The specific embodiment
Below in conjunction with embodiment the present invention is elaborated.
Embodiment 1, at first, gets PLGA and joins in the dichloromethane, and making concentration is 28mgmL
-1PLGA solution, then with 8,9-single-epoxy brevifolin phenol is dissolved in PBS solution, makes 12mgmL
-1Drug solution; Secondly, according to 1: 7 volume ratio drug solution is joined in the PLGA solution, ultrasonic emulsification makes colostrum; At last; Being 2% PVA aqueous solution with this colostrum and mass concentration joins colostrum in the PVA aqueous solution by 1: 11 volume ratio;, stirred 1.5~2.5 hours with 500r/min again after 2~4 hours with the 1500r/min magnetic agitation, dichloromethane is volatilized; Make microsphere solidify 8,9 single-epoxy brevifolin phenol PLGA microspheres.
Embodiment 2, at first, get PLGA and join in the dichloromethane, and making concentration is 30mgmL
-1PLGA solution, then with 8,9-single-epoxy brevifolin phenol is dissolved in PBS solution, makes 11.5mgmL
-1Drug solution; Secondly, according to 1: 8 volume ratio drug solution is joined in the PLGA solution, ultrasonic emulsification makes colostrum; At last; Being 1.5% PVA aqueous solution with this colostrum and mass concentration joins colostrum in the PVA aqueous solution by 1: 9 volume ratio;, stirred 1.5~2.5 hours with 500r/min again after 2~4 hours with the 1500r/min magnetic agitation, dichloromethane is volatilized; Make microsphere solidify 8,9 single-epoxy brevifolin phenol PLGA microspheres.
Embodiment 3, at first, get PLGA and join in the dichloromethane, and making concentration is 25mgmL
-1PLGA solution, then with 8,9-single-epoxy brevifolin phenol is dissolved in PBS solution, makes 15mgmL
-1Drug solution; Secondly, according to 1: 10 volume ratio drug solution is joined in the PLGA solution, ultrasonic emulsification makes colostrum; At last; Being 1% PVA aqueous solution with this colostrum and mass concentration joins colostrum in the PVA aqueous solution by 1: 8 volume ratio;, stirred 1.5~2.5 hours with 500r/min again after 2~4 hours with the 1500r/min magnetic agitation, dichloromethane is volatilized; Make microsphere solidify 8,9 single-epoxy brevifolin phenol PLGA microspheres.
Embodiment 4, at first, get PLGA and join in the dichloromethane, and making concentration is 32mgmL
-1PLGA solution, then with 8,9-single-epoxy brevifolin phenol is dissolved in PBS solution, makes 10mgmL
-1Drug solution; Secondly, according to 1: 5 volume ratio drug solution is joined in the PLGA solution, ultrasonic emulsification makes colostrum; At last; Being 3% PVA aqueous solution with this colostrum and mass concentration joins colostrum in the PVA aqueous solution by 1: 10 volume ratio;, stirred 1.5~2.5 hours with 500r/min again after 2~4 hours with the 1500r/min magnetic agitation, dichloromethane is volatilized; Make microsphere solidify 8,9 single-epoxy brevifolin phenol PLGA microspheres.
Embodiment 5, at first, get PLGA and join in the dichloromethane, and making concentration is 35mgmL
-1PLGA solution, then with 8,9-single-epoxy brevifolin phenol is dissolved in PBS solution, makes 12.5mgmL
-1Drug solution; Secondly, according to 1: 6 volume ratio drug solution is joined in the PLGA solution, ultrasonic emulsification makes colostrum; At last; Being 2% PVA aqueous solution with this colostrum and mass concentration joins colostrum in the PVA aqueous solution by 1: 12 volume ratio;, stirred 1.5~2.5 hours with 500r/min again after 2~4 hours with the 1500r/min magnetic agitation, dichloromethane is volatilized; Make microsphere solidify 8,9 single-epoxy brevifolin phenol PLGA microspheres.
One, microsphere preparation technology's research
The preparation of microsphere receives influence of various factors; These factors are restricting the quality of prepared microsphere jointly; Therefore, through selecting very representative investigation index-envelop rate and drug loading, as the reference index of microspheres quality evaluation; Prepared PLGA microsphere is carried out influence factor's investigation, have very important significance.
1.PLGA kind is to the influence of microsphere envelop rate and drug loading
The condition of choosing ratio, colostrum and the PVA ratio of PLGA concentration, PVA concentration, medicine and PLGA solution and preparing colostrum is investigated the influence of the PLGA of three kinds of different proportion of composing to microsphere envelop rate and drug loading as fixed factor.Each factor and level are seen table 1.
