated alcohol mixed liquor, and the volume ratio of the chloroform and dehydrated alcohol is 11:1.
9. the preparation method of Cabazitaxel protein nano injection according to claim 6, which is characterized in that the ultrasound
Power is 40%, ultrasonic time 8min.
10. the preparation method of Cabazitaxel protein nano injection according to claim 6, which is characterized in that the pH
Value is 8.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111449055A (en) * | 2020-05-23 | 2020-07-28 | 河北康腾生物科技有限公司 | Adipose tissue cryopreservation liquid, preparation method and adipose tissue storage method |
| CN117205305A (en) * | 2018-04-20 | 2023-12-12 | 珠海贝海生物技术有限公司 | Formulations and compositions of cabazitaxel |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101816630A (en) * | 2009-02-26 | 2010-09-01 | 重庆医科大学 | Uricase lipid nanoparticle and preparation method thereof |
| CN103393632A (en) * | 2013-07-26 | 2013-11-20 | 齐鲁制药有限公司 | Cabazitaxel drug composition and preparation method thereof |
| CN103976955A (en) * | 2014-04-21 | 2014-08-13 | 贵州中泰生物科技有限公司 | Human albumin nano-particle and preparation method thereof |
| CN104490797A (en) * | 2014-12-22 | 2015-04-08 | 深圳海王药业有限公司 | Multiphase-stable albumin conjunction type cabazitaxel |
| CN105727303A (en) * | 2014-12-12 | 2016-07-06 | 四川科伦药物研究院有限公司 | Albumin composition highly-carrying cabazitaxel medicine, preparation and preparation method thereof |
| CN106176631A (en) * | 2016-08-12 | 2016-12-07 | 北京蓝贝望生物医药科技股份有限公司 | The freeze-dried composition of antitumor |
| US20170020817A1 (en) * | 2013-12-19 | 2017-01-26 | Luminus Biosciences, Inc. | Solid nanoparticle formulation of microtuble inhibitors with reduced ostwald repening for oral administration |
| CN106580880A (en) * | 2016-12-22 | 2017-04-26 | 深圳海王医药科技研究院有限公司 | Stable cabazitaxel particle redispersible system |
| US20190110993A1 (en) * | 2017-09-12 | 2019-04-18 | Raj Selvaraj | Solid Nanoparticle Formulation of Water Insoluble Pharmaceutical Substances with Reduced Ostwald Ripening |
-
2019
- 2019-06-03 CN CN201910478565.3A patent/CN110075073B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101816630A (en) * | 2009-02-26 | 2010-09-01 | 重庆医科大学 | Uricase lipid nanoparticle and preparation method thereof |
| CN103393632A (en) * | 2013-07-26 | 2013-11-20 | 齐鲁制药有限公司 | Cabazitaxel drug composition and preparation method thereof |
| US20170020817A1 (en) * | 2013-12-19 | 2017-01-26 | Luminus Biosciences, Inc. | Solid nanoparticle formulation of microtuble inhibitors with reduced ostwald repening for oral administration |
| CN103976955A (en) * | 2014-04-21 | 2014-08-13 | 贵州中泰生物科技有限公司 | Human albumin nano-particle and preparation method thereof |
| CN105727303A (en) * | 2014-12-12 | 2016-07-06 | 四川科伦药物研究院有限公司 | Albumin composition highly-carrying cabazitaxel medicine, preparation and preparation method thereof |
| CN104490797A (en) * | 2014-12-22 | 2015-04-08 | 深圳海王药业有限公司 | Multiphase-stable albumin conjunction type cabazitaxel |
| CN106176631A (en) * | 2016-08-12 | 2016-12-07 | 北京蓝贝望生物医药科技股份有限公司 | The freeze-dried composition of antitumor |
| CN106580880A (en) * | 2016-12-22 | 2017-04-26 | 深圳海王医药科技研究院有限公司 | Stable cabazitaxel particle redispersible system |
| US20190110993A1 (en) * | 2017-09-12 | 2019-04-18 | Raj Selvaraj | Solid Nanoparticle Formulation of Water Insoluble Pharmaceutical Substances with Reduced Ostwald Ripening |
Non-Patent Citations (2)
| Title |
|---|
| YANJIE SHAO ET AL.: "Improving cabazitaxel chemical stability in parenteral lipid emulsions using cholesterol", 《EUROPEAN JOURNAL OF PHARMACEUTICAL SCIENCES》 * |
| 曲娜: "卡巴他赛白蛋白纳米粒的制备及抗肿瘤研究", 《中国博士学位论文全文数据库 工程科技Ⅰ辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117205305A (en) * | 2018-04-20 | 2023-12-12 | 珠海贝海生物技术有限公司 | Formulations and compositions of cabazitaxel |
| CN117205305B (en) * | 2018-04-20 | 2024-04-26 | 珠海贝海生物技术有限公司 | Formulations and compositions of cabazitaxel |
| CN111449055A (en) * | 2020-05-23 | 2020-07-28 | 河北康腾生物科技有限公司 | Adipose tissue cryopreservation liquid, preparation method and adipose tissue storage method |
| CN111449055B (en) * | 2020-05-23 | 2021-07-23 | 河北康腾生物科技有限公司 | Adipose tissue cryopreservation liquid, preparation method and adipose tissue storage method |
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