Table 4-1 investigates each factor and the level of PLGA kind
Factor |
Level |
PLGA kind/LA: GA |
65∶35;75∶25;85∶15 |
PLGA concentration/mgmL
-1 |
30 |
PVA concentration/% |
2 |
The colostrum proportion of composing |
1∶7 |
The ratio of colostrum and PVA aqueous solution |
1∶10 |
The condition of preparation colostrum |
Ultrasonic emulsification 2min (ultrasonic 10s, 10s at interval) |
The PLGA kind is seen table 2, Fig. 1 to the investigation result of microspheres quality influence.
Table 2 PLGA kind is to the influence of microsphere envelop rate and drug loading
The PLGA proportion of composing |
Concentration/the mgmL of concentrated solution
-1 |
Envelop rate/% |
Drug loading/% |
Outward appearance |
75∶25 |
0.017 |
92.20 |
5.50 |
The stable homogeneous white emulsion has small amount of precipitate |
85∶15 |
0.029 |
86.09 |
5.06 |
Faint yellow inhomogeneous suspension has a large amount of depositions |
65∶35 |
0.02 |
90.45 |
5.22 |
Faint yellow than homogeneous latex emulsion, small amount of precipitate is arranged |
(poly (lactic-co-glycolic acid) PLGA) is a kind of degradable functional high molecule material to polylactic acid-glycolic guanidine-acetic acid copolymer, and is nontoxic because of having excellent biological compatibility, is widely used in preparing microball prepn.It is to be polymerized at random by two kinds of monomer-lactic acid and hydroxyacetic acid, and different monomer ratio can be prepared dissimilar PLGA, its physical state, and chemical property and palliating degradation degree be the difference along with the difference of compositing monomer ratio all.
This paper selects LA for use: GA=75: 25; LA: GA=85: 15 and LA: GA=65: the PLGA of 35 3 kinds of different proportion of composing is as research material; Envelop rate and drug loading through to its obtained microsphere are investigated; The proportion of composing of discovery PLGA has very big influence to the quality of microsphere, and along with the difference of ratio, the outward appearance of microsphere, drug loading and envelop rate all demonstrate bigger difference.Find as LA: GA=75 through experimentation: in the time of 25, the prepared microsphere of PLGA has higher entrapment and drug loading, and therefore, it is LA: GA=75 that the PLGA proportion of composing is selected in this experiment for use: 25 crystallization is as the host material of parcel microsphere.
2.PVA concentration is to the influence of microsphere envelop rate and drug loading
Ratio and the condition of preparation colostrum of choosing ratio, colostrum and the PVA aqueous solution of PLGA kind, PLGA concentration, medicine and PLGA solution are fixed factor, investigate the influence of the PVA of three kinds of variable concentrations to microsphere envelop rate and drug loading.Each factor and level are seen table 3.
Table 3 is investigated each factor and the level of PVA concentration
Factor |
Level |
PVA concentration/% |
1;2;3 |
PLGA kind/LA: GA |
75∶25 |
PLGA concentration/mgmL
-1 |
30 |
The colostrum proportion of composing |
1∶7 |
The ratio of colostrum and PVA aqueous solution |
1∶10 |
The condition of preparation colostrum |
Ultrasonic emulsification 2min (ultrasonic 10s, 10s at interval) |
PVA concentration is measured the result to the influence of microsphere envelop rate and drug loading and is seen table 4, Fig. 2.
Table 4 PVA concentration is to the influence of microsphere envelop rate and drug loading
PVA concentration/% |
Envelop rate/% |
Drug loading/% |
Outward appearance |
1 |
64.63 |
4.04 |
Yellow homogeneous emulsion |
2 |
92.20 |
5.50 |
White homogeneous emulsion has a small amount of white precipitate |
3 |
80.16 |
5.01 |
Faint yellow suspension has a large amount of white precipitates |
(polyvinyl alcohol is a kind of white or yellow-white powdery granule PVA) to polyvinyl alcohol, is the crystalline polymer material that is formed through alcoholysis by polyvinyl acetate, and is different with alcoholysis degree according to its degree of polymerization, and different character and specification are arranged.The PVA of domestic employing has specifications such as 05-88 and 17-88, and two kinds of filmogens all can be water-soluble.Because the polyvinyl alcohol non-activity does not decompose stable in properties; In digestive tract, be absorbed hardly after the oral administration, 80% polyvinyl alcohol was discharged with stool in 48 hours, therefore; Prepare in the process of microsphere at multi-emulsion method, usually be used as a kind of material relatively more commonly used and be applied to outer aqueous phase.
Outer water is important influence in the process for preparing microsphere with multi-emulsion method, and it is directly connected to the envelop rate and the drug loading of microsphere.This experiment adds the concentration of polyvinyl alcohol only continuous phase concentration is selected in the influence of microsphere envelop rate and drug loading through investigating.The concentration that Fig. 2 reflects PVA all has bigger influence to the envelop rate and the drug loading of microsphere.Through selected above three kinds of PVA concentration are compared discovery, when PVA concentration was 2%, the envelop rate and the drug loading of PLGA microsphere all peaked, and therefore, the PVA aqueous solution of selection 2% is as the outer water of preparation microsphere.
3. different colostrum proportion of composing are to the influence of microsphere envelop rate and drug loading
Choosing the ratio of PLGA kind, PLGA concentration, PVA concentration, colostrum and PVA aqueous solution and the condition of preparation colostrum is fixed factor, investigates the influence of different colostrum ratios to microsphere envelop rate and drug loading.Each factor and level are seen table 5.
Table 5 is investigated each factor and the level of colostrum ratio
Factor |
Level |
The colostrum ratio |
1∶5;1∶7;1∶10 |
PLGA kind/LA: GA |
75∶25 |
PLGA concentration/mgmL
-1 |
30 |
PVA concentration/% |
2 |
The ratio of colostrum and PVA aqueous solution |
1∶10 |
The condition of preparation colostrum |
Ultrasonic emulsification 2min (ultrasonic 10s, 10s at interval) |
Different colostrum proportion of composing are seen table 6 to the influence of microsphere envelop rate and drug loading, Fig. 3.
The different colostrum ratios of table 6 are to the influence of microsphere envelop rate and drug loading
The colostrum ratio |
Envelop rate/% |
Drug loading/% |
Outward appearance |
1∶5 |
60.09 |
5.01 |
Yellow emulsion has a large amount of depositions |
1∶7 |
92.95 |
5.81 |
White homogeneous emulsion has a small amount of white precipitate |
1∶10 |
79.52 |
3.30 |
Faint yellow heterogeneity suspension has deposition |
The amount of the medicine that the size of colostrum proportion of composing directly has influence on pharmaceutical carrier and comprised; Thereby can be directly reflected into the envelop rate of prepared microsphere and the size of drug loading is come up, can see that from Fig. 3 envelop rate and drug loading all demonstrate along with the increase of colostrum proportion of composing and increase earlier the trend that afterwards reduces; When the ratio of drug solution that forms colostrum and PLGA solution is 1: 7; Envelop rate and drug loading all reach maximum, are respectively 92.95%, 5.81%; Therefore, select 1: 7 as the ratio that forms colostrum.
4.PLGA concentration is to the influence of microsphere envelop rate and drug loading
With the ratio of PLGA kind, PVA concentration, colostrum proportion of composing, colostrum and PVA aqueous solution and the condition of preparation colostrum is fixed factor, investigates the influence of different PLGA concentration to microsphere envelop rate and drug loading.Each factor and level are seen table 7.
Table 7 is investigated each factor and the level of PLGA concentration
Factor |
Level |
PLGA concentration/mgmL
-1 |
10;30;50 |
PLGA kind/LA: GA |
75∶25 |
PVA concentration/% |
2 |
The colostrum proportion of composing |
1∶7 |
The ratio of colostrum and PVA aqueous solution |
1∶10 |
The condition of preparation colostrum |
Ultrasonic emulsification 2min (ultrasonic 10s, 10s at interval) |
PLGA concentration is seen table 8 to the influence of microsphere envelop rate and drug loading, Fig. 4.
Table 8 PLGA concentration is to the influence of microsphere envelop rate and drug loading
PLGA concentration/mgmL
-1 |
Envelop rate/% |
Drug loading/% |
Outward appearance |
10 |
41.70 |
7.45 |
White homogeneous emulsion |
30 |
92.20 |
5.50 |
White has a small amount of white precipitate than the homogeneous emulsion |
50 |
74.38 |
2.65 |
Faint yellow heterogeneity suspension |
Fig. 4 reflects that more intuitively PLGA concentration all has remarkable influence to the envelop rate and the drug loading of microsphere, and along with the increase of PLGA concentration, the drug loading of microsphere demonstrates falling tendency, takes the lead in reducing after the increase and seal, as PLGA concentration 30mgmL
-1The time, the envelop rate of microsphere reaches 92.20%, and its drug loading also reaches 5.50%, takes all factors into consideration its influence to envelop rate and drug loading, selects 30mgmL
-1PLGA is as the optimum concentration of this microsphere of preparation.
5. the colostrum method for preparing is to the influence of microsphere envelop rate, drug loading and outward appearance
As fixed factor, each factor and level are seen table 9 with the ratio of PLGA kind, PLGA concentration, PVA concentration, colostrum proportion of composing and colostrum and PVA aqueous solution.
Table 9 is investigated the factor and the level of colostrum method for preparing
Factor |
Level |
PLGA kind/LA: GA |
75∶25 |
PLGA concentration/mgmL
-1 |
30 |
PVA concentration/% |
2 |
The colostrum proportion of composing |
1∶7 |
The ratio of colostrum and PVA aqueous solution |
1∶10 |
Through above fixed factor is set, investigate of the influence of different colostrum method for preparinies to microsphere envelop rate, drug loading and outward appearance, colostrum method for preparing and condition are seen table 10.
Table 10 colostrum method for preparing and condition
The colostrum method for preparing |
Condition |
Ultrasonic |
2min (ultrasonic 10s, 10s at interval) |
High-speed stirred |
10000r·min
-1 |
The colostrum method for preparing is seen table 11 to the influence of microsphere envelop rate, drug loading and outward appearance.
Table 11 colostrum method for preparing is to the influence of microsphere envelop rate, drug loading and outward appearance
The colostrum method for preparing |
Envelop rate/% |
Drug loading/% |
Outward appearance |
Ultrasonic |
92.20 |
5.50 |
White has a small amount of white precipitate than the homogeneous emulsion |
High-speed stirred |
92.95 |
5.81 |
Clarification, homogeneous have a little fluorescence liquid |
The form of the prepared microsphere of different preparation demonstrates bigger difference, the bigger and big or small heterogeneity of ultrasonic method microspheres prepared particle diameter, and the visible larger particles of naked eyes demonstrates the suspension state, and through high speed homogenizer 10000rmin
-1Stir prepared microsphere and demonstrate more uniform pellucidity.Through the envelop rate and the drug loading of two kinds of obtained microspheres of method of comparison, the microsphere that discovery obtains through high-speed stirred, its envelop rate and drug loading all are higher than the envelop rate and the drug loading of the prepared microsphere of ultrasonic method.
Brief summary: comprehensive above analysis and investigation to each factor, select the prescription of optimum condition as the microsphere preparation: 30mgmL
-1PLGA (LA: GA=75: 25); Drug solution and PLGA solution proportion 1: 7; 10000rmin
-1High-speed stirred prepares colostrum; In the aqueous solution of dispersion and 2%PVA, magnetic agitation.
Two, the preparation of best prescription technology microsphere and quality analysis
Through single factor analysis, select 8 of the best, 9-single-epoxy brevifolin phenol microsphere preparation technology prepares 6 batches of microspheres, and preparation technology is following:
Precision is measured a certain amount of 12.5mgmL
-18,9-single-epoxy brevifolin phenol PBS solution, joining concentration according to 1: 7 ratio is 30mgmL
-1PLGA solution in, 10000rmin
-1High-speed stirred makes colostrum, this colostrum is joined in the 2%PVA aqueous solution of 10 times of amounts, and magnetic stirrer is used 1500rmin earlier
-1Stirred 3 hours fast, again magnetic stirring apparatus is transferred to 500rmin
-1, stirring at low speed 2 hours is treated dichloromethane (CH
2Cl
2) volatilize, can obtain solidified microsphere.6 batches of microspheres of preparation under the same condition.
Get according to optimised process make 8, each 1mL of 9-single-epoxy brevifolin phenol PLGA microsphere supernatant liquid filtering liquid joins the upper end of 0.5g sephadex column; Carry out eluting with tri-distilled water,, collect 5~10mL eluent through observing change in color in the gel column; Concentrate, sample introduction, with high effective liquid chromatography for measuring be not wrapped 8; 9-single-epoxy brevifolin phenol, the envelop rate of calculating microsphere.8, the computing formula of 9-single-epoxy brevifolin phenol PLGA microsphere envelop rate is following:
In the formula: M
1---the quality of the single-epoxy brevifolin phenol of adding
M
2---the quality of free single-epoxy brevifolin phenol
M
3---the quality of the PLGA of adding
Prepared 6 batches of microspheres demonstrate stable homogeneous than pellucidity, its envelop rate and drug loading are seen table 12.
Six batches 8 in table 12, the envelop rate and the drug loading of 9-single-epoxy brevifolin phenol PLGA microsphere
Batch |
Envelop rate/% |
Drug loading/% |
1 |
92.75 |
5.51 |
2 |
92.66 |
5.52 |
3 |
92.32 |
5.50 |
4 |
93.04 |
5.54 |
5 |
92.27 |
5.49 |
6 |
93.28 |
5.55 |
Average |
92.72 |
5.52 |
RSD/% |
0.43 |
0.42 |
To prepared 8,9-single-epoxy brevifolin phenol PLGA microsphere is measured its mean diameter through laser particle size analyzer.Particle size distribution is seen Fig. 5.
Six batches of microspheres that make according to optimised process all have higher drug loading and envelop rate, and microspherulite diameter is little and even, observe down in optical microscope to be complete sphere; Good reproducibility; Room temperature held seven days, the basic no change of color, a little white powder appears in the cillin bottle bottom.